Effect of CI-930 [3-(2H)-pyridazinone-4,5-dihydro-6-[4-(1H-imidazolyl) phenyl]-5-methyl-monohydrochloride] and rolipram on human coronary artery smooth muscle cell proliferation.

Experiments were conducted to determine how selective inhibitors of certain cyclic nucleotide phosphodiesterase (PDE) families, namely CI-930 (PDE3 inhibitor; 3-(2H)-pyridazinone-4,5-dihydro-6-[4-(1H-imidazolyl) phenyl]-5-methyl monohydro chloride) and rolipram (PDE4 inhibitor), may affect human coronary artery smooth muscle cell (HCASMC) proliferation. CI-930- and rolipram-inhibitable PDEs accounted for most of the cyclic AMP hydrolyzing activity in HCASMC. Twenty micromolar CI-930 and 20 microM rolipram used individually attenuated proliferation of HCASMC from some, but not all donors, as measured by flow cytometry. The simultaneous addition of 10 microM CI-930 plus 10 microM rolipram caused greater attenuation. This attenuation represented a reduction of the number of cells entering the S phase of the cell cycle and not merely a delay in cell cycle traverse. No statistically significant elevation of cyclic AMP was detected following the addition of either PDE inhibitor individually, but the combination produced significant elevations. It is concluded that CI-930- and rolipram-inhibitable PDE isozymes are expressed in HCASMC and that selective inhibitors of these isozymes can attenuate HCASMC proliferation. The data suggest that selective PDE inhibitors may prevent restenosis in patients following percutaneous transluminal coronary angioplasty because of their effect on HCASMC proliferation, and they may also be useful in retarding the progression of atherosclerosis in individuals at risk. PDE3 and PDE4 inhibitors in combination are more effective than the inhibitors used individually.

Cyclic nucleotide phosphodiesterases and vascular smooth muscle.

At least 30 different phosphodiesterase (PDE) enzymes have now been identified in mammalian tissues and cells, many of which are products of separate genes. These different isoenzyme forms can be subdivided into seven families based on their genetic and functional characteristics. Relatively specific inhibitors are available for at least five of these PDE families. A functional classification based on substrate specificity, regulatory properties, and sensitivity to inhibition by isozyme- and tissue-selective inhibitors can be used in describing the PDEs of vascular smooth muscle. Inhibition of these PDEs, especially with inhibitors of the PDE3 isoform, promotes vascular relaxation, particularly if the preparation of smooth muscle has been preconracted. For the most part, the drugs appear to act directly on smooth muscle; their effects are usually observed in endothelium-denuded preparations. In addition to their cardiotonic properties, many PDE3 inhibitors possess antiplatelet and thrombolytic activities, thereby suggesting the potential benefit of these drugs in treating diseases of the cardiovascular system. Isozyme- and cell-specific drugs have been shown to alter the synthetic state (i.e. proliferative phenotype) of smooth muscle cultures toward the appearance of the contractile phenotype. This suggests the possible use of selective PDE inhibitors to minimize the problem of restenosis seen after angioplasty. The development of novel methods to deliver more potent and selective PDE inhibitors to individual cell types and subcellular locales will lead to new therapeutic uses for this class of drugs in diseases of the cardiovascular system.

Synergistic interactions between selective pharmacological inhibitors of phosphodiesterase isozyme families PDE III and PDE IV to attenuate proliferation of rat vascular smooth muscle cells.

The interaction between selective inhibitors of 3',5'-cyclic-nucleotide phosphodiesterase (PDE) III (cyclic GMP inhibited phosphodiesterase) and selective inhibitors of PDE IV (Ro 20-1724 inhibited phosphodiesterase) to attenuate fetal bovine serum-stimulated incorporation of [3H]thymidine into DNA and cell proliferation was studied in a line (A10) of vascular smooth muscle cells (VSMC). The nonselective PDE inhibitors 3-isobutyl-1-methylxanthine (IBMX) and papaverine attenuated DNA synthesis with EC50 values (16 and 18 microM, respectively) in the same range as their published IC50 values (2-50 and 2-25 microM, respectively) as PDE inhibitors. The selective PDE III inhibitors CI-930 and cilostamide used alone attenuated DNA synthesis with EC50 values (> 300 and 5.3 microM, respectively) that were much higher than published IC50 values (0.15-0.46 and 0.005-0.064 microM, respectively) for inhibition of PDE III. In the presence of the PDE IV inhibitor rolipram (10 microM), their EC50 values were shifted (0.66 and 0.16 microM, respectively) much closer to their respective IC50 values. When the selective PDE IV inhibitors rolipram and Ro 20-1724 were used alone, they attenuated DNA synthesis with EC50 values (111 and > 100 microM, respectively) much higher than their IC50 values (0.6-2.6 and 2-13 microM, respectively) as inhibitors of PDE IV, but 10 microM CI-930 (PDE III inhibitor) shifted their EC50 values (0.56 and 1.5 microM, respectively) much closer to their IC50 values. In experiments that assessed VSMC proliferation using the MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] method, IBMX and papaverine attenuated proliferation with EC50 values (27 and 58 microM, respectively) close to their IC50 values. CI-930 and cilostamide used alone did not cause 50% attenuation of proliferation at the highest concentrations tested (100 and 10 microM, respectively). In the presence of 5 microM rolipram, however, their effects were enhanced greatly with EC50 values (0.86 and 0.23 microM, respectively) that were close to their IC50 values as PDE III inhibitors. Similarly, rolipram and Ro 20-1724 attenuated VSMC proliferation with EC50 values close to their IC50 values in the presence (2.1 and 4.6 microM, respectively) but not in the absence (> 100 and > 10 microM, respectively) of 2 microM CI-930. The interactions between PDE III inhibitors and PDE IV inhibitors to attenuate DNA synthesis and VSMC proliferation were synergistic as determined by the combination index. The data demonstrate that the synergistic interactions that attenuate incorporation of [3H]thymidine into DNA are accompanied by synergistic attenuations of VSMC division.(ABSTRACT TRUNCATED AT 400 WORDS)

Multiple high-affinity cAMP-phosphodiesterases in human T-lymphocytes.

Cyclic nucleotide phosphodiesterases (PDEs) are the only enzymes that inactivate intracellular cyclic AMP (cAMP). Because the functions of T-lymphocytes are modulated by cAMP levels, the isozymes of PDE in these cells are potential targets for new drugs designed to modify the body's immunity through selective alteration of T-lymphocyte PDE activity. Cyclic GMP and 3(2H)-pyridazinone-4,5- dihydro-6-[4-(1H-imidazol-1-yl)phenyl]-5-methyl-monohydrochloride (CI-930) selectively inhibit the catalytic activity of one of the two high affinity cAMP-PDE isozyme families known to occur in mammals, whereas d,l-1,4-[3-butoxy-4-methoxybenzyl]-2-imidazolidinone (Ro 20-1724) selectively inhibits the other. The objectives of this investigation were: (1) to determine whether human T-lymphocytes contain one or both of these pharmacologically distinguishable high-affinity cAMP-PDEs, and (2) to determine the effects of selective inhibitors of these PDEs on lymphocyte blastogenesis. High-affinity cAMP-PDE was found in both the soluble and particulate fractions of T-lymphocyte sonicates. Cyclic GMP and CI-930 inhibited PDE in the particulate fraction better than in the soluble fraction, but the converse was found for Ro 20-1724. CI-930 or Ro 20-1724, used alone, attenuated T-lymphocyte blastogenesis, but neither suppressed it completely. In combination, the same PDE inhibitors caused greater suppression of blastogenesis than either produced alone. The results indicate that human T-lymphocytes contain both CI-930- and Ro 20-1724-inhibitable isozymes. Either of the isozymes can modulate human T-lymphocyte blastogenesis, but inhibition of both isozymes produces synergistic antiblastogenic effects.

High pressure liquid chromatography of cyclic nucleotide phosphodiesterase from purified human T-lymphocytes.

The cyclic nucleotide phosphodiesterase (EC 3.1.4.17) in extracts of purified human peripheral blood T-lymphocytes was examined by ion exchange high pressure liquid chromatography. Four peaks of activity were isolated. The first peak of activity selectively hydrolyzed cyclic GMP. The following 3 peaks of activity (Ia, IIa and IIIa) were selective for cyclic AMP. The selective low Km cyclic AMP-phosphodiesterase inhibitor, Ro 20-1724 (d,1-1,4-[3-butoxy-4-methoxybenzyl]-2-imidazolidinone), did not inhibit the activity in Ia whereas it did inhibit the activity in IIa and IIIa (IC50 = 17 microM). The authors conclude that ion exchange high pressure liquid chromatography described in this communication is a useful method for the isolation of different forms of cyclic nucleotide phosphodiesterase activity from human T-lymphocytes.

Lack of effect of gold salts on acetylcholine or histamine contraction of canine tracheal and bronchial smooth muscle.

In vitro gold salt incubation with proximal and distal canine airway smooth muscle was compared to untreated canine airway muscle with respect to histamine and acetylcholine-induced contraction. Gold chloride and gold sodium thiomalate had no effect on either proximal or distal canine airway smooth muscle agonist-induced contraction. Gold chloride in concentrations greater than 10(-5) M had a direct contractile effect. These results differ from previous studies which showed an inhibition of the histamine response of guinea-pig tracheal smooth muscle following in vitro preincubation with gold salts. This difference suggests that the mechanism(s) of the beneficial effects of long-term gold salt therapy on human asthma cannot be reliably studied with short-term in vitro animal preparations without first assessing species differences with respect to the effects of therapeutic agents. Human airway smooth muscle preparations would be the best means of addressing this question.

Effects of ketotifen on the responsiveness of peripheral blood lymphocyte beta-adrenergic receptors.

The effects of ketotifen therapy on the responsiveness of lymphocyte beta-adrenergic receptors was evaluated by measuring cyclic AMP elevations caused by isoproterenol in cells isolated from patients treated with ketotifen for more than 1 year. Binding of 3H-dihydroalprenolol to beta-receptors was also evaluated. The isoproterenol-induced rise in cyclic AMP relative to each individual's baseline level was greater in patients on current ketotifen therapy than in other asthmatic patients or non-asthmatic subjects. Ketotifen therapy increased the apparent equilibrium dissociation constant for specific 3H-dihydroalprenolol binding to the receptors. Receptor numbers in symptomatic asthma patients on standard drug therapy were decreased. The results indicate that long term ketotifen therapy is associated with increased responsiveness of beta-receptors to stimulation by catecholamines and that this alteration may involve changes in the receptors themselves, their membrane environment, adenylate cyclase or components of the adenylate cyclase coupling system.

Effects of adenosine and theophylline on canine tracheal smooth muscle tone.

The postulated mechanisms by which theophylline induces relaxation of airway smooth muscle include, among others, inhibition of cyclic nucleotide phosphodiesterase(s) and antagonism of adenosine-induced contraction. This latter possibility was examined by investigation of the interaction of theophylline and adenosine in canine tracheal smooth muscle preparations. Adenosine did not alter basal tone i.e. there is no evidence of a contractile response. However, when contraction was induced with methacholine, adenosine caused relaxation. It appears that this relaxation occurred as a consequence of the combination of adenosine with a site within the smooth muscle cell. The prior addition of theophylline (10(-6)-10(-3) M) did not alter adenosine-induced relaxation and in the reverse experiment, the prior addition of adenosine (10(-6)-10(-3) M) did not alter the relaxation produced by theophylline. It is concluded that adenosine relaxes canine tracheal smooth muscle by combination with an intracellular site, rather than a receptor on the cell surface. The hypothesis that theophylline relaxes airways smooth muscle by antagonism of adenosine or that adenosine antagonizes theophylline was not supported by our data.

The mechanism of ergonovine-induced airway smooth muscle contraction.

There have been three articles in the clinical literature of ergonovine maleate-induced bronchospasm. The effect of the alkaloid on isolated canine tracheal smooth muscle was analyzed to investigate the mechanism of ergonovine-induced airway smooth muscle contraction. Both ergonovine and 5-hydroxytryptamine (5HT, serotonin) contracted the smooth muscle preparations with EC50s of 1.35 X 10(-8) mol/L and 5.06 X 10(-7) mol/L, respectively. The maximal contractile response observed with ergonovine was approximately 30% less than that observed with 5HT. Methysergide competitively blocked both ergonovine and 5HT responses with similar calculated pKB values (8.33 against ergonovine and 8.46 against 5HT) and also similar pA2 values determined by Schild plots (8.50 and 8.45, respectively). The relative affinity and efficacy of ergonovine versus 5HT were determined by use of a concentration of the irreversible antagonist, phenoxybenzamine, which partially blocked receptor sites. The calculated affinity of ergonovine was about 16 times higher than that of 5HT. The relative efficacy at EC100 for ergonovine was 0.2, but at EC10 it was 41.9 (5HT efficacy = 1). Ergonovine 10(-9) or 10(-8) mol/L shifted the 5HT dose-response curve to the right without reducing the maximal response, but the shift was nonparallel. Blockade of muscarinic (atropine), alpha 1-adrenergic (prazosin), beta-adrenergic (propranolol), H1 (pyrilamine), or H2 (cimetidine) receptors did not alter ergonovine-induced contraction. These data indicate that ergonovine directly contracts canine tracheal smooth muscle as a result of its combination with 5HT receptors. This effect may result in precipitation of an asthmatic attack in susceptible individuals.

Correlation between inhibition of a cyclic GMP phosphodiesterase and relaxation of canine tracheal smooth muscle.

Inhibition of partially purified cyclic nucleotide phosphodiesterase activity as well as pharmacologically induced relaxation of respiratory airways smooth muscle was examined to determine whether any correlation between these two effects could be found. The phosphodiesterase in extracts of canine tracheal smooth muscle was chromatographed on a DEAE Bio-Gel A column and eluted with a sodium chloride gradient. The peak I activity hydrolyzed cGMP at a higher rate than cAMP although the apparent Km values for these two cyclic nucleotides were relatively close. Comparison of the Ki values for alkylxanthine inhibitors of peak I activity correlated remarkably well with the EC50 values of the same compounds as relaxants of canine tracheal smooth muscle strips. It is concluded that inhibition of the peak I enzyme may cause accumulation of an intracellular pool of cyclic nucleotide and thus produce or contribute to the muscle relaxant effects that were observed.

Selective inhibition by NPT 15392 of lymphocyte cyclic GMP phosphodiesterase.

Cyclic nucleotide phosphodiesterases were measured in mouse spleen and thymus lymphocyte membranes and soluble fractions and in extracts of canine tracheal smooth muscle. The immunostimulant erythro-9(2-hydroxy,3-nonyl) hypoxanthine (NPT 15392) was found to be a potent and relatively selective inhibitor of mouse lymphocyte cyclic GMP phosphodiesterase, with IC50 values 15-180 times greater for cyclic AMP than cyclic GMP phosphodiesterases. The greatest inhibition by NPT 15392 was found using 10 microM substrate, and inhibition was greater in membrane than soluble forms of phosphodiesterase. Spleen soluble enzymes were separated by DEAE Bio-Gel A column into six peaks. A major form of cyclic GMP phosphodiesterase was inhibited effectively by NPT 15392 in a competitive manner (Ki = 50 microM). Cyclic AMP phosphodiesterase activity in the same fraction, but representing only a fifth of the total activity, was also inhibited (Ki = 70 microM). Other soluble enzymes were not affected significantly. Membrane bound enzymes were solubilized and separated into three peaks. One with high affinity for cyclic GMP was strongly inhibited (Ki = 10 microM) by NPT 15392. Inosine and isoprinosine were one-tenth to one-hundredth as effective as NPT 15392 as cyclic nucleotide phosphodiesterase inhibitors. Incubation of mouse splenic lymphocytes with NPT 15392 for 48 hr resulted in enzymes with altered responsiveness to the drug in broken cell assays: inhibition of cyclic GMP hydrolysis was enhanced while that of cyclic AMP hydrolysis was decreased. Among three separated and characterized forms of tracheal smooth muscle phosphodiesterase, NPT 15392 inhibited the low Km cyclic GMP phosphodiesterase 6-10 times more effectively than the other enzymes. These data suggest that the immunopharmacologic activities of NPT 15392 may include specific cyclic GMP phosphodiesterase inhibition as one of several possible mechanisms.

The site of action of Ptychodiscus brevis toxin within the parasympathetic axonal sodium channel h gate in airway smooth muscle.

The red tide toxin produced by Ptychodiscus brevis ( PBTX ) may cause cough, sneezing, and asthma. Previous in vitro studies with isolated canine tracheal smooth muscle demonstrated that PBTX stimulates sodium channels of parasympathetic nerve endings and thus causes a contractile response. The present study investigated the mechanism of the PBTX effect on canine tracheal smooth muscle. Repeated exposure of the muscle strip to PBTX (final concentration 46 micrograms/ml) followed by washout of the toxin resulted in reestablishment of baseline tension but a failure of contraction on further addition of PBTX . However, veratridine and scorpion toxin (SCT), which are voltage-sensitive sodium channel activators, still induced contraction. Furthermore, the contraction caused by veratridine was enhanced by a high dose of PBTX , whereas contraction caused by SCT was not. Responses to veratridine and SCT as well as PBTX (previously reported) were blocked by tetrodotoxin (a sodium channel blocker), while acetylcholine responsiveness remained intact. These results indicate that PBTX receptors in parasympathetic nerves influence Na+ flux at the h gate, that these receptors differ from the veratridine and SCT receptors, and that the conformational change in the receptors induced by PBTX affects the tissue response to veratridine.

The effect of prostaglandin E2 on histamine-stimulated calcium mobilization as a possible explanation for histamine tachyphylaxis in canine tracheal smooth muscle.

Isolated strips of canine tracheal smooth muscle rapidly lost their responsiveness to histamine when placed in a zero calcium Krebs buffer. Responsiveness to acetylcholine, however, was not rapidly lost, and following 120 min of incubation in zero calcium buffer with frequent washes, 10% of the contractile response still remained. The kinetics of each loss of response suggest that primarily a loosely bound source of calcium is mobilized by histamine and a more tightly bound source is mobilized by acetylcholine. Consistent with these data were the effects of the calcium antagonist verapamil. In normal calcium Krebs solution, dose-response curves to histamine were markedly reduced by verapamil while acetylcholine responses were relatively unaffected. In calcium depleted tracheal strips, indomethacin potentiated the calcium dose-response curve, determined by incremental readdition of calcium in the presence of histamine (10(-4) M), with comparatively little effect on the calcium dose-response curve in the presence of acetylcholine (10(-6) M). Also, in indomethacin pretreated tracheal strips, a reduction in the histamine-calcium dose-response curve could be produced by exogenous addition of 2.8 X 10(-9) M and 2.8 X 10(-8) M PGE2. In the acetylcholine-calcium responses there was a significant reduction only at 2.8 X 10(-8) M PGE2. These data suggest that histamine mobilizes primarily a loosely bound, possibly extracellular source of calcium necessary for contraction, and this histamine-stimulated calcium mobilization is sensitive to the effects of PGE2.

Inhibition of a high affinity cyclic AMP phosphodiesterase and relaxation of canine tracheal smooth muscle.

The cyclic nucleotide phosphodiesterase (PDE) activity of canine tracheal smooth muscle (CTS,) was examined. Column chromatography of soluble CTSM-PDE revealed five peaks of activity. One of these peaks (V) was examined further in this study and showed a high affinity for adenosine 3',5'-cyclic monophosphate (Km = 0.63 microM). Seven pharmacological PDE inhibitors were tested for their abilities to inhibit the peak V enzyme and also for their abilities to cause mechanical relaxation of CTSM strips in isolated tissue baths. A strong correlation (P greater than 0.001) between peak V PDE inhibition (-log Ki) and airway muscle relaxation (-log ED50) was found.

Effects of toxin of red tide, Ptychodiscus brevis, on canine tracheal smooth muscle: a possible new asthma-triggering mechanism.

The red tide toxin produced by Ptychodiscus brevis becomes airborne by the thrashing action of the surf and wind and induces cough, rhinorrhea, watery eyes, and sneezing in normal humans and wheezing in asthmatic patients. The mechanism of the contractile response induced by P. brevis toxin (PBTX) was investigated with isolated canine tracheal smooth muscle. Tetrodotoxin and atropine blocked the contractile effect of PBTX, and neostigmine potentiated the contraction. Mepyramine, phentolamine, methysergide, and chlorisondamine did not inhibit the effect of PBTX. This is the first description of a naturally occurring airborne substance that causes smooth muscle contraction by stimulating the axon sodium channels, resulting in the release of acetylcholine at postganglionic parasympathetic efferent nerve endings. The in vitro effect of PBTX on canine tracheal smooth muscle indicates that PBTX is capable of causing respiratory irritation and thus may precipitate an asthmatic attack. It is possible, however, that the mechanism is vivo may also include stimulation of a cough receptor reflex and/or stimulation of sodium channels of afferent vagus nerve fibers. In vitro evidence suggests that isoproterenol, atropine, and verapamil may be used to eliminate or prevent the respiratory symptoms that follow exposure to airborne red tide toxin. The use of high-pressure liquid chromatography separated fractions indicates that the neurotoxic component, not the hemolytic component, is responsible for contractions.

Cyclic nucleotide phosphodiesterase activity in patients with obstructive airways disease.

Cyclic nucleotide phosphodiesterase activity was examined in the pulmonary tissues of patients with no obstructive airways disease, obstructive airways disease responsive to isoproterenol (responders) and obstructive airways disease non-responsive to isoproterenol (non-responders). The phosphodiesterase catalyzed hydrolysis of 0.45 microM levels of adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) was essentially the same in responders as in patients without disease, but was reduced in the non-responders. The results suggest that neither the postulated beta adrenergic receptor-cAMP system defect nor the depressed levels of cAMP reported in patients with bronchodilator-responsive obstruction is caused by an alteration of phosphodiesterase activity.

Effects of ketotifen, a benzocycloheptathiophene, on metacholine- and acetylcholine-induced contractions of canine respiratory smooth muscle.

The relaxant effects of ketotifen on isolated respiratory smooth muscle contracted by methacholine or acetylcholine were examined. Although potent dose-dependent relaxation was observed, it appears that more than 100 times the concentration is required to relax muscle contracted with cholinergic agonists than has been reported for muscle contracted by histamine. This observation offers a possible explanation for the findings of several investigators that dosages of ketotifen administered to patients protect against histamine-induced, but not methacholine- or acetylcholine-induced, bronchospasm.