Role of phosphodiesterase isoenzymes in regulating intracellular cyclic AMP in adenosine-stimulated smooth muscle cells.
Three phosphodiesterase (PDE) isoenzymes were separated by Mono Q h.p.l.c. column chromatography from the soluble fraction of a homogenate of pig aortic smooth muscle cells. The first peak of PDE activity was stimulated by calmodulin in the presence of calcium. The second broad peak contained at least two activities, which were sensitive to inhibition by CI-930 or rolipram respectively. The distribution of total cellular enzyme activity in different subcellular fractions was also determined. The majority (78%) of the total activity was present in the cytosolic fraction, 18% of activity was in a membrane-bound form and 4% of activity was associated with the cytoskeleton. Rolipram-sensitive PDE was present predominantly in the cytosolic fraction, whereas cyclic GMP-inhibited, CI-930-sensitive PDE was evenly distributed between the cytosolic and particulate fractions. All of the calmodulin-dependent PDE activity was found in the soluble fraction. CI-930 and rolipram enhanced, by 2-fold and 3-4-fold respectively, the adenosine-stimulated rise in cellular cyclic AMP level. The increase in cyclic AMP levels due to CI-930 or rolipram was dose-dependent. Removal of adenosine once cyclic AMP had risen resulted in a rapid fall in cyclic AMP levels even in the presence of rolipram and CI-930. M&B 22,948, the calmodulin-dependent PDE inhibitor, caused less than a 25% increase of the adenosine-stimulated cyclic AMP levels by itself, but it contributed substantially to controlling the cyclic AMP levels after the removal of adenosine when used together with CI-930 and rolipram. These phenomena suggested that all three PDE isoenzymes participated in modulating cellular cyclic AMP levels after adenosine stimulation, and that differential importance of the individual isoenzymes depends on cellular cyclic AMP levels.
Xiong
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Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst 01003-4505.
Westhead
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Slakey
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D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D000241:Adenosine / Q000494:pharmacology*; D000818:Animals; D001011:Aorta / Q000166:cytology / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002451:Cell Compartmentation; D002458:Cell Fractionation; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D004305:Dose-Response Relationship, Drug; D006868:Hydrolysis; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology*; D011688:Purinones / Q000494:pharmacology; D011724:Pyridazines / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D020889:Rolipram; D013552:Swine
D002147:Calmodulin; D007527:Isoenzymes; D011688:Purinones; D011724:Pyridazines; D011760:Pyrrolidinones; C042528:4,5-dihydro-6-(4-(imidazol-1-yl)phenyl)-5-methyl-3(2H)-pyridazinone; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; C011145:zaprinast; D020889:Rolipram; D000241:Adenosine; D002118:Calcium