Rat pheochromocytoma cells (PC-12) in culture possess a number of characteristiccs of catecholaminergic neurons including the ability ot synthesize, store, release and take up catecholamines (1,2,3,4,5). These cells provide a useful model system for the study of the interactions of ethanol with the enzymes that synthesize catecholamines as well as the effects of ethanol on the molecular mechanisms related to these phenonmena. Catecholamine synthesis in the PC-12 cells is increased (200 to 300%) after incubation in buffer containing either high potassium (56 mM) or dibutyryl cyclic AMp. Preliminary data in our laboratory indicate that the prior incubation of the PC-12 cells with 80mM ethanol for 30 minutes completely eliminates the increase in catecholamine synthesis produced by dibutyryl cyclic AMP or potassium (56 mM). In order to analyze these observations more completely PC-12 cells will be treated with concentrations of ethanol ranging from 10mM to 100 mM either acutely (5 min to 30 min) or chronically (4 hr to 8 days) and the following aspects of catecholamine synthesis regulation will be evaluated: 1. Cyclic AMP levels. 2. Cyclic AMP-dependent protein kinase activity, 3. In situ turnover of catecholamines measured by the accumulation of 14CO2 + dopamine in the cells after the addition of 14C-tyrosine (carboxyl labelled) to the PC-12 cell media. 4. Tyrosine hydroxylase activity measured in the supernatant fraction of PC-12 cell homogenates. 5. The ability of tyrosine hydroxylase to be phosphorylated in situ by stimuli that activate the enzyme in these cells. In this manner, we hope to provide more complete information on the cellular mechanisms responsible for the interactions of ethanol with the synthesis of catecholamines.
