Characterization of a thromboxane A2/prostaglandin H2 receptor in guinea pig lung membranes using a radioiodinated thromboxane mimetic

Journal Article · Tue Jan 01 04:00:00 EST 1991 · Molecular Pharmacology; (USA)

OSTI ID:6250093

Saussy, Jr, D L; Mais, D E; Dube, G P; Magee, D E; Brune, K A; Kurtz, W L; Williams, C M ^[1]

---

1. Lilly Corporate Center, Indianapolis, IN (USA)

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, three 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).

• OSTI ID: 6250093
• Journal Information: Molecular Pharmacology; (USA), Journal Name: Molecular Pharmacology; (USA) Vol. 39:1; ISSN MOPMA; ISSN 0026-895X
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMALS
AUTONOMIC NERVOUS SYSTEM AGENTS
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BODY
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL COMPOSITION
CONTRACTION
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
GUINEA PIGS
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LUNGS
MAMMALS
MEMBRANE PROTEINS
MEMBRANES
MUSCLES
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANS
PERFUSED ORGANS
PROSTAGLANDINS
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RECEPTORS
RESPIRATORY SYSTEM
RODENTS
SYMPATHOMIMETICS
TRACER TECHNIQUES
VERTEBRATES