The functional investigation of a human adenocarcinoma cell line, stably transfected with the neuropeptide Y Y₁ receptor

Abstract

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 ['251 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 Y1 receptors with selected mutations.

Y4 Receptors Mediate the Inhibitory Responses of Pancreatic Polypeptide in Human and Mouse Colon Mucosa

“…Fresh membranes (20 l, 0.9 Ϯ 0.1 g/l) from HEK293 clones, prepared as outlined by Holliday and Cox (1996), were incubated for 2 h at 22°C in binding buffer [10 mM HEPES, 5 mM KCl, 2.5 mM CaCl 2 , 1.2 mM K 3 PO 4 , 1.2 mM MgSO 4 , 25 mM NaHCO 3 , 0.1 mg/ml bacitracin, and 0.1% bovine serum albumin (BSA), pH 7.4] and 10 pM […”

Control of signalling efficacy by palmitoylation of the rat Y1 receptor

“…In [ 125 I]PYY binding studies (Holliday & Cox, 1996), fresh membrane aliquots were incubated for 120 min at 211C in buffer (4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES) 8 mM, KCl 4 mM, NaHCO 3 20 mM, MgSO 4 1.0 mM, K 3 PO 4 1.0 mM, CaCl 2 2.0 mM, BSA 0.4% w v À1 and bacitracin (0.1 mg ml À1 ); pH 7.4) and [ …”

“…[ ) were prepared in 10 mM N-Tris-(hydroxymethyl)-methyl-2-aminoethane-sulphonic acid (TES) and 0.1 mM phenylmethylsulphonyl fluoride (PMSF; pH 7.6), as described in detail in Holliday & Cox (1996). In [ 125 I]PYY binding studies (Holliday & Cox, 1996), fresh membrane aliquots were incubated for 120 min at 211C in buffer (4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES) 8 mM, KCl 4 mM, NaHCO 3 20 mM, MgSO 4 1.0 mM, K 3 PO 4 1.0 mM, CaCl 2 2.0 mM, BSA 0.4% w v À1 and bacitracin (0.1 mg ml À1 ); pH 7.4) and [ …”

“…To assess the relative efficacies of these nonpalmitoylated receptors in stably transfected Chinese hamster ovary (CHO) K1 cells, we have measured agonist-stimulated guanosine-5 0 -O-(3-thio)triphosphate (GTPg[ 35 S]) binding, a useful technique to directly determine GPCR activation of G proteins (particularly G i ; Williams et al, 1997). In addition, we have adopted a novel functional approach to investigate the Y 1 and Y 1 (C337S) receptors expressed in a human epithelial cell line, by measuring their effects on anion secretion using shortcircuit current (I SC ) techniques (Holliday & Cox, 1996). In contrast to more conventional assays that determine the integrated changes in adenosine 3 0 : 5 0 -cyclic monophosphate (cAMP) production (Kenakin, 1996), I SC responses may be followed in real time to allow indirect measurement of the temporal patterns of Y 1 and Y 1 (C337S) receptor-mediated signalling.…”

Peptidergic Regulation of Intestinal Ion Transport

“…A further potential problem, all too often overlooked in functional studies (though of obvious significance in GI preparations) is the differential stability of agonist peptides, the consequence of which will provide additional variation in the relative orders of agonist potency. For example, we have described an impressive PYY preference (of native and Pro 34 -substituted analogues) for Y 1 receptors stably transfected into HT-29 cells [41] and this PYY preference was still evident (although not as pronounced) in binding assays with HT-29 membranes in the presence of protease inhibitors [42]. It is most likely that preferential degradation of NPY and its Pro 34 analogue by dipeptidyl peptidase IV [43] is responsible for this apparent difference in potency, as this particular peptidase is expressed by HT-29 epithelia [44].…”