Increase In Transport Activity Research Articles

Membrane Particle Distribution in the Sternal Epithelia of the Terrestrial Isopod Porcellio scaber Latr. (Crustacea, Oniscidea) during CaCO3 Deposit Formation and Resorption, a Freeze-Etch Analysis

The anterior sternal epithelium of terrestrial isopods transports cuticular Ca2+ to and from large sternal CaCO3 deposits. We analyzed the anterior and posterior sternal epithelium by the means of the freeze-etch technique and measured the size distribution and density of intramembrane particles (IMPs) during three different molting stages. At least three IMP size classes around 4.5, 7.7, and 9.4 nm can be distinguished on the P-face of the apical and basolateral plasma membrane. An additional size class of around 12.8 nm is restricted to the apical compartment. In the anterior sternal epithelium, the density of these large particles changes by a factor of 1.9 during the molt cycle, suggesting a role in CaCO3 formation and/or resorption. The density of the smaller IMPs rises transiently by a factor of 1.3 in the posterior sternal epithelium only. The IMP density of the basolateral plasma membrane increases significantly by a factor of 1.4 and 1.3 in the anterior and posterior sternal epithelia, respectively. The results indicate that increases in the IMP density contribute to the differentiation to an increased transport activity during the cyclic enlargements of the plasma membrane surface area in the anterior sternal epithelium.

Amino acids are compatible osmolytes for volume recovery after hypertonic shrinkage in vascular endothelial cells.

The response to chronic hypertonic stress has been studied in human endothelial cells derived from saphenous veins. In complete growth medium the full recovery of cell volume requires several hours and is neither associated with an increase in cell K+ nor hindered by bumetanide but depends on an increased intracellular pool of amino acids. The highest increase is exhibited by neutral amino acid substrates of transport system A, such as glutamine and proline, and by the anionic amino acid glutamate. Transport system A is markedly stimulated on hypertonic stress, with an increase in activity roughly proportional to the extent and the duration of the osmotic shrinkage. Cycloheximide prevents the increase in transport activity of system A and the recovery of cell volume. It is concluded that human endothelial cells counteract hypertonic stress through the stimulation of transport system A and the consequent expansion of the intracellular amino acid pool.

Sodium-hydrogen exchange and platelet function.

On stimulation of platelets with agonists, for example, thrombin, a rapid rise in intracellular pH is observed. This alkalinization is mediated by an increase in transport activity of the Na(+)/H(+) exchanger isoform NHE1. In addition to this Na(+)/H(+) exchange mechanism, platelets express bicarbonate/chloride exchangers, which also contribute to pH(i) homeostasis. The main functions of NHE1 in platelets include pH(i) control, volume regulation, and participation in cell signaling. The isoform NHE1 is highly sensitive toward inhibition by EIPA, Hoe694, and Hoe642. The regulation of NHE1 activity is complex and is not completely understood. It includes the MAP kinase cascade, the Ca/calmodulin system, several heterotrimeric G proteins (Galpha12, Galpha13, Galphaq, and Galphai), small G proteins (ras, cdc42, rhoA), and downstream kinases (e.g., p160ROCK). Volume challenges stimulate tyrosine phosphorylation of cytoplasmic proteins, which ultimately activate NHE1. Thrombin, thromboxane, platelet-activating factor, angiotensin II, endothelin, phorbol ester, and Ca(2+) ionophors stimulate NHE1 activity in platelets. Blockade of platelet NHE1 can inhibit platelet activation. With the development of highly specific NHE1 inhibitors, detailed investigation of the relationships between NHE1 activity and platelet activation now becomes feasible.

A Na+-DEPENDENT SYSTEM A AND ASC-INDEPENDENT AMINO ACID TRANSPORT SYSTEM STIMULATED BY GLUCAGON IN RAT HEPATOCYTES

The effects of glucagon on amino acid transport in rat hepatocytes are not fully understood. We examined the effect of this hormone on alanine, serine and cysteine preferring system (system ASC)-mediated amino acid transport in rat hepatocyte monolayers using 2-aminoisobutyric acid (AIB) and l -cysteine. Glucagon induced a time and protein synthesis-dependent stimulation of Na+-dependent alanine preferring system (system A)-independent AIB transport. The glucagon-induced increase in transport activity was not modified by substrate starvation and not related to changes in the intracellular pool of amino acids. Glucagon did not modify system ASC activity measured by l -cysteine. Therefore the transport activity of AIB independent of system A stimulated by glucagon cannot be attributed to system ASC. This suggests a Na+-dependent transport system in rat hepatocytes not identified until now.

Acute and chronic regulation of Na+/K+-ATPase transport activity in the RN22 Schwann cell line in response to stimulation of cyclic AMP production.

Na+/K+-ATPase-dependent Rb+ uptake of RN22 Schwann cells was stimulated by cholera toxin (0.25 microg/ml), forskolin (2 mM), or 8-bromo cAMP (1 mM). At 2 h Rb+ uptake was increased by 162+/-6% (cholera toxin), 151+/-14% (forskolin), and 207+/-15% (8-bromo cAMP). Cholera toxin or 8-bromo cAMP treatment for 12-24 h resulted in a second peak of Na+/K+-ATPase-dependent Rb+ transport activity of 186+/-12 and 265+/-9% of control, respectively. Cholera toxin also transiently stimulated the activity of the Na+, K+, 2Cl- -cotransporter with a peak at 2 h (179+/-9%), returning to basal levels by 24 h. Inhibition of the Na+,K+,2Cl- -cotransporter by bumetanide (0.1 mM) or by reduction of the Na+ gradient (10 mM veratridine treatment) prevented the early peak in ATPase activity but not the second peak. These results indicated that the early transient stimulation of Na+/K+ ATPase activity by cholera toxin was due to an increase in cellular Na+, secondary to stimulation of Na+,K+,2Cl -cotransport activity. Western blot analysis of cellular homogenates and purified membrane fractions showed that the second peak of Rb+ uptake activity was a result of translocation of transport protein from an intracellular microsomal pool to the plasma membrane. Rb+ uptake by dominant negative protein kinase A mutants of the RN22 cell was not stimulated by cholera toxin treatment (acute or chronic) confirming the cAMP/protein kinase A dependency of both acute and long-term regulation of transport activity. In the absence of a change in Michaelis constants or of an increase in total transport protein of cellular homogenates, neither a change in enzyme kinetics nor an increase in de novo synthesis of transport protein could account for the increase in transport activity.

His-65 in the proton-sucrose symporter is an essential amino acid whose modification with site-directed mutagenesis increases transport activity.

The proton-sucrose symporter that mediates phloem loading is a key component of assimilate partitioning in many higher plants. Previous biochemical investigations showed that a diethyl pyrocarbonate-sensitive histidine residue is at or near the substrate-binding site of the symporter. Among the proton-sucrose symporters cloned to date, only the histidine residue at position 65 of AtSUC1 from Arabidopsis thaliana is conserved across species. To test whether His-65 is involved in the transport reaction, we have used site-directed mutagenesis and functional expression in yeast to determine the significance of this residue in the reaction mechanism. Symporters with mutations at His-65 exhibited a range of activities; for example, the H65C mutant resulted in the complete loss of transport capacity, whereas H65Q was almost as active as wild type. Surprisingly, the H65K and H65R symporters transport sucrose at significantly higher rates (increased Vmax) than the wild-type symporter, suggesting His-65 may be associated with a rate-limiting step in the transport reaction. RNA gel blot and protein blot analyses showed that, with the exception of H65C, the variation in transport activity was not because of alterations in steady-state levels of mRNA or symporter protein. Significantly, those symporters with substitutions of His-65 that remained transport competent were no longer sensitive to inactivation by diethyl pyrocarbonate, demonstrating that this is the inhibitor-sensitive histidine residue. Taken together with our previous results, these data show that His-65 is involved in sucrose binding, and increased rates of transport implicate this region of the protein in the transport reaction.

Altered sodium-hydrogen exchange activity is a mechanism for acid-induced hyperproliferation in Barrett's esophagus.

Acid produces a dynamic effect on the cell phenotype of Barrett's esophagus (BE) ex vivo. An acid pulse induces hyperproliferation, whereas continuous acid exposure promotes differentiation. To examine the mechanism for acid pulse-induced hyperproliferation, we studied the Na+/H+ exchanger (NHE), which plays a role in the control of intracellular pH and cell proliferation. NHE was inhibited pharmacologically in endoscopic BE biopsies using amiloride analogs. Cell proliferation was assessed after pulsed or continuous acid exposure using tritiated thymidine incorporation assays and immunohistochemical analysis of proliferating cell nuclear antigen expression. The NHE-dependent intracellular pH response to an acid pulse was examined by pH-sensitive microfluorimetry using a Barrett's adenocarcinoma cell line TE7. NHE inhibition significantly reduced the hyperproliferative acid-pulse effect. Furthermore, the acid-pulse activation of NHE occurred via increased transporter activity (22Na uptake) without any change in NHE-1 protein levels. Inhibition of protein kinase C (PKC), an NHE activator, also reduced the hyperproliferative response. The response of TE7 cells to an acid pulse was similar to that of BE biopsies in terms of cell proliferation and NHE and PKC dependence. Acid-pulse exposure of TE7 cells resulted in intracellular acidification followed by reneutralization to an intracellular pH greater than preacidosis values. We conclude that NHE may mediate the hyperproliferative response of BE to an acid pulse via changes in intracellular pH.

Multiple Signaling Pathways Regulate Cell Surface Expression and Activity of the Excitatory Amino Acid Carrier 1 Subtype of Glu Transporter in C6 Glioma

Neuronal and glial sodium-dependent transporters are crucial for the control of extracellular glutamate levels in the CNS. The regulation of these transporters is relatively unexplored, but the activity of other transporters is regulated by protein kinase C (PKC)- and phosphatidylinositol 3-kinase (PI3K)-mediated trafficking to and from the cell surface. In the present study the C6 glioma cell line was used as a model system that endogenously expresses the excitatory amino acid carrier 1 (EAAC1) subtype of neuronal glutamate transporter. As previously observed, phorbol 12-myristate 13-acetate (PMA) caused an 80% increase in transporter activity within minutes that cannot be attributed to the synthesis of new transporters. This increase in activity correlated with an increase in cell surface expression of EAAC1 as measured by using a membrane-impermeant biotinylation reagent. Both effects of PMA were blocked by the PKC inhibitor bisindolylmaleimide II (Bis II). The putative PI3K inhibitor, wortmannin, decreased L-[3H]-glutamate uptake activity by >50% within minutes. Wortmannin decreased the Vmax of L-[3H]-glutamate and D-[3H]-aspartate transport, but it did not affect Na+-dependent [3H]-glycine transport. Wortmannin also decreased cell surface expression of EAAC1. Although wortmannin did not block the effects of PMA on activity, it prevented the PMA-induced increase in cell surface expression. This trafficking of EAAC1 also was examined with immunofluorescent confocal microscopy, which supported the biotinylation studies and also revealed a clustering of EAAC1 at cell surface after treatment with PMA. These studies suggest that the trafficking of the neuronal glutamate transporter EAAC1 is regulated by two independent signaling pathways and also may suggest a novel endogenous protective mechanism to limit glutamate-induced excitotoxicity.

Thiazolidinediones and their effects on glucose transporters.

Recently, considerable attention has been paid to a promising new family of antidiabetic therapeutic agents, the thiazolidinediones. Several members of this family have been shown to have potent effects to lower hyperglycemia, hyperinsulinemia and hypertriglyceridemia in insulin resistant humans and animals. Even as one member of this family, troglitazone (Rezulin), has become available for clinical use in the US and Japan, we still do not fully understand the mechanisms by which these agents exert their effects. Shimaya and colleagues (1) have employed an insulin resistant model system, the obese Zucker rat, to investigate potential mechanisms of thiazolidinedione action. Treatment of obese animals with a new thiazolidinedione analog, YM268, greatly reduced fasting glucose and insulin levels and improved glucose tolerance. These workers confirmed the reduced glucose transporter 4 (GLUT4) protein expression in adipocytes of obese animals compared with lean littermates and found that YM268 treatment resulted in large increases in GLUT4 protein, along with a generalized increase in membrane protein. They suggest that this change can account, at least in part, for the demonstrated amelioration of insulin resistance after treatment. This, and related work, give rise to two questions. First, what is the ultimate significance of changes in GLUT4 expression in adipose tissue? Secondly, how might thiazolidinediones be acting to regulate glucose transporter expression? Effects of thiazolidinediones on adipose tissue glucose transport activity and transporter expression have been reported since the earliest papers on these agents. A common observation, regardless of the animal model or agent tested, is that glucose transport activity is increased, as is GLUT4 expression (2–6) following treatment. These changes occur together with reductions in fasting glucose and insulin levels, so it is uncertain if the effects on transport/transporters are direct or due to the change in the metabolic environment. Considering the demonstrated abilities of glucose and insulin to regulate glucose transport, it is difficult to separate the cause from the effect. Studies in cultured cell systems, where glucose and insulin levels can be tightly controlled, could help to resolve this issue. 3T3-L1 and 3T3-F442A adipocytes have been treated with a variety of thiazolidinediones, resulting in increases in transport activity and transporter expression (7–11). However, these treatments were performed during fibroblast differentiation into adipocytes and, since the appearance of GLUT4 is one marker of adipocyte differentiation, it is uncertain whether this result is due to augmented differentiation, or a more specific effect on transport which would have greater relevance to the therapeutic situation where adipocytes are already differentiated. Given the results showing that thiazolidinediones alone can drive adipocyte differentiation (7, 11), or augment that due to insulin (7), it is even more difficult to draw definitive conclusions from the majority of these studies. However, it has been shown that troglitazone treatment of 3T3-L1 cells following differentiation into adipocytes resulted in upregulation of basal glucose transport and GLUT1 expression (11). GLUT4 expression was unaltered, further suggesting that changes in GLUT4 may be indirect responses in adipose cells. In even the most obese individual (or animal), the portion of a glucose load utilized in adipose tissue is a modest fraction of the total. It is glucose uptake into muscle that represents the major portion of insulinstimulated glucose uptake and is the most significant site of insulin resistance. Thiazolidinedione treatment of insulin resistant rodents has also been shown to augment glucose uptake into muscle as well as increase GLUT4 expression (2–4, 6, 8, 12–14). Such changes could account, to a greater extent, for increases in whole body glucose disposal and would represent the most significant action of thiazolidinediones. Again, the question remains whether these are direct effects of thiazolidinediones, or due to changes in insulin and glucose levels. Fortunately, data in cultured muscle cells are helpful in resolving this issue. Troglitazone treatment of L6 myocytes (15) and isolated rat cardiac myocytes (16) increased glucose transport and the expression of both GLUT1 and GLUT4. This effect was independent of differentiation because it occurred even in L6 cells that were not differentiated into myotubes (15) or in BC3H-1 myocytes (with pioglitazone) (17) that are incapable of differentiation. It should be noted that the non-differentiated myocytes express only GLUT1 and it is this isoform that is upregulated by thiazolidinedione treatment. We have performed similar European Journal of Endocrinology (1997) 137 610–612 ISSN 0804-4643

Glucose Transporter Isoforms GLUT1 and GLUT3 Transport Dehydroascorbic Acid

Dehydroascorbic acid (DHA) is rapidly taken up by cells and reduced to ascorbic acid (AA). Using the Xenopus laevis oocyte expression system we examined transport of DHA and AA via glucose transporter isoforms GLUT1-5 and SGLT1. The apparent Km of DHA transport via GLUT1 and GLUT3 was 1.1 +/- 0.2 and 1.7 +/- 0.3 mM, respectively. High performance liquid chromatography analysis confirmed 100% reduction of DHA to AA within oocytes. GLUT4 transport of DHA was only 2-4-fold above control and transport kinetics could not be calculated. GLUT2, GLUT5, and SGLT1 did not transport DHA and none of the isoforms transported AA. Radiolabeled sugar transport confirmed transporter function and identity of all cDNA clones was confirmed by restriction fragment mapping. GLUT1 and GLUT3 cDNA were further verified by polymerase chain reaction. DHA transport activity in both GLUT1 and GLUT3 was inhibited by 2-deoxyglucose, D-glucose, and 3-O-methylglucose among other hexoses while fructose and L-glucose showed no inhibition. Inhibition by the endofacial inhibitor, cytochalasin B, was non-competitive and inhibition by the exofacial inhibitor, 4,6-O-ethylidene-alpha-glucose, was competitive. Expressed mutant constructs of GLUT1 and GLUT3 did not transport DHA. DHA and 2-deoxyglucose uptake by Chinese hamster ovary cells overexpressing either GLUT1 or GLUT3 was increased 2-8-fold over control cells. These studies suggest GLUT1 and GLUT3 isoforms are the specific glucose transporter isoforms which mediate DHA transport and subsequent accumulation of AA.

Interaction between taurine and angiotensin II: Modulation of calcium transport and myocardial contractile function

Angiotensin II modulates several aspects of cardiac function, including myocardial contractility, heart rate and myocyte growth. Most of these actions are intimately associated with alterations in calcium transport. Since taurine also modulates calcium transport, we examined possible interactions between taurine and angiotensin II at the level of the major cellular extruder of calcium, the Na−-Ca2+ exchanger. Over a concentration range of 0.5–25 mM, Turne served as an effective inhibitor of angiotensin II-mediated stimulation of the exchanger. An Arrhenius plot of Na+-Ca2+ exchange activity revealed that angiotensin II (2 nM) increased transporter activity by reducing the activation energy of the transport process. Taurine (25 mM) inhibited the angiotensin II effect by partially preventing the reduction in activation energy. However, neither agent significantly altered the transition temperature, ruling out a change in membrane fluidity or an alteration in the rate limiting step of the transporter as a cause of the observed effects. Since the Na+-Ca2+ exchanger plays an important role in the handling of [Ca2+]i by the myocardium, the effect of taurine on angiotensin II's modulation of contractile function was also examined. Hearts perfused with buffer containing angiotensin 11 experienced a slight positive isotropic effect in the absence of taurine but this was converted to a negative inotropic effect in the presence of taurine. The data suggest that Turine inhibits some, but not all of the actions of angiotensin II. The possibility that a phosphorylation event is the site of the angiotensin II-taurine interaction is discussed.

A putative helical domain in the MalK subunit of the ATP-binding-cassette transport system for maltose of Salmonella typhimurium (MalFGK2) is crucial for interaction with MalF and MalG. A study using the LacK protein of Agrobacterium radiobacter as a tool.

The ATP-binding-cassette (ABC) protein LacK of Agrobacterium radiobacter displays high sequence similarity to the MalK subunit of the Salmonella typhimurium maltose-transport system (MalFGK2). We have used LacK as a tool to identify sites of interaction of MalK with the membrane-integral components MalF and MalG. Small amounts of LacK, resulting from the expression of the plasmid-borne lacK gene, proved to be sufficient for partial restoration of growth of a malK strain of S. typhimurium on maltose. LacK failed to substitute for MalK in regulating the expression of maltose-inducible genes but the hybrid complex MalFGLacK2 was sensitive to inducer exclusion. The lacK gene also complemented a ugpC mutant of Escherichia coli to growth on sn-glycerol-3-phosphate as the phosphate source. Partially purified LacK exhibited a spontaneous ATPase activity comparable to that of MalK. A MalK"-'LacK chimeric protein was isolated (by in vivo recombination) in which the N-terminal 140 amino acids of MalK are fused to residues 141-363 of LacK. The protein substituted for MalK in maltose transport considerably better than LacK. Furthermore, random mutagenesis of the plasmid-borne lacK gene yielded three clones that were superior to wild-type lacK in complementing a malK mutation. Single mutations (V114M or L123F) substantially improved the growth of a malK strain on maltose, whereas a double mutation (L123F, S295N) resulted in growth and transport rates that were indistinguishable from those obtained with MalK. In contrast, the introduction of the single change S295N into LacK had no effect but combination with the V114M mutation led to a further twofold increase in transport activity. These results indicate that a putative helical domain in MalK, encompassing residues 89-140, is crucial for a functional, high-affinity interaction with MalF and MalG.

Rapid stimulation of EAAC1-mediated Na+-dependent L-glutamate transport activity in C6 glioma cells by phorbol ester.

C6 glioma cells were used as a model system to study the regulation of EAAC1-mediated Na(+)-dependent L-[3H]glutamate transport. Although a 30-min preincubation with forskolin had no effect on transport activity, preincubation with phorbol 12-myristate 13-acetate (PMA) increased transport activity two- to threefold. PMA caused a time-dependent and concentration-dependent increase in EAAC1-mediated L-[3H]glutamate transport activity. A 2-min preincubation with PMA was sufficient to cause more than a twofold increase in transport activity and the protein synthesis inhibitor cycloheximide had no effect on the increase. These data suggest that this increase is independent of protein synthesis. The EC50 value of PMA for stimulation of transport activity was 80 nM. Kinetic analyses demonstrated that the increase in transport activity was due to a 2.5-fold increase in Vmax with no change in Km. PMA also increased the transport of the nonmetabolizable analogue, D-[3H] aspartate to the same extent. In parallel assays, PMA did not, however, increase Na(+)-dependent glycine transport activity in C6 glioma. The inactive phorbol ester alpha-phorbol 12,13- didecanoate, did not stimulate L-[3H]glutamate transport activity, and the protein kinase C inhibitor chelerythrine blocked the stimulation caused by PMA. Okadaic acid and cyclosporin A, which are phosphatase inhibitors, had no effect on the stimulation of transport activity caused by PMA. The Ca2+ ionophore A23187 did not act synergistically to increase PMA stimulation. In previous studies, PMA caused a rapid increase in amiloride-sensitive Na(+)/H+ transport activity in C6 glioma. In the present study, pre- and coincubation with amiloride had no effect on the stimulation of transport activity caused by PMA. These studies suggest that activation of protein kinase C causes a rapid increase in EAAC1-mediated transport activity. This rapid increase in Na(+)-dependent L-[3H]-glutamate transport activity may provide a novel mechanism for protection against acute insults to the CNS.

The regulation of L-proline transport by insulin-like growth factor-I in human osteoblast-like SaOS-2 cells.

The effect of insulin-like growth factor-I on amino acid transport was studied by measuring the uptake of tritiated L-proline in the cultured human osteoblast-like SaOS-2 cells. The uptake of L-proline was supported by both transport system A, ASC and Gly and by Na+-dependent amino acid transport system A, and by Na+-independent system L. The initial rate of total L-proline uptake as a function of concentration showed saturation and obeyed Michaelis-Menten kinetics with Michaelis constant (Km) and maximum velocity (Vmax) values of 1.87 mM and 8.89 nmol x (mg protein)-1 x (3 min)-1, respectively. Na+-dependent L-proline uptake was significantly stimulated by insulin-like growth factor-I in a time- and concentration-dependent manner. Kinetic analysis showed that insulin-like growth factor-I enhanced transport activity by increasing the Vmax of transport without significant changes in the affinity (Km) of the carrier for the substrate. The increase in transport activity was significantly reduced by cycloheximide. The stimulated increment above basal L-proline uptake was completely inhibited by alpha-(methylamino) isobutyric acid, suggesting that only system A was affected by insulin-like growth factor-I. Na+-dependent L-proline uptake was also stimulated by insulin-like growth factor-II and insulin-like growth factor-I analogues. The insulin-like growth factor-I-stimulated L-proline uptake was inhibited by one of its binding protein, insulin-like growth factor binding protein-4, in a concentration-dependent manner.

Dietary Modulation of Amino Acid Transport in Rat and Human Liver

Specialized diets enriched in the amino acids glutamine and arginine have been shown to benefit surgical patients. In the liver, glutamine supports glutathione biosynthesis, arginine regulates nitric oxide synthesis, and both of these amino acids serve as precursors for ureagenesis, gluconeogenesis, and acute phase protein synthesis. The effects of a diet enriched with glutamine and arginine on hepatic plasma membrane transport activity have not been studied in humans. We hypothesized that feeding supradietary amounts of these nutrients would enhance the activities of the specific carriers which mediate their transmembrane transport in the liver. We fed surgical patients (n= 8) and rats (n= 6) one of three diets: a) a regular diet, b) an enteral liquid diet containing arginine and glutamine, or c) an enteral diet supplemented with pharmacologic amounts of glutamine and arginine. Diets were isocaloric and were administered for 3 days. Hepatic plasma membrane vesicles were prepared from rat liver and from human wedge biopsies obtained at laparotomy. The transport of glutamine and arginine by rat and human vesicles was assayed. Vesicle integrity and functionality were verified by osmolarity plots, enzyme marker enrichments, and time courses. Provision of both a standard enteral liquid diet and one enriched with glutamine and arginine increased the activities of Systems N (glutamine) and y+(arginine) in rat and human liver compared to a control diet. The diet supplemented with glutamine and arginine was the most effective in increasing transport activity. We conclude that the liver responds to diets enriched with specific amino acids by increasing membrane transport activity. This adaptive response provides essential precursors for hepatocytes which may enhance hepatic synthetic functions during catabolic states. This study provides insights into the mechanisms by which enteral nutrition regulates nutrient transport at the cellular level and may provide a biochemical rationale for the use of formulas which are enriched with conditionally essential nutrients.

Na-Pi cotransport in flounder: same transport system in kidney and intestine.

The cloning of a renal Na-Pi contransport in system from winter flounder (P eudopleuronectes americanus) has recently been reported. We used this information to answer the questions 1) what is the distribution of the transport protein along the nephron? and 2) how are renal and intestinal transporters related? The distribution of the flounder NaPi-II protein was tested using two antisera raised against partial sequences (amino acids 1-14 and 388-441) of the transporter. Antibody-specific fluorescence was detected at the basolateral membrane of epithelial cells in the proximal tubular segment PII. Two clones corresponding to the renal Na-Pi cotransporter were isolated from a flounder intestinal cDNA library. Their functional properties were determined using Xenopus laevis oocytes. The apparent affinities for Pi [Michaelis constant (K(m)) = 0.063 mM] and Na (K(m) = 45.3 mM), as well as the pH dependency (increasing transport activity with increasing pH), showed the same characteristics in both intestinal and the renal systems. Sequence analysis revealed that the two intestinal clones were 100% homologous to the renal cDNA, Flounder NaPi-II-specific immunofluorescence was observed predominantly at the apical membrane on intestinal cross sections. We report the cloning and expression of the first intestinal Na-Pi cotransport system. This transporter belongs to the small group of proteins that exhibit the same function in the apical and the basolateral membranes of different cells.

Translocation of GLUT1 Does Not Account for Elevated Glucose Transport in Glucose-deprived 3T3-L1 Adipocytes

Glucose deprivation increases the rate of glucose transport in 3T3-L1 adipocytes in a protein synthesis-dependent fashion. To determine if translocation of either GLUT1 or GLUT4 is responsible for this phenomenon, we adapted existing fractionation procedures toward isolating 3T3-L1 adipocyte membranes. By Western blot analysis of equal protein, GLUT1 was distributed between plasma membranes, high density "microsomal" membranes, and low density "microsomal" membranes isolated from control cells. GLUT4 comigrated with high density and low density membranes. Glucose deprivation for 12 h did not alter the distribution of either GLUT1 or GLUT4, despite an 8-10-fold increase in glucose transport activity in intact cells. Importantly, increased transport activity was retained in plasma membrane vesicles isolated from glucose-deprived cells. These data show for the first time that the increase in transport activity associated with glucose deprivation does not result from the translocation of either of the glucose transporters known to exist in 3T3-L1 adipocytes. As GLUT4 is excluded from the plasma membrane, these data provide evidence for activation of GLUT1.

Glucocorticoid-Dependent Induction of Interleukin-6 Receptor Expression in Human Hepatocytes Facilitates Interleukin-6 Stimulation of Amino Acid Transport

The authors studied the effects of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) on glutamine and alanine transport in isolated human hepatocytes. They also evaluated the role of dexamethasone in modulating this response and its effects on the expression of the plasma membrane high-affinity IL-6 receptor. Animal studies indicate that cytokines are important mediators of the increased hepatic amino acid uptake that occurs during cancer and sepsis, but studies in human tissues are lacking. The control of transport by cytokines and cytokine receptor expression in the liver may provide a mechanism by which hepatocytes can modulate amino acid availability during catabolic disease states. Human hepatocytes were isolated from wedge biopsy specimens and plated in 24-well trays. Interleukin-6 and TNF-alpha, in combination with the synthetic glucocorticoid dexamethasone, were added to hepatocytes in culture, and the transport of radiolabeled glutamine and alanine was measured. Fluorescent-activated cell sorter (FACS) analysis was used to study the effects of dexamethasone on IL-6 receptor number in the well-differentiated human hepatoma HepG2. Both IL-6 and TNF-alpha exerted a small stimulatory effect on alanine and glutamine transport. Dexamethasone alone did not alter transport rates, but pretreatment of cells augmented the effects of both cytokines on carrier-mediated amino acid uptake. Dexamethasone pretreatment and a combination of IL-6 and TNF-alpha resulted in a greater than twofold increase in transport activity. Fluorescent-activated cell sorter analysis demonstrated that dexamethasone induced a threefold increase in the expression of high-affinity IL-6 receptors. Interleukin-6 and TNF-alpha work coordinately with glucocorticoids to stimulate amino acid uptake in human hepatocytes. Dexamethasone exerts a permissive effect on cytokine-mediated increases in transport by increasing IL-6 receptor expression on the cell surface. It is likely that this upregulation of IL-6 receptors "primes" human liver cells for subsequent stimulation by cytokines. The resulting increase in hepatic amino acid transport provides the liver with substrate to support key metabolic pathways during catabolic states.

Induction of High Affinity Glutamate Transport Activity by Amino Acid Deprivation in Renal Epithelial Cells Does Not Involve an Increase in the Amount of Transporter Protein

In renal epithelial cells amino acid deprivation induces an increase in L-Asp transport with a doubling of the Vmax and no change in Km (4.5 micronM) in a cycloheximide-sensitive process. The induction of sodium-depending L-aspartate transport was inhibited by single amino acids that are metabolized to produce glutamate but not by those that do not produce glutamate. The transaminase inhibitor aminooxyacetate in glutamine-free medium caused a decrease in cell glutamate content and an induction of glutamate transport. In complete medium aminooxyacetate neither decreased cell glutamate nor increased transport activity. These results are consistent with a triggering of induction of transport by low intracellular glutamate concentrations. High affinity glutamate transport in these cells is mediated by the excitatory amino acid carrier 1 (EAAC1) gene product. Western blotting using antibodies to the C-terminal region of EAAC1 showed that there is no increase in the amount of EAAC1 protein on prolonged incubation in amino acid-free medium. Conversely, the induction of high affinity glutamate transport by hyperosmotic shock was accompanied by an increase in EAAC1 protein. It is proposed that low glutamate levels lead to the induction of a putative protein that activates the EAAC1 transporter. A model illustrating such a mechanism is described.

Starvation and endotoxin act independently and synergistically to coordinate hepatic glutamine transport.

Because hepatic glutamine transport is markedly enhanced during critical illness, we tested the hypothesis that nutrient starvation and endotoxemia act coordinately to augment transport activity. Fed or starved (48 hours) rats received Escherichia coli endotoxin (LPS, 10 mg/kg of body weight, intraperitoneally) or saline before hepatocyte isolation for measurement of glutamine transport. Hepatocytes were isolated from fed or fasted rats 4 hours after LPS treatment. [3H]glutamine uptake was measured and normalized to cellular protein. Data (mean +/- standard deviation, three separate determinations) were analyzed by Student's t test and analysis of variance. Starvation induced a 1.6-fold increase in glutamine transport, while LPS treatment of fed rats increased transport activity 2.6-fold. Treatment of fasted animals with LPS induced a sixfold increase in glutamine transport. Kinetically, this effect in endotoxemic starved rats was mediated by both an increase in System N Vmax and the induction of a high affinity System A amino acid carrier which transports glutamine. Starvation and endotoxemia regulate hepatocyte glutamine transport independently and synergistically. This hepatic response provides glutamine and other amino acids to support key metabolic pathways in the liver during critical illness.