EFFECT OF HALOTHANE ON CONVENTIONAL PROTEIN-KINASE-C TRANSLOCATION AND DOWN-REGULATION IN RAT CEREBROCORTICAL SYNAPTOSOMES

Protein kinase C (PKC) is a key regulatory enzyme that has been implic ated as a molecular target for the action of general anaesthetics. We have determined the effects of halothane on the translocation and down -regulation of conventional PKC (cPKC) by analysing the subcellular di stribution of PKC activity, [H-3]phorbol-12,13-dibutyrate ([H-3]PDBu) binding and PKC immunoreactivity in intact rat cerebrocortical synapto somes, a subcellular fraction that contains functional nerve terminals . Halothane alone (2.4 vol%) reduced membrane-associated (P<0.05) and increased cytosol (P<0.01) PKC activity, while phorbol-12-myristate, 1 3-acetate (PMA) 0.1 mu mol litre(-1), a metabolically stable activator of PKC, reduced membrane (P<0.01) without altering cytosol PKC activi ty. Halothane and PMA in combination reduced membrane PKC activity to undetectable levels and reduced cytosol PKC activity (P<0.01). Halotha ne alone had no significant effects on the distribution of [H-3]PDBu b inding, while PMA alone significantly reduced both membrane and cytoso l [H-3]PDBu binding (P<0.01). Halothane and PMA in combination reduced membrane and cytosol [H-3]PDBu binding further, but this effect was n ot significantly different from the effect of PMA alone. Experiments u sing isoform-selective antibodies to PKC alpha, PKC beta or PKC gamma demonstrated synergistic interactions between halothane and PMA in pro moting translocation of the three conventional PKC isoforms from the c ytosol to the membrane fraction of synaptosomes and downregulation of their immunoreactivity. Halothane and PMA together reduced cytosol PKC alpha/beta/gamma immunoreactivity significantly more (P<0.05) than PM A alone. Halothane thus has two distinct actions on PKC in synaptosome s: activation of endogenous PKC activity and potentiation of activatio n-induced cPKC translocation and downregulation. These potentially com peting effects may underlie some of the conflicting results obtained w ith halothane on PKC-mediated processes in intact cells.