María J. Peral

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(+)/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 [(14)C]-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 K(m) for creatine of 8.7 microM. 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 (Northerns) 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.

Rat small intestine expresses the reelin–Disabled‐1 signalling pathway

Expression of reelin, reelin receptors [apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (VldlR)] and the Disabled‐1 (Dab1) protein was investigated in rat intestinal mucosa. Intestinal reelin and Dab1 mRNA levels were maximal in the early stages of life, reaching adult levels in 1‐month‐old rats. Expression of reelin mRNA was restricted to fibroblasts, whereas mRNAs of Dab1, ApoER2, VldlR and integrins α3 and β1 were observed in enterocytes, crypts and fibroblasts. Reelin protein was only observed in isolated intestinal fibroblasts and in a cell layer subjacent to the villus epithelium, which seems to be composed of myofibroblasts because it also reacted to α‐smooth muscle actin. The Disabled‐1 and VldlR protein signals were detected in the crypt and villus cells, and they were particularly abundant in the terminal web domain of the enterocytes. The ApoER2 protein signal was detected in the upper half of the villi but not in the crypts. This is the first report showing that rat intestinal mucosa expresses the reelin–Disabled‐1 signalling system.

Developmental decrease in rat small intestinal creatine uptake

Phosphocreatine is an energy buffer and transducer in the heart, the brain and the skeletal muscle. Recently, we have demonstrated the presence of the Na+/Cl-/creatine transporter at the apical membrane of the small intestinal epithelium. Herein the ontogeny and segmental distribution of rat intestinal creatine transport activity are investigated. [14C]-Creatine uptake was measured in the jejunum and ileum of 16 day gestation foetuses, newborn, suckling, weaning, 1-, 2-, 7- and 12-month-old (adult) rats. Creatine content in amniotic fluid, in rat and commercial milk and in rat chow, was measured by HPLC. NaCl-dependent creatine uptake was maximal in newborn rats and, in all the ages tested, higher in the ileum than in the jejunum. In the latter, NaCl-dependent creatine uptake was undetectable after weaning. Kinetic studies revealed that the jejunum and ileum have the same creatine uptake system, and that maturation decreases its Vmax but not the apparent Km. Maintenance of the pups on a commercial milk diet supplemented with creatine prevented the ileal periweaning decline in creatine uptake activity, but not that in the jejunum. In 1-month-old rats, supplementation with creatine increased ileal, but not jejunal, creatine uptake. The results demonstrate for the first time that: (i) creatine uptake along the length of the small intestine is mediated by the same transport system, (ii) the activity of this transport system changes in a specific manner with maturation and (iii) these changes appear to be genetically programmed and controlled by the intestinal creatine content.

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.

Developmental maturation and segmental distribution of rat small intestinal L-carnitine uptake.

Oral L-carnitine supplementation is commonly used in sports nutrition and in medicine; however, there is controversy regarding the mechanisms that mediate intestinal L-carnitine transport. We have previously reported that the Na+/L-carnitine transporter OCTN2 is present in the small intestinal apical membrane. Herein we aimed to find out if this step of intestinal L-carnitine absorption is ontogenically regulated, and if so, to determine the molecular mechanism(s) involved. L-[3H]-Carnitine uptake was measured in the jejunum and ileum of fetuses (E17 and E21), newborn (1 day-old), suckling (15 day-old), weaning (1 month-old) and adult (2 and 6 month-old) Wistar rats. Both, Na+-dependent and Na+-independent L-carnitine uptake rates, normalized to intestinal weight, significantly increased during the late gestation period, and then declined during the suckling period. After weaning, the rate of Na+-dependent L-carnitine uptake is no longer measurable. In E21- fetuses and newborn rats, L-carnitine uptake was higher in the ileum than in the jejunum. The decline in Na+-dependent L-carnitine uptake with maturation was mediated via a decrease in the Vmax of the uptake process with no change in its apparent Km. Semi-quantitative RT-PCR assays showed that OCTN2 mRNA levels were significantly higher in E21-fetuses and newborn rats compared to suckling rats, which were in turn significantly higher than that in adult rats. Neither retardation of weaning nor L-carnitine supplementation prevented the down-regulation of Na+/L-carnitine transport activity. The results demonstrate for the first time that intestinal Na+-dependent L-carnitine uptake activity is under genetic regulation at the transcriptional level.

Prolonged ethanol ingestion increases renal AQP2 and AQP3 expression in adult rats and in their offspring.

This study evaluates the effect of prolonged ethanol ingestion on the renal ability to concentrate urine. Suckling Wistar rats born to mothers given ethanol before and during gestation and suckling periods (ethanol-exposed offspring) were used and the results were compared with those obtained from offspring of dams given diets containing no ethanol. Comparisons were also made between progenitors with or without prolonged ethanol ingestion. Body and kidney weights; arginine-vasopressin (AVP) and aldosterone plasma levels; plasma, urine and renal papillary osmolality; urine outflow; kidney AQP2, AQP3 and AQP4 expression and diencephalon AVP mRNA expression were determined. As compared with control offspring, the ethanol-exposed offspring present i) lower body and kidney weights; ii) lower urine outflow; iii) higher renal AQP2 and AQP3 mRNA; iv) higher renal AQP2 protein content and v) higher urine and renal papillary osmolality. These changes were also observed in the ethanol-treated progenitors, although they were of smaller magnitude. Plasma osmolality, renal AQP4 mRNA, AVP plasma levels and diencephalon AVP mRNA expression were not affected by the ethanol treatment. Plasma levels of aldosterone were only significantly increased in the ethanol-exposed suckling rats. It is concluded that maternal ethanol ingestion before and during gestation and suckling periods affects the renal function of the offspring, up-regulating renal AQP2 expression by an AVP-independent mechanism. Ethanol-treated progenitors manifest similar renal changes, although of lesser magnitude than the offspring.

Proton conductance and intracellular pH recovery from an acid load in chicken enterocytes.

1. Chicken enterocytes present a Na(+)‐independent proton transport mechanism involved in pHi recovery from an acid load. In the current study the nature of this proton transport system is investigated. 2. The pHi of acid‐loaded cells increased when transferred to Na(+)‐free, pH 7.4 buffers, both at 6 and 65 mM extracellular potassium concentration. Addition of nigericin accelerated the rate of cell alkalinization. 3. When acid‐loaded cells were transferred to a Na(+)‐free, pH 6.5 buffer, the cells acidified further, regardless of the extracellular potassium concentration. The addition of nigericin increased the rate of acidification at 6 mM K+ but produced an alkalinization at 65 mM K+. 4. The rate of the Na(+)‐independent regulatory cell alkalinization was inhibited by SCH 28080, DCCD, NBD‐Cl, rotenone or Zn2+. Addition of valinomycin reversed the inhibition induced by SCH 28080, DCCD and NBD‐Cl but not that induced by Zn2+ or rotenone. Zn2+ inhibition was abolished by the metal chelator DTPA. 5. Cytosolic acidification increased the rate of Na(+)‐independent regulatory cell alkalinization. 6. The results suggest that the Na(+)‐independent proton transport system is a Zn(2+)‐sensitive proton‐conducting pathway which is regulated by the cytosolic proton concentration.