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 B2, and prostaglandin F2 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 KD1 = 122 pM and KD2 = 31 nM, and Bmax1 = 124 fmol/mg of protein and Bmax2 = 909 fmol/mg of protein, respectively, was demonstrated on intact HBECs. ET-1 dose-dependently stimulated IP accumulation with EC50 (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 ETA 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 F2 alpha production, suggesting that activation of phospholipase A2 is most likely secondary to IP3-mediated intracellular calcium mobilization because both ET-1-induced IP3 and prostaglandin F2 alpha were inhibited by BQ123. These findings are the first demonstration of ET-1 (ETA-type) receptors linked to phospholipase C and phospholipase A2 activation in HBECs.
Stanimirovic
D B
DB
Stroke Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
Yamamoto
T
T
Uematsu
S
S
Spatz
M
M
D001921:Brain / Q000098:blood supply* / Q000378:metabolism*; D002196:Capillaries / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000096:biosynthesis; D015237:Dinoprost / Q000096:biosynthesis; D065128:Endothelin Receptor Antagonists; D016232:Endothelins / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D010456:Peptides, Cyclic / Q000494:pharmacology; D037342:Pertussis Toxin; D017466:Receptors, Endothelin / Q000378:metabolism*; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D065128:Endothelin Receptor Antagonists; D016232:Endothelins; D007295:Inositol Phosphates; D010456:Peptides, Cyclic; D017466:Receptors, Endothelin; D010566:Virulence Factors, Bordetella; D015237:Dinoprost; D000242:Cyclic AMP; D037342:Pertussis Toxin; D013755:Tetradecanoylphorbol Acetate; C072247:cyclo(Trp-Asp-Pro-Val-Leu)