Molecular mode of inhibition of glycogenolysis in rat liver by the dihydropyridine derivative, BAY R3401: inhibition and inactivation of glycogen phosphorylase by an activated metabolite.

Abstract

The racemic prodrug BAY R3401 suppresses hepatic glycogenolysis. BAY W1807, the active metabolite of BAY R3401, inhibits muscle glycogen phosphorylase a and b. We investigated whether BAY R3401 reduces hepatic glycogenolysis by allosteric inhibition or by phosphatase-catalyzed inactivation of phosphorylase. In gel-filtered liver extracts, racemic BAY U6751 (containing active BAY W1807) was tested for inhibition of phosphorylase in the glycogenolytic (in which only phosphorylase a is active) and glycogen-synthetic (for the evaluation of a:b ratios) directions. Phosphorylase inactivation by endogenous phosphatase was also studied. In liver extracts, BAY U6751 (0.9-36 micromol/l) inhibited glycogen synthesis by phosphorylase b (notwithstanding the inclusion of AMP), but not by phosphorylase a. Inhibition of phosphorylase-a-catalyzed glycogenolysis was partially relieved by AMP (500 micromol/l). BAY U6751 facilitated phosphorylase-a dephosphorylation. Isolated hepatocytes and perfused livers were tested for BAY R3401-induced changes in phosphorylase-a:b ratios and glycogenolytic output. Though ineffective in extracts, BAY R3401 (0.25 micromol/l-0.5 mmol/l) promoted phosphorylase-a dephosphorylation in hepatocytes. In perfused livers exposed to dibutyryl cAMP (100 micromol/l) for maximal activation of phosphorylase, BAY R3401 (125 micromol/l) inactivated phosphorylase by 63% but glucose output dropped by 83%. Inhibition of glycogenolysis suppressed glucose-6-phosphate (G6P) levels. Activation of glycogen synthase after phosphorylase inactivation depended on the maintenance of G6P levels by supplementing glucose (50 mmol/l). We conclude that the metabolites of BAY R3401 suppress hepatic glycogenolysis by allosteric inhibition and by the dephosphorylation of phosphorylase a.