Na+-H+ exchange activity in brush-border membrane vesicles isolated from chick small intestine

This study was undertaken to investigate the presence of a Na+{single bond}H+ antiporter in brush-border membrane vesicles (BBMV) isolated from chick small intestine. An outwardly directed proton gradient (pH 5.5 inside, 7.5 outside) stimulated Na+ uptake into BBMV and resulted in a transient accumulation. No accumulation was observed in the absence of a proton gradient. Voltage clamping the membrane with K+ and valinomycin decreased the Na+ overshoot. Amiloride inhibited pH gradient-driven Na+ uptake in a dose-dependent manner with an IC50 of 44 μM. The relationship between pH gradient-driven Na+ uptake and external Na+ concentration followed simple, saturating Michaelis-Menten kinetics. Eadie-Hofstee analysis of the pH gradient-driven Na+ uptake indicated a single transport system with a Vmax of 33 nmol/mg protein per 15 s and a Km for Na+ of 12 mM. The initial rate of pH-driven Na+ uptake increased as the intravesicular pH decreased, with a Hill coefficient close to 1. These findings indicate that BBMV isolated from chicken small intestine posses a Na+{single bond}H+ exchanger. This exchanger does not appear to be the one involved in cell pH regulation.Dirección General de Investigaciones Científicas y Técnicas PB89-061

Na+-dependent and Na+-independent betaine transport across the apical membrane of rat renal epithelium

The low renal excretion of betaine indicates that the kidney efficiently reabsorbs the betaine filtered by the glomeruli but the mechanisms involved in such a process have been scarcely investigated. We have detected concentrative and non-concentrative betaine transport activity in brush-border membrane vesicles (BBMV) from rat renal cortex and medulla. The concentrative system is the Sodium/Imino-acid Transporter 1 (SIT1) because it is Na+- and Cl--dependent, electrogenic and is inhibited by an anti-SIT1 antibody. Its apparent affinity constant for betaine, Kt, is 1.1 ± 0.5 mM and its maximal transport velocity, Vmax, 0.5 ± 0.1 nmol betaine/mg protein/s. Inhibitors of the Na+/Cl-/betaine uptake are l-proline (75%) and cold betaine, l-carnitine and choline (40-60%). Neither creatine, TEA, taurine, β-alanine, GABA nor glycine significantly inhibited Na+/Cl-/betaine uptake. The non-concentrative betaine transport system is Na+- and H+-independent, electroneutral, with a Kt for betaine of 47 ± 7 μM and a Vmax of 7.8 ± 1 pmol betaine/mg protein/s. Its transport activity is nearly abolished by betaine, followed by L-carnitine (70-80%) and proline (40-50%), but a difference from the Na+/Cl-/betaine transport is that it is inhibited by TEA (approx. 50%) and unaffected by choline. The underlying carrier functions as an antiporter linking betaine entry into the BBMV with the efflux of either l-carnitine or betaine, an exchange unaffected by the anti-SIT1 antibody. As far as we know this is the first work reporting that betaine crosses the apical membrane of rat renal epithelium by SIT1 and by a Na+- and H+-independent transport system.Ministerio de Ciencia y Tecnología BFI2003-00222Junta de Andalucía 2010/BIO-14

Na+/Cl-/creatine transporter activity and expression in rat brain synaptosomes

Creatine is involved in brain ATP homeostasis and it may also act as neurotransmitter. Creatine transport was measured in synaptosomes obtained from the diencephalon and telencephalon of suckling and 2 month-old rats. Synaptosomes accumulate [14C]-creatine and this accumulation was Na+- and Cl--dependent and inhibited by high external K+. The latter suggests that the uptake process is electrogenic. The kinetic study revealed a Km for creatine of 8.7 μM. A 100-fold excess of either non-labelled creatine or guanidinopropionic acid abolished NaCl/creatine uptake, whereas GABA uptake was minimally modified, indicating a high substrate specificity of the creatine transporter. The levels of NaCl/creatine transporter (CRT) activity and those of the 4.2 kb CRT transcript (Northern's) were higher in the diencephalon than in the telencephalon, whereas the 2.7 kb transcript levels were similar in both brain regions and lower than those of the 4.2 kb. These observations suggest that the 4.2 kb transcript may code for the functional CRT. CRT activity and mRNA levels were similar in suckling and adult rats. To our knowledge the current results constitute the first description of the presence of a functional CRT in the axon terminal membrane that may serve to recapture the creatine released during the synapsis.Ministerio de Ciencia y Tecnología 2006-00720European Regional Development Fund (ERDF) BFU2007- 30688-E/BF

D-mannose transport and metabolism in isolated enterocytes

D-mannose transport and metabolism has been studied in enterocytes isolated from chicken small intestine. In the presence of Na+, the mannose taken up by the cells either remains free, is phosphorylated, is catabolized to H2O, or becomes part of membrane components. The mannose remaining free in the cytosol is released when the cells are transferred to an ice bath. The Na+-dependent D-mannose transport is electrogenic and inhibited by ouabain and dinitrophenol; its substrate specificity differs from SGLT-1 transporter. The Glut2 transporter inhibitors phloretin and cytochalasin B added following 30-min mannose uptake reduced the previously accumulated D-mannose, whereas these two agents increased the cell to external medium 3-O-methyl-glucose (3-OMG) concentration ratio. D-mannose efflux rate from preloaded D-[2-3H]-mannose enterocytes is Na+-independent. Phloretin did not affect D-mannose efflux rate, whereas it inhibited that of 3-OMG. Neither mannose uptake nor efflux rate were affected by fructose. It is concluded that part of the mannose taken up by the enterocytes is rapidly metabolized and that enterocytes have two D-mannose transport systems: one is concentrative and Na+-dependent and the other is Na+-independent and passive.Dirección General de Investiagación Científica y Técnica PM99-012

Small and large intestine express a truncated Dab1 isoform that assembles in cell-cell junctions and co-localizes with proteins involved in endocytosis

Disabled-1 (Dab1) is an essential intracellular adaptor protein in the reelin pathway. Our previous studies in mice intestine showed that Dab1 transmits the reelin signal to cytosolic signalling pathways. Here, we determine the Dab1 isoform expressed in rodent small and large intestine, its subcellular location and co-localization with clathrin, caveolin-1 and N-Wasp. PCR and sequencing analysis reveal that rodent small and large intestine express a Dab1 isoform that misses three (Y198, Y200 and Y220) of the five tyrosine phosphorylation sites present in brain Dab1 isoform (canonical) and contains nuclear localization and export signals. Western blot assays show that both, crypts, which shelter progenitor cells, and enterocytes express the same Dab1 isoform, suggesting that epithelial cell differentiation does not regulate intestinal generation of alternatively spliced Dab1 variants. They also reveal that the canonical and the intestinal Dab1 isoforms differ in their total degree of phosphorylation. Immunostaining assays show that in enterocytes Dab1 localizes at the apical and lateral membranes, apical vesicles, close to adherens junctions and desmosomes, as well as in the nucleus; co-localizes with clathrin and with N-Wasp but not with caveolin-1, and in Caco-2 cells Dab1 localizes at cell-to-cell junctions by a Ca2+-dependent process. In conclusion, the results indicate that in rodent intestine a truncated Dab1 variant transmits the reelin signal and may play a role in clathrin-mediated apical endocytosis and in the control of cell-to-cell junction assembly. A function of intestinal Dab1 variant as a nucleocytoplasmic shuttling protein is also inferred from its sequence and nuclear location.Junta de Andalucía CTS 5884Ministerio de Educación y Ciencia AP2007-04201European Molecular Biology Organization ASTF45-201

Ontogeny regulates creatine metabolism in rat small and large intestine

The ontogeny of intestinal CRT, AGAT and GAMT was investigated in foetuses, newborn, suckling, weaning and adult rats. In the colon, CRT mediates creatine transport because it was Na+- and Cl—dependent and inhibited by creatine and GPA. In addition, Northern assays showed two CRT transcripts (2.7-kb and 4.2-kb) and the in situ hybridisation revealed that CRT mRNA is restricted to the colon epithelial cells. The immunohistochemistry revealed that CRT protein was at the apical membrane of colon epithelia. Maturation decreased colonic CRT activity to undetectable levels and increased CRT mRNA abundance. Western assays revealed 57-, 65-, 80- and 116-kDa polypeptides at the intestinal apical membrane. The abundance of the 65-, 80- and 116-kDa polypeptides decreased with age, and that of 57-kDa was only observed in adult rats. The small and large intestine express AGAT and GAMT mRNAs. Maturation decreased AGAT mRNAabundance without affecting that of GAMT. For comparison, renal AGAT mRNAlevels were measured and they were increased with age. The study reports for the first time that: i) the apical membrane of rat colon have an active CRT, ii) development down-regulates CRT activity via post-transcriptional mechanism(s), iii) the intestine might synthesize creatine and iv) intestinal and renal creatine synthesis is ontogenically regulated at the level of AGAT gene expression

Lack of reelin modifies the gene expression in the small intestine of mice

We recently demonstrated that the mucosa of the small intestine of the rat expresses reelin and some components of its signaling system. The current study evaluates whether reelin affects the intestinal gene expression profile using microarray analysis and reeler mice, a natural mutant in which reelin is not expressed. The effect of the mutation on body weight and intestinal morphology is also evaluated. The mutation reduces body and intestinal weight during the first 2 months of age and modifies the morphology of the crypts and villi. For the microarray assays, total RNA was obtained from either isolated epithelial cells or intact small intestine. Of the 45,101 genes present in the microarray the mutation significantly alters the expression of 62 genes in the isolated epithelial cell samples and of 84 in the intact small intestine. The expression of 83% of the genes tested for validation was substantiated by reverse transcriptase polymerase chain reaction. The mutation notably up-regulates genes involved in intestinal metabolism, while it down-regulates genes related with immune response, inflammation, and tumor development. Genes involved in cell proliferation, differentiation, apoptosis, membrane transport and cytoskeleton are also differently expressed in the reeler mice as compared with the control. This is the first report showing that the lack of reelin modifies intestinal morphology and gene expression profile and suggests a role for reelin in intestinal epithelium homeostasis.Junta de Andalucía CTS 588

Reelin-Dab1 signaling system in human colorectal cancer

Reelin is an extracellular matrix protein that plays a critical role in neuronal migration. Here we show that the mucosa of human colon expresses reelin, its receptors ApoER2 and VLDLR, and its effector protein Dab1. Immunohistochemical analyses reveal that reelin expression is restricted to pericryptal myofibroblasts; Dab1 is detected at myofibroblasts, the apical domain of surface epithelial and crypt cells, and a strong linear staining is observed at the basement membrane; VLDLR and ApoER2 are in the cytoplasm of surface epithelium and myofibroblasts, and VLDLR is also detected in the cytoplasm of the crypt cells. Human colorectal cancer downregulates reelin without change in vimentin or N-cadherin mRNA levels. Decreased Reelin mRNA expression is accompanied by decreased HIC1 mRNA levels, increased mRNA levels of ApoER2 and DNMT1, increased reelin hypermethylation and no change in either Cask or TGF-β1 mRNAs, suggesting that reelin repression results from a DNMT1-mediated hypermethylation of the reelin gene promoter. Decreased HIC1 expression may repress reelin transcription via increasing ApoER2 transcription. We conclude that the mucosa of human colon expresses the reelin-Dab1 signaling system and that reelin is repressed in colorectal cancer before epithelial-mesenchymal transition has occurred. The significant down-regulation of reelin expression makes this gene a promising biomarker for colorectal cancers.Junta de Andalucía CTS 588

Hormonal Regulation of chicken intestinal NHE and SGLT-1 activities

The effects of aldosterone and arginine vasotocin (AVT) on intestinal Na/Hexchange (NHE) and Na-sugar cotransport (SGLT-1) activi-ties have been investigated using brush-border membrane ves-icles isolated from Hubbard chicken small and large intestines, and they were compared with those induced by either Nadepletion or dehydration. Na depletion was induced by feeding the chickens with either a low- or a high-Na diet for either 0.5, 1, 2, 4, or 8 days. Ileal and colonic NHE2 activity increased with the duration of the Na depletion, whereas that of intestinal SGLT-1 decreased, reaching a plateau after 2 days of treat-ment. Three-hour incubation of the intestine with aldosterone produced the same effects on NHE activity as does Na deple-tion, without altering SGLT-1 activity. However, 3-h incubation of the intestine with AVT increased intestinal SGLT-1 activity, without affecting intestinal NHE activity. It is concluded that aldosterone regulates apical ileal and colonic NHE2 activity, whereas that of SGLT-1 is regulated by AVT