Human umbilical vein endothelial cells express high affinity neurotensin receptors coupled to intracellular calcium release.

The binding of 125I-neurotensin (NT) to human umbilical vein endothelial cell monolayers was studied. At 20 degrees C, 125I-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). 125I-NT also bound to human aortic endothelial cells with a dissociation 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 and 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, respectively. 125I-NT binding was inhibited by NT analogues with a rank order of potency similar to that characterizing brain high affinity NT binding sites (K0.5, nM): NT8-13 (0.11) > NT (0.35) > acetyl-NT8-13 (1.5) > [Phe11]NT (12) > [D-Tyr11]NT (> 1000). 125I-NT binding was also inhibited by the non-peptide NT antagonist SR 48692 (Ki = 16 nM) but was not affected by levocabastine, an inhibitor of low affinity brain NT binding sites. NT had no effect on cGMP levels in endothelial cells but NT and its analogues increased 45Ca2+ efflux from endothelial cells at nanomolar concentrations with a rank order of potency which was identical to that observed in binding experiments. This effect was inhibited by SR 48692 (IC50 = 8 nM). NT was able to increase phosphoinositide turnover in these cells, and this effect was blocked by SR 48692. The correlation between dissociation constants of NT analogues in binding experiments and IC50 values in 45Ca2+ efflux experiments was very high (r = 0.997) with a slope near unity, indicating that 125I-NT binding sites are functional NT receptors coupled to phosphoinositide hydrolysis and Ca2+ release in human umbilical vein endothelial cells.