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Several Receptors Mediate the Antisecretory Effect of Peptide YY, Neuropeptide Y, and Pancreatic Polypeptide on VIP-Induced Fluid Secretion in the Rat Jejunum In Vivo
Peptides, Vol. 18, No. 4, pp. 551–557, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/97 $17.00 / .00
PII S0196-9781(97)00069-7
Several Receptors Mediate the Antisecretory Effect of Peptide YY, Neuropeptide Y, and Pancreatic Polypeptide on VIP-Induced Fluid Secretion in the Rat Jejunum In Vivo A. SOULI,* J. CHARIOT,* T. VOISIN,* O. PRESSET,* A. TSOCAS,* A. BALASUBRAMANIAM,† M. LABURTHE* AND C. ROZE´*1 *INSERM U410, Faculte´ de Me´decine X. Bichat, BP 416, 75870 Paris Cedex 18, France †University of Cincinnati Medical Center, OH 45267-0558 Received 29 October 1996; Accepted 17 December 1996 SOULI, A., J. CHARIOT, T. VOISIN, O. PRESSET, A. TSOCAS, A. BALASUBRAMANIAM, M. LABURTHE AND C. ROZE´. Several receptors mediate the antisecretory effect of peptide YY, neuropeptide Y, and pancreatic polypeptide on VIP-induced fluid secretion in rat jejunum in vivo. PEPTIDES 18(4) 551–557, 1997.—Several Y receptor subtypes have been cloned and/or pharmacologically characterized that mediate the effects of the regulatory peptides peptide YY (PYY), neuropeptide Y (NPY), and pancreatic polypeptide (PP). These peptides possess antisecretory properties on the intestine. This effect can be blocked in vivo by neural antagonists, suggesting the intervention of neural receptors, although epithelial PYY-preferring receptors have been evidenced on jejunal crypt cells. The purpose of the present experiments was to compare the antisecretory properties in vivo of a series of PYY and NPY derivatives with various affinities for different Y receptor subtypes, in order to determine which subtypes were involved. A model of VIP-stimulated secretion by rat jejunal loops was used. The results were compared with the binding affinities for PYY-preferring receptors determined on rat jejunal crypt cell membranes. Full-length PYY(1–36) was about three times more potent than NPY(1–36), and 10 times more potent than PP in the low dose range. PP, however, had a low efficacy limited to about 50% inhibition of VIP effect. Both Y1 agonists ([Leu 31 ,Pro 34 ]PYY and [Leu 31 ,Pro 34 ]NPY), and Y2 agonists [C-terminal fragments ranging from PYY(3–36) and NPY(3–36) to PYY(22–36) to NPY(22–36)] displayed potent antisecretory properties. PYY derivatives and fragments were always more potent than their respective NPY counterparts. In contrast, Y1 derivatives and PP had very low affinity for the epithelial PYY receptor as measured in vitro by radioreceptor assay. These data suggest that the antisecretory effect of PYY/NPY/PP peptides in vivo involves the effets of several receptors: a Y2-like, PYY-preferring receptor identical to the epithelial receptor, a Y1-like receptor, and a third receptor with high affinity for PP. q 1997 Elsevier Science Inc. Peptide YY Neuropeptide Y Pancreatic polypeptide Truncated analogues Y receptor subtypes
Rat jejunum
been characterized by pharmacological means (namely Y3 , PYYpreferring, unspecific) (2,8,13,25,31,32,49,52). The receptor subtypes involved in the intestinal antisecretory effect of PYY and NPY have been mainly studied in vitro. The fulllength peptides NPY(1–36) and PYY(1–36), which recognize both Y1 and Y2 subtypes, are potent antisecretagogues in the rat jejunum in vitro, with PYY being more potent than NPY (11). In the same species, it was suggested that the jejunal antisecretory effect measured in Ussing chambers was related to Y2 receptors, because C-terminal fragments of NPY, reputed specific of the Y2 subtype, especially NPY(11–36) to NPY(14–36) (10) and PYY(22– 36) (1), were efficient in this preparation.
PEPTIDE YY (PYY), neuropeptide Y (NPY), and pancreatic polypeptide (PP) are closely related endogenous 36-amino acid peptides (47). PYY and NPY are potent inhibitors of gastrointestinal motor and secretory functions (42); they have been shown to inhibit intestinal secretion of electrolytes and water in vitro and in vivo, in different animal species and in man (5,7,9– 12,15,17,24,35,40). Several receptor subtypes for the PYY/NPY/PP family (Y receptors) have been described in different organs and species (4,18,53). Some of them have been cloned (namely, Y1 , Y2 , Y4 Å PP1 , Y5 Å feeding receptor, and Y6 , which is slightly different from Y5 : (3,6,16,19–23,27,28,30,45,54) and others have only
1
Antisecretory effects
Requests for reprints should be addressed to C. Roze´, INSERM U410 Faculte´ de Me´decine X. Bichat, BP 416, 75870 Paris Cedex 18, France.
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In other conditions (crypt enterocyte membranes) ‘‘PYY-preferring’’ receptors, negatively coupled to adenylylcyclase, have been evidenced in the rat small intestine (26,41,49,51). The intervention of other receptors has been suggested in the colon. In rabbit distal colon, the inhibition of vasoactive intestinal peptide (VIP)-stimulated ion transport in vitro involved a novel class of Y receptors with a very similar sensitivity to PP, PYY, NPY, and to the Y1 agonist [Leu 31 ,Pro 34 ]NPY (2). Another new receptor was claimed to be involved in the proabsorptive effect of PYY, NPY, PP, and NPY derivatives in a human colonic epithelial cell line, with an order of potency different from that reported in the rabbit distal colon (13). To summarize the in vitro studies dealing with antisecretory effects of PYY / NPY peptides, the involvement of Y2 and / or PYY-preferring receptors is suggested in the jejunum, whereas other, yet poorly defined receptors might be involved in the colon. To our knowledge, only a single in vivo study, performed in dogs equipped with Thiry-Vella fistulas, has suggested that Y2 receptors were involved in the inhibitory effect of PYY derivatives on intestinal secretion (29). In this study, the proabsorptive effect of a substituted PYY(22–36) analogue was reported after intraluminal administration in the ileum and colon, but not in the jejunum. Neural mechanisms appear to participate in the effects of PYY/NPY peptides in vivo (37,39,44), suggesting the intervention of neural receptors, whereas PYY/NPY receptors are also present on enterocyte membranes. We thus sought to determine whether a single or several subtypes of Y receptors were involved in the PYY/NPY/PP effects on the rat jejunum in vivo. For this purpose we compared the antisecretory effects of PYY(1–36), NPY(1–36), PP, and of fragments or analogues reputed more specific for Y1 ([Leu 31 ,Pro 34 ]PYY, [Leu 31 ,Pro 34 ]NPY) and Y2 receptors [PYY(3–36), PYY(13–36), PYY(22–36), N-aAcPYY(22–36), NPY(3–36), NPY(13–36), NPY(22–36)] on VIP-induced fluid secretion in the rat jejunum. In addition, the in vivo potency of these peptides was compared to their binding affinity in vitro to PYY-preferring receptors in rat jejunal crypt cell membranes. METHOD
In Vivo Experiments Animals. Male Wistar rats weighing 180–200 g (Iffa-Credo, Les Oncins, F 69210 L’Arbresle) were fasted for 24 h with free access to water before the experiments. Peptides. PYY(1–36) (pig, rat), NPY(1–36) (human, rat), PP (human), PYY(3–36), PYY(13–36), NPY(3–36), NPY ( 13 – 36 ) , NPY ( 22 – 36 ) , [ Leu 31 ,Pro 34 ] PYY, [ Leu 31 ,Pro 34 ] NPY, and VIP (human, pig, rat) were purchased from Neosystem, Strasbourg France. PYY(22–36) and N-a-AcPYY(22–36) were generous gifts of A. Balasubramaniam (University of Cincinnati Medical Center, OH). Peptide solutions were prepared in 0.9% NaCl added 0.3% bovine serum albumin (Sigma, A4503) to limit adhesion to injection material. Experimental setup. Rats were anesthetized with pentobarbital (50 mg/kg, IP). The animals were maintained at constant temperature (37 { 0.57C) during the experiment with a heating device. A saphenous vein was cannulated to infuse peptides. The abdomen was opened and a jejunal loop (about 20 cm long) was delimited by two ligatures respectively situated 3 and 23 cm distal to the ligament of Treitz. At time zero (t Å 0), 2 ml of 0.9% saline (prewarmed at 377C) was instilled into the loop with a calibrated syringe (needle 0.4 1 10 mm). Care was taken during the preparation of the loop to protect the marginal artery of the
bowel from damage and to maintain as far as possible the normal anatomical placement of the loop. The jejunal loop was returned to the abdomen, which was then closed. Thirty minutes later, the rats were sacrificed by IV injection of air. The jejunal loop was exteriorized, cut just proximal and distal to the ligatures, and its length was measured. The loop was weighed before and after removing its fluid contents (with light drying between two sheets of filter paper) to determine its fluid contents. Experimental protocol. Intestinal secretion of water and electrolytes was stimulated by VIP (30 mg/kg/h Å 9 nmol/kg/h) IV infused at 2.5 ml/h from t Å 0 to t Å 30 min. PYY, NPY, and their derivatives were administered as a bolus IV injection administered over 1 min, in doses of 3–900 pmol/kg, 15 min before starting VIP infusion. In control groups, intestinal secretion of water was measured in basal conditions (i.e., in rats receiving peptide vehicle alone, 0.9% NaCl added 0.3% bovine serum albumin). Expression of results. The weight of the full (F, mg) and empty loop (E, mg), the loop length (L, cm) and the amount of saline placed in the loop at the beginning of the experiment (2000 ml) allowed to calculate the net water flux (F-E-2000)/L, that was expressed as ml/cm/30 min (assuming that 1 ml saline Å 1 mg). Net absorption is indicated by a negative value and net secretion by a positive value. Results appear in the text and figures as means { 1 SEM of 5 to 13 animals per group. For each peptide the ID50 ( defined as the dose decreasing by 50% the secretory effect of VIP over the basal level ) and its 95% confidence limits were calculated from the regression curve of net water flux on the log of peptide dose, as previously described ( 46 ) . In Vitro Binding Studies Materials. [ 125I]Na (IMS300) was purchased from Amersham (Les Ulis, France). HEPES, PMSF (phenylmethylsulfonyl fluoride), TLCK (N-a-p-tosyl-L-lysine chloromethyl ketone), bacitracin, and the other highly purified chemicals used were purchased from Sigma Chemical Co. (St Louis, MO). BSA (bovine serum albumin, Pentex, Fraction V) was obtained from Miles Laboratories (Elkart, NJ). [ 125I-Tyr 36 ]Monoiodo PYY (referred to as [ 125I]PYY below) was prepared and purified as previously described (49). Preparation of intestinal crypt membranes. Male Wistar rats weighing 250–280 g, fed ad lib, were used in membrane binding experiments. The jejunum was removed after decapitation and crypt cells were separated from villus cells by shaking the everted jejunum for successive periods in a dispersing solution containing EDTA as previously described (48). A cell fraction enriched in crypt cells (containing about 90% of crypt cells) was used to prepare crude membranes. Briefly, crypt cells were homogenized by use of a Waring blendor in 0.25 M sucrose buffered with 10 mM triethanolamine (pH 7.5), containing 5 m M EDTA. After centrifugation at 2,600 1 g for 10 min, the supernatant was centrifuged at 20,000 1 g for 15 min. The resulting pellet was washed in 20 mM HEPES buffer (pH 7.5), repelleted, and stored at 0807C until used (48). Binding of [ 125 I]PYY to membrane-bound receptors. Binding of [ 125I]PYY to membrane preparations was conducted as previously described (48). Briefly, membranes (200 mg protein/ml) were incubated for 120 min at 157C in 250 ml of incubation buffer [20 mM HEPES buffer (pH 7.5), 2% (w/v) BSA, 17 mg/l PMSF, 10 mg/l TLCK, and 100 mg/l bacitracin] containing 0.05 nM [ 125I]PYY (2,200 Ci/mmol) with or without unlabeled PYY or other competing peptides. At the end of the incubation, 150ml aliquots of membranes were mixed with 150 ml ice-cold in-
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FIG. 1. Inhibitory effects of IV bolus injections of Peptide YY, NPY, and PP at different doses (from 3 to 900 pmol/kg) on VIP (30 mg/kg/ h)-induced net water flux in rat jejunum. Positive values correspond to secretion and negative values to absorption. Results are expressed as means { SEM of 5 to 13 animals.
cubation buffer. Bound and free peptides were separated by centrifugation at 20,000 1 g for 10 min, and membrane pellets were washed twice with 10% (w/v) sucrose in 20 mM HEPES buffer (pH 7.5). The radioactivity was then counted with a gamma counter. The nonspecific binding represented about 2% of total binding. All binding data were analyzed using the LIGAND computer program developed by Munson and Rodbard (33). RESULTS
Effect of PYY, NPY, and PP on VIP-Stimulated Fluid Jejunal Secretion PYY(1–36) inhibited in a dose-related manner VIP-stimulated jejunal fluid secretion. In preliminary experiments, two dif-
ferent doses of VIP were used. In basal conditions, the mean water net flux was 046.9 { 3.4 ml/cm/30 min, indicating a basal absorption. After stimulation by 100 mg/kg/h of VIP, the jejunal secretion reached 39 { 2.7 ml/cm/30 min; the inhibition was complete after 300 pmol/kg of PYY and the ID50 was 30 pmol/ kg of PYY (not shown). After stimulation by 30 mg/kg/h of VIP, the jejunal net water flux changed from a basal absorption of 042.2 { 2.9 ml/cm/30 min to a smaller secretion (3.4 { 2.9 ml/cm/30 min); the inhibition was complete after 100 pmol/kg of PYY (Fig. 1) and the ID50 was 6 pmol/kg of PYY (Table 1). The following experiments were then conducted with the smaller VIP dose (30 mg/kg/h), which produced a milder stimulation, mimicking probably a more physiological situation than the larger VIP dose. NPY(1–36) inhibited in a dose-related manner VIP-stimulated jejunal fluid secretion. The inhibition was almost complete at 100 pmol/kg (Fig. 1). The ID50 was 15.6 pmol/kg (Table 1). Compared to PYY, NPY was slightly less potent to inhibit VIPstimulated fluid jejunal secretion. PP also inhibited VIP-stimulated jejunal fluid secretion in a dose-related manner (Fig. 1). However, the inhibition was limited to about 50% of the fluid secretion at the highest doses 300 and 1000 pmol/kg. In its limited range of action PP was less potent than both PYY and NPY. However, when the ID50 was calculated as the PP dose decreasing by 50% the maximal effect of PP (instead of the PP dose decreasing by 50% the effect of VIP), PP appeared as potent as NPY (Table 1). Effect of PYY Derivatives on VIP-Stimulated Fluid Jejunal Secretion All PYY derivatives inhibited VIP-stimulated fluid secretion in a dose-related manner. The decreasing order of potency was: PYY(1–36) ú PYY(3–36) É [Leu 31 ,Pro 34 ]PYY ú PYY(22– 36) ú PYY(13–36) (Fig. 2). The ID50s are indicated in Table 1. Full-length PYY was more potent than any of the other peptides tested and PYY(13–36) was the least potent. N-aAcPYY(22–36) was nearly as potent as PYY(3–36), and several times more potent than PYY(22–36) and PYY(13–36). Two results should be emphasized: 1) the Y2 agonist PYY(3– 36) and the Y1 agonist [Leu 31 ,Pro 34 ]PYY were equipotent; 2)
TABLE 1 ANTISECRETORY POTENCIES OF PYY, NPY, AND THEIR DERIVATIVES IN THE RAT JEJUNUM IN VIVO ID50s Ratio Peptide
PYY(1–36) PYY(3–36) PYY(13–36) PYY(22–36) N-a-Ac PYY(22–36) [Leu31, Pro34]PYY NPY(1–36) NPY(3–36) NPY(13–36) NPY(22–36) [Leu31, Pro34]NPY PP
ID50 (pmol/kg)
95% Confidence Limits
Derivative/Full Length Peptide
Derivative/PYY(1–36)
6.0 13.2 97 55 21.7 15.7 15.6 19.9 É1320 94 100 É200* 11.3†
(3.9–9.3) (7.2–24.2) (60–157) (26–117) (15.4–30.6) (10.6–157) (9.4–25.7) (9.7–40.7) — (55–183) (49–180) — (2.7–47)
1 2.2 16.0 9.2 3.6 2.6 1 1.3 85 6.0 6.4 1 1
1 2.2 16.0 9.2 3.6 2.6 2.6 3.3 218 15.5 16.5 49.5 1.9
* PP was different from other peptides because maximal inhibition reached with any dose of PP was not larger than about 50% (see Fig. 1). † ID50 calculated when considering 50% of maximal PP effect, instead of 50% inhibition of VIP effect.
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FIG. 2. Inhibitory effects of PYY(1–36), PYY(3–36), PYY(13–36), PYY(22–36), and [Leu 31 ,Pro 34 ]PYY on VIP-induced net water flux in rat jejunum, by increasing doses. Each point represents the mean { SEM of 5 to 8 animals.
FIG. 3. Inhibitory effects of NPY(1–36), NPY(3–36), NPY(13–36), NPY(22–36), and [Leu 31 ,Pro 34 ]NPY on VIP-induced net water flux in rat jejunum, by increasing doses. Each point represents the mean { SEM of 5 to 8 animals.
the shorter fragment PYY(22–36) was slightly more potent than PYY(13–36).
36) used did not allow to reach the 50% inhibition allowing to calculate the ID50 . Binding to Rat Jejunal Crypt Cell Membranes In Vitro
Effect of NPY Derivatives on VIP-Stimulated Fluid Jejunal Secretion All NPY derivatives inhibited the VIP-stimulated fluid secretion in a dose-related manner (Fig. 3). The decreasing order of potency was: NPY(1–36) Å NPY(3–36) ú NPY(22–36) Å [Leu 31 ,Pro 34 ]NPY @ NPY(13–36). The ID50 values were, respectively, 15.6, 19.9, 94, 100, and É 1320 pmol/kg (Table 1). NPY(1–36) and the Y2 agonist NPY(3–36) were equipotent, whereas the Y1 agonist [Leu 31 ,Pro 34 ]NPY was about five times less potent. As in the case of PYY fragments, NPY(13–36) was by far the least potent of all NPY derivatives tested, including the shorter peptide NPY(22–36). The largest dose of NPY(13–
Data from the binding experiments are reported in Table 2. PYY(1–36) had the highest affinity for epithelial crypt cell receptors (IC50 Å 0.14 nM). The decreasing order of binding affinities for PYY derivatives was PYY(1–36) ú PYY(3–36) ú PYY(22–36) É PYY(13–36) @ [Leu 31 ,Pro 34 ]PYY. The affinity of NPY(1–36) was lower than that of PYY(1– 36) for epithelial crypt cell receptors. Within the series of NPY derivatives, the decreasing order of binding affinities was NPY(1–36) É NPY(3–36) É NPY(22–36) ú NPY(13–36) @ [Leu 31 ,Pro 34 ]NPY. Within the series of NPY derivatives, this affinity order was similar to the order found for the respective PYY derivatives.
TABLE 2 BINDING OF PYY, NPY, PP, AND THEIR DERIVATIVES TO RAT JEJUNAL CRYPT CELL MEMBRANES IC50s Ratio Peptides
IC50 (nM)
sm (nM)
Derivative/Full Length Peptide
Derivative/PYY(1–36)
PYY(1–36) PYY(3–36) PYY(13–36) PYY(22–36) N-a-Ac PYY(22–36) [Leu31, Pro34]PYY NPY(1–36) NPY(3–36) NPY(13–36) NPY(22–36) [Leu31, Pro34]NPY PP
0.14 0.70 8.9 5.6 0.91 199 4.7 5.0 28.1 5.8 562 ú1000
0.04 0.32 1.21 2.70 0.55 57 1.3 1.8 7.9 2.9 112 —
1.0 5.0 64 40 6.5 1420 1.0 1.1 6.0 1.2 120 ú213
1.0 5.0 64 40 6.5 1420 34 36 201 41 4015 ú7140
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JEJUNAL PYY RECEPTORS IN VIVO Finally, the affinity of PP for crypt cell membranes was very low. Three points should be emphasized among these in vitro data: 1) the Y1-selective agonists [Leu 31 ,Pro 34 ]PYY and [Leu 31 ,Pro 34 ]NPY displayed very low affinities 2) the shorter peptides PYY(22–36) and NPY(22–36) showed better affinities than the respective longer fragments PYY(13–36) and NPY(13–36); 3) PP had a very low affinity for crypt cell membrane receptors. DISCUSSION
The present study was designed to investigate the subtypes of Y receptors involved in the jejunal antisecretory effect of PYY and NPY by comparing the effects of the full-length peptides with different fragments or analogues on VIP-stimulated jejunal secretion in the rat in vivo. The model chosen for the present work was the jejunal ligated loop in situ in anesthetized rats. In a previous article (43), we showed that this model was appropriate to evidence the antisecretory effect of PYY and NPY against VIP- and PGE2-stimulated secretion in the duodenum, jejunum, and ileum. Potency differences, however, occurred between these intestinal segments, so we decided to focus on the mechanisms involved in the jejunum. In this previous work, we used 50-min venous infusions of PYY and NPY, and the comparatively large dose of 100 mg/kg/h of VIP as a stimulant. Because previous articles had claimed that bolus injections of small doses of NPY could act over more than 2 h to inhibit VIP-stimulated intestinal secretion (37) as well as to stimulate duodenal bicarbonate secretion (34), we used bolus injections of PYY and derivatives. Although this protocol might seem inappropriate to study the effect of a peptide with a short half-life, antisecretory effects were clearly evidenced. We also retained in the present study the dose of 30 mg/kg/h of VIP, which seemed to us less ‘‘pharmacological’’ than 100 mg/kg/h, and which allowed us to obtain clear-cut inhibitions with small PYY doses (subtotal inhibition with 30 pmol/kg, ID50 6.0 pmol/kg). In fact, this small dose of VIP mainly blocked the basal absorption rather than stimulated secretion. Y1 and Y2 receptors are pharmacologically well defined and have been cloned (19,21,27,38). Both recognize NPY and PYY but not PP. Y1 receptors recognize the full-length peptides and display a poor affinity for the C-terminal fragments, whereas modified full-length peptides such as [Leu 31 ,Pro 34 ] or [Pro 34 ] are specific Y1 agonists. Y2 receptors are less selective for peptide chain length, and they recognize C-terminal fragments of various lengths: 2–36, 3–36, 13–36, 22–36. A PYY-preferring receptor, binding PYY better than NPY, and poorly PP, negatively coupled to adenylylcyclase, has been described in rat enterocytes ( 26,41,49 ) , in dog adipocytes ( 8 ) , and in a renal epithelial cell line ( 50 ) . Another PYY-preferring receptor has been described in rabbit colon ( 31 ) . In another work using rabbit distal colon in vitro, the proabsorptive effects of PYY, NPY, PP, and the Y1 agonist [ Leu 31 ,Pro 34 ] NPY were similar ( 2 ) , whereas the Y2 agonist PYY ( 13 – 36 ) had no effect, suggesting another receptor subtype. Still different was the potency order of PYY, NPY, PP, and [ Leu 31 ,Pro 34 ] NPY on the human colonic epithelial cell line Col-6 ( 13 ) . Y3 receptors recognize NPY and its derivatives ( 14 ) , human PP, but poorly PYY and avian PP and not at all rat PP ( 53 ) . Y4 / PP1 receptors recognize PP with high affinity and PYY and NPY with very low affinity ( 3,23,30 ) . Recently, a Y5 receptor was cloned from rat hypothalamus ( 22 ) . This receptor, which does not discriminate between NPY, PYY, and PP, is most likely the atypical Y1-like feeding receptor previously described ( 45 ) . Very re-
555 cently a Y6 receptor, close to Y5 , has been cloned from mouse genomic DNA ( 54 ) . An important point is that the jejunal antisecretory effects of NPY and PYY can be suppressed by sigma antagonists in mouse jejunum in vitro (36), and by sigma or a2-adrenoceptor antagonists in rat and human jejunum in vivo (39,44). Thus, PYY peptides may act upon both enterocytes and nerves, and the receptors may be different. Indeed, our present results do not fit within the concept of a single, already described, Y1 or Y2 receptor. The effect of NPY derivatives could to some extent be considered as a Y2-like effect, because NPY(3–36) was as potent as NPY(1–36), and [Leu 31 ,Pro 34 ]NPY was about six times less potent. However, this does not apply to PYY derivatives, because PYY(3–36) and [Leu 31 ,Pro 34 ]PYY, which should discriminate between Y1 and Y2 receptors, were equipotent and were also as potent as NPY(1–36). Thus, Y1 agonists, such as the [Leu 31 ,Pro 34 ] derivatives, and Y2 agonists such as the C-terminal fragments, both displayed appreciable potencies, indicating either that both Y1 and Y2 receptors were involved, or alternatively, that another receptor type was concerned. A major characteristic of the receptors involved in vivo is that they prefer PYY: PYY ( 1 – 36 ) was the most potent molecule tested, and if one compares each PYY fragment or analogue with its NPY counterpart, the potency ratio always favors the PYY derivative. This preference for PYY agrees with the published characteristics of the PYY-preferring receptor preferentially expressed in the crypts of rat small intestine ( 48 ) . The density of these receptors decreases from duodenum to ileum, and they are absent from the cecum and colon ( 26 ) . Similarly, their inhibitory effect on cAMP production has been observed in the rat duodenum, jejunum, and ileum, but not in colon ( 41 ) . In agreement with this distribution, we found that the inhibitory potency of PYY against VIP-induced secretion was greater in the rat duodenum than in the jejunum and ileum ( 43 ) . However, comparing the present in vitro binding data with the in vivo inhibitory potencies indicates that the enterocyte receptor cannot be alone involved in the in vivo effect. Although the potency of the N-terminal fragments of PYY and NPY in vivo is similar to the binding data on enterocyte membranes, a major discrepancy occurs when one considers the binding and effect of the Y1 agonists [ Leu 31 ,Pro 34 ] PYY and [ Leu 31 ,Pro 34 ] NPY. Both of these peptides had a very low affinity for enterocyte membranes ( respectively 1420 and 4015 times lower than PYY, Table 2 ) , whereas they were comparatively potent to inhibit jejunal secretion in vivo ( with a potency which was only 2.6 and 16.5 times lower than PYY, respectively ) . In addition, PYY receptors of enterocyte membranes had very low affinity for PP, whereas low doses of PP displayed distinct inhibitory effects in vivo. Although this inhibitory effect of PP was incomplete and the dose – effect curve was limited to an about 50% inhibition, partial implication of an Y4 / PP1 subtype or another subtype with high affinity for PP has to be considered. Alternatively, a nonspecific receptor for PP, PYY, and NPY such as that described in rabbit colon ( 2 ) or in human colonic cells ( 13 ) could also participate in vivo. Besides the affinity order, another evidence favoring a third receptor subtype mediating the effect of PP is that PP was unable to totally inhibit the VIP effect, whereas all the other derivatives tested in large enough doses could apparently achieve such total inhibition. Presence of this PP receptor subtype may also explain the greater potency of PYY and its fragments in all situations, because PYY also has high affinity for PP receptors ( 20 ) , so that PYY may act both through PYY-preferring and PP receptors.
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Finally, a new characteristic emerging from the present experiments is the nonuniform decrease with chain length of the binding affinity of C-terminal fragments of PYY and NPY. Although one might have expected a progressive decrease of both affinity and potency with decreasing chain length, the 22–36 fragments of PYY and NPY were both more potent in vitro (binding assay) and in vivo (secretion assay) than the 13–36 respective fragments. In addition, the modified peptide N-a-AcPYY(22–36) had both a better in vitro affinity and a greater in vivo potency than PYY(22– 36), thus indicating that the potent antisecretory properties of 22– 36 derivatives (1) were not only related to a longer half-life in vivo, but also to better affinity for at least one of the receptors involved in the antisecretory effect.
As is always the case with in vivo experiments, the present results cannot be completely interpreted in terms of receptor affinity, because differences in peptide half-life may exist between the tested molecules, and may change to some extent the potency order in vivo. Better understanding will come from the use of good subtype-specific antagonists, which, when available, should allow to precise the exact subtypes involved in vivo. In conclusion, we suggest that the potent antisecretory effect of PYY and related peptides on the rat jejunum in vivo is mediated through the occupancy of several receptors: the PYY-preferring receptor described on enterocytes, an Y1-like receptor, and a third receptor with high affinity for PP, which could be Y4 / PP1 and/or Y5 receptor.
REFERENCES 1. Balasubramaniam, A.; Cox, H. M.; Voisin, T.; Laburthe, M.; Stein, M.; Fischer, J. E. Structure–activity studies of peptide YY(22–36): N-a-Ac(Phe 27 )PYY(22–36), a potent antisecretory peptide in rat jejunum. Peptides 14:1011–1016; 1993. 2. Ballantyne, G. H.; Goldenring, J. R.; Fleming, F. X.; Steven, R. J.; Scott, F.; Fielding, L. P.; et al. Inhibition of VIP-stimulated ion transport by a novel Y-receptor phenotype in rabbit distal colon. Am. J. Physiol. 264:G849–G854; 1993. 3. Bard, J. A.; Walker, M. W.; Branchek, T. A.; Weinshank, R. L. Cloning and functional expression of a human Y4 subtype receptor for pancreatic polypeptide, neuropeptide Y, and peptide YY. J. Biol. Chem. 270:26762–26765; 1995. 4. Beck–Sickinger, A. G.; Jung, G. Structure–activity relationships of neuropeptide Y analogues with respect to Y1 and Y2 receptors. Biopolymers (Pept. Sci.) 37:123–142; 1995. 5. Bilchick, A. J.; Hines, O. J.; Adrian, T. E.; Mc Fadden, D. W.; Berger, J. J.; Zinner, M. J.; et al. Peptide YY is a physiological regulator of water and electrolyte absorption in the canine small bowel in vivo. Gastroenterology 105:1441–1448; 1993. 6. Blomqvist, A. G.; Roubos, E. W.; Larhammar, D.; Martens, G. J. Cloning and sequence analysis of a neuropeptide Y/peptide YY receptor Y1 cDNA from Xenopus laevis. Biochim. Biophys. Acta 1261:439–441; 1995. 7. Brown, D. R.; Boster, S. L.; Overend, M. F.; Parsons, A. N.; Treder, B. G. Actions of neuropeptide Y on basal, cyclic AMP-induced and neurally evoked ion transport in porcine distal jejunum. Regul. Pept. 29:31–47; 1990. 8. Castan, I.; Valet, P.; Voisin, T.; Quideau, N.; Laburthe, M.; Lafontan, M. Identification and functional studies of a specific peptide YYpreferring receptor in dog adipocytes. Endocrinology 131:1970– 1976; 1992. 9. Cox, H. M.; Cuthbert, A. W. Neuropeptide Y antagonises secretagogue evoked chloride transport in rat jejunal epithelium. Pflugers Arch. 413:38–42; 1988. 10. Cox, H. M.; Cuthbert, A. W. The effects of neuropeptide Y and its fragments upon basal and electrically stimulated ion secretion in rat jejunum mucosa. Br. J. Pharmacol. 101:247–252; 1990. 11. Cox, H. M.; Cuthbert, A. W.; Hakanson, R.; Wahlestedt, C. The effect of neuropeptide Y and peptide YY on electrogenic ion transport in rat intestinal epithelia. J. Physiol. 398:65–80; 1988. 12. Cox, H. M.; Cuthbert, A. W.; Krstenansky, J. L. The effects of selective amino acid substitution upon neuropeptide Y antisecretory potency in rat jejunum mucosa. Peptides 12:323–327; 1991. 13. Cox, H. M.; Tough, I. R. Functional characterisation of receptors with affinity for PYY, NPY, (Leu 31 ,Pro 34 )NPY and PP in a human colonic epithelial cell line. Br. J. Pharmacol. 116:2673–2678; 1995. 14. Dumont, Y.; Cadieux, A.; Pheng, L. H.; Fournier, A.; St-Pierre, S.; Quirion, R. Peptide YY derivatives as selective neuropeptide Y/ peptide YY Y1 and Y2 agonists devoid of activity for the Y3 receptor sub-type. Mol. Brain Res. 26:320–324; 1994. 15. Eto, B.; Boisset, M.; Eden, P.; Balasubramaniam, A.; Desjeux, J. F. Effects of peptide YY and its analogues on chloride ion secretion in fed and fasted rat jejunum. Peptides 16:1403–1409; 1995.
16. Eva, C.; Keina¨nen, K.; Monyer, H.; Seeburg, P.; Sprengel, R. Molecular cloning of a novel G protein-coupled receptor that may belong to the neuropeptide receptor family. FEBS Lett. 271:80–84; 1990. 17. Friel, D. D.; Miller, R. J.; Walker, M. W. Neuropeptide Y: A powerful modulator of epithelial ion transport. Br. J. Pharmacol. 88:425– 431; 1986. 18. Gehlert, D. R. Subtypes of receptors for neuropeptide Y: Implications for the targeting of therapeutics. Life Sci. 55:551–562; 1994. 19. Gehlert, D. L.; Beavers, L. S.; Johnson, D.; Gackenheimer, S. L.; Schober, D. A.; Gadski, R. A. Expression cloning of a human brain neuropeptide Y Y2 receptor. Mol. Pharmacol. 49:224–228; 1996. 20. Gehlert, D. L.; Schober, D. A.; Beavers, L. S.; Gadski, R. A.; Hoffman, J. A.; Smiley, D. L.; et al. Characterization of the peptide binding requirements for the cloned human pancreatic polypeptide-preferring receptor. Mol. Pharmacol. 50:112–118; 1996. 21. Gerald, C.; Walker, M. W.; Vaysse P. J.; He, C.; Branchek, T. A.; Winshank, R. L. Expression cloning and pharmacological characterization of a human hippocampal neuropeptide Y/peptide YY Y2 receptor subtype. J. Biol. Chem. 270:26758–26761; 1995. 22. Gerald, C.; Walker, M. W.; Criscione, L.; Gustafson, E. L.; Batzi– Hartmann, C.; Smith, K. E.; et al. A receptor subtype involved in neuropeptide-Y-induced food intake. Nature 382:168–171; 1996. 23. Gregor, P.; Millham, M. L.; Feng, Y.; De Carr, L. B.; Mc Caleb, M. L.; Cornfield, L. J. Cloning and characterization of a novel receptor to pancreatic polypeptide, a member of the neuropeptide Y receptor family. FEBS Lett. 381:58–62; 1996. 24. Holzer–Petsche, U.; Petritsch, W.; Hinterleitner, T.; Eherer, A.; Sperk, G.; Krejs, G. J. Effect of neuropeptide Y on jejunal water and ion transport in humans. Gastroenterology 101:325–330; 1991. 25. Huang, S. C.; Tsai, M. F. Receptors for peptide YY and neuropeptide Y on guinea pig pancreatic acini. Peptides 15:405–410; 1994. 26. Laburthe, M.; Chenut, B.; Rouyer-Fessard, C.; Tatemoto, K.; Couvineau, A.; Servin, A.; et al. Interaction of peptide YY with rat intestinal plasma membranes: binding of the radioiodinated peptide. Endocrinology 118:1910–1917; 1986. 27. Larhammer, D.; Blomquist, A. G.; Yee, F.; Jazin, E.; Yoo, H.; Wahlestedt, C. Cloning and functional expression of a human neuropeptide Y/peptide YY receptor of the Y1 type. J. Biol. Chem. 267:10935–10938; 1992. 28. Li, X.; Wu, Y.; North, R. A.; Forte, M. Cloning, functional expression, and developmental regulation of a neuropeptide Y receptor from Drosophila melanogaster. J. Biol. Chem. 267:9–12; 1992. 29. Liu, C. D.; Hines, O. J.; Whang, E. E.; Balasubramaniam, A.; Newton, T. R.; Zinner, M. J.; et al. A novel synthetic analog of peptide YY, BIM-43004, given intraluminally, is proabsorptive. J. Surg. Res. 59:80–84; 1995. 30. Lundell, I.; Blomqvist, A. G.; Berglund, M. M.; Schober, D. A., Johnson, D.; Statnick, M. A., et al. Cloning of a human receptor of the NPY receptor family with high affinity for pancreatic polypeptide and peptide YY. J. Biol. Chem. 270:29123–29128; 1995. 31. Mannon, P. J.; Hernandez, E. J.; Mervin, S. J.; Vigna, S. R.; Taylor, I. L. Characterization of peptide YY receptors in rabbit colonic mucosa. Peptides 14:567–572; 1993.
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JEJUNAL PYY RECEPTORS IN VIVO 32. Mannon, P. J.; Mervin, S. J.; Sheriff-Carter K. D. Characterization of a Y1-preferring NPY/PYY receptor in HT-29 cells. Am. J. Physiol. 267:G901–G907; 1994. 33. Munson, P. J.; Rodbard, D. LIGAND: A versatile computerized approach for characterization of ligand-binding systems. Anal. Biochem. 197:220–239; 1980. 34. Pascaud, X.; Chovet, M.; Roze´ C.; Junien, J. L. Neuropeptide Y and sigma receptor agonists act through a common pathway to stimulate duodenal alkaline secretion in rats. Eur. J. Pharmacol. 231:389–394; 1993. 35. Playford, R. J.; Domin, J.; Beacham, J.; Parmar, K. B.; Tatemoto, K.; Bloom, S. R.; et al. Role of peptide YY in defence against diarrhoea. Lancet 335:1555–1557; 1990. 36. Rivie`re, P. J. M.; Rao, R. K.; Pascaud, X.; Junien, J. L.; Porreca, F. Effects of neuropeptide Y, peptide YY and sigma ligands on ion transport in mouse jejunum. J. Pharmacol. Exp. Ther. 264:1268– 1274; 1993. 37. Rivie`re, P. J. M.; Chovet, M.; Fargeas, M. J.; Pascaud, X.; Junien, J. L. Inhibition par le NPY de la se´cre´tion je´junale induite par le VIP chez le rat; implication de la somatostatine et de relais nerveux. Gastroenterol. Clin. Biol. 19:A102; 1995. 38. Rose, P. M.; Fernandes, P.; Lynch, J. S.; Frazier, S. T.; Fisher, S. M.; Kodukula, K.; et al. Cloning and functional expression of a cDNA encoding a human type 2 neuropeptide Y receptor. J. Biol. Chem. 270:22661–22664; 1995. 39. Roze´, C.; Molis, C.; Fu-Cheng, X.; Gene`ve, J.; Galmiche, J. P. Peptide YY inhibits prostaglandin E2-induced jejunal secretion by a haloperidol sensitive mechanism in normal man [abstr.]. Gastroenterology:110:A35; 1996. 40. Saria, A.; Beubler, E. Neuropeptide Y (NPY) and peptide YY inhibit prostaglandin E2-induced intestinal fluid and electrolyte secretion in the rat jejunum in vivo. Eur. J. Pharmacol. 119:47–52; 1985. 41. Servin, A. L.; Rouyer–Fessard, C.; Balasubramaniam, A. St.-Pierre, S.; Laburthe, M. Peptide-YY and neuropeptide-Y inhibit vasoactive intestinal peptide-stimulated adenosine 3 *,5 *-monophosphate production in rat small intestine: structural requirements of peptides interacting with peptide YY-preferring receptors. Endocrinology 124:692–700; 1989. 42. Sheikh, S. P. Neuropeptide Y and peptide YY: Major modulators of gastrointestinal blood flow and function. Am. J. Physiol. 261:G701– G715; 1991.
557 43. Souli, A.; Fu Cheng, X.; Chariot, J.; Tsocas, A.; Roze´, C. Effets antise´cre´toires du peptide YY et du neuropeptide Y a` trois niveaux de l’intestin greˆle chez le rat. Gastroenterol. Clin. Biol. 20:8–14; 1995. 44. Souli, A.; Presset, O.; Tsocas, A.; Chariot, J.; Roze´, C. Le peptide YY inhibe la se´cre´tion hydroe´lectrolytique je´junale du rat par un me´canisme impliquant des re´cepteurs sigma et/ou adre´nergiques [abstr.]. Gastroenterol. Clin. Biol. 20:379; 1996. 45. Stanley, B. G.; Magdalin, W.; Seirafi, A.; Nguyen, M. M.; Leibowitz, S. F. Evidence for neuropeptide Y mediation of eating produced by food deprivation and for a variant of the Y1 receptor mediating this peptide’s effect. Peptides 13:581–587; 1992. 46. Tallarida, R. J.; Murray, R. B. Manual of pharmacologic calculations with computer programs. New York: Springer–Verlag, 1981:14– 19. 47. Tatemoto, K. Isolation and characterization of peptide YY (PYY), a candidate gut hormone that inhibits pancreatic exocrine secretion. Proc. Natl. Acad. Sci. USA 79:2514–2518; 1982. 48. Voisin, T.; Rouyer–Fessard, C.; Laburthe, M. Distribution of common peptide YY-neuropeptide Y receptor along rat intestinal villuscrypt axis. Am. J. Physiol. 258:G753–G759; 1990. 49. Voisin, T.; Rouyer–Fessard, C.; Laburthe, M. PYY/NPY receptors in small intestine: Characterization, signal transduction and expression during cell differentiation. Ann. NY Acad. Sci. 611:343–346; 1990. 50. Voisin, T.; Bens, M.; Cluzeaud, F.; Vandewalle, A.; Laburthe, M. Peptide YY receptors in the proximal tubule PKSV-PCT cell line derived from transgenic mice. J. Biol. Chem. 268:20547–20554; 1993. 51. Voisin, T.; Couvineau, A.; Rouyer–Fessard, C.; Laburthe, M. Solubilization and hydrodynamic properties of active peptide YY receptor from rat jejunal crypts. Characterization as a Mr 44,000 glycoprotein. J. Biol. Chem. 266:10762–10767; 1991. 52. Wahlestedt, C.; Regunathan, S.; Reis, D. Identification of cultured cells selectively expressing Y1-, Y2-, or Y3-type receptors for neuropeptide Y/peptide YY. Life Sci. 50:PL7–PL12; 1992. 53. Wan, C. P.; Lau, B. H. S. Neuropeptide Y receptor subtypes. Life Sci. 56:1055–1064; 1995. 54. Weinberg, D. H.; Sirinathsinghji, D. J.; Tan, C. P.; Shiao, L. L.; Morin, N.; Rigby, M. R.; et al. Cloning and expression of a novel neuropeptide Y receptor. J. Biol. Chem. 271:16435–16438; 1996.
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Several Receptors Mediate the Antisecretory Effect of Peptide YY, Neuropeptide Y, and Pancreatic Polypeptide on VIP-Induced Fluid Secretion in the Rat Jejunum In Vivo A. SOULI,* J. CHARIOT,* T. VOISIN,* O. PRESSET,* A. TSOCAS,* A. BALASUBRAMANIAM,† M. LABURTHE* AND C. ROZE´*1 *INSERM U410, Faculte´ de Me´decine X. Bichat, BP 416, 75870 Paris Cedex 18, France †University of Cincinnati Medical Center, OH 45267-0558 Received 29 October 1996; Accepted 17 December 1996 SOULI, A., J. CHARIOT, T. VOISIN, O. PRESSET, A. TSOCAS, A. BALASUBRAMANIAM, M. LABURTHE AND C. ROZE´. Several receptors mediate the antisecretory effect of peptide YY, neuropeptide Y, and pancreatic polypeptide on VIP-induced fluid secretion in rat jejunum in vivo. PEPTIDES 18(4) 551–557, 1997.—Several Y receptor subtypes have been cloned and/or pharmacologically characterized that mediate the effects of the regulatory peptides peptide YY (PYY), neuropeptide Y (NPY), and pancreatic polypeptide (PP). These peptides possess antisecretory properties on the intestine. This effect can be blocked in vivo by neural antagonists, suggesting the intervention of neural receptors, although epithelial PYY-preferring receptors have been evidenced on jejunal crypt cells. The purpose of the present experiments was to compare the antisecretory properties in vivo of a series of PYY and NPY derivatives with various affinities for different Y receptor subtypes, in order to determine which subtypes were involved. A model of VIP-stimulated secretion by rat jejunal loops was used. The results were compared with the binding affinities for PYY-preferring receptors determined on rat jejunal crypt cell membranes. Full-length PYY(1–36) was about three times more potent than NPY(1–36), and 10 times more potent than PP in the low dose range. PP, however, had a low efficacy limited to about 50% inhibition of VIP effect. Both Y1 agonists ([Leu 31 ,Pro 34 ]PYY and [Leu 31 ,Pro 34 ]NPY), and Y2 agonists [C-terminal fragments ranging from PYY(3–36) and NPY(3–36) to PYY(22–36) to NPY(22–36)] displayed potent antisecretory properties. PYY derivatives and fragments were always more potent than their respective NPY counterparts. In contrast, Y1 derivatives and PP had very low affinity for the epithelial PYY receptor as measured in vitro by radioreceptor assay. These data suggest that the antisecretory effect of PYY/NPY/PP peptides in vivo involves the effets of several receptors: a Y2-like, PYY-preferring receptor identical to the epithelial receptor, a Y1-like receptor, and a third receptor with high affinity for PP. q 1997 Elsevier Science Inc. Peptide YY Neuropeptide Y Pancreatic polypeptide Truncated analogues Y receptor subtypes
Rat jejunum
been characterized by pharmacological means (namely Y3 , PYYpreferring, unspecific) (2,8,13,25,31,32,49,52). The receptor subtypes involved in the intestinal antisecretory effect of PYY and NPY have been mainly studied in vitro. The fulllength peptides NPY(1–36) and PYY(1–36), which recognize both Y1 and Y2 subtypes, are potent antisecretagogues in the rat jejunum in vitro, with PYY being more potent than NPY (11). In the same species, it was suggested that the jejunal antisecretory effect measured in Ussing chambers was related to Y2 receptors, because C-terminal fragments of NPY, reputed specific of the Y2 subtype, especially NPY(11–36) to NPY(14–36) (10) and PYY(22– 36) (1), were efficient in this preparation.
PEPTIDE YY (PYY), neuropeptide Y (NPY), and pancreatic polypeptide (PP) are closely related endogenous 36-amino acid peptides (47). PYY and NPY are potent inhibitors of gastrointestinal motor and secretory functions (42); they have been shown to inhibit intestinal secretion of electrolytes and water in vitro and in vivo, in different animal species and in man (5,7,9– 12,15,17,24,35,40). Several receptor subtypes for the PYY/NPY/PP family (Y receptors) have been described in different organs and species (4,18,53). Some of them have been cloned (namely, Y1 , Y2 , Y4 Å PP1 , Y5 Å feeding receptor, and Y6 , which is slightly different from Y5 : (3,6,16,19–23,27,28,30,45,54) and others have only
1
Antisecretory effects
Requests for reprints should be addressed to C. Roze´, INSERM U410 Faculte´ de Me´decine X. Bichat, BP 416, 75870 Paris Cedex 18, France.
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In other conditions (crypt enterocyte membranes) ‘‘PYY-preferring’’ receptors, negatively coupled to adenylylcyclase, have been evidenced in the rat small intestine (26,41,49,51). The intervention of other receptors has been suggested in the colon. In rabbit distal colon, the inhibition of vasoactive intestinal peptide (VIP)-stimulated ion transport in vitro involved a novel class of Y receptors with a very similar sensitivity to PP, PYY, NPY, and to the Y1 agonist [Leu 31 ,Pro 34 ]NPY (2). Another new receptor was claimed to be involved in the proabsorptive effect of PYY, NPY, PP, and NPY derivatives in a human colonic epithelial cell line, with an order of potency different from that reported in the rabbit distal colon (13). To summarize the in vitro studies dealing with antisecretory effects of PYY / NPY peptides, the involvement of Y2 and / or PYY-preferring receptors is suggested in the jejunum, whereas other, yet poorly defined receptors might be involved in the colon. To our knowledge, only a single in vivo study, performed in dogs equipped with Thiry-Vella fistulas, has suggested that Y2 receptors were involved in the inhibitory effect of PYY derivatives on intestinal secretion (29). In this study, the proabsorptive effect of a substituted PYY(22–36) analogue was reported after intraluminal administration in the ileum and colon, but not in the jejunum. Neural mechanisms appear to participate in the effects of PYY/NPY peptides in vivo (37,39,44), suggesting the intervention of neural receptors, whereas PYY/NPY receptors are also present on enterocyte membranes. We thus sought to determine whether a single or several subtypes of Y receptors were involved in the PYY/NPY/PP effects on the rat jejunum in vivo. For this purpose we compared the antisecretory effects of PYY(1–36), NPY(1–36), PP, and of fragments or analogues reputed more specific for Y1 ([Leu 31 ,Pro 34 ]PYY, [Leu 31 ,Pro 34 ]NPY) and Y2 receptors [PYY(3–36), PYY(13–36), PYY(22–36), N-aAcPYY(22–36), NPY(3–36), NPY(13–36), NPY(22–36)] on VIP-induced fluid secretion in the rat jejunum. In addition, the in vivo potency of these peptides was compared to their binding affinity in vitro to PYY-preferring receptors in rat jejunal crypt cell membranes. METHOD
In Vivo Experiments Animals. Male Wistar rats weighing 180–200 g (Iffa-Credo, Les Oncins, F 69210 L’Arbresle) were fasted for 24 h with free access to water before the experiments. Peptides. PYY(1–36) (pig, rat), NPY(1–36) (human, rat), PP (human), PYY(3–36), PYY(13–36), NPY(3–36), NPY ( 13 – 36 ) , NPY ( 22 – 36 ) , [ Leu 31 ,Pro 34 ] PYY, [ Leu 31 ,Pro 34 ] NPY, and VIP (human, pig, rat) were purchased from Neosystem, Strasbourg France. PYY(22–36) and N-a-AcPYY(22–36) were generous gifts of A. Balasubramaniam (University of Cincinnati Medical Center, OH). Peptide solutions were prepared in 0.9% NaCl added 0.3% bovine serum albumin (Sigma, A4503) to limit adhesion to injection material. Experimental setup. Rats were anesthetized with pentobarbital (50 mg/kg, IP). The animals were maintained at constant temperature (37 { 0.57C) during the experiment with a heating device. A saphenous vein was cannulated to infuse peptides. The abdomen was opened and a jejunal loop (about 20 cm long) was delimited by two ligatures respectively situated 3 and 23 cm distal to the ligament of Treitz. At time zero (t Å 0), 2 ml of 0.9% saline (prewarmed at 377C) was instilled into the loop with a calibrated syringe (needle 0.4 1 10 mm). Care was taken during the preparation of the loop to protect the marginal artery of the
bowel from damage and to maintain as far as possible the normal anatomical placement of the loop. The jejunal loop was returned to the abdomen, which was then closed. Thirty minutes later, the rats were sacrificed by IV injection of air. The jejunal loop was exteriorized, cut just proximal and distal to the ligatures, and its length was measured. The loop was weighed before and after removing its fluid contents (with light drying between two sheets of filter paper) to determine its fluid contents. Experimental protocol. Intestinal secretion of water and electrolytes was stimulated by VIP (30 mg/kg/h Å 9 nmol/kg/h) IV infused at 2.5 ml/h from t Å 0 to t Å 30 min. PYY, NPY, and their derivatives were administered as a bolus IV injection administered over 1 min, in doses of 3–900 pmol/kg, 15 min before starting VIP infusion. In control groups, intestinal secretion of water was measured in basal conditions (i.e., in rats receiving peptide vehicle alone, 0.9% NaCl added 0.3% bovine serum albumin). Expression of results. The weight of the full (F, mg) and empty loop (E, mg), the loop length (L, cm) and the amount of saline placed in the loop at the beginning of the experiment (2000 ml) allowed to calculate the net water flux (F-E-2000)/L, that was expressed as ml/cm/30 min (assuming that 1 ml saline Å 1 mg). Net absorption is indicated by a negative value and net secretion by a positive value. Results appear in the text and figures as means { 1 SEM of 5 to 13 animals per group. For each peptide the ID50 ( defined as the dose decreasing by 50% the secretory effect of VIP over the basal level ) and its 95% confidence limits were calculated from the regression curve of net water flux on the log of peptide dose, as previously described ( 46 ) . In Vitro Binding Studies Materials. [ 125I]Na (IMS300) was purchased from Amersham (Les Ulis, France). HEPES, PMSF (phenylmethylsulfonyl fluoride), TLCK (N-a-p-tosyl-L-lysine chloromethyl ketone), bacitracin, and the other highly purified chemicals used were purchased from Sigma Chemical Co. (St Louis, MO). BSA (bovine serum albumin, Pentex, Fraction V) was obtained from Miles Laboratories (Elkart, NJ). [ 125I-Tyr 36 ]Monoiodo PYY (referred to as [ 125I]PYY below) was prepared and purified as previously described (49). Preparation of intestinal crypt membranes. Male Wistar rats weighing 250–280 g, fed ad lib, were used in membrane binding experiments. The jejunum was removed after decapitation and crypt cells were separated from villus cells by shaking the everted jejunum for successive periods in a dispersing solution containing EDTA as previously described (48). A cell fraction enriched in crypt cells (containing about 90% of crypt cells) was used to prepare crude membranes. Briefly, crypt cells were homogenized by use of a Waring blendor in 0.25 M sucrose buffered with 10 mM triethanolamine (pH 7.5), containing 5 m M EDTA. After centrifugation at 2,600 1 g for 10 min, the supernatant was centrifuged at 20,000 1 g for 15 min. The resulting pellet was washed in 20 mM HEPES buffer (pH 7.5), repelleted, and stored at 0807C until used (48). Binding of [ 125 I]PYY to membrane-bound receptors. Binding of [ 125I]PYY to membrane preparations was conducted as previously described (48). Briefly, membranes (200 mg protein/ml) were incubated for 120 min at 157C in 250 ml of incubation buffer [20 mM HEPES buffer (pH 7.5), 2% (w/v) BSA, 17 mg/l PMSF, 10 mg/l TLCK, and 100 mg/l bacitracin] containing 0.05 nM [ 125I]PYY (2,200 Ci/mmol) with or without unlabeled PYY or other competing peptides. At the end of the incubation, 150ml aliquots of membranes were mixed with 150 ml ice-cold in-
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FIG. 1. Inhibitory effects of IV bolus injections of Peptide YY, NPY, and PP at different doses (from 3 to 900 pmol/kg) on VIP (30 mg/kg/ h)-induced net water flux in rat jejunum. Positive values correspond to secretion and negative values to absorption. Results are expressed as means { SEM of 5 to 13 animals.
cubation buffer. Bound and free peptides were separated by centrifugation at 20,000 1 g for 10 min, and membrane pellets were washed twice with 10% (w/v) sucrose in 20 mM HEPES buffer (pH 7.5). The radioactivity was then counted with a gamma counter. The nonspecific binding represented about 2% of total binding. All binding data were analyzed using the LIGAND computer program developed by Munson and Rodbard (33). RESULTS
Effect of PYY, NPY, and PP on VIP-Stimulated Fluid Jejunal Secretion PYY(1–36) inhibited in a dose-related manner VIP-stimulated jejunal fluid secretion. In preliminary experiments, two dif-
ferent doses of VIP were used. In basal conditions, the mean water net flux was 046.9 { 3.4 ml/cm/30 min, indicating a basal absorption. After stimulation by 100 mg/kg/h of VIP, the jejunal secretion reached 39 { 2.7 ml/cm/30 min; the inhibition was complete after 300 pmol/kg of PYY and the ID50 was 30 pmol/ kg of PYY (not shown). After stimulation by 30 mg/kg/h of VIP, the jejunal net water flux changed from a basal absorption of 042.2 { 2.9 ml/cm/30 min to a smaller secretion (3.4 { 2.9 ml/cm/30 min); the inhibition was complete after 100 pmol/kg of PYY (Fig. 1) and the ID50 was 6 pmol/kg of PYY (Table 1). The following experiments were then conducted with the smaller VIP dose (30 mg/kg/h), which produced a milder stimulation, mimicking probably a more physiological situation than the larger VIP dose. NPY(1–36) inhibited in a dose-related manner VIP-stimulated jejunal fluid secretion. The inhibition was almost complete at 100 pmol/kg (Fig. 1). The ID50 was 15.6 pmol/kg (Table 1). Compared to PYY, NPY was slightly less potent to inhibit VIPstimulated fluid jejunal secretion. PP also inhibited VIP-stimulated jejunal fluid secretion in a dose-related manner (Fig. 1). However, the inhibition was limited to about 50% of the fluid secretion at the highest doses 300 and 1000 pmol/kg. In its limited range of action PP was less potent than both PYY and NPY. However, when the ID50 was calculated as the PP dose decreasing by 50% the maximal effect of PP (instead of the PP dose decreasing by 50% the effect of VIP), PP appeared as potent as NPY (Table 1). Effect of PYY Derivatives on VIP-Stimulated Fluid Jejunal Secretion All PYY derivatives inhibited VIP-stimulated fluid secretion in a dose-related manner. The decreasing order of potency was: PYY(1–36) ú PYY(3–36) É [Leu 31 ,Pro 34 ]PYY ú PYY(22– 36) ú PYY(13–36) (Fig. 2). The ID50s are indicated in Table 1. Full-length PYY was more potent than any of the other peptides tested and PYY(13–36) was the least potent. N-aAcPYY(22–36) was nearly as potent as PYY(3–36), and several times more potent than PYY(22–36) and PYY(13–36). Two results should be emphasized: 1) the Y2 agonist PYY(3– 36) and the Y1 agonist [Leu 31 ,Pro 34 ]PYY were equipotent; 2)
TABLE 1 ANTISECRETORY POTENCIES OF PYY, NPY, AND THEIR DERIVATIVES IN THE RAT JEJUNUM IN VIVO ID50s Ratio Peptide
PYY(1–36) PYY(3–36) PYY(13–36) PYY(22–36) N-a-Ac PYY(22–36) [Leu31, Pro34]PYY NPY(1–36) NPY(3–36) NPY(13–36) NPY(22–36) [Leu31, Pro34]NPY PP
ID50 (pmol/kg)
95% Confidence Limits
Derivative/Full Length Peptide
Derivative/PYY(1–36)
6.0 13.2 97 55 21.7 15.7 15.6 19.9 É1320 94 100 É200* 11.3†
(3.9–9.3) (7.2–24.2) (60–157) (26–117) (15.4–30.6) (10.6–157) (9.4–25.7) (9.7–40.7) — (55–183) (49–180) — (2.7–47)
1 2.2 16.0 9.2 3.6 2.6 1 1.3 85 6.0 6.4 1 1
1 2.2 16.0 9.2 3.6 2.6 2.6 3.3 218 15.5 16.5 49.5 1.9
* PP was different from other peptides because maximal inhibition reached with any dose of PP was not larger than about 50% (see Fig. 1). † ID50 calculated when considering 50% of maximal PP effect, instead of 50% inhibition of VIP effect.
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FIG. 2. Inhibitory effects of PYY(1–36), PYY(3–36), PYY(13–36), PYY(22–36), and [Leu 31 ,Pro 34 ]PYY on VIP-induced net water flux in rat jejunum, by increasing doses. Each point represents the mean { SEM of 5 to 8 animals.
FIG. 3. Inhibitory effects of NPY(1–36), NPY(3–36), NPY(13–36), NPY(22–36), and [Leu 31 ,Pro 34 ]NPY on VIP-induced net water flux in rat jejunum, by increasing doses. Each point represents the mean { SEM of 5 to 8 animals.
the shorter fragment PYY(22–36) was slightly more potent than PYY(13–36).
36) used did not allow to reach the 50% inhibition allowing to calculate the ID50 . Binding to Rat Jejunal Crypt Cell Membranes In Vitro
Effect of NPY Derivatives on VIP-Stimulated Fluid Jejunal Secretion All NPY derivatives inhibited the VIP-stimulated fluid secretion in a dose-related manner (Fig. 3). The decreasing order of potency was: NPY(1–36) Å NPY(3–36) ú NPY(22–36) Å [Leu 31 ,Pro 34 ]NPY @ NPY(13–36). The ID50 values were, respectively, 15.6, 19.9, 94, 100, and É 1320 pmol/kg (Table 1). NPY(1–36) and the Y2 agonist NPY(3–36) were equipotent, whereas the Y1 agonist [Leu 31 ,Pro 34 ]NPY was about five times less potent. As in the case of PYY fragments, NPY(13–36) was by far the least potent of all NPY derivatives tested, including the shorter peptide NPY(22–36). The largest dose of NPY(13–
Data from the binding experiments are reported in Table 2. PYY(1–36) had the highest affinity for epithelial crypt cell receptors (IC50 Å 0.14 nM). The decreasing order of binding affinities for PYY derivatives was PYY(1–36) ú PYY(3–36) ú PYY(22–36) É PYY(13–36) @ [Leu 31 ,Pro 34 ]PYY. The affinity of NPY(1–36) was lower than that of PYY(1– 36) for epithelial crypt cell receptors. Within the series of NPY derivatives, the decreasing order of binding affinities was NPY(1–36) É NPY(3–36) É NPY(22–36) ú NPY(13–36) @ [Leu 31 ,Pro 34 ]NPY. Within the series of NPY derivatives, this affinity order was similar to the order found for the respective PYY derivatives.
TABLE 2 BINDING OF PYY, NPY, PP, AND THEIR DERIVATIVES TO RAT JEJUNAL CRYPT CELL MEMBRANES IC50s Ratio Peptides
IC50 (nM)
sm (nM)
Derivative/Full Length Peptide
Derivative/PYY(1–36)
PYY(1–36) PYY(3–36) PYY(13–36) PYY(22–36) N-a-Ac PYY(22–36) [Leu31, Pro34]PYY NPY(1–36) NPY(3–36) NPY(13–36) NPY(22–36) [Leu31, Pro34]NPY PP
0.14 0.70 8.9 5.6 0.91 199 4.7 5.0 28.1 5.8 562 ú1000
0.04 0.32 1.21 2.70 0.55 57 1.3 1.8 7.9 2.9 112 —
1.0 5.0 64 40 6.5 1420 1.0 1.1 6.0 1.2 120 ú213
1.0 5.0 64 40 6.5 1420 34 36 201 41 4015 ú7140
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JEJUNAL PYY RECEPTORS IN VIVO Finally, the affinity of PP for crypt cell membranes was very low. Three points should be emphasized among these in vitro data: 1) the Y1-selective agonists [Leu 31 ,Pro 34 ]PYY and [Leu 31 ,Pro 34 ]NPY displayed very low affinities 2) the shorter peptides PYY(22–36) and NPY(22–36) showed better affinities than the respective longer fragments PYY(13–36) and NPY(13–36); 3) PP had a very low affinity for crypt cell membrane receptors. DISCUSSION
The present study was designed to investigate the subtypes of Y receptors involved in the jejunal antisecretory effect of PYY and NPY by comparing the effects of the full-length peptides with different fragments or analogues on VIP-stimulated jejunal secretion in the rat in vivo. The model chosen for the present work was the jejunal ligated loop in situ in anesthetized rats. In a previous article (43), we showed that this model was appropriate to evidence the antisecretory effect of PYY and NPY against VIP- and PGE2-stimulated secretion in the duodenum, jejunum, and ileum. Potency differences, however, occurred between these intestinal segments, so we decided to focus on the mechanisms involved in the jejunum. In this previous work, we used 50-min venous infusions of PYY and NPY, and the comparatively large dose of 100 mg/kg/h of VIP as a stimulant. Because previous articles had claimed that bolus injections of small doses of NPY could act over more than 2 h to inhibit VIP-stimulated intestinal secretion (37) as well as to stimulate duodenal bicarbonate secretion (34), we used bolus injections of PYY and derivatives. Although this protocol might seem inappropriate to study the effect of a peptide with a short half-life, antisecretory effects were clearly evidenced. We also retained in the present study the dose of 30 mg/kg/h of VIP, which seemed to us less ‘‘pharmacological’’ than 100 mg/kg/h, and which allowed us to obtain clear-cut inhibitions with small PYY doses (subtotal inhibition with 30 pmol/kg, ID50 6.0 pmol/kg). In fact, this small dose of VIP mainly blocked the basal absorption rather than stimulated secretion. Y1 and Y2 receptors are pharmacologically well defined and have been cloned (19,21,27,38). Both recognize NPY and PYY but not PP. Y1 receptors recognize the full-length peptides and display a poor affinity for the C-terminal fragments, whereas modified full-length peptides such as [Leu 31 ,Pro 34 ] or [Pro 34 ] are specific Y1 agonists. Y2 receptors are less selective for peptide chain length, and they recognize C-terminal fragments of various lengths: 2–36, 3–36, 13–36, 22–36. A PYY-preferring receptor, binding PYY better than NPY, and poorly PP, negatively coupled to adenylylcyclase, has been described in rat enterocytes ( 26,41,49 ) , in dog adipocytes ( 8 ) , and in a renal epithelial cell line ( 50 ) . Another PYY-preferring receptor has been described in rabbit colon ( 31 ) . In another work using rabbit distal colon in vitro, the proabsorptive effects of PYY, NPY, PP, and the Y1 agonist [ Leu 31 ,Pro 34 ] NPY were similar ( 2 ) , whereas the Y2 agonist PYY ( 13 – 36 ) had no effect, suggesting another receptor subtype. Still different was the potency order of PYY, NPY, PP, and [ Leu 31 ,Pro 34 ] NPY on the human colonic epithelial cell line Col-6 ( 13 ) . Y3 receptors recognize NPY and its derivatives ( 14 ) , human PP, but poorly PYY and avian PP and not at all rat PP ( 53 ) . Y4 / PP1 receptors recognize PP with high affinity and PYY and NPY with very low affinity ( 3,23,30 ) . Recently, a Y5 receptor was cloned from rat hypothalamus ( 22 ) . This receptor, which does not discriminate between NPY, PYY, and PP, is most likely the atypical Y1-like feeding receptor previously described ( 45 ) . Very re-
555 cently a Y6 receptor, close to Y5 , has been cloned from mouse genomic DNA ( 54 ) . An important point is that the jejunal antisecretory effects of NPY and PYY can be suppressed by sigma antagonists in mouse jejunum in vitro (36), and by sigma or a2-adrenoceptor antagonists in rat and human jejunum in vivo (39,44). Thus, PYY peptides may act upon both enterocytes and nerves, and the receptors may be different. Indeed, our present results do not fit within the concept of a single, already described, Y1 or Y2 receptor. The effect of NPY derivatives could to some extent be considered as a Y2-like effect, because NPY(3–36) was as potent as NPY(1–36), and [Leu 31 ,Pro 34 ]NPY was about six times less potent. However, this does not apply to PYY derivatives, because PYY(3–36) and [Leu 31 ,Pro 34 ]PYY, which should discriminate between Y1 and Y2 receptors, were equipotent and were also as potent as NPY(1–36). Thus, Y1 agonists, such as the [Leu 31 ,Pro 34 ] derivatives, and Y2 agonists such as the C-terminal fragments, both displayed appreciable potencies, indicating either that both Y1 and Y2 receptors were involved, or alternatively, that another receptor type was concerned. A major characteristic of the receptors involved in vivo is that they prefer PYY: PYY ( 1 – 36 ) was the most potent molecule tested, and if one compares each PYY fragment or analogue with its NPY counterpart, the potency ratio always favors the PYY derivative. This preference for PYY agrees with the published characteristics of the PYY-preferring receptor preferentially expressed in the crypts of rat small intestine ( 48 ) . The density of these receptors decreases from duodenum to ileum, and they are absent from the cecum and colon ( 26 ) . Similarly, their inhibitory effect on cAMP production has been observed in the rat duodenum, jejunum, and ileum, but not in colon ( 41 ) . In agreement with this distribution, we found that the inhibitory potency of PYY against VIP-induced secretion was greater in the rat duodenum than in the jejunum and ileum ( 43 ) . However, comparing the present in vitro binding data with the in vivo inhibitory potencies indicates that the enterocyte receptor cannot be alone involved in the in vivo effect. Although the potency of the N-terminal fragments of PYY and NPY in vivo is similar to the binding data on enterocyte membranes, a major discrepancy occurs when one considers the binding and effect of the Y1 agonists [ Leu 31 ,Pro 34 ] PYY and [ Leu 31 ,Pro 34 ] NPY. Both of these peptides had a very low affinity for enterocyte membranes ( respectively 1420 and 4015 times lower than PYY, Table 2 ) , whereas they were comparatively potent to inhibit jejunal secretion in vivo ( with a potency which was only 2.6 and 16.5 times lower than PYY, respectively ) . In addition, PYY receptors of enterocyte membranes had very low affinity for PP, whereas low doses of PP displayed distinct inhibitory effects in vivo. Although this inhibitory effect of PP was incomplete and the dose – effect curve was limited to an about 50% inhibition, partial implication of an Y4 / PP1 subtype or another subtype with high affinity for PP has to be considered. Alternatively, a nonspecific receptor for PP, PYY, and NPY such as that described in rabbit colon ( 2 ) or in human colonic cells ( 13 ) could also participate in vivo. Besides the affinity order, another evidence favoring a third receptor subtype mediating the effect of PP is that PP was unable to totally inhibit the VIP effect, whereas all the other derivatives tested in large enough doses could apparently achieve such total inhibition. Presence of this PP receptor subtype may also explain the greater potency of PYY and its fragments in all situations, because PYY also has high affinity for PP receptors ( 20 ) , so that PYY may act both through PYY-preferring and PP receptors.
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Finally, a new characteristic emerging from the present experiments is the nonuniform decrease with chain length of the binding affinity of C-terminal fragments of PYY and NPY. Although one might have expected a progressive decrease of both affinity and potency with decreasing chain length, the 22–36 fragments of PYY and NPY were both more potent in vitro (binding assay) and in vivo (secretion assay) than the 13–36 respective fragments. In addition, the modified peptide N-a-AcPYY(22–36) had both a better in vitro affinity and a greater in vivo potency than PYY(22– 36), thus indicating that the potent antisecretory properties of 22– 36 derivatives (1) were not only related to a longer half-life in vivo, but also to better affinity for at least one of the receptors involved in the antisecretory effect.
As is always the case with in vivo experiments, the present results cannot be completely interpreted in terms of receptor affinity, because differences in peptide half-life may exist between the tested molecules, and may change to some extent the potency order in vivo. Better understanding will come from the use of good subtype-specific antagonists, which, when available, should allow to precise the exact subtypes involved in vivo. In conclusion, we suggest that the potent antisecretory effect of PYY and related peptides on the rat jejunum in vivo is mediated through the occupancy of several receptors: the PYY-preferring receptor described on enterocytes, an Y1-like receptor, and a third receptor with high affinity for PP, which could be Y4 / PP1 and/or Y5 receptor.
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