Fish hepatocyte glycogen phosphorylase - a sensitive indicator for hormonal modulation

The absence of a reproducible method for the assay of glycogen phosphorylas e (GPase) in isolated fish hepatocytes has made the interpretation of hormo ne-induced glycogenolysis data difficult. This study presents such an assay and demonstrates its sensitivity to hormonal activation. The enzyme is ass ayed in the reverse direction using glucose 1-phosphate (G1-P) and glycogen as substrates and uses standard methods for the quantification of the libe rated inorganic phosphate. The assay is highly reproducible, sensitive, and provides an excellent means to follow small and rapid changes in enzyme ph osphorylation status following the addition of hormones. We show for hepato cytes isolated from rockfish (Sebastes caurinus) and brown bullhead (Ameiur us (Ictalurus) nebulosus) that small concentrations of three model hormones , namely epinephrine (catfish), norepinephrine, and prostaglandin E-2 (rock fish), lead to the rapid, concentration and time-dependent conversion of ex isting GPase into the active GPase a form. Some of the enzyme seems to be i mpervious to hormonal activation, as the highest %GPase a never reaches 100 %. We provide evidence that changes in enzyme phosphorylation status provid e a better short-term insight into hormone-dependent activation than estima tes of glucose or some other end products, that usually must accumulate for long periods before detection is possible. Our data also show that GPase i n freshly isolated hepatocytes is already in an activated state and cells s hould be given a period of 'rest' for several hours before hormonal studies involving glycogen breakdown or the cAMP cascade are initiated.