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.
Del RY
S
S
CNR Institute of Clinical Physiology, Pisa, Italy.
Andreassi
M G
MG
Lazzerini
G
G
Iervasi
G
G
Clerico
A
A
Biagini
A
A
Giannessi
D
D
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D016232:Endothelins / Q000378:metabolism*; D006325:Heart Atria; D006352:Heart Ventricles; D006801:Humans; D008566:Membranes / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*
D016232:Endothelins