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

Thromboxane A2 (TXA2) can induce the platelet aggregation and lead to thrombosis. This will cause the low-reflow phenomenon after ischemic stroke and aggravate the damage of brain issues. Therefore, it is potential to develop the drugs inhibiting TXA2 pathway to treat cerebral ischemia.. This study aims to prove the protective effect of N2 (4-(2-(1H-imidazol-1-yl) ethoxy)-3-methoxybenzoic acid) on focal cerebral ischemia and reperfusion injury through platelet aggregation inhibition.. Middle cerebral artery occlusion/reperfusion (MCAO/R) is used as the animal model. Neurological deficit score, Morris water maze, postural reflex test, Limb-use asymmetry test, infarct volume, and water content were performed to evaluate the protective effect of N2 in MCAO/R rats. 9, 11-dieoxy-11α, 9α-methanoepoxyprostaglandin F2α (U46619) or adenosine diphosphate (ADP) was used as the inducer of platelet aggregation.. N2 can improve the motor function, learning and memory ability in MCAO/R rats while reducing the infarct volume. N2 can inhibit TXA2 formation but promote PGI2, and can inhibit platelet aggregation induced by U46619 and ADP. Further, N2 inhibits thrombosis with a minor adverse effect of bleeding than Clopidogrel. In conclusion, N2 can produce the protective effect on MCAO/R brain injury through inhibiting TXA2 formation, platelet aggregation and thrombosis.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Animals; Arteriovenous Shunt, Surgical; Blood Coagulation; Brain; Brain Ischemia; Edema; Enzyme-Linked Immunosorbent Assay; Epoprostenol; Female; Imidazoles; Male; Maze Learning; Platelet Aggregation; Rats; Rats, Sprague-Dawley; Stroke; Thrombosis; Thromboxane A2; Vanillic Acid

In the spontaneously hypertensive rat (SHR) and aging Wistar-Kyoto rats (WKY), acetylcholine releases an endothelium-derived contracting factor (EDCF) produced by endothelial cyclooxygenase-1, which stimulates thromboxane A2 receptors (TP receptors) on vascular smooth muscle. The purpose of the present study was to identify this EDCF by measuring changes in isometric tension and the release of various prostaglandins by acetylcholine. In isolated aortic rings of SHR, U 46619, prostaglandin (PG) H2, PGF2alpha, PGE2, PGD2, prostacyclin (PGI2) and 8-isoprostane, all activate TP receptors of the vascular smooth muscle to produce a contraction (U 46619>>8-isoprostane=PGF2alpha=PGH2>PGE2=PGD2>PGI2). The contractions produced by PGH2 and PGI2 were fast and transient, mimicking endothelium-dependent contractions. PGI2 did not relax isolated aortic rings of WKY and SHR. Acetylcholine evoked the endothelium-dependent release of thromboxane A2, PGF2alpha, PGE2, PGI2 and most likely PGH2 (PGI2>>PGF2alpha>or=PGE2>TXA2>8-isoprostane, PGD2). Dazoxiben abolished the production of thromboxane A2, but did not influence the endothelium-dependent contractions to acetylcholine. The release of PGI2 was significantly larger in the aorta of SHR than in WKY, and the former was more sensitive to the contractile effect of PGI2 than the latter. The inhibition of PGI-synthase was associated with an increase in PGH2 spillover and the enhancement of acetylcholine-induced endothelium-dependent contractions. Thus, in the aorta of SHR and aging WKY, the endothelium-dependent contractions elicited by acetylcholine most likely involve the release of PGI2 with a concomitant contribution of PGH2.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetylcholine; Animals; Aorta, Thoracic; Cyclooxygenase Inhibitors; Endothelium, Vascular; Enzyme Inhibitors; Hypertension; Imidazoles; In Vitro Techniques; Indomethacin; Nitrobenzenes; Prostaglandins; Prostaglandins I; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Salicylates; Sulfonamides; Thromboxane A2; Vasoconstriction; Vasoconstrictor Agents

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

To investigate the nature of the arachidonic acid metabolite involved in the altered reactivity of microvessels of two-kidney, one-clip hypertensive rats and the possible contribution of this product to the elevated blood pressure levels found in two-kidney, one-clip hypertension, mesenteric arterioles either perfused in vitro or studied in vivo were used along with blood pressure determinations. The decreased response to acetylcholine observed was normalized by ridogrel, a thromboxane A2 receptor antagonist, and dazoxiben, a thromboxane A2 synthase inhibitor. The smooth muscle response to nitric oxide, tested with sodium nitroprusside, was unaltered in two-kidney, one-clip hypertensive microvessels. Neither ridogrel nor dazoxiben modified the response to this vasodilator. In contrast, the potentiated response to noradrenaline was corrected by ridogrel and dazoxiben in vitro but not in vivo. Noradrenaline and acetylcholine increased the release of thromboxane A2 from the mesenteric microvessels of two-kidney, one-clip hypertensive rats. Ridogrel and dazoxiben decreased but did not normalize the elevated blood pressure of hypertensive rats. Based on these results, we concluded that: 1) the decreased responsiveness of smooth muscle to acetylcholine resulted from an increase in thromboxane A2 formation rather than a decrease in sensitivity to nitric oxide; 2) thromboxane A2 contributes to the increased noradrenaline response in mesenteric microvessels perfused in vitro while in in vivo other blood borne vasoactive agents may also be involved since the potentiated noradrenaline response was not corrected by inhibiting thromboxane A2 synthesis or receptors; 3) in addition to thromboxane A2, another as yet unidentified factor, may contribute to the elevated blood pressure in two-kidney, one-clip hypertension.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arterioles; Enzyme Inhibitors; Hypertension, Renovascular; Imidazoles; Male; Mesentery; Microcirculation; Nitric Oxide; Nitroprusside; Pentanoic Acids; Perfusion; Pyridines; Rats; Rats, Wistar; Receptors, Prostaglandin; Receptors, Thromboxane; Receptors, Thromboxane A2, Prostaglandin H2; Renal Circulation; Thromboxane A2; Thromboxane-A Synthase; Vascular Resistance; Vasoconstrictor Agents

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

This study investigates the mechanisms involved in kinin-induced contractions in rings of rat portal vein (RPV). Bradykinin (BK), Lys-BK, Met-Lys-BK, Tyr8-BK (TBK) and des-Arg9-BK (DABK) all caused graded contractions in RPV, with the following order of potency (EC50, nanomolar): Met-Lys-BK (0.3) > Lys-BK (0.5) > BK (0.9) > TBK (2.3) >> DABK (46.0). The potency of DABK and maximal contractions for DABK and BK, but not for TBK or NE, increased as a function of in vitro incubation period, reaching the maximum at 4.5 hr. Cycloheximide (a protein synthesis inhibitor, 70 microM), incubated for 4.5 hr, inhibited almost completely the CRCs for DABK and blocked the latter phase of CRCs for BK, not altering contractions induced by U46619 (9,11-dideoxy-9 alpha, 11 alpha-methano-epoxy prostaglandin F2 alpha) (a thromboxane A2/prostaglandin H2-mimetic). Incubation of RPV with D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-BK (HOE 140, a selective B2 receptor antagonist, 0.01-100 nM), caused a parallel rightward displacement of the BK and TBK concentration-response curves (CRCs). Schild plots were linear, yielding pA2 values of 11.4 and 9.3, respectively. The slope for HOE 140 against TBK-induced contractions did not differ from unity (1.23 +/- 0.21), whereas against BK was significantly lesser than unity (0.72 +/- 0.20). The CRCs induced by DABK were not affected by HOE 140 (100 nM). In addition, the CRCs for DABK at 4.5 hr were shifted to the right in a parallel form in the presence of des-Arg9-[Leu8]-BK (a selective B1-receptor antagonist, 1 microM), yielding a pA2 value of 6.7.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Bradykinin; Cycloheximide; Dose-Response Relationship, Drug; Imidazoles; In Vitro Techniques; Indoles; Male; Norepinephrine; Portal Vein; Prostaglandin Endoperoxides, Synthetic; Rats; Rats, Wistar; Receptors, Bradykinin; Thromboxane A2; Vasoconstriction

The aim was to study the effects of endothelin-1 on human placental veins and the role of cyclooxygenase products as mediators of these effects.. Rings of placental veins with and without endothelium were suspended in organ chambers filled with physiologic salt solution. After a period of stabilization at optimal basal tension, isometric tensions in the control group were recorded at increasing concentrations of endothelin-1 (10(-10) to 10(-7) mol/L). Rings in the experimental groups were treated with either indomethacin (cyclooxygenase inhibitor, 10(-5) mol/L), dazoxiben (thromboxane synthetase inhibitor, 10(-4) mol/L), or SQ29548 (thromboxane receptor antagonist, 10(-6) mol/L) before addition of endothelin-1. To demonstrate the presence of functional thromboxane receptors in the rings, contractile responses to U-46619 (10(-9) to 10(-6) mol/L), a thromboxane A2 analog were measured. The effectiveness of SQ29548 blockade was tested in rings treated with SQ29548 (10(-6) mol/L) before addition of U-46619. The concentration-response curves of the treated and control groups were compared with the Student paired t test.. Endothelin-1 in doses of 10(-10) to 10(-7) mol/L caused concentration-dependent contraction of placental veins. Indomethacin significantly reduced the response of veins with endothelium to low endothelin-1 concentrations (10(-9.5) to 10(-9) mol/L), (p < 0.05). However, it had no effect at higher endothelin-1 concentrations or in vessels without endothelium. The presence of functional thromboxane A2 receptors was confirmed by the vasoconstrictor effect of U-46619 and its blockade by treatment with SQ29548. Neither SQ29548 nor the thromboxane A2 synthesis inhibitor dazoxiben significantly influenced the response to endothelin-1.. These results demonstrated that endothelin-1 is a potent vasoconstrictor in the human placental vein. Although functional thromboxane A2 receptors exist in this vessel, endothelin-1's action is independent of thromboxane A2. Prostaglandins may mediate part of the endothelin-1-induced placental vasoconstriction. However, endothelin-1 acts primarily by a direct effect on vascular smooth muscle cells.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adult; Bridged Bicyclo Compounds, Heterocyclic; Endothelins; Endothelium, Vascular; Fatty Acids, Unsaturated; Female; Humans; Hydrazines; Imidazoles; In Vitro Techniques; Indomethacin; Placenta; Pregnancy; Prostaglandin Endoperoxides, Synthetic; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Thromboxane A2; Thromboxane-A Synthase; Vasoconstriction; Vasoconstrictor Agents; Veins

The role of products of cyclooxygenase was investigated in the responses of isolated airways to H2O2. Strips of guinea pig trachea, in some of which the epithelium had been removed mechanically, were suspended in organ chambers, and isometric tension was recorded. Under basal conditions, H2O2 induced indomethacin-sensitive contractions, which were larger in preparations without than in those with epithelium; the difference was abolished by inhibitors of thromboxane synthase or thromboxane A2 receptors. During contractions to acetylcholine, low concentrations of H2O2 induced relaxation in preparations with but had no significant effect in those without epithelium. At higher concentrations of H2O2, the epithelium-dependent relaxation was attenuated but an epithelium-independent relaxation appeared. The epithelium-dependent but not the epithelium-independent responses to H2O2 were blocked by indomethacin. Under basal conditions, prostaglandin E2 (PGE2; < or = 10(-7) M), U-46619, prostaglandin PGF2 alpha (PGF2 alpha), prostaglandin PGD2 (PGD2), and prostacyclin (PGI2) caused contractions. During contractions to acetylcholine, PGE2 induced larger relaxations in preparations with than in those without epithelium. Radioimmunoassay revealed that lower concentrations of H2O2 predominantly increased the release of PGE2 and 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha); in preparations without epithelium, the release of thromboxane B2 was augmented also. At higher concentrations of H2O2, the release of PGE2, PGF2 alpha, PGD2, 6-keto-PGF1 alpha, and thromboxane B2 increased in preparations with and without epithelium. These findings demonstrate that the responses of the guinea pig trachea to H2O2 are mediated mainly by products of cyclooxygenase and that the effects of H2O2 are modulated by the epithelium.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetylcholine; Animals; Bridged Bicyclo Compounds, Heterocyclic; Epithelium; Fatty Acids, Unsaturated; Guinea Pigs; Hydrazines; Hydrogen Peroxide; Imidazoles; In Vitro Techniques; Indomethacin; Isometric Contraction; Male; Muscle Contraction; Muscle, Smooth; Prostaglandin Endoperoxides, Synthetic; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Radioimmunoassay; Receptors, Thromboxane; Thromboxane-A Synthase; Trachea; Vasoconstrictor Agents

When injected i.v. to guinea pigs, the granulocyte secretagog N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) induces bronchoconstriction (BC), lung platelet sequestration and increased transendothelial albumin exchanges in lungs. We evaluated BC and the variations of the lung contents in radiolabeled platelets, erythrocytes and extravascular albumin, as measurements of platelet lung entrapment, reduction of lung blood volume and increase of transendothelial albumin exchanges, respectively. Trimetoquinol, a thromboxane A2 (TXA2)-endoperoxide receptor antagonist, inhibited BC and platelet entrapment by lungs induced by fMLP, but protection was nonspecific because it also suppressed BC by histamine. The specific TXA2 synthetase inhibitor/endoperoxide receptor antagonist ridogrel suppressed BC and reduced lung platelet entrapment, but failed to prevent the increase of extravascular albumin and the decrease of erythrocyte lung contents due to fMLP. Consequently, the fMLP-induced increase of vascular albumin exchanges and reduction of lung blood volume are TXA2-independent. Aspirin prevented BC, but failed to suppress lung platelet entrapment by fMLP, indicating that in vivo platelet activation is not TXA2-dependent, even though the levels of circulating TXB2, the stable metabolite of TXA2, were increased after fMLP concomitantly with that of 6-keto-prostaglandin (PG)F1 alpha, the stable metabolite of PGI2. The ridogrel-treated animals showed reduced blood level of TXB2 and increased levels of 6-keto-PGF1 alpha after fMLP challenge. Blocking the cyclooxygenase pathway with aspirin prevented ridogrel-induced protection against lung platelet sequestration after fMLP, supporting the concept that rechanneling of arachidonate metabolism toward protective prostaglandins accounts for protection by ridogrel.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 6-Ketoprostaglandin F1 alpha; Animals; Arachidonic Acid; Blood Platelets; Bronchoconstriction; Guinea Pigs; Imidazoles; Lung; N-Formylmethionine Leucyl-Phenylalanine; Pentanoic Acids; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Pyridines; Radioimmunoassay; Serum Albumin; Suprofen; Thrombocytopenia; Thromboxane A2; Thromboxane B2; Thromboxane-A Synthase; Tretoquinol

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

We have previously demonstrated that arachidonic acid (AA) and the stable cyclic endoperoxide analogue (U46619) desensitize human platelets at a common site, which is sensitive to endoperoxides/thromboxane receptor antagonists. We now report on the influence of agents which evaluate intracellular levels of platelet adenosine 3',5'-cyclic monophosphate (cAMP) on AA- and U46619-induced platelet desensitization. Prostaglandin E1, prostacyclin, carbacyclin, forskolin or dibutyryl cAMP prevented platelet activation by and desensitization to AA and to U46619 under conditions where the formation of thromboxane B2 was not significantly modified. Inhibition of platelet activation (aggregation and secretion) required a lower increase of the cAMP content than was needed to inhibit desensitization, confirming previous findings that desensitization to and by AA or U46619 are independent from the platelet release reaction. Together, these results indicate that AA-induced desensitization can be modulated by the adenylate cyclase/cAMP system acting at a site distinct from the known mechanisms of Ca2+ sequestration. This site is shared by the AA metabolite responsible for desensitization and by U46619 and is related to their common platelet membrane receptor.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenylyl Cyclases; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Bucladesine; Cyclic AMP; Humans; Imidazoles; In Vitro Techniques; Prostaglandin Endoperoxides, Synthetic; Thromboxane B2

The immunomodulatory effects of thromboxane A2 and prostaglandin E2 on peripheral blood mononuclear leucocytes stimulated with PHA in vitro, and the relationship of this to the time-course of their synthesis in culture, were investigated using prostaglandin E2, a thromboxane A2 synthesis inhibitor (UK37248), a thromboxane A2 mimic (U46619) and a thromboxane A2 receptor blocker (EP045). The inhibitory effect of prostaglandin E2 on PHA-induced human peripheral blood mononuclear leucocyte proliferation diminishes if the addition of PGE2 is delayed. If added 4 hr after a maximum concentration of PHA (5 micrograms/ml), the effect of PGE2 was reduced by 60%. If a submaximal concentration of PHA (1 microgram/ml) was used, the effect of PGE2 was not reduced if added 4 hr later but fell by about 60% after 16 hr. UK37248 moderately inhibited PHA-induced activation while substantially inhibiting thromboxane A2 synthesis and simultaneously enhancing PGE2 synthesis. The enhanced accumulation of PGE2 occurs while sensitivity to PGE2 is dropping. U46619, exogenously applied as a thromboxane A2 mimic, inhibited PHA-induced activation at concentrations that did not significantly alter PGE2 synthesis. EP045, which may modulate the effects of endogenous thromboxane A2 by blocking receptors, did not alter PHA-induced activation. We conclude that thromboxane A2 may have a role in inhibiting PHA-induced activation on the basis of the effect of U46619. However, this study highlights difficulties in utilizing prostaglandin and thromboxane receptor and synthesis inhibitors to examine their endogenous role in the modulation of mitogen-induced activation in vitro. If sensitivity to the purported endogenous substance is limited to the early stages of culture and if only low levels are synthesized at this early stage, then blocking drugs would have little effect.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adult; Dinoprostone; Humans; Imidazoles; Kinetics; Leukocytes; Lymphocyte Activation; Male; Phytohemagglutinins; Prostaglandin Endoperoxides, Synthetic; Prostaglandins E; Prostaglandins, Synthetic; Thromboxane A2

The antiaggregatory and antisecretory effects of two newly developed thromboxane receptor antagonists, BM-13,177 and SQ-29,548, were studied in an in vivo model of platelet activation. Arterial platelet count and whole blood ATP concentrations were measured continuously on-line in the arterial blood of anesthetized rabbits. Injections of collagen decreased peripheral platelet count by 25% of initial value. ATP concentrations increased 50 to 100 nM during collagen challenge. SQ-29,548 and BM-13,177 dose-dependently reduced platelet loss to about 50% of that observed in vehicle treated animals. Injection of arachidonic acid (AA) or 9,11-methanoepoxy-PGH2 resulted in sudden death of the animals associated with a 67 to 69% decrease in platelet count and a marked release of ATP. Pretreatment with SQ-29,548 or BM-13,177 increased survival rates from 0 to 100%, and reduced or totally inhibited ATP secretion and decreases in platelet count. In contrast, the thromboxane synthetase inhibitor, dazoxiben, was effective in inhibiting AA induced sudden death, but was without any effect when 9,11-methanoepoxy-PGH2 was used as the challenging agent. We conclude that SQ-29,548 and BM-13,177 are effective in antagonizing the effects of subsequent conversion to thromboxane A2. BM-13,177 and SQ-29,548 are generally considered as specific antagonists of endoperoxide/thromboxane receptors on platelets and smooth muscles in vitro. Preliminary clinical studies with BM-13,177 showed a marked inhibition of ex vivo platelet aggregation in human volunteers and patients (18, 19). These reports encourage further study of thromboxane receptor antagonists as effective anti-thrombotic drugs in the experimental and clinical setting.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Triphosphate; Animals; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Blood Pressure; Bridged Bicyclo Compounds, Heterocyclic; Collagen; Fatty Acids, Unsaturated; Fibrinolytic Agents; Hydrazines; Imidazoles; Kinetics; Male; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Rabbits; Receptors, Prostaglandin; Receptors, Thromboxane; Sulfonamides; Thromboxane-A Synthase; Thromboxanes

The main cyclooxygenase-dependent arachidonic acid derivatives produced by monocytes and macrophages have been shown to be thromboxane A2 and prostaglandin E2. The immunomodulatory effects of thromboxane A2 were examined using a specific thromboxane synthase inhibitor (dazoxiben), a thromboxane A2 analog (U46619), and a thromboxane A2 receptor blocker (BM13.177). Dazoxiben inhibited lymphocyte proliferation in response to mitogens (PHA and OKT3), but also reoriented cyclic endoperoxide metabolism towards the production of prostaglandin E2. Prostaglandin E2 has been shown previously to inhibit mitogen-induced lymphocyte proliferation. U46619, a stable thromboxane A2 analog, slightly enhanced lymphocyte responses to mitogens in the presence of dazoxiben and in the presence of a cyclooxygenase inhibitor (indomethacin). This occurred at concentrations of U46619 which are probably supraphysiological in view of the short half-life of natural thromboxane A2. Finally, the thromboxane A2 receptor blocker BM13.177 did not have any effect on mitogen-induced lymphocyte proliferation. It is concluded that thromboxane A2 has no or minimal modulatory effects on lymphocyte proliferative responses to mitogens and that the effect of thromboxane A2 synthase inhibition is rather due to reorientation of cyclic endoperoxide metabolism, resulting in increased prostaglandin E2 production.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Antibodies, Monoclonal; Cyclooxygenase Inhibitors; Dinoprostone; Humans; Imidazoles; Indomethacin; Lymphocyte Activation; Lymphocytes; Phytohemagglutinins; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Prostaglandins E; Receptors, Prostaglandin; Receptors, Thromboxane; Sulfonamides; Thromboxane A2; Thromboxane-A Synthase; Thromboxanes

Selective pharmacological blockade of thromboxane-synthase in human platelets by dazoxiben resulted in the reorientation of cyclic-endoperoxides towards PGE2, PGD2 and PGF2 alpha. At concentrations which can be reached when thromboxane-synthase is inhibited, PGE2 (100-500 nM) exerted a marked, concentration-dependent pro-aggregatory effect. This required the formation of endogenous or the addition of exogenous endoperoxides and was prevented by PGD2 or 13-aza-prostanoic acid, a selective antagonist of PGH2/TxA2 receptors. The anti-aggregating effect of PGD2 was evident at concentrations lower than those obtained in dazoxiben-treated platelets. It is proposed that in the absence of TxA2 generation, a combination of endoperoxides and PGE2 may result in normal aggregation. The latter may be inhibited by PGD2. No interference of PGF2 alpha on platelet function could be shown.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Arachidonic Acid; Arachidonic Acids; Aspirin; Blood Platelets; Dinoprost; Dinoprostone; Drug Synergism; Humans; Imidazoles; Oxidoreductases; Platelet Aggregation; Prostaglandin D2; Prostaglandin Endoperoxides; Prostaglandin Endoperoxides, Synthetic; Prostaglandins; Prostaglandins D; Prostaglandins E; Prostaglandins F; Prostanoic Acids; Thromboxane B2; Thromboxane-A Synthase

The mode of action of BM 13.177 (4-[2-(benzenesulfonamido)-ethyl] phenoxyacetic acid), a new anti-aggregating and anti-thrombotic agent, was studied in human washed platelets and citrated PRP. With ASA-treated platelets, BM 13.177 (0.1 - 100 microM) did not inhibit the shape change and the aggregation induced by ADP, serotonin, adrenaline, thrombin, or collagen. Therefore, BM 13.177 is neither an antagonist of ADP, serotonin, adrenaline, thrombin, or collagen nor a common pathway inhibitor like PGE1, or an inhibitor of the platelet interactions during aggregation. However, BM 13.177 (greater than or equal to 0.1 microM) produced a dose-dependent reduction of shape change, aggregation and release of [3H]serotonin induced by the stable PGH2 analogues U 46619 and U 44069 in ASA-treated platelets or ASA-treated citrated PRP. In untreated platelets, BM 13.177 inhibited platelet activation by U 46619 or U 44069 and by exogenous arachidonic acid or by endogenous arachidonic acid mobilized by hydrogen peroxide. Consequently, the ADP- and adrenaline-induced secondary aggregation and [3H]serotonin release in citrated PRP and the major effects of collagen were also inhibited. In washed platelets treated with 10 microM arachidonic acid or 100 microM hydrogen peroxide, the formation of TXB2 was not inhibited by 10 microM BM 13.177. However, the TXB2 formation after stimulation with 1,200 microM hydrogen peroxide was partially reduced by BM 13.177 to the same extent as by PGE1. This reduction may be due to the absence of a secondary release of arachidonic acid from phospholipids if the platelets were prevented from activation by BM 13.177 or PGE1. Arachidonic acid and hydrogen peroxide also induced the shape change, aggregation and release of washed platelets when thromboxane formation was inhibited by dazoxiben. Under these conditions, BM 13.177 was able to abolish the platelet response which was due to accumulating prostaglandin endoperoxides. These results show that BM 13.177 acts as a selective antagonist of TXA2 and prostaglandin endoperoxides. Its inhibitory effect on platelet function does not depend on an inhibition of either the primary release of arachidonic acid or the activities of cyclooxygenase or thromboxane synthetase.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Collagen; Dose-Response Relationship, Drug; Epinephrine; Humans; Hydrogen Peroxide; Imidazoles; Indomethacin; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Prostaglandins E; Serotonin; Sulfonamides; Thromboxane A2; Thromboxane B2; Thromboxanes

Recent studies suggest that platelet activation and subsequent thromboxane (TX) A2 release play important roles in certain coronary syndromes. To further test this possibility, we examined the ability of a selective TXA2-synthetase inhibitor, dazoxiben (UK-37-248), to abolish cyclic flow reductions (CFRs) that occur in experimentally stenosed canine coronary arteries. CFRs, which are characterized by progressive declines in coronary blood flow and interrupted by sudden and usually spontaneous restorations of flow, were produced by placing hard plastic cylindrical constrictors (5 mm long X 4.5 mm outer diameter) on the proximal left anterior descending or circumflex coronary artery in open-chest, anesthetized dogs. Coronary blood flow was measured with pulsed Doppler flow probes placed proximal to the constrictors and regional myocardial blood flow with 15 micron radiolabeled microspheres. CFRs were observed for 1 hr, during which coronary blood flow was monitored continuously. Regional myocardial blood flow was measured before constriction, when coronary blood flow appeared to be at its nadir, and after spontaneous restorations of flow. After 1 hr dazoxiben (2.5 mg/kg iv) or an equal volume of saline was given and coronary blood flow was monitored for another hour. Dazoxiben abolished CFRs completely in 18 of 28 dogs and significantly reduced their frequency in the dogs receiving the drug (10.1 +/- 0.8 vs 3.2 +/- 1.0 per hour [+/- SE]; p less than .001, n = 28). The frequency and magnitude of variations in cyclic blood flow were unchanged after saline (8.8 +/- 0.8 vs 9.0 +/- 1.0 per hour; p = NS, n = 13). The lowest levels of coronary blood flow before and after dazoxiben were 8.6 +/- 2.2% and 48.8 +/- 5.4% of control, respectively (p less than .001, n = 28), whereas this parameter remained unchanged after saline (18.7 +/- 5.7% vs 13.4 +/- 4.1%, respectively; n = 13). The levels of TXB2 and 6-keto-prostaglandin (PG) F1 alpha (stable breakdown products of TXA2 and prostacyclin, respectively) were measured in blood collected from aortic and distal coronary arterial catheters before coronary constriction (control), during CFRs, and after administration of dazoxiben. TXB2 levels measured distal to the stenosis were increased fivefold during CFRs (352 +/- 126 vs 71 +/- 18 pg/ml plasma; p less than .03) and were reduced to preconstriction (control) levels by dazoxiben (57 +/- 12 pg/ml). Aortic TXB2 levels almost doubled with CFRs and also returned to control le

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 6-Ketoprostaglandin F1 alpha; Animals; Coronary Circulation; Coronary Disease; Coronary Vessels; Dogs; Female; Hemodynamics; Imidazoles; Male; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Thromboxane B2

Arachidonic acid (0.2-0.8 mM) retracts clots formed in human citrated platelet-rich plasma by batroxobin. Extracellular calcium ions, but not the secretion of ADP by platelets, are required. AA-induced clot-retraction requires cyclo-oxygenase but not thromboxane synthetase activity since the retraction is inhibited by aspirin but not by selective inhibitors of thromboxane synthesis. The data indicate that endogenous cyclic endoperoxides mediate the retraction. Moreover, intact endoperoxide/thromboxane receptors also seem to be necessary because clot retraction is inhibited by thromboxane receptor antagonists.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Alprostadil; Arachidonic Acid; Arachidonic Acids; Aspirin; Blood Platelets; Clot Retraction; Epoprostenol; Fatty Acids, Unsaturated; Fibrin; Humans; Imidazoles; Prostaglandin Endoperoxides; Prostaglandin Endoperoxides, Synthetic; Prostaglandins D; Prostaglandins E; Prostaglandins H; Prostaglandins, Synthetic; Thromboxane B2

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Cattle; Imidazoles; In Vitro Techniques; Lymphatic System; Muscle Contraction; Muscle, Smooth; Prostaglandin Endoperoxides; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandins H; Sheep; Thromboxane-A Synthase