TY - JOUR
T1 - Characterization of Thromboxane A2/Prostaglandin H2 Binding Sites in Guinea Pig Cardiac Membrane Preparations
AU - Bowling, Nancy
AU - Dubeé, Gregory P.
AU - Kurtz, William L.
AU - Brune, Kellie A.
AU - Saussy, David L.
AU - Dorn, Gerald W.
AU - Mais, Dale E.
PY - 1994/7
Y1 - 1994/7
N2 - Thromboxane A2 (TXA2) and prostaglandin H2 (PGH2) induce platelet aggregation and are potent vasoconstrictors, and they have been implicated in coronary vasospasm and myocardial infarction. The TXA2 mimetic [1S-(1α, 2β (5Z), 3α (1E, 3S*), 4α)]-7-[3-(3-hydroxy-4-(4′-iodophenoxy)-1-butenyl)-7-oxabicy clo-[2.2.1]heptan-2-yl]-5-heptenoic acid (IBOP) was used to characterize binding to microsomal membrane preparations from saline-perfused guinea pig atria (GPA) and ventricles (GPV). [125I]IBOP bound to GPA and GPV in a protein-dependent and saturable manner, although total binding was two-fold greater and non-specific binding was proportionately less in GPA compared to GPV. Analysis of equilibrium binding data indicated one class of binding sites in both GPA and GPV with Kd values of 333 ± 117 and 645 ± 187 pM, respectively, which were in close agreement with kinetically determined Kd values of 226 and 882 pM, respectively. Bmax values of GPA and GPV of 57 ± 5.6 and 24 ± 4.3 fmol/mg protein were significantly different (P < 0.01). Ki values (from IC50s) were determined for various TXA2/PGH2 analogues and prostaglandins in competition binding assays with [125I]IBOP. The rank order for ability to inhibit binding in GPA was U46619 = SQ29548 > I-PTA-OH > PGF2α = PGE2. In GPV, the rank order was U46619 = SQ29548 > PGF2α = I-PTA-OH = PGE2. [125I]IBOP binding to GPA and GPV was completely displaced by the TXA2/PGH2 agonist U46619 and by the TXA2/PGH2 antagonist SQ29548. In contrast, prostaglandins PGF2α and PGE2 were 12.5-480 times less potent than U46619 and SQ29548, and they failed to displace completely [125I]IBOP in either GPA or GPV. [125I]IBOP binding in GPV was likely to a cell type other than ventricular myocyte as no specific binding could be detected in guinea pig isolated cardiomyocyte preparations. In paced guinea pig left atria. 1 μM U46619, 10 nM IBOP and 10 nM isoproterenol each elicited an approximate 35% increase in force of contraction. Responses to IBOP were blocked by 10 μM SQ29548, suggesting that the contractile effects were mediated by TXA2, receptors. Although determination of cell type(s) remains to be identified, these results indicate that specific binding with the TXA2/PGH2 mimetic [125I]IBOP may be quantified in microsomal membrane preparations from guinea pig atria and ventricles, and that these sites may represent the TXA2/PGH2 receptor.
AB - Thromboxane A2 (TXA2) and prostaglandin H2 (PGH2) induce platelet aggregation and are potent vasoconstrictors, and they have been implicated in coronary vasospasm and myocardial infarction. The TXA2 mimetic [1S-(1α, 2β (5Z), 3α (1E, 3S*), 4α)]-7-[3-(3-hydroxy-4-(4′-iodophenoxy)-1-butenyl)-7-oxabicy clo-[2.2.1]heptan-2-yl]-5-heptenoic acid (IBOP) was used to characterize binding to microsomal membrane preparations from saline-perfused guinea pig atria (GPA) and ventricles (GPV). [125I]IBOP bound to GPA and GPV in a protein-dependent and saturable manner, although total binding was two-fold greater and non-specific binding was proportionately less in GPA compared to GPV. Analysis of equilibrium binding data indicated one class of binding sites in both GPA and GPV with Kd values of 333 ± 117 and 645 ± 187 pM, respectively, which were in close agreement with kinetically determined Kd values of 226 and 882 pM, respectively. Bmax values of GPA and GPV of 57 ± 5.6 and 24 ± 4.3 fmol/mg protein were significantly different (P < 0.01). Ki values (from IC50s) were determined for various TXA2/PGH2 analogues and prostaglandins in competition binding assays with [125I]IBOP. The rank order for ability to inhibit binding in GPA was U46619 = SQ29548 > I-PTA-OH > PGF2α = PGE2. In GPV, the rank order was U46619 = SQ29548 > PGF2α = I-PTA-OH = PGE2. [125I]IBOP binding to GPA and GPV was completely displaced by the TXA2/PGH2 agonist U46619 and by the TXA2/PGH2 antagonist SQ29548. In contrast, prostaglandins PGF2α and PGE2 were 12.5-480 times less potent than U46619 and SQ29548, and they failed to displace completely [125I]IBOP in either GPA or GPV. [125I]IBOP binding in GPV was likely to a cell type other than ventricular myocyte as no specific binding could be detected in guinea pig isolated cardiomyocyte preparations. In paced guinea pig left atria. 1 μM U46619, 10 nM IBOP and 10 nM isoproterenol each elicited an approximate 35% increase in force of contraction. Responses to IBOP were blocked by 10 μM SQ29548, suggesting that the contractile effects were mediated by TXA2, receptors. Although determination of cell type(s) remains to be identified, these results indicate that specific binding with the TXA2/PGH2 mimetic [125I]IBOP may be quantified in microsomal membrane preparations from guinea pig atria and ventricles, and that these sites may represent the TXA2/PGH2 receptor.
KW - Cardiac
KW - IBOP
KW - Receptors
KW - SQ29548
KW - Thromboxane
KW - U46619
UR - http://www.scopus.com/inward/record.url?scp=0028183628&partnerID=8YFLogxK
U2 - 10.1006/jmcc.1994.1109
DO - 10.1006/jmcc.1994.1109
M3 - Article
C2 - 7966360
AN - SCOPUS:0028183628
SN - 0022-2828
VL - 26
SP - 915
EP - 923
JO - Journal of Molecular and Cellular Cardiology
JF - Journal of Molecular and Cellular Cardiology
IS - 7
ER -