Phenylephrine-induced Ca2+ oscillations in canine pulmonary artery smooth muscle cells

Modulation of [Ca2+](i) in response to receptor activation is a critical determinant of vascular smooth muscle tone. In this study, we examined the effect of continuous stimulation of alpha(1)-adrenoceptors with phenylephrine (PE) on [Ca2+](i) in single pulmonary artery smooth muscle cells (PASMCs) cultured from explants of canine intrapulmonary artery. Fura 2-loaded PASMCs pretreated with propranolol (5 mu mol/L) were continuously superfused with PE at 37 degrees C on the stage of an inverted fluorescence microscope, and [Ca2+](i) was measured using a dual-wavelength spectrofluorometer. Resting values of [Ca2+](i) were 96+/-4 nmol/L. PE (10 mu mol/L) stimulated oscillations in [Ca2+](i) at a frequency of 1.35+/-0.07/min, which reached a peak [Ca2+](i) of 650+/-26 nmol/L (n=69 cells). The oscillations lasted for >30 minutes and were constant in amplitude and frequency. Both the amplitude and frequency of PE-induced [Ca2+](i) oscillations increased in a dose-dependent (3x10(-8) to 10(-4) mol/L) manner. Pretreatment with the alpha(1)-adrenoceptor antagonist prazosin (50 nmol/L) or removal of extracellular Ca2+ abolished the repetitive [Ca2+](i) oscillations induced by PE. The voltage-operated Ca2+ channel blockers nifedipine (1 mu mol/L) and verapamil (1 mu mol/L) had no effect on the [Ca2+](i) oscillations. In contrast, inhibition of phospholipase C with U73122 (10(-7) to 10(-5) mol/L) attenuated the oscillations in a dose-dependent fashion. The nonselective protein kinase inhibitor staurosporine (10(-9) to 10(-7) mol/L) had a minimal inhibitory effect on the oscillations. Caffeine (30 mmol/L) and thapsigargin (1 mu mol/L) abolished the oscillations, whereas pretreatment with ryanodine (1 to 100 mu mol/L) had no effect. In freshly dispersed PASMCs, PE (10 mu mol/L) induced oscillations in [Ca2+](i) similar to those observed in cultured cells, and patch-clamp experiments revealed oscillations in membrane potential. These results indicate that PE induces [Ca2+](i) oscillations in PASMCs via stimulation of alpha(1)-adrenoceptors coupled to phospholipase C activation. Voltage-operated Ca2+ channels and protein kinases are not required for the oscillations. The requirement for extracellular Ca2+ and intracellular Ca2+ stores indicates that both Ca2+ influx and intracellular Ca2+ release play a role in the maintenance of the oscillations.