Effects of selective inhibitors on cyclic nucleotide phosphodiesterases of rabbit aorta.
In this study three forms of cyclic nucleotide phosphodiesterase (PDE) isolated from rabbit aorta were pharmacologically characterized, and the consequence of selective inhibition of calmodulin-stimulated PDE (CaM-PDE) and cGMP specific PDE (cG-PDE) was evaluated using PDE inhibitors. The cG-PDE (F1) was selectively inhibited by M&B 22948 (IC50 = 0.5 microM) and dipyridamole (IC50 = 7 microM). The cAMP-PDE (cA-PDE, F3) was inhibited more effectively by the cA-PDE inhibitor milrinone than by other PDE inhibitors. The cA-PDE preparation appeared to contain both cG-inhibited PDE and cG-insensitive PDE based on an additive inhibition of the activity by milrinone and SQ 65442, respective inhibitors of these enzymes. Vinpocetine, 8-methoxymethyl isobutylmethylxanthine (8-MeOMeMIX) and M&B 22948 effectively inhibited CaM-PDE (F2). Vinpocetine was a more selective inhibitor of CaM-PDE than M&B 22948 or 8-MeOMeMIX. CaM-PDEs isolated from rabbit aorta and bovine brain exhibited a similar sensitivity to these inhibitors. Seventy-two percent of the cGMP-hydrolyzing activity of this rabbit aortic CaM-PDE preparation was immunoadsorbed to monoclonal antibody (ACC-1) against CaM bound to brain CaM-PDE. Vinpocetine, 8-MeOMeMIX and M&B 22948 at concentrations (30 and 100 microM) which inhibit CaM-PDE greater than 60% increased cGMP but not cAMP levels in l-norepinephrine (NE) preincubated rabbit aortic slices. At concentrations selectively inhibiting cG-PDE, dipyridamole and M&B 22948 increased cGMP levels in untreated slices but failed to increase cGMP levels significantly in NE-treated slices. By contrast, vinpocetine failed to increase cGMP significantly in untreated slices, although it increased cGMP levels in NE or KCl preincubated slices. These data indicate that, in activated (precontracted) aorta, CaM-PDE is a major enzyme, whereas in untreated aorta cG-PDE is a predominant enzyme for the hydrolysis of cGMP. This study also shows a usefulness of selective inhibitors in identifying different forms of PDE and similar drug sensitivities and immunoadsorption of aortic and brain CaM-PDEs by a monoclonal antibody.
Ahn
H S
HS
Department of Pharmacology, Schering-Plough Corp., Bloomfield, NJ 07003.
Crim
W
W
Romano
M
M
Sybertz
E
E
Pitts
B
B
D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000032:analysis* / Q000302:isolation & purification; D000818:Animals; D001011:Aorta / Q000201:enzymology*; D001921:Brain / Q000201:enzymology; D002147:Calmodulin / Q000494:pharmacology; D008297:Male; D020105:Milrinone; D009712:Nucleotides, Cyclic / Q000032:analysis; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D011688:Purinones / Q000494:pharmacology; D011728:Pyridones / Q000494:pharmacology; D011817:Rabbits; D014748:Vinca Alkaloids / Q000494:pharmacology
D002147:Calmodulin; D009712:Nucleotides, Cyclic; D010726:Phosphodiesterase Inhibitors; D011688:Purinones; D011728:Pyridones; D014748:Vinca Alkaloids; C013983:vinpocetine; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; C011145:zaprinast; D020105:Milrinone