Hydrogen sulfide inhibited L-type calcium channels (CaV1.2) via up-regulation of the channel sulfhydration in vascular smooth muscle cells.

Hydrogen sulfide (H2S) exerts different effects on the cardiovascular system by modulating ion channels. The present study was to ascertain whether H2S affects L-type calcium (Ca2+) channels in vascular smooth muscle cells (VSMCs) and the subsequent signaling pathways. Here, CaV1.2 L-type Ca2+ currents (ICa, L) were inhibited by sodium hydrosulfide (NaHS, an H2S donor) in A7r5 cell lines using the whole-cell patch-clamp technique. Then NaHS significantly reduced intracellular Ca2+ concentration ([Ca2+]i) in Bayk8644-stimulated CaV1.2-HEK293 cells by using flow cytometry. However, NaHS did not affect the ryanodine-induced elevation of [Ca2+]i by means of confocal microscopy, ruling out its influence on the intracellular Ca2+ release. In the following, the sulfhydration of L-type Ca2+ channels was determined by Ellman's Test. The results showed that NaHS decreased the number of free sulfhydryls, which was further strengthened by the oxidant sulfhydryl modifier diamide (DM) and significantly counteracted by the reductant sulfhydryl modifier dithiothreitol (DTT). DTT also partly reversed the NaHS-reduced [Ca2+]i in CaV1.2-HEK293 cells. Additionally, NaHS did not change CaV1.2 expression. Furthermore, NaHS increased phosphorylation of PKC and ERK in both a concentration- and a time-dependent manner in VSMCs. Isradipine, L-type Ca2+ channel specific blocker, further increased H2S-induced phosphorylation of PKC and ERK, showing an additive effect with H2S. Therefore, our results suggest that H2S reduced ICa, L & [Ca2+]i and hence influenced the downstream PKC/ERK pathway, which was likely through regulating the sulfhydration of L-type Ca2+ channels in VSMCs.