Adipocyte cyclic nucleotide phosphodiesterase activation by vanadate.

Abstract

The addition of vanadate (Na3VO4) to intact isolated rat adipocytes stimulated cAMP phosphodiesterase activity (Type IV) in the particulate (P2) fraction. Vanadate increased the Vmax of the Type IV phosphodiesterase activity without affecting its apparent substrate affinity. Na3VO4 also stimulated cAMP hydrolysis of cell-free particulate and cytosolic fractions, but this activation required the presence of reduced glutathione (GSH). The mixture of vanadate and glutathione appeared as an emerald green solution (V-GSH complex), which was shown by EPR to contain vanadyl ion. No effect of either GSH or Na3VO4 alone on cell-free particulate cAMP phosphodiesterase activity was observed; however, Na3VO4, alone or in combination with GSH, stimulated cGMP hydrolysis in this subcellular fraction. The V-GSH complex increased the Vmax of the particulate cAMP phosphodiesterase activity without affecting its apparent Km. The activating effect of the complex was rapid in onset, persistent over 30 minutes, and reversible. The EC50 for activation of the particulate cAMP phosphodiesterase was approximately 5 microM Na3VO4 (maintaining the GSH:Na3VO4 molar ratio at 2:1); maximal stimulation was achieved at 0.1 mM Na3VO4. Purified microsomal membranes showed activation similar to that of the P2 fraction, while only a 60% stimulation was observed in purified plasma membranes. The V-GSH complex increased basal insulin-activated Type IV phosphodiesterase activity to a common maximal level. Detergent-solubilized cAMP-phosphodiesterase from the P2 fraction was stimulated 2.5-fold by the V-GSH complex. Limited trypsin treatment of P2 membranes activated cAMP phosphodiesterase and abolished the stimulatory effect of the V-GSH complex. These results are generally consistent with the hypothesis that V-GSH complex activates Type IV phosphodiesterase by an indirect mechanism, which appears to involve predominantly membrane bound components that may be biologically important enzyme regulatory elements.