Tsuyoshi Ishiki

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.

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.

Distinct Activity of Peptide Mimetic Intracellular Ligands (Pepducins) for Proteinase‐Activated Receptor‐1 in Multiple Cells/Tissues

Abstract:  Proteinase‐activated receptor‐1 (PAR1), a G protein–coupled receptor (GPCR) for thrombin, can be activated not only by PAR1‐activating peptides (PAR1APs) based on the N‐terminal cryptic tethered ligand sequence but also by an N‐palmitoylated (Pal) peptide, Pal‐RCLSSSAVANRSKKSRALF‐amide (P1pal‐19), based on the intracellular loop 3 of PAR1, designated pepducin, in human platelets or PAR1‐transfected cells. The present article evaluated the actions of P1pal‐19 and also the shorter peptide, Pal‐RCLSSSAVANRS‐amide (P1pal‐12), known as a possible PAR1 antagonist, in multiple cells/tissues that naturally express PAR1. P1pal‐19 as well as a PAR1AP, TFLLR‐amide, evoked cytosolic Ca2+ mobilization in cultured human lung epithelial cells (A549) and rat gastric mucosal epithelial cells (RGM1). P1pal‐19 and TFLLR‐amide, but not a PAR2‐activating peptide, SLIGRL‐amide, caused delayed prostaglandin E2 formation in RGM1 cells. P1pal‐19, like TFLLR‐amide, produced endothelial NO‐dependent relaxation in rat aorta and epithelial prostanoid‐dependent relaxation in mouse bronchus. The P1pal‐19‐induced relaxation remained constant even after desensitization of PAR1 with TFLLR‐amide in either tissue. P1pal‐19 failed to mimic the contractile effects of TFLLR‐amide in the endothelium‐denuded preparations of rat aorta or superior mesenteric artery and the rat gastric longitudinal smooth muscle strips. P1pal‐12 partially inhibited the vasorelaxation caused by TFLLR‐amide and P1pal‐19, but not SLIGRL‐amide, in the rat aorta. Our data thus indicate that P1pal‐19 is capable of mimicking the effects of PAR1APs in the endothelial and epithelial, but not smooth muscle, cells/tissues, and suggest that P1pal‐12 may act as a PAR1 antagonist in the vascular endothelium.

Rhodanese, but not cystathionine-γ-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.

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.

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.