Reactive sulfur species including sulfides, polysulfides and cysteine hydropersulfide play extensive roles in health and disease, which involve modification of protein functions through the interaction with metals bound to the proteins, cleavage of cysteine disulfide (S-S) bonds and S-persulfidation of cysteine residues. Sulfides over a wide micromolar concentration range enhance the activity of Cav3.2 T-type Ca2+ channels by eliminating Zn2+ bound to the channels, thereby promoting somatic and visceral pain. Cav3.2 is under inhibition by Zn2+ in physiological conditions, so that sulfides function to reboot Cav3.2 from Zn2+ inhibition and increase the excitability of nociceptors. On the other hand, polysulfides generated from sulfides activate TRPA1 channels via cysteine S-persulfidation, thereby facilitating somatic, but not visceral, pain. Thus, Cav3.2 function enhancement by sulfides and TRPA1 activation by polysulfides, synergistically accelerate somatic pain signals. The increased activity of the sulfide/Cav3.2 system, in particular, appears to have a great impact on pathological pain, and may thus serve as a therapeutic target for treatment of neuropathic and inflammatory pain including visceral pain.

Abstract Given possible involvement of the central and peripheral angiotensin system in pain processing, we conducted clinical and preclinical studies to test whether pharmacological inhibition of the angiotensin system would prevent diabetic peripheral neuropathy (DPN) accompanying type 2 diabetes mellitus (T2DM). In the preclinical study, the nociceptive sensitivity was determined in leptin-deficient ob/ob mice, a T2DM model. A clinical retrospective cohort study was conducted, using the medical records of T2DM patients receiving antihypertensives at three hospitals for nearly a decade. In the ob/ob mice, daily treatment with perindopril, an angiotensin-converting enzyme inhibitor (ACEI), or telmisartan, an angiotensin receptor blocker (ARB), but not amlodipine, an L-type calcium channel blocker (CaB), significantly inhibited DPN development without affecting the hyperglycemia. In the clinical study, the enrolled 7464 patients were divided into three groups receiving ACEIs, ARBs and the others (non-ACEI, non-ARB antihypertensives). Bonferroni’s test indicated significantly later DPN development in the ARB and ACEI groups than the others group. The multivariate Cox proportional analysis detected significant negative association of the prescription of ACEIs or ARBs and β-blockers, but not CaBs or diuretics, with DPN development. Thus, our study suggests that pharmacological inhibition of the angiotensin system is beneficial to prevent DPN accompanying T2DM.

Cav3.2, a T-type calcium channel (T-channel) family member, is expressed in the nociceptors and spinal cord, and its activity is largely suppressed by zinc under physiological conditions. In rats, intrathecal and intraplantar administration of a zinc chelator, TPEN, caused T-channel-dependent mechanical hyperalgesia, and the intraplantar, but not intrathecal, TPEN induced Cav3.2 upregulation in the dorsal root ganglion. In mice, intraplantar TPEN also caused mechanical allodynia, which was abolished by T-channel inhibitors or Cav3.2 gene deletion. Together, spinal and peripheral zinc deficiency appears to enhance Cav3.2 activity in the spinal postsynaptic neurons and nociceptors, respectively, thereby promoting pain.

Poly-trans-[ (2-carboxyethyl)germasesquioxane] (Ge-132), an organogermanium, is hydrolyzed to 3-(trihydroxygermyl)propanoic acid (THGP) in aqueous solutions, and reduces inflammation, pain and cancer, whereas the underlying mechanisms remain unknown. Sulfides including H2S, a gasotransmitter, generated from l-cysteine by some enzymes including cystathionine-γ-lyase (CSE), are pro-nociceptive, since they enhance Cav3.2 T-type Ca2+ channel activity expressed in the primary afferents, most probably by canceling the channel inhibition by Zn2+ linked via coordinate bonding to His191 of Cav3.2. Given that germanium is reactive to sulfur, we tested whether THGP would directly trap sulfide, and inhibit sulfide-induced enhancement of Cav3.2 activity and sulfide-dependent pain in mice. Using mass spectrometry and 1H NMR techniques, we demonstrated that THGP directly reacted with sulfides including Na2S and NaSH, and formed a sulfur-containing reaction product, which decreased in the presence of ZnCl2. In Cav3.2-transfected HEK293 cells, THGP inhibited the sulfide-induced enhancement of T-type Ca2+ channel-dependent membrane currents. In mice, THGP, administered systemically or locally, inhibited the mechanical allodynia caused by intraplantar Na2S. In the mice with cyclophosphamide-induced cystitis and cerulein-induced pancreatitis, which exhibited upregulation of CSE in the bladder and pancreas, respectively, systemic administration of THGP as well as a selective T-type Ca2+ channel inhibitor suppressed the cystitis-related and pancreatitis-related visceral pain. These data suggest that THGP traps sulfide and inhibits sulfide-induced enhancement of Cav3.2 activity, leading to suppression of Cav3.2-dependent pain caused by sulfide applied exogenously and generated endogenously.

Irregular regeneration or inappropriate remodeling of the axons of the primary afferent neurons after peripheral nerve trauma could be associated with the development of neuropathic pain. We analyzed the molecular mechanisms for the neuritogenesis and neurite outgrowth caused by prostaglandin E2 (PGE2) in mouse dorsal root ganglion (DRG) neurons, and evaluated their opioid modulation. PGE2 in combination with IBMX, a phosphodiesterase inhibitor, caused neuritogenesis/neurite outgrowth in DRG cells, an effect abolished by a prostanoid EP4, but not EP2, receptor antagonist, and inhibitors of adenylyl cyclase or protein kinase A (PKA). Blockers of T-type Ca2+ channels (T-channels), that are responsible for window currents involving the sustained low-level Ca2+ entry at voltages near the resting membrane potentials and can be functionally upregulated by PKA, inhibited the neuritogenesis/neurite outgrowth caused by PGE2/IBMX or dibutylyl cyclic AMP, a PKA activator, in DRG neurons, an inhibitory effect mimicked by ZnCl2 and ascorbic acid that block Cav3.2, but not Cav3.1 or Cav3.3, T-channels. Morphine and DAMGO, μ-opioid receptor (MOR) agonists, suppressed the neuritogenesis and/or neurite outgrowth induced by PGE2/IBMX in DRG neurons and also DRG neuron-like ND7/23 cells, an effect reversed by naloxone or β-funaltrexamine, a selective MOR antagonist. Our data suggest that the EP4 receptor/PKA/Cav3.2 pathway is involved in the PGE2-induced neuritogenesis/neurite outgrowth in DRG neurons, which can be suppressed by MOR stimulation. We propose that MOR agonists including morphine in the early phase after peripheral nerve trauma might delay the axonal regeneration of the primary afferent neurons but prevent the development of neuropathic pain.

Cav3.2 channels belong to the T-type calcium channel (T-channel) family, i.e., low voltage-activated calcium channels, and are abundantly expressed in the nociceptors, playing a principal role in the development of pathological pain. The channel activity of Cav3.2 is suppressed by zinc under physiological conditions. We thus tested whether dietary zinc deficiency would cause Cav3.2-dependent nociceptive hypersensitivity in mice. In the mice fed with zinc deficient diet for 2 weeks, plasma zinc levels declined by more than half, and mechanical allodynia developed. The dietary zinc deficiency-induced allodynia was restored by T-channel inhibitors or by Cav3.2 gene silencing. These data demonstrate that zinc deficiency induces Cav3.2-dependent nociceptive hypersensitivity in mice, thereby suggesting that pain experienced by patients with diseases accompanied by zinc deficiency (e.g., chronic kidney disease) might involve the increased Cav3.2 activity.

T-type Ca2+ channels (T-channels), particularly Cav3.2 and Cav3.1 isoforms, are promising targets for treating various diseases including intractable pain. Given the potent inhibitory activity of pimozide, an antipsychotic, against T-channels, we conducted structure-activity relationship studies of pimozide derivatives, and identified several compounds including 3a, 3s, and 4 that had potency comparable to that of pimozide in inhibiting T-channels, but little binding affinity to dopamine D2 receptors. The introduction of a phenylbutyl group on the benzoimidazole nuclei of pimozide was considered a key structural modification to reduce the binding affinity to D2 receptors. Those pimozide derivatives potently suppressed T-channel-dependent somatic and visceral pain in mice, without causing any motor dysfunctions attributable to D2 receptor blockade, including catalepsy. The present study thus provides an avenue to develop novel selective T-channel inhibitors available for pain management via the structural modification of existing medicines.

We examined the role of ATP and high mobility group box 1 (HMGB1) in paclitaxel-induced peripheral neuropathy (PIPN). PIPN in mice was prevented by HMGB1 neutralization, macrophage depletion, and P2X7 or P2X4 blockade. Paclitaxel and ATP synergistically released HMGB1 from macrophage-like RAW264.7 cells, but not neuron-like NG108-15 cells. The paclitaxel-induced HMGB1 release from RAW264.7 cells was accelerated by co-culture with NG108-15 cells in a manner dependent on P2X7 or P2X4. Paclitaxel released ATP from NG108-15 cells, but not RAW264.7 cells. Thus, PIPN is considered to involve acceleration of HMGB1 release from macrophages through P2X7 and P2X4 activation by neuron-derived ATP.

Given the role of macrophage-derived high mobility group box 1 (HMGB1) in chemotherapy-induced peripheral neuropathy (CIPN) caused by paclitaxel, we analyzed the role of HMGB1 and macrophages in the CIPN caused by bortezomib, a proteasome-inhibiting chemotherapeutic agent used for the treatment of multiple myeloma. Repeated administration of bortezomib caused CIPN accompanied by early-stage macrophage accumulation in the dorsal root ganglion. This CIPN was prevented by an anti-HMGB1-neutralizing antibody, thrombomodulin alfa capable of accelerating thrombin-dependent degradation of HMGB1, antagonists of the receptor for advanced glycation end-products (RAGE) and C-X-C motif chemokine receptor 4 (CXCR4), known as HMGB1-targeted membrane receptors, or macrophage depletion with liposomal clodronate, as reported in a CIPN model caused by paclitaxel. In macrophage-like RAW264.7 cells, bortezomib as well as MG132, a well-known proteasome inhibitor, caused HMGB1 release, an effect inhibited by caspase inhibitors but not inhibitors of NF-κB and p38 MAP kinase, known to mediate paclitaxel-induced HMGB1 release from macrophages. Bortezomib increased cleaved products of caspase-8 and caused nuclear fragmentation or condensation in macrophages. Repeated treatment with the caspase inhibitor prevented CIPN caused by bortezomib in mice. Our findings suggest that bortezomib causes caspase-dependent release of HMGB1 from macrophages, leading to the development of CIPN via activation of RAGE and CXCR4.

A neuroimmune crosstalk is involved in somatic and visceral pathological pain including inflammatory and neuropathic components. Apart from microglia essential for spinal and supraspinal pain processing, the interaction of bone marrow-derived infiltrating macrophages and/or tissue-resident macrophages with the primary afferent neurons regulates pain signals in the peripheral tissue. Recent studies have uncovered previously unknown characteristics of tissue-resident macrophages, such as their origins and association with regulation of pain signals. Peripheral nerve macrophages and intestinal resident macrophages, in addition to adult monocyte-derived infiltrating macrophages, secrete a variety of mediators, such as tumor necrosis factor-α, interleukin (IL)-1β, IL-6, high mobility group box 1 and bone morphogenic protein 2 (BMP2), that regulate the excitability of the primary afferents. Neuron-derived mediators including neuropeptides, ATP and macrophage-colony stimulating factor regulate the activity or polarization of diverse macrophages. Thus, macrophages have multitasks in homeostatic conditions and participate in somatic and visceral pathological pain by interacting with neurons.

We performed clinical retrospective study in female cancer patients and fundamental experiments in mice, in order to clarify risk factors for paclitaxel-induced peripheral neuropathy (PIPN). In the clinical study, 131 of 189 female outpatients with cancer undergoing paclitaxel-based chemotherapy met inclusion criteria. Breast cancer survivors (n = 40) showed significantly higher overall PIPN (grades 1-4) incidence than non-breast cancer survivors (n = 91). Multivariate sub-analyses of breast cancer survivors showed that 57 years of age or older and endocrine therapy before paclitaxel treatment were significantly associated with severe PIPN (grades 2-4). The age limit was also significantly correlated with overall development of severe PIPN. In the preclinical study, female mice subjected to ovariectomy received repeated administration of paclitaxel, and mechanical nociceptive threshold was assessed by von Frey test. Ovariectomy aggravated PIPN in the mice, an effect prevented by repeated treatment with 17β-estradiol. Repeated administration of thrombomodulin alfa (TMα), known to prevent chemotherapy-induced peripheral neuropathy in rats and mice, also prevented the development of PIPN in the ovariectomized mice. Collectively, breast cancer survivors, particularly with postmenopausal estrogen decline and/or undergoing endocrine therapy, are considered a PIPN-prone subpopulation, and may require non-hormonal pharmacological intervention for PIPN in which TMα may serve as a major candidate.

T-Type Ca2+ channels (T-channels), particularly Cav3.2, are now considered as therapeutic targets for treatment of intractable pain including visceral pain. Among existing medicines, bepridil, a multi-channel blocker, used for treatment of arrhythmia and angina, and pimozide, a dopamine D2 receptor antagonist, known as a typical antipsychotic, have potent T-channel blocking activity. We thus tested whether bepridil and pimozide could suppress visceral pain in mice. Colonic and bladder pain were induced by intracolonic administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) and systemic administration of cyclophosphamide (CPA), respectively. Referred hyperalgesia was assessed by von Frey test, and colonic hypersensitivity to distension by a volume load with intracolonic water injection and spontaneous bladder pain were evaluated by observing nociceptive behaviors in conscious mice. The mice exhibited referred hyperalgesia and colonic hypersensitivity to distension on day 6 after TNBS treatment. Systemic administration of bepridil at 10-20 mg/kg or pimozide at 0.1-0.5 mg/kg strongly reduced the referred hyperalgesia on the TNBS-induced referred hyperalgesia and colonic hypersensitivity to distension. CPA treatment caused bladder pain-like nociceptive behavior and referred hyperalgesia, which were reversed by bepridil at 10-20 mg/kg or pimozide at 0.5-1 mg/kg. Our data thus suggest that bepridil and pimozide, existing medicines capable of blocking T-channels, are useful for treatment of colonic and bladder pain, and serve as seeds for the development of new medicines for visceral pain treatment.

Chemotherapy-induced peripheral neuropathy (CIPN), one of major dose-limiting side effects of first-line chemotherapeutic agents such as paclitaxel, oxaliplatin, vincristine, and bortezomib is resistant to most of existing medicines. The molecular mechanisms of CIPN have not been fully understood. High mobility group box 1 (HMGB1), a nuclear protein, is a damage-associated molecular pattern protein now considered to function as a pro-nociceptive mediator once released to the extracellular space. Most interestingly, HMGB1 plays a key role in the development of CIPN. Soluble thrombomodulin (TMα), known to degrade HMGB1 in a thrombin-dependent manner, prevents CIPN in rodents treated with paclitaxel, oxaliplatin, or vincristine and in patients with colorectal cancer undergoing oxaliplatin-based chemotherapy. In this review, we describe the role of HMGB1 and its upstream/downstream mechanisms in the development of CIPN and show drug candidates that inhibit the HMGB1 pathway, possibly useful for prevention of CIPN.

Overexpression of Cav3.2 T-type Ca2+ channels in L4 dorsal root ganglion (DRG) participates in neuropathic pain after L5 spinal nerve cutting (L5SNC) in rats. The L5SNC-induced neuropathic pain also involves high mobility group box 1 (HMGB1), a damage-associated molecular pattern protein, and its target, the receptor for advanced glycation end-products (RAGE). We thus studied the molecular mechanisms for the L5SNC-induced Cav3.2 overexpression as well as neuropathic pain in rats by focusing on; 1) possible involvement of early growth response 1 (Egr-1), known to regulate transcriptional expression of Cav3.2, and ubiquitin-specific protease 5 (USP5) that protects Cav3.2 from proteasomal degradation, and 2) possible role of HMGB1/RAGE as an upstream signal. Protein levels of Cav3.2 as well as Egr-1 in L4 DRG significantly increased in the early (day 6) and persistent (day 14) phases of neuropathy after L5SNC, while USP5 protein in L4 DRG did not increase on day 6, but day 14. An anti-HMGB1-neutralizing antibody or a low molecular weight heparin, a RAGE antagonist, prevented the development of neuropathic pain and upregulation of Egr-1 and Cav3.2 in L4 DRG after L5SNC. L5SNC increased macrophages accumulating in the sciatic nerves, and the cytoplasm/nuclear ratio of immunoreactive HMGB1 in those macrophages. Our findings suggest that L5SNC-induced Cav3.2 overexpression in L4 DRG and neuropathic pain involves Egr-1 upregulation downstream of the macrophage-derived HMGB1/RAGE pathway, and that the delayed upregulation of USP5 might contribute to the persistent Cav3.2 overexpression and neuropathy.

Given the role of Cav3.2 isoform among T-type Ca2+ channels (T-channels) in somatic and visceral nociceptive processing, we analyzed the contribution of Cav3.2 to butyrate-induced colonic pain and nociceptor hypersensitivity in mice, to evaluate whether Cav3.2 could serve as a target for treatment of visceral pain in irritable bowel syndrome (IBS) patients. Mice of ddY strain, and wild-type and Cav3.2-knockout mice of a C57BL/6J background received intracolonic administration of butyrate twice a day for 3 days. Referred hyperalgesia in the lower abdomen was assessed by von Frey test, and colonic hypersensitivity to distension by a volume load or chemicals was evaluated by counting nociceptive behaviors. Spinal phosphorylated ERK was detected by immunohistochemistry. Cav3.2 knockdown was accomplished by intrathecal injection of antisense oligodeoxynucleotides. Butyrate treatment caused referred hyperalgesia and colonic hypersensitivity to distension in ddY mice, which was abolished by T-channel blockers and/or Cav3.2 knockdown. Butyrate also increased the number of spinal phosphorylated ERK-positive neurons following colonic distension in the anesthetized ddY mice. The butyrate-treated ddY mice also exhibited T-channel-dependent colonic hypersensitivity to intracolonic Na2S, known to enhance Cav3.2 activity, and TRPV1, TRPA1 or proteinase-activated receptor 2 (PAR2) agonists. Wild-type, but not Cav3.2-knockout, mice of a C57BL/6J background, after treated with butyrate, mimicked the T-channel-dependent referred hyperalgesia and colonic hypersensitivity in butyrate-treated ddY mice. Our study provides definitive evidence for an essential role of Cav3.2 in the butyrate-induced colonic pain and nociceptor hypersensitivity, which might serve as a target for treatment of visceral pain in IBS patients.

Cystitis-related bladder pain involves RAGE activation by HMGB1, and increased Cav3.2 T-type Ca2+ channel activity by H2S, generated by upregulated cystathionine-γ-lyase (CSE) in mice treated with cyclophosphamide (CPA). We, thus, investigated possible crosstalk between the HMGB1/RAGE and CSE/H2S/Cav3.2 pathways in the bladder pain development. Bladder pain (nociceptive behavior/referred hyperalgesia) and immuno-reactive CSE expression in the bladder were determined in CPA-treated female mice. Cell signaling was analyzed in urothelial T24 and macrophage-like RAW264.7 cells. The CPA-induced bladder pain was abolished by pharmacological inhibition of T-type Ca2+ channels or CSE, and genetic deletion of Cav3.2. The CPA-induced CSE upregulation, as well as bladder pain was prevented by HMGB1 inactivation, inhibition of HMGB1 release from macrophages, antagonists of RAGE or P2X4/P2X7 receptors, and N-acetylcysteine, an antioxidant. Acrolein, a metabolite of CPA, triggered ATP release from T24 cells. Adenosine triphosphate (ATP) stimulated cell migration via P2X7/P2X4, and caused HMGB1 release via P2X7 in RAW264.7 cells, which was dependent on p38MAPK/NF-κB signaling and reactive oxygen species (ROS) accumulation. Together, our data suggest that CPA, once metabolized to acrolein, causes urothelial ATP-mediated, redox-dependent HMGB1 release from macrophages, which in turn causes RAGE-mediated CSE upregulation and subsequent H2S-targeted Cav3.2-dependent nociceptor excitation, resulting in bladder pain.

HMGB1, a nuclear protein, once released to the extracellular space, promotes somatic and visceral pain signals. We thus analyzed the role of HMGB1 in an intravesical substance P-induced bladder pain syndrome (BPS) mouse model. Intravesical administration of substance P caused referred hyperalgesia/allodynia in the lower abdomen and hindpaw without producing severe urothelial damage, which was prevented by an anti-HMGB1-neutralizing antibody, thrombomodulin α capable of inactivating HMGB1 and antagonists of RAGE or CXCR4. The HMGB1 inactivation or RAGE blockade also reversed the established bladder pain symptoms. HMGB1 and RAGE are thus considered to serve as therapeutic targets for BPS.

High-mobility group box 1 (HMGB1), a nuclear protein, once released to the extracellular space, facilitates pain signals as well as inflammation. Intraplantar or intraspinal application of HMGB1 elicits hyperalgesia/allodynia in rodents by activating the advanced glycosylation end-product specific receptor (receptor for advanced glycation end-products; RAGE) or Toll-like receptor 4 (TLR4). Endogenous HMGB1 derived from neurons, perineuronal cells or immune cells accumulating in the dorsal root ganglion or sensory nerves participates in somatic and visceral pain consisting of neuropathic and/or inflammatory components. Endothelial thrombomodulin (TM) and recombinant human soluble TM, TMα, markedly increase thrombin-dependent degradation of HMGB1, and systemic administration of TMα prevents and reverses various HMGB1-dependent pathological pain. Low MW compounds that directly inactivate HMGB1 or antagonize HMGB1-targeted receptors would be useful to reduce various forms of intractable pain. Thus, HMGB1 and its receptors are considered to serve as promising targets in developing novel agents to prevent or treat pathological pain.

TRPV1 is phosphorylated and functionally upregulated by protein kinases, and negatively regulated by phosphatases including calcineurin. Since the clinical use of calcineurin-inhibiting immunosuppressants is commonly associated with chronic diarrhea, we examined if tacrolimus, a calcineurin inhibitor, promotes TRPV1-dependent colonic hypersensitivity in mice. Intracolonic administration of capsaicin, a TRPV1 agonist, caused referred hyperalgesia in the lower abdomen, an effect prevented by capsazepine, a TRPV1 blocker. Tacrolimus accelerated the intracolonic capsaicin-induced referred hyperalgesia. Similarly, intracolonic capsaicin caused spinal ERK phosphorylation, a marker for nociceptor excitation, an effect promoted by tacrolimus. Thus, tacrolimus may aggravate TRPV1-related colonic pain accompanying irritable bowel syndrome.

We tested whether genetic deletion of Cav3.2 T-type Ca2+ channels abolishes hydrogen sulfide (H2S)-mediated pain signals in mice. In Cav3.2-expressing HEK293 cells, Na2S, an H2S donor, at 100 μM clearly increased Ba2+ currents, as assessed by whole-cell patch-clamp recordings. In wild-type C57BL/6 mice, intraplantar and intracolonic administration of Na2S evoked mechanical allodynia and visceral nociceptive behavior, respectively, which were abolished by TTA-A2, a T-type Ca2+ channel blocker. In Cav3.2-knockout mice of a C57BL/6 background, Na2S caused neither somatic allodynia nor colonic nociception. Our study thus provides definitive evidence for an essential role of Cav3.2 in H2S-dependent somatic and colonic pain.

We tested whether NNC 55-0396 (NNC), a T-type calcium channel (T-channel) blocker, reduces the brain injury caused by middle cerebral artery occlusion and reperfusion (MCAO/R) in mice. NNC, administered i.c.v. before the occlusion, greatly reduced the MCAO/R-induced brain infarct and neurological dysfunctions, although it, given toward the end of occlusion, was less effective. Systemic administration of NNC before the occlusion also attenuated the infarct and neurological dysfunctions. Our data imply that blood-brain-barrier-permeable T-channel blockers such as NNC are capable of reducing MCAO/R-induced brain damage, and that T-channels are involved in neuronal damage induced by ischemia rather than reperfusion.

Bortezomib, a first-line agent for treatment of multiple myeloma, exhibits anticancer activity through proteasome inhibition. However, bortezomib-induced peripheral neuropathy (BIPN) is one of the most serious side effects. Since decreased proteasomal degradation of Cav3.2 T-type calcium channels in the primary afferents is involved in persistent pain, we investigated whether BIPN involves increased protein levels of Cav3.2 in mice. Six repeated i.p. administrations of bortezomib for 12 days developed persistent mechanical allodynia. Systemic administration of novel T-type calcium channel blockers, (2R/S)-6-prenylnaringenin and KTt-45, and of TTA-A2, the well-known blocker, reversed the BIPN. Ascorbic acid, known to block Cav3.2, but not Cav3.1 or 3.3, and silencing of Cav3.2 gene also suppressed BIPN. Protein levels of Cav3.2 in the dorsal root ganglion (DRG) at L4-L6 levels increased throughout days 1-21 after the onset of bortezomib treatment. Protein levels of USP5, a deubiquitinating enzyme that specifically inhibits proteasomal degradation of Cav3.2, increased in DRG on days 3-21, but not day 1, in bortezomib-treated mice. In DRG-derived ND7/23 cells, bortezomib increased protein levels of Cav3.2 and T-channel-dependent currents, as assessed by a patch-clamp method, but did not upregulate expression of Cav3.2 mRNA or USP5 protein. MG-132, another proteasome inhibitor, also increased Cav3.2 protein levels in the cultured cells. Given the previous evidence for USP5 induction following nociceptor excitation, our data suggest that BIPN involves the increased protein levels of Cav3.2 in nociceptors through inhibition of proteasomal degradation of Cav3.2 by bortezomib itself and then by USP5 that is upregulated probably in an activity-dependent manner.

BACKGROUND: Macrophage-derived high mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) protein, plays a key role in the development of chemotherapy-induced peripheral neuropathy (CIPN) caused by paclitaxel in rodents. Endothelial thrombomodulin (TM) promotes thrombin-induced degradation of HMGB1, and TMα, a recombinant human soluble TM, abolishes peripheral HMGB1-induced allodynia in mice. We thus examined whether HMGB1, particularly derived from macrophages, contributes to oxaliplatin-induced neuropathy in mice and analyzed the anti-neuropathic activity of the TM/thrombin system. METHODS: CIPN models were created by the administration of oxaliplatin in mice and rats, and the nociceptive threshold was assessed by von Frey test or paw pressure test. Macrophage-like RAW264.7 cells were stimulated with oxaliplatin in vitro. Proteins were detected and/or quantified by Western blotting, immunostaining, or enzyme-linked immunosorbent assay. RESULTS: Intraperitoneal administration of an anti-HMGB1-neutralizing antibody (AB) at 1 mg/kg prevented the oxaliplatin-induced allodynia in mice and rats. Antagonists of Toll-like receptor (TLR) 4, receptor for advanced glycation end products (RAGE) and CXCR4 among the HMGB1-targeted pro-nociceptive receptors, also mimicked the anti-neuropathic activity of AB in mice. Macrophage accumulation in the sciatic nerve was observed in mice treated with paclitaxel, but not oxaliplatin, and neither macrophage depletion nor inhibitors of macrophage activation affected oxaliplatin-induced allodynia. Oxaliplatin was 10- to 100-fold less potent than paclitaxel in releasing HMGB1 from macrophage-like RAW264.7 cells. Like AB, TMα at 10 mg/kg prevented the oxaliplatin-induced allodynia in mice as well as rats, an effect abolished by argatroban at 10 mg/kg, a thrombin inhibitor. The anti-neuropathic activity of TMα in oxaliplatin-treated mice was suppressed by oral anticoagulants such as warfarin at 1 mg/kg, dabigatran at 75 mg/kg, and rivaroxaban at 10 mg/kg, but not antiplatelet agents such as aspirin at 50 mg/kg and clopidogrel at 10 mg/kg. Repeated administration of the anticoagulants gradually developed neuropathic allodynia and elevated plasma HMGB1 levels in mice treated with a subeffective dose of oxaliplatin. CONCLUSIONS: Our data thus suggests a causative role of HMGB1 derived from non-macrophage cells in oxaliplatin-induced peripheral neuropathy and a thrombin-dependent anti-neuropathic activity of exogenous TMα and, most probably, endogenous TM.

Among voltage-gated Ca2+ channels, T-type Ca2+ channels, which are activated by low voltages, regulate neuronal excitability, spontaneous neurotransmitter release, hormone secretion, etc. and also participate in proliferation of distinct cancer cells. Among three isoforms of T-type Ca2+ channels, Cav3.2 is detectable in 100% of biopsy samples from prostate cancer patients. In general, prostate cancer cells are highly sensitive to androgen deprivation therapy, but often acquire hormone-therapy resistance. The androgen deprivation may trigger neuroendocrine (NE)-like differentiation of some prostate cancer cells. We have analyzed the expression and function of Cav3.2 in human prostate cancer LNCaP cells during NE-like differentiation. NE-like LNCaP cells overexpress Cav3.2 through the CREB/Egr-1 pathway and also cystathionine-γ-lyase (CSE), which generates H2S that enhances the channel activity of Cav3.2. H2S generated by upregulated CSE appears to enhance the activity of upregulated Cav3.2 after the differentiation. The enhanced Cav3.2 activity in NE-like cells may contribute to increased secretion of mitogenic factors essential for androgen-independent proliferation of surrounding prostate cancer cells. It is known that increased extracellular glucose levels enhance Cav3.2 activity through asparagine (N)-linked glycosylation of Cav3.2, which might contribute to diabetic neuropathy. We then found that high glucose accelerates the enhanced channel function and overexpression of Cav3.2 in NE-like LNCaP cells, which might be associated with clinical evidence for diabetes-related poor prognosis of prostate cancer and development of hormone therapy resistance. Thus, Cav3.2 is considered to play a role in the pathophysiology of prostate cancer, and may serve as a therapeutic target.

Since Cav3.2 T-type Ca2+ channels (T-channels) expressed in the primary afferents and CNS contribute to intractable pain, we explored T-channel-blocking components in distinct herbal extracts using a whole-cell patch-clamp technique in HEK293 cells stably expressing Cav3.2 or Cav3.1, and purified and identified sophoraflavanone G (SG) as an active compound from SOPHORAE RADIX (SR). Interestingly, hop-derived SG analogues, (2S)-6-prenylnaringenin (6-PNG) and (2S)-8-PNG, but not naringenin, also blocked T-channels; IC50 (μM) of SG, (2S)-6-PNG and (2S)-8-PNG was 0.68-0.75 for Cav3.2 and 0.99-1.41 for Cav3.1. (2S)-6-PNG and (2S)-8-PNG, but not SG, exhibited reversible inhibition. The racemic (2R/S)-6-PNG as well as (2S)-6-PNG potently blocked Cav3.2, but exhibited minor effect on high-voltage-activated Ca2+ channels and voltage-gated Na+ channels in differentiated NG108-15 cells. In mice, the mechanical allodynia following intraplantar (i.pl.) administration of an H2S donor was abolished by oral or i.p. SR extract and by i.pl. SG, (2S)-6-PNG or (2S)-8-PNG, but not naringenin. Intraperitoneal (2R/S)-6-PNG strongly suppressed visceral pain and spinal ERK phosphorylation following intracolonic administration of an H2S donor in mice. (2R/S)-6-PNG, administered i.pl. or i.p., suppressed the neuropathic allodynia induced by partial sciatic nerve ligation or oxaliplatin, an anti-cancer agent, in mice. (2R/S)-6-PNG had little or no effect on open-field behavior, motor performance or cardiovascular function in mice, and on the contractility of isolated rat aorta. (2R/S)-6-PNG, but not SG, was detectable in the brain after their i.p. administration in mice. Our data suggest that 6-PNG, a hop component, blocks T-channels, and alleviates neuropathic and visceral pain with little side effects.

High mobility group box 1 (HMGB1), a nuclear protein, once released into the extracellular space under pathological conditions, plays a pronociceptive role in redox-dependent distinct active forms, all-thiol HMGB1 (at-HMGB1) and disulfide HMGB1 (ds-HMGB1), that accelerate nociception through the receptor for advanced glycation endproducts (RAGE) and Toll-like receptor 4 (TLR4), respectively. Thrombomodulin (TM), an endothelial membrane protein, and soluble TM, known as TMα, promote thrombin-mediated activation of protein C and also sequester HMGB1, which might facilitate thrombin degradation of HMGB1. The present study aimed at clarifying the role of thrombin in TMα-induced suppression of peripheral HMGB1-dependent allodynia in mice. Thrombin-induced degradation of at-HMGB1 and ds-HMGB1 was accelerated by TMα in vitro. Intraplantar (i.pl.) injection of bovine thymus-derived HMGB1 in an unknown redox state, at-HMGB1, ds-HMGB1 or lipopolysaccharide (LPS), known to cause HMGB1 secretion, produced long-lasting mechanical allodynia in mice, as assessed by von Frey test. TMα, when preadministered i.pl., prevented the allodynia caused by bovine thymus-derived HMGB1, at-HMGB1, ds-HMGB1 or LPS, in a dose-dependent manner. The TMα-induced suppression of the allodynia following i.pl. at-HMGB1, ds-HMGB1 or LPS was abolished by systemic preadministration of argatroban, a thrombin-inhibiting agent, and accelerated by i.pl. co-administered thrombin. Our data clearly indicate that TMα is capable of promoting the thrombin-induced degradation of both at-HMGB1 and ds-HMGB1, and suppresses the allodynia caused by either HMGB1 in a thrombin-dependent manner. Considering the emerging role of HMGB1 in distinct pathological pain models, the present study suggests the therapeutic usefulness of TMα for treatment of intractable and/or persistent pain.

Hydrogen sulfide (H2S) formed by cystathionine-γ-lyase (CSE) enhances the activity of Cav3.2 T-type Ca2+ channels, contributing to the bladder pain accompanying hemorrhagic cystitis caused by systemic administration of cyclophosphamide (CPA) in mice. Given clinical and fundamental evidence for the involvement of the substance P/NK1 receptor systems in bladder pain syndrome (BPS)/interstitial cystitis (IC), we created an intravesical substance P-induced bladder pain model in mice and analyzed the possible involvement of the CSE/Cav3.2 pathway. Bladder pain/cystitis was induced by i.p. CPA or intravesical substance P in female mice. Bladder pain was evaluated by counting nociceptive behavior and by detecting referred hyperalgesia in the lower abdomen and hindpaw. The isolated bladder tissue was weighed to estimate bladder swelling and subjected to histological observation and Western blotting. Intravesical substance P caused profound referred hyperalgesia accompanied by little bladder swelling or edema 6-24 h after the administration, in contrast to i.p. CPA-induced nociceptive behavior/referred hyperalgesia with remarkable bladder swelling/edema and urothelial damage. The bladder pain and/or cystitis symptoms caused by substance P or CPA were prevented by the NK1 receptor antagonist. CSE in the bladder was upregulated by substance P or CPA, and the NK1 antagonist prevented the CPA-induced CSE upregulation. A CSE inhibitor, a T-type Ca2+ channel blocker and gene silencing of Cav3.2 abolished the intravesical substance P-induced referred hyperalgesia. The intravesical substance P-induced pain in mice is useful as a model for nonulcerative BPS, and involves the activation of the NK1 receptor/CSE/H2S/Cav3.2 cascade.

Hydrogen sulfide (H2 S) is generated from l-cysteine by multiple enzymes including cystathionine-γ-lyase (CSE), and promotes nociception by targeting multiple molecules such as Cav 3.2 T-type Ca2+ channels. Bladder pain accompanying cyclophosphamide (CPA)-induced cystitis in mice has been shown to involve the functional upregulation of the CSE/H2 S/Cav 3.2 pathway. Therefore, we investigated whether NF-κB, as an upstream signal of the CSE/H2 S system, contributes to bladder pain in mice with CPA-induced cystitis. Bladder pain-like nociceptive behaviour was observed in CPA-treated mice, and referred hyperalgesia was evaluated by the von Frey test. Isolated bladder weights were assessed to estimate bladder swelling, and protein levels were measured by Western blotting. CPA, administered intraperitoneally, induced nociceptive behaviour, referred hyperalgesia and increased bladder weights in mice. β-Cyano-l-alanine, a reversible selective CSE inhibitor, prevented CPA-induced nociceptive behaviour, referred hyperalgesia, and, in part, increases in bladder weight. CPA markedly increased phosphorylated NF-κB p65 levels in the bladder, an effect that was prevented by pyrrolidine dithiocarbamate (PDTC), an NF-κB inhibitor. PDTC and curcumin, which inhibits NF-κB signals, abolished CPA-induced nociceptive behaviour, referred hyperalgesia and, in part, increases in bladder weight. CPA caused the overexpression of CSE in the bladder, and this was prevented by PDTC or curcumin. The CPA-induced activation of NF-κB signals appeared to cause CSE overexpression in the bladder, contributing to bladder pain and in part swelling, possibly through H2 S/Cav 3.2 signaling. Therefore, NF-κB-inhibiting compounds including curcumin may be useful for the treatment of cystitis-related bladder pain.

Cav3.2 T-type Ca2+ channel activity is suppressed by zinc that binds to the extracellular histidine-191 of Cav3.2, and enhanced by H2S that interacts with zinc. Cav3.2 in nociceptors is upregulated in an activity-dependent manner. The enhanced Cav3.2 activity by H2S formed by the upregulated cystathionine-γ-lyase (CSE) is involved in the cyclophosphamide (CPA)-induced cystitis-related bladder pain in mice. We thus asked if zinc deficiency affects the cystitis-related bladder pain in mice by altering Cav3.2 function and/or expression. Dietary zinc deficiency for 2 weeks greatly decreased zinc concentrations in the plasma but not bladder tissue, and enhanced the bladder pain/referred hyperalgesia (BP/RH) following CPA at 200mg/kg, a subeffective dose, but not 400mg/kg, a maximal dose, an effect abolished by pharmacological blockade or gene silencing of Cav3.2. Acute zinc deficiency caused by systemic N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylendiamine (TPEN), a zinc chelator, mimicked the dietary zinc deficiency-induced Cav3.2-dependent promotion of BP/RH following CPA at 200mg/kg. CPA at 400mg/kg alone or TPEN plus CPA at 200mg/kg caused Cav3.2 overexpression accompanied by upregulation of Egr-1 and USP5, known to promote transcriptional expression and reduce proteasomal degradation of Cav3.2, respectively, in the dorsal root ganglia (DRG). The CSE inhibitor, β-cyano-l-alanine, prevented the BP/RH and upregulation of Cav3.2, Egr-1 and USP5 in DRG following TPEN plus CPA at 200mg/kg. Together, zinc deficiency promotes bladder pain accompanying CPA-induced cystitis by enhancing function and expression of Cav3.2 in nociceptors, suggesting a novel therapeutic avenue for treatment of bladder pain, such as zinc supplementation.

Thrombomodulin (TM), an endothelial protein with anti-coagulant activity, is composed of 5 domains, D1-D5. Recombinant human soluble TM (TMα) consisting of D1-D3, which is generated in CHO cells, suppresses inflammatory and nociceptive signals by inactivating high mobility group box 1 (HMGB1), one of damage-associated molecular patterns. TMα sequesters HMGB1 with the lectin-like D1 and promotes its degradation by thrombin binding to the EGF-like D2. We prepared TM's D123, D1 and D2 by the protein expression system of yeast, and evaluated their effects on HMGB1 degradation in vitro and on the allodynia caused by HMGB1 in distinct redox forms in mice in vivo. TMα and TM's D123, but not D1, promoted the thrombin-dependent degradation of all-thiol (at-HMGB1) and disulfide HMGB1 (ds-HMGB1), an effect mimicked by TM's D2, though to a lesser extent. Intraplantar administration of TMα and TM's D123, but not D1, D2 or D1 plus D2, strongly prevented the mechanical allodynia caused by intraplantar at-HMGB1, ds-HMGB1 or lipopolysaccharide in mice. Our data suggest that, apart from the role of D3, TMα and TM's D123 require both lectin-like D1 capable of sequestering HMGB1 and EGF-like D2 responsible for thrombin-dependent degradation of HMGB1, in abolishing the allodynia caused by exogenous or endogenous HMGB1.

We studied the pronociceptive role of proteinase-activated receptor-2 (PAR2) in mouse bladder. In female mice, intravesical infusion of the PAR2-activating peptide, SLIGRL-amide (SL), caused delayed mechanical hypersensitivity in the lower abdomen, namely 'referred hyperalgesia', 6-24 h after the administration. The PAR2-triggered referred hyperalgesia was prevented by indomethacin or a selective TRPV1 blocker, and restored by a T-type Ca2+ channel blocker. In human urothelial T24 cells, SL caused delayed prostaglandin E2 production and COX-2 upregulation. Our data suggest that luminal PAR2 stimulation in the bladder causes prostanoid-dependent referred hyperalgesia in mice, which involves the activation of TRPV1 and T-type Ca2+ channels.

Voltage-gated calcium channels (VGCCs) are classified into high-voltage-activated (HVA) channels and low-voltage-activated channels consisting of Cav3.1-3.3, known as T ("transient")-type VGCC. There is evidence that certain types of HVA channels are involved in neurogenic inflammation and inflammatory pain, in agreement with reports indicating the therapeutic effectiveness of gabapentinoids, ligands for the α2δ subunit of HVA, in treating not only neuropathic, but also inflammatory, pain. Among the Cav3 family members, Cav3.2 is abundantly expressed in the primary afferents, regulating both neuronal excitability at the peripheral terminals and spontaneous neurotransmitter release at the spinal terminals. The function and expression of Cav3.2 are modulated by a variety of inflammatory mediators including prostanoids and hydrogen sulfide (H2S), a gasotransmitter. The increased activity of Cav3.2 by H2S participates in colonic, bladder and pancreatic pain, and regulates visceral inflammation. Together, VGCCs are involved in inflammation and inflammatory pain, and Cav3.2 T-type VGCC is especially a promising therapeutic target for the treatment of visceral inflammatory pain in patients with irritable bowel syndrome, interstitial cystitis/bladder pain syndrome, pancreatitis, etc., in addition to neuropathic pain.

Transient receptor potential vanilloid-1 (TRPV1) expressed in nociceptors is directly phosphorylated and activated by protein kinase C, and involved in the signaling of pancreatic pain. On the other hand, Ca(v)3.2 T-type Ca2+ channels expressed in nociceptors are functionally upregulated by phosphorylation with protein kinase A and also play a role in pancreatitis-related pain. Calcineurin, a phosphatase, negatively regulates various channel functions including TRPV1, and calcineurin inhibitor-induced pain syndrome by tacrolimus, a calcineurin inhibitor, used as an immunosuppressant, has been a clinical problem. We thus examined the effect of tacrolimus on pancreatitis-related pain in mice. Repeated treatment with cerulein caused referred hyperalgesia accompanying acute pancreatitis, which was unaffected by tacrolimus. Pancreatitis-related symptoms disappeared in 24 h, whereas the referred hyperalgesia recurred following the administration of tacrolimus, which was abolished by the blockers of TRPV1 but not T-type Ca2+ channels. Thus, tacrolimus appears to cause the TRPV1-dependent relapse of pancreatitis-related pain, suggesting the involvement of calcineurin in the termination of pancreatic pain. (C) 2017 S. Karger AG, Basel

We examined the effect of repeated cold (RC) stress on cyclophosphamide (CPA)-induced cystitis/bladder pain in mice, in relation to macrophage activity. CPA, given i.p. at 400 mg/kg, caused bladder pain symptoms accompanying cystitis in both unstressed and RC-stressed mice, which were prevented by the macrophage inhibitor minocycline. A low dose, that is, 200 mg/kg, of CPA still produced bladder pain symptoms in unstressed but not RC-stressed mice. Lipopoly-saccharide-induced cytokine production in peritoneal macrophages from RC-stressed mice was less than that from unstressed mice. Thus, RC stress appears to reduce CPA-induced bladder pain in mice, which may be associated with the decreased macrophage activity. (C) 2017 S. Karger AG, Basel

Extracellular high mobility group box 1 (HMGB1) activates the receptor for advanced glycation end products (RAGE) or Toll-like receptor 4 (TLR4) and forms a heterocomplex with CXCL12 that strongly activates CXCR4, promoting inflammatory and pain signals. In the present study, we investigated the role of HMGB1 in pancreatic pain accompanying cerulein-induced acute pancreatitis in mice. Abdominal referred hyperalgesia accompanying acute pancreatitis occurred within 1 h after 6 hourly injections of cerulein. The anti-HMGB1 neutralizing antibody or recombinant human soluble thrombomodulin (rhsTM), known to inactivate HMGB1, abolished the cerulein-induced referred hyperalgesia, but not pancreatitis itself. Plasma or pancreatic HMGB1 levels did not change, but macrophage infiltration into the pancreas occurred 1 h after cerulein treatment. Minocycline, a macrophage/microglia inhibitor, ethyl pyruvate that inhibits HMGB1 release from macrophages, or liposomal clodronate that depletes macrophages prevented the referred hyperalgesia, but not pancreatitis. Antagonists of RAGE or CXCR4, but not TLR4, strongly suppressed the cerulein-induced referred hyperalgesia, but not pancreatitis. Upregulation of RAGE, CXCR4 and CXCL12, but not TLR4, were detected in the pancreas 1 h after cerulein treatment. Our data suggest that HMGB1 regionally secreted by macrophages mediates pancreatic pain by targeting RAGE and CXCL12/CXCR4 axis in the early stage of acute pancreatitis.

Given that Ca(v)3.2 T-type Ca2+ channels were functionally regulated by asparagine (N)-linked glycosylation, we examined effects of high glucose on the function of Ca(v)3.2, known to regulate secretory function, in neuroendocrine-like differentiated prostate cancer LNCaP cells. High glucose accelerated the increased channel function and overexpression of Ca(v)3.2 during neuroendocrine differentiation, the former prevented by enzymatic inhibition of N-glycosylation and cleavage of N-glycans. Hyperglycemia thus appears to induce N-linked glycosylation-mediated functional upregulation and overexpression of Ca(v)3.2 in neuroendocrine-like differentiated prostate cancer cells. (C) 2017 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Background: Hydrogen sulfide (H2S), a gasotransmitter, is generated from L-cysteine by mainly 3 enzymes, cystathionine-Upsilon-lyase (CSE), cystathionine-beta-synthase, and 3-mercaptopyruvate sulfurtransferase in cooperation with cysteine aminotransferase. The H2S-forming enzymes, particularly CSE, are overexpressed under the pathological conditions such as inflammation, neuronal or neuroendocrine differentiation and cancer development. Given that Ca(v)3.2 T-type Ca2+ channels mediate some of the biological activity of H2S, we focus on the role of the H2S/Ca(v)3.2 pathway in regulating the neuronal and neuroendocrine function. Summary: In the neuronal system, H2S regulates the activity of various ion channels including Ca(v)3.2. Exogenous and endogenous H2S enhances the Ca(v)3.2 channel activity, promoting somatic and visceral pain signaling. The H2S/Ca(v)3.2 pathway also facilitates neuritogenesis or neuronal differentiation. Interestingly, endogenous H2S formed by CSE regulates secretory function by enhancing Ca(v)3.2 channel activity in neuroendocrine-differentiated prostate cancer cells or carotid glomus cells. Key Messages: The H2S/Ca(v)3.2 pathway may serve as therapeutic targets for treatment of intractable pain, neuronal injury, androgen-independent prostate cancer, cardiovascular diseases, etc. (C) 2016 S. Karger AG, Basel

T-type Ca2+ channels (T channels), particularly Ca(v)3.2 among the 3 isoforms, play a role in neuropathic and visceral pain. We thus characterized the effects of RQ-00311651 (RQ), a novel T-channel blocker, in HEK293 cells transfected with human Ca(v)3.1 or Ca(v)3.2 by electrophysiological and fluorescent Ca2+ signaling assays, and also evaluated the antiallodynic/antihyperalgesic activity of RQ in somatic, visceral, and neuropathic pain models in rodents. RQ-00311651 strongly suppressed T currents when tested at holding potentials of -65 similar to - 260 mV, but not -80 mV, in the Ca(v)3.1- or Ca(v)3.2-expressing cells. RQ-00311651 also inhibited high K+-induced Ca2+ signaling in those cells. In mice, RQ, administered intraperitoneally (i.p.) at 5 to 20 mg/kg or orally at 20 to 40 mg/kg, significantly suppressed the somatic hyperalgesia and visceral pain-like nociceptive behavior/referred hyperalgesia caused by intraplantar and intracolonic administration of NaHS or Na2S, H2S donors, respectively, which involve the enhanced activity of Ca(v)3.2 channels. RQ-00311651, given i.p. at 5 to 20 mg/kg, exhibited antiallodynic or antihyperalgesic activity in rats with spinal nerve injury-induced neuropathy or in rats and mice with paclitaxel-induced neuropathy. Oral and i.p. RQ at 10 to 20 mg/kg also suppressed the visceral nociceptive behavior and/or referred hyperalgesia accompanying cerulein-induced acute pancreatitis and cyclophosphamide-induced cystitis in mice. The analgesic and antihyperalgesic/antiallodynic doses of oral and i.p. RQ did not significantly affect the locomotor activity and motor coordination. Together, RQ is considered a state-dependent blocker of Ca(v)3.1/Ca(v)3.2 T channels and may serve as an orally available analgesic for treatment of neuropathic and inflammatory pain including distinct visceral pain with minimum central side effects.

Given that high mobility group box 1 (HMGB1), a nuclear protein, once released to the extracellular space, promotes nociception, we asked if inactivation of HMGB1 prevents or reverses chemotherapy-induced painful neuropathy in rats and also examined possible involvement of Toll-like receptor 4 (TLR4) and the receptor for advanced glycation endproduct (RAGE), known as targets for HMGB1. Painful neuropathy was produced by repeated i.p. administration of paclitaxel or vincristine in rats. Nociceptive threshold was determined by the paw pressure method and/or von Frey test in the hindpaw. Tissue protein levels were determined by immunoblotting. Repeated i.p. administration of the anti-HMGB1-neutralizing antibody or recombinant human soluble thrombomodulin (rhsTM), known to inactivate HMGB1, prevented the development of hyperalgesia and/or allodynia induced by paclitaxel or vincristine in rats. A single i.p. or intraplantar (i.pl.) administration of the antibody or rhsTM reversed the chemotherapy induced neuropathy. A single i.pl. administration of a TLR4 antagonist or low molecular weight heparin, known to inhibit RAGE, attenuated the hyperalgesia caused by i.pl. HMGB1 and also the chemotherapy induced painful neuropathy. Paclitaxel or vincristine treatment significantly decreased protein levels of HMGB1 in the dorsal root ganglia, but not sciatic nerves. HMGB1 thus participates in both development and maintenance of chemotherapy-induced painful neuropathy, in part through RAGE and TLR4. HMGB1 inactivation is considered useful to prevent and treat the chemotherapy-induced painful neuropathy. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

Hydrogen sulfide (H2S), the third gasotransmitter, is endogenously generated by certain H2S synthesizing enzymes, including cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS) from L-cysteine in the mammalian body. Several studies have shown that endogenous and exogenous H2S affects the proliferation of cancer cells, although the effects of H2S appear to vary with cell type, being either promotive or suppressive. In the present study, we determined whether endogenously formed H2S regulates proliferation in human gastric cancer AGS cells. CSE, but not CBS, was expressed in AGS cells. CSE inhibitors, DL-propargylglycine (PPG) and beta-cyano-L-alanine (BCA), significantly suppressed the proliferation of AGS cells in a concentration-dependent manner. CSE inhibitors did not increase lactate dehydrogenase (LDH) release in the same concentration range. The inhibitory effects of PPG and BCA on cell proliferation were reversed by repetitive application of NaHS, a donor of H2S. Interestingly, nuclear condensation and fragmentation were detected in AGS cells treated with PPG or BCA. These results suggest that endogenous H2S produced by CSE may contribute to the proliferation of gastric cancer AGS cells, most probably through anti-apoptotic actions.

We investigated mechanisms for the neuritogenesis caused by prostaglandin E-2 (PGE(2)) or intracellular cyclic AMP (cAMP) in sensory neuron-like ND7/23 cells. PGE(2) caused neuritogenesis, an effect abolished by an EP4 receptor antagonist or inhibitors of adenylyl cyclase (AC) or protein kinase A (PKA) and mimicked by the AC activator forskolin, dibutyryl cAMP (db-cAMP), and selective activators of PKA or Epac. ND7/23 cells expressed both Ca(v)3.1 and Ca(v)3.2 T-type Ca2+ channels (T-channels). The neuritogenesis induced by db-cAMP or PGE(2) was abolished by T-channel blockers. T-channels were functionally upregulated by db-cAMP. The PGE(2)/EP4/cAMP/T-channel pathway thus appears to mediate neuritogenesis in sensory neurons. (C) 2016 Japanese Pharmacological Society. Production and hosting by Elsevier B.V.

Nuclear HMGB1 that contains 3 cysteine residues is acetylated and secreted to the extracellular space, promoting inflammation via multiple molecules such as RAGE and TLR4. We thus evaluated and characterized the redox state-dependent effects of peripheral HMGB1 on nociception. Intraplantar (i.pl.) administration of bovine thymus-derived HMGB1 (bt-HMGB1), all-thiol HMGB1 (at-HMGB1) or disulfide HMGB1 (ds-HMGB1) caused long-lasting mechanical hyperalgesia in mice. The hyperalgesia following i.pl. bt-HMGB1 or at-HMGB1 was attenuated by RAGE inhibitors, while the ds-HMGB1-induced hyperalgesia was abolished by a TLR4 antagonist. Thus, nociceptive processing by peripheral HMGB1 is considered dependent on its redox states. (C) 2016 Japanese Pharmacological Society. Production and hosting by Elsevier B.V.

We examined the effects of intraplantar (i.pl.) administration of NaHS, an H2S donor, known to cause T-type Ca2+ channel (T-channel)-dependent mechanical hyperalgesia, on responsiveness to electric stimulation with 5, 250 and 2000 Hz sine waves (SW) that selectively excites C, A delta and A beta fibers, respectively. NaHS, given i.pl., caused behavioral hypersensitivity to SW stimulation at 5 Hz, but not 250 or 2000 Hz, in rats. NaHS also enhanced phosphorylation of spinal ERK following 5 Hz SW stimulation. Three distinct T-channel blockers abolished the NaHS-induced behavioral hypersensitivity to 5 Hz SW stimulation. Thus, H2S selectively sensitizes C-fiber nociceptors via T-channels. (C) 2016 Japanese Pharmacological Society. Production and hosting by Elsevier B.V.

Neuroendocrine-differentiated prostate cancer cells may contribute to androgen-independent proliferation of surrounding cells through Ca2+-dependent secretion of mitogenic factors. Human prostate cancer LNCaP cells, when neuroendocrine-differentiated, overexpress Ca(v)3.2 T-type Ca2+ channels that contribute to Ca2+-dependent secretion. Given evidence for the acceleration of Ca(v)3.2 activity by hydrogen sulfide (H2S), we examined the roles of the H2S/Ca(v)3.2 pathway and then analyzed the molecular mechanisms of the Ca(v)3.2 overexpression in neuroendocrine-differentiated LNCaP cells. LNCaP cells were differentiated by dibutyryl cyclic AMP. Protein levels and T-type Ca2+ channeldependent currents (T-currents) were measured by immunoblotting and whole-cell pacth-clamp technique, respectively. Spontaneous release of prostatic acid phosphatase (PAP) was monitored to evaluate secretory function. The differentiated LNCaP cells exhibited neurite outgrowth, androgenindependent proliferation and upregulation of mitogenic factors, and also showed elevation of Ca(v)3.2 expression or T-currents. Expression of cystathionine-gamma-Iyase (CSE) and cystathionine-beta-synthase (CBS), H2S-forming enzymes, and spontaneous secretion of PAP increased following the differentiation. The augmented T-currents were enhanced by H2S donors and suppressed by inhibitors of CSE, but not CBS. The PAP secretion was reduced by inhibition of CSE or T-type Ca2+ channels. During differentiation, Egr-1 and REST, positive and negative transcriptional regulators for Ca(v)3.2, were upregulated and downregulated, respectively, and Egr-1 knockdown prevented the Ca(v)3.2 overexpression. Our data suggest that, in neuroendocrine-differentiated LNCaP cells, H2S formed by the upregulated CSE promotes the activity of the upregulated Ca(v)3.2, leading to the elevated secretory functions. The overexpression of Ca(v)3.2 appears to involve upregulation of Egr-1 and downregulation of REST. (C) 2015 Elsevier Inc. All rights reserved.

Hydrogen sulfide (H2S), formed by multiple enzymes, including cystathionine--lyase (CSE), targets Ca(v)3.2 T-type Ca2+ channels (T channels) and transient receptor potential ankyrin-1 (TRPA1), facilitating somatic pain. Pancreatitis-related pain also appears to involve activation of T channels by H2S formed by the upregulated CSE. Therefore, this study investigates the roles of the Ca(v)3.2 isoform and/or TRPA1 in pancreatic nociception in the absence and presence of pancreatitis. In anesthetized mice, AP18, a TRPA1 inhibitor, abolished the Fos expression in the spinal dorsal horn caused by injection of a TRPA1 agonist into the pancreatic duct. As did mibefradil, a T-channel inhibitor, in our previous report, AP18 prevented the Fos expression following ductal NaHS, an H2S donor. In the mice with cerulein-induced acute pancreatitis, the referred hyperalgesia was suppressed by NNC 55-0396 (NNC), a selective T-channel inhibitor; zinc chloride; or ascorbic acid, known to inhibit Ca(v)3.2 selectively among three T-channel isoforms; and knockdown of Ca(v)3.2. In contrast, AP18 and knockdown of TRPA1 had no significant effect on the cerulein-induced referred hyperalgesia, although they significantly potentiated the antihyperalgesic effect of NNC at a subeffective dose. TRPA1 but not Ca(v)3.2 in the dorsal root ganglia was downregulated at a protein level in mice with cerulein-induced pancreatitis. The data indicate that TRPA1 and Ca(v)3.2 mediate the exogenous H2S-induced pancreatic nociception in naive mice and suggest that, in the mice with pancreatitis, Ca(v)3.2 targeted by H2S primarily participates in the pancreatic pain, whereas TRPA1 is downregulated and plays a secondary role in pancreatic nociceptive signaling. (c) 2014 Wiley Periodicals, Inc.

Ca(v)3.2 T-type Ca2+ channels targeted by H2S, a gasotransmitter, participate in cyclophosphamide-induced cystitis and bladder pain. Given that zinc selectively inhibits Ca(v)3.2 among T-channel isoforms and also exhibits antioxidant activity, we examined whether polaprezinc (zinc-L-carnosine), a medicine for peptic ulcer treatment and zinc supplementation, reveals preventive or therapeutic effects on bladder inflammation and/or pain in the mouse with cyclophosphamide-induced cystitis, a model for interstitial cystitis. Systemic administration of cyclophosphamide caused cystitis-related symptoms including increased bladder weight and vascular permeability, and histological signs of bladder edema, accompanied by bladder pain-like nociceptive behavior/referred hyperalgesia. All these symptoms were significantly attenuated by oral preadministration of polaprezinc at 400 mg/kg. The same dose of polaprezinc also prevented the increased malondialdehyde level, an indicator of lipid peroxidation, and protein upregulation of cystathionine-gamma-Iyase. an H2S-generating enzyme, but not occludin, a tight junction-related membrane protein, in the bladder tissue of cyclophosphamide-treated mice. Oral posttreatment with polaprezinc at 30-100 mg/kg reversed the nociceptive behavior/referred hyperalgesia in a dose-dependent manner without affecting the increased bladder weight. Together, our data show that zinc supplementation with polaprezinc prevents the cyclophosphamide-induced cystitis probably through the antioxidant activity, and, like T-channel blockers, reverses the established cystitis-related bladder pain in mice, suggesting novel therapeutic usefulness of polaprezinc. (C) 2015 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

We analyzed signaling mechanisms for prostaglandin E-2 (PGE(2)) production following activation of proteinase-activated receptor-1 (PAR1), a thrombin receptor, in preosteoblastic MC3T3-E1 cells. PAR1 stimulation caused PGE(2) release, an effect suppressed by inhibitors ;of COX-1, COX-2, iPLA(2), cPLA(2), MAP kinases (MAPKs), Src, EGF receptor (EGFR) tyrosine kinase (EGFR-TK) and matrix metalloproteinase (MMP), but not by an intracellular Ca2+ chelator or inhibitors of PI3 kinase, protein kinase C (PKC) and NF-kappa B. PAR1 activation induced phosphorylation of MAPKs and upregulation of COX-2. The phosphorylation of p38 MAPK was suppressed by inhibitors of Src and EGFR-TK. The COX-2 upregulation was dependent on ERK, p38, EGFR-TK, Src, and COX-2 itself. PAR1 activation also induced MEK-dependent phosphorylation of cAMP response element binding protein (CREB). All inhibitors of EP1, EP2, EP3 and EP4 receptors suppressed the PAR1-triggered PGE(2) release. Exogenously applied PGE(2) facilitated PAR1-triggered COX-2 upregulation, but it alone had no effect. Together, the PAR1-mediated PGE(2) production in MC3T3-E1 cells appears to involve iPLA(2) and cPLA(2) for arachidonic acid release, and the MEK/ERK/CREB and Src/MMP/EGFR/p38 pathways for COX-2 upregulation, which is facilitated by endogenous PGE(2) formed by COX-2. These signaling mechanisms might underlie the role of the thrombin/PAR1/PGE(2) system in the early stage of the bone healing.

High mobility group box 1 (HMGB1), one of damage-associated molecular patterns (DAMPs), plays roles in not only inflammation but also processing of somatic pain. Given that no evidence for roles of HMGB1 in visceral pain signaling is available, we asked if HMGB1 participates in bladder pain accompanying cystitis caused by cyclophosphamide in mice, using the anti-HMGB1 neutralizing antibody and recombinant human soluble thrombomodulin (rhsTM) that sequesters HMGB1 and promotes its degradation by thrombin. Cyclophosphamide, administered i.p., caused bladder pain-like nociceptive behavior and referred hyperalgesia accompanying cystitis symptoms including increased bladder weight, an indicator of edema, in mice. The cyclophosphamide-induced bladder pain and referred hyperalgesia, but not increased bladder weight, were prevented by i.p. preadministration of the anti-HMGB1 neutralizing antibody or rhsTM. HMGB1, given i.p., facilitated the bladder pain and referred hyperalgesia caused by a subeffective dose of cyclophosphamide, an effect blocked by rhsTM. In the cyclophosphamide-treated mice, HMGB1 levels greatly decreased in the bladder tissue, particularly in the urothelial cells, but did not change in the plasma. Low molecular weight heparin, known to inhibit the receptor for advanced glycation end products (RAGE), but not lipopolysaccharide from Rhodobacter sphaeroides, an inhibitor of toll-like receptor 4 (TLR4), blocked the cyclophosphamide-induced bladder pain and referred hyperalgesia. Thus, our data indicate involvement of HMGB1 in the cyclophosphamide-induced bladder pain signaling, but not cystitis itself, and suggest that targeting HMGB1 with rhsTM or blocking RAGE might serve as a novel therapeutic strategy for the management of bladder pain. (C) 2013 Elsevier Ltd. All rights reserved.

Hydrogen sulfide (H2S), a gasotransmitter, is formed from L-cysteine by multiple enzymes including cystathionine-gamma-lyase (CSE). We have shown that an H2S donor, NaHS, causes hyperalgesia in rodents, an effect inhibited by knockdown of Ca(v)3.2 T-type Ca2+ channels (T-channels), and that NaHS facilitates T-channel-dependent currents (T-currents) in NG108-15 cells that naturally express Ca(v)3.2. In the present study, we asked if endogenous and exogenous H2S participates in regulation of the channel functions in Ca(v)3.2-transfected HEK293 (Ca(v)3.2-HEK293) cells. DL-Propargylglycine (PPG), a CSE inhibitor, significantly decreased T-currents in Ca(v)3.2-HEK293 cells, but not in NG108-15 cells. NaHS at 1.5 mM did not affect T-currents in Ca(v)3.2-HEK293 cells, but enhanced T-currents in NG108-15 cells. In the presence of PPG, NaHS at 1.5 mM, but not 0.1-0.3 mM, increased T-currents in Ca(v)3.2-HEK293 cells. Similarly, Na2S, another H2S donor, at 0.1-0.3 mM significantly increased T-currents in the presence, but not absence, of PPG in Ca(v)3.2-HEK293 cells. Expression of CSE was detected at protein and mRNA levels in HEK293 cells. Intraplantar administration of Na2S, like NaHS, caused mechanical hyperalgesia, an effect blocked by NNC 55-0396, a T-channel inhibitor. The in vivo potency of Na2S was higher than NaHS. These results suggest that the function of Ca(v)3.2 T-channels is tonically enhanced by endogenous H2S synthesized by CSE in Ca(v)3.2-HEK293 cells, and that exogenous H2S is capable of enhancing Ca(v)3.2 function when endogenous H2S production by CSE is inhibited. In addition, Na2S is considered a more potent H2S donor than NaHS in vitro as well as in vivo. (C) 2014 Elsevier Inc. All rights reserved.

We examined if TRPA1, like TRPV1, contributes to pancreatic nociceptor excitation following proteinase-activated receptor-2 (PAR2) stimulation and to pancreatitis-related pain in mice. A PAR2-activating peptide, infused into the pancreatic duct, caused spinal Fos expression, which was prevented by AP18, a TRPA1 inhibitor. Repeated administration of cerulein caused referred hyperalgesia accompanying pancreatitis, which was reversed by SB366791, a TRPV1 inhibitor, but not AP18. AP18, administered in combination with a subeffective dose of SB366791, significantly suppressed the referred hyperalgesia. Our findings suggest that TRPA1, like TRPV1, mediates PAR2-triggered pancreatic nociception and that TRPA1 in collaboration with TRPV1 latently contributes to pancreatitis-related pain.

Background and Purpose High-mobility group box 1 (HMGB1), a nuclear protein, is actively or passively released during inflammation. Recombinant human soluble thrombomodulin (rhsTM), a medicine for treatment of disseminated intravascular coagulation (DIC), sequesters HMGB1 and promotes its degradation. Given evidence for involvement of HMGB1 in pain signalling, we determined if peripheral HMGB1 causes hyperalgesia, and then asked if rhsTM modulates the HMGB1-dependent hyperalgesia. Experimental Approach Mechanical nociceptive threshold and swelling in rat hindpaw were determined by the paw pressure test and by measuring paw thickness, respectively, and HMGB1 levels in rat hindpaw plantar tissue, dorsal root ganglion (DRG) and serum were determined by Western blotting or elisa. Key Results Intraplantar (i.pl.) administration of HMGB1 rapidly evoked paw swelling and gradually caused hyperalgesia in rats. Systemic administration of rhsTM abolished HMGB1-induced hyperalgesia, and partially blocked paw swelling. LPS, administered i.pl., rapidly produced mild paw swelling, and gradually caused hyperalgesia. The anti-HMGB1 neutralizing antibody abolished LPS-induced hyperalgesia, but partially inhibited paw swelling. rhsTM at a high dose, 10 mg kg-1, prevented both hyperalgesia and paw swelling caused by LPS. In contrast, rhsTM at low doses, 0.001-1 mg kg-1, abolished the LPS-induced hyperalgesia, but not paw swelling. HMGB1 levels greatly decreased in the hindpaw, but not DRG. Serum HMGB1 tended to increase after i.pl. LPS in rats pretreated with vehicle, but not rhsTM. Conclusion and Implications These data suggest that peripheral HMGB1 causes hyperalgesia, and that rhsTM abolishes HMGB1-dependent hyperalgesia, providing novel evidence for therapeutic usefulness of rhsTM as an analgesic. © 2013 The British Pharmacological Society.

Low-voltage-activated T-type Ca2+ channels (T-channels), especially Ca(v)3.2 among the three isoforms (Ca(v)3.1, Ca(v)3.2, and Ca(v)3.3), are now considered to play pivotal roles in processing of pain signals. Ca(v)3.2 T-channels are functionally modulated by extracellular substances such as hydrogen sulfide and ascorbic acid, by intracellular signaling molecules including protein kinases, and by glycosylation. Ca(v)3.2 T-channels are abundantly expressed in both peripheral and central endings of the primary afferent neurons, regulating neuronal excitability and release of excitatory neurotransmitters such as substance P and glutamate, respectively. Functional upregulation of Ca(v)3.2 T-channels is involved in the pathophysiology of inflammatory, neuropathic, and visceral pain. Thus, Ca(v)3.2 T-channels are considered to serve as novel targets for development of drugs for treatment of intractable pain resistant to currently available analgesics.

Hydrogen sulfide (H2S), a gasotransmitter, plays a variety of roles in the mammalian body including the cardiovascular system. Given evidence that H2S donors including NaHS inhibit human platelet aggregation, we examined and characterized the effects of NaFIS on rabbit platelet aggregation and cytosolic Ca2+ mobilization. Rabbit platelet aggregation was determined in platelet-rich plasma (PRP) and washed platelets. Intracellular Ca2+ levels were monitored in Fura2-loaded washed platelets. NaHS prevented rabbit platelet aggregation induced by collagen or ADP, and the effective concentration range of NaHS was 0.1-0.3 mM in PRP and 1-3 mM in washed platelets. In washed platelets, NaHS attenuated cytosolic Ca2+ mobilization induced by collagen or ADP and also reduced platelet aggregation induced by ionomycin, a Ca2+ ionophore. The antiplatelet effect of NaHS was blocked by an adenylyl cyclase inhibitor and enhanced by a phosphodiesterase inhibitor. H2S thus suppresses rabbit platelet aggregation by interfering with both upstream and downstream signals of cytosolic Ca2+ mobilization in a cAMP-dependent manner.

Background and Purpose The Cav3.2 isoform of T-type Ca2+ channels (T channels) is sensitized by hydrogen sulfide, a pro-nociceptive gasotransmitter, and also by PKA that mediates PGE2-induced hyperalgesia. Here we examined and analysed Cav3.2 sensitization via the PGE2/cAMP pathway in NG108-15 cells that express Cav3.2 and produce cAMP in response to PGE2, and its impact on mechanical nociceptive processing in rats. Experimental Approach In NG108-15 cells and rat dorsal root ganglion (DRG) neurons, T-channel-dependent currents (T currents) were measured with the whole-cell patch-clamp technique. The molecular interaction of Cav3.2 with A-kinase anchoring protein 150 (AKAP150) and its phosphorylation were analysed by immunoprecipitation/immunoblotting in NG108-15 cells. Mechanical nociceptive threshold was determined by the paw pressure test in rats. Key Results In NG108-15 cells and/or rat DRG neurons, dibutyryl cAMP (db-cAMP) or PGE2 increased T currents, an effect blocked by AKAP St-Ht31 inhibitor peptide (AKAPI) or KT5720, a PKA inhibitor. The effect of PGE2 was abolished by RQ-00015986-00, an EP4 receptor antagonist. AKAP150 was co-immunoprecipitated with Cav3.2, regardless of stimulation with db-cAMP, and Cav3.2 was phosphorylated by db-cAMP or PGE2. In rats, intraplantar (i.pl.) administration of db-cAMP or PGE2 caused mechanical hyperalgesia, an effect suppressed by AKAPI, two distinct T-channel blockers, NNC 55-0396 and ethosuximide, or ZnCl2, known to inhibit Cav3.2 among T channels. Oral administration of RQ-00015986-00 suppressed the PGE2-induced mechanical hyperalgesia. Conclusion and Implications Our findings suggest that PGE2 causes AKAP-dependent phosphorylation and sensitization of Cav3.2 through the EP4 receptor/cAMP/PKA pathway, leading to mechanical hyperalgesia in rats.

Background and Purpose The Cav3.2 isoform of T-type Ca2+ channels (T channels) is sensitized by hydrogen sulfide, a pro-nociceptive gasotransmitter, and also by PKA that mediates PGE2-induced hyperalgesia. Here we examined and analysed Cav3.2 sensitization via the PGE2/cAMP pathway in NG108-15 cells that express Cav3.2 and produce cAMP in response to PGE2, and its impact on mechanical nociceptive processing in rats. Experimental Approach In NG108-15 cells and rat dorsal root ganglion (DRG) neurons, T-channel-dependent currents (T currents) were measured with the whole-cell patch-clamp technique. The molecular interaction of Cav3.2 with A-kinase anchoring protein 150 (AKAP150) and its phosphorylation were analysed by immunoprecipitation/immunoblotting in NG108-15 cells. Mechanical nociceptive threshold was determined by the paw pressure test in rats. Key Results In NG108-15 cells and/or rat DRG neurons, dibutyryl cAMP (db-cAMP) or PGE2 increased T currents, an effect blocked by AKAP St-Ht31 inhibitor peptide (AKAPI) or KT5720, a PKA inhibitor. The effect of PGE2 was abolished by RQ-00015986-00, an EP4 receptor antagonist. AKAP150 was co-immunoprecipitated with Cav3.2, regardless of stimulation with db-cAMP, and Cav3.2 was phosphorylated by db-cAMP or PGE2. In rats, intraplantar (i.pl.) administration of db-cAMP or PGE2 caused mechanical hyperalgesia, an effect suppressed by AKAPI, two distinct T-channel blockers, NNC 55-0396 and ethosuximide, or ZnCl2, known to inhibit Cav3.2 among T channels. Oral administration of RQ-00015986-00 suppressed the PGE2-induced mechanical hyperalgesia. Conclusion and Implications Our findings suggest that PGE2 causes AKAP-dependent phosphorylation and sensitization of Cav3.2 through the EP4 receptor/cAMP/PKA pathway, leading to mechanical hyperalgesia in rats.

We evaluated the effect of buprenorphine, a mixed agonist for μ-opioid receptors and nociceptin/orphanin FQ peptide (NOP) receptors, in neuropathic rats, using the paw pressure test. Buprenorphine, administered i.p. at 50, but not 20, μg/kg, exhibited naloxone-reversible analgesic activity in naïve rats. In contrast, buprenorphine at 0.5-20 μg/kg produced a naloxone-sensitive antihyperalgesic effect in the L5 spinal nerve-injured neuropathic rats. Intrathecal injection of [N-Phe1]nociceptin(1-13) NH2, a NOP-receptor antagonist, reversed the effect of buprenorphine in neuropathic rats, but not in naïve rats. Together, buprenorphine suppresses neuropathic hyperalgesia by activating NOP and opioid receptors, suggesting its therapeutic usefulness in treatment of neuropathic pain. © The Japanese Pharmacological Society.

BACKGROUND AND PURPOSE Hydrogen sulfide (H2S), generated by enzymes such as cystathionine-?-lyase (CSE) from L-cysteine, facilitates pain signals by activating the Cav3.2 T-type Ca2+ channels. Here, we assessed the involvement of the CSE/H2S/Cav3.2 pathway in cystitis-related bladder pain. EXPERIMENTAL APPROACH Cystitis was induced by i.p. administration of cyclophosphamide in mice. Bladder pain-like nociceptive behaviour was observed and referred hyperalgesia was evaluated using von Frey filaments. Phosphorylation of ERK in the spinal dorsal horn was determined immunohistochemically following intravesical administration of NaHS, an H2S donor. KEY RESULTS Cyclophosphamide caused cystitis-related symptoms including increased bladder weight, accompanied by nociceptive changes (bladder pain-like nociceptive behaviour and referred hyperalgesia). Pretreatment with DL-propargylglycine, an inhibitor of CSE, abolished the nociceptive changes and partly prevented the increased bladder weight. CSE protein in the bladder was markedly up-regulated during development of cystitis. Mibefradil or NNC 550396, blockers of T-type Ca2+ channels, administered after the symptoms of cystitis appeared, reversed the nociceptive changes. Further, silencing of Cav3.2 protein by repeated intrathecal administration of mouse Cav3.2-targeting antisense oligodeoxynucleotides also significantly attenuated the nociceptive changes, but not the increased bladder weight. Finally, the number of cells staining positive for phospho-ERK was increased in the superficial layer of the L6 spinal cord after intravesical administration of NaHS, an effect inhibited by NNC 550396. CONCLUSION AND IMPLICATIONS Endogenous H2S, generated by up-regulated CSE, caused bladder pain and referred hyperalgesia through the activation of Cav3.2 channels, one of the T-type Ca2+ channels, in mice with cyclophosphamide-induced cystitis.

BACKGROUND AND PURPOSE Hydrogen sulfide, a gasotransmitter, facilitates somatic pain signals via activation of Cav3.2 T-type calcium channels in rats. Given evidence for the activation of transient receptor potential ankyrin-1 (TRPA1) channels by H2S, we asked whether TRPA1 channels, in addition to Cav3.2 channels, contribute to the H2S-induced mechanical hyperalgesia and allodynia in mice. EXPERIMENTAL APPROACH Mechanical hyperalgesia and allodynia were evaluated by the von Frey test in mice. Cav3.2 or TRPA1 channels in the sensory neurons were silenced by repeated intrathecal administration of antisense oligodeoxynucleotides in mice. KEY RESULTS Intraplantar administration of NaHS evoked hyperalgesia and allodynia in mice, an effect attenuated or abolished by NNC 550396 or mibefradil, T-type calcium channel blockers, and by ascorbic acid or zinc chloride, known to selectively inhibit Cav3.2 channels, out of the three isoforms of T-type calcium channels. Silencing of Cav3.2 channels in the sensory neurons also prevented the NaHS-induced hyperalgesia and allodynia in mice. The NaHS-induced hyperalgesia and allodynia in mice were significantly suppressed by AP18, a TRPA1 channel blocker, and by silencing of TRPA1 channels in the sensory neurons. CONCLUSIONS AND IMPLICATIONS Mechanical hyperalgesia and allodynia induced by NaHS/H2S required activation of both Cav3.2 and TRPA1 channels in mice.

Luminal hydrogen sulfide (H2S), a gasotransmitter, causes colonic pain / referred hyperalgesia in mice, most probably via activation of T-type Ca2+ channels. Here we analyzed the mechanisms for H2S-induced facilitation of colonic pain signals. Intracolonic administration of NaHS, an H2S donor, evoked visceral pain-like nociceptive behavior and referred hyperalgesia in mice, an effect abolished by NNC 55-0396, a selective T-type Ca2+-channel blocker, or by knockdown of Ca(v)3.2. AP18, a TRPA1 blocker, also prevented the NaHS-induced colonic pain and referred hyperalgesia. These findings demonstrate that H2S-induced colonic pain and referred hyperalgesia require activation of both Ca(v)3.2 and TRPA1 channels in mice.

To clarify the relationship between Helicobacter pylori (H. pylori), a risk factor for gastritis, peptic ulcer and gastric cancer, and proteinase-activated receptors (PARs) that contribute to inflammatory responses, we determined and characterized the biological activity of H. pylori components in the mammalian cells that express PARs. The activity of H. pylori extracts was assessed in distinct cell lines with high expression of PAR1 (RGM1 cells), PAR2 (A549 cells), or PAR2 and PAR4 (HCT-15 cells). A PAR1-activating peptide (AP), but not H. pylori extracts, caused prostaglandin E-2 (PGE(2)) release in RGM1 cells. On the other hand, H. pylori extracts produced release of PGE2 and interleukin-8 (IL-8) in A549 and HCT-15 cells, respectively, as a PAR2-AP did. The activity of H. pylori extracts in A549 cells was not affected by a proteinase inhibitor or exposure to boiling, but abolished by inhibitors of lipopolysaccharide (LPS), IRAK-1/4 or NF-kappa B. The activity of H. pylori extracts in HCT-15 cells was partially suppressed by boiling or the proteinase inhibitor. In rat platelets that express PAR4 and PAR3, like a PAR4-AP, H. pylori extracts induced aggregation when assessed in platelet rich plasma, an effect unaffected by the proteinase inhibitor, but did not cause aggregation of washed rat platelets that responded to the PAR4-AP or thrombin. The present study thus shows the biological activities of H. pylori extracts in A549 and HCT-15 cells or rat platelets, and suggests that they are not mediated by any PAR-activating proteinases, but may involve the other pathogenic factors including LPS.

Hydrogen sulfide (H(2)S), a gasotransmitter, exerts both neurotoxicity and neuroprotection, and targets multiple molecules including NMDA receptors, T-type calcium channels and NO synthase (NOS) that might affect neuronal viability. Here, we determined and characterized effects of NaHS, an H(2)S donor, on cell viability in the primary cultures of mouse fetal cortical neurons. NaHS caused neuronal death, as assessed by LDH release and trypan blue staining, but did not significantly reduce the glutamate toxicity. The neurotoxicity of NaHS was resistant to inhibitors of NMDA receptors, T-type calcium channels and NOS, and was blocked by inhibitors of MEK, but not JNK, p38 MAP kinase, PKC and Src. NaHS caused prompt phosphorylation of ERK and upregulation of Bad, followed by translocation of Bax to mitochondria and release of mitochondrial cytochrome c(1) leading to the nuclear condensation/fragmentation. These effects of NaHS were suppressed by the MEK inhibitor. Our data suggest that the NMDA receptor-independent neurotoxicity of H(2)S involves activation of the MEK/ERK pathway and some apoptotic mechanisms. (C) 2011 Elsevier Inc. All rights reserved.

Hydrogen sulfide (H,S), a gasotransmitter, facilitates pain sensation by targeting Ca(v)3.2 T-type calcium channels. The H(2)S/Ca(v)3.2 pathway appears to play a role in the maintenance of surgically evoked neuropathic pain. Given evidence that chemotherapy-induced neuropathic pain is blocked by ethosuximide, known to block T-type calcium channels, we examined if more selective T-type calcium channel blockers and also inhibitors of cystathionine-gamma-lyase (CSE), a major H(2)S-forming enzyme in the peripheral tissue, are capable of reversing the neuropathic pain evoked by paclitaxel, an anti-cancer drug. It was first demonstrated that T-type calcium channel blockers, NNC 55-0396, known to inhibit Ca(v)3.1, and mibefradil inhibited T-type currents in Ca(v)3.2-transfected HEK293 cells. Repeated systemic administration of paclitaxel caused delayed development of mechanical hyperalgesia, which was reversed by single intraplantar administration of NNC 550396 or mibefradil, and by silencing of Ca(v)3.2 by antisense oligodeoxynucleotides. Systemic administration of DL-propargylglycine and p-cyanoalanine, irreversible and reversible inhibitors of CSE, respectively, also abolished the established neuropathic hyperalgesia. In the paclitaxel-treated rats, upregulation of Ca(v)3.2 and CSE at protein levels was not detected in the dorsal root ganglia (DRG), spinal cord or peripheral tissues including the hindpaws, whereas H(2)S content in hindpaw tissues was significantly elevated. Together, our study demonstrates the effectiveness of NNC 55-0396 in inhibiting Ca(v)3.2, and then suggests that paclitaxel-evoked neuropathic pain might involve the enhanced activity of T-type calcium channels and/or CSE in rats, but not upregulation of Ca(v)3.2 and CSE at protein levels, differing from the previous evidence for the neuropathic pain model induced by spinal nerve cutting in which Ca(v)3.2 was dramatically upregulated in DRG. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Proteinase-activated receptor-1 (PAR I), upon activation, exerts prostanoid-dependent gastroprotection, and increases prostaglandin E-2 (PGE(2)) release through cyclooxygenase-2 (COX-2) upregulation in rat gastric mucosal epithelial RGM1 cells. However, there is a big time lag between the PAR I-triggered PG E2 release and COX-2 upregulation in RGM1 cells; that is, the former event takes 18 h to occur, while the latter rapidly develops and reaches a plateau in 6 h. The present study thus aimed at clarifying mechanisms for the delay of PGE2 release after PAR activation in RGM1 cells. Although a PAR1-activating peptide, TFLLR-NH2, alone caused PGE2 release at 18 h, but not 6 h, TFLLR-NH2 in combination with arachidonic acid dramatically enhanced PGE(2) release even for 1-6 h. TFLLR-NH2 plus linoleic acid caused a similar rapid response. CP-24879, a Delta(5)/Delta(6)-desaturase inhibitor, abolished the PGE(2) release induced by TFLLR-NH2 plus linoleic acid, but not by TFLLR-NH2 alone. The TFLLR-NH2-induced PGE2 release was not affected by inhibitors of cytosolic phospholipase A(2) (cPLA(2)), Ca2+-independent PLA(2) (cPLA2) or secretory PLA(2) (sPLA(2)), but was abolished by their mixture or a pan-PLA(2) inhibitor. Among PLA2 isozymes, mRNA of group IIA sPLA(2) (sPLA(2)-IIA) was upregulated following PAR1 stimulation for 6-18 h, whereas protein levels of PGE synthases were unchanged. These data suggest that the delay of PG E2 release after COX-2 upregulation triggered by PAR1 is due to the poor supply of free arachidonic acid at the early stage in RGM1 cells, and that plural isozymes of PLA2 including sPLA2-IIA may complementarily contribute to the liberation of free arachidonic acid. J. Cell. Biochem. 112: 909-915,2011. (C) 2010 Wiley-Liss, Inc.

Aims: Proteinase-activated receptor-2 (PAR2) and transient receptor potential vanilloid-1 (TRPV1) are co-localized in the primary afferents, and the trans-activation of TRPV1 by PAR2 activation is involved in processing of somatic pain. Given evidence for contribution of PAR2 to pancreatic pain, the present study aimed at clarifying the involvement of TRPV1 in processing of pancreatic pain by the proteinase/PAR2 pathway in mice. Main methods: Acute pancreatitis was created by repeated administration of cerulein in conscious mice, and the referred allodynia/hyperalgesia was assessed using von Frey filaments. Injection of PAR2 agonises into the pancreatic duct was achieved in anesthetized mice, and expression of Fos in the spinal cord was determined by immunohistochemistry. Key findings: The established referred allodynia/hyperalgesia following cerulein treatment was abolished by post-treatment with nafamostat mesilate, a proteinase inhibitor, and with capsazepine, a TRPV1 antagonist, in mice. Injection of trypsin, an endogenous PAR2 agonist, or SLIGRL-NH(2), a PAR2-activating peptide, into the pancreatic duct caused expression of Fos protein in the spinal superficial layers at T8-T10 levels in the mice. The spinal Fos expression caused by trypsin and by SLIGRL-NH(2) was partially blocked by capsazepine, the former effect abolished by nafamostat mesilate. Significance: Our data thus suggest that the proteinase/PAR2/TRPV1 cascade might impact pancreatic pain, in addition to somatic pain, and play a role in the maintenance of pancreatitis-related pain in mice. (C) 2010 Elsevier Inc. All rights reserved.

ヒト肺上皮癌細胞においてクルクミンはCOX-2誘導とAkt依存性NF-κB活性化を抑制することによりPAR2誘起PGE2産生を阻害する

We performed this study to determine if curcumin affects pro-inflammatory responses to activation of proteinase-activated receptor-2 (PAR2) in human pulmonary adenocarcinoma A549 cells. Curcumin completely inhibited the PAR2-triggered prostaglandin E-2 (PGE(2)) production, but notably not interleukin-8 release. Cyclooxygenase-2 (COX-2) upregulation, but not its upstream activation of mitogen-activated protein kinases, caused by PAR2 stimulation was partially inhibited by curcumin. Curcumin inhibited the PAR2-triggered phosphorylation of I-kappa B, an indicator for nuclear factor-kappa B (NF-kappa B) activation, and also its upstream signal Akt, which is known to contribute to PAR2-triggered PGE2 formation, but not COX-2 upregulation. Collectively, curcumin inhibits the PAR2-triggered PGE2 production by suppressing COX-2 upregulation and Akt/NF-kappa B signals in A549 cells.

P>Hydrogen sulfide (H(2)S), a gasotransmitter, induces neuronal differentiation characterized by neuritogenesis and functional up-regulation of high voltage-activated Ca2+ channels, via activation of T-type Ca2+ channels in NG108-15 cells. We thus analyzed signaling mechanisms for the H(2)S-evoked neuronal differentiation. NaHS, a donor for H(2)S, facilitated T-type Ca2+ channel-dependent membrane currents, an effect blocked by ascorbic acid that selectively inhibits Ca(v)3.2 among three T-type channel isoforms. NaHS, applied once at a high concentration (13.5 mM) or repetitively at a relatively low concentration (1.5 mM), as well as ionomycin, a Ca2+ ionophore, evoked neuritogenesis. The neuritogenesis induced by NaHS, but not by ionomycin, was abolished by mibefradil, a T-type Ca2+ channel blocker. PP2, a Src kinase inhibitor, completely suppressed the neuritogenesis caused by NaHS or ionomycin, while it only partially blocked neuritogenesis caused by dibutyryl cAMP, a differentiation inducer. NaHS, but not dibutyryl cAMP, actually caused phosphorylation of Src, an effect blocked by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl, an intracellular Ca2+ chelator, mibefradil or ascorbic acid. The up-regulation of high voltage-activated currents in the cells treated with NaHS was also inhibited by PP2. Together, our data reveal that Src kinase participates in the T-type Ca2+ channel-dependent neuronal differentiation caused by NaHS/H(2)S in NG108-15 cells.

Hydrogen sulfide (H2S) formed from L-cysteine by multiple enzymes including cystathionine-gamma-lyase (CSE) is now considered a gasotransmitter in the mammalian body. Our previous studies have shown that H2S activates/sensitizes Ca(v)3.2 T-type Ca2+ channels, leading to facilitation of somatic and visceral nociception, and that CSE-derived endogenous H2S participates in inflammatory pain. Here, we show novel evidence for involvement of the endogenous H2S-Ca(v)3.2 pathway in neuropathic pain. In the rat subjected to the right L5 spinal nerve cutting (L5SNC), a neuropathic pain model, i.p. administration of DL-propargylglycine (PPG) and beta-cyanoalanine, irreversible and reversible CSE inhibitors, respectively, strongly suppressed the neuropathic hyperalgesia/allodynia. The anti-hyperalgesic effect of PPG was reversed by intraplantar administration of NaHS, a donor for H2S, in the L5SNC rat. Intraplantar administration or topical application of mibefradil, a T-type Ca2+ channel blocker, reversed hyperalgesia in the L5SNC rat. The protein levels of Ca(v)3.2, but not CSE, in the ipsilateral L4, L5 and L6 dorsal root ganglia were dramatically upregulated in the L5SNC rat. Finally, silencing of Ca(v)3.2 in DRG by repeated intrathecal administration of Ca(v)3.2-targeting siRNA significantly attenuated the neuropathic hyperalgesia in the L5SNC rat. In conclusion, our data suggest that Ca(v)3.2 T-type Ca2+ channels in sensory neurons are upregulated and activated/sensitized by CSE-derived endogenous H2S after spinal nerve injury, contributing to the maintenance of neuropathic pain. We thus propose that Ca(v)3.2 and CSE could be targets for the development of therapeutic drugs for the treatment of neuropathic pain. (C) 2010 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Thiazolidinediones, known as peroxisome proliferator-activated receptor-gamma (PPAR gamma) agonists, may modify prostaglandin formation and exert gastroprotective effects. Since activation of proteinase-activated receptor-1 (PAR1) reveals endogenous prostanoid-dependent gastroprotection, we investigated if two thiazolidinediones, ciglitazone and troglitazone, modulate the prostaglandin E-2 (PGE(2)) release caused by activation of PAR1 in normal rat gastric mucosal epithelial RGM1 cells. Ciglitazone dramatically facilitated the PAR1-triggered PGE(2) production and cyclooxygenase-2 (COX-2) upregulation, although it had no effect by itself. In contrast, troglitazone suppressed the PARI-triggered PGE2 production and COX-2 upregulation. Either effect of ciglitazone and troglitazone was resistant to GW9662, a PPAR gamma antagonist. The facilitation of the PGE(2) release by ciglitazone was blocked by inhibitors of MEK, p38 MAP kinase (p38MAPK) and PI3-kinase (PI3K), but not JNK. Nonetheless, ciglitazone failed to enhance the PAR1-triggered phosphorylation of ERK and p38MAPK. In conclusion, ciglitazone and troglitazone, exert opposite effects on the PARI-triggered PGE(2) production and COX-2 upregulation by targeting molecules other than PPAR gamma. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Proteinase-activated receptor-2 (PAR2) triggers upregulation of cyclooxygenase-2 (COX-2) and prostaglandin E-2 (PGE(2)) formation in human alveolar epithelial A549 cells. This COX-2 upregulation appears to involve the Src/epidermal growth factor (EGF) receptor/p38 MAP kinase (p38MAPK) pathway and also the cAMP-response element-binding protein (CREB) pathway. Here, we investigated the roles of nuclear factor-kappa B (NF-kappa B)-related signals in the PAR2-triggered PGE(2) release/COX-2 upregulation in A549 cells. The PAR2-triggered PGE2 release was clearly blocked by an inhibitor of the NF-kappa B pathway. Stimulation of PAR2 actually caused phosphorylation of inhibitor-kappa B, an indicator of NF-kappa B activation, an effect being blocked by inhibitors of MEK, phosphatidylinositol 3-kinase (PI3-kinase), and Akt, but little or not by inhibitors of p38MAPK and JNK. Stimulation of PAR2 also caused phosphorylation of Akt, an effect suppressed by inhibitors of PI3-kinase and MEK. Nonetheless, the PAR2-triggered upregulation of COX-2 was resistant to inhibitors of NF-kappa B, PI3-kinase, and Akt, but was attenuated by inhibitors of MEK and JNK. Stimulation of PAR2 induced phosphorylation of CREB, an effect abolished by an inhibitor of MEK but not inhibitors of p38MAPK and EGF receptor. These findings demonstrate that the MEK / ERK / PI3-kinase / Akt / NF-kappa B pathway is involved in PAR2-triggered PGE2 formation, but not upregulation of COX-2 that is dependent on activation of ERK/CREB and JNK in addition to p38MAPK.

Proteinase-activated receptor-2 (PAR2) triggers upregulation of cyclooxygenase-2 (COX-2) and prostaglandin E-2 (PGE(2)) formation in human alveolar epithelial A549 cells. This COX-2 upregulation appears to involve the Src/epidermal growth factor (EGF) receptor/p38 MAP kinase (p38MAPK) pathway and also the cAMP-response element-binding protein (CREB) pathway. Here, we investigated the roles of nuclear factor-kappa B (NF-kappa B)-related signals in the PAR2-triggered PGE(2) release/COX-2 upregulation in A549 cells. The PAR2-triggered PGE2 release was clearly blocked by an inhibitor of the NF-kappa B pathway. Stimulation of PAR2 actually caused phosphorylation of inhibitor-kappa B, an indicator of NF-kappa B activation, an effect being blocked by inhibitors of MEK, phosphatidylinositol 3-kinase (PI3-kinase), and Akt, but little or not by inhibitors of p38MAPK and JNK. Stimulation of PAR2 also caused phosphorylation of Akt, an effect suppressed by inhibitors of PI3-kinase and MEK. Nonetheless, the PAR2-triggered upregulation of COX-2 was resistant to inhibitors of NF-kappa B, PI3-kinase, and Akt, but was attenuated by inhibitors of MEK and JNK. Stimulation of PAR2 induced phosphorylation of CREB, an effect abolished by an inhibitor of MEK but not inhibitors of p38MAPK and EGF receptor. These findings demonstrate that the MEK / ERK / PI3-kinase / Akt / NF-kappa B pathway is involved in PAR2-triggered PGE2 formation, but not upregulation of COX-2 that is dependent on activation of ERK/CREB and JNK in addition to p38MAPK.

Clinical studies suggest that colonic luminal hydrogen sulfide (H(2)S), produced by sulfate-reducing bacteria or through other pathways, might be involved in the pathogenesis of inflammatory bowel disease (IBD). Nonetheless, this hypothesis has been poorly investigated by basic studies using laboratory animals. We thus focused on two enzymes, cystathionine-gamma-lyase (CSE) that generates H(2)S from L-cysteine, and rhodanese that directly or indirectly detoxifies H(2)S, particularly in relation to the colitis induced by dextran sulfate sodium (DSS) in mice. CSE was a major H(2)S-forming enzyme in colonic and renal homogenates from mice and rats, and the rhodanese activity was also detectable in both tissues. Colitis-related symptoms including decreased body weight gain, diarrhea, hematochezia and shortening of colon length were observed in the mice drinking DSS. Those symptoms were not or only slightly attenuated by repeated administration of a CSE inhibitor. CSE activity and protein levels in the colonic tissue did not notably change in the mice with colitis. In contrast, the activity and protein/mRNA levels of rhodanese in the colon, but not kidney, significantly decreased nearly in parallel with the development of colitis, followed by elevation of rhodanese activity in red blood cells (RBCs). These data show that rhodanese, but not CSE, is associated with DSS-induced colitis in mice, leading to a hypothesis that impaired detoxification of H(2)S due to down-regulation or suppression of colonic rhodanese is involved in IBD. The delayed enhancement of rhodanese activity in RBCs, a possible compensative event, might be available as a disease marker for IBD. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Objective: Hydrogen sulfide (H(2)S) is formed from L-cysteine by multiple enzymes including cystathionine-gamma-lyase (CSE) in mammals, and plays various roles in health and disease. Recently, a pronociceptive role for H(2)S in the processing of somatic pain was identified. Here, the involvement of H(2)S in pancreatic pain is examined. Methods: Anaesthetised rats or mice received an injection of NaHS, a donor for H(2)S, or capsaicin into the pancreatic duct, and the expression of spinal Fos protein was detected by immunohistochemistry. Pancreatitis was created by 6 hourly doses of caerulein in unanaesthetised mice, and pancreatitis-related allodynia/hyperalgesia was evaluated using von Frey hairs. CSE activity and protein levels in pancreatic tissues were measured using the colorimetric method and western blotting, respectively. Results: Either NaHS or capsaicin induced the expression of Fos protein in the superficial layers of the T8 and T9 spinal dorsal horn of rats or mice. The induction of Fos by NaHS but not capsaicin was abolished by mibefradil, a T-type Ca(2+) channel blocker. In conscious mice, repeated doses of caerulein produced pancreatitis accompanied by abdominal allodynia/hyperalgesia. Pretreatment with an inhibitor of CSE prevented the allodynia/hyperalgesia, but not the pancreatitis. A single dose of mibefradil reversed the established pancreatitis-related allodynia/hyperalgesia. Either the activity or protein expression of pancreatic CSE increased after the development of caerulein-induced pancreatitis in mice. Conclusions: The data suggest that pancreatic NaHS/H(2)S most probably targets T-type Ca(2+) channels, leading to nociception, and that endogenous H(2)S produced by CSE and possibly T-type Ca(2+) channels are involved in pancreatitis-related pain.

Hydrogen sulfide (H2S), a gasotransmitter, facilitates membrane currents through T-type Ca2+ channels, and intraplantar (i.pl.) administration of NaHS, a donor of H2S, causes prompt hyperalgesia in rats. In this. context, we asked whether intrathecal (i.t.) administration of NaHS could mimic the hyperalgesic effect of i.pl. NaHS in rats, and then examined if Ca(v)3.2 isoform of T-type Ca2+ channels contributed to the pronociceptive effects of i.t. and i.pl. NaHS. Either i.t. or i.pl. administration of NaHS rapidly decreased nociceptive threshold in rats, as determined by the paw pressure method. The hyperalgesia caused by i.t. and i.pl. NaHS was abolished by co-administration of mibefradil, a pan-T-type Ca2+ channel inhibitor, and also Suppressed by pretreatment with i.t. and i.pl, zinc chloride, known to preferentially inhibit Ca(v)3.2 among T-type Ca2+ channel isoforms, respectively. Repeated i.t. administration of antisense oligodeoxynucleotides (ODNs) targeting rat Cav3.2, but not mismatch ODNs, caused silencing of Ca(v)3.2 protein in the dorsal root ganglia and spinal cord, and then attenuated the hyperalgesia induced by either i.t. or i.pl, NaHS. Our findings thus establish that spinal and peripheral NaHS/H2S activates or sensitizes Ca(v)3.2 T-type Ca2+ channels expressed in the primary afferents and/or spinal nociceptive neurons, leading to sensitization of nociceptive processing and hyperalgesia. (C) 2008 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

We investigated if stimulation of T-type Ca2+ channels with sodium hydrosulfide (NaHS), a donor of hydrogen sulfide (H2S), could cause neuronal differentiation of NG108-15 cells. Like dibutyryl cyclic AMP (db-cAMP), treatment with NaHS at 1.5-13.5 mM for 16 h enhanced neurite outgrowth in a concentration-dependent manner. Synergistic neuritogenic effect was obtained in the cells stimulated with NaHS in combination with db-cAMP at subeffective concentrations. Exposure to NaHS or db-cAMP for 2 days resulted in enhancement of expression of high-voltage-activated currents consisting of N-, P/Q-, L- and also other types, but not of T-type currents. Mibefradil, a pan-T-type channel blocker, abolished the neuritogenesis induced by NaHS, but not by db-cAMP. The NaHS-evoked neuritogenesis was also completely blocked by pretreatment with BAPTA/AM, a chelator of intracellular Ca2+, and by zinc chloride at a concentration known to selectively inhibit Ca(v)3.2 isoform of T-type Ca2+ channels, but not Ca(v)3.1 or Ca(v)3.3. Further, l-ascorbate, recently proven to selectively inhibit Ca(v)3.2, abolished the neuritogenic effect of NaHS, but not db-cAMP. Our data thus demonstrate that NaHS/H2S is a novel inducer of neuronal differentiation in NG108-15 cells, as characterized by neuritogenesis and expression of high-voltage-activated currents, and suggest the involvement of T-type Ca2+ channels, especially Ca(v)3.2.

Proteinase-activated receptor-2 (PAR2) plays pro-inflammatory roles in many organs including the gastrointestinal (GI) tract. To clarify the downstream pro-inflammatory signaling of PAR2 in the GI tract, we examined interleukin-8 (IL-8) release and the underlying cellular signaling following PAR2 stimulation in human colorectal cancer-derived HCT-15 cells and human gastric adenocarcinoma-derived MKN-45 cells. A PAR2-activating peptide, but not a PAR2-inactive scrambled peptide or a PAR1 - activating peptide, caused IL-8 elease in these GI epithelial cells. The PAR2-triggered IL-8 release was suppressed by inhibitors of MEK (U0126) or PI3-kinase (LY294002), and PAR2 stimulation indeed activated the downstream kinases, ERK and Akt. U0126 blocked the phosphorylation of ERK, but not Akt, and LY294002 blocked the phosphorylation of Akt, but not ERK. Together, PAR2 triggers IL-8 release via two independent signaling pathways, MEK/ERK and PI3-kinase/Akt, suggesting a role of PAR2 as a pro-inflammatory receptor in the GI tract. (C) 2008 Elsevier Inc. All rights reserved.

It has been almost a decade since the molecular cloning of all four members of the proteinase-activated receptor ( PAR) family was completed. This unique family of G protein-coupled receptors (GPCRs) mediates specific cellular actions of various endogenous proteinases including thrombin, trypsin, tryptase, etc. and also certain exogenous enzymes. Increasing evidence has been clarifying the emerging roles played by PARs in health and disease. PARs, particularly PAR1 and PAR2, are distributed throughout the gastrointestinal (GI) tract, modulating various GI functions. One of the most important GI functions of PARs is regulation of exocrine secretion in the salivary glands, pancreas and GI mucosal epithelium. PARs also modulate motility of GI smooth muscle, involving multiple mechanisms. PAR2 appears to play dual roles in pancreatitis and related pain, being pro-inflammatory/pro-nociceptive and anti-inflammatory/anti-nociceptive. Similarly, dual roles for PAR1 and PAR2 have been demonstrated in mucosal inflammation/damage throughout the GI tract. There is also fundamental and clinical evidence for involvement of PAR2 in colonic pain. PARs are thus considered key molecules in regulation of GI functions and targets for development of drugs for treatment of various GI diseases.

We investigated possible involvement of three isozymes of prostaglandin E synthase (PGES), microsomal PGES-1 (mPGES-1), mPGES-2 and cytosolic PGES (cPGES) in COX-2-dependent prostaglandin E-2 (PGE(2)) formation following proteinase-activated receptor-2 (PAR2) stimulation in human lung epithelial cells. PAR2 stimulation up-regulated mPGES-1 as well as COX-2, but not mPGES-2 or cPGES, leading to PGE2 formation. The PAR2-triggered up-regulation of mPGES-1 was suppressed by inhibitors of COX-1, cytosolic phospholipase A(2) (cPLA(2)) and MEK, but not COX-2. Finally, a selective inhibitor of mPGES-1 strongly suppressed the PAR2-evoked PGE(2) formation. PAR2 thus appears to trigger specific up-regulation of mPGES-1 that is dependent on prostanoids formed via the MEK/ERK/cPLA(2)/COX-1 pathway, being critical for PGE(2) formation. Copyright (c) 2007 John Wiley & Sons, Ltd.

It has been almost a decade since the molecular cloning of all four members of the proteinase-activated receptor ( PAR) family was completed. This unique family of G protein-coupled receptors (GPCRs) mediates specific cellular actions of various endogenous proteinases including thrombin, trypsin, tryptase, etc. and also certain exogenous enzymes. Increasing evidence has been clarifying the emerging roles played by PARs in health and disease. PARs, particularly PAR1 and PAR2, are distributed throughout the gastrointestinal (GI) tract, modulating various GI functions. One of the most important GI functions of PARs is regulation of exocrine secretion in the salivary glands, pancreas and GI mucosal epithelium. PARs also modulate motility of GI smooth muscle, involving multiple mechanisms. PAR2 appears to play dual roles in pancreatitis and related pain, being pro-inflammatory/pro-nociceptive and anti-inflammatory/anti-nociceptive. Similarly, dual roles for PAR1 and PAR2 have been demonstrated in mucosal inflammation/damage throughout the GI tract. There is also fundamental and clinical evidence for involvement of PAR2 in colonic pain. PARs are thus considered key molecules in regulation of GI functions and targets for development of drugs for treatment of various GI diseases.

Proteinase-activated receptor-2 (PAR2) plays a dual role in the respiratory system, being pro- and anti-inflammatory. In human lung epithelial cells (A549), PAR2 activation causes release of interleukin-8 (IL-8) in addition to prostaglandin E-2 (PGE(2)). In the present study, we thus investigated PAR2-triggered signal transduction pathways causing IL-8 formation in A549 cells. SLIGRL-NH2, a PAR2-activating peptide, but not LSIGRL-NH2,, a scrambled peptide, elicited release of IL-8 from A549 cells for 18 h, as measured by the ELISA method, an effect being suppressed by inhibitors of MEK, JNK, EGF receptor-tyrosine kinase (EGFR-TK), Src, pan-tyrosine kinases and protein kinase C, but not p38 MAP kinase or cyclooxygenase. SLIGRL-NH2, also up-regulated IL-8 at protein and mRNA levels, as determined by Western blotting and RTPCR, respectively. The PAR2-triggered up-regulation of IL-8 protein and mRNA was blocked by an inhibitor of MEK, but not clearly by inhibitors of JNK and EGFR-TK. SLIGRL-NH2 actually phosphorylated JNK as well as ERK, the JNK activation being resistant to inhibitors of Src, pan-tyrosine kinases, protein kinase C and EGFR-TK. Our data suggest that PAR2-triggerd IL-8 formation involves transcriptional upregulation of IL-8 via the MEK-ERK pathway, while the JNK and EGF receptor pathways might rather contribute to a post-transcriptional process for the release of IL-8. (c) 2007 Elsevier B.V. All rights reserved.

Proteinase-activated receptors (PARs), G protein-coupled receptors, play critical roles in the alimentary system. Increasing evidence suggests that endogenous prostaglandins (PGs) mediate some of PARs' gastrointestinal functions. Systemic administration of the PAR1 agonist protects against gastric mucosal injury through PG formation in rats. PGs also appear to contribute, at least in part, to enhancement of gastric mucosal blood flow and suppression of gastric acid secretion by PAR1 activation. There is also evidence for involvement of PGs in modulation of gastrointestinal motility by PAR1 or PAR2. Importantly, modulation of ion transport by PAR1 or PAR2 in the intestinal mucosal epithelium is largely mediated by PGs. Studies using gastric and intestinal mucosal epithelial cell lines imply that the PAR1-triggered formation of PGs involves multiple signaling pathways including Src, EGF receptor trans-activation and activation of MAP kinases. Collectively, a functional linkage of PAR1 and/or PAR2 to PGs is considered important in the gastrointestinal system. © 2007 Springer.

To clarify the presence of cross-talk between H2S and NO, we investigated effect of NaHS, an H2S donor, on activity of recombinant NO synthase (NOS) isoforms. Activity of all nNOS, iNOS and eNOS was inhibited by NaHS (IC50: 0.13-0.21 mM). In contrast, Na2SO3, L-Cysteine and threo-1,4-dimercapto-2,3-butanediol, a reductant, exerted poor inhibition of NOS activity. Increasing concentrations of tetrahydrobiopterin (BH4) reversed the NaHS inhibition of nNOS and eNOS, but not iNOS. Our data thus demonstrate inhibition of three NOS isoforms by NaHS/H2S, and suggest involvement of interaction of NaHS/H2S with BH4 in inhibition of nNOS and eNOS, but not iNOS. (C) 2007 Elsevier Ireland Ltd. All rights reserved.

We investigated effect of hydrogen sulfide (H2S) on oxidative stress-caused cell death in gastric mucosal epithelial cells. In rat normal gastric epithelial RGM1 cells, NaHS, a H2S donor, at 1.5 mM strongly suppressed hydrogen peroxide (H2O2)-caused cell death, while it slightly augmented the H2O2 toxicity at 0.5-1 mM. The protective effect of NaHS was abolished by inhibitors of MEK or JNK, but not of p38 MAP kinase. NaHS at 1.5 mM actually phosphorylated ERK and JNK in RGM1 cells. Glibenclamide, an ATP-sensitive K+ (K-ATP(+)) channel inhibitor, did not affect the protective effect of NaHS, although mRNAs for K(ATP)(+)channel subunits, Kir6.1 and SUR1, were detected in RGM1 cells. In anesthetized rats, oral administration of NaHS protected against gastric mucosal lesion caused by ischemia-reperfusion. These results suggest that NaHS/H2S may protect gastric mucosal epithelial cells against oxidative stress through stimulation of MAP kinase pathways, a therapeutic dose range being very narrow. (C) 2007 Elsevier Ireland Ltd. All rights reserved.

Hydrogen sulfide (H2S), an endogenous gasotransmitter, modulates various biological events such as inflammation in the mammalian body. The present study investigated possible involvement of H2S in peripheral nociceptive processing. Intraplantar (i.pl.) administration of NaHS, a H2S donor, produced prompt hyperalgesia in rats, accompanied by expression of Fos in the spinal dorsal horn. The H2S-evoked hyperalgesia was blocked by 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB), an oxidizing agent, or ethosuximide and mibefradil, T-type Ca2+ channel inhibitors. L-Cysteine, an endogenous source for H2S, given i.pl., also elicited hyperalgesia, an effect being abolished by DL-propargylglycine (PPG) and beta-cyanoalanine (BCA), inhibitors of cystathionine-gamma-lyase, a H2S synthesizing enzyme. PPG and/or BCA partially inhibited the hyperalgesia induced by i.pl. lipopolysaccharide, an effect being reversed by i.pl. NaHS. In the patch-clamp study using undifferentiated NG108-15 cells that express T-type, but not other types, of Ca2+ channels, NaHS enhanced the currents through the T-type channels, an effect being blocked by DTNB. Thus, H2S appears to function as a novel nociceptive messenger through sensitization of T-type Ca2+ channels in the peripheral tissues, particularly during inflammation. (C) 2007 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Camostat mesilate, an orally available proteinase inhibitor, is clinically used for treatment of pancreatitis. Given recent evidence that pancreatic proteinases including trypsin and/or proteinase-activated receptor-2 (PAR2) might be involved in pancreatic pain, we examined if camostat mesilate could suppress spinal Fos expression, a marker for neuronal activation, following specific application of trypsin to the pancreas, and pancreatitis-related referred allodynia. Trypsin, administered into the pancreatic duct, caused delayed expression of Fos proteins in the superficial layer of the bilateral T8 and T9 spinal dorsal horns in rats. The trypsin-induced spinal Fos expression was completely abolished by oral preadministration of camostat mesilate at 300 mg/kg. After hourly repeated (6 times in total) administration of caerulein, mice showed typical symptoms of pancreatitis, accompanied by mechanical allodynia in the upper abdomen (i.e., referred hyperalgesia/allodynia), as assessed by use of von Frey filaments. Camostat mesilate at 100-300 mg/kg, given orally twice before the 1st and 4th doses of cacrulein, abolished the pancreatitis-related abdominal allodynia, while it partially prevented the inflammatory signs. The same doses of camostat mesilate, when administered once after the final dose of caerulein, also revealed significant anti-allodynic effect. These data suggest that camostat mesilate prevents and/or depresses pancreatitis-induced pain and/or referred hyperalgesia/allodynia, in which proteinases including trypsin would play a critical role. (C) 2007 Elsevier Inc. All rights reserved.

Proteinase-activated receptor-1 (PAR1), a thrombin receptor, plays a protective role in gastric mucosa via prostanoid formation. Thus, we studied effects of PAR1 stimulation on prostaglandin E-2 (PGE(2)) formation in rat normal gastric mucosal epithelial RGM1 cells and analyzed the underlying signal transduction mechanisms. The PARI-activating peptide (PAR1-AP) and thrombin increased PGE(2) release from RGM1 cells for 18 h, an effect being suppressed by inhibitors of COX-1, COX-2, MEK, p38 MAP kinase (p38 MAPK), protein kinase C (PKC), Src and EGF receptor-tyrosine kinase (EGFR-TK), but not JNK and matrix metalloproteinase (MMP)/a disintegrin and metalloprotemases (ADAMs). PAR1-AP caused persistent (6 h or more) and transient (5 min) phosphorylation of ERK and p38 MAPK, respectively, followed by delayed reinforcement at 18 h. PAR1-AP up-regulated COX-2 in a manner dependent on MEK and EGFR-TK, but not p38 MAPK. The PARI-mediated persistent ERK phosphorylation was reduced by inhibitors of Src and EGFR-TK. PAR1-AP actually phosphorylated EGF receptors and up-regulated mRNA for heparin-binding-EGF (HB-EGF), the latter effect being blocked by inhibitors of Src, EGFR-TK and MEK. Heparin, an inhibitor for HB-EGF, suppressed PAR1-mediated PGE2 formation and persistent ERK phosphorylation. These results suggest that PAR1 up-regulates COX-2 via persistent activation of MEK/ERK that is dependent on EGFR-TK activation following induction of HB-EGF, leading to PGE(2) formation. In addition, our data also indicate involvement of COX-1, PKC and p38 MAPK in PAR1-triggered PGE(2) formation. PAR1, thus stimulates complex multiple signaling pathways responsible for PGE(2) formation in RGM1 cells. (c) 2006 Elsevier Inc. All rights reserved.

Hydrogen sulfide (H2S), the third gaseous mediator following nitric oxide (NO) and carbon monoxide, modulates various physiological functions in the mammalian body. Here we analyzed and characterized roles for H2S in modulation of smooth muscle motility. In precontracted aortic rings prepared from rats and mice, NaHS, a donor of H2S, induced contraction at low concentrations and relaxation at high concentrations. The NaHS-induced relaxation involved both ATP-sensitive K+ (KATP) channel-dependent and -independent pathways in rat aorta, but was independent of KATP channels in mouse aorta. The NaHS-induced contraction was attributable to direct inhibition of endothelial NO synthase by NaHS. NaHS at high concentrations also caused KATP channel-independent relaxation in airway and gastrointestinal smooth muscle preparations from rats, mice or guinea-pigs. Together, H2S modulates smooth muscle motility through multiple mechanisms, being excitative and suppressive.

Proteinase-activa ted receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, can be activated not only by PARlactivating peptides (PAR1APs) based on the N-terminal cryptic tethered ligand sequence but also by an N-palmitoylated (Pal) peptide, Pal-RCLSSSAVANRSKKSRALF-amide (P1pal-19), based on the intracellular loop 3 of PARI, designated pepducin, in human platelets or PAR1-transfected cells. The present article evaluated the actions of P1pal-19 and also the shorter peptide, Pal-RCLSSSAVANRS-amide (P1pal-12), known as a possible PAR1 antagonist, in multiple cells/tissues that naturally express PAR1. P1pal-19 as well as a PAR1AP, TFLLR-amide, evoked cytosolic Ca2+ mobilization in cultured human lung epithelial cells (A549) and rat gastric mucosal epithelial cells (RGM1). P1pal-19 and TFLLR-amide, but not a PAR2-activating peptide, SLIGRL-amide, caused delayed prostaglandin E-2 formation in RGM1 cells. P1pal-19, like TFLLR-amide, produced endothelial NO-dependent relaxation in rat aorta and epithelial prostanoid-dependent relaxation in mouse bronchus. The P1pal-19-induced relaxation remained constant even after desensitization of PARI with TFLLR-amide in either tissue. P1pal-19 failed to mimic the contractile effects of TFLLR-amide in the endothelium-denuded preparations of rat aorta or superior mesenteric artery and the rat gastric longitudinal smooth muscle strips. P1pal12 partially inhibited the vasorelaxation caused by TFLLR-amide and P1pal-19, but not SLIGRL-amide, in the rat. aorta. Our data thus indicate that P1pal-19 is capable of mimicking the effects of PAR1APs in the endothelial and epithelial, but not smooth muscle, cells/tissues, and suggest that P1pal-12 may act as a PAR1 antagonist in the vascular endothelium.

Bisphosphonates, pyrophosphate analogues, known as inhibitors of bone resorption, appear to cause analgesia in certain clinical painful situations. To detect clinically relevant analgesic property of etidronate, a non-aminobisphosphonate, we examined and characterized its antiallodynic effect in the rat with adjuvant-induced arthritis, in comparison with alendronate, an aminobisphosphonate, as determined by the von Frey test. Repeated systemic administration of etidronate at 10-40 mg/kg/day suppressed the adjuvant-induced mechanical allodynia in rat hindpaw, an effect reaching a plateau in approximately 10 days. Systemic or intraplantar (i.pl.) administration of ATP-sensitive K+ (K-ATP(+)) channel inhibitors, glibenclamide and/or tolbutamide, completely reversed the antiallodynic effect of etidronate within 1 h in the arthritic rats, without affecting the nociceptive scores in naive or arthritic animals that had not received etidronate. Alendronate, administered repeatedly, also revealed similar glibenclamide-reversible antiallodynic effect. In contrast, the antiallodynic effect of repeated systemic indomethacin was resistant to i.pl. glibenclamide in the arthritic rats. Repeated administration of etidronate or alendronate only slightly attenuated the adjuvant-evoked hindpaw edema. Among K-ATP(+) channel subunits, mRNAs for Kir6.1, SUR1, SUR2A and SUR2B were abundant in rat dorsal root ganglia, while Kir6.2 mRNA was poor. Our data demonstrate that repeated etidronate as well as alendronate exhibits antiallodynic activity in arthritic rats, which might be clinically relevant, and suggest involvement of K-ATP(+) channels in the underlying mechanisms. (c) 2006 Elsevier Ltd. All rights reserved.

1 Proteinase-activated receptor-2 (PAR2), a receptor activated by trypsin and tryptase, is abundantly expressed in the gastrointestinal tract including the C-fiber terminal, and might play a role in processing of visceral pain. In the present study, we examined and characterized the roles of PAR2 in pancreatitis-related abdominal hyperalgesia/allodynia in mice. 2 Caerulein, administered i.p. once, caused a small increase in abdominal sensitivity to stimulation with von Frey hairs, without causing pancreatitis, in PAR2-knockout (KO) mice, but not wild-type (WT) mice. 3 Caerulein, given hourly six times in total, caused more profound abdominal hyperalgesia/allodynia in PAR2-KO mice, as compared with WT mice, although no significant differences were detected in the severity of pancreatitis between the KO and WT animals. 4 The PAR2-activating peptide, 2-furoyl-LIGRL-NH2, coadministered repeatedly with caerulein six times in total, abolished the caerulein-evoked abdominal hyperalgesia/allodynia in WT, but not PAR2-KO, mice. Repeated doses of 2-furoyl-LIGRL-NH2 moderately attenuated the severity of caerulein-induced pancreatitis in WT animals. 5 Our data from experiments using PAR2-KO mice provide evidence that PAR2 functions to attenuate pancreatitis-related abdominal hyperalgesia/allodynia without affecting pancreatitis itself, although the PAR2AP applied exogenously is not only antinociceptive but also anti-inflammatory.

Proteinase-activated receptor (PAR)-1 or -2 modulates gastrointestinal transit in vivo. To clarify the underlying mechanisms, we characterized contraction/relaxation caused by TFLLR-NH2 and SLIGRL-NH2, PAR-1- and -2-activating peptides, respectively, in gastric and small intestinal (duodenal, jejunal and ileal) smooth muscle isolated from wild-type and PAR-2-knockout mice. Either SLIGRL-NH2 or TFLLR-NH2 caused both relaxation and contraction in the gastrointestinal preparations from wild-type animals. Apamin, a K+ channel inhibitor, tended to enhance the peptide-evoked contraction in some of the gastrointestinal preparations, whereas it inhibited relaxation responses to either peptide completely in the stomach, but only partially in the small intestine. Indomethacin reduced the contraction caused by SLIGRL-NH2 or TFLLR-NH2 in both gastric and ileal preparations, but unaffected apamin-insensitive relaxant effect of either peptide in ileal preparations. Repeated treatment with capsaicin suppressed the contractile effect of either peptide in the stomach, but not clearly in the ileum, whereas it enhanced the apamin-insensitive relaxant effect in ileal preparations. In any gastrointestinal preparations from PAR-2-knockout mice, SLIGRL-NH2, produced no responses. Thus, the inhibitory component in tension modulation by PAR-1 and -2 involves both apamin-sensitive and -insensitive mechanisms in the small intestine, but is predominantly attributable to the former mechanism in the stomach. The excitatory component in the PAR-1 and -2 modulation may be mediated, in part, by activation of capsaicin-sensitive sensory nerves and/or endogenous prostaglandin formation. Our study thus clarifies the multiple mechanisms for gastrointestinal motility modulation by PAR-1 and -2, and also provides ultimate evidence for involvement of PAR-2. (c) 2005 Elsevier Inc. All rights reserved.

We investigated proteinase-activated receptor-2 (PAR(2))-triggered signal transduction pathways causing increased prostaglandin E-2 (PGE(2)) formation in human lung-derived A549 epithelial cells. The PAR(2) agonist, SLIGRL-NH2 (Ser-Leu-Ile-Gly-Arg-Leu-amide), evoked immediate cytosolic Ca2+ mobilization and delayed (0.5-3 h) PGE(2) formation. The PAR(2)-triggered PGE(2) formation was attenuated by inhibition of the following signal pathway enzymes: cyclooxygenases 1 and 2 (COX-1 and COX-2, respectively), cytosolic Ca2+-dependent phospholipase A(2) (cPLA(2)), the mitogenactivated protein kinases (MAPKs), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) and p38 MAPK, Src family tyrosine kinase, epidermal growth factor (EGF) receptor tyrosine kinase (EGFRK), and protein kinase C (PKC), but not by inhibition of matrix metalloproteinases. SLIGRL-NH2 caused prompt ( 5 min) and transient ERK phosphorylation, blocked in part by inhibitors of PKC and tyrosine kinases but not by an EGFRK inhibitor. SLIGRL-NH2 2 also evoked a relatively delayed (15 min) and persistent (30 min) phosphorylation of p38 MAPK, blocked by inhibitors of Src and EGFRK but not by inhibitors of COX-1 or COX-2. SLIGRL-NH2 elicited a Src inhibitor-blocked prompt (5 min) and transient phosphorylation of the EGFRK. SLIGRL-NH2 up-regulated COX-2 protein and/or mRNA levels that were blocked by inhibition of p38 MAPK, EGFRK, Src, and COX-2 but not MEK-ERK. SLIGRL-NH2 also caused COX-1-dependent up-regulation of microsomal PGE synthase-1 (mPGES-1). We conclude that PAR(2)-triggered PGE(2) formation in A549 cells involves a coordinated up-regulation of COX-2 and mPGES-1 involving cPLA(2), increased cytosolic Ca2+, PKC, Src, MEK-ERK, p38 MAPK, Src-mediated EGF receptor trans-activation, and also metabolic products of both COX-1 and COX-2.

Protease-activated receptors (PARs), a family of G-protein-coupled seven-transmembrane-domain receptors, are activated by proteolytic unmasking of the N-terminal cryptic tethered ligand by certain serine proteases. Among four PAR family members cloned to date, PAR-1, PAR-2, and PAR-4 can also be activated through a non-enzymatic mechanism, which is achieved by direct binding of exogenously applied synthetic peptides based on the tethered ligand sequence, known as PARs-activating peptides, to the body of the receptor. Various peptide mimetics have been synthesized as agonists for PARs with improved potency, selectivity, and stability. Some peptide mimetics and/or nonpeptide compounds have also been developed as antagonists for PAR-1 and PAR-4. PARs are widely distributed in the mammalian body, especially throughout the alimentary systems, and play various roles in physiological/pathophysiological conditions, i.e., modulation of salivary, gastric, or pancreatic glandular exocrine secretion, gastrointestinal smooth muscle motility, gastric mucosal cytoprotection, suppression/facilitation of visceral pain and inflammation, etc. Thus PARs are now, considered novel therapeutic targets, and development of selective agonists and/or antagonists for PARs might provide a novel strategy for the treatment of various diseases that are resistant to current therapeutics.

1 Proteinase-activated receptor-2 (PAR(2)), expressed in capsaicin-sensitive sensory neurons, plays a protective role in gastric mucosa. The present study evaluated gastric mucosal cytoprotective effect of 2-furoyl-LIGRL-NH2, a novel highly potent PAR(2) agonist, in ddY mice and in wild-type and PAR(2)-knockout mice of C57BL/6 background. 2 Gastric mucosal injury was created by oral administration of HCl/ethanol solution in the mice. The native PAR(2)-activating peptide SLIGRL-NH2, administered intraperitoneally (i.p.) at 0.3 - 1 mumol kg(-1) in combination with amastatin, an aminopeptidase inhibitor, but not alone, revealed gastric mucosal protection in ddY mice, which was abolished by ablation of capsaicin-sensitive sensory neurons. 3 I.p. administration of 2-furoyl-LIGRL-NH2 at 0.1 mumol kg(-1), without combined treatment with amastatin, exhibited gastric mucosal cytoprotective activity in ddY mice, the potency being much greater than SLIGRL-NH2 in combination with amastatin. This effect was also inhibited by capsaicin pretreatment. 4 Oral administration of 2-furoyl-LIGRL-NH2 at 0.003 - 0.03 mumol kg(-1) also protected against gastric mucosal lesion in a capsaicin-reversible manner in ddY mice. 5 I.p. 2-furoyl-LIGRL-NH2 at 0.1 - 0.3 mumol kg(-1) caused prompt salivation in anesthetized mice, whereas its oral administration at 0.003 - 1 mumol kg(-1) was incapable of eliciting salivation. 6 In wild-type, but not PAR(2)-knockout, mice of C57BL/6 background, i.p. administration of 2-furoyl-LIGRL-NH2 caused gastric mucosal protection. 7 Thus, 2-furoyl-LIGRL-NH2 is considered a potent and orally available gastric mucosal protective agent. Our data also substantiate a role for PAR2 in gastric mucosal protection and the selective nature of 2-furoyl-LIGRL-NH2.

誘導型NO 合酵素阻害薬ONO.1714 がアセトアミノフェンにより誘起される肝障害に対して抑制的に作用することを証明した。（英文）

麻酔ラットにおいてPAR.1 活性化ペプチドの反復投与によってペプシノーゲン分泌が誘起されることを証明した。（英文）

高血圧ラット（SHRSP）と正常血圧ラット（WKY）の胃底部における一酸化窒素（NO）神経性弛緩反応を比較したところ、SHRSP、WKY 標本間でほとんど差がないことが明らかとなった。（英文）

1. There are known differences in the sensitivity to caffeine between skeletal muscle (soleus) of normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). The present study was performed in order to examine differences in the effects of caffeine on twitch contraction between visceral striated muscle using the outer layer of the oesophagus from WKY rats and stroke-prone SHR (SHRSP). 2. Caffeine, at concentrations ranging from 0.3 to 10 mmol/L, exhibited potentiating effects on twitch contraction in preparations from both WKY rats and SHRSP. The potentiating effect of caffeine was markedly less prominent in preparations from SHRSP compared with preparations from WKY rats. 3. The rate of contraction and relaxation, the time to peak tension and 80% relaxation time were not significantly altered by caffeine at concentrations lower than 3 mmol/L in preparations from either strain. 4. With 10 mmol/L caffeine, the rate of relaxation was markedly reduced and the 80% relaxation time was prolonged, with no significant changes in the rate of contraction, in preparations from WKY rats. These changes were significantly smaller in preparations from SHRSP. 5. The duration of the action potential was greater in preparations from SHRSP than in preparations from WKY rats, although the membrane potential and the amplitude of the action potential were not significantly different between preparations from WKY rats and SHRSP. 6. Caffeine, at 10 mmol/L, prolonged the duration of the action potential in preparations from both strains. The effect of caffeine was not different between preparations from WKY rats and SHRSP. 7. The results of the present study suggest that caffeine augments release of Ca2+ from the sarcoplasmic reticulum (SR) at low concentrations and attenuates Ca2+ re-uptake at 10 mmol/L. Decreased reactivity of SR to caffeine may be a cause of the lesser potentiation of twitch contraction by caffeine in preparations from SHRSP.

The effects Of L-arginine on spontaneous contraction of endothelium-denuded longitudinal preparations of the rat portal vein were studied. L-Arginine increased the frequency of spontaneous contraction concentration-dependently between 10 muM and 1 mM. Changes in contraction amplitude and duration were not remarkable. D-Arginine had a negligible effect on spontaneous contraction. N-omega-nitro-L-arginine (1 mM) did not affect spontaneous contraction or the response to L-arginine. Addition of N-G-monomethyl-L-arginine (1 mM), L-lysine (1 mM) or N-ethymaleimide (0.1 mM) increased the frequency of spontaneous contractions and inhibited the. effect Of L-arginine. Glibenclamide (10 muM) did not affect spontaneous contraction or the response to L-arginine. Spontaneous increase in concentration of intracellular Ca2+, estimated as the ratio of Fura-PE3 fluorescence occurred synchronously with spontaneous contraction. Spontaneous increase in concentration of intracellular Ca2+ occurred more frequently in the presence Of L-arginine (I MM). L-Arginine (1 mM) also increased the number of action potential bursts/min in the longitudinal smooth muscle layer. L-Arginine (I mM) also depolarized cell membranes. This study indicates that L-arginine increases the frequency of spontaneous contraction of longitudinal muscle in the rat portal vein by membrane depolarization through mechanisms that do not involve, nitric oxide or the inhibition of ATP-sensitive K+ channels.

高血圧ラットの血管では内皮依存性弛緩反応の減少がみられるが、カフェインは内皮非依存性の弛緩を引き起こし、この弛緩反応は正常血圧と高血圧ラットの大動脈で差がなかったことから、平滑筋の反応性には異常がないことが示唆された。（英文）

To elucidate whether properties of the sarcoplasmic reticulum are altered, not only in vascular smooth muscle, but also in visceral striated muscle of spontaneously hypertensive rats (SHR), caffeine-induced contractures in oesophageal striated muscle of Wistar Kyoto rats (WKY) and stroke-prone SHR (SHRSP) were compared. In both preparations, 30 mM caffeine induced a contracture with two components. The second component, which was diminished by extracellular Ca2+ removal or Ni2+ but not by verapamil, was much smaller in SHRSP. Both components and differences between WKY and SHRSP coincided with changes in intracellular Ca2+. Although membrane potential was identical between these preparations, caffeine induced slight depolarization only in WKY preparations. Similar depolarization was observed with 10 mM K+, which induced no contraction. It is suggested that the first and the second components of caffeine-induced contracture were induced by Ca2+ released from sarcoplasmic reticulum and by Ca2+ that entered through channels activated by sarcoplasmic reticulum Ca2+ depletion, respectively In SHRSP preparations, Ca2+ from the latter pathway was clearly decreased, although this change is thought not to be related to the initiation of hypertension. These results suggest that Ca2+ handling properties of cell membrane and sarcoplasmic reticulum are generally altered in muscles of SHRSP. (C) 2003 Elsevier Science B.V. All rights reserved.

Endothelium-dependent relaxation (EDR) in the blood vessels of spontaneously hypertensive rats (SHR) and the role of nitric oxide (NO) in the initiation of hypertension are reviewed. EDR was impaired in blood vessels of SHR depending on age and degree of hypertension when compared with those of normotensive rats. The cause of the impairment varied among the type of blood vessels: a decrease in the production of NO and endothelium-derived relaxing factor (EDRF) and an increase in the production of endothelium-derived contracting factor (EDCF) are the main causes of the impairment in large arteries, while a decrease in endothelium-dependent hyperpolarization and increased release of EDCF are the main causes of the impairment in small arteries. Interactions among these endothelium-derived factors and changes in the interactions are also causes of impairment. Superoxide may be involved in the impairment of EDR by destroying NO. The endothelium depresses smooth muscle contraction, including spontaneous tone developed in vascular smooth muscle, and the depressing effect of the endothelium is impaired in the preparations from SHR. The endothelium of blood vessels of SHR are structurally injured as demonstrated by scanning electron microscopy. Antihypertensive treatment prevented these functional and structural changes. Chronic treatment with inhibitors of NO production in normotensive rats impaired EDR and elevated blood pressure. The impairment of EDR is a secondary change due to continued hypertension, and early initiation of antihypertensive therapy is recommended. © 2003 The Pharmaceutical Society of Japan.

ラット食道横紋筋では筋小胞体作用薬により細胞外カルシウム流入が引き起こされ、この流入は正常血圧ラットに比べ高血圧ラット標本において減少していることが示唆された。（英文）

Wistar ラットに L ニトロアルギニン （L NAME） を長期経口投与すると血圧が上昇した。 そのラット動脈の内皮依存性弛緩は非投与ラットに比べ著明に減弱していた。 この減弱は標本に残存した L NAME によるもので投薬による血圧上昇は関与していないと示唆された。 （英文）

1. The mechanisms of oscillatory contraction of arterial smooth muscle in vitro are discussed. 2. The membrane potential and cytoplasmic free Ca2+ concentration in smooth muscle cells oscillate in the presence of agonists. 3. The oscillatory change in the membrane potential of smooth muscle cells is related to Ca2+ release from intracellular stores. 4. Gap junctions between smooth muscle cells play important roles in the synchronized oscillation of the cytoplasmic free Ca2+ concentration in this population of cells. 5. Endothelial cells may increase or decrease the tension oscillation of smooth muscle cells. 6. In arteries from hypertensive rats, an increase in membrane excitability and the number of gap junctions between smooth muscle cells and impaired endothelial function are the main factors responsible for the modulation of tension oscillation.

The membrane hyperpolarization produced by endothelium-derived hyperpolarizing factor (EDHF) is one of the components in the endothelium-dependent relaxations. Different subtype of endothelium receptor is involved in the release of EDHF from that for nitric oxide. Although EDHF has not been identified, it may be related to cytochrome P450. The responses mediated by EDHF have been studied by observation of membrane potential or relaxation which is resistant to inhibitors of nitric oxide synthase and cyclooxygenase. The responses are dependent on potassium gradient across cell membrane, therefore, the response is mediated by potassium channels. The EDHF-mediated hyperpolarization or relaxation is sensitive to charybdotoxin plus apamin, inhibitors of calcium activated potassium channels. The EDHF-mediated response is more remarkable in small resistant arteries than large arteries, therefore, EDHF may play an important role in the regulation of vascular resistance. The EDHF-mediated response is decreased in hypertensive or aged animals. Understanding of EDHF-mediated response will promote development of a new strategy for the treatment of pathophysiological conditions.

1 The effects of cyclopiazonic acid (CPA) and thapsigargin (TG), both of which are known to inhibit sarcoplasmic reticular Ca2+-ATPase, on the mechanical activities, intracellular Ca2+ level and electrical activities of smooth muscle of the carotid artery of stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar Kyoto rats (WKY) were compared. 2 Both CPA and TG induced elevation of tension of the smooth muscle, which was composed of a phasic and a tonic component. The level of tension attained, especially the tonic component, was greater in the preparation from SHRSP. 3 The elevation of tension was associated with an increased intracellular Ca2+ level. Both the elevation of tension and the increase in intracellular Ca2+ were diminished by the removal of extracellular Ca2+ or by the application of verapamil. 4 The resting membrane potential of the preparations from SHRSP were depolarized to a greater extent than those from WKY. CPA depolarized the smooth muscle from both SHRSP and WKY, and the final level was also more depolarized in the preparation from SHRSP. 5 These results indicate that the elevation of tension induced by these drugs is mainly due to increased Ca2+ influx through voltage-dependent Ca2+ channels, and the difference in the action between the preparation from SHRSP and that from WKY can be explained mainly by the changes in the channels. 6 Thus, differences in the action of these drugs on the tension of smooth muscle between preparations from WKY and SHRSP can mainly be explained by the difference in the membrane potential which is related to the difference in voltage-dependent Ca2+ influx.

1. The influence of the passive force on the contraction and endothelium-dependent relaxation in aortae of normotensive Wister Kyoto (WKY) rats and stroke-prone spontaneously hypertensive rats (SHRSP) Here compared. 2. Force changes of endothelium-intact and -removed preparations were measured isometrically by a force-displacement transducer, Endothelium-dependent relaxation was observed by applying acetylcholine to the preparation precontracted in the presence of 5 x 10(-7) mol/L noradrenaline. 3. The preparations showed spontaneously developed tension (tone) that increased with the increase in the passive force. The effect of passive force was greater in preparations from SHRSP. Contraction initiated by noradrenaline was also increased by passive force up to 30 mN, then showed a tendency to decrease. 4. Endothelium-dependent relaxation was depressed as the passive force was increased. Preparations from SHRSP showed impaired endothelium-dependent relaxation and were influenced by passive force to a lesser degree when compared with preparations from WKY rats. 5. Relaxation by sodium nitroprusside was influenced by passive force to a much lesser extent than that by acetylcholine. 6. Indomethacin potentiated endothelium-dependent relaxation and blocked the effect of passive force in both preparations. 7. The difference in relaxation and the effect of passive force is primarily caused by the difference in the release of endothelium-derived contracting factor, which is thought to be a product of the cyclo-oxygenase pathway of the arachidonic acid cascade.

Differences in alpha(2)-adrenoceptor-induced relaxation of the aorta between stroke-prone spontaneously hypertensive rats (SHRSP) and control normotensive Wistar Kyoto rats (WKY) were studied. Changes in the tension of ring preparations of the aortas were measured isometrically. Relaxation was observed in the preparations precontracted in the presence of ONO-11113. a thromboxane A(2) analogue. The alpha(2)-agonist clonidine and UK-14304 induced dose-dependent relaxation in both the WKY and HRP preparations. The relaxation was impaired in the SHRSP preparation. A modified sandwich experiment showed that the relaxing substance from the SHRSP endothelium was decreased. Acetylcholine (ACh) also induced dose-dependent relaxation, and the relaxation was impaired in the SHRSP preparations. alpha(2)-Agonists induced a greater degree of impairment in the relaxation than did ACh. The relaxation induced by alpha(2)-agonists and by ACh was blocked by N-G-nitro-L-arginine (L-NNA). Indomethacin improved the relaxation induced by ACh but not that induced by alpha(2)-agonisrs in the SHRSP aortas. These results suggest that the impairment of relaxation by alpha(2)-agonists in SHRSP is not caused by the increase in the release of endothelium-derived contracting factor (EDCF) but by the reduction in the release of nitric oxide (NO). Alteration of the alpha(2)-adrenoceptors and/or the intracellular mechanism through which NO is synthesized by stimulation of the alpha(2)-adrenoceptors may be the cause of the reduction in relaxation.

1. The influence of the passive force on the contraction and endothelium- dependent relaxation in aortae of normotensive Wistar Kyoto (WKY) rats and stroke-prone spontaneously hypertensive rats (SHRSP) were compared. 2. Force changes of endothelium-intact and -removed preparations were measured isometrically by a force-displacement transducer. Endothelium-dependent relaxation was observed by applying acetylcholine to the preparation precontracted in the presence of 5 x 10-7 mol/L noradrenaline. 3. The preparations showed spontaneously developed tension (tone) that increased with the increase in the passive force. The effect of passive force was greater in preparations from SHRSP. Contraction initiated by noradrenaline was also increased by passive force up to 30 mN, then showed a tendency to decrease. 4. Endothelium-dependent relaxation was depressed as the passive force was increased. Preparations from SHRSP showed impaired endothelium- dependent relaxation and were influenced by passive force to a lesser degree when compared with preparations from WKY rats. 5. Relaxation by sodium nitroprusside was influenced by passive force to a much lesser extent than that by acetylcholine. 6. Indomethacin potentiated endothelium-dependent relaxation and blocked the effect of passive force in both preparations. 7. The difference in relaxation and the effect of passive force is primarily caused by the difference in the release of endothelium-derived contracting factor, which is thought to be a product of the cyclo-oxygenase pathway of the arachidonic acid cascade.

The influences of blood pressure and age of spontaneously hypertensive rats on endothelium dependent tension oscillation of aortic preparation were studied. Rats with different blood pressures, normotensive Wistar Kyoto rats (WKY), spontaneously hypertensive rats (SHR), stroke prone ShR (SHRSP) and malignant type of SHRSP (M SHRSP), were used. The effects of antihypertensive treatment of SHRSP on the tension oscillation were also studied. High doses of noradrenaline induced tension oscillations in endothelium intact preparations of all strains of young rats. The rate of the occurrence of the tension oscillation decreased age dependently. The decrease was faster when the blood pressure of the rats was higher Application of acetylcholine in the presence of noradrenaline induced a relaxation and tension oscillations, both of which were negatively dependent on age anti blood pressure. Antihypertensive treatment of hypertensive rats with hydralazine or captopril prevented a decrease in incidence of the tension oscillation. These influences of age and blood pressure as well as antihypertensive treatments on the tension oscillation resembled those on the endothelium dependent relaxation and are thought to be brought about by functional changes of the endothelium.

Differences in the influences of endothelium derived nitric oxside (NO) on α-agonists-induced contraction in the aortae of spontaneously hypertensive and normotensive rats were studied by blocking NO synthesis with N(G)-nitro- L-arginine (L-NNA). L-NNA potentiated the contraction induced by noradrenaline. The potentiation was smaller in the preparation from stroke prone spontaneously hypertensive rats (SHRSP) than in the preparation from Wistar Kyoto rats (WKY). Similar potentiation was observed in the contraction induced by phenylephrine the potentiation was also smaller in the preparation from SHRSP. α2-agonists, clonidine and UK-14304 induced dose dependent contraction only in the presence of L-NNA. The dose response curves for α2-agonists in SHRSP aorta were different from those in WKY aorta the maximum tension was observed at the concentration of 10-6 M in the preparation from WKY, while the contraction further increased up to 10-4 M in the preparation from WKY. Noradrenaline, clonidine and UK-14304 but not phenyrephrine induced relaxation which was blocked by L-NNA. The relaxation was impaired in the preparation from SHRSP in greater extent than that by acetylcholine. It is suggested that basic or noradrenaline-stimulated NO release from endothelium decreased in the preparation from SHRSP and that α2-adrenoceptor of both the endothelium and smooth muscle may be altered in the preparation from SHRSP.

Differences in noradrenaline-induced tension oscillations between aortae from stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive control Wistar Kyoto rats (WKY) were studied. Endothelium-intact and -removed ring preparations were made from thoracic aorta of SHRSP and WKY, and isometric contraction was observed. Endothelium-intact preparations from WKY showed tension oscillations in response to high concentration of noradrenaline at significantly higher ratio, while only a few endothelium-removed preparation showed tension oscillations in response to low concentration of the drug. Fifteen out of 41 endothelium-removed preparations from SHRSP showed tension oscillations in response to low concentration of noradrenaline but no preparation showed oscillations in response to high concentration of the drug. Seven and 14 out of 42 endothelium-intact preparations from SHRSP showed oscillatory response to low and high concentration of the drug respectively. Acetylcholine-induced relaxation and tension oscillations, both of which being significantly less in the preparation from SHRSP. Both the endothelium-independent tension oscillations and endothelium-dependent tension oscillations were abolished by removal of extracellular Ca or by verapamil. It is suggested that aortic smooth muscle of SHRSP possesses the property which produced tension oscillations, while the tension oscillations of WKY aorta is endothelium-dependent. The opening of voltage-dependent Ca++ channel of smooth muscle cell membrane which generates rhythmic contraction is increased in the aorta of SHRSP, while the release of endothelium-derived factors which induces rhythmic activities of smooth muscle cell is decreased.

An extract from intact spores of Bacillus cereus T having a germination-inducing activity was studied. Two distinct germinative principles were found through dialysis of the extract. One was diffusible through the dialysis membrane and the other was non-diffusible. The activity of the former fraction was inhibited by the addition of 1 mM glyeoletherdiamine-N,N,N',N'-tetraacetic acid (GEDTA), whereas the latter fraction was inactive unless GEDTA was added to the assay system. The diffusible principle maintained the major portion of the activity found in the crude spore extract. By means of high-performance liquid chromatography (HPLC) using a gel permeation chromatography column, 9 fractions were obtained from the deproteinized diffusible fraction. Of those fractions, two fractions (No. 1 and No. 8) were responsible for the germination-inducing activity, but no reconstituted activity was observed unless both fractions No. 1 and No. 8 were added to the assay system. Amino acid analysis of fraction No. 1 revealed that the fraction was rich in free amino acids, especially in alanine. On the other hand, by the use of reverse-phase HPLC and fast atom bombardment mass spectrometry, it was concluded that the effective substance in fraction No. 8 was inosine. Based on these findings, it was suggested that the active substances in fraction No. I might be a free amino acid such as L-alanine and/or Ca2+ and a Ca2+-binding substance.

Spontaneously developed tension (active tone) and intracellular Ca2+ level of aortae from spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP), malignant SHRSP (M-SHRSP) and control Wistar Kyoto rats (WKY) were compared. Systolic blood pressure of WKY, SHR, SHRSP and M-SHRSP was 130mmHg, 200mmHg, 250mmHg and 260mmHg, respectively. Preparations from all strains of spontaneously hypertensive rats exhibited active tone which was abolished by the removal of extracellular Ca2+ or by the application of verapamil. The active tone was greater in the order of aortae from SHR< SHRSP< M-SHRSP. Intracellular Ca2+ level measured by Fura-2 method decreased by the removal of extracellular Ca2+. The degree of the decrease was greater as the blood pressure of the rats increased, indicating the greater elevation of intracellular Ca2+level in the presence of extracellular Ca2+. A correlation was obtained between the active tone, intracellular Ca2+level and blood pressure. Thus, it was demonstrated that the development of the active tone is brought about by the changes in Ca2+ influx of smooth muscle cell membrane and the degree of the change is positively related to the degree of hypertension. © 1993, Japan Society of Smooth Muscle Research. All rights reserved.

Mechanisms of elevation of active tone in arterial smooth muscle of spontaneously hypertensive rats and effects of antihypertensive treatments are reviewed. Spontaneously hypertensive rats are created by Okamoto et al. and classified mainly into three strains, SHR, SHRSP and M-SHRSP. Blood pressure of the rats is higher in this order. The aortic smooth muscle of these rats exhibits elevation of the active tension (tone) under nonstimulated condition. The active tone is greater as the blood pressure of the rats elevated and good correlation between blood pressure and active tone can be obtained. The active tone is sensitive to extracellular Ca and abolished by the removal of extracellular Ca or by the application of Ca-antagonists. It is also associated with increased intracellular Ca level. These results indicate that the active tone is brought about by increase in voltage-dependent Ca channel opening of the cell membrane. Increased voltage-dependent Ca channel opening, i.e. membrane depolarization and increased inward Ca current can be a cause of the elevated tone. Abnormalities in the cellular mechanisms for relaxation of the smooth muscle can also contribute to the elevation of the active tone. Endothelium suppresses the active tone, probably releasing endothelium-derived relaxing factor or hyperpolarizing factor. The suppression of contraction by endothelium is impaired in the blood vessels of spontaneously hypertensive rats, depending on the degree of hypertension. Antihypertensive treatments prevent the development or elevation of the active tone. It also prevents the impairment of function of endothelium. Thus, the maintained hypertension alters functions of smooth muscle membrane and endothelium. The degree and duration of hypertension are thought to be determinant factors of these abnormalities.

Maeda, Y., Yamanaka, R., Sekiguchi, F., Kawabata, A. , 2010, 7, 17-23, Copenhagen, Denmark.

19. 西村幸容、石倉宏恭、松波真帆、篠崎 結、関口富美子、成瀬光栄、喜多村泰輔、明石隆吉、松村謙二、川畑篤史. 第83回 日本薬理学会年会、2010, 3, 16-18, ．

1. 関口富美子, 前田優磨, 松本裕喜, 松波真帆, 西川裕之, 川畑篤史. 、2009, 11, 13, 大津．

近畿大学薬学部・医学部合同学習会におけるPBL教育に関する報告。

4. Kawabata, A., Nishimura, S. 西村幸容, Fukushima, O., Ishikura, H., Takahashi, T., Matsunami, M., Tsujiuchi, T., Sekiguchi, F., Naruse, M., Kamanaka, Y. , 2007, 11, 3-7, Dan Diego, USA.

3. 守行和美、松原かおり、関口富美子、西川裕之、川畑篤史．、2007, 11, 16, 豊中．

9. Kawabata, A., Nishimura, S., Fukushima, O., Ishikura, H., Takahashi, T., Matsunami, M., Sekiguchi, F., Naruse, M. 2007, 8, 28, Nagoya.

33. Kawabata, A., Nagataki, M., Moriyuki, K., Sekiguchi, F., Nishikawa, H. 2006, 1, 25-28, Luxembourg. (Oral presentation)

WKY、SHRSPとも肺動脈圧は、大腿動脈圧に比べ著しい低値を示した。摘出肺動脈の、内皮依存性弛緩には、間に差はなく、低度の肺動脈圧上昇は、内皮依存性の弛緩には依存しないためと考えられた。

2021年度は、ボルテゾミブ、パクリタキセル、オキサリプラチンなどの抗がん薬の副作用として生じる化学療法誘発性末梢神経障害（CIPN）、butyrateやTNBSなどにより誘発される過敏性腸症候群（IBS）モデルにおける内臓痛、２型糖尿病に伴う有痛性末梢神経障害に対して、遺伝子組換えヒト可溶性トロンボモジュリン（トロンボモジュリンアルファ；TMα）が予防的に作用することを検証あるいは証明した。一方、1型糖尿病の末梢神経障害に対してTMαは無効であった。また、抗凝固薬アルガトロバンは、上記痛みモデルにおけるTMαの効果を抑制した。また、TMαの抗CIPN効果にはトロンビン依存性のHMGB1不活性化作用に加えて、プロテインC（PC）およびthrombin-activatable fibrinolysis inhibitor (TAFI)の活性化が関与することを明らかにした。さらに活性化されたPC（APC）とTAFI（TAFIa）の標的分子はそれぞれproteinase-activated receptor 1 (PAR1)および補体アナフィラトキシンC5aであることも突き止めた。また、関西医科大学附属病院薬剤部との共同研究で実施した後ろ向きコホート研究により、2型糖尿病患者において有痛性末梢神経障害の発症率は、抗凝固薬投与患者の方が非投与群よりも有意に高いことを突き止め、内因性トロンビンが恐らく内皮トロンボモジュリン依存性に痛み抑制的に働いているとの仮説を支持する知見が得られた。このように、2021年度の研究により、本課題を進めるための基礎的知見が得られ、2022年度以降の実験計画を円滑に実行するための基盤を整備することができた。

本研究課題では、生体内で数種の合成酵素により産生される硫化水素（H2S）が、抗がん剤による化学療法誘起末梢神経障害（CIPN）の発症および抗がん剤抵抗性獲得に寄与するかを検討することを目的としている。実際に、抗がん剤によるCIPN発症と治療効果の減弱に内因性H2Sの寄与が明らかとなれば、H2S合成酵素阻害薬が抗がん剤との併用投与により、副作用であるCIPNの軽減と抗がん剤の治療効果増大の両面において高い有用性を示すことが期待できる。2020年度は、ヒト多発性骨髄腫（multiple myeloma, MM）由来KMS-11細胞と、MMの治療に使用される抗がん剤ボルテゾミブ（BTZ）に対して耐性を獲得したKMS-11/BTZ細胞を用いて、BTZ非存在下あるいは存在下における細胞増殖におよぼす内因性H2Sの関与について検討を行った。その結果、BTZ非存在下、存在下どちらにおいても、KMS-11およびKMS-11/BTZの細胞増殖はH2S合成酵素のcystathionine-β-synthase（CBS）の阻害薬により顕著に、cystathionine-γ-lyase（CSE）阻害薬により部分的に抑制されたが、CSE阻害薬の効果はKMS-11細胞で小さかった。これら阻害薬による増殖抑制効果はH2S供与体のNa2SあるいはGYY4137により一部減弱した。一方、別のH2S合成酵素である3-mercaptopyruvate sulfur transferase（3-MST）の阻害薬はこれら細胞の増殖にほとんど影響しなかった。これらの結果より、KMS-11およびKMS-11/BTZ細胞の増殖は内因性H2Sにより促進的に調節されており、その産生には主にCBSが寄与していることが示唆された。

RAGEを標的とする難治性疼痛治療薬のシーズを開発するため、神経障害性疼痛および内蔵痛の動物モデルを作成して既存のRAGEの有効性を確認した。ついで、中分子量ヘパリニルフェニルアラニン（MMWHI-F）がRAGEを選択的に遮断し難治性疼痛を抑制することを証明した。さらに、インシリコ解析により同定した低分子化合物がAGE-RAGE bindingを阻害することを証明し、新規RAGE拮抗薬開発のためのシーズになりうることを示唆した。以上、本研究によって高分子および低分子RAGE拮抗薬が痛みの治療に有用であることを明らかにした。

核内蛋白HMGB1はdamage-associated molecular patternsとして、病態時に細胞外に放出される。今回は、癌化学療法誘起神経障害性疼痛および内臓痛におけるHMGB1の役割を解析した。その結果、HMGB1は、RAGE、Toll-like receptor 4、CXCR4などの細胞膜受容体を介して癌化学療法誘起神経障害性疼痛や膵炎に伴う膵臓痛の発症および維持に関与することが明らかとなった。

一次知覚神経のCav3.2 T型カルシウムチャネル（Tチャネル）は、体性痛、内臓痛、神経障害性疼痛などの痛みの発症に寄与することが知られている。本研究では始めに、種々の疼痛モデルを用いて、新規Tチャネル阻害薬RQ-00311651の鎮痛効果を検討し、この化合物が中枢抑制作用の少ない経口投与可能な鎮痛薬となりうることを報告した。また、我々が以前に報告している第5腰神経切断誘起神経障害性疼痛ラットにおける一次知覚神経のCav3.2発現増加に、転写因子Egr-1および脱ユビキチン化酵素USP5の発現増加を介したCav3.2の転写促進およびプロテアソーム分解抑制が関与することを明らかにした。

硫化水素の疼痛情報伝達促進メカニズムにCav3.2 T型カルシウムチャネルが関与することを明らかにした。また、L5脊髄神経切断神経障害性疼痛モデルを作成し、痛覚過敏・アロディニアの保持に内因性硫化水素によるCav3.2の活性化が関与すること、さらに、本モデルではCav3.2の発現誘導がおこっていることを見出し、硫化水素/Cav3.2系が神経因性疼痛の新たな治療標的になりうることを明らかにした。

生体内新規ガス状メッセンジャーとして機能する可能性が示唆されている硫化水素の疼痛情報伝達制御における役割を検討し、以下の知見を得た。 1)硫化水素およびL-システインの投与による痛覚過敏について NaHSをラットの足底内投与することにより、機械的痛覚過敏が誘起されることを見出し、この効果の発現にはT型カルシウムチャネルが関与することを証明した。また、硫化水素の生体内originであるL-システインを足底内投与した場合にも痛覚過敏が認められ、これはL-システインからCSEによって産生される硫化水素によるものであるとの示唆が得られた。一方、NaHSの足底内投与によって、脊髄後角侵害受容ニューロンの活性化が起こることをFos蛋白の発現を指標として証明した。一方、ラットの脊髄内へNaHSを投与した場合にも痛覚過敏の発現を認めている。 2)NG108-15細胞およびマウス脊髄DRGニューロンにおけるT型カルシウムチャネル電流の硫化水素による増強 Whole cell patch clamp法により、NG108-15細胞およびマウス脊髄DRGニューロンにおけるバリウム電流を指標としてT型カルシウムチャネルを介する膜電流が硫化水素によって促進されることを証明した。 3)硫化水素のマウス結腸内投与による内臓痛発現について マウスの結腸内へNaHSを投与することにより内臓痛および関連痛覚過敏が発現するとともに、脊髄後角ではERKリン酸化が誘起されることを証明した。 以上、本研究により、生体内の硫化水素は、痛みの情報伝達における新たなメッセンジャーとして機能していることが明らかとなった。