Angiotensin II (AngII) and elevated extracellular K+ levels [(K+) e] are the primary physiologic regulators of the synthesis and secretion of aldosterone, the hormone responsible for maintaining normal salt balance in the body. Previous studies have indicated that elevated (K+)e stimulates steroidogenesis by depolarizing the glomerulosa cell, thereby activating voltage-dependent Ca2+ channels and increasing Ca2+ influx. This enhanced influx, in turn, activates Ca2+-dependent processes, such as Ca2+/calmodulin-dependent protein kinases. In novel findings we have recently shown that elevated (K+) e also activates phospholipase D (PLD) and increases the phosphorylation of an endogenous protein kinase C (PKC) substrate (myristoylated alanine-rich C kinase substrate or MARCKS). PLD-mediated hydrolysis of membrane phosphatidylcholine generates phosphatidic acid, which can be dephosphorylated to produce diacylglycerol, the natural activator of PKC. Thus, we propose that the elevated (K+)e-induced increase in Ca2+ influx activates PLD to stimulate PKC activity, thereby eliciting MARCKS phosphorylation and sustained aldosterone secretion. Our data also suggest an involvement of PLD in the process of priming. Priming refers to the ability of a pretreatment with AngII to sensitize glomerulosa cells to respond to agents that stimulate Ca2+ influx, including small elevations in (K+) e, with enhanced aldosterone secretion relative to """"""""naive"""""""" unpretreated cells. We have postulated that priming is the result of the observed persistent elevation in PLD-generated diacylglycerol retaining PKC at the plasma membrane, a site at which the enzyme is poised to activate in response to increased Ca2+ influx. Using two-dimensional polyacrylamide gel electrophoretic analysis, proteomics and mass spectrometric identification of phosphorylation sites, PLD activity measurements, overexpression of PLD isoforms, aldosterone radioimmunoassays and western blot analysis, we wish to test the hypothesis that a PLD-generated signal retains PKC at the plasma membrane to result in the induction of priming. We will also define the mechanism of elevated (K+)e-induced PLD activation and examine the hypothesis that this activation stimulates PKC activity, MARCKS phosphorylation and aldosterone secretion. Since priming would allow enhanced aldosterone secretion to multiple AngII or sequential AngII and elevated (K+)e exposures, this research may provide novel insights into normal and pathological changes in steroidogenesis under conditions of altered sodium intake. The complex interactions between AngII and serum K+ levels suggest that abnormalities in the glomerulosa cell response to either agonist, or both, may contribute to the development of some forms of hypertension. ? ?
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