Hydrogen sulfide regulates intracellular pH in rat primary cultured glia cells.

Intracellular pH (pH(i)) plays an important role in the regulation of central nervous system function. In the present study, we examined whether hydrogen sulfide (H(2)S), a recently recognized neuromodulator, regulates pH(i) in rat primary cultured glia cells. pH(i) was measured with a fluorescent sensitive dye, BCECF-AM. Activities of Cl(-)/HCO(3)(-) exchanger and Na(+)/H(+) exchanger were examined by assessing their capacities to load or extrude H(+) upon NH(4)Cl pulse load. We found that NaHS, a H(2)S donor, decreased pH(i) in a concentration-dependent manner ranging from 10 to 200muM in the primary cultured microglia. Blockade of the Cl(-)/HCO(3)(-) exchanger with, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or Na(+)/H(+) exchanger with 5-N-methyl-N-isobutylamiloride (MIA) significantly attenuated the pH(i)-lowering effect of NaHS. Moreover, NaHS significantly increased the activity of Cl(-)/HCO(3)(-) exchanger but inhibited that of Na(+)/H(+) exchanger. The pH regulatory effect of H(2)S was also observed in primary cultured astrocytes, but not in SH-SY5Y neuronal cells. In conclusion, we found for the first time that H(2)S induced intracellular acidification in glia cells via regulation of the activities of Cl(-)/HCO(3)(-) exchanger and Na(+)/H(+) exchanger. The present study may provide new evidence for H(2)S to serve as a neuromodulator and offer a potential approach for the treatment of neurological diseases.

Effect of hydrogen sulfide on intracellular calcium homeostasis in neuronal cells.

Hydrogen sulfide (H(2)S) is now known as a new biological mediator. In the present study, the effects of H(2)S on intracellular calcium ([Ca(2+)](i)) in neuronal SH-SY5Y cells was investigated. In SH-SY5Y neuronal cells, NaHS, a H(2)S donor, concentration-dependently increased [Ca(2+)](i). The H(2)S-induced Ca(2+) elevation was significantly attenuated by EGTA-treated calcium-free Krebs' solution. This elevation was also reduced by antagonists of L-type (verapamil and nifedipine), T-type (mibefradil) calcium channels and N-methyl-d-aspartate receptor (MK-801, AP-5 and ifenprodil). A 90% reduction in H(2)S-induced [Ca(2+)](i) elevation was found in cells pretreated with combination of all three kinds of inhibitors. Depletion of intracellular Ca(2+) store with thapsigargin or cyclopiazonic acid or blockade of ryanodine receptor with ruthenium red significantly attenuated the effect of H(2)S on [Ca(2+)](i). Inhibition of protein kinase A (PKA), phospholipase C (PLC) and protein kinase C (PKC) suppressed the H(2)S-elevated [Ca(2+)](i), suggesting that H(2)S may regulate [Ca(2+)](i) via both PKA and PLC/PKC pathways. In conclusion, it was found in this study that H(2)S increased [Ca(2+)](i) in SH-SY5Y neuronal cells by increasing Ca(2+) influx via plasma membrane and in turn releasing calcium from intracellular calcium store. The findings in the present study provide the direct evidence that H(2)S may serve as a neuromodulator.