The effect of cyclic AMP and cyclic GMP phosphodiesterase inhibitors on the superoxide burst of guinea-pig peritoneal macrophages.

1. The cyclic nucleotide phosphodiesterase (PDE) activity of guinea-pig peritoneal macrophages was partially characterized and the effects of selective and non-selective inhibitors of adenosine 3':5'-cyclic monophosphate (cyclic AMP PDE) and guanosine 3':5'-cyclic monophosphate (cyclic GMP PDE) phosphodiesterases on superoxide generation were investigated using peritoneal macrophages from horse-serum pretreated guinea-pigs. 2. The non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX) and the PDE I/V selective inhibitor, zaprinast, inhibited spontaneous superoxide generation with IC50s of 30.7 +/- 11.3 microM and 145 +/- 17 microM respectively (n = 6 and 5). The concentration-response curves for the PDE IV selective inhibitors rolipram and Ro20-1724 were biphasic; mean maximum inhibitions were 56.9 +/- 5.9% and 66.8 +/- 10.5% respectively at 300 microM, but in 2 out of 6 (rolipram) and 2 out of 5 (Ro20-1724) experiments inhibition was < 50%. The PDE III inhibitor SK&F 94120 was without effect. Spontaneous superoxide generation was reduced 57 +/- 10% by 1 microM prostaglandin E2 (PGE2) and 62.6 +/- 3.76% by 1 microM salbutamol. 3. The increase in superoxide generation elicited by FMLP (10(-9)-10(-5)M) was unaffected by any of the PDE inhibitors studied. Inhibition of FMLP-stimulated superoxide generation by PGE2 was enhanced in the presence of 10 microM IBMX. 4. Macrophages were found to contain a predominantly membrane bound cyclic AMP PDE (90% of total activity) which was unaffected by cyclic GMP or calcium/calmodulin. The cyclic AMP PDE activity in the cytosolic fraction was enhanced in the presence of calcium/calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of a cardiotonic quinolinone derivative Y-20487 on the isoproterenol-induced positive inotropic action and cyclic AMP accumulation in rat ventricular myocardium: comparison with rolipram, Ro 20-1724, milrinone, and isobutylmethylxanthine.

The effect of a new cardiotonic agent Y-20487 [6-(3,6-dihydro-2-oxo-2H-1,3,4-thiadiazin-5-yl)-3,4-dihydro-2(1H)- quinolinone] on cyclic AMP levels of rat ventricular cardiomyocytes and the contractile force of papillary muscles was investigated in comparison with selective cyclic AMP phosphodiesterase (PDE) inhibitors, milrinone (PDE-III selective), rolipram and Ro 20-1724 (PDE-IV selective), and a nonselective inhibitor 3-isobutyl-1-methylxanthine (IBMX). Rolipram and Ro 20-1724 did not elicit cyclic AMP accumulation and positive inotropy, but they potentiated the isoproterenol (ISO)-induced cyclic AMP accumulation more effectively than IBMX. Rolipram was more effective than Ro 20-1724 in enhancing ISO-induced cyclic AMP accumulation but was less effective in enhancing the ISO-induced positive inotropic effect, indicating that these agents produce a differential action on cyclic AMP metabolism and inotropy. Milrinone and Y-20487 elicited cyclic AMP accumulation and positive inotropy by themselves. Whereas milrinone scarcely affected the ISO-induced effects, Y-20487 shifted the concentration-response curve for the positive inotropic effect of ISO to the left to the same extent that IBMX did. Y-20487, however, was much less effective than IBMX in enhancing the ISO-induced cyclic AMP accumulation. The present results indicate that in rat ventricular myocardium, PDE-IV may play a crucial role in breakdown of cyclic AMP generated by beta-adrenoceptor stimulation, whereas other types of PDE isoenzymes, including PDE-III, may be responsible for the cyclic AMP accumulation and direct positive inotropic effect induced by PDE inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

The identification of apparently novel cyclic AMP and cyclic GMP phosphodiesterase activities in guinea-pig tracheal smooth muscle.

Phosphodiesterase (PDE) activities that were capable of hydrolysing cyclic AMP (Km = 6.8 +/- 2 microM) and cyclic GMP (Km = 6.7 +/- 1.6 microM) were isolated from tracheal smooth muscle. These enzyme(s) activities were insensitive to stimulation by calcium/calmodulin and to inhibition by cyclic GMP, rolipram (type IV inhibitor) and siguazodan (type III inhibitor). Zaprinast was a relatively poor inhibitor of both cyclic AMP and cyclic GMP hydrolysis (IC50 = 46 +/- 9 microM and 45 +/- 14 microM respectively). These results suggest that tracheal smooth muscle may contain an apparently novel PDE. However, KCl (30 mM) which facilitates calcium entry in cells, depressed bradykinin-stimulated intracellular cyclic AMP formation, suggesting that the type I PDE may be functionally present. We suggest that considerable caution be exercised in identifying apparently novel PDE isoforms.     

Cardiotonic actions of selective phosphodiesterase inhibitors in rat isolated ventricular cardiomyocytes.

1. The contractile effects of the novel cardiotonic agent HN-10200 (2-[3-methoxy-5-methylsulphinyl-2-thienyl]-1H-imidazo-[4,5-c]-p yri dine hydrochloride), were examined and comparisons made with the responses obtained to a structurally similar compound, sulmazole, and to a number of other compounds which are known to inhibit phosphodiesterase (PDE) isoenzymes with differing selectivities; namely, enoximone (PDE III inhibitor), Ro 20-1724 (PDE IV inhibitor) and 3-isobutyl-1-methylxanthine (non-selective PDE inhibitor). 2. Contractile function, as measured by mechanical shortening, and biochemical systems involving cyclic AMP were investigated in ventricular cardiomyocytes isolated from adult Sprague-Dawley rats (200-250 g). 3. HN-10200 exerted a concentration-dependent (10(-8) M-10(-4) M) positive contractile effect, which was independent of alpha- or beta-adrenoceptor, or histamine receptor stimulation. 4. The efficacies of the contractile responses to the PDE inhibitors were of the order: HN-10200 > IBMX > sulmazole > enoximone and maximum stimulations, which were obtained at concentrations of 10(-4) M, were 54 +/- 4%, 41 +/- 7%, 38 +/- 7% and 26 +/- 5% (mean +/- s.e.) greater than basal levels, respectively (n = 6); the basal value of contractile amplitude (dL), in the absence of PDE inhibitors was 7.39 +/- 0.18% (mean +/- s.e.). Ro 20-1724 did not have any effect on contractile activity. 5. Due to low basal levels of cyclic nucleotides in isolated cells, accumulation of cyclic AMP due to the presence of the PDE inhibitors was detected only when the levels of cyclic nucleotide were enhanced with forskolin (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of theophylline and rolipram on leukotriene C4 (LTC4) synthesis and chemotaxis of human eosinophils from normal and atopic subjects.

1. The effects of the non-selective phosphodiesterase (PDE) inhibitor theophylline and the selective PDE4 inhibitor rolipram on leukotriene C4 (LTC4) synthesis and chemotaxis of complement 5a (C5a)- and platelet-activating factor (PAF)-stimulated human eosinophils obtained from normal and atopic donors were investigated. 2. Eosinophils were purified from peripheral venous blood of normal and atopic subjects by an immunomagnetic procedure to a purity > 99%. Eosinophils were stimulated with PAF (0.1 microM) or C5a 0.1 microM for 15 min and LTC4 was measured by radioimmunoassay (RIA). Eosinophil chemotaxis in response to PAF and C5a was assessed with 48-well microchambers (Boyden). 3. Under these conditions substantial amounts of LTC4 (about 300-1000 pg per 10(6) cells) were only detectable in the presence of indomethacin (0.1-10 microM). To explain this finding it was hypothesized that indomethacin reversed the inhibition of LTC4 synthesis by endogenously synthesized prostaglandins, in particular prostaglandin E2 (PGE2). In fact, eosinophils release 23 pg PGE2 per 10(6) cells following PAF stimulation; this PGE2 synthesis was completely inhibited by indomethacin and readdition of PGE2 inhibited eosinophil LTC4 synthesis (IC50 = 3 nM). The following experiments were performed in the presence of 10 microM indomethacin. 4. Theophylline (IC50 approximately 50 microM) and rolipram (IC50 approximately 0.03-0.2 microM) suppressed PAF- and C5a-stimulated LTC4 synthesis. This PDE inhibitor-induced suppression of LTC4 generation is mediated by activation of protein kinase A, since it was reversed by the protein kinase A inhibitor Rp-8-Br-cyclic AMPS. In addition, exogenous arachidonic acid concentration-dependently (0.3 microM-3 microM) reversed the inhibition of LTC4 synthesis by the PDE inhibitors, indicating that theophylline and rolipram suppress the mobilization of arachidonic acid. The beta 2-adrenoceptor agonist salbutamol inhibited eosinophil LTC4 synthesis (IC50 = 0.08 microM). The combination of salbutamol with theophylline (10 microM) or rolipram (3 nM) appeared to be additive. 5. Theophylline (IC50 approximately 40 microM), rolipram (IC50 approximately 0.02 microM [C5a], approximately 0.6 microM [PAF]) and PGE2 (IC50 approximately 3 nM) inhibited C5a- and PAF-stimulated eosinophil chemotaxis. The combination of PGE2 with theophylline resulted in an additive effect. 6. Both C5a- and PAF-stimulated eosinophil chemotaxis and LTC4 generation were significantly elevated in eosinophils from atopic individuals compared to normal subjects. However, eosinophils from normal and atopic individuals were not different with respect to their total cyclic AMP-PDE and PDE4 isoenzyme activities as well as the potencies of theophylline and rolipram to suppress LTC4 generation and chemotaxis.     

Phosphodiesterase PDE3 blunts the positive inotropic and cyclic AMP enhancing effects of CGP12177 but not of noradrenaline in rat ventricle

1.--The cardiostimulant effects of CGP12177, mediated through a beta(1)-adrenoceptor site with low affinity for (-)-propranolol, are potentiated by the nonselective PDE inhibitor IBMX but the role of PDE isoenzymes is unknown. We studied the effects of the PDE3-selective inhibitor cilostamide (300 nM) and PDE4-selective inhibitor rolipram (1 microM) on the positive inotropic and cyclic AMP-enhancing effects of CGP12177 and noradrenaline in right ventricular strips of rat. 2.--CGP12177 (under (-)-propranolol 200 nM) only increased contractile force in the presence of either cilostamide or rolipram with -logEC(50)M 6.7 (E(max)=23% over basal) and 7.1 (E(max)=50%) respectively. The combination of cilostamide and rolipram caused CGP12177 to enhance contractile force with -logEC(50)M=7.7 and E(max)=178%. 3.--The positive inotropic effects of noradrenaline (-logEC(50)M=6.9) were potentiated by rolipram (-logEC(50)M=7.4) but not by cilostamide (-logEC(50)M=7.0). 4.--In the presence of rolipram and (-)-propranolol, noradrenaline (2 microM) and CGP12177 (10 microM) produced matching inotropic effects but failed to increase cyclic AMP levels. 20 microM (-)-noradrenaline increased cyclic AMP levels, a response further enhanced by rolipram. 5.--Both PDE3 and PDE4 of rat ventricle appear to hydrolyse cyclic AMP generated through the low-affinity beta(1)-adrenoceptor site, thereby preventing inotropic responses of CGP12177. When (-)-noradrenaline interacts with the beta(1)-adrenoceptor, the generated cyclic AMP is hydrolysed only by PDE4, thereby reducing cardiostimulation.     

Endogenous expression of histamine H1 receptors functionally coupled to phosphoinositide hydrolysis in C6 glioma cells: regulation by cyclic AMP.

1. The effects of histamine receptor agonists and antagonists on phospholipid hydrolysis in rat-derived C6 glioma cells have been investigated. 2. Histamine H1 receptor-stimulation caused a concentration-dependent increase in the accumulation of total [3H]-inositol phosphates in cells prelabelled with [3H]-myo-inositol. The rank order of agonist potencies was histamine (EC50 = 24 microM) > N alpha-methylhistamine (EC50 = 31 microM) > 2-thiazolylethylamine (EC50 = 91 microM). 3. The response to 0.1 mM histamine was antagonized in a concentration-dependent manner by the H1-antagonists, mepyramine (apparent Kd = 1 nM) and (+)-chlorpheniramine (apparent Kd = 4 nM). In addition, (-)-chlorpheniramine was more than two orders of magnitude less potent than its (+)-stereoisomer. 4. Elevation of intracellular cyclic AMP accumulation with forskolin (10 microM, EC50 = 0.3 microM), isoprenaline (1 microM, EC50 = 4 nM) or rolipram (0.5 mM), significantly reduced the histamine-mediated (0.1 mM) inositol phosphate response by 37%, 43% and 26% respectively. In contrast, 1,9-dideoxyforskolin did not increase cyclic AMP accumulation and had no effect on the phosphoinositide response to histamine. 5. These data indicate the presence of functionally coupled, endogenous histamine H1 receptors in C6 glioma cells. Furthermore, the results also indicate that H1 receptor-mediated phospholipid hydrolysis is inhibited by the elevation of cyclic AMP levels in these cells.     

Relaxation of guinea-pig trachea by cyclic AMP phosphodiesterase inhibitors and their enhancement by sodium nitroprusside.

1. The effects of agents that elevate either cyclic AMP (the phosphodiesterase (PDE) III inhibitor siguazodan, salbutamol) or cyclic GMP (sodium nitroprusside (SNP)) on the relaxant activity of the PDE IV inhibitor, rolipram, were investigated in carbachol (0.1 microM) precontracted guinea-pig tracheal sheets. 2. Rolipram, siguazodan and SNP caused concentration-related reductions in tone of tissues precontracted with 0.1 microM carbachol (EC50 values 12.5; 2.73 and 0.35 microM respectively). Whilst the concentration-response relationship for the PDE III inhibitor, siguazodan, was monophasic that of the PDE IV inhibitor, rolipram, was biphasic. 3. The relaxant activity of rolipram was markedly enhanced in the presence of 10 microM siguazodan (EC50 < 0.01 microM), 0.1 microM salbutamol (EC50 0.03 microM) and 0.3 microM SNP (EC50 0.03 microM). In contrast, the relaxant activity of siguazodan was unaffected by SNP and only modestly enhanced by rolipram (10 microM) and salbutamol (0.1 microM). 4. The relaxant activity of SNP was enhanced by the PDE V inhibitor SK&F 96231 (30 microM: EC50 0.06 microM) and rolipram (30 microM, EC50 0.08 microM) but was unaffected by 30 microM siguazodan. 5. At concentrations up to 10 microM, neither siguazodan nor rolipram elevated tracheal cyclic AMP levels. However, the combination of 10 microM rolipram and siguazodan caused a two fold increase in the cyclic AMP content (from 2.19 to 4.36 pmol cyclic AMP mg-1 protein). SNP (0.1-10 microM) failed to produce a significant increase in tracheal cyclic AMP levels. At 0.1 microM the effect of SNP on tracheal cyclic AMP levels was significantly (P < 0.05) increased in the presence of rolipram but not siguadozan.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of guinea-pig eosinophil phosphodiesterase activity. Assessment of its involvement in regulating superoxide generation.

Experiments have been performed to characterize guinea-pig peritoneal eosinophil cyclic nucleotide phosphodiesterase (PDE) activity and establish whether it is involved in regulating superoxide (.O2-) generation. Eosinophils were found to contain a predominantly membrane-bound cAMP PDE(s) (92.5 +/- 2.4% of total activity) which was resistant to solubilization with Triton X-100 (1%). This particulate PDE exhibited complex kinetics (Km = 1.3 and 31.4 microM) and was unaffected by cGMP (IC50 greater than 100 microM) or CaCl2 (2 mM) + calmodulin (10 units/mL). Little cGMP PDE activity was detected in either the soluble or particulate fractions. Inhibitors of the Ro-20-1724-inhibited (Type IV) cAMP PDE, namely Ro-20-1724 (IC50 = 0.92 +/- 0.43 microM), rolipram (IC50 = 0.20 +/- 0.04 microM) and denbufylline (IC50 = 0.20 +/- 0.01 microM), potently inhibited the particulate cAMP PDE, as did the non-selective inhibitors trequinsin (IC50 = 0.11 +/- 0.02 microM) and AH-21-132 (IC50 = 2.57 +/- 0.02 microM). Eosinophil cAMP PDE was resistant to SK&F 94120 (IC50 greater than 1000 microM), the cGMP-inhibited (Type III) cAMP PDE inhibitor, and the cGMP PDE (Type I) inhibitor, zaprinast, was only weakly active (IC50 = 35.33 +/- 10.74 microM). .O2- release from resting cells was potently inhibited by rolipram (IC50 = 0.05 +/- 0.03 microM) and denbufylline (IC50 = 0.06 +/- 0.04 microM) but surprisingly, in view of its potent cAMP PDE inhibitory activity, was only weakly decreased by trequinsin (IC50 = 8.0 +/- 2.7 microM). AH-21-132 (IC50 greater than 10 microM), SK&F 94120 (IC50 greater than 10 microM) and zaprinast (IC50 greater than 10 microM) were without effect. Rolipram and denbufylline alone exerted little effect on cAMP in intact cells but, in the presence of 10 microM isoprenaline, potently increased intracellular accumulation (EC50 = 0.45 +/- 0.16 and 0.28 +/- 0.08 microM, respectively). Trequinsin and AH-21-132 only weakly enhanced isoprenaline-stimulated cAMP accumulation. Although it induced a marked rise in cAMP only in the presence of isoprenaline, rolipram (50 microM) alone was able to increase the activity ratio of cAMP-dependent protein kinase from 0.24 to 0.84. The results suggest that Ro-20-1724-inhibited cAMP PDE plays a role in regulating eosinophil .O2- generation. The poor correlation between the PDE inhibitory actions of certain compounds and their effectiveness in elevating cAMP and inhibiting .O2- suggests the existence of a barrier impeding access to the enzyme.     

Effects of SK&F 94120, an inhibitor of cyclic nucleotide phosphodiesterase type III, on human platelets

Elevation of cyclic AMP concentrations in platelets inhibits agonist-induced responses. Pharmacological interventions which could increase the levels of platelet cyclic AMP include activation of the synthesis of cyclic AMP or inhibition of its breakdown. In this study we have investigated the effects of SK&F 94120 on human platelet phosphodiesterase (PDE) activities separated by ion-exchange chromatography, and studied the effects of this agent on platelet responses caused by the agonists collagen, U44069 and ADP. Four PDE activities were identified from human platelet preparations. The PDE activities found comprised a cyclic GMP selective PDE, a Ca2+/calmodulin stimulated PDE, a cyclic GMP stimulated PDE and a "low Km" PDE activity called PDE III by analogy with activities described in other tissues. SK&F 94120 was found selectively to inhibit the "low Km" PDE III activity with an IC50 of 10.8 microM, which is consistent with the effects of this compound on cardiac ventricle PDE activities. Exposure of human platelets to SK&F 94120 produced concentration dependent increases in cyclic AMP, showing that inhibition of PDE III activity alone can cause an increase in the level of platelet cyclic AMP. SK&F 94120 also caused an inhibition of platelet responses to collagen, U44069 and ADP. However, SK&F 94120 was much less effective as an inhibitor of aggregation induced by ADP (IC50 greater than 100 microM) than by collagen (IC50 = 24.1 microM) or by U44069 (IC50 = 1.7 microM). Isobutylmethylxanthine (IBMX), a non-selective PDE inhibitor, was less effective than SK&F 94120 as an inhibitor of platelet responses for the same measured increase in cyclic AMP levels. M&B 22948 and rolipram, inhibitors of PDE I and PDE IV respectively, had no significant effect on platelet responses. These data suggest that selective inhibition of PDE III is the primary mechanism of action of SK&F 94120 as an inhibitor of agonist-induced platelet responses, and that increased cyclic AMP in the pool controlled by PDE III has important consequences on platelet responses. Moreover, these data suggest that some form of compartmentalization of cyclic AMP and/or PDE activity exists in human platelets.     

Inhibition of bronchospasm and ozone-induced airway hyperresponsiveness in the guinea-pig by CDP840, a novel phosphodiesterase type 4 inhibitor.

1. The activity of CDP840, a novel, potent and selective cyclic nucleotide phosphodiesterase type 4 (PDE 4) inhibitor, was evaluated in guinea-pig models (in vitro and in vivo) of bronchospasm, ozone-induced airway hyperresponsiveness (AHR) and non-cholinergic bronchoconstriction. Comparisons were made with (i) other PDE 4 inhibitors: CT1731 (S-enantiomer of CDP840), rolipram, RP73401 and (ii) the clinically used agents salbutamol and theophylline. 2. CDP840 relaxed isolated trachea, under basal tone (EC50 4.5 +/- 1.1 microM) being 17 fold less potent than rolipram (EC50 0.26 +/- 0.13 microM) but attaining the same Emax (83 +/- 6% of the response to 300 microM papaverine). 3. CDP840 relaxed tracheae pre-contracted with carbachol (IC25 39 +/- 9 microM) and histamine (IC25 4 +/- 1 microM) producing monophasic curves. Stereoselectivity was not observed with CT1731 against either carbachol (IC25 33 +/- 11 microM) or histamine (IC25 17 +/- 10 microM). Aminophylline was 1.6 fold (carbachol) and 11 fold (histamine) less potent than CDP840. Rolipram and RP73401 produced tri-phasic relaxation curves but were of similar potency (at the IC25 level) to CDP840 against carbachol (rolipram 18 +/- 5 microM, RP73401 39 +/- 1 microM) whereas against histamine they were approximately 20 fold more potent (rolipram 0.2 +/- 0.1 microM, RP73401 0.2 +/- 0.1 microM). In producing > 30% (carbachol) and > 60% (histamine) relaxation these inhibitors had similar potency and were poor compared to salbutamol. 4. Pre-incubation with CDP840 (10 microM) did not antagonize histamine-induced contraction of isolated trachea; however, it did cause a slight potentiation of the subsequent relaxation to salbutamol (IC50 23 +/- 1 to 15 +/- 2 nM). 5. Pretreatment (1 h) with either CDP840 (1 mg kg-1, i.p. or 3 mg kg-1, i.v.) or rolipram (1 mg kg-1, i.p.) did not bronchodilate or antagonize bronchospasm due to inhaled histamine in anaesthetized, ventilated guinea-pigs. Salbutamol (1 mg kg-1, i.p.) did not bronchodilate but caused a parallel 7 fold rightward shift in the histamine dose-response curve. 6. Stimulation of the vagus nerve in the presence of atropine resulted in a frequency-related bronchoconstriction. CDP840 and rolipram (i.v.) inhibited the response being approximately equipotent (EC50 approximately 10 micrograms kg-1). Neither drug inhibited bronchospasm to inhaled substance P. 7. CDP840 (1-10 micrograms kg-1 i.p.) dose relatedly inhibited ozone-induced bronchoconstriction. CT1731 (1 mg kg-1), rolipram (1 mg kg-1), RP73401 (10 micrograms kg-1) and aminophylline (10 mg kg-1) had no effect. Ozone-induced AHR to inhaled histamine was inhibited by CDP840 in a dose-related manner, 10 micrograms kg-1 abolishing the AHR. This effect was stereoselective as CT1731 was approximately 30 fold less potent than CDP840. Rolipram was approximately 100 fold less potent and RP73401 and aminophylline had no effect. CDP840 was orally active being approximately 10 fold less potent compared to i.p. administration. 8. CDP840 is a poor spasmolytic and anti-spasmogenic agent in response to exogenous mediators; however, it potently inhibits vagally mediated non-cholinergic bronchoconstriction and ozone-induced AHR to histamine. It is possible that regulation of cyclic AMP by PDE 4 contributes to neuronal sensitivity in the airways. Furthermore, CDP840 may suppress AHR without being an overt bronchodilator. Such a profile of activity may have therapeutic benefit in airways diseases such as asthma.     

Control of cyclic AMP levels in primary cultures of human tracheal smooth muscle cells.

1. [3H]-adenosine 3':5'-cyclic monophosphate ([3H]-cyclic AMP) responses were studied in primary cultures of human tracheal smooth muscle cells derived from explants of human trachealis muscle and in short term cultures of acutely dissociated trachealis cells. 2. Isoprenaline induced concentration-dependent [3H]-cyclic AMP formation with an EC50 of 0.2 microM. The response to 10 microM isoprenaline reached a maximum after 5-10 min stimulation and remained stable for periods of up to 1 h. After 10 min stimulation, 1 microM isoprenaline produced a 9.5 fold increase over basal [3H]-cyclic AMP levels. The response to isoprenaline was inhibited by ICI 118551 (10 nM), (apparent KA 1.9 x 10(9) M-1) indicating the probable involvement of a beta 2-adrenoceptor in this response in human cultured tracheal smooth muscle cells. However, with 50 nM ICI 118551 there was a reduction in the maximum response to isoprenaline. Prostaglandin E2 also produced concentration-dependent [3H]-cyclic AMP formation (EC50 0.7 microM, response to 1 microM PGE2 6.4 fold over basal). 3. Forskolin (1 nM - 100 microM) induced concentration-dependent [3H]-cyclic AMP formation in these cells. A 1.6 fold (over basal) response was also observed following stimulation with NaF (10 mM). 4. The nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) (0.1 mM) and the type IV, cyclic AMP selective, phosphodiesterase inhibitor rolipram (0.1 mM) both elevated basal [3H]-cyclic AMP levels by 1.8 and 1.5 fold respectively. IBMX (1-100 microM) and low concentrations of rolipram (< 10 microM), also potentiated the response to 1 microM isoprenaline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the PGE receptor subtype mediating inhibition of superoxide production in human neutrophils.

1. The aims of this study were to characterize the EP receptor subtype mediating the inhibition of superoxide anion generation by formyl methionyl leucine phenylalanine (FMLP)-stimulated human neutrophils, and to test the hypothesis that adenosine 3':5'-cyclic monophosphate (cyclic AMP) is the second messenger mediating the inhibition of the neutrophil by prostaglandin (PG)E2. 2. PGE2 (0.001-10 microM) inhibited FMLP (100 nM)-induced O2-generation from human peripheral blood neutrophils in a concentration-dependent manner, with an EC50 of 0.15 +/- 0.03 microM, and a maximum effect ranging from 36-84% (mean inhibition of 68.7 +/- 2.5%, n = 32). 3. The EP2-receptor agonists, misoprostol, 11-deoxy PGE1, AH13205 and butaprost, all at 10 microM, inhibited O2- generation, causing 95.5 +/- 2.9%, 56.8 +/- 5.2%, 37.1 +/- 6.6% and 18.9 +/- 4.4% inhibition respectively, the latter two being much less effective than PGE2. Similarly, the EP1-receptor agonist, 17-phenyl PGE2 (10 microM), and the EP3/EP1-receptor agonist, sulprostone (10 microM), also inhibited O2- generation, causing 32.2 +/- 7.0% and 15.3 +/- 3.4% inhibition respectively. 4. The non-selective phosphodiesterase inhibitor, isobutyl methylxanthine (IBMX, 0.25 mM) inhibited the FMLP response by 54.5 +/- 5.0%. In addition, IBMX shifted concentration-effect curves for PGE2, misoprostol, 11-deoxy PGE1, butaprost, and AH 13205 to the left, to give EC50s of 0.04 +/- 0.03 (n = 13), 0.07 +/- 0.03 (n = 4), 0.08 +/- 0.03 (n = 4), 0.33 +/- 0.13 (n = 4) and 0.41 +/- 0.2 microM (n = 3) respectively, allowing equieffective concentration-ratios (EECs, PGE2 = 1) of 11.5, 5.3, 50.7 and 12.7 to be calculated.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

1. Rat cultured aortic vascular smooth muscle cells (VSMC) express both cyclic GMP-inhibited cyclic AMP phosphodiesterase (PDE3) and Ro 20-1724-inhibited cyclic AMP phosphodiesterase (PDE4) activities. By utilizing either cilostamide, a PDE3-selective inhibitor, or Ro 20-1724, a PDE4-selective inhibitor, PDE3 and PDE4 activities were shown to account for 15% and 55% of total VSMC cyclic AMP phosphodiesterase (PDE) activity.
2. Treatment of VSMC with either forskolin or 8-bromo-cyclic AMP caused significant concentration- and time-dependent increases in total cellular cyclic AMP PDE activity. Using cilostamide or Ro 20-1724, we demonstrated that both PDE3 and PDE4 activities were increased following forskolin or 8-bromo-cyclic AMP treatment, with a relatively larger effect observed on PDE3 activity. The increase in cyclic AMP PDE activity induced by forskolin or 8-bromo-cyclic AMP was inhibited by actinomycin D or cycloheximide, demonstrating that new mRNA synthesis and protein synthesis were required. An analogue of forskolin which does not activate adenylyl cyclase (1,9-dideoxyforskolin) or an analogue of cyclic GMP (8-bromo-cyclic GMP) did not affect total cyclic AMP PDE activity.
3. Incubation of VSMC with 8-bromo-cyclic AMP for 16 h caused a marked rightward shift in the concentration-response curves for both isoprenaline- and forskolin-mediated activation of adenylyl cyclase. A role for up-regulated cyclic AMP PDE activity in this reduced potency is supported by our observation that cyclic AMP PDE inhibitors (IBMX, cilostamide or Ro 20-1724) partially normalized the effects of isoprenaline or forskolin in treated cells to those in untreated cells.
4. We conclude that VSMC cyclic AMP PDE activity is increased following long-term elevation of cyclic AMP and that increases in PDE3 and PDE4 activities account for more than 70% of this effect. Furthermore, we conclude that increases in cyclic AMP PDE activity contribute to the reduced potency of isoprenaline or forskolin in treated VSMC. These results have implications for long-term use of cyclic AMP PDE inhibitors as therapeutic agents.

     

Differential inhibition of human neutrophil functions. Role of cyclic AMP-specific, cyclic GMP-insensitive phosphodiesterase.

Multiple molecular forms of cyclic nucleotide phosphodiesterase have been characterized in various tissues and cells according to their substrate specificity, intracellular location, and calmodulin dependence. The purpose of this study was to evaluate the possible involvement of different molecular forms of phosphodiesterase in regulating the respiratory burst and lysosomal enzyme release responses of human neutrophils. Treatment with the selective cyclic AMP-specific, cyclic GMP-insensitive phosphodiesterase inhibitors Ro 20-1724 or rolipram, or the nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), resulted in inhibition of respiratory burst stimulated by the chemoattractants formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) (IC50 values: 0.71-17 microM) and complement fragment C5a (IC50 values: 61-93 microM), but did not inhibit phagocytosis-stimulated respiratory burst (less than 10% inhibition at 100 microM). Selective inhibitors of calmodulin-dependent phosphodiesterase (ICI 74,917), calmodulin-insensitive, cyclic GMP-specific phosphodiesterase (M & B 22,948), cyclic GMP-stimulated phosphodiesterase (AR-L 57), or cyclic AMP-specific, cyclic GMP-inhibited phosphodiesterase (amrinone and cilostamide) exhibited little or no inhibitory effect on FMLP- or phagocytosis-stimulated respiratory burst (0-42% inhibition at 100 microM). Regulation of neutrophil activation by phosphodiesterase was also response specific, as Ro 20-1724, rolipram and IBMX were less potent inhibitors of FMLP-induced lysosomal enzyme release (0-14% inhibition at 100 microM). Analysis of human neutrophil preparations confirmed the existence of a cyclic AMP-specific, cyclic GMP-insensitive phosphodiesterase, which was associated with the particulate fraction of the cell. These results demonstrate a role for the cyclic AMP-specific, cyclic GMP-insensitive phosphodiesterase in the regulation of human neutrophil functions, which appears to be both stimulus specific and response specific.     

Adenosine A2B-receptor-mediated cyclic AMP accumulation in primary rat astrocytes.

1. The effects of adenosine receptor agonists and antagonists on the accumulation of cyclic AMP have been investigated in primary cultures of rat astrocytes. 2. Adenosine A2-receptor stimulation caused a concentration-dependent increase in the accumulation of [3H]-cyclic AMP in cells prelabelled with [3H]-adenine. The rank order of agonist potencies was 5'-N-ethylcarboxamidoadenosine (NECA; EC50 = 1 microM) > adenosine (EC50 = 5 microM) > 2-chloroadenosine (EC50 = 20 microM) >> CGS 21680 (EC50 > 10 microM). The presence of 0.5 microM dipyridamole, an adenosine uptake blocker, had no effect on the potency of adenosine. 3. The response to 10 microM NECA was antagonized in a concentration-dependent manner by the non-selective adenosine receptor antagonists, xanthine amine congener (apparent KD = 12 nM), PD 115,199 (apparent KD = 134 nM) and 8-phenyltheophylline (apparent KD = 126 nM). However, the A1-receptor-selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine, had no significant effect on the responses to NECA or 2-chloroadenosine at concentrations up to 1 microM. 4. Stimulation of A1-receptors with the selective agonist, N6-cyclopentyladenosine, did not alter the basal accumulation of [3H]-cyclic AMP but inhibited a forskolin-mediated elevation of [3H]-cyclic AMP accumulation by a maximal value of 42%. This inhibition was fully reversed in the presence of 0.1 microM, 8-cyclopentyl-1,3-dipropylxanthine. 5. The time course for NECA-mediated [3H]-cyclic AMP accumulation was investigated. The results suggest that there is a substantial efflux of cyclic AMP from the cells in addition to the rapid and sustained elevation of intracellular cyclic AMP (5 fold over basal) which was also observed. 6. These data indicate that rat astrocytes in primary culture express an A2B-adenosine receptor coupled positively to adenylyl cyclase. Furthermore, the presence of A1-receptors negatively coupled to adenylyl cyclase appears to have no significant effect on the A2B-receptor-mediated cyclic AMP responses to NECA and 2-chloroadenosine.     

Modulation of carbachol-induced inositol phosphate formation in bovine tracheal smooth muscle by cyclic AMP phosphodiesterase inhibitors.

An investigation was made of a range of agents capable of elevating tissue cyclic AMP levels, or acting as a stable analogue of cyclic AMP, upon carbachol induced inositol phosphate responses in bovine tracheal smooth muscle slices. Whereas the beta 2 adrenoceptor agonist salbutamol (1 microM) and the membrane permeable analogue of cyclic AMP, 8-bromo-cyclic AMP (1 mM) were without effect upon total [3H]inositol phosphate formation induced by carbachol, 3-iso-butyl-1-methylaxanthine (IBMX) (EC50 140 microM), the high Km, cyclic AMP selective phosphodiesterase inhibitor rolipram (EC50 41 microM) and theophylline (EC50 76 microM) all inhibited the inositol phosphate response to low (1 microM) concentrations of carbachol. IBMX (IC50 13 microM), rolipram (IC50 4.6 microM) and theophylline (IC50 180 microM) all relaxed bovine tracheal muscle strips precontracted with methacholine (1 microM). The adenylate cyclase activator forskolin (1 microM), produced a much smaller (10% inhibition) effect upon inositol phosphate formation induced by carbachol. Carbachol (1 microM-1 mM) did not inhibit forskolin induced [3H]cyclic AMP formation. An inhibitor of the cyclic GMP preferring phosphodiesterase isozyme, M&B 22948 (1-100 microM), was without effect upon either carbachol induced inositol phosphate formation or trachealis tone. It is concluded that IBMX, rolipram and theophylline inhibit carbachol stimulated inositol phosphate formation, possibly through a cyclic AMP independent mechanism.     

Effects of selective phosphodiesterase inhibition on cyclic AMP hydrolysis in rat cerebral cortical slices.

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; 10 microM isoprenaline: 14.3 +/- 1.4 pmol mg-1 protein). Addition of a beta-adrenoceptor antagonist to isoprenaline-stimulated cerebral cortical slices caused a rapid decrease in cyclic AMP concentration to basal levels (t1/2: 58 +/- 18 s). 3. Preincubation of slices for 30 min with the phosphodiesterase inhibitors 1-methyl-3-isobutylxanthine, denbufylline, rolipram or Ro20,1724 caused concentration-dependent increases in basal and isoprenaline-stimulated cyclic AMP concentrations and decreased the rate of cyclic AMP hydrolysis measured after addition of a beta-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 Ro20,1724, and is present in cerebrum is of critical importance to the regulation of cyclic AMP concentration in this tissue.