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.

Expression of OCTN2 and OCTN3 in the apical membrane of rat renal cortex and medulla.

Immunological assays and transport measurements in apical membrane vesicles revealed that the apical membrane of rat kidney cortex and medulla presents OCTN2 and OCTN3 proteins and transports L-[(3)H]-carnitine in a Na(+)-dependent and -independent manner. OCTN2 mediates the Na(+)/L-carnitine transport activity measured in medulla because (i) the transport showed the same characteristics as the cortical Na(+)/L-carnitine transporter and (ii) the medulla expressed OCTN2 mRNA and protein. The Na(+)-independent L-carnitine transport activity appears to be mediated by both OCTN2 and OCTN3 since: (i) Na(+)-independent L-carnitine uptake was inhibited by both, anti-OCTN2 and anti-OCTN3 antibodies, (ii) kinetics studies revealed the involvement of a high- and a low-affinity transport systems, and (iii) Western and immunohistochemistry studies revealed that OCTN3 protein is located at the apical membrane of the kidney epithelia. The Na(+)-independent L-carnitine uptake exhibited trans-stimulation by intravesicular L-carnitine or betaine. This trans-stimulation was inhibited by anti-OCTN3 antibody, but not by anti-OCTN2 antibody, indicating that OCTN3 can function as an L-carnitine/organic compound exchanger. This is the first report showing a functional apical OCTN2 in the renal medulla and a functional apical OCTN3 in both renal cortex and medulla.

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 mRNA abundance without affecting that of GAMT. For comparison, renal AGAT mRNA levels 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.

A Na+-dependent D-mannose transporter in the apical membrane of chicken small intestine epithelial cells.

The presence of a Na+/D-mannose cotransporter in brush-border membrane vesicles (BBMV) isolated from chicken small intestine was examined. In the presence of an electrochemical gradient for Na+, but not in its absence, D-mannose was accumulated transiently by the BBMV. D-Mannose uptake into the BBMV was energized by both the membrane potential and the chemical gradient for Na+. The relationship between D-mannose transport and external D-mannose concentration was described by an equation that represented the superposition of a saturable component (Michaelis-Menten constant Km 12.5 microM) and another component unsaturatable up to 80 microM D-mannose. D-Mannose uptake was inhibited by various substances in the following order of potency: D-mannose>>D-glucose>phlorizin>phloretin>D-fructose. For the uptake of alpha-methyl-glucopyranoside the order was D-glucose=phlorizin>>phloretin=D-fructose=D-mannose. The initial rate of D-mannose uptake increased as the extravesicular [Na+] increased, with a Hill coefficient of 1, suggesting that the Na+:D-mannose cotransport stoichiometry is 1:1. It is concluded that the intestinal apical membrane has a saturable, electrogenic and concentration- and Na+-dependent mannose transport mechanism that differs from the sodium-dependent glucose transporter SGLT1.

K+ transport in colonocytes isolated from the chick: effect of anisosmotic buffers.

Potassium transport was measured in isolated chicken colonocytes using 85Rb+ as a tracer for K+. Rb+ was determined by atomic absorption spectrometry. The results revealed that net K+ uptake occurred via at least four mechanisms: (i) Na+,K(+)-ATPase, (ii) K(+)-ATPase, (iii) Na(+)-K(+)-2Cl- cotransport system and (iv) a mechanism(s) which is resistant to both ouabain and bumetanide. The rate of K+(Rb+) efflux is stimulated by the calcium ionophore A23187, inhibited by either quinine, verapamil or Ba2+, and unaffected by either apamin, 3,4-diaminopyridine (3,4-DAP), H2-DIDS or bumetanide. The A23187-induced increase in K+(Rb+) efflux was abolished by apamin. These findings suggest that K+(Rb+) efflux from chicken colonocytes occurs at least in part through Ca(2+)-activated K+ channels. The present results also show that all these K+ transport systems are involved in cell volume regulation. Thus, external hyposmolarity decreased net K+(Rb+) uptake mediated by Na+,K(+)-ATPase, K(+)-ATPase and the Na(+)-K(+)-2Cl- cotransporter and increased K+(Rb+) efflux rate. The opposite was observed under hyperosmotic conditions.

Thyroid hormone regulation of the Na+/glucose cotransporter SGLT1 in Caco-2 cells.

The expression of the Na+/glucose cotransporter (SGLT1) in response to thyroid hormone [3,5,3'-tri-iodo-l-thyronine (T3)] was investigated in the enterocytic model cell line Caco-2/TC7. In differentiated cells, T3 treatment induces an average 10-fold increase in glucose consumption as well as a T3 dose-dependent increase in SGLT1 mRNA abundance. Only cells grown on glucose-containing media, but not on the non-metabolizable glucose analogue alpha-methylglucose (AMG), could respond to T3-treatment. The Vmax parameter of AMG transport was enhanced 6-fold by T3 treatment, whereas the protein abundance of SGLT1 was unchanged. The role of Na+ recycling in the T3-related activation of SGLT1 activity was suggested by both the large increase in Na+/K+ATPase protein abundance and the inhibition, down to control levels, of AMG uptake in ouabain-treated cells. Further investigations aimed at identifying the presence of a second cotransporter that could be expressed erroneously in the colon cancer cell line were unsuccessful: T3-treatment did not modify the sugar-specificity profile of AMG transport and did not induce the expression of SGLT2 as assessed by reverse transcription-PCR. Our results show that T3 can stimulate the SGLT1 cotransport activity in Caco-2 cells. Both transcriptional and translational levels of regulation are involved. Finally, glucose metabolism is required for SGLT1 expression, a result that contrasts with the in vivo situation and may be related to the fetal phenotype of the cells.

Apical ouabain-sensitive K+-activated-ATPase activity in colon and caecum of the chick.

This study sought to investigate the presence and characteristics of K+-ATPase activity in chicken intestinal epithelia. A cytochemical method revealed Na+-independent, ouabain-sensitive, K+-ATPase activity in the apical, but not in the basolateral, membrane of chicken colonic and caecal epithelial cells. K+-ATPase activity was not observed in the small intestine. The measurement of K+-activated pNPPase activity was used to characterize the K+-ATPase activity evidenced by the cytochemical method. In addition, K+ and NH4+, but neither Na+ nor Li+, could activate pNPPase activity in chicken intestinal epithelia. Vanadate abolished ouabain-sensitive, K+-activated pNPPase activity in the three membrane preparations tested, whereas oligomycin and SCH 28080 were without effect. The Km for K+ and the ouabain IC50 values for the apical colonic and caecal K+-activated pNPPase activity were higher than those measured for K+-activated pNPPase activity measured in the basolateral membrane of chicken jejunal enterocytes. The results indicate that the apical membranes of chicken colon and caecum possess Na+-independent, ouabain-sensitive K+-activated-ATPase activity.

PKC activators stimulate H+ conductance in chicken enterocytes.

Chicken enterocytes present a H+-conducting pathway involved in the recovery of intracellular pH (pHi) from an acid load. In the current study we have tested the effect of protein kinase C (PKC) activators on the rate of proton efflux through the H+-conducting pathway. The rate of proton efflux was increased by the addition of 1,2-dioctanoyl-rac-glycerol (DOG) or phorbol 12-myristate 13-acetate (PMA), but it was not affected by the addition of the inactive phorbol ester analogue, 4alpha-phorbol 12, 13-didecanoate. DOG stimulated the process in a dose-dependent manner with a half-maximal effect at 45 microM. Staurosporine and Zn2+ prevented the DOG-dependent increase in the rate of proton efflux. The rate of proton efflux was affected by the pH, and DOG shifted this relationship upward and to the right. These results suggest that the proton-conducting pathway is regulated by PKC.

Glycyl-L-sarcosine transport by ATP-depleted isolated enterocytes from chicks.

Normally energized and ATP-depleted isolated chicken enterocytes have been used to investigate the energetics of intestinal glycyl-L-sarcosine (Gly-Sar) transport, and the results were compared with those obtained for 3-O-methyl-D-glucose (3-OMG) active transport, which is known to be energized by the electrochemical Na+ gradient. The results show that even though Gly-Sar had no effect on Na+ efflux from labeled (22Na) enterocytes, 20 mM L-leucine and 20 mM 3-OMG increased the rate constant of Na+ efflux by 34 and 76%, respectively. In ATP-depleted cells 3-OMG was accumulated in response to experimentally imposed inward Na+ gradients; however, Gly-Sar was not accumulated. In the absence of Na+, an inward proton gradient slightly increased Gly-Sar uptake, although no transient accumulation was observed. An increase in membrane potential by replacement of Cl- by NO3- or by imposition of an outward K+ gradient in the presence of valinomycin stimulated both 3-OMG and Gly-Sar uptake, even in the absence of either H+ or Na+ gradient. When in addition to a K+ diffusion potential, a Na+ gradient was present, 3-OMG transient accumulation was further increased (approximately 2-fold over equilibrium value), and Gly-Sar uptake showed the same pattern as that observed when only membrane potential was present. However, in the presence of an outward K+ diffusion potential and an inward H+ gradient Gly-Sar was stimulated approximately threefold, clearly showing a significant overshoot, whereas 3-OMG was not transiently accumulated.

Role of rat large intestine in reducing diarrhea after 50% or 80% distal small bowel resection.

The effects of intestinal resection on several intestinal parameters have been studied in the large intestine of rats one month after the surgical operation. The results show that both 50% and 80% distal small bowel resection increased net fluid absorption and mucosal permeability and caused expansion of the intercellular spaces of the large intestine. The increase in net fluid absorption was dependent upon the extent of the intestine removed. The cAMP and cGMP content of cecal and colonic mucosa were significantly reduced after jejunoilectomy. Changes in nucleotide levels were dependent on the length of the intestine resected. On the other hand, mucosa Na,K-ATPase specific activity was only increased in the cecum after 80% intestinal resection. The results are discussed in terms of adaptation to prevent diarrhea.

Effects of anisosmotic buffers on K+ transport in isolated chicken enterocytes.

Cells isolated by hyaluronidase incubation from chicken small intestine were used to study the effects of anisosmotic buffers on K+ transport. Hypo-osmolarity (200 mosmol.l-1) reduced both the ouabain-sensitive and the ouabain-resistant, but bumetanide-sensitive, net K+ influx and increased the K+ efflux. The hypo-osmolarity induced K+ efflux was prevented by quinine and unaffected by bumetanide. These results suggest that Ca2+-activated K+ channels may be involved in regulatory volume decrease in chicken enterocytes. Hyperosmotic conditions (400 mosmol.l-1) increased the portion of net K+ influx mediated by the Na+/K+-ATPase and that mediated by the bumetanide-sensitive K+ transport system, and decreased the K+ efflux.

Potassium transport in epithelial cells isolated from small intestine of the chicken.

The transport of potassium has been studied in epithelial cells isolated from chicken small intestine using 86Rb as a tracer for K+. (i) The uptake studies revealed that about 60% of the total K+ net flux is inhibited by ouabain and therefore mediated by the Na+-K+-ATPase. About 20% of the ouabain-insensitive K+ net influx was inhibited by furosemide, bumetanide and by either Na+ or Cl- removal from the incubation solution, suggesting that a Na+/Cl-/K+ cotransport system might be present in chicken enterocytes. (ii) The efflux of K+ was measured from cells preloaded with 86Rb. K+ efflux was inhibited by Ba2+, quinine and verapamil; it was stimulated by A23187, and it was unaffected by 3,4-diaminopyridine. Apamin, that has no effect on basal rates of K+ efflux, abolished the effect of A23187. These findings suggest that K+ efflux appears to occur through Ca2+-activated K+ channels.

Adaptation of electrolytes and fluid transport in rat small and large intestine after distal small bowel resection.

Na+, Cl- and water transport were studied in jejunum, caecum and colon after either 50% or 80% of small bowel resection (SBR). Four weeks after surgery, dry and wet weights, net absorption in vivo of sodium, chloride and water were determined. There was a significant intestinal growth after 50% or 80% SBR except for the colon which only showed increased tissue mass after 80% SBR. Net transport was stimulated both, per organ and per unit mass. In the small intestine and caecum both organ growth and changes in cell function appear to be involved in the adaptive response, regardless the extent of the small intestine resected. In the colon, compensatory growth appear to contribute to the adaptive response only after 80% SBR, whilst the transport function of the colonocytes seems to be stimulated after both types of SBR.