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.

Partial knockdown of G alpha i2 protein is sufficient to abolish the coupling of PYY receptors to biological response in renal proximal tubule cells.

Peptide YY (PYY)-preferring receptors are expressed in the renal proximal tubule cell clone Cl.10 isolated from the PKSV-PCT cell line. They mediate PYY-inhibited cAMP production through coupling with pertussis-sensitive Gi proteins. Previous G alpha i RNA antisense experiments demonstrated the exclusive coupling of the PYY receptor to the Gi2 protein. Here we characterized a clone stably expressing G alpha i2 antisense RNA which exhibited only a partial decrease in G alpha i2 content (#60%) as estimated by Western blot. When compared to control Cl.10 cells, this clone, referred to as Cl.10(t), exhibited: (i) an increase in the dissociation constant of PYY receptors (6.42 vs 0.63 nM); (ii) a complete absence of inhibition of [125I]PYY binding by GTP gamma S and GTP; (iii) the failure of PYY to inhibit basal and forskolin-stimulated cAMP levels; (iv) the failure of PYY to stimulate [35S]GTP gamma S binding to membranes. These findings show that partial knockdown of G alpha i2 expression in Cl.10 cells completely abolish the coupling of PYY receptors to biological response.

G alpha i RNA antisense expression demonstrates the exclusive coupling of peptide YY receptors to G(i)2 proteins in renal proximal tubule cells.

A clone PKSV-PCT Cl.10 referred to as Cl.10 was selected from the PKSV-PCT renal proximal tubule cell line which expressed peptide YY (PYY) receptors (Voisin, T., Bens, M., Cluzeaud, F., Vandewalle, A., and Laburthe, M. (1993) J. Biol. Chem. 268, 20547-20554). In order to identify G(i) protein(s) coupled to PYY receptors, antisense G alpha i protein RNAs were expressed in Cl.10 cells by transfecting the pcDNA3 vector into which were inserted 39 bases of the 5'-noncoding region of G alpha i2 or G alpha i3 used as specific antisense templates. A Cl.10/alpha i2-clone was selected which displayed a drastic decrease (> 90%) of the expression of G alpha i2 without changes of G alpha i3, G alpha s, and G beta subunits (G alpha i1 is not present in Cl.10 cells) as evidenced by Western blots. When compared to untransfected cells, this clone exhibited: (i) an increase in the dissociation constant of PYY receptors (5.3 versus 0.6 nM) identical to that observed in pertussis toxin-treated untransfected cells; (ii) an absence of inhibition of 125I-PYY binding by guanosine 5'-O-(thiotriphosphate) (GTP gamma S); and (iii) the failure of PYY to inhibit cAMP levels and to stimulate [methyl-3H]thymidine incorporation into DNA. A clone was also selected which exhibited a specific decrease (> 80%) of G alpha i3 as compared to untransfected cells. The sensitivity to GTP gamma S and the dissociation constant of PYY receptors as well as PYY-mediated inhibition of cAMP were identical to those observed in untransfected cells. These findings support an exclusive coupling of PYY receptors to G alpha i2.

Galanin receptors in human hypothalamus: biochemical and structural analysis.

Galanin receptors have been characterized in normal human hypothalamus using 125I-galanin binding assays. Competition experiments of porcine 125I-galanin binding to human hypothalamic membranes with native human, porcine and rat galanin (10(-11) M to 10(-8) M) gave comparable results with IC50 close to 0.1 nM. Scatchard analysis indicated one type of high affinity binding sites (Kd = 0.11 nM) with a capacity of 460 fmol/mg protein. Galanin-(1-15) and galanin-(2-29) inhibited tracer binding (IC50 = 1.5 nM), galanin-(3-29) and galanin-(10-29) being inactive. The galanin receptor antagonist, galantide, 10(-14) M to 10(-8) M, also strongly displaced binding of 125I-galanin to the human receptor (IC50 close to 0.15 nM). Guanine nucleotides (from 10(-8) M to 10(-4) M) decreased tracer binding to human membranes by increasing the dissociation of the galanin-receptor complexes. Structural analysis by covalent labelling indicated that the human galanin receptor behaves as a monomeric protein with a molecular mass of 54,000 daltons.

A clonal rat pancreatic delta cell line (Rin14B) expresses a high number of galanin receptors negatively coupled to a pertussis-toxin-sensitive cAMP-production pathway.

Galanin, an ubiquitous neuropeptide, was recently shown to inhibit somatostatin release by the rat islet tumor cell line, Rin-m. By using the clonal pancreatic delta cell line Rin14B, originating from Rin-m cells, we were able to identify the presence of one type of specific galanin-binding site of high affinity (Kd = 1.6 nM; maximal binding capacity = 270 fmol/mg protein) and high specificity for the peptide. Binding of 125I-galanin to these receptors was time-dependent and highly sensitive to guanine nucleotides. Using the cross-linker disuccinimidyl tartrate, covalent linking of the galanin receptor to 125I-galanin in membranes from Rin14B cells, followed by SDS/PAGE analysis of membrane proteins, indicated that the galanin receptor is a protein of 54 kDa. 0.1-100 nM galanin also exerted a marked inhibitory effect on the cAMP-production system under basal conditions, as well as in the presence of the pancreatic peptide glucagon. At a maximal dose, galanin induces a 90-100% decrease of basal and glucagon-stimulated cAMP production levels, with a median inhibition concentration (IC50) of 3 nM galanin. The direct inhibitory effect of galanin on the adenylate cyclase activity in Rin14B cell membranes was also demonstrated (IC50 = 3 nM galanin). The inhibitory effect of galanin on the basal and glucagon-stimulated cAMP production in Rin14B cells was reversed by pertussis toxin. The toxin was also shown to specifically ADP-ribosylate a protein of 41 kDa in membranes from Rin14B cells. Taken together, these data show that the pancreatic delta cell line Rin14B expresses high affinity galanin receptors negatively coupled to a pertussis-toxin-sensitive cAMP-production system.

Galanin inhibits somatostatin release by the rat islet cell tumor in culture, Rin-m.

Using a rat islet cell tumor in culture, Rin-m, we studied the effects of the neuropeptide, galanin, on somatostatin release. Galanin applied to the incubation medium inhibited pancreatic hormone release rapidly and dose dependently with an IC50 at 4 nM and the maximal effect (40% inhibition) was elicited by 100 nM peptide. Pretreatment of Rin-m cells with pertussis toxin abolished the inhibitory effect of galanin on somatostatin release. The results suggest that galanin probably controls the function of the pancreatic delta cell through a pertussis toxin-sensitive pathway.

Galanin receptor in the rat pancreatic beta cell line Rin m 5F. Molecular characterization by chemical cross-linking.

125I-Galanin was cross-linked to receptor in Rin m 5F cell membranes using the bifunctional reagent disuccinimidyl tartarate. Regardless of the presence of reducing agents, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cross-linked galanin-receptor complexes revealed the presence of a radioactive band at Mr 57,000. Excess unlabeled galanin completely inhibited the labeling of the band while other regulatory peptides had no effect. Labeling of the Mr 57,000 complex was abolished by galanin concentration from 10(-9) to 10(-6) M (IC50 = 5 X 10(-9) M). Initial incubation with 125I-galanin in the presence of increasing concentrations of guanyl-5'-yl imidodiphosphate (GMP-P(NH)P) (10(-7) to 10(-4) M) also inhibited the labeling of the Mr 57,000 complex. Moreover, pretreatment of membranes with pertussis toxin before formation of the covalent galanin-receptor complex, dramatically reduced the labeling of the Mr 57,000 species. Covalent Mr 57,000 galanin-receptor complexes solubilized by Triton X-100 bound specifically to wheat germ agglutinin-concanavalin A-, and soybean-coupled Sepharose, supporting the glycoproteic nature of the galanin receptor. Assuming one molecule of 125I-galanin (Mr 3,000) was bound per molecule of protein, these results suggest that the pancreatic galanin receptor is a glycoprotein with a Mr of 54,000 bearing the recognition site for the ligand and which is coupled with a pertussis toxin-sensitive G protein in the plasma membrane.

Structural requirements for galanin interaction with receptors from pancreatic beta cells and from brain tissue of the rat.

The binding activity of several galanin fragments and analogs was measured on specific receptors present in rat brain and the rat pancreatic beta cell line Rin m 5F. In both tissues it was observed that: 1) galanin(3-29), galanin(10-29) and [Ile2]-galanin were ineffective for inhibiting [125I] galanin binding and 2) active peptides had the following rank order of potency: galanin(1-29) greater than [Ac-Trp2]-galanin(2-29) greater than galanin(2-29) greater than galanin(1-15) greater than [Phe2]-galanin greater than [Tyr2]-galanin. It was concluded that the N-terminal portion of galanin is very important for interaction with central or peripheral receptors. The aromatic amino acid in position 2 (Trp in native galanin) plays a crucial role.

Characterization of galanin receptors in the insulin-secreting cell line Rin m 5F: evidence for coupling with a pertussis toxin-sensitive guanosine triphosphate regulatory protein.

The present work characterizes galanin receptors in the insulin-secreting pancreatic beta-cell line Rin m 5F and documents their regulation by guanine nucleotides. Binding of [125I]galanin to cell membranes was found to be temperature dependent, rapid, saturable, reversible, and highly peptide specific. Optimal steady state conditions were achieved after a 60-min incubation at 15 C. The concentration dependence of galanin binding determined by adding increasing concentrations of [125I]galanin indicated that galanin receptors were saturated at 2-3 nM peptide. Scatchard analysis revealed a single class of receptors, with a Kd of 0.3 nM and a binding capacity of 82 fmol/mg protein. Guanyl 5'-yl imidodiphosphate dramatically enhanced the dissociation of bound [125I]galanin. Some guanine nucleotides inhibited [125I]galanin binding to membranes with the following order of potency: guanyl 5'-yl imidodiphosphate greater than GTP = GDP. Other nucleotides had no effect. The effect of the guanine nucleotides was Mg2+ dependent, but Na+ independent, although Mg2+ ions alone (5 mM) slightly enhanced [125I]galanin binding, and Na+ ions alone (100 mM) induced a 60% decrease in the binding. Finally, overnight treatment of Rin m 5F cells with pertussis toxin (0.4 microgram/ml) dramatically reduced [125I]galanin binding to cell membranes. This was related to a 4-fold decrease in receptor affinity, with no change in binding capacity. In conclusion, for the first time evidence of the existence of galanin receptors on functional pancreatic beta-cells is presented. Also, other findings support the fact that galanin receptors are functionally associated with a pertussis toxin-sensitive GTP-binding protein mediating guanine nucleotide control of galanin binding.

Structural requirement for galanin action in the pancreatic beta cell line Rin m 5F.

Galanin fragments were tested for their ability to alter forskolin-stimulated cyclic AMP production and insulin release from Rin m 5F cells. Galanin and its fragments inhibited both events with the following potencies: Gal-(1-29) greater than N-AcGal-(2-29) greater than Gal-(2-29) greater than Gal-(1-15). In contrast, Gal-(3-29), Gal-(10-29) and [Ile2]Gal were inactive. [Phe2]- and [Tyr2]Gal were moderately effective. We conclude that the N-terminal portion of galanin (in particular the aromatic amino acid in position 2) is crucial for activity.

Pancreastatin inhibits insulin release from Rin m 5F cells: reversion by pertussis toxin.

Pancreastatin inhibited carbachol- but not forskolin- or GIP-stimulated insulin release from Rin m 5F cells. The inhibition induced by pancreastatin was dose-dependent, with an ED50 value of 4 nM, and reached a maximum (50% of inhibition) at 10(-7) M peptide. Pretreatment of cells with pertussis toxin abolished the inhibitory effect of pancreastatin on carbachol-induced insulin release. We suggest that pancreastatin exerts a direct inhibitory control on insulin release through a pertussis toxin-sensitive cAMP-independent pathway.

Mechanism of galanin-inhibited insulin release. Occurrence of a pertussis-toxin-sensitive inhibition of adenylate cyclase.

In the insulin-secreting beta cell line Rin m 5F, galanin, a newly discovered ubiquitous neuropeptide, inhibited, by 50%, the stimulation of insulin release induced by gastric inhibitory polypeptide (GIP) or forskolin, i.e. two cAMP-generating effectors. In contrast, it failed to decrease the stimulation of insulin release elicited by either the Ca2+-mobilizing agent, carbamoylcholine, or by dibutyryl-cAMP. Concomitantly, galanin inhibited the GIP- and forskolin-stimulated cAMP production. Furthermore, adenylate cyclase in membranes from Rin m 5F cells was highly sensitive to galanin, which exerted a marked inhibitory effect on the forskolin-stimulated enzyme activity. All these galanin effects were observed at low physiological doses, in the nanomolar range. Overnight treatment of the Rin m 5F cells with pertussis toxin completely abolished the inhibitory effect of galanin on insulin release, cAMP production and adenylate cyclase activity. Moreover, pertussis toxin specifically ADP-ribosylated a 39-kDa protein present in membranes from those cells. Taken together, these data show that the galanin inhibition of insulin release most likely occurs through the inhibition of adenylate cyclase, involving a petussis-toxin-sensitive inhibitory GTP-binding regulatory protein.

Transdifferentiated embryonic neuroretina cells: an in vitro system to study crystallin aggregation process.

Transdifferentiated embryonic quail neuroretina cells synthesize in vitro crystallins (the lens-specific proteins) and form lentoid bodies (structures that mimic lens fiber cells) which also contain crystallins. A comparative study on the size of crystallins is reported in 7-day-old embryonic quail lenses, in 7-day-old embryonic quail transdifferentiated neuroretina cells (normal and MH2 transformed), and in isolated lentoid bodies. Analyses are performed using Superose FPLC in combination with SDS-PAGE and Western blot procedures. In quail lenses, an apparent 560-580-kDa alpha crystallin homopolymer is found and delta crystallin, the major avian lens protein, is detected as a 180-kDa tetramer. beta Crystallins, present in low amount within the 180-kDa peak, are a heterogeneous population composed of subunits of molecular weight identical to those found in chick lenses. In addition, an apparent 46-kDa monomeric delta crystallin is found. Normal and MH2-transformed neuroretina cultures produce an alpha crystallin polymer of lower molecular weight (450 kDa) and delta crystallin in a monomeric or dimeric form. The Western blot pattern of beta crystallins from MH2-transformed neuroretina cultures is strictly identical to that of quail lens beta crystallins. In particular, the beta B1 crystallin, which is specific to lens fiber cell differentiation, and the major beta 25-kDa crystallin are present. However, analysis of isolated lentoid bodies from normal transdifferentiated quail neuroretina cultures showed alpha and delta crystallins of comparable size to those found in lens extract, in particular the delta crystallin in tetrameric form. The lentoid body lens-like structure could favour the crystallin aggregation process.

Crystallin gene expression and lentoid body formation in quail embryo neuroretina cultures transformed by the oncogenic retrovirus Mill Hill 2 or Rous sarcoma virus.

The lens-specific proteins alpha and delta crystallins and lentoid bodies, structures that follow a differentiation pathway similar to that of the lens, regularly appear after 4 to 5 weeks in quail embryo neuroretina monolayer cultures. We have investigated the effects of the avian oncogenic retroviruses Mill Hill 2 and Rous sarcoma virus on this process. Quail embryo neuroretina cells transformed by Mill Hill 2 virus were established into permanent cultures that synthesized alpha and delta crystallins and contained stem cells for the production of lentoid bodies. In contrast, transformation with the Rous sarcoma virus mutant tsNY-68 blocked the appearance of mRNA crystallins, but cytoplasmic alpha and delta crystallin mRNA and alpha crystallin appeared 44 h after a shift to the nonpermissive temperature. However, delta crystallins and lentoid bodies were only present after 7 days. The crystallins of transformed quail neuroretina cultures were immunologically indistinguishable from those of quail lenses and of normal quail embryo neuroretina cultures.

Glutamic acid decarboxylase activity is stimulated in quail retina neuronal cells transformed by Rous sarcoma virus and is regulated by pp60v-src.

Rous sarcoma virus (RSV) stimulates in quail embryo neuro-retina (NR) cultures the specific activity of glutamic acid decarboxylase (GAD), the enzyme responsible for the synthesis of gamma-aminobutyric acid, a major inhibitory neurotransmitter in NR and in central nervous system. In quail embryo NR cultures transformed by ts NY-68, a thermodependent transformation-defective mutant of RSV, stimulation of GAD activity is regulated by pp60v-src, the product of the src gene of RSV. Fibroblasts and myoblasts have a very low GAD activity that is not stimulated after transformation by RSV. Neuronal clones, previously derived from ts NY-68-transformed established NR cell lines, have a high GAD activity which is regulated by pp60v-src, while other clones have a low GAD activity apparently not regulated by pp60v-src. These data indicate that pp60v-src selectively activates the expression of GAD in distinct neuronal cells of quail embryo NR cultures transformed by RSV. GAD activity is also stimulated in NR cells infected with viruses containing v-mil.

A neuronal clone derived from a Rous sarcoma virus-transformed quail embryo neuroretina established culture.

Neuroretina (NR) is an evagination of the central nervous system (CNS) which is composed of photoreceptors, glial (Müller) cells and horizontal, bipolar, amacrine and ganglion neuronal cells. We describe here the usefulness of Rous sarcoma virus (RSV) in the establishment of a neuronal clone from quail embryo neuroretina. When primary cultures of chick and quail embryo neuroretina cells are transformed by RSV, neuronal markers such as ribbon synapses, choline acetyltransferase (CAT) and glutamic acid decarboxylase (GAD) specific activity are present. These RSV-transformed primary cultures can be established into permanent cell lines from which neuronal clones have been isolated. One of them, clone QNR/D, can generate tetrodotoxin(TTX)-inhibitable action potentials on electrical stimulation, has a high GAD activity and binds monoclonal antibodies raised against chick embryo neuroretina. The presence of these neuronal markers suggests that the QNR/D clone is derived from cells of the amacrine or ganglionic lineage. This is the first time that a neuronal cell clone of defined origin has been obtained from the CNS. The neuronal markers of the QNR/D clone are expressed at both the permissive and the non-permissive temperatures for transformation.