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Title: Binding of an ( sup 125 I) labelled thromboxane A2/prostaglandin H2 receptor agonist to baboon platelets

Abstract

To characterize the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor on baboon platelets the binding of (125I)BOP was studied. (125I)BOP bound to washed baboon platelets in a saturable manner. Scatchard analysis of binding isotherms revealed a Kd of 1.12 +/- 0.08 nM and a binding capacity of 54 +/- 5 fmoles/10(8) platelets (326 sites/platelet). Several TXA2/PGH2 agonists and antagonists displaced (125I)BOP from its baboon platelet binding site with a rank order of potency similar to human platelets: I-BOP greater than SQ29548 greater than U46619 = I-PTA-OH greater than PTA-OH. I-BOP aggregated washed baboon platelets with an EC50 of 10 +/- 4 nM. The results indicate that (125I)BOP binds to the TXA2/PGH2 receptor on baboon platelets and that this receptor is similar to its human counterpart.

Authors:
;  [1]
  1. (Univ. of Texas Health Science Center, San Antonio (USA))
Publication Date:
OSTI Identifier:
6828247
Resource Type:
Journal Article
Resource Relation:
Journal Name: Prostaglandins; (USA); Journal Volume: 38:6
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; PROSTAGLANDINS; BIOCHEMICAL REACTION KINETICS; RECEPTORS; CHEMICAL COMPOSITION; BABOONS; BLOOD PLATELETS; IODINE 125; TRACER TECHNIQUES; ANIMALS; BETA DECAY RADIOISOTOPES; BIOLOGICAL MATERIALS; BLOOD; BLOOD CELLS; BODY FLUIDS; DAYS LIVING RADIOISOTOPES; ELECTRON CAPTURE RADIOISOTOPES; INTERMEDIATE MASS NUCLEI; IODINE ISOTOPES; ISOTOPE APPLICATIONS; ISOTOPES; KINETICS; MAMMALS; MATERIALS; MEMBRANE PROTEINS; MONKEYS; NUCLEI; ODD-EVEN NUCLEI; ORGANIC COMPOUNDS; PRIMATES; PROTEINS; RADIOISOTOPES; REACTION KINETICS; VERTEBRATES 550201* -- Biochemistry-- Tracer Techniques

Citation Formats

Dorn, G.W. II, and De Jesus, A.. Binding of an ( sup 125 I) labelled thromboxane A2/prostaglandin H2 receptor agonist to baboon platelets. United States: N. p., 1989. Web. doi:10.1016/0090-6980(89)90047-6.
Dorn, G.W. II, & De Jesus, A.. Binding of an ( sup 125 I) labelled thromboxane A2/prostaglandin H2 receptor agonist to baboon platelets. United States. doi:10.1016/0090-6980(89)90047-6.
Dorn, G.W. II, and De Jesus, A.. 1989. "Binding of an ( sup 125 I) labelled thromboxane A2/prostaglandin H2 receptor agonist to baboon platelets". United States. doi:10.1016/0090-6980(89)90047-6.
@article{osti_6828247,
title = {Binding of an ( sup 125 I) labelled thromboxane A2/prostaglandin H2 receptor agonist to baboon platelets},
author = {Dorn, G.W. II and De Jesus, A.},
abstractNote = {To characterize the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor on baboon platelets the binding of (125I)BOP was studied. (125I)BOP bound to washed baboon platelets in a saturable manner. Scatchard analysis of binding isotherms revealed a Kd of 1.12 +/- 0.08 nM and a binding capacity of 54 +/- 5 fmoles/10(8) platelets (326 sites/platelet). Several TXA2/PGH2 agonists and antagonists displaced (125I)BOP from its baboon platelet binding site with a rank order of potency similar to human platelets: I-BOP greater than SQ29548 greater than U46619 = I-PTA-OH greater than PTA-OH. I-BOP aggregated washed baboon platelets with an EC50 of 10 +/- 4 nM. The results indicate that (125I)BOP binds to the TXA2/PGH2 receptor on baboon platelets and that this receptor is similar to its human counterpart.},
doi = {10.1016/0090-6980(89)90047-6},
journal = {Prostaglandins; (USA)},
number = ,
volume = 38:6,
place = {United States},
year = 1989,
month =
}
  • 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 -more » (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).« less
  • Thromboxane A2 (TXA2) and prostaglandin H2 (PGH2) are potent constrictors of airway smooth muscle and may mediate some of the pulmonary effects of leukotrienes. To date, the TXA2/PGH2 receptor in lung has not been well characterized. In this report, we describe the evaluation of the TXA2/PGH2 receptor in guinea pig lung membranes using the new radiolabeled 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-h eptenoic acid (IBOP). IBOP elicited a dose-dependent contraction of guinea pig lung parenchymal strips (EC50 = 3.03 +/- 0.97 nM, three experiments), which was blocked by the TXA2/PGH2 antagonists SQ29548 (pKB = 7.44 +/- 0.2, threemore » experiments), BM13505 (pKB = 6.29 +/- 0.26, three experiments), and I-PTA-OH (pKB = 5.82 +/- 0.36, three experiments). In radioligand binding studies, the binding of (125I)IBOP to guinea pig lung membranes prepared from perfused lungs was saturable, displaceable, and dependent upon protein concentration. Binding was optimal at pH 6.5 and was enhanced by the addition of mono- and divalent cations. The standard assay buffer was 25 mM 3-(N-morpholino)propanesulfonic acid, pH 6.5, 100 mM NaCl, 5 mM MgCl2. Binding was inhibited by pretreatment with dithiothreitol, N-ethylmaleimide, or beta-mercaptoethanol. Binding was unaffected by the addition of guanine nucleotide analogs at concentrations up to 300 microM. Analysis of the time course of binding of (125)IBOP at 30 degrees yielded k-1 = 0.0447 min-1, k1 = 2.49 x 10(8) M-1 min-1, and Kd = k-1/k1 = 180 pM. Computer analysis of equilibrium binding studies using nonlinear methods (LUNDON-1) revealed a single class of noninteracting binding sites with a Kd of 86.9 +/- 11.9 pM and a Bmax of 81.8 +/- 7.7 fmol/mg of protein (three experiments).« less
  • In this investigation, the role of PG-endoperoxides and TA/sub 2/ in interaction of platelets and a collagen-coated surface was studied. For this purpose, the effects of exogenous AA, of U46619, a stable analog of PG endoperoxides, which simulates the action of PG endoperoxides and TA/sub 2/ on platelets at the receptor level, and of aspirin, a cyclooxygenase inhibitor, on platelet-surface interaction were investigated. The quantity of TA/sub 2/ synthesized in the platelets was determined by measuring accumulation of its stable product TB/sub 2/. After adhesion of the platelets the incubation medium was removed, nonadherent platelets were destroyed by freezing andmore » thawing, and TB/sub 2/ was determined by radioimmunoassay.« less
  • The thromboxane (TX) A{sub 2} receptor (TP) encompasses two alternatively spliced forms, termed the platelet/placental (TP-P) and endothelial (TP-E) type receptors. Experimental evidence suggests that TP activity may be modulated by novel ligands, termed the isoprostanes, that paradoxically act as TP agonists in smooth muscle and TP antagonists in platelet preparations. Here we have investigated whether prototypical isoprostanes 8-iso-prostaglandin (PG)F{sub 2{alpha}} and 8-iso-PGE{sub 2} regulate the activity of TP isoforms expressed in Chinese hamster ovary (CHO) cells using activator protein-1 (AP-1)-luciferase activity as a reporter. AP-1-luciferase activity was increased by a TP agonist [9,11-dideoxy-9{alpha},11{alpha}-methanoepoxy PGF{sub 2{alpha}} (U46619)] in CHO cellsmore » transfected with the human TP-P and TP-E receptors, and this response was fully inhibited by TP antagonists [1S-[1{alpha},2{beta}(Z),3{alpha},5{alpha}]]-7-[3-[[(4-iodophenyl)sulfonyl]amino]-6,6-dimethylbicyclo[3.1.1]hept-2-yl]-5-heptenoic acid (I-SAP) and [1S-[1{alpha},2{alpha}(Z),3{alpha},4{alpha}]]-7-[[2-[(phenylamino) carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1] [hept-2-yl-5-heptenoic acid (SQ 29,548)]. AP-1-luciferase activity was potently (nanomolar concentrations) increased by 8-iso-PGE{sub 2} in CHO TP-P and TP-E cells, and this response was partially inhibited by cotreatment of cells with TP antagonists, whereas 8-iso-PGF{sub 2{alpha}}, was without effect. Cyclooxygenase inhibitors did not abolish 8-iso-PGE{sub 2} mediated AP-1-luciferase activity, indicating that this response is not dependent on de novo TXA{sub 2} biosynthesis. Interestingly, 8-iso-PGE{sub 2}-mediated AP-1-luciferase activity was near maximal in naive cells between 1 and 10 nM concentrations, and this response was not inhibited by TP antagonist or reproduced by agonists for TP or EP{sub 1}/EP{sub 3} receptors. These observations (1) support a role for novel ligands in the regulation of TP-dependent signaling, (2) indicate that TP-P and TP-E couple to AP-1, (3) provide further evidence that isoprostanes function as TP agonists in a cell-type specific fashion, and (4) indicate that additional targets regulated by 8-iso-PGE{sub 2} couple to AP-1.« less
  • The thromboxane A{sub 2} (TXA{sub 2}) receptor (TP) is represented by two alternatively spliced forms, termed the platelet/placental (TP-P) and endothelial (TP-E) type receptors. Experimental evidence suggests that TP isoforms may be regulated by novel ligands termed the isoprostanes, which paradoxically act as TP agonists in smooth muscle and TP antagonists in platelet preparations. Here we have investigated whether prototypical isoprostanes (8-iso-PG{sub 2{sub {alpha}}} and 8-iso-PGE{sub 2}) regulate the activity of TP isoforms expressed in Chinese Hamster Ovary (CHO) cells using activator protein-1 (AP-1)-luciferase activity as a reporter. AP-1-luciferase activity was increased by a TP agonist (U46619) in CHO cellsmore » transfected with the human TP-P and TP-E receptors and this response was fully inhibited by TP antagonists (ISAP, SQ29,548). AP-1-luciferase activity was potently (nM) increased by 8-iso-PGE2 in CHO TP-P and TP-E cells, and this response was partially inhibited by cotreatment of cells with TP antagonists, while 8-iso-PGF{sub 2{sub {alpha}}} was without effect. Cyclooxygenase inhibitors did not abolish 8-iso-PGE{sub 2} mediated AP-1-luciferase activity, indicating that this response is not dependent on de novo TXA2 biosynthesis. Interestingly, 8-iso-PGE{sub 2}-mediated AP-1-luciferase activity was near maximal in naive cells between 1-10 nM concentrations, and this response was not inhibited by TP antagonist or reproduced by agonists for TP or EP1/EP3 receptors. These observations (1) support a role for novel ligands in the regulation of TP-dependent signaling, (2) indicate that TP-P and TP-E couple to AP-1, (3) provide further evidence that isoprostanes function as TP agonists in a cell-type specific fashion, and (4) indicate that additional targets regulated by 8-iso-PGE{sub 2} couple to AP-1.« less