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The functional investigation of a human adenocarcinoma cell line, stably transfected with the neuropeptide Y Y 1 receptor
Author(s) -
Holliday Nicholas D.,
Cox Helen M.
Publication year - 1996
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1996.tb15989.x
Subject(s) - peptide yy , neuropeptide y receptor , vasoactive intestinal peptide , transfection , receptor , cell culture , agonist , endocrinology , medicine , microbiology and biotechnology , pancreatic polypeptide , biology , neuropeptide , clone (java method) , chemistry , biochemistry , hormone , gene , glucagon , genetics
1 The human adenocarcinoma cell line, HT‐29, has been stably transfected with the cDNA sequence for the rat neuropeptide Y (NPY) Y 1 receptor, and three Y1 clones (Y1‐4, Y1‐7 and Y1‐16) have been isolated which express high levels of specific [ 125 I]‐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 EC 50 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 μ m ) 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 EC 50 values (in nM) were 18.6 in Y1‐4, 8.0 in Y1‐7 and 52.5 in Y1‐16. hPP (1 μ m ) produced only small and transient responses in each transfected cell type. 4 The Y 1 receptor agonist, [Leu 31 , Pro 34 ] NPY (1 μ m ) was also effective in the three Y1 cell lines. In the Y1‐7 clone the EC 50 value for the effect of this peptide was 149 nM, 18.6 fold less potent than PYY. 5 PYY and the Y 1 ‐selective non‐peptide antagonist, BIBP 3226 displaced [ 125 I]‐PYY binding from Y1‐7 cell membranes with K i 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 IC 50 of 27.2 nM and 30 nM BIBP 3226 caused a parallel rightward shift on the PYY concentration‐response curve, with an approximate p K B of 8.0. 6 HT‐29 clones stably expressing the Y 1 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 Y 1 ‐specific drugs. This approach will also allow the functional study of NPY Y 1 receptors with selected mutations.