Proteolysis of cyclic AMP phosphodiesterase-II attenuates its ability to be inhibited by compounds which exert positive inotropic actions in cardiac tissue.
Extraction of frozen canine cardiac muscle rendered soluble over 90% of the cyclic AMP phosphodiesterase activity. The residual activity was membrane-bound. Ion exchange chromatography of the soluble activity on DE-52 allowed for the resolution of three distinct cyclic AMP phosphodiesterase fractions termed PDE-I, PDE-II and PDE-III in order of elution from the column by a linear NaCl gradient. The relative ratio of cyclic AMP phosphodiesterase activity exhibited by these three peaks was 1:0.65:0.82 and of cyclic GMP phosphodiesterase activity was 1:0.52:0.05 for PDE-I, PDE-II and PDE-III respectively. PDE-II and PDE-III were further purified by re-chromatography on DE-52. Fractions PDE-II and PDE-III were thermolabile at 50 degrees, decaying as single exponentials with half lives of 180 sec and 77 sec respectively. All three species exhibited non-linear Lineweaver-Burke plots for the hydrolysis of cyclic AMP, exhibiting both high and low affinity components. Hydrolysis of cyclic GMP by all three components obeyed normal kinetics, yielding linear plots. PDE-I was a Ca2+/calmodulin-activated species which exhibited a low Km for both cyclic AMP and cyclic GMP but hydrolysed cyclic GMP with a higher Vmax than for cyclic AMP. PDE-II exhibited a much lower Km for cyclic AMP than for cyclic GMP and a much higher Vmax for the hydrolysis of cyclic AMP. PDE-III exhibited a low Km for both cyclic AMP and cyclic GMP, however, its Vmax for cyclic AMP was about 40-fold higher than for cyclic GMP. Cyclic GMP acted as a potent inhibitor (IC50 = 6.3 microM) of cyclic AMP hydrolysis catalysed by PDE-III but not of the hydrolysis of cyclic AMP by PDE-II (IC50 = 33.2 microM). The phosphodiesterase inhibitors milrinone, CI-930, UK-35,493, carbazeran and buquineran acted as potent inhibitors of cyclic AMP hydrolysis catalysed by both PDE-II and PDE-III enzymes. They did not inhibit PDE-I activity. PDE-II, when prepared in the absence of protease inhibitors exhibited a reduced potency to inhibition by these compounds. Treatment of purified PDE-II with trypsin caused a reduction in enzyme activity and reduced dramatically the sensitivity of PDE-II activity to inhibition by these various compounds. The action of proteolysis in attenuating the inhibitory effect of these compounds on PDE-II was most dramatic with CI-930, milrinone, amrinone, buquineran and UK35,493 and least dramatic with carbazeran and IBMX.(ABSTRACT TRUNCATED AT 400 WORDS)
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Department of Biochemistry, University of Glasgow, Scotland, U.K.
Pyne
N J
NJ
Houslay
M D
MD
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism; D000676:Amrinone / Q000494:pharmacology; D000818:Animals; D002219:Carbamates / Q000494:pharmacology; D002316:Cardiotonic Agents / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D004285:Dogs; D007700:Kinetics; D020105:Milrinone; D009206:Myocardium / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011724:Pyridazines / Q000494:pharmacology; D011728:Pyridones / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism
D002219:Carbamates; D002316:Cardiotonic Agents; D011724:Pyridazines; D011728:Pyridones; D011799:Quinazolines; C031720:carbazeran; C054190:UK 35493; C042528:4,5-dihydro-6-(4-(imidazol-1-yl)phenyl)-5-methyl-3(2H)-pyridazinone; C012675:buquineran; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D010447:Peptide Hydrolases; D014357:Trypsin; D006152:Cyclic GMP; D020105:Milrinone; D000676:Amrinone