ALPHA-2-ADRENERGIC RECEPTORS IN THE HUMAN CELL-LINE, HT29 - CHARACTERIZATION WITH THE FULL AGONIST RADIOLIGAND [H-3] UK-14,304 AND INHIBITION OF ADENYLATE-CYCLASE

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  • 1 January 1985
    • journal article
    • research article
    • Vol. 28  (5) , 422-430
Abstract
We have characterized the .alpha.2-adrenergic receptor in membranes from the human colonic adenocarcinoma cell line, HT29, using the recently introduced .alpha.2-agonist 5-bromo-6-[2-imidazolin-2-yl-amino]quinoxaline ([3H]UK-14,304), two other radioligands, and a series of adrenergic agonists and antagonists. We also investigated .alpha.2-agonist inhibition of HT29 cells adenylate cyclase and reversal of inhibition by .alpha.-adrenergic antagonists. [3H]Yohimbine saturation experiments indicated a single class of sites with a KD of 0.61 nM which agreed with the kinetically determined KD of 0.62 nM. Computer analysis of kinetic and saturation experiments with [3H]UK-14,304 revealed two cases of sites. From the saturation data, one site had high affinity for the radioligand (0.14 nM) and comprised 33% of the total number of sites, whereas other site had lower affinity (6.1 nM). The total number of sites labeled by [3H]UK-14,304 (360 fmol/mg of protein) was approximately equal to the number of sites labeled by [3H]yohimbine (330 fmol/mg), whereas [3H]para-aminoclonidine labeled fewer sites of a single class. Rank order potencies of adrenergic agonists and antagonists obtained from competition binding assays indicated that: (1) the same receptors were labeled by the three radioligands, and (2) the receptors were of the .alpha.2 subtype. UK-14,304 and epinephrine inhibited forskolin- and vasoactive intestinal peptide-stimulated adenylate cyclase in a dose-dependent manner up to 32%. Inhibition of the enzyme was reversed by yohimbine and, less potently, by phentolamine and prazosin in a dose-dependent manner. The HT29 cell line appears to be a useful model system for the investigation of the regulation and mechanism of action of .alpha.2-adrenergic receptors in human tissues.