Inhibition by hydrogen sulfide of rabbit platelet aggregation and calcium mobilization.

Hydrogen sulfide (H2S), a gasotransmitter, plays a variety of roles in the mammalian body including the cardiovascular system. Given evidence that H2S donors including NaHS inhibit human platelet aggregation, we examined and characterized the effects of NaHS on rabbit platelet aggregation and cytosolic Ca(2+) mobilization. Rabbit platelet aggregation was determined in platelet-rich plasma (PRP) and washed platelets. Intracellular Ca(2+) levels were monitored in Fura2-loaded washed platelets. NaHS prevented rabbit platelet aggregation induced by collagen or ADP, and the effective concentration range of NaHS was 0.1-0.3 mM in PRP and 1-3 mM in washed platelets. In washed platelets, NaHS attenuated cytosolic Ca(2+) mobilization induced by collagen or ADP and also reduced platelet aggregation induced by ionomycin, a Ca(2+) ionophore. The anti-platelet effect of NaHS was blocked by an adenylyl cyclase inhibitor and enhanced by a phosphodiesterase inhibitor. H2S thus suppresses rabbit platelet aggregation by interfering with both upstream and downstream signals of cytosolic Ca(2+) mobilization in a cAMP-dependent manner.

Antihyperalgesic effect of buprenorphine involves nociceptin/orphanin FQ peptide-receptor activation in rats with spinal nerve injury-induced neuropathy.

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

AKAP-dependent sensitization of Ca(v) 3.2 channels via the EP(4) receptor/cAMP pathway mediates PGE(2) -induced mechanical hyperalgesia.

BACKGROUND AND PURPOSE: The Ca(v) 3.2 isoform of T-type Ca(2+) channels (T channels) is sensitized by hydrogen sulfide, a pro-nociceptive gasotransmitter, and also by PKA that mediates PGE(2) -induced hyperalgesia. Here we examined and analysed Ca(v) 3.2 sensitization via the PGE(2) /cAMP pathway in NG108-15 cells that express Ca(v) 3.2 and produce cAMP in response to PGE(2) , 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 Ca(v) 3.2 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 PGE(2) increased T currents, an effect blocked by AKAP St-Ht31 inhibitor peptide (AKAPI) or KT5720, a PKA inhibitor. The effect of PGE(2) was abolished by RQ-00015986-00, an EP(4) receptor antagonist. AKAP150 was co-immunoprecipitated with Ca(v) 3.2, regardless of stimulation with db-cAMP, and Ca(v) 3.2 was phosphorylated by db-cAMP or PGE(2) . In rats, intraplantar (i.pl.) administration of db-cAMP or PGE(2) caused mechanical hyperalgesia, an effect suppressed by AKAPI, two distinct T-channel blockers, NNC 55-0396 and ethosuximide, or ZnCl(2) , known to inhibit Ca(v) 3.2 among T channels. Oral administration of RQ-00015986-00 suppressed the PGE(2) -induced mechanical hyperalgesia. CONCLUSION AND IMPLICATIONS: Our findings suggest that PGE(2) causes AKAP-dependent phosphorylation and sensitization of Ca(v) 3.2 through the EP(4) receptor/cAMP/PKA pathway, leading to mechanical hyperalgesia in rats.

Colonic hydrogen sulfide-induced visceral pain and referred hyperalgesia involve activation of both Ca(v)3.2 and TRPA1 channels in mice.

Luminal hydrogen sulfide (H(2)S), a gasotransmitter, causes colonic pain / referred hyperalgesia in mice, most probably via activation of T-type Ca(2+) channels. Here we analyzed the mechanisms for H(2)S-induced facilitation of colonic pain signals. Intracolonic administration of NaHS, an H(2)S donor, evoked visceral pain-like nociceptive behavior and referred hyperalgesia in mice, an effect abolished by NNC 55-0396, a selective T-type Ca(2+)-channel blocker, or by knockdown of Ca(v)3.2. AP18, a TRPA1 blocker, also prevented the NaHS-induced colonic pain and referred hyperalgesia. These findings demonstrate that H(2)S-induced colonic pain and referred hyperalgesia require activation of both Ca(v)3.2 and TRPA1 channels in mice.

Involvement of the endogenous hydrogen sulfide/Ca(v) 3.2 T-type Ca2+ channel pathway in cystitis-related bladder pain in mice.

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.

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

BACKGROUND AND PURPOSE: Hydrogen sulfide, a gasotransmitter, facilitates somatic pain signals via activation of Ca(v)3.2 T-type calcium channels in rats. Given evidence for the activation of transient receptor potential ankyrin-1 (TRPA1) channels by H(2)S, we asked whether TRPA1 channels, in addition to Ca(v)3.2 channels, contribute to the H(2)S-induced mechanical hyperalgesia and allodynia in mice. EXPERIMENTAL APPROACH: Mechanical hyperalgesia and allodynia were evaluated by the von Frey test in mice. Ca(v)3.2 or TRPA1 channels in the sensory neurons were silenced by repeated intrathecal administration of antisense oligodeoxynucleotides in mice. KEY RESULTS: Intraplantar administration of NaHS evoked hyperalgesia and allodynia in mice, an effect attenuated or abolished by NNC 55-0396 or mibefradil, T-type calcium channel blockers, and by ascorbic acid or zinc chloride, known to selectively inhibit Ca(v)3.2 channels, out of the three isoforms of T-type calcium channels. Silencing of Ca(v)3.2 channels in the sensory neurons also prevented the NaHS-induced hyperalgesia and allodynia in mice. The NaHS-induced hyperalgesia and allodynia in mice were significantly suppressed by AP18, a TRPA1 channel blocker, and by silencing of TRPA1 channels in the sensory neurons. CONCLUSIONS AND IMPLICATIONS: Mechanical hyperalgesia and allodynia induced by NaHS/H(2)S required activation of both Ca(v)3.2 and TRPA1 channels in mice.

Topical application of disodium isostearyl 2-O-L-ascorbyl phosphate, an amphiphilic ascorbic acid derivative, reduces neuropathic hyperalgesia in rats.

BACKGROUND AND PURPOSE: Ca(v) 3.2 T-type calcium channels, targeted by H(2) S, are involved in neuropathic hyperalgesia in rats and ascorbic acid inhibits Ca(v) 3.2 channels. Therefore, we evaluated the effects of intraplantar (i.pl.) administration of ascorbic acid or topical application of disodium isostearyl 2-O-L-ascorbyl phosphate (DI-VCP), a skin-permeable ascorbate derivative on hyperalgesia induced by NaHS, an H(2) S donor, and on neuropathic hyperalgesia. EXPERIMENTAL APPROACH: In rats mechanical hyperalgesia was evoked by i.pl. NaHS, and neuropathic hyperalgesia was induced by L5 spinal nerve cutting (L5SNC) or by repeated administration of paclitaxel, an anti-cancer drug. Dermal ascorbic acid levels were determined colorimetrically. KEY RESULTS: The NaHS-evoked Ca(v) 3.2 channel-dependent hyperalgesia was inhibited by co-administered ascorbic acid. Topical application of DI-VCP, but not ascorbic acid, prevented the NaHS-evoked hyperalgesia, and also increased dermal ascorbic acid levels. Neuropathic hyperalgesia induced by L5SNC or paclitaxel was reversed by i.pl. NNC 55-0396, a selective T-type calcium channel blocker, ascorbic acid or DI-VCP, and by topical DI-VCP, but not by topical ascorbic acid. The effects of i.pl. ascorbic acid and topical DI-VCP in the paclitaxel-treated rats were characterized by the faster onset and greater magnitude, compared with their effects in the L5SNC rats. Dermal ascorbic acid levels in the hindpaw significantly decreased after paclitaxel treatment, but not L5SNC, which was reversed by topical DI-VCP. CONCLUSIONS AND IMPLICATIONS: Ascorbic acid, known to inhibit Ca(v) 3.2 channels, suppressed neuropathic hyperalgesia. DI-VCP ointment for topical application may be of benefit in the treatment of neuropathic pain.

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

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 300mg/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.

The proteinase/proteinase-activated receptor-2/transient receptor potential vanilloid-1 cascade impacts pancreatic pain in mice.

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.

Phosphorylation of ERK in the spinal dorsal horn following pancreatic pronociceptive stimuli with proteinase-activated receptor-2 agonists and hydrogen sulfide in rats: evidence for involvement of distinct mechanisms.

Noxious stimuli cause prompt phosphorylation of extracellular signal-regulated kinase (ERK) in the spinal dorsal horn that contributes to facilitation of pain sensation and is often used as an immediate marker for excitation of spinal neurons following somatic and colonic nociception. Here we asked whether two distinct pronociceptive stimuli with proteinase-activated receptor-2 (PAR2) agonists and hydrogen sulfide (H(2)S) in the pancreas cause phosphorylation of ERK in the spinal dorsal horn and also examined involvement of their possible downstream signaling molecules, transient receptor potential vanilloid-1 (TRPV1) and T-type Ca(2+) channels, respectively. Capsaicin (a TRPV1 agonist), trypsin (an endogenous PAR2 agonist), SLIGRL-NH(2) (a PAR2-activating peptide), and NaHS (an H(2)S donor) were infused into the pancreatic duct in anesthetized rats, and phosphorylated ERK in the spinal cord was detected by immunohistochemistry. Intraductal administration of capsaicin and trypsin caused prompt phosphorylation of ERK in the superficial layers of T9, but not T5 or T12, spinal dorsal horn. SLIGRL-NH(2) and NaHS, administered in the same manner, also produced ERK phosphorylation in the corresponding spinal regions. Mibefradil, a T-type Ca(2+) channel blocker, abolished the phosphorylation of ERK caused by intraductal NaHS but not SLIGRL-NH(2). In contrast, capsazepine, an inhibitor of TRPV1, suppressed the phosphorylation of ERK caused by intraductal SLIGRL-NH(2) but not NaHS. Our data thus demonstrate that pancreatic pronociceptive stimuli with PAR2 agonists and H(2)S cause ERK phosphorylation in the spinal dorsal horn, through activation of TRPV1 and T-type Ca(2+) channels, respectively, and that those two pronociceptive pathways are independent of each other.

Rhodanese, but not cystathionine-gamma-lyase, is associated with dextran sulfate sodium-evoked colitis in mice: a sign of impaired colonic sulfide detoxification?

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.

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

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 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 Ca(2+) channels with sodium hydrosulfide (NaHS), a donor of hydrogen sulfide (H(2)S), 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 Ca(2+), and by zinc chloride at a concentration known to selectively inhibit Ca(v)3.2 isoform of T-type Ca(2+) channels, but not Ca(v)3.1 or Ca(v)3.3. Further, L-ascorbate, recently proven to selectively inhibit Ca(v)3.2, abolished the neuritogenic effect of NaHS, but not db-cAMP. Our data thus demonstrate that NaHS/H(2)S 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 Ca(2+) channels, especially Ca(v)3.2.

The proteinase inhibitor camostat mesilate suppresses pancreatic pain in rodents.

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

Antiallodynic effect of etidronate, a bisphosphonate, in rats with adjuvant-induced arthritis: involvement of ATP-sensitive K+ channels.

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

Colonic hyperalgesia triggered by proteinase-activated receptor-2 in mice: involvement of endogenous bradykinin.

Intracolonic (i.col.) administration of the PAR2-activating peptide (PAR2AP) SLIGRL-NH2 slowly develops visceral hypersensitivity to i.col. capsaicin in ddY mice. Thus, we further analyzed roles of PAR2 in colonic hypersensitivity, using the novel potent PAR2AP, 2-furoyl-LIGRL-NH2 and PAR2-knockout (KO) mice. In ddY mice, i.col. 2-furoyl-LIGRL-NH2 produced delayed (6 h later) facilitation of capsaicin-evoked visceral nociception, an effect being much more potent than SLIGRL-NH2. Such effects were mimicked by i.col. trypsin. In wild-type (WT), but not PAR2-KO, mice of C57BL/6 background, i.col. PAR2 agonists caused delayed facilitation of sensitivity to capsaicin. The PAR2-triggered visceral hypersensitivity was abolished by a bradykinin B2 receptor antagonist, HOE-140. Our data thus provide ultimate evidence for role of PAR2 in colonic hypersensitivity, and suggest involvement of the bradykinin-B2 pathway.

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-NH(2), 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-NH(2) 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.