Characterization of indolidan- 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 gradient (fractions F1 to Fv1). These fractions were characterized by their 45Ca2+ uptake and release properties, [3H] ryanodine binding [used as sarcoplasmic reticulum (SR) markers] and their [3H]nitrendipine binding (as a T-system marker). The solubilized canine and human SR-enriched membranes were subjected to high performance liquid chromatography and the PDE forms were then analyzed for their kinetic properties and drug sensitivies. In human SR, a notable amount of PDE I hydrolyzing both cAMP and cGMP was characterized; however, its stimulation by calmodulin was reduced. Two selective cAMP-PDE forms were identified in the canine and human cardiac SR-enriched fractions. The major form presents the characteristics of PDE III: an apparent Km value of 0.29 and 0.35 microM in canine and human cardiac SR, respectively, potent inhibition by cGMP and AAL 05 > cilostamide > Cl 930 > indolidan, and insensitivity to rolipram. The other form displays the properties of PDE IV: an apparent Km value of 1.4 and 1.3 microM in canine and human cardiac SR respectively, potent inhibition by rolipram and poorly sensitive to inhibition by PDE III inhibitors. The PDE IV distribution in canine SR suggests that this form is mostly associated with the FII fraction enriched in sarcolemmal membranes. In contrast, PDE III assessed by its indolidan sensitivity and [3H]LY186126 binding is associated with the microsomal membranes enriched in vesicles derived from T-tubule and junctional SR membranes. Because these membranes are directly involved in controlling excitation-contraction coupling, such PDE location enhances the physiologic relevance to study their implication in regulating cardiac contraction.
Laboratoire de Pharmacologie Cellulaire et Moléculaire, CNRS URA 600, Université Louis Pasteur de Strasbourg, Illkirch, France.
C
C
Lugnier
B
B
Muller
A
A
Le Bec
C
C
Beaudry
E
E
Rousseau
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D000818:Animals; D002316:Cardiotonic Agents / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004285:Dogs; D006801:Humans; D066298:In Vitro Techniques; D007211:Indoles / Q000378:metabolism / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology*; D000078183:Oxindoles; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011724:Pyridazines / Q000378:metabolism / Q000494:pharmacology*; D011760:Pyrrolidinones / Q000494:pharmacology*; D020889:Rolipram; D012519:Sarcoplasmic Reticulum / Q000201:enzymology
D002316:Cardiotonic Agents; D007211:Indoles; D000078183:Oxindoles; D010726:Phosphodiesterase Inhibitors; D011724:Pyridazines; D011760:Pyrrolidinones; C050325:indolidan; C059371:LY 186126; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D020889:Rolipram