Selective inhibition of a high affinity type IV cyclic AMP phosphodiesterase in bovine trachealis by AH 21-132. Relevance to the spasmolytic and anti-spasmogenic actions of AH 21-132 in the intact tissue.
The ability of a papaverine-derived bronchodilator, AH 21-132, to inhibit cyclic nucleotide hydrolysis and to increase the cAMP content and the activity of cAMP-dependent protein kinase (A-kinase) was evaluated in bovine tracheal smooth muscle (BTSM) and related to the mechanical effects elicited by this compound in vitro. AH 21-132 (100 nM-1 mM) produced a concentration-related relaxation of BTSM pre-contracted with methacholine (MCh) that was subject to marked functional antagonism. AH 21-132 (100 microM) also displayed anti-spasmogenic activity preventing the generation of tone induced by low, but not high, concentrations of MCh. Neither the spasmolytic nor anti-spasmogenic effects of AH 21-132 were antagonized by the beta 2-adrenoceptor blocking drug ICI 118551 (50 nM). Three Ca(2+)- and calmodulin-independent cyclic nucleotide phosphodiesterases (PDE) were resolved from the soluble fraction of BTSM homogenates by Q-Sepharose anion exchange chromatography. These PDEs were identified by kinetic and inhibitor sensitivity criteria as the Type II (cGMP-stimulated), Type IV (Ro 20-1724-inhibited) and Type V (cGMP-specific) isoenzymes. A small amount (approximately 5%) of a Type III PDE seemed to be present but this was not identified with certainty. AH 21-132 selectively inhibited Type IV PDE in a competitive manner with an IC50 and KI of 3.7 and 2.7 microM, respectively. AH 21-132 similarly increased the cAMP content (from 5.3 to 23.1 pmol/mg protein after 1 mM AH 21-132) and activated A-kinase (from 29.6% to 53.5% after 1 mM AH 21-132) in intact BTSM over the same concentration range at which this compound influenced tone. In addition, AH 21-132 in high concentrations (greater than 100 microM), while exerting no direct effect on A-kinase itself, markedly potentiated (ca. four-fold at 3 mM AH 21-132) the ability of cAMP to activate A-kinase without affecting the affinity of cAMP for this enzyme. It is concluded that the spasmolytic and anti-spasmogenic effects of AH 21-132 in BTSM may be related, in part, to its ability to inhibit Type IV PDE, increase the intracellular cAMP content and so activate A-kinase. A cyclic nucleotide-dependent mechanism is therefore proposed. In addition, the ability of AH 21-132 to augment cAMP-dependent phosphorylation in a cell-free system, when Type IV PDE is inhibited fully, provides the possibility that the observed relaxation elicited by high concentrations of AH 21-132, while cAMP-dependent, does not require any further increase in the intracellular cAMP concentration.
Giembycz
M A
MA
Department of Thoracic Medicine, National Heart and Lung Institute, Royal Brompton Hospital, London, U.K.
Barnes
P J
PJ
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D000818:Animals; D002417:Cattle; D002474:Cell-Free System / Q000187:drug effects / Q000201:enzymology; D002852:Chromatography, Ion Exchange; D000242:Cyclic AMP / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D016210:Methacholine Chloride / Q000037:antagonists & inhibitors; D009126:Muscle Relaxation / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects; D009287:Naphthyridines / Q000494:pharmacology*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism; D012995:Solubility; D014132:Trachea / Q000187:drug effects* / Q000201:enzymology / Q000502:physiology
D007527:Isoenzymes; D009287:Naphthyridines; D010726:Phosphodiesterase Inhibitors; D016210:Methacholine Chloride; C044843:AH 21-132; D000242:Cyclic AMP; D011494:Protein Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases