Mechanism Of Inhibition Of Bis(7)-tacrine On GABA-activated Current In Cultured Rat Hippocampal Neurons And The Modulation Of Extracellular PH

Part 1Mechanism of bis(7)-tacrine inhibition of GABA-activated current incultured rat hippocampal neuronsBis(7)-tacrine is a novel dimeric acetylcholinesterase inhibitor derived from tacrine thatshows promise for the treatment of Alzheimer's disease.We have previously reported thatbis(7)-tacrine inhibits GABAA receptors.In the present study we investigated themechanism of bis(7)-tacrine inhibition of GABA_A receptor function using whole-cellpatch-clamp recording in cultured rat hippocampal neurons.Bis(7)-tacrine produced agradual decline of GABA-activated current to a steady-state,but this was not an indication of use-dependence,as the gradually-declining component could be eliminated by exposureto bis(7)-tacrine prior to GABA application.In addition,bis(7)-tacrine inhibition did notrequire the presence of agonist,and GABA-activated current recovered completely frominhibition by bis(7)-tacrine in the absence of agonist.The slow onset of inhibition bybis(7)-tacrine was not apparently due to an action at an intracellular site,as inclusion of 25μM bis(7)-tacrine in the recording pipette did not alter inhibition by 2.5μM bis(7)-tacrineapplied externally.Bis(7)-tacrine shifted the GABA concentration-response curve to theright in a parallel manner and the pA₂ value estimated from a Schild plot was 5.7.Bis(7)-tacrine increased the time constant of activation of GABA-gated ion channelswithout affecting the time constants of deactivation or desensitization.These resultssuggest that bis(7)-tacrine is a competitive GABA_A receptor antagonist with slow onset andoffset kinetics.The competitive inhibition of GABA receptors by bis(7)-tacrine couldcontribute to its ability to enhance memory. Part 2The modulation of extracellular pH on inhibition of bis(7)-tacrine onGABA-activated current in cultured rat hippocampal neuronsPH in brain may changes during physiological and pathophysiological conditions.Under acidic conditions,proton inhibits NMDA-activated current in cultured rathippocampal neurons.Our previous study reported that bis(7)-tacrine proton-dependentlyinhibits NMDA-activated current in cultured rat hippocampal neurons.In this study wereported that bis(7)-tacrine is a competitive GABA_A receptor antagonist with slow onsetand offset kinetics.However,whether pH could modulate the inhibition of bis(7)-tacrine onGABA-activated current in cultured rat hippocampal neurons have not been fully elucidated.So we further investigated the effect of extracelluar pH on GABA-activated current incultured hippocampal neurons and observed the modulation of pH on the inhibition ofbis(7)-tacrine using the whole-cell patch clamp recording.The results show that,in 70%(63/90) of tested cells,at acidic conditions,proton inhibited GABA-activated current inconcentration-dependent mannar.At alkaline conditions,it has an opposite effect.However,in 30% (27/90) of tested cells,proton has no apparent effect.At pH range from 6.0 to 8.8,the inhibitory effect of bis(7)-tacrine decreased dramatically with the increase ofextracellular pH.At pH 6.0,the concentration-response curve of bis(7)-tacrine is shiftedleftwards with the IC₅₀ value decreased from 5.61±0.45μM (at pH 7.4) to 1.79±0.12μM.In addition,bis(7)-tacrine shifts the proton inhibition curve rightwards.These resultsindicate that bis(7)-tacrine proton-dependently inhibits GABA-activated current in culturedrat hippocampal neurons.In addition,bis(7)-tacrine sensitizes GABA_A receptor to protoninhibition.This is beneficial for therapeutic effects of bis(7)-tacrine under acidic conditions associated with some pathophysiological conditions such as ischemia.