INFLUENCE OF ACUTE AND CHRONIC ETHANOL TREATMENT ON MUSCARINIC RESPONSES AND RECEPTOR EXPRESSION IN CHINESE-HAMSTER OVARY CELLS

The influence of ethanol on the muscarinic receptor-mediated release o f inositol phosphate from Chinese hamster ovary (CHO) cells stably tra nsfected with one of the five subtypes of muscarinic acetylcholine rec eptor was determined. In CHO cells expressing M3 muscarinic receptors (CHO-M3), carbamylcholine increased muscarinic receptor-induced releas e of inositol phosphate by 150-350% following a 15-min incubation with an EC50 of approximate to 30 mu M. Maximal responses were obtained wi th 1 mM carbamylcholine, while responses to 10 mM carbamylcholine were somewhat less than maximal. Preincubation with atropine for 10 min in hibited the response with an IC50 of approximate to 30 nM. CHO cells t ransfected with M1, M3, and M5 receptors displayed a similar pattern o f activity; CHO cells transfected with M2 and M4, as well as untransfe cted cells, were unresponsive to carbamylcholine. Ethanol acutely inhi bited the response of CHO-M3 cells to carbamylcholine by 15% at 18 mM and by 47% at 180 mM (the highest concentration examined). CHO-M3 cell s were incubated with 50 mM ethanol for 48 hr. This treatment did not. affect the number of cells or their protein content (113 pg/cell). Th e expression of M3 muscarinic receptors (determined using [H-3]N-methy lscopolamine) increased from 1.34 +/- 0.23 to 1.75 +/- 0.16 pmol/mg pr otein (P < 0.05). In contrast, carbamylcholine-stimulated release of i nositol phosphate was depressed by 40-70% in four experiments. Concent ration-response analyses indicated a non competitive inhibitory mechan ism. This dissociation of muscarinic receptor expression and muscarini c signaling suggests a compensatory increase in receptor expression in response to chronic inhibition of muscarinic signaling by ethanol. (C ) 1997 Elsevier Science Inc.