Molecular Pharmacology, Vol 13, 1060-1075, Copyright © 1977 by the American Society for Pharmacology and Experimental Therapeutics

Evidence for Alpha Adrenergic Activation of Phosphorylase and Inactivation of Glycogen Synthase in Rat Adipocytes

Effects of Alpha and Beta Adrenergic Agonists and Antagonists on Glycogen Synthase and Phosphorylase

¹ University of Virginia School of Medicine, Department of Pharmacology, Charlottesville, Virginia 22903

The effects of alpha and beta adrenergic agonists and antagonists on the percentages of phosphorylase a and glycogen synthase I activities were investigated in isolated rat adipocytes. (-)-Epinephrine, (-)-norepinephrine, and (-)-isoproterenol increased the concentration of adenosine cyclic 3',5'-monophosphate (cAMP) and the percentage of phosphorylase a activity in a dose-dependent manner. Isoproterenol was about 10 times more potent than epinephrine or norepinephrine with respect to these increases as well as to decreases in the percentage of glycogen synthase I activity. Although all three agents decreased synthase I activity to the same extent, the maximal effects of epinephrine and norepinephrine on phosphorylase a activity were approximately 25% greater than the maximal effect of isoproterenol. In the presence of the alpha adrenergic antagonists phentolamine, phenoxybenzamine, and dihydroergotamine, the maximal effect of norepinephrine on increasing the percentage of phosphorylase a was reduced to that of isoproterenol. All three alpha adrenergic antagonists potentiated the ability of norepinephrine to increase the concentration of cAMP. The maximal effect of (-)-phenylephrine on phosphorylase a activity was less than the maximal effect of isoproterenol. When cells were incubated with isoproterenol plus phenylephrine, phosphorylase a activity was increased to levels observed with epinephrine or norepinephrine. Methoxamine also increased phosphorylase a activity, and the effects of isoproterenol and methoxamine on phosphorylase were additive. Incubation of cells with 5 mM dibutyryl cAMP decreased synthase I activity and increased phosphorylase a activity to the level observed with isoproterenol. When dibutyryl cAMP was added to cells together with isoproterenol or epinephrine, no further increase in phosphorylase a activity over that obtained with the catecholamines alone was observed. However, when cells were incubated with phenylephrine plus dibutyryl cAMP, the percentage of phosphorylase a activity was increased to that observed with epinephrine. (-)-Propranolol completely blocked the rise in cAMP observed following incubation of cells with epinephrine, norepinephrine, or phenylephrine; yet an increase in phosphorylase a activity and a decrease in synthase I activity were still observed. Incubation of cells with 20 µM propranolol together with 1 µM phenoxybenzamine did not reverse the decrease in synthase I activity produced by 200 milliunits/ml of adrenocorticotrophic hormone, but completely abolished the effect of 200 µM phenylephrine. Incubation of cells with methoxamine (1-10 µM) decreased glycogen synthase I activity. Phentolamine (20 µM) completely blocked this effect of methoxamine, as well as the ability of methoxamine to increase phosphorylase a activity.

Note:
ACKNOWLEDGMENTS We wish to thank Dr. Gary Brooker for allowing us to use the facilities and reagents of his laboratory in the performance of the radioimmunoassay for cAMP, and both Dr. Brooker and Jeffrey F. Harper for teaching us the techniques involved in this assay. We thank Dr. James C. Garrison for helpful suggestions during the course of this work. Technical assistance in the enzyme assays was provided by Mark Sullivan and Howard Froehlich. We also thank Dr. Yoram Oron, Dr. Alfred G. Gilman, and Dr. Theodore Rall for their critical reviews of the manuscript.

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