Atsufumi Kawabata

Hydrogen sulfide as a novel nociceptive messenger

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

The protease-activated receptor-2 agonist induces gastric mucus secretion and mucosal cytoprotection

Protease-activated receptor-2 (PAR-2), a receptor activated by trypsin/tryptase, modulates smooth muscle tone and exocrine secretion in the salivary glands and pancreas. Given that PAR-2 is expressed throughout the gastrointestinal tract, we investigated effects of PAR-2 agonists on mucus secretion and gastric mucosal injury in the rat. PAR-2-activating peptides triggered secretion of mucus in the stomach, but not in the duodenum. This mucus secretion was abolished by pretreatment with capsaicin, which stimulates and ablates specific sensory neurons, but it was resistant to cyclo-oxygenase inhibition. In contrast, capsaicin treatment failed to block PAR-2-mediated secretion from the salivary glands. Intravenous calcitonin gene-related peptide (CGRP) and neurokinin A markedly elicited gastric mucus secretion, as did substance P to a lesser extent. Specific antagonists of the CGRP1 and NK2, but not the NK1, receptors inhibited PAR-2-mediated mucus secretion. Pretreatment with the PAR-2 agonist strongly prevented gastric injury caused by HCl-ethanol or indomethacin. Thus, PAR-2 activation triggers the cytoprotective secretion of gastric mucus by stimulating the release of CGRP and tachykinins from sensory neurons. In contrast, the PAR-2-mediated salivary exocrine secretion appears to be independent of capsaicin-sensitive sensory neurons.

Effect of topical administration of l‐arginine on formalin‐induced nociception in the mouse: a dual role of peripherally formed NO in pain modulation

1 We investigated the effects of intraplantar (i.pl.) administration of l‐arginine and NG‐nitro‐l‐arginine methyl ester (l‐NAME) on formalin‐induced behavioural nociception in the mouse. 2 l‐ but not d‐arginine, at 0.1 − 1 μg per paw, coadministered with i.pl. formalin, enhanced the second‐but not the first‐phase nociceptive responses, whereas it was without significant effects at 3 μg per paw, and conversely, produced antinociception at 10 μg per paw, resulting in a bell‐shaped dose‐response curve. 3 l‐NAME at 0.1 − 1 μg per paw, when administered i.pl., exhibited antinociceptive activity in the second phase in a dose‐dependent manner, although its d‐enantiomer produced no effect. 4 An antinociceptive dose (1 μg per paw) of l‐NAME (i.pl.) considerably reduced the increase in second‐phase nociception elicited by low doses (1 μg per paw) of i.pl. l‐arginine. The second‐phase nociception decrease induced by a large dose (10 μg per paw) of i.pl. l‐arginine was markedly reversed by i.pl. l‐NAME at 0.1 μg per paw, raising it to a level above that of the control (formalin only). 5 These results suggest that peripheral NO plays a dual role in nociceptive modulation, depending on the tissue level, inducing either nociceptive or antinociceptive responses.

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.

Increased vascular permeability by a specific agonist of protease-activated receptor-2 in rat hindpaw

The present study examined the effect of intraplantar (i.pl.) administration of a selective agonist of protease‐activated receptor (PAR)‐2, SLIGRL‐NH2(PP6‐NH2), on vascular permeability in rat hindpaw. PP6‐NH2, administered i.pl. at 10–100 nmol per paw, enhanced vascular permeability and caused oedema formation in rat hindpaw. SLIGRL (PP6‐OH) and trypsin, by i.pl. administration, also elicited an increase in vascular permeability, although i.pl. administration of the mixture of constituent amino acids of PP6‐OH at an equivalent dose did not. The PP6‐NH2‐induced increase in vascular permeability was abolished by repeated pretreatment with compound 48/80 to deplete bioactive amines in mast cells. These findings suggest that the activation of PAR‐2 induces acute inflammation, at least partially, via mast cell degranulation in rat hindpaw.

L-arginine exerts a dual role in nociceptive processing in the brain: involvement of the kyotorphin-Met-enkephalin pathway and NO-cyclic GMP pathway.

1 Intracerebroventricular (i.c.v.) administration of l‐arginine (l‐Arg), at 10–100 μg per mouse, produced antinociception in mice, as assessed by the tail flick test; this antinociception was reversed by pretreatment (s.c.) with naltrindole (NTI), a δ‐selective opioid antagonist, and by co‐administered l‐leucyl‐l‐arginine (Leu‐Arg), a kyotorphin (endogenous Met‐enkephalin releaser) receptor antagonist. 2 l‐NG‐nitroarginine methyl ester (l‐NAME), a NO synthase inhibitor, but not d‐NG‐nitroarginine methyl ester, given i.c.v. at 3–10 μg per mouse, exhibited antinociceptive activity that was resistant to naloxone (s.c.), NTI (s.c.) and Leu‐Arg (i.c.v.). 3 The l‐NAME (i.c.v.)‐induced antinociception was not reversed by l‐Arg (i.c.v.), which was antinociceptive by itself, but was abolished by combined injection of l‐Arg plus Leu‐Arg (i.c.v.) or by l‐Arg (i.c.v.) after NTI (s.c.). 4 Methylene blue (MB), a soluble guanylate cyclase inhibitor, at 0.1–1 μg per mouse, produced antinociception by i.c.v. administration. The antinociception induced by MB (i.c.v.) or l‐NAME (i.c.v.) was reversed by co‐administered dibutyryl cyclic GMP. 5 These findings suggest that l‐Arg plays a dual role in nociceptive processing in the brain, being antinociceptive via the kyotorphin‐Met‐enkephalin pathway and nociceptive via the NO‐cyclic GMP pathway.

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.

Proteinase-activated receptor-2 (PAR-2): regulation of salivary and pancreatic exocrine secretion in vivo in rats and mice.

Proteinase‐activated receptor‐2 (PAR‐2) is expressed throughout the gastrointestinal tract including the pancreas, and may be involved in digestive functions. The aim of our study was to evaluate a potential role for PAR‐2 in regulating salivary and pancreatic exocrine secretion in vivo. PAR‐2‐activating peptides (PAR‐2‐APs), but not selective PAR‐1‐APs, administered intravenously, increased salivary secretion in the mouse or rat; this effect of the PAR‐2‐APs was unaffected by atropine, phentolamine, propranolol or indomethacin. Secretion (amylase) by rat parotid gland slices in vitro was also stimulated by PAR‐2‐APs and trypsin, but not by activation of other PARs. PAR‐2‐APs, administered to rats in vivo, caused a prompt effect on pancreatic exocrine secretion. PAR‐2 mRNA, known to be present in pancreatic tissue, was also detected in parotid tissue. Our results indicate that in addition to a potential role in regulating cardiovascular and respiratory functions, PAR‐2 may also play a general role in vivo for the direct regulation of glandular exocrine secretion.

Peripheral PAR-2 triggers thermal hyperalgesia and nociceptive responses in rats.

Protease-activated receptor-2 (PAR-2), a member of the G protein-coupled, seven trans-membrane domain receptor family, is activated by trypsin/tryptase and present in various tissues including the primary sensory neurons, playing a role in development of neurogenic inflammation. The present study examined if activation of peripheral PAR-2 could modulate nociception in the rat. Expression of mRNA for PAR-2 was confirmed in the L4–6 dorsal root ganglia, but not spinal cord. The PAR-2-activating peptide SLIGRL-NH2 administered by the intraplantar (i.pl.) route, produced thermal, but not mechanical, hyperalgesia in the rat, although the PAR-2-inactive control peptide LSIGRL-NH2 had no effect. Not only the PAR-2-activating but also inactive peptides elicited nociceptive behavior (licking/biting) in the intact rats, whereas only the former peptide produced such behavior in the rats that had received repeated administration of compound 48/80 for mast cell depletion. These data provide novel evidence that activation of peripheral PAR-2 is pro-nociceptive, producing thermal hyperalgesia and also triggering pain sensation, by itself, independently of mast cell degranulation.

Upregulation of Cav3.2 T-type calcium channels targeted by endogenous hydrogen sulfide contributes to maintenance of neuropathic pain

&NA; Hydrogen sulfide (H2S) formed from l‐cysteine by multiple enzymes including cystathionine‐&ggr;‐lyase (CSE) is now considered a gasotransmitter in the mammalian body. Our previous studies have shown that H2S activates/sensitizes Cav3.2 T‐type Ca2+ channels, leading to facilitation of somatic and visceral nociception, and that CSE‐derived endogenous H2S participates in inflammatory pain. Here, we show novel evidence for involvement of the endogenous H2S–Cav3.2 pathway in neuropathic pain. In the rat subjected to the right L5 spinal nerve cutting (L5SNC), a neuropathic pain model, i.p. administration of dl‐propargylglycine (PPG) and &bgr;‐cyanoalanine, irreversible and reversible CSE inhibitors, respectively, strongly suppressed the neuropathic hyperalgesia/allodynia. The anti‐hyperalgesic effect of PPG was reversed by intraplantar administration of NaHS, a donor for H2S, in the L5SNC rat. Intraplantar administration or topical application of mibefradil, a T‐type Ca2+ channel blocker, reversed hyperalgesia in the L5SNC rat. The protein levels of Cav3.2, but not CSE, in the ipsilateral L4, L5 and L6 dorsal root ganglia were dramatically upregulated in the L5SNC rat. Finally, silencing of Cav3.2 in DRG by repeated intrathecal administration of Cav3.2‐targeting siRNA significantly attenuated the neuropathic hyperalgesia in the L5SNC rat. In conclusion, our data suggest that Cav3.2 T‐type Ca2+ channels in sensory neurons are upregulated and activated/sensitized by CSE‐derived endogenous H2S after spinal nerve injury, contributing to the maintenance of neuropathic pain. We thus propose that Cav3.2 and CSE could be targets for the development of therapeutic drugs for the treatment of neuropathic pain.