ABSTRACT
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.
Subject(s)
Cell Adhesion Molecules, Neuronal/deficiency , Extracellular Matrix Proteins/deficiency , Intestine, Small/metabolism , Nerve Tissue Proteins/deficiency , Serine Endopeptidases/deficiency , Transcriptome , Animals , Cell Adhesion Molecules, Neuronal/genetics , Extracellular Matrix Proteins/genetics , Gene Expression Profiling , Intestinal Mucosa/metabolism , Intestine, Small/anatomy & histology , Mice , Mice, Inbred C57BL , Nerve Tissue Proteins/genetics , Oligonucleotide Array Sequence Analysis , Organ Size , Reelin Protein , Serine Endopeptidases/geneticsABSTRACT
The current work investigates whether creatine metabolism is involved in renal adaptation to dehydration. Wistar rats were either deprived of water or induced to drink water abundantly during 60 h. Cortical and medullar mRNA levels of Na(+)/Cl(-)/creatine transporter (CRT), l-arginine: glycine amidinotransferase (AGAT), guanidinoacetate methyltransferase (GAMT) and of the tonicity sensitive genes coding for aquaporin 2, Na(+)/Cl(-)/betaine transporter and glucocorticoid-inducible kinase were measured by real-time PCR assays. The activity of the CRT and that of Na(+)/alpha-methyl-glucose transporter were evaluated in renal brush-border membrane vesicles. In water loaded animals, the mRNA levels of AGAT and CRT, and the activity of the CRT were greater in the cortex than in the medulla. GAMT mRNA levels were of similar magnitude and lower than those of AGAT mRNA. Dehydration decreased cortical and medullar AGAT and CRT mRNA levels and CRT activity and it did no affect GAMT mRNA abundance. These decreases were creatine specific because dehydration increased Na(+)/alpha-methyl-glucose transporter activity and the mRNA abundance of aquaporin 2, Na(+)/Cl(-)/betaine transporter and glucocorticoid-inducible kinase. In conclusion, this is the first report showing that: i) the kidneys express significant amounts of GAMT mRNA, ii) dehydration down-regulates the expression of AGAT gene and iii) dehydration down-regulates CRT gene expression and activity.
Subject(s)
Antidiuretic Agents/pharmacology , Creatine/metabolism , Kidney/drug effects , Kidney/metabolism , Amidinotransferases/biosynthesis , Amidinotransferases/genetics , Amidinotransferases/metabolism , Animals , Creatine/blood , Creatine/urine , Drinking/drug effects , Drinking/physiology , Guanidinoacetate N-Methyltransferase/biosynthesis , Guanidinoacetate N-Methyltransferase/genetics , Guanidinoacetate N-Methyltransferase/metabolism , Kidney Medulla/drug effects , Kidney Medulla/metabolism , Male , Rats , Rats, WistarABSTRACT
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.
Subject(s)
Carnitine/metabolism , Cell Membrane/metabolism , Epithelial Cells/metabolism , Kidney Tubules/metabolism , Organic Cation Transport Proteins/metabolism , Animals , Antibodies, Blocking/pharmacology , Betaine/metabolism , Biological Transport, Active/physiology , Cell Membrane/ultrastructure , Epithelial Cells/ultrastructure , Immunohistochemistry , Kidney Tubules/ultrastructure , Male , Mice , Organic Cation Transport Proteins/antagonists & inhibitors , Organic Cation Transport Proteins/genetics , RNA, Messenger/metabolism , Rats , Rats, Wistar , Sodium/metabolism , Solute Carrier Family 22 Member 5 , Transport Vesicles/metabolism , Transport Vesicles/ultrastructureABSTRACT
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 alpha3 and beta1 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 alpha-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.
Subject(s)
Adaptor Proteins, Signal Transducing/biosynthesis , Cell Adhesion Molecules, Neuronal/biosynthesis , Extracellular Matrix Proteins/biosynthesis , Intestine, Small/metabolism , Nerve Tissue Proteins/biosynthesis , Receptors, Lipoprotein/biosynthesis , Serine Endopeptidases/biosynthesis , Animals , Caco-2 Cells , Humans , Intestinal Mucosa/metabolism , Intestine, Small/growth & development , LDL-Receptor Related Proteins , RNA, Messenger/metabolism , Rats , Receptors, LDL/biosynthesis , Reelin Protein , Signal TransductionABSTRACT
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 (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.
Subject(s)
Chlorides/metabolism , Diencephalon/metabolism , Membrane Transport Proteins/biosynthesis , Sodium/metabolism , Synaptosomes/metabolism , Telencephalon/metabolism , Age Factors , Animals , Animals, Suckling , Biological Transport , Cell Membrane/metabolism , Creatine/metabolism , Kinetics , Membrane Potentials , Membrane Transport Proteins/genetics , Osmolar Concentration , RNA, Messenger/biosynthesis , Rats , Rats, WistarABSTRACT
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.
Subject(s)
Aging/metabolism , Creatine/metabolism , Intestine, Large/metabolism , Intestine, Small/metabolism , Amidinotransferases/biosynthesis , Animals , Animals, Newborn , Animals, Suckling , Blotting, Northern , Blotting, Western , Creatine/administration & dosage , Creatine/pharmacokinetics , Energy Metabolism , Guanidinoacetate N-Methyltransferase/biosynthesis , Immunohistochemistry , Intestinal Absorption , Intestine, Large/embryology , Intestine, Large/growth & development , Intestine, Small/embryology , Intestine, Small/growth & development , Membrane Transport Proteins/biosynthesis , Rats , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Creatine plays a role in energy storage and transport/shuttle of high-energy phosphate in heart, brain, retina, testis and skeletal muscle. These tissues take creatine from the plasma via a 2Na(+)/1Cl(-)/1creatine cotransporter (CRT). We have previously demonstrated that renal apical membrane presents a 2Na(+)/1Cl(-)/1creatine cotransport activity. The goal of this study was to determine whether this transporter is ontogenically regulated. Na(+)/Cl(-)/creatine transport activity was evaluated by measuring [(14)C]-creatine uptake into renal brush-border membrane vesicles. CRT mRNA expression was measured by Northern and real-time PCR assays. E20 foetuses, newborn, suckling, weaning and adult (2- and 8-month-old) Wistar rats were used. The results revealed that neither the vesicular volume, the binding of creatine to the brush-border membrane vesicles, nor the purity of the brush-border membrane vesicle preparations was affected by maturation. Fetal and neonatal kidneys contained a creatine transporter that was qualitatively indistinguishable from that in the adult: it was concentrative, Na(+)- and Cl(-)-dependent, electrogenic and inhibited by guanidinopropionic acid. Maturation increased this transport activity by increasing the maximal rate of transport (V(max)) without significantly changing the apparent K(m). Northern analysis revealed two transcripts for CRT of 2.7 kb and 4.2 kb in all the ages tested. Northern and real-time PCR assays showed that, as seen with NaCl-dependent creatine transport activity, maturation increased CRT mRNA expression. This study reports for the first time that: (i) an apical renal Na(+)/Cl(-)/creatine cotransporter is already active in rat foetuses and (ii) development regulates Na(+)/Cl(-)/creatine cotransport activity by increasing the density and/or turnover of the transporters.
Subject(s)
Chlorides/metabolism , Creatine/metabolism , Kidney/metabolism , Membrane Transport Proteins/metabolism , Sodium/metabolism , Age Factors , Animals , Animals, Newborn , Guanidines/pharmacology , Kidney/ultrastructure , Membrane Potentials , Microvilli/metabolism , Polymerase Chain Reaction , Propionates/pharmacology , Rats , Rats, Wistar , Up-RegulationABSTRACT
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.
Subject(s)
Aging/metabolism , Creatine/administration & dosage , Ileum/metabolism , Jejunum/metabolism , Animals , Biological Transport, Active/physiology , Creatine/metabolism , Rats , Rats, WistarABSTRACT
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-[(3)H]-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 V(max) of the uptake process with no change in its apparent K(m). 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.
Subject(s)
Aging/metabolism , Carnitine/pharmacokinetics , Intestine, Small/embryology , Intestine, Small/metabolism , Organic Cation Transport Proteins/metabolism , Sodium/metabolism , Administration, Oral , Animals , Animals, Newborn , Carnitine/administration & dosage , Metabolic Clearance Rate , Rats , Rats, Wistar , Solute Carrier Family 22 Member 5 , Tissue DistributionABSTRACT
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, gamma-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, gamma-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.
Subject(s)
Carnitine/metabolism , Cell Membrane/metabolism , Enterocytes/metabolism , Membrane Proteins/metabolism , Organic Cation Transport Proteins/metabolism , Sodium/metabolism , Animals , Biological Transport, Active , Cell Polarity , Chickens , Enterocytes/cytology , Intestine, Small/cytology , Intestine, Small/metabolism , Membrane Proteins/genetics , Mice , Organic Cation Transport Proteins/genetics , Radioligand Assay , Rats , Transport Vesicles/metabolismABSTRACT
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.
Subject(s)
Aquaporins/metabolism , Central Nervous System Depressants/toxicity , Ethanol/toxicity , Fetus/drug effects , Kidney/drug effects , Kidney/metabolism , Aldosterone/blood , Animals , Aquaporin 2 , Aquaporin 3 , Aquaporin 4 , Aquaporins/genetics , Arginine Vasopressin/blood , Body Weight/drug effects , Diencephalon/metabolism , Female , Male , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, WistarABSTRACT
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. [(14)C] 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 K(m) for creatine of 29 microM. [(14)C] creatine uptake was efficiently antagonized by non-labelled creatine, guanidinopropionic acid and cyclocreatine. More distant structural analogues of creatine, such as GABA, choline, glycine, beta-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.
Subject(s)
Chlorides/metabolism , Intestine, Small/metabolism , Membrane Transport Proteins/metabolism , Sodium/metabolism , Animals , Blotting, Northern , Blotting, Western , Chickens , Chlorides/pharmacology , Cloning, Molecular , Creatine/pharmacokinetics , DNA, Complementary/genetics , Energy Metabolism , Enterocytes/metabolism , Humans , Ilium/metabolism , Immunohistochemistry , In Situ Hybridization , Kinetics , Male , Membrane Potentials/physiology , Membrane Transport Proteins/genetics , Ouabain/pharmacology , Rats , Rats, Wistar , Sodium/pharmacology , Time Factors , Tissue DistributionABSTRACT
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.
Subject(s)
Biological Transport, Active/physiology , Carnitine/pharmacokinetics , Enterocytes/metabolism , Intestine, Small/metabolism , Microvilli/metabolism , Organic Cation Transport Proteins , Animals , Biological Transport, Active/drug effects , Carrier Proteins , Cell-Free System , Cells, Cultured , Chickens , Enterocytes/chemistry , Enterocytes/cytology , Enterocytes/drug effects , Hydrogen-Ion Concentration , In Situ Hybridization, Fluorescence/methods , Intestine, Small/chemistry , Intestine, Small/cytology , Intestine, Small/drug effects , Membrane Potentials/physiology , Membrane Proteins , Microvilli/chemistry , Microvilli/drug effects , Microvilli/ultrastructure , Models, Biological , Molecular Probe Techniques , Protein Binding , Sensitivity and Specificity , Sodium/pharmacology , Solute Carrier Family 22 Member 5 , Temperature , Transport Vesicles/metabolismABSTRACT
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.
Subject(s)
Intestinal Mucosa/physiology , Intestine, Small/physiology , Kidney Cortex/physiology , Mannose/metabolism , Microvilli/physiology , Sodium/metabolism , Animals , Biological Transport , Cell Membrane/metabolism , Cell Membrane/physiology , Jejunum/physiology , Kinetics , Male , Membrane Potentials , Methylglucosides/metabolism , Microvilli/metabolism , Osmolar Concentration , Rats , Rats, WistarABSTRACT
Malpighian tubules (MT) of Rhodnius prolixus transport fluid at very high rates. To identify whether aquaporins (AQPs) are present in the MT of R. prolixus, total ribonucleic acid (RNA) was isolated from MT and used in a reverse transcription, polymerase chain reaction (RT-PCR), with two degenerate primers to highly conserved regions of the members of the AQPs family. A deoxyribonucleic acid (DNA) fragment of 370 bp was amplified; its sequence revealed a novel protein, representing a new member of the major intrinsic protein (MIP) family. The complementary DNA (cDNA) sequence of this new MIP protein was cloned by using RNA from MT and the rapid amplification of cDNA ends (RACE) technique. The cDNA had 1133 bp and the largest open reading frame coded for a protein of 286 amino acids, named R. prolixus major intrinsic protein (Rp-MIP). The hydrophobicity profile of the amino acid sequence predicts six transmembrane domains. Northern blot analysis of MT RNA showed a single transcript of about 1-1.3 kb for Rp-MIP. RT-PCR of single isolated MT and in situ hybridization analysis showed Rp-MIP transcripts in both proximal and distal segments. Expression of Rp-MIP in Xenopus laevis oocytes doubled the osmotic water permeability Pf, indicating that Rp-MIP may function as an aquaporin protein in the MT of the insect and thus may participate in urine formation in R. prolixus.
Subject(s)
Insect Proteins/analysis , Malpighian Tubules/chemistry , Rhodnius , Amino Acid Sequence , Animals , Aquaporins/genetics , Base Sequence , Cell Membrane/chemistry , Chemical Phenomena , Chemistry, Physical , Cloning, Molecular , DNA/analysis , DNA/chemistry , DNA, Complementary/chemistry , DNA, Complementary/genetics , In Situ Hybridization , Insect Proteins/chemistry , Insect Proteins/genetics , Molecular Sequence Data , Open Reading Frames , Phylogeny , RNA/isolation & purification , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Sequence AlignmentABSTRACT
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.
Subject(s)
Epithelial Cells/metabolism , Intestinal Mucosa/metabolism , Mannose/pharmacokinetics , Membrane Glycoproteins/metabolism , Monosaccharide Transport Proteins/metabolism , Sodium/metabolism , Animals , Carbon Radioisotopes , Chickens , Fructose/pharmacokinetics , Glucose/pharmacokinetics , Ileum/metabolism , Intestinal Mucosa/cytology , Jejunum/metabolism , Kinetics , Membrane Potentials/physiology , Methylglucosides/pharmacokinetics , Microvilli/metabolism , Osmolar Concentration , Patch-Clamp Techniques , Phloretin/pharmacokinetics , Phlorhizin/pharmacokinetics , Sodium-Glucose Transporter 1 , TritiumABSTRACT
The effects of aldosterone and arginine vasotocin (AVT) on intestinal Na(+)/H(+) exchange (NHE) and Na(+)-sugar cotransport (SGLT-1) activities have been investigated using brush-border membrane vesicles isolated from Hubbard chicken small and large intestines, and they were compared with those induced by either Na(+) depletion 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 treatment. Three-hour incubation of the intestine with aldosterone produced the same effects on NHE activity as does Na(+) depletion, 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.
Subject(s)
Aldosterone/pharmacology , Intestinal Mucosa/metabolism , Membrane Glycoproteins/metabolism , Monosaccharide Transport Proteins/metabolism , Sodium-Hydrogen Exchangers/metabolism , Vasotocin/pharmacology , Aldosterone/blood , Animals , Biological Transport/drug effects , Chickens , Colon/metabolism , Diet, Sodium-Restricted , Glucose/metabolism , Ileum/metabolism , Intestines/drug effects , Intestines/ultrastructure , Jejunum/metabolism , Kinetics , Microvilli/drug effects , Microvilli/metabolism , Sodium/metabolism , Sodium, Dietary/administration & dosage , Sodium-Glucose Transporter 1ABSTRACT
We have performed a detailed in situ hybridization study of the distribution of aquaporin-4 messenger RNA in the CNS. Contrary to expectation, we demonstrate that aquaporin-4 is ubiquitously expressed in the CNS. Strong hybridization labeling was detected in multiple olfactory areas, cortical cells, medial habenular nucleus, bed nucleus of the stria terminalis, tenia tecta, pial surface, pontine nucleus, hippocampal formation and multiple thalamic and hypothalamic areas. A low but significant hybridization signal was found, among others, in the choroid plexus of the lateral ventricles, ependymal cells, dorsal raphe and cerebellum. Overall, a preferential distribution of aquaporin-4 messenger RNA-expressing cells was evident in numerous periventricular organs. From the distribution study, the presence of aquaporin-4 messenger RNA-expressing cells in neuronal layers was evident in neuronal layers including the CA1 -CA3 hippocampal pyramidal cells, granular dentate cells and cortical cells. Further evidence of neuronal expression comes from the semicircular arrangement of aquaporin-4 messenger RNA-expressing cells in the bed nucleus of the stria terminalis and medial habenular nucleus exhibiting Nissl-stained morphological features typical of neurons. Combined glial fibrillary acidic protein immunohistochemistry and aquaporin-4 messenger RNA in situ hybridization demonstrated that aquaporin-4 messenger RNA is expressed by glial fibrillary acidic protein-lacking cells. We conclude that aquaporin-4 messenger RNA is present in a collection of structures typically involved in the regulation of water and sodium intake and that aquaporin-4 water channels could be the osmosensor mechanism responsible for detecting changes in cell volume by these cells.
Subject(s)
Aquaporins/genetics , Paraventricular Hypothalamic Nucleus/chemistry , Water-Electrolyte Balance/genetics , Animals , Aquaporin 4 , Brain Stem/chemistry , Brain Stem/cytology , Gene Expression/physiology , Glial Fibrillary Acidic Protein/analysis , In Situ Hybridization , Mesencephalon/chemistry , Mesencephalon/cytology , Neuroglia/chemistry , Neuroglia/physiology , Neurons/chemistry , Neurons/physiology , Olfactory Pathways/chemistry , Olfactory Pathways/cytology , Paraventricular Hypothalamic Nucleus/cytology , Purkinje Cells/chemistry , RNA, Messenger/analysis , Rats , Rats, Wistar , Third Ventricle/chemistry , Third Ventricle/cytologyABSTRACT
Astrocytes and aquaporin-4 (AQP4) play a significant role in brain ion homeostasis. Consequently the regulation of AQP4 mRNA in the CNS after different neurological insults was of interest. A single intrastriatal injection of ringer or quinolinic acid strongly induced AQP4 mRNA in the striatum, specially at the core of the lesion. Colocalization studies demonstrated that AQP4 mRNA induction was restricted to hypertrophic astrocytes. The extent of striatal AQP4 mRNA induction did not correlate with neuronal degeneration, but it did with extravasation of Evans blue dye as a marker of BBB disruption. Distant lesions were additionally induced by either 6-OHDA or a knife cut in the medial forebrain bundle (MFB). The former, but not the latter, induced a high AQP4 mRNA expression in the lesioned substantia nigra. However, axotomy of the MFB induced a high AQP4 mRNA expression at the lesion site. We conclude that the induction of AQP4 mRNA expression is related to disruption of the blood-brain barrier and under brain edema conditions this water channel plays a key role in the reestablishment of the brain osmotic equilibrium.
Subject(s)
Aquaporins/biosynthesis , Astrocytes/metabolism , Brain Edema/metabolism , Brain Injuries/metabolism , Animals , Aquaporin 4 , Aquaporins/physiology , Axotomy , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/physiology , Brain Injuries/chemically induced , Brain Injuries/physiopathology , Corpus Striatum/drug effects , Corpus Striatum/metabolism , Corpus Striatum/pathology , Female , Glial Fibrillary Acidic Protein/biosynthesis , Immunohistochemistry , In Situ Hybridization , Oxidopamine/toxicity , Quinolinic Acid/toxicity , RNA, Messenger/biosynthesis , Rats , Rats, Wistar , Substantia Nigra/drug effects , Substantia Nigra/metabolism , Substantia Nigra/pathology , Up-Regulation/drug effects , Up-Regulation/physiology , Water-Electrolyte Balance/drug effectsABSTRACT
Since specific proteins responsible for water transport (aquaporins, AQPs) have been identified in a great variety of tissues, we decided to study the presence of AQP3 in the gastrointestinal tract (GIT) of Wistar rats. Poly(A+) RNA was purified from the mucosa of the stomach, jejunum, ileum and colon, and gross detection of AQP3 mRNA was done by Northern blot analysis. In situ hybridization studies were carried out to precisely localize the distribution of this transcript. Sections of the different tissues were hybridized with @400-bp [35S]riboprobes. The results presented here demonstrate that AQP3 is expressed throughout the GIT, with its expression in the colon and ileum greater than that in the stomach. Immunohistochemistry experiments, using a polyclonal antibody against AQP3, revealed that AQP3 protein is present at the basolateral membrane of the epithelial cells lining the villus tip of the small intestine and colon. The finding of AQP3 in the intestinal epithelia strongly suggests that this protein functions as a pathway for water transport in this epithelium.