Fig. 3

Activation of Cav3.2 T-type calcium channel by γ-cystathionase (CTH) /H₂S signaling in MSC-H cells. MSC-H cells had fourfold higher H₂S level in the culture supernatant than MSC-C cells. The extracellular H₂S level was reduced in siH2 and siH3 cells with HMGB1 knock-down. When MSC-H cells were treated with 10 mmol/L PAG to inhibit CTH activity, H₂S level in the culture supernatant was greatly decreased (a). Intracellular H₂S level was assayed by using WSP-5 probes which reacted with intracellular H₂S to emit green fluorescence. The fluorescent intensity was significantly higher in MSC-H cells than MSC-C cells, suggesting a remarkable increase of intracellular H₂S in MSC-H cells. The intracellular H₂S diminished when MSC-H cells was treated with 10 mmol/l PAG or subjected to HMGB1 knock-down as shown in siH2 and siH3 cells (b). The expression of CTH was increased in MSC-H cells but inhibited by HMGB1 knock-down as shown in siH2 and siH3 cells. Cystathionine-β-synthase (CBS) remained at a low level in all study groups regardless of HMGB1 overexpression and knock-down (c). Blockage of CTH by 10 mmol/l PAG reduced the fluorescent intensity of Rhod-3 probes without change of CACNA1H expression (d), suggesting a pivotal role of CTH on regulation of intracellular free calcium in MSC-H cells. The asterisk indicated a P value < 0.05