CHARACTERIZATION OF MEMBRANE-BOUND CYCLIC-NUCLEOTIDE PHOSPHODIESTERASES FROM BOVINE AORTIC SMOOTH-MUSCLE

This study reports the isolation and characterization of cyclic nucleotide phosphodiesterases (PDEs) associated with membrane fraction in comparison to cytosolic forms from bovine aorta. DEAE-Sephacel chromatography of a solubilized membrane fraction from a homogenate, prepared under isotonic conditions in the presence of protease inhibitors, yielded one major peak of PDE activity that specifically hydrolyzed cAMP and was not stimulated by calmodulin: It appeared to contain two subtypes of PDE. The first subtype belonged to the cyclic GMP (cGMP)-inhibited PDE family, (PDE III): It had an apparent K(m) value of 0.4-mu-M and was potently inhibited by cGMP, LY186126, and cilostamide. The second was a rolipram-sensitive PDE form (PDE IV) that had an apparent K(m) value for cAMP hydrolysis of 1.1-mu-M, was selectively inhibited by rolipram and denbufylline, and was insensitive to cGMP. These two forms had kinetic and pharmacologic profiles similar to those resolved by DEAE-Sephacel from the cytosolic fraction (105,000 g supernatant). In addition, DEAE-Sephacel chromatography of the cytosolic fraction revealed another peak of PDE activity that could be resolved with high-performance liquid chromatography (HPLC) into a calmodulin-sensitive form that preferentially hydrolyzed cGMP (PDE I) and a calmodulin-insensitive form that specifically hydrolyzed cGMP (PDE V). The presence of a PDE III in vascular smooth muscle that exhibited similarities to the cGMP-inhibited PDE from cardiac tissues, the target of several new cardiotonic agents, suggests that a single mechanism of action may account for the cardiotonic and vasodilating properties of PDE III inhibitors.