Role of cyclic nucleotide phosphodiesterase isozymes in intact canine trachealis.

The role of individual cyclic nucleotide phosphodiesterase (PDE) isozymes in regulating cAMP and cGMP content in intact canine trachealis was examined using isozyme-selective and nonselective PDE inhibitors. The inhibitors used in this study were characterized previously [Mol. Pharmacol. 37:206-214 (1990)] and included: 1) zaprinast, an inhibitor (Ki = 0.1 microM) of the cGMP-specific PDE (cAMP Km = 135 microM; cGMP Km = 4 microM); 2) SK&F 94120, an inhibitor (Ki = 7 microM) of the cGMP-inhibited PDE (cAMP Km = 0.3 microM; cGMP Km = 8 microM); 3) Ro 20-1724, an inhibitor (Ki = 5 microM) of the cAMP-specific PDE (cAMP Km = 4 microM; cGMP Km = 40 microM); and 4) 3-isobutyl-1-methylxanthine (IBMX), a nonselective PDE inhibitor (IC50 = 1-30 microM). In addition to the aforementioned isozymes, canine trachealis contains a Ca2+/calmodulin-stimulated PDE (cAMP Km = 1 microM; cGMP Km = 2 microM) and a GMP-stimulated PDE (cAMP Km = 93 microM; cGMP Km = 60 microM), for which selective inhibitors are not available. Isolated canine trachealis strips were contracted with methacholine and exposed to various concentrations of PDE inhibitors, before being relaxed by the cumulative addition of isoproterenol, an adenylate cyclase activator, or sodium nitroprusside, a guanylate cyclase activator. At the completion of the concentration-response studies, tissues were flash-frozen and assayed for cyclic nucleotide content. Neither isoproterenol-induced relaxation nor cAMP accumulation was altered by zaprinast, but both of these responses were potentiated by pretreatment of tissues with either SK&F 94120 or Ro 20-1724. The effects of SK&F 94120 and Ro 20-1724 were additive, and the combination of SK&F 94120, Ro-1724, and IBMX had no greater effect on the responses to isoproperenol than did either IBMX alone or the combination of SK&F 94120 plus Ro 20-1724. In contrast, zaprinast potentiated sodium nitroprusside-induced relaxation and cGMP accumulation, whereas neither SK&F 94120 nor Ro 20-1724 altered these responses. IBMX produced a greater potentiation than did zaprinast, and the combination of zaprinast and IBMX had a greater effect than either agent alone. The results of this study suggest that the cGMP-inhibited and cAMP-specific PDEs are responsible for cAMP hydrolysis in intact canine trachealis, whereas cGMP hydrolysis is mediated by the cGMP-specific PDE as well as the Ca2+/calmodulin-stimulated PDE and/or the cGMP-stimulated PDE.     

The effects of theophylline and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (ro 20-1724) on protein secretion from rat parotid gland

1 The effects of two chemically distinct cyclic nucleotide phosphodiesterase (PDE) inhibitors on protein secretion from superfused rat parotid gland were studied.2 In the presence of 1.0 mM Ca(2+), Ro 20-1724 (10 muM), an imidazolidinone derivative, increased the secretory response to isoprenaline 100% and the isoprenaline-dependent accumulation of adenosine cyclic 3',5'-monophosphate (cyclic AMP) 300-400%. At this concentration Ro 20-1724 alone did not cause protein secretion, accumulation of cyclic AMP or significantly inhibit PDE activity in cell-free preparations from parotid gland.3 In the absence of added Ca(2+) and in the presence of 1.0 mM EGTA, Ro 20-1724 inhibited the secretory response to isoprenaline 65% while increasing isoprenaline-dependent cyclic AMP accumulation 200%.4 In the presence of Ca(2+), theophylline (10 mM) stimulated protein secretion but did not cause the accumulation of cyclic AMP. When combined with isoprenaline the rate of secretion was greater than the sum of the effects of the individual drugs but there was no effect of theophylline on the isoprenaline-dependent accumulation of cyclic AMP.5 Theophylline-stimulated protein secretion is increased by omitting Ca(2+) from the superfusion medium without any detectable change in cyclic AMP accumulation. Under these conditions Ro 20-1724 inhibits theophylline-stimulated protein secretion and the maximum rate of protein secretion in the presence of isoprenaline and theophylline is no greater than that seen with either agent alone.6 It is concluded that the theophylline effects do not result from inhibition of PDE. It is suggested that the primary action of both drugs on parotid gland acinar cells is to alter the distribution of intracellular Ca(2+). Ro 20-1724 may also inhibit Ca(2+)/calmodulin activated enzymes such as PDE.     

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.

     

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.     

The phosphodiesterase 3 inhibitor cilostamide enhances inotropic responses to glucagon but not to dobutamine in rat ventricular myocardium

The effects of phosphodiesterase (PDE) inhibitors (1-3) on tissue cAMP concentrations and the inotropic responses to dobutamine and glucagon were investigated in electrically driven right ventricular strips of the rat heart. Dobutamine (0.3-100 microM) produced a concentration-dependent positive inotropic effect which was not affected by 50 nM (+/-)-1-(2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy)-3-((1-methylethyl)amino)-2-butanol hydrochloride (ICI 118551), a beta2-receptor antagonist, but was virtually abolished by 0.3 microM (+/-)-2-hydroxy-5-(2-((2-hydroxy-3-(4-(1-methyl-4-(trifluoromethyl)-1H-imidazol-2-l)phenoxy)propyl)amino)ethoxy)-benzamide methanesulfonate (CGP 20712A), a beta1-receptor antagonist. Glucagon (0.01-1 microM) also enhanced the contractility of the preparation in a concentration-dependent way. Selective inhibitors of PDE 1 8-methoxymethyl-3-isobutyl-1-methylxantine (MIMX, 1 muM), PDE 2 erythro-9-[2-hydroxy-3-nonyl]adenine (EHNA, 1 microM) and PDE 3 cilostamide (0.1 microM) did not affect basal contractility. Cilostamide increased the positive inotropic effects of glucagon but not those of dobutamine. MIMX and EHNA did not alter the effects of either dobutamine or glucagon. Dobutamine (3 microM), but not glucagon (0.1 microM), increased tissue levels of cAMP. 1 microM of MIMX or EHNA were devoid of effects and failed to alter the effects of dobutamine and glucagon on cAMP. Cilostamide (0.1 microM) did not increase the effects of dobutamine but caused glucagon to enhance cAMP. The pharmacological and biochemical data presented in this study can be explained quantitatively by a cell compartment model in which PDE 3 appears to be colocalized with the contractile machinery responsible for the effects of glucagon but not those of dobutamine. Neither PDE 1 nor PDE 2 appears to regulate the inotropic effects of dobutamine and glucagon in rat ventricular myocardium.     

Inhibition of pig aortic smooth muscle cell DNA synthesis by selective type III and type IV cyclic AMP phosphodiesterase inhibitors.

Foetal calf serum (FCS) and platelet-derived growth factor (PDGF)-stimulated incorporation of [3H]thymidine into pig aortic smooth muscle cell (ASMC) DNA was decreased by agents that either stimulated the synthesis (forskolin) or inhibited the breakdown (3-isobutyl-1-methylxanthine, IBMX) of cAMP. FCS-stimulated incorporation of [3H]thymidine into DNA was also reduced by selective inhibitors of cAMP-specific phosphodiesterase (PDE IV) (Ro-20-1724, rolipram) and cGMP-inhibited cAMP PDE (PDE III) (SK&F 94836). IBMX, Ro-20-1724, rolipram and SK&F 94836 enhanced forskolin inhibition of DNA synthesis. Alone, rolipram was a relatively weak inhibitor of FCS-induced ASMC DNA synthesis (IC25 greater than 20 microM); however, in the presence of a threshold concentration of SK&F 94836 (20 microM), the potency of rolipram increased (IC25 = 4 microM), suggesting synergy in the actions of PDE III and PDE IV inhibitors. SK&F 94836 and rolipram elicited 30% and 37%, respectively, reductions in FCS-induced ASMC proliferation and potentiated the inhibitory actions of forskolin. PDE III and PDE IV inhibitors alone, exerted minimal effects on ASMC cAMP levels after a short term (10 min) or long-term (2 or 24 hr) exposure, but enhanced forskolin-induced accumulation of cAMP. ASMC spontaneously released cAMP into the extracellular medium, a process that was increased by forskolin. PDE III and PDE IV inhibitors had no effect alone on cAMP extrusion but enhanced the effect of forskolin. Exposure of ASMC to forskolin or SK&F 94836 for 15 min increased the activity ratio (AR) of cAMP-dependent protein kinase from 0.05 to 0.17 and 0.23, respectively. Ro-20-1724, alone, did not affect cAMP-dependent protein kinase but enhanced the stimulatory effect of forskolin (AR = 0.37) and SK&F 94836 (AR = 0.27). Agents that increased cGMP synthesis (glycerol trinitrate, atrial natriuretic factor) or decreased its hydrolysis by selectively inhibiting cGMP-specific PDE (PDE V) (zaprinast) exerted no effects on FCS- or PDGF-stimulated [3H]thymidine incorporation into DNA either alone or in combination. The cytosolic fraction of pig ASMC contained four cyclic nucleotide PDEs which were categorized as PDE V, Ca2+/calmodulin-stimulated PDE (PDE I), PDE III and PDE IV. PDE I and III activities were also associated with the particulate fraction. The results demonstrate that inhibitors of PDEs III and IV alone or in combination with forskolin, reduce ASMC DNA synthesis and proliferation, through an action likely to involve elevation of intracellular cAMP. In contrast, inhibition of cGMP hydrolysing PDE subtypes (I and V) exerted no effect on DNA synthesis in this cell type.     

Subtype-specific roles of cAMP phosphodiesterases in regulation of atrial natriuretic peptide release

cAMP is known to control the release of atrial natriuretic peptide. To define the roles of cyclic nucleotide phosphodiesterase subtypes in the regulation of atrial natriuretic peptide (ANP) release, experiments were done with perfused beating rabbit atria. Phosphodiesterase 3 subtype-specific inhibitors, milrinone and cilostamide, inhibited myocytic ANP release with a concomitant increase in cAMP efflux. Similarly, trequinsin, another phosphodiesterase 3 inhibitor, decreased ANP release. A phosphodiesterase 4 subtype-specific inhibitor, rolipram, did not significantly change ANP release but increased AMP efflux. Also, 4-[(3-butoxy-4-methoxyphenyl)methyl]-2-imidazolidinone (Ro 20-1724), another phosphodiesterase 4 inhibitor, did not significantly change ANP release. The cAMP efflux was higher in the atrium treated with rolipram than in the atrium treated with milrinone or cilostamide. The data show that the cAMP pool, which is metabolized by phosphodiesterase 3, but not phosphodiesterase 4, is closely related to the basal regulation of atrial ANP release. The results suggest that intracellular cAMP is compartmentalized in the regulation of atrial ANP release, and that the release is controlled by a phosphodiesterase subtype-specific mechanism.     

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)     

Regulation of adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in UMR-106 osteoblast-like cells: role of cAMP-phosphodiesterase and cAMP efflux.

The present study aimed to define the role of adenosine 3',5'-cyclic monophosphate (cAMP)-phosphodiesterase (PDE) activity and the possible involvement of cAMP efflux on parathyroid hormone (PTH)-stimulated intracellular cAMP accumulation in cultured osteoblast-like UMR-106 cells. Treatment of the cells with 10 nM PTH (1-84) rapidly increased the level of intracellular cAMP. PTH stimulation also increased the cAMP efflux rate. The efflux of cAMP could only account for a minor part of the decrease in intracellular cAMP. Six peaks of cAMP-hydrolyzing PDE activity were separated by Q-Sepharose chromatography. The first peak to elute was stimulated by Ca2+/calmodulin and provided less than 2% of the total eluted cAMP-PDE activity. The second peak, providing less than 4% of the cAMP-PDE activity, was stimulated 3-fold by 4 microM cyclic GMP (cGMP) and was sensitive to the PDE2 isoenzyme-selective inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA). The third peak, providing less than 10% of the cAMP-PDE activity, was insensitive to rolipram, EHNA, Ca2+/calmodulin, and cGMP. Peaks 4, 5 and 6 were sensitive to rolipram (IC50 < 0.1 microM) and provided approximately 85% of the total cAMP-hydrolyzing activity. It is concluded that cAMP-PDE activity in UMR-106 cells plays a major role in the control of intracellular cAMP accumulation, whereas only moderate amounts of cAMP are extruded from the cells through cAMP efflux. The main cAMP-hydrolyzing PDE isozyme is cAMP-specific/rolipram-sensitive. Ca2+/calmodulin-stimulated PDE, cGMP-stimulated PDE, and presently unidentified cAMP-specific/rolipram-insensitive PDE are also present in UMR-106 cells.     

Regulation of calcium current by low-Km cyclic AMP phosphodiesterases in cardiac cells.

The voltage-gated Ca2+ current (ICa) in cardiac myocytes is regulated by cAMP-dependent phosphorylation. Although the regulation of ICa via mechanisms involving modulation of cAMP synthesis is well understood, the regulation of cAMP degradation has been less thoroughly investigated. The goal of the present study was to investigate the participation of different subclasses of cAMP phosphodiesterase (PDE) in regulating cAMP-dependent phosphorylation of Ca2+ channels in frog ventricular myocytes. Cardiomyocytes were isolated enzymatically and mechanically and were patch-clamped using the whole-cell configuration of the patch-clamp technique. The effects of various low-Km cAMP PDE inhibitors on ICa were examined. None of the inhibitors tested [milrinone, indolidan, 1-methyl 3-isobutyl xanthine (MIX), rolipram, or Ro 20-1724] were able to elevate ICa in the absence of elevated cAMP, although they all increased ICa in the presence of submaximal levels of cAMP. This result suggests that these compounds do not act directly on Ca2+ channels but rather modulate cAMP degradation. Half-maximal effects were observed with 1.4 microM milrinone and 3.4 microM MIX. Milrinone was effective when applied from either the extracellular or intracellular surface, whereas MIX was effective only when applied from the extracellular solution. In the presence of internal cGMP, which stimulates the cGMP-stimulated PDE, the low-Km cAMP PDE inhibitors had no effect on ICa, whereas high concentrations of MIX, which inhibit the cGMP-stimulated PDE, increased ICa. This would support the hypothesis that cGMP-stimulated PDE either has a much stronger capacity to hydrolyze cAMP or is more efficiently coupled to Ca2+ channels than the low-Km cAMP PDEs.     

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)     

Selective inhibition of cyclic nucleotide phosphodiesterases of human, bovine and rat aorta

Cyclic nucleotide phosphodiesterase (PDE) activity from the 105,000 g supernatant of human, bovine and rat aorta smooth muscle cells was resolved by DEAE-trisacryl chromatography into three major forms showing similar properties in each species. In addition to the two PDE forms previously characterized in vascular tissues (a cAMP-PDE and a calmodulin-dependent PDE), a cGMP-PDE, insensitive to calmodulin, was isolated and characterized in the aorta of the three species. Each isolated PDE form was differently inhibited by various chemical compounds, and these compounds produced effects on cyclic nucleotide levels in isolated rat aorta which could be expected from their inhibitory effect on isolated PDE forms. At concentrations non-selectively inhibiting the three isolated PDE forms (including the calmodulin-dependent one), IBMX (3-isobutyl-1-methylxanthine) and trequinsin markedly and dose-dependently increased both cAMP and cGMP aorta levels (up to 7-fold, in presence of 500 microM IBMX). By contrast selective inhibitors of cGMP-PDE or cAMP-PDE could only induce a moderate elevation (by 1.5-3-fold) in cGMP or cAMP levels, respectively. In the case of M&B 22,948, a highly specific and potent inhibitor of cGMP-PDE, a concentration-dependent increase in tissue cGMP levels was produced by concentrations (in the microM range) active in inhibiting the isolated enzyme. In the case of selective cAMP-PDE inhibitors (rolipram and Ro 20-1724), however, a significant increase in aorta cAMP content was induced only in the presence of drug concentrations which were much higher (200 and 500 microM, respectively) than those inhibiting the isolated enzyme (IC50:5 and 18 microM, respectively). Inhibitors of both cGMP-PDE and cAMP-PDE (dipyridamole, cilostamide and its derivative AAL 05) produced the same moderate effects as did the combination of a selective cGMP-PDE inhibitor and a selective cAMP-PDE inhibitor on the levels of both cGMP and cAMP. These results show that the three forms of PDE isolated from aortic smooth muscle retain properties that they exhibit in the tissue and which are similar in the three species examined, including man. They suggest that each form participates in a specific manner to the regulation of cAMP and cGMP concentrations in aorta smooth muscle cells.     

Phosphodiesterases do not limit β1-adrenoceptor-mediated sinoatrial tachycardia: evidence with PDE3 and PDE4 in rabbits and PDE1–5 in rats

The mammalian heart expresses at least five phosphodiesterases (PDE1–5). Catecholamines produce surges of inotropically relevant cAMP through β₁-adrenoceptor stimulation. cAMP is mainly hydrolysed by PDE3 and/or PDE4 thereby blunting contractility. Basal sinoatrial beating rate in mouse, rat, piglet and rabbit sinoatrial cells is reduced by PDE3 and/or PDE4 through hydrolysis of cAMP. However, in rodents, the tachycardia elicited by catecholamines through production of cAMP by β-adrenoceptor activation is not controlled by PDE3 and PDE4, despite a blunting effect of PDE3 or/and PDE4 on basal sinoatrial beating, but it is unknown whether PDE3 limits catecholamine-evoked tachycardia in the rabbit. Since rabbit sinoatrial cells are an important model for pacemaker research, we investigated whether the positive chronotropic effects of (?)-noradrenaline on spontaneously beating right atria of the rabbit are potentiated by inhibition of PDE3 with cilostamide (300 nM). We also studied the sinoatrial effects of the PDE4 inhibitor rolipram (10 μM) and its influence on the responses to (?)-noradrenaline. For comparison, we investigated the influence of cilostamide and rolipram on the positive inotropic responses to (?)-noradrenaline on rabbit left atria and right ventricular papillary muscles. Cilostamide and concurrent cilostamide?+?rolipram, but not rolipram alone, increased sinoatrial rate by 15% and 31% of the effect of (?)-isoprenaline (200?µM) but the PDE inhibitors did not significantly change the chronotropic potency of (?)-noradrenaline. In contrast in papillary muscle, the positive inotropic effects of (?)-noradrenaline were potentiated 2.4-, 2.6- and 44-fold by cilostamide, rolipram and concurrent cilostamide?+?rolipram, respectively. In left atrium, the positive inotropic effects of (?)-noradrenaline were marginally potentiated by cilostamide, as well as potentiated 2.7- and 32-fold by rolipram and by concurrent cilostamide and rolipram respectively. To compare the influence of PDE1–5 on basal sinoatrial rate and (?)-noradrenaline-evoked tachycardia, we investigated on rat right atria the effects of selective inhibitors. The PDE4 inhibitor rolipram and non-selective inhibitor isobutyl-methylxanthine caused tachycardia with –logEC₅₀s of 7.2 and 5.0 and E _max of 18% and 102% of (?)-isoprenaline, respectively. Rolipram did not change the chronotropic potency of (?)-noradrenaline. At high concentrations (10–30?µM), the PDE1, PDE3 and PDE5 inhibitors 8-methoxymethyl-3-isobutyl-1-methylxanthine, cilostamide and sildenafil, respectively, caused marginal tachycardia but did not significantly change the chronotropic potency of (?)-noradrenaline. The PDE2-selective inhibitor erythro-9-[2-hydroxy-3-nonyl]adenine caused marginal bradycardia at 30?µM and tended to reduce the chronotropic potency of (?)-noradrenaline. Rabbit PDE3 reduces basal sinoatrial rate. Although PDE4 only marginally reduces rate, under conditions of PDE3 inhibition, it further reduces sinoatrial rate. Both PDE3 and PDE4 control atrial and ventricular positive inotropic effects of (?)-noradrenaline. In contrast, neither PDE3 nor PDE4 limit the sinoatrial tachycardia induced by (?)-noradrenaline. In the rat, only PDE4, but not PDE1, PDE2, PDE3 and PDE5, reduces basal sinoatrial rate. None of the five rat PDEs limits the (?)-noradrenaline-evoked tachycardia. Taken together, these results confirm and expand evidence for our proposal that the cAMP-compartment modulating basal sinoatrial rate, controlled by PDE3 and/or PDE4, is different from the PDE-resistant cAMP compartment involved in β₁-adrenoceptor-mediated sinoatrial tachycardia.     

Cyclic adenosine 3'',5''-monophosphate and beta-effects in rat isolated superior cervical ganglia

Isoprenaline (0.01-1 microM) increased the amount of cyclic adenosine 3',5'-monophosphate (cyclic AMP) in rat isolated superior cervical ganglia by up to 10 times after 10 min application. Cyclic AMP levels returned to control values after 20 min washing. Salbutamol, in concentrations (1-100 microM) that depolarized the ganglion and facilitated submaximal transmission, did not significantly raise ganglionic cyclic AMP levels. The action of isoprenaline was antagonized by butoxamine (apparent KI approximately equal to 0.14 microM) and weakly by practolol (apparent KI approximately equal to 9.1 microM). The effect of 0.1 microM isoprenaline was also inhibited 94% by 100 microM of the adenylate cyclase inhibitor, 9-(tetrahydro-2-furyl)adenine (SQ 22,536). Exogenous dibutyryl cyclic AMP did not replicate the effects of isoprenaline on ganglionic d.c. potentials or submaximal transmission. The phosphodiesterase inhibitors theophylline, isobutylmethylxanthine or 4-(3,4-dibutoxybenzyl)-2-imidazolidinone (Ro 20-1724) did not potentiate these electrical responses to isoprenaline. The adenylate cyclase inhibitor, SQ 22,536, did not inhibit the electrical responses to isoprenaline. It is concluded that available evidence does not support the view that the ganglion depolarization or facilitation of submaximal transmission in rat isolated ganglia produced by isoprenaline are likely to be mediated by cyclic AMP.     

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

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

Characterization and selective inhibition of cyclic nucleotide phosphodiesterase isozymes in canine tracheal smooth muscle

Cyclic nucleotide phosphodiesterases (PDEs) from canine trachealis were characterized with respect to their kinetic properties, sensitivity to selective inhibitors, and subcellular distribution. Extracts from whole tissue homogenates were applied to DEAE-Sepharose anion exchange columns and eluted with a linear sodium acetate gradient. Three major peaks of PDE activity were resolved. The first (PDE I), which eluted at 0.2 M sodium acetate, was applied to a calmodulin (CaM)-Sepharose affinity column and resolved into CaM-insensitive and CaM-sensitive PDEs. The CaM-insensitive isozyme (PDE Ia) had apparent Km values of 135 microM (cAMP) and 4 microM (cGMP) and was potently inhibited by zaprinast (Ki = 0.1 microM). The CaM-sensitive isozyme (PDE Ic) had apparent Km values of 1 microM (cAMP) and 2 microM (cGMP) and was inhibited by zaprinast with an apparent Ki of 35 microM. The second peak of activity (PDE II) from the anion exchange column eluted at 0.3 M sodium acetate and had apparent Km values of 93 microM (cAMP) and 60 microM (cGMP). The enzyme displayed positive cooperativity with respect to the hydrolysis of cAMP (nH = 1.7). Low concentrations of cGMP (0.1-1 microM) reduced cooperativity (nH = 1.1) and increased the hydrolysis of 1 microM cAMP. The third peak of activity from the anion exchange column eluted at 0.6 M sodium acetate and displayed anomalous kinetics that suggested the presence of two isozymes. This was supported by the observation that enzyme activity was only partially inhibited by SK&F 94120 or Ro 20-1724 but was abolished by the combination of the two PDE inhibitors. Subsequent studies confirmed the existence of two isozymes. The first, PDE III, had apparent Km values of 0.3 microM (cAMP) and 8 microM (cGMP) and was inhibited by cGMP (IC50 = 0.1 microM), SK&F 94120 (Ki = 7.8 microM), and SK&F 94836 (Ki = 0.4 microM). The second, PDE IV, had apparent Km values of 4 microM (cAMP) and 40 microM (cGMP) and was inhibited by Ro 20-1724 (Ki = 5.2 microM) and rolipram (Ki = 0.5 microM) but not by cGMP. Assessment of the 100,000 x g soluble and particulate PDE activity revealed that all five isozymes were present in the soluble fraction, but only four isozymes (PDEs Ia, Ic, III, and IV) were present in the particulate fraction. These results indicate that five distinct PDE isozyme exist in canine trachealis and that these isozymes differ in their kinetic characteristics, sensitivity to activators and inhibitors, and subcellular distribution.     

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)     

Bronchodilator and anti-inflammatory activities of glaucine: In vitro studies in human airway smooth muscle and polymorphonuclear leukocytes.

1. Selective phosphodiesterase 4 (PDE4) inhibitors are of potential interest in the treatment of asthma. We examined the effects of the alkaloid S-(+)-glaucine, a PDE4 inhibitor, on human isolated bronchus and granulocyte function. 2. Glaucine selectively inhibited PDE4 from human bronchus and polymorphonuclear leukocytes (PMN) in a non-competitive manner (Ki=3.4 microM). Glaucine displaced [3H]-rolipram from its high-affinity binding sites in rat brain cortex membranes (IC50 approximately 100 microM). 3. Glaucine inhibited the spontaneous and histamine-induced tone in human isolated bronchus (pD2 approximately 4.5). Glaucine (10 microM) did not potentiate the isoprenaline-induced relaxation but augmented cyclic AMP accumulation by isoprenaline. The glaucine-induced relaxation was resistant to H-89, a protein kinase A inhibitor. Glaucine depressed the contractile responses to Ca2+ (pD'2 approximately 3.62) and reduced the sustained rise of [Ca2+]i produced by histamine in cultured human airway smooth muscle cells (-log IC50 approximately 4.3). 4. Glaucine augmented cyclic AMP levels in human polymorphonuclear leukocytes challenged with N-formyl-Met-Leu-Phe (FMLP) or isoprenaline, and inhibited FMLP-induced superoxide generation, elastase release, leukotriene B4 production, [Ca2+]i signal and platelet aggregation as well as opsonized zymosan-, phorbol myristate acetate-, and A23187-induced superoxide release. The inhibitory effect of glaucine on superoxide generation by FMLP was reduced by H-89. 5. In conclusion, Ca2+ channel antagonism by glaucine appears mainly responsible for the relaxant effect of glaucine in human isolated bronchus while PDE4 inhibition contributes to the inhibitory effects of glaucine in human granulocytes. The very low PDE4/binding site ratio found for glaucine makes this compound attractive for further structure-activity studies.     

Inhibitory effects of flavonoids on phosphodiesterase isozymes from guinea pig and their structure-activity relationships

The structure-activity relationships of flavonoids with regard to their inhibitory effects on phosphodiesterase (PDE) isozymes are little known. The activities of PDE1-5 were measured by a two-step procedure using cAMP with [(3)H]-cAMP or cGMP with [(3)H]-cGMP as substrates. In the present results, PDE1, 5, 2, and 4 isozymes were partially purified from guinea pig lungs in that order, and PDE3 was from the heart. The IC(50) values of PDE1-5 were greater than those reported previously for the reference drugs, vinpocetin, EHNA, milrinone, Ro 20-1724, and zaprinast, by 5-, 5-, 7-, 5-, and 3-fold, respectively. As shown in Table 2, luteolin revealed non-selective inhibition of PDE1-5 with IC(50) values in a range of 10-20 microM, as did genistein except with a low potency on PDE5. Daidzein, an inactive analogue of genistein in tyrosine kinase inhibition, showed selective inhibition of PDE3 with an IC(50) value of around 30 microM, as did eriodictyol with an IC(50) value of around 50 microM. Hesperetin and prunetin exhibited more-selective inhibition of PDE4 with IC(50) values of around 30 and 60 microM, respectively. Luteolin-7-glucoside exhibited dual inhibition of PDE2/PDE4 with an IC(50) value of around 40 microM. Diosmetin more-selectively inhibited PDE2 (IC(50) of 4.8 microM) than PDE1, PDE4, or PDE5. However, biochanin A more-selectively inhibited PDE4 (IC(50) of 8.5 microM) than PDE1 or PDE2. Apigenin inhibited PDE1-3 with IC(50) values of around 10-25 microM. Myricetin inhibited PDE1-4 with IC(50) values of around 10-40 microM. The same was true for quercetin, but we rather consider that it more-selectively inhibited PDE3 and PDE4 (IC(50) of < 10 microM). In conclusion, it is possible to synthesize useful drugs through elucidating the structure-activity relationships of flavonoids with respect to inhibition of PDE isozymes at concentrations used in this in vitro study.     

Study of the mechanisms involved in the vasorelaxation induced by (-)-epigallocatechin-3-gallate in rat aorta

This study investigated several mechanisms involved in the vasorelaxant effects of (-)-epigallocatechin-3-gallate (EGCG). EGCG (1 microM-1 mM) concentration dependently relaxed, after a transient increase in tension, contractions induced by noradrenaline (NA, 1 microM), high extracellular KCl (60 mM), or phorbol 12-myristate 13-acetate (PMA, 1 microM) in intact rat aortic rings. In a Ca2+ -free solution, EGCG (1 microM-1 mM) relaxed 1 microM PMA-induced contractions, without previous transient contraction. However, EGCG (1 microM-1 mM) did not affect the 1 microM okadaic acid-induced contractions. Removal of endothelium and/or pretreatment with glibenclamide (10 microM), tetraethylammonium (2 mM) or charybdotoxin (100 nM) plus apamin (500 nM) did not modify the vasorelaxant effects of EGCG. In addition, EGCG noncompetitively antagonized the contractions induced by NA (in 1.5 mM Ca2+ -containing solution) and Ca2+ (in depolarizing Ca2+ -free high KCl 60 mM solution). In rat aortic smooth muscle cells (RASMC), EGCG (100 microM) reduced increases in cytosolic free Ca2+ concentration ([Ca2+]i) induced by angiotensin II (ANG II, 100 nM) and KCl (60 mM) in 1.5 mM CaCl2 -containing solution and by ANG II (100 nM) in the absence of extracellular Ca2+. In RASMC, EGCG (100 microM) did not modify basal generation of cAMP or cGMP, but significantly reversed the inhibitory effects of NA (1 microM) and high KCl (60 mM) on cAMP and cGMP production. EGCG inhibited the enzymatic activity of all the cyclic nucleotide PDE isoenzymes present in vascular tissue, being more effective on PDE2 (IC50 approximately 17) and on PDE1 (IC50 approximately 25). Our results suggest that the vasorelaxant effects of EGCG in rat aorta are mediated, at least in part, by an inhibition of PDE activity, and the subsequent increase in cyclic nucleotide levels in RASMC, which, in turn, can reduce agonist- or high KCl concentration-induced increases in [Ca2+]i.