Abstract. 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.

BACKGROUND AND PURPOSEThe Cav3.2 isoform of T-type Ca 2+ channels (T channels) is sensitized by hydrogen sulfide, a pro-nociceptive gasotransmitter, and also by PKA that mediates PGE2-induced hyperalgesia. Here we examined and analysed Cav3.2 sensitization via the PGE2/cAMP pathway in NG108-15 cells that express Cav3.2 and produce cAMP in response to PGE2, and its impact on mechanical nociceptive processing in rats. EXPERIMENTAL APPROACHIn NG108-15 cells and rat dorsal root ganglion (DRG) neurons, T-channel-dependent currents (T currents) were measured with the whole-cell patch-clamp technique. The molecular interaction of Cav3.2 with A-kinase anchoring protein 150 (AKAP150) and its phosphorylation were analysed by immunoprecipitation/immunoblotting in NG108-15 cells. Mechanical nociceptive threshold was determined by the paw pressure test in rats. KEY RESULTSIn NG108-15 cells and/or rat DRG neurons, dibutyryl cAMP (db-cAMP) or PGE2 increased T currents, an effect blocked by AKAP St-Ht31 inhibitor peptide (AKAPI) or KT5720, a PKA inhibitor. The effect of PGE2 was abolished by RQ-00015986-00, an EP4 receptor antagonist. AKAP150 was co-immunoprecipitated with Cav3.2, regardless of stimulation with db-cAMP, and Cav3.2 was phosphorylated by db-cAMP or PGE2. In rats, intraplantar (i.pl.) administration of db-cAMP or PGE2 caused mechanical hyperalgesia, an effect suppressed by AKAPI, two distinct T-channel blockers, NNC 55-0396 and ethosuximide, or ZnCl2, known to inhibit Cav3.2 among T channels. Oral administration of RQ-00015986-00 suppressed the PGE2-induced mechanical hyperalgesia. CONCLUSION AND IMPLICATIONSOur findings suggest that PGE2 causes AKAP-dependent phosphorylation and sensitization of Cav3.2 through the EP4 receptor/cAMP/PKA pathway, leading to mechanical hyperalgesia in rats. AbbreviationsAKAP, A-kinase anchoring protein; AKAPI, AKAP St-Ht31 inhibitor peptide; AUC, area under the curve;

Hydrogen sulfide (H 2 S), a gasotransmitter, plays a variety of roles in the mammalian body including the cardiovascular system. Given evidence that H 2 S 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 antiplatelet effect of NaHS was blocked by an adenylyl cyclase inhibitor and enhanced by a phosphodiesterase inhibitor. H 2 S thus suppresses rabbit platelet aggregation by interfering with both upstream and downstream signals of cytosolic Ca 2+ mobilization in a cAMP-dependent manner.

Abstract.We evaluated the effect of buprenorphine, a mixed agonist for -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, g/kg, exhibited naloxone-reversible analgesic activity in naïve rats. In contrast, buprenorphine at 0.5 -20 g/kg produced a naloxone-sensitive antihyperalgesic effect in the L5 spinal nerve-injured neuropathic rats. Intrathecal injection of [N-Phe 1 ]nociceptin(1-13)NH 2 , 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.