Regulation of prostacyclin and prostaglandin E(2) receptor mediated responses in adult rat dorsal root ganglion cells, in vitro.
ABSTRACT: 1. Primary cultures of adult rat dorsal root ganglia (DRG) were prepared to examine the properties of prostacyclin (IP) receptors and prostaglandin E(2) (EP) receptors in sensory neurones. 2. IP receptor agonists, cicaprost and iloprost, stimulated adenylyl cyclase activity with EC(50) values of 22 and 28 nM, respectively. Prostaglandin E(1) (PGE(1)) and prostaglandin E(2) (PGE(2)) were 7 fold less potent than cicaprost and iloprost, with PGE(2) displaying a lower maximal response. 3. Adenylyl cyclase activation by iloprost, PGE(1) and PGE(2), but not by forskolin, was highly dependent on DRG cell density. Although the potency of iloprost and PGE(2) for stimulating adenylyl cyclase was unchanged, their maximal responses were significantly increased at low cell density. 4. Both IP and EP(2/4) receptors could be down-regulated by agonist pretreatment, however the presence of cyclo-oxygenase (COX) inhibitors did not prevent this apparent down-regulation of IP and EP(2/4) receptors at high DRG cell densities. 5. Stimulation of adenylyl cyclase by the neuropeptide calcitonin gene-related peptide was also decreased at high DRG cell density, whereas the responses to beta-adrenoceptor agonists were increased at high DRG cell density. 6. Addition of nerve growth factor (NGF), or the addition of anti-neurotrophin antibodies during the 5-day culture of DRG cells, had no effect on IP receptor-mediated responses. 7. These results indicate that G(s)-coupled receptors involved in nociception are regulated in a variable manner in adult rat sensory neurones, and that this cell density-dependent regulation may be agonist-independent for IP and EP(2/4) receptors.
Project description:1. Our study shows that the prostacyclin analogues AFP-07 and cicaprost are moderately potent agonists for prostanoid EP(4) receptors, in addition to being highly potent IP(1) receptor agonists. Both activities were demonstrated on piglet and rabbit saphenous veins, which are established EP(4) preparations. 2. On piglet saphenous vein, PGE(2) was 6.1, 24, 96, 138, 168 and 285 times respectively more potent than AFP-07, cicaprost, PGI(2), iloprost, carbacyclin and TEI-9063 in causing relaxation. Another prostacyclin analogue taprostene did not induce maximum relaxation (21 - 74%), and did not oppose the action of PGE(2). The EP(4) receptor antagonist AH 23848 (30 microM) blocked relaxant responses to PGE(2) (dose ratio=8.6+/-1.3, s.e.mean) to a greater extent than cicaprost (4.9+/-0.7) and AFP-07 (3.8+/-0.8), had variable effects on TEI-9063-induced relaxation (3.7+/-1.5), and had no effect on taprostene responses (<2.0). 3. On rabbit saphenous vein, AH 23848 blocked the relaxant actions of PGE(2), AFP-07, cicaprost, iloprost and carbacyclin to similar extents. 4. AFP-07, cicaprost and TEI-9063 showed high IP(1) relaxant potency on piglet carotid artery, rabbit mesenteric artery and guinea-pig aorta, with AFP-07 confirmed as the most potent IP(1) agonist reported to date. AH 23848 did not block cicaprost-induced relaxation of piglet carotid artery. EP(3) contractile systems in these preparations can confound IP(1) agonist potency estimations. 5. Caution is urged when using AFP-07 and cicaprost to characterize IP(1) receptors in the presence of EP(4) receptors. Taprostene may be a lead to a highly selective IP(1) receptor agonist.
Project description:1. Chinese hamster ovary (CHO) cells were transiently transfected with the mouse prostacyclin (mIP) receptor to examine IP agonist-mediated stimulation of [(3)H]-cyclic AMP and [(3)H]-inositol phosphate production. 2. The prostacyclin analogues, cicaprost, iloprost, carbacyclin and prostaglandin E(1), stimulated adenylyl cyclase activity with EC(50) values of 5, 6, 25 and 95 nM, respectively. These IP agonists also stimulated the phospholipase C pathway with 10 - 40 fold lower potency than stimulation of adenylyl cyclase. 3. The non-prostanoid prostacyclin mimetics, octimibate, BMY 42393 and BMY 45778, also stimulated adenylyl cyclase activity, with EC(50) values of 219, 166 and 398 nM, respectively, but failed to stimulate [(3)H]-inositol phosphate production. 4. Octimibate, BMY 42393 and BMY 45778 inhibited iloprost-stimulated [(3)H]-inositol phosphate production in a non-competitive manner. 5. Activation of the endogenously-expressed P(2) purinergic receptor by ATP led to an increase in [(3)H]-inositol phosphate production which was inhibited by the non-prostanoid prostacyclin mimetics in non-transfected CHO cells. Prostacyclin analogues and other prostanoid receptor ligands failed to inhibit ATP-stimulated [(3)H]-inositol phosphate production. 6. A comparison between the IP receptor-specific non-prostanoid ONO-1310 and the structurally-related EP(3) receptor-specific agonist ONO-AP-324, indicated that the inhibitory effect of non-prostanoids was specific for those compounds known to activate IP receptors. 7. The non-prostanoid prostacyclin mimetics also inhibited phospholipase C activity when stimulated by constitutively-active mutant Galpha(q)RC, Galpha(14)RC and Galpha(16)QL transiently expressed in CHO cells. These drugs did not inhibit adenylyl cyclase activity when stimulated by the constitutively-active mutant Galpha(s)QL. 8. These results suggest that non-prostanoid prostacyclin mimetics can specifically inhibit [(3)H]-inositol phosphate production by targeting G(q/11) and/or phospholipase C in CHO cells, and that this effect is independent of IP receptors.
Project description:We tested hypothesis that activation of the prostacyclin (PGI<sub>2</sub>) receptor (IP receptor) signaling pathway in cerebral microvessels plays an important role in the metabolism of amyloid precursor protein (APP). In human brain microvascular endothelial cells activation of IP receptor with the stable analogue of PGI<sub>2</sub>, iloprost, stimulated expression of amyloid precursor protein and a disintegrin and metalloprotease 10 (ADAM10), resulting in an increased production of the neuroprotective and anticoagulant molecule, soluble APP? (sAPP?). Selective agonist of IP receptor, cicaprost, and adenylyl cyclase activator, forskolin, also enhanced expression of amyloid precursor protein and ADAM10. Notably, in cerebral microvessels of IP receptor knockout mice, protein levels of APP and ADAM10 were reduced. In addition, iloprost increased protein levels of peroxisome proliferator-activated receptor ? (PPAR?) in human brain microvascular endothelial cells. PPAR?-siRNA abolished iloprost-augmented protein expression of ADAM10. In contrast, GW501516 (a selective agonist of PPAR?) upregulated ADAM10 and increased production of sAPP?. Genetic deletion of endothelial PPAR? (ePPAR?<sup>-/-</sup>) in mice significantly reduced cerebral microvascular expression of ADAM10 and production of sAPP?. In vivo treatment with GW501516 increased sAPP? content in hippocampus of wild type mice but not in hippocampus of ePPAR?<sup>-/-</sup> mice. Our findings identified previously unrecognized role of IP-PPAR? signal transduction pathway in the production of sAPP? in cerebral microvasculature.
Project description:1. The prostanoid receptor(s) on human airways smooth muscle (HASM) cells that mediates the inhibitory effect of PGE(2) on interleukin (IL)-1 beta-induced granulocyte/macrophage colony-stimulating factor (GM-CSF) release has been classified. 2. IL-1 beta evoked the release of GM-CSF from HASM cells, which was suppressed by PGE(2), 16,16-dimethyl PGE(2) (nonselective), misoprostol (EP(2)/EP(3)-selective), ONO-AE1-259 and butaprost (both EP(2)-selective) with pIC(50) values of 8.61, 7.13, 5.64, 8.79 and 5.43, respectively. EP-receptor agonists that have selectivity for the EP(1)-(17-phenyl-omega-trinor PGE(2)) and EP(3)-receptor (sulprostone) subtypes as well as cicaprost (IP-selective), PGD(2), PGF(2 alpha) and U-46619 (TP-selective) were poorly active or inactive at concentrations up to 10 microM. 3. AH 6809, a drug that can be used to selectively block EP(2)-receptors in HASM cells, antagonised the inhibitory effect of PGE(2), 16,16-dimethyl PGE(2) and ONO-AE1-259 with apparent pA(2) values of 5.85, 6.09 and 6.1 respectively. In contrast, the EP(4)-receptor antagonists, AH 23848B and L-161,982, failed to displace to the right the concentration-response curves that described the inhibition of GM-CSF release evoked by PGE(2) and ONO-AE1-259. 4. Inhibition of GM-CSF release by PGE(2) and 8-Br-cAMP was abolished in cells infected with an adenovirus vector encoding an inhibitor protein of cAMP-dependent protein kinase (PKA) but not by H-89, a purported small molecule inhibitor of PKA. 5. We conclude that prostanoid receptors of the EP(2)-subtype mediate the inhibitory effect of PGE(2) on GM-CSF release from HASM cells by recruiting a PKA-dependent pathway. In addition, the data illustrate that caution should be exercised when using H-89 in studies designed to assess the role of PKA in biological processes.
Project description:<h4>Background and purpose</h4>In patients with pulmonary hypertension (PH) associated with lung disease and/or hypoxia (Group III), decreased pulmonary vascular tone and tissue hypoxia is therapeutically beneficial. PGE<sub>2</sub> and PGI<sub>2</sub> induce potent relaxation of human bronchi from non-PH (control) patients via EP<sub>4</sub> and IP receptors, respectively. However, the effects of PGE<sub>2</sub> /PGI<sub>2</sub> and their mimetics on human bronchi from PH patients are unknown. Here, we have compared relaxant effects of several PGI<sub>2</sub> -mimetics approved for treating PH Group I with several PGE<sub>2</sub> -mimetics, in bronchial preparations derived from PH Group III and control patients.<h4>Experimental approach</h4>Relaxation of bronchial muscle was assessed in samples isolated from control and PH Group III patients. Expression of prostanoid receptors was analysed by western blot and real-time PCR, and endogenous PGE<sub>2</sub> , PGI<sub>2</sub> , and cAMP levels were determined by ELISA.<h4>Key results</h4>Maximal relaxations induced by different EP<sub>4</sub> receptor agonists (PGE<sub>2</sub> , L-902688, and ONO-AE1-329) were decreased in human bronchi from PH patients, compared with controls. However, maximal relaxations produced by PGI<sub>2</sub> -mimetics (iloprost, treprostinil, and beraprost) were similar for both groups of patients. Both EP<sub>4</sub> and IP receptor protein and mRNA expressions were significantly lower in human bronchi from PH patients. cAMP levels significantly correlated with PGI<sub>2</sub> but not with PGE<sub>2</sub> levels.<h4>Conclusion and implications</h4>The PGI<sub>2</sub> -mimetics retained maximal bronchodilation in PH Group III patients, whereas bronchodilation induced by EP<sub>4</sub> receptor agonists was decreased. Restoration of EP<sub>4</sub> receptor expression in airways of PH Group III patients with respiratory diseases could bring additional therapeutic benefit.
Project description:BACKGROUND AND PURPOSE: Neonatal pulmonary hypertension (PPHN) is characterized by pulmonary vasoconstriction, due in part to dysregulation of the thromboxane prostanoid (TP) receptor. Hypoxia induces TP receptor-mediated hyperresponsiveness, whereas serine phosphorylation mediates desensitization of TP receptors. We hypothesized that prostacyclin (IP) receptor activity induces TP receptor phosphorylation and decreases ligand affinity; that TP receptor sensitization in hypoxic myocytes is due to IP receptor inactivation; and that this would be reversible by the cAMP-specific phosphodiesterase inhibitor milrinone. EXPERIMENTAL APPROACH: We examined functional regulation of TP receptors by serine phosphorylation and effects of IP receptor stimulation and protein kinase A (PKA) activity on TP receptor sensitivity in myocytes from neonatal porcine resistance pulmonary arteries after 72?h hypoxia in vitro. Ca(2+) response curves to U46619 (TP receptor agonist) were determined in hypoxic and normoxic myocytes incubated with or without iloprost (IP receptor agonist), forskolin (adenylyl cyclase activator), H8 (PKA inhibitor) or milrinone. TP and IP receptor saturation binding kinetics were measured in presence of iloprost or 8-bromo-cAMP. KEY RESULTS: Ligand affinity for TP receptors was normalized in vitro by IP receptor signalling intermediates. However, IP receptor affinity was compromised in hypoxic myocytes, decreasing cAMP production. Milrinone normalized TP receptor sensitivity in hypoxic myocytes by restoring PKA-mediated regulatory TP receptor phosphorylation. CONCLUSIONS AND IMPLICATIONS: TP receptor sensitivity and EC(50) for TP receptor agonists was regulated by PKA, as TP receptor serine phosphorylation by PKA down-regulated Ca(2+) mobilization. Hypoxia decreased IP receptor activity and cAMP generation, inducing TP receptor hyperresponsiveness, which was reversed by milrinone.
Project description:In previous studies investigating cross-talk of signalling between prostaglandin (PG)E(2) receptor (EP) and the TPalpha and TPbeta isoforms of the human thromboxane (TX)A(2) receptor (TP), 17-phenyl trinor PGE(2)-induced desensitization of TP receptor signalling through activation of the AH6809 and SC19220-sensitive EP(1) subtype of the EP receptor family, in a cell-specific manner. Here, we sought to further investigate that cross-talk in human erythroleukaemic (HEL) 92.1.7 cells.Specificity of 17-phenyl trinor PGE(2) signalling and its possible cross-talk with signalling by TPalpha/TPbeta receptors endogenously expressed in HEL cells was examined through assessment of agonist-induced inositol 1,4,5-trisphosphate (IP)(3) generation and intracellular calcium ([Ca(2+)](i)) mobilization.While 17-Phenyl trinor PGE(2) led to activation of phospholipase (PL)Cbeta to yield increases in IP(3) generation and [Ca(2+)](i), it did not desensitize but rather augmented that signalling in response to subsequent stimulation with the TXA(2) mimetic U46619. Furthermore, the augmentation was reciprocal. Signalling by 17-phenyl trinor PGE(2) was found to occur through AH6809- and SC19920-insensitive, Pertussis toxin-sensitive, G(i)/G(betagamma)-dependent activation of PLCbeta. Further pharmacological investigation using selective EP receptor subtype agonists and antagonists confirmed that 17-phenyl trinor PGE(2)-mediated signalling and reciprocal cross-talk with the TP receptors occurred through the EP(3), rather than the EP(1), EP(2) or EP(4) receptor subtype in HEL cells.The EP(1) and EP(3) subtypes of the EP receptor family mediated intermolecular cross-talk to differentially regulate TP receptor-mediated signalling whereby activation of EP(1) receptors impaired or desensitized, while that of EP(3) receptors augmented signalling through TPalpha/TPbeta receptors, in a cell type-specific manner.
Project description:Amyloid-beta (Abeta) peptides, generated by the proteolysis of beta-amyloid precursor protein by beta- and gamma-secretases, play an important role in the pathogenesis of Alzheimer disease. Inflammation is also important. We recently reported that prostaglandin E(2) (PGE(2)), a strong inducer of inflammation, stimulates the production of Abeta through EP(2) and EP(4) receptors, and here we have examined the molecular mechanism. Activation of EP(2) and EP(4) receptors is coupled to an increase in cellular cAMP levels and activation of protein kinase A (PKA). We found that inhibitors of adenylate cyclase and PKA suppress EP(2), but not EP(4), receptor-mediated stimulation of the Abeta production. In contrast, inhibitors of endocytosis suppressed EP(4), but not EP(2), receptor-mediated stimulation. Activation of gamma-secretase was observed with the activation of EP(4) receptors but not EP(2) receptors. PGE(2)-dependent internalization of the EP(4) receptor was observed, and cells expressing a mutant EP(4) receptor lacking the internalization activity did not exhibit PGE(2)-stimulated production of Abeta. A physical interaction between the EP(4) receptor and PS-1, a catalytic subunit of gamma-secretases, was revealed by immunoprecipitation assays. PGE(2)-induced internalization of PS-1 and co-localization of EP(4), PS-1, and Rab7 (a marker of late endosomes and lysosomes) was observed. Co-localization of PS-1 and Rab7 was also observed in the brain of wild-type mice but not of EP(4) receptor null mice. These results suggest that PGE(2)-stimulated production of Abeta involves EP(4) receptor-mediated endocytosis of PS-1 followed by activation of the gamma-secretase, as well as EP(2) receptor-dependent activation of adenylate cyclase and PKA, both of which are important in the inflammation-mediated progression of Alzheimer disease.
Project description:The intermolecular cross-regulation mediated by the prostanoid IP-receptor (IP)/EP(1) receptor (EP(1)) agonists PGI(2) and 17 phenyl trinor PGE(2) on TP receptor (TP) signalling within platelets was compared to that which occurs to the individual TPalpha and TPbeta receptors over-expressed in human embryonic kidney (HEK) 293 cells. Ligand mediated TP receptor activation was monitored by analysing mobilization of intracellular calcium ([Ca(2+)](i)) following stimulation with the selective thromboxane (TX) A(2) mimetic U46619. Consistent with previous studies, in platelets, PGI(2) acting through endogenous IP receptors completely inhibited U46619-mediated TP receptor signalling in a protein kinase (PK) A-dependent, PKC-independent manner. In HEK 293 cells, PGI(2), acting through endogenous AH6809 sensitive EP(1) rather than IP receptors, and the selective EP(1) receptor agonist 17 phenyl trinor PGE(2) antagonized U46619-mediated signalling by both TPalpha and TPbeta receptors in a PKC-dependent, PKA-independent manner. The maximum response induced by either ligand was significantly (P<0.005) greater for the TPalpha receptor than the TPbeta receptor, pointing to possible physiologic differences between the TP isoforms, although the potency of each ligand was similar for both TP receptors. TP(Delta328), a truncated variant of TP receptor lacking the C-tail sequences unique to TPalpha or TPbeta receptors, was not sensitive to EP(1) receptor-mediated regulation by PGI(2) or 17 phenyl trinor PGE(2) In conclusion, these data confirm that TPalpha and TPbeta receptors are subject to cross regulation by EP(1) receptor signalling in HEK 293 cells mediated by PKC at sites unique to the individual TP receptors and that TPalpha receptor responses are significantly more reduced by EP(1) receptor regulation than those of the TPbeta receptor.
Project description:Rhythmical transient constrictions of the lymphatic vessels provide the means for efficient lymph drainage and interstitial tissue fluid balance. This activity is critical during inflammation, to avoid or limit oedema resulting from increased vascular permeability, mediated by the release of various inflammatory mediators. In this study, we investigated the mechanisms by which prostaglandin E(2) (PGE(2)) and prostacyclin modulate lymphatic contractility in isolated guinea pig mesenteric lymphatic vessels.Quantitative RT-PCR was used to assess the expression of mRNA for enzymes and receptors involved in the production and action of PGE(2) and prostacyclin in mesenteric collecting lymphatic vessels. Frequency and amplitude of lymphatic vessel constriction were measured in the presence of these prostaglandins and the role of their respective EP and IP receptors assessed.Prostaglandin E(2) and prostacyclin decreased concentration-dependently the frequency, without affecting the amplitude, of lymphatic constriction. Data obtained in the presence of the EP(4) receptor antagonists, GW627368x (1 microM) and AH23848B (30 microM) and the IP receptor antagonist CAY10441 (0.1 microM) suggest that PGE(2) predominantly activates EP(4), whereas prostacyclin mainly stimulates IP receptors. Inhibition of responses to either prostaglandin with H89 (10 microM) or glibenclamide (1 microM) suggested a role for the activation of protein kinase A and ATP-sensitive K(+) channels.Our findings characterized the inhibition of lymphatic pumping induced by PGE(2) or prostacyclin in guinea pig mesenteric lymphatics. This action is likely to impair oedema resolution and to contribute to the pro-inflammatory actions of these prostaglandins.