The purpose of this project is to determine second messenger pathways involved in receptor regulation of neurotransmitter levels. Adrenal medullary cells were used as a model neuronal system to study calcium-mobilizing receptors and their effects on protein kinase C and short- and long-term enkephalin and catecholamine 2 secretion. Changes in intracellular calcium were studied using Fura2, (4 5)Ca2+ influx was used to study the contribution of extracellular calcium, and [3H]phorbol dibutyrate binding was used to follow protein kinase C translocation. Nicotine and membrane depolarization produced a pronounced increase in intracellular calcium and short-term secretion, and was surprisingly effective in stimulating PKC translocation. Extracellular calcium was required for these effects. In contrast, angiotensin II and other phospholipase C-linked receptor agonists produced a transient increase in intracellular calcium regardless of extracellular calcium concentration. Single cell studies suggest that the apparent transient response reflects oscillations above and below the basal calcium level, which continues for at least an hour. These oscillations are dependent on extracellular calcium as is short-term secretion. Angiotensin II stimulates a conotoxin-sensitive calcium influx which may be distinct from the angiotensin effect on phospholipase C. The relative roles of calcium influx, intracellular calcium mobilization and protein kinase C activation in long-term secretion and gene regulation are presently being studied. Previous work from this lab has demonstrated that splanchnic innervation exerts a predominantly inhibitory effect on enkephalin and catecholamine gene transinjection; inhibitory effects on Ca-i responses can be seen under some conditions with GABA, histamine and ATP. Possible mechanisms of action of these inhibitory agonists are being examined. In addition to providing further insight into the mechanisms of action of different receptor agonists, these studies should lead to a better understanding of how second messenger signals are integrated for long term effects.
