ABSTRACT
Previous studies have suggested that activation of calcium-phospholipid-dependent protein kinase (PKC) enhances benzodiazepine (BZD)- and pentobarbital (PB)- mediated potentiation of alpha(1)beta(1)gamma(2) GABA(A) receptors (GABA(A)-Rs). To delineate the underlying mechanism(s), voltage-clamp recordings were performed on recombinant alpha(1)beta(1)gamma(2) GABA(A) receptors functionally expressed in Xenopus laevis oocytes. GABA(A)-Rs were tested for their sensitivity to diazepam and PB before and after incubation in phorbol 12-myristate 13-acetate (PMA). PMA (25 nM) significantly attenuated the GABA(A) current (p<0.05, n=12-19) up to 90%. PMA treatment, however, did not alter the sensitivity to diazepam or pentobarbital. Similar results were obtained with recombinant alpha(1)beta(2)gamma(2) GABA receptors. These data suggest that PKC activation does not alter the allosteric modulation of GABA(A)-Rs by benzodiazepines and barbiturates and is consistent with the observation from other studies in oocytes that PMA decreases the amplitude of the GABA-activated currents via receptor internalization rather than modification of receptor kinetics.
Subject(s)
Barbiturates/pharmacology , Benzodiazepines/pharmacology , Protein Kinase C/metabolism , Receptors, GABA-A/drug effects , Receptors, GABA-A/metabolism , Allosteric Regulation/drug effects , Animals , Cells, Cultured , Diazepam/pharmacology , Enzyme Activation/drug effects , GABA Modulators/pharmacology , Hypnotics and Sedatives/pharmacology , Membrane Potentials/drug effects , Oocytes/cytology , Oocytes/metabolism , Patch-Clamp Techniques , Pentobarbital/pharmacology , Protein Subunits , Rats , Recombinant Proteins/drug effects , Recombinant Proteins/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Xenopus laevisABSTRACT
Previous studies suggest that diazepam (DZP) increases the desensitization rate of GABA(A) receptors, although this effect could simply be a consequence of the DZP-induced increase in GABA sensitivity rather than a direct modulation of desensitization kinetics. To distinguish these two possibilities, voltage clamp recordings were performed on rat alpha1beta2gamma2 GABA(A) receptors expressed in Xenopus laevis oocytes. Complete GABA concentration-response relationships were obtained in the absence and presence of 1 microM DZP and the observed shift in GABA sensitivity (approximately 2.5-fold) was used to adjust GABA and GABA plus DZP to the same level of activation. In this case, DZP had no significant effect on either the rate of onset or recovery from desensitization. This suggests that the apparent effect of DZP on the rate of desensitization is secondary to the increase in GABA sensitivity and not due to a direct effect on the process of desensitization.
Subject(s)
Benzodiazepines/pharmacology , GABA Modulators/pharmacology , Receptors, GABA-A/drug effects , Animals , Chloride Channels/drug effects , Chloride Channels/metabolism , Diazepam/pharmacology , Kinetics , Patch-Clamp Techniques , Rats , Receptors, GABA-A/biosynthesis , Transcription, Genetic , Xenopus laevisABSTRACT
Mescaline (3,4,5-trimethoxyphenylethylamine; MES) and its analogs, anhalinine (ANH) and methylenemescaline trimer (MMT) were investigated, using sciatic-sartorius preparations of the frog and cortical tissue from the rat. The effects of MES and its analogs were examined with respect to muscle twitch, resting membrane potential and nicotinic receptor binding. Mescaline and its analogs (10-100 microM) blocked both directly and neurally evoked twitches but their effects on neurally evoked twitches were greater than those on directly evoked twitches. Mescaline, ANH and MMT decreased amplitude of the miniature endplate and endplate potentials, decreased acetylcholine (ACh) quantal content, hyperpolarized the resting membrane potential and prolonged duration of the action potential. They did not significantly displace the binding of [125I]-alpha-bungarotoxin (alpha-BTX) to nicotinic receptors, at concentrations which blocked neuromuscular transmission. These results suggest that MES and its analogs inhibit cholinergic neuromuscular transmission by blocking release of ACh; they also affect K+ conductance.
