BINDING CHARACTERISTICS OF ENDOTHELIN ISOFORMS (ET-1, ET-2, AND ET-3) IN VASCULAR SMOOTH-MUSCLE CELLS

The existence of distinct endothelin (ET) receptor subtypes has been reported in several tissues. In the present study, we investigated the binding characteristics of the three endothelin isoforms to cultured rat aortic smooth muscle cells. [I-125]ET-1, [I-125]ET-2, and [I-125]ET-3 bound to an apparent single class of binding sites with apparent dissociation constants (K(d)) of 111, 123, and 1410 pM and binding capacities (B(max)) of 54.1, 46.0, and 7.9 fmol/10(6) cells, respectively. The binding of the three radiolabeled endothelin isoforms was equally inhibited by ET-1 and ET-2. ET-3 was more effective in competing with [I-125]ET-3 than with [I-125]ET-1 or [I-125]ET-2. In contrast to ET-1 and ET-2, the binding of ET-3 was reversible. Furthermore, 18 h of pre-exposure of the cells to 1 nM ET-1 or ET-2 decreased the ET-1 binding capacity, whereas ET-3 (10 nM) was ineffective. ET-3 binding characteristics were not affected by pretreatment of the cells with any of the endothelin isoforms. These results suggest the presence of two distinct endothelin cells. The ET-1 and ET-2 preferring receptor (80-85%), sensitive to downregulation or internalization, elicits an irreversible binding. The second subtype (15-20%) binds the three endothelin isoforms with the same affinity in a reversible manner, and is insensitive to downregulation or internalization.