Expression and regulation of leptin receptor proteins in afferent and efferent neurons of the vagus nerve.

Leptin, the product of the ob gene, plays a key role in the regulation of food intake via a cross-talk between hypothalamic leptin receptors and neuropeptides that affect feeding behaviour. Recent studies have shown a synergistic interaction between leptin and cholecystokinin (CCK) leading to suppression of food intake, which involves CCK-1 receptors and capsaicin-sensitive vagal fibres. In this study, we have investigated the presence of leptin receptors in afferent and efferent neurons of the vagus nerve. By using reverse transcription-polymerase chain reaction, mRNAs encoding long (Ob-Rb) and short (Ob-Ra) leptin receptor isoforms were detected in the rat nodose ganglion, which contains the cell bodies of the vagal afferent neurons. Western blot analysis confirmed the presence of leptin receptor-immunoreactive proteins in extracts from the vagal trunk. Immunohistochemistry showed the presence of leptin receptors and the leptin-induced transcription factor STAT3 in the cytoplasm of nodose ganglion cells. In cervical vagal segments, levels of leptin receptor protein displayed physiological regulation, with decreased amounts after feeding and increased levels after food restriction. In addition, leptin receptor and STAT3 immunoreactivities were detected in neurons of the nucleus of tractus solitarius (NTS) and the dorsal motor nucleus of the vagus nerve (DMNX) by immunofluorescence histochemistry. Furthermore, direct double-labelling demonstrated colocalization of Ob-Rb and STAT3 immunoreactivities in cholinergic vagal efferent cell bodies of the DMNX. It is speculated that vagal leptin receptors, apart from being activated by adipocyte-derived leptin, may also be influenced by leptin produced by the stomach. This may explain the synergistic action of leptin and CCK on neuronal activity in the NTS and on food intake.

The H3 receptor is involved in cholecystokinin inhibition of food intake in rats.

We investigated the peripheral effects of an H3-receptor agonist and an H3-receptor antagonist (R)alpha-methylhistamine (Ralpha-MeHA) and thioperamide, respectively, on basal feeding and the CCK8-induced inhibition of food intake in rat. Intraperitoneal injection of thioperamide reduced food intake in a dose-dependent manner with maximal inhibition (35%, P<0.01 vs saline) at 3 mg/kg. (R)alpha-MeHA (0.3-3 mg/kg i.p.), an H3-receptor agonist alone had no effect on feeding but reversed the thioperamide-induced inhibition of food intake in a dose-dependent manner. The maximal feeding inhibitory dose of thioperamide (3 mg.kg i.p) increased by 40% and 22 % (P<0.01 vs saline) brain and stomach histamine contents, respectively. Histamine (0.3 - 6 mg/kg i.p.) and CCK-8 (3 - 30 microg/kg i.p) also inhibited food intake in a dose-dependent manner. Inhibition was 20% to 40% for histamine and 40% to 80% (P<0.01 vs saline) for CCK8. CCK-8 inhibition of feeding was increased by thioperamide and prevented by (R)alpha-MeHA in a dose-dependent way. In addition, CCK-8 did not reduce food intake if rats were pretreated with pyrilamine or ranitidine postsynaptic H1- and H2-receptor antagonists respectively. Our data suggest that the H3-receptor is involved in basal feeding. They also suggest that CCK satiety depends upon the release of histamine which acts on the H2- and H1-receptors, the final mediators of this effect.

Human apolipoprotein A-IV reduces gastric acid secretion and diminishes ulcer formation in transgenic mice.

We have investigated the involvement of human apolipoprotein A-IV (apoA-IV) in gastric acid secretion and ulcer formation in recently generated apoA-IV transgenic mice. Compared to control littermates, transgenic animals showed a gastric acid secretion decreased by 43-77% whereas only slight variations were observed in the different cell population densities within the gastric mucosa. In addition, no variation in gastrin levels was observed. Transgenics were protected against indomethacin-induced ulcer formation, with lesions diminishing by 45 to 64% compared to controls. These results indicate that endogenous apoA-IV expression can regulate gastric acid secretion and ulcer development.

Physiological role of cholecystokinin B/gastrin receptor in leptin secretion.

In the present study, we investigated whether cholecystokinin (CCK) or its structurally related peptide gastrin participates in long term regulation of adipocyte leptin secretion. The levels of circulating leptin observed after 2 and 6 h of refeeding in 18-h fast rats were significantly lowered by injection of the specific gastrin/CCK-B receptor antagonist YM022 at doses that did not affect feeding behavior. Moreover, in normally fed animals, circulating leptin was markedly decreased by chronic injection of YM022 (from 4 +/- 0.6 to 2.1 +/- 0.5 ng/ml). Consistent with these observations, YM022 treatment decreased leptin messenger RNA (mRNA) levels and increased the leptin content in rat epididymal fat tissue. Rat adipocytes exclusively contain gastrin/CCK-B receptor mRNA, but not CCK-A receptor mRNA. Furthermore, adipocyte membranes bound [125I]CCK-8 in a saturable manner, with kinetics consistent with a single class of high affinity sites with a Kd of 0.2 nM. These data argue for a physiological role for the CCK-B/gastrin receptor in adipocyte leptin regulation. We therefore propose that gastrin is involved in long term regulation of leptin expression and secretion in rat fat tissues through activation of an adipocyte gastrin/CCK-B receptor.

The stomach is a source of leptin.

The circulating peptide leptin, which is the product of the ob gene, provides feedback information on the size of fat stores to central Ob receptors that control food intake and body-weight homeostasis. Leptin has so far been reported to be secreted only by adipocytes and the placenta. Here we show that leptin messenger RNA and leptin protein are present in rat gastric epithelium, and that cells in the glands of the gastric fundic mucosa are immunoreactive for leptin. The physiological function of this previously unsuspected source of leptin is unknown. However, both feeding and administration of CCK-8 (the biologically active carboxy-terminal end of cholecystokinin) result in a rapid and large decrease in both leptin cell immunoreactivity and the leptin content of the fundic epithelium, with a concomitant increase in the concentration of leptin in the plasma. These results indicate that gastric leptin may be involved in early CCK-mediated effects activated by food intake, possibly including satiety.

YM022, a highly potent and selective CCKB antagonist inhibiting gastric acid secretion in the rat, the cat and isolated rabbit glands.

We investigated the effects of the novel CCKB/gastrin antagonist YM022 on gastric acid secretion in vivo and in vitro, compared to CI-988 and L365,260 as reference antagonists. In the anaesthetized rat, pentagastrin-induced stimulation of gastric acid secretion was dose-dependently and up to 100% inhibited by i.v. administration of YM022 with an ID50 of 0.009 +/- 0.0006 mumol/kg h in comparison to 0.6 +/- 0.03 and 3.40 +/- 0.05 mumol/kg h for CI-988 and L-365,260, respectively. In the gastric fistula cat, i.v. administration of YM022 produced a similar inhibitory effect with an ID50 of 0.02 mumol/kg in comparison to 1.6 and 2.5 mumol/kg for CI-988 and L-365,260, respectively. Furthermore, bolus injection of 0.6 mumol/kg YM022 produced 100% inhibition within 30 min and 85% inhibition was still observed after 3 h. In the isolated rabbit gastric glands, CCK8-stimulated 14C-aminopyrine uptake was inhibited according to the following rank order of potency: YM022 (IC50 = 0.0012 microM) > > CI-988 (IC50 = 0.2 microM) > > L365,260 (IC50 = 2.8 microM). Unlike with L365,260, no influence of CI-988 and YM022 on histamine-stimulated acid output was shown in this study. Thus, YM022 is a highly potent and selective gastric CCKB/gastrin receptor antagonist and has a long-lasting inhibitory effect on gastric acid secretion.

Characterization of a beta 3-adrenoceptor stimulating gastrin and somatostatin secretions in rat antrum.

The beta 3-adrenoceptor (beta 3-AR) agonist SR-58611A {ethyl-[(7s)-7-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]5, 6,7,8-tetrahydronaphth-2-yl]oxyacetate hydrochloride} stimulated somatostatin and gastrin releases in isolated rat gastric antral epithelial cells. Stimulation was a concentration-dependent process with 50% effective concentrations of 2.7 +/- 1.1 and 3.8 +/- 1.9 nM compared with 209 +/- 71 and 230 +/- 51 nM for isoproterenol, respectively. It was inhibited by selective beta-AR antagonists with the following rank order of potency: SR-59230A 3-(2-ethylphenoxy)1-[(1S)-1,2,3,4-tetrahydronaphth- 1-ylamino]-(2S)-2-propranol oxalate; beta 3-AR antagonist > ICI-118551[erythro-(+/-)-1-(7-methylindan-4-yloxy)-3- isopropylaminobutan-2-ol-hydrochloride; beta 2-AR antagonist > CGP-20712A[(+/-)-[2-(3-carbarmoyl-4-hydroxyphenoxy)-et hyl- amino]-3-[4 (1-methyl-4-trifluoromethyl-2-imidazolyl)-phenoxy]- 2-propranol; beta 1-AR antagonist]. Furthermore, specific binding of 125I-cyanopindolol to the isolated cells was demonstrated and was displaced by the beta-AR antagonists according to the same rank order of potency and with apparent dissociation constants consistent with the 50% inhibitory concentrations for SR-58611A-stimulated somatostatin and gastrin releases. In addition, the presence of beta 3-AR mRNA was detected by reverse transcriptase polymerase chain reaction. These findings provide the first evidence for a gastric beta 3-AR mediating catecholamine stimulation of gastrin and somatostatin releases from antral cells.

Role of N- and C-terminal substituents on the CCK-B agonist-antagonist pharmacological profile of Boc-Trp-Phg-Asp-Nal-NH2 derivatives.

Among the CCK derivatives, the tetrapeptide Boc-Trp-Phg-Asp-Nal-NH2 (1) behaves as a short potent CCK-B agonist which led to the development of an efficient peptidase-resistant CCK-B antagonist by bismethylation of its terminal CONH2 group. Further modifications of the N- and C-terminal moieties of 1 have been performed and are described in this paper, together with the pharmacological profile of the novel synthetized compounds. Introduction of more bulky substituents than NalNH2 on the C-terminal part decreased the CCK-B receptor binding affinity. In the series of N-protected tetrapeptides X30-Phg31-Asp32-Nal33-N(CH3)2, the Boc-substituent was shown to be optimal among the N-protecting groups Boc, 2Adoc, propionyl or acetyl when X = Trp. On the other hand, when X = alpha MeTrp, its optimal N-protecting group was 2Adoc and its configuration was preferentially D. In the newly synthesized compounds, 13: 2Adoc-D-alpha MeTrp-Phg-Asp-NalN(CH3)2 and 16: 2Adoc-D-alpha MeTrp-Phg-Asp-NalNH2 had the best CCK-B receptor affinities (KI = 3.5 and 3.4 nM, respectively) and were selected for further biological evaluation. Interestingly, when tested for their capacity to influence inositol phosphate formation, induced by CCK8 in CHO cells transfected with the rat CCK-B receptor, compound 13 behaved as a full CCK-B antagonist with an IC50 value of 18 +/- 1 nM, being as potent as the antagonist L-365,260 and PD-134,308 (IC50 values respectively, 39 +/- 17 and 30 +/- 2 nM), whereas compound 16 was found to behave as a partial CCK-B agonist. Indeed 16 behaved as an antagonist on the firing rate of rat CA1 hippocampal neurons and acted as an agonist in the pentagastrin stimulated gastric acid secretion (EC50 = 12 nmol/kg) in anesthetized rats. Compound 13 in contrast, was found to inhibit the pentagastrin action at a dose (ID50 = 0.56 mumol/kg) similar to the potent antagonist PD-134,308 (ID50 = 0.4 mumol/kg). The antagonist/agonist properties of compounds 13 and 16 show that both N- and C-terminal substituents modulate the pharmacological properties in the Boc-CCK4 derivatives presented here.

Helicobacter pylori stimulates gastric acid secretion via platelet activating factor.

Platelet activating factor (PAF) is a phospholipid mediator known as potent ulcerogenic agent but its physiological role is still unknown in the gastrointestinal tract. Lyso PAF the immediate PAF procursor has no deleterious effect in the gastrointestinal tract. We have previously reported that lyso PAF is produced by gastric mucosa in basal condition and in response to gastrin in healthy men. Helicobacter pylori metabolises lyso PAF to produce PAF. The aim was to study the effect of PAF on the gastric acid secretion. Isolated rabbit glands were used as a model and acid secretion was assessed by (14C) Aminopyrine (AP) uptake. PAF and histamine stimulated AP accumulation time- and dose-dependently. PAF-induced AP accumulation was supressed by omeprazole and Fura 2. BN50727 a specific PAF antagonist inhibited PAF-induced AP accumulation. The presence of a PAF receptor transcript was investigated by reverse transcriptase polymerase chain reaction (RT-PCR) from total mRNA using two primers in which oligonucleotides were synthetized from the human leucocyte PAF receptor cDNA. A single RT-PCR band of the transcript with expected size was detected in the crude isolated cell fraction. These results and others from our laboratory suggest that PAF stimulates gastric acid secretion via specific receptor on the parietal cells and H. pylori produces PAF which may induce mucosal injury directly or indirectly via acid pathway.

Regulation of platelet-activating factor production in gastric epithelial cells.

The authors have previously reported that platelet-activating factor (PAF), a phospholipid mediator with potent proinflammatory activities, is produced in the gastric mucosa and stimulates gastric acid secretion in humans and animals. In the present study they used the human gastric tumour cells HGT1 (clone 6) to examine whether PAF production is regulated by neuromediators. PAF was extracted by ethanol and assayed by the washed platelet aggregation test. HGT1 cells produced PAF spontaneously (110 +/- 20 pg 10(6) cells). The addition of vasoactive intestinal peptide (VIP; 10(-9) to 10(-7) mol L(-1)) or of histamine (10(-5) to 10(-3) mol L(-1)) increased PAF production by three- to fivefold, while the addition of carbachol (10(-7) to 10(-4) mol L(-1)) increased PAF production up to sevenfold. PAF production was also increased up to 10- to 13-fold, in a dose- and time-dependent manner, by the addition of calcium and two- to threefold by the addition of phorbol myristate acetate (PMA; 10(-7) to 10(-5) mol L(-1)). However, the addition of dibutyryl cyclic AMP (dBcAMP; 10(-6) to 10(-4) mol L(-1) was without any effect. This is the first report showing PAF production by gastric epithelial cells in response to histamine, VIP and carbachol. Furthermore, the findings are consistent with a central role of calcium in this production. The results of this study, together with those of previous studies from the authors' laboratory, support the hypothesis that PAF is a physiological mediator of gastric acid secretion.

H3-receptor activation inhibits cholinergic stimulation of acid secretion in isolated rabbit fundic glands.

We previously reported evidence for H3-receptor inhibition of cholinergic stimulation of acid secretion by isolated rabbit gastric glands. Because this inhibition was unsensitive to H2 antagonists, we postulated that the parietal cell should bear a H3-receptor. In the present study, we investigated the effects of M1-M3 muscarinic receptors antagonists on carbachol- and thioperamide-induced acid secretion (14CAP uptake) by isolated rabbit gastric glands. Furthermore, we examined whether H3-receptor ligands could affect [3H]-N-methylscopolamine binding to the isolated rabbit parietal cells. Both carbachol and thioperamide concentration-dependently stimulated 14CAP uptake in the glands with maximal responses being achieved for 100 microM and 0.1 microM, respectively. These stimulations were concentration-dependently inhibited by the H3-receptor agonists R(alpha)-methylhistamine and imetit. Maximal inhibitions did not exceed 60% for 1 microM. The muscarinic receptor antagonists, hexa-sila-difenidol p-fluoro analog (M3), pirenzepine (M1) and gallamine (M2) inhibited carbachol-induced 14CAP uptake with IC50 of 50 nM, 10 microM and >> 100 microM, respectively. Thioperamide-induced 14CAP uptake was also inhibited by hexa-sila-difenidol p-fluoro analog and pirenzepine with IC50 of 90 nM and 12 microM, respectively; whereas gallamine had no effect. [3H]-N-methylscopolamine binding to isolated parietal cells was inhibited by atropine and pFHHSiD with IC50 of 15 nM and 132 nM, respectively. Neither R(alpha)-MeHA nor thioperamide did affect this binding although a H3-receptor inhibitory effect was observed on carbachol-induced 14CAP uptake by the cells. These data support that H3-receptor activation inhibits M3-mediated cholinergic stimulation of acid secretion through mechanisms operating downstream to the receptors sites.

Platelet-activating factor stimulates gastric acid secretion in isolated rabbit gastric glands.

We examined the effect of platelet-activating factor (PAF) on gastric acid secretion by isolated rabbit gastric glands as determined by [14C]aminopyrine ([14C]AP) uptake. PAF, histamine, and carbachol time- and concentration-dependently stimulated [14C]AP uptake, with estimated half-maximal effective concentrations of 60 pM, 0.25 microM, and 0.1 microM, respectively. PAF-induced [14C]AP uptake was inhibited by the specific PAF antagonists BN-50727 and SR-27417 and by the proton pump inhibitors omeprazole and lansoprazole. However, the H2-receptor antagonist famotidine had no effect. Buffering extracellular Ca2+ by ethylene glycol-bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid resulted in a shift to the right of the time-course effect of PAF without altering the maximal response, whereas buffering intracellular Ca2+ by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid 2-acetoxymethyl ester, as well as blocking Ca2+ channels by verapamil, inhibited PAF-induced [14C]AP uptake. Intracellular Ca2+ concentration in isolated rabbit gastric glands, as measured by fura 2-acetoxymethyl ester, concentration-dependently increased in response to PAF, to a maximum of 1.5-fold for 0.1 microM. These results suggest that PAF stimulates gastric acid secretion via specific receptors activating intracellular Ca2+ mobilization, which could be located on the parietal cells.

H3-receptor regulation of vascular gastrin and somatostatin releases by the isolated rat stomach.

We have studied the effects of the H3-receptor agonist (R) alpha-methylhistamine [(R) alpha-MeHA] and the H3-receptor antagonist thioperamide (Thiop) on basal- and carbachol-stimulated vascular gastrin release (GR) and somatostatin release (SR) by the isolated rat stomach. Carbachol dose-dependently stimulated and inhibited GR and SR, respectively. Maximal stimulation of GR (500 +/- 112 percent of basal; p < .01), and maximal inhibition of SR (-62 +/- 9 percent under basal; p < .01) were obtained with 1 micron carbachol. Neither (R)alpha-MeHA nor Thiop, up to 10 microns, affected GR. However, SR was dose-dependently enhanced by Thiop (25 +/- 8 percent for 10 microns). Carbachol stimulation of GR was strongly inhibited by Thiop (30 +/- 7 percent for 100 nM and 73 +/- 14 percent for 1 microgram), whereas it was potentiated by (R)alpha-MeHA. Carbachol inhibition of SR was reversed by Thiop and (R)alpha-MeHA. However, the reversal effect of (R)alpha-MeHA was prevented by the CCKB/gastrin receptor antagonist PD134308. These results support H3-receptor regulation of basal and cholinergically-stimulated GR and SR.

Neurotensin and oxyntomodulin-(30-37) potentiate PYY regulation of gastric acid and somatostatin secretions.

The present study was designed to investigate, in cats provided with both a gastric fistula and a denervated fundic Heidenhain pouch, the effect of peptide YY (PYY) on pentagastrin-stimulated gastric acid and somatostatin secretions and to determine whether neurotensin (NT) and the COOH-terminal octapeptide of oxyntomodulin [Oxm-(30-37)] would modify these secretions. Intravenous infusion of PYY (0.1 nmol.kg-1.h-1), NT (15 nmol.kg-1.h-1), or Oxm-(30-37) (60 nmol.kg-1.h-1) did not affect basal acid secretion. However, they significantly inhibited pentagastrin-stimulated gastric acid output up to 50% (P < 0.01) in the main stomach. Furthermore, they significantly increased gastric somatostatin release by +750, +1,700, and +600% over basal level (P < 0.01) for (in nmol.kg-1.h-1) 0.1 PYY, 15 NT, and 60 Oxm-(30-37), respectively. On the other hand, the effects of 0.1 nmol.kg-1.h-1 PYY were potentiated by subthreshold doses of NT (5 nmol.kg-1.h-1) or Oxm-(30-37) (15 nmol.kg-1.h-1). These findings suggest that there could be a cooperation between the three peptides in the intestinal regulation of gastric secretions.

Possible mediation by luminal somatostatin of bombesin-induced satiety in the cat.

Intravenous bombesin produced a dose-related stimulation of luminal gastric somatostatin output and a concomitant dose-dependent inhibition of food intake in the gastric fistula cat. Maximal food intake inhibition was observed at 1,280 pmol.kg-1.h-1 and corresponded to 65 +/- 7% (P less than 0.01). These effects of bombesin were dose dependently abolished by the specific bombesin-receptor antagonist, [Leu13-psi(CH2NH)-Leu14]bombesin. Furthermore, intragastric administration of somatostatin-14, at doses corresponding to those found in the gastric lumen in response to intravenously administered bombesin, significantly inhibited the first 30 min of food intake. This administration had however no effect on total (daily) food intake. We therefore suggest that luminal gastric somatostatin could at least account for bombesin-induced short-term satiety.

Pharmacological characterization of histamine H3 receptors in isolated rabbit gastric glands.

The effects of the specific H3 agonist (R)-alpha-methylhistamine (alpha-MeHA) and the specific H3 antagonist thioperamide were examined on histamine release and acid secretion [( 14C]-aminopyrine (AP) accumulation) by isolated rabbit gastric glands. Thioperamide significantly enhanced basal histamine release from the glands (+50% at 30 min for 10(-7) M thioperamide; P less than 0.01), and this increase was prevented by alpha-MeHA. Histamine-elicited AP accumulation was increased by 18% (P less than 0.05) by 10(-7) M thioperamide and decreased by 70% (P less than 0.01) by 10(-6) M of the H2 antagonist ranitidine. Thioperamide alone significantly enhanced AP accumulation in a dose-dependent manner, whereas alpha-MeHA had no effect of its own on this accumulation. Thioperamide stimulation of basal AP accumulation was not modified by ranitidine but was 50% decreased by alpha-MeHA. Furthermore, carbachol-induced AP accumulation was decreased by alpha-MeHA and increased by thioperamide; the latter effect was not blocked by ranitidine. These findings support that H3 receptors pharmacologically distinct from H2 receptors are involved in the regulation of histamine-stimulated acid secretion. They further suggest that these gastric H3 receptors occur in the gastric glands as 1) H3 autoreceptors located on the histamine-secreting cells and acting to downregulate histamine release from these cells and 2) H3 (or H3-like) receptors located on the parietal cell and regulating in a negative manner the acid secretory process.

Cholecystokinin-A receptor mediation of food intake in cats.

The selective cholecystokinin (CCK)-B-receptor agonist and antagonist, BC 264 and L 365260, respectively, and the CCK-A-receptor antagonist, L 364718, were used to investigate the possible involvement of different classes of CCK receptors in the control of food intake induced by exogenous CCK octapeptide (CCK-8) in the cat with gastric fistula. Intravenous infusion of CCK-8 dose dependently inhibited milk intake under sham-feeding conditions, maximal inhibition reaching 52 +/- 7% (P less than 0.001) with 0.88 nmol.kg-1.h-1. L 364718 prevented this inhibition, whereas L 365260 was ineffective over the dose range tested. The reversal effect of L 364718 on 0.88 nmol.kg-1.h-1 CCK-8-induced inhibition of milk intake was observed at doses as low as 0.44 nmol.kg-1.h-1. The selective CCK-B-receptor agonist, BC 264, in doses ranging from 0.88 to 7 nmol.kg-1.h-1, had no effect on milk intake under sham-feeding conditions, although it dose dependently stimulated gastric acid output. Furthermore, neither L 364718 nor L 365260 (88 nmol.kg-1.h-1 iv) stimulated milk intake when given in the absence of CCK-8. We conclude that exogenous CCK-8 causes satiety in the cat through activation of peripheral CCK-A receptors.