The functional investigation of a human adenocarcinoma cell line, stably transfected with the neuropeptide Y Y1 receptor.
1. The human adenocarcinoma cell line, HT-29, has been stably transfected with the cDNA sequence for the rat neuropeptide Y (NPY) Y1 receptor, and three Y1 clones (Y1-4, Y1-7 and Y1-16) have been isolated which express high levels of specific [125I]-PYY binding. We have studied the functional responses or lack of responses to peptide YY (PYY) and its analogues in the three transfected clones and HT-29 wild type (wt) cells. 2. Vasoactive intestinal polypeptide (VIP) produced long-lasting increases in short-circuit current (SCC) in both HT-29 wt cells and the Y1 clones. VIP EC50 values were 8.4-11.7 nM in all four cases. The elevation in SCC after a maximal concentration of VIP (30 nM) was significantly greater in Y1-7 cells than in either HT-29 wt epithelia or the other Y1 cell lines. 3. PYY (100 nM) and human pancreatic polypeptide (hPP; 1 microM) were ineffective in HT-29 wt cells under either basal or stimulated conditions. In contrast, basolateral additions of PYY reduced both basal and VIP-stimulated SCC in all three Y1 clones. After VIP, the PYY EC50 values (in nM) were 18.6 in Y1-4, 8.0 in Y1-7 and 52.5 in Y1-16 hPP (1 microM) produced only small and transient responses in each transfected cell type. 4. The Y1 receptor agonist, [Leu31, Pro34] NPY (1 microM) was also effective in the three Y1 cell lines. In the Y1-7 clone the EC50 value for the effect of this peptide was 149 nM, 18.6 fold less potent than PYY. 5. PYY and the Y1-selective non-peptide antagonist, BIBP 3226 displaced [125I]-PYY binding from Y1-7 cell membranes with Ki values of 2.0 and 3.1 nM respectively. In the Y1-7 clone, BIBP 3226 fully inhibited the reductions in VIP-stimulated SCC induced by 30 nM PYY, with an IC50 of 27.2 nM and 30 nM BIBP 3226 caused a parallel rightward shift on the PYY concentration-response curve, with an approximate pKB of 8.0. 6. HT-29 clones stably expressing the Y1 receptor therefore show responses to PYY and its analogues that are characteristic of that subtype, and the Y1-7 clone in particular will be useful in the assessment of novel Y1-specific drugs. This approach will also allow the functional study of NPY Yi receptors with selected mutations.
Holliday
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ND
Epithelial Research Unit, Royal College of Surgeons of England, London.
Cox
H M
HM
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D003000:Clonidine / Q000494:pharmacology; D019073:HT29 Cells / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D006801:Humans; D007457:Iodine Radioisotopes; D009478:Neuropeptide Y / Q000031:analogs & derivatives / Q000494:pharmacology; D010191:Pancreatic Polypeptide / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D019894:Peptide YY; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology; D017476:Receptors, Neuropeptide Y / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D013004:Somatostatin / Q000494:pharmacology; D013268:Stimulation, Chemical; D014162:Transfection; D014407:Tumor Cells, Cultured; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology
D000316:Adrenergic alpha-Agonists; C092926:BIBP 3226; D007457:Iodine Radioisotopes; D009478:Neuropeptide Y; D010446:Peptide Fragments; D010455:Peptides; D017476:Receptors, Neuropeptide Y; C097212:neuropeptide Y-Y1 receptor; D019894:Peptide YY; C062259:neuropeptide Y, Leu(31)-Pro(34)-; D014660:Vasoactive Intestinal Peptide; D013004:Somatostatin; D010191:Pancreatic Polypeptide; D001120:Arginine; D003000:Clonidine