A novel approach for the study of thromboxane A2 and prostaglandin H2 receptors using an 125I-labeled ligand.
Previous studies of eicosanoid receptors have utilized 3H-labeled ligands. However, 125I has a higher theoretical specific activity (approximately 2000 Ci/mmole) than 3H (29 Ci/mmole), which provides a potential advantage for 125I ligand binding studies when the receptor density is low. Since eicosanoids do not possess an easily iodinatable structure (e.g. a phenol or imidazole ring), it is not feasible to directly incorporate 125I into the molecule. The thromboxane A2/prostaglandin H2 receptor antagonist, cis-7-(2-p-hydroxyphenylethanolaminocyclopentyl)-heptanoic acid (cis-APO), was synthesized to test the concept that it could be labeled with 125I and used as a ligand for binding studies. cis-APO is a structural analog of 13-azaprostanoic acid, a TXA2/PGH2 antagonist [G. C. Le Breton, D. L. Venton, S. E. Enke and P. V. Halushka, Proc. natn. Acad. Sci. U.S.A. 76, 4097 (1979)], in which the omega aliphatic chain was substituted with 2-p-hydroxyphenylethanol, which contains a phenolic group. [127I]cis-APO was synthesized by insertion of 127I (stable isotope) into the phenolic portion of the molecule under alkaline conditions. [125I]-cis-APO was synthesized via insertion of 125I (unstable isotope) into the molecule in the presence of chloramine T. cis-APO inhibited human platelet aggregation induced by the thromboxane mimetic U46619 [C. Malmsten, Life Sci. 18, 169 (1976)]. The IC50 for cis-APO was 6.4 +/- 0.7 microM and for [127I]-cis-APO was 9.8 +/- 1.3 microM (P less than 0.001). [125I]-cis-APO binding to a human platelet membrane preparation at 4 degrees was time and protein concentration dependent, saturable, and reduced or abolished by trypsin or boiling respectively. The Kd for iodo-cis-APO determined at equilibrium using a Scatchard analysis was 1.48 microM and the maximum binding capacity was 18.7 pmoles/mg protein. The forward rate constant (k+1) was 2.3 X 10(3) M-1 s-1 and the dissociation constant (k-1) was 2.12 X 10(-3) s-1. The Kd determined from k-1/k+1 was 0.92 microM. These observations show that the omega side chain of eicosanoid analogs can be substituted with a phenolic group, iodinated, and retain biological activity. These molecules may then be utilized to study thromboxane A2 or prostaglandin H2 receptors.
Halushka
P V
PV
MacDermot
J
J
Knapp
D R
DR
Eller
T
T
Saussy
D L
DL
Mais
D
D
Blair
I A
IA
Dollery
C T
CT
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D001792:Blood Platelets / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D010974:Platelet Aggregation / Q000187:drug effects; D011450:Prostaglandin Endoperoxides, Synthetic / Q000037:antagonists & inhibitors; D011869:Radioligand Assay; D011956:Receptors, Cell Surface / Q000032:analysis*; D011982:Receptors, Prostaglandin / Q000032:analysis* / Q000187:drug effects; D017482:Receptors, Thromboxane; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D013237:Stereoisomerism
D007457:Iodine Radioisotopes; D011450:Prostaglandin Endoperoxides, Synthetic; D011956:Receptors, Cell Surface; D011982:Receptors, Prostaglandin; D017482:Receptors, Thromboxane; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid