©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Human Umbilical Vein Endothelial Cells Express High Affinity Neurotensin Receptors Coupled to Intracellular Calcium Release (*)

(Received for publication, August 16, 1994; and in revised form, October 18, 1994)

Paul Schaeffer Marie-Claude Laplace Pierre Savi Anne-Marie Pflieger Danielle Gully Jean-Marc Herbert (§)

From the From Sanofi Recherche, 195 Route d' Espagne, 31036 Toulouse, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The binding of I-neurotensin (NT) to human umbilical vein endothelial cell monolayers was studied. At 20 °C, I-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-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 times 10^6M s and 3.5 times 10 s, respectively. I-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): NT (0.11) > NT (0.35) > acetyl-NT (1.5) > [Phe]NT (12) > [D-Tyr]NT (>1000). I-NT binding was also inhibited by the non-peptide NT antagonist SR 48692 (K = 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 Ca 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 (IC = 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 IC values in Ca efflux experiments was very high (r = 0.997) with a slope near unity, indicating that I-NT binding sites are functional NT receptors coupled to phosphoinositide hydrolysis and Ca release in human umbilical vein endothelial cells.


INTRODUCTION

The tridecapeptide neurotensin (NT) (^1)(1) has been shown to act as a neurotransmitter in the central nervous system, and it has been suggested that it acts as a local hormone in peripheral tissues(2) . Peripheral NT is localized essentially in the endocrine N-cells of intestinal mucosa(3, 4) . Plasma NT levels in humans which increase after food intake (2) may modulate peristaltic intestinal contractions(5) . Depending on the location, NT is able to contract or relax intestinal muscle either directly through interaction with smooth muscle cells or indirectly through the activation of neurotransmitter release from intramural nerve endings(2) . These conclusions are based on indirect evidence based on the blockade of neurotransmitter release by tetrodotoxin. However, binding studies using iodinated NT derivatives(6, 7, 8) , as well as electrophysiological experiments on isolated smooth muscle cells(9) , provide convincing evidence that some effects of NT are due to direct interaction of the peptide with intestinal smooth muscle cell receptors.

In contrast to the intestinal effects of NT, the mechanisms of the cardiovascular effects of this peptide are much less well characterized, and it is still unclear if NT acts directly or indirectly on vascular cells. Indeed, intravenous infusion of NT produces species-dependent systemic as well as regional cardiovascular effects(1, 10, 11, 12, 13) . The effects of NT have also been studied in isolated organ experiments, where NT contracted rat portal vein by a histamine-independent mechanism(14) , but had no contractile activity on rabbit pulmonary artery(15) , rat aorta(16) , or dog carotid arteries(17) . In these latter experiments, an endothelium-dependent relaxing effect could be demonstrated(17) , suggesting that NT may induce smooth muscle relaxation through a direct interaction with endothelial cells via an activation of NO synthase. Subcutaneous injection of NT also produces an increase in cutaneous vascular permeability which can be attributed to either a direct effect of NT on vascular endothelial cells or an indirect histamine-related mechanism (18) .

Despite these numerous in vivo and in vitro pharmacological studies, no NT binding sites have been described on vascular smooth muscle or endothelial cells yet. NT binding to mast cells has been reported(19, 20) , but the low affinity and specificity of these binding sites raise several questions concerning their potential implication in histamine release(2) . We hereby describe the existence and characterization of high affinity NT receptors on human umbilical vein and aortic endothelial cells, suggesting that some of the cardiovascular effects of NT might be due to direct interaction of NT with endothelial cells.


EXPERIMENTAL PROCEDURES

Materials

(3-[I]Iodotyrosyl-3)NT (I-NT, specific activity, 2000 Ci/mmol), [myo-^3H]inositol (100 Ci/mmol), and CaCl(2) (specific activity, 10-40 Ci/mg) were from Amersham Corp. (Les Ulis, France). RPMI 1640 medium and PBS were from Biochrom KG (Poly Labo, Strasbourg, France). Fetal calf serum and human fibronectin were from Boehringer Mannheim (Meylan, France). Heparin, endothelial cell growth supplement, bacitracin, 1,10-phenanthroline, and NT and its fragments were from Sigma (Saint-Quentin Fallavier, France). SR 48692 was synthesized at Sanofi Recherche (Toulouse, France)(21) . AG1-X8 resin and chromatography columns were from Bio-Rad (Ivry sur Seine, France). HAECs were obtained from Clonetics (TEBU, Paris, France). HUVECs were from the American Type Culture Collection (Rockville, MD). Oligonucleotides primers and oligoprobe were generous gifts of D. Shire (Sanofi Recherche, Labège, France).

Cell Culture

HUVECs were routinely cultured in 75-cm^2 flasks coated with human fibronectin (5 µg/cm^2) in RPMI 1640 medium containing 10% fetal calf serum, 100 IU penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 100 µg/ml heparin, and 30 µg/ml endothelial cell growth supplement. For experiments, cells were detached by trypsin/EDTA (0.02-0.05%) and seeded in fibronectin-coated 24-well plates (binding experiments), 35-mm Petri dishes (Ca efflux experiments), or 60-mm dishes (phosphoinositide turnover experiments) and used at confluence. Culture conditions for HAECs were identical to those used for HUVECs.

Binding Experiments

I-NT binding experiments were performed on cell monolayers. Medium was aspirated, and cells washed two times with PBS and incubated with 1 ml of binding buffer containing, 140 mM NaCl, 5 mM KCl, 1.8 mM CaCl(2), 3.6 mM MgCl(2), 2 mg/ml bovine serum albumin, 1 mM glucose, 40 mg/liter bacitracin, 1 mM 1,10-phenanthroline, and 25 mM Hepes/Tris, pH 7.4, in the presence of I-NT and the tested compounds. Preliminary experiments showed that, at 20 °C, equilibrium was reached after 1 h of incubation. At the end of the incubation period, the buffer was aspirated, and the cells were washed two times with ice-cold PBS. Cells were then digested with 0.1 N NaOH for 2 h, and the resulting solution counted in a counter. Results for equilibrium binding experiments, kinetic experiments, and binding inhibition studies were analyzed by a nonlinear regression program(22) .

Ca Efflux Experiments

Cell monolayers in 35-mm dishes were incubated overnight with CaCl(2) (10 µCi/ml). The experiment was started by removal of the medium and replacement by physiological salt solution (PSS, composition: 145 mM NaCl, 5 mM KCl, 1 mM MgCl(2), 1 mM CaCl(2), 5.6 mM glucose, 1 g/liter bovine serum albumin, 100 mg/liter bacitracin, 1 mM 1,10-phenanthroline, 5 mM Hepes/NaOH, pH 7.4) devoid of CaCl(2). The solution was removed every 30 s and replaced by fresh PSS at 37 °C. Radioactivity in the solution was determined by scintillation counting. Results were expressed as fractional rate (R) of Ca efflux:

where Q(i) = quantity of Ca lost from the cells during time period i and Q(c) = quantity of Ca in the cells at the end of the experiment. Ca efflux was stimulated by addition of NT to the washing solution after eight medium changes. When the effects of antagonists were studied, they were present from the beginning of the experiment, i.e. for 4 min before the addition of NT. For concentration-effect relationships and inhibition experiments, Ca efflux during the 1st min of stimulation was calculated by summation and corrected by subtracting base-line efflux. Data from several experiments were pooled and analyzed together by fitting the sigmoidal equation to the data by nonlinear regression thus determining EC values and their standard errors (23) using the program Sigmaplot (Jandel Scientific, Erkrath, Germany).

Measurement of Phosphoinositide Turnover

Confluent cell monolayers in 60-mm dishes were incubated for 72 h in normal culture medium containing 5 µCi/ml of [myo-^3H]inositol. Medium was then aspirated, and the cell monolayers were washed twice with PBS and incubated for 30 min with PSS containing 20 mM LiCl. Cells were then stimulated in the same medium with different concentrations of NT and antagonist for an additional 30 min at 37 °C. At the end of the incubation period, buffer was aspirated, and the cells were extracted with an ice-cold 0.1 N methanol/HCl (50/50) solution for 30 min. Extracts were then neutralized with 1 M Na(2)CO(3), and [^3H]inositol monophosphate was separated as described by Berridge et al.(24) using columns containing 1 ml of AG1-X8 resin.

Analysis of NT Receptor mRNA, RT-PCR of NT Receptor mRNA

RNA was extracted from confluent HUVECs with the Glassmax RNA MicroIsolation kit (Life Technologies, Inc., Eragny, France) according to the manufacturer's procedure and quantified on a GeneQuant spectrophotometer (Pharmacia Biotech Europe, Saint Quentin en Yvelines, France). RT-PCR was performed as described by Kawasaki(25) . First strand cDNA was synthesized from 0.5 µg of RNA, using a SuperScript preamplification system (Life Technologies, Inc.) with oligo(dT) as a primer. Samples were then treated with 2 units of RNase H and amplified by 35 repeated cycles at 94 °C for 1 min, 50 °C for 2 min, and 72 °C for 2 min. The PCR reaction mixture (total volume 50 µl) contained 2 units of Taq DNA polymerase (Perkin-Elmer, Saint Quentin en Yvelines, France) with 10 pmol of the following primers: 5`-CAG GTC AAC ACC TTC ATG TC-3` and 5`-ACT GCT CAT CCG AGA TGT AG-3`. These primers spanned a 269-bp portion of the NT cDNA between bases 1083 and 1352(26) . Blank (control) was carried out in the same conditions in samples in which RNA was omitted.

Southern Blot

Amplified products were subjected to electrophoresis on 2% agarose gel and visualized under UV light, after ethidium bromide staining. Resolved amplicons were then transferred overnight onto a nitrocellulose membrane (Biodyne B, Pall, Saint Germain en Laye, France) in 0.4 N NaOH, and hybridization was performed as follows. The membrane was rinsed twice in 2 times sodium saline citrate buffer and soaked in 5 ml of hybridization buffer (1% gelatin, 0.5% casein, 0.5 M NaCl, 0.1 M Tris-HCl, pH 8.8, 0.1% Tween 20) in an hybridization incubator (model 400, Robbins Scientific, Sunnyvaley, CA) for 15 min, at 42 °C. HRP-labeled oligonucleotide probe (100 ng) was added, and the incubation was carried out for 30 min at 42 °C. The probe (5`-ACT GCT CAT CCG AGA TGT AG-3`) was designed to hybridize a sequence corresponding to bases 1222-1241 of the human NT receptor cDNA. It was coupled at the 5` end with HRP according to the method of Bouaboula et al.(27) . The membrane was rinsed twice in 10 ml of 0.1 times sodium saline citrate, 0.1% SDS for 20 min at 42 °C, and the hybridized probe was revealed with the ECL detection kit on hyperfilm HP films (Amersham Corp., Les Ulis, France).


RESULTS

As shown in Fig. 1A, I-NT binding to HUVEC monolayers at 20 °C was saturable, with a nonspecific binding representing less than 20% of total binding at all concentrations. The Scatchard plot (Fig. 1A, inset) was linear, indicative of a single class of non-interacting binding sites. From several experiments, the mean dissociation constant (K(d)) was found to be of 0.23 ± 0.08 nM and the binding site density (B(max)) represented 5500 ± 1300 sites/cell (n = 3). Binding studies carried out at 37 °C gave results which were identical to those from experiments performed at 20 °C, with K(d) = 0.40 ± 0.23 nM and B(max) = 5800 ± 1500 sites/cell (n = 2). Further experiments were therefore carried out at 20 °C in order to study binding at NT affinities close to the physiological range while minimizing internalization and degradation of NT which is likely to occur at 37 °C. At this temperature, NT binding sites could also be detected in HAECs, where K(d) = 0.6 ± 0.26 nM and B(max) = 32,000 ± 1,700 sites/cell (n = 3, not shown).


Figure 1: I-NT binding to HUVEC monolayers. A, equilibrium binding experiments. Confluent cells were incubated for 1 h at 20 °C with increasing concentrations of I-NT in the presence or absence of 1 µM of unlabeled NT. The specific binding (circle) was determined as the difference between total (bullet ) and nonspecific binding (box). Results are from one experiment representative of a total of three experiments performed in triplicate. Inset, Scatchard plot of the specific binding, for this particular experiment K= 0.12 nM and B(max) = 3600 sites/cell. B, association kinetics of I-NT binding. Confluent cells were incubated at 20 °C with I-NT (100 pM) and the reaction was terminated at different time points by two rapid washes with ice-cold PSS. Nonspecific binding was determined in the presence of 1 µM of unlabeled NT and was substracted from total binding to give specific binding at each time point. Data are the mean of four experiments performed in triplicate. The solid line represents the fit of a monoexponential function to the data, allowing to determine k. C, dissociation of I-NT binding to HUVECs. After 1 h of incubation with 100 pMI-NT, 1 µM of unlabeled NT (circle) or of SR 48692 (bullet) was added and the reaction terminated at different time points by two rapid washes with PSS. Data are the mean of three experiments performed in triplicate. The solid line represents the fit of a monoexponential function to the data, which allowed for the calculation of k.



The kinetics of I-NT binding indicated that the association of I-NT (100 pM) was complete after 1 h of incubation and remained stable for up to 2 h. As shown in Fig. 1B, the mean data were well described by a monoexponential equation corresponding to a pseudo-first order association reaction, with k = 0.031 min (t = 22 min). Dissociation of I-NT could be induced by either unlabeled NT or SR 48692, a specific non-peptide antagonist of high affinity NT receptors, and was identical in both cases (Fig. 1C). Dissociation was rapid, with a half-life of 33 min, corresponding to a dissociation rate constant (k) of 3.5 times 10 s. A small percentage of I-NT (around 20%) did not dissociate and remained associated to the cells even after 3 h of incubation. The association rate constant k was found to be 1.6 times 10^6M s. Calculating the dissociation constant K(d) = k/k gave a value of 0.22 nM, identical to the K(d) value determined in equilibrium binding experiments.

As shown in Fig. 2, NT and its analogues inhibited I-NT binding in a monophasic manner, slope factors being close to unity. As the concentration of I-NT used (100 pM) was less than half of the K(d) value of NT, the concentrations of compounds giving 50% of inhibition of I-NT binding (K(0.5)) can be considered equal within experimental error to the affinities of the compounds for I-NT binding sites. The following rank order of potency was determined (K(0.5) ± S.E. nM, n = 2-3): NT (0.11 ± 0.06), NT (0.35 ± 0.05), acetyl-NT (1.5 ± 0.7), [Phe]NT (12 ± 5). [D-Tyr]NT was inactive up to 10 µM. I-NT binding could also be inhibited by SR 48692, with a K(0.5) value of 16 ± 4 nM. The rank order of potency of NT analogues and the inhibition by SR 48692 show that endothelial cell NT binding sites were very similar to rat brain NT receptors(21) . In rat and mouse brain, two different classes of I-NT binding sites have been distinguished using the antihistaminergic compound levocabastine which specifically inhibited I-NT binding to low affinity sites(28, 29) . However, in endothelial cell monolayers levocabastine had no effect on I-NT binding up to a concentration of 10 µM (not shown), suggesting that HUVEC NT receptors were of the high affinity type.


Figure 2: Effect of NT analogues and SR 48692 on I-NT binding to HUVECs. HUVEC monolayers were incubated with I-NT (100 pM) and unlabeled NT (circle), NT (bullet), acetyl-NT (), [Phe]NT (box), and SR 48692 () for 1 h at 20 °C. Results, expressed as percent of specific I-NT binding remaining in the presence of the compounds, are from one representative experiment performed in triplicate. The solid lines represent a fit of the sigmoidal equation to the data.



The effect of several peptides, which, like NT, release histamine from mast cells, was assessed on I-NT binding to endothelial cells: neither substance P, nor bradykinin or other peptides like angiotensin II, vasoactive intestinal peptide, or corticotropin-releasing factor had any effect on I-NT binding to HUVECs at 10 µM (not shown).

The presence of NT receptors in HUVECs was visualized after RT-PCR amplification of the NT receptor mRNA extracted from HUVECs which showed only one 269-bp single band after electrophoresis (Fig. 3A). As shown in Fig. 3B, this band was identified as a NT receptor mRNA-derived amplicon by Southern blotting, whereas no amplification products could be detected in the control samples. NT has been shown to increase phosphoinositide turnover and activate intracellular free Ca release in HT29 colonic cancer cells (30) and rat NT receptor transfected Chinese hamster ovary cells(31) , whereas it increased cGMP levels and activated NO synthesis in N1E-115 neuroblastoma cells(32, 33, 34) . In HUVECs, we were unable to demonstrate an increase in cGMP levels induced by NT (3-100 nM), whereas the calcium ionophore A23187 (1 µM) increased cGMP levels more than 5-fold from 0.28 to 1.6 pmol/10^6 cells (not shown). However, NT was able to modulate Ca homeostasis in these cells. As shown in Fig. 4, NT induced a strong but transient increase in Ca efflux in HUVEC monolayers which had been prelabeled with Ca overnight. The maximal increase as well as the kinetics of Ca efflux were dose-dependent. At high concentrations of NT, the maximal efflux was reached after 30 s of incubation, whereas at lower concentrations it took 1 min to attain the peak response (Fig. 4). As shown in the inset of Fig. 4, the concentration-effect relationship of NT was steep, with a threshold around 1 nM of NT, and a maximal effect was reached at 10 nM NT. This maximal effect represented a loss of around 15% of the total intracellular Ca during 1 min of stimulation. To determine whether the functional effects of NT analogues were related to their potency as inhibitors of I-NT binding, these compounds were tested. As shown in Fig. 5, all NT analogues with the exception of the very low affinity compound [D-Tyr]NT were able to induce Ca efflux from HUVECs. The rank order of potency was identical to the order determined in binding experiments (EC ± S.E. nM, n = 4): NT (1.3 ± 0.23), NT (4 ± 1.5), acetyl-NT (10 ± 5), [Phe]NT (90 ± 20). Levocabastine had no effect up to a concentration of 10 µM. A good correlation was obtained between binding data and the functional response of NT analogues (r = 0.997) with a slope value not very far from unity (0.88). This observation strongly suggested that the I-NT binding sites were involved in the activation of Ca efflux in HUVECs. To further demonstrate the specificity of the stimulatory effect of NT, the effect of the antagonist SR 48692 on NT-induced Ca release was determined. SR 48692 inhibited Ca efflux with an IC value of 8 ± 1.2 nM, a value close to its IC value in binding experiments. NT has been shown to increase the turnover of phosphoinositide in HT29 cells(30) , and the effect of NT on Ca efflux suggests that the peptide may also stimulate phosphoinositide turnover in endothelial cells. Indeed, as shown in Fig. 6, NT increased phosphatidyl turnover about 2-fold in a concentration range similar to that active in HT29 cells and identical to the concentrations increasing Ca efflux in endothelial cells. Like for Ca efflux, this effect of NT was inhibited in a dose-dependent manner by the antagonist SR 48692 (Fig. 6, inset).


Figure 3: Detection of the NT receptor mRNA in HUVEC. cDNA derived from HUVEC mRNA was amplified by RT-PCR, and electrophoresis was carried out as described under ``Experimental Procedures.'' Gel was stained for 6 min with ethidium bromide (1 µg/ml) and photographed under UV light (A). Hybridization was performed after transfer onto a nitrocellulose membrane and HRP-labeled probe was detected (B). Lane 1, standards; lane 2, control samples; lane 3, PCR amplification product from HUVECs.




Figure 4: Effect of NT on Ca efflux in HUVECs. Ca-labeled cell monolayers were washed every 30 s with PSS and Ca in the washing solution determined by scintillation counting. The horizontal bar denotes the presence of saline (&cjs1730;) or NT (0.1 nM, ; 1 NM, down triangle; 3 NM, up triangle; 10 NM, box; 100 NM, circle). RESULTS ARE THE MEAN OF AT LEAST FOUR DETERMINATIONS ON DIFFERENT BATCHES OF CELLS. Inset, CONCENTRATION-EFFECT RELATIONSHIP OF THE EFFECT OF NT ON CAEFFLUX. RESULTS REPRESENT THE CAEFFLUX DURING THE 1ST MIN OF STIMULATION WITH NT. Error barsSHOW THE S.E. OF THE DATA. THE solid lineREPRESENTS A FIT OF THE SIGMOIDAL EQUATION TO THE DATA.




Figure 5: Effect of NT analogues on Ca




Figure 6: Effect of NT on phosphoinositide metabolism in HUVECs. Cell monolayers were incubated for 30 min with different concentration of NT, and inositol monophosphate (IP1) accumulation was determined as described under ``Experimental Procedures.'' Results are expressed as percent increase of the control value and are the mean of four determinations performed in triplicate. Error bars represent the S.E. Inset, inhibitory effect of SR 48692, 10 nM (full bar) and 1 µM (shaded bar) on NT, 100 nM (empty bar)-stimulated inositol monophosphate accumulation. SR 48692 was present for 30 min before the addition of NT.




DISCUSSION

This work shows for the first time the existence of high affinity functional NT binding sites in human endothelial cells of aortic and venous origin. Up to now, NT binding sites have been reported only in brain and gastrointestinal smooth muscle tissues of different species. With the exception of the low affinity binding sites of rat brain, which were inhibited by levocabastine(28, 29) , these binding sites appeared remarkably similar, with the same rank order of potency for different NT analogues. Only in mast cells could a different type of NT receptor exist: NT binding in these cells is inhibited by bradykinin at low concentrations(20) , but because of the low affinity of NT binding in these cells (K(d) = 150 nM) it is not clear whether these binding sites are NT receptors or are representative of the interaction of NT with other membrane proteins (such as G-proteins) involved in the secretion of histamine(2) . A similar remark applies to macrophage NT binding sites, which also bound substance P(35) . At the present time, three types of NT binding sites can be considered: the high affinity, brain-type NT receptor, the low affinity brain receptor and the low affinity mastocyte or macrophage NT binding sites. The NT binding sites on endothelial cells described in this study resemble the high affinity brain receptors by several different criteria: (i) a very high affinity for NT and a rank order of potency for NT analogues similar to the one observed in the rat brain, with NT significantly more active than NT and [Phe]NT; (ii) no inhibition by levocabastine, differentiating it from low affinity brain sites; (iii) no inhibition by different histamine-releasing peptides such as bradykinin and substance P, unlike mastocyte binding sites; and (iv) a single-band hybridization of endothelial cell mRNA with a human brain NT receptor probe. Together, these results suggest that HUVECs express a NT receptor very similar to the high affinity type expressed in electrically excitable cells such as neurons and smooth muscle cells. Furthermore NT binding sites were found on aortic endothelial cells from adult humans, suggesting that these NT receptors are not an idiosyncrasy of fetal endothelial cells from umbilical vein, but are a common feature of endothelial cells of human origin.

The NT binding sites in endothelial cells were functionally coupled to intracellular Ca release, and different NT analogues acted as agonists with the same rank order of potency as that observed in binding experiments. However, although the K(0.5) values determined in binding experiments and the EC values from Ca efflux experiments were closely correlated, EC values were about 10-fold higher than the K(0.5) values. A similar discrepancy has already been observed in HT29 cells, where the IC value of NT for stimulation of Ca efflux was 2 nM, whereas the dissociation constant of NT in binding experiments was 0.27 nM(30) . As in HT29 cells, this was probably due to the short incubation times used in Ca efflux experiments compared to those used in the binding studies.

NT stimulated phosphoinositide turnover in HUVECs, suggesting that the effect on Ca efflux was a consequence of the production of inositol 1,4,5-trisphosphate and subsequent stimulation of Ca release from intracellular stores. Thus, results from binding studies and second messenger determinations show that HUVECs express functional NT receptors which are coupled to phosphoinositide turnover and intracellular Ca release. These receptors are blocked by SR 48692 at low nanomolar concentrations, allowing this compound to be used to probe the role of these receptors in vitro as well as in vivo. This is a relevant problem because, up to now, the vascular effects of NT injection, in particular hypotension and increase in membrane permeability, have essentially been attributed to an indirect action through release of histamine from mastocytes or interaction with innervating terminations. The detection of functional NT binding sites in HUVECs raises the possibility of a direct action of NT on endothelial cells of the cardiovascular system. Possible consequences of endothelial NT receptor activation could be (i) the release of vasorelaxating substances such as NO and (ii) the increase of vascular endothelial permeability. Increases on intracellular free Ca, implicated both in the release of NO from endothelial cells (36) and in the changes of endothelial monolayer permeability induced by different compounds(37) , strengthen this hypothesis.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Haemobiology Research Dept., Sanofi Recherche, 195 Route d'Espagne, 31036 Toulouse, France. Tel: 33-62-14-23-61; Fax: 33-62-14-22-86.

(^1)
The abbreviations used are: NT, neurotensin; PBS, phosphate-buffered saline; HAEC, human aortic endothelial cell; HUVEC, human umbilical vein endothelial cell; HRP, horseradish peroxidase; PCR, polymerase chain reaction; RT, reverse transcription.


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