CHARACTERIZATION OF INDOLIDAN-SENSITIVE AND ROLIPRAM-SENSITIVE CYCLIC-NUCLEOTIDE PHOSPHODIESTERASES IN CANINE AND HUMAN CARDIAC MICROSOMAL FRACTIONS

The distribution of phosphodiesterase (PDE) activities was studied in canine cardiac microsomal fractions separated by sucrose density gradi ent (fractions F(I) to F(VI)). These fractions were characterized by t heir Ca-45(2+) uptake and release properties, [H-3] ryanodine binding [used as sarcoplasmic reticulum (SR) markers] and their [H-3]nitrendip ine binding (as a T-system marker). The solubilized canine and human S R-enriched membranes were subjected to high performance liquid chromat ography and the PDE forms were then analyzed for their kinetic propert ies and drug sensitivities. In human SR, a notable amount of PDE I hyd rolyzing both cAMP and cGMP was characterized; however, its stimulatio n by calmodulin was reduced. Two selective cAMP-PDE forms were identif ied in the canine and human cardiac SR-enriched fractions. The major f orm presents the characteristics of PDE III: an apparent K(m) value of 0.29 and 0.35 muM in canine and human cardiac SR, respectively, poten t inhibition by cGMP and AAL 05 > cilostamide > CI 930 > indolidan, an d insensitivity to rolipram. The other form displays the properties of PDE IV: an apparent K(m) value of 1.4 and 1.3 muM in canine and human cardiac SR respectively, potent inhibition by rolipram and poorly sen sitive to inhibition by PDE III inhibitors. The PDE IV distribution in canine SR suggests that this form is mostly associated with the F(II) fraction enriched in sarcolemmal membranes. In contrast. PDE III asse ssed by its indolidan sensitivity and [H-3]LY186126 binding is associa ted with the microsomal membranes enriched in vesicles derived from T- tubule and junctional SR membranes. Because these membranes are direct ly involved in controlling excitation-contraction coupling, such PDE l ocation enhances the physiologic relevance to study their implication in regulating cardiac contraction.