title: The identification of a new cyclic nucleotide phosphodiesterase activity in human and guinea-pig cardiac ventricle. Implications for the mechanism of action of selective phosphodiesterase inhibitors.
abstract: Four cyclic nucleotide phosphodiesterase (PDE) activities were separated from low-speed supernatants of homogenates of human cardiac ventricle by DEAE-Sepharose chromatography, and designated PDE I-PDE IV in order of elution with an increasing salt gradient. PDE I was a Ca2+/calmodulin-stimulated activity, and PDE II was an activity with a high Km for cyclic AMP which was stimulated by low concentrations of cyclic GMP. Human ventricle PDE III had Km values of 0.14 microM (cyclic AMP) and 4 microM (cyclic GMP), and showed simple Michaelis-Menten kinetics with both substrates. PDE IV is a previously unrecognized activity in cardiac muscle, the human enzyme having Km values of 2 microM (cyclic AMP) and 50 microM (cyclic GMP). PDE III and PDE IV were not activated by cyclic nucleotides or calmodulin. Four PDE activities were also isolated from guinea-pig ventricle, and had very similar kinetic properties. By gel filtration, the Mr of PDE III was 60,000, and that of PDE IV 45,000. The drug SK&F 94120 selectively and competitively inhibited PDE III with a Ki value of 0.8 microM (human), showing simple hyperbolic inhibition kinetics. Rolipram (Schering ZK 62711) and Ro 20-1724 (Roche), which have previously been reported to inhibit PDE III-like activities strongly, were shown to be weak inhibitors of human and guinea-pig PDE III enzymes (Ki values greater than 25 microM), but potent inhibitors of PDE IV [Ki values 2.4 microM (Rolipram) and 3.1 microM (Ro 20-1724) with human PDE IV]. The inhibition in all cases demonstrated simple hyperbolic competition. These observations suggest that the previously reported complex inhibition of PDE III-type activities from cardiac muscle was caused by incomplete separation of the PDE III from other enzymes, particularly PDE IV.
mesh_terms: D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D002852:Chromatography, Ion Exchange; D006168:Guinea Pigs; D006352:Heart Ventricles / Q000201:enzymology; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D011719:Pyrazines / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D020889:Rolipram
chemical_list: D007527:Isoenzymes; D011719:Pyrazines; D011760:Pyrrolidinones; C048350:5-(4-acetamidophenyl)pyrazin-2(1H)-one; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; D020889:Rolipram