FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1991676972> ?p ?o ?g. }

• W1991676972 endingPage "3413" @default.
• W1991676972 startingPage "3409" @default.
• W1991676972 abstract "The binding of 125I-neurotensin (NT) to human umbilical vein endothelial cell monolayers was studied. At 20°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 × 106M−1 s-1 and 3.5 × 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 (12Sumners C. Phillips M.I. Richard E.M. Hypertension. 1982; 4: 888-893Crossref PubMed Scopus (39) Google Scholar) > [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. The binding of 125I-neurotensin (NT) to human umbilical vein endothelial cell monolayers was studied. At 20°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 × 106M−1 s-1 and 3.5 × 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 (12Sumners C. Phillips M.I. Richard E.M. Hypertension. 1982; 4: 888-893Crossref PubMed Scopus (39) Google Scholar) > [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. The tridecapeptide neurotensin (NT) ( 1The abbreviations used are: NTneurotensinPBSphosphate-buffered salineHAEChuman aortic endothelial cellHUVEChuman umbilical vein endothelial cellHRPhorseradish peroxidasePCRpolymerase chain reactionRTreverse transcription. )(1Carraway R.E. Leeman S.E. J. Biol. Chem. 1973; 248: 6854-6861Abstract Full Text PDF PubMed Google Scholar) 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(2Kitabgi P. Checler F. Mazella J. Vincent J.P. Rev. Clin. Basic Pharmacol. 1985; 5: 397-486PubMed Google Scholar). Peripheral NT is localized essentially in the endocrine N-cells of intestinal mucosa(3Sundler F. Alumets J. Hakansson R. Carraway R.E. Leeman S.E. Histochemistry. 1977; 53: 24-34Crossref Scopus (41) Google Scholar, 4Helmstaedter V. Feurle G.E. Forssmann W.G. Cell Tissue Res. 1977; 184: 445-452PubMed Google Scholar). Plasma NT levels in humans which increase after food intake (2Kitabgi P. Checler F. Mazella J. Vincent J.P. Rev. Clin. Basic Pharmacol. 1985; 5: 397-486PubMed Google Scholar) may modulate peristaltic intestinal contractions(5Buhner S. Ehrlein H.J. Can. J. Physiol. Pharmacol. 1989; 67: 1534-1539Crossref PubMed Scopus (6) Google Scholar). 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(2Kitabgi P. Checler F. Mazella J. Vincent J.P. Rev. Clin. Basic Pharmacol. 1985; 5: 397-486PubMed Google Scholar). These conclusions are based on indirect evidence based on the blockade of neurotransmitter release by tetrodotoxin. However, binding studies using iodinated NT derivatives(6Kitabgi P. Kwan C.Y. Fox J.E. Vincent J.P. Peptides. 1984; 5: 917-923Crossref PubMed Scopus (32) Google Scholar, 7Ahmad S. Berezin I. Vincent J.P. Daniel E.E. Biochim. Biophys. Acta. 1987; 896: 224-238Crossref PubMed Scopus (30) Google Scholar, 8Amadh S. Daniel E.E. Peptides. 1991; 12: 623-629Crossref PubMed Scopus (10) Google Scholar), as well as electrophysiological experiments on isolated smooth muscle cells(9Komori S. Matsuoka T. Kwon S.C. Takewaki T. Ohashi H. Br. J. Pharmacol. 1992; 107: 790-796Crossref PubMed Scopus (10) Google Scholar), provide convincing evidence that some effects of NT are due to direct interaction of the peptide with intestinal smooth muscle cell receptors. neurotensin phosphate-buffered saline human aortic endothelial cell human umbilical vein endothelial cell horseradish peroxidase polymerase chain reaction reverse transcription. 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(1Carraway R.E. Leeman S.E. J. Biol. Chem. 1973; 248: 6854-6861Abstract Full Text PDF PubMed Google Scholar, 10Quirion R. Rioux F. Regoli D. St-Pierre S. Life Sci. 1980; 27: 1889-1895Crossref PubMed Scopus (52) Google Scholar, 11Keourac R. Rioux F. St-Pierre S. Life Sci. 1981; 28: 2477-2487Crossref PubMed Scopus (21) Google Scholar, 12Sumners C. Phillips M.I. Richard E.M. Hypertension. 1982; 4: 888-893Crossref PubMed Scopus (39) Google Scholar, 13Bachelard H. Gardiner S.M. Kemp P.A. Bennett T. Br. J. Pharmacol. 1992; 105: 191-201Crossref PubMed Scopus (12) Google Scholar). The effects of NT have also been studied in isolated organ experiments, where NT contracted rat portal vein by a histamine-independent mechanism(14Rioux, F., Quirion, R., Leblanc, M. A., Regoli, D., St-Pierre, S., Life Sci., 27, 259–267.Google Scholar), but had no contractile activity on rabbit pulmonary artery(15Obara H. Kusunoki M. Mori M. Mikawa K. Iwai S. Peptides. 1989; 10: 241-243Crossref PubMed Scopus (16) Google Scholar), rat aorta(16Di Paola E. Richelson E. Eur. J. Pharmacol. 1990; 175: 279-283Crossref PubMed Scopus (17) Google Scholar), or dog carotid arteries(17D'Orléans-Juste P. Dion S. Mizrahi J. Regoli D. Eur. J. Pharmacol. 1985; 114: 9-21Crossref PubMed Scopus (152) Google Scholar). In these latter experiments, an endothelium-dependent relaxing effect could be demonstrated(17D'Orléans-Juste P. Dion S. Mizrahi J. Regoli D. Eur. J. Pharmacol. 1985; 114: 9-21Crossref PubMed Scopus (152) Google Scholar), 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 (18Carraway R.E. Cochrane D.E. Salmonsen R. Muraki K. Boucher W. Peptides. 1991; 12: 1105-1111Crossref PubMed Scopus (28) Google Scholar). 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(19Lazarus L.H. Perrin M.H. Brown M.R. J. Biol. Chem. 1977; 252: 7174-7179Abstract Full Text PDF PubMed Google Scholar, 20Lazarus L.H. Perrin M.H. Brown M.R. Rivier J.E. J. Biol. Chem. 1977; 252: 7180-7183Abstract Full Text PDF PubMed Google Scholar), but the low affinity and specificity of these binding sites raise several questions concerning their potential implication in histamine release(2Kitabgi P. Checler F. Mazella J. Vincent J.P. Rev. Clin. Basic Pharmacol. 1985; 5: 397-486PubMed Google Scholar). 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. (3-[125I]Iodotyrosyl-3)NT (125I-NT, specific activity, 2000 Ci/mmol), [myo-3H]inositol (100 Ci/mmol), and 45CaCl2 (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)(21Gully D. Canton M. Boigegrain R. Jeanjean F. Poncelet M Gueudet C. Heaulme M. Brouard A. Pelaprat D. Labbé-Jullié C. Mazella J. Soubrié P. Maffrand J.P. Rostène W. Kitabgi P. Le Fur G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 65-69Crossref PubMed Scopus (400) Google Scholar). 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). HUVECs were routinely cultured in 75-cm2 flasks coated with human fibronectin (5 μg/cm2) 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 (45Ca efflux experiments), or 60-mm dishes (phosphoinositide turnover experiments) and used at confluence. Culture conditions for HAECs were identical to those used for HUVECs. 125I-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 CaCl2, 3.6 mM MgCl2, 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 125I-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 γ counter. Results for equilibrium binding experiments, kinetic experiments, and binding inhibition studies were analyzed by a nonlinear regression program(22Munson P.J. Rodbard D. Anal. Biochem. 1980; 107: 220-239Crossref PubMed Scopus (7772) Google Scholar). Cell monolayers in 35-mm dishes were incubated overnight with 45CaCl2 (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 MgCl2, 1 mM CaCl2, 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 45CaCl2. 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 45Ca efflux: Ri=QiQc+∑n=ilastQn where Qi = quantity of 45Ca2+ lost from the cells during time period i and Qc = quantity of 45Ca2+ in the cells at the end of the experiment. 45Ca2+ 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, 45Ca2+ 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 EC50 values and their standard errors (23De Léan A. Munson P.J. Rodbard D. Am. J. Physiol. 1978; 235: E97-E102Crossref PubMed Google Scholar) using the program Sigmaplot (Jandel Scientific, Erkrath, Germany). 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 Na2CO3, and [3H]inositol monophosphate was separated as described by Berridge et al.(24Berridge M. Dawson R.M.C. Downes C. Heslop J.P. Irvine R. Biochem. J. 1983; 212: 473-482Crossref PubMed Scopus (1541) Google Scholar) using columns containing 1 ml of AG1-X8 resin. 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(25Kawasaki E.S. Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. PCR Protocols. Academic Press, San Diego, CA1990: 21-38Crossref Google Scholar). 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(26Vita N. Laurent P. Lefort S. Chalon P. Dumont X. Kaghad M. Gully D. Le Fur G. Ferrara P. Caput D. FEBS Lett. 1993; 317: 139-142Crossref PubMed Scopus (229) Google Scholar). Blank (control) was carried out in the same conditions in samples in which RNA was omitted. 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 × 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.(27Bouaboula M. Legoux P. Pésségué B. Delpech B. Dumont X. Piechaczyk M. Casellas P. Shire D. J. Biol. Chem. 1992; 267: 21830-21838Abstract Full Text PDF PubMed Google Scholar). The membrane was rinsed twice in 10 ml of 0.1 × 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). As shown in Fig. 1A, 125I-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 (Kd) was found to be of 0.23 ± 0.08 nM and the binding site density (Bmax) 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 Kd = 0.40 ± 0.23 nM and Bmax = 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 Kd = 0.6 ± 0.26 nM and Bmax = 32,000 ± 1,700 sites/cell (n = 3, not shown). The kinetics of 125I-NT binding indicated that the association of 125I-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 kobs = 0.031 min-1 (t1/2 = 22 min). Dissociation of 125I-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−1) of 3.5 × 10-4 s−1. A small percentage of 125I-NT (around 20%) did not dissociate and remained associated to the cells even after 3 h of incubation. The association rate constant k+1 was found to be 1.6 × 106M−1 s−1. Calculating the dissociation constant Kd = k−1/k+1 gave a value of 0.22 nM, identical to the Kd value determined in equilibrium binding experiments. As shown in Fig. 2, NT and its analogues inhibited 125I-NT binding in a monophasic manner, slope factors being close to unity. As the concentration of 125I-NT used (100 pM) was less than half of the Kd value of NT, the concentrations of compounds giving 50% of inhibition of 125I-NT binding (K0.5) can be considered equal within experimental error to the affinities of the compounds for 125I-NT binding sites. The following rank order of potency was determined (K0.5± S.E. nM, n = 2-3): NT8-13 (0.11 ± 0.06), NT (0.35 ± 0.05), acetyl-NT8-13 (1.5 ± 0.7), [Phe11]NT (12 ± 5). [D-Tyr11]NT was inactive up to 10 μM. 125I-NT binding could also be inhibited by SR 48692, with a K0.5 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(21Gully D. Canton M. Boigegrain R. Jeanjean F. Poncelet M Gueudet C. Heaulme M. Brouard A. Pelaprat D. Labbé-Jullié C. Mazella J. Soubrié P. Maffrand J.P. Rostène W. Kitabgi P. Le Fur G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 65-69Crossref PubMed Scopus (400) Google Scholar). In rat and mouse brain, two different classes of 125I-NT binding sites have been distinguished using the antihistaminergic compound levocabastine which specifically inhibited 125I-NT binding to low affinity sites(28Schotte A. Leysen J.E. Laduron P.M. Naunyn-Schmiedebergs Arch. Pharmacol. 1986; 333: 400-405Crossref PubMed Scopus (147) Google Scholar, 29Mazella J. Chabry J. Kitabgi P. Vincent J.P. J. Biol. Chem. 1988; 263: 144-149Abstract Full Text PDF PubMed Google Scholar). However, in endothelial cell monolayers levocabastine had no effect on 125I-NT binding up to a concentration of 10 μM (not shown), suggesting that HUVEC NT receptors were of the high affinity type. The effect of several peptides, which, like NT, release histamine from mast cells, was assessed on 125I-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 125I-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 Ca2+ release in HT29 colonic cancer cells (30Bozou J.C. Rochet N. Magnaldo I. Vincent J.P. Kitabgi P. Biochem. J. 1989; 264: 871-878Crossref PubMed Scopus (99) Google Scholar) and rat NT receptor transfected Chinese hamster ovary cells(31Watson M.A. Yamada M. Yamada M. Cusack B. Veverka K. Bolden-Watson C. Richelson E. J. Neurochem. 1992; 59: 1967-1970Crossref PubMed Scopus (49) Google Scholar), whereas it increased cGMP levels and activated NO synthesis in N1E-115 neuroblastoma cells(32Gilbert J.A. Richelson E. Eur. J. Pharmacol. 1984; 99: 245-246Crossref PubMed Scopus (79) Google Scholar, 33Amar S. Mazella J. Checler F. Kitabgi P. Vincent J.P. Biochem. Biophys. Res. Commun. 1985; 129: 117-125Crossref PubMed Scopus (48) Google Scholar, 34Förstermann U. Gorsky L.D. Pollock J.S. Ishii K. Schmidt H.H.H.W. Heller M. Murad F. Mol. Pharmacol. 1990; 38: 7-13PubMed Google Scholar). 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/106 cells (not shown). However, NT was able to modulate Ca2+ homeostasis in these cells. As shown in Fig. 4, NT induced a strong but transient increase in 45Ca2+ efflux in HUVEC monolayers which had been prelabeled with 45Ca2+ overnight. The maximal increase as well as the kinetics of 45Ca2+ 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 45Ca2+ during 1 min of stimulation. To determine whether the functional effects of NT analogues were related to their potency as inhibitors of 125I-NT binding, these compounds were tested. As shown in Fig. 5, all NT analogues with the exception of the very low affinity compound [D-Tyr11]NT were able to induce 45Ca2+ efflux from HUVECs. The rank order of potency was identical to the order determined in binding experiments (EC50± S.E. nM, n = 4): NT8-13 (1.3 ± 0.23), NT (4 ± 1.5), acetyl-NT8-13 (10 ± 5), [Phe11]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 125I-NT binding sites were involved in the activation of 45Ca2+ efflux in HUVECs. To further demonstrate the specificity of the stimulatory effect of NT, the effect of the antagonist SR 48692 on NT-induced 45Ca2+ release was determined. SR 48692 inhibited 45Ca2+ efflux with an IC50 value of 8 ± 1.2 nM, a value close to its IC50 value in binding experiments. NT has been shown to increase the turnover of phosphoinositide in HT29 cells(30Bozou J.C. Rochet N. Magnaldo I. Vincent J.P. Kitabgi P. Biochem. J. 1989; 264: 871-878Crossref PubMed Scopus (99) Google Scholar), and the effect of NT on 45Ca2+ 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 45Ca2+ efflux in endothelial cells. Like for 45Ca2+ efflux, this effect of NT was inhibited in a dose-dependent manner by the antagonist SR 48692 (Fig. 6, inset).Figure 4:Effect of NT on 45Ca2+ efflux in HUVECs. 45Ca2+-labeled cell monolayers were washed every 30 s with PSS and 45Ca2+ in the washing solution determined by scintillation counting. The horizontal bar denotes the presence of saline (⬢) or NT (0.1 nM, ⋄; 1 NM, ▿; 3 NM, ▵; 10 NM, □; 100 NM, ○). RESULTS ARE THE MEAN OF AT LEAST FOUR DETERMINATIONS ON DIFFERENT BATCHES OF CELLS. Inset, CONCENTRATION-EFFECT RELATIONSHIP OF THE EFFECT OF NT ON 45CA2+EFFLUX. RESULTS REPRESENT THE 45CA2+EFFLUX 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5:Effect of NT analogues on 45Ca2+View Large Image Figure ViewerDownload Hi-res image Download (PPT)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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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(28Schotte A. Leysen J.E. Laduron P.M. Naunyn-Schmiedebergs Arch. Pharmacol. 1986; 333: 400-405Crossref PubMed Scopus (147) Google Scholar, 29Mazella J. Chabry J. Kitabgi P. Vincent J.P. J. Biol. Chem. 1988; 263: 144-149Abstract Full Text PDF PubMed Google Scholar), 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(20Lazarus L.H. Perrin M.H. Brown M.R. Rivier J.E. J. Biol. Chem. 1977; 252: 7180-7183Abstract Full Text PDF PubMed Google Scholar), but because of the low affinity of NT binding in these cells (Kd = 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(2Kitabgi P. Checler F. Mazella J. Vincent J.P. Rev. Clin. Basic Pharmacol. 1985; 5: 397-486PubMed Google Scholar). A similar remark applies to macrophage NT binding sites, which also bound substance P(35Bar-Shavit Z. Terry S. Blumberg S. Goldman R. Neuropeptides. 1982; 2: 325-335Crossref Scopus (45) Google Scholar). 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 NT8-13 significantly more active than NT and [Phe11]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 Ca2+ release, and different NT analogues acted as agonists with the same rank order of potency as that observed in binding experiments. However, although the K0.5 values determined in binding experiments and the EC50 values from 45Ca2+ efflux experiments were closely correlated, EC50 values were about 10-fold higher than the K0.5 values. A similar discrepancy has already been observed in HT29 cells, where the IC50 value of NT for stimulation of 45Ca2+ efflux was 2 nM, whereas the dissociation constant of NT in binding experiments was 0.27 nM(30Bozou J.C. Rochet N. Magnaldo I. Vincent J.P. Kitabgi P. Biochem. J. 1989; 264: 871-878Crossref PubMed Scopus (99) Google Scholar). As in HT29 cells, this was probably due to the short incubation times used in 45Ca2+ efflux experiments compared to those used in the binding studies. NT stimulated phosphoinositide turnover in HUVECs, suggesting that the effect on 45Ca2+ efflux was a consequence of the production of inositol 1,4,5-trisphosphate and subsequent stimulation of Ca2+ 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 Ca2+ 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 Ca2+, implicated both in the release of NO from endothelial cells (36He P. Pagakis S.N. Curry F.E. Am. J. Physiol. 1990; 258: H1366-H1374PubMed Google Scholar) and in the changes of endothelial monolayer permeability induced by different compounds(37Moncada S. Palmer R.M.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar), strengthen this hypothesis." @default.
• W1991676972 created "2016-06-24" @default.
• W1991676972 creator A5010860742 @default.
• W1991676972 creator A5012194201 @default.
• W1991676972 creator A5026199225 @default.
• W1991676972 creator A5057526959 @default.
• W1991676972 creator A5060778764 @default.
• W1991676972 creator A5087669796 @default.
• W1991676972 date "1995-02-01" @default.
• W1991676972 modified "2023-09-27" @default.
• W1991676972 title "Human Umbilical Vein Endothelial Cells Express High Affinity Neurotensin Receptors Coupled to Intracellular Calcium Release" @default.
• W1991676972 cites W1483907253 @default.
• W1991676972 cites W1573974844 @default.
• W1991676972 cites W1593792753 @default.
• W1991676972 cites W1607957872 @default.
• W1991676972 cites W1802980406 @default.
• W1991676972 cites W1967303569 @default.
• W1991676972 cites W1971111446 @default.
• W1991676972 cites W1979012883 @default.
• W1991676972 cites W1982961466 @default.
• W1991676972 cites W1997747246 @default.
• W1991676972 cites W2000249139 @default.
• W1991676972 cites W2005669075 @default.
• W1991676972 cites W2007539277 @default.
• W1991676972 cites W2008710572 @default.
• W1991676972 cites W2010044058 @default.
• W1991676972 cites W2018723879 @default.
• W1991676972 cites W2021551491 @default.
• W1991676972 cites W2035778895 @default.
• W1991676972 cites W2043813981 @default.
• W1991676972 cites W2046605145 @default.
• W1991676972 cites W2062034250 @default.
• W1991676972 cites W2068196787 @default.
• W1991676972 cites W2073384618 @default.
• W1991676972 cites W2077730943 @default.
• W1991676972 cites W2082922238 @default.
• W1991676972 cites W2085566481 @default.
• W1991676972 cites W2167817965 @default.
• W1991676972 cites W2218739829 @default.
• W1991676972 cites W2223822401 @default.
• W1991676972 cites W2885428316 @default.
• W1991676972 cites W97769542 @default.
• W1991676972 doi "https://doi.org/10.1074/jbc.270.7.3409" @default.
• W1991676972 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/7852427" @default.
• W1991676972 hasPublicationYear "1995" @default.
• W1991676972 type Work @default.
• W1991676972 sameAs 1991676972 @default.
• W1991676972 citedByCount "29" @default.
• W1991676972 countsByYear W19916769722014 @default.
• W1991676972 countsByYear W19916769722015 @default.
• W1991676972 countsByYear W19916769722016 @default.
• W1991676972 countsByYear W19916769722017 @default.
• W1991676972 countsByYear W19916769722018 @default.
• W1991676972 countsByYear W19916769722020 @default.
• W1991676972 crossrefType "journal-article" @default.
• W1991676972 hasAuthorship W1991676972A5010860742 @default.
• W1991676972 hasAuthorship W1991676972A5012194201 @default.
• W1991676972 hasAuthorship W1991676972A5026199225 @default.
• W1991676972 hasAuthorship W1991676972A5057526959 @default.
• W1991676972 hasAuthorship W1991676972A5060778764 @default.
• W1991676972 hasAuthorship W1991676972A5087669796 @default.
• W1991676972 hasBestOaLocation W19916769721 @default.
• W1991676972 hasConcept C118303440 @default.
• W1991676972 hasConcept C126322002 @default.
• W1991676972 hasConcept C134018914 @default.
• W1991676972 hasConcept C170493617 @default.
• W1991676972 hasConcept C181080969 @default.
• W1991676972 hasConcept C185592680 @default.
• W1991676972 hasConcept C202751555 @default.
• W1991676972 hasConcept C2777411675 @default.
• W1991676972 hasConcept C2778976297 @default.
• W1991676972 hasConcept C519063684 @default.
• W1991676972 hasConcept C55493867 @default.
• W1991676972 hasConcept C71924100 @default.
• W1991676972 hasConcept C79879829 @default.
• W1991676972 hasConcept C86803240 @default.
• W1991676972 hasConcept C95444343 @default.
• W1991676972 hasConceptScore W1991676972C118303440 @default.
• W1991676972 hasConceptScore W1991676972C126322002 @default.
• W1991676972 hasConceptScore W1991676972C134018914 @default.
• W1991676972 hasConceptScore W1991676972C170493617 @default.
• W1991676972 hasConceptScore W1991676972C181080969 @default.
• W1991676972 hasConceptScore W1991676972C185592680 @default.
• W1991676972 hasConceptScore W1991676972C202751555 @default.
• W1991676972 hasConceptScore W1991676972C2777411675 @default.
• W1991676972 hasConceptScore W1991676972C2778976297 @default.
• W1991676972 hasConceptScore W1991676972C519063684 @default.
• W1991676972 hasConceptScore W1991676972C55493867 @default.
• W1991676972 hasConceptScore W1991676972C71924100 @default.
• W1991676972 hasConceptScore W1991676972C79879829 @default.
• W1991676972 hasConceptScore W1991676972C86803240 @default.
• W1991676972 hasConceptScore W1991676972C95444343 @default.
• W1991676972 hasIssue "7" @default.
• W1991676972 hasLocation W19916769721 @default.
• W1991676972 hasOpenAccess W1991676972 @default.
• W1991676972 hasPrimaryLocation W19916769721 @default.
• W1991676972 hasRelatedWork W1258457027 @default.
• W1991676972 hasRelatedWork W1985697105 @default.

• next