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.     

Binding and activity of the prostacyclin receptor (IP) agonists, treprostinil and iloprost, at human prostanoid receptors: treprostinil is a potent DP1 and EP2 agonist

The prostacyclin analogues, iloprost and treprostinil are extensively used in treating pulmonary hypertension. Their binding profile and corresponding biochemical cellular responses on human prostanoid receptors expressed in cell lines, have now been compared. Iloprost had high binding affinity for EP1 and IP receptors (Ki 1.1 and 3.9 nM, respectively), low affinity for FP, EP3 or EP4 receptors, and very low affinity for EP2, DP1 or TP receptors. By contrast, treprostinil had high affinity for the DP1, EP2 and IP receptors (Ki 4.4, 3.6 and 32 nM, respectively), low affinity for EP1 and EP4 receptors and even lower affinity for EP3, FP and TP receptors. In functional assays, iloprost had similar high activity in elevating cyclic AMP levels in cells expressing the human IP receptor and stimulating calcium influx in cells expressing EP1 receptors (EC50 0.37 and 0.3 nM, respectively) with the rank order of activity on the other receptors comparable to the binding assays. As with binding studies, treprostinil elevated cyclic AMP with a similar high potency in cells expressing DP1, IP and EP2 receptors (EC50 0.6, 1.9 and 6.2 nM, respectively), but had low activity at the other receptors. Activation of IP, DP1 and EP2 receptors, as with treprostinil, can all result in vasodilatation of human pulmonary arteries. However, activation of EP1 receptors can provoke vasoconstriction, and hence may offset the IP-receptor mediated vasodilator effects of iloprost. Treprostinil may therefore differ from iloprost in its overall beneficial pulmonary vasorelaxant profile and other pharmacological actions, especially in diseases where the IP receptor is down-regulated.     

Prostacyclin receptor-independent inhibition of phospholipase C activity by non-prostanoid prostacyclin mimetics.

1. Chinese hamster ovary (CHO) cells were transiently transfected with the mouse prostacyclin (mIP) receptor to examine IP agonist-mediated stimulation of [(3)H]-cyclic AMP and [(3)H]-inositol phosphate production. 2. The prostacyclin analogues, cicaprost, iloprost, carbacyclin and prostaglandin E(1), stimulated adenylyl cyclase activity with EC(50) values of 5, 6, 25 and 95 nM, respectively. These IP agonists also stimulated the phospholipase C pathway with 10 - 40 fold lower potency than stimulation of adenylyl cyclase. 3. The non-prostanoid prostacyclin mimetics, octimibate, BMY 42393 and BMY 45778, also stimulated adenylyl cyclase activity, with EC(50) values of 219, 166 and 398 nM, respectively, but failed to stimulate [(3)H]-inositol phosphate production. 4. Octimibate, BMY 42393 and BMY 45778 inhibited iloprost-stimulated [(3)H]-inositol phosphate production in a non-competitive manner. 5. Activation of the endogenously-expressed P(2) purinergic receptor by ATP led to an increase in [(3)H]-inositol phosphate production which was inhibited by the non-prostanoid prostacyclin mimetics in non-transfected CHO cells. Prostacyclin analogues and other prostanoid receptor ligands failed to inhibit ATP-stimulated [(3)H]-inositol phosphate production. 6. A comparison between the IP receptor-specific non-prostanoid ONO-1310 and the structurally-related EP(3) receptor-specific agonist ONO-AP-324, indicated that the inhibitory effect of non-prostanoids was specific for those compounds known to activate IP receptors. 7. The non-prostanoid prostacyclin mimetics also inhibited phospholipase C activity when stimulated by constitutively-active mutant Galpha(q)RC, Galpha(14)RC and Galpha(16)QL transiently expressed in CHO cells. These drugs did not inhibit adenylyl cyclase activity when stimulated by the constitutively-active mutant Galpha(s)QL. 8. These results suggest that non-prostanoid prostacyclin mimetics can specifically inhibit [(3)H]-inositol phosphate production by targeting G(q/11) and/or phospholipase C in CHO cells, and that this effect is independent of IP receptors.     

Differential effects of prostacyclin and iloprost in the isolated carotid artery of the guinea-pig

The effects on membrane potential of prostacyclin and iloprost were compared in smooth muscle cells of the guinea pig carotid artery. Both prostacyclin and iloprost induced hyperpolarization of the smooth muscle cells. In the presence of (3R)-3-(4-fluorophenyl-sulfonamido)-1,2,3,4-tetrahydro-9-carbazolepropanoic acid (Bay U3405), an antagonist of TP receptors, the response to iloprost was unaffected while that to prostacyclin was increased. Iloprost-induced hyperpolarizations were abolished by glibenclamide while those to prostacyclin were either not affected, or converted to either depolarization or to rhythmic electrical activity. The latter effects of prostacyclin were abolished by Bay U3405. After removal of the endothelium, iloprost and prostacyclin produced hyperpolarizations similar to those observed in control blood vessels. However, in the presence of glibenclamide, prostacyclin produced only depolarizations inhibited by Bay U3405. These results suggest that iloprost activates IP receptors and K(ATP) channels in smooth muscle. In contrast, prostacyclin produces additional endothelium-dependent and -independent effects via activation of TP receptors.     

Iloprost down-regulates the expression of the growth regulatory gene Cyr61 in human vascular smooth muscle cells

Prostacyclin and its mimetics have repeatedly been shown to act antiatherogenic and to inhibit neointima formation in several animal models of vascular injury. Treatment of human vascular smooth muscle cells with the prostacyclin mimetic iloprost (100 nm) drastically reduces expression of Cyr61, encoding the growth-regulatory cystein-rich angiogenic protein, without affecting the degradation rate of Cyr61 mRNA. Thrombin-induced Cyr61 expression was inhibited completely in the presence of iloprost. It is concluded that vasoprotective actions of prostacyclin in vivo may in part be due to inhibition of expression of the growth regulatory gene Cyr61 at sites of vascular lesions.     

Functional and ligand binding studies suggest heterogeneity of platelet prostacyclin receptors.

1. This study describes attempts to compare prostacyclin (IP-) receptors in human, pig, horse, rabbit and rat platelets and in circular muscle of human, rabbit and dog mesenteric and pig gastroepiploic arteries. Three stable prostacyclin analogues, iloprost, cicaprost and 6a-carba-prostacyclin (6a-carba-PGI2) and a prostaglandin endoperoxide analogue EP 157 (previously shown to mimic prostacyclin on human platelets) were used. 2. Our main conclusion is that prostacyclin receptors on human, pig and horse platelets are similar in nature, but distinct from those on rabbit and rat platelets. Functional studies (inhibition of aggregation) showed that iloprost and cicaprost always had similar potencies whereas 6a-carba PGI2 was much more potent than EP 157 on rabbit and rat platelets (300 and 1000 fold on a molar basis) compared with human, pig and horse platelets (2, 7 and 7 fold respectively). Measurement of initial rates of cyclic AMP production confirmed these orders of potency. 3. Although pig platelets were quite sensitive to inhibition by EP 157 (threshold = 10 nM in some experiments), maximal inhibition of aggregation was not always achieved (20 microM). EP 157 also produced only small elevations of cyclic AMP and inhibited rises in cyclic AMP induced by iloprost. It is possible that EP 157 has a lower efficacy than iloprost at the IP-receptor and on pig platelets it can sometimes act as a partial agonist. 4. Human, pig and horse platelet membranes bound [3H]-iloprost at 30 degrees C and this binding was inhibited by the four prostanoids. On human and pig membranes the order of potency was cicaprost = iloprost greater than 6a-carba PGI2 greater than EP 157. The order of potency may be similar on horse platelet membranes, but the analysis is complicated by the presence of a second component of [3H]-iloprost binding that is inhibited by iloprost and 6a-carba PGI2 but not by cicaprost. This binding may be due to the presence of an EP1-receptor, since iloprost and 6a-carba PGI2 but not cicaprost are known to have potent EP1-receptor agonist actions on smooth muscle preparations. IC50 values for cicaprost inhibition on human, pig and horse membranes were 110, 90 and 165 nM respectively. The need for IP-receptor radioligands of greater specificity is apparent from these studies. 5. Minimal binding of [3H]-iloprost to rabbit and rat platelet membranes was obtained at 30 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pro-migratory actions of the prostacyclin receptor in human breast cancer cells that over-express cyclooxygenase-2

Metastasis is the major cause of death in cancer patients. Elevated expression of cyclooxygenase-2 (COX-2) is observed in many human cancers and over-production of downstream prostaglandins (PGs) has been shown to stimulate metastasis. A role for increased PGE₂ production has been proposed, but whether other PGs contribute is currently unclear. In this study the pro-migratory actions of individual PGs were evaluated in MDA-MB-468 breast cancer cells that stably over-expressed COX-2 (MDA-COX-2 cells); cell migration was quantified using 3D-matrigel droplet assays. Inhibition of the prostacyclin and PGE synthases, but not alternate prostanoid synthases, prevented the increase in MDA-COX-2 cell migration produced by arachidonic acid (AA); direct treatment of cells with the stable prostacyclin analogue cicaprost also promoted migration. Pharmacological antagonism and knockdown of the IP receptor decreased cell migration, while antagonists of the alternate DP, EP2, FP, and TP prostanoid receptors were inactive. In support of these findings, activation of the IP receptor also enhanced migration in the MDA-MB-468, MDA-MB-231 and A549 cell lines, and IP receptor knock-down in MDA-COX-2 cells decreased the expression of a number of pro-migratory genes. In further studies, the prostacyclin/IP receptor and PGE₂/EP4 receptor pathways were found to be functionally independent and the inhibition of phosphatidylinositol 3-kinase (PI3K) and p38 mitogen-activated protein kinase (MAPK) selectively impaired the IP-receptor-dependent migration in MDA-COX-2 cells. Taken together, the prostacyclin/IP/PI3K-p38 MAPK axis has emerged as a novel pro-migratory pathway in breast cancer cells that over-express COX-2. This information could be utilized in novel treatment strategies to minimize tumor metastasis.     

Preservation of Gi coupling of a chimeric EP3/I-type prostaglandin (IP) receptor.

For the EP3 subtype of prostaglandin E receptors, different C-terminal splice variants are known, which are coupled to distinct heterotrimeric GTP-binding proteins (G-proteins). To test the hypothesis that the C-terminal domain is essential for the G-protein-coupling specificity of the EP3 receptor, we exchanged the carboxyl-terminal tail of a porcine Gi-coupled EP3 receptor isoform for the corresponding C-terminal part of a Gs-coupled prostaglandin receptor. The porcine EP3 receptor was truncated at a lysine (K350) residue at the end of the seventh transmembrane region, representing the splicing site of the different EP3 receptor isoforms. The wild-type C-terminus (37 amino acids) was substituted by the C-terminal tail (89 amino acids) of the human I-type prostaglandin receptor (hIP-R). The G-protein coupling of the resulting chimeric receptor protein was studied in transfected Chinese hamster ovary (CHO) cells. Stimulation of the chimeric receptor protein with the EP3 receptor-specific agonist M&B 28.767 did not increase adenosine 3',5'-cyclic monophosphate (cAMP) formation but did reduce the forskolin-stimulated cAMP formation, indicating Gi coupling. Furthermore, the chimeric receptor did not show constitutive activity as demonstrated for the C-terminally truncated EP3 receptor. Thus, coupling specificity of the EP3 receptor is not exclusively mediated by the carboxyl-terminal tail, and constitutive activity of a C-terminally truncated EP3 receptor can be suppressed by the hIP-R C-terminus.     

Cloned human EP1 prostanoid receptor pharmacology characterized using radioligand binding techniques

Prostaglandins such as prostaglandin E2 (PGE2) interact with EP-class prostanoid receptors including EP1, EP2, EP3 and EP4 subtypes. We have conducted a detailed pharmacological characterization of the binding of [3H]-PGE2 to recombinant human EP1 prostanoid receptors expressed in human embryonic kidney (HEK-293) cells using a broad panel of natural and synthetic prostanoids. The receptor displayed high affinity (Kd = 16.0 +/- 0.69 nM; n = 3) for [3H]-PGE2, and was expressed at high levels (Bmax =3.69 +/- 0.30 pmol (mg protein)(-1)) in cell membranes of HEK-293 cells. Specific binding constituted 97.5 +/- 1.4% (n = 12) of the total binding. In competition assays, the rank order of affinities of natural prostanoids for the receptor was PGE2 > PGE1 > PGF2 > PGI2 > PGD2. PGE2 was more effective than PGE1 at displacing bound [3H]-PGE2 (Ki for PGE2 = 14.9 +/- 2.2 nM; Ki for PGE1 = 165 +/- 29 nM). The affinities of enprostil (Ki = 14.5 +/- 3.1 nM) and 17-phenyl-omega-trinor-PGE2 (Ki = 7.3 +/- 2.7 nM) for the receptor were quite similar to that of PGE2, while that of sulprostone (Ki = 137 + 13 nM) more closely resembled PGE1. Some compounds historically classified as specific for DP prostanoid receptors bound with relatively high affinity to the recombinant human EP1 receptor (e.g. ZK118182 (K = 73.4 +/- 8.6 nM) and ZK110841 (K = 166 +/- 20 nM)). All FP (e.g. travoprost acid, fluprostenol), IP (iloprost) and TP (SQ29548) receptor-specific ligands exhibited low affinity (Ki > or = 1 microM).     

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.     

Glial cells isolated from dorsal root ganglia express prostaglandin E(2) (EP(4)) and prostacyclin (IP) receptors

Isolated cells from adult rat dorsal root ganglia (DRG) are frequently used as a model system to study responses of primary sensory neurons to nociceptor sensitizing agents such as prostaglandin E(2) and prostacyclin, which are presumed to act only on the neurons in typical mixed cell cultures. In the present study, we evaluated the expression of prostaglandin E(2) (EP(4)) and prostacyclin (IP) receptors in cultures of mixed DRG cells and in purified DRG glia. We show here that EP(4) and IP receptor agonists stimulated adenylyl cyclase activity in both mixed DRG cells and in purified DRG glia, and that these responses were specifically inhibited by EP(4) and IP receptor antagonists, respectively. The presence of EP(4) and IP receptors in DRG glia was further confirmed by the expression of EP(4) and IP receptor immunoreactivity and mRNA. With the increasing awareness of neuron-glial interactions within intact DRG and the use of isolated DRG cells in the study of mechanisms underlying nociception, it will be essential to consider the role played by EP(4) and IP receptor-expressing glial cells when evaluating prostanoid-induced sensitization of DRG neurons.     

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.     

Protein kinase A-dependent coupling of mouse prostacyclin receptors to Gi is cell-type dependent

The ability of the prostacyclin (IP) receptor agonist cicaprost to activate Gs-, Gq/11- and Gi-mediated cell signalling pathways has been examined in Chinese hamster ovary (CHO) cells and human embryonic kidney 293 (HEK 293) cells expressing the cloned human (hIP) or mouse (mIP) prostacyclin receptor, and compared with data from NG108-15 and SK-N-SH cells that endogenously express rat/mouse and human IP receptors, respectively. Cicaprost stimulated [3H]cyclic AMP production with EC50 values of 1.5-22 nM, and stimulated [3H]inositol phosphate production (EC50 values 49-457 nM) in all but the SK-N-SH cells. Cicaprost failed to inhibit forskolin-stimulated [3H]cyclic AMP production in any of these cell lines. Therefore, although both human and mouse IP receptors couple to Gs and Gq/11-mediated signalling pathways in a cell type-dependent manner, we could find no evidence for IP receptor coupling to Gi.     

Activation of adenylyl cyclase by endogenous G(s)-coupled receptors in human embryonic kidney 293 cells is attenuated by 5-HT(7) receptor expression

Human 5-hydroxytryptamine(7) (5-HT(7)) receptors display characteristics shared with receptors believed to form a tight physical coupling with G protein in the absence of ligand. Some receptors apparently preassociated with G(i/o) and G(q/11) are reported to inhibit the signaling of other similarly coupled G protein-coupled receptors by limiting their access to activate a common G protein pool. Therefore, we determined whether 5-HT(7) receptor expression was sufficient to limit signaling of endogenously expressed G(s)-coupled receptors in human embryonic kidney (HEK) 293 cells. Using the ecdysone-inducible expression system, which allows for the titration of increasing receptor density in the same clonal cell line, we compared the effects of 5-HT(4(b)) and 5-HT(7(a,b,d)) receptor expression on adenylyl cyclase (AC) stimulation by the endogenous G(s)-coupled beta-adrenergic (betaAR) and prostanoid EP (EPR) receptors. betaAR- and EPR-stimulated AC activity was attenuated by 5-HT(7) receptor expression in both membrane preparations and intact HEK293 cells. betaAR- and EPR-stimulated AC activity was unaffected by expression of the G(s)-coupled 5-HT(4) receptor. The mechanism of this heterologous desensitization seems independent of protein kinase A activation, nor does it occur at the level of G protein activation because 1) betaAR- and EPR-stimulated AC activity was not restored to control values when Galpha(s) was overexpressed; and 2) beta(1)AR and beta(2)AR activation of Galpha(s) was unaffected by the expression of 5-HT(7) receptors. In addition, overexpression of AC isoforms was unable to rescue betaAR- and EPR-stimulated AC activity. Therefore, 5-HT(7) receptors probably limit access and/or impede activation of AC by betaAR and EP receptors. Although the 5-HT(7) receptor may preassociate with G protein and/or AC, the mechanism of this heterologous desensitization remains elusive.     

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.     

Partial agonism of taprostene at prostanoid IP receptors in vascular preparations from guinea-pig, rat, and mouse

This study investigates whether incomplete relaxation of vascular smooth muscle preparations induced by the prostacyclin analogue taprostene is due to partial agonism at prostanoid IP receptors. In the presence of the prostanoid EP4 receptor antagonist AH 23848, 3 microM taprostene induced 45% relaxation of phenylephrine-contracted guinea-pig saphenous vein rings and displaced log concentration-response curves for the prostacyclin analogues AFP-07, TEI-9063, and cicaprost to the right, parallel to their predicted addition curves. In contrast, taprostene interacted additively with prostaglandin E2 (PGE2), ONO-AE1-259 (selective EP2 agonist), and acetylcholine. Similarly, on rat tail artery contracted with phenylephrine, 3 microM taprostene (20% relaxation) opposed AFP-07- but not PGE2-induced relaxation. However, under U-46619-induced tone (AH 23848 absent), taprostene antagonized AFP-07 and cicaprost more than TEI-9063, suggesting that the latter has more than one relaxation mechanism. The presence of a sensitive EP3 contractile system in mouse aorta interfered with IP receptor-mediated relaxation. By generating tone with phenylephrine and the potent EP3 agonist sulprostone, it was possible to show that 3 microM taprostene (15% relaxation) selectively opposed relaxations induced by AFP-07, TEI-9063, and cicaprost. Our experiments indicate that taprostene is a partial agonist at prostanoid IP receptors, and may be a lead to an IP receptor antagonist.     

Bell-shaped curves for prostaglandin-induced modulation of adenylate cyclase: two mutually opposing effects

Each of the natural prostanoid is at least one order of magnitude more potent for its specific receptor (DP, EP, FP, IP and TP) than any of the other prostanoids. However, they are able to interact also with one or more of the other classes of prostanoid receptors. The concentration-response curves for modulation of adenylate cyclase activity in rabbit mesenteric artery smooth muscle cells by different prostaglandins are not always monotonic, i.e. simple sigmoidal curves in logarithmic scale, but they are often biphasic. Prostacyclin, iloprost and prostaglandin E(1) showed a convex bell-shaped curve, i.e. adenylate cyclase activity is stimulated at lower concentrations and inhibited at higher concentrations, while the curve of prostaglandin E(2) showed a concave bell-shaped curve, i.e. adenylate cyclase is inhibited at lower concentrations and stimulated at higher concentrations. By selectively inhibiting one of the transduction mechanisms present in mesenteric smooth muscle cells, we have demonstrated that the observed responses to these prostanoids are likely due to two mutually opposing effects. Thus, the data previously published by our laboratory on a prostacyclin analog, 5(Z)-carbacyclin, might be reinterpreted more correctly in the light of this new possibility.     

Adenylyl cyclase type 6 overexpression selectively enhances β-adrenergic and prostacyclin receptor-mediated inhibition of cardiac fibroblast function because of colocalization in lipid rafts

Cardiac fibroblasts produce and degrade extracellular matrix and are critical in regulating cardiac remodeling and hypertrophy. Fibroblasts are activated by factors such as transforming growth factor beta and inhibited by agents that elevate 3',5'-cyclic adenosine monophosphate (cAMP) levels. cAMP signal generation and response is known to be compartmentalized in many cell types in part through the colocalization of receptors and specific adenylyl cyclase isoforms in lipid rafts and caveolae. The present study sought to define the localization of key G protein-coupled receptors with adenylyl cyclase type 6 (AC6) in lipid rafts of rat cardiac fibroblasts and to determine if this colocalization was functionally relevant. We found that cardiac fibroblasts produce cAMP in response to agonists for beta-adrenergic (isoproterenol), prostaglandin EP(2) (butaprost), adenosine (adenosine-5'-N-ethylcarboxamide, NECA), and prostacyclin (beraprost) receptors. Overexpression of AC6 increased cAMP production stimulated by isoproterenol and beraprost but not by butaprost or NECA. A key function of fibroblasts is the production of collagen. Isoproterenol- and beraprostmediated inhibition of collagen synthesis was also enhanced by AC6 overexpression, while inhibition by butaprost and NECA were unaltered. Lipid raft fractions from cardiac fibroblasts contain the preponderance of beta-adrenergic receptors and AC6 but exclude EP(2) receptors. While we could not determine the localization of native prostacyclin receptors, we were able to determine that epitope-tagged prostanoid IP receptors (IPR) expressed in COS7 cells did localize, in part, in lipid raft fractions. These findings indicate that IP receptors are expressed in lipid rafts and can activate raft-localized AC isoforms. AC6 is completely compartmentized in lipid raft domains where it is activated solely by coresident G protein-coupled receptors to regulate cardiac fibroblast function.     

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.