ENDOTHELIN RECEPTORS IN CULTURED ADULT-RAT CARDIAC FIBROBLASTS

Objectives: Endothelins, released by vascular endothelial cells, are known growth promoters of various mesenchymal cells that contribute to stromal protein accumulation. Whether endothelins could contribute to myocardial fibrosis depends, in part on whether cardiac fibroblasts have endothelin receptors. The identification and binding characteristics of endothelin-1 and endothelin-3 and their ET(A) and ET(B) receptor subtypes in cultured adult rat cardiac fibroblasts represented study objectives. Methods: Cultured rat cardiac fibroblasts (passages 5-10) grown until confluence were used to study radioligand binding assays, receptor subtypes, association and dissociation, effects of agonist and antagonist on binding kinetics, and affinity cross Linking. Results: Binding association of I-125-endothelin-1 and I-125-endothelin-3 was rapid, specific, and saturable within 60 minutes. The dissociation of receptor bound I-125-endothelin-1 was slow and partially reversible (30%-40%), suggesting more than one class of endothelin receptors, whereas the dissociation of I-125-endothelin-3 was time dependent and reversible. Competitive displacement with unlabelled endothelin-1, endothelin-3, endothelin-receptor nonselective sarafotoxin (S6b), and ET(A) receptor selective antagonist FED-3512-PI were used to identify receptor subtypes. Displacement of I-125-endothelin-1 by cold endothelin-1, resulted in a low affinity, high binding site (IC50 5.4 X 10(-9) M; 3.6 x 10(4) binding sites.cell(-1)) and a high affinity, low binding site (IC50 4.2 X 10(-4) M; 11 830 binding sites.cell(-1)). With I-125-endothelin-1 the IC50 s for sarafotoxin, endothelin-3, and FED-3512-PI were 1.8 X 10(-10) 1.7 X 10(-9), and 3.7 X 10(-9) M, respectively; for I-125-endothelin-3 these IC(50)s were 2.28 x 10(-11), 1.9 x 10(-10), and 1.7 x 10(-9) M, respectively. Endothelin receptor subunits of 53, 37, 34, and 24 kDa were identified by affinity cross linking. Conclusion: Endothelin-1 and endothelin-3 binding and ET(A) and ET(B) receptor subtypes are present in cardiac fibroblasts with ET(B) predominant. The presence of these receptors support the hypothesis that endothelins may regulate cardiac fibroblast function.