ETHANOL INHIBITS MUSCARINIC RECEPTOR-STIMULATED PHOSPHOINOSITIDE METABOLISM AND CALCIUM MOBILIZATION IN RAT PRIMARY CORTICAL CULTURES

In recent years, it has been hypothesized that muscarinic receptor-sti mulated phosphoinositide (PI) metabolism may represent a relevant targ et for the developmental neurotoxicity of ethanol. Age-, brain region- , and receptor-specific inhibitory effects of ethanol on this system h ave been found, both in vitro and after in vivo administration. As a d irect consequence of this action, alterations of calcium homeostasis w ould be expected, through alterations of inositol trisphosphate format ion, which mediates intracellular calcium mobilization. In the present study, the effects of ethanol (50-500 mM) on carbachol-stimulated PI metabolism and free intracellular calcium levels were investigated in rat primary cortical cultures, by measuring release of inositol phosph ates and utilizing the two calcium probes fluo-3 and indo-1 on an ACAS (Adherent Cell Analysis and Sorting) Laser Cytometer. Ethanol exerted a concentration-dependent inhibition of carbachol-stimulated PI metab olism. In addition, ethanol's inhibitory effect paralleled the tempora l development of the muscarinic receptor signal transduction system, w ith the strongest inhibition (25-50%) occurring when maximal stimulati on by carbachol occurs (days 5-7). Ethanol also exerted a concentratio n-dependent decrease in free intracellular calcium levels following ca rbachol 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. The inhibitory effects of ethano l seemed dependent upon preincubation time, in that a longer preincuba tion (30 min) with the lowest dose (50 mM), showed almost the same dec rease in responding cell number and reduction in spike amplitude in re sponding cells, as a shorter incubation (10 min) with the highest etha nol dose (500 mM). The specificity of the response to carbachol was de monstrated by blocking the response with 10 mu M atropine. Moreover, e xperiments with carbachol in calcium-free buffer with 1 mM EGTA indica ted that the initial calcium spike was due to intracellular calcium mo bilization from intracellular stores. Since calcium is believed to pla y important roles in cell proliferation and differentiation, these res ults support the hypothesis that this intracellular signal-transductio n pathway may be a target for ethanol, contributing to its development al neurotoxicity.