Propranolol, a β-adrenergic receptor antagonist, also inhibits phosphatidate phosphohydrolase, the enzyme that converts phosphatidic acid into diacylglycerol. This latter effect has prompted recent use of propranolol in studies examining the importance of diacylglycerol and phosphatidic acid in cellular signalling events. Here, we show that propranolol is also an inhibitor of protein kinase C. At concentrations ≥ 20 μM, propranolol reduced [3H]phorbol dibutyrate binding (IC50 = 200 μM) and phorbol myristate acetate-stimulated superoxide anion release (IC50 = 130 μM) in human neutrophils. Scatchard analysis showed that propranolol lowers the number of phorbol diester binding sites without significantly affecting their affinity. In vitro kinetic analysis, performed in a mixed micellar assay with protein kinase C purified from human neutrophils, suggested a competitive inhibition of propranolol with the cofactor phosphatidylserine. Complex kinetic patterns were observed with respect to diacylglycerol and ATP, approximating competitive and noncompetitive inhibition, respectively. Taken together, these results suggest that the drug interacts at the level of the regulatory domain of the enzyme. Fifty % inhibition occurred at ∼150 μM propranolol. Similar levels of inhibition were obtained using exogenous (histone) and endogenous (p47-phox, a NADPH oxidase component) substrates. Protein kinase C-α and protein kinase C-β two protein kinase C isozymes present in human neutrophils, were inhibited by propranolol in a comparable manner. In the range of concentrations tested (30-1000 μM), neither cAMP-dependent protein kinase nor neutrophil protein tyrosine kinases were affected. The racemic form of propranolol and the (+) and the (-) stereoisomers were equally active, and other β-adrenergic receptor antagonists (pindolol) and agonists (isoproterenol) were inactive. This suggests that the inhibitory action of propranolol on protein kinase C is related to the amphipathic nature of the drug rather than to its β-adrenergic receptor blocking ability. Analogs of propranolol were synthesized and found to be more potent protein kinase C inhibitors, with IC50 values in the 10-20 μM range. We conclude that the ability of propranolol to inhibit both protein kinase C and PA phosphohydrolase complicates interpretation of results when this drug is used in signal transduction studies. In addition, propranolol may be a useful prototype for the synthesis of new protein kinase C inhibitors.
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - Oct 5 1992|
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