The Prostacyclin Analogue Iloprost Modulates CXCL10 in Systemic Sclerosis.

The prostacyclin analogue iloprost is used to treat vascular alterations and digital ulcers, the early derangements manifesting in systemic sclerosis (SSc), an autoimmune disease leading to skin and organ fibrosis. Bioindicator(s) of SSc onset and progress are still lacking and the therapeutic approach remains a challenge. The T helper 1 (Th1) chemokine interferon (IFN)γ-induced protein 10 (IP-10/CXCL10) associates with disease progression and worse prognosis. Endothelial cells and fibroblasts, under Th1-dominance, release CXCL10, further enhancing SSc's detrimental status. We analyzed the effect of iloprost on CXCL10 in endothelial cells, dermal fibroblasts, and in the serum of SSc patients. Human endothelial cells and dermal fibroblasts activated with IFNγ/Tumor Necrosis Factor (TNF)α, with/without iloprost, were investigated for CXCL10 secretion/expression and for intracellular signaling cascade underlying chemokine release (Signal Transducer and Activator of Transcription 1, STAT1; Nuclear Factor kappa-light-chain-enhancer of activated B cells, NF-kB; c-Jun NH2-terminal kinase, JNK: Phosphatidyl-Inositol 3-kinase (PI3K)/protein kinase B, AKT; Extracellular signal-Regulated Kinase 1/2, ERK1/2). CXCL10 was quantified in sera from 25 patients taking iloprost, satisfying the American College of Rheumatology (ACR)/European Alliance of Associations for Rheumatology (EULAR) 2013 classification criteria for SSc, and in sera from 20 SSc sex/age-matched subjects without therapy, previously collected. In human endothelial cells and fibroblasts, iloprost targeted CXCL10, almost preventing IFNγ/TNFα-dependent cascade activation in endothelial cells. In SSc subjects taking iloprost, serum CXCL10 was lower. These in vitro and in vivo data suggest a potential role of iloprost to limit CXCL10 at local vascular/dermal and systemic levels in SSc and warrant further translational research aimed to ameliorate SSc understanding/management.

Superiority of cilostazol among antiplatelet FDA-approved drugs against COVID 19 Mpro and spike protein: Drug repurposing approach.

Coronavirus disease 2019 (COVID 19) was first identified in Wuhan, China near the end of 2019. To date, COVID-19 had spread to almost 235 countries and territories due to its highly infectious nature. Moreover, there is no vaccine or Food and Drug Administration (FDA)-approved drug. More time is needed to establish one of them. Consequently, the drug repurposing approach seems to be the most attractive and quick solution to accommodate this crisis. In this regard, we performed molecular docking-based virtual screening of antiplatelet FDA-approved drugs on the key two viral target proteins: main protease (Mpro ) and spike glycoprotein (S) as potential inhibitor candidates for COVID-19. In the present study, 15 antiplatelet FDA-approved drugs were investigated against the concerned targets using the Molecular Docking Server. Our study revealed that only cilostazol has the most favorable binding interaction on Mpro (PDB ID: 6LU7) and cilostazol, iloprost, epoprostenol, prasugrel, and icosapent ethyl have a higher binding affinity on spike glycoprotein (S) (PDB ID: 6VYB) compared with recent anti-CoVID-19. Therefore, cilostazol is a promising FDA drug against COVID-19 by inhibiting both Mpro and S protein. The insights gained in this study may be useful for quick approach against COVID-19 in the future.

Anti-inflammatory effects of the prostacyclin analogue iloprost in an in vitro model of inflamed human dental pulp cells.

Iloprost's anti-inflammatory effects on human dental pulp stem cells (HDPCs) are currently unknown. We hypothesized that iloprost could downregulate the expression of inflammatory-related genes and protein in an inflamed HDPC in vitro model. To induce inflammation, the HDPCs were treated with a cocktail of interleukin-1 beta, interferon-gamma, and tumour necrosis alpha, at a ratio of 1:10:100. Iloprost (10-6  M) was then added or not to the cultures. Interleukin-6 (IL-6) and interleukin-12 (IL-12) mRNA expression were assessed by real-time polymerase chain reaction. IL-6 protein expression was assessed by enzyme-linked immunosorbent assay. The results were analysed using one-way ANOVA or the Kruskal-Wallis test. The cytokine cocktail induced more robust IL-6 expression than LPS treatment. Iloprost slightly, yet significantly, downregulated IL-6 and IL-12 mRNA expression. These findings suggest that iloprost might be used as a beneficial component in vital pulp therapy.

Adenosine diphosphate-induced aggregation is enhanced in platelets obtained from patients with thrombotic primary antiphospholipid syndrome (t-PAPS): Role of P2Y12 -cAMP signaling pathway.

BACKGROUND: Thrombotic antiphospholipid syndrome (t-PAPS) is characterized by arterial, venous, or microvascular occlusions, which are explained, in part, by the presence of antiphospholipid (aPL) antibodies. Although there is much evidence indicating that isolated aPL antibodies increase the activity of platelets obtained from healthy volunteers, platelet function in t-PAPS has not been as widely studied. OBJECTIVE: To evaluate platelet reactivity in t-PAPS patients. METHODS: Platelet aggregation, protein expression, and cyclic nucleotide levels were carried out in platelet rich plasma (PRP) or washed platelets (WPs) obtained from t-PAPS or healthy volunteers. RESULTS: ADP-induced aggregation was significantly higher in PRP obtained from t-PAPS than obtained from the control. The protein expression of P2Y12 receptor and Gs alpha was significantly higher and lower, respectively in WPs from t-PAPS patients. In PRP incubated with iloprost or sodium nitroprusside, the residual platelet reactivity induced by ADP was still higher in PRP from t-PAPS than from the control. Lower intracellular levels of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) were observed in unstimulated PRP from t-PAPS patients. The protein expression of soluble guanylate cyclase subunits and phosphodiesterases types 3 and 5 did not differ. The antiplatelet activity of ticagrelor was similar between the groups and cilostazol significantly potentiated this response. Isolated aPL antibodies obtained from t-PAPS patients potentiated ADP-induced aggregation in healthy platelets but did not affect the inhibitory responses induced by iloprost or sodium nitroprusside. CONCLUSIONS: The overexpression of P2Y12 receptor, accompanied by lower levels of cAMP and cGMP levels produced greater amplitude of ADP aggregation in platelets from t-PAPS patients.

Prostaglandin E2 protects murine lungs from bleomycin-induced pulmonary fibrosis and lung dysfunction.

Prostaglandin E(2) (PGE(2)) is a lipid mediator that is produced via the metabolism of arachidonic acid by cyclooxygenase enzymes. In the lung, PGE(2) acts as an anti-inflammatory factor and plays an important role in tissue repair processes. Although several studies have examined the role of PGE(2) in the pathogenesis of pulmonary fibrosis in rodents, results have generally been conflicting, and few studies have examined the therapeutic effects of PGE(2) on the accompanying lung dysfunction. In this study, an established model of pulmonary fibrosis was used in which 10-12-wk-old male C57BL/6 mice were administered a single dose (1.0 mg/kg) of bleomycin via oropharyngeal aspiration. To test the role of prostaglandins in this model, mice were dosed, via surgically implanted minipumps, with either vehicle, PGE(2) (1.32 µg/h), or the prostacyclin analog iloprost (0.33 µg/h) beginning 7 days before or 14 days after bleomycin administration. Endpoints assessed at 7 days after bleomycin administration included proinflammatory cytokine levels and measurement of cellular infiltration into the lung. Endpoints assessed at 21 days after bleomycin administration included lung function assessment via invasive (FlexiVent) analysis, cellular infiltration, lung collagen content, and semiquantitative histological analysis of the degree of lung fibrosis (Ashcroft method). Seven days after bleomycin administration, lymphocyte numbers and chemokine C-C motif ligand 2 expression were significantly lower in PGE(2)- and iloprost-treated animals compared with vehicle-treated controls (P < 0.05). When administered 7 days before bleomycin challenge, PGE(2) also protected against the decline in lung static compliance, lung fibrosis, and collagen production that is associated with 3 wk of bleomycin exposure. However, PGE(2) had no therapeutic effect on these parameters when administered 14 days after bleomycin challenge. In summary, PGE(2) prevented the decline in lung static compliance and protected against lung fibrosis when it was administered before bleomycin challenge but had no therapeutic effect when administered after bleomycin challenge.

Bronchodilation induced by PGE2 is impaired in Group III pulmonary hypertension.

BACKGROUND AND PURPOSE: 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. PGE2 and PGI2 induce potent relaxation of human bronchi from non-PH (control) patients via EP4 and IP receptors, respectively. However, the effects of PGE2 /PGI2 and their mimetics on human bronchi from PH patients are unknown. Here, we have compared relaxant effects of several PGI2 -mimetics approved for treating PH Group I with several PGE2 -mimetics, in bronchial preparations derived from PH Group III and control patients. EXPERIMENTAL APPROACH: 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 PGE2 , PGI2 , and cAMP levels were determined by ELISA. KEY RESULTS: Maximal relaxations induced by different EP4 receptor agonists (PGE2 , L-902688, and ONO-AE1-329) were decreased in human bronchi from PH patients, compared with controls. However, maximal relaxations produced by PGI2 -mimetics (iloprost, treprostinil, and beraprost) were similar for both groups of patients. Both EP4 and IP receptor protein and mRNA expressions were significantly lower in human bronchi from PH patients. cAMP levels significantly correlated with PGI2 but not with PGE2 levels. CONCLUSION AND IMPLICATIONS: The PGI2 -mimetics retained maximal bronchodilation in PH Group III patients, whereas bronchodilation induced by EP4 receptor agonists was decreased. Restoration of EP4 receptor expression in airways of PH Group III patients with respiratory diseases could bring additional therapeutic benefit.

Transcriptional inhibition of protease-activated receptor-1 expression by prostacyclin in human vascular smooth muscle cells.

OBJECTIVE: Stimulation of protease-activated receptor-1 (PAR-1) by thrombin causes vascular smooth muscle cell (SMC) mitogenesis and has been implicated in the vascular response to injury. Vascular injury is also associated with enhanced formation of PGE2 and PGI2 (prostacyclin). This study investigates whether PGI2 and PGE2 modify the expression of PAR-1 and the cellular response to thrombin in human SMC. METHODS AND RESULTS: The PGI2-mimetic iloprost (1 to 100 nmol/L) attenuated mRNA, total protein, and cell surface expression of PAR-1. This was associated with inhibition of thrombin-induced mitogenesis and migration. Comparable inhibition of PAR-1 expression was observed with the selective IP-receptor agonist cicaprost, the adenylyl cyclase activator forskolin, the phosphodiesterase inhibitor isobutylmethylxanthine and the PKA activator dibutyryl-cAMP. Similar effects of PGE2 required micromolar concentrations. The specific PKA-inhibitor Myr-PKI prevented PAR-1 downregulation by iloprost. The potential role of Rho family GTPases in PAR-1 regulation was also investigated. Iloprost decreased Rac1 mRNA and the Rac1 inhibitor NSC23766 mimicked the inhibitory effects of iloprost on PAR-1 protein--but not mRNA. The Rho kinase inhibitor Y27632 did not influence PAR-1 expression. CONCLUSIONS: IP-receptor agonists may limit the mitogenic actions of thrombin in human SMC by downregulating PAR-1 via modulation of cAMP-/PKA- and Rac1-dependent signaling pathways.

The effect of the farnesyl protein transferase inhibitor SCH66336 on isoprenylation and signalling by the prostacyclin receptor.

Like Ras, farnesylation of the IP (prostacyclin receptor) is required for its efficient intracellular signalling, and hence the IP represents a potential target for inhibition by FTIs [FTase (farnesyl protein transferase) inhibitors]. Herein, the effect of SCH66336 on the isoprenylation and function of the human and mouse IPs overexpressed in human embryonic kidney 293 cells, and by the IP endogenously expressed in human erythroleukaemia cells, was investigated. SCH66336 yielded concentration-dependent decreases in IP-mediated cAMP generation (IC50 0.27-0.62 nM), [Ca2+]i mobilization (IC50 26.6-48.3 nM) and IP internalization, but had no effect on signalling by the non-isoprenylated beta2 adrenergic receptor or b isoform of the TP (prostanoid thromboxane A2 receptor). Additionally, SCH66336 impaired IP-mediated crossdesensitization of TPa signalling (IC50 56.1 nM) and reduced farnesylation of the molecular chaperone protein HDJ-2 (IC50 3.1 nM). To establish whether farnesylation of the IP is inhibited and/or whether its 'CaaX motif' might undergo alternative geranylgeranylation in the presence of SCH66336, a series of chimaeric Ha (Harvey)-Ras fusions were generated by replacing its CaaX motif (-CVLS) with that of the IP (-CSLC) or, as controls, of Ki (Kirsten)-Ras 4B (-CVIM) or Rac 1 (-CVLL). Whereas SCH66336 had no effect on Ha-RasCVLL isoprenylation in vitro or in whole cells, it supported alternative geranylgeranylation of Ha-RasCVIM, but completely impaired isoprenylation of both Ha-RasCVLS and Ha-RasCSLC. These data confirm that the -CSLC motif of the IP is a direct target for inhibition by the FTI SCH66336, and in the presence of strong FTase inhibition, the IP does not undergo compensatory geranylgeranylation

[3H]iloprost and prostaglandin E2 compete for the same receptor site on cardiac sarcolemmal membranes.

We have previously demonstrated that high-affinity PGE receptors are present on purified cardiac sarcolemmal (SL) membrane from bovine heart (Lopaschuk et al. (1989) Circ. Res. 65, 538-545). In this study we determined whether PGI2 receptors are also present on the cardiac SL membrane. Due to the extreme lability of prostacyclin (PGI2) under physiological conditions, the PGI2 analogue, Iloprost was substituted for PGI2. 3H-Iloprost specifically bound to two sites on the SL membrane; one of high affinity (Kd = 0.3 nM, Bmax = 97.0 fmol/mg SL), and one of lower affinity (Kd = 20.6 nM, Bmax = 1589 fmol/mg SL). Competition studies demonstrated that the concentrations of PGE2 and PGE1 necessary to displace 50% of the specific binding of 20 nM [3H]Iloprost on cardiac SL were 15-fold lower than the concentrations of unlabelled Iloprost necessary to displace 50% of binding. In contrast, a 15-fold higher concentration of unlabelled Iloprost was needed to displaced 50% of specific binding of 2 nM [3H]PGE2 compared to the concentrations of PGE1 or PGE2 required to displace 50% of [3H]PGE2 binding. In summary, our results indicate that a prostacyclin receptor is present on the cardiac sarcolemmal membrane, and that PGI2 competes for the same receptor site as PGE2.

Cyclic AMP produces desensitization of prostacyclin and adenosine A2 receptors in hybrid cell lines but does not affect Gs function.

Prostacyclin and adenosine A2 receptors stimulate adenylate cyclase activity in the related somatic hybrid cell lines NG108-15 and NCB20. The role of cAMP in the desensitization of these receptors has been examined. Pretreatment for 17 h with forskolin or 8-bromo-cAMP had the same effect in both cell lines. There was no change in the response to sodium fluoride or forskolin, suggesting that the function of Gs and adenylate cyclase were unaffected by increased levels of cAMP. Receptor responses were affected however; the maximum response to N-ethylcarboxamidoadenosine (an A2 receptor agonist) was reduced by 30-40%, there was a small but consistent shift to the right of the dose-response curve for iloprost (a stable analogue of prostacyclin) and [3H]iloprost binding studies revealed a loss of prostacyclin receptors. However, the loss of receptor responsiveness was much smaller than that which occurs following pretreatment with prostacyclin or adenosine A2 receptor agonists (Keen et al. (1989) Biochem. Pharmacol. 38, 3827-3833; Kelly et al. (1990) Br. J. Pharmacol. 99, 309-316) suggesting that cAMP may not play a major role in agonist mediated desensitization.

Induction of prostaglandin I(2) receptor by tumor necrosis factor alpha in osteoblastic MC3T3-E1 cells.

Mouse osteoblastic cells MC3T3-E1 produced prostaglandin E(2) via the reaction of cyclooxygenase-2 enzyme induced by tumor necrosis factor alpha (TNFalpha). Originally, the mRNA level for prostaglandin I(2) receptor (IP) was low in the cells. However, the addition of TNFalpha brought about a marked increase in the IP mRNA with a lag of about 3 h up to an about 8-fold higher level for 24 h. In addition, the induction of IP was supported by a binding experiment of [(3)H]iloprost (a stable analogue of prostaglandin I(2)). The amount of iloprost bound to the TNFalpha-stimulated cell membranes increased to a saturation level around 30 nM. Dexamethasone, cycloheximide and cyclooxygenase inhibitor suppressed the IP mRNA induction. The finding with the latter two compounds suggested a TNFalpha-dependent de novo synthesis of a protein, which is involved in the IP mRNA induction and may be attributed partially to the induced cyclooxygenase-2.

Expression of prostacyclin receptors in luteinizing hormone-releasing hormone immortalized neurons: role in the control of hormone secretion.

PGs of the E series are involved in the control of LHRH secretion. The present experiments were conducted to clarify whether PGI2 (prostacyclin) might be also involved in such a control, using multiple methodological approaches on immortalized LHRH-secreting neurons. A RT-PCR procedure to detect mouse PGI2 receptor (IP) messenger RNA was first applied, and the results obtained showed the presence of a specific transcript in two cell lines of immortalized LHRH neurons (GT1-1 and GN11 cell lines). Receptor binding assays on membrane preparations from GT1-1 cells showed the presence of a single specific and saturable class of binding sites (Kd = 4.6 nM; 10,000 sites/cell) for [3H]iloprost, a stable analog of PGI2. Competition experiments showed that the binding sites labeled by [3H]iloprost possess the pharmacological characteristics of IP receptors. In functional studies, PGI2 and its analogs, iloprost and cicaprost, were able to stimulate LHRH release from the GT1-1 cells with elevated potencies (EC50 = 0.6-4.3 nM); PGE1 was only slightly less active (EC50 = 28.5 nM), whereas PGE2, considered the major PG involved in LHRH secretion, was poorly effective (EC50 = 921 nM). The relative potencies (EC50) of these compounds in stimulating the intracellular accumulation of cAMP were in line with their LHRH-releasing activities. In conclusion, these results indicate that immortalized LHRH-secreting neurons express IP receptors through which PGI2 may exert relevant effects on LHRH release.

Cloning and expression of a cDNA for the human prostacyclin receptor.

A functional cDNA for the human prostacyclin receptor was isolated from a cDNA library of CMK cells, a human megakaryocytic leukaemia cell line. The cDNA encodes a protein consisting of 386 amino acid residues with seven putative transmembrane domains and a deduced molecular weight of 40,956. [3H]Iloprost specifically bound to the membrane of CHO cells stably expressing the cDNA with a Kd of 3.3 nM. This binding was displaced by unlabelled prostanoids in the order of iloprost = cicaprost >> carbacyclin > prostaglandin E1 (PGE1) > STA2. PGE2, PGD2 and PGF 2 alpha did not inhibit it. Iloprost in a concentration-dependent manner increased the cAMP level and generated inositol trisphosphate in these cells, indicating that this human receptor can couple to multiple signal transduction pathways.

Nonprostanoid prostacyclin mimetics. 5. Structure-activity relationships associated with [3-[4-(4,5-diphenyl-2-oxazolyl)-5- oxazolyl]phenoxy]acetic acid.

cis-[3-[2-(4,5-Diphenyl-2-oxazolyl)ethenyl]phenoxy]acetic acid (3) was previously identified as a nonprostanoid prostacyclin (PGI2) mimetic that potently inhibits ADP-induced aggregation of human platelets with an IC50 of 0.18 microM. As part of an effort to further explore structure-activity relationships for this class of platelet inhibitor and to provide additional insight into the nonprostanoid PGI2 mimetic pharmacophore, the effect of constraining the cis-olefin moiety of 3 into various ring systems was examined. Incorporation of the cis-olefin of 3 into either an oxazole (26) or an unsubstituted pyrazole (35) heterocycle provided compounds that are equipotent with progenitor 3. However, the oxazole 11f, which is isomeric with 26, inhibits ADP-induced human platelet aggregation in vitro with an IC50 of 0.027 microM, 6-fold more potent than 3, 26, or 35. These results suggest that the central oxazole ring of 11f is functioning as more than a simple scaffold that provides optimal stereodefinition for interaction with the PGI2 receptor. The nitrogen atom of the central heterocycle of 11f is postulated to engage in hydrogen-bond formation with a donor moiety in the PGI2 receptor protein, an interaction not available to 26 due to the markedly different topology. In support of this contention, the crystal structures of 11f and 26 contain strong intermolecular hydrogen bonds between the carboxylic acid hydrogen atom and the nitrogen atom of the central oxazole ring. Although 11f and 26 are exact isosteres and could, in principle, adopt the same molecular packing arrangement in the solid state, this is not the case, and the intermolecular hydrogen-bonding interactions in 11f and 26 are accommodated by entirely different molecular packing arrangements. Incorporation of the olefin moiety of 3 into a benzene ring provided a compound, 40, over 60-fold weaker with an IC50 of 11.1 microM. The affinities of 11f, 26, 31, 32, and 40 for the human platelet PGI2 receptor, determined by displacement of [3H]iloprost, correlated with inhibition of platelet function. The solid-state structures of 11f, 26, 31, 32, and 40 were determined and revealed that the more potent compounds 11f and 26 adopt a relatively planar overall topography. In contrast, the central phenyl ring and the phenoxy ring of the weakly active compound 40 are rotated by 53 degrees from planarity. The chemical shifts of the protons of the phenoxy rings of 3, 11f, 18, 26, 31, 32, and 40 suggest that in solution 3, 11f, 18, and 26 adopt a planar conformation while 40 does not.(ABSTRACT TRUNCATED AT 400 WORDS)

Local delivery of platelets with encapsulated iloprost to balloon injured pig carotid arteries: effect on platelet deposition and neointima formation.

Local delivery of a drug to the arterial wall during angioplasty is an approach which might reduce the incidence of occlusive events such as thrombosis and restenosis, without the risk of systemic side effects. By exploiting their natural primary haemostatic properties, platelets, with encapsulated drugs, can be targeted to a vessel wall injury site and act as a depot for sustained release. The platelet plasma membrane can be reversibly permeabilised by high voltage, short duration electrical pulses (electroporation). Drugs will diffuse into porated platelets and become trapped on resealing. We have studied the effects of autologous platelets, electroloaded with the stable prostacyclin analogue, iloprost on platelet deposition and neointima formation in a pig carotid angioplasty model. Iloprost loaded or control platelets were delivered locally and immediately to the balloon injured site using a double balloon delivery catheter. Acute platelet deposition was measured using 111-Indium, and neointima formation at 21 days post angioplasty was assessed by morphometric analysis. In pigs treated with iloprost loaded platelets, platelet deposition on the artery at 2 hours post injury was dramatically reduced (to approximately monolayer coverage), when compared with arteries from pigs treated with control platelets. In pigs with deeply injured arteries, i.e. with extensively ruptured internal elastic lamina (IEL), platelet deposition was reduced by 88% compared with control arteries (118 +/- 20 x 10(6)/cm vs. 14 +/- 2 x 10(6)/cm, means +/- SI, 2P < 0.001). In minimally injured arteries (IEL intact) a 65% reduction in platelet deposition was observed (55 +/- 24 x 10(6)/cm vs. 19 +/- 3 x 10(6)/cm. 2P < 0.002). A high concentration of free iloprost, delivered to the angioplasty site, with control platelets, had far less effect on platelet deposition, substantiating the advantage of platelet encapsulation. At 21 days post injury, morphometry of the carotid arteries after treatment with iloprost loaded platelets showed significant reductions in intimal area and intimal/medial ratios in minimally injured vessels (P < 0.05) as compared with vessels from pigs treated with control platelets. With deeply injured vessels, the mean differences (control vs. treated) for the same morphometric parameters were not significant. This novel approach of electro-encapsulating drugs within autologous platelets, and using them as highly biocompatible and biodegradable drug targeting vehicles might, with the appropriate choice of encapsulated agent, have potential for reducing the incidence of occlusion after angioplasty and thrombolysis procedures.

Desensitization of adenylate cyclase responses following exposure to IP prostanoid receptor agonists. Homologous and heterologous desensitization exhibit the same time course.

Pretreatment of NG108-15 cells with 0.03-25 microM prostaglandin E1 (PGE1) produced decreases in the maximal stimulation of adenylate cyclase activity produced by iloprost, N-ethylcarboxamidoadenosine and sodium fluoride. The rate of desensitization to all three agents was dependent on the concentration of PGE1 used, but at each concentration of PGE1 the rate of loss of responsiveness to each agent was the same, suggesting that the decreases in responsiveness may be mediated by a single process. Functional desensitization was accompanied by a decrease in the specific binding of [3H]iloprost, consistent with a 75-80% decrease in IP receptor number, with no change in the coupling of the remaining IP receptors to G protein. At each concentration of PGE1 used, the times taken for half maximal decreases in receptor number and functional responsiveness were similar, suggesting that IP receptor down-regulation is a relatively early event in desensitization. IP receptor down-regulation could be inhibited partially by 100 microM chloroquine, suggesting that lysosomal breakdown of receptors may be occurring.

Thiopental and ether anaesthesia decrease the prostaglandin receptor density of the rat liver.

Male Sprague-Dawley rats were killed either by decapitation or during anaesthesia with thiopental or diethylether by aortectomy. Livers were removed and liver plasma membranes were prepared using standard techniques. Direct binding experiments with 3H-PGE1 and 3H-iloprost revealed heterogeneity of the binding sites (high and low affinity binding sites), whereas 3H-PGE2 demonstrated only high affinity binding to the liver. The highest binding capacity for all radioligands was found for livers after decapitation. Livers obtained during anaesthesia showed a significantly (p less than 0.05 to p less than 0.001) lower binding capacity and binding affinity for 3H-PGE1, 3H-PGE2 and 3H-iloprost. The reduction in binding activity was more pronounced in livers obtained during inhalation than thiopental anaesthesia. Specific binding amounted to 82.1 +/- 7% for 3H-PGE2, 75.3 +/- 9% for 3H-PGE1 and 78.9 +/- 8% for iloprost in livers obtained after decapitation. In livers obtained during anaesthesia specific prostaglandin binding was significantly (p less than 0.01) decreased, again being more pronounced during inhalation than thiopental anaesthesia. These results suggest that some anaesthetics interfere with prostaglandin receptors of the liver.

Pharmacology of functional endogenous IP prostanoid receptors in NCB-20 cells: comparison with binding data from human platelets.

The objective of these studies was to characterize the effects of a broad range of prostanoid agonists upon the stimulation of cAMP production in National Cancer Bank (NCB-20; mouse neuroblastoma/hamster brain hybridoma) cells. The pharmacology of these functional responses in NCB-20 cells was compared with that of the classic endogenous IP receptor present on human platelets using [3H]-iloprost binding techniques. In both assay systems, agonists from the IP prostanoid class exhibited the highest affinities and functional potencies. Specific prostanoids exhibited the following rank order of potency (EC50 +/- SEM) in stimulating cAMP production in the NCB-20 cells: carbaprostacyclin (4.3 +/- 0.9 nM) = PGI2 (6.6 +/-1.5 nM) > iloprost (75+/-13 nM) > 11-deoxy PGE, (378+/-138 nM) > misoprostol (1,243+/-48) > PGE2 (3020+/-700 nM) > ZK-118182 (7265+/-455 nM). Iloprost wasthe most potent compound in the human platelet binding assay while prostanoidsfromthe DPand EP receptor classes showed modest affinity. These studies provide functional and binding information for a broad range of both natural and synthetic prostanoid receptor ligands at the endogenous IP receptor in two different cell types.

Guanine nucleotide sensitivity of [3H]iloprost binding to prostacyclin receptors.

A component of the displaceable binding of the stable prostacyclin analogue, [3H]iloprost, to membranes from human platelets and the somatic hybrid cell lines NG108-15 and NCB20, was inhibited by guanine nucleotides. The order of potency of a range of nucleotides for this effect was GTP gamma S greater than GppNHp greater than GTP greater than GDP = GMP; ATP, UTP and CTP were ineffective at concentrations up to 1 mM. In the presence of 100 microM GppNHp, iloprost binding curves were displaced to the right of curves obtained in the absence of guanine nucleotide, and their Hill slopes were greater. This was consistent with a conversion of a minor population of high affinity agonist binding sites to lower affinity sites in the presence of guanine nucleotides. These effects of guanine nucleotides on the binding of the agonist ligand [3H]iloprost were consistent with an interaction with a G protein coupled receptor.

Gastric prostaglandin E2 receptors are the common antisecretory target of mucosal prostanoids.

The gastric mucosa produces all principal prostaglandin (PG) types, but receptor binding studies in this tissue have as yet been performed exclusively with [3H]PGE2. Therefore we compared the binding of different 3H-labelled prostanoids to fundic mucosal plasma membranes from the porcine stomach. Binding sites for [3H]PGE2, [3H]iloprost and [3H]PGF2 alpha had similar nanomolar dissociation constants with high affinities for unlabelled PGE2. Iloprost and PGF2 alpha were 10- and 100-fold less potent competitors with Hill slopes near unity in all cases. In further [3H]PGE2 competition studies the affinities of prostanoid ligands with selectivity for different PG receptor types correlated closely with their respective antisecretory potencies, as tested by [14C]aminopyrine uptake in isolated porcine parietal cells. We conclude that parietal cell PGE2 receptors are the common antisecretory target for all prostanoid types in the porcine stomach. There was no evidence for other mucosal PG receptors possibly involved in acid secretion.