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.

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.

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.

'VASPFix' for measurement of VASP phosphorylation in platelets and for monitoring effects of P2Y12 antagonists.

Vasodilator-stimulated phosphoprotein (VASP) is phosphorylated and dephosphorylated consequent to increases and decreases in cyclic nucleotide levels. Monitoring changes in VASP phosphorylation is an established method for indirect measurement of cyclic nucleotides. Here we describe the use of an innovative cocktail, VASPFix, which allows sensitive and reproducible measurement of phosphorylated VASP (VASP-P) in a simple, single-step procedure using cytometric bead technology. Frozen VASPFix-treated samples are stable for at least six months prior to analysis. We successfully used VASPFix to measure VASP-P in platelets in both platelet-rich plasma and blood in response to compounds that increase (dibutyryl cAMP, adenosine, iloprost, PGE1) and decrease (ADP, PGE1) cAMP, and to determine the effects of certain receptor antagonists on the results obtained. The change in VASP-P brought about by adding ADP to PGE1-stimulated platelets is a combination of the effect of ADP at the P2Y12 receptor and of PGE1 at both IP and EP3 receptors. For iloprost-stimulated platelets EP3 receptors are not involved. A procedure in which iloprost, ADP and VASPFix were used to determine effectiveness of clopidogrel and prasugrel in patients was compared with an established commercial procedure that uses PGE1 and ADP; the latter produced higher platelet reactivity values that were the result of PGE1 interacting with platelet EP3 receptors. We conclude that VASPFix can be used both as a research tool and for clinical investigations and provides better specificity for P2Y12 receptor inhibition. The latter confers a distinct advantage over existing methods used to monitor effects of P2Y12 antagonists on platelet function.

Diamide decreases deformability of rabbit erythrocytes and attenuates low oxygen tension-induced ATP release.

Exposure of erythrocytes to reduced oxygen (O(2)) tension activates the heterotrimeric G-protein Gi, resulting in the accumulation of cyclic AMP (cAMP) and release of ATP. The mechanism by which exposure of erythrocytes to reduced O(2) tension activates Gi is not known. Here we investigate the hypothesis that, in rabbit erythrocytes, ATP release in response to exposure to reduced O(2) tension is linked to erythrocyte membrane deformability. If this hypothesis is correct, then decreasing the deformability of the erythrocyte membrane should decrease the release of ATP in response to reduced O(2) tension. We report that treating erythrocytes with diamide, a compound that decreases erythrocyte deformability, inhibits low O(2) tension-induced ATP release. Treating erythrocytes with diamide does not, however, interfere with cAMP accumulation or ATP release in response to a direct activator of Gi (mastoparan 7) or in response to receptor-mediated activation of Gs (the prostacyclin analog, iloprost). These results demonstrate that diamide (100 micromol/L) does not directly inhibit the signaling pathways for ATP release from rabbit erythrocytes and support the hypothesis that low O(2) tension-induced ATP release from these cells is linked to membrane deformability.

Targeting of the prostacyclin specific IP1 receptor in lungs with molecular conjugates comprising prostaglandin I2 analogues.

Molecular conjugates comprising targeting ligands hold great promise for site-specific gene delivery to distant tumors and individual organs including the lung. Here we show that prostaglandin I2 analogues can be used to improve gene transfer efficiency of polyethylenimine (PEI) gene vectors on bronchial and alveolar epithelial cells in vitro and lungs of mice in vivo. Prostacyclin (IP1) receptor expression was confirmed in pulmonary epithelial cell lines by western blot. Iloprost (ILO) and treprostinil (TRP), two prostaglandin I2 analogues, were conjugated to fluorescein-labeled BSA (FLUO-BSA) and compared for IP1 receptor binding/uptake in different lung cell lines. Binding of FLUO-BSA-ILO was 2-4-fold higher than for FLUO-BSA-TRP and could be specifically inhibited by free ILO and IP1 receptor antagonist CAY10449. Internalization of FLUO-BSA-ILO was confirmed by confocal microscopy. Molecular conjugates of PEI and ILO (PEI-g-ILO) were synthesized with increasing coupling degree (F(ILO) (ILO:PEI) = 2, 5, 8, 16) and analyzed for DNA binding, particle formation and transfection efficiency. At optimized conditions (N/P 4, F(ILO) = 5), gene expression using PEI-g-ILO was significantly up to 46-fold higher than for PEI gene vectors and specifically inhibited by CAY10449. Gene expression in the lungs of mice after aerosol delivery was 14-fold higher with PEI-g-ILO F(ILO) = 5 than for PEI. We suggest that targeting of IP1 receptor using ILO represents a promising approach to improve pulmonary gene transfer.

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

Prostacyclin is an autocrine regulator in the contraction of oviductal smooth muscle.

BACKGROUND: It was recently discovered that prostacyclin constituted 40-50% of prostaglandins (PG) produced by minced human oviduct. It is well established that prostacyclin relaxes vascular smooth muscle, but whether oviductal smooth muscle synthesizes prostacyclin and whether its contraction is affected by prostacyclin remain unclear. METHODS: Smooth muscle microdissected from human oviducts was used for the study. The expression of prostacyclin synthase (PGIS) and prostacyclin receptor (IP) was confirmed by Western blot analysis. Metabolites of [(3)H]PGH(2) were analysed for prostacyclin. Functional coupling of IP to adenyl cyclase was assessed by the accumulation of intracellular cAMP upon prostacyclin challenge. The presence of saturable, specific binding sites for prostacyclin was confirmed by binding assay. The identity of IP was further confirmed by RT-PCR and nucleotide sequence analysis. Finally, the effects of prostacyclin on muscle contraction were studied. RESULTS: Human oviductal smooth muscle expresses functionally active PGIS and IP. The IP expressed is the same as that cloned from human lung tissue. The ED(50) of prostacyclin to increase intracellular cAMP was 16 nmol/l. Prostacyclin dose-dependently decreased the amplitude of muscle contraction. CONCLUSIONS: Human oviductal smooth muscle produces prostacyclin, which, in turn, decreases its contractility. Prostacyclin may regulate embryo transport.

Impaired receptor binding and activation associated with a human prostacyclin receptor polymorphism.

The human prostacyclin receptor (hIP) is a seven transmembrane-spanning G-protein-coupled receptor that plays an important role in vascular homeostasis. Recent genetic analyses (SNP database, NCBI) have revealed the first two polymorphisms within the coding sequence, V25M and R212H. Here we present structure-function characterizations of these polymorphisms at physiological pH (7.4) and at an acidic pH (6.8) that would be encountered during stress such as renal, respiratory, or heart failure. Through a series of competition binding and G-protein activation assays (measured by cAMP production), we determined that the V25M polymorph exhibited agonist binding and G-protein activation similar to wild-type receptor at normal pH (7.4). However, the R212H variant demonstrated a significant decrease in binding affinity at lower pH (R212H at pH 7.4, K(i) = 2.2 +/- 1.2 nm; pH 6.8 K(i) = 45.6 +/- 12.0 nm). The R212H polymorph also exhibited abnormal activation at both pH 7.4 and pH 6.8 (pH 7.4, R212H EC(50) = 2.8 +/- 0.5 nm versus wild-type hIP EC(50) = 0.5 +/- 0.1 nm; pH 6.8, R212H EC(50) = 3.2 +/- 1.6 nm versus wild-type hIP EC(50) = 0.5 +/- 0.2 nm). Polymorphisms of the human prostacyclin receptor potentially may be important predictors of disease progress during biological stressors such as acidosis in which urgent correction of bodily pH may be required to restore normal hemostasis and vasodilation. This study provides the mechanistic basis for further research into genetic risk factors and pharmacogenetics of cardiovascular disease associated with hIP.

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.

Characterization of chimeric prostacyclin/prostaglandin D(2) receptors.

The functional activity of two chimeric mouse prostacyclin/prostaglandin D(2) (IP/DP) receptors, in which the carboxyl-terminal region of the IP receptor was progressively replaced by that of the DP receptor, was examined in Chinese hamster ovary (CHO) cells. The order of potency of prostaglandin D(2), prostaglandin E(2) and the IP receptor agonists cicaprost, iloprost and BMY 45778 (3-[4-(4, 5-diphenyl-2-oxazolyl)-5-oxazolyl]phenoxy]acetic acid) to stimulate cyclic AMP production was identical for the IP(N-VII)/DP(C), IP(N-V)/DP(VI-C) and wild-type IP receptors. IP(N-VII)/DP(C) receptor-expressing cells showed increases in basal adenylate cyclase activity, agonist potency and coupling efficiency. In addition, the intrinsic activity of the partial IP receptor agonists BMY 45778 and PGE(2) was significantly increased in IP(N-VII)/DP(C) receptor-expressing cells. Therefore, substitution of just the carboxyl-terminal tail of the IP receptor by that of the DP receptor appears to result in a chimeric IP/DP receptor with all the properties of a constitutively-active receptor.

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.

The prostacyclin receptor is isoprenylated. Isoprenylation is required for efficient receptor-effector coupling.

The prostacyclin receptor (IP), a G protein-coupled receptor, mediates the actions of the prostanoid prostacyclin and its mimetics. IPs from a number of species each contain identically conserved putative isoprenylation CAAX motifs, each with the sequence CSLC. Metabolic labeling of human embryonic kidney (HEK) 293 cells stably overexpressing the hemagluttinin epitope-tagged IP in the presence of [(3)H]mevalonolactone established that the mouse IP is isoprenylated. Studies involving in vitro assays confirmed that recombinant forms of the human and mouse IP are modified by carbon 15 farnesyl isoprenoids. Disruption of isoprenylation, by site-directed mutagenesis of Cys(414) to Ser(414), within the CAAX motif, abolished isoprenylation of IP(SSLC) both in vitro and in transfected cells. Scatchard analysis of the wild type (IP) and mutant (IP(SSLC)) receptor confirmed that each receptor exhibited high and low affinity binding sites for [(3)H]iloprost, which were not influenced by receptor isoprenylation. Whereas stable cell lines overexpressing IP generated significant agonist (iloprost and cicaprost)-mediated increases in cAMP relative to nontransfected cells, cAMP generation by IP(SSLC) cells was not significantly different from the control, nontransfected HEK 293 cells. Moreover, co-expression of the alpha (alpha) subunit of Gs generated significant augmentations in cAMP by IP but not by IP(SSLC) cells. Whereas IP also demonstrated significant, dose-dependent increases in [Ca(2+)](i) in response to iloprost or cicaprost compared with the nontransfected HEK 293 cells, mobilization of [Ca(2+)](i) by IP(SSLC) was significantly impaired. Co-transfection of cells with either Galpha(q) or Galpha(11) resulted in significant augmentation of agonist-mediated [Ca(2+)](i) mobilization by IP cells but not by IP(SSLC) cells or by the control, HEK 293 cells. In addition, inhibition of isoprenylation by lovastatin treatment significantly reduced agonist-mediated cAMP generation by IP in comparison to the nonisoprenylated beta(2) adrenergic receptor or nontreated cells. Hence, isoprenylation of IP does not influence ligand binding but is required for efficient coupling to the effectors adenylyl cyclase and phospholipase C.

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.

Selective activation of a chimeric Gi1/Gs G protein alpha subunit by the human IP prostanoid receptor: analysis using agonist stimulation of high affinity GTPase activity and [35S]guanosine-5'-O-(3-thio)triphosphate binding.

A FLAG-tagged form of the human IP prostanoid receptor was expressed stably in HEK 293 cells. This bound [3H]iloprost with high affinity and stimulated cAMP production when exposed to agonist. Iloprost produced weak stimulation of GTPase activity and [35S]guanosine-5'-O-(3-thio)triphosphate binding in membranes of these cells. Pretreatment of cells with pertussis toxin did not modify iloprost-mediated stimulation, but this was blocked by cholera toxin. The effects of iloprost were not increased by coexpression of either Gsalpha or Gi1alpha. In contrast, coexpression of a chimeric G protein alpha subunit in which the carboxyl-terminal six amino acids of Gi1alpha were altered to those of Gsalpha resulted in robust stimulation by iloprost. Because the chimeric G protein alpha subunit (Gi1/Gs6alpha) is not a substrate for either pertussis or cholera toxin, pretreatment of cells coexpressing the IP prostanoid receptor and Gi1/Gs6alpha with a mixture of these toxins resulted in resolution of the signal derived from activation of the chimeric G protein. Agonist-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate binding and GTPase activity assays are the most commonly used strategies to examine interactions between G protein-coupled receptors and G proteins. These usually are not appropriate for receptors such as the IP prostanoid receptor that interact with G proteins with low rates of guanine nucleotide exchange and hydrolysis. Chimeric G proteins such as Gi1/Gs6alpha that allow appropriate receptor contacts to be converted to the higher nucleotide turnover rates typical of the Gi family G proteins can overcome this and offer a novel means to examine agonist function at such receptors.

Prostacyclin receptor desensitization is a reversible phenomenon in human platelets.

BACKGROUND: Long-term exposure of platelets to endogenous or exogenous prostacyclin or its analogues might result in desensitization of the platelet prostacyclin receptor in vitro and in vivo accompanied by a loss in receptor density on the platelet surface and a reduced sensitivity toward the inhibitory effects of prostacyclins. However, the reversibility of this process in platelets has not yet been investigated. METHODS AND RESULTS: Human platelets desensitized by the chemically stable prostacyclin analogue iloprost showed a significant reduction in [3H]-iloprost binding sites that was reversed by saponin permeabilization. This indicates functionally active internalized prostacyclin receptors. To assess whether the internalized prostacyclin receptors recycle to the cell surface after withdrawal of the agonist, iloprost sensitivity and prostacyclin receptor binding properties of iloprost (30 nmol/L, 2 hours) desensitized platelets incubated in iloprost-free autologous plasma were investigated. While desensitized platelets showed a significant increase in IC50 for iloprost inhibition of thrombin-induced platelet aggregation, serotonin release, and p-selectin expression and a reduced iloprost-stimulated cAMP formation, platelet iloprost sensitivity was restored 3 hours after iloprost withdrawal. In addition, the significant reduction in Bmax and the increase in K(D) of prostacyclin receptors in desensitized platelets as revealed by [3H]-iloprost binding studies also returned to the initial values. CONCLUSIONS: These results indicate that prostacyclin receptors internalized during short-term desensitization are not degraded but can be recycled rapidly to the platelet surface in a functionally active form after withdrawal of the agonist.

Affinity for [3H]iloprost binding sites and cAMP synthesis activity of a 3-oxa-methano prostaglandin I1 analog, SM-10906, in human platelets and endothelial cells.

SM-10902 ((+)-methyl [2-[(2R,3aS,4R,5R,6aS)-octahydro-5-hydroxy-4-[(E)-(3S,5S)-3- hydroxy-5-methyl-1-nonenyl]-2-pentalenyl]ethoxy]acetate) and its free acid, SM-10906 are new stable 3-oxa-methano prostaglandin (PG) I1 analogs. Their affinities for [3H]iloprost and [3H]PGE2 binding sites in human platelets and human umbilical vascular endothelial cells were compared with those of the PGI2 analog iloprost, PGE1 and PGE2 by the radioligand binding assay method. The cyclic AMP (cAMP) synthesis activity of these drugs were also determined in human umbilical vascular endothelial cells. We found that SM-10906 apparently displaced [3H]iloprost binding to the membrane fractions in those cells since the pKi values were 6.30 in platelets, 7.52 in vein endothelial cells and 6.31 in the arterial endothelial cells. The pKi values of SM-10906 for [3H]PGE2 binding sites were significantly lower than those obtained for [3H]iloprost binding. SM-10902, which is a prodrug of SM-10906, showed low affinity for [3H]iloprost binding sites in those cells. SM-10906 also dose-dependently enhanced the cAMP level in the vascular endothelial cells. Thus, these findings indicate that SM-10906 binds to [3H]iloprost binding sites and exhibits pharmacological functions such as an anti-platelet action and a cytoprotective action in endothelial cells through the elevation of intracellular cAMP contents.

7,7-Difluoroprostacyclin derivative, AFP-07, a highly selective and potent agonist for the prostacyclin receptor.

Recently, we cloned cDNAs for the prostacyclin receptor (IP) and the four mouse PGE receptor subtypes, EP1, EP2, EP3, and EP4, and established Chinese hamster ovary cells that stably express each receptor. We examined the agonist potency and selectivity of AFP-07, a 7,7-difluoroprostacyclin derivative, compared with widely used stable prostacyclin analogue, iloprost, using the cells expressing each cloned receptor. AFP-07 strongly displaced the [3H] iloprost binding to the IP receptor-expressing cell membranes, the half maximal concentration for the displacement being 3 nM, which was one order lower than that of iloprost. AFP-07 concentration-dependently stimulated cAMP formation in the IP-expressing cells, the half-maximal concentration for the stimulation being 10 pM, which was one order lower than that of iloprost. On the other hand, AFP-07 showed lower affinity for EP1, EP2, EP3, and EP4 than PGE2, but iloprost had the same affinity as PGE2 for the EP1, These results demonstrate that AFP-07 is a potent and highly selective agonist for the IP receptor.

[3-[4-(4,5-Diphenyl-2-oxazolyl)-5-oxazolyl]phenoxy]acetic acid (BMY 45778) is a potent non-prostanoid prostacyclin partial agonist: effects on platelet aggregation, adenylyl cyclase, cAMP levels, protein kinase, and iloprost binding.

[3-[4-(4,5-diphenyl-2-oxazolyl)-5-oxazolyl]phenoxy]acetic acid (BMY 45778) inhibits human (IC50 = 35 nM), rabbit (136 nM) and rat (1.3 microM) platelet aggregation. This compound activates adenylyl cyclase (ED50 = 6-10 nM) and stimulates GTPase in human platelet membrane preparations. The potency (EC50) of BMY 45778 stimulating adenylyl cyclase is comparable to iloprost. However, maximal stimulation of GTPase by BMY 45778 is approximately half the iloprost-stimulated activity, and BMY 45778 limits the GTPase stimulation by iloprost suggesting that BMY 45778 is a partial agonist at the IP receptor. BMY 45778 completely prevents [3H]]Iloprost binding to platelet membranes (IC50 = 7 nM). In whole platelets, BMY 45778 causes elevation of platelet cAMP levels (cAMP content doubles at 13 nM) and activation of the cAMP-dependent protein kinase (cAMP-protein kinase ratio is twice basal at 2 nM). BMY 45778 treatment of whole platelets also desensitizes the adenylyl cyclase activation by iloprost. These results indicate that BMY 45778, which is structurally different from prostacyclin and most prostacyclin agonists, acts by stimulating prostacyclin (IP) receptors.

Agonist-dependent phosphorylation of an epitope-tagged human prostacyclin receptor.

An epitope-tagged human prostacyclin receptor (HAhIP) was constructed and stably transfected into human embryonic kidney 293 cells. The receptor exhibited high (Kd = 0.4 +/- 0.08 nM, Bmax = 0.7 +/- 0.2 pmol/mg protein; n = 4) and low (Kd = 75 +/- 27.4 nM, Bmax = 7.1 +/- 3.6 pmol/mg protein; n = 4) affinity for iloprost and coupled to both cAMP (EC50 = 0.1 +/- 0.03 nM) and inositol phosphate (EC50 = 43.1 +/- 10 nM) production. The receptor resolved on SDS-polyacrylamide gel electrophoresis as a broad complex with a molecular mass of 44-62 kDa and is glycosylated and phosphorylated. Stimulation of transfected cells with iloprost induced a rapid time- and concentration-dependent phosphorylation of HAhIP. Pretreatment of cells with a protein kinase C (PKC) inhibitor (GF109203X; 5 microM) abolished basal phosphorylation and dramatically reduced iloprost-induced HAhIP phosphorylation. A protein kinase A (PKA) inhibitor (H89) was largely ineffective under the same conditions. HAhIP phosphorylation was stimulated by receptor-dependent (thrombin, 2 units/ml) or receptor-independent (phorbol 12-myristate 13-acetate, 5 microM) PKC activation; both were abolished by pretreatment of cells with GF109203X. In contrast, receptor-independent (forskolin (5 microM) or dibutyryl cAMP (1 microM)) activation of PKA did not induce HAhIP phosphorylation. These results indicate that the human prostacyclin receptor may be regulated by agonist-dependent phosphorylation. This appears to be mediated, in part, by activation of PKC but not by PKA.