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Center for Anesthesiology Research, Division of Anesthesiology and Critical Care Medicine, The Cleveland Clinic Foundation, Cleveland, Ohio 44195
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
1-adrenoreceptor activation in PASMCs.
1-adrenoreceptor activation
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