Search

Calcium/phospholipid-dependent protein kinase (protein kinase C, PKC) has been suggested to play a role in the sensitivity of gamma-aminobutyrate type A (GABAA) receptors to ethanol. We tested a line of null mutant mice that lacks the gamma isoform of PKC (PKC gamma) to determine the role of this brain-specific isoenzyme in ethanol sensitivity. We found that the mutation reduced the amount of PKC gamma immunoreactivity in cerebellum to undetectable levels without altering the levels of the alpha, beta I, or beta II isoforms of PKC. The mutant mice display reduced sensitivity to the effects of ethanol on loss of righting reflex and hypothermia but show normal responses to flunitrazepam or pentobarbital. Likewise, GABAA receptor function of isolated brain membranes showed that the mutation abolished the action of ethanol but did not alter actions of flunitrazepam or pentobarbital. These studies show the unique interactions of ethanol with GABAA receptors and suggest protein kinase isoenzymes as possible determinants of genetic differences in response to ethanol.

Ethanol enhancement of GABAA receptor function has been found in some, but not all, studies. These results suggest the existence of ethanol-sensitive and -resistant receptors that may differ in subunit composition, although methodological differences (e.g., 36Cl- flux versus membrane currents) could also contribute to the different results. To examine these possibilities, we used mouse L(tk-) cells stably transfected with alpha 1 + beta 1 or alpha 1 + beta 1 + gamma 2L GABAA receptor subunit DNAs and compared 36Cl- flux with whole-cell, patch-clamp measurements of GABAA receptor function. Both techniques detected a similar modulation of the GABA receptor by ethanol, flunitrazepam, and pentobarbital. The potentiating action of ethanol required the gamma-subunit and was maximal at a concentration of 10 mM. Similar ethanol potentiation was obtained with brief (20 msec) or long (2 sec) applications of GABA. Analysis of data obtained from individual cells expressing alpha 1 beta 1-gamma 2L subunits showed considerable variability in sensitivity to ethanol, particularly with concentrations of 30 and 100 mM. Ethanol potentiated GABA action if the cells were grown on coverslips coated with polylysine, but had no effect on GABAA receptors of cells grown on uncoated coverslips. Thus, ethanol action was influenced by the growth matrix. Taken together, these data indicate that a gamma-subunit is necessary, but not sufficient, for ethanol sensitivity in this cell system. We suggest that posttranslational processing, particularly receptor phosphorylation, may also be important and that stably transfected cells will be useful in elucidating these events.

The Meyer-Overton hypothesis, predicting that the potency of an anesthetic correlates with its affinity for lipid, is a cornerstone of modern anesthetic theory. Several halogenated compounds were recently found to deviate from this prediction, whereas others did not. We tested the abilities of enflurane and five of these compounds to potentiate gamma-aminobutyric acid (GABA)A receptor responses in Xenopus oocytes expressing alpha 1 beta 2 or alpha 1 beta 2 gamma 2S GABAA receptors. Enflurane and the anesthetic 1-chloro-1,2,2-trifluorocyclobutane (F3) strongly potentiated chloride currents produced by 5 microM GABA with both alpha 1 beta 2 and alpha 1 beta 2 gamma 2S receptors. This potentiation decreased as the GABA concentration was raised. The transitional compound (less potent than predicted by its lipid solubility) 2-bromoheptafluoropropane produced modest enhancement, whereas three nonanesthetics (neither causing anesthesia in vivo nor decreasing the requirement for known anesthetics), 1,2-dichlorohexafluorocyclobutane, 2-chloroheptafluoropropane, and 2,3-chlorooctafluorobutane, did not affect GABAA receptor currents. Although all five compounds were predicted to be anesthetics by the Meyer Overton hypothesis, only F3 behaved as an anesthetic in vivo and only F3 markedly potentiated GABAA receptor responses in oocytes. These results strongly implicate the GABAA receptor in general anesthesia. Fluorescence polarization studies showed that anesthetics (enflurane and F3), but not nonasthetics (1,2 dichlorohexafluorocyclobutane and 2,3-chlorooctafluorobutane) disordered membrane lipids. Thus, for the compounds studied actions on both GABAA receptor function and lipid order distinguish between anesthetics and nonanesthetics.

Microtubules are present at postsynaptic densities in brain and are proposed to be involved in anchoring neurotransmitter receptor clusters at postsynaptic membranes. However, the influence of microtubules on gamma-aminobutyric acidA (GABAA) receptors has not been studied. Microtubule-affecting agents were tested for their actions on GABAA receptor function, by measuring muscimol-stimulated chloride uptake into cerebral cortical microsacs and proteoliposomes and GABA-mediated currents in Xenopus laevis oocytes expressing GABAA receptors. Colchicine, nocodazole, vinblastine, and taxol inhibited muscimol-stimulated chloride uptake. beta- and gamma-lumicolchicine did not inhibit GABAA ergic function. Colchicine decreased the potency of muscimol, a GABA agonist, to stimulate chloride uptake without affecting the specific binding of [3H]flunitrazepam or t-[35S]butylbicyclophosphorothionate to the GABAA receptor, or the allosteric modulation of binding of these ligands by muscimol. The function of purified GABAA receptors reconstituted in proteoliposomes, a preparation not containing microtubule components, was not affected by colchicine. In contrast to the results seen in human monocytes by other investigators, we found that colchicine decreased, rather than increased, protein kinase A activity in cortical microsacs. Thus, protein kinase A modulation of the GABAA receptor is not a likely mechanism for the actions of colchicine. We propose that microtubule-depolymerizing agents inhibit GABAA ergic function by disrupting the interaction of GABAA receptors with microtubules.

Both 5-HT3 receptor antagonists and benzodiazepine receptor ligands have effects on anxiety, and alter the behavioral action of ethanol. For these reasons, we tested the ability of several 5-HT3 receptor antagonists to inhibit the ligand binding and function of the gamma-aminobutyric acidA/benzodiazepine receptor Cl- channel complex of mouse brain membranes. MDL 72222 (1-a-H-3-a-5-aH-optropan-3yl-3,5-dichlorobenzoate) and LY 278584 (1-methyl-N-(8-methyl-8-azabicyclo[3.2.1.]oct-3-yl)-1H-indazole-3- carboxamide) inhibited [3H]flunitrazepam binding with Ki values of approximately 20 microM; ICS 205-930 (3 alpha-tropanyl-1H-indole-3-carboxylic acid ester) was more potent with a Ki of 0.8 microM. ICS 205-930 (50 microM) had no effect on [3H]muscimol binding. ICS 205-930, MDL 72222, and LY 278584 all inhibited the binding of [35S]TBPS (tert-butylbicyclophosphorothionate) with Ki values of approximately 10 microM and reduced muscimol-dependent 36Cl- flux into mouse cortical microsacs by 30-45% at a concentration of 10 microM. ICS 205-930, MDL 72222, and LY 278584 (at micromolar concentrations) reduced GABA-gated chloride currents studied in Xenopus oocytes expressing human alpha 1 beta 1 gamma 2S GABAA receptor subunits. ICS 205-930 differed from the other two 5-HT3 receptor antagonists in that it induced a biphasic effect on GABA-gated currents: at concentrations from 0.1 to 5 microM it potentiated GABA responses, whereas at higher concentrations (50-100 microM) it produced inhibition. The stimulatory action induced by ICS 205-930 was due to interaction at the benzodiazepine recognition site because expression of the gamma 2 subunit was required and Ro 15-1788 (1 microM) completely prevented the potentiation caused by ICS 205-930. Thus, several 5-HT3 receptor antagonists inhibit benzodiazepine binding and affect GABAA receptor function. These actions are most pronounced for ICS 205-930 and likely involve direct affects on the GABA/benzodiazepine complex rather than interactions with 5-HT3 receptors.

An analogue of colchicine, beta-lumicolchicine, does not bind tubulin or disrupt microtubules. However, this compound is not pharmacologically completely inactive. beta-Lumicolchicine was found to competitively inhibit [3H]flunitrazepam binding and to enhance muscimol-stimulated 36Cl- uptake in mouse cerebral cortical microsacs. It also markedly potentiated GABA responses in Xenopus oocytes expressing human alpha 1 beta 2 gamma 2S, but not alpha 1 beta 2, GABAA receptor subunits; this potentiation was reversed by the benzodiazepine receptor antagonist flumazenil. These results strongly suggest a direct effect of beta-lumicolchicine on the GABAA receptor/chloride channel complex and caution that it possesses pharmacological effects, despite its inability to disrupt microtubules. Furthermore, beta-lumicolchicine is structurally unrelated to benzodiazepines or quinolines and may provide a novel approach to the synthesis of ligands for this receptor.

The greater sensitivity of long-sleep (LS), as compared with short-sleep (SS), mice to ethanol is due in part to differences in GABAA receptor function in specific brain regions. To determine if differences in subunit composition of GABAA receptors contribute to this differential sensitivity, we measured alpha 1 and gamma 2 subunit mRNAs with Northern analysis and in situ hybridization and gamma 2S, gamma 2L and alpha 6 subunit mRNAs with polymerase chain reaction (PCR) amplification. No differences in mRNAs in whole brain were apparent by Northern analysis. In situ hybridization revealed that alpha 1 and gamma 2 subunit mRNAs were co-localized in many brain regions but that they still had distinct patterns of hybridization. However, the few differences observed between LS and SS mice in the levels of hybridization for these subunits did not show a regional distribution consistent with ethanol sensitivity differences. Similar ratios of gamma 2L, and gamma 2S subunit mRNAs were found in LS and SS mouse cerebral cortex and hippocampus, and both mouse lines expressed essentially only gamma 2L subunit mRNA in cerebellum. mRNA for the alpha 6 subunit was detected only in cerebellum and also was qualitatively similar between LS and SS mice. Studies of muscimol-stimulated 36Cl- uptake by cortical membrane vesicles confirmed earlier findings that ethanol does not enhance function of GABAA receptors in SS mice when assayed at 30 degrees C. However, at 34 degrees C ethanol did increase this function in SS mice although the enhancement remained greater in LS mice. These functional results, together with the results showing similar levels of alpha 1, gamma 2S, gamma 2L and alpha 6 subunits in LS and SS mice, suggest that the ethanol-insensitivity of SS mouse GABAA receptors cannot be due solely to lack of subunits required for ethanol action and further suggest that differences in catalytic mechanisms affecting post-translational processing may account for some genetic differences in ethanol sensitivity of GABAA receptors.

The effects of protein kinase C (PKC) activators on gamma-aminobutyric acidA (GABAA) receptor function were studied by two-electrode voltage-clamp in Xenopus oocytes expressing brain mRNA or subunit cDNAs and in isolated mouse brain cerebellar membrane vesicles (microsacs), using 36Cl- uptake. Both oocytes and microsacs showed transient (desensitizing) and sustained (nondesensitizing) GABAA receptor responses. In oocytes expressing brain mRNA, the PKC activator phorbol myristoyl acetate (PMA), but not the inactive analog phorbol 12-monomyristate, inhibited both transient and sustained GABA-gated chloride currents. The inhibition by PMA was concentration dependent, with an EC50 of approximately 5 nM, and resulted in a decrease in the efficacy, but not the potency, of GABA. Additionally, PMA inhibited GABA-gated chloride currents in oocytes expressing alpha 1 beta 1 gamma 2L subunit cDNAs. The effect of PMA on recombinant receptors was significantly antagonized by PKC inhibitory peptide (PKCI). In the microsac preparation, the PKC activators (-)-7-octylindolactam V and PMA inhibited the sustained phase of 36Cl- flux without altering the transient phase. The action of PMA was blocked by kinase inhibitors and by depletion of Mg-ATP and was mimicked by protein phosphatase inhibitors. These results demonstrate that activation of PKC inhibits GABAA receptor function, and the results from the microsac experiments suggest that PKC-dependent phosphorylation preferentially inactivates a nondesensitized form or state of the receptor.

To determine whether genetic differences in development of ethanol dependence are related to changes in gamma-aminobutyric acidA (GABAA) receptor function, we measured 36Cl- uptake by brain cortical membrane vesicles from withdrawal seizure prone and withdrawal seizure resistant (WSP/WSR) mice treated chronically with ethanol. Muscimol-stimulated chloride flux was not different between WSP and WSR mice before or after ethanol treatment. Also, augmentation of muscimol action by flunitrazepam or inhibition of muscimol action by the inverse agonists Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a]- [1,4]benzodiazepine-3-carboxylate) and methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) was not different for ethanol-naive WSP and WSR mice. However, chronic ethanol administration enhanced the inhibitory actions of DMCM and Ro 15-4513 on membranes from WSP but not WSR mice. Conversely, chronic ethanol treatment attenuated the action of flunitrazepam on membranes from WSR but not WSP mice, suggesting that the actions of benzodiazepine agonists and inverse agonists are under separate genetic control. These genetic differences in actions of DMCM and Ro 15-4513 indicate that sensitization to benzodiazepine inverse agonists produced by chronic ethanol treatment may be related to development of withdrawal seizures and suggest that differences in the GABA/benzodiazepine receptor complex represent alleles that have segregated during the selection of the WSP/WSR mice.

Effects of ethanol in vitro on membrane vesicles (microsacs prepared from mouse cerebral cortex) were evaluated by monitoring 36Cl- influx. Different assay parameters were tested to determine increased or decreased action of ethanol on GABA-activated chloride channels. The ability of 30 mM ethanol to augment 36Cl- flux was seen at 0 degrees C, in the absence of GABA ("direct" action of ethanol), and at 34 degrees C in the presence of GABA, using two different assay procedures. Picrotoxin blocked the direct effects of ethanol (at 0 degrees C) suggesting GABAa involvement. Endogenous GABA in the medium surrounding the microsacs was assayed at different temperatures both in the presence and absence of GABA and ethanol. The direct effect of ethanol did not appear to involve the action of endogenous GABA. In addition to temperature effects on the assay, time of membrane storage also influenced ethanol action. Microsacs stored on ice for 2 hours or more lost their ability to respond to ethanol but not to GABA, pentobarbital or flunitrazepam. When these drugs were tested on membranes from mice that had been sacrificed by cervical dislocation as opposed to decapitation, ethanol did not augment GABA-stimulated chloride flux. The method of sacrifice did not influence the response to GABA, pentobarbital or flunitrazepam.

The sensitivity to tetracycline of 345 bacterial isolates from periodontal lesions was determined. Most species of bacteria, including those thought to be involved in the initiation and progress of destructive periodontal disease, were inhibited in vitro by tetracycline concentrations of 4 to 8 micrograms/ml. This concentration is equivalent to crevicular fluid levels of tetracycline at dosages of 1 gm/day. These data indicate that tetracycline is inhibitory at levels achieved in crevicular fluid for bacteria currently implicated in destructive periodontal disease.