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  • Title: In vitro study of alpha 2-adrenoceptor turnover and metabolism using the adenocarcinoma cell line HT29.
    Author: Paris H, Taouis M, Galitzky J.
    Journal: Mol Pharmacol; 1987 Nov; 32(5):646-54. PubMed ID: 2891026.
    Abstract:
    We have utilized the adenocarcinoma cell line HT29 as an in vitro model to investigate the turnover and the metabolism of the alpha 2-adrenoceptor. The biosynthesis rate of the receptor was studied in postconfluent HT29 cells, when its density expressed as fmol/mg of cell membrane protein is constant, by following the recovery of the receptor binding capacity after blockade with the non-reversible alpha-adrenergic antagonist benextramine. Study of the inhibition of [3H]yohimbine and [3H]UK-14,304 binding showed that benextramine was a more potent antagonist at alpha 2-adrenoceptor than phenoxybenzamine. The incubation of intact HT29 cells for 30 min in the presence of 10(-5) M benextramine irreversibly blocked more than 95% of the alpha 2-adrenoceptors and totally suppressed the inhibitory effect of UK-14,304 on cyclic AMP production. The blockade appeared specific, since benextramine effects were prevented by alpha 2-adrenergic agents. Moreover, neither vasoactive intestinal polypeptide responsiveness nor other tested aspects of the regulation of the adenylate cyclase was altered by the treatment. Study of the time course of receptor recovery after irreversible blockade indicated that alpha 2-adrenoceptors reappeared in the cells with a monoexponential kinetic. The linearization of the repopulation curve obtained with the labeled antagonist [3H]yohimbine allowed the determination of the rate constant for receptor degradation (k = 0.0268 +/- 0.0025 hr-1) and the rate of receptor synthesis (6.91 +/- 0.64 fmol/mg of cell membrane protein/hr) corresponding to the synthesis of about 500 receptors/cell/hr. The alpha 2-adrenoceptor half-life was 26 +/- 3 hr. Measurement of the biological effects associated to the alpha-adrenoceptor stimulation during the course of receptor recovery indicated a relationship between the number of cell receptors and the percentage of inhibition of the cyclic AMP accumulation induced by forskolin. The receptor reappearance was totally inhibited by either actinomycin or cycloheximide or tunicamycin, showing that the recovery corresponded to de novo synthesized receptor and giving indirect evidence for the glycoproteic nature of the alpha 2-adrenoceptor. Deprivation for glucose or glutamine also impeded the recovery process; by contrast, addition of UK-14,304 or clonidine did not interfere, indicating that the expression of the alpha 2-adrenoceptor is not subject to homologous regulation in the HT29 cell.
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