Maho Matsunami

Luminal hydrogen sulfide plays a pronociceptive role in mouse colon

Objective: Given recent evidence that hydrogen sulfide (H2S), a gasotransmitter, promotes somatic pain through redox modulation of T-type Ca2+ channels, the roles of colonic luminal H2S in visceral nociceptive processing in mice were examined. Methods: After intracolonic administration of NaHS, an H2S donor, visceral pain-like behaviour and referred abdominal allodynia/hyperalgesia were evaluated. Phosphorylation of extracellular signal-regulated protein kinase (ERK) in the spinal dorsal horn was determined immunohistochemically. The whole-cell recording technique was used to evaluate T-type Ca2+ currents (T-currents) in cultured dorsal root ganglion (DRG) neurons. Results: Like capsaicin, NaHS, administered intracolonically at 0.5–5 nmol per mouse, triggered visceral nociceptive behaviour accompanied by referred allodynia/hyperalgesia in mice. Phosphorylation of ERK in the spinal dorsal horn was detected following intracolonic NaHS or capsaicin. The behavioural effects of intracolonic NaHS were abolished by a T-type channel blocker or an oxidant, but not inhibitors of L-type Ca2+ channels or ATP-sensitive K+ (KATP) channels. Intraperitoneal NaHS at 60 μmol/kg facilitated intracolonic capsaicin-evoked visceral nociception, an effect abolished by the T-type channel blocker, although it alone produced no behavioural effect. In DRG neurons, T-currents were enhanced by NaHS. Conclusions: These findings suggest that colonic luminal H2S/NaHS plays pronociceptive roles, and imply that the underlying mechanisms might involve sensitisation/activation of T-type channels probably in the primary afferents, aside from the issue of the selectivity of mibefradil.

Hyperalgesia induced by spinal and peripheral hydrogen sulfide: Evidence for involvement of Cav3.2 T-type calcium channels

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

Hydrogen sulfide as a novel mediator for pancreatic pain in rodents

Objective: Hydrogen sulfide (H2S) is formed from l-cysteine by multiple enzymes including cystathionine-γ-lyase (CSE) in mammals, and plays various roles in health and disease. Recently, a pronociceptive role for H2S in the processing of somatic pain was identified. Here, the involvement of H2S in pancreatic pain is examined. Methods: Anaesthetised rats or mice received an injection of NaHS, a donor for H2S, or capsaicin into the pancreatic duct, and the expression of spinal Fos protein was detected by immunohistochemistry. Pancreatitis was created by 6 hourly doses of caerulein in unanaesthetised mice, and pancreatitis-related allodynia/hyperalgesia was evaluated using von Frey hairs. CSE activity and protein levels in pancreatic tissues were measured using the colorimetric method and western blotting, respectively. Results: Either NaHS or capsaicin induced the expression of Fos protein in the superficial layers of the T8 and T9 spinal dorsal horn of rats or mice. The induction of Fos by NaHS but not capsaicin was abolished by mibefradil, a T-type Ca2+ channel blocker. In conscious mice, repeated doses of caerulein produced pancreatitis accompanied by abdominal allodynia/hyperalgesia. Pretreatment with an inhibitor of CSE prevented the allodynia/hyperalgesia, but not the pancreatitis. A single dose of mibefradil reversed the established pancreatitis-related allodynia/hyperalgesia. Either the activity or protein expression of pancreatic CSE increased after the development of caerulein-induced pancreatitis in mice. Conclusions: The data suggest that pancreatic NaHS/H2S most probably targets T-type Ca2+ channels, leading to nociception, and that endogenous H2S produced by CSE and possibly T-type Ca2+ channels are involved in pancreatitis-related pain.

Inhibition of T-type calcium channels and hydrogen sulfide-forming enzyme reverses paclitaxel-evoked neuropathic hyperalgesia in rats.

Hydrogen sulfide (H₂S), a gasotransmitter, facilitates pain sensation by targeting Ca(v)3.2 T-type calcium channels. The H₂S/Ca(v)3.2 pathway appears to play a role in the maintenance of surgically evoked neuropathic pain. Given evidence that chemotherapy-induced neuropathic pain is blocked by ethosuximide, known to block T-type calcium channels, we examined if more selective T-type calcium channel blockers and also inhibitors of cystathionine-γ-lyase (CSE), a major H₂S-forming enzyme in the peripheral tissue, are capable of reversing the neuropathic pain evoked by paclitaxel, an anti-cancer drug. It was first demonstrated that T-type calcium channel blockers, NNC 55-0396, known to inhibit Ca(v)3.1, and mibefradil inhibited T-type currents in Ca(v)3.2-transfected HEK293 cells. Repeated systemic administration of paclitaxel caused delayed development of mechanical hyperalgesia, which was reversed by single intraplantar administration of NNC 55-0396 or mibefradil, and by silencing of Ca(v)3.2 by antisense oligodeoxynucleotides. Systemic administration of dl-propargylglycine and β-cyanoalanine, irreversible and reversible inhibitors of CSE, respectively, also abolished the established neuropathic hyperalgesia. In the paclitaxel-treated rats, upregulation of Ca(v)3.2 and CSE at protein levels was not detected in the dorsal root ganglia (DRG), spinal cord or peripheral tissues including the hindpaws, whereas H(2)S content in hindpaw tissues was significantly elevated. Together, our study demonstrates the effectiveness of NNC 55-0396 in inhibiting Ca(v)3.2, and then suggests that paclitaxel-evoked neuropathic pain might involve the enhanced activity of T-type calcium channels and/or CSE in rats, but not upregulation of Ca(v)3.2 and CSE at protein levels, differing from the previous evidence for the neuropathic pain model induced by spinal nerve cutting in which Ca(v)3.2 was dramatically upregulated in DRG.

Gastrointestinal roles for proteinase-activated receptors in health and disease

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

Hydrogen sulfide‐induced mechanical hyperalgesia and allodynia require activation of both Cav3.2 and TRPA1 channels in mice

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.

Suppression of pancreatitis‐related allodynia/hyperalgesia by proteinase‐activated receptor‐2 in mice

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

ONO-8130, a selective prostanoid EP1 receptor antagonist, relieves bladder pain in mice with cyclophosphamide-induced cystitis.

&NA; Given the previous evidence for involvement of prostanoid EP1 receptors in facilitation of the bladder afferent nerve activity and micturition reflex, the present study investigated the effect of ONO‐8130, a selective EP1 receptor antagonist, on cystitis‐related bladder pain in mice. Cystitis in mice was produced by intraperitoneal administration of cyclophosphamide at 300 mg/kg. Bladder pain‐like nociceptive behavior and referred hyperalgesia were assessed in conscious mice. Phosphorylation of extracellular signal‐regulated kinase (ERK) in the L6 spinal cord was determined by immunohistochemistry in anesthetized mice. Cyclophosphamide treatment caused bladder pain‐like nociceptive behavior and referred hyperalgesia accompanying cystitis symptoms, including increased bladder weight and vascular permeability and upregulation of cyclooxygenase‐2 in the bladder tissue. Oral preadministration of ONO‐8130 at 0.3–30 mg/kg strongly prevented both the bladder pain‐like behavior and referred hyperalgesia in a dose‐dependent manner, but had slight effect on the increased bladder weight and vascular permeability. Oral ONO‐8130 at 30 mg/kg also reversed the established cystitis‐related bladder pain. Intravesical administration of prostaglandin E2 caused prompt phosphorylation of ERK in the L6 spinal cord, an effect blocked by ONO‐8130. Our findings strongly suggest that the prostaglandin E2/EP1 system participates in processing of cystitis‐related bladder pain, and that EP1 antagonists including ONO‐8130 are useful for treatment of bladder pain, particularly in interstitial cystitis. Prostaglandin E2 contributes to cystitis‐related bladder pain via EP1 receptors in mice, indicating possible therapeutic usefulness of selective EP1 antagonists.

Involvement of the endogenous hydrogen sulfide/Cav3.2 T-type Ca2+ channel pathway in cystitis-related bladder pain in mice

BACKGROUND AND PURPOSE Hydrogen sulfide (H2S), generated by enzymes such as cystathionine‐γ‐lyase (CSE) from L‐cysteine, facilitates pain signals by activating the Cav3.2 T‐type Ca2+ channels. Here, we assessed the involvement of the CSE/H2S/Cav3.2 pathway in cystitis‐related bladder pain.

Hydrogen sulfide evokes neurite outgrowth and expression of high-voltage-activated Ca2+ currents in NG108-15 cells: involvement of T-type Ca2+ channels

We investigated if stimulation of T‐type Ca2+ channels with sodium hydrosulfide (NaHS), a donor of hydrogen sulfide (H2S), could cause neuronal differentiation of NG108‐15 cells. Like dibutyryl cyclic AMP (db‐cAMP), treatment with NaHS at 1.5–13.5 mM for 16 h enhanced neurite outgrowth in a concentration‐dependent manner. Synergistic neuritogenic effect was obtained in the cells stimulated with NaHS in combination with db‐cAMP at subeffective concentrations. Exposure to NaHS or db‐cAMP for 2 days resulted in enhancement of expression of high‐voltage‐activated currents consisting of N‐, P/Q‐, L‐ and also other types, but not of T‐type currents. Mibefradil, a pan‐T‐type channel blocker, abolished the neuritogenesis induced by NaHS, but not by db‐cAMP. The NaHS‐evoked neuritogenesis was also completely blocked by pretreatment with BAPTA/AM, a chelator of intracellular Ca2+, and by zinc chloride at a concentration known to selectively inhibit Cav3.2 isoform of T‐type Ca2+ channels, but not Cav3.1 or Cav3.3. Further, l‐ascorbate, recently proven to selectively inhibit Cav3.2, abolished the neuritogenic effect of NaHS, but not db‐cAMP. Our data thus demonstrate that NaHS/H2S is a novel inducer of neuronal differentiation in NG108‐15 cells, as characterized by neuritogenesis and expression of high‐voltage‐activated currents, and suggest the involvement of T‐type Ca2+ channels, especially Cav3.2.