Search

Aims Prostaglandin E2 (PGE2) has been shown to mediate anti-ischaemic effects and cardiomyocyte hypertrophy and there is evidence for an involvement of the prostaglandin EP3-receptor subtype. This study focuses on the EP3-mediated hypertrophic action and investigates intracellular signalling pathways of the EP3-receptor subtype in the murine heart. Methods and results Cardiac function was analyzed in vivo by magnetic resonance imaging (MRI) in transgenic (tg) mice with cardio-specific overexpression of the EP3 receptor in comparison with wild-type (wt) mice. Left ventricular (LV) function was determined in isolated perfused hearts subjected to 60 min of zero-flow ischaemia and 45 min of reperfusion. Calcineurin activity and nuclear activity of nuclear factor of activated T-cells (NFAT) were determined by a modified malachite green assay and ELISA, respectively. Extracellular matrix compounds were analyzed by RT–PCR and histology. MRI indicated a significant increase in end-diastolic and end-systolic volume in tg hearts. LV ejection fraction was severely decreased in tg hearts while the relative LV mass was significantly increased. In Langendorff perfused hearts, EP3-receptor overexpression resulted in a marked blunting of the ischaemia-induced increase in LV end-diastolic pressure and creatine kinase release. Analysis of EP3-receptor-mediated signalling revealed significantly increased calcineurin activity and nuclear activity of NFAT in tg hearts. Moreover, elevated mRNA levels of collagen types I and III as well as the collagen-binding proteoglycans biglycan and decorin were detected in tg hearts. Conclusion EP3-receptor-mediated signalling results in a significant anti-ischaemic action and activation of the pro-hypertrophic calcineurin signalling pathway, suggesting the involvement of the EP3 subtype in both PGE2-mediated cardioprotection as well as cardiac hypertrophy.

Background: Cyclooxygenase-2 (COX-2), the inducible isoform of the cyclooxygenases, is upregulated in various inflammatory renal diseases and responsible for prostaglandin formation. As prostaglandins are known to influence cell adhesion processes, we investigated the effect of COX-2 inhibition on the expression of αv integrins, which are also enhanced in renal diseases and control the adherence between the endothelium and the extracellular matrix (ECM) in the glomerulus. Methods: Healthy female Wistar rats and animals with previously induced passive Heymann nephritis (PHN) received either 5 mg/kg body weight/day celecoxib or a placebo. After 28 days, renal cortical mRNA expression of COX-2 and αv integrin subunits was determined. Results: Rats with PHN showed a significant 1.7-fold increase in renal cortical mRNA expression of αv integrin subunits. Treatment with celecoxib increased cortical αv integrin mRNA expression 2.2-fold (p < 0.05) in healthy animals and 4.0-fold (p < 0.05) in rats with PHN, but lowered COX-2 mRNA expression in rats with PHN to 0.8-fold (p < 0.05). An inverse correlation between the expression of COX-2 and αv integrins in rats with PHN was demonstrated. Conclusions: It is suggested that COX-2-derived prostaglandins suppress the expression of αv integrins. This implies a previously unknown role for COX-2 in chronic inflammation in the kidney.

Migration and proliferation of medial smooth muscle cells (SMC) in the arterial intima contributes to the development of atherosclerotic plaques and restenotic processes after coronary angioplasty. Prostacyclin (PGI 2 )-mediated stimulation of cyclic adenosine 3′5′-monophosphate (cAMP) signaling is believed to be important for maintaining SMC in a quiescent state. In order to identify new cellular targets of PGI 2 /cAMP action, we have used microarray screening to examine changes in the transcriptional profile in human vascular SMC in response to exposure to the stable PGI 2 mimetic iloprost. We have identified 83 genes with significantly altered expression after iloprost (100 nM) exposure for 6 hr. Fifty-one genes were upregulated, among them stanniocalcin precursor (18.8±2.7), zinc finger transcription factor (7.8±2.0), hyaluronan synthase 2 (6.8±1.8), cyclooxygenase 2 (4.7±0.8), dual specific phosphatase (3.9±0.5) and vascular endothelial growth factor (2.3±0.4). Thirty-two genes were reduced, among them cystein-rich angiogenic protein (−14.9±1.3), monocyte chemotactic protein 1 (−7.4±1.1) and plasminogen activator inhibitor PAI-1 (−4.5±0.5). By means of semi-quantitative RT-PCR, time-courses of gene expression were established. The present study identified genes not hitherto recognized to be targets of PGI 2 action, providing further insight into its cAMP-mediated effects on SMC growth, migration and matrix secretion.

Prostacyclin and its mimetics have repeatedly been shown to act antiatherogenic and to inhibit neointima formation in several animal models of vascular injury. Treatment of human vascular smooth muscle cells with the prostacyclin mimetic iloprost (100 nm) drastically reduces expression of Cyr61, encoding the growth-regulatory cystein-rich angiogenic protein, without affecting the degradation rate of Cyr61 mRNA. Thrombin-induced Cyr61 expression was inhibited completely in the presence of iloprost. It is concluded that vasoprotective actions of prostacyclin in vivo may in part be due to inhibition of expression of the growth regulatory gene Cyr61 at sites of vascular lesions.

Prostaglandin-endoperoxide synthase-2 (PGH-synthase) or cyclooxygenase-2 (COX-2) is inducible by a variety of stimuli, e.g. inflammatory mediators, growth factors and hormones and is believed to be responsible for the majority of inflammatory prostanoid production. Moreover, it has been demonstrated that COX-2 contributes substantially to prostacyclin-synthesis in patients with atherosclerosis. In this study, we demonstrate an up-regulation of COX-2 mRNA, protein and product formation by the prostacyclin-mimetic iloprost in human vascular smooth muscle cells (hSMC). COX-2 mRNA expression was induced transiently between 1 and 6 hr and returned to basal levels after 16 hr of iloprost stimulation. COX-2 protein was induced concomitantly between 3 and 6 hr of iloprost stimulation. This was accompanied by an increase in PGI 2 formation. Forskolin, a direct activator of adenylyl cyclase, and dibutyryl cAMP, a cell-permeable cAMP analogue-induced COX-2 mRNA, suggesting a cAMP-dependent COX-2 expression in hSMC. Iloprost-induced COX-2 protein expression and PGI 2 formation was synergistically elevated by co-stimulation with the phorbolester PMA (phorbol-12-myristate-13-acetate). It is concluded, that the observed up-regulation of COX-2 with subsequent release of newly synthesized PGI 2 and the synergistic effect of iloprost and phorbolester on PGI 2 formation provide a positive feedback of prostaglandins on their own synthesizing enzyme. This might be important for control of hSMC proliferation, migration and differentiation as well as inhibition of platelet aggregation.

Background — Cyclooxygenase-2 (COX-2) plays a key role in human inflammatory disorders such as vascular inflammation. COX-2 promoter activity is induced by proinflammatory mediators, but the role of cyclic adenosine monophosphate response element (CRE) in promoter stimulation remains unclear. Methods and Results — Transient transfection of a 0.9-kb COX-2 promoter fragment bearing CRE mutation abrogated COX-2 promoter activity induced by proinflammatory mediators in human endothelial cells and fibroblasts. Dual mutations of CRE and an upstream CCAAT/enhancer binding protein (C/EBP) site did not have an additional effect. Binding of CREB-2, ATF-2, USF-2, and c-Jun transactivators to a wild-type and CRE-mutated oligonucleotide was analyzed by a novel DNA-binding assay. CREB-2 and ATF-2 in nuclear extracts of unstimulated endothelial cells bound to CRE, whereas USF-2 and c-Jun or c-Fos bound to non-CRE sites. CREB-2 and c-Fos binding was increased by phorbol 12-myristate 13-acetate but not tumor necrosis factor-α. The binding assay and chromatin immunoprecipitation revealed binding of P300 coactivator to the COX-2 promoter region. Conclusions — CRE plays an obligatory role in COX-2 promoter activation by diverse stimuli. CREB-2 and ATF-2 bound to CRE serve as an anchor for P300 interaction with upstream transactivators and downstream transcription machinery.

Abstract The action of prostacyclin, prostaglandin E 1 (PGE 1 ), and their mimetics on myocardial function includes changes in contractility, electrophysiological properties, and protection from injury caused by transient myocardial ischemia. This study was undertaken to investigate the basic properties of myocardial E-type prostaglandin (EP) receptors. Ligand binding studies using an enriched preparation of sarcolemmal membranes prepared from pig hearts revealed a single class of binding sites for [ 3 H]PGE 1 , with a K d of 3.7 nmol/L and a B max of 92 fmol/mg protein. Competition experiments indicated highest affinity for EPs, suggesting an EP receptor. In addition, the EP receptor subtype–selective agonists sulprostone (EP 1 and EP 3 ) and M&B 28.767 (EP 3 ) were active, suggesting the presence of an EP 3 receptor subtype. PGE 1 stimulated sarcolemmal GTPase and inhibited sarcolemmal adenylyl cyclase activity, indicating EP 3 receptor coupling to an inhibitory G protein (G i ). Additional in vivo experiments showed that intracoronary infusion of PGE 1 (1 nmol/min) decreased isoprenaline-stimulated left ventricular contractile activity without altering systemic vascular resistance. This inhibition of β-adrenergic effects is compatible with the known myocardial anti-ischemic action of prostaglandins. Further experiments examined EP 3 receptor density and G-protein coupling in sarcolemma from ischemic and reperfused ischemic myocardium. In anesthetized open-chest minipigs, occlusion of the left anterior descending coronary artery for 60 minutes increased EP 3 receptor density by 50%, whereas receptor affinity was unchanged. This upregulation was prevented by pretreatment with colchicine (2 mg/kg IV), indicating microtubule-dependent receptor externalization. Northern hybridization showed comparable EP 3 receptor mRNA expression in control and ischemic myocardium. The increase of receptor protein was reversed during 60 minutes of reperfusion. G-protein coupling proved to be intact in ischemic and reperfused ischemic myocardial tissue, as shown by preserved GTP-γ-S–induced decrease of [ 3 H]PGE 1 binding. These data demonstrate for the first time that myocardial receptors for PGE 1 belong to the EP 3 subtype. The properties of this receptor include inhibition of adenylyl cyclase and upregulation during regional myocardial ischemia, suggesting an involvement in the anti-ischemic activity of E- and I-type prostaglandins.

Background— The generation of prostaglandin E 2 (PGE 2 ) is significantly increased in acute myocardial ischemia and reperfusion. PGE 2 , in addition to other prostaglandins, protects the reperfused ischemic myocardium. It has been hypothesized that this cardioprotection is mediated by E-type prostaglandin receptors of the G i -coupled EP 3 subtype. Methods and Results— We tested this hypothesis by generating transgenic (tg) mice with cardiospecific overexpression of the EP 3 receptor. According to ligand binding, a 40-fold overexpression of the EP 3 receptor was achieved in membranes prepared from tg hearts compared with wild-type (wt) littermates. In isolated cardiomyocytes from tg mice, the forskolin-induced rise in cAMP was markedly attenuated, indicating coupling of the overexpressed EP 3 receptor to inhibitory G proteins (G i ) with constitutive receptor activity. There was no evidence for EP 3 receptor coupling to G q -mediated protein kinase C signaling. Isolated hearts from tg and wt mice were subjected to 60 minutes of no-flow ischemia and 45 minutes of reperfusion. In tg hearts, ischemic contracture was markedly delayed compared with wt hearts, and the ischemia-induced increase in left ventricular end-diastolic pressure was reduced by 55%. Creatine kinase and lactate dehydrogenase release was significantly decreased by 85% and 73%, respectively, compared with wt hearts. Conclusions— Constitutive prostaglandin EP 3 receptor signaling exerts a protective effect on cardiomyocytes, which is probably G i mediated and results in a remarkable attenuation of myocardial injury during ischemia and reperfusion. Cardioprotective actions of E-type prostaglandins may be mediated by this receptor subtype.

Migration of vascular smooth cells from the media to the intima essentially contributes to neointima formation after percutaneous transluminal angioplasty and stent implantation. The stable prostacyclin mimetic iloprost has been shown to inhibit neointima formation in experimental restenosis, but it is currently unknown whether this may be caused by an antimigratory effect. Hence, the present study analyses (i) the influence of G(s)-coupled prostacyclin (IP) receptors on cell migration and (ii) verifies whether EP(3) receptors with opposite (i.e., G(i)) coupling may conversely stimulate cell migration. In a modified Boyden chamber model, it was shown that iloprost dose-dependently inhibits the migration of primary human arterial smooth muscle cells, which constitutively express the IP receptor. On the other hand, human arterial smooth muscle cell migration was stimulated by the EP(3) receptor agonist M&B 28.767. To independently study the effects of these receptors, IP or EP(3) receptors were stably overexpressed in chinese hamster ovary cells (CHO-IP and CHO-EP(3)). Chemotaxis of CHO cells transfected with G(s)-coupled IP receptors was concentration-dependently inhibited by iloprost (2-100 nM), while there was no effect of iloprost on mock-transfected CHO. By contrast, CHO-cells that overexpressed EP(3) receptors showed a significant, concentration dependent (1-100 nM) increase of cell migration in presence of the selective EP(3) agonist M&B 28.767. It is concluded that the prostacyclin mimetic iloprost inhibits vascular cell migration, which probably depends on a G(s)-mediated increase of intracellular cAMP. EP(3) receptors conversely stimulate CHO migration.

Background: Nitric oxide (NO) is an important mediator of inflammatory processes, including macrophage-mediated cellular host defense, and is found to be increased in peritonitis. The ability of human mononuclear cells to contribute to the NO production by expression of active inducible NO synthase (iNOS) is still discussed controversely. Aims: This study was designed to investigate the influence of prostacyclin receptor (IP receptor) activation on iNOS expression and NO formation in human peripheral blood monocytes. Method and Results: Using reverse transcriptase-polymerase chain reaction, we demonstrated that human monocytes express high levels of IP receptor mRNA. Stimulation of monocytes with the IP receptor selective agonist cicaprost (100 nM) significantly induced cellular cyclic adenosine monophosphate formation, indicating functional coupling of the receptor to Gs. Treatment of cells with lipopolysaccharide (LPS)/interferon gamma (IFN-γ) further enhanced the IP receptor mRNA expression 2.7 ± 0.1-fold above basal levels (n = 6). Analysis of iNOS expression revealed barely detectable mRNA levels in unstimulated monocytes which were increased 3.75 ± 0.3-fold (n = 5) after costimulation with 1 µg/ml LPS and 250 U/ml INF-γ for 16 h. Further increases of iNOS mRNA expression (9.4 ± 0.9-fold above basal, n = 5) were obtained, if the monocytes were costimulated with 1 µg/ml LPS, 250 U/ml INF-γ, and 100 nM cicaprost for 16 h. Measurement of the NO generation correlated with the polymerase chain reaction data: treatment of cells with 1 µg/ml LPS plus 250 U/ml INF-γ increased the NO2 production to 2.6 µM, being above the basal level of 2.0 µM, as determined in the cell culture medium. Additional treatment with 100 nM cicaprost further significantly increased the NO2 production to 3.43 µM. Conclusions: An IP receptor mediated increase in cyclic adenosine monophosphate formation plays an important role in enhancing LPS/IFN-γ-induced iNOS expression in human monocytes/macrophages and may, therefore, contribute to the increased production of NO during peritonitis.

Phosphorylation of the human prostacyclin (PGI2) receptor (hIP-R) by diacylglycerol-regulated protein kinase C (PKC) has been reported to be responsible for its rapid desensitization in HEK293 cells. In this study we demonstrate, that human fibroblasts reveal a much slower hIP-R desensitization kinetics, which was neither affected by stimulation nor inhibition of PKC by either phorbol 12-myristate-13-acetate or GF-109203X suggesting a different cellular mechanism. Although agonist-promoted sequestration of a C-terminally green fluorescent protein-tagged hIP-R was demonstrated, it did not account for the long-term desensitization. Concanavalin A did not abolish, but accelerated receptor desensitization kinetics. Resensitization of hIP-R involved receptor recycling and/or de novo synthesis of receptor protein, depending on the duration of prior desensitization. This is the first study investigating the mechanisms of hIP-R desensitization in intact human cells naturally expressing hIP-R. Our data suggest, that a hitherto unknown mechanism of hIP-R long-term desensitization, which is independent of receptor phosphorylation by conventional and novel type PKC isoforms or endocytosis, is a key event in regulating the cellular responsiveness to PGI2.

The contents of prostaglandins in seminal plasma from a total of 73 men were evaluated. The subjects were grouped as follows: normospermic men, patients with impaired motility, patients with small untreated varicocele and patients with impaired motility and Kallikrein therapy. Sperm density, morphology and motility were examined. High performance reversed phase liquid chromatography (HPLC) in combination with specific radioimmunoassays were used for the determination of PGE2, PGI2 and PGF2 alpha. There was a significant difference (p less than 0.025; F-test) between the PGI2 concentrations in patients with impaired motility (5.6 +/- 1.4 pg/mg protein) and normal men (8.8 +/- 3.7 pg/mg protein). PGE2 and PGF2 alpha were significantly different in patients with varicocele (p less than 0.025, F-test). Wide ranges of prostaglandins occurred in the Kallikrein-group with no significant differences. We conclude that: a) PGI2 is an additional prostaglandin compound in seminal plasma, b) its measurement may not be useful as diagnostic parameter in subfertile men and c) Kallikrein has no influence on the prostaglandin content in seminal plasma and other seminal parameters as motility, motility index and sperm counts.