This application focusses on the Ca channels and on mechanisms of exocytosis found in chromaffin cells. In the animal, chromaffin cells in the adrenal medulla secrete catecholamines and several neuropeptide hormones. Secretion is triggered by Ca2+ entering the cells through voltage-gated Ca channels. Chromaffin cells possess three different types of Ca channels. One type, the facilitation Ca channel, is not activated by single brief depolarizations. This channel is normally silent; it does not contribute to electrical activity or to Ca2+ influx. Facilitation Ca channels are activated by many depolarizations to physiological potentials, or by depolarizations to very positive potentials. Recruitment of these channels may involve a novel form of channel regulation, voltage- dependent phosphorylation. All three types of Ca channels found in chromaffin cells are capable of triggering secretion; facilitation Ca channels are most efficient. Secretion produced by facilitation Ca channels is larger, faster, and has a shorter latency when compared to release produced by the other channels types, even when the Ca current is smaller. By simultaneously measuring Ca currents, [Ca2+] and secretion (with capacitance), we hope to determine the optimal conditions for triggering release by each type of Ca channel. We will model secretion for each type of Ca channel present in chromaffin cells.

Chromaffin cells store and secrete either epinephrine or norepinephrine (some cells may secrete both). In a few preliminary studies norepinephrine containing cells were found to have no facilitation Ca channels. Using voltammetric techniques to identify chromaffin cells as either epinephrine or norepinephrine secreting, we will verify this finding. Norepinephrine cells will be studied extensively to better understand catecholamine secretion from cells that presumably have no facilitation Ca channels. All the Ca channels in norepinephrine cells will be tested for efficacy in promoting secretion. These data will be compared to secretion data from epinephrine containing cells that have facilitation Ca channels. These studies may expose alterations in secretion resulting from differences in the Ca channels present.

Ca currents recorded in chromaffin cells using the perforated patch whole- cell configuration are different than currents recorded with whole-cell patch clamp configuration with low EGTA in the pipette. This is an important point as almost all capacitance studies secretion have used whole-cell recording conditions with low EGTA. We will study secretion from chromaffin cells using perforated whole-cell recording conditions. Given our preliminary results I feel it's crucial to study secretion with the intracellular constituents intact. Finally, we will determine whether secretion can be modulated by neurotransmitters, while keeping Ca currents and [Ca2+]i constant. If modulation is found, we will try and elucidate the mechanism.

R01GM043560

1991-04-01

1999-08-31