Functional characterization of endothelin receptors on cultured brain capillary endothelial cells of the rat.

Abstract This report describes the effects of endothelins (ET-1 and ET-3) on ion transport systems expressed on cultured rat brain capillary endothelial cells (RBEC) and includes investigation of pharmacological properties of ET receptors, their reactivity and induction of signal transduction pathways. ET-1 stimulated IP 3 formation and Ca 2+ uptake with half-maximal effective concentrations ( ec 50 ) of 0.68 and 0.93 nM, respectively; the effects of ET-3 on these responses were much weaker. ET-1-stimulated IP 3 formation and Ca 2+ uptake were inhibited by an ET A antagonist (BQ123) and a phospholipase C (PLC) inhibitor (U73122), indicating the presence of ET A receptors coupled to PLC. ET-1 stimulated K + efflux (through a quinine-sensitive mechanism) and K + uptake (through both ouabain-sensitive and bumetanide-sensitive mechanisms) with ec 50 of 0.59 and 0.68 nM, respectively. The potencies of ET-3 on these responses were considerably lower than those of ET-1. By contrast, ET-1 or ET-3 stimulated Na + uptake with similarly high potencies ( ec 50 = 0.80 and 1.89 nM, respectively) through EIPA (a Na + /H + exchange inhibitor)-sensitive mechanisms. ET-stimulated K + efflux, K + uptake and Na + uptake activities were all inhibited by BQ123 (but not by BQ788), suggesting the involvement of ET A (and not ET B ) receptors in all these responses. ET-1 stimulated K + uptake and efflux were inhibited by either U73122 or an intracellular Ca 2+ chelator, suggesting that these two responses were mediated via PLC. In contrast, ET stimulation of Na + uptake was unaffected by PLC inhibition or intracellular Ca 2+ chelation. These data suggest the presence of two distinct subtypes of ET A receptors on RBEC; one appears to be a typical ET A receptor which is coupled to PLC and has higher binding affinity for ET-1 than ET-3. The other (ET A -like) receptor is similarly activated by ET-1 and ET-3 with high potencies but is independent of PLC. This possibility was further confirmed by the [ 125 I]ET-1 binding studies demonstrating the presence of high- and low-affinity ET-3 binding sites.