Oxytocin is expressed throughout the human gastrointestinal tract

BACKGROUND/AIM: Several studies have described that oxytocin exerts stimulatory or inhibitory effects on gut functions. Recently, mRNA for oxytocin and its receptor was found throughout the entire human gastrointestinal (GI) tract. The aim of this study was to examine the cellular localization and distribution of the corresponding proteins. MATERIAL AND METHODS: Full-thickness biopsies from 24 patients, covering the entire GI tract, were collected during operations at the Department of Surgery in Malm? and Lund. The biopsies were taken from non_affected margins. The biopsies were fixed by immersion, rinsed in buffered sucrose, and kept frozen at 70 degrees C. Indirect immunofluorescence with primary antibodies to oxytocin and its receptor was used. RESULTS: Oxytocin was expressed in nerve cell bodies and nerve fibres in the myenteric and submucous ganglia all along the GI tract. Immunoreactive nerve cell bodies in myenteric ganglia predominated in the proximal (antrum and duodenum) and distal gut, while those in the submucous ganglia were more numerous in the ileum and colon. The oxytocin receptor was not detectable by two different antibodies in any tissue in the GI tract. CONCLUSION: Oxytocin is expressed in the myenteric and submucous ganglia and nerve fibres along the entire human GI tract. The role for oxytocin in the physiology and pathophysiology of the bowel remains to be settled.     

Comparative expression of hexose transporters (SGLT1, GLUT1, GLUT2 and GLUT5) throughout the mouse gastrointestinal tract

Hexose transporters play a pivotal role in the absorption of food-derived monosaccharides in the gastrointestinal tract. Although a basic knowledge of the hexose transporters has already been gained, their detailed distribution and comparative intensities of expression throughout the gastrointestinal tract have not been fully elucidated. In this study, we quantitatively evaluated the expression of SGLT1, GLUT1, GLUT2, and GLUT5 by in situ hybridization and real-time PCR techniques using a total of 28 segments from the gastrointestinal tract of 9-week-old mice. GLUT2 and GLUT5 mRNA expressions were detected predominantly from the proximal to middle parts of the small intestine, showing identical expression profiles, while SGLT1 mRNA was expressed not only in the small intestine but also in the large intestine. Notably, GLUT1 mRNA was expressed at a considerable level in both the stomach and large intestine but was negligible in the small intestine. Immunohistochemistry demonstrated the polarized localization of hexose transporters in the large intestine: SGLT1 on the luminal surface and GLUT1 on the basal side of epithelial cells. The present study provided more elaborate information concerning the localization of hexose transporters in the small intestine. Furthermore, this study revealed the significant expression of glucose transporters in the large intestine, suggesting the existence of the physiological uptake of glucose in that location in mice.     

Constitutive expression and induction of CYP1B1 mRNA in the mouse.

Previous studies appeared to indicate that CYP1B1 was not constitutively expressed in mouse liver. In our laboratory, we demonstrated using aromatic hydrocarbon-responsive receptor knock-out (AHR-(-)/-) mice that both piperonyl butoxide (PBO) and acenaphtyhlene (ACN) are AHR-independent inducers of murine CYP1A2 and CYP1B1 mRNA. In the current study, we demonstrate both constitutive levels and induction of CYP1B1 in mouse liver. The induction of CYP1B1 mRNA by PBO or ACN was higher in DBA/2 (Ahrd) than in C57BL/6 (Ahrb-1) mice, while 3-methylcholanthrene induced CYP1B1 more in C57BL/6 than in DBA/2 mice. These results suggest that CYP1B1 may also be induced by more than one mechanism. In addition, constitutive expression of CYP1B1 was detected in liver, kidney, and lung of untreated C57BL/6 mice. There was no gender difference in CYP1B1 expression; however, in C57BL/6 mice, the kidney contained less CYP1B1 than either liver or lung.     

Two alternatively spliced mouse urokinase receptor mRNAs with different histological localization in the gastrointestinal tract

Two mouse urokinase-type plasminogen activator receptor (muPAR) cDNAs were isolated: muPAR1 is homologous to the human urokinase-type plasminogen activator receptor while muPAR2 codes for a 199 residue protein sharing the first 133 residues with muPAR1. Mouse genomic DNA sequencing indicates that the two different mRNAs arise by alternative splicing. In situ hybridization showed differential expression of the two mRNAs in mouse gastric mucosa. muPAR1 mRNA is located in luminal epithelial cells situated close to urokinase-type plasminogen activator-producing connective tissue cells of the lamina propria, pointing to plasmin generation controlled by the cooperation of different cells that may play a role in the release of gastric epithelial cells. muPAR2 mRNA is expressed in the basal epithelial cells, and the deduced protein sequence includes the receptor ligand binding domain, but omits the region involved in glycolipid-mediated membrane anchoring, suggesting that muPAR2 may code for a secreted uPA binding protein.     

Expression and cellular localization of the mRNA for the 25-kDa heat-shock protein in the mouse

The 25-kDa heat-shock protein (Hsp25) is a member of the small heat-shock protein family but its function remains largely unknown. In the present study we examined the expression and cellular localization of Hsp25 mRNA in mice under physiological, unstressed conditions using Northern blot and in situ hybridization analyses with specific oligonucleotide probes. At the organ level, high amounts of Hsp25 mRNA were detected in the oesophagus, skin,eye, stomach, lung and urinary bladder, with moderate amounts in the heart, skeletal muscle, aorta, adrenal gland, ovary, testis, uterus, large intestine, and thymus. At the cellular level, intense to moderate signals for Hsp25 mRNA were localized in the muscle cells of smooth, heart and skeletal types, in the epithelial cells of stratified squamous and transitional types and of the oviduct, in the steroid endocrine cells of the adrenal cortex and corpus luteum, as well as in the spermatocytes of the testis. In contrast, the signal was scarcely detectable in the nervous tissues, lymphatic tissues, the columnar epithelial cells of the digestive tract, or the parenchymal cells of the liver, pancreas and kidney. These results suggest some significant role for Hsp25 in distinct populations of mouse cells under physiological conditions.     

The protein chaperone Ssa1 affects mRNA localization to the mitochondria

Many nuclear-transcribed mRNAs encoding mitochondrial proteins are localized near the mitochondrial outer membrane. A yet unresolved question is whether protein synthesis is important for transport of these mRNAs to their destination. Herein we present a connection between mRNA localization in yeast and the protein chaperone Ssa1. Ssa1 depletion lowered mRNA association with mitochondria while its overexpression increased it. A genome-wide analysis revealed that Ssa proteins preferentially affect mRNAs encoding hydrophobic proteins, which are expected targets for these protein chaperones. Importantly, deletion of the mitochondrial receptor Tom70 abolished the impact of Ssa1 overexpression on mRNAs encoding Tom70 targets. Taken together, our results suggest a role for Ssa1 in mediating localization of nascent peptide-ribosome-mRNA complexes to the mitochondria, consistent with a co-translational transport process.     

Expression and localization of aquaporins in rat gastrointestinal tract

A family of water-selective channels, aquaporins (AQP), has beendemonstrated in various organs and tissues. However, the localizationand expression of the AQP family members in the gastrointestinal tracthave not been entirely elucidated. This study aimed to demonstrate theexpression and distribution of several types of the AQP family and tospeculate on their role in water transport in the rat gastrointestinal tract. By RNase protection assay, expression of AQP1-5 and AQP8 was examined in various portions through the gastrointestinal tract.AQP1 and AQP3 mRNAs were diffusely expressed from esophagus to colon,and their expression was relatively intense in the small intestine andcolon. In contrast, AQP4 mRNA was selectively expressed in the stomachand small intestine and AQP8 mRNA in the jejunum and colon.Immunohistochemistry and in situ hybridization demonstrated cellularlocalization of these AQP in these portions. AQP1 was localized onendothelial cells of lymphatic vessels in the submucosa and laminapropria throughout the gastrointestinal tract. AQP3 was detected on thecircumferential plasma membranes of stratified squamous epithelialcells in the esophagus and basolateral membranes of cardiac glandepithelia in the lower stomach and of surface columnar epithelia in thecolon. However, AQP3 was not apparently detected in the smallintestine. AQP4 was present on the basolateral membrane of the parietalcells in the lower stomach and selectively in the basolateral membranesof deep intestinal gland cells in the small intestine. AQP8 mRNAexpression was demonstrated in the absorptive columnar epithelial cellsof the jejunum and colon by in situ hybridization. These findings mayindicate that water crosses the epithelial layer through these waterchannels, suggesting a possible role of the transcellular route forwater intake or outlet in the gastrointestinal tract.     

Expression of stress inducible protein 1 (Stip1) in the mouse testis

Phthalate esters are considered endocrine disruptors that interfere with the endocrine balance and development of the mammalian testis. Mono-2-ethylhexyl phthalate (MEHP), the active metabolite of the ubiquitously used plasticizer di-2-ethylhexyl phthalate (DEHP), acts upon Sertoli cells as initial target. By subtractive cDNA libraries we identified genes deregulated as response to MEHP in primary cultures of mouse Sertoli cells. The expression of mouse stress inducible protein 1 (Stip1) was detected as upregulated as a result of MEHP exposure. Stip1 is a cochaperone protein that is homologous to the human heat shock cognate protein 70 (hsc70)/heat shock protein 90 (hsp90)-organizing protein (Hop). To assess the presence and localization of Stip1 in mouse testis and its potential role in stress defense, we studied the expression pattern of the Stip1 protein by immunohistochemistry and of the mRNA by in situ hybridization. Both the protein and the mRNA of Stip1 were mainly found in the cytoplasm of all types of spermatogonia and spermatocytes up till zygotene, the expression decreased during late pachytene and was very weak in diplotene spermatocytes and round spermatids. Interestingly, this expression pattern resembled the pattern of stress sensitivity of spermatogenic cells in that the most sensitive cell types show the weakest expression of Stip1. This suggests an important role for Stip1 in the ability of germ cells to survive in stress conditions including high temperatures.     

Identification and localization of soluble sulfotransferases in the human gastrointestinal tract

Soluble SULTs (sulfotransferases) are important in the regulation of messenger molecules and the elimination of xenobiotics. However, sulfo-conjugation of various substrates can also lead to the formation of reactive metabolites that may induce cancer and cause other damage. The aim of the present study was to identify the SULT forms expressed in the human gastrointestinal tract, especially the colon and rectum (common sites for cancer), and to determine their cellular localization. Normal colonic or rectal tissue, resected with tumours, was obtained from 39 subjects. For comparison, we additionally studied one to four samples from stomach, jejunum, ileum, cecum and liver. SULTs were detected by immunoblotting, immunohistochemistry and measurement of enzyme activities. SULT1A1, 1A3 and 1B1 were found in all parts of the gastrointestinal tract, often exceeding levels in liver (where these forms were present at high, undetectable and low levels respectively). They were predominantly localized in differentiated enterocytes. SULT1E1 and 2A1 were only detected in liver, jejunum, ileum and cecum. SULT1C1 was readily found in stomach, but was negligible elsewhere. SULT1A2 was present at low levels in individual samples. The remaining forms were not detected with the limitation that only high levels could be recognized with the antisera used. In conclusion, SULTs are abundant in the gastrointestinal tract of man. We suspect that they are involved in the presystemic elimination of bioactive food-borne components, including aglycones released by gut microbiota, as well as the bioactivation of some procarcinogens.     

The ultrastructural localization and quantitation of cholinergic receptors at the mouse motor endplate

Summary The snake venom toxin, -bungarotoxin, is known to bind specifically to the acetylcholine receptor at skeletal muscle endplates. In this study, tritiated -bungarotoxin has been used in conjunction with electron-microscope autoradiography to visualize and enumerate acetylcholine receptor sites at the neuromuscular junctions of the mouse diaphragm. From an analysis of the grain distribution, the receptor sites appear to be located specifically on the postjunctional membrane. The density there is about 8,500/² of membrane surface. For comparison purposes, cholinesterases and related active centers were labeled using [³H] diisopropylfluorophosphate; they were shown to be at this same concentration over the synaptic membranes (or along the cleft). The 11 relationship of the receptors to the cholinesterase type of site, found previously to hold in studies on whole endplates, is also true at the ultrastructural level in this case. In fact, this 11 relationship is believed to be a characteristic of the postsynaptic membranes of endplates in other muscles and other vertebrates.Based on the constant density value thus arrived at, the total surface areas of postsynaptic and of presynaptic membranes are at once obtained from the known total numbers of these sites per endplate, available from previous studies in this laboratory. Examples of such synaptic surface area values are given. These values are only reliable for a given muscle type if the approximate fiber size is defined.     

The yeast Cbk1 kinase regulates mRNA localization via the mRNA-binding protein Ssd1

The mRNA-binding protein Ssd1 is a substrate for the Saccharomyces cerevisiae LATS/NDR orthologue Cbk1, which controls polarized growth, cell separation, and cell integrity. We discovered that most Ssd1 localizes diffusely within the cytoplasm, but some transiently accumulates at sites of polarized growth. Cbk1 inhibition and cellular stress cause Ssd1 to redistribute to mRNA processing bodies (P-bodies) and stress granules, which are known to repress translation. Ssd1 recruitment to P-bodies is independent of mRNA binding and is promoted by the removal of Cbk1 phosphorylation sites. SSD1 deletion severely impairs the asymmetric localization of the Ssd1-associated mRNA, SRL1. Expression of phosphomimetic Ssd1 promotes polarized localization of SRL1 mRNA, whereas phosphorylation-deficient Ssd1 causes constitutive localization of SRL1 mRNA to P-bodies and causes cellular lysis. These data support the model that Cbk1-mediated phosphorylation of Ssd1 promotes the cortical localization of Ssd1-mRNA complexes, whereas Cbk1 inhibition, cellular stress, and Ssd1 dephosphorylation promote Ssd1-mRNA interactions with P-bodies and stress granules, leading to translational repression.     

Distribution, quantitation, and origin of immunoreactive neuropeptide Y in the human gastrointestinal tract

A radioimmunoassay for measurement of immunoreactive neuropeptide Y has been developed using antiserum from a rabbit (221) immunized with porcine neuropeptide Y. Antibody 221 has been characterized for both sensitivity and specificity. To determine the distribution of neuropeptide Y in the human gastrointestinal tract, fresh tissue specimens were separated by microdissection into the muscularis externa and the mucosa-submucosa. To examine the origin of neuropeptide Y in human colon, specimens of aganglionic and ganglionic colon were obtained from patients with Hirschsprung's disease. Immunoreactive neuropeptide Y in human gut was present in highest concentrations in the muscularis externa of the stomach and in lowest concentrations in the muscularis externa of the ileum and descending colon. Neuropeptide Y in the stomach was present in higher concentrations in the muscularis externa than in the mucosa-submucosa, but in the descending colon there were lower concentrations of neuropeptide Y in the muscularis externa than in the mucosa-submucosa. In Hirschsprung's disease, concentrations of neuropeptide Y were increased in aganglionic colon in both the muscularis externa and the mucosa-submucosa, compared to corresponding layers from proximal ganglionic colon. Extracts of the gastric muscularis externa and the colonic mucosa-submucosa were separated by C18 reverse-phase high-performance liquid chromatography. One major immunoreactive species was identified by radioimmunoassay which eluted in a position similar to synthetic human neuropeptide Y. These results demonstrated both regional and layer differences in concentrations of neuropeptide Y in human gut. Increased concentrations of neuropeptide Y in aganglionic colon from Hirschsprung's disease most likely result from enlargement of neuropeptide Y-containing extrinsic nerve fibers in both the mucosa-submucosa and the muscularis externa.     

Acute sodium arsenite administration induces pulmonary CYP1A1 mRNA, protein and activity in the rat

Modulation of the cytochrome P450 (CYP) monooxygenase system (P450) by arsenite was investigated in male, adult Sprague-Dawley rats treated with a single dose (75 micromol/kg, sc) of sodium arsenite (As3+). Total CYP content and P450-dependent 7-pentoxyresorufin O-pentylation (PROD) and 7-ethoxyresorufin O-deethylation (EROD) activities of liver microsomes decreased maximally (33, 35, and 50% of control, respectively) 1 day after As3+ treatment. Maximum decreases of CYP content and P450 catalytic activities corresponded with maximum increases of microsomal heme oxygenase (HO) activity and with increased total plasma bilirubin concentrations. EROD activity increased maximally in lung (300%) 5 days after a single dose of As3+. Lung CYP1A1 mRNA and protein levels also increased maximally 5 days after treatment. A small but significant increase in EROD activity (65%) was observed in lung microsomes 24 h following a 1 h infusion of bilirubin (7.5 mg/kg) into rats. However, administration of bilirubin to the lung via intratracheal injection (0.25 and 2.5 mg/kg) did not increase CYP1A1 monooxygenase activity or mRNA. This study demonstrates that P450 is modulated in an isozyme (CYP1A1 vs CYP2B1/2) selective manner in rat lung after acute As3+ administration. Administration of bilirubin, a potential aryl hydrocarbon receptor (AHR) ligand, by infusion or intratracheal instillation did not upregulate pulmonary CYP1A1 at the mRNA level under our treatment conditions.     

Persistence of colicinogenic Escherichia coli in the mouse gastrointestinal tract

Background  

The ability of a bacterial strain to competitively exclude or displace other strains can be attributed to the production of narrow spectrum antimicrobials, the bacteriocins. In an attempt to evaluate the importance of bacteriocin production for Escherichia coli strain residence in the gastrointestinal tract, a murine model experimental evolution study was undertaken.     

CYP4A mRNA, protein, and product in rat lungs: novel localization in vascular endothelium.

The vasodilatory effect of 20-hydroxyeicosatetraenoic acid (20-HETE) on lung arteries is opposite to the constrictor effect seen in cerebral and renal vessels. These observations raise questions about the cellular localization of 20-HETE-forming isoforms in pulmonary arteries and other tissues. Using in situ hybridization, we demonstrate for the first time CYP4A (a family of cytochrome P-450 enzymes catalyzing formation of 20-HETE from the substrate arachidonic acid) mRNA in pulmonary arterial endothelial and smooth muscle cells, bronchial smooth muscle and bronchial epithelial cells, type I epithelial cells, and macrophages in adult male rat lungs. Moreover, we detect CYP4A protein in rat pulmonary arteries and bronchi as well as cultured endothelial cells. Finally, we identify endogenously formed 20-HETE by using fluorescent HPLC techniques, as well as the capacity to convert arachidonic acid into 20-HETE in pulmonary arteries, bronchi, and endothelium. These data show that 20-HETE is an endogenous product of several pulmonary cell types and is localized to tissues that optimally position it to modulate physiological functions such as smooth muscle tone or electrolyte flux.