Characterization of a Y1-preferring NPY/PYY receptor in HT-29 cells.
Equilibrium binding studies showed that butyrate-treated HT-29 cells express a high-affinity 125I-labeled peptide YY (125I-PYY) binding site with a dissociation constant of 0.32 +/- 0.12 nM (mean +/- SE, n = 4). This site was Y1 preferring because neuropeptide Y (NPY) and the Y1-selective agonist [Leu31,Pro34]NPY were equipotent to PYY at displacing 125I-PYY; PYY-(13-36) and pancreatic polypeptide were > 1,000- and 10,000-fold less potent at displacing the radioligand. PYY and [Leu31,Pro34]NPY inhibited forskolin-stimulated adenosine 3',5'-cyclic monophosphate production 63% and 48%, respectively, with a half-maximal inhibitory concentration between 0.1 and 1.0 nM. PYY and [Leu31,Pro34]NPY had no effect on release of intracellular calcium alone or on the increase in intracellular calcium concentration caused by carbachol or neurotensin. Northern blot analysis of poly(A)+ RNA from HT-29 cells demonstrated a single transcript of 2.5 kb that hybridized to a human Y1-receptor cDNA probe. Sequence analysis of a reverse transcription-polymerase chain reaction product amplified with primers based on human Y1-receptor cDNA confirmed that these cells contained mRNA encoding the human Y1 receptor. These studies show that butyrate-treated HT-29 cells constitutively express the Y1-preferring NPY/PYY receptor and Y1 mRNA and provide a new model for studies of PYY-regulated epithelial cell function and tissue-specific expression of the human Y1-receptor gene.
Division of Gastroenterology, Duke University Medical Center, Durham, North Carolina 27710.
P J
PJ
Mannon
S J
SJ
Mervin
K D
KD
Sheriff-Carter
D001665:Binding Sites; D015152:Blotting, Northern; D002087:Butyrates / Q000494:pharmacology; D020148:Butyric Acid; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000037:antagonists & inhibitors; D005768:Gastrointestinal Hormones / Q000494:pharmacology; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D019894:Peptide YY; D010455:Peptides / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011964:Receptors, Gastrointestinal Hormone / Q000145:classification* / Q000378:metabolism*; D017476:Receptors, Neuropeptide Y / Q000145:classification* / Q000378:metabolism*; D014158:Transcription, Genetic
D002087:Butyrates; D005768:Gastrointestinal Hormones; D010455:Peptides; D011964:Receptors, Gastrointestinal Hormone; D017476:Receptors, Neuropeptide Y; C082172:peptide YY receptor; D019894:Peptide YY; D020148:Butyric Acid; D000242:Cyclic AMP; D002118:Calcium