Phosphoinositide metabolism and exocytosis in adrenal chromaffin cells.

Abstract

This thesis investigates the regulation of phosphoinositide metabolism in adrenal chromaffin cells and examines its relationship to exocytosis. Chapter II: Nicotinic receptor agonists, muscarinic agonists, and depolarization with elevated K$\sp{+}$ all increased the formation of ($\sp3$H) inositol phosphates in intact cells. The effects of variations in extracellular Ca$\sp{2+}$ and organic Ca$\sp{2+}$ channel antagonists indicated that the activation of phospholipase C by nicotinic agonists and depolarization resulted from Ca$\sp{2+}$ influx. Phospholipase C activation by Ca$\sp{2+}$ influx was a direct effect which did not result from hormone release. Chapter III: In digitonin-permeabilized cells, micromolar Ca$\sp{2+}$ and GTP$\tau$S stimulated the formation of inositol phosphates. Guanine nucleotides modulated the Ca$\sp{2+}$-sensitivity of phospholipase C activity. Ca$\sp{2+}$ (but not GTP$\tau$S) also caused an increase in labeled PIP and PIP$\sb2$ levels. ATP supported PIP and PIP$\sb2$ synthesis but did not activate phospholipase C. Ca$\sp{2+}$ and ATP acted synergistically to enhance the subsequent response to GTP$\tau$S. Thus, polyphosphoinositide synthesis is important in determining the amplitude of the signals produced by phospholipase C. The differential regulation of polyphosphoinositide levels and phospholipase C activity by Ca$\sp{2+}$, GTP$\tau$S, and ATP may lead to synergistic interactions between these agents. Chapter IV: Incubation of permeabilized cells with a PI-specific phospholipase C (PI-PLC) hydrolyzed over 70% of cellular PI; PIP and PIP$\sb2$ levels also declined, reflecting degradation by phosphatases. Cells permeabilized in the absence of ATP displayed a similar loss of PIP and PIP$\sb2$. Ca$\sp{2+}$-dependent secretion was potentiated by ATP. PI-PLC treatment selectively inhibited the ATP-dependent component of secretion. The inhibition was not caused by diacylglycerol, IP$\sb1$, or protein kinase C. Activation of the endogenous G-protein-linked polyphosphoinositide-phospholipase C also inhibited secretion under conditions which prevented polyphosphoinositide resynthesis. These findings suggest that secretion requires the presence of PIP and/or PIP$\sb2$. Because phospholipase C activity was not correlated with secretion, the polyphosphoinositide requirement for secretion probably does not arise from the role of these lipids as phospholipase C substrates.