Neurotensin stimulates inositol trisphosphate-mediated calcium mobilization but not protein kinase C activation in HT29 cells. Involvement of a G-protein.
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)
Bozou
J C
JC
Centre de Biochimie, CNRS, Université de Nice, Faculté de Sciences, Parc Valrose, France.
Rochet
N
N
Magnaldo
I
I
Vincent
J P
JP
Kitabgi
P
P
D000230:Adenocarcinoma; D000634:Aminoquinolines; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003110:Colonic Neoplasms; D004075:Diglycerides / Q000378:metabolism; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D009496:Neurotensin / Q000378:metabolism / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism*; D018028:Receptors, Neurotensin; D017981:Receptors, Neurotransmitter / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism*
D000634:Aminoquinolines; D004075:Diglycerides; D005456:Fluorescent Dyes; D018028:Receptors, Neurotensin; D017981:Receptors, Neurotransmitter; D012265:Ribonucleotides; D009496:Neurotensin; D015759:Ionomycin; D015544:Inositol 1,4,5-Trisphosphate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; C035739:Quin2; D002118:Calcium