Prostacyclin receptor-independent inhibition of phospholipase C activity by non-prostanoid prostacyclin mimetics.
ABSTRACT: 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:Prostacyclins are extensively used to treat pulmonary arterial hypertension (PAH), a life-threatening disease involving the progressive thickening of small pulmonary arteries. Although these agents are considered to act therapeutically via the prostanoid IP receptor, treprostinil is the only prostacyclin mimetic that potently binds to the prostanoid EP? receptor, the role of which is unknown in PAH. We hypothesised that EP? receptors contribute to the anti-proliferative effects of treprostinil in human pulmonary arterial smooth muscle cells (PASMCs), contrasting with selexipag, a non-prostanoid selective IP agonist. Human PASMCs from PAH patients were used to assess prostanoid receptor expression, cell proliferation, and cyclic adenosine monophosphate (cAMP) levels following the addition of agonists, antagonists or EP? receptor small interfering RNAs (siRNAs). Immunohistochemical staining was performed in lung sections from control and PAH patients. We demonstrate using selective IP (RO1138452) and EP? (PF-04418948) antagonists that the anti-proliferative actions of treprostinil depend largely on EP? receptors rather than IP receptors, unlike MRE-269 (selexipag-active metabolite). Likewise, EP? receptor knockdown selectively reduced the functional responses to treprostinil but not MRE-269. Furthermore, EP? receptor levels were enhanced in human PASMCs and in lung sections from PAH patients compared to controls. Thus, EP? receptors represent a novel therapeutic target for treprostinil, highlighting key pharmacological differences between prostacyclin mimetics used in PAH.
Project description:Prostacyclin and thromboxane mediate opposing cardiovascular effects through their receptors, the prostacyclin receptor (IP) and thromboxane receptor (TP). Individuals heterozygous for an IP variant, IP(R212C), displayed exaggerated loss of platelet IP responsiveness and accelerated cardiovascular disease. We examined association of IP(R212C) into homo- and heterodimeric receptor complexes and the impact on prostacyclin and thromboxane biology.Dimerization of the IP, IP(R212C), and TPalpha was examined by bioluminesence resonance energy transfer in transfected HEK293 cells. We observed an equal propensity for formation of IPIP homodimers and IPTPalpha heterodimers. Compared with the IP alone, IP(R212C) displayed reduced cAMP generation and increased endoplasmic reticulum localization but underwent normal homo- and heterodimerization. When the IP(R212C) and IP were coexpressed, a dominant negative action of the variant was evident with enhanced wild-type IP localization to the endoplasmic reticulum and reduced agonist-dependent signaling. Further, the TPalpha activation response, which was shifted from inositol phosphate to cAMP generation following IPTPalpha heterodimerization, was normalized when the TPalpha instead dimerized with IP(R212C).IP(R212C) exerts a dominant action on the wild-type IP and TPalpha through dimerization. This likely contributes to accelerated cardiovascular disease in individuals carrying 1 copy of the variant allele.
Project description:Prostacyclin mimetics (PMs) are effective for the treatment of pulmonary arterial hypertension (PAH). However, their clinical use may be limited by their adverse events. This study aims to quantify the different PM adverse events (AEs) with regard to their selectivity towards the prostacyclin (IP) receptor and their administrative routes. The study included randomised, placebo-controlled trials comparing iloprost, beraprost, treprostinil, and selexipag to placebo (published 2002–2016). We report the group efficacy differences between treatment and placebo by weighted and standardised mean difference. The probability of adverse events was determined by the odds ratio (OR). Of the 14 randomised clinical trials involving 3518 PAH patients, outcome and adverse event data were meta-analysed by drug type and route of administration. Prostacyclin mimetics comparison demonstrated a more significant discontinuation of the IP-selective agonist, selexipag, due to an adverse event (OR = 2.2; 95% CI: 1.5, 3.3). Compared to placebo, site pain associated with subcutaneously administered treprostinil was the most significant likely adverse event (OR = 17.5; 95% CI: 11.1, 27.1). Parenteral PMs were associated with fewer adverse effects overall. The overall efficacy of PMs to improve 6-minute walk distance by 16.3 meters was significant (95% CI: 13.0, 19.7). Decreases in pulmonary vascular resistance index (SMD = -5.5; 95% CI: -10.1, -0.9; I² = 98%) and mean pulmonary arterial pressure (SMD = -1.0; 95% CI: -2.6, -0.7; I² = 99%) in treatment groups were found to be significant. Adverse event profiles varied in response to administration route and PM type but were not negated by use of a selective IP agonist. Prostacyclin mimetics exposure to non-target IP receptors may underpin some AEs reported.
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: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:G protein-coupled receptors (GPCRs) interact with heterotrimeric G proteins and initiate a wide variety of signaling pathways. The molecular nature of GPCR-G protein interactions in the clinically important thromboxane A2 (TxA(2)) receptor (TP) and prostacyclin (PGI(2)) receptor (IP) is poorly understood. The TP activates its cognate G protein (Gαq) in response to the binding of thromboxane, while the IP signals through Gαs in response to the binding of prostacyclin. Here, we utilized a combination of approaches consisting of chimeric receptors, molecular modeling, and site-directed mutagenesis to precisely study the specificity of G protein coupling. Multiple chimeric receptors were constructed by replacing the TP intracellular loops (ICLs) with the ICL regions of the IP. Our results demonstrate that both the sequences and lengths of ICL2 and ICL3 influenced G protein specificity. Importantly, we identified a precise ICL region on the prostanoid receptors TP and IP that can switch G protein specificities. The validities of the chimeric technique and the derived molecular model were confirmed by introducing clinically relevant naturally occurring mutations (R60L in the TP and R212C in the IP). Our findings provide new molecular insights into prostanoid receptor-G protein interactions, which are of general significance for understanding the structural basis of G protein activation by GPCRs in basic health and cardiovascular disease.
Project description:The prostanoid prostacyclin, or prostaglandin I2, plays an essential role in many aspects of cardiovascular disease. The actions of prostacyclin are mainly mediated through its activation of the prostacyclin receptor or, in short, the IP. In recent studies, the cytoplasmic carboxy-terminal domain of the IP was shown to bind several PDZ domains of the multi-PDZ adaptor PDZK1. The interaction between the two proteins was found to enhance cell surface expression of the IP and to be functionally important in promoting prostacyclin-induced endothelial cell migration and angiogenesis. To investigate the interaction of the IP with the first PDZ domain (PDZ1) of PDZK1, we generated a nine residue peptide (KK(411)IAACSLC(417)) containing the seven carboxy-terminal amino acids of the IP and measured its binding affinity to a recombinant protein corresponding to PDZ1 by isothermal titration calorimetry. We determined that the IP interacts with PDZ1 with a binding affinity of 8.2 µM. Using the same technique, we also determined that the farnesylated form of carboxy-terminus of the IP does not bind to PDZ1. To understand the molecular basis of these findings, we solved the high resolution crystal structure of PDZ1 bound to a 7-residue peptide derived from the carboxy-terminus of the non-farnesylated form of IP ((411)IAACSLC(417)). Analysis of the structure demonstrates a critical role for the three carboxy-terminal amino acids in establishing a strong interaction with PDZ1 and explains the inability of the farnesylated form of IP to interact with the PDZ1 domain of PDZK1 at least in vitro.
Project description:Prostacyclin analogs, used to treat idiopathic pulmonary arterial hypertension (IPAH), are assumed to work through prostacyclin (IP) receptors linked to cyclic AMP (cAMP) generation, although the potential to signal through peroxisome proliferator-activated receptor-? (PPAR?) exists.IP receptor and PPAR? expression may be depressed in IPAH. We wished to determine if pathways remain functional and if analogs continue to inhibit smooth muscle proliferation.We used Western blotting to determine IP receptor expression in peripheral pulmonary arterial smooth muscle cells (PASMCs) from normal and IPAH lungs and immunohistochemistry to evaluate IP receptor and PPAR? expression in distal arteries.Cell proliferation and cAMP assays assessed analog responses in human and mouse PASMCs and HEK-293 cells. Proliferative rates of IPAH cells were greater than normal human PASMCs. IP receptor protein levels were lower in PASMCs from patients with IPAH, but treprostinil reduced replication and treprostinil-induced cAMP elevation appeared normal. Responses to prostacyclin analogs were largely dependent on the IP receptor and cAMP in normal PASMCs, although in IP(-/-) receptor cells analogs inhibited growth in a cAMP-independent, PPAR?-dependent manner. In IPAH cells, antiproliferative responses to analogs were insensitive to IP receptor or adenylyl cyclase antagonists but were potentiated by a PPAR? agonist and inhibited (? 60%) by the PPAR? antagonist GW9662. This coincided with increased PPAR? expression in the medial layer of acinar arteries.The antiproliferative effects of prostacyclin analogs are preserved in IPAH despite IP receptor down-regulation and abnormal coupling. PPAR? may represent a previously unrecognized pathway by which these agents inhibit smooth muscle proliferation.
Project description:Prostacyclin and its prostacyclin receptor, the I Prostanoid (IP), play essential roles in regulating hemostasis and vascular tone and have been implicated in a range cardio-protective effects but through largely unknown mechanisms. In this study, the influence of cholesterol on human IP [(h)IP] gene expression was investigated in cultured vascular endothelial and platelet-progenitor megakaryocytic cells. Cholesterol depletion increased human prostacyclin receptor (hIP) mRNA, hIP promoter-directed reporter gene expression, and hIP-induced cAMP generation in all cell types. Furthermore, the constitutively active sterol-response element binding protein (SREBP)1a, but not SREBP2, increased hIP mRNA and promoter-directed gene expression, and deletional and mutational analysis uncovered an evolutionary conserved sterol-response element (SRE), adjacent to a known functional Sp1 element, within the core hIP promoter. Moreover, chromatin immunoprecipitation assays confirmed direct cholesterol-regulated binding of SREBP1a to this hIP promoter region in vivo, and immunofluorescence microscopy corroborated that cholesterol depletion significantly increases hIP expression levels. In conclusion, the hIP gene is directly regulated by cholesterol depletion, which occurs through binding of SREBP1a to a functional SRE within its core promoter. Mechanistically, these data establish that cholesterol can regulate hIP expression, which may, at least in part, account for the combined cardio-protective actions of low serum cholesterol through its regulation of IP expression within the human vasculature.
Project description:Of the known prostanoid receptors, human eosinophils express the prostaglandin D2 (PGD2) receptors DP1 [also D-type prostanoid (DP)] and DP2 (also chemoattractant receptor homologous molecule, expressed on Th2 cells), the prostaglandin E2 receptors EP2 and EP4, and the prostacyclin (PGI2) receptor IP. Prostanoids can bind to either one or multiple receptors, characteristically have a short half-life in vivo, and are quickly degraded into metabolites with altered affinity and specificity for a given receptor subtype. Prostanoid receptors signal mainly through G proteins and naturally activate signal transduction pathways according to the G protein subtype that they preferentially interact with. This can lead to the activation of sometimes opposing signaling pathways. In addition, prostanoid signaling is often cell-type specific and also the combination of expressed receptors can influence the outcome of the prostanoid impulse. Accordingly, it is assumed that eosinophils and their (patho-)physiological functions are governed by a sensitive prostanoid signaling network. In this review, we specifically focus on the functions of PGD2, PGE2, and PGI2 and their receptors on eosinophils. We discuss their significance in allergic and non-allergic diseases and summarize potential targets for drug intervention.