Inhibition by reproterol of cAMP PDE in intact mastocytoma P-815 cells

In vitro studies in rat mastocytes and human monocytes suggested that reproterol (a selective beta(2)-adrenoceptor agonist with a theophylline moiety) exerts anti-inflammatory actions through inhibition of cyclic AMP (cAMP) PDE activity. Thus, reproterol was tested for its ability to inhibit cAMP PDE in cultured mouse mastocytoma P-815 cells. cAMP PDE activity was measured in intact cells by spectrofluorometry using the fluorescent substrate 2'-O-anthraniloyl cAMP. Reproterol was more potent than theophylline to inhibit cAMP PDE (pIC(50)=4.28+/-0.25 vs. 3.16+/-0.05). This contrasted with disrupted cells, where the PDE inhibitory potency of reproterol was low (pIC(50)=2.85+/-0.03) and similar to that of theophylline (pIC(50)=2.66+/-0.19). No cAMP PDE inhibition was found with other beta(2)-agonists tested (fenoterol, salbutamol, salmeterol and formoterol). Finally, the selective PDE inhibitors calmidazolium (100 nM), milrinone (5 microM) and rolipram (50 microM) inhibited cAMP PDE activity by approximately 20, 30 and 25% respectively. In conclusion, reproterol potently and non-specifically inhibited intracellular cAMP phosphodiesterases in intact mastocytoma cells. This can explain the previously reported beta(2)-adrenoceptor-independent anti-inflammatory actions of reproterol in vitro. Further studies are required to define the anti-inflammatory potential of reproterol in asthma.     

Characterization of the phosphodiesterase (PDE) pattern of in vitro-generated human dendritic cells (DC) and the influence of PDE inhibitors on DC function

During differentiation of human monocytes (CD14(+)/CD1a(-)) to CD14(-)/CD1a(+)dendritic cells (DC), a drastic decrease in PDE4 activity was observed, while activities of PDE1 and PDE3 substantially increased. DC released tumour necrosis factor-alpha (TNF) in response to lipopolysaccharide (LPS) challenge, which was abolished both by dexamethasone and the cyclic AMP-elevating drugs db-cAMP and PGE(2). In addition, rolipram, at PDE4-selective concentrations, blocked TNF release by 37 +/- 5% (P<0.05 vs. control). The PDE3 inhibitor motapizone only marginally influenced TNF synthesis, but a synergistic inhibitory effect was noted in combination with rolipram. Qualitatively, similar inhibitory effects were observed in DC-stimulated T cell responses. Motapizone, lacking efficacy when used alone, increased the effect of rolipram in blocking CD4(+)T lymphocyte proliferation in response to antigen (Ag) (tetanus toxoid, TT; keyhole limpet hemocyanin, KLH) presented by DC and in allogeneic mixed leukocyte reactions (MLR). However, in these coculture systems the T cells rather than the DC seem to be the major target cells of PDE-inhibitor action. In summary, PDE inhibitors can affect DC function directly as demonstrated by blocking TNF release and their efficacy reflects the changes in the PDE activity profile during differentiation from their monocyte precursors. These results together with the known efficacy of PDE3/4 inhibitors in T cells support the concept of combined PDE3/4 inhibitors for asthma therapy.     

Phosphodiesterase inhibitors and forskolin Up-regulate arginase activity in rabbit alveolar macrophages

Alveolar macrophages (AMsmall ef, Cyrillic) express considerable arginase activity which can be modulated by various mediators. As inhibitors of phosphodiesterase (PDE) play an increasing role in the treatment of chronic inflammatory and obstructive airway disease, we tested whether PDE inhibitors affect arginase activity in AMsmall ef, Cyrillic. Isolated rabbit AMsmall ef, Cyrillic were cultured for 20 h in the absence or presence of bacterial lipopolysaccharides (LPS) and/or different test substances. Thereafter arginase activity was determined by measuring the formation of [(3)H]-L-ornithine during 1 h incubation with [(3)H]-L-arginine. Lipopolysaccharide-enhanced (0. 01-5 microg/ml) maximal arginase activity by about 2.5-fold. The non-selective PDE inhibitor IBMX and the PDE4 selective inhibitor rolipram (each up to 30 microM) caused a 2.4-fold increase in arginase activity, and these effects were additive to those of LPS. The PDE3-selective inhibitor siguazodan had only marginal effects. Forskolin (10 microM) also enhanced arginase activity in the absence and presence of LPS. The effect of forskolin was almost prevented by cycloheximide (30 microM) and largely attenuated by the protein kinase A inhibitor KT 5720 (300 nM). In conclusion, inhibition of the cAMP-specific PDE4, like direct activation of adenylyl cyclase, causes an up-regulation of arginase activity in rabbit AMsmall ef, Cyrillic.     

Phosphodiesterase and cyclic adenosine monophosphate-dependent inhibition of T-lymphocyte chemotaxis.

There is abundant evidence for T-lymphocyte recruitment into the airways in allergic inflammatory responses. This study has tested the hypothesis that T-cell chemotaxis induced by platelet-activating factor (PAF) and human recombinant interleukin-8 (hrIL-8) can be attenuated by inhibition of phosphodiesterase activity and raised intracellular 3',5'-cyclic adenosine monophosphate (cAMP) levels. This study used theophylline, a nonselective phosphodiesterase (PDE) inhibitor, and rolipram, a selective PDE4 inhibitor, to study the effect of PDE inhibition on T-cell chemotaxis. The beta2-adrenoceptor agonist, salbutamol, the adenylyl cyclase activator, forskolin, and the cAMP analogue, dibutyryl cAMP (db-cAMP), were used to demonstrate a role for raised cAMP levels. T-cells were obtained from 10 atopic asthmatics, and the phenotype of migrating cells was examined by flow cytometry. Theophylline caused an inhibition of both PAF-and hrIL-8-induced chemotaxis (mean+/-SEM maximum inhibition at 1 mM: 73+/-4% and 48+/-8% for hrIL-8 and PAF, respectively) that was not specific for the CD4+, CD8+, CD45RO+ or CD45RA+ T-cell subsets. T-cell chemotaxis was more sensitive to treatment with rolipram whose effect was already significant from 0.1 microM on hrIL-8-induced chemotaxis. Both a low concentration of salbutamol (0.1 mM) and forskolin (10 microM) potentiated the inhibitory effect of a low concentration of theophylline (25 microM) on responses to PAF but not to hrIL-8. Finally, T-cell chemotaxis was also inhibited by db-cAMP. It is concluded that attenuation of T-cell chemotaxis to two chemoattractants of relevance to asthma pathogenesis can be achieved via phosphodiesterase inhibition and increased intracellular 3', 5'-cyclic monophosphate using drugs active on cyclic nucleotide phosphodiesterase. This action may explain the anti-inflammatory effects of theophylline and related drugs in asthma.     

Allergen challenge alters lymphocyte phosphodiesterase activity in horses with heaves

Heaves is an allergic airway disease in horses characterised by reversible airway obstruction, bronchial hyperresponsiveness and airway inflammation associated with a Th(2) response. Cyclic nucleotide-dependent signalling pathways can regulate lymphocyte function. In this study, we examined lymphocyte PDE activity comparing horses with heaves to healthy control animals. Total PDE activity and the effects of isoenzyme selective inhibitors were measured before, 5 and 24 h after the start of a 7 h allergen challenge. Allergen challenge had no effect on either total cAMP PDE activity or its inhibition by the PDE4 selective inhibitor, rolipram, and the non-selective PDE inhibitor, theophylline. In contrast, the PDE3 selective inhibitor, quazinone, caused significantly greater inhibition of cAMP PDE activity before challenge in the heaves susceptible group. Additionally, total cGMP PDE activity was significantly lower 24 h after the start of challenge in the heaves affected group (11+/-2 and 21+/-3 pmol/min/mg for heaves and control animals, respectively) and the PDE5 selective inhibitor, zaprinast, caused significantly less inhibition in the heaves group at this time point. The functional significance of these findings was explored by examining the effect of PDE3, PDE4 and PDE5 selective inhibitors on mitogen-induced mononuclear cell proliferation before and 24 h after the start of allergen challenge. Proliferation decreased after challenge in the heaves group (stimulation index=328+/-110 and 200+/-72 before and after challenge, respectively) whilst remaining constant in the control group (stimulation index=161+/-13 and 183+/-45 before and after challenge, respectively). However, all three PDE inhibitors caused a similar amount of inhibition at each time point and the effect of a combination of a PDE3 and a PDE5 inhibitor was simply additive in both groups. These results suggest differences in the control of lymphocyte PDE activity in horses with heaves.     

The effect of selective phosphodiesterase isoenzyme inhibition on neutrophil function in vitro

Neutrophil-derived proteases such as neutrophil elastase (NE) and matrix metalloproteinase (MMP) are implicated in the pathogenesis of Chronic Obstructive Pulmonary Disease (COPD). In this study, the effects of selective phosphodiesterase (PDE) inhibition on NE and MMP-9 release, as well as Myeloperoxidase (MPO) activity and integrin-mediated neutrophil adhesion to human umbilical vein endothelial cells (HUVECs), were investigated. Human neutrophils were treated with PDE inhibitors (10(-11)-10(-4)M) in the absence and presence of TNF-alpha (tumour necrosis factor) (100 U ml(-1)) for 30 min, prior to fMLP activation. After 45 min, the cells were removed and NE, MPO and MMP-9 release assessed. In the adhesion studies, the neutrophils were radio-labelled with 51Cr, stimulated and immediately transferred to cultured HUVEC monolayers for 30 min, prior to assessment of adhesion. TNF-alpha (100 U ml(-1)) acted synergistically with fMLP in stimulating azurophil degranulation with respect to both MPO activity (P<0.01) and NE release (P<0.01). In contrast, an additive effect was observed with TNF-alpha and fMLP with regard to MMP-9 release and neutrophil adhesion to HUVECs. The PDE4 inhibitors, roflumilast, roflumilast N-oxide, cilomilast and rolipram significantly suppressed MPO, NE and MMP-9 release in both the presence and absence of TNF-alpha (P<0.05; n=6-10) and also reduced neutrophil adhesion to HUVECs. In contrast, milrinone, a PDE3 inhibitor and the non-selective PDE inhibitor, theophylline did not inhibit azurophil degranulation under any of the experimental conditions. These data provide further evidence that selective PDE4 isoenzyme inhibitors can inhibit neutrophil degranulation, effects not shared by PDE3 inhibitors or theophylline.     

Pharmacology of a new cyclic nucleotide phosphodiesterase type 4 inhibitor,V11294

V11294 is a new cyclic nucleotide phosphodiesterase type 4 (PDE4) inhibitor of the rolipram class. In this report we present the pharmacological profile of V11294. V11294 inhibited PDE4 isolated from human lung with IC(50) 405 nM, compared to 3700 nM for rolipram. In contrast, V11294 inhibition of human PDE3 and PDE5 occurred only at concentrations greater than 100,000 nM. Like rolipram, V11294 inhibited PDE4D more potently than other PDE4 subtypes. V11294, when incubated with human anticoagulated whole blood in vitro, or administered to mice, caused increased cAMP concentration, consistent with inhibition of PDE4. V11294 inhibited lectin-induced proliferation and lipopolysaccharide-induced TNFalpha synthesis by human adherent monocytes in vitro and inhibited lipopolysaccharide-induced TNFalpha synthesis in mice. V11294 caused relaxation of guinea pig isolated trachea and inhibited allergen-induced bronchoconstriction and eosinophilia in guinea pigs at doses of 1 and 3 mg/kg, p.o. In ferrets, V11294 was not emetogenic at doses up to 30 mg/kg, p.o., despite plasma concentration reaching 10-fold the IC(50) for PDE4. In contrast, rolipram induced severe retching and vomiting at 10 mg/kg, p.o. In conclusion, V11294 is an orally active PDE4 inhibitor that exhibits antiinflammatory activity in vitro, and in vivo at doses that are not emetogenic.     

Expiratory flows and airway inflammation in elderly asthmatic patients

Asthma in the elderly is often underrecognized and suboptimally treated, resulting in an increased morbidity and mortality. The characteristics of asthma-related bronchitis and its optimal treatment remain to be determined in this population. We aimed to compare lung function and airway inflammation in elderly and younger asthmatic subjects. Data from two induced sputum databases were analyzed in three groups of asthmatic subjects (18-30 y, n = 136; 31-59 y, n = 385; 60-72 y, n = 172) and one group of healthy elderly subjects (60-89 y, n = 16). Expiratory flows and induced sputum cell counts were analyzed. Airway obstruction was more marked in elderly asthmatics compared with healthy elderly or younger asthmatic subjects (p < 0.01). An increase in sputum neutrophils and a decrease in macrophages and lymphocytes were observed in elderly asthmatics (p < 0.0001). Neutrophil percentages significantly increased with asthma severity in the young and the middle-aged groups, while they remained similar in elderly asthmatics regardless of asthma severity (p < 0.05). Neutrophil percentages weakly correlated with the dose of ICS in all asthmatics (r = 0.17, p < 0.0001). Age and dose of ICS were independent predictors of neutrophil percentage in asthmatic subjects in a regression model (R(2) = 0.12). Asthma in the elderly is associated with a more marked airway obstruction and sputum neutrophilia. Both age and the dose of corticosteroids need to be considered in the interpretation of the clinical relevance of sputum neutrophil count.     

Mitigation of Stress-Induced Gastric Mucosal Lesions by a Specific Type IV Phosphodiesterase Inhibitor

Inhibition of type IV phosphodiesterase (PDE4) activity is beneficial in various inflammations. However, the effect of phosphodiesterase inhibitors on the development of stress-induced gastric mucosal lesions has not been reported. In the present study, we examined the effect of a specific PDE4 inhibitor (rolipram) on stress-induced gastric mucosal lesions. Rats were exposed to water-immersion stress with or without pretreatment with rolipram. Ulcer index and myeloperoxidase activity of the gastric mucosa were evaluated. Gastric mucosal lesions and mucosal myeloperoxidase activity were suppressed by treatment with rolipram without acid suppression. The effect of intraperitoneal administration of 2.5 mg/kg rolipram on suppression of mucosal lesions was almost equal to that of treatment with 200 mg/kg cimetidine. We demonstrated that a specific PDE4 inhibitor has a potent anti-ulcer effect presumably mediated by an increment in intracellular cAMP in inflammatory cells, in which this enzyme is abundantly and specifically expressed.     

The novel phosphodiesterase 4 inhibitor, CI-1044, inhibits LPS-induced TNF-alpha production in whole blood from COPD patients

Chronic obstructive pulmonary disease (COPD) is a common, progressive respiratory disease that causes great morbidity and mortality despite treatment. Tumor necrosis factor alpha (TNF-alpha) plays a central role as a pro-inflammatory cytokine in COPD. TNF-alpha release is markedly inhibited by phosphodiesterase type 4 (PDE4) inhibitors that have proven efficacious in COPD clinical trials. The aim of this study was to compare the in vitro activities of the novel selective PDE4 inhibitors CI-1044 compared to well-known PDE4 inhibitors, rolipram and cilomilast, and to the glucocorticoid dexamethasone at reducing lipopolysaccharide (LPS)-induced TNF-alpha release in whole blood from COPD patients and healthy subjects. In the whole blood from COPD patients pre-incubation with PDE4 inhibitors or dexamethasone resulted in a dose-dependent inhibition of LPS-induced TNF-alpha release with IC(50) values of 1.3+/-0.7, 2.8+/-0.9 microM, higher to 10 microM and lesser than 0.03 microM for CI-1044, rolipram, cilomilast and dexamethasone, respectively. We observed a similar inhibition in the whole blood from healthy volunteers with, however, higher IC(50) values. These results indicate that CI-1044 inhibits in vitro LPS-induced TNF-alpha release in whole blood from COPD patients better than rolipram and cilomilast and suggested that it could be a useful anti-inflammatory therapy in COPD.     

Inhibition of PDE4 phosphodiesterase activity induces growth suppression, apoptosis, glucocorticoid sensitivity, p53, and p21(WAF1/CIP1) proteins in human acute lymphoblastic leukemia cells

Glucocorticoids are integral to successful treatment of childhood acute lymphoblastic leukemia (ALL) and other lymphoid malignancies. A large body of data indicates that in various model systems, elevation of cyclic adenosine monophosphate (cAMP) can potentiate glucocorticoid response, although this has not been well evaluated as a potential leukemia treatment. Although cAMP analogs have been studied, little data exist regarding the potential toxicity to leukemia cells of pharmacologic elevation of cAMP levels in leukemic blasts. Using MTT assays of cell proliferation on CEM ALL cells, we found that aminophylline and other nonspecific phosphodiesterase (PDE) inhibitors suppress cell growth. This effect is replicated by the PDE4-specific PDE inhibitor rolipram, but not by specific inhibitors of the PDE1 or PDE3 classes. We found that PDE inhibitors cause increased dexamethasone sensitivity and a synergistic effect with the adenylyl cyclase activator forskolin. We observed several important cellular characteristics associated with this treatment, including elevation of cAMP, induction of p53 and p21(WAF1/CIP1) proteins, G(1) and G(2)/M cell cycle arrest, and increased apoptosis. Sensitivity to forskolin and rolipram is shared by at least 2 pediatric ALL cell lines, CEM and Reh cells. Some cell lines derived from adult-type lymphoid malignancies also show sensitivity to this treatment. These findings suggest that PDE inhibitors have therapeutic potential in human ALL and characterize the molecular mechanisms that may be involved in this response.     

Comparison of the effects of cilostazol and milrinone on cAMP-PDE activity,intracellular cAMP and calcium in the heart

We investigated the basis for the difference in the cardiotonic effects of the PDE3 inhibitors cilostazol and milrinone in the rabbit heart. Cilostazol displayed greater selectivity than milrinone for inhibition of cAMP-PDE activity in microsomal vs cytosolic fractions from rabbit heart. This difference was due to the inhibition of significantly less cytosolic cAMP-PDE activity by cilostazol compared to milrinone. A combination of cilostazol (>15 M) and the PDE4 selective inhibitor, rolipram (5 M), inhibited levels of cytosolic cAMP-PDE activity similar to those inhibited by milrinone on its own. This suggested that milrinone inhibited PDE4 in addition to PDE3 activity. In isolated rabbit cardiomyocytes, milrinone (>10 M) caused greater elevations in intracellular cAMP and calcium than cilostazol. In the presence of rolipram, however, the cAMP and calcium elevating effects of cilostazol and milrinone were similar. Therefore, in rabbit heart, partial inhibition of PDE4 by milrinone contributed to greater increases in cardiomyocyte cAMP and calcium levels than cilostazol. PDE4 activity in failing human heart was lower than in rabbit heart and there was no significant difference in the inhibition of human cytosolic cAMP-PDE by cilostazol and milrinone. Our results suggest that in normal rabbit heart inhibition of PDE4 by milrinone may partly contribute to the greater cardiotonic effect of milrinone when compared to cilostazol. However, the lower level of PDE4 activity in failing human heart suggests that factors other than inhibition of PDE4 by milrinone may contribute to differences in cardiotonic action when compared to cilostazol.     

Selenium, glutathione peroxidase and superoxide dismutase in maltese asthmatic patients: effect of glucocorticoid administration

Oxidative processes, mediated by free radical chemistry, are recognized to contribute significantly to the inflammatory pathology of bronchial asthma. This study analysed the degree of defence against reactive oxygen species in Maltese, asthmatic patients and in normal individuals, by measuring plasma selenium concentration, erythrocyte glutathione peroxidase (GSH-Px) activity and erythrocyte superoxide dismutase (SOD) activity, in order to determine their antioxidant status. The effect of glucocorticoids on the status of these antioxidants in patients was also investigated. The measurement of antioxidant status was carried out both in mild (n = 22) and severe (n = 37) asthmatics, as well as in healthy controls (n = 49). The same antioxidant profile was then investigated in a group of 16 severe asthmatics following treatment for 4 weeks with inhaled beclomethasone dipropionate (750 micrograms twice daily), and in a second group of 16 patients suffering from severe asthma, following 2-weeks treatment with oral prednisolone (15 mg daily during the first week and 10 mg daily during the second). No statistically significant difference was found in the plasma selenium concentrations and erythrocyte glutathione peroxidase activities between patients and controls. Both mild and severe asthmatics, however, exhibited a statistically significant lower erythrocyte superoxide dismutase activity than normal subjects (mild asthmatics: 62.9 (2.9) SOD 525 U/ml, severe asthmatics: 60.6 (1.9) SOD 525 U/ml, normal: 68.5 (1.1) SOD 525 U/ml, P < 0.01). Inhaled beclomethasone dipropionate exerted no effect on this antioxidant profile, while prednisolone caused a significant increase in plasma selenium concentration over pretreatment values (pretreatment: 118.3 (4.4) ng/ml, post-treatment: 138.1 (4.6 ng/ml, P < 0.01). It is thus suggested that asthmatic patients in Malta might be more susceptible to superoxide-induced damage than normal individuals. The reason for the prednisolone-induced augmentation of plasma selenium could not be determined from this study. It is postulated that the drug may decrease the excretion rate of the element, and may thus exert a positive antioxidant effect in individuals of established low selenium status.     

Regulation by phosphodiesterase isoforms of protein kinase A-mediated attenuation of myocardial protein kinase D activation

Protein kinase D (PKD) targets several proteins in the heart, including cardiac troponin I (cTnI) and class II histone deacetylases, and regulates cardiac contraction and hypertrophy. In adult rat ventricular myocytes (ARVM), PKD activation by endothelin-1 (ET1) occurs via protein kinase Cε and is attenuated by cAMP-dependent protein kinase (PKA). Intracellular compartmentalisation of cAMP, arising from localised activity of distinct cyclic nucleotide phosphodiesterase (PDE) isoforms, may result in spatially constrained regulation of the PKA activity that inhibits PKD activation. We have investigated the roles of the predominant cardiac PDE isoforms, PDE2, PDE3 and PDE4, in PKA-mediated inhibition of PKD activation. Pretreatment of ARVM with the non-selective PDE inhibitor isobutylmethylxanthine (IBMX) attenuated subsequent PKD activation by ET1. However, selective inhibition of PDE2 [by erythro-9-(2-hydroxy-3-nonyl) adenine, EHNA], PDE3 (by cilostamide) or PDE4 (by rolipram) individually had no effect on ET1-induced PKD activation. Selective inhibition of individual PDE isoforms also had no effect on the phosphorylation status of the established cardiac PKA substrates phospholamban (PLB; at Ser16) and cTnI (at Ser22/23), which increased markedly with IBMX. Combined administration of cilostamide and rolipram, like IBMX alone, attenuated ET1-induced PKD activation and increased PLB and cTnI phosphorylation, while combined administration of EHNA and cilostamide or EHNA and rolipram was ineffective. Thus, cAMP pools controlled by PDE3 and PDE4, but not PDE2, regulate the PKA activity that inhibits ET1-induced PKD activation. Furthermore, PDE3 and PDE4 play redundant roles in this process, such that inhibition of both isoforms is required to achieve PKA-mediated attenuation of PKD activation.     

Evidence of oxidative stress in asthma and COPD: potential inhibitory effect of theophylline

To evaluate the potential inhibitory effect of theophylline on the pulmonary oxidative stress in asthma and chronic obstructive pulmonary disease (COPD), we concomitantly measured the blood levels of theophylline, a non-selective phosphodiesterase (PDE) inhibitor and lipid peroxides as an index of oxidative stress. The plasma levels of lipid peroxides were significantly elevated in patients with asthma (3.48 +/- 0.11 nmol ml(-1); mean +/- SEM; n=21, P<0.01), non- or ex-smoking patients with COPD (3.55 +/- 0.11 nmol ml(-1); n = 20, P<0.01), and current-smoking patients with COPD (3.53 +/- 0.15 nmol ml(-1); n = 15, P<0.01), respectively, as compared to those of non-smoking controls (3.02 +/- 0.08 nmol ml(-1); n = 19). There was a significant negative correlation between the plasma level of lipid peroxides and the forced expiratory volume in 1 sec (FEV1)% of forced vital capacity in these subjects (r = -0.304; n = 75, P < 0.01). In asthmatics, there was a significant negative correlation between the plasma level of lipid peroxides and the serum level of theophylline (r = -0.495; n = 18, P<0.05). These results suggest that there may be increased oxidative stress in patients with asthma and COPD, and indicate that oxidative stress could possibly attribute to the pathophysiology of asthma and COPD in leading to airflow obstruction and that theophylline could potentially inhibit oxidative stress in the process of bronchopulmonary inflammation in asthmatics.     

Phosphodiesterase 4 inhibition synergizes with relaxin signaling to promote decidualization of human endometrial stromal cells

The decidualization of endometrial stromal cells in the secretory phase of the menstrual cycle is an essential prerequisite for the implantation of a blastocyst. This profound differentiation process is accompanied by sustained elevated intracellular cAMP concentrations in vivo. Primary cell cultures of endometrial stromal cells decidualize by treatment with cAMP-elevating agents in vitro. Our previous findings indicated that the cAMP-degrading activities of phosphodiesterases (PDE) and signaling of the peptide hormone relaxin are coupled in human endometrial stromal cells. In the present study we have chosen a pharmacological approach to test whether relaxin binding and PDE inhibition cooperate to induce decidualization. Measurement of PDE activity and relaxin-stimulated cAMP accumulation in the presence of diverse PDE inhibitors identified PDE4 and PDE8 as the principal PDE isoforms involved in human endometrial stromal cells. The PDE4 inhibitor rolipram was most effective in elevating intracellular cAMP concentrations and synergizing with relaxin to achieve maximal in vitro decidualization, as determined by measurement of the expression of the decidual marker genes for prolactin and IGF-binding protein-1 and measurement of prolactin secretion. Gene expression for PDE4D and PDE4C was significantly up-regulated during in vitro decidualization. Treatment of cell cultures with the protein kinase A inhibitor H89 revealed a minor role for protein kinase A-mediated positive feedback control of PDE4 activity in human endometrial stromal cells, consistent with sustained elevated cAMP essential for decidualization in vitro. These findings introduce the new idea of clinically applying the combination of a specific PDE4 inhibitor with an effector such as relaxin, thereby offering an alternative nonsteroidal luteal phase support for the endometrium to encourage endometrial development and implantation in subfertile women undergoing assisted reproductive technology procedures.     

Rolipram, salbutamol and prostaglandin E2 suppress TNFalpha release from human monocytes by activating Type II cAMP-dependent protein kinase

The extent to which cAMP-dependent protein kinase (PKA) mediates the inhibitory effects of cAMP-elevating drugs on tumour necrosis factor (TNF) alpha release from lipopolysaccharide (LPS)-stimulated human monocytes is equivocal. Here, we have investigated the role of this kinase by exploiting the ability of certain novel cAMP analogues to inhibit or activate PKA and the recently described cAMP-guanine nucleotide-exchange factors (GEFs). Pre-treatment of monocytes with Rp-8-Br-cAMPS, a selective inhibitor of Type I PKA that has no effect on basal or stimulated Rap1 (a downstream effector of cAMP-GEFs) activity, potentiated LPS-induced TNFalpha output but had little or no effect on the suppression of this cytokine effected by rolipram (a PDE4 inhibitor), prostaglandin (PG) E2 and salbutamol (a beta2-adrenoceptor agonist). In contrast, Rp-8-pCPT-cAMPS, which selectively blocks Type II PKA with only weak activity against Rap1, significantly antagonised or abolished the inhibitory effect of these cAMP-elevating agents. Pre-treatment of monocytes with 8-pCPT-2'-O-Me-cAMPS, a potent activator of cAMP-GEFs, failed to suppress TNFalpha output at concentrations known to profoundly activate Rap1. Collectively, these results indicate that cAMP-elevating drugs suppress TNFalpha release from LPS-stimulated human monocytes by activating PKA independently of cAMP-GEFs. Furthermore, by using phosphorothioate cAMP analogue PKA inhibitors we provide evidence that the Type II PKA isoenzyme is functionally the most important.     

Control of renin secretion from rat juxtaglomerular cells by cAMP-specific phosphodiesterases

We tested the hypothesis that cGMP stimulates renin release through inhibition of the cAMP-specific phosphodiesterase 3 (PDE3) in isolated rat juxtaglomerular (JG) cells. In addition, we assessed the involvement of PDE4 in JG-cell function. JG cells expressed PDE3A and PDE3B, and the PDE3 inhibitor trequinsin increased cellular cAMP content, enhanced forskolin-induced cAMP formation, and stimulated renin release from incubated and superfused JG cells. Trequinsin-mediated stimulation of renin release was inhibited by the permeable protein kinase A antagonist Rp-8-CPT-cAMPS. PDE4C was also expressed, and the PDE4 inhibitor rolipram enhanced cellular cAMP content. Dialysis of single JG cells with cAMP in whole-cell patch-clamp experiments led to concentration-dependent, biphasic changes in cell membrane capacitance (C(m)) with a marked increase in C(m) at 1 micromol/L, no net change at 10 micromol/L, and a decrease at 100 micromol/L cAMP. cGMP also had a dual effect on C(m) at 10-fold higher concentration compared with cAMP. Trequinsin, milrinone, and rolipram mimicked the effect of cAMP on C(m). Trequinsin, cAMP, and cGMP enhanced outward current 2- to 3-fold at positive membrane potentials. The effects of cAMP, cGMP, and trequinsin on C(m) and cell currents were abolished by inhibition of protein kinase A with Rp-cAMPs. We conclude that degradation of cAMP by PDE3 and PDE4 contributes to regulation of renin release from JG cells. Our data provide evidence at the cellular level that stimulation of renin release by cGMP involves inhibition of PDE3 resulting in enhanced cAMP formation and activation of the cAMP sensitive protein kinase.