15-hydroxy-11-alpha-9-alpha-(epoxymethano)prosta-5-13-dienoic-acid and sulprostone

Although prostanoids are known to be involved in regulation of the spontaneous beating rate of cultured neonatal rat cardiomyocytes, the various subtypes of prostanoid receptors have not been investigated in detail. In our experiments, prostaglandin (PG)F(2α) and prostanoid FP receptor agonists (fluprostenol, latanoprost and cloprostenol) produced a decrease in the beating rate. Two prostanoid IP receptor agonists (iloprost and beraprost) induced first a marked drop in the beating rate and then definitive abrogation of beating. In contrast, the prostanoid DP receptor agonists (PGD(2) and BW245C) and TP receptor agonists (U-46619) produced increases in the beating rate. Sulprostone (a prostanoid EP(1) and EP(3) receptor agonist) induced marked increases in the beating rate, which were suppressed by SC-19220 (a selective prostanoid EP(1) antagonist). Butaprost (a selective prostanoid EP(2) receptor agonist), misoprostol (a prostanoid EP(2) and EP(3) receptor agonist), 11-deoxy-PGE(1) (a prostanoid EP(2), EP(3) and EP(4) receptor agonist) did not alter the beating rate. Our results strongly suggest that prostanoid EP(1) receptors are involved in positive regulation of the beating rate. Prostanoid EP(1) receptor expression was confirmed by western blotting with a selective antibody. Hence, neonatal rat cardiomyocytes express both prostanoid IP and FP receptors (which negatively regulate the spontaneous beating rate) and prostanoid TP, DP(1) and EP(1) receptors (which positively regulate the spontaneous beating rate).

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Animals, Newborn; Blotting, Western; Cells, Cultured; Cloprostenol; Dibenz(b,f)(1,4)oxazepine-10(11H)-carboxylic acid, 8-chloro-, 2-acetylhydrazide; Dinoprostone; Dose-Response Relationship, Drug; Epoprostenol; Hydantoins; Iloprost; Latanoprost; Myocytes, Cardiac; Prostaglandin D2; Prostaglandins F, Synthetic; Rats; Rats, Sprague-Dawley; Receptors, Prostaglandin; Receptors, Prostaglandin E, EP1 Subtype; Receptors, Thromboxane

Atherosclerosis has an important inflammatory component. Macrophages accumulating in atherosclerotic arteries produce prostaglandin E(2) (PGE(2)), a main inflammatory mediator. Platelets express inhibitory receptors (EP(2), EP(4)) and a stimulatory receptor (EP(3)) for this prostanoid. Recently, it has been reported in ApoE(-/-) mice that PGE(2) accumulating in inflammatory atherosclerotic lesions might contribute to atherothrombosis after plaque rupture by activating platelet EP(3), and EP(3) blockade has been proposed to be a promising new approach in anti-thrombotic therapy. The aim of our investigation was to study the role of PGE(2) in human atherosclerotic plaques on human platelet function and thrombus formation. Plaque PGE(2) might either activate or inhibit platelets depending on stimulation of either EP(3) or EP(4), respectively. We found that the two EP(3)-antagonists AE5-599 (300 nM) and AE3-240 (300 nM) specifically and completely inhibited the synergistic effect of the EP(3)-agonist sulprostone on U46619-induced platelet aggregation in blood. However, these two EP(3)-antagonists neither inhibited atherosclerotic plaque-induced platelet aggregation, GPIIb/IIIa exposure, dense and alpha granule secretion in blood nor reduced plaque-induced platelet thrombus formation under arterial flow. The EP(4)-antagonist AE3-208 (1-3 μM) potentiated in combination with PGE(2) (1 μM) ADP-induced aggregation, demonstrating that PGE(2) enhances platelet aggregation when the inhibitory EP(4)-receptor is inactivated. However, plaque-induced platelet aggregation was not augmented after platelet pre-treatment with AE3-208, indicating that plaque PGE(2) does not stimulate the EP(4)-receptor. We found that PGE(2) was present in plaques only at very low levels (15 pg PGE(2)/mg plaque). We conclude that PGE(2) in human atherosclerotic lesions does not modulate (i.e. stimulate or inhibit) atherothrombosis in blood after plaque rupture.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Abortifacient Agents, Nonsteroidal; Animals; Apolipoproteins E; Blood Platelets; Carotid Stenosis; Dinoprostone; Female; Humans; Male; Mice; Mice, Knockout; Naphthalenes; Phenylbutyrates; Plaque, Atherosclerotic; Platelet Aggregation; Platelet Glycoprotein GPIIb-IIIa Complex; Receptors, Prostaglandin E, EP3 Subtype; Receptors, Prostaglandin E, EP4 Subtype; Rupture, Spontaneous; Thrombosis; Vasoconstrictor Agents

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Blood Platelets; Cells, Cultured; Citrates; Collagen; Dinoprostone; Drug Synergism; Hirudins; Humans; Inflammation; Platelet Aggregation; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP3 Subtype; Vasoconstrictor Agents

In previous studies investigating cross-talk of signalling between prostaglandin (PG)E(2) receptor (EP) and the TPalpha and TPbeta isoforms of the human thromboxane (TX)A(2) receptor (TP), 17-phenyl trinor PGE(2)-induced desensitization of TP receptor signalling through activation of the AH6809 and SC19220-sensitive EP(1) 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.. Specificity of 17-phenyl trinor PGE(2) signalling and its possible cross-talk with signalling by TPalpha/TPbeta receptors endogenously expressed in HEL cells was examined through assessment of agonist-induced inositol 1,4,5-trisphosphate (IP)(3) generation and intracellular calcium ([Ca(2+)](i)) mobilization.. While 17-Phenyl trinor PGE(2) led to activation of phospholipase (PL)Cbeta to yield increases in IP(3) generation and [Ca(2+)](i), it did not desensitize but rather augmented that signalling in response to subsequent stimulation with the TXA(2) mimetic U46619. Furthermore, the augmentation was reciprocal. Signalling by 17-phenyl trinor PGE(2) was found to occur through AH6809- and SC19920-insensitive, Pertussis toxin-sensitive, G(i)/G(betagamma)-dependent activation of PLCbeta. Further pharmacological investigation using selective EP receptor subtype agonists and antagonists confirmed that 17-phenyl trinor PGE(2)-mediated signalling and reciprocal cross-talk with the TP receptors occurred through the EP(3), rather than the EP(1), EP(2) or EP(4) receptor subtype in HEL cells.. The EP(1) and EP(3) subtypes of the EP receptor family mediated intermolecular cross-talk to differentially regulate TP receptor-mediated signalling whereby activation of EP(1) receptors impaired or desensitized, while that of EP(3) receptors augmented signalling through TPalpha/TPbeta receptors, in a cell type-specific manner.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Calcium; Cell Line; Cell Line, Tumor; Dinoprostone; Enzyme Activation; GTP-Binding Protein alpha Subunits; Humans; Inositol 1,4,5-Trisphosphate; Phospholipase C beta; Protein Isoforms; Receptor Cross-Talk; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP1 Subtype; Receptors, Prostaglandin E, EP3 Subtype; Receptors, Thromboxane A2, Prostaglandin H2; Signal Transduction

Differentiation induction as a therapeutic strategy has, so far, the greatest impact in hematopoietic malignancies, most notably leukemia. Diosgenin is a very interesting natural product because, depending on the specific dose used, its biological effect is very different in HEL (human erythroleukemia) cells. For example, at 10 microM, diosgenin induced megakaryocytic differentiation, in contrast to 40 microM diosgenin, which induced apoptosis in HEL cells previously demonstrated using sedimentation field-flow fractionation (SdFFF). The goal of this work focused on the correlation between cyclooxygenase-2 (COX-2) and thromboxane synthase (TxS) and megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, the technique of SdFFF, having been validated in our models, was used in this new study as an analytical tool that provided us with more or less enriched differentiated cell fractions that could then be used for further analyses of enzyme protein expression and activity for the first time. In our study, we showed the implication of COX-2 and TxS in diosgenin-induced megakaryocytic differentiation in HEL cells. Furthermore, we showed that the analytical technique of SdFFF may be used as a tool to confirm our results as a function of the degree of cell differentiation.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Cattle; Cell Differentiation; Cell Line, Tumor; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Dinoprostone; Diosgenin; Fractionation, Field Flow; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Erythroblastic, Acute; Megakaryocytes; Microsomes; Platelet Membrane Glycoprotein IIb; Ploidies; Thromboxane-A Synthase

The present study examined the hypothesis that prostaglandin E2 (PGE2) through activation of prostaglandin E (EP) receptor contributes to endothelium-dependent contractions.. Western blotting revealed that the protein expression of EP1 receptor was significantly down-regulated in the aorta of the spontaneously hypertensive rat (SHR), but there was no significant difference in the expression of EP2, EP4, and total EP3 receptors between preparations of Wistar Kyoto rats (WKY) and SHR. Isometric tension studies showed that low concentrations of PGE2 caused endothelium-dependent relaxations in WKY but not in aortas of the SHR. High concentrations of PGE2 evoked contractions predominately through the activation of thromboxane-prostanoid (TP) receptors in the WKY, but involves the dual activation EP and TP receptors in the SHR. SQ29,548, BAYu3405 and Terutroban (TP receptor antagonists), and AH6809 (non-selective EP receptor antagonist) abolished, while SC19220 (preferential EP1 receptor antagonist) did not inhibit endothelium-dependent contractions. Both SC19220 and AH6809 significantly inhibited contractions to U46619 (TP receptor agonist).. The present study demonstrates that the contraction caused by PGE2 in the SHR aorta is dependent on the activation of EP1 and TP receptors, but that endothelium-dependent contractions do not require the former. Thus, PGE2 is unlikely to be an endothelium-derived contracting factor in this artery. The ability of AH6809 to inhibit endothelium-dependent contractions can be attributed to its partial antagonism at TP receptors. Nevertheless, the impairment of PGE2-mediated relaxation may contribute to endothelial dysfunction in the aorta of the SHR.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Aorta, Thoracic; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Carbazoles; Dibenz(b,f)(1,4)oxazepine-10(11H)-carboxylic acid, 8-chloro-, 2-acetylhydrazide; Dinoprostone; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelium, Vascular; Fatty Acids, Unsaturated; Hydrazines; Hypertension; Immunohistochemistry; Naphthalenes; Phenylephrine; Potassium Chloride; Propionates; Prostaglandin Antagonists; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP1 Subtype; Receptors, Thromboxane; Sulfonamides; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Xanthones

The cyclooxygenase-prostanoid pathway regulates myometrial contractility through activation of prostanoid receptors on uterine smooth muscles. However, the possible expression of prostanoid receptors on autonomic nerves cannot be excluded completely. The aim of the present study was to clarify the presence of neural prostanoid receptors on adrenergic nerves in the porcine uterine longitudinal muscle. In [(3)H]-noradrenaline-loaded longitudinal muscle strips of porcine uterus, electrical field stimulation (EFS) evoked [(3)H]-noradrenaline release in a stimulation frequency-dependent manner. The EFS-evoked release was completely abolished in Ca(2+)-free (EGTA, 1mM) incubation medium and by tetrodotoxin or omega-conotoxin GVIA, suggesting that [(3)H]-noradrenaline was released from neural components. The EFS-evoked [(3)H]-noradrenaline release was significantly enhanced by treatment with indomethacin. In the presence of indomethacin, PGE(2) and PGF(2alpha), but not PGD(2), inhibited the EFS-evoked [(3)H]-noradrenaline release. Of synthetic prostanoid receptor agonists examined, both U46619 (TP) and sulprostone (EP(1)/EP(3)) decreased the EFS-evoked [(3)H]-noradrenaline release in a concentration-dependent manner, while fluprostenol (FP), BW245C (DP) and butaprost (EP(2)) were almost ineffective. SQ29548 (TP receptor antagonist) blocked the effect of U46619, but SC19220 (EP(1) receptor antagonist) did not change the inhibition by sulprostone or PGE(2). Double immunofluorescence staining using protein gene product 9.5, tyrosine hydroxylase, EP(3) receptor and TP receptor antibodies suggested the localization of EP(3) or TP receptors on adrenergic nerves in the porcine uterus. These results indicated that neural EP(3) and TP receptors are present on adrenergic nerves of the porcine uterine longitudinal muscle. Endogenous prostanoid produced by cyclooxygenase can regulate noradrenaline release in an inhibitory manner through activation of these neural prostanoid receptors.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Alprostadil; Animals; Dinoprost; Dinoprostone; Electric Stimulation; Female; In Vitro Techniques; Microscopy, Confocal; Microscopy, Fluorescence; Myometrium; Neurons; Norepinephrine; Prostaglandin D2; Prostaglandins; Prostaglandins F, Synthetic; Receptors, Androgen; Receptors, Prostaglandin; Swine

This study investigates the pronounced synergism between the weak contractile action of prostaglandin E(2) (PGE(2)) and strong actions of phenylephrine, U-46619 and K(+) on rat isolated femoral artery. The potency ranking for synergism was SC-46275 (prostanoid receptor agonist selectivity: EP(3)>>EP(1))=sulprostone (EP(3)>EP(1))>17-phenyl PGE(2) (EP(1)>EP(3)). The novel EP(3) antagonist L-798106 (0.2-1microM) blocked the enhanced action of sulprostone (pA(2)=7.35-8.10), while the EP(1) antagonist SC-51322 (1microM) did not (pA(2)<6.0). Matching responses to priming agent and priming agent/sulprostone were similarly suppressed by nifedipine (300nM) and the selective Rho-kinase inhibitors H-1152 (0.1-1microM) and Y-27632 (1-10microM). Our findings implicate an EP(3) receptor in the prostanoid component of contractile synergism. While the synergism predominantly operates through a Ca(2+) influx-Rho-kinase pathway, the EP(3) receptor does not necessarily transduce via Rho-kinase.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Alprostadil; Animals; Dinoprostone; Drug Interactions; Drug Synergism; Femoral Artery; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Male; Nifedipine; Phenylephrine; Potassium; Prostaglandins F, Synthetic; Protein Serine-Threonine Kinases; Rats; Rats, Sprague-Dawley; Receptors, Prostaglandin; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP1 Subtype; Receptors, Prostaglandin E, EP3 Subtype; rho-Associated Kinases; Sensitivity and Specificity; Sulfonamides; Vasoconstrictor Agents

We have investigated the actions of various prostanoid receptor agonists on an isolated preparation of the ferret cervical vagus using a grease-gap extracellular recording technique. The potency ranking for depolarization was BW245C (5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl) hydantoin; DP-selective, EC50=0.14 microM)>prostaglandin E2 (nonselective EP agonist)>U-46619 (11alpha, 9alpha-epoxymethano-15S-hydroxyprosta-5Z,13E-dienoic acid; TP agonist)>prostaglandin F2alpha (FP receptor agonist). Sulprostone (EP1/EP3-selective), fluprostenol (FP-selective) and cicaprost and iloprost (both IP-selective) had minimal effects. It is likely that DP, EP2/EP4 and TP receptors are present on the vagal fibres of the ferret.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Biguanides; Dinoprost; Dinoprostone; Dose-Response Relationship, Drug; Electrophysiology; Epoprostenol; Ferrets; Hydantoins; Iloprost; In Vitro Techniques; Male; Prostaglandins; Prostaglandins F, Synthetic; Serotonin; Vagus Nerve

1. A variety of prostanoids were examined for their ability to alter the periarterial nerve stimulation-induced release of noradrenaline (NA) and neuropeptide Y immunoreactive compounds (NPY-ir) from the perfused mesenteric arterial bed of the rat. 2. Periarterial nerve stimulation (16 Hz) increased the overflow of NA, NPY-ir and perfusion pressure. 3. The prostacyclin (PGI2) analogues, carbaPGI2 and cicaprost both produced a concentration-dependent attenuation of the nerve stimulation-induced increase in NA, NPY-ir overflow and perfusion pressure. 4. The prostaglandin (PG) analogue PGE2 attenuated the evoked increase in NPY-ir overflow as well as a modest decrease in NA. 5. PGE1, sulprostone and iloprost attenuated the nerve stimulation-induced increase in NA overflow but not NPY-ir. 6. Neither PGF2alpha nor the thromboxane A2 analogue U46619 altered the evoked increase in NA or NPY-ir overflow. 7. The results support the view that sympathetic co-transmitter release can be differentially modulated by paracrine/autocrine mediators at sympathetic neuroeffector junctions.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Alprostadil; Animals; Dinoprost; Dinoprostone; Electric Stimulation; Epoprostenol; Iloprost; Male; Mesenteric Arteries; Neuropeptide Y; Norepinephrine; Perfusion; Prostaglandins; Radioimmunoassay; Rats; Rats, Sprague-Dawley

1. Since the role of prostanoid receptors in intestinal peristalsis is largely unknown, the peristaltic motor effects of some prostaglandin (DP, EP, IP), thromboxane (TP) and leukotriene (LT) receptor agonists and antagonists were investigated. 2. Propulsive peristalsis in fluid-perfused segments from the guinea-pig small intestine was triggered by a rise of the intraluminal pressure and recorded via the intraluminal pressure changes associated with the peristaltic waves. Alterations of distension sensitivity were deduced from alterations of the peristaltic pressure threshold and modifications of peristaltic performance were reflected by modifications of the amplitude, maximal acceleration and residual baseline pressure of the peristaltic waves. 3. Four categories of peristaltic motor effects became apparent: a decrease in distension sensitivity and peristaltic performance as induced by the EP1/EP3 receptor agonist sulprostone and the TP receptor agonist U-46619 (1-1000 nM); a decrease in distension sensitivity without a major change in peristaltic performance as induced by PGD(2) (3-300 nM) and LTD(4) (10-100 nM); a decrease in peristaltic performance without a major change in distension sensitivity as induced by PGE(1), PGE(2) (1-1000 nM) and the EP1/IP receptor agonist iloprost (1-100 nM); and a decrease in peristaltic performance associated with an increase in distension sensitivity as induced by the EP2 receptor agonist butaprost (1-1000 nM). The DP receptor agonist BW-245 C (1-1000 nM) was without effect. 4. The peristaltic motor action of sulprostone remained unchanged by the EP1 receptor antagonist SC-51089 (1 micro M) and the DP/EP1/EP2 receptor antagonist AH-6809 (30 micro M), whereas that of U-46619 and LTD(4) was prevented by the TP receptor antagonist SQ-29548 (10 micro M) and the cysteinyl-leukotriene(1) (cysLT(1)) receptor antagonist tomelukast (10 micro M), respectively. 5. These observations and their pharmacological analysis indicate that activation of EP2, EP3, IP, TP and cysLT(1) receptors, but not DP receptors, modulate intestinal peristalsis in a receptor-selective manner, whereas activation of EP1 seems to be without influence on propulsive peristalsis. In a wider perspective it appears as if the effect of prostanoid receptor agonists to induce diarrhoea is due to their prosecretory but not peristaltic motor action.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Alprostadil; Animals; Bridged Bicyclo Compounds, Heterocyclic; Dinoprostone; Dose-Response Relationship, Drug; Fatty Acids, Unsaturated; Female; Guinea Pigs; Hydantoins; Hydrazines; Iloprost; In Vitro Techniques; Intestine, Small; Leukotriene Antagonists; Leukotriene D4; Male; Oxazepines; Peristalsis; Prostaglandin D2; Prostaglandins A; Receptors, Leukotriene; Receptors, Prostaglandin; Xanthenes; Xanthones; Yohimbine

The effects of various prostanoids on the growth, melanogenesis and dendrification of cultured iridal melanocytes were studied. Iridal melanocytes were isolated and cultured with medium supplemented with cAMP elevating agents and basic fibroblast growth factor (bFGF) (complete medium). The iridal melanocytes were plated into multiple well plates and cultured with complete medium or various deleted media with or without various prostanoids at different concentrations. After 6 days, the numbers of cells and dendrites were counted and melanin content was measured and compared with controls. Prostaglandin E(2), an EP(2)receptor agonist (AH 13205) and AGN 192093 (thromboxane mimetic) stimulated growth, melanogenesis and dendrification of cultured iridal melanocytes in cAMP-deleted medium. A mixed EP(1)and EP(3)receptor agonist (sulprostone), a EP(4)receptor agonist (ONO-AE1-329), IP receptor agonists (cicaprost or iloprost) and a TP receptor agonist (U-46619) showed no effect. Prostaglandin D(2)showed stimulating effects. However, these stimulating effects could not be blocked by the addition of a DP receptor antagonist (BW A868C). Furthermore, a DP receptor agonist (BW 245C) showed no effects, indicating that the effect of prostaglandin D(2)may involve receptors other than the DP receptor subtype. The present study indicates that: (1) among various EP receptor agonists, only an EP(2)receptor agonist has stimulating effects on iridal melanocytes; (2) DP, IP and TP receptor agonists do not have stimulating effects; and (3) the mechanisms of action of prostaglandin D(2)and AGN 192093 need further study.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adult; Cell Count; Cells, Cultured; Cyclic AMP; Dinoprostone; Epoprostenol; Fibroblast Growth Factor 2; Humans; Hydantoins; Iris; Melanins; Melanocytes; Prostaglandin Antagonists; Prostanoic Acids; Receptors, Prostaglandin

1. To characterize the prostanoid receptors (TP, FP, EP(1) and/or EP(3)) involved in the vasoconstriction of human pulmonary veins, isolated venous preparations were challenged with different prostanoid-receptor agonists in the absence or presence of selective antagonists. 2. The stable thromboxane A(2) mimetic, U46619, was a potent constrictor agonist on human pulmonary veins (pEC(50)=8.60+/-0.11 and E(max)=4.61+/-0.46 g; n=15). The affinity values for two selective TP-antagonists (BAY u3405 and GR32191B) versus U46619 were BAY u3405: pA(2)=8.94+/-0.23 (n=3) and GR32191B: apparent pK(B)=8.25+/-0.34 (n=3), respectively. These results are consistent with the involvement of TP-receptor in the U46619 induced contractions. 3. The two EP(1)-/EP(3)- agonists (17-phenyl-PGE(2) and sulprostone) induced contraction of human pumonary veins (pEC(50)=8.56+/-0.18; E(max)=0.56+/-0.24 g; n=5 and pEC(50)=7.65+/-0.13; E(max)=1.10+/-0.12 g; n=14, respectively). The potency ranking for these agonists: 17-phenyl-PGE(2) > sulprostone suggests the involvement of an EP(1)-receptor rather than EP(3). In addition, the contractions induced by sulprostone, 17-phenyl-PGE(2) and the IP-/EP(1)- agonist (iloprost) were blocked by the DP-/EP(1)-/EP(2)-receptor antagonist (AH6809) as well as by the EP(1) antagonist (SC19220). 4. PGF(2alpha) induced small contractions which were blocked by AH6809 while fluprostenol was ineffective. These results indicate that FP-receptors are not implicated in the contraction of human pulmonary veins. 5. These data suggest that the contractions induced by prostanoids involved TP- and EP(1)-receptors in human pulmonary venous smooth muscle.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Biphenyl Compounds; Carbazoles; Culture Techniques; Dibenz(b,f)(1,4)oxazepine-10(11H)-carboxylic acid, 8-chloro-, 2-acetylhydrazide; Dinoprostone; Dose-Response Relationship, Drug; Endothelium, Vascular; Female; Heptanoic Acids; Humans; Iloprost; Male; Middle Aged; Muscle Contraction; Muscle, Smooth, Vascular; Prostaglandin Antagonists; Prostaglandins F, Synthetic; Pulmonary Veins; Receptors, Prostaglandin; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP1 Subtype; Receptors, Prostaglandin E, EP3 Subtype; Receptors, Thromboxane; Sulfonamides; Vasoconstriction; Xanthenes; Xanthones

Contraction of guinea-pig isolated aorta induced by the prostaglandin E analogue sulprostone (1-400 nM) has a lower maximum response (40%) than that of phenylephrine or U-46619 (TP-receptor agonist). A prostanoid EP3-receptor subtype is involved based on agonist potency ranking: equi-effective molar ratios (EMR) are sulprostone (EC50 approximately equal to 23 nM) 1.0, SC-46275 0.11, misoprostol 2.2, gemeprost 3.3, PGE2 5.4, 17-phenyl PGE2 6.0, GR-63799 8.9. GR-63799, which contains a bulky ester group, is relatively more potent on neuronal EP3 preparations than on the aorta. ONO-AP-324, a relative of the non-prostanoid prostacyclin mimetic series, behaves as an EP3 partial agonist on the aorta, inhibiting sulprostone responses but acting synergistically (in a similar manner to sulprostone) with phenylephrine; it may be a useful pharmacological tool for studying EP3-receptors. Sulprostone contractions are markedly suppressed in zero-Ca2+ bathing fluid containing either 2 mM EDTA or 50 microM EGTA, and by Cd2+ (500 microM), but are usually unaffected by nifedipine (0.3 microM) and verapamil (4.44 microM). Influx of Ca2+, but not through L-type Ca2+-channels, appears to be the major contractile mechanism. The guinea-pig aorta is a valuable addition to the vascular EP3 preparations available and may increase our knowledge of the mechanisms whereby Gi-coupled receptors mediate vasoconstriction (c.f. 5-HT1B/D- and alpha2-receptors). The possibility of certain EP3 agonists distinguishing EP3-receptor isoforms is discussed.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetates; Animals; Aorta, Thoracic; Benzhydryl Compounds; Calcium; Dinoprostone; Guinea Pigs; In Vitro Techniques; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Oxytocics; Pulmonary Artery; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP3 Subtype; Vas Deferens; Vasoconstrictor Agents

Prostaglandins (PGs) have been implicated in the regulation of vasopressin (VP) and oxytocin (OT) release in response to various stimuli. To examine the site and mechanism of actions of PGs, we studied effects of PGE2 and PG-receptor agonists on supraoptic nucleus (SON) neurones of rat hypothalamic slice preparations using extracellular recording and whole-cell patch-clamp techniques. PGE2 modulated the electrical activity of more than 80% of the neurones studied. The effects of PGE2 on both phasic and non-phasic neurones were mostly excitatory, and dose-dependent. The effects of PGE2 were mimicked by PGF2alpha or the FP agonist, fluprostenol, whereas PGD2 or the selective EP, IP or TP agonist was less effective or had no effect. The effects of PGE2 were unaffected by the EP1 antagonist, SC-51322, but reduced to 80% of control by the EP1/FP/TP antagonist, ONO-NT-012, which reduced the effects of fluprostenol to 32% of control. Moreover, some neurones responsive to PGE2 did not respond to fluprostenol. Patch-clamp analysis in SON slice preparations revealed that PGE2 at 10(-6) M depolarized the membrane potential by 3.9+/-0.3 mV from the resting membrane potential of -58.4+/-2.2 mV in the current-clamp mode. In the voltage-clamp mode, PGE2 induced inward currents at a holding potential of -70 or -80 mV, while it did not affect spontaneous excitatory postsynaptic currents. PGE2 induced currents also in dissociated SON neurones and the reversal potential of the currents was -35.5+/-0.9 mV, which was similar to that of currents induced by fluprostenol. These results suggest that SON neurones possess at least two types of PG receptors, FP receptors and EP receptors of a subclass different from EP1, EP2, or EP3, and that activation of these receptors leads to the opening of nonselective cation channels, membrane depolarization and increase of the action potential discharge.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Bridged Bicyclo Compounds; Dinoprost; Dinoprostone; Dose-Response Relationship, Drug; Excitatory Postsynaptic Potentials; In Vitro Techniques; Male; Neurons; Oxazepines; Patch-Clamp Techniques; Prostaglandin D2; Prostaglandins, Synthetic; Rats; Rats, Wistar; Receptors, Prostaglandin; Styrenes; Supraoptic Nucleus

1. The influence of endogenously produced prostanoids on active transepithelial Ca2+ transport and cAMP formation was investigated in immunodissected rabbit kidney connecting and cortical collecting tubule cells grown to confluency on permeable supports. 2. The cyclo-oxygenase inhibitor indomethacin dose-dependently (IC50 = 18 nM) reduced the net apical-to-basolateral Ca2+ transport by 57%. Inhibition was reversed in medium obtained from monolayers incubated in the absence of indomethacin. 3. HPLC analysis following incubation with 14C-labelled arachidonic acid revealed the presence of a wide variety of radiolabelled prostanoids in both the apical and basolateral media. These findings are compatible with the endogenous production and subsequent release of stimulatory prostanoids. 4. The inhibitory action of indomethacin was reversed by the addition of the prostanoids PGE1, PGE2 and PGA2, but not PGD2, PGF2 alpha, the stable PGI2 analogue cicaprost or the thromboxane A2 mimetic U-46619. PGE2 stimulated transepithelial Ca2+ transport dose dependently (EC50 = 3 nM), irrespective of the compartment of which it was added. The stimulatory effect of PGE2 was paralleled by increased cAMP formation, suggesting the apical and basolateral presence of stimulatory prostanoid receptors EP2 and/or EP4. 5. Sulprostone, an analogue selective for EP1 and EP3 receptors, inhibited transepithelial Ca2+ transport in indomethacin-treated monolayers only when applied basolaterally, suggesting the exclusive presence of inhibitory EP receptors on the basolateral membrane. 6. The percentage by which parathyroid hormone and arginine vasopressin increased both transepithelial Ca2+ transport and cAMP formation was dramatically increased in indomethacin-inhibited cells as compared with control cells, demonstrating that indomethacin unmasks the actions of these hormones to their full extent.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arachidonic Acid; Arginine Vasopressin; Biological Transport; Calcium; Cells, Cultured; Chromatography, High Pressure Liquid; Cyclic AMP; Cyclooxygenase Inhibitors; Dinoprostone; Eicosanoids; Epoprostenol; Indomethacin; Kidney Tubules; Models, Biological; Parathyroid Hormone; Prostaglandin Endoperoxides, Synthetic; Prostaglandins; Rabbits; Receptors, Prostaglandin; Thromboxane A2; Virulence Factors, Bordetella

[3H]PGE2 and [3H]PGF2 alpha were shown to bind with similar binding capacity and dissociation constants to bovine aorta endothelial cells. The similarity in the binding parameters suggests that both agonists may bind to the same binding site. Displacement of [3H]PGE2 performed with PGE2, PGF2 alpha or U-46619, a thromboxane agonist, shows that all three prostanoids displaced the bound [3H]PGE2 with comparable potency (IC50 = 10(-7) M). These results indicated that the three different prostanoids, which serve as specific agonists to different prostanoid receptors, also compete for the same binding site in bovine endothelial cells with similar affinity. Comparison of the displacement of [3H]PGE2 or [3H]PGF2 alpha by a number of prostaglandin agonists and antagonists further supports the notion that the natural prostanoids bind with similar affinities to the same binding site. Thus, sulprostone, an EP1/EP3 agonist, displaced bound [3H]PGE2 and [3H]PGF2 alpha with IC50 of about 10(-7) M. On the other hand, thromboxane antagonists (BAY u-3405 and GR-32191B), EP1 specific antagonist (SC-19220) EP1/DP antagonist (AH-6809) and iloprost, a stable prostacyclin agonist, failed to displace bound [3H]PGE2 or [3H]PGF2 alpha at a concentration range of 10(-9)-10(-6) M. Gradual increase of sodium fluoride (NaF), a general activator of G binding proteins, or incubation of permeabilized cells with GTP gamma S resulted in a decrease in [3H]PGE2 binding, suggesting that the binding site represents a low-affinity common prostanoid receptor which, similar to other prostanoid receptors, is probably coupled with G binding proteins.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Aorta; Binding Sites; Biphenyl Compounds; Carbazoles; Cattle; Cells, Cultured; Dibenz(b,f)(1,4)oxazepine-10(11H)-carboxylic acid, 8-chloro-, 2-acetylhydrazide; Dinoprostone; Endothelium, Vascular; Epoprostenol; Heptanoic Acids; Iloprost; Prostaglandin Endoperoxides, Synthetic; Prostaglandins; Sulfonamides; Thromboxane A2; Thromboxanes; Xanthenes; Xanthones

We wished to determine whether any evidence indicates that the ductus arteriosus has prostanoid receptors coupled to contractile pathways and whether the sensitivity of the ductus to the dilator effect of prostaglandin E2 (PGE2) was inhibited by other prostanoids. Rings of ductus arteriosus were isolated from fetal New Zealand White rabbits (28 days of gestation) and mounted in vitro. In the presence of 1 microM indomethacin, the vessel was relaxed with either 300 nM forskolin or 10 nM PGE2, and cumulative concentration-contraction response curves to several synthetic prostanoids were obtained with or without a receptor antagonist when available. The vessel was also precontracted with 1 microM indomethacin and 25 mM K+ in 13-14.5 kPa O2, and cumulative concentration-relaxation response curves to PGE2 were obtained with and without addition of prostanoids. In 300 nM forskolin, both U46619 and sulprostone caused concentration-dependent contractions of the ductus in the nanomolar range (EC50 values, i.e., the interpolated molar concentration of the drug causing 50% of its own eventual maximum response of 33 and 42 nM, respectively). Responses to GR63799X and PGF2 alpha were complicated by the fact that these agonists caused relaxation at high concentrations (> or = 30 nM). The response to U46619 was shifted to the right by the thromboxane receptor antagonist EP 092. In 10 nM PGE2, U46619, sulprostone, and GR63799X elicited similar contractile responses, whereas PGF2 alpha had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Analysis of Variance; Animals; Colforsin; Dinoprost; Dinoprostone; Dose-Response Relationship, Drug; Ductus Arteriosus; Female; In Vitro Techniques; Indomethacin; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Pregnancy; Prostaglandin Endoperoxides, Synthetic; Prostaglandins E, Synthetic; Rabbits; Receptors, Prostaglandin; Thromboxane A2; Thromboxanes; Vasoconstrictor Agents

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Biphenyl Compounds; Cyclic AMP; Dinoprostone; Dose-Response Relationship, Drug; Histamine; In Vitro Techniques; Muscle Relaxation; Muscle, Smooth, Vascular; Prostaglandin Endoperoxides, Synthetic; Prostaglandins; Receptors, Prostaglandin E; Saphenous Vein; Swine; Thromboxane A2; Vasoconstrictor Agents; Venae Cavae

1. The 16-phenoxy prostaglandin E analogue sulprostone consistently potentiates primary aggregation waves induced by adenosine 5'-diphosphate (ADP), PAF and 11,9-epoxymethano PGH2 (U-46619) in platelet-rich plasma from human donors. The effect is not blocked by the TP-receptor antagonists, EP 092 and GR 32191. The high potency of sulprostone (threshold concentration = 4-10 nM) and the weak block of sulprostone potentiation by the EP1-receptor antagonist, AH 6809 (pA2 = 4.3) suggest the involvement of EP3-receptors as opposed to EP1- or EP2-subtypes. 2. Eight prostaglandin E (PGE) analogues were compared against sulprostone for their effects on PAF-induced aggregation in human platelet-rich plasma (PRP) in the presence of GR 32191 and the DP-receptor antagonist, BW A868C. PGE2 and 11-deoxy PGE2-1-alcohol showed evidence of both potentiating and inhibitory actions and butaprost showed only inhibitory activity at high concentrations. The remaining analogues always elicited potentiation, with the following potency ranking: sulprostone = 16,16-dimethyl PGE2 > MB 28767 > misoprostol > GR 63779X = 17-phenyl-omega-trinor PGE2. The results again indicate that EP3- rather than EP1- or EP2-receptors are involved. However, relative potentiating potency could be affected by differences in plasma protein binding and the very high sensitivity of the human platelet to prostacyclin (IP)-receptor-mediated inhibition (IC50 for the specific IP-receptor agonist cicaprost = 0.8 nM). 3. On human washed platelet suspensions the PGE analogues, with the exception of butaprost,inhibited the rise in adenosine 3':5'-cyclic monophosphate (cyclic AMP) induced by cicaprost (8 nM).PGE2 produced a monophasic inhibition curve (IC50 = 5.4 nM, 92% inhibition at 600 nM). The potency ranking was 16,16-dimethyl PGE2> sulprostone>MB 28767 = PGE2> misoprostol> GR 63778X>17-phenyl-w-trinor PGE2> 1 1-deoxy PGE2-1-alcohol. AH 6809 inhibited the effect of sulprostone and 17-phenyl-c-trinor PGE2 with pA2 values of 5.75 and 5.32 respectively; these values are at least one log unit lower than those found for EP1-receptor block in smooth muscle.4. There is a statistically significant correlation between IC50 values for the PGE analogues on the human platelet cyclic AMP assay and the guinea-pig vas deferens (standard EP3 preparation): slope =1.00, r = 0.80, P <0.05. However the correlation is far from ideal and GR 63779X in particular has a lower potency in the cyclic AMP assay. At this time we suggest th

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Dinoprostone; Drug Synergism; Humans; Platelet Activating Factor; Platelet Aggregation; Prostaglandin Antagonists; Prostaglandin Endoperoxides, Synthetic; Prostaglandins E, Synthetic; Xanthenes; Xanthones

Stimulation of DP, but not TP or FP, prostanoid receptors has previously been shown to reduce intraocular pressure (IOP) in rabbits. However the role of EP receptors (EP1, EP2, and EP3 subtypes) has not been studied extensively. Sulprostone, RS-61565, and RS-20216 have been studied for effects on rabbit IOP, and their prostanoid-receptor profiles characterized. The data suggest that the EP3, but not EP2, FP, or TP activity of these agonists correlated with the intraocular hypotensive effects. Moreover, RS-20216 lowered IOP at a dose of 5 micrograms for up to 12 hr after administration. In contrast to PGE1 and PGE2, which elicited both hyper- and hypotensive responses, sulprostone, RS-61565, and RS-20216 elicited only a hypotensive responses with no signs of ocular irritation. Thus stimulation of the EP3 receptor results in a lowering of IOP in rabbits. Compounds specific for this receptor subtype may act as novel therapeutic agents for the treatment of glaucoma.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Alprostadil; Analysis of Variance; Animals; Dinoprostone; Female; Guinea Pigs; Intraocular Pressure; Male; Molecular Structure; Muscle Contraction; Prostaglandin Endoperoxides, Synthetic; Prostaglandins E, Synthetic; Prostaglandins, Synthetic; Rabbits; Rats; Rats, Inbred Strains; Receptors, Prostaglandin; Receptors, Prostaglandin E; Receptors, Thromboxane; Tonometry, Ocular