COS-7 CELLS STABLY TRANSFECTED TO EXPRESS THE HUMAN ET(B)-RECEPTOR PROVIDE A USEFUL SCREEN FOR ENDOTHELIN RECEPTOR ANTAGONISTS

Endothelin acts via specific membrane-bound receptors through signal t ransduction pathways that include increases in intracellular free calc ium and inositol triphosphate generation. Two endothelin receptors hav e been cloned. The ET(A) receptor is ET-1 selective, and the ET(B) rec eptor is isopeptide nonselective. Both receptor subtypes are widely di stributed throughout the body, although ET(A) receptors predominate in vascular smooth muscle, whereas ET(B) receptors predominate in the br ain. The presence of mixed receptor subtypes makes functional screenin g of subtype-specific analogues difficult. A eukaryotic expression vec tor was constructed by inserting the cloned coding region of the human ET(B) receptor downstream from the Rous sarcoma promoter. COS-7 cells were transfected with this construct, and cell lines were isolated wi th stably integrated copies of the relevant gene. One line, 1C7, was s hown to specifically bind I-125-ET-1. Scatchard analysis indicated a K (d) value of 8.8 pM and a B(max) value of 1.02 pM/mg. ET-1 stimulated phosphoinositide hydrolysis in a dose-dependent manner, as did ET-3, s arafotoxin 6c, and [1,3,13,15Ala]ET-1, whereas BQ123, a selective ET(A ) receptor antagonist, did not inhibit the action of ET-1. The transfe cted receptor stimulates phosphoinositide (PI) hydrolysis via a pertus sis-sensitive pathway. Pretreatment of the membrane from 1C7 cells wit h dithio-bis-nitrobenzoic acid (DTNB) a negatively charged, nonpenetra ting agent capable of oxidizing sulfhydryl groups, and N-ethyl-maleimi de (NEM), a penetrating agent that causes irreversible alkylation of s ulfhydryl groups, significantly reduces B(max) but has no effect on K( d). In whole cells, DTNB pretreatment abolishes the ability of ET-I to stimulate PI hydrolysis.