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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.

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.

The percutaneous absorption of a fentanyl (FEN)-patch is affected by various external factors including the volume of sebum secretion, which causes changes in the skin surface environment. In this study, we prepared a lard-based sebum-like secretion (SLS), and applied it to investigate the effect of different skin surface conditions on the drug penetration of a FEN-patch. In vitro work to test drug release using the Franz diffusion cell indicated that drug release was significantly suppressed by treatment with 5% SLS, which is equivalent to the amount of daily human sebum secretion. Conversely, in ex vivo experiments using rat skin, the amount of FEN that accumulated in the skin tissue of the 5% SLS-treated rats was higher in comparison with the non-SLS treated group. Furthermore, in vivo experiments indicated that the plasma FEN concentration in rats treated with the FEN-patch was significantly increased by treatment with 5% SLS. These results suggest that the sebum affected the release, accumulation, and absorption of FEN from the FEN-patch, and the FEN concentration in the blood was reflected by the balance of the suppression of drug release and the enhancement of drug accumulation in the skin with SLS.

Mohs paste is useful for controlling exudates from wounds and infections and is used to treat patients with inoperable skin tumors. Unfortunately, Mohs paste is difficult to preserve because its viscosity and stickiness increase dramatically immediately after preparation, resulting in decreased usability. In this study, the combined use of cryopreservation and kneading was shown to improve long-term storage of Mohs paste. At 25°C, Mohs pastes solidified rapidly, and viscosity reached approximately 700 Pa·s 5 h after preparation. In contrast, cryopreservation at -20°C attenuated hardening of Mohs pastes, and kneading also decreased viscosity. The viscosity of Mohs pastes cotreated with cryopreservation and kneading after 7 months of storage was

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. Keywords: Tacrolimus, Colonic pain, TRPV1

Background: Accurate prognosis in terminal cancer patients is useful to improve their quality of life and also to decide the cessation of fluid administration. Nonetheless, few prognostic indicator...

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.

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.

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.

Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting adverse reaction in cancer patients treated with several cytotoxic anticancer agents including paclitaxel. Duloxetine, an antidepressant known as a serotonin-noradrenalin reuptake inhibitor, is the only agent that has moderate evidence for the use to treat painful CIPN. The present retrospective cohort study aimed to analyze risk factors for paclitaxel-induced peripheral neuropathy (PIPN), and investigate ongoing prescription drug use for PIPN in Japan. Female breast and gynecologic cancer patients who underwent paclitaxel-based chemotherapy at a single center in Japan between January 2016 and December 2019 were enrolled in this study. Patients’ information obtained from electronic medical records were statistically analyzed to test possible risk factors on PIPN diagnosis. Patients’ age, total paclitaxel dose, the history of female hormone-related diseases, hypertension and body mass index (BMI), but not additional platinum agents, were significantly associated with increased PIPN diagnosis. Drugs prescribed for PIPN included duloxetine, pregabalin, mecobalamin and Goshajinkigan, a polyherbal medicine, regardless of poor evidence for their effectiveness against CIPN, and were greatly different between breast and gynecologic cancer patients diagnosed with PIPN at the departments of Surgery and Gynecology, respectively. Thus, older age, greater total paclitaxel dose, the history of estrogen-related diseases, hypertension and BMI are considered risk factors for PIPN in paclitaxel-based chemotherapy of female cancer patients. It appears an urgent need to establish a guideline of evidence-based pharmacotherapy for PIPN.

Cystitis-related bladder pain involves RAGE activation by HMGB1, and increased Cav3.2 T-type Ca2+ channel activity by H2S, generated by upregulated cystathionine-&gamma, 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-&kappa, B 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.

Given our recent evidence for the role of high mobility group box 1 (HMGB1) in chemotherapy-induced peripheral neuropathy (CIPN) in rats, we examined the origin of HMGB1 and the upstream and downstream mechanisms of HMGB1 release involved in paclitaxel-induced neuropathy in mice. Paclitaxel treatment developed mechanical allodynia in mice, as assessed by von Frey test, which was prevented by an anti-HMGB1-neutralizing antibody or thrombomodulin alfa capable of inactivating HMGB1. RAGE or CXCR4 antagonists, ethyl pyruvate or minocycline, known to inhibit HMGB1 release from macrophages, and liposomal clodronate, a macrophage depletor, prevented the paclitaxel-induced allodynia. Paclitaxel caused upregulation of RAGE and CXCR4 in the dorsal root ganglia and macrophage accumulation in the sciatic nerve. In macrophage-like RAW264.7 cells, paclitaxel evoked cytoplasmic translocation of nuclear HMGB1 followed by its extracellular release, and overexpression of CBP and PCAF, histone acetyltransferases (HATs), known to cause acetylation and cytoplasmic translocation of HMGB1, which were suppressed by ethyl pyruvate, N-acetyl- l -cysteine, an anti-oxidant, and SB203580 and PDTC, inhibitors of p38 MAP kinase (p38MAPK) and NF-κB, respectively. Paclitaxel increased accumulation of reactive oxygen species (ROS) and phosphorylation of p38MAPK, NF-κB p65 and I-κB in RAW264.7 cells. In mice, N-acetyl- l -cysteine or PDTC prevented the paclitaxel-induced allodynia. Co-culture of neuron-like NG108-15 cells or stimulation with their conditioned medium promoted paclitaxel-induced HMGB1 release from RAW264.7 cells. Our data indicate that HMGB1 released from macrophages through the ROS/p38MAPK/NF-κB/HAT pathway participates in the paclitaxel-induced peripheral neuropathy in mice, and unveils an emerging therapeutic avenue targeting a neuroimmune crosstalk in CIPN.

Since 6-prenylnaringenin (6-PNG) was recently identified as a novel T-type calcium channel blocker with the IC50 value around 1 µM, a series of flavanone derivatives were designed, synthesized and subsequently evaluated for T-channel-blocking activity in HEK293 cells transfected with Cav3.2 T-type channels using a patch-clamp technique. As a result, several new flavanones blocked Cav3.2-dependent T-currents more potently than 6-PNG. In the synthesized compounds, 6-(3-ethylpent-2-enyl)-5,7-dihydroxy-2-(2-hydroxyphenyl)chroman-4-one 8j, 6-(3-ethylpent-2-enyl)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one 11b, 6-(2-cyclopentylideneethyl)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one 11d, and 6-(2-Cyclopentylethyl)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one 12c were more potent blocker than 6-PNG with the IC50 value of 0.39, 0.26, 0.46, and 0.50 µM, respectively. Among the above four derivatives, the compound 8j provided the best result in the in vivo experiments; i.e. systemic administration of 8j at the minimum dose completely restored neuropathic pain induced by partial sciatic nerve ligation in mice.

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.

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.

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.

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. Keywords: Hydrogen sulfide, Cav3.2 T-type calcium channel, Pain

Background: Hydrogen sulfide (H2S), a gasotransmitter, is generated from L-cysteine by mainly 3 enzymes, cystathionine-γ-lyase (CSE), cystathionine-β-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 Cav3.2 T-type Ca2+ channels mediate some of the biological activity of H2S, we focus on the role of the H2S/Cav3.2 pathway in regulating the neuronal and neuroendocrine function. Summary: In the neuronal system, H2S regulates the activity of various ion channels including Cav3.2. Exogenous and endogenous H2S enhances the Cav3.2 channel activity, promoting somatic and visceral pain signaling. The H2S/Cav3.2 pathway also facilitates neuritogenesis or neuronal differentiation. Interestingly, endogenous H2S formed by CSE regulates secretory function by enhancing Cav3.2 channel activity in neuroendocrine-differentiated prostate cancer cells or carotid glomus cells. Key Messages: The H2S/Cav3.2 pathway may serve as therapeutic targets for treatment of intractable pain, neuronal injury, androgen-independent prostate cancer, cardiovascular diseases, etc.

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.

Cav3.2 T-type Ca2+ channels are expressed in the primary afferents and play a pronociceptive role. The activity of Cav3.2 is enhanced by H2S, a gasotransmitter, and suppressed by ascorbic acid (vitamin C) through metal-catalyzed oxidation of the Zn2+-binding His191 in Cav3.2. Since rodents, but not humans, are capable of synthesizing ascorbic acid, the present study examined the role of ascorbic acid in nociceptive processing, using the mice lacking GNL/SMP30, an enzyme essential for ascorbic acid biosynthesis. Intraplantar and intracolonic administration of NaHS, an H2S donor, caused somatic allodynia and referred hyperalgesia, respectively, and repeated treatment with paclitaxel produced neuropathic allodynia in wild-type mice, all of which were suppressed by ascorbic acid or T-type Ca2+ channel blockers. Dietary ascorbic acid restriction caused dramatic decreases in plasma and tissue ascorbic acid levels in GNL/SMP30-knockout, but not wild-type, mice. The ascorbic acid restriction enhanced the somatic and visceral hypersensitivity following intraplantar and intracolonic NaHS, respectively, and paclitaxel-induced neuropathy in GNL/SMP30-knockout mice, while it had no such effect in wild-type mice. Together, our data unveil the critical role of ascorbic acid in regulating somatic and visceral pain sensitivity and support accumulating clinical evidence for the usefulness of ascorbic acid in pain management.

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 Ca 2+ 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.

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.

Hydrogen sulfide (H2S) is considered the third gasotransmitter following nitric oxide (NO) and carbon monoxide (CO) in the mammalian body including the brain, heart, blood vessels, liver, kidney, pancreas, lung, gastrointestinal tract and reproductive organs. H2S is formed endogenously from L-cysteine by multiple enzymes, such as cystathionine-γ-lyase, cystathionine-β-synthase and 3-mercaptopyruvate sulfurtransferase in combination with cysteine aminotransferase, and participates in a variety of biological events through a number of target molecules. Exogenous and/or endogenous H2S enhances the activity of T-type Ca(2+) channels in NG108-15 cells and isolated dorsal root ganglion neurons that abundantly express Cav3.2, and in Cav3.2-transfected HEK293 cells. Cav3.2 mediates not only the H2S-induced enhancement of pain signals in nociceptor neurons, but also neuronal differentiation characterized by neuritogenesis and functional upregulation of high voltage-activated Ca(2+) channels in NG108-15 cells. In this review, we focus on the functional modulation by H2S of primarily Cav3.2 T-type Ca(2+) channels and the molecular mechanisms underlying the H2S-induced neuronal differentiation.

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.

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 (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.

The rodents exposed to repeated cold stress according to a specific schedule, known as specific alternation of rhythm in temperature (SART), exhibit autonomic imbalance, and is now used as an experimental model of fibromyalgia. To explore the susceptibility of SART-stressed animals to novel acute stress, we tested whether exposure of mice to SART stress for 1 week alters the extent of acute restraint stress-induced hyperthermia. Mice were subjected to 7-d SART stress sessions; i.e., the mice were alternately exposed to 24 and 4°C at 1-h intervals during the daytime (09:00-16:00) and kept at 4°C overnight (16:00-09:00). SART-stressed and unstressed mice were exposed to acute restraint stress for 20-60 min, during which rectal temperature was monitored. Serum corticosterone levels were measured before and after 60-min exposure to restraint stress. SART stress itself did not alter the body temperature or serum corticosterone levels in mice. Acute restraint stress increased the body temperature and serum corticosterone levels, both responses being greater in SART-stressed mice than unstressed mice. The enhanced hyperthermic responses to acute restraint stress in SART-stressed mice were significantly attenuated by SR59230A, a β3 adrenoceptor antagonist, but unaffected by diazepam, an anxiolytic, mifepristone, a glucocorticoid receptor antagonist, or indomethacin, a cyclooxygenase inhibitor. These results suggest that SART stress enhances the susceptibility of mice to acute restraint stress, characterized by increased hyperthermia and corticosterone secretion, and that the increased hyperthermic responses to acute stress might involve accelerated activation of sympathetic β3 adrenoceptors, known to regulate non-shivering thermogenesis in the brown adipose tissue.

Given that Cav3.2 T-type Ca2+ channels were functionally regulated by asparagine (N)-linked glycosylation, we examined effects of high glucose on the function of Cav3.2, known to regulate secretory function, in neuroendocrine-like differentiated prostate cancer LNCaP cells. High glucose accelerated the increased channel function and overexpression of Cav3.2 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 Cav3.2 in neuroendocrine-like differentiated prostate cancer cells.

Hydrogen sulfide (H(2)S), formed by multiple enzymes, including cystathionine-γ-lyase (CSE), targets Ca(v)3.2 T-type Ca(2+) 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 H(2)S 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 H(2)S 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 H(2)S-induced pancreatic nociception in naive mice and suggest that, in the mice with pancreatitis, Ca(v)3.2 targeted by H(2)S primarily participates in the pancreatic pain, whereas TRPA1 is downregulated and plays a secondary role in pancreatic nociceptive signaling.

Hydrogen sulfide (H2S), a gasotransmitter, is formed from l -cysteine by multiple enzymes including cystathionine-γ-lyase (CSE). We have shown that an H2S donor, NaHS, causes hyperalgesia in rodents, an effect inhibited by knockdown of Cav3.2 T-type Ca2+ channels (T-channels), and that NaHS facilitates T-channel-dependent currents (T-currents) in NG108-15 cells that naturally express Cav3.2. In the present study, we asked if endogenous and exogenous H2S participates in regulation of the channel functions in Cav3.2-transfected HEK293 (Cav3.2-HEK293) cells. dl -Propargylglycine (PPG), a CSE inhibitor, significantly decreased T-currents in Cav3.2-HEK293 cells, but not in NG108-15 cells. NaHS at 1.5 mM did not affect T-currents in Cav3.2-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 Cav3.2-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 Cav3.2-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 Cav3.2 T-channels is tonically enhanced by endogenous H2S synthesized by CSE in Cav3.2-HEK293 cells, and that exogenous H2S is capable of enhancing Cav3.2 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.

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.

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.

Low-voltage-activated T-type Ca2+ channels (T-channels), especially Cav3.2 among the three isoforms (Cav3.1, Cav3.2, and Cav3.3), are now considered to play pivotal roles in processing of pain signals. Cav3.2 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. Cav3.2 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 Cav3.2 T-channels is involved in the pathophysiology of inflammatory, neuropathic, and visceral pain. Thus, Cav3.2 T-channels are considered to serve as novel targets for development of drugs for treatment of intractable pain resistant to currently available analgesics. Keywords:: Cav3.2, T-type calcium channel, neuropathy, visceral pain, hydrogen sulfide

Lipopolysaccharide (LPS) induces hyperthermia accompanied by various other systemic inflammatory symptoms. The rodents exposed to repeated cold (RC) stress according to a specific schedule are useful as experimental models for autonomic imbalance or fibromyalgia. It is now proven that RC-stressed mice exhibit tolerance to LPS, we examined thermal responses to LPS challenge in RC-stressed mice by monitoring core temperature using the telemetry system. Systemic administration of LPS caused bimodal hyperthermic responses in RC-stressed and unstressed mice. The magnitude of the LPS-induced hyperthermia was greater in RC-stressed mice than in unstressed mice. The RC stress-induced enhancement of hyperthermic responses to LPS was abolished by pretreatment with diclofenac, which is a cyclooxygenase (COX) inhibitor. LPS did not significantly increase COX-2 protein levels in the lung or hypothalamus of RC-stressed or unstressed mice. RC stress did not alter baseline serum corticosterone levels or their increases in response to LPS challenge. These results suggest that RC stress enhances the susceptibility of mice to LPS challenge, leading to greater prostanoid-dependent hyperthermia, which might contribute to tolerance to LPS in RC-stressed mice.

Hydrogen sulfide (H2S), the third gasotransmitter, is endogenously generated by certain H2S synthesizing enzymes, including cystathionine-γ-lyase (CSE) and cystathionine-β-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 β-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.

Background and purpose Hydrogen sulfide (H(2) S), generated by enzymes such as cystathionine-γ-lyase (CSE) from L-cysteine, facilitates pain signals by activating the Ca(v) 3.2 T-type Ca(2+) channels. Here, we assessed the involvement of the CSE/H(2) S/Ca(v) 3.2 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 H(2) S 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 55-0396, blockers of T-type Ca(2+) channels, administered after the symptoms of cystitis appeared, reversed the nociceptive changes. Further, silencing of Ca(v) 3.2 protein by repeated intrathecal administration of mouse Ca(v) 3.2-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 55-0396. Conclusion and implications Endogenous H(2) S, generated by up-regulated CSE, caused bladder pain and referred hyperalgesia through the activation of Ca(v) 3.2 channels, one of the T-type Ca(2+) 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.