Overexpression of gastric leptin precedes adipocyte leptin during high-fat diet and is linked to 5HT-containing enterochromaffin cells.

OBJECTIVES: In obesity, while hyperleptinemia highly correlates with excess fat mass, the status of gastric leptin remains unknown. Here, we investigated the expression of leptin in stomach biopsies of obese humans and analyzed the temporal changes of gastric leptin expression in response to diet-induced obesity and its impact on 5-hydroxytryptamine (5HT)-producing cells. METHODS: Enterochromaffin (EC) cells and expression of leptin, PAX4 (critical factor for EC specification), tryptophane hydroxylase-1 (TPH1, the peripheral rate-limiting enzyme for 5HT) and 5HT were examined by immunofluorescence, quantitative real-time PCR, radioimmunoassay, respectively, in stomach and duodenum biopsies from 19 obese and 14 normo-weighed individuals, and in mucosa scrapings from C57Bl6/J diet-induced obese mice, leptin-deficient ob/ob mice and intestine-specific leptin receptor isoform B-deficient mice. RESULTS: Gastric mucosa of obese subjects displays an increased expression of leptin (LEP mRNA by fivefold and protein by twofold, P<0.01), TPH1 ((1.75-2.73, 95% confidence interval (CI)) vs (0.38-0.67, 95% CI); P<0.01) and PAX4 ((1.33-2.11, 95%CI) vs (0.62-0.81, 95% CI); P<0.01) as compared with normo-weighed individuals. In diet-induced obese mice, the overexpressions of gastric leptin, antral Pax4, Tph1 and increased EC cell number occurred before the onset of obesity and hyperleptinemia (reflect of adipocyte leptin production). In addition, leptin deficiency was associated with reduced Pax4 mRNA, whereas oral leptin treatment enhanced both Tph1 and Pax4 mRNA. Finally, mice with an intestine-specific deletion of leptin signaling exhibit significant decrease in duodenal mucosa 5HT content. CONCLUSIONS: These data demonstrate that gastric leptin is upregulated in obese individuals. RESULTS from high-fat diet mice showed that overexpression of gastric leptin that is linked to gut '5HT pathway' occurred before the onset of obesity and expansion of fat mass. This may be relevant in the pathophysiology of obesity.

Leptin regulates sugar and amino acids transport in the human intestinal cell line Caco-2.

AIM: Studies in rodents have shown that leptin controls sugars and glutamine entry in the enterocytes by regulating membrane transporters. Here, we have examined the effect of leptin on sugar and amino acids absorption in the human model of intestinal cells Caco-2 and investigated the transporters involved. METHODS: Substrate uptake experiments were performed in Caco-2 cells, grown on plates, in the presence and the absence of leptin, and the expression of the different transporters in brush border membrane vesicles was analysed by Western blot. RESULTS: Leptin inhibited 0.1 mm α-methyl-D-glucoside uptake after 5 or 30 min treatment and decreased SGLT1 protein abundance in the apical membrane. Uptake of 20 µm glutamine and 0.1 mm phenylalanine was also inhibited by leptin, indicating sensitivity to the hormone of the Na(+) -dependent neutral amino acid transporters ASCT2 and B(0) AT1. This inhibition was accompanied by a reduction in the transporters expression at the brush border membrane. Leptin also inhibited 1 mm proline and ß-alanine uptake in Na(+) medium at pH 6, conditions for optimal activity of the H(+) -dependent neutral amino acid transporter PAT1. In this case, abundance of PAT1 in the brush border membrane after leptin treatment was not modified. Interestingly, leptin inhibitory effect on ß-alanine uptake was reversed by the PKA inhibitor H-89 suggesting involvement of PKA pathway in leptin's regulation of PAT1 activity. CONCLUSION: These data show in human intestinal cells that leptin can rapidly control the activity of physiologically relevant transporters for rich-energy molecules, that is, D-glucose (SGLT1) and amino acids (ASCT2, B(0) AT1 and PAT1).

Metformin-induced regulation of the intestinal D-glucose transporters.

Metformin is an orally administered drug that lowers blood glucose and improves insulin sensitivity in patients with non insulin-dependent diabetes. Although the antihyperglycemic effect of metformin has been extensively studied, its cellular mechanism(s) of action (including the effect on enterocyte) remains to be defined. This study was designed to examine the effect of metformin on glucose transporters in enterocyte. Na(+)-dependent glucose transporter-1 (SGLT-1) activity was followed as glucose-induced short-circuit current (Isc) in Ussing chambers. The effect of metformin (10 micromol/L, 3 min) on transmural glucose transport was studied in isolated rat jejunal loops. Its impact on abundance of transporters SGLT-1 and GLUT2 in jejunal brush border membranes (BBM) and its effect on the phosphorylation of AMP-activated protein kinase (AMPK) alpha2 subunit was studied by western blot. Acute effect of metformin was also measured in vivo by oral glucose tolerance test (OGTT). Metformin markedly inhibited glucose-induced Isc (approximately 77%) after mucosal addition. In addition, metformin reduced the glucose-induced abundance of SGLT-1 in BBM and increased those of GLUT2, concomitantly increasing the phosphorylation of intracellular AMPKalpha2. This effect of metformin was also observed using non-metabolizable sugar alpha3-O-methyl glucose. Transmural glucose transport measured in vitro was increased by 22% under metformin. Finally, oral metformin markedly increased glucose tolerance in OGTT. In conclusion, metformin slightly increases intestinal glucose absorption by inducing a re-distribution of glucose transporters in BBM through AMPK control in enterocyte. In addition to its action to other splanchnic tissues, this could constitute a peripheral signal contributing to the beneficial effect of metformin on glucose tolerance.

Functional characterization and expression of PBR in rat gastric mucosa: stimulation of chloride secretion by PBR ligands.

Previous studies have demonstrated that gastric mucosa contained high levels of the polypeptide diazepam binding inhibitor, the endogenous ligand of the peripheral-type benzodiazepine receptor (PBR). However, the expression and function of this receptor protein in these tissues have not been investigated. Immunohistochemistry identified an intense PBR immunoreactivity in the mucous and parietal cells of rat gastric fundus and in the mucous cells of antrum. Immunoelectron microscopy revealed the mitochondrial localization of PBR in these cells. Binding of isoquinoline PK 11195 and benzodiazepine Ro5-4864 to gastric membranes showed that fundus had more PBR-binding sites than antrum, displaying higher affinity for PK 11195 than Ro5-4864. In a Ussing chamber, PK 11195 and Ro5-4864 increased short-circuit current (I(sc)) in fundic and antral mucosa in a concentration-dependent manner in the presence of GABA(A) and central benzodiazepine receptor (CBR) blockers. This increase in I(sc) was abolished after external Cl(-) substitution and was sensitive to chloride channels or transporter inhibitors. PK 11195-induced chloride secretion was also 1) sensitive to verapamil and extracellular calcium depletion, 2) blocked by thapsigargin and intracellular calcium depletion, and 3) abolished by the mitochondrial pore transition complex inhibitor cyclosporine A. PK 11195 had no direct effect on H(+) secretion, indicating that it stimulates a component of Cl(-) secretion independent of acid secretion in fundic mucosa. These data demonstrate that mucous and parietal cells of the gastric mucosa express mitochondrial PBR functionally coupled to Ca(2+)-dependent Cl(-) secretion, possibly involved in the gastric mucosa protection.

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

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

Differential upregulation of cellular adhesion molecules at the sites of oxidative stress in experimental acute pancreatitis.

BACKGROUND: Severe acute pancreatitis (AP)(2) is associated with exaggerated leukocyte adherence and activation. Endothelial cellular adhesion molecules (CAMs) can be induced by cytokines, but also directly by oxygen free radicals (OFRs), mediated by nuclear factor kappa-B (NF-kappa B). We investigated the behavior of inducible CAMs in relation to pancreatic oxidative stress. Our novel modification of cerium capture histochemistry (reaction of OFRs with cerium produces laser reflective Ce perhydroxide precipitates) combined with reflectance confocal laser scanning microscopy (CLSM) allows the histological codemonstration of in vivo OFR production and immunolabeled CAMs, or NF-kappa B. METHODS: Taurocholate AP was induced in rats; sham operated and normal animals served as controls. To achieve in situ, in vivo reaction of cerium with OFRs, animals were perfused with CeCl(3) solution at different time points (1, 2, 8, 24 h) and then sacrificed. E-selectin, P-selectin, ICAM-1, VCAM, and NF-kappa B p65 were labeled by immunofluorescence (IF) on frozen sections of cerium perfused pancreata. IF and Ce perhydroxide reflectance were simultaneously detected by CLSM. Pancreatic gene expression of the same CAMs was quantified by competitive RT-PCR (MIMIC internal control). RESULTS: Control pancreata showed negligible reflectance and minimal CAM expression. Early (1, 2 h) AP samples were characterized by intense, heterogeneous acinar OFR production, strong P-selectin, and increasing ICAM expression, with nuclear translocation of p65, histologically all colocalizing with the areas of acinar oxidative stress. Adherent polymorphonuclear leukocytes (PMNs) displayed weak OFR formation. Later (8, 24 h), a slowly declining P-selectin, but persisting ICAM-1 expression, was paralleled by widespread adherence of PMNs producing surprisingly large amounts of OFRs. VCAM and E-selectin showed a mild increase at 24 h. CAM gene activation was in good correlation with the protein expression. CONCLUSIONS: The early acinar oxidative stress is colocalized with NF-kappa B activation, preferential P-selectin, and ICAM upregulation in this AP model. Subsequently, adherent, activated PMNs become the major source of OFRs, thereby contributing to tissue damage.

Cerium-based histochemical demonstration of oxidative stress in taurocholate-induced acute pancreatitis in rats. A confocal laser scanning microscopic study.

Direct in vivo histological detection of oxygen-derived free radicals (OFRs) in inflammatory conditions is not fully resolved. We report an application of cerium histochemistry (in which capture of OFRs by Ce atoms results in laser-reflectant cerium-perhydroxide precipitates) combined with reflectance confocal laser scanning microscopy (CLSM) to demonstrate the evolution of oxidative stress in taurocholate-induced acute pancreatitis (AP) in rats. Animals were perfused with CeCl(3) in vivo and cryostat sections of pancreata were studied by CLSM. Vascular endothelium was immunolabeled for PECAM-1. OFR production by isolated polymorphonuclear leukocytes (PMNs) incubated in vitro with CeCl(3) was quantified by image analysis. In the pancreas, strong OFR-derived cerium reflectance signals were seen in acinar cells at 1-2 hr, capillaries and small venules were frequently engorged by cerium precipitates, and adherent PMNs presented weak intracellular reflectance signals. At 8-24 hr, acinar cell OFR production decreased, whereas adherent/transmigrated PMNs displayed abundant intra- and pericellular reflectance. PECAM-1 expression was unchanged. PMNs from ascites or blood showed significant (p<0.01) time-dependent OFR production, plateauing from 2 hr. The modified cerium capture/CLSM method allows the co-demonstration of in vivo oxidative stress and cellular structures labeled with fluorescent markers. In vivo oxidative stress was shown histologically for the first time in experimental AP.

Isolation and characterization of surfactant-like particles in rat and human colon.

The source of a phospholipid-rich layer recovered from the surface of the mammalian colon has been obscure. This report describes the isolation of a low-density membrane from the surface of rat and human colons (d = 1.07-1.08 g/ml), with a low cholesterol-to-phospholipid ratio and phosphatidylcholine as its major phospholipid. Electron microscopy shows unilamellar and partially coiled membranes. Compared with microvillous membranes isolated from underlying mucosa, this extracellular membrane is enriched for tissue-unspecific alkaline phosphatase and surfactant protein A. It does not contain small intestinal marker proteins (intestinal alkaline phosphatase and sucrase-isomaltase). The human membrane contains only traces of the colonic microvillous membrane marker, carcinoembryonic antigen. Antiserum against the rat colonic membrane does not recognize colonic microvillous membrane or small intestinal surfactant-like particle proteins. Antiserum against human colonic membrane identifies one protein in the surfactant-like particle from the adjacent small intestine and two proteins in the colonic microvillous membrane. These data show that the colonocyte microvillous membrane is covered by another membrane with a different protein composition. Enrichment for surfactant protein A suggests that this colonic membrane is another example of a surfactant-like particle sharing proteins with pulmonary surfactant.

Expression of hydrophilic surfactant proteins by mesentery cells in rat and man.

Human peritoneal dialysis effluent (PDE) contains a phosphatidylcholine-rich compound similar to the surfactant that lines lung alveoli. This material is secreted by mesothelial cells. Lung surfactant is also characterized by four proteins essential to its function. After having long been considered as lung-specific, some of them have been found in gastric and intestinal epithelial cells. To explore further the similarity between lung and peritoneal surfactants, we investigated whether mesothelial cells also produce surfactant proteins. We used rat transparent mesentery, human visceral peritoneum biopsies and PDE. Surfactant proteins were searched for after one- and two-dimensional SDS/PAGE and Western blotting. On a one-dimensional Western blot, bands at 38 and 66 kDa in rat mesentery, and at 38 and 66 kDa in human peritoneal mesothelial cells (in vivo and in vitro) and PDE, corresponded to monomeric and dimeric forms of lung surfactant protein A (SP-A). On two-dimensional Western blots, the 32 and 38 kDa spots in mesentery and PDE localized at the acidic pH appropriate to the SP-A monomer's isoelectric point. SP-D was also identified at the same 43 kDa molecular mass as in lung. SP-B was not detected in mesenteric samples. Expression of SP mRNA species was also assessed by reverse transcriptase-PCR, which was performed with specific primers of surfactant protein cDNA sequences. With primers of SP-A and SP-D, DNA fragments of the same size were amplified in lung and mesentery, indicating the presence of SP-A and SP-D mRNA species. These fragments were labelled by appropriate probes in a Southern blot. No amplification was obtained for SP-B. These results show that mesentery cells produce SP-A and SP-D, although they are of embryonic origin (mesodermal) and are different from those of the lung and digestive tract (endodermal) that secrete these surfactants.

Antibody against pulmonary surfactant protein A recognizes proteins in intestine and swim bladder of the freshwater fish, carp.

An antibody raised against rat pulmonary surfactant protein A (SP-A) bound on Western blots to proteins present in intestinal mucosa and in swim bladder, but not in gills of the carp. The fish protein(s) revealed by the antibody exhibited an electrophoretical behavior similar to that of rat SP-A with a characteristic triplet in the 30- to 35-kDa range. It therefore appears that proteins immunologically very close to mammalian SP-A are present in modern fish which evolved from ancestors that never possessed lungs. The association of SP-A with phospholipid-rich surfactant-like materials appears as a phylogenetically old feature.

Dibutyryl cyclic adenosine monophosphate and forskolin alter the paracellular pathway in cultured corneal endothelial cells.

PURPOSE: This study describes the effects of the cyclic adenosine monophosphate (cAMP) pathway on the tight junctional barrier of the corneal endothelium, which plays a critical role in maintaining the corneal stroma in an underhydrated, transparent state. METHODS: Subcultured bovine corneal endothelial cells grown on filters were used to study the effects of dibutyryl-cAMP and forskolin on transendothelial electrical resistance and [3H]inulin flux. The tight junction-associated protein ZO-1 (zonula occludens protein-1) and F-actin were visualized by indirect immunofluorescence, and the ultrastructural organization of junctional complexes was studied by freeze-fracture electron microscopy. RESULTS: Cells formed a continuous monolayer of closely apposed hexagonal-type cells separated by a discontinuous belt of tight junctions with a transendothelial electrical resistance of 20.8 +/- 0.6 omega.cm2. Dibutyryl-cAMP (10(-4) M) and forskolin (10(-5) M) increased cell cAMP, significantly decreased the transendothelial resistance by 54% and 43%, respectively, and increased the flux of [3H]inulin from the apical to the basal side of the cells by 56% and 40%, respectively. Both agents also induced condensation of F-actin at the cell borders without any marked changes in the immunostaining of ZO-1 that delineated cell peripheries. However, freeze-fracture studies showed that dibutyryl-cAMP and forskolin induced dispersion of the tight junction network. CONCLUSIONS: These data suggest that activation of the cAMP-dependent pathway, leading to structural changes of the tight junctional network, may modulate the passive fluxes mediated by the paracellular pathway of the corneal endothelial barrier.

Evidence for apical Na+/H+ exchanger in bovine main pancreatic duct.

The finding of a high PCO2 in basally secreted pancreatic juice of man and dog raises the hypothesis of proton secretion from ductal epithelial cells presumably through a Na+/H+ exchanger. To test this possibility, H+ luminal secretion and Na+ movements were measured in vitro on samples of bovine pancreatic ducts mounted in Ussing-type chambers. The rate of luminal acidification measured by the pH stat method, using bicarbonate-free media gassed with 100% O2, reached 2.75 muEq/cm2/hr. Proton secretion was blocked in the presence of 1 nM amiloride or in the absence of Na+ (replaced by choline) in the mucosal solution. Study of transepithelial 22Na fluxes in short-circuited tissue, bathed on both sides by control Ringer solution, gassed by 95% O2-5% CO2 demonstrated a net sodium transport from the mucosal to the interstitial side of the duct (net 22Na flux = 3.23 +/- 0.8 muEq/cm2/hr). This net sodium transport was electroneutral and blocked by mucosal amiloride (0.5-1 mM/liter) or by interstitial ouabain (1 mM/liter). These results are consistent with the existence of a Na+/H+ exchanger on the luminal side of the bovine main pancreatic duct.

Immunoreactive substance P and calcitonin-gene-related peptide (CGRP) in rat milk and in human milk and infant formulas.

This study examined the presence of substance P and calcitonin-gene-related peptide (CGRP) immunoreactivities in various milks and infant formulas. Rat milk was obtained from lactating dams between parturition and weaning (0, 2, 5, 10, 15, and 20 d postpartum). Samples of human milk were obtained from seven multiparous, nonsmoking white women, and newborn infant formulas were purchased from local stores. Substance P and CGRP were measured by competitive enzyme immunoassay using acetylcholinesterase-peptide conjugates as tracers. In rats, substance P and CGRP were below detectable concentrations in amniotic fluid from the last day of gestation. In contrast, in milk the concentrations of substance P and CGRP-like immunoreactivities were high on the first day of lactation (3.1 +/- 0.2 and 23.1 +/- 1.5 micrograms/L, respectively), then dropped after day 2 (1.6 +/- 0.7 and 7.5 +/- 0.4 microgram/L, respectively) and remained fairly constant until weaning. Significant concentrations of substance P and CGRP were found in human milk (129.2 +/- 27 ng/L and 4.5 +/- 0.7 microgram/L, respectively, at 15 wk), but substance P or CGRP could not be detected in any of the formulas tested. These data show that milk contains high concentrations of immunoreactive substance P and CGRP. In rats the absence of peptides in amniotic fluid suggests that there is a flood of peptides into the gastrointestinal tract of neonates when suckling is initiated. Significant concentrations of substance P and CGRP in human milk but not in infant formulas may therefore have physiologic implications for neonatal nutrition.

Pulmonary surfactant protein A (SP-A) is expressed by epithelial cells of small and large intestine.

Surfactant protein A (SP-A) is the most abundant protein associated with phospholipids in pulmonary surfactant. There are several lines of evidence that pulmonary and gastrointestinal epithelium produce closely related surface-active materials, although the presence of SP-A in gastrointestinal tract has so far not been reported. Indirect immunofluorescence experiments using different antibodies raised against rat pulmonary SP-A showed that some jejunal and colonic but not gastric epithelial cells positively stained for SP-A. Analysis of the proteins in cell lysates from rat small intestine and colon studied by Western blot revealed several immuno-reactive bands, including the characteristic triplet of 26-, 32-, and 38-kDa monomeric proteins, less strongly labeled than in lung cells, and higher molecular mass forms of 66 and 120 kDa also present in lung cells. The 66- and 120-kDa bands displayed the expected isoelectric pH of SP-A after two-dimensional electrophoresis. Alkylation induced conversion of the 120-kDa form (almost completely) and the 66-kDa form (partly) into the 26-38-kDa monomeric species. The presence of SP-A mRNA in rat stomach, small intestine, and colon was then searched for by conventional cDNA/reverse transcriptase-polymerase chain reaction. Products of appropriate size (372 base pairs) identical to that of pulmonary tissue were amplified in small intestine and colon but not in stomach or in other tissues used as controls. Cloning and sequencing of rat colon SP-A cDNA revealed the same sequence as the one reported for rat lung SP-A. Furthermore, analysis of the transcriptional initiation site of SP-A gene in colon by anchored-polymerase chain reaction showed that transcription was initiated at the same site in both colon and lung. These data, which demonstrate that small intestine and colon express SP-A constitutively and that this protein is present in some epithelial cells, extend the concept of intestinal surfactant and underline its close relationships to pulmonary surfactant.

[Surfactants in the digestive tube]. / Les surfactants dans le tube digestif.

Epithelia of the gastrointestinal tract both synthesize and secrete surfactant-like materials. Like the pulmonary surfactant, these materials contain not only phospholipids, but also surfactant apoproteins, regarded until recently as specific products of the bronchoalveolar epithelium. Presented here are the various roles, most of them still speculative, of these phospholipids-containing products in the gastro-intestinal tract.

Milk-stimulated PGE2 production by isolated gastric cells: a possible role in the inhibition of histamine-induced acid secretion.

The effects of a milk diet on gastric acid secretion of rats fed raw bovine milk for 4 days were examined using dispersed gastric cells. Parietal cell acid secretion was estimated by the accumulation of 14C-aminopyrine (AP), an index of secretory function. Basal AP accumulation was significantly increased (60%) by the milk diet. There was a marked upward shift in the dose-response curve of histamine (HA; 10(-8) to 10(-3) M) in milk-fed rats, indicating enhanced sensitivity of parietal cell-H2 receptor to exogenous HA. In contrast, the dose-dependent inhibition of HA-induced AP accumulation by prostaglandin (PG) E2 was significantly reduced, indicating that the parietal cells of milk-fed rats were less sensitive to exogenous PGE2. The PGE2 content of bovine milk was low (less than 20 pg/ml), but the production of endogenous PGE2 by the gastric cells was dramatically increased by the milk diet and exhibited maximal control production rate in the presence of 10 microM arachidonic acid. The increased responsiveness to histamine and the decreased responsiveness to PGE2 indicated that the milk diet induced low histamine and high PGE2 availability in the vicinity of the parietal cell basolateral membrane. This regulation, which involves stimulation of PGE2 production in the gastric mucosa, may underly the inhibition of acid secretion observed in vivo in chronically milk-fed adult rats.

Relationship between maternal milk PGE2 and gastric acid secretion in newborn rats.

We previously demonstrated that in rats gastric acid secretion declines after birth and drops steeply on day 12 of life. In the present study, we investigated the part played in this decline by prostaglandin E2 (PGE2) from maternal milk. PGE2 content was first measured in the milk of untreated dams 0, 1, 5, 10, 12, 15, and 18 days after parturition. PGE2 levels were high during the first 5 days (123.5-200.5 pg/ml), declined significantly between days 10 and 15 (56.6-85.4 pg/ml; P less than 0.05), and dropped to 18.4 pg/ml on day 18. We also found that depleting milk of PGE2 prevented drop of acid secretion in 12-day-old suckling rats. Injecting lactating dams with indomethacin significantly reduced milk PGE2 content by 65% vs. milk of untreated dams. Surprisingly, administration of sesame oil, the indomethacin vehicle to the dams, increased milk PGE2 content by 182%. In the pups of the indomethacin-treated dams, acid secretion did not drop. On the contrary, in vivo basal and histamine-induced acid output rose markedly by 40 and 50%, respectively, and in vitro the net movements of 36Cl and 22Na measured in the isolated stomach indicated that active Cl- secretion had resumed. Mucosal PGE2 did not appear to be significantly involved in early development of acid secretion because administration of indomethacin to pups from untreated dams did not significantly modify the secretion measured on day 12. Data indicate that maternal milk depletion of PGE2 prevents the drop of gastric acid secretion previously observed in 12-day-old pups and suggest that in infant rats maternal PGE2 plays a physiological part in regulating acid secretion.

Inhibition by cow's milk of histamine-induced gastric acid secretion in the rat.

The effects of a 4-day milk diet on basal and histamine-induced gastric acid secretion were examined in vivo in adult rats. Rats were fed on raw cow's milk only. Their acid secretion was measured in the perfused stomach under basal conditions or after administration of varying doses of histamine. In milk-fed rats, basal secretion rose slightly compared with control secretion (1.86 +/- 0.09 vs. 1.52 +/- 0.06 microEq H+/10 min; p less than 0.01), but maximal secretion in response to noncumulative doses of histamine (range: 7.0-20.0 mg/kg) was inhibited by 57%. The degree of this inhibition was similar to that observed in control rats with an equimolar dose of H2-antagonist cimetidine. These results indicate that in the adult rat, chronic administration of cow's milk strongly inhibits histamine-stimulated acid output.

Development of acid secretory function in the rat stomach: sensitivity to secretagogues and corticosterone.

Gastric acid secretion was studied in anesthetized rats from day 6 of the postnatal period up to the time of weaning. Basal H+ secretion was detected from day 6 in the first group studied (2.4 +/- 0.2 muEq of H+/10 min/100 g of body weight, BW) and remained constant up to the time of weaning (day 18: 2.5 +/- 0.2 muEq of H+/10 min/100 g of body weight) except for the period between days 10 and 12, when it fell significantly (1.5 +/- 0.06 muEq H+/10 min/100 g of BW on day 12). Both histamine H2 receptor sensitivity and intracellular transduction mechanism activities were evaluated by studying the secretory responses to histamine, impromidine (an H2 receptor agonist), cimetidine (an H2 receptor antagonist), forskolin (a direct adenylate cyclase activator), and dibutyryl (db) cAMP (an analogue of cAMP, the intracellular messenger mediating the response to histamine). The effects of pentagastrin and carbachol were also determined. The secretory responses obtained on days 6, 8, and 18 were similar and represented about threefold increases over basal secretion for all the secretagogues used. After weaning on day 20, both the basal secretion and the response to secretagogues were significantly increased compared with those of unweaned animals. On day 12, the responses were always weaker than on both days 8 and 18. Injection of 1 mg/kg of corticosterone 21 acetate daily from day 8 resulted on day 12 in a basal secretion and a response to histamine equivalent to those measured in 18-day-old pups not injected.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigen absorption by the jejunal epithelium of children with cow's milk allergy.

To establish if intestinal permeability to exogenous antigens is involved in cow's milk allergy (CMA) in infants, 33 children 1 to 24 months old (18 controls and 15 with CMA) were tested for intestinal permeability to the protein marker horseradish peroxidase (HRP). Jejunal biopsies were performed either during the initial period of diagnosis, at the mean (and SE) age of 3 +/- 1 months, and/or 1 yr later, at the age of 13 +/- 2 months, just before and after a milk challenge. A small fragment of the biopsy was studied for histology and the remainder was mounted in an Ussing chamber for simultaneous measurement of mucosal to serosal transport of HRP in its intact and degraded forms and electrical parameters including short-circuit current and conductance. No modification in HRP absorption was noted in control children aged from 2 months to 11 yr, indicating that gut closure probably occurred earlier in life. During the initial period of CMA, transepithelial HRP fluxes were significantly higher, about 8-fold, in children with CMA (intact HRP flux = 48.5 +/- 15.2, 95% confidence interval, 11.2 to 85.7 versus 5.9 +/- 1.2, 95% confidence interval 2.9 to 8.3, in control children). In addition, short-circuit current was increased but conductance was unchanged. After several months on a milk-free diet, HRP flux and short-circuit current returned to control values. Just after the milk challenge and independently of the clinical issue, a slight rise in HRP permeability was observed but it was not significant and remained within control values.(ABSTRACT TRUNCATED AT 250 WORDS)