Kinase-dependent activation of voltage-gated Ca~(2+) channels by ET-1 in pulmonary arterial myocytes during chronic hypoxia

摘要

Exposure to chronic hypoxia (CH) causes pulmonary hypertension. The vasoconstrictor endothelin-1 (ET-1) is thought to play a role in the development of hypoxic pulmonary hypertension. In pulmonary arterial smooth muscle cells (PASMCs) from chronically hypoxic rats, ET-1 signaling is altered, with the ET-1-induced change in intracellular calcium concentration (△[Ca~(2+)]_i occurring through activation of voltage-dependent Ca~(2+) channels (VDCC) even though ET-l-in-duced depolarization via inhibition of K~+ channels is lost. The mechanism underlying this response is unclear. We hypothesized that activation of VDCCs by ET-1 following CH might be mediated by protein kinase C (PKC) and/or Rho kinase, both of which have been shown to phosphorylate and activate VDCCs. To test this hypothesis, we examined the effects of PKC and Rho kinase inhibitors on the ET-1-induced △[Ca~(2+)]_i in PASMCs from rats exposed to CH (10% O_2, 3 wk) using the Ca~(2+)-sensitive dye fura 2-AM and fluorescent microscopy techniques. We found that staurosporine and GF109203X, inhibitors of PKC, and Y-27632 and HA 1077, Rho kinase inhibitors, reduced the ET-1-induced A[Ca~(2+)]i by >70%. Inhibition of tyrosine kinases (TKs) with genistein or tyrphostin A23, or combined inhibition of PKC, TKs, and Rho kinase, reduced the A[Ca~(2+)]_i to a similar extent as inhibition of either PKC or Rho kinase alone. The ability of PKC or Rho kinase to activate VDCCs in our cells was verified using phorbol 12-myristate 13-acetate and GTP--γ-S. These results suggest that following CH, the ET-1-induced A[Ca~(2+)]i in PASMCs occurs via Ca~(2+) influx through VDCCs mediated primarily by PKC, TKs, and Rho kinase.