Alpha 2-adrenergic receptors in the human cell line, HT29. Characterization with the full agonist radioligand [3H]UK-14,304 and inhibition of adenylate cyclase.
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 cell 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 classes 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 the 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: the same receptors were labeled by the three radioligands, and 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.
Turner
J T
JT
Ray-Prenger
C
C
Bylund
D B
DB
D000230:Adenocarcinoma / Q000032:analysis*; D000067956:Adenylyl Cyclase Inhibitors; D000316:Adrenergic alpha-Agonists / Q000378:metabolism*; D000068438:Brimonidine Tartrate; D002460:Cell Line; D003000:Clonidine / Q000031:analogs & derivatives / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D003110:Colonic Neoplasms / Q000032:analysis*; D004305:Dose-Response Relationship, Drug; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D011810:Quinoxalines / Q000378:metabolism*; D011869:Radioligand Assay; D011942:Receptors, Adrenergic, alpha / Q000032:analysis*; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology; D015016:Yohimbine / Q000378:metabolism
D000067956:Adenylyl Cyclase Inhibitors; D000316:Adrenergic alpha-Agonists; D011810:Quinoxalines; D011942:Receptors, Adrenergic, alpha; D005576:Colforsin; D015016:Yohimbine; D014660:Vasoactive Intestinal Peptide; D000068438:Brimonidine Tartrate; C016986:apraclonidine; D006160:Guanosine Triphosphate; D008274:Magnesium; D003000:Clonidine