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

Twenty four healthy men were treated with GR32191, a thromboxane receptor antagonist with a long duration of action, in a double blind placebo-controlled crossover study. Platelet aggregation in response to a thromboxane (TX) mimetic (U46619) was studied turbidometrically using platelet rich plasma (PRP) prepared 12 h after dosing (80 mg po) and 1.5 h after a second dose (40 mg po). To determine whether the long lasting inhibition caused by GR32191 is associated with persistent inhibitory activity in plasma (from residual drug or from an active metabolite), platelet poor plasma (PPP) from treated subjects was mixed with PRP from placebo treated controls. 12 h after dosing this caused 40-80% inhibition, consistent with the plasma concentration of GR32191. Inhibition of U46619 in PRP from GR32191 treated subjects mixed with PPP from controls was even greater (essentially 100%). We conclude that the prolonged activity of GR32191 is due in part to a reduction in available thromboxane receptors and in part to its persistence in plasma for longer than had previously been appreciated.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adolescent; Adult; Biphenyl Compounds; Blood Platelets; Double-Blind Method; Heptanoic Acids; Humans; Male; Platelet Aggregation; Platelet Aggregation Inhibitors; Prostaglandin Endoperoxides, Synthetic; Random Allocation; Receptors, Prostaglandin; Receptors, Thromboxane; Thromboxane A2; Thromboxane B2

1 The effect of single, serially increasing, intravenous doses of a specific thromboxane receptor blocking drug, vapiprost, upon platelet aggregation induced ex vivo by the thromboxane A2 mimetic, U-46619, was examined in 12 healthy males. 2 Subjects received either 1 (n = 1 subject), 2 (n = 6), 3 (n = 2), or 4 (n = 3) administrations of vapiprost within the dose range 0.125 to 16 mg and, in random order, placebo on separate study days at intervals of at least 48 h. 3 All doses of vapiprost produced an immediate antagonism of U-46619-induced platelet aggregation in whole blood. Both the magnitude and duration of the rightward displacement of the concentration-effect curves increased with dose. Although lower doses produced parallel displacements of these curves, with the higher doses the maximum response to U-46619 was reduced such that 50% platelet aggregation was not achieved. After the 16 mg dose of vapiprost, virtually complete suppression of platelet aggregation (up to a concentration of 30 microM) was seen. This degree of inhibition was maintained for 2 h after dosing, following which there was a gradual return to pre-dose U-46619 sensitivity over the next 12 to 24 h. U-46619-induced platelet aggregation was unaffected by placebo. 4 Across the dose range, vapiprost was rapidly cleared from plasma, with an elimination half-life of 69-84 min and a plasma clearance of 514-721 ml min-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adult; Biphenyl Compounds; Blood Pressure; Electrocardiography; Half-Life; Heptanoic Acids; Humans; Infusions, Intravenous; Male; Peak Expiratory Flow Rate; Platelet Aggregation; Platelet Aggregation Inhibitors; Prostaglandin Endoperoxides, Synthetic; Pulse; Receptors, Prostaglandin; Receptors, Thromboxane

The effects of GR32191, a novel, specific, thromboxane A2 receptor-blocking drug, on platelet aggregation induced by U-46619 and adenosine diphosphate (ADP) ex vivo, have been examined in healthy male subjects. In two placebo-controlled studies (the first, a single-dose, crossover study, and the second, a group comparative, multiple-dose study), concentration-effect curves for both agonists were constructed in whole blood by counting platelets electronically before and after dosing. Single oral doses of 0.125 and 0.25 mg/kg (four subjects) and 0.5 and 1.0 mg/kg (four subjects) produced dose-related shifts to the right in U-46619 concentration-effect curves. The elimination half-life of GR32191 (up to 8 hours postdrug) was approximately 2 hours. Multiple dosing with GR32191, 17.5 mg (equivalent to 0.25 mg/kg for a 70 kg subject), three times daily (three subjects) or every 12 hours (six subjects), resulted in a cumulative inhibitory effect on U-46619 platelet aggregation in the apparent absence of a build-up of plasma concentrations of GR32191. Primary platelet aggregation induced by ADP was unaffected by GR32191. Multiple dosing had no effect on lancet bleeding time, and no drug-related changes were seen in routine laboratory hematologic and biochemical screens. GR32191 was well tolerated, although a subject with a history of drug-induced rectal bleeding reported the same symptom while taking GR32191.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Adult; Biphenyl Compounds; Dose-Response Relationship, Drug; Heptanoic Acids; Humans; Male; Platelet Aggregation; Platelet Aggregation Inhibitors; Prostaglandin Endoperoxides, Synthetic; Receptors, Prostaglandin; Receptors, Thromboxane; Single-Blind Method; Thromboxane A2

1. GR32191B is a potent and selective thromboxane receptor antagonist. Glyceryl trinitrate (GTN) also inhibits platelet function in vitro. Both have been reported to prolong bleeding time. Since they may be used together in patients with coronary artery disease, we have investigated the possibility of an interaction between them. 2. Twenty-four healthy male volunteers were treated with GR32191B using a double-blind placebo-controlled crossover design. Bleeding times were determined before and after GR32191B or placebo and during co-administration of GTN. Plasma GR32191B concentration was measured. Platelet aggregation in response to adenosine diphosphate and to a thromboxane mimetic, U46619, was measured ex vivo. Urinary excretion of thromboxane (TX)B2 and 2,3-dinor-TXB2 was determined in 24 h urine samples. 3. Twelve hours after GR32191B (80 mg; p.o.), the plasma concentration was 36.6 +/- 2.7 nM (mean +/- s.e. mean) and bleeding time was increased by 66%. Ninety minutes after a second dose (40 mg; p.o.) the plasma concentration was 431.9 +/- 23.6 nM but bleeding time was not further prolonged. 4. Responses of platelets to U46619 were selectively antagonised following GR32191B. Urinary excretion rates of TXB2 and 2,3-dinor TXB2 were similar after treatment with GR32191B or placebo. 5. GTN (1 mg sub-lingually) had no significant effect on bleeding time 90-100 min following either placebo or GR32191B. 6. We conclude that GR32191B, in a dose that causes profound yet specific blockade of thromboxane receptors, causes only a modest prolongation of bleeding time that is unaffected by a therapeutic dose of GTN. This may prove advantageous when GR32191B is used in patients with ischaemic heart disease.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adolescent; Adult; Biphenyl Compounds; Bleeding Time; Blood Pressure; Chromatography, High Pressure Liquid; Double-Blind Method; Drug Interactions; Heart Rate; Heptanoic Acids; Humans; Male; Nitroglycerin; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Function Tests; Prostaglandin Endoperoxides, Synthetic; Receptors, Prostaglandin; Receptors, Thromboxane

1. Dilatation of the cerebral vasculature is recognised to be involved in the pathophysiology of migraine. Furthermore, elevated levels of prostaglandin E(2) (PGE(2)) occur in the blood, plasma and saliva of migraineurs during an attack, suggestive of a contributory role. In the present study, we have characterised the prostanoid receptors involved in the relaxation and contraction of human middle cerebral arteries in vitro. 2. In the presence of indomethacin (3 microm) and the TP receptor antagonist GR32191 (1 microM), PGE(2) was found to relax phenylephrine precontracted cerebral arterial rings in a concentration-dependent manner (mean pEC(50) 8.0+/-0.1, n=5). 3. Establishment of a rank order of potency using the EP(4)>EP(2) agonist 11-deoxy PGE(1), and the EP(2)>EP(4) agonist PGE(1)-OH (mean pEC(50) of 7.6+/-0.1 (n=6) and 6.4+/-0.1 (n=4), respectively), suggested the presence of functional EP(4) receptors. Furthermore, the selective EP(2) receptor agonist butaprost at concentrations <1 microM failed to relax the tissues. 4. Blockade of EP(4) receptors with the EP(4) receptor antagonists AH23848 and EP(4)A caused significant rightward displacements in PGE(2) concentration-response curves, exhibiting pA(2) and pK(B) values of 5.7+/-0.1, n=3, and 8.4, n=3, respectively. 5. The IP receptor agonists iloprost and cicaprost relaxed phenylephrine precontracted cerebral arterial rings (mean pEC(50) values 8.3+/-0.1 (n=4) and 8.1+/-0.1 (n=9), respectively). In contrast, the DP and FP receptor agonists PGD(2) and PGF(2 alpha) failed to cause appreciable relaxation or contraction at concentrations of up to 30 microm. In the absence of phenylephrine contraction and GR32191, the TP receptor agonist U46619 caused concentration-dependent contraction of cerebral artery (mean pEC(50) 7.4+/-0.3, n=3). 6. These data demonstrate the presence of prostanoid EP(4) receptors mediating PGE(2) vasodilatation of human middle cerebral artery. IP receptors mediating relaxation and TP receptors mediating contraction were also functionally demonstrated.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Aged; Aged, 80 and over; Biphenyl Compounds; Dinoprostone; Dose-Response Relationship, Drug; Female; Heptanoic Acids; Humans; Iloprost; In Vitro Techniques; Indomethacin; Male; Middle Aged; Middle Cerebral Artery; Muscle Contraction; Muscle, Smooth, Vascular; Phenylephrine; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP2 Subtype; Receptors, Prostaglandin E, EP4 Subtype; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

Several prostanoids were investigated for a potential to induce emesis in Suncus murinus. The TP receptor agonist 11alpha,9alpha-epoxymethano-15S-hydroxyprosta-5Z,13E-dienoic acid (U46619) induced emesis at doses as low as 3 microg/kg, i.p. but the DP receptor agonist 5-(6-Carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl) hydantoin (BW245C) was approximately 1000 times less potent. The emetic action of U46619 (300 microg/kg, i.p.) was antagonized significantly by the TP receptor antagonist, vapiprost (P<0.05). EP (prostaglandin E(2), 17-phenyl-omega-trinor prostaglandin E(2), misoprostol and sulprostone), FP (prostaglandin F(2alpha) and fluprostenol) and IP (iloprost and cicaprost) receptor agonists failed to induce consistent emesis at doses up to 300-1000 microg/kg, i.p. Fluprostenol reduced nicotine (5 mg/kg, s.c.)-but not copper sulphate (120 mg/kg, intragastric)-induced emesis; the other inconsistently emetic prostanoids were inactive to modify drug-induced emesis. The results indicate an involvement of TP and possibly DP and FP receptors in the emetic reflex of S. murinus.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Biphenyl Compounds; Copper Sulfate; Dose-Response Relationship, Drug; Female; Heptanoic Acids; Hydantoins; Injections, Intraperitoneal; Injections, Subcutaneous; Intubation, Gastrointestinal; Male; Nausea; Nicotine; Prostaglandins; Prostaglandins F, Synthetic; Reaction Time; Receptors, Thromboxane; Shrews; Time Factors; Vomiting

Arachidonic acid (AA) is a potent inducer of platelet aggregation in vitro; this activity is due to its conversion to biologically active metabolites, prostaglandin (PG) endoperoxides and thromboxane A2 (TxA2). PG endoperoxides and TxA, are thought to act on the same receptor; however, at least two isoforms of this receptor have been identified. The aim of our work was to clarify whether endoperoxides and TxA2 activate the same or different receptor subtypes to induce aggregation and calcium movements in human platelets. AA-induced aggregation and calcium rises were still detectable in platelets preincubated with thromboxane synthase inhibitors, which suppress TxA2 formation and induce PGH2 accumulation, suggesting that PG endoperoxides can activate platelets. Exogenously added PGH2 was able to induce aggregation and calcium rises. Pretreatment of platelets with GR32191B or platelet activating factor, which desensitize one of the two receptor subtypes identified in platelets, did not prevent calcium rises induced by endogenously generated or by exogenouly added PGH2, indicating that TxA2 and PG endoperoxides share the same receptor subtype(s) to activate platelets. HEK-293 cells overexpressing either of the two thromboxane receptor isoforms cloned to date (TPalpha and TPbeta) and identified in human platelets, stimulated with PGH2, or with the stable endoperoxide analog U46619, formed inositol phosphates. These data show that endoperoxides and TXA2 mediate their effects on platelets acting on both, and the same, receptor isoform(s).

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Aspirin; Biphenyl Compounds; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Calcium Signaling; Cells, Cultured; Enzyme Inhibitors; Fatty Acids, Unsaturated; Heptanoic Acids; Humans; Hydrazines; Imidazoles; Inositol Phosphates; Kidney; Methacrylates; Phenylacetates; Platelet Activating Factor; Platelet Activation; Prostaglandin H2; Prostaglandins H; Protein Isoforms; Receptors, Thromboxane; Recombinant Fusion Proteins; Sulfonamides; Thromboxane A2; Thromboxane B2; Thromboxane-A Synthase

This study was undertaken to investigate the vascular actions (contraction and relaxation) of the F(2)-isoprostane metabolites 15-keto-15-F(2t)-IsoP, 2,3-dinor-15-F(2t)-IsoP, and 2,3-dinor-5,6-dihydro -15-F(2t)-IsoP in comparison with 15-F(2t)-IsoP on the rat thoracic aorta. 15-keto-15-F(2t)-IsoP induced a vasoconstriction in a concentration-dependent manner with a pD(2) value of 5.80 +/- 0.05, whereas 2,3-dinor-15-F(2t)-IsoP and 2,3-dinor-5,6-dihydro-15-F(2t)-IsoP had no effect. The parent compound 15-F(2t)-IsoP was more potent (pD(2) value: 6.46 +/- 0.1). Endothelium removal had no influence on the contraction to 15-keto-15-F(2t)-IsoP. GR32191 (a TP-receptor antagonist) concentration-dependently inhibited the contraction induced by 15-keto-15-F(2t)-IsoP, with a significant decrease in the E(max) values for GR32191 10(-7) M. Pretreatment with 2,3-dinor-15-F(2t)-IsoP and 2,3-dinor-5,6-dihydro-15-F(2t)-IsoP induced no alteration of 15-F(2t)-IsoP concentration-response curves. In contrast, 15-keto-15-F(2t)-IsoP pretreatment competitively inhibited the response to 15-F(2t)-IsoP. When concentration ratios of EC(50) values were used, a Schild regression of this data was linear with a slope of 0.974 and a pA(2) value of 6.13. 15-keto-15-F(2t)-IsoP at high concentrations caused a weak concentration-dependent relaxation of rat aorta rings contracted with U46619 (3.10(-8) M) that was not modified in the absence of endothelium. In contrast, 2,3-dinor-15-F(2t)-IsoP and 2,3-dinor-5,6-dihydro-15-F(2t)-IsoP induced no vasodilation. In conclusion, among the F(2)-isoprostane metabolites, 2,3-dinor-15-F(2t)-IsoP and 2,3-dinor-5,6-dihydro-15-F(2t)-IsoP did not cause vasorelaxation or vasoconstriction on the rat thoracic aorta. In contrast, 15-keto-15-F(2t)-IsoP mediates contraction through activation of TP-receptors, probably as a partial agonist, and induces a weak endothelium-independent relaxation at high concentrations.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Aorta, Thoracic; Biphenyl Compounds; Dinoprost; Endothelium, Vascular; F2-Isoprostanes; Heptanoic Acids; In Vitro Techniques; Male; Muscle, Smooth, Vascular; Potassium Chloride; Prostaglandin Antagonists; Prostaglandins F, Synthetic; Rats; Rats, Wistar; Receptors, Prostaglandin; Receptors, Thromboxane; Vasoconstriction; Vasoconstrictor Agents; Vasodilation

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

Isoprostaglandin F(2alpha) type-III (formerly known as 8-iso-prostaglandin F(2alpha)) is produced in large quantities in vivo in clinical situations associated with oxidant stress such as atherosclerosis, hypercholesterolemia, and myocardial reperfusion. Isoprostaglandin F(2alpha) type-III may alter smooth muscle and platelet functions. The aim of this study was to evaluate the effects of isoprostaglandin F(2alpha) type-III on isolated human internal mammary arteries, and to characterise the signalling underlying mechanisms. In organ baths, concentration-dependent contractions of human internal mammary arteries were obtained in response to isoprostaglandin F(2alpha) type-III stimulation. The responses to isoprostaglandin F(2alpha) type-III were inhibited in a concentration-dependent manner by the thromboxane A(2) receptor antagonist, GR 32191 ([1R-[1 alpha(Z), 2beta,3beta,5 alpha(+)-7-[[1, 1'-biphenyl)-4-yl]methoxy]-3-hydroxy-2-(1-piperidinyl) cyclo pentyl]-4-4heptanoic acid], hydrochloride), 3x10(-9) to 3x10(-7) M). However, this effect was associated with a decreased maximal contraction. AH 6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid, 10(-6) to 3x10(-5) M), an EP(1)-DP receptor antagonist had no effect on isoprostaglandin F(2alpha) type-III-induced contractions. The maximal responses to isoprostaglandin F(2alpha) type-III were significantly reduced in the presence of the cyclooxygenase inhibitor indomethacin (10(-5) M) (E(max): 147+/-20% vs. 213+/-19% in control group, P<0.05). Isoprostaglandin F(2alpha) type-III stimulated thromboxane B(2) release (5.7-fold increase) from human internal mammary arteries. Baicaleine, a non-specific lipoxygenase inhibitor, (10(-4) M) and AA 861 (2,3,5-trimethyl-6-(12-hydroxy-5, 10-dodecadiynyl)-1,4 benzoquinone), a 5-lipoxygenase inhibitor (10(-5) M) did not affect isoprostaglandin F(2alpha) type-III response. In conclusion, this study shows that (1) isoprostaglandin F(2alpha) type-III is a vasoconstrictor in human internal mammary arteries, with a potency equivalent to prostaglandin F(2alpha), (2) the contractions induced by isoprostaglandin F(2alpha) type-III are mediated by TP receptor but not EP(1)-DP-receptor activation, (3) thromboxane A(2) but not cysteinyl leukotrienes production is involved in the vascular effects of isoprostaglandin F(2alpha) type-III. Isoprostaglandin F(2alpha) type-III, produced at sites of free radical generation, may play an important role in internal mammary artery spasm in situatio

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adult; Aged; Benzoquinones; Biphenyl Compounds; Dinoprost; Dose-Response Relationship, Drug; F2-Isoprostanes; Female; Flavanones; Flavonoids; Heptanoic Acids; Humans; In Vitro Techniques; Leukotrienes; Lipoxygenase Inhibitors; Male; Mammary Arteries; Middle Aged; Prostaglandin Antagonists; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP1 Subtype; Receptors, Thromboxane; Thromboxane B2; Vasoconstriction; Vasoconstrictor Agents; Xanthenes; Xanthones

The newly revived coronary bypass graft, the radial artery (RA), is more spastic than the internal mammary artery. Thromboxane A2 is a potent vasoconstrictor for arterial grafts. This study was therefore designed to determine whether the specific thromboxane A2 (TP) receptor antagonist, GR32191B, is effective in inhibition of prostanoid or nonprostanoid receptors in the RA.. The effect of GR32191B was studied in human RA segments, taken from coronary bypass patients, in organ chambers. Two effects of GR32191B were tested: (1) the relaxation induced by GR32191B in the RA precontracted with the TP receptor agonists U46619 and PGF2alpha or nonprostanoid vasoconstrictors (noradrenaline [NA], angiotensin II [AII], and K+ ) and (2) the inhibitory effect of GR32191B on TP receptor agonists and nonprostanoid vasoconstrictors.. In U46619 (10 nm, n=7) and PGF2alpha (1 microm, n=7) precontracted RA, GR32191B induced 100% relaxation (10-100 microm ) but not after precontraction with nonprostanoid stimuli (5.8% for K+, 25 mm, n=6, 24.4% for NA, 3 microm, n=8, and 53.2% for AII, 3 nm, n=5) (P<0.001). Treatment with GR32191B (30 nm ) significantly depressed the contraction with U46619 (from 160.1+/-11.0% to 116.8+/-13.1%, P<0. 05) or PGF2alpha (from 91.3+/-12.3% to 42.2+/-9.2%, P<0.01). The contraction was further abolished by 3 microm GR32191B. However, GR32191B at 3 microm did not significantly inhibit the contraction induced by either NA, AII, or K+.. GR32191B is a highly potent and specific TP receptor antagonist for the human RA. It may be particularly useful in inhibiting TXA2-mediated vasoconstriction and therefore in reducing the complications related to vasospasm in this graft.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Angiotensin II; Biphenyl Compounds; Coronary Artery Bypass; Dinoprost; Heptanoic Acids; Humans; In Vitro Techniques; Muscle, Smooth, Vascular; Norepinephrine; Potassium; Prostaglandin Antagonists; Radial Artery; Thromboxane A2; Vasoconstrictor Agents

Interleukin-1beta has been reported to induce airway hyperresponsiveness in several animal models. In this study, we have investigated whether interleukin-1beta was able to potentiate the contractions of human isolated small bronchi (internal diameter < or = 1 mm) provoked by a specific tachykinin NK1 receptor agonist, [Sar9,Met(O2)11]substance P. Pre-incubation of human isolated small bronchi with interleukin-1beta (10 ng/ml, in Krebs-Henseleit solution, at 21 degrees C for 15 h) potentiated the contractile response to [Sar9,Met(O2)11]substance P. It also increased the [Sar9,Met(O2)11]substance P-induced release of thromboxane B2, the stable metabolite of thromboxane A2. Indomethacin (10(-6) M), a non-specific cyclooxygenase inhibitor, or GR 32191 ((1R-(1alpha(Z)),2beta,3beta,5alpha))-(+)-7-(5-(((1,1' -biphenyl)-4-yl)-methoxy)-3-hydroxy-2-(1-piperidinyl)cyclopentyl)-4-hept enoic acid, hydrochloride) (10(-6) M), a prostanoid TP-receptor antagonist, blocked the contractions induced by [Sar9,Met(O2)11]substance P both in control experiments and after interleukin-1beta pre-treatment, indicating that prostanoids and thromboxane receptors are directly implicated in the [Sar9,Met(O2)11]substance P-induced contractile response. The thromboxane mimetic U-46619 (10(-8)-10(-6) M) (9,11-dideoxy-11alpha,9alpha-epoxymethano-prostaglandin F2alpha)-induced contractions of human isolated small bronchi were not enhanced by interleukin-1beta pre-treatment, suggesting that no up-regulation of thromboxane receptors occurred. Furthermore, the cyclooxygenase-2 inhibitor CGP 28238 (6-(2,4-difluorophenoxy)-5-methyl-sulfonylamino-1-indanon e) (10(-6) M) had no direct effect on [Sar9,Met(O2)11]substance P-provoked contractions, but inhibited the interleukin-1beta-induced potentiation of [Sar9,Met(O2)11]substance P response. In conclusion, our results show that interleukin-1beta pre-treatment is able to potentiate the contractions of isolated human small bronchi provoked by [Sar9,Met(O2)11]substance P both by increasing prostanoid synthesis and by inducing a cyclooxygenase-2 pathway.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Aged; Anti-Inflammatory Agents, Non-Steroidal; Biphenyl Compounds; Bronchi; Cyclooxygenase Inhibitors; Dose-Response Relationship, Drug; Drug Hypersensitivity; Female; Heptanoic Acids; Humans; In Vitro Techniques; Indans; Indomethacin; Interleukin-1; Male; Muscle Contraction; Muscle, Smooth; Prostaglandin Antagonists; Receptors, Tachykinin; Substance P; Thromboxane B2; Time Factors; Vasoconstrictor Agents

This study compares the effects of threshold concentrations of endothelin-1 in isolated rat basilar arteries with those in mesenteric arterial branches and investigates the mechanisms of inhibitory and potentiating endothelin-1-effects. In basilar arteries, endothelin-1 reduces the contractions induced by 5-hydroxytryptamine (5-HT), by the thromboxane A2 agonist U46619, and by vasopressin. The inhibitory effect of endothelin-1 on the contraction induced by 5-HT is abolished by deendothelialization, by the endothelin ET(B) receptor antagonist RES 701-1, by indomethacin, or by glibenclamide. In mesenteric arteries, endothelin-1 potentiates the contractile effects of 5-HT, U46619, and vasopressin. The potentiation of the contractile effect induced by 5-HT is only somewhat modified by deendothelialization, but abolished by the thromboxane A2 receptor antagonists GR32191 and ridogrel. U46619 potentiates the 5-HT-effect in mesenteric arteries. Thus, though the contractile endothelin ET(A) receptors were not blocked, threshold concentrations of endothelin-1 inhibited contractile effects in the rat basilar artery via activation of endothelial ET(B) receptors. Prostaglandins and ATP-sensitive K+ channels are involved in this inhibitory action. In contrast, endothelin-1 potentiates contractile actions in mesenteric arteries via the release of endogeneous thromboxane A2 from non-endothelial cells. The study points out the completely different role of the endothelium in combined effects of endothelin-1 between cerebral and mesenteric arteries.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Basilar Artery; Biphenyl Compounds; Drug Synergism; Endothelin Receptor Antagonists; Endothelin-1; Glyburide; Heptanoic Acids; Indomethacin; Male; Mesenteric Arteries; Muscle Contraction; Muscle, Smooth, Vascular; Pentanoic Acids; Peptides, Cyclic; Pyridines; Rats; Rats, Sprague-Dawley; Serotonin; Vasoconstriction; Vasopressins

1. 5-HT and the prostanoid TP receptor agonists, U46619 and I-BOP, constricted the human umbilical artery with pEC50 values of 7.3+/-0. 2, 6.7+/-0.1, and 7.3+/-0.2, respectively. The selective TP receptor antagonist, GR32191 (0.1 microM), shifted the concentration-effect curves to U46619 and I-BOP to the right, but had no effect on the response to 5-HT. 2. The natural prostaglandins, PGF2alpha and PGE2, caused concentration-dependent contraction with pEC50 values of 5.2+/-0.2 and 4.9+/-0.2, respectively. PGD2 was a partial agonist with a pEC50 of 5.24+/-0.03. GR32191 (0.1 microM) inhibited the responses to all of these compounds suggesting that they produce contraction by acting at TP receptors. 3. Sulprostone failed to elicit contraction in the human umbilical artery at concentrations up to 4.4 microM suggesting the absence of EP1 and EP3 receptors. Despite this, 17-phenyltrinor PGE2 and GR63799 both induced contraction at concentrations above 1 microM, but the effects were sensitive to GR32191 (0.1 microM). 4. Fluprostenol had no effect on the human umbilical artery at concentrations up to 17 microM suggesting the absence of FP receptors. Cloprostenol was ineffective in two tissues, but caused contraction in one tissue at the highest concentration tested (1.7 microM). However, this response was abolished in the presence of GR32191 (0.1 microM). 5. The effects of four TP receptor antagonists were assessed by global non-linear regression analysis. GR32191, SQ29548, SQ30741, and ICI192605 competitively inhibited responses to U46619 with pKb values of 8.0+/-0.1, 7.6+/-0.1, 7.0+/-0. 2 and 8.1+/-0.1, respectively. 6 These results suggest that the human umbilical artery functionally expresses TP receptors, but not EP1, EP2 or FP receptors.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Dinoprost; Dinoprostone; Fatty Acids, Unsaturated; Female; Heptanoic Acids; Humans; In Vitro Techniques; Isometric Contraction; Pregnancy; Prostaglandin Antagonists; Receptors, Prostaglandin; Serotonin; Umbilical Arteries; Vasoconstriction; Vasoconstrictor Agents

Glibenclamide, like other hypoglycemic sulfonylurea derivatives, is a potent blocker of ATP-regulated K+ channels. In addition, it is reported to inhibit prostanoid-induced contractions of isolated vascular smooth muscle from different animal species. We investigated the effect of glibenclamide on the thromboxane A2-mimetic U-46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy-prostaglandin F2alpha)-induced contractions in human isolated internal mammary arteries and saphenous veins. In the two vascular preparations, glibenclamide (3, 10 and 30 microM) caused a concentration-dependent shift to the right of the U-46619 contraction-response curve with a reduction, at the highest concentrations, in the maximal responses. This inhibitory effect appears selective for thromboxane A2-induced contractions since glibenclamide (30 microM) did not alter the contraction of internal mammary arteries in response to norepinephrine and of saphenous veins in response to 5-hydroxytryptamine (5-HT) and endothelin-1. However, glibenclamide reduced the endothelin-1-induced contraction in internal mammary arteries. The endothelin-1-induced contractions were similarly inhibited by GR 32191 ([1R-[1alpha(Z),2beta,3beta,5alpha]]-(+)-7-[5-([1,1'-b iphenyl]-4-ylmethoxy)-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-++ +heptonoic acid, a thromboxane A2 receptor antagonist. These results suggest that glibenclamide also reduced the endothelin-1-induced contractions by inhibiting a thromboxane A2 receptor-mediated component of the contraction elicited by this peptide. In conclusion, glibenclamide clearly appears to exert a specific inhibitory influence on prostanoid-induced contractions in human internal mammary arteries and saphenous veins.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Biphenyl Compounds; Dose-Response Relationship, Drug; Endothelin-1; Glyburide; Heptanoic Acids; Humans; Hypoglycemic Agents; Mammary Arteries; Muscle Contraction; Muscle, Smooth, Vascular; Norepinephrine; Prostaglandin Antagonists; Receptors, Thromboxane; Saphenous Vein; Serotonin; Thromboxane A2; Vasoconstrictor Agents

1. Cumulative concentration-response curves (CRC) to prostaglandin E1 (PGE1), PGE2, PGD2 and PGF2alpha (0.01-30 microM) and to the thromboxane A2 (TXA2) receptor agonist U-46619 (0.01-30 microM) were constructed in human isolated detrusor muscle strips both in basal conditions and during electrical field stimulation. 2. All the agonists tested contracted the detrusor muscle. The rank order of agonist potency was: PGF2alpha > U-46619 > PGE2 whereas weak contractile responses were obtained with PGD2 and PGE1. Any of the agonists tested was able to induce a clear plateau of response even at 30 microM. 3. The selective TXA2 antagonist, GR 32191B (vapiprost), antagonized U-46619-induced contractions with an apparent pK(B) value of 8.27+/-0.12 (n = 4 for each antagonist concentration). GR 32191B (0.3 microM) did not antagonize the contractile responses to PGF2alpha and it was a non-surmountable antagonist of PGE2 (apparent pK(B) of 7.09+/-0.04; n = 5). The EP receptor antagonist AH 6809 at 10 microM shifted to the right the CRC to U-46619 (apparent pK(B) value of 5.88+/-0.04; n = 4). 4. Electrical field stimulation (20 Hz, 70 V, pulse width 0.1 ms, trains of 5 s every 60 s) elicited contractions fully sensitive to TTX (0.3 microM) and atropine (1 microM). U-46619 (0.01-3 microM) potentiated the twitch contraction in a dose-dependent manner and this effect was competitively antagonized by GR 32191B with an estimated pK(B) of 8.54+/-0.14 (n = 4 for each antagonist concentration). PGF2alpha in the range 0.01-10 microM (n = 7), but not PGE2 and PGE1 (n = 3 for each), also potentiated the twitch contraction of detrusor muscle strips (23.5+/-0.3% of KCl 100 mM-induced contraction) but this potentiation was unaffected by 0.3 microM GR 32191B (n = 5). 5. Cumulative additions of U-46619 (0.01-30 microM) were without effect on contractions induced by direct smooth muscle excitation (20 Hz, 40 V, 6 ms pulse width, trains of 2 s every 60 s, in the presence of TTX 1 microM; n = 3). Moreover, pretreatment of the tissue with 0.3 microM U-46619 did not potentiate the smooth muscle response to 7 microM bethanecol (n = 2). 6. We concluded that TXA2 can induce direct contraction of human isolated urinary bladder through the classical TXA2 receptor. Prostanoid receptors, fully activated by PGE2 and PGF2alpha are also present. All these receptors are probably located post-junctionally. The rank order of agonist potency and the fact that GR 32191B, but not AH6809, antagonized respon

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Alprostadil; Biphenyl Compounds; Dinoprost; Dinoprostone; Electric Stimulation; Heptanoic Acids; Humans; In Vitro Techniques; Muscle Contraction; Muscle, Smooth; Prostaglandin Antagonists; Prostaglandin D2; Receptors, Prostaglandin; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP1 Subtype; Receptors, Prostaglandin E, EP2 Subtype; Receptors, Thromboxane; Thromboxane A2; Urinary Bladder; Xanthenes; Xanthones

F2-isoprostanes are prostaglandin (PG) isomers produced in vivo through free radical-catalyzed peroxidation of arachidonic acid, which may affect platelet function. The current study investigated the effects of 8-epiprostaglandin F2alpha (8-epi-PGF2alpha) on critical events of platelet activation. A dose-dependent increase in platelet adhesion to fibrinogen- and plasma-coated microwells by 8-epi-PGF2alpha (1 to 1000 nmol/L) was observed when resting platelets (plasma from 1.3+/-0.2% to 5.5+/-0.2%, EC50 of 48 nmol/L; fibrinogen from 3.3+/-0.3% to 6.4+/-0.2%, EC50 of 35 nmol/L; mean+/-SEM, n=8, P<0.001) and thrombin-stimulated human platelets were used. The expression of the adhesion molecule glycoprotein IIb/IIIa was increased by 10 to 1000 nmol/L 8-epi-PGF2alpha in resting platelets (from 64.8+/-2.1% to 83.9+/-1.3%; n=5, P<0.01) and in stimulated platelets. The secretion of the glycoprotein GMP-140 increased only in the presence of both thrombin and 10 to 1000 nmol/L 8-epi-PGF2alpha (from 48.5+/-3.1% to 63.1+/-2.0%, P<0.05). The antiaggregatory effects of both the NO donor NOR-3 (basal, 21.4+/-4.6%; with 8-epi-PGF2alpha, 30.8+/-6.9%; n=14, P<0.05) and endothelial cells that release NO (basal, 18.5+/-4.6%; with 8-epi-PGF2alpha, 30.7+/-5.3%; n=15, P<0.001) were also reduced. All of these effects were prevented by the thromboxane receptor antagonist GR32191 but not affected by acetylsalicylic acid. An increase in free intracellular calcium concentration, measured with the use of fura 2, was observed with 8-epi-PGF2alpha. In conclusion, F2-isoprostanes may participate in oxidative injury by inducing platelet activation and by reducing the antiplatelet activity of NO: increased platelet adhesiveness and expression of the fibrinogen receptor are induced by nanomolar amounts of 8-epi-PG-F2alpha. Platelet secretion and aggregation can also be induced in the presence of platelet agonists.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adult; Biphenyl Compounds; Blood Platelets; Calcium; Cells, Cultured; Dinoprost; Dose-Response Relationship, Drug; Drug Interactions; Endothelium, Vascular; F2-Isoprostanes; Heptanoic Acids; Humans; Nitric Oxide; P-Selectin; Platelet Adhesiveness; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIIb-IIIa Complex; Receptors, Thromboxane; Vasoconstrictor Agents

The ability of the thromboxane A2 receptor antagonist, GR32191 ([1R-[1alpha(Z),2beta3beta,5alpha]]-7-[5-[[(1,1'-biphe nyl)-4-yl]methoxy]-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-heptenoic acid), and the sulphonylurea, glibenclamide, to antagonise contractions to the thromboxane A2 mimetic, U46619 ((15S)-hydroxy-11alpha,9alpha(epoxymethano)prosta-5Z, 13E-dienoic acid), were assessed in rat and guinea-pig isolated large (aorta) and small (mesentery and coronary) arteries. U46619 concentration-response curves were constructed in the absence and presence of GR32191 and glibenclamide and pKB values calculated. GR32191 caused significant rightward shifts in U46619 concentration-response curves and was a more potent antagonist in guinea-pig vessels (pKB approximately 9.4) than rat arteries (pKB approximately 7.9). Conversely, glibenclamide failed to inhibit contractions to U46619 in guinea-pig vessels but antagonised responses to U46619 in rat aorta (pKB = 6.1) and mesenteric artery (pKB = 6.3). In combination, GR32191 and glibenclamide caused a shift in the concentration-effect curve to U46619 in rat aorta that was additive. These results suggest that glibenclamide can discriminate between species differences in thromboxane A2 receptors and may exert its inhibitory effect upon U46619-mediated contractions at the level of the thromboxane A2 receptor.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Aorta; Biphenyl Compounds; Dose-Response Relationship, Drug; Drug Interactions; Glyburide; Guinea Pigs; Heptanoic Acids; In Vitro Techniques; Male; Rats; Rats, Inbred WKY; Receptors, Thromboxane; Species Specificity; Thoracic Arteries; Vasoconstriction; Vasoconstrictor Agents

We investigated the pharmacological characteristics of Z-335 ((+/-)-sodium[2-[4-(chlorophenylsulfonylaminomethyl)indan-5-yl]ace tate monohydrate), a new indan derivative. Z-335 inhibited the specific binding of [3H]SQ-29548 to human platelets and guinea pig platelet membranes. The IC50 values of Z-335 for human platelets and guinea pig platelet membranes were 29.9 +/- 3.1 nM with a slope of 1.09 +/- 0.05 and 32.5 +/- 1.7 nM with a slope of 1.07 +/- 0.02, respectively. Z-335 inhibited thromboxane A2 receptor-mediated human and guinea pig platelet aggregation in vitro and oral administration of this drug to guinea pigs inhibited U-46619- and collagen-induced platelet aggregation for 24 h. Z-335 dose-dependently prevented the occurrence of U-46619-induced pulmonary thromboembolism in mice and the protective effect of this drug (0.3 and 3 mg/kg, p.o.) lasted for 24 h. These results strongly suggest that Z-335 is a potent, orally active and long-lasting thromboxane A2 receptor antagonist, which may be useful as an antiplatelet drug.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Biphenyl Compounds; Cilostazol; Drug Interactions; Fibrinolytic Agents; Guinea Pigs; Heptanoic Acids; Humans; Indans; Male; Mice; Mice, Inbred ICR; Phenylacetates; Platelet Aggregation; Platelet Aggregation Inhibitors; Pulmonary Embolism; Receptors, Thromboxane; Sulfonamides; Tetrazoles; Vasoconstrictor Agents

Lipopolysaccharide (LPS) and interleukin (IL)-1beta have been reported to induce airway hyperresponsiveness in several animal models. This study investigated the effect of LPS or IL-1beta on bradykinin-induced human isolated bronchi contraction. LPS (100 ng x mL(-1) for 3-6 h) and IL-1beta (3x10(-10) and 3x10(-9) M for 20 min to 3 h) time-dependently potentiated bradykinin-induced contraction. This contraction was abolished, as in control experiments, by indomethacin (10(-6) M) or by the thromboxane (Tx) receptor antagonist GR 32191 but not by the cyclo-oxygenase-2 inhibitor, CGP28238. In contrast, the Tx mimetic U46619-induced contraction of human bronchi was not enhanced IL-1beta pretreatment. In the presence of GR 32191 (10(-6) M), bradykinin induced a prostanoid dependent relaxation that was not significantly modified by IL-1beta pretreatment. Determination of prostanoids in the organ bath fluid showed that bradykinin induced TxB2, the stable metabolite of TxA2, and 6-keto prostaglandin F1alpha, the stable metabolite of PGI2, release. Only TxA2 release was potentiated by IL-1beta. Taken together our results suggest that interleukin-1beta (1-3 h)-induced potentiation of the effect of bradykinin is linked to an increased activity of thromboxane synthase and, in turn, to increased thromboxane synthesis.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 6-Ketoprostaglandin F1 alpha; Aged; Biphenyl Compounds; Bradykinin; Bronchi; Bronchial Hyperreactivity; Bronchoconstriction; Cyclooxygenase Inhibitors; Dose-Response Relationship, Drug; Heptanoic Acids; Humans; In Vitro Techniques; Indomethacin; Interleukin-1; Lipopolysaccharides; Middle Aged; Receptors, Thromboxane; Thromboxanes

1. In isolated ring preparations of the rabbit saphenous vein, prostaglandin E2 (PGE2) caused well-defined, stable and concentration-dependent relaxations of KCl contracted tissues with a mean potency (p[A50]) of 9.39. 2. The prostanoid EP-receptor agonists, PGE1, 11-deoxy PGE1, 16,16-dimethyl PGE2 and misoprostol were all full agonists in this preparation. The EP2-receptor selective agonists, butaprost and AH13205, and the EP1/EP3-receptor selective agonist, sulprostone, also relaxed this tissue but were at least 300 times less potent than PGE2. 3. Prostaglandin D2 (PGD2), the DP-receptor agonist, BW245C, and the IP-receptor agonist, cicaprost, caused concentration-dependent relaxations of the rabbit saphenous vein but were at least 60 times less potent than PGE2. 4. The selective EP4-receptor antagonist, AH23848B (30 microM), antagonized the PGE2 concentration-effect (E/[A]) curves yielding a pA2 estimate of 4.96. The EP1/DP-receptor antagonist, AH6809 (10 microM), had no effect on the location of PGE2 E/[A] curves. 5. The stable thromboxane A2-mimetic, U46619, elicited concentration-dependent contractions of the rabbit saphenous vein (p[A50] = 8.01) however, PGE2 and prostaglandin F2 alpha (PGF2 alpha) were unable to produce a contractile response. The response to U46619 was competitively antagonized by the TP-receptor antagonist, GR32191B, yielding a pKB estimate of 7.08. 6. In the rabbit isolated ear artery, PGE2, misoprostol and AH13205 relaxed tissues pre-contracted with phenylephrine. PGE2 (p[A50] = 7.04) and misoprostol were equipotent, whereas AH13205 was some 40 fold less potent. AH23848B (30 microM) and AH6809 (1 and 10 microM) caused no significant shift in the location of PGE2 E/[A] curves. 7. These data suggest that the rabbit isolated saphenous vein contains prostanoid, EP-, DP-, IP- and TP-receptors. Based on antagonist affinity information and agonist potency orders, the rabbit saphenous vein contains an inhibitory prostanoid EP-receptor different from that in the rabbit ear artery, but comparable to the recently described EP4-receptor.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arteries; Biphenyl Compounds; Dinoprostone; Dose-Response Relationship, Drug; Ear; Heptanoic Acids; In Vitro Techniques; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Phenylephrine; Prostaglandin Endoperoxides, Synthetic; Prostaglandins E; Rabbits; Receptors, Prostaglandin E; Saphenous Vein; Thromboxane A2; Vasoconstrictor Agents

[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 previously demonstrated that the bradykinin-induced contraction of human isolated small bronchi is inhibited by indomethacin, capsaicin (N-methyl-N-6-nonenamide) and ruthenium red but not by tachykinin receptor antagonists. The thromboxane A2 receptor (TP receptor) antagonist GR32191 ((1R-(1 alpha(Z),2 beta,3 beta,5 alpha))-(+)-7-(5-(((1,1'-biphenyl)-4-yl)- methoxy)-3-hydroxy-2-(1-piperidinyl)cyclopentyl)-4-heptenoic acid, hydrochloride) (10(-10) to 10(-8) M) dose dependently inhibited the effect of bradykinin, suggesting the mediation of the TP receptor in the action of bradykinin. With higher concentrations of GR32191 (10(-7) and 10(-6) M) bradykinin induced a relaxation which was inhibited by indomethacin and by the bradykinin B2 receptor antagonist Hoe 140 (D-Arg0[Hyp3,Thi-5,D-Tic7,Oic8]bradykinin). The thromboxane A2 synthase inhibitor dazoxiben (4-(-2-(1H-imidazol-1-yl)ethoxy) benzoic acid hydrochloride) 10(-6) M inhibited the bradykinin-induced contraction, suggesting that thromboxane A2 was involved in TP receptor stimulation. The thromboxane A2 mimetic U-46619 (9,11-dideoxy-11 alpha,9 alpha-epoxy-methano-prostaglandin F2 alpha)-induced contraction of human distal bronchi was not inhibited by capsaicin and ruthenium red. Our data suggest that bradykinin contracts human isolated small bronchi through thromboxane A2 release. The inhibitory effect of ruthenium red and capsaicin on the bradykinin response may be due to inhibition of thromboxane A2 release or arachidonic mobilisation.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adult; Aged; Biphenyl Compounds; Bradykinin; Bronchi; Capsaicin; Dose-Response Relationship, Drug; Heptanoic Acids; Humans; Imidazoles; Indomethacin; Male; Middle Aged; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Prostaglandin Endoperoxides, Synthetic; Receptors, Thromboxane; Ruthenium Red; Thromboxane A2; Thromboxane-A Synthase; Vasoconstrictor Agents

Arterial grafts have been demonstrated to be very effective for coronary artery bypass surgery. Thromboxane A2 (TXA2) is a potent vasoconstrictor for arterial grafts. To determine whether the specific TXA2 (TP) receptor antagonist GR32191B is effective in inhibition of prostanoid or nonprostanoid receptors, we studied the effect of GR32191B in human internal mammary artery (IMA) segments, taken from coronary bypass patients, in organ chambers. In IMA precontracted with U46619 (10 nM, n = 7), prostaglandin F2 alpha (PGF2 alpha 1 microM, n = 7), or potassium chloride (K+ 25 microM, n = 6). GR32191B induced 100.0 +/- 0, 97.86 +/- 2.14, or 45.87 +/- 7.63% relaxation. In other experiments, one IMA ring taken from each patient was used as a control and others from the same patient were allocated to other groups treated with different concentrations of GR32191B [3-300 nM for U46619, 30 nM-3 microM for PGF2 alpha, 300 nM-100 microM for K+, 3 microM norepinephrine (NE), and 3 microM for 5-hydroxytryptamine (5-HT)] for 1 h before concentration-contraction curves to these vasoconstrictors (expressed as percentage of K(+)-induced contraction force) were established. Treatment with GR32191B (300 nM) significantly decreased the contraction induced by U46619 (from 306.4 +/- 22.1 to 61.9 +/- 24.9%, p < 0.01) and that induced by PGF2 alpha (from 208.2 +/- 13.5 to 1.4 +/- 1.4%, p < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Biphenyl Compounds; Coronary Artery Bypass; Dinoprost; Dose-Response Relationship, Drug; Female; Heptanoic Acids; Humans; In Vitro Techniques; Male; Mammary Arteries; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Norepinephrine; Potassium Chloride; Prostaglandin Antagonists; Prostaglandin Endoperoxides, Synthetic; Receptors, Prostaglandin; Receptors, Thromboxane; Serotonin; Serotonin Receptor Agonists; Thromboxane A2; Vasoconstrictor Agents

1. This study was undertaken to characterize pharmacologically the prostanoid receptor subtypes mediating constriction and relaxation of human isolated uterine artery. 2. U-46619 was a potent constrictor agonist on human uterine artery (EC50 [95% CL] = 3.5 [1.8-6.7] nM). Prostaglandin E2 (PGE2), PGF2 alpha, PGD2 and PGI2 only weakly constricted the uterine artery, being at least 100 times less potent than U-46619. The PGE2 and PGI2 constrictor effects may be modified by the potent dilator effects of these compounds. A number of agonists which show selectivity for FP-, DP- and EP-receptors including ICI 81008, BW 245C, sulprostone, rioprostil and butaprost, failed to cause any constriction at concentrations up to 30 microM. 3. Constrictor responses induced by all agonists tested were reduced or abolished by the TP-receptor blocking drugs, GR 32191 and EP 092. pA2 estimates for both antagonists versus U-46619 were 8.50, values which are consistent with their affinities at TP-receptors. 4. In preparations pre-constricted with phenylephrine (1 microM) both PGI2 and PGE2 were potent relaxant agonists. The selective IP-receptor agonists, cicaprost and iloprost, also dilated human uterine artery and were approximately 10 fold more potent than PGI2. The EP2-receptor agonists, butaprost and rioprostil and the selective DP-agonist, BW 245C, were at least 100 fold weaker than PGI2 and PGE2 suggesting that neither DP- nor EP2 receptors were involved. 5. We conclude that TP-receptors mediate constriction, whereas IP- and possibly EP4-receptors mediate relaxation of human uterine artery.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Arteries; Biphenyl Compounds; Epoprostenol; Female; Heptanoic Acids; Humans; In Vitro Techniques; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Prostaglandin Antagonists; Prostaglandin Endoperoxides, Synthetic; Receptors, Prostaglandin; Thromboxane A2; Uterus; Vasoconstrictor Agents

The powerful vasoconstrictor autacoid thromboxane A2 (TxA2) has pathological roles in many diseases including pre-eclampsia or pregnancy induced hypertension (PIH). Adenosine and other purines are released by tissues during ischemia as occurs in the utero-placental circulation during PIH. These substances, particularly adenosine, may modulate TxA2 constrictor responses. We therefore characterized TxA2 receptors in the umbilical artery in vitro using the competitive antagonist GR32191. Also examined was the Ca2+ channels' involvement in adenosine-induced inhibition of TxA2 vasoconstriction. Results showed that TxA2 receptors on umbilical arteries are identical to those present in platelets, the placenta and umbilical vein. Adenosine was found to inhibit equally constriction involving either voltage or receptor operated Ca2+ channels.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine; Adolescent; Adult; Biphenyl Compounds; Calcium Channels; Drug Interactions; Female; Heptanoic Acids; Humans; Muscle, Smooth, Vascular; Nifedipine; Prostaglandin Endoperoxides, Synthetic; Receptors, Thromboxane; Thromboxane A2; Umbilical Arteries; Vasoconstriction; Vasoconstrictor Agents

1. Cumulative administrations of U46619, a thromboxane A2 analogue, and prostaglandin (PG) F2 alpha produced concentration-dependent contractions of isolated dog renal arterial preparations, which were significantly and concentration-dependently inhibited by vapiprost. 2. A bolus administration of U46619 or PGF2 alpha produced sustained contracture of these preparations, which was concentration-dependently relaxed by cumulative vapiprost. 3. Results indicate that vapiprost inhibits U46619- and PGF2 alpha-induced dog renal arterial contractions through antagonism for so-called TP receptors.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Biphenyl Compounds; Dinoprost; Dogs; Dose-Response Relationship, Drug; Female; Heptanoic Acids; In Vitro Techniques; Male; Prostaglandin Endoperoxides, Synthetic; Receptors, Thromboxane; Renal Artery; Thromboxane A2; Vasoconstriction; Vasoconstrictor Agents

Two TxA2/PGH2 receptor binding sites linked to different effector systems have recently been identified. Since plasmenylethanolamine represents the major phospholipid reservoir of arachidonic acid (AA) in resting human platelets, we assessed the differential role of these binding sites on plasmenylethanolamine hydrolysis by phospholipase A2 activity upon platelet activation by determining the generation of the corresponding [3H]lysoplasmenylethanolamine. Ethanolamine-containing phospholipids in platelets were pre-labelled with [3H]ethanolamine prior to platelet stimulation with U46619 (1 microM), a TxA2 mimetic, in the presence or absence of S-145, an antagonist of the low affinity TxA2/PGH2 receptor. Labelled platelets were also treated with the TxA2/PGH2 receptor antagonist, GR32191B, prior to washing (which blocks the low affinity site of the receptor) and subsequent stimulation. The above conditions provided for blockage of platelet aggregation but not shape change with U46619. The rise in [3H]lysoplasmenylethanolamine accumulation (170% of unstimulated controls) with U46619 as the agonist was inhibited in platelets pre-treated with S-145 and in platelets washed from GR32191B. Similar findings were also obtained for [3H]lysophosphatidylethanolamine accumulation. The present results indicate that the TxA2-dependent activation of plasmenylethanolamine cleavage by phospholipase A2 in intact human platelets is predominantly linked to the low affinity site of the TxA2/PGH2 receptor and may be important for platelet aggregation but not shape change.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Binding Sites; Biphenyl Compounds; Blood Platelets; Bridged Bicyclo Compounds; Fatty Acids, Monounsaturated; Heptanoic Acids; Humans; Lysophospholipids; Phospholipases A; Phospholipases A2; Plasmalogens; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Receptors, Prostaglandin; Receptors, Thromboxane; Receptors, Thromboxane A2, Prostaglandin H2; Thromboxane A2

A range of prostanoid agonists were tested for activity on isolated ring preparations of piglet saphenous vein. The selective TxA2-mimetic (TP-receptor agonist), U-46619, contracted the preparation in a concentration-related fashion. These contractions were inhibited by the TP-receptor blocking drug, GR32191B, producing a pA2 of 7.8 (slope = 1.6). Prostanoid-induced relaxant responses were studied on preparations which had been pre-contracted using an EC60 concentration of phenylephrine (mean EC60 = 0.97 microM), in the presence of GR32191B (1 microM), to block contractile TP-receptors. Under these conditions, PGD2, PGE2, PGF2 alpha, PGI2, and U-46619, all caused concentration-related relaxation. PGE2 was the most potent agonist (EC50 = 0.23nM), whereas, all of the other agonists were at least 1,000-fold weaker, providing strong evidence for the presence of inhibitory EP-receptors. The selective synthetic EP-agonists, sulprostone (EP1/EP3) and AH13205X (EP2), were next tested for relaxant activity. While both compounds caused concentration-related relaxant activity, they were respectively 6,000 and 11,000-fold less potent than PGE2. The potent TP-receptor blocking drugs, AH22921X and AH23848B, were both weak antagonists of PGE2 but not isoproterenol-induced relaxant responses of piglet saphenous vein in a concentration-related fashion. These two compounds had pA2 values against PGE2 of 5.3 and 5.4 respectively, with regression slopes not significantly different from unity. In contrast, neither compound at a concentration of 30 microM had any antagonist activity against prostanoid-induced effects on guinea-pig fundus (EP1), rabbit ear artery (EP2) or guinea-pig vas deferens (EP3). In conclusion, the piglet saphenous vein contains TP-receptors mediating smooth muscle contraction, and a PGE2-specific (EP) receptor mediating relaxation. The inhibitory EP-receptor does not appear to be of the EP1, EP2 or EP3-subtypes, and appears therefore to be a novel subtype which we tentatively term EP4, and the potent TP-receptor blocking drugs, AH22921X and AH23848B, appear to be weak, but specific EP4-receptor blocking drugs.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Biphenyl Compounds; Guinea Pigs; Heptanoic Acids; In Vitro Techniques; Male; Muscle Contraction; Muscle Relaxation; Prostaglandin Endoperoxides, Synthetic; Prostaglandins; Rabbits; Receptors, Prostaglandin; Saphenous Vein; Swine; Thromboxane A2

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. The inactivity of sulprostone (EP3/EPj receptor selective) and the low potency of AH1 3205 (EP2-receptor selective) suggest that the rat trachea contains an atypical EP-receptor that does not conform to the current classification system.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Biphenyl Compounds; Dinoprostone; Heptanoic Acids; In Vitro Techniques; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Prostaglandin Endoperoxides, Synthetic; Prostaglandins E, Synthetic; Prostanoic Acids; Rats; Rats, Sprague-Dawley; Receptors, Prostaglandin E; Thromboxane A2; Trachea

1. The interaction of the specific thromboxane (TP-) receptor blocking drug, [3H]-GR32191 with human intact platelets and platelet membranes has been investigated in vitro. 2. On intact platelets, association of specific [3H]-GR32191 binding at 37 degrees C was biphasic, with an initial rapid component and a slower secondary phase. Dissociation experiments indicated displacement from two sites with t1/2 values of 8.1 and 65.6 minutes. Kd values derived from the kinetic rate constants for the rapid onset/offset and slow onset/offset phases were 0.4 and 0.5 nM respectively. 3. Competition binding of [3H]-GR32191 and GR32191 on intact platelets gave an IC50 of 2.3 nM. Scatchard analysis indicated a single class of binding site with a Kd of 2.2 nM. Further analysis of the data yielded a Hill slope of -1.0 again indicating an interaction at a single binding site. Saturation binding experiments gave a similar estimate of the Kd value for [3H]-GR32191 to that obtained from competition binding experiments. A possible explanation for the biphasic interaction of the GR32191 in intact platelets may lie in restriction of its access to and egress from a population of TP-receptors. 4. In platelet membranes at 37 degrees C, specific [3H]-GR32191 binding was complete within 5 min with a calculated association rate constant of 3.2 x 10(8) M-1 min-1. Dissociation of [3H]-GR32191 was relatively slow, with measurable specific binding persisting for > 40 min. Analysis of these data yielded a t1/2 of 17.7 min and a dissociation rate constant of 0.04 min-1 and indicated dissociation from a single site. The ti for dissociation appeared to be related to the contact time of platelet membranes with [3H]-GR32191.Derivation of a Kd from the kinetic rate constants gave a value of 0.13 nM.5. Competition binding of [3H]-GR32191 and GR32191 to platelet membranes gave an IC50 value of 3.5 nM. Scatchard analysis of these data indicated a single binding site with a Kd of 2.1 nM. Saturation binding experiments with [3H]-GR32191 yielded similar IC50 and Kd values to those from competition experiments.6. In further competition binding experiments, the TP-receptor agonists U-46619, STA2, EP171 and 9,1 1-azo PGH2 and antagonists SQ29,548, BM 13.177 and EP092 all competed with specific [3H]-GR32191 binding on intact platelets and, where determined, on platelet membranes. All compounds fully displaced specific [3H]-GR32191 binding. However, where tested, the ICso values for a particular compound w

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Binding, Competitive; Biphenyl Compounds; Blood Platelets; Cell Membrane; Heptanoic Acids; Humans; In Vitro Techniques; Platelet Aggregation; Platelet Aggregation Inhibitors; Prostaglandin Endoperoxides, Synthetic; Radioligand Assay; Receptors, Thromboxane; Vasoconstrictor Agents

It has previously been shown that human umbilical artery (HUA) smooth muscle produces thromboxane A2 in response to increasing oxygen levels and that this thromboxane promotes contraction. To investigate the intracellular action of thromboxane A2, strips of HUA longitudinal smooth muscle were permeabilized using alpha-toxin from the bacterium Staphylococcus aureus. This treatment rendered the surface membrane permeable to low-molecular-weight substances but left functional thromboxane A2 receptors. Tension measurements were used to investigate the effect of the stable thromboxane A2 analogue, U-46619, on the Ca2+ sensitivity of smooth muscle contractile proteins. U-46619 (1 nM to 1 microM) potentiated submaximal Ca(2+)-activated force (generated by [Ca2+], 50 nM to 3 microM) but not maximal Ca(2+)-activated force (generated by [Ca2+], 10-100 microM). The specific thromboxane A2 receptor antagonist, GR-32191B (1 microM), inhibited the action of U-46619 (0.1 microM). The potentiation of submaximal Ca(2+)-activated force produced by the muscle in response to U-46619 (0.1 microM) was antagonized by guanosine 5'-O-(2-thiodiphosphate) (1 mM), the nonhydrolyzable analogue of GDP, and mimicked by guanosine 5'-O-(3-thiotriphosphate) (100 microM), the nonhydrolyzable analogue of GTP. These results suggest that U-46619 acts via the previously identified thromboxane A2 receptor to promote Ca2+ sensitivity of tension production in HUA smooth muscle. Furthermore, this effect appears to be mediated via a G protein.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Biphenyl Compounds; Calcium; Drug Synergism; Guanosine 5'-O-(3-Thiotriphosphate); Heptanoic Acids; In Vitro Techniques; Prostaglandin Endoperoxides, Synthetic; Thromboxane A2; Umbilical Arteries; Vasoconstriction

Regional vascular responses to the thromboxane A2 analogue U46619 and effects of the selective thromboxane receptor blocking drug vapiprost on these responses were examined in anesthetized dogs. Hemodynamic responses to U46619 (0.5 micrograms/kg into the left atrium), norepinephrine (NE, 0.3 microgram/kg, i.v.) and angiotensin II (AII, 30 or 60 ng/kg, i.v.) were periodically tested before and after administration of vapiprost (10, 30 or 100 micrograms/kg, i.v.) or its vehicle. In the absence of vapiprost, U46619 increased total peripheral (TPR), vertebral (VR), coronary (CR) and renal (RR) vascular resistance by 60.1 +/- 4.7%, 33.6 +/- 4.9%, 15.3 +/- 1.3% and 120.8 +/- 17.4%, respectively, indicating that vasoconstrictor responses to U46619 were most prominent in the renal vascular bed as compared to those in the vertebral or coronary vasculatures. Vapiprost as well as the vehicle did not affect the base-line hemodynamics. However, vapiprost apparently inhibited the U46619-induced vasoconstriction in all measured vascular beds in a dose-related manner without attenuating vasoconstrictor responses to NE compared to the inhibitions of VR and CR. These results demonstrate that there was a regional difference both in the vasoconstrictor responses to U46619 and in the blocking effects of vapiprost, and indicate that vapiprost is a potent and selective antagonist for thromboxane receptors in vivo.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Anesthesia; Angiotensin II; Animals; Biphenyl Compounds; Blood Pressure; Dogs; Female; Heptanoic Acids; Male; Norepinephrine; Prostaglandin Endoperoxides, Synthetic; Receptors, Prostaglandin; Regional Blood Flow; Vasoconstrictor Agents

Effects of the new thromboxane A2 antagonist vapiprost (SN-309, GR-32191B, CAS 85505-64-2) on isolated canine blood vessels were investigated. U46619 ((15S)-hydroxy-11a, 9a-(epoxymethano) prosta-5Z, 13E-dienoic acid) 10(-10)-10(-6) mol/l, a thromboxane A2 analogue, produced concentration-dependent contractions of oblong or ring preparations isolated from basilar, coronary, mesenteric and femoral arteries. Vapiprost 10(-8) and 10(-7) mol/l significantly and concentration-dependently shifted the concentration-contraction curves for U46619 of these arteries to the right. The pA2 values were 8.80 +/- 0.09 in basilar arteries, 8.67 +/- 0.12 in coronary arteries, 8.86 +/- 0.05 in mesenteric arteries and 9.01 +/- 0.07 in femoral arteries. On the other hand, oblong or ring preparations of basilar, coronary, mesenteric and femoral arteries showed sustained contractile responses to KCl 3 x 10(-2) mol/l, U46619 10(-7) mol/l or prostaglandin (PG) F2 alpha 10(-5) mol/l. Norepinephrine (NE) 3 x 10(-5) mol/l also produced sustained contractions in mesenteric and femoral arterial preparations, but not in basilar and coronary arterial preparations. Vapiprost 10(-10)-3 x 10(-6) mol/l relaxed these four arterial preparations constricted with U46619 10(-7) mol/l and PGF 2 alpha 10(-5) mol/l in a concentration-dependent fashion, but hardly affected them constricted with KCl 3 x 10(-2) mol/l. NE 3 x 10(-5) mol/l-induced contractures of mesenteric and femoral arterial preparations were not influenced by any concentrations of vapiprost. Results indicate that vapiprost has an antagonistic action on a so-called TP-receptor and/or a vasoconstrictive prostaglandin(s)-receptor and thus produces vasorelaxation.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Biphenyl Compounds; Dinoprost; Dogs; Female; Heptanoic Acids; In Vitro Techniques; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Norepinephrine; Potassium Chloride; Prostaglandin Endoperoxides, Synthetic; Receptors, Prostaglandin; Thromboxane A2; Vasoconstrictor Agents

To investigate possible subclasses of thromboxane receptors in vascular and airways smooth muscles, we evaluated activities of five structurally different competitive thromboxane receptor antagonists (i.e., SQ 29,548 [( 1S-[1 alpha, 2 alpha(5Z), 3 alpha, 4 alpha]]-7-[3- [2- [(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1] hept-2-yl]-5-heptenoic acid), L655,240 (3-[1-(4-chlorobenzyl)-5- fluoro-3-methylindol-2yl]2,2-dimethyl propanoic acid), BM 13,505 (4-[2-[[(4-chlorophenyl)sulfonyl]amino]ethyl]benzeneacetic acid), GR 32,191 [( 1R-[1 alpha (Z), 2 beta, 3 beta, 5 alpha]]-(+)-7- [5-[[(1, 1'-biphenyl)-4-yl]methoxy]-3-hydroxy-2-(1- piperidinyl)cyclopentyl]-4-heptenoic acid hydrochloride] and SQ 30,741 [1S- [1 alpha,2 alpha(5Z),3 alpha,4 alpha]]- 7[[[[[( oxaheptyl)amino]acetyl]amino]methyl]- 7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid)] in trachea, aorta and portal vein from both rats and guinea pigs. Schild plots and drug receptor dissociation constants (KB) were determined for each antagonist in each tissue using U-46,619 as the agonist. Rank orders of potency were identical in rat aorta, rat trachea and rat portal vein (SQ 29,548 greater than L 655,240 greater than BM 13,505 greater than GR 32,191 greater than SQ 30,741), with calculated KB values ranging from 0.5 to 20 nM. Rank orders of potency in guinea pig trachea and guinea pig portal vein were the same (GR 32,191 greater than SQ 29,548 greater than SQ 30,741 greater than L 655,240 greater than BM 13,505), with KB values ranging from 0.1 to 30 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Drug Antagonism; Fatty Acids, Unsaturated; Heptanoic Acids; Hydrazines; Indoles; Male; Muscle, Smooth, Vascular; Phenylacetates; Prostaglandin Endoperoxides, Synthetic; Rats; Receptors, Prostaglandin; Receptors, Thromboxane; Species Specificity; Sulfonamides; Thromboxane A2

1. Preincubation of guinea-pig tracheal smooth muscle with leukotriene E4 (LTE4) in vitro increased its subsequent responsiveness to histamine. 2. LTE4 pretreatment of guinea-pig tracheal strips did not affect the subsequent responsiveness to either the contractile agents, carbachol and KCl, or to the relaxant beta-adrenoceptor agonist, isoprenaline. 3. LTE4-induced airway histamine hyperresponsiveness was blocked by indomethacin (5 microM), GR32191 (3 microM), atropine (1 microM) and tetrodotoxin (1 microM). 4. U46619, a stable thromboxane A2-analogue, at a non-contractile concentration of 4 nM, increased tracheal smooth muscle sensitivity to histamine. 5. Both LTE4 and U46619 pretreatment increased the contractile response of tracheal smooth muscle to electrical field stimulation. 6. Preincubation of human bronchial spirals with LTE4 in vitro increased its subsequent responsiveness to histamine. 7. LTE4-induced histamine hyperresponsiveness of human bronchus was inhibited by GR32191 (3 microM) and atropine (1 microM). 8. It is proposed that LTE4 induces guinea-pig airway smooth muscle hyperresponsiveness to histamine via a facilitation of cholinergic neurotransmission, which is dependent upon the secondary generation of prostanoid mediator(s) acting on TP-receptors situated on cholinergic nerve terminals. In addition, it is suggested that LTE4 may induce histamine hyperresponsiveness of human bronchus in vitro by a similar mechanism as to that seen in guinea-pig central airway smooth muscle.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Aged; Animals; Atropine; Biphenyl Compounds; Bronchi; Carbachol; Electric Stimulation; Guinea Pigs; Heptanoic Acids; Histamine; Humans; In Vitro Techniques; Indomethacin; Leukotriene E4; Male; Middle Aged; Muscle Contraction; Muscle, Smooth; Potassium Chloride; Prostaglandin Endoperoxides, Synthetic; SRS-A; Trachea

Studies of the hierarchies of agonist and antagonist affinity for the prostaglandin (PG)H2/thromboxane (Tx)A2 receptor have been performed to establish whether distinct receptor subtypes exist in platelets and vascular smooth muscle cells (VSMC). They have yielded conflicting results. The pattern of homologous desensitization of phospholipase C activation and [Ca++i] increase induced by the PGH2/TxA2 agonist U46619 in rat aortic SMC was similar to that previously observed in human platelets: rapid desensitization of both responses followed by a delayed loss of binding sites from the cell membrane. Recently, the pattern of receptor inactivation by the antagonist ligand, GR 32191, has identified two subtypes in platelets. GR 32191 binds reversibly (GRr) to a site that mediates platelet shape change and an increase [Ca++i] and irreversibly (GRirr) to a site linked to phospholipase C activation and aggregation. In contrast to platelets, studies of ligand dissociation only identified GRr sites in rat aortic SMC and GR 32191 failed to inactivate PGH2/TxA2 receptors as detected by the PGH2/TxA2 receptor antagonist, [3H]SQ 29548. Inhibition of U46619-induced contraction of both rat aortic and human saphenous vein was competitive, consistent with the absence of GRirr sites in VSMC. Platelet activating factor, which heterologously desensitizes U46619-evoked phospholipase C activation in platelets, had no such effect in VSMC. The biochemical events attendant to PGH2/TxA2 receptor desensitization are similar in SMC and platelets. However, both the pattern of receptor inactivation by GR 32191 and of heterologous desensitization by PAF, suggest that VSMC lack the receptor subtype that transduces aggregation of platelets.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Binding, Competitive; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cells, Cultured; Fatty Acids, Unsaturated; Heptanoic Acids; Hydrazines; Male; Muscle Contraction; Muscle, Smooth, Vascular; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Rats; Rats, Inbred Strains; Receptors, Prostaglandin; Receptors, Thromboxane; Receptors, Thromboxane A2, Prostaglandin H2; Thromboxane A2

A selective thromboxane A2 (TXA2) receptor blocking agent, vapiprost, was orally administered to healthy male Japanese volunteers to investigate the pharmacokinetic and pharmacodynamic properties. The time-profile of vapiprost concentration in plasma was determined and the effects of the drug on platelet aggregation in platelet-rich plasma (PRP) induced by a stable TXA2 receptor agonist U-46619, adenosine diphosphate (ADP) and collagen, and platelet aggregation in whole blood induced by U-46619 ex vivo were simultaneously examined and compared. In the single-dose study (5, 10, and 20 mg/man) the plasma concentrations of the drug were fitted well to a one-compartment open model with a first-order absorption. The area under plasma concentration-time curve (AUC) and maximum plasma concentration (Cmax) showed dose-related increases, whereas the mean elimination half-lives (t1/2) remained approximately constant within the range of 0.99-1.1 hour. The drug was hardly recovered unchanged in urine. The platelet aggregation in PRP induced by collagen or U-46619 and the secondary aggregation by ADP were inhibited; that induced by U-46619 was the most specifically and completely inhibited at 2 hours after administration of any dose. The duration for maintaining the significant inhibition tended to depend on the dose and ranged from 24 to 36 hours after administration, which was much longer than expected from the plasma concentration of drug. The time-profile of inhibiting whole blood platelet aggregation that was induced by U-46619 was almost parallel to that of platelet aggregation in PRP by the same aggregant. The bleeding time was slightly prolonged 2 and 8 hours after administrations of 10 and 20 mg, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Administration, Oral; Adult; Biphenyl Compounds; Collagen; Drug Administration Schedule; Heptanoic Acids; Humans; Male; Platelet Aggregation; Platelet Aggregation Inhibitors; Prostaglandin Endoperoxides, Synthetic; Receptors, Prostaglandin; Receptors, Thromboxane; Thromboxane A2

1. The effects of selective thromboxane antagonists and a thromboxane synthase inhibitor on the contraction to 9,11-dideoxy-11 alpha,9 alpha-epoxymethano-prostaglandin F2 alpha (U46619) and oxygen in the human umbilical artery (HUA) were examined. The effect of the antagonists on contractions to both 5-hydroxytryptamine (5-HT) and 5-carboxamidotryptamine (5-CT) were also examined. 2. U46619 (0.3 nM-10 microM) contracted the HUA. This contraction was antagonized by two selective thromboxane receptor antagonists EP092 (10 nM-1 microM) and GR32191B (10 nM-1 microM). The contraction was not affected by the selective thromboxane synthase inhibitor, dazoxiben (10 nM-1 microM). 3. When the oxygen tension was increased from 16 mmHg to 120 mmHg, the HUA transiently contracted. Both thromboxane antagonists inhibited this contraction in a concentration-dependent manner with 1 microM almost completely abolishing the response (the oxygen-induced contraction of the control preparation normally increases with a second exposure to 120 mmHg oxygen). 4. In low (16 mmHg) oxygen, responses to both 5-HT and 5-CT were unaffected by both thromboxane receptor antagonists at concentrations up to 1 microM. In high oxygen (120 mmHg) responses to both 5-HT and 5-CT were biphasic in nature, with an additional initial high sensitivity phase, which was abolished by a cyclo-oxygenase inhibitor. In high oxygen, EP092 and GR32191B blocked this initial phase in a concentration-dependent manner, returning sensitivity to 5-HT and 5-CT to that seen in low oxygen. 5. The thromboxane synthase inhibitor, dazoxiben, at concentrations greater than 10 nm inhibited the contraction to 120 mmHg oxygen and at 1 microM, dazoxiben almost abolished the response. In low oxygen, the response to 5-HT was unaffected by dazoxiben at concentrations up to 10 microM. In high oxygen, the initial phase of the contraction to 5-HT was inhibited by concentrations greater than 10 nm, with no effect on the maximum response. 6. The results show that thromboxane receptor antagonism or blockade of thromboxane synthesis selectively attenuates oxygen-induced contractions and those responses to 5-HT and 5-CT which are dependent on high oxygen for their expression. This suggests that the contractions caused by high oxygen tension, and the enhancement of the contractile effects of low concentrations of 5-HT and 5-CT in the presence of high oxygen tension are mediated by endogenously released thromboxane A2.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Biphenyl Compounds; Female; Heptanoic Acids; Humans; Imidazoles; In Vitro Techniques; Muscle Contraction; Muscle, Smooth, Vascular; Oxygen; Pregnancy; Prostaglandin Endoperoxides, Synthetic; Prostaglandins, Synthetic; Serotonin; Thromboxane-A Synthase; Thromboxanes; Umbilical Arteries

The vasoconstrictor eicosanoid thromboxane plays an important role in the pathogenesis of several renal diseases. As an autacoid, its local release alters blood flow and induces platelet aggregation. We report a direct stimulatory effect of thromboxane on extracellular matrix protein production and gene expression in vitro. Treatment of two cell types, differentiated mouse teratocarcinoma cells (F9+) and human glomerular mesangial cells, with two different thromboxane analogues resulted in increased production of components of the extracellular matrix including fibronectin and the basement membrane proteins laminin and type IV collagen. These responses to thromboxane were not the result of a mitogenic effect of thromboxane nor the result of an increase in total cellular protein. The increased production of extracellular matrix proteins was, at least in part, due to an increase in the steady-state level of mRNA for these genes. Furthermore, the effect of thromboxane was markedly inhibited by cotreatment with a thromboxane-receptor antagonist. These results suggest a new potential role for thromboxane as a mediator of the sclerotic and fibrotic responses to injury.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adult; Animals; Biphenyl Compounds; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cell Division; Cell Line; Cells, Cultured; Collagen; DNA Replication; Extracellular Matrix Proteins; Fatty Acids, Unsaturated; Fibronectins; Glomerular Mesangium; Heptanoic Acids; Humans; Kinetics; Laminin; Mice; Prostaglandin Endoperoxides, Synthetic; RNA, Messenger; Teratoma; Thromboxanes; Thymidine

Thromboxane A2 (TXA2) induces platelet shape change, secretion, and aggregation. Using a novel TXA2/prostaglandin endoperoxide receptor antagonist, [1r-[1 alpha(Z),2 beta,3 beta,5 alpha]]-(+)-7-[5-[[(1,1'- biphenyl)-4-yl]methoxy]-3-hydroxy-2-(1-piperidinyl) cyclopentyl]-4-heptenoic acid hydrochloride (GR32191), we demonstrate that these responses are mediated by at least two receptor-effector systems. GR32191 non-competitively inhibited platelet aggregation to the TXA2 mimetics, (15S)-hydroxy-11,9-(epoxymethano) prostadienoic acid (U46619) and [1S-(1 alpha,2 beta(5Z),3 alpha (1E,-3S), 4 alpha)]-7-[3-(3-hydroxy-4-(p-iodophenoxy)-1-butenyl)7- oxabicyclo[2.2.1]hept-2yl]-5-heptenoic acid by binding irreversibly to a TXA2/prostaglandin endoperoxide receptor. Dissociation of [3H]GR32191 from human platelets demonstrated two specific binding sites, one which was rapidly dissociating and a site to which binding was essentially irreversible. Stimulation by U46619 of platelets incubated with GR32191 and subsequently washed to expose the reversible binding site failed to aggregate or to secrete [3H]5-hydroxy-tryptamine; formation of inositol phosphates and activation of protein kinase C were markedly suppressed. In contrast, platelet shape change and calcium stimulation remained at 90% of control. Furthermore, stimulation of the reversible binding site with U46619 induced aggregation in the presence of ADP, demonstrating its functional importance in amplifying the response to other agonists. These data suggest that TXA2 mediates platelet activation through at least two receptor-effector systems; one linked to phospholipase C activation, resulting in platelet aggregation and secretion and a second site mediating an increase in cytosolic calcium and platelet shape change.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Biphenyl Compounds; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Edetic Acid; Fatty Acids, Unsaturated; Heptanoic Acids; Humans; Hydrazines; In Vitro Techniques; Indomethacin; Kinetics; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Receptors, Prostaglandin; Receptors, Thromboxane; Structure-Activity Relationship; Thrombin; Thromboxane A2; Thromboxanes

Electrical field stimulation of dog isolated basilar artery produced neurogenically mediated contractions which were unaffected by phentolamine (1 microM), atropine (1 microM), ketanserin (1 microM) or methiothepin (0.1 microM). Responses were abolished by GR32191 (1-10 nM), BM 13.177 (0.1-10 microM) or flurbiprofen (0.5 microM) and markedly attenuated by dazoxiben (1-10 microM). Removal of the endothelium by Triton X-100-perfusion did not modify the magnitude of contractions to electrical stimulation and GR32191 still abolished the responses. GR32191 (1-10 nM) did not modify neurogenically mediated contraction of rabbit ear artery or potassium chloride-induced contraction of dog basilar artery. The results suggest that electrical field stimulation of dog basilar artery causes contractions which are mediated via a cyclo-oxygenase product with characteristics similar to thromboxane. This thromboxane-like substance is not endothelial in origin, nor released by contraction of the cerebrovascular smooth muscle per se and is therefore derived from a subendothelial, possibly neuronal, source.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Basilar Artery; Biphenyl Compounds; Dinoprost; Dogs; Electric Stimulation; Endothelium, Vascular; Female; Heptanoic Acids; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth, Vascular; Neurons; Phentolamine; Prostaglandin Endoperoxides, Synthetic; Rabbits; Substance P; Thromboxanes

In this study we investigated the effect of the selective and potent thromboxane A2 (TxA2) receptor antagonist GR32191 on smooth muscle contraction induced by the TxA2 analogue U46619, prostaglandin (PG) D2, PGF2 alpha, and methacholine (MCh) in guinea pig airways in vitro and the airways response provoked by inhaled PGD2 and MCh in asthmatic subjects in vivo. GR32191 antagonized competitively the contractile responses of all three prostanoids to a similar degree but had no effect on MCh-induced contractions. In asthmatic subjects GR32191, in a single oral dose of 80 mg, did not affect base-line airway caliber or MCh-induced broncho-constriction but caused significant inhibition of PGD2-induced bronchoconstriction, displacing the concentration-response curves to the right by greater than 10-fold. The effect of the same oral dose of GR32191 on allergen-induced immediate bronchoconstriction was subsequently investigated in allergic asthmatic subjects. In individual subjects, GR32191 inhibited to varying degrees the overall bronchoconstrictor response, with the maximum effect occurring between 10 and 30 min after allergen challenge. These studies suggest that prostanoids contribute to the immediate bronchoconstriction induced by inhaled allergen in allergic asthmatics, and that this effect is mediated by stimulation of a thromboxane receptor.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adolescent; Adult; Animals; Asthma; Biphenyl Compounds; Bronchi; Dinoprost; Guinea Pigs; Heptanoic Acids; Histamine; Humans; Male; Methacholine Compounds; Prostaglandin D2; Prostaglandin Endoperoxides, Synthetic; Receptors, Prostaglandin; Receptors, Thromboxane; Thromboxane A2

1. The thromboxane A2 (TP)-receptor blocking activity and specificity of action of GR32191 ([1R-[1 alpha(Z),2 beta,3 beta,5 alpha]]-(+)-7-[5-([1,1'-biphenyl] -4-ylmethoxy)-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-heptoni c acid has been evaluated in human platelets and various smooth muscle preparations, both vascular and non-vascular, from a range of species including man. 2. Utilising a platelet counting method to assess aggregation the drug was found to antagonise, in a surmountable manner, human platelet aggregation produced by the TP-receptor agonists, U-46619, EP171 and SQ26655, in whole blood and physiological buffer, with pA2 values of approximately 8.3 and 8.7 in the two media respectively. In the presence of GR32191 the rate of aggregation induced by U-46619 was slowed. 3. The effect of GR32191 upon U-46619-induced platelet shape change and aggregation in platelet-rich plasma was evaluated utilising a turbidometric technique. Both shape change and aggregation were antagonised by GR32191. At relatively high concentrations of the drug a slowing of aggregation and shape change to U-44619 was seen and an unsurmountable antagonism became apparent. 4. The action of GR32191 upon human platelets was specific with platelet aggregation induced by adenosine 5'-diphosphate, platelet activating factor, vasopressin and adrenaline and the inhibitory effects of prostacylin (PGI2), prostaglandin D2 (PGD2) and N-ethylcarboxamide-adenosine (NECA) being unaffected by concentrations of the drug as high as 10 microM. Furthermore, at concentrations of up to 100 microM, the drug itself produced no shape change or aggregation, of human platelets. 5. GR32191 also specifically and potently antagonised in a competitive, surmountable manner the contractile actions of U-46619 upon human vascular smooth muscle and antagonised U-46619-induced contractions of vascular and airways smooth muscle preparations from rat, dog, guinea-pig and rabbit with varying potency. This is discussed in terms of possible heterogeneity of TP-receptors. 6. GR32191 therefore represents a highly potent and specific TP-receptor blocking drug. This profile of action, coupled to its long duration of effect in man described elsewhere, make it an ideal drug tool for elucidating the physiological and pathophysiological role of thromboxane A2.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Aorta, Thoracic; Biphenyl Compounds; Blood Platelets; Dogs; Guinea Pigs; Heptanoic Acids; Humans; In Vitro Techniques; Male; Muscle, Smooth; Muscle, Smooth, Vascular; Platelet Aggregation; Platelet Aggregation Inhibitors; Prostaglandin Endoperoxides, Synthetic; Pulmonary Artery; Rabbits; Rats; Receptors, Prostaglandin; Receptors, Thromboxane

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Biphenyl Compounds; Collagen; Fatty Acids, Monounsaturated; Heptanoic Acids; Humans; In Vitro Techniques; Pentanoic Acids; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Pyridines; Thromboxane-A Synthase; Valerates