Capacitative Ca2+ entry and tyrosine kinase activation in canine pulmonary arterial smooth muscle cells

Abstract

We investigated the role of capacitative Ca2+ entry and tyrosine kinase activation in mediating phenylephrine (PE)-induced oscillations in intracellular free Ca2+ concentration ([Ca2+](i)) in canine pulmonary arterial smooth muscle cells (PASMCs). [Ca2+](i) was measured as the 340- to 380-nm ratio in individual fura 2-loaded PASMCs. Resting [Ca2+](i) was 96 ± 4 nmol/l. PE (10 μmol/l) stimulated oscillations in [Ca2+](i), with a peak amplitude of 437 ± 22 nmol/l and a frequency of 1.01 ± 0.12/min. Thapsigargin (1 μmol/l) was used to deplete sarcoplasmic reticulum (SR) Ca2+ after extracellular Ca2+ was removed. Under these conditions, a nifedipine-insensitive, sustained increase in [Ca2+](i) (140 ± 7% of control value) was observed when the extracellular Ca2+ concentration was restored; i.e., capacitative Ca2+ entry was demonstrated. Capacitative Ca2+ entry also refilled SR Ca2+ stores. Capacitative Ca2+ entry was attenuated (32 ± 3% of control value) by 50 μmol/l of SKF-96365 (a nonselective Ca2+-channel inhibitor). Tyrosine kinase inhibition with tyrphostin 23 (100 μmol/l) and genistein (100 μmol/l) also inhibited capacitative Ca2+ entry to 63 ± 12 and 85 ± 4% of control values, respectively. SKF-96365 (30 μmol/l) attenuated both the amplitude (15 ± 7% of control value) and frequency (50 ± 21% of control value) of PE-induced Ca2+ oscillations. SKF-96365 (50 μmol/l) abolished the oscillations. Tyrphostin 23 (100 μmol/l) also inhibited the amplitude (17 ± 7% of control value) and frequency (45 ± 9% of control value) of the oscillations. Genistein (30 μmol/l) had similar effects. Both SKF-96365 and tyrphostin 23 attenuated PE-induced contraction in isolated pulmonary arterial rings. These results demonstrate that capacitative Ca2+ entry is present and capable of refilling SR Ca2+ stores in canine PASMCs and may be involved in regulating PE-induced Ca2+ oscillations. A tyrosine kinase is involved in the signal transduction pathway for α1-adrenoreceptor activation in PASMCs.