Catecholamine regulation of human erythrocyte membrane protein kinase

Abstract

The effect of catecholamines on membrane-associated protein kinase in the mature human erythrocyte was investigated. Protein kinase activity was assayed after isolation of membranes from intact erythrocytes incubated with and without catecholamines. Activation of the enzyme is expressed as the ratio of the extent of phosphorylation of exogenous protein substrate in the absence to that in the presence of 2.5 μM cyclic AMP (cAMP). The potent β-adrenergic agonist, (-)isoproterenol (2 μM), (-)epinephrine (10 μM) and (-)norepinephrine (10μM) stimulated the cAMP-dependent protein kinase in membranes, 38 ± 7%, 31 ± 6%, and 30 ± 6%, respectively. Maximal stimulation of membrane protein kinase by 10 μM (-)epinephrine was obtained ≃30 min after initiation of the incubation of erythrocytes with the hormone. The concentrations of (-)catecholamines that gave half-maximal stimulation of the membrane protein kinase were 0.17 μM for isoproterenol, 0.35 μM for epinephrine, and 0.63 μM for norepinephrine. The membrane protein kinase response to β-adrenergic agonists was found to be stereospecific. The stimulation of membrane protein kinase by 10 μM (-)epinephrine was inhibited by the β-adrenergic antagonist, (-)propranolol with EC50 = 0.60 μM, and the inhibition of agonist stimulation of the cAMP-dependent protein kinase by propranolol was stereospecific. These studies suggest that a functional β-adrenergic receptor exists in the mature human erythrocyte.