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

BACKGROUND AND PURPOSE: Beraprost 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 APPROACH: Vascular 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 RESULTS: Esuberaprost 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 IMPLICATIONS: Stereoisomer 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.

Update on pulmonary arterial hypertension pharmacotherapy.

Pulmonary artery hypertension (PAH) refers to several subgroups of disease in which the mean pulmonary artery pressure (mPAP) is elevated to more than 25 mm Hg, pulmonary artery wedge pressure (PAWP) ≤ 15 mmHg, and an elevated pulmonary vascular resistance (PVR) > 3 Wood units as confirmed by right heart catheterization. The prevalence and geographic distribution of PAH vary depending on the type and etiology of the disease. Despite enormous efforts in the research and development of therapeutic agents in the last twenty years, the disease remains relatively incurable and the overall prognosis remains guarded. Median survival for an untreated patient is 2.8 years. In the last three decades, there have been dramatic advances in understanding the molecular mechanisms and signaling pathways involved in the disease, resulting in emerging new treatment strategies. In the following pages, we will review currently approved treatments for PAH, as well as a new generation of investigational drugs.

1-Methylnicotinamide protects against liver injury induced by concanavalin A via a prostacyclin-dependent mechanism: A possible involvement of IL-4 and TNF-α.

We have recently demonstrated that concanavalin A (Con A)-induced hepatitis is associated with the release of endogenous 1-methylnicotinamide (MNA). Here we study the mechanism by which exogenous MNA alleviates Con A-induced liver inflammation and injury in vivo. The involvement of prostacyclin (PGI2) in hepatoprotective action of MNA (30-100 mg kg(-1); i.v.) was studied by the use of IP receptor antagonist RO3244794 (10 mg kg(-1); p.o.) given prior to Con A (5-20 mg kg(-1); i.v.). Liver damage was assessed by measurements of: liver specific transaminases in plasma (alanine aminotransferase; aspartate aminotransferase); cytokines release (IL-4, IFN-γ and TNF-α); liver histopathology; and 24h survival rates. Additionally, the effect of a stable analog of prostacyclin (carbaprostacyclin) on IL-4, IFN-γ and TNF-α production by isolated spleen lymphocytes in response to Con A was analyzed. MNA diminished Con A-induced rise in liver specific transaminases, alleviated histopathological injury and improved 24h survival rates, the latter effect in a degree comparable with the pretreatment of animals with dexamethasone (0.5 mg kg(-1); i.p.). MNA inhibited also a rise in IL-4 and TNF-α concentration in plasma measured 2 h after Con A administration, while IFN-γ was less affected. The effects of MNA were reversed by pretreatment with IP antagonist RO3244794. In isolated spleen lymphocytes, carbaprostacyclin profoundly decreased production of IL-4, the effect on TNF-α was modest with no effect on IFN-γ production. In conclusion, MNA attenuated Con A-induced hepatitis by a prostacyclin-dependent mechanism involving the inhibition of lymphocytes-derived IL-4 and the inhibition of Kuppfer-cells derived TNF-α.

Beraprost sodium, a stable prostacyclin analogue, elicits dilation of isolated porcine retinal arterioles: roles of eNOS and potassium channels.

PURPOSE: Prostacyclin (PGI2) is usually described as an endoEDRFsthelium-derived relaxing factor, but the vasoreactivity to PGI2 in the retinal arterioles and the underlying mechanisms are not fully understood. We examined the effects of PGI2 on the retinal microcirculation using beraprost sodium (BPS), a stable PGI2 analogue, and the signaling mechanisms involved in this vasomotor activity. METHODS: Porcine retinal arterioles were isolated, cannulated, and pressurized without flow in vitro. Video microscopic techniques recorded the diametric responses to BPS. RESULTS: Beraprost sodium elicited dose-dependent (0.1 pM-0.1 µM) vasodilation of the retinal arterioles that was abolished by the PGI2 receptor (IP) antagonist CAY10441. Beraprost sodium-induced vasodilation decreased by 50% after the endothelium was removed and was inhibited by the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) comparable with denudation. Inhibition of soluble guanylyl cyclase by 1H-1,2,4-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and blockage of protein kinase A (PKA) by Rp-8-Br-cAMPS were comparable to L-NAME. Beraprost sodium-induced vasodilation was also inhibited by the nonselective potassium channel inhibitor, tetraethylammonium, and the adenosine triphosphate-sensitive potassium (KATP) channel blocker, glibenclamide. Residual vasodilation in the presence of glibenclamide decreased further with subsequent application of ODQ. CONCLUSIONS: Beraprost sodium, a stable PGI2 analogue, causes vasodilation of the retinal arterioles mediated via the IP receptor. The current findings suggest that BPS elicits endothelium-dependent and -independent dilation of the retinal arterioles mediated by NO induced by activation of PKA in the endothelium and the KATP channel activation in the vascular smooth muscle, respectively.

Existing drugs and agents under investigation for pulmonary arterial hypertension.

Pulmonary arterial hypertension is a progressive and debilitating disorder with an associated high morbidity and mortality rate. Significant advances in our understanding of the epidemiology, pathogenesis, and pathophysiology of pulmonary hypertension have occurred over the past several decades. This has allowed the development of new therapeutic options in this disease. Today, our selection of therapeutic modalities is broader, including calcium channel blockers, prostanoids, endothelin receptor antagonists, phosphodiesterase inhibitors, and soluble guanylate cyclase stimulators, but the disease remains fatal. This underscores the need for a continued search for novel therapies. Several potential pharmacologic agents for the treatment of pulmonary arterial hypertension are under clinical development and some promising results with these treatments have been reported. These agents include rho-kinase inhibitors, long-acting nonprostanoid prostacyclin receptor agonists, tyrosine protein kinase inhibitors, endothelial nitric oxide synthase couplers, synthetically produced vasoactive intestinal peptide, antagonists of the 5-HT2 receptors, and others. This article will review several of these promising new therapies and will discuss the current evidence regarding their potential benefit in pulmonary arterial hypertension.

Differential actions of the prostacyclin analogues treprostinil and iloprost and the selexipag metabolite, MRE-269 (ACT-333679) in rat small pulmonary arteries and veins.

The prostacyclin (IP) receptor agonists, treprostinil, iloprost and the selexipag metabolite, MRE-269 (ACT-333679) were evaluated in rat distal pulmonary blood vessels. Small pulmonary arteries and veins were pre-contracted with the thromboxane mimetic, U46619 (25 and 100nM, respectively), and relaxation determined with and without IP receptor antagonists, RO1138452 and RO3244794. In arteries, treprostinil was a more potent vasorelaxant than iloprost, while the efficacy of iloprost was greater. In pulmonary arteries, treprostinil-induced relaxation was essentially abolished by both IP antagonists (1µM), while responses to iloprost were partially inhibited. Both treprostinil and iloprost were equipotent, prominently relaxing pulmonary veins with responses being similarly and partially sensitive to IP antagonists. In contrast, RO1138452 failed to inhibit relaxations to MRE-269 in either pulmonary arteries or veins, suggesting no involvement of typical IP receptors. Thus, rat pulmonary tissues cannot be considered appropriate to assess classical IP receptors using the proposed highly selective non-prostanoid agonist MRE-269, contrasting with the IP receptor agonism profile of prostacyclin analogues, iloprost and treprostinil.

Cyclooxygenase-2-derived prostacyclin regulates arterial thrombus formation by suppressing tissue factor in a sirtuin-1-dependent-manner.

BACKGROUND: Selective inhibitors of cyclooxygenase (COX)-2 increase the risk of myocardial infarction and thrombotic events, but the responsible mechanisms are not fully understood. METHODS AND RESULTS: We found that ferric chloride-induced arterial thrombus formation was significantly greater in COX-2 knockout compared with wild-type mice. Cross-transfusion experiments excluded the likelihood that COX-2 knockout platelets, despite enhanced aggregation responses to collagen and thrombin, are responsible for increased arterial thrombus formation in COX-2 knockout mice. Importantly, we observed that COX-2 deletion decreased prostacyclin synthase and production and peroxisome proliferator-activated receptor- and sirtuin-1 (SIRT1) expression, with consequent increased upregulation of tissue factor (TF), the primary initiator of blood coagulation. Treatment of wild-type mice with a prostacyclin receptor antagonist or a peroxisome proliferator-activated receptor-δ antagonist, which predisposes to arterial thrombosis, decreased SIRT1 expression and increased TF activity. Conversely, exogenous prostacyclin or peroxisome proliferator-activated receptor-δ agonist completely reversed the thrombotic phenotype in COX-2 knockout mice, restoring normal SIRT1 levels and reducing TF activity. Furthermore, inhibition of SIRT1 increased TF expression and activity and promoted generation of occlusive thrombi in wild-type mice, whereas SIRT1 activation was sufficient to decrease abnormal TF activity and prothrombotic status in COX-2 knockout mice. CONCLUSIONS: Modulation of SIRT1 and hence TF by prostacyclin/peroxisome proliferator-activated receptor-δ pathways not only represents a new mechanism in controlling arterial thrombus formation but also might be a useful target for therapeutic intervention in the atherothrombotic complications associated with COX-2 inhibitors.

The COX-2/PGI2 receptor axis plays an obligatory role in mediating the cardioprotection conferred by the late phase of ischemic preconditioning.

BACKGROUND: Pharmacologic studies with cyclooxygenase-2 (COX-2) inhibitors suggest that the late phase of ischemic preconditioning (PC) is mediated by COX-2. However, nonspecific effects of COX-2 inhibitors cannot be ruled out, and the selectivity of these inhibitors for COX-2 vs. COX-1 is only relative. Furthermore, the specific prostaglandin (PG) receptors responsible for the salubrious actions of COX-2-derived prostanoids remain unclear. OBJECTIVE: To determine the role of COX-2 and prostacyclin receptor (IP) in late PC by gene deletion. METHODS: COX-2 knockout (KO) mice (COX-2(-/-)), prostacyclin receptor KO (IP(-/-)) mice, and respective wildtype (WT, COX-2(+/+) and IP(+/+)) mice underwent sham surgery or PC with six 4-min coronary occlusion (O)/4-min R cycles 24 h before a 30-min O/24 h R. RESULTS: There were no significant differences in infarct size (IS) between non-preconditioned (non-PC) COX-2(+/+), COX-2(-/-), IP(+/+), and IP(-/-) mice, indicating that neither COX-2 nor IP modulates IS in the absence of PC. When COX-2(-/-) or IP(-/-) mice were preconditioned, IS was not reduced, indicating that the protection of late PC was completely abrogated by deletion of either the COX-2 or the IP gene. Administration of the IP selective antagonist, RO3244794 to C57BL6/J (B6) mice 30 min prior to the 30-min O had no effect on IS. When B6 mice were preconditioned 24 h prior to the 30-min O, IS was markedly reduced; however, the protection of late PC was completely abrogated by pretreatment of RO3244794. CONCLUSIONS: This is the first study to demonstrate that targeted disruption of the COX-2 gene completely abrogates the infarct-sparing effect of late PC, and that the IP, downstream of the COX-2/prostanoid pathway, is a key mediator of the late PC. These results provide unequivocal molecular genetic evidence for an essential role of the COX-2/PGI2 receptor axis in the cardioprotection afforded by the late PC.

Molecular mechanisms regulating the vascular prostacyclin pathways and their adaptation during pregnancy and in the newborn.

Prostacyclin (PGI(2)) is a member of the prostanoid group of eicosanoids that regulate homeostasis, hemostasis, smooth muscle function and inflammation. Prostanoids are derived from arachidonic acid by the sequential actions of phospholipase A(2), cyclooxygenase (COX), and specific prostaglandin (PG) synthases. There are two major COX enzymes, COX1 and COX2, that differ in structure, tissue distribution, subcellular localization, and function. COX1 is largely constitutively expressed, whereas COX2 is induced at sites of inflammation and vascular injury. PGI(2) is produced by endothelial cells and influences many cardiovascular processes. PGI(2) acts mainly on the prostacyclin (IP) receptor, but because of receptor homology, PGI(2) analogs such as iloprost may act on other prostanoid receptors with variable affinities. PGI(2)/IP interaction stimulates G protein-coupled increase in cAMP and protein kinase A, resulting in decreased [Ca(2+)](i), and could also cause inhibition of Rho kinase, leading to vascular smooth muscle relaxation. In addition, PGI(2) intracrine signaling may target nuclear peroxisome proliferator-activated receptors and regulate gene transcription. PGI(2) counteracts the vasoconstrictor and platelet aggregation effects of thromboxane A(2) (TXA(2)), and both prostanoids create an important balance in cardiovascular homeostasis. The PGI(2)/TXA(2) balance is particularly critical in the regulation of maternal and fetal vascular function during pregnancy and in the newborn. A decrease in PGI(2)/TXA(2) ratio in the maternal, fetal, and neonatal circulation may contribute to preeclampsia, intrauterine growth restriction, and persistent pulmonary hypertension of the newborn (PPHN), respectively. On the other hand, increased PGI(2) activity may contribute to patent ductus arteriosus (PDA) and intraventricular hemorrhage in premature newborns. These observations have raised interest in the use of COX inhibitors and PGI(2) analogs in the management of pregnancy-associated and neonatal vascular disorders. The use of aspirin to decrease TXA(2) synthesis has shown little benefit in preeclampsia, whereas indomethacin and ibuprofen are used effectively to close PDA in the premature newborn. PGI(2) analogs have been used effectively in primary pulmonary hypertension in adults and have shown promise in PPHN. Careful examination of PGI(2) metabolism and the complex interplay with other prostanoids will help design specific modulators of the PGI(2)-dependent pathways for the management of pregnancy-related and neonatal vascular disorders.

Endothelium-dependent mechanisms of the vasodilatory effect of the endocannabinoid, anandamide, in the rat pulmonary artery.

Endocannabinoids exhibit vasodilatory properties and reduce blood pressure in vivo. However, the influence and mechanism of action of the prominent endocannabinoid, anandamide (AEA), in pulmonary arteries are not known. The present study determined the vascular response to AEA in isolated rat pulmonary arteries. AEA relaxed rat pulmonary arteries that were pre-constricted with U-46619. This relaxation was reduced by the following conditions:removal of the endothelium; in KCl pre-constricted preparations; in the presence of the potassium channel (K(Ca)) blockers, tetraethylammonium and the combination of charybdotoxin and apamin, and the prostacyclin receptor antagonist, RO1138452. Inhibitors of cyclooxygenase (indomethacin), nitric oxide (NO) synthase (N(G)-nitro-l-arginine methyl ester) and fatty acid amide hydrolase (URB597) alone or in combination diminished AEA-induced relaxation in endothelium-intact vessels. The remaining experiments were performed in the presence of URB597 to eliminate the influence of AEA metabolites. Antagonists of the endothelial cannabinoid receptor (CB(x)), O-1918 and cannabidiol, attenuated the AEA-induced response. Antagonists of CB(1), CB(2) and TRPV1 receptors, AM251, AM630 and capsazepine, respectively, did not modify the AEA-induced response. A reference activator of CB(x) receptors, abnormal cannabidiol, mimicked the receptor-mediated AEA effects. The present study demonstrated that AEA relaxed rat pulmonary arteries in an endothelium-dependent fashion via the activation of the O-1918-sensitive CB(x) receptor and/or prostacyclin-like vasoactive products of AEA. One or both of these mechanisms may involve K(Ca) or the NO pathway.

Prostacyclin receptor in tumor endothelial cells promotes angiogenesis in an autocrine manner.

Molecules highly expressed in tumor endothelial cells (TEC) are important for specific targeting of these cells. Previously, using DNA microarray analysis, we found that the prostacyclin receptor (IP receptor) gene was upregulated in TEC compared with normal endothelial cells (NEC). Although prostacyclin is implicated in re-endothelialization and angiogenesis, its role remains largely unknown in TEC. Moreover, the effect of the IP receptor on TEC has not been reported. In the present study we investigated the function of the IP receptor in TEC. The TEC were isolated from two types of human tumor xenografts in nude mice, while NEC were isolated from normal counterparts. Prostacyclin secretion levels in TEC were significantly higher than those in NEC, as shown using ELISA. Real-time RT-PCR showed that the IP receptor was upregulated in TEC compared with NEC. Furthermore, migration and tube formation of TEC were suppressed by the IP receptor antagonist RO1138452. Immunohistostaining showed that the IP receptor was specifically expressed in blood vessels of renal cell carcinoma specimens, but not in glomerular vessels of normal renal tissue. These findings suggest that the IP receptor is a TEC-specific marker and might be a useful therapeutic target.

Pharmacological and expression profile of the prostaglandin I(2) receptor in the rat craniovascular system.

Activation of the trigeminal nerve terminals around cerebral and meningeal arteries is thought to be an important patho-mechanism in migraine. Vasodilatation of the cranial arteries may also play a role in increasing nociception. Prostaglandin I(2) (PGI(2)) is capable of inducing a headache in healthy volunteers, a response that is likely to be mediated by the prostaglandin I(2) receptor (IP). This study investigates the functional and molecular characteristics of the IP receptor in the rat craniovascular system. In the closed cranial window model, iloprost, an IP receptor agonist, dilated the rat middle meningeal artery (MMA) (E(max)=170%±16%; pED(50)=6.5±0.2) but not the rat cerebral artery (CA) in vivo. The specific antagonist of the IP receptor, CAY10441, significantly blocked the iloprost-induced response dose-dependently, with the highest dose attenuating iloprost (1µgkg(-1)) induced dilatations by 70% (p<0.05). CAY10441 did not have any effect on the prostaglandin E(2)-induced vasodilatory response, thus suggesting no interaction with EP(2) and EP(4) receptors. IP receptor mRNA transcripts and protein were present in meningeal as well as in cerebral rat vasculature, and localized the IP receptor protein to the smooth vasculature of the cranial arteries (MMA, MCA and basilar artery). Together, these results demonstrate that the IP receptor mediates the dilatory effect of PGI(2) in the cranial vasculature in rats. Antagonism of this receptor might be of therapeutic relevance in acute migraine treatment.

Disruption of COX-2 and eNOS does not confer protection from cardiovascular failure in lipopolysaccharide-treated conscious mice and isolated vascular rings.

It was hypothesized that a serial stimulation of vascular cyclooxygenase-2 (COX-2) with subsequent activation of endothelial nitric oxide synthase (eNOS) is responsible for decrease in blood pressure, cardiac performance, and vascular reactivity in endotoxemia caused by LPS. The hypothesis was tested in catheterized, conscious, freely moving, wild-type mice and mice (C57BL/6J background) with targeted deletion of COX-2 and eNOS that were given an intravenous LPS bolus (2 mg/kg, 055:B5). In vitro studies were performed on murine aorta rings. LPS caused a concomitant decrease in mean arterial blood pressure (MAP) and heart rate (HR) that was significant after 3 h and was sustained throughout the experiment (8 h). The LPS-induced changes in MAP and HR were not different from control in COX-2(-/-) and eNOS(-/-) mice. A prostacyclin receptor antagonist (BR5064) blocked the hypotensive effect of a prostacyclin agonist (beraprost), but did not attenuate the LPS-induced decrease in MAP and HR. LPS decreased eNOS and neuronal NOS mRNA abundances in several organs, while inducible NOS mRNA was enhanced. In aortic rings, LPS suppressed α(1)-adrenoceptor-mediated vascular tone. Inhibition of COX-2 activity (NS 398), disruption of COX-2, endothelium removal, or eNOS deletion (eNOS(-/-)) did not improve vascular reactivity after LPS, while the NO synthase blockers 1400W and N(G)-nitro-l-arginine methyl ester prevented loss of tone. COX-2 and eNOS activities are not necessary for LPS-induced decreases in blood pressure, heart rate, and vascular reactivity. Inducible NOS activity appears crucial. COX-2 and eNOS are not obvious therapeutic targets for cardiovascular rescue during gram-negative endotoxemic shock.

Inhibitory effect of schisandrin on spontaneous contraction of isolated rat colon.

This study examined the effect of schisandrin, one of the major lignans isolated from Schisandra chinensis, on spontaneous contraction in rat colon and its possible mechanisms. Schisandrin produced a concentration-dependent inhibition (EC50=1.66 µM) on the colonic spontaneous contraction. The relaxant effect of schisandrin could be abolished by the neuronal Na+ channel blocker tetrodotoxin (1 µM) but not affected by propranolol (1 µM), phentolamine (1 µM), atropine (1 µM) or nicotine desensitization, suggesting possible involvement of non-adrenergic non-cholinergic (NANC) transmitters released from enteric nerves. N(ω)-nitro-l-arginine methyl ester (100-300 µM), a nitric oxide synthase inhibitor, attenuated the schisandrin response. The role of nitric oxide (NO) was confirmed by an increase in colonic NO production after schisandrin incubation, and the inhibition on the schisandrin responses by soluble guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-α]-quinoxalin-1-one (1-30 µM). Non-nitrergic NANC components may also be involved in the action of schisandrin, as suggested by the significant inhibition of apamin on the schisandrin-induced responses. Pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt hydrate (100 µM), a selective P2 purinoceptor antagonist, markedly attenuated the responses to schisandrin. In contrast, neither 8-cyclopentyl-1,3-dipropylxanthine, an antagonist for adenosine A1 receptors, nor chymotrypsin, a serine endopeptidase, affected the responses. All available results have demonstrated that schisandrin produced NANC relaxation on the rat colon, with the involvement of NO and acting via cGMP-dependent pathways. ATP, but not adenosine and VIP, likely plays a role in the non-nitrergic, apamin-sensitive component of the response.

Selective prostacyclin receptor agonism augments glucocorticoid-induced gene expression in human bronchial epithelial cells.

Prostacyclin receptor (IP-receptor) agonists display anti-inflammatory and antiviral activity in cell-based assays and in preclinical models of asthma and chronic obstructive pulmonary disease. In this study, we have extended these observations by demonstrating that IP-receptor activation also can enhance the ability of glucocorticoids to induce genes with anti-inflammatory activity. BEAS-2B bronchial epithelial cells stably transfected with a glucocorticoid response element (GRE) luciferase reporter were activated in a concentration-dependent manner by the glucocorticoid dexamethasone. An IP-receptor agonist, taprostene, increased cAMP in these cells and augmented luciferase expression at all concentrations of dexamethasone examined. Analysis of the concentration-response relationship that described this effect showed that taprostene increased the magnitude of transcription without affecting the potency of dexamethasone and was, thus, steroid-sparing in this simple system. RO3244794, an IP-receptor antagonist, and oligonucleotides that selectively silenced the IP-receptor gene, PTGIR, abolished these effects of taprostene. Infection of BEAS-2B GRE reporter cells with an adenovirus vector encoding a highly selective inhibitor of cAMP-dependent protein kinase (PKA) also prevented taprostene from enhancing GRE-dependent transcription. In BEAS-2B cells and primary cultures of human airway epithelial cells, taprostene and dexamethasone interacted either additively or cooperatively in the expression of three glucocorticoid-inducible genes (GILZ, MKP-1, and p57(kip2)) that have anti-inflammatory potential. Collectively, these data show that IP-receptor agonists can augment the ability of glucocorticoids to induce anti-inflammatory genes in human airway epithelial cells by activating a cAMP/PKA-dependent mechanism. This observation may have clinical relevance in the treatment of airway inflammatory diseases that are either refractory or respond suboptimally to glucocorticoids.

Cyclooxygenase and thromboxane/prostaglandin receptor contribute to aortic endothelium-dependent dysfunction in aging female spontaneously hypertensive rats.

Cyclooxygenase (COX)-derived vasoconstrictory prostanoids contribute to impaired endothelium-dependent vasorelaxation in aging male (m) spontaneously hypertensive rats (SHR); however, vasomotor responses in aging female (f) SHR and sex differences in aging SHR are unknown. Examining mechanisms governing dysfunction in aging fSHR will contribute to understanding sex-dependent vascular complications in advanced hypertension. Aortic endothelium-dependent relaxation dose responses (ACh) of 16- and 30-wk-old mSHR and fSHR and normotensive Wistar-Kyoto rats were examined in the absence (no drug control) and presence of COX inhibition [indomethacin (Indo)] and thromboxane/prostaglandin receptor inhibition (SQ-29548). No drug control-treated 16-wk mSHR exhibited considerable blunting of the peak relaxation response to ACh (e.g., 77 +/- 4% relaxation to 10(-5) mol/l) vs. Wistar-Kyoto controls (89 +/- 6%), and greater dysfunction occurred in 30-wk mSHR (63 +/- 2%). Interestingly, ACh relaxations of fSHR were unimpaired at 16 wk (101 +/- 2% to 10(-5) mol/l), but blunted in 30 wk (76 +/- 4%). Indo and SQ-29548 restored robust ACh vasorelaxation in all groups (e.g., 113 +/- 3 and 112 +/- 3%, respectively, in Indo- and SQ-29548-treated 30-wk fSHR). Aortic COX-1 protein expression was elevated by 75% in 30-wk vs. 16-wk fSHR, whereas group-averaged ACh-stimulated aortic PGI(2) release (assessed as 6- keto-PGF(1alpha)) was 30% greater in 30-wk vs. 16-wk fSHR (9,926 +/- 890 vs. 7,621 +/- 690 pg.ml(-1).mg dry wt(-1)), although this did not reach significance (P = 0.0758). Dramatic deterioration of endothelium-dependent vasomotor function in fSHR across this age range involves COX and thromboxane/prostaglandin receptor, supporting a mechanism of impairment similar to that which occurs in aging mSHR.

Vascular actions of the prostacyclin receptor antagonist BAY 73-1449 in the portal hypertensive rat.

We have investigated the actions of the postacyclin receptor antagonist BAY 73-1449 on shunt vessel development and shunt flow in the portal vein ligated portal hypertensive Wistar rat in vivo. BAY 73-1449 (0.1-1 mg/kg), given intravenously, did not significantly reduce mesenteric inflow, but significantly reduced splenic shunt vessel outflow, compared to the effects of vehicle, in anaesthetized portal vein ligated rats as measured by shunt vessel conductance. There were no differences between portal vein ligated animals treated, beginning just before portal vein ligation, with vehicle for 7 days and animals treated for 7 days with BAY 73-1449 (1-5 mg/kg, s.c.) in the degree of porto-systemic shunting, as measured by the radioactive microsphere technique in anaesthetized rats. Portal pressure was similar in animals treated with vehicle or BAY 73-1449. It is concluded that the prostacyclin receptor antagonist BAY 73-1449 can acutely reduce shunt vessel blood flow in portal hypertensive rats.

Effects of cyclooxygenase inhibition on canine coronary artery blood flow and thrombosis.

This study was designed to determine the effect of inhibitors of cyclooxygenase (COX)-1, COX-2, and the nonselective COX inhibitor naproxen on coronary vasoactivity and thrombogenicity under baseline and lipopolysaccharide (LPS)-induced inflammatory conditions. We hypothesize that endothelial COX-1 is the primary COX isoform in the canine normal coronary artery, which mediates arachidonic acid (AA)-induced vasodilatation. However, COX-2 can be induced and overexpressed by inflammatory mediators and becomes the major local COX isoform responsible for the production of antithrombotic prostaglandins during systemic inflammation. The interventions included the selective COX-1 inhibitor SC-560 (0.3 mg/kg iv), the selective COX-2 inhibitor nimesulide (5 mg/kg iv), or the nonselective COX inhibitor naproxen (3 mg/kg iv). The selective prostacyclin (IP) receptor antagonist RO-3244794 (RO) was used as an investigational tool to delineate the role of prostacyclin (PGI(2)) in modulating vascular reactivity. AA-induced vasodilatation of the left circumflex coronary artery was suppressed to a similar extent by each of the COX inhibitors and RO. The data suggest that AA-induced vasodilatation in the normal coronary artery is mediated by a single COX isoform, the constitutive endothelial COX-1, which is reported to be susceptible to COX-2 inhibitors. The effect of the COX inhibitors on thrombus formation was evaluated in a model of carotid artery thrombosis secondary to electrolytic-induced vessel wall injury. Pretreatment with LPS (0.5 mg/kg iv) induced a systemic inflammatory response and prolonged the time-to-occlusive thrombus formation, which was reduced in the LPS-treated animals by the administration of nimesulide. In contrast, neither SC-560 nor naproxen influenced the time to thrombosis in the animals pretreated with LPS. The data are of significance in view of reported adverse cardiovascular events observed in clinical trials involving the use of selective COX-2 inhibitors, thereby suggesting that the endothelial constitutive COX-1 and the inducible vascular COX-2 serve important functions in maintaining vascular homeostasis.

A new class of analgesic agents toward prostacyclin receptor inhibition: synthesis, biological studies and QSAR analysis of 1-hydroxyl-2-substituted phenyl-4,4,5,5-tetramethylimidazolines.

By studying the structural similarity of analgesic imidazolines and 2-phenylnitronyl nitroxides, 20 1-hydroxyl-2-substituted phenyl-4,4,5,5-tetramethylimidazolines (2a-t) were newly synthesized as selective antagonists of prostacyclin receptor (IP receptor). In the in vivo tail-flick assay, 2a-t (dose, 0.13 mmol/kg) receiving mice showed increased pain thresholds ranging from 20.52+/-7.25% to 90.94+/-11.97%, which were significantly higher than that ranged from 12.27+/-9.56% to 17.71+/-7.00% shown by normal saline (NS) receiving mice. In the in vivo tail bleeding assay, 2a-t (dose, 1.30 mmol/kg) receiving mice gave a bleeding time ranging from 116.3+/-8.0 s to 119.6+/-7.1 s, and NS receiving mice gave a bleeding time ranging from 116.7+/-7.5s to 119.1+/-8.7s, which were at a substantially equal level. These observations imply that no bleeding risk occurred even when 10-fold dose of analgesic assay was used. In the in vitro vasorelaxation assay, it was found that when the aortic strip contracted by noradrenaline (NE, final concentration, 10(-7) M) was treated with the solution of 2a-t in NS (final concentration, 5 x 10(-3) M) only lower percentage inhibitions ranged from 6.63+/-2.72% to 46.28+/-2.63% were recorded. Relatively higher concentration of 2a-t (5 x 10(-3) M) and relatively lower percentage inhibitions (13 of 20 less than 23.27+/-3.47%) suggest that 2a-t exhibit few vasodilation activity. To shed some light on the potential analgesic mechanisms of 2a-t, moreover, a QSAR analysis was carried out by using the multiple linear regression method. Taken altogether, the current study confirms that as selective antagonist of IP receptor 1-hydroxyl-2-substituted phenyl-4,4,5,5-tetramethylimidazoline may be a promising lead compound of analgesic agent without cardiovascular and bleeding side effects.

Effects of the selective prostacyclin receptor antagonist RO3244019 on the micturition reflex in rats.

PURPOSE: We investigated the role of prostacyclin on afferent modulation of the micturition reflex using the novel selective prostacyclin receptor antagonist RO3244019 in rat models of bladder function. MATERIALS AND METHODS: The effects of RO3244019 on urodynamic parameters were evaluated in 3 rat models. In the anesthetized isovolumetric bladder contraction and the volume induced micturition reflex (Refill) models the effects of RO3244019 and chronic capsaicin desensitization were compared. In the citric acid induced detrusor overactivity model the effects of RO3244019 and the cyclooxygenase inhibitor indomethacin were evaluated. RESULTS: In the isovolumetric bladder contraction model RO3244019 dose dependently decreased bladder contraction frequency with a mean +/- SEM maximum decrease of 72.2% +/- 4.3% at 3.16 mg/kg. RO3244019 also dose dependently increased the micturition threshold in the Refill model with a maximum increase of 86.9% +/- 19.1% at 3.0 mg/kg. In animals that were chronically treated with capsaicin bladder contraction frequency was decreased by 88.9% in the isovolumetric bladder contraction model and micturition threshold was increased by 68.1% in the Refill model relative to sham treated rats. RO3244019 (3.0 mg/kg) further increased the micturition threshold in capsaicin treated animals by 53.7% +/- 18.1% from baseline. In the citric acid induced detrusor overactivity model citric acid decreased the voiding interval to 28.5% of baseline. This effect was reversed by RO3244019 (73.0% +/- 6.4%) and indomethacin (97.7% +/- 5.5%) at 3.0 mg/kg compared to vehicle (55.0% +/- 4.1%). CONCLUSIONS: The prostacyclin receptor antagonist RO3244019 decreased bladder contraction frequency and increased micturition threshold in the anesthetized isovolumetric bladder contraction and Refill models, respectively, and increased the micturition voiding interval in the conscious citric acid induced detrusor overactivity model. Additionally, RO3244019 remained effective for increasing the micturition threshold in the Refill model even following chronic capsaicin desensitization. Taken together these data suggest that prostacyclin may have a facilitory role in the micturition reflex by modulating the threshold for activation of capsaicin sensitive and insensitive bladder sensory afferents.