IDENTIFICATION, CHARACTERIZATION AND FUNCTIONAL-ROLE OF PHOSPHODIESTERASE ISOZYMES IN HUMAN AIRWAY SMOOTH-MUSCLE

In the present study phosphodiesterase (PDE) isozymes in human airway smooth muscle were isolated, identified and characterized, and the fun ctional roles of these isozymes in intact bronchi were evaluated by us ing isozyme-selective PDE inhibitors. PDE isozymes in human trachealis were isolated by using a combination of DEAE-Sepharose and calmodulin -Sepharose affinity chromatography, and were identified based upon the ir kinetic characteristics as well as their sensitivity to allosteric modulators and isozyme-selective PDE inhibitors. By using this approac h, six distinct isozymes were identified: two calmodulin-stimulated PD Es (PDE I(alpha) and PDE I(beta)), cyclic GMP (cGMP)-stimulated PDE (P DE II), cGMP-inhibited PDE (PDE III), cyclic AMP cAMP)-specific PDE (P DE IV) and cGMP-specific PDE (PDE V). PDEs III and IV were the major c AMP-hydrolyzing enzymes present, whereas PDEs I(alpha), I(beta) and V accounted for most of the cGMP-hydrolytic activity. In carbachol-preco ntracted small (<0.5-2 mm diameter) or large (4-15 mm diameter) human bronchus, zaprinast (10 nM-30 muM), the selective PDE V inhibitor, was without marked relaxant activity (<13%), whereas rolipram (30 muM), t he selective PDE IV inhibitor, produced approximately 25% relaxation i n both preparations. Siguazodan was a significantly more effective rel axant than zaprinast or rolipram in large bronchus, producing a maximu m relaxation of 77 +/- 15% at a concentration of 30 muM, whereas in sm all bronchus 30 muM siguazodan elicited 20 +/- 6% relaxation. Similar results were obtained in large bronchi contracted with leukotriene (LT ) D4 (0.1 muM). The ability of isozyme-selective PDE inhibitors to pot entiate agonist-induced relaxation was studied in LTD4-contracted larg e bronchi. Siguazodan (10 muM), but not rolipram (10 muM) or zaprinast (10 muM), potentiated the relaxant response in LTD4-contracted large bronchus to isoproterenol, a beta adrenoceptor agonist thought to indu ce relaxation via a cAMP-mediated mechanism. In contrast, zaprinast (1 0 muM), but not siguazodan (10 muM), potentiated relaxation induced by sodium nitroprusside, a nitrovasodilator that relaxes airway smooth m uscle via a cGMP-mediated mechanism. The most striking observation fro m functional studies was that the combination of rolipram and siguazod an produced a much greater relaxation of small or large human bronchi than either agent alone, indicating an interaction between PDE III and PDE IV inhibitors that was at least additive and, in some cases, syne rgistic. The data indicate that the human trachealis contains represen tatives of all five known families of PDE isozymes, and suggests that a dual PDE III/IV inhibitor may be a more effective bronchodilator tha n selective PDE III or PDE IV inhibitors.