Noncompetitive antagonism of bupivacaine on the neuronal nicotinic acetylcholine receptors in rat hippocampus

Abstract

Neuronal nicotinic acetylcholine receptors (nAChRs) in the brain, especially in the hippocampus, are thought to play a crucial role in the physiology of learning and memory. Characterization of the neuronal nAChRs by molecular, immunological and physiological techniques has revealed that their molecular structures, physiology and pharmacology are highly diverse and different from those of the muscle nAChR. However, little is known at this time about noncompetitive antagonism of these nAChRs. The purpose of this study was to characterize the interaction of bupivacaine, an open channel blocker of muscle nAChR, with the neuronal nAChRs in cultured hippocampal neurons. Using whole-cell and single-channel modes of the patch-clamp technique, the interactions of bupivacaine with the neuronal nAChRs were studied under different experimental conditions. Whole-cell currents with two different types of decay kinetics could be induced by ACh (300 muM). Currents that were fast-decaying were highly sensitive to blockade by the competitive nicotinic antagonist methyllycaconitine (MLA) at 10 nM. Currents having a slow decay were only partially blocked by MLA. Both the fast and slowly decaying ACh-induced whole-cell currents were blocked by bupivacaine, but with different sensitivity. Open-channel blockade by the drug was evidenced by a substantial decrease in the decay time constants of slowly decaying whole-cell currents and was confirmed by a shortening of the lifetime of ACh-activated single channels. The weak voltage dependence of the reduction of the decay time constant of the slowly decaying current suggests that the bupivacaine-binding site(s) responsible for open-channel blockade is(are) probably located near the orifice of the receptor ion channel. The non-linear relationship of this blockade with increasing concentration of bupivacaine indicated two distinct blocking rates, and suggested a closed-channel interaction of the drug at higher concentration, which was supported by the bupivacaine-induced decrease in the probability of a channel being opened. The blockade of the slowly decaying current by bupivacaine at high concentration was use dependent. Bupivacaine decreased the peak amplitude but did not change the decay time constant of the fast decaying ACh-induced currents, suggesting that a closed-channel blockade, but not open-channel blockade was taking place. This closed-channel blockade was time dependent and was noncompetitive. The neuronal nAChRs were more sensitive than NMDA, kainate, quisqualate, GABA, or glycine receptors to the action of bupivacaine.