Alanine Transport Research Articles

Alanine and leucine transport in unfertilized pig oocytes and early blastocysts

Amino acid transport is facilitated by specific transporters within the plasma membrane of the cell. In mouse oocytes and cleavage-stage conceptus Na(+)-dependent L-alanine and L-leucine transport are nearly undetectable. Sodium-dependent transport via system B0,+ in the mouse conceptus increases greatly between the 8-cell and blastocyst stages. By contrast, data presented here for the pig show that L-alanine and L-leucine transport is mainly Na(+)-dependent in the oocyte; this Na(+)-dependent component of transport becomes undetectable by the blastocyst stage. The Na(+)-dependent component of transport in oocytes is inhibited by BCH (2-aminoendo-bicyclo[2.2.1] hexane-2-carboxylic acid) and L-lysine and thus could be a form of system B0,+. In both oocytes and blastocysts Na(+)-independent L-leucine transport is inhibited by BCH, which is consistent with the presence of system L. The dramatic decrease in Na(+)-dependent amino acid transport activity could occur in pig conceptuses in association with the onset of RNA synthesis during the 4-cell stage. Regardless of the precise time during development at which it occurs, however, this dramatic, developmentally regulated decrease in Na(+)-dependent alanine and leucine transport activity contrasts sharply with the large increase in Na(+)-dependent system B0,+ activity that occurs during preimplantation development of murine conceptuses. Elucidation of the molecular mechanisms by which these changes occur should contribute to an understanding of regulation of gene expression during early development.

Heterogeneity of L-alanine transport systems in brush-border membrane vesicles from rat placenta during late gestation.

The placental uptake of L-alanine was studied by using purified brush-border membrane vesicles from rat trophoblasts. Saturation curves were carried out at 37 degrees C in buffers containing 100 mM (zero-trans)-NaSCN, -NaCl, -KSCN, -KCl, or -N-methyl-D-glucamine gluconate. The uncorrected uptake results were fitted by non-linear regression analysis to an equation involving one diffusional component either one or two saturable Michaelian transport terms. In the presence of NaCl, two distinct L-alanine transport systems were distinguished, named respectively System 1 (S-1; Vm1 about 760 pmol/s per mg of protein; KT1 = 0.5 mM) and System 2 (S-2; Vm2 about 1700 pmol/s per mg; KT2 = 9 mM). In contrast, in the presence of K+ (KCl = KSCN) or in the absence of any alkali-metal ions (N-methyl-D-glucamine gluconate), only one saturable system was apparent, which we identify as S-2. When Na+ is present, S-1, but not S-2, appears to be rheogenic, since its maximal transport capacity significantly increases in the presence of an inside-negative membrane potential, created either by replacing Cl- with the permeant anion thiocyanate (NaSCN > NaCl) or by applying an appropriate K+ gradient and valinomycin. alpha-(Methylamino)isobutyrate (methyl-AIB) appears to be a substrate of S-1, but not of S-2. For reasons that remain to be explained, however, methyl-AIB inhibits S-2. We conclude that S-1 represents a truly Na(+)-dependent mechanism, where Na+ behaves as an obligatory activator, whereas S-2 cannot discriminate between Na+ and K+, although its activity is higher in the presence of alkali-metal ions than in their absence (Na+ = K+ > N-methyl-D-glucammonium ion). S-2 appears to be fully developed 2 days before birth, whereas S-1 undergoes a capacity-type activation between days 19.5 and 21.5 of gestation, i.e. its apparent Vmax. nearly doubles, whereas its KT remains constant.

Brush border membrane proteins in experimental Fanconi's syndrome induced by 4-pentenoate and maleate.

Fanconi's syndrome was investigated using brush border membrane (BBM) vesicles isolated from dog kidney. Sodium-dependent uptake of glucose, phosphate, and amino acids and protein phosphorylation were studied in BBM isolated from normal and from 4-pentenoate- and maleate-treated animals. The time course of D-glucose and phosphate uptake, in BBM vesicles, remained unchanged, indicating that both treatments had no effect on carrier properties, and that permeabilities to these substrates and to sodium were not modified. Furthermore, sodium-dependent transport of alanine, phenylalanine, proline, glycine, and glutamate into vesicles remained unaltered by either treatment. 4-Pentenoate treatment caused modifications of the phosphorylation pattern of BBM proteins: the phosphorylation of two proteins (61 and 74 kDa) was increased and that of two others (48 and 53 kDa) was decreased. Maleate treatment caused an increase in the phosphorylation for the same 61-kDa protein, which was also affected by 4-pentenoate treatment, suggesting that phosphorylation of this protein could be related to a mechanism involved in both 4-pentenoate- and maleate-induced Fanconi's syndrome. These changes were also observed in the presence of sodium fluoride and L-bromotetramisole, indicating that the modification of phosphorylation was not due to a difference in phosphatase activities. These results suggest that Fanconi's syndrome induced by 4-pentenoate or maleate is not caused by an inhibition of BBM Na(+)-dependent transport systems. Our results also suggest that protein phosphorylation may play an important role in the molecular defect involved in Fanconi's syndrome.

Effects of starvation on valine and alanine transport across the intestinal mucosal border in sea bass, Dicentrarchus labrax.

Kinetics of intestinal transport of L-alanine and L-valine (substrates of the A-system and the L-system, respectively, in mammals) across the brush-border membrane in sea bass, Dicentrarchus labrax, were studied on intact mucosa using a short-term uptake technique. When fish were starved for 4-8 weeks, total influx (mucosa-to-cell) of valine fell owing to disappearance or modification of the diffusion component. The maximum influx rate of saturable component increased but its affinity (reflected by the Michaelis constant) decreased. Alanine transport by Na(+)-dependent and diffusion pathways was unchanged after starvation. Fasting also induced an almost 20% decrease in the length of intestinal microvilli.

Amino acid deprivation leads to the emergence of System A activity and the synthesis of a specific membrane glycoprotein in the bovine renal epithelial cell line NBL-1.

1. Amino acid deprivation of confluent monolayers of the bovine renal epithelial cell line NBL-1 causes a stimulation of Na(+)-dependent alanine transport. 2. This stimulation is mediated by a protein-synthesis-dependent induction of 2-(methylamino)isobutyric acid (methyl-AIB)-sensitive alanine transport activity (System A), which was not previously present in these cells. 3. Induction was prevented by the addition of methyl-AIB, alanine or glutamine. 4. Tunicamycin prevented the induction of alanine transport activity. 5. Induction of System A activity was accompanied by incorporation of [3H]mannose into a single membrane protein band of molecular mass 113-140 kDa. 6. These results are consistent with the possibility that induced System A activity in confluent NBL-1 cells is mediated by the synthesis of a 113-140 kDa membrane glycoprotein.

The bovine renal epithelial cell line NBL-1 expresses a broad specificity Na+-dependent neutral amino acid transport system (System B°) similar to that in bovine renal brush border membrane vesicles

(1) In the bovine renal epithelial cell line NBL-1, transport of alanine and glutamine is highly Na+-dependent while the transport of leucine and phenylalanine is also stimulated by Na+ although to a much lesser extent. (2) Na+-dependent alanine transport is insensitive to inhibition by methyl AIB, lysine and glutamate but is inhibited by a range of other neutral amino acids. (3) Inhibition of Na+-dependent alanine transort by glutamine, phebylalanine and leucine is competitive indicating that these amino acids are transported on a common carrier. (4) Amino acid transport in these cells appears to be localised preferentially on the basolateral membrane. (5) The results are consistent with the presence in confluent cells of a broad-specificity Na+-dependent neutral amino acid transport system (System B°) similar to that in bovine BBMV. An Na+-independent system with high activity similar to System L is also present. (6) It is argued that previous results in the literature are consistent with the occurrence of this broad-specificity system in other renal cell lines.

Changes in alanine and glutamine transport during rat red blood cell maturation.

Alanine and glutamine transport have been studied during red blood cell maturation in the rat. Kinetic parameters of Na(+)-dependent L-alanine transport were: Km 0.43 and 1.88 mM and Vmax 158 and 45 nmoles/ml ICW/min for reticulocytes and erythrocytes, respectively. During red cell maturation in the rat there is a loss of capacity and affinity of the system ASC for L-alanine transport. The values for Na(+)-dependent L-glutamine transport in reticulocytes were Km 0.51 mM and Vmax 157 nmoles/ml ICW/min. On the other hand, a total loss of L-glutamine transport mediated by both N and ASC systems is demonstrated in mature red cells. This seems to indicate that during rat red cell maturation the system N disappears. Furthermore, the system ASC specificity in mature cells changes, and glutamine enters the red cell by non-mediated diffusion processes.

Inhibition of sodium-dependent transport systems in LLC-PK1 cells by metabolites of ifosfamide.

Ifosfamide (IF) is an alkylating cytostatic derived from nitrogen mustard. In addition to its well-known urotoxic effects (hemorrhagic cystitis), several cases of Fanconi syndrome following IF therapy have been reported. No information is available concerning the pathomechanisms of this tubulotoxicity. We used the permanent renal epithelial cell line LLC-PK1 in order to investigate whether major metabolites of IF (i.e. 4-OH-IF, acrolein and chloracetyldehyde) induced the transport defects most frequently detected after IF therapy in vivo. LLC-PK1 cells of passages 162-177, grown in plastic culture dishes, were used in a confluent state. Sodium-dependent and independent fluxes of l-[3H]alanine and of D-[3H]glucose were determined by standard techniques. Activities of marker enzymes of apical and basolateral membranes, of mitochondria and of endoplasmic reticulum were determined in cell homogenates. IF itself has no detectable effect on fluxes of l-alanine and D-glucose in LLC-PK1 cells. The IF metabolite 4-OOH-IF induces a clear inhibition of sodium-dependent fluxes of both substrates after a 24-hour exposure of cells to 100 mumol/l of 4-OOH-IF. Chloracetaldehyde induces a biphasic response of sodium-dependent fluxes of l-alanine with increased uptake rates at low concentrations (< 200 mumol/l) and with a short incubation time, while higher concentrations and long exposure of the cells leads to a reduction in sodium coupled transport. Glucose transport is affected in a comparable way, however, in contrast to alanine transport, chloracetaldehyde also stimulates sodium-independent fluxes of glucose. Acrolein is the most toxic substance tested. It severely damages cell monolayers at concentrations beyond 75 mumol/l. Sodium-coupled glucose and alanine transport is inhibited by acrolein at concentrations higher than 50 mumol/l. Sodium-coupled glucose transport is more sensitive to all metabolites tested than alanine transport. While acrolein strongly affects both transport systems, marker enzymes of the apical plasma membrane, i.e. alkaline phosphatase and leucine amino-peptidase, are not significantly inhibited, suggesting a specificity of the toxic effect for the transport proteins. We conclude that LLC-PK1 cells represent a good model for further investigation of the pathogenesis of Fanconi syndrome after IF therapy. Sodium-dependent transport systems are more sensitive to acrolein than other cell surface proteins.

Changes in alanine transport in plasma membrane vesicles from rat liver during the early stages of diethylnitrosamine-induced hepatocarcinogenesis

The transport of L-alanine, a natural substrate of system A, across liver plasma membrane vesicle preparations was modified during the early stages of rat DENA hepatocarcinogenesis. Kinetic studies indicated an increase of the Vmax, with normal Km values, at 30 h in rats undergoing a partial hepatectomy. Normal Vmax and drastically reduced Km values were present using membrane preparations from liver tissue showing enzyme-altered hyperplastic foci and/or preneoplastic nodules. The results suggest that alanine transport is differently affected by initiating and promoting stimuli during rat DENA hepatocarcinogenesis. The changes of the Vmax could be related to the promoting effect of partial hepatectomy on cell proliferation whereas the changes of the affinity constant (Km) could be the result of intrinsic modifications of the transporter in initiated cells.

Functional and structural changes in the jejunum of the rat following cysteamine and stress-induced duodenal ulcer.

The effects of cysteamine and stress-induced duodenal ulcer on the functional and structural properties of the rat jejunum were investigated. The absorptive capacity of the jejunum was determined using alanine as the permeant solute and the single-pass perfusion technique. A statistically significant decrease (p < 0.01) in alanine absorption was observed after 8 h and 3 days of duodenal ulcer induction by stress and cysteamine respectively. However, alanine transport measured 7 days after cysteamine or stress ulcer induction showed no significant change from control values. Cysteamine and stress-induced duodenal ulcer did not show any significant change in water absorption across the jejunum when measured after 8 h, 3 and 7 days of ulcer induction. Microscopically, the jejunum of rats with 3-day cysteamine-induced ulcer exhibited diffuse type of apical derangements with excessive swelling of the villi and progressive degenerative changes. No such changes were noticed on the 7th day nor in the jejunum of the rats with stress-induced duodenal ulcer. The results suggest that cysteamine-induced duodenal ulcer produces an inhibition in the absorptive capacity of the jejunum which is time-dependent and reversible.

Impaired alanine uptake by basolateral liver plasma membrane vesicles from rats consuming ethanol

Chronic ethanol consumption reduces alanine transport by rat basolateral liver plasma membrane (blLPM) vesicles; however, the mechanism for this effect remains uncertain. It may be related to the ethanol-induced changes in blLPM fluidity and lipid composition; alternatively ethanol might reduce the number of transporters in the blLPM. To investigate the effect of blLPM fluidity and lipid composition on Na(+)-dependent alanine uptake these parameters were altered in vitro. Increasing the blLPM fluidity had no effect on Na(+)-dependent alanine uptake by blLPM vesicles or the activity of amino acid transport systems, A and ASC. Because ethanol is known to reduce the blLPM cholesterol content, the influence of altering blLPM cholesterol on alanine transport by these membranes was investigated next. Neither an increase nor a decrease of the cholesterol content of the blLPM altered Na(+)-dependent alanine uptake or the activity of system A or ASC. Finally, the influence of chronic ethanol consumption on the specific binding of [3H]alanine to blLPM was studied. The dissociation constant for alanine binding to blLPM from ethanol-fed rats and their pair-fed controls was similar (1.9 +/- 0.2 vs. 2.0 +/- 0.3 mM); however, the maximal binding capacity for alanine was significantly (P less than 0.05) lower in the blLPM from ethanol-fed rats (316 +/- 53 pmol/mg protein) compared with their pair-fed controls (527 +/- 79 pmol/mg protein). These studies do not support the hypothesis that ethanol-induced changes in blLPM fluidity are responsible for the impaired alanine transport; they do suggest that ethanol may reduce the

ΔpH-Dependent Amino Acid Transport into Plasma Membrane Vesicles Isolated from Sugar Beet (Beta vulgaris L.) Leaves

Proton-coupled aliphatic, neutral amino acid transport was investigated in plasma membrane vesicles isolated from sugar beet (Beta vulgaris L., cv Great Western) leaves. Two neutral amino acid symport systems were resolved based on inter-amino acid transport competition and on large variations in the specific activity of each porter in different species. Competitive inhibition was observed for transport competition between alanine, methionine, glutamine, and leucine (the alanine group) and between isoleucine, valine, and threonine (the isoleucine group). The apparent K(m) and K(i) values were similar for transport competition among amino acids within the alanine group. In contrast, the kinetics of transport competition between these two groups of amino acids did not fit a simple competitive model. Furthermore, members of the isoleucine group were weak transport antagonists of the alanine group. These results are consistent with two independent neutral amino acid porters. In support of that conclusion, the ratio of the specific activity of alanine transport versus isoleucine transport varied from two- to 13-fold in plasma membrane vesicles isolated from different plant species. This ratio would be expected to remain relatively stable if these amino acids were moving through a single transport system and, indeed, the ratio of alanine to glutamine transport varied less than twofold. Analysis of the predicted structure of the aliphatic, neutral amino acids in solution shows that isoleucine, valine, and threonine contain a branched methyl or hydroxyl group at the beta-carbon position that places a dense electron cloud close to the alpha-amino group. This does not occur for the unbranched amino acids or those that branch further away, e.g. leucine. We hypothesize that this structural feature of isoleucine, valine, and threonine results in unfavorable steric interactions with the alanine transport system that limits their flux through this porter. Hydrophobicity and hydrated volumes did not account for the observed differences in transport specificity.

Adaptive regulation of alanine transport in hepatic plasma membrane vesicles from the endotoxin-treated rat

The mechanisms by which hepatic alanine consumption is increased during endotoxemia were investigated to gain further insight into the altered amino acid metabolism which characterizes critical illness. Rats were studied 12 hr after receiving endotoxin (ENDO) or saline. Hepatic alanine delivery was determined in vivo and hepatic alanine content was measured. Hepatocyte transport activity was studied by evaluation of [ 3H]-alanine accumulation in hepatocyte plasma membrane vesicles (HPMVs). Vesicle integrity was demonstrated by electron microscopy and a 14-fold enrichment in 5′-nucleotidase. Endotoxin treatment resulted in a state of hyperalaninemia and a threefold increase in hepatic alanine delivery (2.79 ± 0.17 μmole/100 g body weight/min in controls vs 8.13 ± 0.98 in ENDO animals; P < 0.001). Data from HPMVs revealed the presence of a high- and low-affinity component of alanine transport. Endotoxin treatment resulted in a 30% decrease in the V max of the high-affinity transport component (3355 ± 177 pmole/mg protein/10 sec in controls vs 2338 ± 270 in the ENDO group; P < 0.05). Concomitant with the observed changes in alanine delivery and transport activity, endotoxin treatment resulted in a 56% rise in hepatic alanine content (2.53 ± 0.29 μmole/g liver in controls vs 3.95 ± 0.23 in ENDO; P < 0.005). These data indicate that the accelerated hepatic alanine consumption which occurs during endotoxemia is primarily the result of increased hepatic substrate delivery. Despite the resultant repression of transport activity, delivery begins to outdistance the metabolic capability of the hepatocyte to utilize alanine and intracellular alanine levels rise.

Adaptive regulation of brush-border amino acid transport in a chronic excluded jejunal limb

We examined the alterations in brush-border glutamine transport that occurred in a surgically defunctionalized jejunal limb excluded from mucosal food contact. Dogs were surgically prepared with Roux-en-Y gastrojejunostomies to permit same-intestine comparisons of glutamine transport and glutaminase activity in jejunal segments that were in incontinuity or excluded for a 6-mo period. Transport of glutamine, alanine, and glucose was measured in brush-border membrane vesicles prepared from each intestinal section; membrane marker enzymes were enriched to the same degree in incontinuity and excluded portions. The Na(+)-dependent glutamine cotransport apparent Km was the same in the excluded (779 +/- 63 microM) and incontinuity (873 +/- 105 microM) limbs. However, the Jmax for Na(+)-independent glutamine transport in the incontinuity jejunum (158.7 +/- 15.7 pmol.mg protein-1.s-1) was double that in the excluded limb (71.2 +/- 4.6 pmol.mg protein-1.s-1). Na(+)-dependent carrier-mediated glutamine transport rates were lower than the Na(+)-dependent system, but Na(+)-independent kinetic parameters were not significantly different in incontinuity vs. excluded limbs (Jmax 7.9 +/- 0.6 pmol.mg protein-1.s-1; Km 140 +/- 20 microM). Similarly, the passive diffusion permeability coefficient was the same for both excluded and incontinuity jejunal limbs (22.7 +/- 0.9 nl.mg protein-1.s-1). Mucosal glutaminase enzyme activity was increased by 28% in the incontinuity limb (4.32 +/- 0.21 vs. 3.36 +/- 0.35 mumol.mg protein-1.h-1; P less than 0.02). Transport rates of alanine and glucose were also diminished in the excluded limb (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Fluoride selectively stimulates Na-dependent phosphate transport in osteoblast-like cells.

The influence of fluoride (F) on the transport of Pi was investigated in the osteoblast-like cell line UMR-106. Exposure of cells to F induced a dose-related stimulation of the Na-coupled Pi transport. Pi transport was significantly increased 6 h after 1 mM F incubation, with maximal response observed at 24 h (F 38.0 +/- 2.3, vehicle 19.8 +/- 1.2 pmol.micrograms DNA-1.4 min-1; P less than 0.001). Na-dependent alanine transport was not changed by F. The selective effect of F on Pi transport was not associated with changes in adenosine 3',5'-cyclic monophosphate, cell proliferation, or alkaline phosphatase activity. However, it was completely blunted by inhibiting translational processes with cycloheximide. Furthermore, F enhanced the stimulatory effect on Pi transport of various mitogens such as fetal calf serum, insulin, and insulin-like growth factor I. In conclusion, F can selectively enhance the activity of the Pi transport system present in the plasma membrane of UMR 106 osteoblast-like cells by a mechanism that probably involves newly synthetized proteins.

Brush Border Transport of Glutamine and Other Substrates During Sepsis and Endotoxemia

The effects of severe infection on luminal transport of amino acids and glucose by the small intestine were investigated. Studies were done in endotoxin-treated rats and in septic patients who underwent resection of otherwise normal small bowel. In rats the kinetics of the brush border glutamine transporter and the glutaminase enzyme were examined. In patients the effects of severe infection on the transport of glutamine, alanine, leucine, and glucose were studied. Transport was measured using small intestinal brush border membrane vesicles that were prepared by Mg++ aggregation/differential centrifugation. Uptake of radiolabeled substrate was measured using a rapid mixing/filtration technique. Vesicles demonstrated 15-fold enrichments of enzyme markers, classic overshoots, transport into an osmotically active space, and similar 2-hour equilibrium values. The sodium-dependent pathway accounted for nearly 90% of total carrier-mediated transport. Kinetic studies on rat jejunal glutaminase indicated a decrease in activity as early as 2 hours after endotoxin secondary to a decrease in enzyme affinity for glutamine (Km = 2.23 +/- 0.20 mmol/L [millimolar] in controls versus 4.55 +/- 0.67 in endotoxin, p less than 0.03), rather than a change in Vmax. By 12 hours the decrease in glutaminase activity was due to a decrease in Vmax (222 +/- 36 nmol/mg protein/min in controls versus 96 +/- 16 in endotoxin, p less than 0.03) rather than a significant change in Km. Transport data indicated a decrease in sodium-dependent jejunal glutamine uptake 12 hours after endotoxin secondary to a 35% reduction in maximal transport velocity (Vmax = 325 +/- 12 pmol/mg protein/10 sec in controls versus 214 +/- 8 in endotoxin, p less than 0.0001) with no change in Km (carrier affinity). Sodium-dependent glutamine transport was also decreased in septic patients, both in the jejunum (Vmax for control jejunum = 786 +/- 96 pmol/mg protein/10 sec versus 417 +/- 43 for septic jejunum, p less than 0.01) and in the ileum (Vmax of control ileum = 1126 +/- 66 pmol/mg protein/10 sec versus 415 +/- 24 in septic ileum, p less than 0.001) The rate of jejunal transport of alanine, leucine, and glucose was also decreased in septic patients by 30% to 50% (p less than 0.01). These data suggest that there is a generalized down-regulation of sodium-dependent carrier-mediated substrate transport across the brush border during severe infection, which probably occurs secondary to a decrease in transporter synthesis or an increase in the rate of carrier degradation.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation ofRaja erinacea basolateral liver plasma membranes: Characterization of lipid composition and fluidity

Developing a method for isolating skate (Raja erinacea) basolateral liver plasma membranes, as well as characterizing the lipid composition and fluidity of these membranes, was the primary purpose of this study. Membranes were isolated using self-generating Percoll gradients. Marker enzyme studies indicate that this preparation is highly enriched in the basolateral domain of the liver plasma membrane and largely free of contamination by intracellular organelles or canalicular membranes. Further, these membranes contain the agency responsible for Na(+)-dependent alanine transport. This finding indicates that this membrane preparation will be useful for the study of skate liver plasma membrane transport processes. The lipid composition and fluidity (as assessed by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene) of the skate basolateral liver plasma membrane shows little variation among preparations. Further, DPH anisotropy plotted as a function of temperature yields a straight line (r = 0.99) which indicates that there is no lipid phase change in these membranes from 4 degrees to 37 degrees C. The membrane preparation does contain substantial phospholipase A2 activity. The function of this enzyme is, in part, to modify membrane lipid composition and fluidity in response to temperature variations; therefore, this finding suggests that in situ lipid metabolizing enzymes may play a central role in the adaptation of skate basolateral liver plasma membranes to changes in the ambient temperature.

Selective stimulation of brush border glutamine transport in the tumor-bearing rat

Intestinal extraction of circulating glutamine across the basolateral membrane is diminished in the tumor-bearing rat (TBR). This study was designed to investigate the effects of progressive malignant growth on brush border glutamine transport in order to gain further insight into the adaptive/regulatory changes in intestinal glutamine metabolism that occur in the tumor-bearing rat. Fischer 344 rats (225 ± 5 g) were implanted with fibrosarcoma cells and were studied at various time points after implantation when the tumors comprised 7%, 20%, and 29% of total body weight. Control and tumor-bearing rats were pair-fed throughout the study. Jejunal brush border membrane vesicles (BBMVs) were prepared by magnesium aggregation/differential centrifugation and transport of radioactively labeled l-glutamine, l-leucine, l-alanine, and d-glucose by BBMVs was measured using a Millipore filtration technique. BBMVs were enriched 15-fold in alkaline phosphatase, indicating brush border vesicle purity. Uptake of all substrates occurred into an osmotically active space, exhibited overshoots, and had similar 1-hr equilibrium values. The rate of glutamine uptake by BBMVs from all tumor-bearing rats was significantly greater than controls, regardless of tumor size. The increase in transport activity was not due to a change in carrier affinity but rather to an increase in maximal transport velocity. In rats with small tumors (7% of body weight), the V max was 431 ± 40 pmole/mg protein/10 sec compared to 259 ± 30 in control animals ( P < 0.01). In marked contrast, the mean transport of alanine was diminished in BBMVs from TBR (31 ± 3 pmole/mg protein/10 sec in TBR vs 23 ± 2 in controls, P < 0.05). There was no significant difference in glucose and leucine transport between control and TBR. The results suggest that the upregulation in brush border transport that occurs in this tumor model may be specific for glutamine, indicating the high cellular priority and requirement for glutamine. Such adaptive regulation in brush border transport activity in tumor-bearing animals has not been previously described.

Sodium-dependent phosphate and alanine transports but sodium-independent hexose transport in type II alveolar epithelial cells in primary culture

Inorganic phosphate, amino acids and sugars are of obvious importance in lung metabolism. We investigated sodium-coupled transports with these organic and inorganic substrates in type II alveolar epithelial cells from adult rat after one day in culture. Alveolar type II cells actively transported inorganic phosphate and alanine, a neutral amino acid, by sodium-dependent processes. Cellular uptakes of phosphate and alanine were decreased by about 80% by external sodium substitution, inhibited by ouabain (30 and 41%, respectively) and displayed saturable kinetics. Two sodium-phosphate cotransport systems were characterized: a high-affinity one (apparent K m = 18 μM) with a V max of 13.5 nmol/mg protein per 10 min and a low-affinity one (apparent K m = 126 μM) with a V max of 22.5 nmol/mg protein per 10 min. Alanine transport had an apparent K m of 87.9 μM and a V max of 43.5 nmol/mg protein per 10 min. By contrast, cultured alveolar type II cells did not express sodium-dependent hexose transport. Increasing time in culture decreased V max values of the two phosphate transport systems in day 4 while sodium-dependent alanine uptake was unchanged. This study demonstrated the existence of sodium-dependent phosphate and amino acid transports in alveolar type II cells similar to those documented in other epithelial cell types. These sodium-coupled transports provide a potent mechanism for phosphate and amino acid absorption and are likely to play a role in substrate availability for cellular metabolism and in regulating the composition of the alveolar subphase. The decrease in phosphate uptake with time in culture is parallel to decrease in surfactant synthesis reported in cultured alveolar type II cells, suggesting that phosphate availability for surfactant synthesis may be accomplished by a sodium-dependent phosphate uptake.

Regulation of basolateral membrane potential after stimulation of Na+ transport in proximal tubules

We have previously shown that stimulation of apical Na-coupled glucose and alanine transport produces a transient depolarization of basolateral membrane potential (Vbl) in rabbit proximal convoluted tubule (PCT, S1 segment). The present study is aimed at understanding the origin of the membrane repolarization following the initial effect of addition of luminal cotransported solutes. Luminal addition of 10-15 mM L-alanine produced a rapid and highly significant depolarization of Vbl (20.3 +/- 1.1 mV, n = 15) which was transient and associated with an increase in the fractional K+ conductance of the basolateral membrane (tK) from 8 to 29% (P less than 0.01, n = 6). Despite the significant increase in tK, the repolarization was only slightly reduced by the presence of basolateral Ba2+ (2 mM, n = 6) or quinine (0.5 mM, n = 5). The repolarization was greatly reduced in the presence of 0.1 mM 4-acetamino-4'isothiocyamostilbene-2,2'-disulfonic acid (SITS) and blunted by bicarbonate-free solutions. Intracellular pH (pHi) determined with the fluorescent dye 2',7'-bis-2-carboxyethyl-5(and -6)-carboxyfluorescein (BCECF), averaged 7.39 +/- 0.02 in control solution (n = 9) and increased to 7.50 +/- 0.03 in the first 15 sec after the luminal application of alanine. This was followed by a significant acidification averaging 0.16 +/- 0.01 pH unit in the next 3 min. In conclusion, we believe that, contrary to other leaky epithelia, rabbit PCT can regulate its basolateral membrane potential not only through an increase in K+ conductance but also through a cellular acidification reducing the basolateral HCO3- exit through the electrogenic Na-3(HCO3) cotransport mechanism.