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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.

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

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.

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.

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.

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.

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.

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.

Neuroendocrine-differentiated prostate cancer cells may contribute to androgen-independent proliferation of surrounding cells through Ca(2+)-dependent secretion of mitogenic factors. Human prostate cancer LNCaP cells, when neuroendocrine-differentiated, overexpress Cav3.2 T-type Ca(2+) channels that contribute to Ca(2+)-dependent secretion. Given evidence for the acceleration of Cav3.2 activity by hydrogen sulfide (H2S), we examined the roles of the H2S/Cav3.2 pathway and then analyzed the molecular mechanisms of the Cav3.2 overexpression in neuroendocrine-differentiated LNCaP cells. LNCaP cells were differentiated by dibutyryl cyclic AMP. Protein levels and T-type Ca(2+) channel-dependent 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, androgen-independent proliferation and upregulation of mitogenic factors, and also showed elevation of Cav3.2 expression or T-currents. Expression of cystathionine-γ-lyase (CSE) and cystathionine-β-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 Ca(2+) channels. During differentiation, Egr-1 and REST, positive and negative transcriptional regulators for Cav3.2, were upregulated and downregulated, respectively, and Egr-1 knockdown prevented the Cav3.2 overexpression. Our data suggest that, in neuroendocrine-differentiated LNCaP cells, H2S formed by the upregulated CSE promotes the activity of the upregulated Cav3.2, leading to the elevated secretory functions. The overexpression of Cav3.2 appears to involve upregulation of Egr-1 and downregulation of REST.

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.

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.

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.

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.

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, 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.

Luminal hydrogen sulfide (H(2)S) causes colonic pain and referred hyperalgesia in mice through activation of T-type Ca(2+) channels. To test a hypothesis that H(2)S might chelate and remove endogenous Zn(2+) that inhibits the Ca(v)3.2 isoform of T-type Ca(2+) channels, facilitating visceral nociception, we asked if intracolonic (i.col.) administration of Zn(2+) chelators mimics H(2)S-induced visceral nociception. Visceral nociceptive behavior and referred abdominal allodynia/hyperalgesia were determined after i.col. administration of NaHS, a donor for H(2)S, or Zn(2+) chelators in mice. Phospholylation of extracellular signal-regulated protein kinase (ERK) in the spinal cord was analyzed by immunohistochemistry. The visceral nociceptive behavior and referred abdominal allodynia/hyperalgesia caused by i.col. NaHS in mice were abolished by i.col. preadministration of zinc chloride (ZnCl(2)), known to selectively inhibit Ca(v)3.2, but not Ca(v)3.1 or Ca(v)3.3, isoforms of T-type Ca(2+) channels, and by i.p. preadministration of mibefradil, a pan-T-type Ca(2+) channel blocker. Two distinct Zn(2+) chelators, N,N,N',N'-tetrakis(2-pyridylmethyl)ehylenediamine (TPEN) and dipicolinic acid, when administered i.col., mimicked the NaHS-evoked visceral nociceptive behavior and referred abdominal allodynia/hyperalgesia, which were inhibited by mibefradil and by NNC 55-0396, another T-type Ca(2+) channel blocker. Like i.col. NaHS, i.col. TPEN caused prompt phosphorylation of ERK in the spinal dorsal horn, an effect blocked by mibefradil. Removal of luminal Zn(2+) by H(2)S and other Zn(2+) chelators thus produces colonic pain through activation of T-type Ca(2+) channels, most probably of the Ca(v)3.2 isoform. Hence, endogenous Zn(2+) is considered to play a critical role in modulating visceral pain.

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 agonists 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.

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.

We investigated effect of hydrogen sulfide (H(2)S) on oxidative stress-caused cell death in gastric mucosal epithelial cells. In rat normal gastric epithelial RGM1 cells, NaHS, a H(2)S donor, at 1.5mM strongly suppressed hydrogen peroxide (H(2)O(2))-caused cell death, while it slightly augmented the H(2)O(2) toxicity at 0.5-1mM. The protective effect of NaHS was abolished by inhibitors of MEK or JNK, but not of p38 MAP kinase. NaHS at 1.5mM 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/H(2)S may protect gastric mucosal epithelial cells against oxidative stress through stimulation of MAP kinase pathways, a therapeutic dose range being very narrow.

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 PAR1-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 metalloproteinases (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 PAR1-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 PGE 2 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.

Receptor-activating peptides for protease-activated receptors (PARs) 1 or 2 enhance gastric mucosal blood flow (GMBF) and protect against gastric mucosal injury in rats. We thus examined and characterized the effects of PAR-1 and PAR-2 agonists on the isometric tension in isolated rat gastric artery. The agonists for PAR-2 or PAR-1 produced vasodilation in the endothelium-intact arterial rings, which was abolished by removal of the endothelium. The mechanisms underlying the PAR-2- and PAR-1-mediated relaxation involved NO, endothelium-derived hyperpolarizing factor (EDHF) and prostanoids, to distinct extent, as evaluated by use of inhibitors of NO synthase, cyclo-oxygenase and Ca2+-activated K+ channels. The EDHF-dependent relaxation responses were significantly attenuated by gap junction inhibitors. These findings demonstrate that endothelial PAR-1 and PAR-2, upon activation, dilate the gastric artery via NO and prostanoid formation and also EDHF mechanisms including gap junctions, which would enhance GMBF.

We evaluated if ONO-1714, known as an inducible nitric oxide synthase (iNOS) inhibitor, could inhibit neuronal NOS (nNOS) and exert antinociception. ONO-1714 potently inhibited both crude rat cerebellar NOS and recombinant human nNOS in vitro. Systemic ONO-1714 at 1–10 mg/kg suppressed carrageenan-induced thermal hyperalgesia in rats, an effect being equivalent to the antinociception caused by l -NAME or 7-nitroindazole at 25 mg/kg. The same doses of ONO-1714 also caused hypertension. Intrathecal (i.t.) ONO-1714 potently reduced the hyperalgesia, the effective dose range (0.2–0.6 μg/rat) being much lower than the antinociceptive dose (150 μg/rat) of i.t. l -NAME. Thus, ONO-1714 is considered a potent inhibitor of nNOS in addition to iNOS. The distinct relative antinociceptive activities of systemic and i.t. ONO-1714 are attributable to its possible poor blood–brain barrier permeability.

Overproduction of nitric oxide (NO) in the liver has been implicated as an important event in endotoxin shock and in other models of hepatic inflammation and injury. The present study was undertaken to evaluate the effect of ONO-1714, a potent and specific inhibitor of inducible NO synthase (iNOS), on acetaminophen-induced hepatotoxicity in the rats. Oral administration of ONO-1714 dose-dependently inhibited NOx (NO2- and NO3-) accumulation in rat plasma after lipopolysaccharide (LPS) treatment. Intraperitoneal acetaminophen at 1 g/kg caused damage to the centrilobular regions of the liver and increase in serum alanine and aspartate transaminase (ALT and AST, respectively) levels accompanied by elevated plasma NOx levels after 24 h. Oral administration of ONO-1714 at 10 and 100 microg/kg dose-dependently reduced the acetaminophen-induced hepatic tissue damage and the increases in serum ALT and AST levels. ONO-1714 also blocked the increase in plasma NOx concentrations. These findings demonstrate that oral ONO-1714, an iNOS inhibitor, protects against acetaminophen-evoked hepatic inflammation/injury, strongly suggesting that NO produced by iNOS plays a key role in the pathogenesis of this drug-induced hepatotoxicity.

Protease-activated receptor-2, a G protein-coupled receptor activated by serine proteases such as trypsin, tryptase and coagulation factors VIIa and Xa, modulates pancreatic and salivary exocrine secretion. In the present study, we examined the distribution of PAR-2 in the pancreas and parotid gland, and characterized the PAR-2-mediated secretion of amylase by these tissues in vivo. Immunohistochemical analyses using the polyclonal antibody against rat PAR-2 clearly showed abundant expression of PAR-2 in rat pancreatic and parotid acini. The PAR-2 agonist SLIGRL-NH2, administered intraperitoneally (i.p.) at 1-10 micromol/kg and 1.5-15 micromol/kg, in combination with amastatin, an aminopeptidase inhibitor, facilitated in vivo secretion of pancreatic and salivary amylase in a dose-dependent manner, respectively, in the mouse. The PAR-2-mediated secretion of pancreatic amylase was abolished by pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor. The secretion of salivary amylase in response to the PAR-2 agonist at a large dose, 15 micromol/kg, but not at a smaller dose, 5 micromol/kg, was partially reduced by L-NAME. Pretreatment with capsaicin for ablation of the sensory neurons did not modify the PAR-2-mediated secretion of pancreatic and salivary amylase in the mouse. In conclusion, our study demonstrates expression of PAR-2 in rat pancreatic acini as well as parotid acini and indicates that nitric oxide participates in the PAR-2-mediated in vivo secretion of pancreatic amylase, and, to a certain extent, of salivary amylase, although capsaicin-sensitive sensory neurons, known to be activated by PAR-2, are not involved in the evoked pancreatic or salivary amylase secretion.

Protease-activated receptor-2 (PAR-2), expressed in sensory neurons, triggers thermal hyperalgesia, nociceptive behavior and spinal Fos expression in rats. In the present study, we examined if the nociceptive processing by PAR-2 is mediated by trans-activation of capsaicin receptors. The thermal hyperalgesia following an intraplantar (i.pl.) administration of the PAR-2-activating peptide SLIGRL-NH2 was completely abolished by the capsaicin receptor antagonist capsazepine. In contrast, neither the nociceptive behavior nor spinal Fos expression in response to i.pl. SLIGRL-NH2 were attenuated by capsazepine. Our data imply that trans-activation of capsaicin receptors by PAR-2 might be involved in the PAR-2-triggered thermal hyperalgesia, but not nociception.

Protease-activated receptor-2 (PAR-2) and/or effector cell protease receptor-1 (EPR-1) may mediate the direct cellular actions of coagulation factor Xa in some cultured cell lines. The present study examined if factor Xa could actually evoke relaxation through either of these receptor systems in isolated rat aorta. Factor Xa at 8.5-85 nM, like the PAR-2-activators trypsin and SLIGRL-NH(2), produced nitric oxide-dependent relaxation in the precontracted aortic rings. PAR-2 desensitization abolished relaxation responses to factor Xa as well as trypsin in the rings. The factor Xa interepidermal growth factor synthetic peptide L(83)FTRKL(88)(G)-NH(2), known to block factor Xa binding to EPR-1, failed to inhibit factor Xa-evoked relaxation in the preparations. Our findings provide evidence that factor Xa evokes relaxation by activating PAR-2, but independently of EPR-1, in the rat aorta. The factor Xa-PAR-2 pathway might thus contribute to the severe hypotension during sepsis, in which multiple coagulation factors including factor X would become activated and PAR-2 would be induced.

Urokinase type plasminogen activator (uPA) may influence brain pathophysiology after injury. We studied disruption of the blood-brain barrier (BBB) and changes in the vasculature after a brain stab wound in uPA-deficient, uPA receptor-deficient, and PA inhibitor-1 (PAI-1) deficient mice. The extravasation of immunoglobulin was greater in PAI-1 deficient mice; less pronounced in uPA-deficient mice; similar to controls in uPA receptor-deficient mice. Vasculatures in the wound proliferated in PAI-1 deficient mice. Our study shows that uPA affects BBB disruption. PA enhances angiogenesis after brain injury.

We developed a convenient and specific method for the determination of mucin-type glycoproteins using galactose oxidase and horseradish peroxidase on the basis of the contents of galactosyl and N -acetylgalactosaminyl residues in glycoproteins. Galactose and galactosamine residues released from glycoproteins after hydrolysis were oxidized with galactose oxidase and subsequently the resultant hydrogen peroxide was determined by a combination of horseradish peroxidase and 3-( p -hydroxyphenyl) propionic acid as a fluorogenic substrate. The contents of galactose/galactosamine residues in N - and O -glycans, as determined by the galactose oxidase-peroxidase method, were in good agreement with those described in the previous reports. We applied the present method to determine mucin-type glycoproteins secreted from rat gastric mucosa by stimulation with misoprostol, a prostaglandin E 1 analogue in vivo. Thus, the galactose oxidase-peroxidase method is useful for the determination of mucin-type glycoproteins in biological materials.

Activation of protease-activated receptor (PAR)-1 or PAR-2 elicits inflammation most probably via mast cell degranulation in vivo. The present study aimed at characterizing PARs in rat peritoneal mast cells (PMC). Messenger RNA for PAR-1, but not for PAR-2, was detected in PMC. Thrombin, the PAR-1 agonist SFLLR-NH2 or the PAR-2 agonist SLIGRL-NH2 failed to induce histamine release from PMC. Surprisingly, the PAR-2-inactive control peptide LSIGRL-NH2 triggered histamine release from PMC. Thus, PAR-1, but not PAR-2, are expressed in PMC, whereas neither PAR-1 nor PAR-2 are considered to be involved in degranulation of PMC. LSIGRL-NH2 does not appear to be appropriate as a control peptide for PAR-2 in inflammation studies.

The relationship between the accumulation of platinum in the cerebral cortex following cisplatin administration and injury to the blood-brain barrier after lipopolysaccharide (LPS) treatment was investigated. The appearance of intravenously injected fluorescein in the brain was significantly increased 10-24 h after LPS treatment, the effect being dose-dependent. Platinum was detectable in the cerebral cortex of cisplatin-treated mice 24 h after LPS treatment, but not without LPS treatment. In mice pretreated with α-tocopherol, LPS administration did not significantly augment fluorescein penetration into the brain, whereas pretreatment with either allopurinol or ascorbic acid did not modify the LPS-induced increase in fluorescein penetration. In contrast, platinum in the cerebral cortex after cisplatin administration was still detectable in the allopurinol-, ascorbic acid-, and α-tocopherol-pretreated groups, and the levels of platinum in these groups were not significantly different from those in the group treated with LPS only. Administration of superoxide dismutase (SOD), but not of catalase, tended to inhibit the penetration of fluorescein. Both SOD and catalase significantly lowered platinum content in the cerebral cortex following cisplatin administration in mice treated with LPS. Thus, free radicals may injure the blood-brain barrier in mice challenged with LPS, and allow cisplatin to penetrate into the cerebral cortex, resulting in platinum accumulation.

Kyotorphin, an endogenous [Met5]enkephalin-releasing antinociceptive dipeptide (L-Tyr-L-Arg), is formed by kyotorphin synthetase from its constituent amino acids, L-Tyr and L-Arg, in the brain in an ATP-Mg(2+)-dependent manner. To elucidate the physiological role of kyotorphin in organs other than the brain, we examined the activity of kyotorphin synthetase in the rat adrenal glands and spinal cord. By Sephacryl S-300 gel-filtration chromatography of the soluble extracts from both the organs, the enzyme activity forming immunoreactive kyotorphin from L-Tyr and L-Arg in the presence of ATP and MgCl2 was detected in the fractions with the molecular mass of 200-300 kDa, being drastically reduced by the omission of ATP and MgCl2 from the reaction medium. The Km values of the partially purified adrenal and spinal kyotorphin synthetase for L-Tyr, L-Arg, ATP, and MgCl2 were close to those of the brain enzyme. The activity of adrenal kyotorphin synthetase was inhibited by some L-Arg analogues. NG-nitro-L-arginine methyl ester, alpha-methyl-L-ornithine and D-Arg, but not by NG-nitro-L-arginine and N-iminoethyl-L-ornithine. In the crude soluble extracts from the adrenal glands and spinal cord, kyotorphin was formed by kyotorphin synthetase, and also by the enzymatic processing of the precursor proteins, in the presence of physiological concentrations of L-Tyr and L-Arg in addition to ATP and MgCl2. Thus, kyotorphin synthetase resembling that in the brain is present in the rat adrenal glands and spinal cord. The present findings may predict a functional role of the L-Arg-kyotorphin pathway in these organs.

Protease-activated receptor-2 (PAR-2) is abundantly expressed in gastric mucosal chief cells, facilitating pepsinogen secretion. In the present study, we investigated whether PAR-1, a thrombin receptor, could modulate pepsinogen secretion in rats. The PAR-1-activating peptide TFLLR-NH(2) as well as the PAR-2-activating peptide SLIGRL-NH(2), administered i.v. repeatedly at 1-h intervals, significantly increased gastric pepsinogen secretion over 2-4 h (after two to four doses). In contrast, the control peptide FTLLR-NH(2), given in the same manner, had no such effect. Thus, PAR-1, like PAR-2, might function to facilitate pepsinogen secretion, suggesting a novel role of the thrombin-PAR-1-pathway in the stomach.

Abnormal hemostatic profiles indicating hemorrhagic tendency have been reported in rodents exposed to prolonged fluctuation in ambient temperature, known as SART (specific alternation of rhythm in temperature)-stressed animals. In this study, investigation was made of possible involvement of oxygen-derived free radicals in the development of stress-induced hemostatic alteration. SART-stressed rats and mice exhibited marked decrease in platelet count, fibrinogen level and factor VIII:C activity. Superoxide dismutase, when administered s.c. twice a day to mice for 7 days of stress exposure, inhibited the above alterations. Catalase given in the same manner, had essentially the same effect, though to a lesser extent. Allopurinol administered orally once daily during stress reduced stress-induced thrombocytopenia, but caused considerable increase in fibrinogen and factor VIII:C activity in stressed and unstressed mice. Lipid peroxide significantly increased in the heart but not in the plasma following stress exposure in rats and mice. Active oxygens would thus appear to be, at least partially, involved in the development of abnormal hemostasis induced by SART stress.

L-Tyrosine methyl ester (L-Tyr-OMe) produces naloxone-reversible antinociception in mice. This effect was characterized in the present study. L-Tyr-OMe administered s.c. at doses of 100-400 mg/kg elicited dose-dependent antinociception, which was antagonized by s.c. and i.c.v. naloxone at 1 mg/kg and 10 ng/mouse, respectively, but not by i.t. injection at 10 ng/mouse. The antinociceptive effect of systemic L-Tyr-OMe was also inhibited by naltrindole (0.1 mg/kg s.c.), a selective delta-opioid receptor antagonist, but was resistant to L-leucyl-L-arginine (3 micrograms/mouse i.c.v.), a kyotorphin (L-tyrosyl-L-arginine) receptor antagonist. This effect was reduced by i.t., but not by i.c.v., phentolamine at 0.1-1 microgram/mouse, while methysergide (10 microgram/mouse i.t.) and p-chlorophenylalanine (500 mg/kg s.c.) did not have such an inhibitory effect. L-Tyr-OMe, given i.c.v. and i.t. at 0.1-10 microgram/mouse, also produced dose-dependent antinociception, the latter effect being naloxone-reversible but resistant to i.t. phentolamine. These results suggest that L-Tyr-OMe (s.c.)-induced antinociception is mediated by central delta-opioid receptors and by the bulbo-spinal noradrenergic system, but not by the central 'kyotorphinergic' system, and that L-Tyr-OMe, when given i.t., produces antinociception via spinal opioidergic, but not noradrenergic systems.

Platelet hypoaggregability has been reported in rats exposed to a chronic form of environmental stress induced by long-lasting fluctuation in air temperature, known as SART (specific alternation of rhythm in temperature) stress. This study examines functional characteristics of platelets from stressed rats in more detail. Exposure to stress reduced aggregation and ATP release in platelets stimulated with collagen, as determined using platelet-rich plasma (PRP). The resting levels of ATP but not ADP in platelets from stressed rats were lower than those from unstressed ones. Collagen-induced release and resting level of serotonin also decreased in platelets from stressed rats. In contrast, stress failed to cause hypoaggregability of washed platelets. Circulating platelet aggregates were detected in stressed rats. From these data, SART stress appears to cause intravascular activation of platelets in spite of in vitro hypofunctions. Alteration in plasma milieu may be associated with stress-induced platelet hypofunctions in PRP.

A prolongation of bleeding time accompanied by thrombocytopenia and abnormalities in coagulation-fibrinolysis systems has been observed in laboratory animals exposed to a chronic form of environmental stress induced by severe fluctuations of air temperature, known as SART (specific alternation of rhythm in temperature) stress. In order to clarify the hemostatic profile under SART stress in more detail, the present study examined platelet aggregability in vitro as well as in vivo in stressed rats. During exposure to stress, thrombocytopenia developed from day 5, and remained up to at least day 14. In vitro aggregation of platelets stimulated by ADP or collagen was markedly decreased in stressed rats, compared with unstressed rats. Furthermore, stressed rats exhibited in vivo hypoaggregability of platelets, as estimated by the magnitude of the drop in circulating platelet counts following intravenous injection of ADP and collagen. Protein and cholesterol content in platelets remained constant after stress exposure. These results indicate that SART-stressed rats exhibit platelet dysfunctions in addition to thrombocytopenia. Considering the previous findings, the hemostatic system under SART stress appears to show a general tendency toward hemorrhage.

Kyotorphin (KTP), an antinociceptive dipeptide (Tyr-Arg), is formed by KTP synthetase from L-Tyr and L-Arg in the brain. We examined the effects of various L-Arg analogues on immunoreactive KTP (iKTP) formation by KTP synthetase purified partially from rat brain. The NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), but not NG-nitro-L-arginine and N-iminoethyl-L-ornithine, suppressed iKTP formation by KTP synthetase from 1 mM of L-Arg and L-Tyr, the IC50 value being 2.33 mM. Similarly, alpha-methyl-L-ornithine (alpha-MO) inhibited KTP synthetase, the IC50 value being 2.51 mM. D-Arg at high concentrations also exhibited a weak inhibitory effect. Kinetic experiments indicated that the inhibition by L-NAME and alpha-MO of KTP synthetase is competitive. Thus, these L-Arg analogues appear to act as the competitive inhibitor of KTP synthetase.

H2S, a gasotransmitter, is formed by multiple enzymes including cystathionine-gamma-lyase (CSE). H2S enhances function of Cav3.2 T-type calcium channels in NG108-15 cells, isolated dorsal root ganglion neurons and Cav3.2-transfected HEK293 cells, and the H2S/Cav3.2 pathway plays a role in somatic and visceral pain signaling. H2S also causes Cav3.2-dependent neuronal differentiation characterized by neuritogenesis in NG108-15 cells. Interestingly, androgen-sensitive human prostate cancer LNCaP cells exhibit neuroendocrine-like differentiation following elevation of intracellular cyclic AMP levels or androgen deprivation, and the differentiated cells abundantly express Cav3.2 responsible for calcium-dependent secretion of proliferation factors. We have found that neuroendocrine-like differentiation causes upregulation of CSE as well as Cav3.2, and endogenous H2S formed by CSE facilitates Cav3.2 function and then secretory responses in LNCaP cells. Expression of Egr1 at an early stage appears to be involved in transcriptional upregulation of Cav3.2. Our study thus suggest that H2S formed by upregulated CSE regulates secretory function via activation of upregulated Cav3.2 in neuroendocrine-like differentiated LNCaP cells, contributing to androgen-independent proliferation of surrounding cells, and the CSE/H2S/Cav3.2 pathway might serve as targets for pharmacological interventions for preventing acquisition of hormone therapy resistance of prostate cancer.

H2S induces somatic and visceral hyperalgesia via Cav3.2 T-type Ca2+ channels (T-channels) and also TRPA1 channels. Interestingly, ascorbic acid (AA) selectively inhibits Cav3.2, and AA administration inhibits H2S-induced hyperalgesia and neuropathic pain. Here we examined effect of AA deficiency on H2S-evoked somatic or visceral hyperalgesia, using SMP30/GNL knockout (KO) mice that are incapable of synthesizing AA and dependent on dietary AA. After weaning, the KO mice were fed on an AA-deficient diet and had free access to water containing sufficient (1.5 g/L) AA [AA (+)] or only minimum AA (0.0375 g/L) [AA (−)] for seven weeks. The AA content dramatically decreased in the brain, spinal cord, plasma, hindpaw and colon from AA (−) KO mice, but not AA (+) KO mice, although AA deficiency was not induced by dietary control in wild-type (WT) mice. In WT mice, intraplantar (i.pl.) and intracolonic (i.col.) administration of NaHS, an H2S donor, caused hyperalgesia in the hindpaw and lower abdomen, respectively, as assessed by von Frey test, which was blocked by T-channel inhibitors or AA. In AA (−) KO mice, i.pl. and i.col. NaHS produced greater somatic and colonic pronociceptive activities, respectively, as compared with AA (+) KO mice and WT mice. In contrast, AA deficiency did not alter the pronociceptive effects of ally isothiocyanate, a TRPA1 agonist, in KO mice. Together, AA appears to modulate H2S-induced somatic/visceral pain most probably by limiting Cav3.2 function (1489).

Hydrogen sulfide (H 2 S) is a gasotransmitter synthesized by multiple enzymes including cystathionine-gamma-lyase (CSE) in the mammalian body. H 2 S donors cause neuronal differentiation characterized by neuritogenesis via activation of Ca v 3.2 T-type Ca 2+ channels (T-channels) in NG108-15 cells. Recently, we have shown that the cAMP/PKA pathway induces prompt phosphorylation and sensitization of Ca v 3.2 in NG108-15 cells. Thus, we investigated roles of the endogenous H 2 S/Ca v 3.2 pathway in the cAMP-dependent neuritogenesis in NG108-15 cells. Neuritogenesis was produced by stimulation with dibutyryl cAMP (db-cAMP) or Na 2 S, an H 2 S donor, for 16 h. The db-cAMP-induced neuritogenesis was suppressed by inhibitors of PKA, CSE, and T-channels, while the Na 2 S-induced neuritogenesis was reduced by inhibitors of T-channels, but not PKA. Stimulation with db-cAMP for 6 h caused functional and protein upregulation of Ca v 3.2, but did not alter CSE protein expression. A CSE inhibitor suppressed the persistently enhanced T-currents by 6-h db-cAMP stimulation, although it did not affect the rapid facilitation of T-currents by 10-min db-cAMP stimulation. Our data suggest that the cAMP/PKA pathway does not only cause rapid phosphorylation-mediated Ca v 3.2 sensitization, but also long-lasting functional facilitation of Ca v 3.2 through protein upregulation and of the CSE/H 2 S pathway that further enhances Ca v 3.2 function, resulting in neuritogenesis and possibly neuronal differentiation in NG108-15 cells.