Section 9
Chapter 8,606

Endothelin-1 receptor binding and cellular signal transduction in cultured human brain endothelial cells

Stanimirovic, D.B.; Yamamoto, T.; Uematsu, S.; Spatz, M.

Journal of Neurochemistry 62(2): 592-601


ISSN/ISBN: 0022-3042
PMID: 8294922
DOI: 10.1046/j.1471-4159.1994.62020592.x
Accession: 008605193

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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-2alpha 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 (IP-3) 0.79 nM, = 0.79 nM, whereas ET-3 was ineffective. The order of potency for displacing ET-1 from high-affinity binding sites (ET-1 gt ET-2 gt sarafotoxin S6b gt 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-2alpha 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-2alpha 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.

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