Determination of endothelin binding sites in bovine atrium and ventricle membranes: a model for binding studies in human cardiac tissue.

The characterization of the binding sites of endothelin (ET) could constitute a useful tool to better understand its role in different pathophysiological conditions in human myocardial tissue. Crude membrane fractions were prepared from bovine atrium and ventricle for binding-assay assessment; membranes were also prepared from portions of atrium or ventricle < 100 mg each, a size comparable with human surgical fragments, and Scatchard analysis was performed from each single fragment. Portions of cardiac tissue were homogenized in 10 volumes of Tris/HCl buffer, 50 mM, pH 7.4, containing protease inhibitors; the supernatant was centrifuged at 50,000 x g for 30 min and the pellet stored at -80 degrees C. 125-I-ET-1 binding was performed by using 50 micrograms of protein and scalar concentrations of radioligand (8-200 pM), at 37 degrees C for 4 h and was stopped by filtration through a glass-fibre filter. The specific binding was saturable (at 200 pM of 125-I-ET-1 for 100 micrograms/ml of protein); the equilibrium was reached in 4 h at 37 degrees C at a radioligand concentration of 150 pM; a similar pattern, but a lower binding was obtained at 4 degrees C and 25 degrees C; the binding linearly increased with protein concentrations from 12.5 to 150 micrograms, and saturated at higher concentration. Non-specific binding was determined in presence of 0.1 microM unlabelled ET-1 and was about 2% of the total radioactivity added, for 125-I-ET-1 ranging from 8 to 200 pM, at 100 micrograms/ml of protein. By Scatchard analysis Kd proved to be 20.7 +/- 3.9 and 19.9 +/- 0.6 pM, Bmax 42.4 +/- 12.7 and 59.3 +/- 13.0 fmol/mg of protein, for atrium and ventricle respectively. Differential inhibition of binding was observed for the three ET isoforms; IC50 were similar for atrium and ventricle and proved to be 50 nM for ET-1 and ET-2 and > 200 nM for ET-3. This latter finding and the Scatchard analysis suggest the presence of a single class of binding sites in bovine cardiac membranes. No significant differences in absolute values of Kd and Bmax were found when cardiac membranes were obtained by small tissue fragments.