Characterization of [ 125 I]‐PD164333, an ET A selective non‐peptide radiolabelled antagonist, in normal and diseased human tissues

We have synthesized a new low molecular weight, non‐peptide radioligand, [ 125 I]‐PD164333, an analogue of the orally active butenolide antagonists of the endothelin ET A receptor. Analysis of saturation binding assays demonstrated that [ 125 I]‐PD164333 bound with high affinity to a single population of receptors ( n 3 individuals ±s.e.mean) in human aorta ( K D =0.26±0.08 n M ; B max =8.8±3.95 fmol mg ‐1 protein), left ventricle from the heart ( K D =0.16±0.02 n M ; B max =34.2± 3.02 fmol mg ‐1 protein) and kidney (K D =1.24±0.16 n M ; B max =125.3±35.07 fmol mg ‐1 protein). In each case Hill slopes were close to unity. In kinetic experiments, the binding of [ 125 I]‐PD164333 to ET A receptors in sections of heart was time‐dependent and rapid at 23°C. The data were fitted to a one site model, with an association rate constant ( K 1 of 2.66±0.213×10 8 M ‐1 min ‐1 , and a half‐time for association of 11 min. The binding was reversible at 23°C: analysis of the data indicated [ 125 I]‐PD164333 dissociated from a single site, with a dissociation rate constant of 0.0031±0.0004 min ‐1 , a half‐time for dissociation of 216 min and a K D calculated from these kinetic data of 0.01 n M . Unlabelled PD164333 inhibited the binding of [ 125 I]‐ET‐1 to left ventricle (which expresses both subtypes) in a biphasic manner with a K D ET A of 0.99±0.32 n M and K D ET B of 2.41±0.22 μ M , giving a selectivity of 2500 fold. ET A ‐selective ligands competed monophasically for [ 125 I]‐PD164333 binding in left ventricle, a one site fit was preferred to a two site model giving similar nanomolar affinities: BQ123, K D =3.93 ±0.18 n M ; FR139317 K D =3.53±0.69 n M . In contrast, the ET B selective agonists, BQ3020 and sarafotoxin S6c (1 μ M ) did not inhibit binding. In human isolated saphenous vein, unlabelled PD164333 was a functional antagonist, producing parallel rightward shifts of the endothelin‐1 (ET‐1) concentration‐response curve (pA 2 =8.84) and a slope of unity. In the human brain, autoradiography revealed high levels of [ 125 I]‐PD164333 binding to the pial arteries of the cerebral cortex and to the numerous smaller intercerebral vessels penetrating the underlying grey and white matter. Conduit and resistance vessels contributing to the control of blood pressure from the heart, kidney, lungs and adrenal also displayed high densities of binding. In diseased vessels, binding of [ 125 I]‐PD164333 was confined to the medial layer of both coronary arteries with advanced at