Potential roles for calcium-sensing receptor (CaSR) and transient receptor potential ankyrin-1 (TRPA1) in murine anorectic response to deoxynivalenol (vomitoxin).

Food contamination by the trichothecene mycotoxin deoxynivalenol (DON, vomitoxin) has the potential to adversely affect animal and human health by suppressing food intake and impairing growth. In mice, the DON-induced anorectic response results from aberrant satiety hormone secretion by enteroendocrine cells (EECs) of the gastrointestinal tract. Recent in vitro studies in the murine STC-1 EEC model have linked DON-induced satiety hormone secretion to activation of calcium-sensing receptor (CaSR), a G-coupled protein receptor, and transient receptor potential ankyrin-1 (TRPA1), a TRP channel. However, it is unknown whether similar mechanisms mediate DON's anorectic effects in vivo. Here, we tested the hypothesis that DON-induced food refusal and satiety hormone release in the mouse are linked to activation of CaSR and TRPA1. Oral treatment with selective agonists for CaSR (R-568) or TRPA1 (allyl isothiocyanate (AITC)) suppressed food intake in mice, and the agonist's effects were suppressed by pretreatment with corresponding antagonists NPS-2143 or ruthenium red (RR), respectively. Importantly, NPS-2143 or RR inhibited both DON-induced food refusal and plasma elevations of the satiety hormones cholecystokinin (CCK) and peptide YY3-36 (PYY3-36); cotreatment with both antagonists additively suppressed both anorectic and hormone responses to DON. Taken together, these in vivo data along with prior in vitro findings support the contention that activation of CaSR and TRPA1 contributes to DON-induced food refusal by mediating satiety hormone exocytosis from EEC.

Peptide YY3-36 and 5-hydroxytryptamine mediate emesis induction by trichothecene deoxynivalenol (vomitoxin).

Deoxynivalenol (DON, vomitoxin), a trichothecene mycotoxin produced by Fusarium sp. that frequently occurs in cereal grains, has been associated with human and animal food poisoning. Although a common hallmark of DON-induced toxicity is the rapid onset of emesis, the mechanisms for this adverse effect are not fully understood. Recently, our laboratory has demonstrated that the mink (Neovison vison) is a suitable small animal model for investigating trichothecene-induced emesis. The goal of this study was to use this model to determine the roles of two gut satiety hormones, peptide YY3-36 (PYY3-36) and cholecystokinin (CCK), and the neurotransmitter 5-hydroxytryptamine (5-HT) in DON-induced emesis. Following ip exposure to DON at 0.1 and 0.25mg/kg bw, emesis induction ensued within 15-30min and then persisted up to 120min. Plasma DON measurement revealed that this emesis period correlated with the rapid distribution and clearance of the toxin. Significant elevations in both plasma PYY3-36 (30-60min) and 5-HT (60min) but not CCK were observed during emesis. Pretreatment with the neuropeptide Y2 receptor antagonist JNJ-31020028 attenuated DON- and PYY-induced emesis, whereas the CCK1 receptor antagonist devezapide did not alter DON's emetic effects. The 5-HT3 receptor antagonist granisetron completely suppressed induction of vomiting by DON and the 5-HT inducer cisplatin. Granisetron pretreatment also partially blocked PYY3-36-induced emesis, suggesting a potential upstream role for this gut satiety hormone in 5-HT release. Taken together, the results suggest that both PYY3-36 and 5-HT play contributory roles in DON-induced emesis.

A role for metalloendopeptidases in the breakdown of the gut hormone, PYY 3-36.

Peptide YY(3-36) (PYY(3-36)) is a gut hormone that acts on Y2 receptors to reduce appetite. Obese humans are sensitive to the anorectic effects of PYY(3-36) and display a blunted postprandial rise in PYY(3-36). Bariatric surgery results in increased circulating PYY-immunoreactivity, which appears to play a role in postoperative weight loss. The utility of PYY(3-36) as an antiobesity treatment is limited by its short circulating half-life. Insight into the mechanisms by which PYY(3-36) is degraded may aid design of long-acting PYY(3-36) analogues or enzyme inhibitor therapies. We aimed to investigate the role of metalloendopeptidases in PYY(3-36) degradation and determine whether modulation of these enzymes enhanced PYY(3-36) plasma levels and bioactivity in vivo. Degradation and resultant cleavage products of PYY(3-36) were characterized after incubation with neprilysin and meprin ß and with a kidney brush border preparation in vitro. Specific metalloendopeptidase inhibitors were coadministered with PYY(3-36) to mice and subsequent PYY(3-36) plasma levels and bioactivity determined. Meprin ß cleaves PYY(3-36) at multiple conserved acidic sites. Blocking the actions of meprin ß prevents the degradative effect of kidney brush borders on PYY(3-36). In mice, pretreatment with actinonin significantly prolonged the anorectic effect of PYY(3-36) and maintained higher PYY(3-36) plasma levels than treatment with PYY(3-36) alone. These studies suggest that inhibiting the degradation of PYY(3-36) using specific inhibitor therapies and/or the design of analogues resistant to cleavage by meprins may be useful to antiobesity therapeutics.

Ghrelin attenuates the inhibitory effects of glucagon-like peptide-1 and peptide YY(3-36) on food intake and gastric emptying in rats.

Ghrelin stimulates, while glucagon-like peptide-1 (GLP-1) and peptide YY(3-36) [PYY(3-36)] inhibit, food intake and gastric emptying in rats. We determined the dose-dependent effects of a 3-h intravenous infusion of ghrelin at dark onset on food intake in freely feeding rats, and on the inhibitory effects of intravenous infusion of GLP-1 and PYY(3-36) on food intake and gastric emptying. Ghrelin (150 pmol x kg(-1) x min(-1)) stimulated food intake by 28% during the infusion period primarily by increasing meal frequency; doses of 15 and 50 pmol x kg(-1) x min(-1) had no effect. GLP-1 (15 pmol x kg(-1) x min(-1)) inhibited food intake by 35-54%; coinfusion of ghrelin at 50 and 150 pmol x kg(-1) x min(-1) attenuated this effect by 60 and 64%, respectively. PYY(3-36) (15 pmol x kg(-1) x min(-1)) inhibited food intake by 32%; ghrelin at 15 and 50 pmol x kg(-1) x min(-1) attenuated this effect by 54 and 74%, respectively. A 20-min intravenous infusion of ghrelin (15-150 pmol x kg(-1) x min(-1)) attenuated GLP-1-and PYY(3-36)-induced inhibition of gastric emptying of saline by 6-29%. Thus, intravenous infusion of ghrelin during the early dark period stimulates food intake in freely feeding rats by increasing meal frequency, and similar doses of ghrelin attenuate gastric emptying and feeding responses to GLP-1 and PYY(3-36). These results suggest that ghrelin may stimulate food intake in part by attenuating the inhibitory effects of GLP-1 and PYY(3-36) on gastric emptying and food intake.

Functional consequences of neuropeptide Y Y 2 receptor knockout and Y2 antagonism in mouse and human colonic tissues.

1 Neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) differentially activate three Y receptors (Y(1), Y(2) and Y(4)) in mouse and human isolated colon. 2 The aim of this study was to characterise Y(2) receptor-mediated responses in colon mucosa and longitudinal smooth muscle preparations from wild type (Y(2)+/+) and knockout (Y(2)-/-) mice and to compare the former with human mucosal Y agonist responses. Inhibition of mucosal short-circuit current and increases in muscle tone were monitored in colonic tissues from Y(2)+/+ and Y(2)-/- mice+/-Y(1) ((R)-N-[[4-(aminocarbonylaminomethyl)phenyl)methyl]-N(2)-(diphenylacetyl)-argininamide-trifluoroacetate (BIBO3304) or Y(2) (S)-N(2)-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6H)-oxodibenz[b,e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl]cyclopentyl]acetyl]-N-[2-[1,2-dihydro-3,5(4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]-argininamide (BIIE0246) antagonists. 3 Predictably, Y(2)-/- tissues were insensitive to Y(2)-preferred agonist PYY(3-36) (

Neuropeptide Y and peptide YY inhibit excitatory synaptic transmission in the rat dorsal motor nucleus of the vagus.

Pancreatic polypeptides (PPs) such as neuropeptide Y (NPY) and peptide YY (PYY) exert profound, vagally mediated effects on gastrointestinal (GI) motility and secretion. Whole-cell patch clamp recordings were made from brainstem slices containing identified GI-projecting rat dorsal motor nucleus of the vagus (DMV) neurons to determine the mechanism of action of PPs. Electrical stimulation of nucleus tractus solitarii (NTS) induced excitatory postsynaptic currents (EPSCs) that were reduced in a concentration-dependent manner by NPY and PYY (both at 0.1-300 nM) in 65 % of the neurons. An increase in the paired-pulse ratio without changes in the postsynaptic membrane input resistance or EPSC rise and decay time suggested that the effects of PPs on EPSCs were due to actions at presynaptic receptors. The Y1 and Y2 receptor selective agonists [Leu31,Pro34]NPY and NPY(3-36) (both at 100 nM) mimicked the inhibition of NPY and PYY on the EPSC amplitude. The effects of 100 nM NPY, but not PYY, were antagonized partially by the Y1 receptor selective antagonist BIBP3226 (0.1 micro M). In addition, the inhibition of the EPSC amplitude induced by NPY, but not PYY, was attenuated partially by pretreatment with the alpha2 adrenoceptor antagonist yohimbine (10 micro M), and occluded partially by the alpha2 adrenoceptor agonist UK14,304 (10 micro M) as well as by pretreatment with reserpine. Pretreatment with a combination of BIBP3226 and yohimbine almost completely antagonized the NPY-mediated effects on EPSCs. Contrary to the inhibition of EPSCs, perfusion with PPs had no effect on the amplitude of inhibitory postsynaptic currents (IPSCs) and a minimal effect on a minority of DMV neurons. Differences in the receptor subtypes utilized and in the mechanism of action of NPY and PYY may indicate functional differences in their roles within the circuitry of the dorsal vagal complex (DVC).

PYY regulates pancreatic exocrine secretion through multiple receptors in the awake rat.

Peptide YY (PYY) is one of several regulatory peptides reported to modulate pancreatic secretion. PYY circulates in two forms, PYY1-36 and PYY3-36, and binds to multiple receptor subtypes. We sought to determine if PYY1-36 or PYY3-36 regulates neurally mediated pancreatic secretion through the Y1, Y2, and/or Y5 receptor subtypes. Experiments were conducted in awake, surgically recovered rats. In order to determine the effects of the PYYs on basal pancreatic secretion, either PYY1-36, [Pro34] PYY1-36 (a Y1/Y5 agonist), or PYY3-36 (a Y2/Y5 agonist) were infused for 40 min at doses of 0, 12.5, 25, or 50 pmol/kg/hr while measuring pancreatic juice volume and protein. PYY1-36 increased pancreatic protein secretion at 25 and 50 pmol/kg/hr (P < 0.05) in a dose-dependent manner (P < 0.001, R2 = 0.990). The Y2/Y5 receptor agonist PYY3-36 significantly inhibited pancreatic juice volume and protein at 12.5 and 25 pmol/kg/hr, but stimulated protein secretion at higher doses (P < 0.001, R2 = 0.995). The Y1/Y5 agonist, [Pro34] PYY1-36, had no significant effect on basal pancreatic exocrine secretion. Therefore, PYY1-36, PYY3-36 and [Pro34] PYY1-36 produced different, dose-dependent changes on basal pancreatic exocrine secretion. Inhibition of pancreatic secretion by circulating PYY1-36 and PYY3-36 are primarily mediated by the Y2 receptor. Since [Pro34] PYY1-36 did not change pancreatic secretion, it can be concluded that circulating PYY1-36 or PYY3-36 does not modulate pancreatic secretion through the Y1 or Y5 receptors. Since the stimulatory effects of PYY1-36 on pancreatic secretion could not be explained by the actions of PYY3-36 or [Pro34] PYY1-36 on Y1 or Y2 receptors, and since PYY1-36 fails to bind to Y3 or Y4 receptors, we also conclude that PYY1-36 may stimulate pancreatic secretion in a dose-dependent mechanism through a PYY receptor subtype different from Y1, Y2, Y3, Y4 or Y5.

Pathways and receptors involved in peptide YY induced contraction of rat proximal colonic muscle in vitro.

BACKGROUND: Peptide YY (PYY) is involved in the regulation of several gut functions, including secretion and motility. It exerts its effects through a family of six receptors, commonly named the Y receptor family. AIMS: To characterise the effects of PYY on strips of rat proximal colon in vitro, and to determine the pathways and receptors involved. METHODS: Contractions of strips removed from the muscle layer of rat proximal colon were recorded under isometric conditions, using PYY, Y receptor agonists and antagonists, and nerve blockers. Reverse transcription-polymerase chain reaction was also performed to detect the presence of mRNA coding for Y receptors. Finally, smooth muscle cells were isolated to estimate the cell length and intracellular Ca(2+) concentration in the presence and absence of PYY. RESULTS: PYY, neuropeptide Y (NPY), pancreatic polypeptide (PP) and [Leu31,Pro34]NPY induced a dose dependent contraction of strips from proximal colon. Tetrodotoxin partially inhibited the PYY and NPY induced contractions, and strongly inhibited the PP induced contraction. Specific antagonists showed the involvement of cholinergic nicotinic receptors and NK1 receptor. BIBP 3226, a specific Y1 antagonist, did not modify the colonic smooth muscle response to PYY, whereas blocking L-type Ca(2+) channels with D-600 abolished its effects. Moreover, PYY induced an increase in intracellular Ca(2+) concentration, associated with a reduction in cell length. mRNA encoding Y1 and Y4 receptors were detected in the muscle strips. CONCLUSIONS: These findings suggest that PYY stimulates colonic contractile activity in vitro through (a) a nervous Y4 dependent pathway and (b) a pathway involving a potential new receptor on myocytes.

Antisecretory effect of peptide YY through neural receptors in the rat jejunum in vitro.

Basal short circuit current (Isc) was measured in stripped rat jejunum after addition of neural antagonists and of peptide YY (PYY). Basal Isc was slightly (by 10-21%) but significantly inhibited by tetrodotoxin, hexamethonium, idazoxan, and the sigma antagonist BMY 14,802. PYY (10(-7) M) reduced basal Isc by approximately 54%. This inhibition was unchanged by hexamethonium but reduced by 44-68% in the presence of tetrodotoxin, idazoxan, haloperidol, BMY 14,802, and atropine. The Y2 agonist pYY(3-36) was more potent than the Y1 agonist (Leu31,Pro34)PYY. In conclusion, PYY reduces basal Isc in rat jejunum in part through a neural mechanism involving muscarinic receptors, alpha2 adrenoceptors, and sigma receptors and, in part, through a direct effect on enterocytes. The PYY effect seems mainly carried out through Y2-receptor activation.

Characterization of rabbit kidney and brain pancreatic polypeptide-binding neuropeptide Y receptors: differences with Y1 and Y2 sites in sensitivity to amiloride derivatives affecting sodium transport.

Sites sensitive to human and rat pancreatic polypeptides (hPP and rPP) accounted for more than 30% of the specific binding of [125I](Leu31,Pro34) human peptide YY (LP-PYY) in particulates from rabbit kidney cortex, and about 10% of the specific binding in membranes from rabbit hypothalamus. The binding of [125I]hPP or [125I]rPP showed a high-affinity displacement with either hPP, rPP, LP-PYY, neuropeptide Y or peptide YY (Ki below 50 pM for all), while being quite insensitive to Y2-selective ligands. The PP binding had a high sensitivity to alkali cations and inhibitors of phospholipase C, very similar to that of LP-PYY binding 'masked' by excess cold hPP. However, as different from the Y1-like LP-PYY binding, but similar to the binding of the Y2-selective ligand [125I]human peptide YY(3-36) (hPYY(3-36)), the PP binding showed a low sensitivity to guanosine polyphosphates. The PP binding was much more sensitive to N5-substituted amiloride inhibitors of Na+ transport than the binding of LP-PYY, or that of hPYY(3-36). The inhibition of PP binding by N5-substituted amilorides was not enhanced by guanine nucleotides or by phospholipase C blockers. However, pairing of N5-substituted amilorides disproportionately increased the inhibition of hPP binding. Thus, in rabbit kidney or hypothalamus, the high-affinity PP-responding sites share some of the basic properties of the Y1 and Y2 sites, but are distinguished from both by a high sensitivity to compounds affecting sodium transport. These PP/NPY receptors could associate with membrane structures involved in the control of ion balance and osmotic responses.

Intestinal fat-induced inhibition of meal-stimulated gastric acid secretion depends on CCK but not peptide YY.

Fat in small intestine decreases meal-stimulated gastric acid secretion and slows gastric emptying. CCK is a mediator of this inhibitory effect (an enterogastrone). Because intravenously administered peptide YY (PYY) inhibits acid secretion, endogenous PYY released by fat may also be an enterogastrone. Four dogs were equipped with gastric, duodenal, and midgut fistulas. PYY antibody (anti-PYY) at a dose of 0.5 mg/kg or CCK-A receptor antagonist (devazepide) at a dose of 0.1 mg/kg was administered alone or in combination 10 min before the proximal half of the gut was perfused with 60 mM oleate or buffer. Acid secretion and gastric emptying were measured. We found that 1) peptone-induced gastric acid secretion was inhibited by intestinal fat (P < 0.0001), 2) inhibition of acid secretion by intestinal fat was reversed by CCK-A receptor antagonist (P < 0.0001) but not by anti-PYY, and 3) slowing of gastric emptying by fat was reversed by CCK-A antagonist (P < 0. 05) but not by anti-PYY. We concluded that inhibition of peptone meal-induced gastric acid secretion and slowing of gastric emptying by intestinal fat depended on CCK but not on circulating PYY.

Short-chain fatty acids modify colonic motility through nerves and polypeptide YY release in the rat.

Short-chain fatty acids (SCFAs) are recognized as the major anions of the large intestinal content in humans, but their effect on colonic motility is controversial. This study explores the colonic motor effect of SCFAs and their mechanisms in the rat. Colonic motility (electromyography) and transit time (plastic markers) were measured in conscious rats while SCFAs were infused into the colon, either alone or after administration of neural antagonists or immunoneutralization of circulating polypeptide YY (PYY). SCFA-induced PYY release was measured by RIA and then simulated by infusing exogenous PYY. Intracolonic infusion of 0.4 mmol/h SCFAs had no effect, whereas 2 mmol/h SCFAs reduced colonic motility (36 +/- 3 vs. 57 +/- 4 spike bursts/h with saline, P < 0.05) by decreasing the ratio of nonpropulsive to propulsive activity. This resulted in an increased transit rate (P < 0.01). Neither alpha-adrenoceptor blockade nor nitric oxide synthase inhibition prevented SCFA-induced motility reduction. Intraluminal procaine infusion suppressed the SCFA effect, indicating that a local neural mechanism was involved. SCFA colonic infusion stimulated PYY release in blood. Immunoneutralization of circulating PYY abolished the effect of SCFAs on colonic motility, whereas exogenous PYY infusion partly reproduced this effect. SCFAs modify colonic motor patterns in the rat and increase transit rate; local nerve fibers and PYY are involved in this effect.

Octreotide inhibition of flushing and colonic motor dysfunction in carcinoid syndrome.

OBJECTIVES: Previous studies showed increased plasma motilin and substance P concentrations and accelerated motor function in the small bowel and colon in patients with carcinoid diarrhea. Octreotide is beneficial in patients with carcinoid syndrome. Our hypothesis was that octreotide inhibits accelerated motility and gut neuropeptides in carcinoid syndrome. METHODS: In 12 patients with metastatic carcinoid syndrome, we investigated the effect of octreotide 50 microg s.c. t.i.d (n = 6) or placebo (n = 6) on postprandial symptoms, GI transit, colonic motility, and circulating levels of selected circulating peptides and amines. RESULTS: Octreotide reduced postprandial flushing (p = 0.03) but not pain. Octreotide significantly retarded overall colonic transit and proximal colonic emptying (p < 0.05); it tended to prolong small bowel transit time (p = 0.13) and to reduce postprandial colonic tone (p = 0.08) compared with placebo. Octreotide also reduced circulating levels of peptide YY, neurotensin, vasoactive intestinal polypeptide, and substance P but had no effect on plasma motilin, neuropeptide Y, calcitonin gene-related peptide, or histamine after meal ingestion. CONCLUSION: Octreotide ameliorates gut motor dysfunctions that characterize carcinoid diarrhea; the potential role of specific antagonism of serotonin, substance P, and vasoactive intestinal polypeptide alone or in combination with agents that inhibit their release in carcinoid diarrhea deserves further study.

Neural modulation of the antisecretory effect of peptide YY in the rat jejunum.

The endocrine and neural peptide, peptide YY, inhibits intestinal secretion of water and electrolytes in several animal species and in man. Peptide YY receptors have been evidenced on isolated rat jejunal crypt cells, but neural receptors are also likely to participate in the antisecretory effect of peptide YY in vivo. The aim of the present study was to investigate the mechanisms of the peptide YY effect on vasoactive intestinal peptide (VIP)-stimulated jejunal net water flux in the rat. Antagonist experiments using several drugs affecting neurally mediated processes were done for the purpose. A small peptide YY dose (10 pmol/kg) inhibited significantly (P < 0.005) the jejunal net water flux produced by 30 microg/kg per h of VIP. The inhibitory effect of peptide YY was suppressed, or strongly and significantly reduced, by tetrodotoxin, hexamethonium, lidocaine, idazoxan and BMY14,802 (51-(4-fluorophenyl)-4-(-4-(5-fluoro-2pyrimidinyl)-1-piperazinyl)- 1-butanol), whereas devazepide and L-NAME (L-omega-N-arginine methyl ester) had no effect. These results suggest that peptide YY inhibits VIP-stimulated jejunal net water flux in vivo through a neural mechanism implicating the participation of nicotinic synapses, alpha2-adrenoceptors and sigma receptors.