15-hydroxy-11-alpha-9-alpha-(epoxymethano)prosta-5-13-dienoic-acid and 7-(3-(3-hydroxy-4-(4--iodophenoxy)-1-butenyl)-7-oxabicyclo(2.2.1)heptan-2-yl)-5-heptenoic-acid

Chronic rhinosinusitis without nasal polyps (CRSsNP) is a common chronic disease and the etiology remains unclear. Thromboxane A2 (TXA2) participates in platelet aggregation and tissue inflammation. In this study, the CXCL1/8 chemokine and TXA2-TP receptor expression in the CRSsNP mucosa was investigated.. Immunohistochemistry, chemokine release assay by ELISA, RT-PCR, Real-time PCR, Western blotting, pharmacological and siRNA knockdown analysis were applied in the CRSsNP tissue specimen and cultured nasal mucosa-derived fibroblasts.. The immunohistochemistry results indicated that CXCL1 and CXCL8 were highly expressed in the CRSsNP mucosa compared with the controls; however, the TP receptors were expressed in both mucosa. Therefore, U46619 and IBOP, a TXA2 analog and TP agonist, were used to explore the role of TP activation in CXCL1/8 expression; both of these induced CXCL1/8 mRNA and protein expression in CRSsNP mucosa-derived fibroblasts. U46619 phosphorylated PI-3K, cyclic AMP (cAMP)/PKA, PKC, and cAMP response element (CREB). Activation of cAMP/PKA, PKC, and CREB was the major pathway for cxcl1/8 gene transcription. Pharmacological and siRNA knockdown analyses revealed that activation of cAMP/PKA and PKCμ/PKD pathways were required for CREB phosphorylation and PKA/C crosstalked with the PI-3K pathway.. Our study provides the first evidence for abundant TP receptor and CXCL1/8 expression in human CRSsNP mucosa and for TXA2 stimulation inducing CXCL1/8 expression in nasal fibroblasts primarily through TP receptor, cAMP/PKA, PKCμ/PKD, and CREB-related pathways.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Bridged Bicyclo Compounds, Heterocyclic; Case-Control Studies; Cells, Cultured; Chemokine CXCL1; Cyclic AMP Response Element-Binding Protein; Fatty Acids, Unsaturated; Fibroblasts; Interleukin-8; Nasal Mucosa; Phosphatidylinositol 3-Kinases; Protein Kinase C; Receptors, Thromboxane A2, Prostaglandin H2; Rhinitis; Second Messenger Systems; Sinusitis; Thromboxane A2

Patent ductus arteriosus (PDA) is a common life-threatening complication among premature infants. Although cyclooxygenase inhibitors are frequently used to treat PDA, as they inhibit the synthesis of prostaglandin E(2), the most potent vasodilator in the ductus arteriosus (DA), their efficacy is often limited. As thromboxane A(2) (TXA(2)) induces vascular contraction via the TXA(2) receptor (TP), we hypothesized that TP stimulation would promote DA closure.. To measure the inner diameter of the vessels, a rapid whole-body freezing method was used.. Injection of the selective TP agonists U46619 and I-BOP constricted the fetal DA at embryonic day 19 (e19) and e21 in a dose-dependent manner. Of note, U46619 also exerted a vasoconstrictive effect on two different types of postnatal PDA models: premature PDA and hypoxia-induced PDA. We also found that U46619 constricted the ex vivo DA ring to a greater extent than it constricted the ex vivo aorta. Furthermore, we found that U46619 at lower concentrations (up to 0.05 mg/g of body weight) had a minimal vasoconstrictive effect on other vessels and did not induce microthrombosis in the pulmonary capillary arteries.. Low-dose TP stimulation constricts the DA with minimal adverse effects at least in rat neonates and our results could point to an alternative potent vasoconstrictor for PDA.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Analysis of Variance; Animals; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Capillaries; Cryopreservation; Dose-Response Relationship, Drug; Ductus Arteriosus; Ductus Arteriosus, Patent; Fatty Acids, Unsaturated; Fetus; Pulmonary Alveoli; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction; Receptors, Thromboxane A2, Prostaglandin H2; Vasoconstriction

The thromboxane receptor (TPr) and multiple TPr ligands, including thromboxane A(2) (TxA(2)) and prostaglandin H(2), are elevated during vascular and atherothrombotic diseases. How TPr stimulation causes vascular injury remains poorly defined. This study was conducted to investigate the mechanism by which TPr stimulation leads to vascular injury.. Exposure of bovine aortic endothelial cells to either [1S-(1α,2β(5Z),3α(1E,3R),4α]-7-[3-(3-hydroxy-4-(d'-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1] heptan-2-yl]-5'-heptenoic acid (IBOP) or U46619, 2 structurally related TxA(2) mimetics, for 24 hours markedly increased the release of superoxide anions (O(2)(·-)) and peroxynitrite (ONOO(-)) but reduced cyclic GMP, an index of nitric oxide bioactivity. IBOP also significantly suppressed activity of endothelial nitric oxide synthase (eNOS), increased enzyme-inactive eNOS monomers, and reduced levels of tetrahydrobiopterin, an essential eNOS cofactor. IBOP- and U46619-induced increases in O(2)(·-) were accompanied by the membrane translocation of the p67(phox) subunit of NAD(P)H oxidase. Pharmacological or genetic inhibition of either NAD(P)H oxidase or TPr abolished IBOP-induced O(2)(·-) formation. Furthermore, TPr activation significantly increased protein kinase C-ζ (PKC-ζ) in membrane fractions and PKC-ζ phosphorylation at Thr410. Consistently, PKC-ζ inhibition abolished TPr activation-induced membrane translocation of p67(phox) and O(2)(·-) production. Finally, exposure of isolated mouse aortae to IBOP markedly increased O(2)(·-) in wild-type but not in those from gp91(phox) knockout mice.. We conclude that TPr activation via PKC-ζ-mediated NAD(P)H oxidase activation increases both O(2)(·-) and ONOO(-), resulting in eNOS uncoupling in endothelial cells.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Apoptosis; Biopterins; Bridged Bicyclo Compounds, Heterocyclic; Cattle; Cell Survival; Cells, Cultured; Cyclic GMP; Cytochrome P-450 Enzyme System; Endothelial Cells; Enzyme Activation; Enzyme Inhibitors; Fatty Acids, Unsaturated; Intramolecular Oxidoreductases; Mice; Mice, Inbred C57BL; Mice, Knockout; NADPH Oxidases; Nitric Oxide; Nitric Oxide Synthase Type III; Oxidative Stress; Peroxynitrous Acid; Phosphoproteins; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Protein Transport; Receptors, Immunologic; Receptors, Thromboxane A2, Prostaglandin H2; RNA Interference; Signal Transduction; Superoxides; Time Factors; Tyrosine

This study was conducted to elucidate the molecular mechanisms of thromboxane A2 receptor (TP)-induced insulin resistance in endothelial cells. Exposure of human umbilical vein endothelial cells (HUVECs) or mouse aortic endothelial cells to either IBOP or U46619, two structurally related thromboxane A(2) mimetics, significantly reduced insulin-stimulated phosphorylation of endothelial nitric-oxide synthase (eNOS) at Ser(1177) and Akt at Ser(473). These effects were abolished by pharmacological or genetic inhibitors of TP. TP-induced suppression of both eNOS and Akt phosphorylation was accompanied by up-regulation of PTEN (phosphatase and tension homolog deleted on chromosome 10), Ser(380)/Thr(382/383) PTEN phosphorylation, and PTEN lipid phosphatase activity. PTEN-specific small interference RNA restored insulin signaling in the face of TP activation. The small GTPase, Rho, was also activated by TP stimulation, and pretreatment of HUVECs with Y27632, a Rho-associated kinase inhibitor, rescued TP-impaired insulin signaling. Consistent with this result, pertussis toxin abrogated IBOP-induced dephosphorylation of both Akt and eNOS, implicating the G(i) family of G proteins in the suppressive effects of TP. In mice, high fat diet-induced diabetes was associated with aortic PTEN up-regulation, PTEN-Ser(380)/Thr(382/383) phosphorylation, and dephosphorylation of both Akt (at Ser(473)) and eNOS (at Ser(1177)). Importantly, administration of TP antagonist blocked these changes. We conclude that TP stimulation impairs insulin signaling in vascular endothelial cells by selectively activating the Rho/Rho-associated kinase/LKB1/PTEN pathway.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; AMP-Activated Protein Kinase Kinases; AMP-Activated Protein Kinases; Animals; Base Sequence; Biomimetic Materials; Bridged Bicyclo Compounds, Heterocyclic; Cells, Cultured; Dietary Fats; Endothelial Cells; Fatty Acids, Unsaturated; Humans; Insulin; Ligands; Male; Mice; Mice, Inbred C57BL; Nitric Oxide Synthase Type III; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Receptors, Thromboxane A2, Prostaglandin H2; rho-Associated Kinases; RNA, Small Interfering; Signal Transduction

Thromboxane A2 receptor (TPr) stimulation induces cellular hypertrophy in vascular smooth muscle cells (VSMCs); however, regulation of VSMC hypertrophy remains poorly understood. Here we show that TPr stimulation activates AMP-activated kinase (AMPK), which in turn limits TPr-induced protein synthesis in VSMCs. Exposure of cultured VSMCs to either TPr agonists, IBOP and U46619, or exogenous hydrogen peroxide (H2O2) caused time- and dose-dependent AMPK activation, as evidenced by increased phosphorylation of both AMPK-Thr172 and acetyl-coenzyme A carboxylase-Ser79, a downstream enzyme of AMPK, whereas SQ29548, a selective TPr antagonist, significantly attenuated TPr-enhanced AMPK activation. In parallel, both IBOP and U46619 significantly increased the production of reactive oxygen species such as H2O2. Furthermore, adenoviral overexpression of catalase (an H2O2 scavenger) abolished, whereas superoxide dismutase (which catalyzes H2O2 formation) enhanced, IBOP-induced AMPK activation, suggesting that TPr-activated AMPK was mediated by H2O2. Consistently, exposure of VSMCs to either TPr agonists or exogenous H2O2 dose-dependently increased the phosphorylation of LKB1 (at serines 428 and 307), an AMPK kinase, as well as coimmunoprecipitation of AMPK with LKB1. In addition, direct mutagenesis of either Ser428 or Ser307 of LKB1 into alanine, like the kinase-dead LKB1 mutant, abolished both TPr-stimulated AMPK activation and coimmunoprecipitation. Finally, genetic inhibition of AMPK significantly accentuated IBOP-enhanced protein synthesis, whereas adenoviral overexpression of constitutively active AMPK abolished IBOP-enhance protein synthesis in VSMCs. We conclude that TPr stimulation triggers reactive oxygen species-mediated LKB1-dependent AMPK activation, which in return inhibits cellular protein synthesis in VSMCs.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenoviridae; AMP-Activated Protein Kinase Kinases; AMP-Activated Protein Kinases; Animals; Bridged Bicyclo Compounds, Heterocyclic; Catalase; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Activation; Fatty Acids, Unsaturated; Hydrogen Peroxide; Hypertrophy; Multienzyme Complexes; Muscle, Smooth, Vascular; Mutagenesis, Site-Directed; Mutation, Missense; Myocytes, Smooth Muscle; Oxidants; Phosphorylation; Protein Biosynthesis; Protein Serine-Threonine Kinases; Rats; Receptors, Thromboxane A2, Prostaglandin H2; Superoxide Dismutase; Transduction, Genetic; Vasoconstrictor Agents

Because sulfonylureas, such as glibenclamide, are used to treat Type 2 diabetes and because this disease is associated with various cardiovascular complications that may be mediated by thromboxane (TX), this study was designed to characterize the role of glibenclamide on TX-mediated contractions in isolated ring segments of bovine coronary arteries and rabbit aortas. A series of TXA(2) analogs [9,11 Dideoxy-9alpha, 11alpha-methanoepoxy prostaglandin F(2alpha) (U46619), [1S-(1alpha, 2beta(5Z),3alpha(1E, 3R*),4alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo [2.2.1]heptan-2-yl]-5-heptenoic acid (I-BOP), carbocyclic TXA(2) (CTA(2)) and 9,11-dideoxy-9alpha,11alpha-epoxymethano prostaglandin F(2alpha) (U44069)], endothelin and phenylephrine contracted both types of blood vessels. Glibenclamide (10 microM) inhibited the contraction to each of the TX agonists but had no effect on endothelin- or phenylephrine-induced contractions. We hypothesized that this effect was due to a direct effect to block the vascular smooth muscle cell TX receptor. Receptor binding studies were performed in rabbit vascular smooth muscle cells and indicated that glibenclamide (10 microM) inhibited (125)I-BOP binding by more than 80%. The inhibition constants or K(i) for glibenclamide was 0.53 microM. These studies provide the first evidence that the ability of glibenclamide to inhibit TX-mediated contractions occurs independent of the vascular K(ATP) channel and is, instead, mediated by the blockade of the vascular TX receptor.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Triphosphate; Animals; Aorta, Thoracic; Bridged Bicyclo Compounds, Heterocyclic; Cattle; Coronary Vessels; Fatty Acids, Unsaturated; Glyburide; In Vitro Techniques; Male; Muscle, Smooth, Vascular; Potassium Channels; Prostaglandin Endoperoxides, Synthetic; Rabbits; Radioligand Assay; Receptors, Thromboxane; Thromboxane A2; Vasoconstriction; Vasoconstrictor Agents

Vascular endothelial growth factor (VEGF) is an important patho-physiological mediator of angiogenesis. VEGF-induced endothelial cell (EC) migration and angiogenesis often occur in complicated environments containing multiple agents capable of modifying the response. Thromboxane (TX) A2 is released from multiple cell types and is a prime mediator of pathogenesis of many vascular diseases. Human EC express both TXA2 receptor (TP) isoforms; however, the effects of individual TP isoforms on VEGF-induced EC migration and angogenesis are unknown. We report here that the TXA2 mimetic [1S-(1alpha, 2beta(5Z), 3alpha(1E, 3R), 4alpha]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxab icyclo-[2.2.1]heptan-2yl]-5'-heptenoic acid (IBOP) (100 nmol/L) is a potent antagonist (IC50 30 nmol/L) of VEGF-induced EC migration and differentiation. TPbeta, but not TPalpha, expression is required for the inhibition of VEGF-induced migration and angiogenesis. IBOP costimulation suppressed nitric oxide (NO) release from VEGF-treated EC through decreased activation of Akt, eNOS, and PDK1. TPbeta costimulation also ablated the increase in focal adhesion formation in response to VEGF. This mechanism was characterized by decreased recruitment of focal adhesion kinase (FAK) and vinculin to the alpha(v)beta3 integrin and reduced FAK and Src activation in response to VEGF. Addition of NO donors together with transfection of a constitutively active Src construct could circumvent the blockade of VEGF-induced migration by TP; however, neither intervention alone was sufficient. Thus, TP stimulation appears to limit angiogenesis, at least in part, by inhibiting the pro-angiogenic cytokine VEGF. These data further support a role for antagonism of TP activation in enhancing the angiogenic response in tissues exposed to elevated TXA2 levels in which revascularization is important.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Bridged Bicyclo Compounds, Heterocyclic; Capillaries; Cell Movement; Endothelial Cells; Endothelium, Vascular; Fatty Acids, Unsaturated; Focal Adhesions; Humans; Hydrazines; Neovascularization, Physiologic; Nitric Oxide; Nitric Oxide Donors; Phosphorylation; Protein Isoforms; Protein Kinases; Protein Processing, Post-Translational; Rats; Receptors, Thromboxane A2, Prostaglandin H2; Recombinant Proteins; Signal Transduction; Transfection; Umbilical Veins; Vascular Endothelial Growth Factor A

We report herein a novel class of thromboxane receptor (TP receptor) antagonists modeled on unstable natural lipids that we identified several years ago, the hepoxilins. These antagonists have been rendered chemically and biologically more stable than the natural compounds through structural modification by chemical synthesis. We demonstrate that the analogs inhibit the aggregation of human platelets in vitro evoked by the thromboxane receptor agonists, I-BOP ([1S-[1alpha,2alpha(Z),3beta(1E,3S*),4alpha]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabi-cyclo[2.2.1]hept-2-yl]5-heptenoic acid) and U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy-prosta-5Z,13E-dien-1-oic acid). The most potent of the analogs described, PBT-3 [10(S)-hydroxy-11,12-cyclopropyl-eicosa-5Z,8Z,14Z-trienoic acid methyl ester], has an IC(50) versus aggregation by I-BOP = 0.6 x 10(-7) M and versus U46619 = 7 x 10(-7) M, representing one of the most potent anti-aggregating substances so far described. PBT-3 also inhibits thromboxane formation and aggregation evoked by collagen with an IC(50) = 8 x 10(-7) M. Other PBT (hepoxilin cyclopropane) analogs so far tested were 5- to 10-fold less active, and the native hepoxilins were about 500-fold less active. Neither PBT-3 nor the other analogs inhibited 12-lipoxygenase, phospholipase A(2), or cyclooxygenase 1 or 2, and weakly stimulated adenyl cyclase (threshold stimulation at 10(-7) M and little selectivity for each of the PBT compounds). TP antagonism by PBT-3 was further demonstrated in receptor binding studies through use of (125)I-BOP, where the IC(50) for PBT-3 was 8 x 10(-9) M, approximately 16-fold less than for I-BOP itself. These findings identify a new mode of action of PBT-3 and other related analogs as primarily TP antagonists. These studies identify a new family of compounds useful in further development as novel therapeutics for thromboxane-mediated diseases.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 8,11,14-Eicosatrienoic Acid; Arachidonate 12-Lipoxygenase; Bridged Bicyclo Compounds, Heterocyclic; Cyclic AMP; Cyclooxygenase 1; Cyclooxygenase 2; Dose-Response Relationship, Drug; Fatty Acids, Unsaturated; Humans; In Vitro Techniques; Isoenzymes; Membrane Proteins; Phospholipases A; Platelet Aggregation; Prostaglandin-Endoperoxide Synthases; Receptors, Thromboxane; Thromboxane-A Synthase; Vasoconstrictor Agents

Thromboxane (TX) A2 effects in the kidneys include contraction of glomerular mesangial cells and intrarenal vascular tissue. A kidney cDNA encoding a TX receptor expressed in rat renal glomeruli and rat renal arterial smooth muscle cells has been reported. However, TXA2 receptors in human kidneys have not been documented. The purpose of this study was to identify and characterize TXA2 receptors in glomeruli and intrarenal arteries isolated from human kidneys. Normal kidneys, not used for transplant because of technical reasons, were kept at -70 degrees C and used for research purposes. The glomeruli and intrarenal arteries were isolated from renal cortical tissue by a mechanical sieving technique. The equilibrium dissociation constant and receptor number were determined by nonlinear analysis of binding inhibition data. The data were generated in radioreceptor assays using [125I]-BOP, a stable analog of TXA2. The dissociation constants (mean +/- SEM) for binding of I-BOP to human glomeruli and intrarenal arterial membranes were 6.6 +/- 1.1 nM (n = 7) and 20 +/- 6 nM (n = 7), respectively (p < 0.05). The receptor number was 311 +/- 91 fmol/mg protein (n = 7) in glomeruli and 74 +/- 16 fmol/mg protein (n = 7) in intrarenal arterial membranes (p < 0.04). The order of specificity of TXA2 analogs for [125I]-BOP binding sites was similar in glomeruli and in arterial membranes and was I-BOP > or = U46619 > or = pinane TXA2 > or = carbocyclic TXA2 > or = PGH2. These findings provide direct evidence for the presence of specific, high-affinity [125I]-BOP binding sites in human renal glomeruli and extraglomerular vascular tissue. These data also indicate that the human binding sites have higher affinity for the TXA2 agonist I-BOP than for PGH2.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Bicyclic Monoterpenes; Binding, Competitive; Bridged Bicyclo Compounds, Heterocyclic; Fatty Acids, Unsaturated; Humans; Iodine Radioisotopes; Kidney Cortex; Kidney Glomerulus; Ligands; Muscle, Smooth, Vascular; Receptors, Thromboxane; Renal Artery; Thromboxane A2; Vasoconstrictor Agents

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

1. Cumulative concentration-effect curves for the selective prostanoid TP receptor agonist, U46619, were constructed in strips of human non-pregnant myometrium grouped according to tissue excision site (top, lateral wall, lower uterine segment, sub-serosal or sub-endometrial), tissue orientation (strips cut either parallel or perpendicular to the serosa) and donor menstrual status (proliferative or secretory phase). 2. U46619 was excitatory in all tissues. There was no significant difference in either pEC50 or maximum response between groups (P<0.05). The range of pEC50 values was 6.8+/-0.1 (lateral wall, proliferative phase, n=5) to 7.1+/-0.3 (lateral wall, secretory phase, n=5). The range of maximum response values was 0.9+/-0.8 N cm-2 (lateral wall, proliferative phase, n=5) to 3.1+/-1.0 N cm-2 (lateral wall, secretory phase, n=5). 3. Saturation binding analyses were conducted using the radiolabelled TP receptor agonist, [125I]-BOP. Binding parameters were estimated for membranes prepared from human non-pregnant myometrium excised from the lateral wall and grouped according to donor menstrual status. 4. There were no significant differences in the mean pKd and [R]tot values for [125I]-BOP binding between the two groups (proliferative phase: pKd=8.3+/-0.3, [R]tot=412+/-319 fmol mg protein-1, n=5; secretory phase: pKd=8.5+/-0.4, [R]tot=369+/-192 fmol mg protein-1, n=6; P<0.05). 5. These data indicate that U46619-mediated responses in human non-pregnant myometrium are not influenced by tissue excision site, tissue orientation or donor menstrual status and that [125I]-BOP binding is not influenced by donor menstrual status. This suggests that the TP receptor population is homogeneous throughout the human non-pregnant myometrium, and not subject to hormonal regulation.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adult; Bridged Bicyclo Compounds, Heterocyclic; Fatty Acids, Unsaturated; Female; Humans; In Vitro Techniques; Iodine Radioisotopes; Kinetics; Menstrual Cycle; Middle Aged; Myometrium; Pregnancy; Receptors, Thromboxane; Uterine Contraction; Vasoconstrictor Agents

This study investigates thromboxane A2-induced cell signaling and mitogenesis of bovine coronary artery smooth muscle cells. The thromboxane mimetic U 46619 [(15S)-hydroxy-11,9-(epoxymethano) prosta-5Z,13E-dienoic acid] (10 microM) stimulated [Ca2+]i signals, phosphorylation of MAP kinase (mitogen-activated protein kinase), and expression of c-fos mRNA in smooth muscle cells. In contrast, no stimulation of DNA synthesis or cell proliferation by U 46619 was observed. However, platelet-derived growth factor-BB (20 ng/ml)-induced mitogenesis was potentiated by U 46619. Similar results were obtained with I-BOP [1S-(1 alpha,2 beta(5Z),3 alpha(1E,3R*), 4 alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo [2.2.1] heptan-2-yl]-5-heptenoic acid]. These potentiating effects were abrogated by a specific thromboxane receptor antagonist, suggesting that the potentiation of platelet-derived growth factor-BB-induced smooth muscle cell mitogenesis by U 46619 and I-BOP was mediated by thromboxane receptors. It is concluded that thromboxane A2 generated by blood platelets at the site of vessel injury induces cell signaling in smooth muscle cells but acts as a mitogen only in the presence of growth factor(s).

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cattle; Cell Division; Cells, Cultured; Coronary Vessels; DNA; Fatty Acids, Unsaturated; Mitogens; Muscle, Smooth, Vascular; Phosphorylation; Platelet-Derived Growth Factor; Prostaglandin Endoperoxides, Synthetic; Proto-Oncogene Proteins c-fos; Signal Transduction; Thromboxane A2; Vasoconstrictor Agents

Pregnancy is associated with the reduction of vascular sensitivity to vasoconstrictor compounds. We have examined whether pregnancy in rabbits induces hyposensitivity of the pulmonary vascular system to U-46619. Anesthetized, mechanically ventilated nonpregnant (NP; n = 7) and late-pregnant (P; n = 7) rabbits were studied. The intravenous injection of 0.03, 0.1, and 0.3 microgram/kg U-46619 led to a dose-dependent elevation of mean pulmonary arterial pressure (MPAP) in NP rabbits from a baseline value of 15 +/- 1 to 22 +/- 1 mgHg. There was no significant MPAP response to intravenous administration of U-46619 in P rabbits. The pulmonary arterial pressure response of isolated, ventilated, and buffer-perfused lungs of P rabbits was also blunted (P < 0.001 vs. NP). Pulmonary arterial membrane binding of [125I]BOP, another thromboxane (Tx)A2 analog, indicated 48 +/- 16 fmol receptors/mg protein in P rabbits and 193 +/- 48 fmol receptors/mg protein in NP samples (P < 0.025). Receptor affinity [1/dissociation constant (KD)] was also lower in the tissue of P rabbits (P < 0.01 vs. NP). The urinary excretion of the stable TxA2 metabolite 11-dehydro-TxB2 was lower in P than in NP rabbits (P < 0.02), which made homologous desensitization an unlikely explanation for the changes of vascular TxA2 receptors. These results show that, in late gestation, rabbit pulmonary vascular sensitivity to U-46619 is reduced simultaneously with, and as a possible consequence of, downregulation of specific receptors.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arteries; Bridged Bicyclo Compounds, Heterocyclic; Fatty Acids, Unsaturated; Female; Hemodynamics; In Vitro Techniques; Pregnancy; Pregnancy, Animal; Prostaglandin Endoperoxides, Synthetic; Pulmonary Circulation; Rabbits; Receptors, Cell Surface; Reference Values; Thromboxane A2; Urine; Vasoconstrictor Agents

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Bridged Bicyclo Compounds, Heterocyclic; Carbazoles; Fatty Acids, Unsaturated; Female; Humans; Hydrazines; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Muscle, Smooth, Vascular; Myometrium; Receptors, Thromboxane; Sulfonamides; Thromboxane A2; Umbilical Arteries

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Blood Platelets; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Diethyl Pyrocarbonate; Fatty Acids, Monounsaturated; Fatty Acids, Unsaturated; Humans; Hydrazines; In Vitro Techniques; Iodine Radioisotopes; Kinetics; Radioligand Assay; Receptors, Prostaglandin; Receptors, Thromboxane; Receptors, Thromboxane A2, Prostaglandin H2

8-epi-Prostaglandin (PG) F2alpha may be formed by cyclooxygenases 1 and 2 or by a free radical catalyzed process as an isoprostane. Concentrations of 8-epi-PGF2alpha in the range 1 nM to 1 microM induce a dose-dependent increase in platelet shape change, in calcium release from intracellular stores [Ca2+]iand in inositol phosphates; it also causes irreversible platelet aggregation, dependent on thromboxane generation, when incubated with subthreshold concentrations of ADP, thrombin, collagen, and arachidonic acid. Much higher concentrations of 8-epi-PGF2alpha (10-20 microM) alone induce weak, reversible aggregation. Although these effects are prevented by pharmacological thromboxane receptor antagonists, they are unlikely to be mediated by thromboxane receptors. Thus, 8-epi-PGF2alpha does not compete for binding at the stably expressed placental or endothelial isoforms of the thromboxane receptor or for binding of thromboxane ligands to human platelets. Furthermore, the response to 8-epi PGF2alpha exhibits structural specificity versus 8-epi PGF3alpha and PGF2alpha. Concentrations in the range that evoke its effects on platelets do not desensitize the aggregation response stimulated by thromboxane or PGH2 analogs. Unlike primary prostaglandins, which are rapidly metabolized to inactive products, 8-epi PGF2alpha circulates in plasma. However, the systemic concentrations found in healthy volunteers (median 48 pmol/liter) and in patients with hepatic cirrhosis (median 147 pmol/liter), a syndrome of oxidant stress in vivo, fall well below those which modulate platelet function. 8-Epi PGF2alpha may amplify the response to platelet agonists in syndromes where oxidant stress and platelet activation coincide. Despite blockade by thromboxane antagonists, 8-epi PGF2alpha does not activate either of the thromboxane receptor isoforms described in platelets. Activation of a distinct receptor would be consistent with the enzymatic formation of 8-epi PGF2alpha by cyclooxygenases. However, incidental activation of such a receptor by systemic concentrations of 8-epi PGF2alpha is unlikely to occur, even in syndromes of excessive free radical generation in vivo.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Arachidonic Acid; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cell Line; Collagen; Dinoprost; F2-Isoprostanes; Fatty Acids, Unsaturated; Humans; In Vitro Techniques; Inositol Phosphates; Kidney; Kinetics; Pertussis Toxin; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Radioligand Assay; Receptors, Thromboxane; Recombinant Proteins; Thrombin; Thromboxane A2; Transfection; Virulence Factors, Bordetella

We have used both functional and binding studies to fully characterize the prostanoid TP receptor in the myometrium from nonpregnant human donors. Both U-46,619 and I-BOP produced concentration-dependent contraction of human myometrial strips in vitro (pEC50 = 6.9 +/- 0.6; and 7.8 +/- 0.5, respectively). U-46,619-induced contractions were attenuated by the TP receptor antagonists: ICI 192,605 (pKB = 9.2 +/- 0.3); ICI D1,542 (pKB = 9.1 +/- 0.3); L670,596 (pKB = 8.6 +/- 0.3); GR 32,191 (pKB = 8.6 +/- 0.2); SQ 29,548 (pKB = 8.2 +/- 0.5); ONO 3,708 (pKB = 8.1 +/- 0.3) and BM 13,505 (pKB = 7.4 +/- 0.2). The binding of [125I]-BOP to human myometrial membranes was saturable, selective and displaceable. Equilibrium binding of [125I]-BOP identified one class of sites, Kd = 3.4 nM (pKd = 8.7 +/- 0.4) and a maximum binding of 323.1 +/- 361.5 fmol/mg protein. The addition of the nonhydrolyzable GTP analog GTP gamma S (100 microM) to the assay had no effect on [125I]-BOP binding. The Kd determined kinetically was 4.1 +/- 0.2 nM. TP receptor antagonists competed for [125I]-BOP binding: ICI D1,542 (pIC50 = 8.3 +/- 0.4); L670,596 (pIC50 = 7.9 +/- 0.1); ICI 192,605 (pIC50 = 7.2 +/- 0.1); ONO 3,708 (pIC50 = 7.2 +/- 0.04); SQ 29,548 (pIC50 = 7.2 +/- 0.1); GR 32,191 (pIC50 = 7.0 +/- 0.2); BM 13,505 (pIC50 = 6.8 +/- 0.1). The rank order of potency for the seven TP receptor antagonists in displacing [125I]-BOP from its binding site was correlated (r = 0.75) with the rank order of potency in inhibiting U-46,619-induced contraction of myometrial strips. Ligands selective for other prostanoid receptors were unable to significantly displace [125I]-BOP binding. These results are consistent with the notion that the human myometrial TP receptor is pharmacologically similar to the low affinity TP receptor in human platelets.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adult; Binding, Competitive; Bridged Bicyclo Compounds, Heterocyclic; Dose-Response Relationship, Drug; Fatty Acids, Unsaturated; Female; Guanosine 5'-O-(3-Thiotriphosphate); Humans; In Vitro Techniques; Middle Aged; Myometrium; Prostaglandin Endoperoxides, Synthetic; Receptors, Thromboxane; Thromboxane A2

Whole cell voltage clamp recordings were used to investigate the effects of thromboxane A2 (TXA2) agonists on the voltage-dependent Ca2+ currents in rat hippocampal CA1 neurons. TXA2 agonists [1S-[1 alpha, 2 beta(5Z), 3 alpha(1E, 3S*)4 alpha ]]-7-[3-[3-hydroxy-4-(4'-iodophenoxy)-1-butenyl]-7-oxabicyclo [2,2,1]heptan-2-yl]-5-heptenoic acid (I-BOP) and U-46619, reversibly suppressed the whole cell Ca2+ currents in a concentration-dependent manner. The effect was blocked by specific TXA2 receptor antagonist, SQ-29548. I-BOP as well as U-46619 inhibited both omega-conotoxin GVIA (CgTx)-sensitive and nimodipine sensitive Ca2+ currents but had no effect on CgTx/nimodipine insensitive Ca2+ currents. The I-BOP and U-46619 inhibition of Ca2+ currents was blocked by internal dialysis of hippocampal neurons with specific protein kinase C (PKC) inhibitors, NPC-15437 and PKC inhibitor-(19-36). Pretreatment of hippocampal neurons with either 5 micrograms/ml pertussis toxin (PTX) or 5 micrograms/ml cholera toxin (CTX) did not significantly affect the suppression of the Ca2+ currents by I-BOP and U-46619. Dialyzing with 1 mM guanosine 5'-O-(3-thiotriphosphate) or 1 mM GDP significantly attenuated the I-BOP or U-46619 action. These results demonstrate that TXA2 agonists inhibit both CgTx- and nimodipine-sensitive Ca2+ currents but not CgTx/nimodipine-insensitive currents in rat hippocampal CA1 neurons via a PTX- and CTX-insensitive G protein-coupled activation of the PKC pathway.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Baclofen; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Calcium Channel Blockers; Calcium Channels; Cells, Cultured; Cyclic AMP; Enzyme Activation; Fatty Acids, Unsaturated; GTP-Binding Proteins; Hippocampus; Ion Channel Gating; Membrane Potentials; Nimodipine; omega-Conotoxin GVIA; Patch-Clamp Techniques; Peptides; Prostaglandin Endoperoxides, Synthetic; Protein Kinase C; Rats; Rats, Sprague-Dawley; Receptors, Thromboxane; Thromboxane A2

We have cloned a rat kidney thromboxane A2 receptor (TP) cDNA. This receptor was shown to be functional in that the thromboxane A2 mimetics, U46619 and I-BOP, elicited calcium transients in Xenopus oocytes and HEK293 cells expressing the TP receptor, respectively. Comparison of the affinities of the rat and human TP sites for the agonist radiolgand [125 I] BOP showed that the rat TP site has about a ten-fold higher affinity for this drug (KD approximately 0.5 vs. 4.4 nM) while the affinities of the two sites for other compounds (U46619, I-PTH-OH) were the same. Our results are significant in that they identify a cloned TP as having a picomolar affinity for [125 I] BOP.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Amino Acid Sequence; Animals; Binding, Competitive; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cells, Cultured; Cloning, Molecular; Electrophysiology; Fatty Acids, Unsaturated; Hydrazines; Kidney; Male; Molecular Sequence Data; Oocytes; Prostaglandin Endoperoxides, Synthetic; Rats; Rats, Sprague-Dawley; Receptors, Thromboxane; Sequence Alignment; Sequence Analysis; Thromboxane A2; Xenopus laevis

In this study, we first tested the hypothesis that the previously demonstrated circulatory failure and thrombocytopenia induced by intracaval administration of thromboxane A2 (TxA2) analogues in nonpregnant (NP) rabbits [G. Losonczy, I. Mucha, J. DiPirro, J. Sweeney, G. Brown, J. Brentjens, and R. Venuto. Am. J. Physiol. 265 (Regulatory Integrative Comp. Physiol. 34): R772-R780, 1993] could be avoided if the compounds were given instead into the aortic arch. Conscious New Zealand White rabbits received bolus injections of U-46619 (5-20 micrograms) through a previously implanted catheter threaded into the aortic arch. Indeed, mean arterial pressure (MAP) rose modestly, and thrombocytopenia did not develop. Next, we compared the blood pressure responses of pregnant (P) rabbits with those of NP rabbits to intra-aortic U-46619 and I-BOP, because they had been found to be resistant to both the hypotensive and platelet aggregatory effects of intracaval U-46619. Resting blood pressure was lower in P than in NP rabbits (74 +/- 3 vs. 95 +/- 2 mmHg), but showed a greater increase in response to U-46619. For example, following a 20-micrograms dose blood pressure rose 20 +/- 0.3 mmHg in P vs. 12 +/- 2.1 mmHg in NP rabbits (P < 0.02). Similar results were obtained with the second TxA2 analogue I-BOP. Pregnancy-induced enhancement of blood pressure elevation may be the consequence of peripheral vasoconstriction, which was not seen in NP rabbits. Thus the actions of TxA2 analogues U-46619 and I-BOP are markedly influenced by the route of administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Aorta; Blood Pressure; Bridged Bicyclo Compounds, Heterocyclic; Fatty Acids, Unsaturated; Female; Heart Rate; Injections, Intra-Arterial; Platelet Count; Pregnancy; Pregnancy, Animal; Prostaglandin Endoperoxides, Synthetic; Rabbits; Thromboxane A2; Thromboxanes

8-Epi-prostaglandin F2 alpha (8-epi-PGF 2 alpha) is a nonenzymatic, free radical-catalyzed peroxidation product of arachidonic acid that has potent biological activity, including contraction of vasculature and inhibition of aggregation induced by thromboxane (TX) A2 mimetics. In the present study, we demonstrate that 8-epi-PGF2 alpha could inhibit platelet aggregation induced by the TX mimetics U46619 and I-BOP as well as low-dose collagen but not thrombin or the primary wave of aggregation caused by high-dose ADP. The secondary (TX-dependent) wave of aggregation induced by high-dose ADP, however, was not affected. This suppression was dose dependent where 3.6 and 3.3 microM of 8-epi-PGF2 alpha caused 50% inhibition of platelet aggregation induced by U46619 and I-BOP, respectively, whereas 10 microM caused approximately 72% inhibition of collagen-induced aggregation. In contrast, 8-epi-PGF2 alpha significantly potentiated reversible platelet aggregation in response to low-dose ADP. These results indicate that 8-epi-PGF2 alpha has partial agonist activity. 9 alpha,11 beta-PGF2, a structural isomer of 8-epi-PGF2 alpha, inhibited platelet aggregation induced by collagen, high- and low-dose ADP and thrombin, demonstrating marked differences between structural isomers where 9 alpha,11 beta-PGF2 inhibited platelet aggregation induced by TX-dependent as well as TX-independent stimuli. In addition to platelet aggregation, we performed competition-binding assays on washed human platelets using [125I]BOP to further investigate the interaction of 8-epi-PGF2 alpha and 9 alpha,11 beta-PGF2 with TXA2/PGH2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adult; Binding, Competitive; Blood Platelets; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Dinoprost; Fatty Acids, Unsaturated; Humans; In Vitro Techniques; Platelet Aggregation Inhibitors; Prostaglandin Endoperoxides, Synthetic; Prostaglandins; Prostaglandins H; Receptors, Prostaglandin; Receptors, Thromboxane; Receptors, Thromboxane A2, Prostaglandin H2; Thromboxane A2

Prostaglandin (PG) I2 elicits a biphasic concentration-response curve in rat aorta: lower concentrations elicit relaxation, whereas at higher concentrations, the relaxation is reversed. The purpose of this study was to investigate 1) the nature of the receptors that mediate these effects and 2) whether the relaxant efficacy of PGI2 is decreased at higher PGI2 concentrations by PGI2-induced contraction. PGI2 (1 microM), the stable PGI2 analogue carbacyclin (1 microM), and PGE1 (3 microM) induced maximal relaxations of 55, 40, and 63%, respectively, of norepinephrine-contracted aorta, whereas higher concentrations of PGI2, carbacyclin, and PGE1 reversed the relaxation. The thromboxane (Tx) A2-PGH2 receptor antagonist, SQ-29548, abolished the reversal of the PGI2-, carbacyclin-, and PGE1-induced relaxation, and maximal relaxations to PGI2, carbacyclin, and PGE1 increased to 73, 85, and 89% of the norepinephrine contraction, respectively, with 50% effective concentrations of 0.16, 0.43, and 0.83 microM, respectively. PGE2 and PGD2 did not induce relaxation in the presence or absence of SQ-29548. PGI2 and carbacyclin displaced the TxA2-PGH2 receptor ligand 1S-[1 alpha,2 beta(5Z),3 alpha(1E,3S),4 alpha]-7-(3-[3-hydroxy-4-(p- [125I]iodophenoxy)-1-butenyl]7-oxabicyclo[2.2.1]hept-2-yl]-5- heptenoic acid from cultured rat aorta smooth muscle cells with concentrations of competing ligand that displaced 50% of the specifically bound radioligand from its binding site of 6.0 and 2.3 microM, respectively. These results suggest that 1) PGI2 induces relaxation through a PGI2-PGE1 receptor, and 2) higher concentrations of PGI2 act at the TxA2-PGH2 receptor to decrease PGI2-induced relaxation.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cells, Cultured; Epoprostenol; Fatty Acids, Unsaturated; Hydrazines; Male; Muscle, Smooth, Vascular; Prostaglandin Endoperoxides, Synthetic; Prostaglandins; Rats; Rats, Sprague-Dawley; Receptors, Thromboxane; Thromboxane A2; Vasoconstriction; Vasoconstrictor Agents; Vasomotor System

Rat glomerular mesangial cells were used to investigate mechanisms of thromboxane A2 (TxA2) receptor regulation in the kidney. Exposure of mesangial cells to the TxA2 agonist U-46619 for 10 min reduced subsequent TxA2-induced increases in inositol phosphates and intracellular Ca2+ levels by approximately 70%. This loss of receptor responsiveness could be blocked by the TxA2 receptor antagonist SQ-29548 and was reversible after removal of agonist from the incubation medium. Radioligand binding studies using the TxA2 agonist [125I]BOP suggested that exposure of mesangial cells to U-46619 for 10 min reduced TxA2 receptor responsiveness without a loss of receptor sites from plasma membrane fractions of the cell, although the density of mesangial cell TxA2 receptors was decreased by approximately 60% after more prolonged exposure of mesangial cells to thromboxane agonists. Both desensitization to U-46619 and loss of TxA2 binding sites could be attenuated by the protein kinase C (PKC) inhibitors staurosporine, sphingosine, or H-7, and TxA2 receptor responsiveness was reduced in cells incubated with phorbol esters before stimulation with thromboxane agonists. We conclude that 1) agonist-specific decreases in TxA2 receptor responsiveness may involve initial uncoupling of the receptor from its effector systems, followed by a loss of TxA2 receptor sites from plasma membrane fractions of the cell, and 2) PKC may be involved in these processes.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Binding Sites; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Cells, Cultured; Fatty Acids, Unsaturated; Glomerular Mesangium; Hydrazines; Inositol Phosphates; Intracellular Membranes; Prostaglandin Endoperoxides, Synthetic; Protein Kinase Inhibitors; Rats; Rats, Inbred Strains; Receptors, Thromboxane; Thromboxane A2; Thromboxanes

Human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors are linked to phosphoinositide-specific phospholipase C (PI-PLC) via a G protein tentatively identified as a member of the Gq class. In contrast, platelet thrombin receptors appear to activate PI-PLC via other unidentified G proteins. Platelets from most dogs are TXA2 insensitive (TXA2-); i.e., they do not aggregate irreversibly or secrete although they bind TXA2, but they respond normally to thrombin. In contrast, a minority of dogs have TXA2-sensitive (TXA2+) platelets that are responsive to TXA2. To determine the mechanism responsible for TXA2- platelets, we evaluated receptor activation of PI-PLC. Equilibrium binding of TXA2/PGH2 receptor agonists, [125I]BOP and [3H]U46619, and antagonist, [3H]SQ29,548, revealed comparable high-affinity binding to TXA2-, TXA2+, and human platelets. U46619-induced PI-PLC activation was impaired in TXA2- platelets as evidenced by reduced (a) phosphorylation of the 47-kD substrate of protein kinase C, (b) phosphatidic acid (PA) formation, (c) rise in cytosolic calcium concentration, and (d) inositol 1,4,5 trisphosphate (IP3) formation, while thrombin-induced PI-PLC activation was not impaired. GTPase activity stimulated by U46619, but not by thrombin, was markedly reduced in TXA2- platelets. Antisera to Gq class alpha subunits abolished U46619-induced GTPase activity in TXA2-, TXA2+, and human platelets. Direct G protein stimulation by GTP gamma S yielded significantly less PA and IP3 in TXA2- platelets. Immunotransfer blotting revealed comparable quantities of Gq class alpha-subunits in all three platelet types. Thus, TXA2- dog platelets have impaired PI-PLC activation in response to TXA2/PGH2 receptor agonists secondary to G protein dysfunction, presumably involving a member of the Gq class.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Blood Platelets; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Dogs; Enzyme Activation; Fatty Acids, Unsaturated; GTP Phosphohydrolases; GTP-Binding Proteins; Humans; Inositol 1,4,5-Trisphosphate; Phosphatidic Acids; Phosphorylation; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Protein Kinases; Receptors, Thromboxane; Serotonin; Thromboxane A2; Type C Phospholipases

We compared the hemodynamic actions of U-46619, a stable thromboxane A2 (TxA2) prostaglandin H2 (PGH2) analogue, in nonpregnant (NP) rabbits with those observed in late pregnant (P) rabbits. An intravenous injection of U-46619 (10 micrograms) to each of eight NP chronically instrumented rabbits (mean body weight 3.4 kg) induced an immediate (1 min) and reversible fall of cardiac output (CO, 66%) and mean arterial pressure (MAP, 41%, both P < 0.01). P rabbits (n = 6, mean body weight 3.8 kg), however, responded with an elevation of MAP (5%, P < 0.02) upon intravenous injection of the drug (10 micrograms), while CO remained unchanged. The fall of CO in NP rabbits was associated with the temporary disappearance of a fraction of circulating platelets between the superior vena cava and the aortic arch. The number of platelets at 30 and 60 s after U-46619 was reduced (P < 0.05) by 14 and 20% respectively in the aortic blood, whereas caval platelet counts were unchanged until 90 s (-6%, P < 0.05). In contrast, intraaortic administration of this drug (10 micrograms) to NP rabbits resulted in neither thrombocytopenia nor hypotension. U-46619 (10-30 micrograms i.v.) caused no decrease in platelet count in the aorta of P rabbits. In vitro, U-46619-induced aggregation of platelets harvested from P rabbits was also blunted (P < 0.001). This could not be attributed to reduced affinity or number of platelet thromboxane receptors. The data indicate that U-46619 induces a fall of arterial pressure simultaneous with intravascular platelet aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Blood Platelets; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Fatty Acids, Unsaturated; Female; Hemodynamics; Platelet Aggregation; Platelet Count; Pregnancy; Pregnancy, Animal; Prostaglandin Endoperoxides, Synthetic; Rabbits; Receptors, Thromboxane; Reference Values

Desensitization of platelet thromboxane (TX)A2/prostaglandin (PG)H2 receptors was induced by incubating platelet-rich plasma with the stable PGH2 analog 11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619) (1 microM). Iloprost, a stable prostacyclin analog, was included in the incubation to prevent platelet activation. The TXA2 mimetic, [1S-1 alpha,2 beta(5Z), 3 alpha(1E,3S*), 4 alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo - [2.2.1]heptan-2-yl]-5-heptenoic acid (I-BOP), was used to induce platelet aggregation, shape change and increases in intracellular free calcium. The EC50 values for I-BOP-induced rise in intracellular free calcium (control = 10.2 +/- 1.5 nM; desensitized = 79.4 +/- 22.4 nM, n = 6, P less than .05), aggregation (control = 15.8 +/- 2.4 nM; desensitized = 51.7 +/- 11.9 nM; P less than .05, n = 5) and shape change (control = 172 +/- 37 pM; desensitized = 350 +/- 60 pM; P less than .05, n = 7) were increased by the preincubation with U46619. Aggregation responses to thrombin and the calcium ionophore, ionomycin, were unaltered by the preincubation with U46619. Equilibrium binding studies at pH 7.4 revealed a decrease in the number of binding sites for the receptor antagonist 9,11-dimethylmethano-11,12- methano-16(3-iodo-4-hydroxyphenyl)-13,14-dihydro-13-aza-15 alpha beta-omega- tetranor-TXA2 [125I]PTA-OH) (control = 3246 +/- 509 sites/platelet, desensitized = 2198 +/- 324 sites/platelet, n = 6, P less than .05) without a change in affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Binding Sites; Binding, Competitive; Blood Platelets; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Fatty Acids, Unsaturated; Humans; Iloprost; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Receptors, Prostaglandin; Receptors, Thromboxane; Vasoconstrictor Agents

The thromboxane (TXA2) receptors on rat and guinea-pig lung strips were compared using TXA2 agonists and TXA2 receptor antagonists. On rat lung strip several TXA2 mimetics were full agonists whilst the primary prostanoids lacked agonist activity. On guinea-pig lung strip the same agonists displayed markedly different efficacies. Both preparations contained homogeneous populations of TXA2 receptors as evidenced by BAY u3405 giving comparable pA2 values against four TXA2 mimetics. However, the observed pA2's of nine different TXA2 receptor antagonists, determined against U46619, did not correlate between the two preparations. These results point to the existence of TXA2 receptor subtypes.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Carbazoles; Dose-Response Relationship, Drug; Fatty Acids, Unsaturated; Guinea Pigs; In Vitro Techniques; Lung; Male; Muscle Contraction; Muscle, Smooth; Prostaglandin Endoperoxides, Synthetic; Rats; Rats, Sprague-Dawley; Receptors, Thromboxane; Sulfonamides; Thromboxanes

The effects of changes in pH on the binding of agonists and antagonists to the human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor were determined. Competition binding studies were performed with the TXA2/PGH2 mimetic [1S-1 alpha,2 beta (5Z), 3 alpha(1E,3R*),4 alpha)]-7-[3-(3-hydroxy-4'-iodophenoxy)-1-buteny) 7-oxabicyclo-[2.2.1]-heptan-2-yl]-5-heptenoic acid ([125I]BOP). The pH optimum for binding of [125I] BOP to washed human platelets was broad with a range of pH 4-6 in contrast to that of the TXA2/PGH2 receptor antagonist 9,11-dimethyl-methano-11,12-methano-16-(3-iodo-4-hydroxyl)-13-aza-15 alpha,beta-omega-tetranorthromboxane A2 ([125I]PTA-OH) which was 7.4. Scatchard analysis of [125I]BOP binding in washed platelets at pH 7.4, 6.0, and 5.0 revealed an increase in affinity (Kd = 1.16 +/- 0.06, 0.64 +/- 0.09, and 0.48 +/- 0.05 nM, respectively) and an increase in the number of receptors (Bmax = 2807 +/- 415, 5397 +/- 636, and 7265 +/- 753 sites/platelet, respectively). The potency of I-BOP to induce shape change in washed platelets at pH 6.0 was also significantly increased from an EC50 value of 0.34 +/- 0.016 nM at pH 7.4 to 0.174 +/- 0.014 nM at pH 6.0 (n = 6, p less than 0.05). In contrast, the EC50 value for thrombin was unaffected by the change in pH. In competition binding studies with [125I]BOP, the affinity of the agonists U46619 and ONO11113 were increased at pH 6.0 compared to 7.4. In contrast, the affinity of the TXA2/PGH2 receptor antagonists I-PTA-OH, SQ29548, and L657925 were either decreased or unchanged at pH 6.0 compared to 7.4. Diethyl pyrocarbonate and N-bromosuccinimide, reagents used to modify histidine residues, reversed the increase in affinity of [125I]BOP at pH 6.0 to values equivalent to those at pH 7.4. In solubilized platelet membranes, the effects of NBS were blocked by coincubation with the TXA2/PGH2 mimetic U46619. The results suggest that agonist and antagonist binding characteristics are different for the TXA2/PGH2 receptor and that histidine residue(s) may play an important role in the binding of TXA2/PGH2 ligands to the receptor.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Blood Platelets; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Bromosuccinimide; Carbazoles; Cell Membrane; Diethyl Pyrocarbonate; Fatty Acids, Unsaturated; Histidine; Humans; Hydrazines; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Ligands; Platelet Activation; Prostaglandin Endoperoxides, Synthetic; Protein Binding; Receptors, Prostaglandin; Receptors, Thromboxane; Receptors, Thromboxane A2, Prostaglandin H2; Solubility; Thromboxane A2

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

Stable synthetic mimetics of thromboxane (TX) A2 and prostaglandin (PG) H2 have been synthesized and reported to stimulate platelets and vascular smooth muscle. The synthetic agonists induce aggregation of isolated platelets and contraction of vascular tissue. The tritiated agonists [3H]U46619 and [3H]U44069 have been used in radioligand binding studies to characterize platelet and vascular smooth muscle TXA2/PGH2 receptors, but have limited usefulness due to their low specific activities and variable specific binding. In an attempt to overcome these problems, we have synthesized a stable, high affinity, 125I-radiolabeled TXA2/PGH2 receptor agonist, [1S-(1 alpha, 2 beta (5Z), 3 alpha(1E,3S*), 4 alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo - [2.2.1]heptan-2-yl]-5-heptenoic acid (I-BOP). I-BOP induced shape change, increased intracellular free calcium concentrations and aggregated isolated human platelets (EC50 = 0.21 +/- 0.05 nM, n = 3; 4.1 +/- 1.1 nM, n = 4; 10.8 +/- 3 nM, n = 9, respectively). The kinetically determined Kd was 1.02 +/- 0.33 nM (kobs = 0.19 +/- 0.05 min-1, k-1 = 0.097 +/- 0.02 min-1, k1 = 0.119 +/- 0.03 min-1 M, n = 4). Equilibrium binding studies of [125I]BOP to isolated human platelets indicated one class of high affinity sites, Kd = 2.2 +/- 0.3 nM and a maximum binding of 0.028 +/- 0.002 x 10(-12) mol/10(7) platelets (1699 +/- 162 sites/platelet, n = 9).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Bridged-Ring Compounds; Fatty Acids, Unsaturated; Humans; Iodine Radioisotopes; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Radioligand Assay; Receptors, Prostaglandin; Receptors, Thromboxane; Receptors, Thromboxane A2, Prostaglandin H2