RESUMEN
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.
Asunto(s)
Mucosa Intestinal/metabolismo , Receptores de la Hormona Gastrointestinal/clasificación , Receptores de la Hormona Gastrointestinal/metabolismo , Receptores de Neuropéptido Y/clasificación , Receptores de Neuropéptido Y/metabolismo , Sitios de Unión , Northern Blotting , Butiratos/farmacología , Ácido Butírico , Calcio/metabolismo , Línea Celular , AMP Cíclico/antagonistas & inhibidores , Hormonas Gastrointestinales/farmacología , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Membranas Intracelulares/metabolismo , Péptido YY , Péptidos/farmacología , Reacción en Cadena de la Polimerasa , Transcripción GenéticaRESUMEN
Quantitative receptor autoradiography localized a high-affinity binding site for 125I-peptide YY to the mucosa in rabbit distal colon. Scatchard binding analysis revealed a single-affinity binding site (KD = 0.29 nM) with binding specificity similar to other peptide YY-preferring receptors (peptide YY > or = neuropeptide Y >> pancreatic polypeptide). Radioligand binding studies using colonic mucosal membranes confirmed high-affinity peptide YY binding sites (KD = 0.26 nM) with time, temperature, and protein dependence, as well as saturability characteristic of receptor-ligand binding. Selective peptide analogues showed a subpopulation of these 125I-peptide YY binding sites to resemble the Y1-type neuropeptide Y receptor. Peptide YY may exert local antisecretory effects on the colonic epithelium via these binding sites.
Asunto(s)
Hormonas Gastrointestinales/metabolismo , Mucosa Intestinal/química , Péptidos/metabolismo , Receptores de la Hormona Gastrointestinal , Animales , Autorradiografía , Colon/química , Densitometría , Femenino , Técnicas In Vitro , Radioisótopos de Yodo , Péptido YY , Conejos , Ensayo de Unión Radioligante , Receptores de Superficie Celular/análisisRESUMEN
The neuropeptide Y (NPY) receptor was solubilized from rat brain membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS). The binding of 125I-NPY to CHAPS extracts was protein, time, and temperature dependent. Unlabeled NPY and the related peptides peptide YY (PYY) and pancreatic polypeptide inhibited 125I-NPY binding to solubilized receptors with relative potencies similar to those seen with membrane-bound receptors: NPY greater than PYY much greater than pancreatic polypeptide. Scatchard analysis of equilibrium binding data showed the CHAPS extracts to contain a single population of binding sites with a KD of 3.6 +/- 0.4 nM (mean +/- SEM) and a Bmax of 5.0 +/- 0.2 pmol/mg of protein. In addition the 125I-NPY binding to the soluble receptor was not inhibited by guanosine-5'-O-(3-thiotriphosphate), in contrast to the GTP sensitivity displayed by the membrane-bound receptor. Gel filtration chromatography using Sepharose 6B revealed a single peak of binding activity corresponding to a Mr of approximately 67,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after chemical cross-linking revealed a single band at Mr 62,000. After solubilization and gel chromatography a 50- to 100-fold purification of the NPY receptor was obtained.