Effect of halothane on conventional protein kinase C translocation and down-regulation in rat cerebrocortical synaptosomes

Abstract

Protein kinase C (PKC) is a key regulatory enzyme that has been implicated 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 distribution of PKC activity, [3H]phorbol-12,13-dibutyrate ([3H]PDBu) binding and PKC immunoreactivity in intact rat cerebrocortical synaptosomes, 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, 13-acetate (PMA) 0.1 mumol litre-1, a metabolically stable activator of PKC, reduced membrane (P < 0.01) without altering cytosol PKC activity. Halothane and PMA in combination reduced membrane PKC activity to undetectable levels and reduced cytosol PKC activity (P < 0.01). Halothane alone had no significant effects on the distribution of [3H]PDBu binding, while PMA alone significantly reduced both membrane and cytosol [3H]PDBu binding (P < 0.01). Halothane and PMA in combination reduced membrane and cytosol [3H]PDBu binding further, but this effect was not significantly different from the effect of PMA alone. Experiments using isoform-selective antibodies to PKC alpha, PKC beta or PKC gamma demonstrated synergistic interactions between halothane and PMA in promoting translocation of the three conventional PKC isoforms from the cytosol to the membrane fraction of synaptosomes and down-regulation of their immunoreactivity. Halothane and PMA together reduced cytosol PKC alpha/beta/gamma immunoreactivity significantly more (P < 0.05) than PMA alone. Halothane thus has two distinct actions on PKC in synaptosomes: activation of endogenous PKC activity and potentiation of activation-induced cPKC translocation and down-regulation. These potentially competing effects may underlie some of the conflicting results obtained with halothane on PKC-mediated processes in intact cells.

The dopaminergic innervation of the pigeon telencephalon: distribution of DARPP-32 and co-occurrence with glutamate decarboxylase and tyrosine hydroxylase

“…Aliquots (100 µg of total protein determined by the bicinchoninic acid method 89 with bovine serum albumin as standard) were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on gels cast from 13% (w/v) acrylamide and transferred to 0.2-µm nitrocellulose membranes (Schlicher and Scheull) as described. 35 Immunoblotting with anti-bovine DARPP-32 antibody C24-6a 36 at a 1:500 dilution was performed as described 35 using prestained molecular weight markers (Amersham).…”

Effects of Different Types of Anesthetic Agents on Cellular Protein Kinase C-γ Dynamics in Mouse Brain

“…The tissue was sonicated 3 times for 10 s in 1: 10 (w/v) homogenization buffer: 50 mmol/l 3-(N-morpholino)propanesulfonic acid (pH 7.6), 2 mmol/l dithiothreitol, 3 mmol/l Mg(CH 3 COO) 2 , 0.1 mmol/l sodium orthovanadate, 0.1 mmol/l phenylmethylsulfonyl fluoride, 20 g/ml leupeptin, 10 g/ml pepstatin, 5 g/ml aprotinin, and 0.32 mol/l sucrose on ice. The homogenates were centrifuged at 800 g for 15 min at 4 ° C, and then the supernatant was centrifuged at 9,200 g for 15 min at 4 ° C. The resulting crude synaptosomal (membrane) fraction (P2) was sonicated for 10 s in homogenization buffer containing 0.1% Triton X-100, and the supernatant was centrifuged at 165,000 g for 1 h at 2 ° C in a TL100.2 rotor.…”

“…Thus, PKC is an attractive molecular target for the synaptic action of general anesthetics [1] . PKCs are classified into the following three subtypes: classical PKC (c-PKC), novel PKC, and atypical PKC [2] . c-PKC is an important element in signal transduction pathways regulated by calcium.…”

Halothane facilitates the translocation of GRK-2 and phosphorylation of ß2- adrenergic receptor in rat synaptosomes

“…2 0 Thus, it is often utilized for the study of protein phosphorylation in the synapse. 21,22 In addition, synaptosomal ß-adrenergic receptor could be an experimental model for studying the effect of anesthetics on phosphorylation of the G-protein coupled receptors. In the present study, we focused on phosphorylation of the ß2-adrenergic receptor, which has been extensively studied by a molecular biological approach for a decade.…”

“…21,22 They demonstrated that halothane 2.4% facilitated translocation of conventional PKC from cytosol to membrane and down-regulation in rat cerebrocortical synaptosomes. In their experiment, the simultaneous presence of phorbol ester was required for the action.…”