1 The aims of the present study were to characterize the cyclic nucleotide phosphodiesterase (PDE) isoenzyme activities present in human bronchi and to examine the ability of selective isoenzyme inhibitors to relax histamine and methacholine precontracted preparations of human bronchi. 2 Three separations of pooled human bronchial tissue samples were performed. Ion-exchange chromatography showed that the soluble fraction of human bronchial preparations contains PDE I, II, III, IV and V isoenzyme activities. Multiple forms of PDE I and PDE IV were observed and PDE IV was the main cyclic AMP hydrolytic activity. 3 3-Isobutyl-l-methylxanthine (IBMX) non-selectively inhibited all separated isoenzyme activities. Zaprinast selectively inhibited PDE V, but also effectively inhibited one of the two PDE I isoforms identified. The PDE IV selective inhibitors rolipram and RO-201724, inhibited the PDE IV activities as did the dual PDE III/IV inhibitor, Org 30029. Org 9935, a PDE III selective inhibitor, potently attenuated part of the PDE IV activity peak in one of three separations performed, indicating that some PDE III activity may co-elute with PDE IV under the experimental conditions employed. 4 PDE IV-selective (rolipram), PDE III-selective (Org 9935) and dual PDE III/IV (Org 30029) inhibitors were effective relaxants of human bronchial smooth muscle. The PDE V/PDE I inhibitor, zaprinast was relatively ineffective. 5 The present study demonstrates in human bronchi, as in animal airways smooth muscle, that inhibitors of PDE III, PDEIV and dual PDE III/IV have potentially useful bronchodilator activity and are worthy of further consideration as anti-asthma drugs.
The species dependent variation in the cardiotonic activity of selective cyclic nucleotide phosphodiesterase (PDE) isoenzyme inhibitors was examined by comparing the inotropic and PDE inhibitory effects of Org 30029 (N-hydroxy-5,6-dimethoxy-benzo[b]thiophene-2-carboximidamide HCl), 3-isobutyl-1-methyl-xanthine (IBMX), milrinone and rolipram in rat and rabbit ventricular myocardium. The relative activities of PDE isoenzymes in rat and rabbit cardiac ventricle were also examined to assess the role of the different PDE subtypes in modulating contractile force in the two species. In rabbit papillary muscles, IBMX, Org 30029 and milrinone increased contractile force whilst rolipram was inactive. The rank order of potency of the active compounds was Org 30029 greater than IBMX greater than milrinone. Only Org 30029 and IBMX produced significant positive inotropic responses in rat papillary muscles, milrinone and rolipram being inactive. However, large positive inotropic responses were obtained in rat papillary muscles when milrinone and rolipram were tested in combination. In rabbit papillary muscles, the positive inotropic action of milrinone was markedly potentiated by rolipram. Four main types of PDE (I, II, III, IV) isoenzymes were resolved, by DEAE-sepharose or Mono-Q ion-exchange chromatography, from both rat and rabbit cardiac ventricular tissue. In rabbit, Ca2+/calmodulin dependent PDE (PDE I) and cyclic GMP inhibited PDE (PDE III) were the dominant cAMP activities. In contrast, cyclic GMP stimulated PDE (PDE II), PDE III and cGMP insensitive PDE (PDE IV) represented the main cAMP activities in rat cardiac ventricle. The inhibitory effects of Org 30029, IBMX, milrinone and rolipram on PDE isoenzymes from rat and rabbit cardiac ventricle were essentially similar.(ABSTRACT TRUNCATED AT 250 WORDS)
1 The profile of cyclic nucleotide phosphodiesterase (PDE) isoenzymes and the relaxant effects of isoenzyme selective inhibitors were examined in bovine tracheal smooth muscle. The compounds examined were the non-selective inhibitor 3-isobutyl-1-methylxanthine (IBMX), zaprinast (PDE V selective), milrinone and Org 9935 (4,5-dihydro-6-(5,6-dimethoxy-benzo[b]thien-2-yl)-5-methyl-1(2H)-pyridazinone; both PDE III selective), rolipram (PDE IV selective) and Org 30029 (N-hydioxy-5,6-dimethoxy-benzo[b]-thiophene-2-carboximidamide HCl a dual PDE III/IV inhibitor). 2 Ion exchange chromatography showed three main peaks of PDE activity. The first peak was stimulated by Ca2+/calmodulin (PDE I), the adenosine 3': 5'-cyclic monophosphate (cyclic AMP) hydrolytic activity of the second peak was stimulated by guanosine 3':5'-cyclic monophosphate (cyclic GMP) (PDE II) whilst that of the third peak was not significantly modified by any regulator (PDE IV). Calmodulin affinity chromatography revealed the additional presence of cyclic GMP-specific PDE (PDE V) in the first peak. A clearly distinct peak of cyclic GMP-inhibited PDE (PDE III) was not observed. However, Org 9935 inhibited the third activity peak more effectively in the presence, than in the absence, of rolipram (3 ,umol 1-1), indicating the presence of PDE III activity. 2) did not completely relax preparations at concentrations up to 100 pImol I-. Org 30029 (pD2 values; histamine 6.2 + 0.1, methacholine 5.4 + 0.1) was a more effective relaxant than can be explained on the basis of PDE IV inhibition alone. 5 We conclude that bovine tracheal smooth muscle contains five distinct PDE isoenzymes. PDE IV appears to be more important in the modulation of tissue function than PDE III and PDE V.
Beecham-Wulfing Res. Labs., D3212 Gronau (Leine), F.R.G.1 Denbufylline has been examined for its ability to inhibit cyclic nucleotide phosphodiesterase isoenzymes from rat cardiac ventricle and cerebrum, as well as for its affinity for adenosine A1 and A2 receptors and the re-uptake site. For comparison, SK&F 94120, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX) were examined as phosphodiesterase inhibitors whilst N6-cyclohexyladenosine, R(-)-N6-(2-phenylisopropyl)-adenosine, 5'-N-ethylcarboxamido-adenosine, 2-nitrobenzylthioinosine, theophylline and IBMX were examined for their affinity for adenosine binding sites. 2 This investigation confirmed the presence of four phosphodiesterase activities in rat cardiac ventricle; in rat cerebrum only three were present.3 Denbufylline selectively inhibited one form of Ca2"-independent, low Km cyclic AMP phosphodiesterase. The form inhibited was one of two present in cardiac ventricle and the sole one in cerebrum. This form was not inhibited by cyclic GMP. The inotropic agent SK&F 94120 selectively inhibited the form of cyclic AMP phosphodiesterase which was inhibited by cyclic GMP present in cardiac ventricle. Theophylline and IBMX were relatively non-selective phosphodiesterase inhibitors.4 Denbufylline was a less potent inhibitor of ligand binding to adenosine receptors than of cyclic AMP phosphodiesterase. This contrasted with theophylline, which had a higher affinity for adenosine receptors, and IBMX which showed no marked selectivity. Denbufylline, theophylline and IBMX all had a low affinity for the adenosine re-uptake site. 5 Denbufylline is being developed as an agent for the therapy of multi-infarct dementia. The selective inhibition of a particular low Km cyclic AMP phosphodiesterase may account for the activity of this compound.
I The positive inotropic and chronotropic responses to sympathomimetic amines were examined in guinea-pig isolated atria.2 The order of potency measured from EC3o values was isoprenaline > orciprenaline > salbutamol > fenoterol > terbutaline. Terbutaline and salbutamol were partial agonists on rate and together with orciprenaline and fenoterol also on tension responses. 3 Functional antagonism by carbachol caused a rightwards shift of the dose-response curve and depression of the maximum response. The rate maxima for orciprenaline, fenoterol and terbutaline were above that of isoprenaline. All the tension maxima were below isoprenaline. 4 Dissociation constants (KA) and relative efficacies (er) were determined by analogy with irreversible antagonism. 5 The relative orders of affinity (KA) were isoprenaline > orciprenaline > fenoterol > salbutamol > terbutaline. Affinities were identical on rate and tension. 6 The relative efficacies were all greater than isoprenaline for rate responses. On tension they were the same or less than isoprenaline. 7 The implications of these results are discussed, in particular the fact that a partial agonist has a greater efficacy than a full agonist.
1 The effects of selective inhibition of phosphodiesterase activities on the concentration and rate of hydrolysis of adenosine 3': 5' cyclic-monophosphate (cyclic AMP) in rat cerebral cortical slices has been studied. 2 Isoprenaline caused a rapid, concentration-dependent increase in cyclic AMP concentration to new steady-state levels (basal: 7.1 + 0.7; 10M isoprenaline: 14.3 + 1.4 pmolmg'1 protein). Addition of a 13-adrenoceptor antagonist to isoprenaline-stimulated cerebral cortical slices caused a rapid decrease in cyclic AMP concentration to basal levels (t 12: 58 + 18 s). 3 Preincubation of slices for 30min with the phosphodiesterase inhibitors 1-methyl-3-isobutylxanthine, denbufylline, rolipram or Ro2O 1724 caused concentration-dependent increases in basal and isoprenalinestimulated cyclic AMP concentrations and decreased the rate of cyclic AMP hydrolysis measured after .addition of a P-adrenoceptor antagonist. However, SKF 94120 and zaprinast had none of these effects.4 The results are discussed with respect to previous studies of phosphodiesterase isozymic activities isolated from cerebrum and it is suggested that the Ca2+/calmodulin-independent, low Km cyclic AMP phosphodiesterase isozyme, which is selectively inhibited by denbufylline, rolipram and Ro2O 1724, and is present in cerebrum is of critical importance to the regulation of cyclic AMP concentration in this tissue.
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