Phosphorylase Activity in Rat Liver and Skeletal Muscle after Catecholamines.

The tune course of liver phosphorylase activation after catecholamine administration and the relative abilities of several adrenergic blocking agents to block catecholamine-induced phosphorylase activation in liver and skeletal muscle was studied in intact anesthetized rats. Subcutaneously injected epinephrine and norepinephrine increased the liver phosphorylase activity, the peak effect occurring 10 min after injection. The liver phosphorylase levels were somewhat reduced 30 min after injection. Isoproterenol administration did not increase the level of rat liver phosphorylase. Blood glucose changes after catecholamine treatment did not mirror the changes in liver phosphorylase activity. Complete blockade of the liver phosphorylase response to epinephrine was observed with the a-adrenergic blocking agent ergotamine. Phenoxybenzamine and two fi-adrenergic blocking agents, dichloroisoproterenol and nethalide, gave partial blockade. These data indicate the difficulty in the classification of the liver metabolic receptor. The activation by epinephrine of skeletal muscle phosphorylase appears to be affected only by /3-adrenergic blocking agents. THE blood glucose elevation, liver phosphorylase activation, and glycogenolysis induced by the catecholamines have been extensively investigated.lp2 Although the phosphorylase response has been reported consistently for liver slices in vitro, an effect in z’iro after catecholamine administration has been both confirmed and denied.3-5 The effect of adrenergic blocking agents on liver phosphorylase activation after catecholamine administration has not been clarified.6 Phosphorylase activation in skeletal muscle after catecholamine administration is well documented;‘,* however, no data could be found demonstrating the nature of this response after adrenergic blocking agents. The present investigation will be concerned with the time-response relationships of phosphorylase activation and blood glucose levels in rat liver after the administration of L-epinephrine, L-norepinephrine, and DL-isoproterenol and the susceptibility of epinephrine-induced activation of liver and skeletal muscle phosphorylase to adrenergic blockade. METHODS White female rats (180 to 200 g, Holtzman strain) were used throughout the investigation. Rats were maintained on food at all times. Anesthesia was produced by the intraperitoneal injection of hexobarbital sodium (150 to 180 mg/kg) 10 min before sacrifice or catecholamine infusion. * Supported by a contract between The University of Michigan and the Office of Naval Research, NONR1224-27. t Present address: Dept. of Pharmacology, Washington Univ., School of Medicine, St. Louis, MO., U.S.A. 1407 I408 K. R. HORNBKOO~( and T. M. BKODY Commercial solutions of L-epinephrine (Adrenalin. Parke-Davis and Co). or crystailine L-epinephrine, L-norepinephrine (Levophed, Winthrop Lab.), and DL-isoproterenol (Isuprel, Winthrop Lab.) were diluted in a 0.9:; NaCI-0.1 y,,, NaNSO,, solution to obtain the proper col~celltratio~l for injection. The time.response curves \vere determined after subcutaneous injection of the catecholamine (500 t~,g/kg). The intravenous infusion of L-epinephrine (0.25 pg/kg.min--l for IO min) \vas accomplished a5 described previously.” The tissues nere excised rapidly from anesthetized animals and frozen in beakers of isopentane cooled in alcohol-dry ice. The liver was excised before the skeletal muscle in all experiments. Phosphorylase and glycogen were assayed as previously reported.!’ The liver sample was diluted such that an equivalent of 5 mg wet \\eight of liver (I :200 dilution) was assayed. The incubation time for the liver assay \\a3 15 min. All liver phosphorylase determinations were carried out in the presence of I mM adenosine monophosphate. The data are expressed as micromoles inorganic phosphate liberated per gram wet weight of liver.lninLIte i incubation time. The skeletal muscle phosphorylase was assayed both with and without the addition of I mM adenosine monophosphate and the data are expressed as per cent phosphorylase CI.!’ Blood glucose was determined by the method of Nelson. I0 Blood was obtained from a tail vein by means of a Unopette” (generously supplied by Dr. Horace W. Gerardc. Esso Research Lab. and Mr. J. Lucker, Becton, Dickinson and Co.). This allowed the accurate sampling of 13 ~1 of blood which was then diluted and deproteinized. Blood glucose is expressed as a per cent increase over the daily control level. Over-all mean blood glucose control levels were 92.7 3-7 g/l00 ml (mean ~:I, standard error, N 27). Dichloroisoproterenol (DCI), obtained from the Aldrich Chemical Co., and nethalide (ICI-38, l74), generously supplied by Dr. J. W. Black. were injected subcutaneously 30 min before ~at~cholamine admi~tistration. Ergotamine tartrate (Gynergcn, Sandoz) was injected subcutaneously (1.25 mg/kg) 30 min before catecholamine infusion. Phenoxybenzamine (Dibenzyline, Smith Kline & French Labs.) Mas injected intraperitoneally (5 mg/kg and 20 mg,/kg) 2 to 3 hr before catechotamine adi~~illistr~~t~on. Hexal~~et~~o~~iu~~ chloride ( IO mgikg intraperitoneally) was adl~lillistered 30 min before catecholamine infusion.