Abstract:
Ethanol is known to directly influence the developing nervous system, the neuronal membrane being a primary site of action. In recent years, it has been hypothesized that the muscarinic receptor-stimulated phosphoinositide (PI) metabolism may represent a relevant target for the developmental neurotoxicity of ethanol. Age, brain rgion, and receptor-specific inhibitory effects of ethanol on this system have been found, both in vitro and after in vivo administration. As a direct consequence of this actionn, alterations of calcium homeostasis would be expected, through alterations of inositol triphophate formation, which mediates intracellular calcium mobilization. In the present study, the effects of ethanol (50-500 mM) on carbachol-0stimulated PI metabolism and free intracellular calcium levels were investigated in rat primary cortical cultures, bu measuring release of inositol phosphates and utilizing the two calcium probes FLUO-3 and INDO-1 on an ACAS (Adherent Cell Analysis and Sorting) Laser Cytometer. Ethanol exerted a dose-dependent inhibition of carbachol (1 mM)-stimulated PI metabolism. In addition, ethanol's inhibitory effect paralleled the temporal development of the muscarinic receptor signal transduction system, with the strongest inhibition occuring when maximal stimulation with 1 mM carbachol occurs (days 5-7). Ethanol also exerted a dose-dependent decrease in free intracellular calcium levels following carbachol stimulation. Both initial calcium spike amplitude, seen in all responsive cells, as well as the total number of cells responding to carbachol, were decreased by ethanol in a dose-dependent manner. The inhibitory effects of ethanol seemed dependent upon preincubation time, in that a longer preincubation (30 min) with the lowest dose (50 mM), showed almost the same decrease in responding cell number and reduction in spike amplitude in responding cells, as a shorter incubation (10 min) with the highest ethanol dose (500 mM). The specificity of the response to carbachol was demonstrated by blocking the response with 10 uM atrophine. Moreover, experiments with carbachol in calcium-free buffer (with 1mM EGTA) indicated that the initial calcium spike was due to intracellular calcium mobilization from intracellular stores. Since calcium is believed to play important roles in cell proliferation and differentiation, these results support the hypothesis that this intracellular signal-transduction pathway may be a likely target for the developmental neurotoxicity of ethanol.