Isozyme-selective Phosphodiesterase Inhibitors Research Articles

Phosphodiesterase Isozymes Involved in Regulation of Secretion in Isolated Mouse Stomach in Vitro

(+/-)-(E)-4-Ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide] (NOR-3), a nitric-oxide (NO) donor, is known to increase HCO(3)(-) secretion in rat stomachs, intracellularly mediated by cGMP; yet, there is no information about the phosphodiesterase (PDE) isozyme involved in this process. We examined the effects of various isozyme-selective PDE inhibitors on the secretion of HCO(3)(-) in the mouse stomach in vitro and the type(s) of PDE isozymes involved in the response to NO. The gastric mucosa of DDY mice was stripped of the muscle layer and mounted on an Ussing chamber. HCO(3)(-) secretion was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. NOR-3, 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), and various PDE inhibitors were added to the serosal side. Vinpocetine (PDE1 inhibitor) or zaprinast (PDE5 inhibitor) was also added serosally 30 min before NOR-3 or 8-Br-cGMP. Both NOR-3 and 8-Br-cGMP stimulated HCO(3)(-) secretion in a dose-dependent manner, and the response to NOR-3 was significantly inhibited by methylene blue. Likewise, the secretion induced by NOR-3 or 8-Br-cGMP was significantly attenuated by 6-((2S,3S)-3-(4-chloro-2-methylphenylsulfonylaminomethyl)-bicyclo(2.2.2)octan-2-yl)-5Z-hexenoic acid (ONO-8711), the PGE receptor (EP)1 antagonist, as well as indomethacin and potentiated by both vinpocetine and zaprinast at doses that had no effect by themselves on the basal secretion, whereas other subtype-selective PDE inhibitors had no effect. NOR-3 increased the mucosal PGE(2) content in a methylene blue-inhibitable manner. These results suggest that NO stimulates gastric HCO(3)(-) secretion mediated intracellularly by cGMP and modified by both PDE1 and PDE5, and this response is finally mediated by endogenous PGE(2) via the activation of EP1 receptors.

Phosphodiesterase isozymes involved in regulation of HCO 3− secretion in isolated mouse duodenum in vitro

We examined the effects of various isozyme-selective PDE inhibitors on HCO 3 − secretion in the mouse duodenum in vitro and investigated which type(s) of phosphodiesterase (PDE) isozymes are involved in the response to PGE 2 and NO. The duodenal mucosa of male DDY mice was stripped of the muscle layer and mounted on an Ussing chamber, and HCO 3 − secretion was measured at pH 7.0 by a pH-stat method using 2 mM HCl. Both PGE 2 and NOR-3 (NO donor) increased HCO 3 − secretion in the mouse duodenum in vitro, and the response to PGE 2 was inhibited by both EP3 and EP4 antagonists but not EP1 antagonist, while that to NOR-3 was inhibited by methylene blue. IBMX, a nonselective PDE inhibitor, significantly increased basal HCO 3 − secretion and potentiated the responses to both PGE 2 and NOR-3. Likewise, vinpocetine (PDE1 inhibitor) and cilostamide (PDE3 inhibitor) also increased the basal secretion at high doses and potentiated the HCO 3 − response to PGE 2 at doses that had no effect by themselves on the basal secretion. By contrast, the HCO 3 − stimulatory action of NOR-3 was significantly potentiated by vinpocetine but not cilostamide. Inhibitors of other PDE subtypes had no effect on the HCO 3 − secretion under basal or stimulated conditions. Both PDE1 and PDE3 mRNAs were expressed in the duodenal mucosa. These results suggested that PDE1 and PDE3 are involved in the regulation of duodenal HCO 3 − secretion and that the response to PGE 2 is associated with both PDE1 and PDE3, while the response to NO is mainly modulated by PDE1.

Efficacy and Selectivity of Phosphodiesterase-Targeted Drugs in Inhibiting Photoreceptor Phosphodiesterase (PDE6) in Retinal Photoreceptors

Phosphodiesterase (PDE) inhibitors are important therapeutic agents, but their effects on photoreceptor PDE (PDE6) and photoreceptor cells are poorly understood. The potency and selectivity of various classes of PDE inhibitors on purified rod and cone PDE6 and on intact rod outer segments (ROS) were characterized. The inhibition constant (K(i)) of isozyme-selective PDE inhibitors was determined for purified rod and cone PDE6. Perturbations of cGMP levels in isolated ROS suspensions by PDE inhibitors were quantitated by a cGMP enzyme-linked immunoassay. Most PDE5-selective inhibitors were excellent PDE6 inhibitors. Vardenafil, a potent PDE5 inhibitor (K(i) = 0.2 nM), was the most potent PDE6 inhibitor tested (K(i) = 0.7 nM). Zaprinast was the only drug that inhibited PDE6 more potently than did PDE5. PDE1-selective inhibitors were equally effective in inhibiting PDE6. In intact ROS, PDE inhibitors elevated cGMP levels, but none fully inhibited PDE6. Their potency for elevating cGMP levels in ROS was much lower than their ability to inhibit the purified enzyme. Competition between PDE5/6-selective drugs and the inhibitory gamma-subunit for the active site of PDE6 is proposed to reduce the effectiveness of drugs at the enzyme-active site. Several classes of PDE inhibitors inhibit PDE6 equally as well as the PDE family to which they are targeted. In intact ROS, high PDE6 concentrations, binding of the gamma-subunit to the active site, and calcium feedback mechanisms attenuate the effectiveness of PDE inhibitors to inhibit PDE6 and disrupt the cGMP signaling pathway during visual transduction.

Phyllodulcin, a constituent of "Amacha", inhibits phosphodiesterase in bovine adrenocortical cells.

Phyllodulcin, a constituent of "Amacha", inhibits phosphodiesterase in bovine adrenocortical cells.

Recent perspectives in the design of antiasthmatic agents.

Asthma is a common respiratory disorder. It can no longer be viewed as a reversible airway obstruction but should instead be considered primarily as an inflammatory illness that has bronchial hyperreactivity and bronchospasm as its result. There are several potential benefits as well as limitations of the currently available antiasthmatic agents such as anticholinergics, beta 2-selective agonists, methylxanthines, corticosteroids, or mast cell stabilizers. Recent trends in the design of new antiasthmatic agents include isozyme selective phosphodiesterase inhibitors, inhibitors of the biosynthesis of interleukin-4 and IL-4 antagonists, lipoxygenase and leukotriene inhibitors, thromboxane A2 receptor antagonists, potassium channel openers and monoclonal antibodies.

Ca 2+/calmodulin-dependent cyclic GMP phosphodiesterase activity in granule neurons and astrocytes from rat cerebellum

Cyclic GMP (cGMP) formation induced by agonist stimulation of Ca 2+/calmodulin-dependent nitric oxide (NO) synthase type I is known to occur in both granule cell and astrocyte cultures from rat cerebellum. Here we show that in these same cells cGMP is predominantly hydrolyzed by a Ca 2+/calmodulin-dependent phosphodiesterase. At 10 μM cGMP, Ca 2+ (25 μM) stimulated basal (Ca 2+-independent) phosphodiesterase activity about 6 times in granular neurons and 15 times in astrocytes. The calmodulin antagonist calmidazolium blocked the Ca 2+-dependent phosphodiesterase activity and exogenous calmodulin increased 3–4-fold the stimulatory potency of Ca 2+ in both cell types (EC 50 values 1.26±0.20 and 1.50±0.42 μM in the absence and 0.38±0.11 and 0.39±0.14 μM in the presence of 1 μM calmodulin, for neurons and astrocytes, respectively). In both cell types K m values for cGMP at 25 μM Ca 2+ were similar (1.72±0.20 and 1.92±0.09 μM) and phosphodiesterase activities were inhibited by isozyme-selective phosphodiesterase inhibitors with potencies analogous to those described for Ca 2+/calmodulin-phosphodiesterases or phosphodiesterase type 1 isoforms in other preparations. The nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxantine (IBMX) effectively blocked the Ca 2+/calmodulin-phosphodiesterase activity in granule cell and astrocyte extracts (IC 50 values at 1 μM cGMP: 31±10 μM and 46±6 μM, respectively), in contrast to the apparent inability of this compound to inhibit the Ca 2+-dependent activity reported in whole brain extracts. These results demonstrate that comparable phosphodiesterase type 1 activities are found in the cytosols of cerebellar granule cells and astrocytes and suggest that these activities may play an important role in controlling cGMP levels in cells where the Ca 2+-dependent NO synthase type I is stimulated. © 1997 Elsevier Science B.V. All rights reserved.

Effects of nonselective and isozyme selective cyclic nucleotide phosphodiesterase inhibitors on antigen-induced cytokine gene expression in peripheral blood mononuclear cells.

Cyclic nucleotide phosphodiesterase (PDE) enzymes may participate in regulation of the inflammatory response through their effects on second messengers. In the present study, we have investigated the role of nonselective and isozyme selective PDE inhibitors in altering the antigen-driven cytokine gene expression of peripheral blood mononuclear cells (PBMCs) from atopic individuals. Ragweed and tetanus toxoid were used as model antigens. The nonselective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the selective PDE4 inhibitor, rolipram, markedly suppressed interleukin-5 (IL-5) and interferon gamma (IFN gamma) gene expression in both antigen-driven systems. Gene expression for IL-4 was unaffected by these agents in the ragweed-driven system. Message for IL-4 could not be detected in the tetanus toxoid-driven system, despite the use of a quantitative, competitive reverse transcription-polymerase chain reaction (RT-PCR) assay sensitive to less than 10 fg of target template. The PDE3 inhibitor, siguazodan, was ineffective in downregulating gene expression for the proinflammatory cytokines assayed; when used in combination with the PDE4 inhibitor, the PDE3 inhibitor provided no increase in efficacy over that seen with the PDE4 inhibitor alone. Gene expression for the A and B isoforms of the PDE4 in PBMCs was unaffected by antigen stimulation or treatment with the PDE4 inhibitor; however, differences in expression of these two isoforms were apparent when a variety of immune cell lines were studied. These data support the hypothesis that the primary anti-inflammatory target for PDE inhibition in PBMCs is the PDE4. Furthermore, the expression of various isoforms of this enzyme may differ between immune cell types. Finally, PDE4 isoform expression in PBMCs is independent of treatment with an isozyme selective inhibitor.

Intratracheal administration of phosphodiesterase III inhibitor attenuates bronchoconstriction in cats: a preliminary report.

The effects of intratracheal administration of MKS 492, a selective phosphodiesterase (PDE) III inhibitor, were studied in five anesthetized bronchoconstricted cats. The animals were challenged by four repeated doses of intratracheal methacholine (67 micrograms/kg), and the degree of bronchoconstriction was assessed from increases in respiratory system resistance (Rrs). All animals demonstrated good bronchoconstrictive responses (i.e., 86-99% increases in Rrs) to methacholine without tachyphylaxis. On a separate day, the cats received the same four doses of methacholine after being pretreated with either intratracheal saline or three different doses of MKS 492 (0.17, 1.7, and 17 micrograms/kg). The increases in Rrs with 1.7 micrograms/kg [52.6 +/- 8.4% (SE)] and 17 micrograms/kg of MKS 492 (44.4 +/- 10.1%) were smaller than those with saline pretreatment (88.1 +/- 16.8%) (P < 0.05). There were no treatment-associated changes in mean arterial pressure or heart rate during administration of MKS 492. We conclude that intratracheal MKS 492 effectively reduced methacholine-induced bronchoconstriction in a dose-dependent fashion without substantial systemic effects. These preliminary results suggest that inhalation of isozyme-selective PDE inhibitors may deserve consideration for clinical trials provided that more extensive preclinical investigations justify such trials.

Modulation of antigen- and mitogen-induced proliferative responses of peripheral blood mononuclear cells by nonselective and isozyme selective cyclic nucleotide phosphodiesterase inhibitors.

Cyclic nucleotide phosphodiesterase (PDE) enzymes are felt to play a role in the regulation of inflammatory responses through their effects on cAMP. In this study, we investigated the effects of nonselective and isozyme selective PDE inhibitors on the proliferative responses of peripheral blood mononuclear cells (PBMCs) to ragweed (RW, a Th2 stimulus), tetanus toxoid (TT, a Th1 stimulus), and phytohemagglutinin in ragweed-allergic patients. The nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) produced nearly identical concentration-dependent inhibition for PBMCs cultured with either Ag (RW and TT) or mitogen (phytohemagglutinin). Neither the type V inhibitor, zaprinast, nor the type III inhibitor, siguazodan, was effective at inhibiting antigen- or mitogen-driven proliferative responses of human PBMCs. The type IV inhibitor, rolipram, was unique in its ability to inhibit the RW-driven proliferative response and, to a lesser extent, the TT-driven proliferative response. However, rolipram was ineffective at inhibiting the mitogen-driven proliferative response. Only with a combination of type III and type IV inhibitors could the efficacy on the TT response be made to approximate that of the type IV inhibitor alone in the RW-driven system. Although the efficacies of IBMX and rolipram were identical in the RW-driven system, the IC50 value of the latter was 10-fold lower, similar to the difference noted in the TT-driven system between IBMX alone and the combination of rolipram with siguazodan. Dose-response curves generated by using the D- or L-isomers of rolipram were not appreciably different from each other or from the curve generated with racemic rolipram. The addition of supraphysiologic doses of human rIL-2 and rIL-4 was unable to counteract the inhibitory effect of IBMX or rolipram. These data support the hypothesis that modulation of proliferation of PBMCs by selective PDE inhibitors varies in sensitivity with the type of stimulus used, and that the type IV PDE exerts the predominant cAMP-associated regulatory effect on allergen-driven proliferation. Finally, the inhibitory effect induced by rolipram is independent of its stereochemistry and cannot be exclusively attributed to deficits in IL-2 or IL-4.

Effects of isozyme-selective phosphodiesterase inhibitors on eosinophil infiltration in the guinea-pig lung

The effects of selective phosphodiesterase isozyme inhibitors on eosinophil infiltration induced by antigen challenge or by exposure to an aerosol of platelet-activating factor (PAF) were investigated in the guinea-pig. Pretreatment 24 h and 3 h before antigen challenge or PAF exposure with theophylline (100 mg/kg), rolipram (5 mg/kg) and Ro 20-1724 (30 mg/kg) significantly reduced the increase in eosinophil numbers in bronchoalveolar lavage fluid. In contrast, milrinone, SK&F 94–120 and zaprinast were ineffective against antigen-induced eosinophil recruitment. Theophylline, rolipram and Ro 20-1724 also significantly reduced the release of eosinophil peroxidase into the bronchoalveolar lavage fluid. It is suggested that inhibitors of type IV phosphodiesterase have anti-inflammatory effects in the airways and may be useful in the treatment of asthma.

Amrinone attenuates airway constriction during halothane anesthesia.

Phosphodiesterase (PDE) inhibitors should, in theory, be useful in patients with both cardiac and reactive airway disease who require anesthesia. The effects of the PDE inhibitor, amrinone, on the release of endogenous catecholamines and on airway reactivity to aerosol methacholine challenge were evaluated during thiopental/fentanyl and halothane anesthesia in five mongrel dogs. Responses to methacholine aerosol challenge (0.03, 0.075, 0.15, 0.30, 0.75, and 3.0 mg/ml) were measured during four conditions: thiopental/fentanyl anesthesia, thiopental/fentanyl anesthesia with amrinone infusion, halothane anesthesia, and halothane anesthesia with amrinone infusion. Increase in pulmonary resistance (RL) and decreases in dynamic compliances (Cdyn) were calculated for each methacholine dose. Plasma epinephrine and norepinephrine concentrations were measured during thiopental/fentanyl anesthesia with amrinone infusion. Before aerosol challenge, baseline RL and Cdyn did not differ in the four groups. Amrinone significantly attenuated the pulmonary response to methacholine during both thiopental/fentanyl and halothane anesthesia. Plasma catecholamine concentrations did not increase during amrinone infusion. Amrinone attenuates methacholine-induced airway responses even during halothane anesthesia. These data indicate that isozyme-selective PDE inhibitors hold promise for the perioperative treatment of bronchospasm.

Effects of isozyme selective phosphodiesterase inhibitors on bovine tracheal smooth muscle tone

Effects of isozyme selective phosphodiesterase inhibitors on bovine tracheal smooth muscle tone

The future role of isozyme selective phosphodiesterase inhibitors in asthma

The future role of isozyme selective phosphodiesterase inhibitors in asthma

Interactions Between Isoprenaline, Sodium Nitroprusside, and Isozyme-Selective Phosphodiesterase Inhibitors on ADP-Induced Aggregation and Cyclic Nucleotide Levels in Human Platelets

The interactions between isoprenaline, sodium nitroprusside, and the isozyme-selective phosphodiesterase inhibitors OPC 3911 ("cAMP specific") and zaprinast ("cGMP specific") on platelet aggregation induced by adenosine diphosphate (ADP) and on levels of cAMP and cGMP were studied. Isoprenaline at 10(-6)M diminished aggregation by 28%, and this effect was enhanced by 10(-7)-10(-6)M OPC 3911. Neither 10(-6) M isoprenaline nor 10(-7)M OPC 3911 elevated cAMP, but in combination they caused a significant rise in cAMP (27% above the basal level), accompanying the synergistic functional inhibition, without affecting cGMP levels. Sodium nitroprusside at 10(-5) M diminished aggregation by 39%, elevated cGMP levels (81-110%), but also caused a statistically significant increase in cAMP (21-32%), and enhanced the effects of 10(-6)M isoprenaline on cAMP levels. Zaprinast at 10(-5) M caused a modest inhibition of aggregation by 20%, and a small increase in cGMP (20%), and it clearly enhanced the effects of 10(-5)M sodium nitroprusside on both cGMP and cAMP levels, but not on aggregation. The cAMP-increasing effect of sodium nitroprusside might be a consequence of a cGMP-mediated inhibition of the "low-Km cGMP-inhibited phosphodiesterase" that is also inhibited by OPC 3911. The effects of all of the drugs on ADP-induced aggregation seem to depend more on their effect on cAMP levels than on the levels on cGMP.

Effect of isozyme selective phosphodiesterase inhibitors on bovine tracheal smooth muscle tone

Effect of isozyme selective phosphodiesterase inhibitors on bovine tracheal smooth muscle tone

Characterization and pharmacological relevance of high affinity binding sites for [3H]LY186126, a cardiotonic phosphodiesterase inhibitor, in canine cardiac membranes.

[3H]LY186126, an analogue of the cardiotonic agent indolidan, was shown to bind reversibly and with high affinity (Kd = 4 nM) to a single class of binding sites within canine myocardial vesicles. Binding site density measured in various cardiac membrane fractions correlated well with Ca2+-ATPase activity (r = 0.94; p less than 0.01), but not with Na+,K+-ATPase or azide sensitive ATPase, indicating a localization of these sites within sarcoplasmic reticulum membranes. Divalent cations were required for binding and displayed the following order of activation: Zn2+ greater than Mn2+ greater than Mg2+ greater than Ca2+. Differential activation of [3H]LY186126 binding by various divalent cations was due to alterations in binding site density, rather than affinity. cGMP and selective inhibitors of type IV membrane-bound phosphodiesterase (SR-PDE), for example, indolidan, milrinone, imazodan, and enoximone, selectively displaced bound [3H]LY186126 caffeine, theophylline, and rolipram were relatively impotent as inhibitors of radiolabel binding. Kd values from displacement curves were highly correlated with IC50 values for inhibition of SR-PDE (r = 0.92; p less than 0.001). In addition, Kd values correlated well with published ED50 values for increases in cardiac contractility in pentobarbital-anesthetized dogs (r = 0.94; p less than 0.001). The results support the hypothesis that [3H]LY186126 labels the pharmacological receptor for the class of positive inotropic agents characterized as isozyme-selective phosphodiesterase inhibitors. Furthermore, the data suggest that the identity of the site labeled by [3H]LY186126 is SR-PDE, the type IV isozyme of cardiac phosphodiesterase located in the sarcoplasmic reticulum.

Action of mediators on airway smooth muscle: functional antagonism as a mechanism for bronchodilator drugs.

The beta-adrenoceptor agonists have become the cornerstone of bronchodilator therapy. These agents are "functional" or "physiologic" antagonists that actively relax airway smooth muscle through a cyclic-AMP (cAMP)-mediated decrease in myoplasmic Ca2+ content. Hence, unlike specific receptor antagonists, the sympathomimetics should reverse bronchoconstriction regardless of the mediator(s) involved. Indeed, one of the primary beneficial attributes of beta-adrenoceptor agonists is their inhibitory activity against a wide range of bronchoconstrictors. As successful as the sympathomimetic bronchodilators have been, they are not without liabilities. These liabilities include: 1) cardiovascular and skeletal muscle side effects, 2) an inherent subsensitivity of the patient population to beta-adrenoceptor agonists, 3) the development of tolerance, and 4) loss of efficacy during severe asthmatic episodes. These limitations are not specific for individual agents but are shared by all beta-adrenoceptor agonists. A significant improvement in the pharmacotherapy of asthma would be obtained by identifying novel bronchodilators devoid of one or more of the aforementioned liabilities. The development of isozyme-selective phosphodiesterase (PDE) inhibitors is one promising approach toward this goal. Interest in PDE inhibition as a therapeutic target has been renewed by the realization that PDEs exist in multiple isoforms and that the distribution of these isoforms varies significantly among tissues. This information, coupled with the recent synthesis of PDE inhibitors selective for several of the isozymes, raises the possibility of breeding organ-selectivity into this class of compounds. Results from preliminary experiments with isozyme-selective PDE inhibitors have helped to identify appropriate drug targets in airway smooth muscle. These early studies suggest that the synthesis of novel isozyme-selective PDE inhibitors not only may provide tools with which to understand the biologic function of various PDE isozymes, but may also lead to the development of improved therapeutic agents.