The peptide YY-preferring receptor mediating inhibition of small intestinal secretion is a peripheral Y(2) receptor: pharmacological evidence and molecular cloning.

A peptide YY (PYY)-preferring receptor [PYY > neuropeptide Y (NPY)] was previously characterized in rat small intestinal crypt cells, where it mediates inhibition of fluid secretion. Here, we investigated the possible status of this receptor as a peripheral Y(2) receptor in rats. Typical Y(2) agonists (PYY(3-36), NPY(3-36), NPY(13-36), C2-NPY) and very short PYY analogs (N-alpha-Ac-PYY(22-36) and N-alpha-Ac-PYY(25-36)) acting at the intestinal PYY receptor were tested for their ability to inhibit the binding of (125)I-PYY to membranes of rat intestinal crypt cells and of CHO cells stably transfected with the rat hippocampal Y(2) receptor cDNA. Similar PYY preference was observed and all analogs exhibited comparable high affinity in both binding assays. The same held true for the specific Y(2) antagonist BIIE0246 with a K(i) value of 6.5 and 9.0 nM, respectively. BIIE0246 completely abolished the inhibition of cAMP production by PYY in crypt cells and transfected CHO cells. Moreover, the antagonist 1) considerably reversed the PYY-induced reduction of short-circuit current in rat jejunum mucosa in Ussing chamber and 2) completely abolished the antisecretory action of PYY on vasoactive intestinal peptide (VIP)-induced fluid secretion in rat jejunum in vivo. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) experiments showed that Y(2) receptor transcripts were present in intestinal crypt cells (3 x 10(2) molecules/100 ng RNA(T)) with no expression in villus cells, in complete agreement with the exclusive binding of PYY in crypt cells. Finally, a full-length Y(2) receptor was cloned by RT-PCR from rat intestinal crypt cells and also from human small intestine. We conclude that the so-called PYY-preferring receptor mediating inhibition of intestinal secretion is a peripheral Y(2) receptor.

Functional and molecular properties of the human recombinant Y4 receptor: resistance to agonist-promoted desensitization.

After stable transfection of Chinese hamster ovary cells with the human Y4 receptor, clone 29 was isolated and studied for receptor properties. The following data were obtained: 1) one class of binding site was identified by analysis of (125)I-human pancreatic polypeptide (hPP) binding to cell membranes with a K(d) value of 0. 26 nM and a B(max) value of 1.44 pmol/mg protein; 2) the K(i) values for inhibition of (125)I-hPP binding by hPP, human peptide YY (hPYY), human neuropeptide Y (hNPY), and analogs were hPP (0.7 nM) < rat PP (47 nM) < hPYY (94 nM) < h[Leu(31)-Pro(34)]NPY (124 nM) << hNPY = porcine NPY(13-36) = rat D-[Trp(32)]NPY (>1 microM); 3) cross-linking experiments using (125)I-hPP identified a single M(r) 60,000 glycosylated Y4 receptor; and 4) the natural peptides hPP, hPYY, and hNPY inhibited forskolin-stimulated cAMP production in clone 29 cells with EC(50) values of 0.56 nM, 218 nM, and >1 microM, respectively. The inhibitory effect of hPP was abolished when cells were incubated with pertussis toxin, indicating a pertussis toxin-sensitive G(i) protein-mediated event. 5) Exposure of cells to 10 nM hPP for 24 h resulted in the absence of modification of binding capacity (1.38 versus 1.44 pmol/mg protein in control cells) or affinity (0.31 versus 0.26 nM in control cells); there also was no modification in the potency and efficacy of hPP in inhibiting forskolin-stimulated cAMP. Immunofluorescence indicated that the Y4 receptor was not internalized within the cells after 24-h treatment with 10 nM hPP. These data support that Y4 receptors are resistant to agonist-promoted desensitization and internalization. Clone 29 cells provide a valuable tool to further characterize the pharmacological aspects of human Y4 receptor.

Identification and distribution of mRNA encoding the Y1, Y2, Y4, and Y5 receptors for peptides of the PP-fold family in the rat intestine and colon.

Peptide YY (PYY), neuropeptide Y (NPY) and pancreatic polypeptide (PP) are structurally related peptides which have potent antisecretory effects in small and/or large intestines. Receptors mediating these effects are still unknown with the exception of a PYY-preferring receptor expressed in small intestinal crypts. In the present study, expression of recently cloned Y1, Y2, and Y5 receptors which have similar affinity for PYY and NPY and Y4 receptors which have a high affinity for PP was investigated in gut by RT-PCR analysis. The data show that all Y receptors are expressed in small intestine and/or colon but with specific distributions. Y1 receptors are only expressed in nonepithelial colonic tissue, whereas Y2 and Y4 receptors are present in both epithelial and nonepithelial tissue of the small or large intestine. In contrast, Y5 receptor expression appears to be restricted to epithelial crypts of the small intestine and nonepithelial tissue of colon. Sequencing of PCR products showed 100% identity with the corresponding sequences of the cloned Y1, Y4, or Y5 receptors. The PCR product obtained with Y2 primers from rat crypt cells showed 84% identity with the cloned human Y2 receptor. These data indicate a wide distribution of Y receptors in small intestine and colon. They also suggest that Y1, Y2, Y4, and Y5 receptors may be responsible for still unexplained effects of PYY, NPY, or PP on secretion in small and large intestines.

Pharmacological profile of the rat intestinal crypt peptide YY receptor vs. the recombinant rat Y5 receptor.

Peptide YY and neuropeptide Y have potent antisecretory effects in rat small intestine. Scatchard analysis of [125I]peptide YY binding revealed a 10-fold higher concentration of receptors in rat jejunal crypt cells than in villus cells and no detectable receptors in colonic epithelium. Reverse transcription polymerase chain reaction analysis of neuropeptide Y Y5 receptor mRNA indicated that they are mainly expressed in rat jejunal crypts with very few or no expression in villus cells and colon epithelium, respectively. In order to determine whether neuropeptide Y Y5 receptors could represent the intestinal crypt receptor for peptide YY and neuropeptide Y, the ability of peptide YY, neuropeptide Y, pancreatic polypeptide and analogues to inhibit [125I]peptide YY binding to membrane prepared from rat crypt cells and COS-7 cells (African green monkey kidney cells) transfected with the rat neuropeptide Y Y5 receptor cDNA was tested. It appeared that several analogues displayed different inhibition constants (Ki) in the two binding assays, more especially N-alpha-acetyl-peptide YY-(22-36) which was 1200 x more potent in the crypt cell binding assay than in the recombinant neuropeptide Y Y5 receptor binding assay. These data support that the intestinal crypt peptide YY receptor is not a Y5 receptor. reserved.