Chronic ethanol exposure enhances signaling through muscarinic receptors expressed by cRNA injection in Xenopus oocytes: implications for mechanism of action.

The molecular mechanisms underlying the cerebral symptoms of ethanol withdrawal syndrome are poorly understood. In addition to ethanol's effect on GABA and NMDA receptors, ethanol affects muscarinic acetylcholine signaling. This interaction has attracted attention because of the importance of muscarinic signaling in consciousness. Chronic ethanol exposure increases muscarinic receptor binding. Increased transcription of receptor message has been suggested as the underlying mechanism, but this hypothesis has not been tested directly. Therefore, we studied the effects of ethanol on muscarinic signaling in a model that bypasses transcription of muscarinic receptor genes. We expressed rat m1 muscarinic receptors by cRNA microinjection in Xenopus oocytes. Cells were voltage-clamped at -70 mV and effects of prolonged (24, 48, and 72 hr) exposure to ethanol (25, 50, and 100 mM) on methylcholine-induced calcium-activated Cl- currents were determined. Effects of prolonged ethanol exposure on currents induced by stimulation of lysophosphatidate receptors, direct G protein activation, or inositol trisphosphate receptor activation were studied as well. Prolonged ethanol exposure enhanced methylcholine (or lysophosphatidate-)-induced currents in a time- and concentration-dependent manner. Thus, enhanced muscarinic gene transcription is not required for ethanol enhancement of muscarinic signaling. Lack of ethanol effect on inositol trisphosphate-induced signaling suggests that intracellular signaling systems downstream of phospholipase C are not involved. In contrast, currents induced by direct G protein stimulation were enhanced significantly. Therefore, one potential site of ethanol's action on muscarinic signaling is upregulation of the associated G protein or enhancement of its functioning.

Synergistic inhibition of lysophosphatidic acid signaling by charged and uncharged local anesthetics.

UNLABELLED: We investigated the mechanism of benzocaine (permanently uncharged) and QX314 (permanently charged) inhibition of lysophosphatidic acid (LPA) signaling. To determine their site of action, we studied effects of these drugs, alone and in combination, on LPA-induced Ca2+-dependent Cl currents (I(Cl(Ca))) in Xenopus oocytes. After 10 min exposure to benzocaine, QX314 (10(-6)-10(-2) M), or both, we measured effects on I(Cl(Ca)) induced by LPA (with and without protein kinase [PKC] activation/inhibition) and on I(Cl(Ca)) induced by the intracellular injection of IP3 and GTPgammaS. LPA application to oocytes resulted in I(Cl(Ca)) (50% effective concentration approximately 10(-8) M). Both anesthetics inhibited LPA signaling concentration-dependently (50% inhibitory concentration [IC50] benzocaine 0.9 mM, QX314 0.66 mM). The combination acted synergistically (IC50 benzocaine 0.097 mM/QX314 0.048 mM). Intracellular signaling pathways were not affected. This study shows that benzocaine and QX314 inhibit LPA signaling and act synergistically, which is most easily explained by the existence of two different binding sites. Lack of inhibition of IP3 or GTPgammaS-induced I(Cl(Ca)) identifies the receptor as a target. Activation of PKC can be excluded as a potential mechanism. IMPLICATIONS: Lysophosphatidic acid may play a role in wound healing, and its signaling is inhibited by local anesthetics. We identified the membrane receptor as the local anesthetic site of action and showed that charged (QX314) and uncharged (benzocaine) local anesthetics inhibit lysophosphatidic acid signaling synergistically, which can be explained by the presence of different binding sites.