sq-29548 and 7-(3-(3-hydroxy-4-(4--iodophenoxy)-1-butenyl)-7-oxabicyclo(2.2.1)heptan-2-yl)-5-heptenoic-acid

Thromboxane (TX) A2 is released from multiple cell types and is a prime mediator of the pathogenesis of many vascular events, including angiogenesis. Endothelial cells express TXA2 receptors (TP) but the effects of TP stimulation on angiogenesis remain controversial. In this study, we show that stimulation of endothelial cell TP impairs ligand-induced FGF receptor internalization and consequently abrogates FGF-2-induced endothelial cell migration in vitro and angiogenesis in vivo. Prevention of FGF-2-induced angiogenesis was associated with expression of the TPbeta isoform. The deficit in FGFR1 internalization was mediated through activation of TPbeta preventing the FGF-2-mediated decrease in p53 expression, thus enhancing thrombospondin-1 (TSP-1) release from EC and reducing FGFR1 internalization. Once released TSP-1 interacted with the alpha(v)beta3 integrin on the EC surface. On stimulation, FGFR1 and alpha(v)beta3 were found to associate in a complex. We determined that complex formation was important for receptor internalization as conditions that inhibit FGFR1 internalization, such as inappropriate ligation of alpha(v)beta3 by either TSP-1 or a neutralizing antibody, disrupted the complex. These results establish a novel role for isoform specific regulation of angiogenesis by TP, provide the first functional significance for the existence of two TP isoforms in humans, and clarify the mechanism by which TP signaling regulates FGFR1 kinetics and signaling.

Topics: Bridged Bicyclo Compounds, Heterocyclic; Cell Cycle; Cell Movement; Endocytosis; Endothelial Cells; Endothelium, Vascular; Fatty Acids, Unsaturated; Fibroblast Growth Factor 2; Humans; Hydrazines; Inflammation; Integrin alphaVbeta3; Ischemia; Ligands; Neovascularization, Physiologic; Protein Isoforms; Receptor Protein-Tyrosine Kinases; Receptor, Fibroblast Growth Factor, Type 1; Receptors, Fibroblast Growth Factor; Receptors, Thromboxane A2, Prostaglandin H2; Thrombospondin 1; Thromboxane A2; Transcription, Genetic; Tumor Suppressor Protein p53

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

A single gene encodes the human thromboxane receptor (TP), of which there are two identified splice variants, alpha and beta. Both isoforms are rapidly phosphorylated in response to thromboxane agonists when overexpressed in human embryonic kidney 293 cells; this phenomenon is only slightly altered by inhibitors of protein kinase C. Pharmacological studies have defined two classes of TP in human platelets; sites that bind the agonist I-BOP with high affinity support platelet shape change. Low affinity sites, which irreversibly bind the antagonist GR 32191, transduce platelet activation and aggregation. Isoform-specific antibodies permitted detection of TPalpha, but not TPbeta, from human platelets, although mRNA for both isoforms is present. A broad protein band of 50-60 kDa, reflecting the glycosylated receptor, was phosphorylated upon activation of platelets for 2 min with I-BOP. This was a rapid ( approximately 30 s) and transient (maximum, 2-4 min) event and was inhibited by TP antagonists. Both arachidonic acid and low concentrations of collagen stimulated TPalpha phosphorylation, which was blocked by cyclooxygenase inhibition or TP antagonism. Blockade of the low affinity TP sites with GR 32191 prevented I-BOP-induced TPalpha phosphorylation. This coincided with agonist-induced platelet aggregation and activation but not shape change. Also, activation of these sites with the isoprostane iPF2alpha-III induced platelet shape change but not TPalpha phosphorylation. Heterologous TP phosphorylation was observed in aspirin-treated platelets exposed to thrombin, high concentrations of collagen, and the calcium ionophore A 23187. Both homologous and heterologous agonist-induced phosphorylation of endogenous TPalpha was blocked by protein kinase C inhibitors. TPalpha was the only isoform detectably translated in human platelets. This appeared to correspond to the activation of the low affinity site defined by the antagonist GR 32191 and not activated by the high affinity agonist, iPF2alpha-III. Protein kinase C played a more important role in agonist-induced phosphorylation of native TPalpha in human platelets than in human embryonic kidney 293 cells overexpressing recombinant TPalpha.

Topics: Arachidonic Acid; Aspirin; Biphenyl Compounds; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Electrophoresis, Polyacrylamide Gel; Fatty Acids, Unsaturated; Heptanoic Acids; Humans; Hydrazines; Phosphorylation; Platelet Activation; Platelet Aggregation Inhibitors; Protein Kinase C; Receptors, Thromboxane; Thrombin; Thromboxane A2

Previous subtyping of thromboxane A2 (TXA2) receptors in platelets and vascular smooth muscle cells was based on pharmacological criteria. Two distinct carboxy-terminal splice variants for TXA2 receptors exist and they couple to several different G protein alpha subunits including Galpha13, but it has not been established whether either or both isoforms interact with and signal through it. We sought to determine: (1) which TXA2 receptor isoforms exist in vascular smooth muscle, (2) if Galpha13 is present in vascular smooth muscle and (3) if Galpha13 interacts with either or both of the two TXA2 receptor isoforms as determined by changes in ligand binding properties and generation of intracellular signals. Both TXA2 receptor isoforms and Galpha13 were found in vascular smooth muscle cells. Both the alpha and beta isoforms of the TXA2 receptors were transiently transfected with or without Galpha13 into COS-7 (radioligand binding assays) or CHO cells (agonist induced Na+/H+ exchange). Co-expression of each receptor isoform with Galpha13 significantly (P<0.05) increased the affinity of each receptor for the two agonists, I-BOP and ONO11113, and decreased the affinity of the receptor for the antagonists, SQ29,548 and L657,925. I-BOP stimulated Na+/H+ exchange in vascular smooth muscle cells. Co-expression of Galpha13 with each TXA2 receptor isoform in CHO cells resulted in a significant (P<0.04) agonist induced increase in Na+/H+ exchange compared to cells not transfected with Galpha13. The results support the possibility that the previous classification of TXA2 receptor subtypes based on pharmacological criteria reflect unique interactions with specific G protein alpha subunits.

Topics: Animals; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Cell Line; CHO Cells; Cloning, Molecular; COS Cells; Cricetinae; DNA, Complementary; Fatty Acids, Unsaturated; GTP-Binding Proteins; Hydrazines; Muscle, Smooth, Vascular; Protein Isoforms; Receptors, Thromboxane; Transfection

The two most extensively characterized thromboxane/prostaglandin endoperoxide (TP) receptors, from human platelets and rat vascular smooth muscle, exhibit thromboxane agonist [15-(1alpha,2beta(5Z), 3alpha-(1E,3S), 4alpha)]-7-[3-hydroxy-4-(p-iodophenoxy)-1-butenyl-7-oxabi cyclohepteno ic acid (I-BOP) binding affinities that differ by an order of magnitude, rat TP having the higher affinity. We utilized this difference in I-BOP affinity to identify structural determinants of TP receptor heterogeneity. No significant difference was found in the rank order of affinities for a series of thromboxane receptor ligands to bind to cloned human TPalpha versus rat TP, indicating that these represent species homologs, not distinct TP subtypes. Structural determinants for observed differences in I-BOP binding Kd were localized by creating chimeric human/rat TP followed by mutational substitution of specific critical amino acids. Initially, seven chimeric receptors with splice sites in transmembranes 1, 2, 4, or 7 were constructed and expressed in HEK293 cells for analysis of ligand binding properties. Substitution of any part except the carboxyl tail of the human TP into the rat TP resulted in a receptor with I-BOP binding affinity intermediate between the two. Analysis of chimeras in which only the extracellular amino terminus and a portion of transmembrane 1 were switched localized the determinant of high affinity binding to the region between amino acids 3 and 40. Using this chimera, amino acids in the human portion (extracellular amino terminus and part of transmembrane 1) were replaced with analogous amino acids from rat TP to regain high affinity I-BOP binding. Only when amino acid Val37 and either Val36 or Ala40 were reverted to their respective rat TP counterparts (Ala36, Leu37, and Gly40, respectively) was high affinity I-BOP binding recovered. The mechanism for the increased I-BOP affinity may be the lengthening of the amino acid side chain at position 37, thus extending this group further into the putative I-BOP binding pocket, with compensatory shortening of side chains in spatially adjacent amino acids.

Topics: Amino Acid Sequence; Animals; Bridged Bicyclo Compounds, Heterocyclic; Fatty Acids, Unsaturated; Humans; Hydrazines; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Rats; Receptors, Prostaglandin; Receptors, Thromboxane; Recombinant Fusion Proteins; Structure-Activity Relationship

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

A thromboxane A2 receptor cDNA was isolated from a human placenta library by polymerase chain reaction (PCR) and was expressed in insect (Sf21) cells using baculovirus system. The recombinant receptor exhibited [3H]-SQ29548 and [125I]-BOP binding activities with Kd values of 1.01 +/- 0.09 nM and 1.63 +/- 0.23 nM, respectively. The receptor binding activity was inhibited by dithiothreitol in a time- and concentration-dependent manner, indicating the involvement of disulfide linkage in ligand binding. The role of the four conserved cysteinyl residues in ligand binding was further examined by site-directed mutagenesis. Each of the four cysteinyl residues was respectively mutated to a serine residue. C102S, C105S, and C183S mutants exhibited no ligand binding activity although successful expression was achieved as revealed by immunoblot analysis, whereas C257S mutant retained most of the binding activity. Homology analysis of all prostanoid receptors indicates that Cys-105 (first extracellular loop) and Cys-183 (second extracellular loop) are conserved and are presumed to form a disulfide bond for receptor stability as suggested by the inhibition of ligand binding by dithiothreitol reduction. Loss of binding activity by C102S mutant revealed that the sulfhydryl group of Cys-102 must play an essential role in ligand binding. Molecular modeling proposed that the Ser-201 is involved in interacting with TXA2 by forming hydrogen bonding. Point mutations of both Ser-201 and a conserved Ser-255 did not affect the ligand binding specificity and affinity for [3H]-SQ29548, but have significantly altered Kd values for [125I]-BOP. These results indicate that various cysteinyl and serine residues of thromboxane A2 receptor may play different roles in ligand binding.

Topics: Amino Acid Sequence; Animals; Baculoviridae; Base Sequence; Binding Sites; Bridged Bicyclo Compounds, Heterocyclic; Cell Line; Cloning, Molecular; Cysteine; Disulfides; DNA Primers; DNA, Complementary; Fatty Acids, Unsaturated; Humans; Hydrazines; Hydrogen Bonding; Kinetics; Ligands; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Point Mutation; Polymerase Chain Reaction; Receptors, Thromboxane; Recombinant Proteins; Sequence Homology, Amino Acid; Serine; Spodoptera

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

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

The isoprostanes are nonenzymatically generated prostanoids synthesized in vivo in humans and rats through reactions catalyzed by free oxygen radicals. 8-Epi-prostaglandin F2 alpha (8-epi-PGF2 alpha), an F2-isoprostane, is a potent smooth muscle constrictor. A thromboxane A2 (TxA2) receptor antagonist, SQ 29548, blocks renal vasoconstriction during 8-epi-PGF2 alpha administration in rats. With the use of cultured rat aortic smooth muscle cells, we found specific binding sites for [3H]SQ 29548 and for [125I]BOP, a TxA2 agonist. Both ligands were displaced from these binding sites by 8-epi-PGF2 alpha, although with significantly lesser potency than nonlabeled SQ 29548, I-BOP, or U-46619, a TxA2 agonist. In contrast, 8-epi-PGF2 alpha stimulated inositol 1,4,5-trisphosphate production and DNA synthesis in these cells with significantly greater potency than any TxA2 agonist, effects only partially inhibited by SQ 29548. In human TxA2 receptor cDNA-transfected cells, competition by 8-epi-PGF2 alpha for specific [3H]SQ 29548 binding was negligible. Thus 8-epi-PGF2 alpha probably exerts its biological actions in vascular smooth muscle through activation of receptor sites related to but distinct from TxA2 receptors. The existence of such binding sites suggests novel avenues for investigation into the biology of TxA2 and of free radical-mediated tissue injury.

Topics: Animals; Binding, Competitive; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Transformed; Cells, Cultured; Dinoprost; DNA; Fatty Acids, Unsaturated; Hydrazines; Inositol 1,4,5-Trisphosphate; Male; Muscle, Smooth, Vascular; Rats; Receptors, Thromboxane; Transfection

Thromboxane (TX) A2 has been implicated as an important pathophysiologic mediator of a variety of cardiovascular diseases. Monocytes synthesize TXA2 and it modulates their function. This study sought to characterize monocyte TXA2 receptors. Radioligand binding studies were performed on membranes prepared from equine peripheral blood monocytes using [125I]BOP, a TXA2 receptor agonist. [125I]BOP bound to a single class of binding sites (Kd = 1.0 +/- 0.3 nM and Bmax = 389 +/- 191 fmol/mg protein; n = 5). Several TXA2 receptor agonists and antagonists competed for binding with [125I]BOP. I-BOP produced a concentration-dependent inhibition of endotoxin-induced tumor necrosis factor (TNF) activity (IC50 = 9.6 +/- 2.5 nM; n = 5). In contrast to its effects in platelets and vascular smooth muscle, I-BOP significantly increased cAMP formation in monocytes (EC50 = 22 +/- 3.6 nM; n = 4). The TXA2 receptor antagonists SQ29548 (5.6 microM) and L657925 (0.13 microM) significantly blocked I-BOP-stimulated cAMP formation but did not block 250 nM prostaglandin E2-stimulated cAMP formation. These data support the presence of a TXA2 receptor in equine peripheral blood monocytes. Activation of this receptor results in suppression of endotoxin-induced TNF formation and stimulation of cAMP production. Increased cAMP production after receptor activation suggests that this receptor may represent a unique subclass of TXA2 receptors.

Topics: Animals; Binding, Competitive; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Carbazoles; Cell Membrane; Cell Separation; Cyclic AMP; Fatty Acids, Unsaturated; Horses; Hydrazines; Iodine Radioisotopes; Kinetics; Monocytes; Radioligand Assay; Receptors, Thromboxane; Tumor Necrosis Factor-alpha

8-iso-PGE2, an E2-isoprostane, decreased GFR and RPF dose-dependently in rats, but with lesser potency than 8-epi-PGF2 alpha, an F2-isoprostane. This effect was abolished by SQ29,548. 8-iso-PGE2 displaced [3H]SQ29,548 or [125I]BOP binding in aortic smooth muscle cells with the affinity rank order of SQ29,548 > = I-BOP > U46,619 > 8-iso-PGE2 > PGF2 alpha, while it activated phospholipase C with a potency greater than those of I-BOP or U46,619 and lesser than that of 8-epi-PGF2 alpha. We concluded that 8-iso-PGE2 is a renal vasoconstrictor linked to phosphoinositide metabolism. Its vascular smooth muscle contractile actions are likely mediated through activation of putative thromboxane A2 receptor-like "isoprostane receptors."

Topics: Animals; Aorta, Thoracic; Binding, Competitive; Blood Pressure; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cells, Cultured; Dinoprostone; Enzyme Activation; Fatty Acids, Unsaturated; Glomerular Filtration Rate; Hydrazines; Inositol 1,4,5-Trisphosphate; Isomerism; Isoprostanes; Kidney; Kinetics; Male; Muscle, Smooth, Vascular; Phospholipases; Rats; Rats, Sprague-Dawley; Receptors, Prostaglandin; Receptors, Prostaglandin E; Receptors, Thromboxane; Regional Blood Flow

Thromboxane A2 (TxA2) stimulates contraction of glomerular mesangial cells. However, mesangial cell TxA2 receptors have not been previously characterized. We therefore investigated TxA2 binding and TxA2-associated signal transduction pathways in rat glomerular mesangial cells using the specific thromboxane receptor agonist (1S-[1 alpha,2 beta(5Z),3 alpha-(1E,3S)4 alpha])-7-(3-[3-hydroxy-4-(p- iodophenoxy)-1-butenyl]7-oxabicyclo[2.2.1]hept-2-yl)-5-heptenoic acid (IBOP). In these cells, [125I]BOP binding was saturable, displaceable, and of high affinity. Scatchard analysis revealed a single class of binding sites with a dissociation constant (Kd) of 293 pM and a maximal density of binding sites (Bmax) of 33 fmol/mg protein. Specific binding was inhibited by the thromboxane agonist (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U-46619) [inhibitor dissociation constant (Ki) = 297 nM] and the TxA2 receptor antagonists SQ 29548 (Ki = 1 nM) and (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 (GR 32191) (Ki = 92 nM). Binding was also highly specific for thromboxane because prostaglandin E2 (Ki = 16 microM) and the inactive thromboxane metabolite, TxB2 (Ki = 41 microM), were approximately 1,000-fold less potent at inhibiting binding. IBOP stimulated phosphatidylinositol hydrolysis with an effective concentration of drug that produces 50% of the maximal response of 229 pM, which correlated well with the equilibrium Kd and enhanced phosphorylation of an acidic 80-kDa protein substrate for protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Binding, Competitive; Biphenyl Compounds; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cells, Cultured; Dose-Response Relationship, Drug; Fatty Acids, Unsaturated; Heptanoic Acids; Hydrazines; Inositol Phosphates; Intracellular Signaling Peptides and Proteins; Kidney Glomerulus; Kinetics; Membrane Proteins; Myristoylated Alanine-Rich C Kinase Substrate; Phosphorylation; Protein Kinase C; Proteins; Rats; Signal Transduction; Thromboxanes

Binding and function of BMS 180,291 ([(+)1S-(1 alpha,2 alpha,3 alpha,4 alpha)]-2-[[3-[4-[(n-pentylamino)carbonyl]-2-oxazolyl]-7- oxabicyclo[2.2.1] hept-2-yl]methyl]benzenepropanoic acid]) in human platelets was examined. Kinetic determination of [3H]BMS 180,291 binding produced ligand-receptor association and dissociation rates of 1.4 x 10(7) +/- 0.2 M-1 x min-1 (n = 5) and 0.04 +/- 0.005 min-1 (n = 5), respectively. The resultant Kd was 3.1 +/- 1.1 nM (n = 5). Saturation binding analysis in platelet membranes was consistent with a single class of [3H]BMS 180,291 binding sites with a Kd of 3.6 +/- 0.19 nM (n = 4) and a binding site maxima (Bmax) of 2099.1 +/- 70.3 fmol/mg of protein (n = 4). Specific [3H]BMS 180,291 binding was inhibited by thromboxane A2/endoperoxide receptor antagonists and agonists with a rank order of potency of: BMS 180,291 > or = SQ 29,548 = I-BOP race 15-(1 alpha,2 beta(5Z), 3 alpha(1E,3S),4 alpha) d7-[3-(3-hydroxy-4-(p-iodophenoxy)-1-butenyl)-7- oxabicyclo[2.2.1]hept-2-yl]5-heptenoic acid) > or = BM 13,505 > or = SQ 30,741 = U 44,609 > U 46,619 >> BM 13,177. Prostaglandin E2 and prostacyclin did not appreciably inhibit the specific binding of [3H]BMS 180,291. BMS 180,291 (10 nM-5 microM) shifted the I-BOP-induced platelet shape change curve to the right in a parallel manner without reduction of the maximal response (KB = 13 +/- 3.5 nM; pA2 = 8 +/- 0.2; slope = -1.0 +/- 0.05), whereas 30 nM drug decreased the maximal I-BOP-induced platelet aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Biphenyl Compounds; Blood Platelets; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Fatty Acids, Unsaturated; Heptanoic Acids; Humans; Hydrazines; In Vitro Techniques; Kinetics; Oxazoles; Platelet Aggregation; Propionates; Receptors, Thromboxane

Topics: Animals; Blood Platelets; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Fatty Acids, Unsaturated; Guinea Pigs; Hydrazines; Lung; Muscle, Smooth, Vascular; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandins H; Rats; Receptors, Prostaglandin; Receptors, Thromboxane; Receptors, Thromboxane A2, Prostaglandin H2; Thromboxane A2

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