CYCLIC-NUCLEOTIDE PHOSPHODIESTERASE ISOZYMES IN RAT MESANGIAL CELLS

We characterized cyclic nucleotide phosphodiesterases isolated from ra t mesangial cells and assessed their roles in regulating cellular cycl ic nucleotide levels. Three peaks of phosphodiesterase activity were e luted by a linear sodium acetate gradient from a Q Sepharose column lo aded with the mesangial cell extract. The first peak activity was stim ulated by Ca2+-calmodulin and inhibited by calmodulin-stimulated phosp hodiesterase inhibitors but not by a selective cGMP specific phosphodi esterase V inhibitor. The second, minor activity peak was stimulated b y cyclic GMP and inhibited by EHNA [erythro-9-(2-hydroxy-3-nonyl)adeni ne], a selective inhibitor of cyclic GMP-stimulated phosphodiesterase II. The last peak activity was not inhibited by cyclic GMP but selecti vely inhibited by rolipram [4-(3-cyclopentyloxy-4-methoxyphenyl)-2] or Ro 20-1724 [4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone], inhibito rs of cyclic AMP specific, cyclic GMP insensitive phosphodiesterase IV . Based on their order of chromatographic elution, kinetic properties and sensitivity to allosteric agents and inhibitors, the peak 1, 2 and 3 correspond to phosphodiesterase I, II and IV. The basal cyclic GMP level was raised more effectively by selective inhibitor of phosphodie sterase I than phosphodiesterase II. In contrast, the atrial natriuret ic factor-induced cyclic GMP elevation was potentiated more effectivel y by selective inhibitors of phosphodiesterase II than phosphodiestera se I. The forskolin-induced cyclic AMP increase was greatly potentiate d by selective phosphodiesterase IV inhibitors but not by other phosph odiesterase inhibitors. These data suggest that phosphodiesterase I an d II are responsible for cyclic GMP hydrolysis whereas phosphodiestera se IV is mainly responsible for cyclic AMP hydrolysis. The relative im portance of phosphodiesterase I and II varied with the intracellular c yclic GMP concentration.