ENDOTHELIN RECEPTORS IN CULTURED ADULT-RAT CARDIAC FIBROBLASTS

Objectives: Endothelins, released by vascular endothelial cells, are k nown 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 ha ve endothelin receptors. The identification and binding characteristic s of endothelin-1 and endothelin-3 and their ET(A) and ET(B) receptor subtypes in cultured adult rat cardiac fibroblasts represented study o bjectives. Methods: Cultured rat cardiac fibroblasts (passages 5-10) g rown until confluence were used to study radioligand binding assays, r eceptor 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 r apid, specific, and saturable within 60 minutes. The dissociation of r eceptor bound I-125-endothelin-1 was slow and partially reversible (30 %-40%), suggesting more than one class of endothelin receptors, wherea s the dissociation of I-125-endothelin-3 was time dependent and revers ible. Competitive displacement with unlabelled endothelin-1, endotheli n-3, endothelin-receptor nonselective sarafotoxin (S6b), and ET(A) rec eptor selective antagonist FED-3512-PI were used to identify receptor subtypes. Displacement of I-125-endothelin-1 by cold endothelin-1, res ulted 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-endoth elin-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-12 5-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, 3 4, and 24 kDa were identified by affinity cross linking. Conclusion: E ndothelin-1 and endothelin-3 binding and ET(A) and ET(B) receptor subt ypes are present in cardiac fibroblasts with ET(B) predominant. The pr esence of these receptors support the hypothesis that endothelins may regulate cardiac fibroblast function.