Rat Intestinal Tract Research Articles

Montmorillonite adsorbs creatinine and accelerates creatinine excretion from the intestine

This study aims to evaluate the sorption by montmorillonite of creatinine and the accelerating effect of montmorillonite on creatinine excretion from the intestine. The sorption of montmorillonite was observed in vitro. Also, rat intestinal tract and blood vessels were perfused circularly with perfusate with or without creatinine, respectively, to study the promotion of creatinine diffusion from the blood vessel to the intestine and the inhibition of creatinine absorption in the intestinal tract. The effect of decreasing the serum concentration of creatinine was studied in an acute hypercreatininaemia mouse model. The concentration of creatinine was determined by the basic picric acid method. Montmorillonite adsorbed creatinine markedly in the simulated intestinal solution in a concentration-dependent manner. The sorption-time curve of montmorillonite with creatinine showed that the sorption was fast. The adsorption rate reached a maximum in 10 min. The pH of the solution influenced the sorption, the rate of which was higher at a low pH than at a high pH. Creatinine could diffuse from the blood vessel to the intestine and was reabsorbed in the intestine. Montmorillonite promoted the diffusion and inhibited the absorption. Montmorillonite decreased the serum creatinine level of hypercreatininaemia mice prepared by injecting creatinine intraperitoneally. Montmorillonite adsorbs creatinine and accelerates its excretion from the intestine.

Formulation optimization and in situ absorption in rat intestinal tract of quercetin-loaded microemulsion

A new microemulsion system has been developed to increase the solubility and oral absorption of quercetin, a poorly water-soluble drug. The formulation of quercetin-loaded microemulsion was optimized by a simplex lattice experiment design. The optimized microemulsion formulation consisted of oil (7%, w/w), surfactant (48%, w/w), and cosurfactant (45%, w/w). Under this condition, the mean droplet diameter of microemulsion was 38.9 nm and solubility of quercetin in the microemulsion was 4.138 mg/ml. The in situ absorption property of quercetin-loaded microemulsion in rat intestine was studied and the results showed there was significant difference in absorption parameters such as K a, t 1/2 and uptake percentages between microemulsion and micelle solution containing quercetin. The study on absorption percentage in different regions of rat intestine attested that the colon had the best permeability, followed by ileum, duodenum in order. It can be concluded that microemulsion can improve the solubility and oral absorption of quercetin, a poorly water-soluble drug.

Self-micelle formation and the incorporation of lipid in the formulation affect the intestinal absorption of Panax notoginseng

The purpose of this research is to evaluate the effect of self-micelle formation and incorporation of lipid in the formulation on absorption of ginsenosides Rg1 and Rb1 from intestinal tract in rats. Ginsenosides Rg1 and Rb1 were extracted from Panax notoginseng saponins (PNS). The critical micellar concentration (CMC) of PNS in deionzied water was determined to be 0.339 mg/ml. At normal physiological ionic strengths, PNS was salted out from the solution above the CMC. The particle size of the micelle grows as PNS concentration increases. By in situ injection to a closed loop of the rat jejunum, AUC 0–6 h obtained after administration of low concentration solution (12 mg/ml) was 3.61 times for ginsenoside Rg1 and 3.84-folds for ginsenoside Rb1 compared with high concentration solution (120 mg/ml). The release rate of ginsenosides in aqueous medium was too slow to complete in 24 h, especially for Rb1. The data suggested that the self-micelle formation tendency in ginsenosides might prevent them from permeation or absorption through the cell membrane of gastrointestinal (GI) tract. To inhibit the formation of micelles, lipid was incorporated in the PNS formulation. The intraduodenal bioavailability in rats showed that the bioavailability was enhanced remarkably relative to the aqueous solution. AUC 0−∞ of ginsenoside Rg1 and Rb1 in the lipid-based formulation were 207.52 ± 53.95 and 1961.72 ± 686.60 μg ml −1 h, compared with 7.87 ± 2.85 and 148.58 ± 36.73 μg ml −1 h, respectively from its aqueous solution. These findings suggested a new strategy to increase the absorption of amphiphilic saponins.

Quantification of Bifidobacterium spp. and Lactobacillus spp. in rat fecal samples by real-time PCR

The microbiota of the rat intestinal tract constitutes a complex ecosystem of microorganisms. We have developed a real-time quantitative PCR assay based on genus-specific 16S rDNA primers and 3' minor groove binder (MGB) probes for accurate detection and quantification of a wide range of Bifidobacterium spp. (30 species) and Lactobocillus spp. (15 species) in rat fecal samples. Real-time PCR detection of serially diluted DNA isolated from reference strains of Bifidobacterium longum and Lactobacillus acidophilus was linear for cell counts ranging from 10(6) to 10 cells per PCR assay. The method proved applicable to the detection of Bifidobacterium spp. and Lactobacillus spp. at concentrations down to 10 CFU per PCR, corresponding to 5 x 10(4) CFU/g feces. The inter-extract reproducibility was high, with a coefficient of variation ranging from 0.24% to 1.07% for the Bifidobacterium assay and from 0.05% to 1.28% for the Lactobacillus assay. We conclude that real-time PCR is a very sensitive and precise technique for extensive quantitative evaluation of gut Bifidobacterium spp. and Lactobacillus spp. Thus, the approach used here to detect and quantify bacteria with group-specific primers should contribute to further studies of the composition and dynamics of the rat intestinal microbiota.

Screening for in vivo metabolites of isovalertatin family oligosaccharides in rats by liquid chromatography coupled to electrospray ionization tandem mass spectrometry

Liquid chromatography/electrospray ionization tandem mass spectrometry (LC/MS(n)) was used to identify trace levels of in vivo metabolites after the administration of isovalertatin M23 or isovalertatin D23 to rats. The biosamples of urine, feces, and ileum incubation were pre-treated by solid-phase extraction (SPE), and then chromatographed with a reversed-phase C8 column with acetonitrile/1.5 mM aqueous ammonia (18:82, v/v) as the mobile solvent. The parent drug and the possible metabolites were identified by two independent qualitative parameters, retention time and collision-induced dissociation product ions. Nine and seven metabolites were successfully characterized from biosamples after administration of isovalertatins M23 and D23, respectively, to rats. The metabolism seemed to take place in the rat intestinal tract, and metabolic pathways were identified including isovaleryl de-esterification and hydrolysis of alpha-glucose units located either at the reducing or the non-reducing terminus.

Pharmacokinetics of sildenafil after intravenous and oral administration in rats: Hepatic and intestinal first-pass effects

Pharmacokinetics of sildenafil after intravenous and oral administration at various doses and first-pass effect at 30 mg/kg were evaluated in rats. After intravenous administration (10, 30, and 50 mg/kg), the dose-normalized AUC values were proportional to intravenous doses studied. However, after oral administration (10, 30, and 100 mg/kg), the dose-normalized AUC values increased significantly with increasing doses, possibly due to saturation of metabolism of sildenafil in rat intestinal tract. After oral administration (30 mg/kg), approximately 0.626% was not absorbed and F was 14.6%. The AUC after intragastric administration was significantly smaller (71.4% decrease) than that after intraportal administration, however, the values were not significantly different between intragastric and intraduodenal administration. The above data suggested that intestinal first-pass effect of sildenafil was approximately 71% of oral dose in rats. The AUC values after intraportal administration were significantly smaller (49% decrease) than that after intravenous administration. This suggested that hepatic first-pass effect of sildenafil after absorption into the portal vein was approximately 49% of oral dose in rats (approximately 49% was equivalent to approximately 13.7% of oral dose). The low F of sildenafil at a dose of 30 mg/kg in rats could be mainly due to considerable intestinal first-pass effect.

Effects of portal triad clamping on nitric oxide synthase positive neurons in myenteric plexus of rat intestinal tract

目的:研究肝门阻断后大鼠肠道肌间神经丛内一氧化氮合酶(NOS)神经元表达的变化,以探讨肝门阻断对肠道功能影响的神经机制.方法:SD大鼠分成实验组(根据阻断时间分为肝门阻断20 min组、40 min组及60 min组)和对照组,取各组相同部位的小肠和结肠,制作肠肌间神经丛标本行NADPH-d组化染色,观察和比较各组NOS阳性神经元的分布密度和染色情况.结果:与对照组比较,实验组小肠(28.89±5.49,32.22±7.03,36.89±8.58 vs 21.78±4.56,P＜0.01)和结肠(34.22±8.82,39.39±8.91,44.61±9.94 vs 25.94±5.59,P＜0.01)的肠肌间神经丛内NOS阳性神经元数量增多,胞体大而染色深;实验组之间比较,肝门阻断60 min组的NOS阳性神经元数量多于肝门阻断20 min组(小肠:36.89±8.58 vs 28.89±5.49;结肠:44.61±9.94 vs 34.22±8.82,P＜0.05),肝门阻断60 min组和40 min组的NOS阳性神经元胞体较大,染色较深,但节间束NOS阳性神经纤维稀少.结论:肝门阻断会影响或损伤肠神经系统的NO能神经,这可能是术后肠道运动功能障碍发生的神经机制.

Phosphatidylinositol increases HDL-C levels in humans

Studies have shown that phosphatidylinositol (PI) can stimulate reverse cholesterol transport by enhancing the flux of cholesterol into HDL and by promoting the transport of high density lipoprotein-cholesterol (HDL-C) to the liver and bile. The goal of this study was to determine the safety and therapeutic value of PI after oral administration to normolipidemic human subjects. We performed a randomized 2 week study in 16 normolipidemic subjects. Subjects received either 2.8 or 5.6 g of PI, with or without food. PI was well tolerated by all subjects. PI significantly affected the levels of HDL-C and triglyceride in the plasma of subjects receiving PI with food. The lower dose showed a 13% increase in HDL-C, whereas the high dose showed an increase of 18% over the 2 week period. Both low- and high-dose groups showed significant increases in plasma apolipoprotein A-I. The high dose of PI also decreased plasma triglycerides by 36% in the fed subjects. These data suggest that after only 2 weeks, PI may have a comparable therapeutic value to niacin, with negligible side effects.

Colonization of the neonatal rat intestinal tract from environmental exposure to the anaerobic bacterium Oxalobacter formigenes.

Oxalobacter formigenes, an anaerobic bacterium that inhabits the mammalian gastrointestinal tract, has an important symbiotic relationship with its vertebrate hosts by regulating oxalic acid homeostasis. Epidemiological studies of O. formigenes colonization in man have shown that colonization occurs in young children, that every child can become colonized naturally, that >20% lose colonization during adolescence or as adults and that stable colonization can be disrupted by antibiotic use or changes in diet, greatly affecting subsequent health. As O. formigenes is a fastidious anaerobe that seldom re-colonizes adults, the question arises as to how initial colonization occurs. To investigate this question, non-colonized female laboratory rats were placed on diets high in oxalate and were colonized by oesophageal gavage with O. formigenes either before or after being impregnated. Faecal specimens from their offspring were tested for the presence of O. formigenes. Although the bacterium was first detected in a few neonates as early as 7 days post-partum, colonization of all the offspring did not occur until after weaning. In each case, the offspring were colonized with the bacterial strain carried by their mothers. To determine whether O. formigenes colonization occurs vertically or horizontally, newborn rats were placed with foster mothers that were either non-colonized or colonized with an O. formigenes strain different from that of their natural mothers. Colonization occurred temporally in a manner similar to natural colonization but all offspring became colonized only with the O. formigenes strain of the foster mothers. These data indicate that intestinal colonization occurs horizontally, but does not answer the question of how O. formigenes survives the aerobic environment in order to be transmitted.

Steroids in the intestinal tract of rats are affected by dietary-fibre-rich barley-based diets.

The aim of the present study was to investigate the influence of dietary-fibre (DF)-rich barley-based diets on bile acids (BA) and neutral sterols (NS) in the intestinal tract of rats. For this purpose, young male Wistar rats (n 50; ten per group) weighing about 67 g were fed either a barley-free diet (control group) or diets containing 500 g barley meal extrudates/kg or a barley meal-Novelose mixture (groups A-D) for 6 weeks. These barley products contained 7-24 g resistant starch/100 g and 7-12 g (1 --> 3),(1 --> 4)-beta-glucan/100 g. More steroids were transported towards the lower parts of the intestinal tract when higher concentrations of macromolecular DF were present in the diets (P < 0.001). Tauroconjugated and primary BA dominated in the contents of the small intestine. Intense enzymic conversion of BA occurred in the caecum and colon. The fermentation of DF affected indirectly the amount of formed secondary BA. The main BA present in the caecal contents were muricholic acids, hyodeoxycholic acid and cholic acid. The BA spectrum in the colonic contents was different from that in the caecum. A higher concentration of NS appeared in the intestinal contents of the groups fed the barley-based diets than in the controls (P < 0.005). The microbial conversion of cholesterol to coprostanol, cholestanone and coprostanone was influenced by the amount and composition of the DF in the gut. DF in the diet may affect the concentration and spectrum of steroids in the intestinal tract. The results are relevant for the discussion of mechanisms behind the cholesterol-lowering effects of DF.

In vivo production of hydroxyl radical by enterococcus faecalis colonizing the intestinal tract using aromatic hydroxylation

Enterococcus faecalis is an intestinal commensal that produces extracellular superoxide (O2•−) through autoxidation of membrane-associated demethylmenaquinone. To assess free radical production by E. faecalis in vivo, intestinal tracts of rats were colonized using wild-type E. faecalis or a mutant strain with attenuated O2•− production. Ex vivo electron paramagnetic resonance spin trapping study of colonic contents (mean ± SD) showed 1.4 ± 1.5 and 0.094 ± 0.24 μM 5,5-dimethyl-1-pyrroline-N-oxide-hydroxyl radical adduct/gm stool for rats colonized with wild-type and mutant strains, respectively (p = .002). In vivo hydroxyl radical production was further assayed by aromatic hydroxylation using phenyl N-tert-butylnitrone (PBN) and d-phenylalanine. Hydroxylated PBN and d-phenylalanine products were recovered from stool (μM/gm colonic contents/109 colony forming units) and urine (μM/h/ml), respectively, and quantified using electrochemical detection. Hydroxylated (OH) PBNs and isomeric tyrosines (hydroxylated phenylalanine) were significantly increased (mean ± SD) for rats colonized with wild-type E. faecalis (2-OH PBN, 63 ± 58; 3-OH PBN, 63 ± 84; ortho-tyrosine, 31 ± 27; meta-tyrosine, 17 ± 14) compared to the mutant strain (2-OH PBN, 2.5 ± 7.3 (p < .001); 3-OH PBN, 3.9 ± 12.3 (p = .01); ortho-tyrosine, 1.9 ± 6.0 (p < .001); meta-tyrosine, 1.5 ± 3.4 (p = .03)). Similar differences were observed following in vitro incubations of these bacteria with aromatic targets. These results confirm in vivo production of hydroxyl radical by E. faecalis colonizing the intestine, and indicate this bacterium may be a potent source of oxidative stress on the intestinal epithelium.

The Degree of Methylation Influences the Degradation of Pectin in the Intestinal Tract of Rats and In Vitro

We investigated the degradation, metabolism, fate, and selected effects of pectin in the intestinal tract of rats. Conventional and germfree rats were fed for 3 wk diets containing 6.5% pectin (degree of methylation 34.5, 70.8 and 92.6%, respectively) or pectin-free diets. Pectin passes the small intestine as a macromolecule. The molecular weight distribution of pectins isolated from intestinal contents of germfree rats were unaffected by diet. No or very little galacturonan was found in cecum, colon or feces of most of the conventional rats. In colon contents of some conventional rats, di- and trigalacturonic acid were present. Total anaerobic and Bacteroides counts were greater in groups fed pectin. The concentration of short-chain fatty acids (SCFA) was higher in cecum and feces in all pectin-fed groups. With increasing degree of methylation, the formation rate of SCFA decreased in the cecum of conventional rats. During in vitro fermentation of pectin with fecal flora from rats, unsaturated oligogalacturonic acids appeared as intermediate products. Low-methoxyl pectin was fermented faster than high-methoxyl pectins in vivo and in vitro. Pectin-fed rats had greater ileum, cecum and colon weights. We conclude that structural parameters of pectin influence its microbial degradation in the intestinal tract.

Thrittene, Homologous with Somatostatin-28(1–13), Is a Novel Peptide in Mammalian Gut and Circulation

Preprosomatostatin is a gene expressed ubiquitously among vertebrates, and at least two duplications of this gene have occurred during evolution. Somatostatin-28 (S-28) and somatostatin-14 (S-14), C-terminal products of prosomatostatin (ProS), are differentially expressed in mammalian neurons, D cells, and enterocytes. One pathway for the generation of S-14 entails the excision of Arg13-Lys14 in S-28, leading to equivalent amounts of S-28((1-12)). Using an antiserum (F-4), directed to the N-terminal region of S-28 that does not react with S-28((1-12)), we detected a peptide, in addition to S-28 and ProS, that was present in human plasma and in the intestinal tract of rats and monkeys. This F-4 reacting peptide was purified from monkey ileum; and its amino acid sequence, molecular mass, and chromatographic characteristics conformed to those of S-28((1-13)), a peptide not described heretofore. When extracts of the small intestine were measured by RIA, there was a discordance in the ratio of peptides reacting with F-4 and those containing the C terminus of ProS, suggesting sites of synthesis for S-28((1-13)) distinct from those for S-14 and S-28. This was supported by immunocytochemistry, wherein F-4 reactivity was localized in gastrointestinal (GI) endocrine cells and a widespread plexus of neurons within the wall of the distal gut while immunoreactivity to C-terminal domains of S-14 and S-28 in these neurons was absent. Further, F-4 immunoreactivity persisted in similar GI endocrine cells and myenteric neurons in mice with a targeted deletion of the preprosomatostatin gene. We believe that these data suggest a novel peptide produced in the mammalian gut, homologous with the 13 residues of the proximal region of S-28 but not derived from the ProS gene. Pending characterization of the gene from which this peptide is derived, its distribution, and function, we have designated this peptide as thrittene. Its localization in both GI endocrine cells and gut neurons suggests that thrittene may function as both a hormone and neurotransmitter.

Distribution and properties of neutral ceramidase activity in rat intestinal tract.

Ceramide plays an important role in regulating cell proliferation and apoptosis. Recent studies indicate that generation of ceramide in the intestine from sphingomyelin hydrolysis may be implicated in colon cancer development. The enzymes that catalyze the further hydrolysis of ceramide in the intestine have, however, not been well investigated. Our data reveal the existence of a ceramidase (EC 3.5.1.23) in rat intestinal mucosa with an optimal pH of 7.0. One milligram of mucosal protein is able to hydrolyze 44.0+/-9.6 nmol of ceramide in 1 hr. The activity is low in the proximal duodenum and increases to a plateau in the proximal jejunum. The activity is then similar throughout the small intestine, until it declines in the distal part of ileum. Some activity is also detectable in the colon. The activity increases slightly in the presence of monomeric bile salt concentrations and sharply at the critical micellar concentration. Similar patterns were observed for both primary (taurocholate) and secondary (taurodeoxycholate) bile salts. The addition of Triton X-100 enhances the ceramidase activity at optimal bile salt concentration. The reaction is linear with time for the first 20 min and the hydrolytic rate declines slowly thereafter. Finally, the activity shows a considerable resistance against tryptic degradation, as 71% of the ceramidase activity remained when the homogenates were preincubated with high concentrations of trypsin. Intestinal mucosa also has a ceramide synthesis activity, with a distribution pattern generally paralleling ceramide hydrolysis activity. In conclusion, intestinal neutral ceramidase has a distinct distribution pattern and bile salt dependence, which enables it to collaborate with intestinal sphingomyelinase in hydrolysis of sphingomyelin.

Food viscosity as determinant for adaptive growth responses in rat intestine: long-term feeding of different hydroxyethyl celluloses

Carbohydrate gelling agents can be regarded as being representative for the soluble and viscous fractions of dietary fibre. Their dietary concentration affects the consistency of the ingested food as well as the dilution of nutrients and energy. By feeding hydroxyethyl cellulose (HEC) differing in molecular mass, and thus in its viscosity properties, only the consistency of the diet was modified. Three HEC (of low (LV), medium (MV) and high viscosity (HV)) were employed in a 6-week feeding study with female rats to evaluate the effect of the viscosity on adaptive responses of intestinal growth variables. Each of the HEC was added in three increasing concentrations (8, 16, and 32%, w/w) to a fibre-free control diet to yield nine test groups besides a fibre-free and an additional, fibre-rich, cereal-based control group. Except for the highest concentration of the high viscosity product (32% HV-HEC), the dilution of the energy density of the diet was almost completely compensated by an increased food intake. With the same exception, energy utilisation was not impaired and, therefore, body-weight gains in the test groups were not significantly different from that in the control. Most other changes, e.g. increases in small intestinal length, mucosal DNA content, caecal and colonic weight, not only depended on the dietary concentration but also on the viscosity of HEC in a manner that either increasing the viscosity at a given dietary concentration or increasing the dietary concentration at a given viscosity led to the same results. These findings clearly prove the important role of the viscosity of the lumen content, as a mere physico-chemical factor, in determining adaptative growth responses in the intestinal tract of rats.

DRA expression and brush border membrane anion exchange characteristics in rat and rabbit duodenum

Backround and Aims: Duodenal bicarbonate secretion in the resting state is in part mediated by an apical anion exchanger whose molecular nature is unknown. Recent data suggest that the dra gene, which is defective in congenital chlorid diarrhea, encodes an anion exchanger which, in oocytes, mediates sulfate as well as Cl uptake. We now investigated if the DRA gene product may be the apical anion exchanger in the duodenum. Methods: To test this hypothesis, we cloned rat and rabbit DRA cDNA fragments and studied the relative expression levels of DRA and NHE3 (Na+/H+ exchanger isoform 3) in the different segments of the rat and rabbit intestine by Northern analysis and semiquantitative PCR. We then isolated rat and rabbit duodenal and jejunal brush border membrane vesicles and studied 36Clund 35S0~uptake into Cl, HCOj, SO~and gluconate-loaded vesicles in the absence and presence of a pH-gradient (to distinguish SO~from HS04 transport) and the absence and presence of DIDS. We also examined HC03secretion in stripped rat and rabbit intestinal mucosa in the Ussing-chamber. Results: DRA was highly expressed in rat and rabbit intestinal tract, with the expression pattern colon>duodenum>ileum>jejunum in the rat and ileumc-colon> duodenum in the rabbit, and clearly differed from the expression pattern for NHE3 which was colon>ileum> jejunum>duodenum in both species. This excess DRA over NHE3 expression in the small intestine but not the proximal colon may explain why the resting state (absorbing) small intestine in the Ussing-chamber displays electroneutral HC03secretion whereas the proximal colon acidifies the luminal perfusate under resting conditions. Both rat and rabbit BBMvs displayed DIDS-sensitive CIICI-, HC03-/Ct, SO~-/Cl, and CIISO~exchange, findings compatible with DRA characteristics. Anion affinity for the internal and external anion binding sites differed somewhat between rat and rabbit but were Cl >HCOj in both species. Summary and Conclusions: The high duodenal DRA expression levels and the fact that the transport characteristics of rat and rabbit BBM anion exchange corresponds with those found in oocytes and Hek293 cells suggests that DRA is the likely transport protein which mediates electroneutral HCOj secretion in the duodenum.

Immunolabelling of the rat intestinal tract with antibodies specific to the long form of the 5-hydroxytryptamine 3 receptor

The mouse 5-hydroxytryptamine 3 (5-HT 3) type of serotonin receptors is expressed as two forms, 5-HT 3R-A L and 5-HT 3R-A S, generated by alternative splicing of its primary transcript, that differ by a stretch of six amino acids in the second intracellular loop domain. Because this six-amino acid region contains a putative phosphorylation site that may be important for the function and/or regulation of 5HT 3R-A L receptor, specifically, we developed polyclonal antibodies as appropriate tools for studies relevant to this question. Antibodies against a 20-amino acid peptide corresponding to the sequence of 5-HT 3R-A L at the level of this six-amino acid region were obtained as soon as one month after injection of this synthetic peptide to rabbits. Immunocytochemistry with these antibodies led to a strong positive labelling of plasma membrane, reticulum and Golgi apparatus of COS-7 cells expressing cloned murine 5-HT 3R-A L, whereas COS-7 cells expressing similar levels of 5-HT 3R-A S exhibited only a very weak labelling. Immunoblots of fusion proteins combining glutathion-S-transferase and the second cytoplasmic loop of 5-HT 3R-A L or 5-HT 3R-A S revealed a c. 20-fold selectivity of the antibodies for the first, long form, as evaluated by densitometric analysis of enhanced chemiluminescence detection. Similarly, immunoblots of COS-7 cells transfected with cloned 5-HT 3 receptors showed that the anti-peptide antibodies detected a band at 53,000 mol. wt only in cells transfected with 5-HT 3R-A L. Under optimal conditions, antibodies immunoprecipitated 52% of 5-HT 3R-A L, but only 11% of 5-HT 3R-A S, solubilized from COS-7 cells transfected with the respective encoding plasmids. In the rat, no immunoautoradiographic labelling by the anti-peptide antibodies could be detected in brain structures which had previously been described to express preferentially a short form of the 5-HT 3 receptor. In contrast, a strong immunolabelling was found in the intestinal mucosa, especially in the rat fetus (at the 17th embryonic day), suggesting the possible participation of the 5-HT 3R-A L isoform in the development of this tissue. These results show that specific antibodies are useful tools for the visualization of the least abundant 5-HT 3 receptor isoform in rat tissue.

Immunocytochemical localization of the NPY/PYY Y1 receptor in enteric neurons, endothelial cells, and endocrine-like cells of the rat intestinal tract.

Neuropeptide Y (NPY) and peptide YY (PYY) are structurally related peptides that are considered to mediate inhibitory actions on gastrointestinal motility, secretion, and blood flow. Several receptor subtypes for these peptides have been identified and the Y1, Y2, Y4/PP1, Y5, and Y5/PP2/Y2b receptors have been cloned. In this article we report the immunocytochemical localization of the Y1 receptor to myenteric and submucosal nerve cell bodies, endothelial cells, and scattered endocrine-like cells of rat intestinal tract. Moreover, double immunofluorescence demonstrates that subpopulations of the Y1 receptor-positive nerve cell bodies are immunopositive for NPY, vasoactive intestinal polypeptide, and nitric oxide synthase. In part, such co-localizations were made possible by use of peroxidase-mediated deposition of tyramide, which permitted use of antisera derived from the same species. Our observations suggest the existence of multiple neuronal, endothelial, and endocrine target sites for NPY and PYY and that some of the actions of these regulatory peptides can be mediated by vasoactive intestinal peptide and nitric oxide synthase.

Ferritin gene expression and transferrin receptor activity in intestine of rats with varying iron stores.

Expression of transferrin receptor and ferritin genes has been shown previously to be under transcriptional and posttranscriptional regulation, the latter being reciprocally regulated according to cellular iron levels. This study examined transferrin receptor function and ferritin gene expression along the crypt-villus axis of the intestinal tract in rats with varying iron stores. Altered iron stores were produced by feeding a control diet and diets low or high in iron (2% carbonyl iron) for 8-10 wk. Expression and activity of the ferritin genes were assessed by in situ hybridization and immunohistochemical localization, respectively. Transferrin receptor activity was determined by the uptake of intravenously injected transferrin-bound iron and was shown to increase with the level of iron loading. In all iron status groups, ferritin mRNA was seen at highest levels in the epithelial cells of the crypt and macrophages within the lamina propria and at lower levels in villus epithelial cells. In all groups, ferritin protein was not seen in the crypt region but was seen with increasing staining in the apical two-thirds of the villus cells of control and iron-loaded, but not iron-deficient, rats. Ferritin staining increased with iron loading. We conclude that in undifferentiated crypt cells ferritin genes are transcribed, but the message is not translated. After differentiation, these genes appear to be controlled posttranscriptionally by cellular iron stores.

Localization and capacity of sphingomyelin digestion in the rat intestinal tract

Dietary sphingomyelin (SM) undergoes sequential cleavage to ceramide and sphingosine in the intestine. A distinctive intestinal sphingomyelinase (SMase) with alkaline pH-optimum was earlier identified by us. The activity was highest in middle and lower small intestine, but its role in SM digestion has not been clarified. In this study we examined the extension and capacity of SM digestion in vivo. After feeding rats 0.2, 6.6, or 32 μmol SM containing 2 μCi 3H-sphingosine-labeled milk SM ( 3H-SM), radioactivity was analyzed in intestinal contents and tissues 2, 4, and 8 hr later. The proportion of radioactivity in the contents of small intestine increased with the dose of SM; 9% of given dose with 0.2 μmol, 34% with 6.6 μmol, and 71% with 32 μmol, respectively after 2 hr. Lowest tissue radioactivity was found in duodenum and proximal jejunum and highest in distal jejunum and proximal ileum. Three to twenty one percent of radioactivity in the intestinal tissue was in ceramide, the proportion varying with the dose given, region of the intestine, and time after administration. After administration of 6.6 or 32 μmol SM, significant amounts of intact SM and ceramide was found in intestinal contents, colon, and excreted faeces. Colon was exposed to ceramide derived from exogenous SM in amounts that were rather proportional to the dose of SM fed. SM digestion is thus a process extending over the whole intestine and occurring mainly in the middle and lower parts of the small intestine. The site of digestion coincides with the distribution of the alkaline SMase, indicating that this enzyme catalyzes the first step in the digestion. The extension and limited capacity of the SM digestion leads to an exposure of the lower small intestine and colon to SM and sphingolipid metabolites.