Insulin regulation of protein phosphorylation in hepatocytes. Studies using two effectors: amiloride and natural aliphatic polyamines.
The effects of amiloride and of natural aliphatic polyamines on basal and hormone-stimulated protein phosphorylations in hepatocytes were studied. Cells isolated from adult rats were incubated in suspension with (32P)-orthophosphate, in the absence or presence of the effectors at varying concentrations and for different times; hepatocytes were then exposed to various hormones for 10 min. Phosphoproteins contained in total cell lysates were analyzed by one- and two-dimensional gel electrophoresis and autoradiography. Amiloride and spermine (the most effective amine) decreased the basal level of phosphorylation of proteins of 46, 34 and 22 kDal, and increased that of 18 kDal and 93 kDal proteins. These effects were maximal with external concentrations of 1 mM and 7.5-10 mM amiloride and spermine, respectively. They were detectable after a lag period of about 10 min and reached a plateau after 45 min. Pretreatment of cells with these effectors almost completely prevented stimulation of the phosphorylation of the 46 and 34 kDal proteins by insulin. In contrast, the effects of vasopressin on the same proteins were only partly inhibited, whereas those of glucagon appeared largely unaffected. The major effect observed in intact cells (i.e., decreased phosphorylation) could be reproduced in a cell-free system where no kinase activity persisted. Amiloride or spermine added directly to cell extracts strongly accelerated the dephosphorylation of 46 kDal protein and also of the 61 kDal protein identified as pyruvate kinase. Furthermore, restoration of the activity of this enzyme occurred concomitantly with dephosphorylation of the 61 kDal protein, an observation supporting the notion that amiloride and spermine could activate a phosphoprotein phosphatase.
D000584:Amiloride / Q000494:pharmacology*; D000818:Animals; D002474:Cell-Free System; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004793:Enzyme Reactivators; D005934:Glucagon / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008970:Molecular Weight; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011073:Polyamines / Q000494:pharmacology*; D011770:Pyruvate Kinase / Q000378:metabolism; D051381:Rats; D013096:Spermine / Q000494:pharmacology; D014667:Vasopressins / Q000494:pharmacology
D004793:Enzyme Reactivators; D007328:Insulin; D010750:Phosphoproteins; D011073:Polyamines; D014667:Vasopressins; D013096:Spermine; D000584:Amiloride; D005934:Glucagon; D011770:Pyruvate Kinase