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

Author:

Hamada Hiroshi1,Damron Derek S.1,Hong Sung Jin1,Van Wagoner David R.1,Murray Paul A.1

Affiliation:

1. From the Center for Anesthesiology Research, Division of Anesthesiology and Critical Care Medicine, The Cleveland (Ohio) Clinic Foundation.

Abstract

Abstract Modulation of [Ca 2+ ] 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 α 1 -adrenoceptors with phenylephrine (PE) on [Ca 2+ ] 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 [Ca 2+ ] i was measured using a dual-wavelength spectrofluorometer. Resting values of [Ca 2+ ] i were 96±4 nmol/L. PE (10 μmol/L) stimulated oscillations in [Ca 2+ ] i at a frequency of 1.35±0.07/min, which reached a peak [Ca 2+ ] 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 [Ca 2+ ] i oscillations increased in a dose-dependent (3×10 −8 to 10 −4 mol/L) manner. Pretreatment with the α 1 -adrenoceptor antagonist prazosin (50 nmol/L) or removal of extracellular Ca 2+ abolished the repetitive [Ca 2+ ] i oscillations induced by PE. The voltage-operated Ca 2+ channel blockers nifedipine (1 μmol/L) and verapamil (1 μmol/L) had no effect on the [Ca 2+ ] 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 [Ca 2+ ] i similar to those observed in cultured cells, and patch-clamp experiments revealed oscillations in membrane potential. These results indicate that PE induces [Ca 2+ ] i oscillations in PASMCs via stimulation of α 1 -adrenoceptors coupled to phospholipase C activation. Voltage-operated Ca 2+ channels and protein kinases are not required for the oscillations. The requirement for extracellular Ca 2+ and intracellular Ca 2+ stores indicates that both Ca 2+ influx and intracellular Ca 2+ release play a role in the maintenance of the oscillations.

Publisher

Ovid Technologies (Wolters Kluwer Health)

Subject

Cardiology and Cardiovascular Medicine,Physiology

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