Recent evidence has suggested that membrane ion channels which regulate the flux of important monovalent and divalent ions across the cell membrane may be particularly sensitive to the actions of ethanol and mediate the development of functional tolerance and potent pharmacological actions of ethanol an mediate the development of functional tolerance and physical dependence following chronic exposure. Recent studies have show that there are multiple types (L,N,T) of calcium channels in brain which exhibit different sensitivities to blockade by organic calcium channel blockers. An unequal distribution of multiple calcium channel subtypes throughout the brain may account for brain regional differences in calcium channel blockade observed with ethanol. the specific calcium channel subtype(s) which are blocked by ethanol is not know and attempts to characterize these effects are complicated by the heterogeneous nature of brain tissue. The experiments described in this study are designed to investigate the effects of ethanol on different types of voltage-sensitive calcium channels which can be expressed in cultured rat adrenal medullary chromaffin cells and PC- 12 cells. These cells are good models for the study of calcium-mediated transmitter release processes and appear to possess only the L-type of calcium channel. However, treatment of these cells in culture with nerve growth factor (NGF) results in the expression of a second type of channel which resembles the N-type observed in nerves. Studies will be conducted to characterize the sensitivity of these different channel types to the inhibitory actions of ethanol and will be correlated with changes in the density of calcium channels following NGF treatment. Measurement of fast- phase calcium entry(45Ca 2+), intracellular free calcium (Fura-2 spectroscopy) and endogenous catecholamine release (high performance liquid chromatography) will be made from NGF treated and untreated cells. Changes in calcium channel density following NGF treatment will be measured using radioligand binding techniques. The effects of acute and chronic ethanol exposure on the binding parameters of these cell types will be investigated in order to correlate changes in calcium channel density with functional changes in ion flux, regulation of intracellular free calcium and catecholamine release following NGF treatment. These experiments are designed to study the action of ethanol on multiple types of voltage- sensitive calcium channels which can be expressed in cell culture and should yield valuable insights regarding the molecular mechanisms underlying the complex actions of ethanol on neuronal function.
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