Hydrogen sulfide (H(2)S), an endogenous gasotransmitter, modulates various biological events such as inflammation in the mammalian body. The present study investigated possible involvement of H(2)S in peripheral nociceptive processing. Intraplantar (i.pl.) administration of NaHS, a H(2)S donor, produced prompt hyperalgesia in rats, accompanied by expression of Fos in the spinal dorsal horn. The H(2)S-evoked hyperalgesia was blocked by 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB), an oxidizing agent, or ethosuximide and mibefradil, T-type Ca(2+) channel inhibitors. L-Cysteine, an endogenous source for H(2)S, given i.pl., also elicited hyperalgesia, an effect being abolished by DL-propargylglycine (PPG) and beta-cyanoalanine (BCA), inhibitors of cystathionine-gamma-lyase, a H(2)S 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 Ca(2+) channels, NaHS enhanced the currents through the T-type channels, an effect being blocked by DTNB. Thus, H(2)S appears to function as a novel nociceptive messenger through sensitization of T-type Ca(2+) channels in the peripheral tissues, particularly during inflammation.

These findings suggest that colonic luminal H(2)S/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.

We investigated proteinase-activated receptor-2 (PAR 2 )-triggered signal transduction pathways causing increased prostaglandin E 2 (PGE 2 ) formation in human lung-derived A549 epithelial cells. The PAR 2 agonist, SLIGRL-NH 2 (Ser-Leu-Ile-Gly-Arg-Leu-amide), evoked immediate cytosolic Ca 2ϩ mobilization and delayed (0.5-3 h) PGE 2 formation. The PAR 2 -triggered PGE 2 formation was attenuated by inhibition of the following signal pathway enzymes: cyclooxygenases 1 and 2 (COX-1 and COX-2, respectively), cytosolic Ca 2ϩ -dependent phospholipase A 2 (cPLA 2 ), the mitogenactivated protein kinases (MAPKs), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) and p38 MAPK, Src family tyrosine kinase, epidermal growth factor (EGF) receptor tyrosine kinase (EGFRK), and protein kinase C (PKC), but not by inhibition of matrix metalloproteinases. SLIGRL-NH 2 caused prompt (5 min) and transient ERK phosphorylation, blocked in part by inhibitors of PKC and tyrosine kinases but not by an EGFRK inhibitor. SLIGRL-NH 2 also evoked a relatively delayed (15 min) and persistent (30 min) phosphorylation of p38 MAPK, blocked by inhibitors of Src and EGFRK but not by inhibitors of COX-1 or COX-2. SLIGRL-NH 2 elicited a Src inhibitor-blocked prompt (5 min) and transient phosphorylation of the EGFRK. SLIGRL-NH 2 up-regulated COX-2 protein and/or mRNA levels that were blocked by inhibition of p38 MAPK, EGFRK, Src, and COX-2 but not MEK-ERK. SLIGRL-NH 2 also caused COX-1-dependent up-regulation of microsomal PGE synthase-1 (mPGES-1). We conclude that PAR 2 -triggered PGE 2 formation in A549 cells involves a coordinated up-regulation of COX-2 and mPGES-1 involving cPLA 2 , increased cytosolic Ca 2ϩ , PKC, Src, MEK-ERK, p38 MAPK, Src-mediated EGF receptor trans-activation, and also metabolic products of both COX-1 and COX-2.Proteinase-activated receptors (PARs), a family of G protein-coupled seven-trans-membrane domain receptors, consisting of PARs 1 to 4, are now known to mediate a variety of intracellular signaling and subsequent cellular events caused by specific extracellular proteinases (Hollenberg

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 (NaHS), a donor of H 2 S causes prompt hyperalgesia, an effect being abolished by mibefradil, an inhibitor of T-type Ca 2+ channels Kawabata 2008). Our electrophysiological evidence has also demonstrated that NaHS actually enhances membrane currents through T-type channels in NG108-15 cells, neuroblastoma · glioma hybrid cells, as well as mouse dorsal root ganglion neurons Kawabata 2008).Neuroblast differentiation into neurons is judged by neurite outgrowth and synapse formation, and can be characterized by changes in electrophysiological properties, i.e. later appearance of high-voltage-activated (HVA) Ca 2+ currents after the first appearance of T-type Ca 2+ currents (Gottmann et al. 1988;Goodwin et al. 1989;McCobb et al. 1989;Chemin et al. 2002). NG108-15 cells are widely used in studies on neuronal development and differentiation, and known to be abundant in T-type channels but not HVA channels, unless differentiated (Nirenberg et al. 1983;Chemin et al. 2002). NG108-15 cells, when stimulated with dibutyryl cyclic AMP (db-cAMP), develop neuron-like properties, revealing neurite outgrowth, synapse formation and functional expression of HVA Ca 2+ currents (Kleinman et al. 1988;Han et al. 1991;Kasai and Neher 1992;Taussig et al. 1992;Chemin et al. 2002). Chemin et al. (Chemin et al. 2002(Chemin et al. , 2004 have shown that blockade of T-type channels, particularly of Ca v 3.2 (a 1H ) isoform, partially inhibits db-cAMP-evoked neuritogenesis and abolishes concomitant HVA Ca 2+ current expression in NG108-15 cells. Since H 2 S is capable of facilitating T-type currents, as mentioned above, it is likely that H 2 S might cause and/or promote neuronal differentiation in NG108-15 cells. Thus, in the present study, we asked if NG108-15 cells treated with NaHS, a donor for H 2 S, reveal neuron-like properties, by examining neurite outgrowth and expression of HVA Ca 2+ currents. Materials and methodsCell culture and assessment of neurite outgrowth NG108-15 cells were cultured in high glucose-containing Dulbecco's Modified Eagle's Medium (Sigma, St. Louis, MO, USA) supplemented with 0.1 mM hypoxanthine, 1 lM aminopterin, 16 lM thymidine, 50 U/mL penicillin, 50 lg/mL streptomycin and 10% fetal calf serum (...

J. Neurochem. (2010) 114, 512–519. Abstract Hydrogen sulfide (H2S), a gasotransmitter, induces neuronal differentiation characterized by neuritogenesis and functional up‐regulation of high voltage‐activated Ca2+ channels, via activation of T‐type Ca2+ channels in NG108‐15 cells. We thus analyzed signaling mechanisms for the H2S‐evoked neuronal differentiation. NaHS, a donor for H2S, facilitated T‐type Ca2+ channel‐dependent membrane currents, an effect blocked by ascorbic acid that selectively inhibits Cav3.2 among three T‐type channel isoforms. NaHS, applied once at a high concentration (13.5 mM) or repetitively at a relatively low concentration (1.5 mM), as well as ionomycin, a Ca2+ ionophore, evoked neuritogenesis. The neuritogenesis induced by NaHS, but not by ionomycin, was abolished by mibefradil, a T‐type Ca2+ channel blocker. PP2, a Src kinase inhibitor, completely suppressed the neuritogenesis caused by NaHS or ionomycin, while it only partially blocked neuritogenesis caused by dibutyryl cAMP, a differentiation inducer. NaHS, but not dibutyryl cAMP, actually caused phosphorylation of Src, an effect blocked by 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid acetoxymethyl, an intracellular Ca2+ chelator, mibefradil or ascorbic acid. The up‐regulation of high voltage‐activated currents in the cells treated with NaHS was also inhibited by PP2. Together, our data reveal that Src kinase participates in the T‐type Ca2+ channel‐dependent neuronal differentiation caused by NaHS/H2S in NG108‐15 cells.