ENDOTHELIN-1 RECEPTOR-BINDING AND CELLULAR SIGNAL-TRANSDUCTION IN CULTURED HUMAN BRAIN ENDOTHELIAL-CELLS

The kinetic properties of endothelin-1 (ET-1) binding sites and the production of inositol phosphates (IPs; IP1, IP2, IP3), cyclic AMP, thromboxane B-2, and prostaglandin F-2 alpha induced by various endothelins (ET-1, ET-2, ET-3, and sarafotoxin S6b) were examined in endothelial cells derived from human brain microvessels (HBECs). The presence of both high- and low-affinity binding sites for ET-1 with K-D1 = 122 pM and K-D2 = 31 nM, and B-max1 = 124 fmol/mg of protein and B-max2 = 909 fmol/mg of protein, respectively, was demonstrated on intact HBECs. ET-1 dose-dependently stimulated IP accumulation with EC(50) (IP3) = 0.79 nM, whereas ET-3 was ineffective. The order of potency for displacing ET-1 from high-affinity binding sites (ET-1 > ET-2 > sarafotoxin S6b > ET-3) correlated exponentially with the ability of respective ligands to induce IP3 formation. ET-1-induced IP3 formation by HBEC was inhibited by the ET(A) receptor antagonist, BQ123. The protein kinase C activator phorbol myristate ester dose-dependently inhibited the ET-1-stimulated production of IPs, whereas pertussis toxin was ineffective. Cyclic AMF production by HBECs was enhanced by both phorbol myristate ester and ET-1, and potentiated by combined treatment with ET-1 and phorbol myristate ester. Data indicate that protein kinase C plays a role in regulating the ET-1-induced activation of phospholipase C, whereas interaction of different messenger systems may regulate ET-1-induced accumulation of cyclic AMP. ET-1 also stimulated endothelial prostaglandin F-2 alpha production, suggesting that activation of phospholipase A(2) is most likely secondary to IP3-mediated intracellular calcium mobilization because both ET-1-induced IP3 and prostaglandin F-2 alpha were inhibited by BQ123. These findings are the first demonstration of ET-1 (ET,-type) receptors linked to phospholipase C and phospholipase A(2) activation in HBECs.