Differential effects of calyculin A and okadaic acid on the glucose-induced regulation of glycogen synthase and phosphorylase activities in cultured hepatocytes.
The effects of the phosphatase inhibitors calyculin A and okadaic acid were investigated to determine the roles of protein phosphatases type 1 and 2A in the regulation of the activities of glycogen synthase and phosphorylase by glucose in a primary culture of hepatocytes. Glycogen synthesis, as measured by the incorporation of labelled glucose into glycogen, was inhibited in a dose-dependent manner by calyculin A (IC50 = 2.2 nM) and okadaic acid with (IC50 = 14 nM). Glucose-induced activation of glycogen synthase was inhibited by calyculin A and okadaic acid with IC50 values of 3.7 nM and 90 nM, respectively. Phosphorylase was simultaneously activated by these inhibitors with calyculin A again being more active (P < 0.001) than okadaic acid. The differing potencies (P < 0.001) of these inhibitors on the activities of glycogen synthase and phosphorylase were also observed with varying concentrations of glucose (5.6-60 mM) in the medium and at different incubation periods upto 120 min. It has been previously shown that both inhibitors inhibit protein phosphatase-2A with equal potency and calyculin A is a more potent inhibitor of protein phosphatase-1 than okadaic acid. Heat- and proteinase-treated cytosolic fractions from hepatocytes incubated with calyculin A and okadaic acid showed similar differential inhibitory activities towards purified types 1 and 2-A protein phosphatases. Hence, these data provide further evidence that protein phosphatase type-1 plays a major role in the control of glycogen synthesis by regulating the activities of glycogen synthase and phosphorylase.
Pugazhenthi
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Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.
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B
Gali
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Khandelwal
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D000818:Animals; D002478:Cells, Cultured / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004988:Ethers, Cyclic / Q000494:pharmacology*; D005947:Glucose / Q000494:pharmacology; D006003:Glycogen / Q000096:biosynthesis; D006006:Glycogen Synthase / Q000037:antagonists & inhibitors*; D008099:Liver / Q000187:drug effects*; D008297:Male; D008387:Marine Toxins; D019319:Okadaic Acid; D010080:Oxazoles / Q000494:pharmacology*; D006005:Phosphorylases / Q000037:antagonists & inhibitors*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015854:Up-Regulation
D004988:Ethers, Cyclic; D008387:Marine Toxins; D010080:Oxazoles; D019319:Okadaic Acid; C059041:calyculin A; D006003:Glycogen; D006005:Phosphorylases; D006006:Glycogen Synthase; D005947:Glucose