M.L. Calonge

Human, rat and chicken small intestinal Na+‐Cl−‐creatine transporter: functional, molecular characterization and localization

In spite of all the fascinating properties of oral creatine supplementation, the mechanism(s) mediating its intestinal absorption has(have) not been investigated. The purpose of this study was to characterize intestinal creatine transport. [14C]Creatine uptake was measured in chicken enterocytes and rat ileum, and expression of the creatine transporter CRT was examined in human, rat and chicken small intestine by reverse transcription‐polymerase chain reaction, Northern blot, in situ hybridization, immunoblotting and immunohistochemistry. Results show that enterocytes accumulate creatine against its concentration gradient. This accumulation was electrogenic, Na+‐ and Cl−‐dependent, with a probable stoichiometry of 2 Na+: 1 Cl−: 1 creatine, and inhibited by ouabain and iodoacetic acid. The kinetic study revealed a Km for creatine of 29 μm. [14C]Creatine uptake was efficiently antagonized by non‐labelled creatine, guanidinopropionic acid and cyclocreatine. More distant structural analogues of creatine, such as GABA, choline, glycine, β‐alanine, taurine and betaine, had no effect on intestinal creatine uptake, indicating a high substrate specificity of the creatine transporter. Consistent with these functional data, messenger RNA for CRT was detected only in the cells lining the intestinal villus. The sequences of partial clones, and of the full‐length cDNA clone, isolated from human and rat small intestine were identical to previously cloned CRT cDNAs. Immunological analysis revealed that CRT protein was mainly associated with the apical membrane of the enterocytes. This study reports for the first time that mammalian and avian enterocytes express CRT along the villus, where it mediates high‐affinity, Na+‐ and Cl−‐dependent, apical creatine uptake.

Functional characterization of intestinal L-carnitine transport.

The carnitine transporter OCTN2 is responsible for the renal reabsorption of filtered L-carnitine. However, there is controversy regarding the intestinal L-carnitine transport mechanism(s). In this study, the characteristics of L-carnitine transport in both, isolated chicken enterocytes and brush-border membrane vesicles (BBMV) were studied. In situ hybridization was also performed in chicken small intestine. Chicken enterocytes maintain a steady-state L-carnitine gradient of 5 to 1 and 90% of the transported L-carnitine remains in a readily diffusive form. After 5 min, L-Carnitine uptake into BBMV overshot the equilibrium value by a factor of 2.5. Concentrative L-carnitine transport is Na+-, membrane voltage-and pH-dependent, has a high affinity for L-carnitine (Km 26 - 31 microM ) and a 1:1 Na+: L-carnitine stoichiometry. L-Carnitine uptake into either enterocytes or BBMV was inhibited by excess amount of cold L-carnitine > D-carnitine = acetyl-L-carnitine = gamma-butyrobetaine > palmitoyl-L-carnitine > betaine > TEA, whereas alanine, histidine, GABA or choline were without significant effect. In situ hybridization studies revealed that only the cells lining the intestinal villus expressed OCTN2 mRNA. This is the first demonstration of the operation of a Na+/L-carnitine cotransport system in the apical membrane of enterocytes. This transporter has properties similar to those of OCTN2.

OCTN3: A Na+-independent L-carnitine transporter in enterocytes basolateral membrane

L‐carnitine transport has been measured in enterocytes and basolateral membrane vesicles (BLMV) isolated from chicken intestinal epithelia. In the nominally Na+‐free conditions chicken enterocytes take up L‐carnitine until the cell to medium L‐carnitine ratio is 1. This uptake was inhibited by L‐carnitine, D‐carnitine, γ‐butyrobetaine, acetylcarnitine, tetraethylammonium (TEA), and betaine. L‐3H‐carnitine uptake into BLMV showed no overshoot, and it was (i) Na+‐independent, (ii) trans‐stimulated by intravesicular L‐carnitine, and (iii) cis‐inhibited by TEA and cold L‐carnitine. L‐3H‐carnitine efflux from L‐3H‐carnitine preloaded enterocytes was also Na+‐independent, and trans‐stimulated by L‐carnitine, D‐carnitine, γ‐butyrobetaine, acetylcarnitine, TEA, and betaine. Both, uptake and efflux of L‐carnitine were inhibited by verapamil and unaffected by either extracellular pH or palmitoyl‐L‐carnitine. RT‐PCR with specific primers for the mouse OCTN3 transporter revealed the existence of OCTN3 mRNA in mouse intestine, which was confirmed by in situ hybridization studies. Immunohystochemical analysis showed that OCTN3 protein was mainly associated with the basolateral membrane of rat and chicken enterocytes, whereas OCTN2 was detected at the apical membrane. In conclusion, the results demonstrate for the first time that (i) mammalian small intestine expresses OCTN3 mRNA along the villus and (ii) that OCTN3 protein is located in the basolateral membrane. They also suggest that OCTN3 could mediate the passive, Na+ and pH‐independent L‐carnitine transport activity measured in the three experimental conditions.

Na(+)-dependent D-mannose transport at the apical membrane of rat small intestine and kidney cortex.

The presence of a Na(+)/D-mannose cotransport activity in brush-border membrane vesicles (BBMV), isolated from either rat small intestine or rat kidney cortex, is examined. In the presence of an electrochemical Na(+) gradient, but not in its absence, D-mannose was transiently accumulated by the BBMV. D-Mannose uptake into the BBMV was energized by both the electrical membrane potential and the Na(+) chemical gradient. D-Mannose transport vs. external D-mannose concentration can be described by an equation that represents a superposition of a saturable component and another component that cannot be saturated up to 50 microM D-mannose. D-Mannose uptake was inhibited by D-mannose >> D-glucose>phlorizin, whereas for alpha-methyl glucopyranoside the order was D-glucose=phlorizin >> D-mannose. The initial rate of D-mannose uptake increased as the extravesicular Na(+) concentration increased, with a Hill coefficient of 1, suggesting that the Na(+):D-mannose cotransport stoichiometry is 1:1. It is concluded that both rat intestinal and renal apical membrane have a concentrative, saturable, electrogenic and Na(+)-dependent D-mannose transport mechanism, which is different from SGLT1.

Effect of dehydration on apical Na+-H+ exchange activity and Na+-dependent sugar transport in brush-border membrane vesicles isolated from chick intestine

Abstract The current work examines the effect of 4 days of water deprivation on Na+-H+ exchange and Na+-sugar cotransport systems in brush-border membrane vesicles isolated from either the jejunum, ileum or the colon of the chick. Apical Na+-H+ exchange activity was evaluated by measuring the pH-gradient-dependent Na+ uptake. The contribution of the Na+-H+ exchangers NHE2 and NHE3 to total Na+-H+ exchange activity was evaluated from their sensitivity to the amiloride-related drug HOE694. Dehydration increased plasma aldosterone levels from 12 to 70 pg/ml and also the activities of both Na+-H+ exchange and Na+-dependent sugar transport in the three intestinal regions tested. Na+-independent sugar transport was not modified by 4 days of water deprivation. In the ileum and colon the increase in Na+-H+ exchange activity was due to an increase in NHE2 activity, whereas in the jejunum it was due to an increase in both NHE2 and NHE3. Since we have previously reported that chronic Na+ depletion increases plasma aldosterone levels and NHE2 activity in ileum and colon, decreased small and large intestine Na+-sugar cotransport activity and had no effect on jejunal apical Na+-H+ exchange activity, it can be concluded that: (1) aldosterone does not regulate intestinal Na+-dependent sugar transport, and (2) the regulation of jejunal Na+-H+ exchange activity differs from that of either the ileum or the colon.

Dab1 and reelin participate in a common signal pathway that controls intestinal crypt/villus unit dynamics

The myofibroblasts placed underneath the epithelium of the rodent small intestine express reelin, and the reelin absence modifies both the morphology and the cell renewal processes of the crypt–villus unit. In the developing central nervous system, the reelin effects are mediated by the disabled‐1 (Dab1) protein. The present work explores whether Dab1 mediates the reelin control of the crypt–villus unit dynamics by examining in the mouse small intestine the consequences of the absence of (i) Dab1 (scrambler mutation) on crypt–villus unit cell renewal processes and morphology and (ii) reelin (reeler mutation) on the intestinal expression of Dab1.

Na+-HCO3− cotransporter and intracellular pH regulation in chicken enterocytes

The current studies examine the presence of the Na+-HCO3− cotransporter in chicken enterocytes and its role in cytosolic pH (pHi) regulation. The pH-sensitive dye 2′,7′-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF) was used to monitor pHi. Under resting conditions, pHi was 7.25 in solutions buffered with bis(2-hydroxyethyl)-1-piperazine ethanesulphonic acid (HEPES) and 7.17 in those buffered with HCO3−. Removal of external Na+ decreased pHi and readdition of Na+ rapidly increased pHi towards the control values. These Na+-dependent changes were greater in HCO3−than in HEPES-buffered solutions. In HCO3−- free solutions the Na+-dependent changes in pHi were prevented by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and unaffected by 4,4′-diisothiocyanatostilbene disulphonic acid (H2-DIDS). In the presence of HCO3−, the Na+-induced changes in pHi were sensitive to both EIPA and H2-DIDS. In the presence of EIPA, cells partially recovered from a moderate acid load only when both Na+ and HCO3−were present. This pHi recovery, which was EIPA resistant, and dependent on Na+ and HCO3−, was inhibited by H2-DIDS and occurred at equal rates in both Cl−-containing and Cl−-free solutions. Kinetic analysis of the rate of HCO3−- and Na+- dependent pHi recovery from an acid load as a function of the Na+ concentration revealed first-order kinetics with a Michaelis constant, Km, of 11 mmol/l Na+. It is concluded that in HCO3/− buffered solutions both the Na+/H+ exchanger and the Na+-HCO3− cotransporter participate in setting the resting pHi in isolated chicken enterocytes and help the recovery from acid loads.

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‐[3H]‐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. J. Cell. Physiol. 223: 451–459, 2010.

Dab2, Megalin, Cubilin and Amnionless Receptor Complex Might Mediate Intestinal Endocytosis in the Suckling Rat

We previously proposed that Dab2 participates in the endocytosis of milk macromolecules in rat small intestine. Here we investigate the receptors that may mediate this endocytosis by studying the effects of age and diet on megalin, VLDLR, and ApoER2 expression, and that of age on the expression of cubilin and amnionless. Of megalin, VLDLR and ApoER2, only the megalin expression pattern resembles that of Dab2 previously reported. Thus the mRNA and protein levels of megalin and Dab2 are high in the intestine of the suckling rat, down‐regulated by age and up‐regulated by milk diet, mainly in the ileum. Neither age nor diet affect ApoER2 mRNA levels. The effect of age on VLDLR mRNA levels depends on the epithelial cell tested but they are down‐regulated by milk diet. In the suckling rat, the intestinal expressions of both cubilin and amnionless are similar to that of megalin and megalin, cubilin, amnionless and Dab2 co‐localize at the microvilli and in the apical endocytic apparatus. Co‐localization of Dab2 with ApoER2 and VLDLR at the microvilli and in the apical endocytic apparatus is also observed. This is the first report showing intestinal co‐localization of: megalin/cubilin/amnionless/Dab2, VLDLR/Dab2 and ApoER2/Dab2. We conclude that the megalin/cubilin/amnionless/Dab2 complex/es participate in intestinal processes, mainly during the lactation period and that Dab2 may act as an adaptor in intestinal processes mediated by ApoER2 and VLDLR. J. Cell. Biochem. 115: 510–522, 2014.

Ontogeny of Na+/l-carnitine transporter and of γ-trimethylaminobutyraldehyde dehydrogenase and γ-butyrobetaine hydroxylase genes expression in rat kidney

The kidney synthesizes L-carnitine and reabsorbs it via the Na(+)/L-carnitine cotransporter OCTN2. This study investigates the ontogeny of OCTN2, gamma-trimethylaminobutyraldehyde dehydrogenase (TMABA-DH) and gamma-butyrobetaine hydroxylase (BBH) in rat kidneys. Foetuses, newborn, suckling, weaning and adult rats were used. The apical membranes of foetal and newborn rat kidneys express OCTN2 transport activity, which is up-regulated by age. Maturation significantly increased the V(max) of this transport system without changing the apparent K(t), which excludes a maturation-related expression of different transporter isoforms. Northern analysis showed a 3.7kb transcript for OCTN2 in all the ages tested. Northern and RT-PCR assays revealed that maturation increased renal expression of OCTN2 mRNA. Foetuses express TMABA-DH mRNA and this expression increased during postnatal life. BBH mRNA, however, was detected during the suckling period onwards and its abundance was not changed significantly by maturation. This study reports for the first time that, in rat kidneys: (i) an apical OCTN2 transporter is active in rat foetuses, (ii) ontogeny up-regulates OCTN2 activity by increasing the density and/or turnover of the transporters, (iii) the maturation-related changes in OCTN2 are in part mediated by transcriptional mechanism(s) and (iv) the expression of both, TMABA-DH and BBH mRNA is ontogenically regulated. Some of these results were published as an abstract (García-Delgado et al., 2003).