Specific Phosphodiesterase Inhibitors Research Articles

Phosphodiesterase inhibitors for persistent pulmonary hypertension of the newborn: a review.

Persistent pulmonary hypertension of the newborn (PPHN) is a complex syndrome with multiple causes, with an incidence of 0.43-6.8/1,000 live births and a mortality of 10-20%. Survivors have high morbidity in the forms of neurodevelopmental and audiological impairment, cognitive delays, hearing loss, and a high rate of rehospitalization. The optimal approach to the management of PPHN remains controversial. Inhaled nitric oxide (iNO) is currently regarded as the gold standard therapy, but with as many as 30% of cases failing to respond, has not proven to be the single magic bullet. Given the complex pathophysiology of the disease, any such magic bullet is unlikely. A number of recent studies have suggested a role for specific phosphodiesterase (PDE) inhibitors in the management of PPHN. Sildenafil, a specific PDE5 inhibitor, appears the most promising of such agents. We aim to review the current status and limitations of iNO and the potential of PDE inhibitors in the management of PPHN. The reasons why caution is warranted before specific PDE5 inhibitors like sildenafil are labelled as potential magic bullets for PPHN will be discussed. The need for randomized-controlled trials to determine the safety, efficacy, and long-term outcome following treatment with sildenafil in PPHN is emphasized.

Protection by sildenafil and theophylline of lead acetate-induced oxidative stress in rat submandibular gland and saliva.

The role of oxidative stress in lead toxicity has been proposed in many organs, however, no study has been performed in the salivary glands, which are important parts of the gastrointestinal tract with a high implication in health of the whole body. Recently, it has been proposed that increasing the levels of cGMP and cAMP in the cells may protect from the neurotoxicity of lead. The objective of this study was to determine the ability of lead acetate to produce oxidative stress in rat submandibular as the main salivary gland of the body and to study the role of pretreatment by specific phosphodiesterase inhibitors in the prevention of oxidative stress. Lead acetate (100 mg/kg), alone or in combination with theophylline (25 mg/kg) and sildenafil (5 mg/kg), was administered intraperitoneally to rats. After 2 hours and under general anaesthesia, the submandibular gland ducts were cannulated intraorally using microcannula, and pure saliva was collected for 30 min using pilocarpine (8 mg/kg) as a secretagogue. The submandibular glands were then isolated free under surgery. Oxidative stress in the gland and pure saliva were evaluated measuring lipid peroxidation (thiobarbituric acid reactive substances assay), total thiol groups content and total antioxidant capacity (the ferric reducing ability assay). Results showed significant oxidative stress in the gland and secretions as indicated by increased lipid peroxidation, decreased total antioxidant capacity and thiol group levels. The use of cAMP and cGMP phosodiesterase inhibitors, theophylline and sildenafil, prevented lead-induced increased lipid peroxidation and also protected from decreased thiol groups content and total antioxidant power of the gland and secretions. The same trend of effects was observed in gland and saliva. It is concluded that lead toxicity is mediated through oxidative stress in salivary glands, while increasing intracellular cAMP and cGMP levels may prevent lead-induced oxidative stress.

Bovine cumulus cell-oocyte gap junctional communication during in vitro maturation in response to manipulation of cell-specific cyclic adenosine 3',5'-monophosophate levels.

In the growing follicle, communication between the oocyte and its surrounding follicular cells is essential for normal oocyte and follicular development. Maturation of the fully grown oocyte in vivo is associated with the loss of cumulus cell-oocyte gap junctional communication, preventing entry of meiotic-modulating factors such as cAMP into the oocyte. We have previously shown that oocyte and cumulus cell cAMP levels can be independently regulated using inhibitors of cell-specific phosphodiesterase (PDE) isoenzymes. The objectives of this study were to examine the effects of cell type-specific PDE inhibitors on the maintenance of cumulus cell-oocyte gap junction communication (GJC) and oocyte meiotic progression. Cumulus-oocyte complexes (COCs) were aspirated from antral follicles of abattoir-derived ovaries. Cumulus cell-oocyte GJC during oocyte maturation was quantified using the fluorescent dye, calcein-AM. COCs were cultured in the presence of specific PDE inhibitors, milrinone (an oocyte PDE3 inhibitor) or rolipram (a cumulus cell PDE4 inhibitor), and were pulsed with calcein-AM to allow dye transfer between the two cell types. Following cumulus cell removal, fluorescence in denuded oocytes was measured by microphotometry, and meiotic progression was assessed. In control COCs, dye transfer from cumulus cells to the oocyte fell progressively from 0 to 9 h, after which oocyte-cumulus cell GJC was completely lost. Loss of GJC was significantly attenuated (P < 0.05) during this time in response to treatment with milrinone and rolipram. Forskolin maintained GJC at the initial 0 h level until 3-4 h of culture, whereas treatment with milrinone and forskolin together actually increased the level of dye transfer above that in COCs treated with forskolin alone. Importantly, all treatments that prolonged GJC also delayed meiotic resumption, with meiosis generally resuming when fluorescence had fallen to approximately 40% of initial levels. These results, together with our previous studies, demonstrate that treatments that maintain or elevate cAMP levels in cumulus cells, oocytes, or both result in prolonged oocyte-cumulus cell communication and delayed meiotic resumption.

Increasing intracellular cAMP and cGMP inhibits cadmium-induced oxidative stress in rat submandibular saliva

The effect of cadmium on induction of oxidative stress in rat submandibular saliva and protective role of increasing intracellular cAMP and cGMP by use of specific phosphodiesterase inhibitors, theophylline and sildenafil were investigated. Pure submandibular saliva was collected intraorally by micro polyethylene cannula from anaesthetized rats using pilocarpine as secretagogue. Acute administration of cadmium (10 mg/kg) caused significant oxidative stress by increasing lipid peroxidation by-products (thiobarbituric reactive substances, TBARS) and decreasing total thiols and total antioxidant power of the saliva. Concurrent therapy of rats by theophylline (25 mg/kg) and sildenafil (5 mg/kg) prevented cadmium-induced oxidative stress in saliva. Theophylline and sildenafil inhibited cadmium-induced increase in lipid peroxidation and decrease in total thiols and antioxidant power. It is concluded that cadmium administration results in oxidative stress in rat submandibular saliva, which can be protected by concurrent administration of specific cyclic nucleotide phosphodiesterase inhibitors.

Rolipram Inhibits Polarization and Migration of Human T Lymphocytes

Phosphodiesterase inhibitors possess anti-inflammatory and immunomodulatory properties and seem to have a great potential in the treatment of inflammatory skin diseases; however, an overall study on the effects of specific phosphodiesterase inhibitors, such as rolipram on the processes involved in the extravasation of lymphoid cells has not been performed. In this work we have assessed the effect of rolipram on the adhesion, polarization, and migration of normal human T lymphocytes. We found that low concentrations of rolipram were able to inhibit significantly the adhesion of T cells to the beta1 and beta2 integrin ligands vascular cell adhesion molecule-1 and intercellular adhesion molecule-1. Rolipram also interfered with the activation of integrins, and significantly inhibited the homotypic aggregation of T lymphocytes induced by anti-beta1 and anti-alpha4 integrin chain monoclonal antibodies. In addition, rolipram had a downregulatory effect on the activation of T cells, and significantly diminished the expression of the activation antigens CD69, CD25, and CD98 induced by phytohemagglutinin. Finally, this drug inhibited the polarization and transendothelial migration of T lymphocytes induced by the chemokine CXCL12 (SDF-1) and the chemotactic cytokine interleukin-15. The results indicate that rolipram, at low concentrations, exerts an important anti-inflammatory and immunomodulatory effect, and suggest that this selective phosphodiesterase inhibitor may be an effective tool for the therapy of immune-mediated diseases.

Hemodynamic effects of sildenafil in patients with stable ischemic heart disease

Background: Recently the new specific phosphodiesterase-5 inhibitor sildenafil was introduced into therapy for erectile dysfunction. The hemodynamic effects of sildenafil may be potentially hazardous for patients with cardiac disease. Sildenafil has been reported to augment the hypotensive effects of nitrates. There is sparse information regarding the systemic and pulmonary hemodynamic effects of a single oral dose of sildenafil in patients with stable angina. Methods: Male patients referred for coronary angiography with diagnosis of chronic stable angina were enrolled in this study to assess the acute hemodynamic effects of sildenafil. Patients receiving long-acting or sublingual nitrates for the last 6 h before the study were excluded. Hemodynamic measurement were taken during right and left heart catheterization in the basal state and 60 min after 50 mg of oral sildenafil. Results: Twelve patients (age 53±7 years) were studied. All had stable angina CCS class II or III. Four had previous myocardial infarction. By coronary angiography, seven patients had at least one coronary artery with >70% stenosis, four had at least one with 50–70% stenosis, and one had only intimal irregularities. There were no significant effects of sildenafil on systemic or pulmonary arterial pressure, left ventricle endiastolic pressure, cardiac output, and systemic or pulmonary vascular resistance ( P>0.05 for all). No adverse events were observed. Conclusion: A single oral dose of sildenafil had no significant hemodynamic effect in supine patients with stable angina. Isolated administration of sildenafil does not appear to be associated to adverse cardiovascular effects.

Selective pulmonary vasodilation in acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is characterized by a marked maldistribution of pulmonary perfusion in favor of nonventilated, atelectatic areas of the lungs, and it is the main cause of pulmonary right-to-left shunting and hypoxemia. Therapeutic interventions to selectively influence pulmonary perfusion in ARDS became feasible with the introduction of inhaled nitric oxide, which provided a means not only to reduce pulmonary hypertension, but also to improve matching of ventilation to perfusion and, thus, hypoxemia. Clinical studies in ARDS subsequently demonstrated that the combination of inhaled nitric oxide with other interventions, such as positive end-expiratory pressure and prone positioning, yielded beneficial and additive effects on arterial oxygenation. Although the available randomized, controlled trials of this novel concept have so far failed to show an improved outcome in ARDS, inhaled nitric oxide is a clinically valuable option for the treatment of severe refractory hypoxemia in ARDS, and largely promoted the concept of selective pulmonary vasodilation in intensive care practice. Currently, aerosolization of various vasodilators, in particular prostaglandins, is under evaluation in models of acute lung injury and human ARDS. Ongoing research aims to augment the effectiveness of vasodilators with specific inhibitors of phosphodiesterases or by combination with intravenous vasoconstrictors. Consequently, several alternative ways to selectively modulate pulmonary vascular tone in patients with ARDS may be available in the near future. Cost-benefit analysis of these therapeutic options will largely determine their future perspective.

Opposing facilitatory and inhibitory modulation of glutamate release elicited by cAMP production in cerebrocortical nerve terminals (synaptosomes)

Activation of cAMP-protein kinase A (PKA) is widely reported to facilitate synaptic transmission. Here, we examined the presynaptic loci of PKA action using isolated nerve terminals (synaptosoms). The adenylyl cyclase (AC) activator, forskolin, failed to have any effect on 4-aminopyridine (4-AP)-evoked glutamate release, when added alone. However, in the presence of the alkylxanthine, IBMX, forskolin strongly facilitated glutamate release. This potentiation of release was blocked by the PKA inhibitors Rp-cAMPS and H7. Given that IBMX has dual activity, antagonizing adenosine receptors as well as inhibiting cAMP phosphodiesterase, we examined the effect of a selective adenosine A 1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and RO20-1724, a specific phosphodiesterase inhibitor. Both unmasked the forskolin-mediated modulation of glutamate release. Conversely, the adenosine analogue, N 6-cyclohexyladenosine (CHA), reversed the facilitation induced by forskolin+RO20-1724. Adenosine A 1 receptor activation, therefore, appears to curtail cAMP/PKA-induced potentiation of glutamate release. Looking at the targets for cAMP/PKA-mediated potentiation of glutamate release, while synaptosomal excitability was only marginally increased, basal and 4-AP-evoked-increases in [Ca 2+] c were substantially enhanced by forskolin+IBMX. Moreover, glutamate release elicited by Ca 2+-ionophore (ionomycin)-induced Ca 2+-entry was facilitated by forskolin+IBMX. cAMP/PKA-mediated facilitation of glutamate release may therefore involve modulation of Ca 2+-entry, as well as downstream events controlling synaptic vesicle recruitment and exocytosis.

Subthreshold doses of nebulized prostacyclin and rolipram synergistaically protect against lung ischemia-reperfusion.

Pulmonary edema caused by increased microvascular permeability is an important feature of lung ischemia-reperfusion (I/R) injury. We investigated the impact of co-aerosolized prostaglandin (PG)I(2) and the 3',5-cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase inhibitor rolipram on microvascular leakage following I/R injury. Buffer-perfused rabbit lungs were exposed to 270 minutes of warm ischemia while anoxic ventilation and a positive intravascular pressure were maintained. On reperfusion, a massive increase of the capillary filtration coefficient and severe edema formation were noted, whereas microvascular pressures displayed only minor changes. Short-time aerosolization of subthreshold doses of either rolipram (33 microg) or PGI(2) (2.6 microg) at the beginning of ischemia did not attenuate the leakage response, whereas the co-aerosolization of both agents largely blocked any permeability increase and edema formation, independent of hemodynamic effects. The same was true when the co-aerosolization was undertaken before onset of ischemia. Similarly, the intravascular administration of rolipram and PGI(2) showed a synergistic reduction of I/R-induced vascular leak but demanded 10-fold higher doses. Intravascular release of cAMP was markedly enhanced on combined PGI(2)-rolipram administration but depended on the mode of delivery of these agents. Low doses of aerosolized prostacyclin and rolipram synergistically protect against severe lung I/R injury and can be used independently of lung perfusion. This strategy may be suitable for an improvement of organ preservation in lung transplantation including early management of non-heart-beating donors.

The specific type-4 phosphodiesterase inhibitor mesopram alleviates experimental colitis in mice.

Mesopram, a specific inhibitor of type-4 phosphodiesterase, decreases the synthesis of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). In the present study, we investigated the effect of mesopram in dextran sulfate sodium (DSS)-induced murine colitis. In the preventive model, colitis was induced by DSS simultaneously with the application of mesopram in BALB/c mice. In the therapeutic model, colitis was induced in BALB/c mice by DSS over 7 days. At day 8, DSS was discontinued, and treatment was started. Mesopram was applied intraperitoneally or orally. The clinical score was calculated daily during the course of each study. Post mortem, colon length, histologic score, and expression of TNF-alpha and IFN-gamma in colons were determined. In the preventive model, mesopram significantly reduced the maximal clinical score, decreased colon shortening, and the histologic score. A dose finding study, using the preventive model, showed that most clinical and post mortem benefit was achieved with 50 mg/kg mesopram compared with 2 and 10 mg/kg. In the therapeutic model, i.p. mesopram treatment led to a significant reduction of clinical score. Both, i.p. and p.o. mesopram significantly reversed DSS-induced colon shortening and reduced the ex vivo colonic production of IFN-gamma. We conclude that the specific type-4 phosphodiesterase inhibitor mesopram ameliorates murine colitis both in a preventive and a therapeutic setting.

Endothelial responses of the aorta from adrenomedullin transgenic mice and knockout mice.

Adrenomedullin (AM) is a potent vascular wall-derived vasorelaxing peptide which induces the release of nitric oxide (NO). To explore the role of endogenous AM in vascular function, we examined the effects of acetylcholine (ACh), AM, and AM receptor antagonists [AM (22-52), and calcitonin gene-related peptide (CGRP) (8-37)] on the isometric tension of aortic rings isolated from AM transgenic (TG) and knockout (KO) mice and wild type littermates (WT). ACh and AM caused a dose-dependent reduction of the isometric tension of aortic rings, but the degree of vasodilatation was smaller in TG than in KO or WT (% delta tension [10(-6) mol/l ACh]: KO -69 +/- 10%, WT -39 +/- 8%, TG -29 +/- 1%, p < 0.01). On the other hand, N(G)-nitro-L-arginine methyl ester, an NO synthase inhibitor, induced greater vasoconstriction in TG (% delta tension 10(-5)mol/l: KO +78 +/- 16%, WT +99 +/- 27%, TG +184 +/- 20%, p < 0.01), whereas E-4021, a cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase inhibitor, caused greater vasodilation in TG mice. Both AM antagonists increased tension in TG to a greater extent than in KO or WT mice (% delta tension [10(-6) mol/l CGRP (8-37)]: KO +24 +/- 5%, WT +51 +/- 6%, TG +75 +/- 7%, p < 0.01). Endothelial denudation of the aorta diminished the vasoconstriction caused by the AM antagonists. In conclusion, the amounts of AM expressed in the aortic endothelium influenced baseline NO release. AM antagonists increased vascular tone in WT as well as in TG, suggesting that endogenous AM plays a physiological role in the regulation of aortic tone.

Attenuation of cAMP accumulation in adult rat cardiac fibroblasts by IL-1beta and NO: role of cGMP-stimulated PDE2.

Treatment of cultured adult rat cardiac fibroblasts with interleukin-1beta (IL-1beta) induces the inducible nitric oxide synthase (iNOS) expression, increases nitric oxide (NO) and cGMP production, and attenuates cAMP accumulation in response to isoproterenol by ~50%. Reduced cAMP accumulation is due to NO production: the effect is mimicked by NO donors and prevented by N(G)-monomethyl-L-arginine, an NOS inhibitor. Effects of NO are not restricted to the beta-adrenergic response; the response to forskolin is similarly diminished. NO donors only slightly (12%) decrease forskolin-stimulated adenylyl cyclase (AC) activity in cardiac fibroblast plasma membranes, suggesting that the main effect of NO is not a direct one on AC. An inhibitor of soluble guanylyl cyclase inhibits the effects of IL-1beta and NO donors; inhibition of cGMP-dependent protein kinase is without effect. 3-Isobutyl-1-methylxanthine, a nonspecific phosphodiesterase (PDE) inhibitor, and erythro-9-(2-hydroxy-3-nonyl)adenine, a specific inhibitor of the cGMP-stimulated PDE (PDE2), completely restore cAMP accumulation in sodium nitroprusside-treated fibroblasts and largely reverse the attenuated response in IL-1beta-treated fibroblasts. Although NO reportedly acts by reducing AC activity in some cells, in cardiac fibroblasts NO production decreases cAMP accumulation largely by the cGMP-mediated activation of PDE2.

The Effect of Phosphodiesterase Inhibition on Gallbladder Motility in Vitro

Background. Coffee consumption decreases the risk of gallstone disease. The proposed motility effects of caffeine, a phosphodiesterase (PDE) inhibitor, are unknown. The aim of our study was to determine the effect of caffeine and specific PDE inhibitors on gallbladder motility in vitro.Methods. Gallbladder muscle strips from opossums were attached to force transducers. Baseline tone, electrical field stimulation (EFS)-induced nitric oxide-mediated off responses, and changes in the cholecystokinin (CCK)-8 dose–response relationship were measured. Caffeine; vinpocetine (VIN), type I PDE inhibitor; erythro-9-[2-hydroxy-3-nonyl]adenine HCl (EHNA), type II PDE inhibitor; zarderverine (ZARD), type III/IV PDE inhibitor; Ro 20-1724, type IV PDE inhibitor; and zaprinast (ZAP), type V PDE inhibitor were added to the tissue baths.Results. Caffeine and all specific PDE inhibitors decreased baseline tone. Caffeine, VIN, EHNA, ZARD, Ro 20-1724, and ZAP decreased the EFS-induced off response. Caffeine (10−5 M) and EHNA increased the CCK-8 dose–response contractions. This effect was not inhibited by atropine. Caffeine increased the tissue levels of cyclic 3′,5′-guanosine monophosphate but not cyclic 3′,5′-adenosine monophosphate. Caffeine decreases baseline tone and the off response via type I–V PDE pathways. Caffeine also increases CCK-induced gallbladder contractions via type II PDE pathways.Conclusion. These effects may be a mechanism that contributes to the decreased gallstone formation with caffeine consumption.

Different effects of specific phosphodiesterase inhibitors on oxidative stress during coronary autoregulation in isolated rat heart

Different effects of specific phosphodiesterase inhibitors on oxidative stress during coronary autoregulation in isolated rat heart

Calcium-independent and cAMP-dependent Modulation of Soluble Guanylyl Cyclase Activity by G Protein-coupled Receptors in Pituitary Cells

It is well established that G protein-coupled receptors stimulate nitric oxide-sensitive soluble guanylyl cyclase by increasing intracellular Ca(2+) and activating Ca(2+)-dependent nitric-oxide synthases. In pituitary cells receptors that stimulated adenylyl cyclase, growth hormone-releasing hormone, corticotropin-releasing factor, and thyrotropin-releasing hormone also stimulated calcium signaling and increased cGMP levels, whereas receptors that inhibited adenylyl cyclase, endothelin-A, and dopamine-2 also inhibited spontaneous calcium transients and decreased cGMP levels. However, receptor-controlled up- and down-regulation of cyclic nucleotide accumulation was not blocked by abolition of Ca(2+) signaling, suggesting that cAMP production affects cGMP accumulation. Agonist-induced cGMP accumulation was observed in cells incubated in the presence of various phosphodiesterase and soluble guanylyl cyclase inhibitors, confirming that G(s)-coupled receptors stimulated de novo cGMP production. Furthermore, cholera toxin (an activator of G(s)), forskolin (an activator of adenylyl cyclase), and 8-Br-cAMP (a permeable cAMP analog) mimicked the stimulatory action of G(s)-coupled receptors on cGMP production. Basal, agonist-, cholera toxin-, and forskolin-stimulated cGMP production, but not cAMP production, was significantly reduced in cells treated with H89, a protein kinase A inhibitor. These results indicate that coupling seven plasma membrane-domain receptors to an adenylyl cyclase signaling pathway provides an additional calcium-independent and cAMP-dependent mechanism for modulating soluble guanylyl cyclase activity in pituitary cells.

Differential effects of specific phosphodiesterase isoenzyme inhibitors on bovine oocyte meiotic maturation.

The differential regulation of cAMP levels within the oocyte and somatic (cumulus) cell compartments of the bovine follicle, and the subsequent regulation of oocyte meiotic maturation was examined through specific cell-type localisation of phosphodiesterases (PDEs). Selective PDE inhibitors were used to modulate cAMP levels in each of the two follicular compartments and to examine their effects on oocyte meiotic maturation. Ovaries were obtained from an abattoir and cumulus-oocyte complexes (COC) were aspirated from antral follicles into culture medium supplemented with 4 mg/ml BSA and 2mM 3-isobutyl-1-methylxanthine (IBMX). COC, denuded oocytes (DO), or mural granulosa cells (MGC) were cultured either with or without forskolin or FSH, in the presence of specific PDE inhibitors; either milrinone (PDE3 inhibitor), cilostamide (PDE3 inhibitor), or rolipram (PDE4 inhibitor). COC/DO cultures were assessed for meiotic progression and cAMP content, and MGC for cAMP production. The type 3 PDE inhibitor, but not the type 4, prevented spontaneous meiotic maturation and elevated intraoocyte cAMP in cultured denuded oocytes. In contrast, the type 4 PDE inhibitor had no effect on the oocyte, but elevated mural granulosa and cumulus cell cAMP production. The results of this study indicate that specific PDE subtypes are differentially localised within the two compartments of the bovine follicle-the type 3 PDE in the oocyte and the type 4 PDE in the granulosa cells. In addition, oocyte cAMP levels are primarily regulated in bovine oocytes by its degradation by PDE, whereas granulosa cell cAMP levels are controlled mainly by active adenylate cyclase, with both sources able to participate in oocyte meiotic regulation.

Benefit of vaginal sildenafil citrate in assisted reproduction therapy

Benefit of vaginal sildenafil citrate in assisted reproduction therapy

Characterisation of cyclic nucleotide phosphodiesterase isoforms in the media layer of the main pulmonary artery

Cyclic nucleotides are involved in the control of pulmonary vascular tone. In the present study, we measured the cyclic nucleotide specific phosphodiesterase (PDE) activity in the media of bovine isolated main pulmonary artery (MPA). Total cAMP- and cGMP-PDE activities were measured in microsomal and cytosolic fractions. Both cyclic nucleotides were hydrolysed in these subcellular fractions at consistently higher rate in the cytosolic than in the microsomal fraction. Using different classes of PDE modulator, at least four PDE isoforms (PDE1, 3, 4 and 5) were identified in these fractions. PDE3 (cilostamide-sensitive), PDE4 (rolipram-sensitive) and PDE5 (zaprinast- and DMPPO-sensitive) isoforms appeared as the main isozymes implicated in the cAMP and cGMP hydrolytic activities. Calcium–camodulin stimulated PDE activity (PDE1) was mainly present in the cytosolic fraction. PDE2, although present, had a lower hydrolytic activity since addition of its specific inhibitor, erythro-9-(2-hydroxy-3nonyl)adenine (EHNA), to a combination of inhibitors of PDE3, 4 and 5 produced no further significant reduction in the enzymatic activity. Resolution of PDE activities from the cytosolic fraction using anion exchange chromatography confirmed this finding. Functional experiments performed in endothelium-denuded rings of rat MPA revealed that all specific PDE inhibitors used relaxed precontracted vascular smooth muscle preparations in a concentration-dependent manner. The rank order of potency was cilostamide >zaprinast>rolipram>>EHNA. The present study demonstrates the presence in the smooth muscle cells-containing layer of MPA of PDE1, 3, 4 and 5 isoforms and suggests that PDE3, 4 and 5 are the main enzymes involved in the control of vascular tone.

Lysophosphatidic acid stimulates proliferation of cultured smooth muscle cells from human BPH tissue: sildenafil and papaverin generate inhibition.

The endogenous substance lysophosphatidic acid (LPA) has been found to generate proliferation of cultured smooth muscle cells (SMC). Therefore, the effect of LPA on human benign prostate hyperplasia (BPH) could be of interest. The proliferative effect of LPA on cultured human prostatic SMC from specimens obtained at trans-urethral resection of the prostate (TURP) because of BPH, was analyzed by [3H]-thymidine and [35S]-methionine incorporation. In addition, LPA stimulated BPH SMC were treated with papaverin, forskolin, sildenafil or zaprinast, well known to increase the intracellular level of cAMP or cGMP. LPA produced a dose-dependent increase in BPH SMC, both regarding DNA- and protein-synthesis with EC50 values of 3 and 10 microM, respectively. Furthermore, both papaverin, a general phosphodiesterase inhibitor regarding cAMP hydrolyzes, and forskolin, an adenylyl cyclase stimulating agent, inhibited the LPA-stimulated DNA replication in a dose dependent manner with IC50 = 2.5, and 0.35 microM, respectively. cGMP increasing agents, such as the NO-donors SIN-1 and SNAP, produced a weak anti-proliferative response. However, both phosphodiesterase 5 inhibitors sildenafil (Viagra) and zaprinast efficiently blocked DNA replication. In addition, when the protein synthesis was examined, we found that the LPA response was significantly inhibited by forskolin and papaverin. The major conclusion of this investigation is that the endogenous serum component LPA, is able to promote human BPH SMC growth. In addition, our study indicates that cyclic nucleotides can inhibit this effect. Future clinical studies will be needed to determine if different specific phosphodiesterase inhibitors per se or in combination could represent a new therapeutic possibility for the treatment of BPH.

Effect of type 3 and type 4 phosphodiesterase inhibitors on the maintenance of bovine oocytes in meiotic arrest.

The use of broad-spectrum inhibitors first suggested that phosphodiesterases (PDEs) are involved in the maturation of bovine oocytes. Modulation of individual PDE families is now possible with the use of newly developed type-specific PDE inhibitors. This study evaluated the role of type 3- and type 4-specific PDE inhibitors on the meiotic arrest of bovine cumulus-oocyte complexes (COCs) and denuded oocytes (DOs). It also evaluated the role of these specific inhibitors on meiotic arrest when COCs are incubated in the presence or absence of theca cell monolayers. Bovine COCs were aspirated from ovaries collected at the abattoir. Denuded oocytes and COCs were incubated for 12 h in culture medium alone or culture medium containing the type 3 PDE inhibitors cilostamide (10 and 20 microM) or milrinone (10 and 50 microM) or the type 4 PDE inhibitor rolipram (10 and 50 microM). Oocytes were then fixed and classified according to the status of nuclear maturation. Cumulus-oocyte complexes were coincubated with untreated theca cell monolayers or theca cell monolayers treated with the different specific PDE inhibitors. Bovine COCs or DOs incubated in culture medium resumed meiosis, but supplementation of the culture medium with the PDE3 inhibitors cilostamide or milrinone resulted in meiotic arrest. On the other hand, supplementation of the culture medium with rolipram did not prevent oocyte maturation. Furthermore, PDE3 inhibitors, but not PDE 4 inhibitors, had an additive effect on the inhibitory action of theca cell monolayers on oocyte maturation. These data support the hypothesis that inhibition of PDE3 prevents the meiotic resumption of bovine oocytes, whereas inhibition of PDE4 does not block oocyte maturation even under normally inhibitory conditions. The additive effect of PDE3 inhibitors on the ability of theca cells to maintain bovine oocytes in meiotic arrest suggests that type 3 PDE has an important role in meiotic resumption of bovine oocytes.