Activation of endogenous protein kinase C by halothane in synaptosomes

Abstract

Background: Protein kinase C is a signal transducing enzyme that is an important regulator of multiple physiologic processes and a potential molecular target for general anesthetic actions. However, the results of previous studies of the effects of general anesthetics on protein kinase C activation in vitro have been inconsistent. Methods: The effects of halothane on endogenous brain protein kinase C activation were analyzed in isolated rat cerebrocortical nerve terminals (synaptosomes) and in synaptic membranes. Protein kinase C activation was monitored by the phosphorylation of MARCKS, a specific endogenous substrate. Results: Halothane stimulated basal Ca 2+- dependent phosphorylation of MARCKS (M(r) = 83,000) in lysed synaptic membranes (2.1-fold; P < 0.01) and in intact synaptosomes (1.4-fold; P < 0.01). The EC 50 for stimulation of MARCKS phosphorylation by halothane in synaptic membranes was 1.8 vol%. A selective peptide protein kinase C inhibitor, but not a protein phosphatase inhibitor (okadaic acid) or a peptide inhibitor of Ca 2+/calmodulin-dependent protein kinase II, another Ca 2+-dependent signal transducing enzyme, blocked halothane-stimulated MARCKS phosphorylation in synaptic membranes. Halothane did not affect the phosphorylation of synapsin 1, a synaptic vesicle-associated protein substrate for Ca 2+/calmodulin-dependent protein kinase II and cyclic AMP- dependent protein kinase, in synaptic membranes or in intact synaptosomes subjected to KCl-evoked depolarization. However, halothane stimulated synapsin 1 phosphorylation evoked by ionomycin (a Ca 2+ ionophore that permeabilizes membranes to Ca 2+ in intact synaptosomes. Conclusions: Halothane acutely stimulated basal protein kinase C activity in synaptosomes when assayed with endogenous nerve terminal substrates, lipids, and protein kinase C. This effect appeared to be selective for protein kinase C, because two other structurally similar second messenger-regulated protein kinases were not affected. Direct determinations of anesthetic effects on endogenous protein kinase C activation, translocation, and/or down-regulation are necessary to determine the ultimate effect of anesthetics on the protein kinase C signaling pathway in intact cells.