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Endothelin‐1 Receptor Binding and Cellular Signal Transduction in Cultured Human Brain Endothelial Cells
Author(s) -
Stanimirovic Danica B.,
Yamamoto Toshifumi,
Uematsu Sumio,
Spatz Maria
Publication year - 1994
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1994.62020592.x
Subject(s) - protein kinase c , pertussis toxin , endothelins , second messenger system , phospholipase c , phorbol , activator (genetics) , signal transduction , biology , receptor , microbiology and biotechnology , inositol phosphate , endothelin 1 , g protein , inositol , biochemistry
The kinetic properties of endothelin‐1 (ET‐1) binding sites and the production of inositol phosphates (IPs; IP 1 , IP 2 , IP 3 ), cyclic AMP, thromboxane B 2 , and prostaglandin F 2α 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 p M and K D2 = 31 n M , 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 (IP 3 ) = 0.79 n M , 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 IP 3 formation. ET‐1‐induced IP 3 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 AMP 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 2α production, suggesting that activation of phospholipase A 2 is most likely secondary to IP 3 ‐mediated intracellular calcium mobilization because both ET‐1‐induced IP 3 and prostaglandin F 2α were inhibited by BQ123. These findings are the first demonstration of ET‐1 (ET A ‐type) receptors linked to phospholipase C and phospholipase A 2 activation in HBECs.