Isoforms of cyclic nucleotide phosphodiesterase PDE3 and their contribution to cAMP hydrolytic activity in subcellular fractions of human myocardium.
Three isoforms of PDE3 (cGMP-inhibited) cyclic nucleotide phosphodiesterase regulate cAMP content in different intracellular compartments of cardiac myocytes in response to different signals. We characterized the catalytic activity and inhibitor sensitivity of these isoforms by using recombinant proteins. We determined their contribution to cAMP hydrolysis in cytosolic and microsomal fractions of human myocardium at 0.1 and 1.0 microm cAMP in the absence and presence of Ca(2+)/calmodulin. We examined the effects of cGMP on cAMP hydrolysis in these fractions. PDE3A-136, PDE3A-118, and PDE3A-94 have similar K(m) and k(cat) values for cAMP and are equal in their sensitivities to inhibition by cGMP and cilostazol. In microsomes, PDE3A-136, PDE3A-118, and PDE3A-94 comprise the majority of cAMP hydrolytic activity under all conditions. In cytosolic fractions, PDE3A-118 and PDE3A-94 comprise >50% of the cAMP hydrolytic activity at 0.1 microm cAMP, in the absence of Ca(2+)/calmodulin. At 1.0 microm cAMP, in the presence of Ca(2+)/calmodulin, activation of Ca(2+)/calmodulin-activated (PDE1) and other non-PDE3 phosphodiesterases reduces their contribution to <20% of cAMP hydrolytic activity. cGMP inhibits cAMP hydrolysis in microsomal fractions by inhibiting PDE3 and in cytosolic fractions by inhibiting both PDE3 and PDE1. These findings indicate that the contribution of PDE3 isoforms to the regulation of cAMP hydrolysis in intracellular compartments of human myocardium and the effects of PDE3 inhibition on cAMP hydrolysis in these compartments are highly dependent on intracellular [Ca(2+)] and [cAMP], which are lower in failing hearts than in normal hearts. cGMP may amplify cAMP-mediated signaling in intracellular compartments of human myocardium by PDE3-dependent and PDE3-independent mechanisms.
Hambleton
Ryan
R
Cardiology Section, Veterans Affairs Salt Lake City Health Care System, Salt Lake City, Utah 84148, USA.
Krall
Judith
J
Tikishvili
Eliso
E
Honeggar
Matthew
M
Ahmad
Faiyaz
F
Manganiello
Vincent C
VC
Movsesian
Matthew A
MA
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling; D000242:Cyclic AMP / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism / Q000494:pharmacology; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009206:Myocardium / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011994:Recombinant Proteins; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; C517287:PDE3A protein, human; D006152:Cyclic GMP