ABSTRACT
Human fibroblasts in primary cell culture were studied for their ability to bind to endothelin (ET), a 21-amino acid peptide with profound vasoconstricting properties. When 125I-labeled ET-1 was incubated with confluent orbital fibroblasts in the presence of increasing concentrations of unlabeled ligand, a single class of binding site was defined with a dissociation constant of 1.42 x 10(-8) M and a maximal binding capacity of 9.1 x 10(-10) mol/micrograms protein. ET-3 was a substantially less potent competitor for 125I-ET-1 binding sites than was unlabeled ET-1. Dermal fibroblasts demonstrated approximately 75% less ET-1 saturation binding activity, on a cellular protein basis, than did those from the orbit. Orbital fibroblasts responded to ET-1 (10(-9) M) with a rapid and transient increase in the free concentration of intracellular Ca2+ ([Ca2+]i) as assessed by monitoring acetoxymethyl ester of fura 2 fluorescence intensity. Rechallenge with the peptide elicited a substantially attenuated response than that seen after the initial treatment. There was no consistent effect of ET-1 on [Ca2+]i in dermal cultures. ET-3 failed to influence [Ca2+]i in either type of fibroblast. It would appear that orbital fibroblasts bind and respond to ET in a manner distinct from that observed in dermal fibroblasts, raising the possibility that the peptide may have site-specific actions in orbital connective tissue.
