The vasoactive endothelins have been detected in the cerebrospinal fluid of patients with various cerebrovascular disorders and implicated in predisposing vascular diseases (hypertension, vasospasm). Many of the vascular reactivities under normal and pathologic conditions are mediated by specific receptors. Therefore, the kinetic properties of endothelin-1 (ET-1) binding sites and the production of inositol phosphates (IP1, IP2, IP3), cAMP, thromboxane B2 (TxB2), and prostaglandin F2alpha (PGF2alpha) induced by various endothelins (ET-1, ET-2, ET-3, and sarafotoxin S6b) were examined in endothelial cells derived from human brain microvessels (HBEC). 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 protein and B(Max2) = 909 fmol/mg protein, respectively, was demonstrated on intact HBEC. ET-1 dose-dependently stimulated inositol phosphate (IP) accumulation with EC50(IP3) = 0.79 nM, while ET-3 was ineffective. The order of potency for displacing ET-1 from high affinity binding sites (IC50 was ET-1 is greater than ET-2 is greater than sarafotoxin S6b is greater than ET-3) correlated exponentially with the ability of the respective ligands to induce IP3 formation. The protein kinase C (PKC) activator phorbol myristate ester (PMA) dose-dependently blocked the ET-1 stimulated production of IPs, while pertussis toxin (Ptx) was ineffective. cAMP production by HBEC was enhanced by both PMS and ET-1, and potentiated by combined treatment with ET-1 and PMA. Data indicate that PKC plays a role in regulation of ET-1-induced activation of phospholipase C (PLC), while interaction of different messenger systems may regulate ET-1-induced accumulation of cAMP. ET-1 stimulated endothelial TxB2 and PGF2alpha production, suggesting that activation of phospholipase A2 (PLA2) is most likely secondary to IP3-mediated intracellular calcium mobilization, These findings are the first demonstration of ET-1(ETA-type) receptors linked to PLC and PLA2 activation in HBEC.
