HUMAN UMBILICAL VEIN ENDOTHELIAL-CELLS EXPRESS HIGH-AFFINITY NEUROTENSIN RECEPTORS COUPLED TO INTRACELLULAR CALCIUM-RELEASE

The binding of I-125-neurotensin (NT) to human umbilical vein endothel ial cell monolayers was studied. At 20 degrees C, I-125-NT bound to a single class of binding sites with a dissociation constant of 0.23 +/- 0.08 nM and a binding site density of 5500 +/- 1300 sites/cell (n = 3 ). I-125-NT also bound to human aortic endothelial cells with a dissoc iation constant of 0.6 +/- 0.26 nM and a binding site density of 32000 +/- 1700 sites/cell. Association and dissociation kinetics were of a pseudo-first order ana gave association and dissociation rate constant values of 1.6 x 10(6) M(-1) s(-1) and 3.5 x 10(-4) s(-1), respectivel y. I-125-NT binding was inhibited by NT analogues with a rank order of potency similar to that characterizing brain high affinity NT binding sites (K-0.5, nM): NT8-13 (0.11) > NT (0.35) > acetyl-NT8-13 (1.5) > [Phe(11)]NT (12) > [D-Tyr(11)]NT (> 1000). I-125-NT binding was also i nhibited by the non-peptide NT antagonist SR 48692 (K-i = 16 nM) but w as not affected by levocabastine, an inhibitor of low affinity brain N T binding sites. NT had no effect on cGMP levels in endothelial cells but NT and its analogues increased Ca-45(2+) efflux from endothelial c ells at nanomolar concentrations with a rank order of potency which wa s identical to that observed in binding experiments. This effect was i nhibited by SR 48692 (IC50 = 8 nM). NT was able to increase phosphoino sitide turnover in these cells, and this effect was blocked by SR 4869 2. The correlation between dissociation constants of NT analogues in b inding experiments and IC50 values in Ca-45(2+) efflux experiments was very high (r = 0.997) with a slope near unity, indicating that I-125- NT binding sites ard functional NT receptors coupled to phosphoinositi de hydrolysis and Ca2+ release in human umbilical vein endothelial cel ls.