Polarity of neutral amino acid transport and characterization of a broad specificity transport activity in a kidney epithelial cell line, MDCK

The Madin-Darby canine kidney (MDCK) cell line was investigated with respect to the cellular polarity of amino acid transport in early confluent versus late confluent cultures. Early confluent cultures could take up amino acids from the apical and the basolateral sides of the cell layer via amino acid transport Systems A, ASC, and L. However, in late confluent cultures the activities of Systems A and L were clearly localized to the basolateral surface of the cell monolayer. In addition to the presence of systems A, ASC, and L, a novel activity, measurable under conditions used for quantitating System ASC, was found to be active in the apical membrane of these cells. This transporter, termed System G (for general), recognized basic and neutral amino acids with high affinity and acidic amino acids with lower affinity. System G exhibited broad substrate specificity, strict cation specificity, and a broad pH optimum with maximal activity at acidic pH. The activity of System G was relatively low after growth in serum-containing medium but was induced in a defined medium. Induction of System G activity was dependent upon the presence of prostaglandin E1. The broad substrate specificity, low pH optimum, and Na+ dependence suggest that System G may function in apical membranes as an energy-dependent transport route during reabsorption of amino acids from the kidney tubule lumen.     

Amino acid transport in kidney epithelial cell line (MDCK): characteristics of Na+/amino acid symport in membrane vesicles and basolateral localization in cell monolayers

Na+-stimulated amino acid transport was investigated in MDCK kidney epithelial cell monolayers and in isolated membrane vesicles. When transport polarity was assessed in confluent polarized epithelial cell monolayers cultured on Nucleopore filters and mounted between two lucite chambers, Na+-stimulated transport of 2-(methylamino)isobutyric acid (MeAIB), a substrate specific for the A system, was predominantly localized on the basolateral membrane. Na+-stimulated amino acid transport activity was maximal in subconfluent cultures, and was substantially reduced after confluence. A membrane vesicle preparation was isolated from confluent MDCK cell cultures which was enriched in Na+-stimulated MeAIB transport activity and Na+,K+,ATPase activity, a basolateral marker, but was not enriched in apical marker enzyme activities or significantly contaminated by mitochondria. Na+-coupled amino acid transport activity assayed in vesicles exhibited a marked dependence on external pH, with an optimum at pH 7.4. The pattern of competitive interactions among neutral amino acids was characteristic of A system transport. Na+-coupled MeAIB and AIB transport in vesicles was electrogenic, stimulated by creation of an interior-negative membrane potential. The Na+ dependence of amino acid transport in vesicles suggested a Na+ symport mechanism with a 1:1 stoichiometry between Na+ and amino acid.     

Hormonal regulation of hepatic amino acid transport

The transport of 2-aminoisobutyric acid (AIB) into liver tissue was increased by both insulin and glucagon. We have now shown that these hormones do not stimulate the same transport system. Glucagon, possibly via cAMP, increased the hepatic uptake of AIB by a mechanism which resembled system A. This glucagon-sensitive system could be monitored by the use of the model amino acid MeAIB. In contrast, the insulin-stimulated system exhibited little or no affinity for MeAIB and will be referred to as system B. On the basis of other reports that the hepatic transport of AIB is almost entirely Na⁺ dependent and the present finding that the uptake of 2-aminobicyclo [2,2,1] heptane-2-carboxylic acid (BCH) was not stimulated by either hormone, we conclude that system B is Na⁺ dependent. Furthermore, insulin added to the perfusate of livers from glucagon-pretreated donors suppressed the increase in AIB or MeAIB uptake. Depending upon the specificities of systems A and B, both of which are unknown for liver tissue, the insulin/glucagon ratio may alter the composition of the intracellular pool of amino acids.     

Effects of 5-azacytidine, sodium butyrate, and phorbol esters on amino acid transport system A in a kidney epithelial cell line, MDCK: evidence for multiple mechanisms of regulation

Neutral amino acid transport by system A was investigated in the epithelial cell lines MDCK and MDCK-T1. The latter line is a chemically induced, oncogenically transformed line derived from MDCK. Inducers of differentiation, sodium butyrate and 5-azacytidine, and a tumor promoter, TPA, were used as probes to delineate pathways of regulation involved in system A response to a variety of physiological conditions and agents. Azacytidine, an inhibitor of DNA methylation, and butyrate, an enhancer of histone acetylation, inhibited expression of system A, had little effect on system ASC, and slightly stimulated system L. Inhibition of system A expression by butyrate and azacytidine occurred under different conditions. Increases in system A activity due to amino acid starvation or transformation were inhibited by butyrate but not by azacytidine. Repressed system A activity, normally observed in the presence of high levels of amino acids, was more sensitive to azacytidine than to butyrate. The tumor promoter, TPA, stimulated system A activity in MDCK cells under normal growth conditions but did not stimulate activity in amino acid-starved MDCK cells or in MDCK-T1 cells. Stimulation of system A activity by TPA was prevented by prior exposure to butyrate but not to azacytidine. These results suggest 1) that system A expression observed in growing amino-acid-repressed MDCK cells is modulated by an azacytidine-sensitive mechanism and 2) that the elevated expression of system A activity induced by amino acid starvation, by chemical transformation to MDCK-T1, and by TPA is modulated by a butyrate-sensitive mechanism.     

Development of cell surface polarity in the epithelial Madin-Darby canine kidney (MDCK) cell line.

The development of surface polarity has been studied in the epithelial Madin-Darby canine kidney (MDCK) cell line by examining two basolateral markers: a monoclonal antibody against a 58-kd protein and [35S]methionine uptake. The surface distribution of these markers was followed after plating the cells on coverslips or nitrocellulose filters. In subconfluent monolayers the apical surface of many cells was stained with the anti-58-kd antibody. Clearing of the apical surface occurred first after confluency had been reached in cells grown on coverslips. Similarly, in cells grown on filters the basolateral 58-kd protein disappeared from the apical surface concomitantly with the development of a measurable electrical resistance over the cell monolayer. The uptake of [35S]methionine was measured from both sides of filter-grown cells and began to polarize early after seeding, reaching a value of greater than 98% basolateral in the fully polarized monolayer. These results emphasize that the development of surface polarity in MDCK cells is a gradual process, and that extensive cell-cell contacts seem to be required for complete surface polarization.     

Adaptive regulatory control of System A transport activity in a kidney epithelial cell line (MDCK) and in a transformed variant (MDCK-T1)

Adaptive regulatory control of System A activity was investigated using MDCK cells and a chemically induced, oncogenic transformant of MDCK cells, MDCK-T1. Within 7 hours after transfer to an amino-acid-deficient medium, A activity of subconfluent MDCK cells had maximally derepressed, but this activity in confluent MDCK cells and in subconfluent transformed cells showed little capacity for derepression. Amino-acid-starved, subconfluent MDCK cells were used to study trans-inhibition and repression of A activity by individual amino acids. Trans-inhibition and repression were defined as the cycloheximide-insensitive and cycloheximide-sensitive components, respectively, of the total inhibition. Trans-inhibition correlated well with substrate affinity, but repression did not. Trans-inhibition and repression were further characterized using alpha-(methylamino) isobutyric acid (mAIB), a trans-inhibitor, and glutamate, an effective repressor. The apparent initial T 1/2 for inhibition by mAIB in the presence of cycloheximide was 0.5 hours, while that for repression by glutamate was 4.7 hours. Half-maximal inhibition by mAIB and repression by glutamate occurred at approximately 0.02 mM and 0.07 mM, respectively. Reversal of trans-inhibition by methionine occurred in the presence of cycloheximide within 1-4 hours after removal of methionine. The A system of the transformed MDCK-T1 cells showed elevated activity, little capacity for derepression, resistance to repression by amino acids, but retention of sensitivity to trans-inhibition. Kinetic analysis of mAIB uptake indicated that the A system of MDCK-T1 cells has become kinetically more complex in a manner which resembled amino-acid-starved rather than amino-acid-fed MDCK cells. These results suggest that the A system of MDCK-T1 cells has become resistant to adaptive regulatory control.     

Regulation of amino acid transport in the renal epithelial cell line NBL-1

Summary The activities of the transport systems A, B° and X_AG- are induced by various forms of stress in renal epithelial cells. Amino acid deprivation induces System A and X_AG- in a protein-synthesis dependent process. In the case of System X_AG- evidence is presented that induction of transport does not involve an increase in the amount of mRNA for the transporter or of the amount of transport protein. Preliminary evidence for the existence of a novel glycoprotein which is induced in parallel to the induction of these transport systems is presented. It is suggested that the induction of amino acid transport proteins and of some of the so-called stress proteins may be triggered by a common molecular mechanism.     

Hormonal regulation of amino acid transport system N in primary cultures of rat hepatocytes

The transport of histidine and glutamine via system N in cultured hepatocytes was found to be subject to hormonal control. This long-term regulation showed the following characteristics. The transport capacity for histidine and glutamine (system N) increased slowly in response to the combination of dexamethasone and insulin to about 4-fold that of controls after 18-30 h. A similar time course was found for the stimulation of system N (2.5-fold) by dexamethasone and glucagon. In contrast the uptake of alpha-aminoisobutyric acid (system A) was rapidly stimulated 3-fold by dexamethasone and insulin and 5-fold by dexamethasone and glucagon within 3-6 h but decreased towards control rates after 24 h of cultivation in minimal essential medium. Dexamethasone, insulin and glucagon each stimulated glutamine uptake about 2-fold in cultures maintained in W/AB 77 medium, while the combination of dexamethasone with either glucagon or insulin resulted in a 3-4-fold increase. Dexamethasone was most effective at about 0.1 microM. Higher concentrations were less efficient. Insulin reached its optimal effect at concentrations above 1 microM. Kinetic analysis revealed that the increased capacity of glutamine transport in response to hormones was due to an increase in Vmax, while Km was essentially unchanged. The hormone-induced stimulation of system N was prevented by cycloheximide. The induced uptake of glutamine was inhibited by excess amounts of asparagine and histidine but not of alpha-methylaminoisobutyric acid or cysteine. These results clearly differentiate the hormonal regulation of system N from that of system A.     

The acidic amino acid transport system of the baby hamster kidney cell line BHK21-C13

The uptake of L-glutamate into BHK21-C13 cells in culture has been studied. This amino acid appears to be transported via a relatively high affinity, low capacity, Na+-dependent transport system capable of the rapid accumulation of substrate amino acids. Kinetic studies of the inhibition of L-glutamate uptake has provided information as to the substrate and the molecular configuration required for transport via the glutamate transport system. This system exhibited marked substrate specificity and was only capable of transporting L-glutamate and aspartate and certain closely related acidic amino acid analogues.     

Cationic amino acid transport by two renal epithelial cell lines: LLC-PK1 and MDCK cells

LLC-PK1 and MDCK cells take up cationic amino acids (lysine and arginine) by a specific sodium independent transport system. Uptake is inhibited by ornithine in LLC-PK1 and MDCK cells either in the presence or absence of sodium and by glutamine or homoserine in MDCK cells in the presence of sodium. Trans-stimulation of uptake occurs in the presence of intracellular cationic amino acids. Experiments with valinomycin or with different extracellular potassium concentrations suggest that uptake is dependent on the membrane potential of these cells. These transport features are similar to those previously ascribed to a transport system denominated y+ in other cells. Further experiments suggested that this carrier system is localised to the basolateral membrane in each cell type.     

Activation of amino acid diffusion by a volume increase in cultured kidney (MDCK) cells

Summary When MDCK cells are cultured in MEM, they maintain a high concentration of three amino acids: glutamate (25mm), taurine (19 mm) and glycine (9 mm). With incubation of the cells in hypotonic media, the contents of these amino acids measured by HPLC are reduced in different time courses: taurine decreases most rapidly, followed by glutamate and glycine. All these losses are Na⁺ independent. To determine the transport mechanism activated by the hypotonic media, increasing external concentrations reaching 60 mm for nine different amino acids in Na⁺-free media were tested separately. For the five neutral (zwitterionic) amino acids, taurine, glycine, alanine, phenylalanine and tryptophan, cell contents increased linearly with external concentrations in hypotonic media, whereas in isotonic media only a slight rise was observed. The two anionic amino acids, glutamate and aspartate, were also increased linearly with their external concentrations in hypotonic media, but the changes were lower than those found for neutral amino acids. The presence of a negative membrane potential was responsible for this behavior since, using a K⁺ hypotonic medium which clamps the potential to zero, the glutamate content was found to increase linearly with an amplitude similar to the one observed for neutral amino acid. When external concentrations of two cationic amino acids, arginine and lysine, were increased in hypotonic media, only a small change, similar to that in isotonic media, was observed. These results indicate that a diffusion process for neutral and anionic amino acids is activated by a volume increase and it is suggested that an anion channel is involved.     

Cortisol stimulates system A amino acid transport and SNAT2 expression in a human placental cell line (BeWo)

Both placental system A activity and fetal plasma cortisol concentrations are associated with intrauterine growth retardation, but it is not known if these factors are mechanistically related. Previous functional studies using hepatoma cells and fibroblasts produced conflicting results regarding the regulation of system A by cortisol. Using the b30 BeWo choriocarcinoma cell line, we investigated the regulation of system A by cortisol. System A function was analyzed using methyl amino isobutyric acid (MeAIB) transcellular transport studies. Transporter expression [system A transporter (SNAT)1/2] was studied at the mRNA and protein levels using Northern and Western blotting, respectively. Localization was carried out using immunocytochemistry. The [(14)C]MeAIB transfer rate across BeWo monolayers after preincubation with cortisol for 24 h was significantly increased compared with control. This was associated with a relocalization of the SNAT2 transporter at lower cortisol levels and significant upregulation of mRNA and protein expression levels at cortisol levels >1 microM. This is the first study to show functional and molecular regulation of system A by cortisol in BeWo cells. It is also the first study to identify which system A isoform is regulated. These results suggest that cortisol may be involved in upregulation of system A in the placenta to ensure sufficient amino acid supply to the developing fetus.     

Accumulation of sulfoglycolipids in hyperosmosis-resistant clones derived from the renal epithelial cell line MDCK (Madin-Darby canine kidney cell).

1. Two clones (osmR-A and osmR-B) resistant to hyperosmotic media of 700 and 800 mosmol/l, respectively, were selected from Madin-Darby canine kidney (MDCK) cells. 2. When cultured in isosmotic medium (300 mosmol/l), the concentration of galactosyl sulfatide and lactosyl sulfatide in these hyperosmosis-resistant clones was 3.4-5.9 times higher than in the wild-type MDCK. The rate of incorporation of [35S]sulfate into sulfolipids of osmR-A and osmR-B was 1.9-6.7 times higher than MDCK. 3. The stimulation of incorporation into sulfolipids by hyperosmotic culture was completely inhibited by cycloheximide. The pulse-chase studies indicated decreased turnover rate of sulfolipids in osmR-A.     

System A amino acid transport in a rat submandibular ductal cell line

1. Neutral amino acid transport was studied in an established cell line derived from rat submandibular glands, RSMTx. 2. The greatest portion of alpha-amino isobutyrate (AIB) transport is mediated by system A. This component is Na+ dependent, pH sensitive, markedly inhibited by methyl AIB and enhanced 2-5-fold by amino acid depletion. 3. Evidence for the presence of other neutral amino acid transport systems, presumably ASC and L, was also found in these cells.     

The adaptive regulation of amino acid transport system A is associated to changes in ATA2 expression

The activity of transport system A for neutral amino acids is adaptively stimulated upon amino acid starvation. In cultured human fibroblasts this treatment causes an increase in the expression of the ATA2 system A transporter gene. ATA2 mRNA increase and transport stimulation are suppressed by system A substrates, but they are unaffected by other amino acids. Supplementation of amino acid-starved cells with substrates of system A causes a decrease in both ATA2 mRNA and system A transport activity. These results suggest a direct relationship between ATA2 expression and system A transport activity.     

Transepithelial transport in cell culture:d-Glucose transport by a pig kidney cell line (LLC-PK1)

Summary The pig kidney cell line LLC-PK₁ cultured on a collagen coated membrane filter formed a continuous sheet of oriented asymmetrical epithelial cells joined by occluding junctions. A transepithelial electrical potential (PD) and short-circuit current (SCC) were dependent on the presence of Na and sugar in the apical bathing solution. In the presence of 5.5mm d-glucose, a PD of 2.8 mV, apical surface negative, a SCC of 13 A cm^–2 and transepithelial resistance of 211 ohm·cm² were recorded. The SCC was promptly reduced by the addition of phlorizin to the apical bath but unaffected when placed in the basolateral bath. The effect on SCC of various sugars was compared by the concentrations required for half-maximal SCC: 0.13mm -methyl-d-glucoside, 0.28mm d-glucose, 0.65mm -methyl-d-glucoside, 0.77mm 6-deoxy-d-glucose, 4.8mm d-galactose, and 29mm 3-O-methyl-glucose. When [Na] was reduced, the concentration ofd-glucose required for half-maximal SCC increased. Isotopically labeled³H and¹⁴Cd-glucose were used to simultaneously determine bidirectional fluxes; a resultant net apical-to-basolateral transport was present and abolished by phlorizin. The transported isotope cochromatographed with labeledd-glucose, indicating negligible metabolism of transported glucose. The pig kidney cell line, LLC-PK₁, provides a cell culture model for the investigation of mechanisms of transepithelial glucose transport.     

The role of system A for neutral amino acid transport in the regulation of cell volume

System A is a secondary active, sodium dependent transport system for neutral amino acids. Strictly coupled with Na,K-ATPase, its activity determines the size of the intracellular amino acid pool, through a complex network of metabolic reaction and exchange fluxes. Many hormones and drugs affect system A activity in specific cell models or tissues. In all the cell models tested thus far the activity of the system is stimulated by amino acid starvation, cell cycle progression, and the incubation under hypertonic conditions. These three conditions produce marked alterations of cell volume. The stimulation of system A activity plays an important role in cell volume restoration, through an expansion of the intracellular amino acid pool. Under normal conditions, system A substrates represent a major fraction of cell compatible osmolytes, organic compounds that exert a protein stabilizing effect. It is, therefore, likely that the activation of system A represents a portion of a more complex response triggered by exposure to stresses of various nature. Since system A transporters have been recently cloned, the molecular bases of these regulatory mechanisms will probably be elucidated in a short time.     

Hormonal regulation of amino acid transport and cAMP production in monolayer cultures of rat hepatocytes

The effects of insulin, glucagon or Dexamethasone (DEX) and of glucagon with insulin or DEX were examined on the uptake of 2-amino [1-¹⁴C]isobutyric acid (AIB) and N-Methyl-2-amino [1-¹⁴C]isobutyric acid (NMe AIB) in monolayer cultures of rat hepatocytes. Insulin and glucagon stimulated the uptake of both the amino acids and DEX inhibited it, showing that all three of these hormones regulate the A system (the sodium-dependent system that permits the transport of NMe AIB) for amino acid transport in these cultures. Experiments investigating the transport of aminocyclopentane-1-carboxylic acid, 1- [carboxyl-¹⁴C] in the presence of excess AIB or in the absence of sodium showed that insulin had no effect on the activity of the L system (the sodium-independent system that prefers leucine). Experiments on the uptake of AIB in the presence of excess NMe AIB showed insulin had no effect on the transport activity of the ASC system (the sodium-dependent system that does not transport NEe AIB). Insulin concentrations ranging from 0.1 nM to 100 nM did not antagonize the stimulatory effect of optimum or suboptimum concentrations of glucagon on the uptake of either AIB or NMe AIB. Similarly, glucagon did not antagonize the stimulatory effect of optimum or suboptimum concentrations of insulin on the uptake of both the amino acids. The combined effect of insulin and glucagon was additive on the rate as well as the cumulative uptake of both AIB and NMe AIB. DEX alone inhibited the transport of both AIB and NMe AIB by about 25%, while glucagon caused a 2–3-fold increase; however, the addition of glucagon to cultures containing DEX caused a 7–8-fold increase in the uptake of both AIB and NMe AIB when compared to cultures containing DEX alone. The effect of insulin on the levels of cAMP was also investigated. Insulin had no effect on the cAMP levels in cultures treated or untreated with optimum or suboptimum concentrations of glucagon.