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 (...

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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.

Background: Hydrogen sulfide (H₂S), a gasotransmitter, is generated from L-cysteine by mainly 3 enzymes, cystathionine-γ-lyase (CSE), cystathionine-β-synthase, and 3-mercaptopyruvate sulfurtransferase in cooperation with cysteine aminotransferase. The H₂S-forming enzymes, particularly CSE, are overexpressed under the pathological conditions such as inflammation, neuronal or neuroendocrine differentiation and cancer development. Given that Ca_v3.2 T-type Ca²⁺ channels mediate some of the biological activity of H₂S, we focus on the role of the H₂S/Ca_v3.2 pathway in regulating the neuronal and neuroendocrine function. Summary: In the neuronal system, H₂S regulates the activity of various ion channels including Ca_v3.2. Exogenous and endogenous H₂S enhances the Ca_v3.2 channel activity, promoting somatic and visceral pain signaling. The H₂S/Ca_v3.2 pathway also facilitates neuritogenesis or neuronal differentiation. Interestingly, endogenous H₂S formed by CSE regulates secretory function by enhancing Ca_v3.2 channel activity in neuroendocrine-differentiated prostate cancer cells or carotid glomus cells. Key Messages: The H₂S/Ca_v3.2 pathway may serve as therapeutic targets for treatment of intractable pain, neuronal injury, androgen-independent prostate cancer, cardiovascular diseases, etc.

Given that Ca3.2 T-type Ca channels were functionally regulated by asparagine (N)-linked glycosylation, we examined effects of high glucose on the function of Ca3.2, known to regulate secretory function, in neuroendocrine-like differentiated prostate cancer LNCaP cells. High glucose accelerated the increased channel function and overexpression of Ca3.2 during neuroendocrine differentiation, the former prevented by enzymatic inhibition of N-glycosylation and cleavage of N-glycans. Hyperglycemia thus appears to induce N-linked glycosylation-mediated functional upregulation and overexpression of Ca3.2 in neuroendocrine-like differentiated prostate cancer cells.

Helicobacter pylori (H. pylori) is involved in the pathogenesis of gastric ulcer and cancer, and might secret proteinases. Given emerging roles of proteinase‐activated receptors (PARs) in the gastric mucosa, we investigated the correlation between expression of PARs and H. pylori infection in the stomach isolated from gastric cancer patients. The extent of H. pylori infection was evaluated as regional protein levels of ureases A and B, and analyzed in relation to expression levels of PAR1 and PAR2 in the corresponding regions. In the cancer area, expression of PAR1, but not PAR2, was positively correlated with protein levels of either urease, whereas no such a correlation was observed in the non‐cancer area. There was no significant difference in levels of any proteins between the non‐cancer and cancer areas. Together, expression of PAR1, but not PAR2, appears to be associated with H. pylori infection in gastric cancer tissues. It is likely that the increased expression of PAR1 might be involved in the development of H. pylori‐related gastric cancer.