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 IP3 formation and Ca2+ uptake with half-maximal effective concentrations (EC50) of 0.68 and 0.93 nM, respectively; the effects of ET-3 on these responses were much weaker. ET-1-stimulated IP3 formation and Ca2+ uptake were inhibited by an ETA antagonist (BQ123) and a phospholipase C (PLC) inhibitor (U73122), indicating the presence of ETA 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 EC50 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 (EC50 = 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 ETA (and not ETB) receptors in all these responses. ET-1 stimulated K+ uptake and efflux were inhibited by either U73122 or an intracellular Ca2+ chelator, suggesting that these two responses were mediated via PLC. In contrast, ET stimulation of Na+ uptake was unaffected by PLC inhibition or intracellular Ca2+ chelation. These data suggest the presence of two distinct subtypes of ETA receptors on RBEC; one appears to be a typical ETA receptor which is coupled to PLC and has higher binding affinity for ET-1 than ET-3. The other (ETA-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 [125I]ET-1 binding studies demonstrating the presence of high- and low-affinity ET-3 binding sites.