Capacitative Ca(2+) entry and tyrosine kinase activation in canine pulmonary arterial smooth muscle cells.
We investigated the role of capacitative Ca(2+) entry and tyrosine kinase activation in mediating phenylephrine (PE)-induced oscillations in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in canine pulmonary arterial smooth muscle cells (PASMCs). [Ca(2+)](i) was measured as the 340- to 380-nm ratio in individual fura 2-loaded PASMCs. Resting [Ca(2+)](i) was 96 +/- 4 nmol/l. PE (10 micromol/l) stimulated oscillations in [Ca(2+)](i), with a peak amplitude of 437 +/- 22 nmol/l and a frequency of 1.01 +/- 0.12/min. Thapsigargin (1 micromol/l) was used to deplete sarcoplasmic reticulum (SR) Ca(2+) after extracellular Ca(2+) was removed. Under these conditions, a nifedipine-insensitive, sustained increase in [Ca(2+)](i) (140 +/- 7% of control value) was observed when the extracellular Ca(2+) concentration was restored; i.e., capacitative Ca(2+) entry was demonstrated. Capacitative Ca(2+) entry also refilled SR Ca(2+) stores. Capacitative Ca(2+) entry was attenuated (32 +/- 3% of control value) by 50 micromol/l of SKF-96365 (a nonselective Ca(2+)-channel inhibitor). Tyrosine kinase inhibition with tyrphostin 23 (100 micromol/l) and genistein (100 micromol/l) also inhibited capacitative Ca(2+) entry to 63 +/- 12 and 85 +/- 4% of control values, respectively. SKF-96365 (30 micromol/l) attenuated both the amplitude (15 +/- 7% of control value) and frequency (50 +/- 21% of control value) of PE-induced Ca(2+) oscillations. SKF-96365 (50 micromol/l) abolished the oscillations. Tyrphostin 23 (100 micromol/l) also inhibited the amplitude (17 +/- 7% of control value) and frequency (45 +/- 9% of control value) of the oscillations. Genistein (30 micromol/l) had similar effects. Both SKF-96365 and tyrphostin 23 attenuated PE-induced contraction in isolated pulmonary arterial rings. These results demonstrate that capacitative Ca(2+) entry is present and capable of refilling SR Ca(2+) stores in canine PASMCs and may be involved in regulating PE-induced Ca(2+) oscillations. A tyrosine kinase is involved in the signal transduction pathway for alpha(1)-adrenoreceptor activation in PASMCs.
Doi
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Center for Anesthesiology Research, Division of Anesthesiology and Critical Care Medicine, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Damron
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Horibe
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Murray
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PA
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D004285:Dogs; D004553:Electric Conductivity; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D009991:Oscillometry; D009994:Osmolar Concentration; D010656:Phenylephrine / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism*; D015203:Reproducibility of Results; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology
D000316:Adrenergic alpha-Agonists; D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D010656:Phenylephrine; D019284:Thapsigargin; D011505:Protein-Tyrosine Kinases; D002118:Calcium