Regulation of prostacyclin and prostaglandin E(2) receptor mediated responses in adult rat dorsal root ganglion cells, in vitro

1. Primary cultures of adult rat dorsal root ganglia (DRG) were prepared to examine the properties of prostacyclin (IP) receptors and prostaglandin E(2) (EP) receptors in sensory neurones. 2. IP receptor agonists, cicaprost and iloprost, stimulated adenylyl cyclase activity with EC(50) values of 22 and 28 nM, respectively. Prostaglandin E(1) (PGE(1)) and prostaglandin E(2) (PGE(2)) were 7 fold less potent than cicaprost and iloprost, with PGE(2) displaying a lower maximal response. 3. Adenylyl cyclase activation by iloprost, PGE(1) and PGE(2), but not by forskolin, was highly dependent on DRG cell density. Although the potency of iloprost and PGE(2) for stimulating adenylyl cyclase was unchanged, their maximal responses were significantly increased at low cell density. 4. Both IP and EP(2/4) receptors could be down-regulated by agonist pretreatment, however the presence of cyclo-oxygenase (COX) inhibitors did not prevent this apparent down-regulation of IP and EP(2/4) receptors at high DRG cell densities. 5. Stimulation of adenylyl cyclase by the neuropeptide calcitonin gene-related peptide was also decreased at high DRG cell density, whereas the responses to beta-adrenoceptor agonists were increased at high DRG cell density. 6. Addition of nerve growth factor (NGF), or the addition of anti-neurotrophin antibodies during the 5-day culture of DRG cells, had no effect on IP receptor-mediated responses. 7. These results indicate that G(s)-coupled receptors involved in nociception are regulated in a variable manner in adult rat sensory neurones, and that this cell density-dependent regulation may be agonist-independent for IP and EP(2/4) receptors.     

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.     

Characterization of the prostaglandin E2 sensitive (EP)-receptor in the rat isolated trachea.

1. Using a range of natural and synthetic prostanoid receptor agonists and antagonists, we have shown that the rat isolated trachea contains a heterogeneous population of prostaglandin receptor sub-types mediating both relaxation and contraction of the smooth muscle. Prostaglandin E2 (PGE2) elicits smooth muscle relaxation of pre-contracted preparations, the responses being well defined, with a mean potency (p[A50]) of 7.81 +/- 0.05. 2. 11-deoxy PGE1 16,16-dimethyl PGE2 and misoprostol were all full agonists at this receptor, whilst AH13205 was a low potency agonist, and sulprostone was inactive. 3. The EP1 receptor antagonist, AH6809 (5 microM), and the selective DP receptor antagonist, BW A868C (0.1 microM), had no significant effect on the concentration-effect (E/[A]) curves to PGE2. 4. The putative EP4-receptor antagonist, AH23848B, produced non-competitive antagonism of the PGE2 response curves; pA2 values of 5.07 +/- 0.15 and 5.24 +/- 0.19 were obtained at concentrations of 30 microM and 100 microM respectively. 5. The synthetic thromboxane A2 mimetic, U46619, caused smooth muscle contractions, with a mean p[A50] of 6.90 +/- 0.11. This response was antagonized by the TP receptor antagonist, GR32191B, yielding a mean pA2 of 8.31. 6. At concentrations of 1 microM and above, prostaglandin D2 (PGD2) and the IP-receptor agonist, cicaprost, generally elicited concentration-dependent relaxations of the rat trachea. Prostaglandin F2 alpha (PGF2 alpha) was without affinity or efficacy. 7. These data suggest that the rat isolated trachea contains EP-receptors, TP-receptors, and few, if any DP, IP or FP-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential regulation of phosphorylation of the cAMP response element-binding protein after activation of EP2 and EP4 prostanoid receptors by prostaglandin E2

The EP2 and EP4 prostanoid receptors are G-protein-coupled receptors whose activation by their endogenous ligand, prostaglandin (PG) E2, stimulates the formation of intracellular cAMP. We have previously reported that the stimulation of cAMP formation in EP4-expressing cells is significantly less than in EP2-expressing cells, despite nearly identical levels of receptor expression (J Biol Chem 277:2614-2619, 2002). In addition, a component of EP4 receptor signaling, but not of EP2 receptor signaling, was found to involve the activation of phosphatidylinositol 3-kinase (PI3K). In this study, we report that PGE2 stimulation of cells expressing either the EP2 or EP4 receptor results in the phosphorylation of the cAMP response element binding protein (CREB) at serine-133. Pretreatment of cells with N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H-89), an inhibitor of protein kinase A (PKA), attenuated the PGE2-mediated phosphorylation of CREB in EP2-expressing cells, but not in EP4-expressing cells. Pretreatment of cells with wortmannin, an inhibitor of PI3K, had no effects on the PGE2-mediated phosphorylation of CREB in either EP2- or EP4-expressing cells, although it significantly increased the PGE2-mediated activation of PKA in EP4-expressing cells. However, combined pretreatment with H-89 and wortmannin blocked PGE2-mediated phosphorylation in EP2-expressing cells as well as in EP2-expressing cells. PGE2-mediated intracellular cAMP formation was not affected by pretreatment with wortmannin, or combined treatment with wortmannin and H-89, in either the EP2- or EP4-expressing cells. These findings suggest that PGE2 stimulation of EP4 receptors, but not EP2 receptors, results in the activation of a PI3K signaling pathway that inhibits the activity of PKA and that the PGE2-mediated phosphorylation of CREB by these receptors occurs through different signaling pathways     

EP4 receptor mediation of prostaglandin E2-stimulated mucus secretion by rabbit gastric epithelial cells

Prostaglandin (PG) E receptors are divided into four subtypes (EP1-EP4). We investigated the EP receptor subtype involved in PGE2-stimulated mucus secretion by rabbit gastric epithelial cells. Northern blot analysis revealed that epithelial cells express EP3 and EP4 receptor mRNAs, but neither EP1 nor EP2 receptor mRNAs were detected. PGE2, 11-deoxy-PGE1 (an EP3/EP4/EP2 agonist) and 16,16-dimethyl-PGE2 (an EP3/EP2/EP4 agonist) concentration-dependently promoted mucus secretion. In contrast, 17-phenyl-PGE2 (an EP3/EP1 agonist), sulprostone (an EP3/EP1 agonist), and butaprost (an EP2 agonist) failed to stimulate secretion. The effective concentrations of PGE2, 11-deoxy-PGE1, and 16,16-dimethyl-PGE2 were associated with their affinities for the EP4 receptor. In addition, PGE2, 11-deoxy-PGE1, and 16,16-dimethyl-PGE2 increased cyclic AMP (cAMP) production, but the other prostanoids had no effect. SQ22536 [9-(tetrahydro-2'-furyl)adenine; an adenylate cyclase inhibitor] inhibited both the increased cAMP production and mucus secretion induced by PGE2, 11-deoxy-PGE1, and 16,16-dimethyl-PGE2. H-89 (N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinoline sulfonamide; a protein kinase A inhibitor) also abolished the stimulatory effects of the prostanoids on mucus secretion, but calphostin C (a protein kinase C inhibitor) did not. These results indicate that PGE2 promotes mucus secretion by rabbit gastric epithelial cells, mediated through EP4 receptor stimulation and the subsequent activation of protein kinase A.     

Ca2+ signals evoked by histamine H1 receptors are attenuated by activation of prostaglandin EP2 and EP4 receptors in human aortic smooth muscle cells

Background and Purpose

Histamine and prostaglandin E₂ (PGE₂), directly and via their effects on other cells, regulate the behaviour of vascular smooth muscle (VSM), but their effects on human VSM are incompletely resolved.

Experimental Approach

The effects of PGE₂ on histamine-evoked changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and adenylyl cyclase activity were measured in populations of cultured human aortic smooth muscle cells (ASMCs). Selective ligands of histamine and EP receptors were used to identify the receptors that mediate the responses.

Key Results

Histamine, via H₁ receptors, stimulates an increase in [Ca²⁺]_i that is entirely mediated by activation of inositol 1,4,5-trisphosphate receptors. Selective stimulation of EP₂ or EP₄ receptors attenuates histamine-evoked Ca²⁺ signals, but the effects of PGE₂ on both Ca²⁺ signals and AC activity are largely mediated by EP₂ receptors.

Conclusions and Implications

Two important inflammatory mediators, histamine via H₁ receptors and PGE₂ acting largely via EP₂ receptors, exert opposing effects on [Ca²⁺]_i in human ASMCs.     

Agonist-induced phosphorylation by G protein-coupled receptor kinases of the EP4 receptor carboxyl-terminal domain in an EP3/EP4 prostaglandin E(2) receptor hybrid.

Prostaglandin E(2) receptors (EP-Rs) belong to the family of heterotrimeric G protein-coupled ectoreceptors with seven transmembrane domains. They can be subdivided into four subtypes according to their ligand-binding and G protein-coupling specificity: EP1 couple to G(q), EP2 and EP4 to G(s), and EP3 to G(i). The EP4-R, in contrast to the EP3beta-R, shows rapid agonist-induced desensitization. The agonist-induced desensitization depends on the presence of the EP4-R carboxyl-terminal domain, which also confers desensitization in a G(i)-coupled rEP3hEP4 carboxyl-terminal domain receptor hybrid (rEP3hEP4-Ct-R). To elucidate the possible mechanism of this desensitization, in vivo phosphorylation stimulated by activators of second messenger kinases, by prostaglandin E(2), or by the EP3-R agonist M&B28767 was investigated in COS-7 cells expressing FLAG-epitope-tagged rat EP3beta-R (rEP3beta-R), hEP4-R, or rEP3hEP4-Ct-R. Stimulation of protein kinase C with phorbol-12-myristate-13-acetate led to a slight phosphorylation of the FLAG-rEP3beta-R but to a strong phosphorylation of the FLAG-hEP4-R and the FLAG-rEP3hEP4-Ct-R, which was suppressed by the protein kinase A and protein kinase C inhibitor staurosporine. Prostaglandin E(2) stimulated phosphorylation of the FLAG-hEP4-R in its carboxyl-terminal receptor domain. The EP3-R agonist M&B28767 induced a time- and dose-dependent phosphorylation of the FLAG-rEP3hEP4-Ct-R but not of the FLAG-rEP3beta-R. Agonist-induced phosphorylation of the FLAG-hEP4-R and the FLAG-rEP3hEP4-Ct-R were not inhibited by staurosporine, which implies a role of G protein-coupled receptor kinases (GRKs) in agonist-induced receptor phosphorylation. Overexpression of GRKs in FLAG-rEP3hEP4-Ct-R-expressing COS-7 cells augmented the M&B28767-induced receptor phosphorylation and receptor sequestration. These findings indicate that phosphorylation of the carboxyl-terminal hEP4-R domain possibly by GRKs but not by second messenger kinases may be involved in rapid agonist-induced desensitization of the hEP4-R and the rEP3hEP4-Ct-R.     

Activation of IP and EP(3) receptors alters cAMP-dependent cell migration

Migration of vascular smooth cells from the media to the intima essentially contributes to neointima formation after percutaneous transluminal angioplasty and stent implantation. The stable prostacyclin mimetic iloprost has been shown to inhibit neointima formation in experimental restenosis, but it is currently unknown whether this may be caused by an antimigratory effect. Hence, the present study analyses (i) the influence of G(s)-coupled prostacyclin (IP) receptors on cell migration and (ii) verifies whether EP(3) receptors with opposite (i.e., G(i)) coupling may conversely stimulate cell migration. In a modified Boyden chamber model, it was shown that iloprost dose-dependently inhibits the migration of primary human arterial smooth muscle cells, which constitutively express the IP receptor. On the other hand, human arterial smooth muscle cell migration was stimulated by the EP(3) receptor agonist M&B 28.767. To independently study the effects of these receptors, IP or EP(3) receptors were stably overexpressed in chinese hamster ovary cells (CHO-IP and CHO-EP(3)). Chemotaxis of CHO cells transfected with G(s)-coupled IP receptors was concentration-dependently inhibited by iloprost (2-100 nM), while there was no effect of iloprost on mock-transfected CHO. By contrast, CHO-cells that overexpressed EP(3) receptors showed a significant, concentration dependent (1-100 nM) increase of cell migration in presence of the selective EP(3) agonist M&B 28.767. It is concluded that the prostacyclin mimetic iloprost inhibits vascular cell migration, which probably depends on a G(s)-mediated increase of intracellular cAMP. EP(3) receptors conversely stimulate CHO migration.     

Prostaglandin E(2) increases cyclic AMP and inhibits endothelin-1 production/secretion by guinea-pig tracheal epithelial cells through EP(4) receptors

Prostaglandin E(2) (PGE(2)) increased adenosine 3' : 5'-cyclic monophosphate (cyclic AMP) formation in tracheal epithelial cells and concomitantly decreased the production/secretion of immunoreactive endothelin (irET). Naturally occurring prostanoids and selective and non-selective EP receptor agonists showed the following rank order of potency in stimulating cyclic AMP generation by epithelial cells: PGE(2) (EP-selective)>16,16-dimethyl PGE(2) (EP-selective)>11-deoxy PGE(2) (EP-selective)>iloprost (IP/EP(1)/EP(3)-selective), butaprost (EP(2)-selective), PGD(2) (DP-selective), PGF(2alpha) (FP-selective). The lack of responsiveness of the latter prostanoids indicated that the prostanoid receptor present in these cells is not of the DP, FP, IP, EP(1), EP(2) or EP(3) subtype. Pre-incubating the cells with the selective TP/EP(4)-receptor antagonists AH23848B and AH22921X antagonized the PGE(2)-evoked cyclic AMP generation. This suggested that EP(4) receptors mediate PGE(2) effects. However, in addition to any antagonistic effects at EP(4)-receptors, both compounds, to a different extent, modified cyclic AMP metabolism. The selective EP(1), DP and EP(2) receptor antagonist (AH6809) failed to inhibit PGE(2)-evoked cyclic AMP generation which confirmed that the EP(2) receptor subtype did not contribute to the change in cyclic AMP formation in these cells. The PGE(2)-induced inhibition of irET production by guinea-pig tracheal epithelial cells was due to cyclic AMP generation and activation of the cyclic AMP-dependent protein kinase since this effect was reverted by the cyclic AMP antagonist Rp-cAMPS. These results provide the first evidence supporting the existence of a functional prostaglandin E(2) receptor that shares the pharmacological features of the EP(4)-receptor subtype in guinea-pig tracheal epithelial cells. These receptors modulate cyclic AMP formation as well as ET-1 production/secretion in these cells.     

Comparison of agonist-induced internalization of the human EP2 and EP4 prostaglandin receptors: role of the carboxyl terminus in EP4 receptor sequestration

Prostaglandin E(2) (PGE(2)) couples to stimulation of adenylyl cyclase through two distinct G protein-coupled receptors designated EP2 and EP4. Although they have similar affinities for PGE(2), the EP(2) and EP4 receptors have distinct structural characteristics. EP2 is a 358-amino-acid protein with short third intracellular loop and C-terminal domains, whereas EP4 consists of 488 amino acids with a long third intracellular loop and a long cytoplasmic tail. The ability of the HA epitope-tagged receptors to undergo PGE(2)-induced internalization was examined by enzyme-linked immunosorbent assay and immunofluorescence microscopy after expression in human embryonic kidney 293 cells. The EP2 receptor did not internalize, whereas the EP4 receptor underwent rapid internalization. Truncation of the EP4 receptor after amino acid 350, which removes 138 residues, abolished internalization. Truncation after amino acid 369 markedly attenuated internalization, whereas truncation after amino acid 383 had little effect. Serine and threonine residues in the region 350 to 383 were mutated to determine their role in internalization. The mutants S370-382A, a full-length receptor containing six serine-to-alanine mutations in the region 370 to 382, and S354-369A, containing four serine mutations and one threonine mutation in the region 350 to 370, both internalized to the same extent as the wild-type. A further mutant, designated S354-382A, containing amino acid substitutions S354A, S359A, S364A, S366G, T369A, S370A, S371A, S374A, S377A, S379A, and S382A, also internalized to the same extent as the wild-type. We conclude that the C terminus of the EP4 receptor is involved in internalization; however, serine and threonine residues do not seem to be involved.     

Inhibition of prostaglandin E(2) production by taiwanin C isolated from the root of Acanthopanax chiisanensis and the mechanism of action

Five lignans, l-sesamin, savinin, helioxanthin, taiwanin C, and cis-dibenzylbutyrolactone, were isolated from the root of Acanthopanax chiisanensis (Araliaceae), a Korean medicinal plant, and their inhibitory effects on the production of prostaglandin (PG) E(2) stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) in rat peritoneal macrophages were examined. Among the five lignans, taiwanin C was the most potent (IC(50)=0.12 microM), followed by helioxanthin, cis-dibenzylbutyrolactone, and savinin. l-Sesamin had no effect. Taiwanin C showed no inhibitory effect on the TPA-induced release of radioactivity from [3H]arachidonic acid-labeled macrophages, nor did it inhibit the expression of cyclooxygenase (COX)-2 protein induced by TPA. However, the activities of isolated COX-1 and COX-2 were inhibited by taiwanin C (IC(50)=1.06 and 9.31 microM, respectively), reflecting the inhibition of both COX-1- and COX-2-dependent PGE(2) production in the cell culture system. These findings suggest that the mechanism of action of taiwanin C in the inhibition of PGE(2) production is the direct inhibition of COX enzymatic activity.     

Characterization of [3H]-prostaglandin E2 binding to prostaglandin EP4 receptors expressed with Semliki Forest virus

1. The human prostaglandin EP₄ receptor has been expressed by use of the Semliki Forest virus system.
2. In cell membranes [³H]-prostaglandin E₂ ([³H]-PGE₂) bound to a high affinity site with a K_d of 1.12±0.3 nM and a B_max of 3.1±0.3 pmol mg⁻¹ protein.
3. In competition studies the rank order of potency for prostaglandins was PGE₂  = PGE₁  ≈#62;  PGF_2α =PGI₂.
4. The binding of [³H]-PGE₂ to cell membranes was inhibited by approximately 60% by the addition of guanylnucleotides, suggesting that this proportion of the receptors was G-protein coupled.
5. [³H]-PGE₂ binding was increased by greater than 200% by the addition of divalent cations, with little change in the IC₅₀ of PGE₂.
6. In saturation studies removal of divalent cations and addition of GTPγS resulted in a 65% reduction in the B_max with no change in the K_d. These results are consistent with the ligand labelling two states of the receptor R*, a high affinity state and R*G, a high affinity G protein coupled state.

     

Distinction between relaxations induced via prostanoid EP(4) and IP(1) receptors in pig and rabbit blood vessels

1. Our study shows that the prostacyclin analogues AFP-07 and cicaprost are moderately potent agonists for prostanoid EP(4) receptors, in addition to being highly potent IP(1) receptor agonists. Both activities were demonstrated on piglet and rabbit saphenous veins, which are established EP(4) preparations. 2. On piglet saphenous vein, PGE(2) was 6.1, 24, 96, 138, 168 and 285 times respectively more potent than AFP-07, cicaprost, PGI(2), iloprost, carbacyclin and TEI-9063 in causing relaxation. Another prostacyclin analogue taprostene did not induce maximum relaxation (21 - 74%), and did not oppose the action of PGE(2). The EP(4) receptor antagonist AH 23848 (30 microM) blocked relaxant responses to PGE(2) (dose ratio=8.6+/-1.3, s.e.mean) to a greater extent than cicaprost (4.9+/-0.7) and AFP-07 (3.8+/-0.8), had variable effects on TEI-9063-induced relaxation (3.7+/-1.5), and had no effect on taprostene responses (<2.0). 3. On rabbit saphenous vein, AH 23848 blocked the relaxant actions of PGE(2), AFP-07, cicaprost, iloprost and carbacyclin to similar extents. 4. AFP-07, cicaprost and TEI-9063 showed high IP(1) relaxant potency on piglet carotid artery, rabbit mesenteric artery and guinea-pig aorta, with AFP-07 confirmed as the most potent IP(1) agonist reported to date. AH 23848 did not block cicaprost-induced relaxation of piglet carotid artery. EP(3) contractile systems in these preparations can confound IP(1) agonist potency estimations. 5. Caution is urged when using AFP-07 and cicaprost to characterize IP(1) receptors in the presence of EP(4) receptors. Taprostene may be a lead to a highly selective IP(1) receptor agonist.     

Intermolecular cross-talk between the prostaglandin E2 receptor (EP)3 subtype and thromboxane A2 receptor signalling in human erythroleukaemic cells

Background and purpose:

In previous studies investigating cross-talk of signalling between prostaglandin (PG)E₂ receptor (EP) and the TPα and TPβ isoforms of the human thromboxane (TX)A₂ receptor (TP), 17-phenyl trinor PGE₂-induced desensitization of TP receptor signalling through activation of the AH6809 and SC19220-sensitive EP₁ subtype of the EP receptor family, in a cell-specific manner. Here, we sought to further investigate that cross-talk in human erythroleukaemic (HEL) 92.1.7 cells.

Experimental approach:

Specificity of 17-phenyl trinor PGE₂ signalling and its possible cross-talk with signalling by TPα/TPβ receptors endogenously expressed in HEL cells was examined through assessment of agonist-induced inositol 1,4,5-trisphosphate (IP)₃ generation and intracellular calcium ([Ca²⁺]_i) mobilization.

Key results:

While 17-Phenyl trinor PGE₂ led to activation of phospholipase (PL)Cβ to yield increases in IP₃ generation and [Ca²⁺]_i, it did not desensitize but rather augmented that signalling in response to subsequent stimulation with the TXA₂ mimetic U46619. Furthermore, the augmentation was reciprocal. Signalling by 17-phenyl trinor PGE₂ was found to occur through AH6809- and SC19920-insensitive, Pertussis toxin-sensitive, G_i/G_βγ-dependent activation of PLCβ. Further pharmacological investigation using selective EP receptor subtype agonists and antagonists confirmed that 17-phenyl trinor PGE₂-mediated signalling and reciprocal cross-talk with the TP receptors occurred through the EP₃, rather than the EP₁, EP₂ or EP₄ receptor subtype in HEL cells.

Conclusions and Implications:

The EP₁ and EP₃ subtypes of the EP receptor family mediated intermolecular cross-talk to differentially regulate TP receptor-mediated signalling whereby activation of EP₁ receptors impaired or desensitized, while that of EP₃ receptors augmented signalling through TPα/TPβ receptors, in a cell type-specific manner.     

Inhibition of prostaglandin E2 production by platycodin D isolated from the root of Platycodon grandiflorum.

Platycodin D, isolated from the root of Platycodon grandiflorum A. DC. (Campanulaceae) suppressed prostaglandin E2 production at 10 and 30 microM in rat peritoneal macrophages stimulated by the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA). Platycodin D3 and oleanolic acid showed no effect at these concentrations. Western blot analysis revealed that the induction of COX-2 protein by TPA was inhibited by platycodin D in parallel with the inhibition of prostaglandin E2 production. Platycodin D showed no direct effect on COX-1 and COX-2 activities. TPA-induced release of [3H]arachidonic acid from pre-labeled macrophages was also not inhibited by platycodin D.     

Some direct and reflex cardiovascular actions of prostacyclin (PGI2) and prostaglandin E2 in anaesthetized dogs.

1 The aim of the study was to determine the mechanism of the hypotension and bradycardia produced by prostacyclin (PGI2). 2 Haemodynamic studies were carried out in nineteen open-chest beagle dogs anaesthetized with chloralose. PGI2 was infused intravenously or into the left atrium. 3 Infusions of PGI2 either intravenously or into the left atrium equally reduced arterial pressure and total peripheral resistance but bradycardia was greater after infusion into the left atrium. 4 Comparison of effects of PGI2 with those of prostaglandin E2 (PGE2) showed that although left atrial infusions both reduced aortic pressure and total peripheral resistance, PGE2 always increased heart rate, cardiac output and maximum acceleration. 5 Similar effects were observed with sodium nitroprusside except that it always caused tachycardia and reduced stroke volume. 6 Atropine (0.05 or 1 mg/kg i.v.) reduced or reversed the bradycardia induced by PGI2 but its hypotensive effects were reduced only after 1 mg/kg atropine. After vagotomy changes in cardiac output, stroke volume and maximum acceleration were increased, the hypotensive effects of PGI2 were reduced and the bradycardia was reversed; effects induced by PGE2 were not significantly altered. 7 The hypotension induced by prostacyclin is due to two components, a direct relaxation of vascular smooth muscle and a reflex, non-cholinergic vasodilatation. The bradycardia is reflex in nature and is partially mediated by the vagus pathway.     

Piglet saphenous vein contains multiple relaxatory prostanoid receptors: evidence for EP4, EP2, DP and IP receptor subtypes

Prostaglandin E(2) produced endothelium-independent relaxation of phenylephrine- and 5-HT-contracted piglet saphenous vein (PSV; pEC(50)=8.6+/-0.2; n=6).The prostanoid EP(4) receptor antagonist GW627368X (30-300 nM) produced parallel rightward displacement of PGE(2) concentration-effect (E/[A]) curves (pK(b)=9.2+/-0.2; slope=1). Higher concentrations of GW627368X did not produce further rightward shifts, revealing the presence of non-EP(4) prostanoid receptors.In all, 18 other prostanoid receptor agonists relaxed PSV in a concentration-related manner. Relative potencies of agonists most sensitive to 10 muM GW627368X (and therefore predominantly activating EP(4) receptors) correlated well with those at human recombinant EP(4) receptors in human embryonic kidney (HEK-293) cells (r(2)=0.74). In the presence of 10 microM GW627368X, the rank order of agonist relative potency matched that of the human recombinant EP(2) receptor in Chinese hamster ovary cells (r(2)=0.72). Iloprost, cicaprost and PGI(2) relaxed PSV maximally and were antagonised by 10 microM GW627368X, demonstrating that they were full EP(4) receptor agonists. Residual responses to these compounds in the presence of GW627368X suggested the presence of IP receptors.BW245C relaxed PSV maximally (pEC(50)=6.8+/-0.1). In the presence of 10 microM GW627368X, BW245C produced biphasic E/[A] curves (phase one pEC(50)=6.6; alpha=24%; phase two pEC(50)=5.1; alpha=112%). Phase two was antagonised by the DP receptor antagonist BW A868C (1 microM), demonstrating that BW245C is an agonist at DP and EP4 receptors.We conclude that PSV contains EP(4), EP(2), DP and IP receptors; IP receptor agonists are also porcine EP(4) receptor agonists.     

Treatment in vivo with Ph CL28A alters prostaglandin E2, prostacyclin and leukotriene C4 metabolism in rat isolated lung

We have studied the effects of a potent inhibitor of prostaglandin (PG) catabolism, Ph CL28A, given in vivo, on PG metabolism in rat perfused lungs, isolated at different times after treatment. Two doses of Ph CL28A were used, 10 and 30 mg/kg, and lungs isolated at 1, 2 and 4h after a single injection (i.p.). The catabolism of exogenous PGE₂ was decreased for up to 2h after injection. Synthesis of PGI₂ using exogenous arachidonic acid, was increased by treatment with Ph CL28A. Stimulation of the turnover of endogenous arachidonic acid with the calcium ionophore A23187 led to the synthesis of LTC₄ as well as PGI₂. Treatment in vivo with Ph CL28A, decreased the output of LTC₄ but increased that of PGI₂. However in the presence of indomethacin, the perfused lung did not form any PGI₂ and the output of LTC₄ was still inhibited by Ph CL28A. We conclude that the inhibition of LT formation in lung by Ph CL28A was not due to the increased production of PGI₂. These two properties, inhibition of LT synthesis and potentiation of PGI₂ formation, may synergize in vivo to give anti-inflammatory activity.     

Lack of interaction between prostaglandin E2 receptor subtypes in regulating adenylyl cyclase activity in cultured rat dorsal root ganglion cells

The hyperalgesic response to prostaglandin E2 (PGE2) is thought to be mediated by activation of the cAMP/protein kinase A pathway in primary sensory neurones. The aim of this study was to investigate the relative contribution of different PGE2 (EP) receptor subtypes to the overall activity of adenylyl cyclase in adult rat isolated dorsal root ganglion (DRG) cells, in vitro. PGE2 and the prostanoid EP4 receptor agonist ONO-AE1-329 increased [3H]cAMP production with EC50 values of 500 nM and 70 nM, respectively, and showed similar efficacies. No combination of prostanoid EP1, EP2, EP3 or EP4 receptor selective agonists produced synergistic increases in [3H]cAMP. The prostacyclin mimetic cicaprost increased [3H]cAMP production with an EC50 value of 42 nM and produced a significantly greater maximal response compared with PGE2. No evidence for prostanoid EP3 receptor-dependent inhibition of adenylyl cyclase activity could be obtained to account for the relatively weak effect of PGE2 compared with prostacyclin receptor agonists. Interestingly, sulprostone (prostanoid EP3/EP1 receptor agonist) caused a Rho-kinase-dependent retraction of neurites, suggesting an alternative role for prostanoid EP3 receptors in DRG cells. In conclusion, PGE2 mediated increases in adenylyl cyclase activity in primary sensory neurones is likely to be mediated by activation of prostanoid EP4 receptors, and is not under inhibitory control by prostanoid EP3 receptors.     

Effects of sulphinpyrazone and aspirin on prostaglandin I2 (prostacyclin) synthesis by endothelial cells.

Synthesis of prostaglandin I2, (PGI2, prostacyclin) by vascular endothelium (assayed by the ability of cultured endothelial cells to inhibit platelet aggregation) was inhibited by aspirin. At 100 mumol/l aspirin completely blocked measurable PGI2 production, but endothelial cells had substantially recovered their ability to synthesize PGI2 24 h after removal of the drug. In contrast, the effect of 1 mmol/l aspirin was still evident 24 h after drug withdrawal. Sulphinpyrazone also inhibited PGI2 synthesis, but was about 100 fold less potent than aspirin, and the effect of the drug was lost within 24 h of its addition, even when endothelial cells were left in contact with the drug during this period.