Cell volume affects glycogen phosphorylase activity in fish hepatocytes

The activity of glycogen phosphorylase (GPase) in the active a-form (GPase a) is dependent on the hydration state of hepatocytes. We establish that GPase a catalysis in catfish (Ameiurus nebulosus) hepatocytes is a function of medium osmolarity and that a linear relationship exists between GPase a activity and osmolarity between 254 mosmol l^–1 and 478 mosmol l^–1. Exposure of isolated hepatocytes to hyperosmotic media increases enzyme activity up to 7-fold, indicative of covalent phosphorylation. GPase activation associated with cell shrinkage peaks within 10 min of exposure. The average degree of activation (2.7-fold-increase of GPase a) is only slightly less than in hepatocytes exposed to glucagon (3.1-fold-increase) under isosmotic conditions; with glucagon, the maximum is reached within 2 min. Phosphorylation status remains elevated during the entire 40 min experimental period; cells do not undergo regulatory volume increase (RVI) during this period and do not regain pre-exposure volume. We interpret the increased GPase a activity as an inherent response to hyperosmotic stress, likely brought about by molecular crowding. Activation of the enzyme results in increased glucose production from endogenous glycogen. Glucose is not retained in the liver cells, but may act as an oxidative substrate in extrahepatic tissues for the increased metabolic demand of ion regulation. Protein kinase A or intracellular Ca²⁺ make apparently small contributions to the activation of GPase, leaving us to speculate on alternate routes of enzyme activation. Conversely, hepatocyte swelling in hyposmotic medium leads to significant decreases in GPase a activity and curtailed glucose output. A minimum is attained in 10 min, and pre-insult rates are re-established within 40 min, somewhat lagging behind readjustment in cell volume by regulatory volume decrease (RVD). We conclude that cell swelling and subsequent RVD do not signify stress to the cells and metabolic demand may be decreased under cell swelling conditions. Alteration of GPase phosphorylation with extracellular osmolarity appears to be a general phenomenon, since we also find it in hepatocytes of another freshwater catfish (Clarias batrachus) and a marine scorpaenid (Sebastes caurinus).Abbreviations BAPTA 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid - BSA bovine serum albumin - cAMP adenosine 3',5-cyclic monophosphate - GPase glycogen phosphorylase - MDH malate dehydrogenase - MHM modified Hanks medium - PKA c-AMP dependent protein kinase A - 8-Br-Rp-cAMPS 8-Bromo-Rp-3',5'-cyclic adenosine monophosphorothioate - RT room temperature - RVD regulatory volume decrease - RVI regulatory volume increaseCommunicated by L.C.-H. Wang