Glucagon and the Ca2+-linked hormones angiotensin II, norepinephrine, and vasopressin stimulate the phosphorylation of distinct substrates in intact hepatocytes

Recent studies have demonstrated that angiotensin II, catecholamines, and vasopressin can stimulate the phosphorylation of hepatic cytosolic proteins via a Ca2+-linked cyclic AMP-independent mechanism. The present study used high resolution, two-dimensional gel electrophoresis to determine if the proteins phosphorylated in response to the Ca2+-linked hormones were distinct from those affected by glucagon acting via the cyclic AMP-dependent pathway. Intact hepatocytes labeled with 32P]PO4(3-) were stimulated with glucagon, angiotensin II, l-norepinephrine, and vasopressin and over 100 phosphorylated proteins resolved by two-dimensional electrophoresis and autoradiography. Six important enzymes known to be regulated through covalent modification were positively identified, including phosphorylase, phosphofructokinase, pyruvate kinase, fructose-6-phosphate, 2-kinase, phenylalanine hydroxylase, and fructose-1,6-bisphosphatase. Computer analysis of the autoradiograms from control and hormone-treated cells demonstrated that glucagon increased the phosphorylation state of 12 phosphoproteins and reduced the phosphorylation of one protein with a Mr = 21,000 and a pI = 5.9. The Ca2+-linked hormones stimulated the phosphorylation of 7 phosphoproteins and also reduced the phosphorylation state of the 21,000-dalton protein. Angiotensin II, l-norepinephrine, and vasopressin had equivalent effects on protein phosphorylation. There were six protein substrates uniquely affected by glucagon and one phosphoprotein uniquely stimulated by the Ca2+-linked hormones. Seven substrates were affected by stimulation of the cell with either glucagon or the Ca2+-linked hormones. These results demonstrate that, while there is overlap in the substrates affected by glucagon and the Ca2+-linked hormones, each pathway is able to affect the phosphorylation of unique substrates. This finding suggests that the two types of hormones may have some distinct effects on hepatic function.U