Beta-adrenergic receptor-mediated phospholipase C activation independent of cAMP formation in turkey erythrocyte membranes.
The effect of the beta-adrenergic receptor agonist isoproterenol on guanine nucleotide-dependent phospholipase C (PLC) activity was examined in turkey erythrocyte membranes prepared from [3H]inositol-labeled turkey erythrocytes. In the presence of guanosine 5'-(gamma-thiotriphosphate) (GTP[S]) isoproterenol caused a dose-dependent stimulation of [3H]inositol phosphate ([3H]InsP) formation. The activation of PLC by GTP[S] occurred after an initial lag period of 1-2 min and was followed by a sustained rate of [3H]InsP formation which remained linear for 4-5 min. Isoproterenol decreased the lag period for GTP[S]-induced [3H]InsP formation and increased PLC activity at all time points following this lag. Consequently, isoproterenol shifted the dose-response curve for GTP[S] to the left (10-fold) and increased the maximal response. The EC50 value for isoproterenol-induced activation of PLC was 104 +/- 17 nM. Isoproterenol also potentiated GTP-dependent PLC activity but was ineffective in stimulating the enzyme in the presence of AIF4-. The PLC activation by isoproterenol was completely inhibited by propanolol and atenolol but was unaffected by prazosin or yohimbine. Although GTP[S] and isoproterenol could increase cAMP formation in this membrane preparation, the isoproterenol-induced stimulation of PLC occurred in the absence of ATP and was independent of cAMP formation. Furthermore, addition of cAMP, 8-bromo-cAMP, forskolin, or either the regulatory or catalytic subunits of cAMP-dependent protein kinase failed to stimulate [3H]InsP formation and had no effect on the responses elicited by GTP[S] and isoproterenol. Isoproterenol also stimulated [3H]InsP2 and [3H]InsP3 production in intact erythrocytes. Cholera toxin had no effect on [3H]InsP formation in the intact cells under conditions where it stimulated cAMP accumulation. In addition, the activation of PLC by GTP[S] and isoproterenol was unaffected in membranes prepared from cholera toxin-treated erythrocytes. These data demonstrate that stimulation of turkey erythrocyte beta-adrenergic receptors by isoproterenol results in a direct activation of guanine nucleotide-dependent PLC.
Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
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Rooney
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Hager
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Thomas
D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000818:Animals; D000242:Cyclic AMP / Q000096:biosynthesis*; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007295:Inositol Phosphates / Q000096:biosynthesis / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000378:metabolism*; D014422:Turkeys; D010738:Type C Phospholipases / Q000378:metabolism*
D000319:Adrenergic beta-Antagonists; D007295:Inositol Phosphates; D011943:Receptors, Adrenergic, beta; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP; D010738:Type C Phospholipases; D007545:Isoproterenol