Phospholipid regulation of a cyclic AMP-specific phosphodiesterase (PDE4) from U937 cells.

The regulation of phosphodiesterase-4 (PDE4) by various phospholipids was explored using PDE4s partially purified from U937 cells. Preincubation (5 min, 4 degrees C) of the large molecular weight PDE4 denoted "Peak 2 PDE4" with mixed phosphatidic acids (PAs) produced a 2-fold increase in its Vmax without changing its Km (approximately 2 microM) for cyclic AMP. This "activation" was not limited to PAs with specific fatty acid substituents: Synthetic PAs containing saturated and/or unsaturated fatty acids 16-20 carbons long produced similar effects. Lysophosphatidic acids (LPAs) and phosphatidylserines (PSs) also induced PDE4 activation, whereas phosphatidylcholines (PCs), phosphatidylethanolamines (PEs) and diacylglycerol did not. Antibodies to a peptide region near the PDE4 catalytic site specifically inhibited PA-induced activation. The data demonstrate that anionic phospholipids can act as non-essential activators of a leukocyte PDE4, and suggest biochemical crosstalk between phospholipid-dependent and cyclic AMP-dependent signalling pathways in human leukocytes.

Emetic, central nervous system, and pulmonary activities of rolipram in the dog.

Rolipram was characterized for its emetic, behavioral, cardiovascular and pulmonary activities in dogs, to assess its systemic pharmacology and potential bronchodilatory selectivity. At doses > or = 0.1 mg/kg i.v., rolipram induced emesis, anxiety, and stepping behaviors in conscious dogs, and increased heart rate and cardiac contractility in anesthetized instrumented dogs not treated with a beta-adrenoceptor antagonist. Cardiovascular effects in anesthetized dogs were apparently related to rolipram's central nervous system activities, in that they were associated with a reversal pentobarbital-induced anesthesia and could be ablated by pentobarbital supplementation. Rolipram's reversal of anesthesia was confirmed in uninstrumented dogs, where rolipram shortened pentobarbital sleep time while increasing heart and respiratory rates. After intragastric administration, rolipram exhibited greater emetic potency (100% emesis at 0.1 mg/kg p.o.) and lesser bronchodilatory potency (ED50 = 0.04 mg/kg i.d.) than after i.v. administration. The data demonstrate that rolipram is a potent bronchodilator that produces central nervous system effects only at higher doses when administered i.v. to the dog. Administered intragastrically, however, the bronchodilatory selectivity of rolipram is reduced presumably as a result of the activation of emetic reflexes at sites within the gastrointestinal tract.

Rolipram inhibition of phosphodiesterase-4 activation.

Rolipram inhibited U937 cell phosphodiesterase-4 in either the presence or absence of saturating (100 micrograms/ml) phosphatidic acid in an apparently phospholipid-independent manner, exhibiting similar kinetics (Ki values = 0.41 and 0.59 microM, respectively). At low concentrations (10 and 100 nM), however, rolipram caused a rightward shift of the phosphatidic acid concentration-response curve for phosphodiesterase-4 activation, suppressing activation by up to 70%. Maximum inhibition of phosphodiesterase-4 activation occurred at phosphatidic acid concentrations of 5-40 micrograms/ml. The results suggest that rolipram is capable of inhibiting phosphodiesterase-4 by both phospholipid-dependent and phospholipid-independent mechanisms.

Phosphodiesterase-IV inhibition, respiratory muscle relaxation and bronchodilation by WAY-PDA-641.

The ability of 1-[3-(cyclopentyloxy)-4-methoxyphenyl]ethanone (E)-O-(aminocarbonyl) oxime) (WAY-PDA-641) to inhibit cyclic AMP-metabolizing phosphodiesterases (PDEs) and to relax respiratory muscle was explored as part of a program to identify a PDE-IV inhibitor for potential use in the treatment of asthma. WAY-PDA-641 was identified as a preferential inhibitor of PDE-IV, possessing 36 times greater potency versus canine trachealis PDE-IV than PDE-III (IC50, 4.2 x 10(-7) M and 1.5 x 10(-5) M, respectively). The classification of WAY-PDA-641 as a preferential PDE-IV inhibitor was supported in radioligand binding studies, which demonstrated that 10 microM WAY-PDA-641 did not displace ligands from a large number of receptors, and in functional studies, which used isolated guinea pig tracheal rings. Under conditions in which tracheal rings were made sensitive to the relaxant effects of PDE-IV or PDE-III inhibitors, WAY-PDA-641 induced relaxation with IC50S of 2.6 x 10(-8) M (PDE-IV) and 3.2 x 10(-5) M (PDE-III). Moreover, PDE-IV inhibitory concentrations of WAY-PDA-641 significantly potentiated the relaxant effects of albuterol. WAY-PDA-641 reversed tracheal contractions induced by prostaglandin F2 alpha, leukotriene D4 or histamine in a biphasic manner consistent with its activity as a preferential PDE-IV inhibitor. The IC50S for reversal of each spasmogen were similar, which confirmed that WAY-PDA-641 is a functional antagonist of respiratory muscle contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and stabilization of large molecular weight PDE-IVs from U937 cells.

Cytosolic cyclic nucleotide phosphodiesterases (PDEs) from human (promonocytic) U937 cells were rapidly resolved by DEAE-Sepharose CL-6B anion exchange chromatography into two major peaks of cAMP-specific activity possessing average Kms of 1.70 microM (Peak 1) and 1.65 microM (Peak 2). Both peaks were predominantly PDE-IV, but possessed molecular weights higher than those generally reported for partially purified PDE-IVs. Storage of Peak 2 for 24 h at 4 degrees C resulted in a doubling of its Vmax and an apparent decrease in its molecular weight. Activation of Peak 2 PDE-IV was prevented when the sodium acetate concentration in its buffer was reduced by dilution immediately following isolation. Although the relevance of this activation to cellular regulation of PDE-IV is undefined, the isolation and stabilization of PDE-IV in its large molecular weight form will be critical to future investigations of PDE-IV regulation.

Anti-leukotriene effects of WY-50,295 tromethamine in isolated guinea pig pulmonary tissues.

The abilities of WY-50,295 tromethamine, a 5-lipoxygenase inhibitor, to inhibit antigen-induced leukotriene (LT) release from guinea pig lung fragments, and to prevent LTD4 or antigen-induced contraction of isolated guinea pig tracheal muscle were compared with the activities of zileuton and MK-886 (two selective 5-lipoxygenase inhibitors), and LY171883 (a LTD4 receptor antagonist). In fragmented guinea pig lung, WY-50,295 tromethamine inhibited antigen-induced LT release with an IC50 of 0.63 microM, and was 4.6- and 5.2-fold more potent than zileuton and MK-886, respectively. WY-50,295 tromethamine differed from these 5-lipoxygenase inhibitors, however, in that WY-50,295 tromethamine competitively antagonized LTD4-induced tracheal contractions (pA2 = 6.06) at concentrations that inhibited LT release. LY171883 was an effective LTD4 receptor antagonist (pA2 = 6.96), that only inhibited antigen-induced LT release at higher concentrations (IC50 = 7.9 microM). WY-50,295 tromethamine almost completely inhibited antigen-induced leukotriene-dependent tracheal contractions, whereas high concentrations of zileuton, MK-886, or LY171883 produced only partial inhibition. This partial inhibition was likely to result from 'breakthrough' 5-lipoxygenase activity, because combinations of zileuton plus MK-886 or zileuton plus LY171883, were more effective than zileuton, MK-886, or LY171883 alone. The greater efficacy of WY-50,295 tromethamine in the antigen-challenged guinea pig trachea is likely to result from its combined abilities to prevent LT biosynthesis and block LTD4 receptors.

Effects of rolipram and CI-930 on IL-2 mRNA transcription in human Jurkat cells.

Interleukin-2 (IL-2) is a major mediator of immunologic responses involved in many chronic inflammatory diseases. We have investigated the effects of rolipram, a PDE-IV inhibitor, and CI-930, a PDE-III inhibitor, on IL-2 gene expression in the Jurkat human T cell line. The immunosuppressant cyclosporin A (CsA) was included as a positive control. Jurkat cells were stimulated with 1 microgram/ml phytohemagglutinin (PHA) and 50 ng/ml phorbol 12-myristate, 13-acetate (PMA) for 6 h, and mRNA was analyzed using reverse transcription and polymerase chain reaction (RT/PCR). IL-2 transcription was greatly inhibited by 1 microM CsA, whereas neither 10 microM rolipram nor 10 microM CI-930 had any effect on steady-state levels of IL-2 mRNA. Therefore, PDE inhibitors do not affect synthesis of IL-2 mRNA in this model of activated T cells. This is of interest given that these agents inhibit the proliferation of primary T cells. For murine splenocytes stimulated by 2.5 micrograms/ml concanavalin A (Con A), rolipram had an IC50 of 0.09 microM and CI-930 an IC50 of 4.4 microM. These concentrations are below those at which IL-2 mRNA synthesis was shown to be unaffected. Therefore, the mechanism by which inhibitors of PDE-III and PDE-IV affect T cell proliferation is not likely to involve suppression of IL-2 mRNA transcription.

Differential regulation of TNF-alpha and IL-1beta production from endotoxin stimulated human monocytes by phosphodiesterase inhibitors.

The effect of selective PDE-I (vinpocetine), PDE-III (milrinone, CI-930), PDE-IV (rolipram, nitroquazone), and PDE-V (zaprinast) isozyme inhibitors on TNF-alpha and IL-1beta production from LPS stimulated human monocytes was investigated. The PDE-IV inhibitors caused a concentration dependent inhibition of TNF-alpha production, but only partially inhibited IL-1beta at high concentrations. High concentrations of the PDE-III inhibitors weakly inhibited TNF-alpha, but had no effect on IL-1beta production. PDE-V inhibition was associated with an augmentation of cytokine secretion. Studies with combinations of PDE isozyme inhibitors indicated that PDE-III and PDE-V inhibitors modulate rolipram's suppression of TNF production in an additive manner. These data confirm that TNF-alpha and IL-1beta production from LPS stimulated human monocytes are differentially regulated, and suggest that PDE-IV inhibitors have the potential to suppress TNF levels in man.

Bronchial vs. cardiovascular activities of selective phosphodiesterase inhibitors in the anesthetized beta-blocked dog.

This study was undertaken to determine whether isozyme-specific inhibitors of cAMP-selective phosphodiesterases (PDEs) induce bronchodilation without the cardiovascular side effects known to be produced by nonselective PDE inhibitors. The abilities of PDE inhibitors to reverse the bronchoconstriction induced by serotonin in a beta-blocked anesthetized dog were compared simultaneously with their effects on cardiac contractile force (+dP/dt), heart rate and blood pressure. Aminophylline and enprofylline, two antiasthma drugs with nonselective PDE inhibitory activity, produced dose-dependent (ED50S = 2.3 and 0.58 mg/kg, respectively) and complete (95-100%) bronchodilation accompanied by profound increases in cardiac force, tachycardia and decreases in blood pressure. Similar effects were observed with forskolin, a potent adenylate cyclase activator. Imazodan and CI-930 (inhibitors of the cGMP-inhibitable cAMP-selective PDE isozyme designated PDE-III) induced pulmonary and cardiovascular effects virtually identical to the nonselective PDE inhibitors, but with greater potency (ED50S = 0.02 and 0.04 mg/kg). In contrast, rolipram and Ro 20-1724 (inhibitors of a cGMP-insensitive cAMP-selective PDE isoform commonly designated PDE-IV) induced bronchodilation in the dog (ED50S = 0.007 and 0.63 mg/kg, respectively) without causing significant changes in cardiac force or heart rate, even after predosing dogs with forskolin. However, rolipram and Ro 20-1724 were less efficacious than the other inhibitors in that they induced only partial (50-60%) bronchodilation. The results suggest that canine respiratory muscle tension is regulated by both PDE-III and PDE-IV. Inhibitors of PDE-IV produce bronchodilation in the described model with minimal concomitant cardiac side effects.(ABSTRACT TRUNCATED AT 250 WORDS)

WY-50, 295 tromethamine: an orally active 5-lipoxygenase inhibitor with anti-allergic activity.

WY-50,295 tromethamine inhibited antigen-induced peptidoleukotriene (pLT) release from fragmented sensitized guinea pig lung (IC50 = 0.63 microM), antagonized LTD4-induced contractions of isolated guinea pig trachea (pA2 = 6.06), and suppressed antigen-induced contraction of sensitized guinea pig trachea over the 0.1-10 microM concentration range. In vivo, WY-50,295 tromethamine inhibited LTD4-induced bronchoconstriction (ED50 = 1.3 mg/kg i.v. and 6.6 mg/kg p.o.) and antigen-induced bronchoconstriction (ED50 = 2.5 mg/kg i.v. and 7.3 mg/kg p.o.) in anesthetized guinea pigs. Peak activity vs antigen was noted at 4-6 h after oral dosing and remained significant through 18 h. These studies demonstrate that WY-50,295 tromethamine possesses the complimentary actions of 5-LO inhibition and LTD4 receptor antagonism.

Evidence that prostaglandins can contract the rat aorta via a novel protein kinase C-dependent mechanism.

The effects of prostaglandin (PG) F2 alpha and PGB2 on isolated rat aortic strips were studied in calcium-free 1 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid buffer to explore the mechanisms of PG-induced smooth muscle contraction. In the absence of extracellular calcium, PGF2 alpha and PGB2 induced sustained contractions that were similar to those induced by activators of protein kinase C (PKC) or norepinephrine. These sustained contractions were apparently induced via a pharmacomechanical coupling mechanism because they could be elicited repeatedly in the absence of extracellular calcium and were not affected by changes in buffer concentrations of potassium, magnesium or phosphate. They depended on intracellular but not extracellular calcium because they were reversed by nitroprusside but not by nifedipine, verapamil or diltiazem. Contractions induced by combining either PG with 12-O-tetradecanoyl-phorbol-13-acetate or mezerein were consistent with the suggestion that PG-induced contractions were induced via a PKC-dependent mechanism. Moreover, both PGF2 alpha- and PGB2-induced contractions were inhibited noncompetitively by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine at concentrations known to inhibit PKC but not myosin light chain kinase. These data, combined with those published from other laboratories, suggest that PGF2 alpha and PGB2 induce contraction of the rat aorta, at least in part, by activating PKC. However, PG-induced contractions did not require enhanced phosphoinositide hydrolysis and apparently did not involve the mobilization of intracellular calcium by inositol 1,4,5-trisphosphate. The data thus suggest that PGs activate PKC via a novel mechanism.

Structure-activity relationships of 1H-indole-7-carboxamides as a novel series of selective alpha 1-adrenoceptor agonists.

The present isolated tissue study was designed to quantitate the alpha-adrenoceptor agonist activity of AY- 30,191 (5-(1-hydroxy-2-amino-ethyl)-1H-indole-7-carboxamide) and a series of related compounds. AY-30,191 induced contractions in the rabbit aorta, which were blocked by prazosin. In the rat vas deferens, while clonidine inhibited the electrically induced twitch response, AY-30,191 caused a prazosin-sensitive augmentation. In the dog saphenous vein, rauwolscine was less effective than the combination of rauwolscine and prazosin in inhibiting the contractions induced by AY-30,191. Pretreatment of the dog saphenous vein with phenoxybenzamine reduced the response to AY-30,191. The addition of rauwolscine to phenoxybenzamine-treated tissues had no effect on the contractions to AY-30,191 remaining after phenoxybenzamine treatment. These results suggest that AY-30,191 is a selective alpha 1-adrenoceptor agonist. Optimal alpha 1-adrenoceptor agonist activity in the 1H-indole-7-carboxamide series was seen in compounds in which a) the indole ring and the ethylamine side chain were intact; b) the indole nitrogen was unsubstituted; and c) the carboxamide was present at the 7-position in the indole ring. Removal of the carboxamide decreased alpha 1-adrenoceptor activity and, more importantly, resulted in a loss of alpha 1-adrenoceptor selectivity. Replacement of the carboxamide in the 7 position with methanesulfonamide resulted in a decrease in activity but a retention of alpha 1-adrenoceptor selectivity, whereas the dimethylamino analog was nonselective and the phosphoramidic acid diethylester analog was inactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the beta-adrenoceptor affinity and selectivity of cetamolol, atenolol, betaxolol, and ICI-118,551.

The objective of the present study was to compare the quantitative differences in the beta 1- vs. beta 2-adrenoceptor affinity and selectivity of cetamolol and its enantiomers to the reference compounds atenolol, betaxolol, and ICI-118,551, using isolated tissues obtained from the dog, guinea pig, and rat. Cetamolol antagonized the beta-adrenoceptor-mediated responses induced by isoproterenol, epinephrine, norepinephrine, and salbutamol, in tissues from both the dog and guinea pig, in a concentration-dependent manner. For a given tissue, the beta-adrenoceptor antagonist activity of cetamolol (measured as a pA2 or pKB value) was independent of the agonist used. In the dog tissues, cetamolol was more potent at inhibiting responses in the coronary artery (beta 1-adrenoceptors) than in the saphenous vein (beta 2-adrenoceptors). In the guinea pig tissues, the potency of cetamolol was approximately the same in the trachea (mixed beta 1- and beta 2-adrenoceptors) and atria (predominately beta 1-adrenoceptors), but lower in the soleus muscle (beta 2-adrenoceptors). Studies with the S-(-) and R-(+) enantiomers of cetamolol demonstrated that the S-(-) enantiomer was approximately 100-fold more potent at beta 1-adrenoceptors than the R-(+) enantiomer. In rat brain, cetamolol displaced [3H]-dihydroalprenolol bound to homogenates of cortex (beta 1-adrenoceptor binding sites) and cerebellum (beta 2-adrenoceptor binding sites). The potency of cetamolol at beta 1-adrenoceptors was found to be similar to that of betaxolol but greater than that of atenolol. However, the magnitude of the beta 1-adrenoceptor selectivity displayed by atenolol and betaxolol was greater than that displayed by cetamolol. In contrast, ICI-118,551 was found to possess potent and selective affinity for beta 2-adrenoceptors.

Intracellular mechanisms of contraction in the hypertensive and normotensive rat aortas.

The roles of intracellular mechanisms of contraction were compared in aortas from genetically hypertensive and normotensive rats by investigating the contractile activities of prostaglandin F2 alpha (PGF2 alpha) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in the absence of extracellular calcium. PGF2 alpha induced a greater amount of tension in aortic strips from hypertensive than normotensive rats. Furthermore, in hypertensive but not normotensive rat aortas, PGF2 alpha- and TPA-induced contractions were additive and were capable of sustaining a near maximum degree of muscle activation. Therefore, the intracellular contractile mechanisms in hypertensive vascular tissue may differ from those in normotensive tissue.

Interaction of neutrophils with vascular smooth muscle: identification of a neutrophil-derived relaxing factor.

Experiments were designed to study the interaction of rat peritoneal neutrophils with the vascular smooth muscle of the rat aorta. Rings of aorta, suspended in 10-ml organ chambers containing a physiologic salt solution, were precontracted with phenylephrine. Neutrophils (1 X 10(5) -4 X 10(7) cells/organ chamber) caused a cell number-dependent relaxation of the rat aorta that was augmented by superoxide dismutase (100 U/ml) or changing the oxygen content from 95 to 21%. The neutrophil-induced smooth muscle relaxation occurred in rings with and without endothelium and in rings precontracted with increasing concentrations of phenylephrine, prostaglandin F2 alpha or KCI. Catalase (1000 U/ml) and mannitol (1 X 10(-3) M) did not block the neutrophil-induced relaxation, whereas phenazine methosulfate (1 X 10(-5) M), hydroquinone (3 X 10(-5) M) and methylene blue (1 X 10(-5) M) reversed the neutrophil-induced relaxation. Pre-exposure of endothelium-rubbed rings to neutrophils (2 X 10(7) cells/organ chamber; 15 min) depressed the subsequent concentration-response curve to phenylephrine but augmented the relaxation induced by the phosphodiesterase inhibitor zaprinast (1 X 10(-5) M). The effluent from a column restraining the neutrophils induced a relaxation of endothelium-rubbed aortic rings that was prevented by methylene blue (1 X 10(-5) M). These results demonstrate that rat neutrophils release a factor that has a pharmacologic profile similar to that previously reported for the relaxing factor released from the vascular endothelium.

Co-regulation of tracheal tone by cyclic AMP- and cyclic GMP-dependent mechanisms.

The regulation of guinea pig tracheal muscle tone by cyclic AMP-dependent and cyclic GMP-dependent relaxant mechanisms was investigated by studying the tracheal relaxant activities of forskolin, nitroprusside, N6-2'-O-dibutyryl-cyclic AMP and 8-bromoguanosine-cyclic GMP. In carbachol (3 X 10(-6) M)-contracted isolated tracheal rings, N6-2'-O-dibutyryl-cyclic AMP and 8-bromoguanosine-cyclic GMP each caused biphasic relaxation responses, which consisted of an acute relaxation followed by a sustained but lesser degree of relaxation. The biphasic nature of this response is suggested to result from a functional counter-balancing of cyclic nucleotide-dependent relaxant mechanisms and the contractile mechanisms stimulated by carbachol. The sensitivity of carbachol-contracted tracheal rings to forskolin and nitroprusside (activators of adenylate and guanylate cyclase, respectively) was generally not influenced by N6-2'-O-dibutyryl-cyclic AMP or 8-bromoguanosine-cyclic GMP in concentrations that induced up to 50% relaxation of the trachea. Furthermore, the partial relaxation of tracheal tension with one cyclic nucleotide analog did not alter the sensitivity of the tracheal rings to the other. These results demonstrate that cyclic AMP- and cyclic GMP-dependent mechanisms induce relaxations of the trachea that are functionally additive, each neither potentiating nor depressing the effects of the other. In the presence of 3 X 10(-6) M carbachol, the effectiveness of cyclic AMP- and cyclic GMP-dependent relaxant mechanisms appears to be fixed, and independent of the amount of active tension being maintained by the tracheal muscle itself.

In vitro isolated tissue studies with atiprosin (AY-28,228): a new antihypertensive compound.

Using in vitro isolated tissue and binding studies we have defined a receptor activity profile for atiprosin. The most prominent action of the compound was significant alpha-adrenoceptor antagonist activity (pA2 = 8.11) that was less potent than prazosin (pA2 = 8.78) but more potent than either ketanserin (pA2 = 7.34) or indoramin (pA2 = 7.85). In addition, atiprosin was selective for alpha 1-adrenoceptors, since the compound was over 100-fold less potent as an antagonist at alpha 2-adrenoceptors (pA2 = 6.04). Atiprosin also displayed selective serotonin 5-HT2-receptor antagonist activity similar to that seen with ketanserin, but with a lesser potency (pA2 atiprosin, 6.87; ketanserin, 8.61). Although atiprosin also displayed significant histamine H1-antagonist activity (pA2 = 7.32), it was less potent as an H1-antagonist than as an alpha 1-adrenoceptor antagonist. In contrast, both indoramin and ketanserin displayed H1-antagonist activity (pA2 8.77 and 8.83, respectively) greater than their respective alpha 1-adrenoceptor antagonist activities. Atiprosin had little or no activity at either beta 1- or beta 2-adrenoceptors, muscarinic or histamine H2-receptors, nor any marked ability to produce smooth muscle relaxation either by a direct effect or via calcium entry blockade.