Cytosolic free calcium oscillates in single bovine adrenal glomerulosa cells in response to angiotensin II stimulation

Abstract

The characteristics of the change in cytosolic free Ca2+ concentration ([Ca2+]i) in response to agonist stimulation were studied in individual cells of the bovine adrenal zona glomerulosa. Following digestion and dispersion, the cells were loaded with the fluorescent Ca2+ indicator fura-2. The cells adhered to Concanavalin A-coated coverslips and were studied using dual excitation wavelength microfluorimetry. In this procedure individual cells under constant perfusion are visualized by microscopy and excited with light alternating rapidly between 340 and 380 nm. The ratio of fluorescence (F) emitted from the cell (F340/F380) correlates directly with [Ca2+]i. Continuous stimulation with angiotensin II (AII; 10 nmol/l) resulted in a brisk transient rise in [Ca2+]i within 8 s of application of the stimulus. In 50% of cells studied, this initial peak was followed by a series of oscillations in [Ca2+]i lasting up to 13 min, with an average period of 33.0 +/- 5.9 (S.E.M.) seconds. [Ca2+]i did not return to prestimulation levels and, subsequent to the oscillatory phase, the [Ca2+]i remained increased for several minutes. Upon removal of extracellular Ca2+ the oscillations ceased almost immediately although [Ca2+]i remained increased. However, in Ca2+-free medium, a single peak of [Ca2+]i still occurred in response to AII. Cells remained refractory to restimulation over a 15-min period. In contrast, stimulation with K+ (8 mmol/l) rapidly increased [Ca2+]i to a level similar to that induced by AII but without inducing oscillations. Moreover, the effect lasted only while K+ was present and was highly reproducible over multiple stimulations during a 15-min period. These results corroborate, at the single cell level, the known action of AII of causing release of intracellular Ca2+, but reveal a more complex mechanism of action on Ca2+ influx than previously recognized, possibly invoking a role for a putative second messenger-operated membrane Ca2+ channel.