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Background Recent evidence is consistent with the view that volatile anesthetics interact directly with excitable membrane-bound channel proteins. If these agents interact directly with chiral centers in the neuronal cell membrane, then their effects should be stereoselective. Using rat brain membranes enriched in gamma-aminobutyric acid type A (GABAA) receptors, we investigated the hypothesis that the permeability response of this well-characterized central nervous system channel protein to stereoisomers of isoflurane is stereoselective. Methods Rat brain synaptic microvesicles were prepared by differential centrifugation. Agonist-stimulated 36Cl- flux through membrane-bound GABAA receptors was assayed in the presence of (+)- and (-)-isoflurane and compared with control conditions. Results Both isomers increased the potency and efficacy of GABA; however, (+)-isoflurane was significantly more potent and efficacious than the (-)-isomer. For example, the (+)-isomer (140 microM) reduced the median effective concentration of GABA from 12.7 +/- 1.0 to 5.4 +/- 0.5 microM, whereas the (-)-isomer reduced it to 9.6 +/- 1.0 microM (P < 0.001). The (+)-isomer also was 1.6 times as potent as the (-)-isomer in augmenting 5 microM GABA-gated flux (79 +/- 11 vs. 130 +/- 17 microM, respectively; P = 0.01). In addition, the (+)-isomer produced significantly greater maximal enhancement of flux (9.4 +/- 0.4 vs. 7.0 +/- 0.3 nmol.mg-1.3 s-1; P < 0.001). Conclusions Isoflurane's effects on GABA-gated chloride flux were stereoselective. This result supports direct interaction with a stereoselective site, possibly the GABAA channel protein itself, rather than a nonspecific perturbation of the surrounding membrane lipid. In addition, these findings, from a functional assay using mammalian brain, agree with recent observations in invertebrate ion channels and mammalian neuronal cell cultures.

Central nervous system protein kinases are the intracellular effectors for many of the signal transduction pathways essential to neurotransmission. Although the in vitro activity of at least one of these important enzymes, protein kinase C, is diminished by therapeutic concentrations of ethanol and other central depressants, the relationship of this effect to intoxication in vivo is not known. If intoxication by ethanol involves central protein kinase inhibition, then other inhibitors of these enzymes should enhance ethanol's intoxicating potency. To test this hypothesis, we compared the median effective concentrations of ethanol and two other n-alkanols for loss-of-righting reflex in Rana pipiens tadpoles pretreated with staurosporine and in untreated controls. Alkanol concentrations were confirmed by gas chromatography and staurosporine concentrations by ultraviolet absorbance spectrophotometry. Results obtained with 650 animals demonstrate that pretreatment with staurosporine concentrations in the nanomolar range significantly decrease the median effective concentration for ethanol (56% of control; p < 0.001), butanol (38% of control; p < 0.001), and octanol (59% of control; p < 0.001). This finding supports that central protein kinase inhibition may be involved in the acute intoxicating effects of ethanol and other n-alkanols.

Protein kinase C, the intracellular effector for the inositol phosphate-mediated signal transduction pathway, plays a key role in neurotransmission in the central nervous system. Although the in vitro activity of protein kinase C is inhibited by therapeutic concentrations of volatile anesthetics, the relation of this effect to in vivo obtundation has not been established. If obtundation by volatile anesthetics involves protein kinase C inhibition, then an inhibitor of this enzyme should decrease the anesthetic requirement. To test this hypothesis, we compared the EC50s of halothane and diethylether for loss of the righting reflex in Rana pipiens tadpoles pretreated with Staurosporine and in untreated controls. Anesthetic concentrations were confirmed by gas chromatography and Staurosporine concentrations by ultraviolet absorbance spectropho-tometry. Results obtained in more than 1000 animals indicated that pretreatment with Staurosporine concentrations in the nanomolar range significantly decreased the EC50 for both halothane (68% of control; P < 0.035) and diethylether (41% of control; P < 0.001). This finding implies that protein kinase C inhibition may play a role in general anesthetic-induced obtundation.

Theiler's virus (TV)-infected mice were treated with antithymocyte serum (ATS), cyclophosphamide or pepstatin (a protease inhibitor) to determine the effect on demyelination. When ATS and cyclophosphamide were begun at the time of infection there was significantly less demyelination at 2.5-3.5 weeks than in pepstatin or non-treated infected controls. When immunosuppression was continued for 5 weeks, or when it was not started until 5 weeks post-infection, no significant decrease in demyelination was seen compared to controls. The findings indicate that timing of immunosuppression is critical in determining the extent of TV demyelination. Such demyelination may occur by different mechanisms that are active at different times. The "bystander effect' may be important in early demyelination, but late demyelination may be due to other causes, such as oligodendrocyte lytic infection.