Characterization of cyclic nucleotide phosphodiesterases from cultured bovine aortic endothelial cells.
Experiments were carried out in order to isolate and characterize the cyclic nucleotide phosphodiesterase activities in primary and low passages of cultured bovine aortic endothelial cells. The subcellular characterization of the cyclic nucleotide hydrolytic activity showed that both cAMP and cGMP hydrolytic activities were predominant in the cytosolic rather than the particulate fraction of the endothelial cell homogenate. At a low substrate concentration (0.25 microM), the major hydrolytic activity was for cAMP while at a high concentration (20 microM) it was for both cAMP and cGMP. Both cAMP and cGMP hydrolytic activities were insensitive to calmodulin. Cytosolic cyclic nucleotide phosphodiesterase activity was resolved into two distinct phosphodiesterase forms using HPLC. The first eluted form was designated cGS-PDE: it hydrolysed both cAMP and cGMP and its cAMP hydrolytic activity was markedly enhanced by the presence of cGMP. The second form was designated cAMP-PDE: it selectively hydrolysed cAMP. The cytosolic cAMP-PDE was inhibited by micromolar concentrations of cAMP-PDE inhibitors such as trequinsin, rolipram, dipyridamole or papaverine. The cGS-PDE was inhibited by micromolar concentrations of trequinsin, dipyridamole and papaverine and was insensitive to rolipram, except for the hydrolysis of cAMP which was inhibited in the micromolar range. Both the cAMP-PDE and the cGS-PDE were relatively insensitive to the selective cGMP-PDE inhibitor, zaprinast which was about 750-fold less potent on endothelial PDEs than on smooth muscle cGMP-PDE. The identification of selective and specific PDE inhibitors of the different PDE forms may allow a better understanding of the regulation and the role of cyclic nucleotides in endothelial cells.
Lugnier
C
C
Laboratoire de Pharmacologie Cellulaire et Moléculaire, CNRS URA 600, Université Louis Pasteur de Strasbourg, Illkirch, France.
Schini
V B
VB
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001011:Aorta; D002417:Cattle; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004730:Endothelium, Vascular / Q000201:enzymology*; D007700:Kinetics; D013347:Subcellular Fractions / Q000201:enzymology
D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D006152:Cyclic GMP