title: Kinase-dependent activation of voltage-gated Ca2+ channels by ET-1 in pulmonary arterial myocytes during chronic hypoxia.
abstract: 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 (Δ[Ca(2+)](i)) occurring through activation of voltage-dependent Ca(2+) channels (VDCC) even though ET-1-induced 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 Δ[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 Δ[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 Δ[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 Δ[Ca(2+)](i) in PASMCs occurs via Ca(2+) influx through VDCCs mediated primarily by PKC, TKs, and Rho kinase.
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, 5501 Hopkins Bayview Circle, Baltimore, MD 21224, USA.
authors.forename: Trevor
authors.initials: T
authors.lastname: Luke
authors.forename: Julie
authors.initials: J
authors.lastname: Maylor
authors.forename: Clark
authors.initials: C
authors.lastname: Undem
authors.forename: J T
authors.initials: JT
authors.lastname: Sylvester
authors.forename: Larissa A
authors.initials: LA
authors.lastname: Shimoda
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism; D020013:Calcium Signaling; D002908:Chronic Disease; D019332:Endothelin-1 / Q000378:metabolism* / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives; D015870:Gene Expression; D006976:Hypertension, Pulmonary / Q000209:etiology / Q000378:metabolism / Q000503:physiopathology; D000860:Hypoxia / Q000150:complications / Q000378:metabolism* / Q000503:physiopathology; D015640:Ion Channel Gating / Q000187:drug effects; D008297:Male; D008856:Microscopy, Fluorescence; D032342:Muscle Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D054460:rho-Associated Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D015220:Calcium Channels; D019332:Endothelin-1; D005456:Fluorescent Dyes; D047428:Protein Kinase Inhibitors; C049925:fura-2-am; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D002118:Calcium; D016257:Fura-2