Reduced airway hyperresponsiveness by phosphodiesterase 3 and 4 inhibitors in guinea-pigs

The aim of the present study was to compare the effects of selective phosphodiesterase (PDE) 3, 4 and 5 inhibitors on antigen-induced airway hyperresponsiveness in sensitized guinea-pigs. When the sensitized guinea-pigs were orally pre-treated with the selective PDE4 inhibitor, Ro 20-1724 (30 mg/kg), and studied 48h after OA, a significant reduction (P<0.01) of the leftward shift of the dose-response curve to ACh was noted, whereas it was ineffective at the lower dose (10 mg/kg). Administration of the selective PDE3 inhibitor, milrinone (30 mg/kg) also elicited a significant reduction (P<0.01) of the airway hyperresponsiveness, whereas the PDE5 inhibitor zaprinast (30 mg/kg) was ineffective. These results show that both PDE3 and PDE4 inhibitors are able to inhibit the antigen-induced airway hyperresponsiveness in sensitized guinea-pigs and support the potential utility of selective PDE inhibitors in the treatment of asthma.     

The identification of a new cyclic nucleotide phosphodiesterase activity in human and guinea-pig cardiac ventricle. Implications for the mechanism of action of selective phosphodiesterase inhibitors.

Four cyclic nucleotide phosphodiesterase (PDE) activities were separated from low-speed supernatants of homogenates of human cardiac ventricle by DEAE-Sepharose chromatography, and designated PDE I-PDE IV in order of elution with an increasing salt gradient. PDE I was a Ca2+/calmodulin-stimulated activity, and PDE II was an activity with a high Km for cyclic AMP which was stimulated by low concentrations of cyclic GMP. Human ventricle PDE III had Km values of 0.14 microM (cyclic AMP) and 4 microM (cyclic GMP), and showed simple Michaelis-Menten kinetics with both substrates. PDE IV is a previously unrecognized activity in cardiac muscle, the human enzyme having Km values of 2 microM (cyclic AMP) and 50 microM (cyclic GMP). PDE III and PDE IV were not activated by cyclic nucleotides or calmodulin. Four PDE activities were also isolated from guinea-pig ventricle, and had very similar kinetic properties. By gel filtration, the Mr of PDE III was 60,000, and that of PDE IV 45,000. The drug SK&F 94120 selectively and competitively inhibited PDE III with a Ki value of 0.8 microM (human), showing simple hyperbolic inhibition kinetics. Rolipram (Schering ZK 62711) and Ro 20-1724 (Roche), which have previously been reported to inhibit PDE III-like activities strongly, were shown to be weak inhibitors of human and guinea-pig PDE III enzymes (Ki values greater than 25 microM), but potent inhibitors of PDE IV [Ki values 2.4 microM (Rolipram) and 3.1 microM (Ro 20-1724) with human PDE IV]. The inhibition in all cases demonstrated simple hyperbolic competition. These observations suggest that the previously reported complex inhibition of PDE III-type activities from cardiac muscle was caused by incomplete separation of the PDE III from other enzymes, particularly PDE IV.     

Analgesic and anti-inflammatory effects of phosphodiesterase inhibitors

The aim of the present study was to investigate the role of phosphodiesterase (PDE) enzyme inhibitors namely rolipram and theophylline in pain and inflammation in experimental animals. Rolipram, a selective PDE IV inhibitor and theophylline a nonspecific PDE inhibitor exerted dose dependent analgesic and anti-inflammatory effect against acetic acid-induced writhing in mice and carrageenan-induced paw edema in rats, respectively. Nimesulide (1, 2 mg/kg) produced significant anti-inflammatory effect. Further, nimesulide (0.5 mg/kg) potentiated analgesic effect of rolipram but it failed to modulate the anti-inflammatory effect of PDE inhibitors. Present study suggests that PDE enzymes might be playing a role in nociceptive and inflammatory responses in animals.     

Preliminary identification and role of phosphodiesterase isozymes in human basophils.

We attempted to identify and establish the role of cyclic nucleotide phosphodiesterase (PDE) isozymes in human basophils by using standard biochemical techniques as well as describing the effects of isozyme-selective and nonselective inhibitors of PDE. The nonselective PDE inhibitors, theophylline and 3-isobutyl-1-methylxanthine, inhibited anti-IgE-induced release of histamine and leukotriene C4 (LTC4) from basophils. This inhibition was accompanied by elevations in cAMP levels. Rolipram, an inhibitor of the low Km cAMP-specific PDE (PDE IV), inhibited the release of both histamine and LTC4 from activated basophils and increased cAMP levels in these cells. In contrast, mediator release from basophils was not inhibited by either siguazodan or SK&F 95654, inhibitors of the cGMP-inhibited PDE (PDE III) or zaprinast, an inhibitor of the cGMP-specific PDE (PDE V). SK&F 95654 failed to elevate basophil cAMP in these experiments whereas zaprinast induced significant increases in cAMP content. The inhibitory effect of rolipram on mediator release was potentiated by siguazodan or SK&F 95654, but not by zaprinast. SK&F 95654 also enhanced the ability of rolipram to increase cAMP content. Forskolin, a direct activator of adenylate cyclase, inhibited IgE-dependent release of mediators from basophils and increased cAMP levels in these cells. These effects were enhanced by rolipram, but not by SK&F 95654 or zaprinast. The cell permeant analog of cAMP, dibutyryl cAMP, inhibited mediator release from these cells, a property not shared by either dibutyryl-cGMP or sodium nitroprusside, an activator of soluble guanylate cyclase. The presence of both PDE III and PDE IV was confirmed by partially purifying and characterizing PDE activity in broken cell preparations. Overall, these data lend support to the hypothesis that cAMP inhibits mediator release from basophils and suggest that the major PDE isozyme responsible for regulating cyclic AMP content in these cells is PDE IV, with a minor contribution from PDE III. However, the finding that zaprinast caused increases in cAMP without inhibiting mediator release indicates that cAMP accumulation is not invariably linked to an inhibition of basophil activation.     

Interactions of methylxanthines, nonxanthine phosphodiesterase inhibitors, and calcium with morphine in the guinea pig myenteric plexus.

In an earlier study, theophylline was shown to antagonize the morphine-induced inhibition of electrically induced contractions of the longitudinal muscle-myenteric plexus preparation from the guinea pig ileum. In the present study, acetylcholine (ACh) released from the myenteric plexus was measured directly using a radioenzymatic assay. Theophylline antagonized the morphine-induced inhibiton of ACh release. A similar antagonism was also observed with caffeine and 3-isobutyl-l-methylxanthine (IBMX). All three methylxanthines also increased ACh release. The nonxanthine phosphodiesterase (PDE) inhibitors 4-(3-butoxy-4-methoxy)-2-imidazolidinone (Ro 20-1724) and l-ethyl-4-isopropylidenehydrazino-1 H-pyrozolo(3,4-b)-pyridine-5-carboxylate, ethylester, HCl (SQ 20,009) generally did not antagonize the morphine-induced inhibiton of ACh release. The PDE inhibitor SQ 20,009 but not Ro 20-1724, enhanced the release of ACh. Both high calcium concentration and the divalent cation ionophore A23187 antagonized the inhibitory action of morphine on ACh release. These observations suggest that alteration in calcium fluxes rather than the inhibiton of PDE mediate the methylxanthine-induced antagonism of morphine in this preparation.     

Type III phosphodiesterase plays a necessary role in the growth-promoting actions of insulin, insulin-like growth factor-I, and Ha p21ras in Xenopus laevis oocytes.

Three phosphodiesterase (PDE) type III inhibitors were tested and found to inhibit Xenopus oocyte maturation induced by insulin with apparent IC50 values of 2.2 +/- 0.2 microM Cl-930, 25 +/- 3 microM imazodan (Cl-914), and 786 +/- 237 microM piroximone (MDL 19,205). The same rank order of potencies was observed for inhibition of insulin-like growth factor-I (IGF-I)-induced oocyte maturation, with IC50 values of 5.5 +/- 0.9 microM Cl-930, 54 +/- 4 microM imazodan, and 1190 +/- 395 microM piroximone. Oocyte maturation induced by microinjection of Ha p21ras was also inhibited by pretreatment of oocytes with Cl-930 or imazodan, with IC50 values of 4.3 +/- 1.2 and 59 +/- 4 microM, respectively. Progesterone-induced maturation was not affected by PDE III inhibitor action; and, neither type IV PDE inhibitors (Ro 20, 1724 or rolipram) nor dipyridamole (a type V PDE inhibitor) inhibited cell division induced by IGF-I or microinjected Ha p21ras. In addition, while insulin-stimulated oocyte PDE activity measured in vivo after microinjection of 200 microM [3H] cAMP was inhibited by nonselective and type III-specific drugs (with IC50 values of 4.2 +/- 1.8 microM Cl-930 and 26 +/- 6 microM imazodan), type IV and type V inhibitors did not inhibit hormone-stimulated enzyme activity. This pharmacological evidence demonstrates a necessary role for PDE III in insulin-, IGF-I-, and p21ras-induced meiotic cell division in Xenopus laevis oocytes.     

Effects of phosphodiesterase 3,4,5 inhibitors on hepatocyte cAMP levels,glycogenolysis, gluconeogenesis and suceptibility to a mitochondrial toxin

Various phosphodiesterase (PDE) 3,4 and 5 inhibitors have been compared with glucagon for their effectiveness at increasing hepatocyte cAMP, glycogenolysis and gluconeogenesis. Preincubation of isolated hepatocytes with PDE 3 and 4 inhibitors (50 M) for 2 h induced significant increases in cellular cAMP level. The order of effectiveness was: glucagon (78%), V11294A (42%), rolipram (40%), milrinone (36%), CDP-840 (33%), R₀ 20-1724 (31%), papaverine (27%), isobutylmethylxanthine (28%), isoliquiritigenin (25%), theophylline (22%), and amrinone (22%). The PDE 5 inhibitors dipyridamol and sildenafil had only a slight effect on cAMP levels. Glucose formation was increased as a result of increased glycogenolysis in the following order of effectiveness: glucagon (89%), V11294A (63%), rolipram (61%), milrinone (50%), CDP-840 (46%), R₀ 20-1724 (45%), sildenafil (34%), dipyridamol (31%), papaverine (30%), isobutylmethylxanthine (29%), theophylline (20%), amrinone (20%), and isoliquiritigenin (20%). Rolipram and milrinone, selective PDE 4 and PDE 3 inhibitors respectively, stimulated the gluconeogenesis of alanine, lactate + pyruvate, or fructose in hepatocytes isolated from fasted rats. On the other hand, selective cGMP specific phospodiesterase inhibitors, sildenafil and dipyridamol inhibited alanine-induced gluconeogenesis. All PDE inhibitors increased hepatocyte susceptibility to cyanide toxicity (3–4 fold) which was prevented by fructose whereas PDE 5 inhibitors did not significantly increase hepatocyte susceptibility.     

Coexpression of human cAMP-specific phosphodiesterase activity and high affinity rolipram binding in yeast.

Studies by various investigators have demonstrated that the low Km, cAMP-specific phosphodiesterase (PDE IV) is selectively inhibited by a group of compounds typified by rolipram and Ro 20-1724. In addition to inhibiting the catalytic activity of PDE IV, rolipram binds to a high affinity binding site present in brain homogenates. Although it has been assumed that the high affinity rolipram-binding site is PDE IV, no direct evidence has been produced to support this assumption. The present studies were undertaken to determine whether the rolipram-binding site is coexpressed with PDE IV catalytic activity in Saccharomyces cerevisiae genetically engineered to express human recombinant monocytic PDE IV (hPDE IV). Expressing hPDE IV cDNA in yeast resulted in a 20-fold increase in PDE activity that was evident within 1 h of induction and reached a maximum by 3-6 h. The recombinant protein represented hPDE IV as judged by its immunoreactivity, molecular mass (approximately 88 kDa), kinetic characteristics (cAMP Km = 3.1 microM; cGMP Km greater than 100 microM), sensitivity to rolipram (Ki = 0.06 microM), and insensitivity to siguazodan (PDE III inhibitor) and zaprinast (PDE V inhibitor). Saturable, high affinity [3H] (R)-rolipram-binding sites (Kd = 1.0 nM) were coexpressed with PDE activity, indicating that both binding activity and catalytic activity are properties of the same protein. A limited number of compounds were tested for their ability to inhibit hPDE IV catalytic activity and compete for [3H](R)-rolipram binding. Analysis of the data revealed little correlation (r2 = 0.35) in the structure-activity relationships for hPDE IV inhibition versus competition for [3H] (R)-rolipram binding. In fact, certain compounds (e.g. (R)-rolipram Ro 20-1724) possessed a 10-100-fold selectivity for inhibition of [3H] (R)-rolipram binding over hPDE IV inhibition, whereas others (e.g. dipyridamole, trequinsin) possessed a 10-fold selectivity for PDE inhibition. Thus, although the results of these studies demonstrate that hPDE IV activity and high affinity [3H](R)-rolipram binding are properties of the same protein, they do not provide clear cut evidence linking the binding site with the PDE inhibitory activity of rolipram and related compounds.     

The Therapeutic Profile of Rolipram,PDE Target and Mechanism of Action as a Neuroprotectant following Spinal Cord Injury

The extent of damage following spinal cord injury (SCI) can be reduced by various neuroprotective regimens that include maintaining levels of cyclic adenosine monophosphate (cyclic AMP), via administration of the phosphodiesterase 4 (PDE4) inhibitor Rolipram. The current study sought to determine the optimal neuroprotective dose, route and therapeutic window for Rolipram following contusive SCI in rat as well as its prominent PDE target and putative mechanism of protection. Rolipram or vehicle control (10% ethanol) was given subcutaneously (s.c.) daily for 2 wk post-injury (PI) after which the preservation of oligodendrocytes, neurons and central myelinated axons was stereologically assessed. Doses of 0.1 mg/kg to 1.0 mg/kg (given at 1 h PI) increased neuronal survival; 0.5 mg to 1.0 mg/kg protected oligodendrocytes and 1.0 mg/kg produced optimal preservation of central myelinated axons. Ethanol also demonstrated significant neuronal and oligo-protection; though the preservation provided was significantly less than Rolipram. Subsequent use of this optimal Rolipram dose, 1.0 mg/kg, via different routes (i.v., s.c. or oral, 1 h PI), demonstrated that i.v. administration produced the most significant and consistent cyto- and axo- protection, although all routes were effective. Examination of the therapeutic window for i.v. Rolipram (1.0 mg/kg), when initiated between 1 and 48 h after SCI, revealed maximal neuroprotection at 2 h post-SCI, although the protective efficacy of Rolipram could still be observed when administration was delayed for up to 48 h PI. Importantly, use of the optimal Rolipram regimen significantly improved locomotor function after SCI as measured by the BBB score. Lastly we show SCI-induced changes in PDE4A, B and D expression and phosphorylation as well as cytokine expression and immune cell infiltration. We demonstrate that Rolipram abrogates SCI-induced PDE4B1 and PDE4A5 production, PDE4A5 phosphorylation, MCP-1 expression and immune cell infiltration, while preventing post-injury reductions in IL-10. This work supports the use of Rolipram as an acute neuroprotectant following SCI and defines an optimal administration protocol and target for its therapeutic application.     

The pharmacodynamic action of the cyclic AMP phosphodiesterase inhibitor rolipram on prolactin producing rat pituitary adenoma (GH4C1) cells

Rolipram (4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone) represents a new class of specific low K_m cAMP phosphodiesterase (PDE) inhibitors. This compound enhances basal, hormone- and forskolin-elicited cAMP accumulation in prolactin (PRL) producing rat pituitary adenoma (GH₄C₁) cells in culture (ED₅₀=5·10^–8 M). This effect is due to a selective inhibition of the low K_m cAMP PDE (type III), since neither basal nor hormone-stimulated adenylate cyclase (AC) nor the Ca²⁺/calmodulin-dependent PDE were affected by rolipram. The drug enhanced vasoactive intestinal polypeptide (VIP)-stimulated PRL-secretion, while thyroliberin (TRH)- and 12-0-tetradecanoyl phorbol-13-acetate (TPA)-elicited PRL egress were slightly reduced indicating a cAMP-mediated reduction of protein kinase C (PK-C) mediated PRL release. Interestingly, inhibition of PRL secretion by somatostatin (SRIH) was completely suppressed suggesting cAMP-mediated inactivation of some GTP-binding protein(s) of the _i family (G _i2 orG _k). Rolipram did not affect phosphoinositide metabolism (i.e. IP₃ accumulation), neither acutely nor after long term administration. Rolipram, like the cAMP PDE inhibitor Ro 20–1724, did not influence AC and PDE I, but dose-dependently inhibited PDE III activity.Long term incubation of GH₄C₁ cells with rolipram in the presence of noradrenaline (NA) exerted a marginal decrease of -receptor number, AC activation and cAMP accumulation, while Ro 20–1724 brought about a marked down-regulation and desensitization of the AC complex.In summary, rolipram selectively interacts with PDE III in rat pituitary adenoma cells in culture and does not result in -adrenoceptor AC downregulation. These features are not shared by the other drugs tested.     

Differential inhibition of cyclic AMP and cyclic GMP hydrolysis in rat renal cortex.

Adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP) metabolism in rat renal cortex was examined. Athough the cyclic AMP and cyclic GMP phosphodiesterases are similarly distributed between the soluble and particulate fractions following differential centrifugation, their susceptibility to inhibition by theophylline, dl-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724), and 1-methyl-3-isobutylxanthine (MIX) are quite different. Ro 20-1724 selectively inhibited both renal cortical-soluble and particulate cyclic AMP degradation, but had little effect on cyclic GMP hydrolysis. Theophylline and MIX effectively inhibited degradation of both cyclic nucleotides, with MIX the more potent inhibitor. Effects of these agents on the cyclic AMP and cyclic GMP content of cortical slices corresponded to their relative potency in broken cell preparations. Thus, in cortical slices, Ro 20-1724 (2 mm) had the least effect on basal (without agonist), carbamylcholine, and NaN₃-stimulated cyclic GMP accumulation, but markedly increased basal and (parathyroid hormone) PTH-mediated cyclic AMP accumulation, MIX (2 mm) which was as effective as Ro 20-1724 in potentiating basal and PTH-stimulated increases in cyclic AMP also mediated the greatest augmentation of basal, carbamylcholine, and NaN₃-stimulated accumulation of cyclic GMP. By contrast, theophylline (10 mm) which was only 12% as effective as Ro 20-1724 in increasing the total slice cyclic AMP content in the presence of PTH was much more effective than Ro 20-1724 in potentiating carbamylcholine and NaN₃-mediated increases in cyclic GMP. These results demonstrate selective inhibition of cyclic nucleotide phosphodiesterase activities in the rat renal cortex and support the possibility of multiple cyclic nucleotide phosphodiesterases in this tissue. Furthermore, both cyclic nucleotides appear to be rapidly degraded in the renal cortex.     

Inhibitors of phosphodiesterase III block stimulation of Xenopus laevis oocyte ribosomal S6 kinase activity by insulin-like growth factor-I.

Insulin-like growth factor-I (IGF-I) stimulated Xenopus laevis oocyte ribosomal S6 kinase activity 5- to 10-fold, with an apparent EC50 of 0.8 +/- 0.1 nM after 90 min of hormone treatment. IGF-I-stimulated enzyme activity was inhibited by treatment of oocytes with nonselective phosphodiesterase (PDE) inhibitors, with apparent IC50 values of 2 +/- 1 microM papaverine, 20 +/- 2 microM isobutylmethylxanthine, and 128 +/- 16 microM theophylline. Type III PDE inhibitors also inhibited IGF-I-stimulated S6 kinase activity with IC50 values of 9.7 +/- 0.3 microM Cl-930 and 84 +/- 23 microM imazodan (Cl-914). These drugs apparently affected an intracellular molecular event leading to activation of S6 kinase, since Cl-930 prevented IGF-I-stimulation of S6 kinase, but had no direct inhibitory effect when added to the S6 kinase enzyme assay mixture. While hormone-stimulated S6 kinase activity was inhibited by isobutylmethylxanthine (nonselective PDE inhibitor) and Cl-930 (PDE III inhibitor), Ro 20, 1724 and rolipram (PDE IV inhibitors) and dipyridamole (PDE V inhibitor) had no significant effect on activated enzyme levels. The time course for IGF-I stimulation of oocyte S6 kinase displayed a small early peak of activity approximately 0.15-0.4 time required for 50% of cell population to display white spots (GVBD50) and a second major increase in activity at 0.6-0.7 GVBD50 that was sustained until meiotic maturation was complete. The second wave of enzyme activation was inhibited by Cl-930, but the early increase was not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic nucleotide-mediated regulation of vascular smooth muscle cell cyclic nucleotide phosphodiesterase activity

Cultured rat aortic vascular smooth muscle cells (VSMC) express both cGMP- inhibited cAMP phosphodiesterase (PDE-3) and Ro,20-1724-inhibited cAMP phosphodiesterase (PDE-4) activities. Utilizing a PDE-3-selective inhibitor (cilostamide) and a PDE-4-selective inhibitor (Ro,20-1724), PDE-3 and PDE-4 activities were shown to account for 15 and 55% of total VSMC cAMP phosphodiesterase (PDE) activity. Incubations of VSMC with either forskolin or 8-bromo-cAMP caused a concentration- and time-dependent increase in total cellular cAMP PDE activity. In these cells, both PDE-3 and PDE-4 activities were increased, with a relatively larger effect observed on PDE-3 activity. Similar incubations with an activator of soluble guanylyl cyclase (sodium nitroprusside) or with 8-bromo-cGMP did not increase cAMP PDE activity. cAMP-induced increases in cAMP PDE activity were inhibited with actinomycin D or cycloheximide, demonstrating that new mRNA and protein synthesis were required. We conclude that VSMC cAMP PDE activity is elevated following long-term elevation of cAMP, and that increases in PDE-3 and PDE-4 activities account for more than 70% of this increase. These results may have implications for long-term use of cAMP PDE inhibitors as therapeutic agents.     

Characterization of human, dog and rabbit corpus cavernosum type 5 phosphodiesterases

Human, dog and rabbit corpus cavernosum type 5 phosphodiesterases (PDE5) were isolated and their characteristics were compared. The three enzymes showed Km values of 0.8, 2.1 and 2.3 uM, respectively. They exhibited similar pH-dependence with optimal pH being 7.5. They required Mg++ for activity and the activity was suppressed by high concentrations of Zn++ (0.1-1 mM). Sildenafil potently inhibited the three enzymes with IC50 values of 3.6, 1.7 and 3.0 nM, respectively. Dipyridamole and IBMX (3-isobutyl-1-methylxanthine) each also inhibited the three enzymes with similar, albeit lower, potencies (IC50 about 1.1 and 5.7 uM, respectively). However, zaprinast exhibited a significantly higher potency against the rabbit enzyme (IC50 53 nM) than against the human and dog PDE5s (IC5s 332 and 217 nM, respectively). Thus, the corpus cavernosum PDE5s are very similar among the various species with the only significant difference being their sensitivity to zaprinast. Human platelet PDE5 was also characterized by comparison with the corpus cavernosum enzyme. The platelet enzyme exhibited a Km, pH-, Mg++- and Zn++-dependence, and sensitivity to sildenafil and zaprinast very similar to those of the corpus cavernosum PDE5. However, compared with corpus cavernosum PDE5, the platelet enzyme exhibited higher sensitivity to dipyridamole and IBMX (IC50 0.46 and 1.8 uM, respectively). This study shows that despite similar kinetics and enzymatic properties, corpus cavernosum PDE5s from different species, and corpus cavernosum and platelet PDE5s, can have differential sensitivity to pharmacological inhibitors.     

Differential effects of Ro 20-1724 and isobutylmethylxanthine on the basal force of contraction and beta-adrenoceptor-mediated response in the rat ventricular myocardium.

Effects of Ro 20-1724, a selective inhibitor of soluble cGMP-insensitive type IV phosphodiesterase, on the force and cAMP levels were compared with those of 3-isobutyl-1-methylaxanthine, a non-selective inhibitor, in the rat ventricular myocardium. Ro 20-1724 scarcely affected the basal force of contraction and cAMP levels, whereas it enhanced the positive intropic effect and cAMP accumulation induced by isoproterenol more effectively than 3-isobutyl-1-methylxanthine. These results imply that inhibition of the soluble cGMP-insensitive type IV PDE by Ro 20-1724 may be crucially involved in the regulation of myocardial contractility through the interaction with cAMP generation in the rat ventricular myocardium.     

Differential actions of phosphodiesterase inhibitors on secretin- and vasoactive intestinal peptide-induced increases in chief cell cAMP

The actions of three different phosphodiesterase inhibitors, theophylline, 3-isobutyl-1-methylxanthine (IBMX) and Ro 20-1724 (Ro), on cellular cAMP and pepsinogen secretion from dispersed chief cells prepared from guinea pig stomach were examined. The relative order of potency for increasing cAMP and pepsinogen secretion was Ro greater than IBMX greater than theophylline. Ro, the most efficacious agent, caused a 17-fold increase in basal cAMP and a similar augmentation of the increase in cAMP caused by secretin or vasoactive intestinal peptide (VIP). Differential actions of these agents on the dose-response curves for secretin- and VIP-induced increases in cAMP suggest that chief cell receptors for these peptides are coupled to pools of cAMP that are acted upon by heterogeneous phosphodiesterases with varying sensitivities to inhibitors. Moreover, Ro, a selective inhibitor of low Km cAMP-specific phosphodiesterases, is the most potent and efficacious agent tested in this cell system.     

Evaluation of the pentose phosphate pathway from 14CO2 data. Fallibility of a classic equation when applied to non-homogeneous tissues.

Two cyclic nucleotide phosphodiesterase (PDE) activities were identified in pig aortic endothelial cells, a cyclic GMP-stimulated PDE and a cyclic AMP PDE. Cyclic GMP-stimulated PDE had Km values of 367 microM for cyclic AMP and 24 microM for cyclic GMP, and low concentrations (1 microM) of cyclic GMP increased the affinity of the enzyme for cyclic AMP (Km = 13 microM) without changing the Vmax. This isoenzyme was inhibited by trequinsin [IC50 (concn. giving 50% inhibition of substrate hydrolysis) = 0.6 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 0.6 microM for cyclic GMP hydrolysis] and dipyridamole (IC50 = 5 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 3 microM for cyclic GMP hydrolysis). Cyclic AMP PDE exhibited a Km of 2 microM for cyclic AMP and did not hydrolyse cyclic GMP. This activity was inhibited by trequinsin (IC50 = 0.2 microM), dipyridamole (IC50 = 6 microM) and, selectively, by rolipram (IC50 = 3 microM). Inhibitors of cyclic GMP PDE (M&B 22948) and of low Km (Type III) cyclic AMP PDE (SK&F 94120) only weakly inhibited the two endothelial PDEs. Incubation of intact cells with trequinsin and dipyridamole induced large increases in cyclic GMP, which were completely blocked by LY-83583. Rolipram, SK&F 94120 and M&B 22948 did not significantly influence cyclic GMP accumulation. Dipyridamole enhanced the increase in cyclic GMP induced by sodium nitroprusside. Cyclic AMP accumulation was stimulated by dipyridamole and trequinsin with and without forskolin. Rolipram, although without effect alone, increased cyclic AMP in the presence of forskolin, whereas M&B 22948 and SK&F 94120 had no effects on resting or forskolin-stimulated levels. These results suggest that cyclic GMP-stimulated PDE regulates cyclic GMP levels and that both endothelial PDE isoenzymes contribute to the control of cyclic AMP.     

Tyrosine kinase-independent inhibition of cyclic-AMP phosphodiesterase by genistein and tyrphostin 51.

The phosphodiesterase activity in the HT4.7 neural cell line was pharmacologically characterized, and phosphodiesterase isozyme 4 (PDE4) was found to be the predominant isozyme. The Km for cAMP was 1-2 microM, indicative of a "low Km" phosphodiesterase, and the activity was inhibited by PDE4-selective inhibitors rolipram and Ro20-1724, but not PDE3- or PDE2-selective inhibitors. Calcium, calmodulin, and cGMP, regulators of PDE1, PDE2, and PDE3, had no effect on cAMP hydrolysis. The protein tyrosine kinase inhibitor, genistein, inhibited HT4.7 cAMP phosphodiesterase activity by 85-95% with an IC50 of 4 microM; whereas daidzein, an inactive structural analog of genistein, had little effect on phosphodiesterase activity. This is a common pharmacological criterion used to implicate the regulation by a tyrosine kinase. However, genistein still inhibited phosphodiesterase activity with a mixed pattern of inhibition even when ion-exchange chromatography was used to partially purify phosphodiesterase away from the tyrosine kinase activity. Moreover, tyrphostin 51, another tyrosine kinase inhibitor, was found to also inhibit partially purified phosphodiesterase activity noncompetitively. These data suggest that HT4.7 phosphodiesterase activity is dominated by PDE4 and can be regulated by genistein and tyrphostin 51 by a tyrosine kinase-independent mechanism.     

Interactions of formylmethionyl-leucyl-phenylalanine, adenosine, and phosphodiesterase inhibitors in human monocytes. Effects on superoxide release, inositol phosphates and cAMP

Cessation of the fMLF-induced burst of human monocyte superoxide release was associated with a rise in cAMP. This was not due to inhibition of phosphodiesterase (PDE), the major form of which was the PDE IV isozyme. The action of burst inhibitors did not correlate with cAMP levels: Rolipram, a PDe IV inhibitor, increased cAMP 6-fold, with minimal effects on the burst; whereas theophylline increased cAMP less than 2-fold but decreased the burst to less than half. Although theophylline and the adenylate cyclase activator, adenosine, inhibited fMLF-induced superoxide release, they did not inhibit production of inositol phosphates. Thus, these studies on inhibition of superoxide release implicated neither cAMP nor inositol phosphates.     

NT-702 (parogrelil hydrochloride, NM-702), a novel and potent phosphodiesterase inhibitor, improves reduced walking distance and lowered hindlimb plantar surface temperature in a rat experimental intermittent claudication model

NT-702 (parogrelil hydrochloride, NM-702), 4-bromo-6-[3-(4-chlorophenyl)propoxy]-5-[(pyridin-3-ylmethyl)amino]pyridazin-3(2H)-one hydrochloride, a novel phosphodiesterase (PDE) inhibitor synthesized as a potent vasodilatory and antiplatelet agent, is being developed for the treatment of intermittent claudication (IC) in patients with peripheral arterial disease. We assessed the efficacy of NT-702 in an experimental IC model as compared with cilostazol and additionally investigated the pharmacological property in vitro and ex vivo. NT-702 selectively inhibited PDE3 (IC(50)=0.179 and 0.260 nM for PDE3A and 3B) more potently than cilostazol (IC(50)=231 and 237 nM for PDE3A and 3B) among recombinant human PDE1 to PDE6. NT-702 inhibited in vitro human platelet aggregation induced by various agonists (IC(50)=11 to 67 nM) and phenylephrine-induced rat aortic contraction (IC(50)=24 nM). Corresponding results for cilostazol were 4.1 to 17 microM and 1.0 microM, respectively. NT-702 (3 mg/kg or more) significantly inhibited ex vivo rat platelet aggregation after a single oral dose. For cilostazol, 300 mg/kg was effective. In a rat femoral artery ligation model, NT-702 at 5 and 10 mg/kg repeated oral doses twice a day (BID) for 13 days significantly improved the reduced walking distance while the lowered plantar surface temperature was improved at 2.5 mg/kg and more. Cilostazol also improved the walking distance and surface temperature at 300 mg/kg BID but significant difference was only observed for surface temperature on day 8. These results suggest that NT-702 can be expected to have therapeutic advantage for IC.