Ethanol has been reported to alter synaptic transmission and neurosecretion; however, the mechanisms involved in these actions are poorly understood. Neurosecretory mechanisms and the actions of ethanol on those mechanisms were studied in cell lines in tissue culture. In the mouse pituitary cell line, AtT-20, somatostatin inhibits both the secretion of ACTH and the cytosolic calcium rise evoked by secretagogues. We studied the effect of somatostatin on membrane calcium current in these cells using the whole-cell patch-clamp technique. Calcium current activated rapidly at potentials positive to -40 mV, peaked in 6-7 ms, and inactivated slowly. Somatostatin (0.01 to 1 micromolar) decreased the calcium current by 30% during voltage steps eliciting maximal current amplitude. The results suggest that the somatostatin-induced inhibition of ACTH secretion may result from the reduction of peak calcium current by somatostatin. Neurosecretory mechanisms were also investigated in the rat chromaffin cell line, PC12. We studied the effect of muscarinic agonists on intracellular calcium, phosphoinositide metabolism, and transmitter release. Addition of the muscarinic agonists methacholine or muscarine caused an increase in intracellular free calcium levels. The increase in intracellular calcium was blocked by atropine; however, removal of extracellular calcium did not block the response. In addition, methacholine increased cellular levels of inositol triphosphate and stimulated the release of norepinephrine. These results indicate that PC12 cells provide a model for investigating the interrelationship between phosphatidylinositol metabolism, intracellular free calcium, and secretion. The effects of ethanol are being tested on the secretory mechanism in these two cell lines. The significance of the project derives from the fact that characterization of synaptic and neurosecretory mechanisms and the actions of ethanol on those mechanisms should increase our understanding of the cellular basis of ethanol's actions in the nervous and endocrine systems.
