The central hypothesis of this project is that cytosolic free calcium concentration (Cai) is the major regulator of aldosterone biosynthesis and secretion. In the first grant period, investigation of Cai in adrenal zona glomerulosa (ZG) was extended to the single cell level. These studies precisely quantitated the extent of Cai changes that occur during secretagogue stimulation. Elevation of extracellular K+ in its physiological range produces a sustained rise in Cai that is probably a consequence of the activation of voltage-dependent Ca2+ channels. On the other hand, the Cai response to angiotensin II (AngII) exhibits a spatially non-uniform and temporally oscillating pattern. Despite the complexity of the Cai responses, our results from parallel studies comparing the temporal characteristics of aldosterone secretion and Cai have further strengthen the Ca2+ hypothesis for aldosterone regulation. The experiments in the present proposal will employ single cell Cai measurement and plasma membrane, patch-clamp electrophysiology to address the following Specific Aims: (1) To determine if cytosolic Ca2+ mobilization mechanisms underlie the oscillatory pattern of ZG cell Cai stimulated by AngII. (2) To determine the role of plasma membrane potential and voltage- and receptor operated ion channels in controlling or modulating the Cai oscillations. (3) To investigate the mechanisms responsible for the restoration of Cai to basal levels following a stimulated rise. Abnormal regulation of aldosterone secretion has been reported in a substantial fraction of the hypertensive population, particularly in response to AngII infusion. The mechanisms responsible for these alterations in hormonal secretion are unknown. We have recently documented that ZG cells from the spontaneously hypertensive rat (SHR) have an elevated resting Cai and a blunted calcium response to AngII and K+ application, suggesting that a sustained increase in Cai may be casually related to the altered steroid response to secretagogues. These findings provide the basis for the last Specific Aim: (4) To test the hypothesis that the pathophysiological consequences of the hypertensive state result, in part, from effects on basal and secretagogue-modified Cai levels in single ZG cells.
