Prostanoid IP Research Articles

Steroid-Induced Ocular Hypertension in Mice Is Differentially Reduced by Selective EP2, EP3, EP4, and IP Prostanoid Receptor Agonists.

We tested five chemically and metabolically stable prostaglandin (PG) receptor agonists in a mouse model of dexamethasone-induced ocular hypertension (OHT). Whilst all compounds significantly (p < 0.05, ANOVA) lowered intraocular pressure (IOP) after twice-daily bilateral topical ocular dosing (5 µg/dose) over three weeks, the time course and magnitude of the responses varied. The onset of action of NS-304 (IP-PG receptor agonist) and rivenprost (EP4-PG receptor agonist) was slower than that of misoprostol (mixed EP2/EP3/EP4-PG receptor agonist), PF-04217329 (EP2-PG receptor agonist), and butaprost (EP2-PG receptor agonist). The rank order of IOP-lowering efficacies aligned with the onset of actions of these compounds. Peak IOP reductions relative to vehicle controls were as follows: misoprostol (74.52%) = PF-04217329 (74.32%) > butaprost (65.2%) > rivenprost (58.4%) > NS-304 (55.3%). A literature survey indicated that few previously evaluated compounds (e.g., latanoprost, timolol, pilocarpine, brimonidine, dorzolamide, cromakalim analog (CKLP1), losartan, tissue plasminogen activator, trans-resveratrol, sodium 4-phenyl acetic acid, etc.) in various animal models of steroid-induced OHT were able to match the effectiveness of misoprostol, PF-04217329 or butaprost. Since a common feature of the latter compounds is their relatively high affinity and potency at the EP2-PG receptor sub-type, which activates the production of intracellular cAMP in target cells, our studies suggest that drugs selective for the EP2-PG receptor may be suited to treat corticosteroid-induced OHT.

Role of Prostaglandins in Nitric Oxide-Induced Glial Cell-Mediated Vasodilation in Rat Retina.

We previously identified that NO derived from neuronal cells acts on glial cells and causes vasodilation in the healthy rat retina via the release of epoxyeicosatrienoic acids (EETs) and prostaglandins (PGs) by activation of the arachidonic acid cascade. However, it is not clear which PG types are involved in these responses. The aim of the present study was to identify prostanoid receptors involved in glial cell-derived vasodilation induced by NO in rat retina. Male Wistar rats were used to examine the effects of intravitreal pretreatment with indomethacin, a cyclooxygenase inhibitor; PF-04418948, a prostanoid EP2 receptor antagonist; and CAY10441, a prostanoid IP receptor antagonist, on the changes in the retinal arteriolar diameter induced by intravitreal administration of NOR3, an NO donor. Retinal arteriolar diameters were measured using ocular fundus images captured with a high-resolution digital camera in vivo. The increase in the retinal arteriolar diameter induced by intravitreal injection of NOR3 was significantly suppressed by intravitreal pretreatment with indomethacin and PF-04418948, but not by CAY10441. The dose of PF-04418948 and CAY10441 injected intravitreally in the present study significantly reduced the increase in the retinal arteriolar diameter induced by prostaglandin E2 (PGE2) and prostaglandin I2 (PGI2), respectively. These results suggest that activation of the arachidonic acid cascade and subsequent stimulation of prostanoid EP2 receptors are involved in rat retinal vasodilatory responses evoked by NO-induced glial cell stimulation. Therefore, glial cell-derived PGE2, similar to EETs, may play an important role in retinal vasodilatory mechanisms.

A transcriptomics-guided drug target discovery strategy identifies receptor ligands for lung regeneration.

Currently, there is no pharmacological treatment targeting defective tissue repair in chronic disease. Here, we used a transcriptomics-guided drug target discovery strategy using gene signatures of smoking-associated chronic obstructive pulmonary disease (COPD) and from mice chronically exposed to cigarette smoke, identifying druggable targets expressed in alveolar epithelial progenitors, of which we screened the function in lung organoids. We found several drug targets with regenerative potential, of which EP and IP prostanoid receptor ligands had the most profound therapeutic potential in restoring cigarette smoke–induced defects in alveolar epithelial progenitors in vitro and in vivo. Mechanistically, we found, using single-cell RNA sequencing analysis, that circadian clock and cell cycle/apoptosis signaling pathways were differentially expressed in alveolar epithelial progenitor cells in patients with COPD and in a relevant model of COPD, which was prevented by prostaglandin E2 or prostacyclin mimetics. We conclude that specific targeting of EP and IP receptors offers therapeutic potential for injury to repair in COPD.

Characterisation of cough evoked by inhaled treprostinil and treprostinil palmitil

Cough is induced by inhaled prostacyclin analogues including treprostinil (TRE), and, at higher doses, treprostinil palmitil (TP), a prodrug of TRE. In this report, we have investigated mechanisms involved in TRE- and TP-induced cough, using a dry powder formulation of TP (TPIP) to supplement previous data obtained with an aqueous suspension formulation of TP (TPIS).Experiments in guinea pigs and rats investigated the prostanoid receptor subtype producing cough and whether it involved activation of sensory nerves in the airways and vasculature. Experiments involved treatment with prostanoid, tachykinin and bradykinin receptor antagonists, a cyclooxygenase inhibitor and TRE administration to the isolated larynx or intravenously.In guinea pigs, cough with inhaled TRE (1.23 µg·kg−1) was not observed with an equivalent dose of TPIP and required higher inhaled doses (12.8 and 35.8 µg·kg−1) to induce cough. TRE cough was blocked with IP and tachykinin NK1 receptor antagonists but not with EP1, EP2, EP3, DP1 or bradykinin B2 antagonists or a cyclooxygenase inhibitor. TRE administered to the isolated larynx or intravenously in rats produced no apnoea or swallowing, whereas citric acid, capsaicin and hypertonic saline had significant effects.The mechanisms inducing cough with inhaled TRE likely involves the activation of prostanoid IP receptors on jugular C-fibres in the tracheobronchial airways. Cough induced by inhaled dry powder and nebulised formulations of TP occurs at higher inhaled doses than TRE, presumably due to the slow, sustained release of TRE from the prodrug resulting in lower concentrations of TRE at the airway sensory nerves.

Involvement of Gi protein-dependent BKCa channel activation in β2-adrenoceptor-mediated dilation of retinal arterioles in rats.

Circulating catecholamines contribute to the regulation of retinal vascular tone. Our previous studies have demonstrated that the activation of large-conductance Ca2+-activated K+ (BKCa) channels is involved in the β2-adrenoceptor-mediated dilation of retinal arterioles in rats. The present study aimed to examine the role of Gi protein in the β2-adrenoceptor-mediated activation of BKCa channels in the retinal arterioles. Images of in vivo rat ocular fundi were captured, and the diameters of retinal arterioles were measured. Systemic blood pressure and heart rate were recorded continuously. Intravenous infusion of formoterol (0.01-0.3 μg/kg/min), a β2-adrenoceptor agonist, increased the diameter of retinal arterioles but decreased mean arterial pressure in a dose-dependent manner. Intravitreal injection of iberiotoxin (20 pmol/eye), an inhibitor of BKCa channels, significantly attenuated the formoterol-induced dilation of retinal arterioles. Similar results were obtained when salbutamol (0.03-3 μg/kg/min), another β2-adrenoceptor agonist, was used instead of formoterol. However, iberiotoxin had no significant effect on retinal vasodilator responses to intravenous infusion of denopamine (1-30 μg/kg/min; a β1-adrenoceptor agonist), CL316243 (0.3-10 μg/kg/min; a β3-adrenoceptor agonist), prostaglandin I2 (0.03-10 μg/kg/min; a prostanoid IP receptor agonist), and forskolin (1-10 μg/kg/min; an adenylyl cyclase activator). Intravitreal injection of pertussis toxin (66 ng/eye; a Gi protein inhibitor) significantly attenuated the dilation of retinal arterioles induced by formoterol but not by denopamine and CL316243. In the presence of pertussis toxin, iberiotoxin had no inhibitory effect on formoterol-induced dilation of retinal arterioles. These results suggest that stimulation of β2-adrenoceptors dilates retinal arterioles through pertussis toxin-sensitive Gi protein-dependent activation of BKCa channels in rats in vivo.

Pharmacology of the single isomer, esuberaprost (beraprost-314d) on pulmonary vascular tone, IP receptors and human smooth muscle proliferation in pulmonary hypertension

Background and purposeBeraprost is a prostacyclin analogue and IP receptor agonist which is approved to treat pulmonary arterial hypertension (PAH) in Asia. The beraprost-314d isomer (esuberaprost) is one of four stereoisomers contained within the racemic mixture of beraprost. The pharmacological profile of esuberaprost is now evaluated to determine how stereoisomer separation affects its potency and mode of action in functional assays. Experimental approachVascular tone was assessed using wire myography in rat and human distal pulmonary arteries (PAs) pre-contracted with U46619 (100 nM). HEK-293 cells stably expressing the human IP receptor (HEK-293-IP) and pulmonary arterial smooth muscle cells (PASMCs) derived from PAH patients were used to assess cyclic AMP (cAMP) generation and cell proliferation, respectively. Key resultsEsuberaprost relaxed rat PAs with a 5-fold greater potency compared with beraprost, and effects were strongly inhibited by RO3244794 (IP receptor antagonist) or L-NAME (NO synthase inhibitor). Esuberaprost caused EP3 receptor-dependent vasoconstriction at high concentrations ≥ 1000 nM, but contractions were 50% lower compared to beraprost. In HEK-293-IP cells, esuberaprost was 26-fold more potent (EC50 0.4 nM) at increasing cAMP than beraprost. In human PASMCs, esuberaprost was 40-fold more potent than beraprost at inhibiting cell proliferation (EC50 3 nM versus 120 nM), contrasting the 5-fold potency difference for cAMP elevation. Antiproliferative effects of esuberaprost appeared more dependent on NO than on the IP receptor. In PAs from patients with pulmonary hypertension, esuberaprost, caused some relaxation whereas beraprost instead produced a weak contraction. Conclusions and implicationsStereoisomer separation of beraprost has a significant effect on the pharmacology of the individual isomer, esuberaprost, identified in vitro as a highly potent prostanoid IP receptor agonist.

The Role of the K2P Channels TASK‐1, TREK‐1 and TREK‐2 in the Use of Treprostinil Therapy in Pulmonary Arterial Hypertension

IntroductionPulmonary arterial hypertension (PAH) is a progressive and ultimately fatal disease affecting ~6 people per million, per year (1). Recent studies have reported mutations in the two‐pore domain potassium (K2P) channel TASK‐1 (KCNK3) giving rise to PAH (2). Treprostinil is a stable prostacyclin analogue often administered as a therapy in PAH, to keep blood vessels open. Its mode of action is thought to occur through an interaction with prostanoid receptors, DP2, EP2 and IP (3). Patients undergoing continuous subcutaneous treprostinil infusion can often exhibit severe pain at the site of delivery (4). In this study we investigate the action of treprostinil on TASK‐1 and on two other K2P channels, TREK‐1 (KCNK2) and TREK‐2 (KCNK10), which regulate the excitability of sensory neurons and are implicated in pain (5).MethodUsing the whole‐cell patch‐clamp technique, currents were measured through human TASK‐1, TREK‐1 and TREK‐2 channels transiently expressed in tsA‐201 cells. TASK‐1 was also co‐expressed with prostanoid receptors (IP, DP2 and EP2). Data are expressed as mean ± SEM (n = cells) of current measured at −40 mV and mean % change ± SEM (n = cells), with statistical analysis performed using a paired t‐test.ResultsAcute application of treprostinil (1 μM) on cells expressing TASK‐1 channels had no effect on current (control: 234 ± 80 pA; treprostinil: 240 ± 53 pA, n = 5, p &gt; 0.05). Co‐expression of TASK‐1 with various prostanoid receptors, followed by acute application of treprostinil (1 μM) resulted in varying effects on TASK‐1 current. In cells expressing WT TASK‐1 and IP receptors, the average TASK‐1 current significantly decreased by 50% in the presence of treprostinil (control: 333 ± 82 pA; treprostinil (1 μM): 173 ± 46 pA, n = 6, p &lt; 0.05). Whilst co‐expression of TASK‐1 with EP2 receptors saw a 30% increase in TASK‐1 current (control: 294 ± 65 pA; treprostinil (1 μM): 368 ± 78 pA, n = 8, p &lt; 0.05). Co‐expression of TASK‐1 with DP2 receptors saw no significant change (3%) in TASK‐1 current (control: 387 ± 45 pA; treprostinil (1 μM): 398 ± 54 pA, n = 5, p &gt; 0.05). Interestingly, treprostinil had a potent inhibitory effect on TREK‐1 (80 ± 8%, n = 5, p &lt; 0.05) and TREK‐2 channels (59 ± 9%, n = 5, p &lt; 0.05) in the absence of prostanoid receptors.ConclusionIn this study, acutely applied treprostinil did not appear to have a direct action on the TASK‐1 channels, themselves, instead its effects appeared to occur through the activation of prostanoid receptors and their associated signalling pathways. Conversely, treprostinil demonstrated a direct inhibitory effect on TREK‐1 and TREK‐2 channels. This effect of treprostinil may explain infusion site pain, a common side‐effect of patients on subcutaneous treatment (4). Acute enhancement of TREK channel activity at the infusion site could therefore help to reduce discomfort in PAH patients.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Prostanoid Receptor Antagonist Effects on Intraocular Pressure, Supported by Ocular Biodisposition Experiments.

Since all prostanoid receptors affect intraocular pressure (IOP) and endogenous prostanoids are found in ocular tissues, the pressor effects of prostanoid antagonists were comprehensively evaluated. The absence of effects of most of these antagonists was not entirely anticipated. To ensure no false-negative results, ocular biodisposition studies were conducted. Monkeys with laser-induced ocular hypertension were used to study antagonist effects on IOP. Ocular biodisposition of each antagonist was assessed in rabbits, with LC/MS/MS analyses of tissue extracts and blood. EP1, EP2, EP3, EP4, FP, IP, and TP prostanoid receptor antagonists did not affect IOP, even at a high 1% dose. These studies were followed by ocular biodisposition studies. Striking differences in ocular tissue bioavailability were observed, which were independent of solubility. Only the EP1 antagonist SC-51322 failed to penetrate sufficiently to be bioavailable in the aqueous humor and ciliary body/iris. This obliged testing an alternative EP1 antagonist, namely ONO-8713, to reliably conclude that an EP1 antagonist does not alter IOP. These antagonist studies provided no evidence for individual endogenous prostanoids exerting a meaningful role in regulating IOP. They do reaffirm the critical importance of studying ocular bioavailability for confirming negative data. Large differences among the antagonists in anterior segment and even ocular surface tissue biodisposition were observed in rabbits. It appears from these monkey studies, supported by rabbit ocular bioavailability data, that an absence of drug effect in the eye cannot be adequately substantiated without determination of ocular pharmacokinetics.

Stimulation of fat storage by prostacyclin and selective agonists of prostanoid IP receptor during the maturation phase of cultured adipocytes.

We have previously shown that cultured adipocytes have the ability to biosynthesize prostaglandin (PG) I2 called alternatively as prostacyclin during the maturation phase by the positive regulation of gene expression of PGI synthase and the prostanoid IP receptor. To clarify how prostacyclin regulates adipogenesis, we investigated the effects of prostacyclin and the specific agonists or antagonists for the IP receptor on the storage of fats during the maturation phase of cultured adipocytes. Exogenous PGI2 and the related selective agonists for the IP receptor including MRE-269 and treprostinil rescued the storage of fats attenuated by aspirin, a cyclooxygenase inhibitor. On the other hand, selective antagonists for IP such as CAY10441 and CAY10449 were effective to suppress the accumulation of fats as GW9662, a specific antagonist for peroxisome proliferator-activated receptor (PPAR)γ. Thus, pro-adipogenic action of prostacyclin can be explained by the action mediated through the IP receptor expressed at the maturation stage of adipocytes. Cultured adipocytes incubated with each of PGI2 and MRE-269 together with troglitazone, an activator for PPARγ, exhibited additively higher stimulation of fats storage than with either compound alone. The combined effect of MRE-269 and troglitazone was almost abolished by co-incubation with GW9662, but not with CAY10441. Increasing concentrations of troglitazone were found to reverse the inhibitory effect of CAY10441 in a dose-dependent manner while those of MRE-269 failed to rescue adipogenesis suppressed by GW9662, indicating the critical role of the PPARγ activation as a downstream factor for the stimulated adipogenesis through the IP receptor. Treatment of cultured adipocytes with cell permeable stable cAMP analogues or forskolin as a cAMP elevating agent partly restored the inhibitory effect of aspirin. However, excess levels of cAMP stimulated by forskolin attenuated adipogenesis. Supplementation with H-89, a cell permeable inhibitor for protein kinase A (PKA), had no effect on the promoting action of PGI2 or MRE-269 along with aspirin on the storage of fats, suggesting that the promotion of adipogenesis mediated by the IP receptor does not require the PKA activity.

The Prostacyclin Analog Treprostinil Inhibits Ano1-Encoded Ca2+-Activated Cl− Channels and Mouse Pulmonary Arterial Tone Through Stimulation of cAMP-Dependent Signaling Pathway

Ca2+-activated Cl- channels (CaCCs) encoded by the gene Tmem16a or Anoctamin1 (ANO1) produce membrane depolarization and contraction of vascular smooth muscle cells (VSMCs) when stimulated by endogenous vasoconstrictors acting on Gq-protein coupled receptors. Treprostinil (TPL) is one of several prostacyclin analogs clinically used to treat patients diagnosed with pulmonary arterial hypertension (PAH). TPL reduces pulmonary arterial tone in PAH patients by binding to the IP prostanoid receptor. Activation of this Gs-coupled receptor leads to elevation in cAMP levels causing vasorelaxation by reducing intracellular [Ca2+]i. In this study, we examined whether TPL could exert vasorelaxation of mouse pulmonary arteries (mPA) by inhibiting, at least in part, ANO1-encoded CaCCs. TPL dose-dependently inhibited the contraction of mPA elicited by 10 μM 5-HT (IC50 ≈ 500 nM). The specific ANO1 inhibitor T16Ainh-A01 also dose-dependently inhibited the 5-HT-induced contraction of mPA but had no effect on the KCl-induced contraction (85.4 mM) at concentrations ≤ 10 μM. 200 nM TPL produced a rightward shift of the dose-response curve to T16AInh-A01, suggesting that TPL may inhibit CaCCs. Consistent with this hypothesis, Ca2+-activated Cl- currents (elicited by 1 μM free Ca2+ in the pipette solution) recorded in HEK-293 cells transfected with mouse ANO1 were significantly reduced by TPL (1 μM), or the adenylate cyclase activator Forskolin (10 μM). These preliminary results suggest that elevation of cAMP levels (perhaps through a phosphorylation step involving Protein Kinase A) may down-regulate ANO1-encoded CaCCs and attenuate the depolarization and vasoconstriction triggered by 5-HT. Our findings also suggest that part of the beneficial effects of TPL in PAH patients might be attributable to an interaction with ANO1 channels.

Involvement of prostaglandin I2 in nitric oxide-induced vasodilation of retinal arterioles in rats

The soluble guanylyl cyclase/cGMP system plays an important role in the vasodilator response to nitric oxide (NO) in various vascular beds. However, in rat retinal arterioles, the cyclooxygenase-1/cAMP-mediated pathway contributes to the vasodilator effects of NO, although the specific prostanoid involved remains to be elucidated. In the present study, we investigated the role of prostaglandin I2 and its receptor (prostanoid IP receptor) system in NO-induced vasodilation of rat retinal arterioles in vivo. Fundus images were captured using a digital camera that was equipped with a special objective lens. Changes in diameter of retinal arterioles were assessed. The NO donor (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR3) increased the diameter of retinal arterioles but decreased systemic blood pressure in a dose-dependent manner. Treatment of rats with indomethacin, a non-selective cyclooxygenase inhibitor, markedly attenuated the retinal vasodilator, but not depressor responses to NOR3. The prostanoid IP receptor antagonist 4,5-dihydro-N-[4-[[4-(1-methylethoxy)phenyl]methyl]phenyl]-1H-imadazol-2-amine (CAY10441), and the prostaglandin I2 synthase inhibitor 9α,11α-azoprosta-5Z,13E-dien-1-oic acid (U-51605), both showed similar preventive effects against the NOR3-induced retinal vasodilator response. Neither CAY10441 nor U-51605 showed any significant effects on the depressor response to NOR3. NOR3 enhanced the release of prostaglandin I2 from cultured human retinal microvascular endothelial cells and the NOR3-induced prostaglandin I2 release was almost completely abolished by the cyclooxygenase-1 inhibitor SC-560, but not by the cyclooxygenase-2 inhibitor NS-398. However, NOR3 did not increase the release of prostaglandin I2 from human intestinal microvascular endothelial cells. These results suggest that NO exerts its dilatory effect via cyclooxygenase-1/prostaglandin I2/prostanoid IP receptor signaling mechanisms in the retinal vasculature.

Assessing the agonist profiles of the prostacyclin analogues treprostinil and naxaprostene, particularly their DP1 activity

In this study, the inhibitory profiles of the prostacyclin analogues treprostinil and naxaprostene on several isolated smooth muscle preparations have been investigated. Treprostinil was an agonist for prostanoid DP1, EP2 and IP receptors, but not EP4 receptors; its DP1 potency was only 3–4 times less than PGD2 itself. Naxaprostene was much more selective for IP receptors and tended towards partial agonism. Treprostinil is a 13,14-dihydro analogue and the role of conformation around C12–15 in controlling agonist specificity is debated; the synthesis of new analogues is proposed and possible clinical usage discussed. In terms of selective prostanoid antagonists employed, BW-A868C/MK-0524 (DP1), ACA-23 (EP2) and GW-627368 (EP4) were found fit for purpose. However, the IP antagonist RO-1138452 was compromised by α1 and α2-adrenoceptor-mediated contractile activity on rat tail artery and anti-muscarinic activity on mouse trachea. There is a need for IP receptor antagonists with better selectivity and higher affinity.

Mechanisms of endothelium-dependent relaxation evoked by anandamide in isolated human pulmonary arteries

Endocannabinoids contract, relax or do not affect vessels with different calibre and tone in the pulmonary circulation in four species. The aim of the present study was to determine the mechanisms involved in the anandamide-induced relaxation of human pulmonary arteries (hPAs). Studies were performed in the isolated hPAs pre-constricted with the prostanoid TP receptor agonist, U-46619. To detect fatty acid amide hydrolase (FAAH) expression, Western blots were used. Anandamide concentration dependently relaxed the endothelium-intact hPAs pre-constricted with U-46619. The anandamide-induced relaxation was virtually abolished by removal of the endothelium and strongly attenuated by inhibitors of cyclooxygenases (indomethacin, COX-1/COX-2, and nimesulide, COX-2), nitric oxide synthase (N G-nitro-l-arginine methyl ester) given separately or in combination, FAAH (URB597), and the prostanoid IP receptor antagonist, RO1138452. The anandamide-evoked relaxation in the endothelium-intact vessels was attenuated in KCl pre-constricted preparations or by the inhibitor of large-conductance Ca2+-activated K+ channels, iberiotoxin. In experiments performed in the presence of URB597 to exclude effects of anandamide metabolites, the antagonist of the endothelial cannabinoid receptor, O-1918, diminished the anandamide-evoked relaxation whereas the antagonists of cannabinoid CB1, CB2 and vanilloid TRPV1 receptors, AM251, SR144528 and capsazepine, respectively, had no effect. Western blot studies revealed the occurrence of FAAH protein in the hPAs. The present study shows that anandamide breakdown products, cyclooxygenase pathways, nitric oxide, potassium channels and the O-1918-sensitive cannabinoid receptor play a role in the anandamide-induced relaxation of the hPAs with intact endothelium.

New Insights into Structural Determinants for Prostanoid Thromboxane A2 Receptor- and Prostacyclin Receptor-G Protein Coupling

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.

Prostanoid Receptors Involved in Regulation of the Beating Rate of Neonatal Rat Cardiomyocytes

Although prostanoids are known to be involved in regulation of the spontaneous beating rate of cultured neonatal rat cardiomyocytes, the various subtypes of prostanoid receptors have not been investigated in detail. In our experiments, prostaglandin (PG)F2α and prostanoid FP receptor agonists (fluprostenol, latanoprost and cloprostenol) produced a decrease in the beating rate. Two prostanoid IP receptor agonists (iloprost and beraprost) induced first a marked drop in the beating rate and then definitive abrogation of beating. In contrast, the prostanoid DP receptor agonists (PGD2 and BW245C) and TP receptor agonists (U-46619) produced increases in the beating rate. Sulprostone (a prostanoid EP1 and EP3 receptor agonist) induced marked increases in the beating rate, which were suppressed by SC-19220 (a selective prostanoid EP1 antagonist). Butaprost (a selective prostanoid EP2 receptor agonist), misoprostol (a prostanoid EP2 and EP3 receptor agonist), 11-deoxy-PGE1 (a prostanoid EP2, EP3 and EP4 receptor agonist) did not alter the beating rate. Our results strongly suggest that prostanoid EP1 receptors are involved in positive regulation of the beating rate. Prostanoid EP1 receptor expression was confirmed by western blotting with a selective antibody. Hence, neonatal rat cardiomyocytes express both prostanoid IP and FP receptors (which negatively regulate the spontaneous beating rate) and prostanoid TP, DP1 and EP1 receptors (which positively regulate the spontaneous beating rate).

G PROTEIN‐COUPLED RECEPTORS

The invention provides human G-protein coupled receptors (GCREC) and polynucleotides which identify and encode GCREC. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with aberrant expression of GCREC.

Role of prostanoid IP and EP receptors in mediating vasorelaxant responses to PGI 2 analogues in rat tail artery: Evidence for G i/o modulation via EP 3 receptors

Prostanoid IP receptors coupled to Gs are thought to be the primary target for prostacyclin (PGI 2) analogues. However, these agents also activate prostanoid EP 1–4 receptor subtypes to varying degrees, which are positively (EP 2/4) or negatively (EP 3) coupled to adenylate cyclase through Gs or Gi, respectively. We investigated the role of these receptors in modulating relaxation to PGI 2 analogues cicaprost, iloprost and treprostinil in pre-contracted segments of rat tail artery. Prostanoid IP (RO1138452), EP 4 (GW627368X), EP 3 (L-798106), EP 1–3 (AH6809), and EP 1 (SC-51322) receptor antagonists were used to determine each receptor contribution. The role of G i/o was investigated using pertussis toxin (PTX), while dependence on cAMP was determined using adenylate cyclase (2′5′dideoxyadenosine, DDA) and protein kinase A (2′-O-monobutyryladenosine- 3′,5′-cyclic monophosphorothioate, Rp- isomer, Rp-2′-O-MB-cAMPS) inhibitors, and by measurement of tissue cAMP. All analogues caused relaxation which was significantly ( P < 0.01) inhibited by RO1138452; with maximum response to cicaprost, iloprost and treprostinil reduced by 51%, 66% and 37%, respectively. GW627368X had no effect when used alone, but in combination with RO1138452, caused a rightward shift of the curves for cicaprost and iloprost but not treprostinil. PTX treatment potentiated relaxation to all 3 analogues ( P < 0.01), as did L798106 and AH6809 but not SC-51322. Basal cAMP levels were higher in PTX-treated tissues and DDA- and Rp-2'-O-MB-cAMPs--sensitive responses increased to analogue concentrations < 0.1 μM. In conclusion, prostanoid EP 3 receptors via G i/o negatively modulate prostanoid IP receptor-mediated relaxation to cicaprost, iloprost and treprostinil. However, other pathways contribute to analogue-induced vasorelaxation, the nature of which remains unclear for treprostinil.

Localization of prostanoid receptors in the mouse inner ear

Conclusion: EP4, EP2, and IP prostanoid receptors exert an otoprotective function and FP may be important for fluid homeostasis in the inner ear. Objective: To investigate the expression of prostanoid receptors in the normal mouse inner ear. Methods: CBA/J mice were used in this study. The localization of prostanoid receptors, i.e. DP, EP1, EP2, EP3, EP4, FP, IP, and TP, in the inner ear, i.e. the cochlea, vestibular end organs, endolymphatic sac, was studied by immunohistochemical techniques. Results: The EP4, IP, and FP prostanoid receptors were found to be abundantly distributed in many inner ear structures, i.e. stria vascularis, inner and outer hair cells, spiral ganglion cells, vestibular sensory and ganglion cells, and the endolymphatic sac. EP2 and EP3 are also localized in the inner ear whereas DP, EP1, and TP are only weakly expressed.

553 Molecular effects of anti-angiogenic therapy in breast cancer xenografts

Prostacyclin analogues have the potential to be effective agents in a new generation of drug-eluting stents by virtue of prostanoid IP receptor mediated anti-proliferative effects on smooth muscle cells. However, prostanoid IP receptor mediated vessel relaxation is reduced at elevated analogue concentrations. The mechanisms underlying this loss of activity are unclear, and its influence on the anti-proliferative potential of these compounds remains to be determined. A classical organ bath approach was used to examine the functional response of the rabbit iliac artery to the prostacyclin analogues, AFP-07 and cicaprost. Selective receptor antagonists for prostanoid IP (RO-1138452), EP1 (SC-51322) and EP3 (L-798106) receptors were used to characterise the receptors involved. The effects of these agents on proliferation ([3H]-thymidine incorporation) of rabbit iliac artery smooth muscle cells stimulated by foetal calf serum were then studied. AFP-07 gave a bell-shaped log concentration–response curve consisting of prostanoid IP receptor mediated relaxation followed by reversal at higher concentrations. SC-51322 and L-798106 potentiated this relaxation, although only L-798106 completely removed the contractile element. The prostanoid EP3 receptor agonist, sulprostone, produced constriction, which was attenuated by L-798106. RO-1138452 blocked the inhibitory action of AFP-07 and cicaprost on proliferation, implicating an involvement of prostanoid IP receptors. L-798106 had no effect on the anti-proliferative effect of cicaprost, but reduced the effect of AFP-07. Non-selective activation of prostanoid EP3 receptors (and possibly prostanoid EP1 receptors) compromises the relaxant activity of prostacyclin analogues, although it does not reduce the anti-proliferative capacity of these compounds in the model studied.

Activation of prostanoid EP receptors by prostacyclin analogues in rabbit iliac artery: Implications for anti -restenotic potential

Prostacyclin analogues have the potential to be effective agents in a new generation of drug-eluting stents by virtue of prostanoid IP receptor mediated anti-proliferative effects on smooth muscle cells. However, prostanoid IP receptor mediated vessel relaxation is reduced at elevated analogue concentrations. The mechanisms underlying this loss of activity are unclear, and its influence on the anti-proliferative potential of these compounds remains to be determined. A classical organ bath approach was used to examine the functional response of the rabbit iliac artery to the prostacyclin analogues, AFP-07 and cicaprost. Selective receptor antagonists for prostanoid IP (RO-1138452), EP 1 (SC-51322) and EP 3 (L-798106) receptors were used to characterise the receptors involved. The effects of these agents on proliferation ([ 3H]-thymidine incorporation) of rabbit iliac artery smooth muscle cells stimulated by foetal calf serum were then studied. AFP-07 gave a bell-shaped log concentration–response curve consisting of prostanoid IP receptor mediated relaxation followed by reversal at higher concentrations. SC-51322 and L-798106 potentiated this relaxation, although only L-798106 completely removed the contractile element. The prostanoid EP 3 receptor agonist, sulprostone, produced constriction, which was attenuated by L-798106. RO-1138452 blocked the inhibitory action of AFP-07 and cicaprost on proliferation, implicating an involvement of prostanoid IP receptors. L-798106 had no effect on the anti-proliferative effect of cicaprost, but reduced the effect of AFP-07. Non-selective activation of prostanoid EP 3 receptors (and possibly prostanoid EP 1 receptors) compromises the relaxant activity of prostacyclin analogues, although it does not reduce the anti-proliferative capacity of these compounds in the model studied.