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. 1989 Dec 15;264(3):871–878. doi: 10.1042/bj2640871

Neurotensin stimulates inositol trisphosphate-mediated calcium mobilization but not protein kinase C activation in HT29 cells. Involvement of a G-protein.

J C Bozou 1, N Rochet 1, I Magnaldo 1, J P Vincent 1, P Kitabgi 1
PMCID: PMC1133666  PMID: 2559720

Abstract

It has previously been shown that neurotensin binds to high-affinity receptors in the adenocarcinoma HT29 cell line, and that receptor occupancy leads to inositol phosphate formation. The present study was designed to investigate further the effects of neurotensin on calcium mobilization and protein kinase C (PKC) activation in HT29 cells, and to assess the role of GTP-binding proteins (G-proteins) in the neurotensin response. Direct measurements of cytosolic Ca2+ variations using the fluorescent indicator quin 2 showed that neurotensin (0.1-1 microM) elicited Ca2+ transients in HT29 cells. These transients occurred after the neurotensin-stimulated formation of Ins(1,4,5)P3, as measured by means of a specific radioreceptor assay. In addition, the peptide induced a decrease in the 45Ca2+ content of cells previously equilibrated with this isotope. The peptide effect was rapid, long-lasting and concentration-dependent, with an EC50 of 2 nM. Phorbol 12-myristate 13-acetate (PMA) inhibited by 50% the neurotensin effects on both intracellular Ca2+ and inositol phosphate levels. The inhibition by PMA was abolished in PKC-depleted cells. Pertussis toxin had no effect on either the Ca2+ or inositol phosphate responses to neurotensin. Epidermal growth factor (EGF) receptors which are present in HT29 cells have been shown to be down-regulated through phosphorylation by PKC in a variety of systems. Here, PMA markedly (70-80%) inhibited EGF binding to HT29 cells. Scatchard analysis revealed that PMA abolished the high-affinity component of EGF binding, an effect that was totally reversed in PKC-depleted cells. In contrast, neurotensin slightly (10-20%) inhibited EGF binding to HT29 cells, and its effect was only partly reversed by PKC depletion. Neurotensin had no detectable effect on sn-1,2-diacylglycerol levels in HT29 cells, as measured by a specific and sensitive enzymic assay. In membranes prepared from HT29 cells, monoiodo[125I-Tyr3]neurotensin bound to a single population of receptors with a dissociation constant of 0.27 nM. Sodium and GTP inhibited neurotensin binding in a concentration-dependent manner. Maximal inhibition reached 80% with Na+ and 35% with GTP.IC50 values were 20 mM and 0.2 microM for Na+ and GTP respectively. Li+ and K+ were less effective than Na+ and the effects of GTP were shared by GDP and guanosine-5'-[beta gamma- imido]triphosphate but not by ATP. Scatchard analysis of binding data indicated that Na+ and GTP converted the high-affinity neurotensin-binding sites into lower affinity binding sites. The properties of the effects of Na+ and GTP on neurotensin-receptor interactions are characteristic of those receptors which interact with G-proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

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Selected References

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  1. Abdel-Latif A. A. Calcium-mobilizing receptors, polyphosphoinositides, and the generation of second messengers. Pharmacol Rev. 1986 Sep;38(3):227–272. [PubMed] [Google Scholar]
  2. Amar S., Kitabgi P., Vincent J. P. Activation of phosphatidylinositol turnover by neurotensin receptors in the human colonic adenocarcinoma cell line HT29. FEBS Lett. 1986 May 26;201(1):31–36. doi: 10.1016/0014-5793(86)80565-8. [DOI] [PubMed] [Google Scholar]
  3. Amar S., Kitabgi P., Vincent J. P. Stimulation of inositol phosphate production by neurotensin in neuroblastoma N1E115 cells: implication of GTP-binding proteins and relationship with the cyclic GMP response. J Neurochem. 1987 Oct;49(4):999–1006. doi: 10.1111/j.1471-4159.1987.tb09986.x. [DOI] [PubMed] [Google Scholar]
  4. Berridge M. J. Inositol trisphosphate and diacylglycerol as second messengers. Biochem J. 1984 Jun 1;220(2):345–360. doi: 10.1042/bj2200345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bishop W. R., Bell R. M. Attenuation of sn-1,2-diacylglycerol second messengers. Metabolism of exogenous diacylglycerols by human platelets. J Biol Chem. 1986 Sep 25;261(27):12513–12519. [PubMed] [Google Scholar]
  6. Bozou J. C., Amar S., Vincent J. P., Kitabgi P. Neurotensin-mediated inhibition of cyclic AMP formation in neuroblastoma N1E115 cells: involvement of the inhibitory GTP-binding component of adenylate cyclase. Mol Pharmacol. 1986 May;29(5):489–496. [PubMed] [Google Scholar]
  7. Brown K. D., Blay J., Irvine R. F., Heslop J. P., Berridge M. J. Reduction of epidermal growth factor receptor affinity by heterologous ligands: evidence for a mechanism involving the breakdown of phosphoinositides and the activation of protein kinase C. Biochem Biophys Res Commun. 1984 Aug 30;123(1):377–384. doi: 10.1016/0006-291x(84)90424-8. [DOI] [PubMed] [Google Scholar]
  8. Canonico P. L., Sortino M. A., Speciale C., Scapagnini U. Neurotensin stimulates polyphosphoinositide breakdown and prolactin release in anterior pituitary cells in culture. Mol Cell Endocrinol. 1985 Oct;42(3):215–220. doi: 10.1016/0303-7207(85)90051-6. [DOI] [PubMed] [Google Scholar]
  9. Checler F., Amar S., Kitabgi P., Vincent J. P. Catabolism of neurotensin by neural (neuroblastoma clone N1E115) and extraneural (HT29) cell lines. Peptides. 1986 Nov-Dec;7(6):1071–1077. doi: 10.1016/0196-9781(86)90136-1. [DOI] [PubMed] [Google Scholar]
  10. Fehlmann M., Morin O., Kitabgi P., Freychet P. Insulin and glucagon receptors of isolated rat hepatocytes: comparison between hormone binding and amino acid transport stimulation. Endocrinology. 1981 Jul;109(1):253–261. doi: 10.1210/endo-109-1-253. [DOI] [PubMed] [Google Scholar]
  11. Goedert M., Pinnock R. D., Downes C. P., Mantyh P. W., Emson P. C. Neurotensin stimulates inositol phospholipid hydrolysis in rat brain slices. Brain Res. 1984 Dec 3;323(1):193–197. doi: 10.1016/0006-8993(84)90288-9. [DOI] [PubMed] [Google Scholar]
  12. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  13. Kikkawa U., Nishizuka Y. The role of protein kinase C in transmembrane signalling. Annu Rev Cell Biol. 1986;2:149–178. doi: 10.1146/annurev.cb.02.110186.001053. [DOI] [PubMed] [Google Scholar]
  14. Kitabgi P., Checler F., Mazella J., Vincent J. P. Pharmacology and biochemistry of neurotensin receptors. Rev Clin Basic Pharm. 1985 Jul-Dec;5(3-4):397–486. [PubMed] [Google Scholar]
  15. Kitabgi P. Effects of neurotensin on intestinal smooth muscle: application to the study of structure-activity relationships. Ann N Y Acad Sci. 1982;400:37–55. doi: 10.1111/j.1749-6632.1982.tb31559.x. [DOI] [PubMed] [Google Scholar]
  16. Kitabgi P., Poustis C., Granier C., Van Rietschoten J., Rivier J., Morgat J. L., Freychet P. Neurotensin binding to extraneural and neural receptors: comparison with biological activity and structure--activity relationships. Mol Pharmacol. 1980 Jul;18(1):11–19. [PubMed] [Google Scholar]
  17. Lopez-Rivas A., Rozengurt E. Vasopressin rapidly stimulates Ca2+ efflux from intracellular pool in quiescent Swiss 3T3 cells. Am J Physiol. 1984 Sep;247(3 Pt 1):C156–C162. doi: 10.1152/ajpcell.1984.247.3.C156. [DOI] [PubMed] [Google Scholar]
  18. Memo M., Carboni E., Trabucchi M., Carruba M. O., Spano P. F. Dopamine inhibition of neurotensin-induced increase in Ca2+ influx into rat pituitary cells. Brain Res. 1985 Nov 18;347(2):253–257. doi: 10.1016/0006-8993(85)90184-2. [DOI] [PubMed] [Google Scholar]
  19. Mendoza S. A., Schneider J. A., Lopez-Rivas A., Sinnett-Smith J. W., Rozengurt E. Early events elicited by bombesin and structurally related peptides in quiescent Swiss 3T3 cells. II. Changes in Na+ and Ca2+ fluxes, Na+/K+ pump activity, and intracellular pH. J Cell Biol. 1986 Jun;102(6):2223–2233. doi: 10.1083/jcb.102.6.2223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pandol S. J., Schoeffield M. S., Fimmel C. J., Muallem S. The agonist-sensitive calcium pool in the pancreatic acinar cell. Activation of plasma membrane Ca2+ influx mechanism. J Biol Chem. 1987 Dec 15;262(35):16963–16968. [PubMed] [Google Scholar]
  21. Preiss J., Loomis C. R., Bishop W. R., Stein R., Niedel J. E., Bell R. M. Quantitative measurement of sn-1,2-diacylglycerols present in platelets, hepatocytes, and ras- and sis-transformed normal rat kidney cells. J Biol Chem. 1986 Jul 5;261(19):8597–8600. [PubMed] [Google Scholar]
  22. Prescott S. M., Majerus P. W. Characterization of 1,2-diacylglycerol hydrolysis in human platelets. Demonstration of an arachidonoyl-monoacylglycerol intermediate. J Biol Chem. 1983 Jan 25;258(2):764–769. [PubMed] [Google Scholar]
  23. Rodbell M. The role of hormone receptors and GTP-regulatory proteins in membrane transduction. Nature. 1980 Mar 6;284(5751):17–22. doi: 10.1038/284017a0. [DOI] [PubMed] [Google Scholar]
  24. Sadoul J. L., Mazella J., Amar S., Kitabgi P., Vincent J. P. Preparation of neurotensin selectively iodinated on the tyrosine 3 residue. Biological activity and binding properties on mammalian neurotensin receptors. Biochem Biophys Res Commun. 1984 May 16;120(3):812–819. doi: 10.1016/s0006-291x(84)80179-5. [DOI] [PubMed] [Google Scholar]
  25. Spiegel A. M. Signal transduction by guanine nucleotide binding proteins. Mol Cell Endocrinol. 1987 Jan;49(1):1–16. doi: 10.1016/0303-7207(87)90058-x. [DOI] [PubMed] [Google Scholar]
  26. Tsien R. Y., Pozzan T., Rink T. J. Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J Cell Biol. 1982 Aug;94(2):325–334. doi: 10.1083/jcb.94.2.325. [DOI] [PMC free article] [PubMed] [Google Scholar]

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