Phenylephrine-Induced Ca 2+ Oscillations in Canine Pulmonary Artery Smooth Muscle Cells

Abstract

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 α₁-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 μmol/L) were continuously superfused with PE at 37°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 μmol/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 (3×10⁻⁸ to 10⁻⁴ mol/L) manner. Pretreatment with the α₁-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 μmol/L) and verapamil (1 μmol/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 μmol/L) abolished the oscillations, whereas pretreatment with ryanodine (1 to 100 μmol/L) had no effect. In freshly dispersed PASMCs, PE (10 μmol/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 α₁-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.