Regulation of the neurotensin receptor and intracellular calcium mobilization in HT29 cells.
Regulation of the neurotensin receptor-inositol phosphate-intracellular Ca2+ ((Ca2+]i) pathway was studied in HT29 cells. Preincubation with neurotensin or phorbol 12-myristate, 13-acetate decreased the number of cell surface neurotensin receptors and neurotensin-induced increases of inositol trisphosphates and [Ca2+]i. The phorbol 12-myristate, 13-acetate-(43 +/- 1% at 1 microM) but not the neurotensin (65 +/- 9% at 10 nM)-induced decrease in receptors was blocked by staurosporine. The decrease in cell surface receptors was accompanied by a 55 +/- 7% shift of specifically bound 125I-neurotensin from the plasma membrane fraction to the light vesicle fraction of sucrose density gradients if a 37 degrees C incubation step was included. The time course for desensitization of [Ca2+]i mobilization was more rapid (maximal at approximately 1 min) than for loss of receptors (maximal at 45 min). After a 5-min exposure to neurotensin, the cell surface receptor number rapidly returned to control levels in the absence of agonist, but [Ca2+]i sensitivity to neurotensin recovered only partially. Incubation with carbachol, ATP or phorbol 12-myristate, 13-acetate desensitized the subsequent [Ca2+]i response to neurotensin. These results demonstrate a polyphosphoinositide-[Ca2+]i pathway in HT29 cells stimulable by neurotensin and other agents. The results from pre-incubation studies indicate that the neurotensin receptor-signaling pathway is homologously and heterologously regulated. Finally, differences in time courses for loss and recovery of cell surface receptors and desensitization of the [Ca2+]i response, as well as the lack of effect on 125I-neurotensin binding of other agonists that desensitize the neurotensin [Ca2+]i response, suggest that receptor internalization alone does not account for desensitization of the system.
Turner
J T
JT
Department of Pharmacology, School of Medicine, University of Missouri-Columbia.
James-Kracke
M R
MR
Camden
J M
JM
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D009496:Neurotensin / Q000378:metabolism / Q000494:pharmacology*; D011869:Radioligand Assay; D018028:Receptors, Neurotensin; D017981:Receptors, Neurotransmitter / Q000187:drug effects* / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D018028:Receptors, Neurotensin; D017981:Receptors, Neurotransmitter; D009496:Neurotensin; D015544:Inositol 1,4,5-Trisphosphate; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium