Abstract
Angiotensin II, catecholamines, and vasopressin can stimulate the phosphorylation of 10 hepatic cytosolic proteins via a Ca2+-linked, cyclic AMP-independent mechanism. To explore the role of known Ca2+-sensitive protein kinases in this response, [32P]PO4(3-)-labeled hepatocytes were stimulated with various agonists, the cytoplasmic proteins were separated on two-dimensional gels, and the resulting autoradiographs were computer analyzed. The role of phosphorylase kinase was examined using hepatocytes from gsd/gsd rats which are deficient in this enzyme. The phosphorylation state of phosphorylase was not increased by glucagon, angiotensin II, or vasopressin in hepatocytes from the gsd/gsd animals. The phosphorylation state of all other substrates was changed by glucagon or the Ca2+-linked hormones to the same extent in gsd/gsd hepatocytes as in normal Wistar controls, suggesting that phosphorylase kinase plays a restricted role in the hormone response. The role of the Ca2+- and phospholipid-sensitive protein kinase (protein kinase C) was examined by stimulating hepatocytes with phorbol esters which are thought to activate protein kinase C by substituting for diacylglycerol. Phorbol esters increased the phosphorylation state of 3 of the 10 substrates affected by angiotensin II or vasopressin, but did not stimulate Ca2+ fluxes in hepatocytes. Treatment of hepatocytes with the Ca2+ ionophore A23187 mimicked the effect of the Ca2+-linked hormones on the phosphorylation of the other 7 substrates. The results demonstrate that at least three Ca2+-sensitive protein kinases are involved in the response of hepatocytes to Ca2+-linked hormones. Since these kinases can be activated independently by phorbol esters or A23187, the results imply that hormones such as vasopressin generate two intracellular messengers, diacylglycerol and Ca2+ ion.
Highlights
Angiotensin 11, catecholamines, and vasopressin can 11,vasopressin, and al-adrenergic agonists stimulate different stimulate the phosphorylation of 10 hepatic cytosolic membrane events including phosphatidylinositol turnover
These observations suggest that hepatocytes contain proteinkinases responsive to a variety of intracellular messengers but that the substrate specificity of these enzymes overlaps to a significant extent [17, 18]
Ca2+-linkedhormones to the same extent in gsdlgsd gests that theeffect of glucagon on the phosphorylation state hepatocytes as in normal Wistar controls, suggesting of hepatic enzymes is mediated by the cyclic AMP-dependent that phosphorylase kinase plays a restricted role in the protein kinase [16, 19,20,21]
Summary
Cellular messenger, cyclic AMP [1,2,3]. Ngiotensin Preparation of Hepatocytes, Incubation Procedures, and Sample. For AM-19952 and University of Virginia Diabetes Center Grant AM- protein phosphorylation experiments, the hepatocytes were resus-. The costs of publication of this article were defrayed in part by the payment of page charges. This article must be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Recipient of Research Career Development AwardAM-00491 from the National Institutes of Health
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