Vol. zyx 23. zy No. zy 5 May zy IWY Chronic Ethanol Exposure Enhances Signaling Through Muscarinic Receptors Expressed by cRNA Injection in Xenopus Oocytes: Implications for Mechanism of Action Christian W. Honemann, Andrea Wong, John zyxwv A. M. Arledge, and Marcel E. Durieux The molccular mcchanisms underlying thc ccrebral symptoms of ethanol withdrawal syndrome are poorly undcrstocid. In addition to ethanol's cffcct on GABA and NMDA reccptors, ethanol affects mus- carinic acetylcholine signaling. This interaction has attracted attention because of the importance of muscarinic signaling in consciousness. Chronic cthanol cxposure increases muscarinic receptor binding. Incrcascd transcription of rcccptor message has been suggested as the underlying mcchanism, but this hypothcsis has not been tcstcd directly. Thcrcforc. we studied the effects of ethanol on muscarinic signaling in a model that bypasses transcription of muscarinic rcccptor gencs. We cxpresscd rat ml muscarinic receptors by cRNA microinjection in zyxwvutsrqp Xerzopiis oocytcs. Cells wcrc voltage-clampcd at -70 mV and effects of prolonged (24. 48, and 72 hr) cxposurc to ethanol (25. 50, and 100 mM) on methylcholinc-induced calcium-activated C1~ currcnts were dctcrmincd. Effects of prolonged ethanol exposure on currents induced by stimulation of lysophosphatidatc rcccptors. direct G protein activation, or inositol trisphosphatc rcccptor activation wcrc studied as well. Prolonged cthanol cxposure cnhanccd niethylcholine (or lysopliosphatidatc-)-induced currents in a time- and concentration-dcpendcnt manner. Thus, enhanced muscarinic gcnc transcription is not required for ethanol cnhanccmcnt of muscarinic signaling. Lack of ethanol cffcct on inositol trisphosphatc-induccd signaling suggests that intracellular signaling systems downstream of' phospholipase C arc not involved. In contrast, currcnts induced by dircct G protein stim- ulation were enhanced significantly. Thcrcforc. onc potential site of cthanol's action on muscarinic signal- ing is uprcgulation of thc associatcd G protein or cnhancemcnt of its functioning. Key Words: Chronic Ethanol Exposure, ml Muscarinic Acctylcholiire Reccptor, G Protcin, Intracellular Signaling. LTHOUGH THE mechanisms of ethanol's profound A effects on cerebral functioning remain largely unex- plained,' several sites of action of potential relevance have been identified. These include ionotropic receptors, such as the GABA,2-4 and NMDA glutamate'-' receptors, as well as metabotropic receptors, such as muscarinic acetylcholine receptors."-I2 Particularly, the latter have received significant attention recently, as selective antagonism of CNS muscarinic signal- ing induces symptoms similar to some of those observed during acute ethanol intoxication. The muscarinic antago- nist scopolamine, for example, is clinically used as a seda- Frurii tlic Deparlinent zyxwvutsrqp of Ancsdieuiology, Uiiit~c~rsify of ViQ'riia, Charlottrs- Recei\~tl for puhlicatiori Jitrie 17. zyxwvutsrqp 1998; occrpted March 3, 1999. This stirrly was suppurid by tlic Natiotiril lnstitutes of Heulth (Grant GMS52387 to M. E. D.) arid Innovativc~ Mediziriisclie Forsclzung, Westfiilisclie Wilheb~is-Urri~cr:viriit, Miinstcr, Gemiony (Grant Hij-1-6-I1/98-27 to C, H! H.) Reprint rerpests: Marcel E. Diwiew M. D.. 1)epcirtnierit of Anesthesiology, UniLwsify of' Virginia HSC, P.0. Box 10010, C'harlottesvillc: VA 22906-0010; F m (80.1) 982-3 161; E-nicril: rned2p ~i.~~itgiiiia. tdic vilk Virginia. Coliyriglit zyxwvutsrqpo 0 I999 by tlrc) Research Society on Alcoliolistn. .4kwliol C'liti 1.i-p Krv, Vol 23. No 5, 1'140: pp 791-7W tive and amnestic agent. In addition, Katner et al.I3 re- ported that muscarinic receptor signaling within the pedunculopontine nucleus and ventral tegmental area reg- ulates ethanol drinking behavior. Acute ethanol exposure has been reported to inhibit muscarinic ~ignaling.'"'~ A modulatory role for muscarinic signaling in adaptation to chronic ethanol use has been suggested: prolonged eth- anol exposure has been shown to enhance muscarinic re- ceptor binding in human neuroblastoma (although this was not observed in rat brainI6). If muscarinic receptor expression is enhanced during chronic ethanol treatment, ethanol withdrawal after prolonged use would be antici- pated to lead to relative CNS muscarinic overstimulation. Indeed, the CNS symptoms of alcohol withdrawal syn- drome include many similar to those observed with Aman- ita rnuscaria toxicity, including irritability, confusion, rest- lessness, hallucinations, and convulsions. " The high incidence and great clinical relevance of chronic ethanol use and ethanol withdrawal make it impor- tant to understand in detail if and how prolonged ethanol exposure enhances muscarinic signaling. Several groups, including Larsson et al." and Hu et a1." reported that 791