Identification of a H+/glucose and galactose symporter gene glt from Xanthomonas oryzae pv. oryzae

We identified a glucose and galactose transporter gene from the plant-pathogenic bacterium Xanthomonas oryzae pv. oryzae. Sequence analysis indicated that the gene, named glt, encoded a polypeptide of 592 amino acid residues and the product was significantly homologous with members of the Na+/glucose cotransporter (SGLT) family from mammalian and bacterial origin, especially with vSGLT from Vibrio parahaemolyticus (50% identity). GLT functioned as a glucose and galactose transporter in an Escherichia coli mutant deficient in glucose and galactose transport activity. A protonophore inhibited the transport activity, suggesting that GLT is a H+-coupled glucose/galactose symporter.     

Developmentally regulated 75-kilodalton protein expressed in LLC-PK1 cultures is a component of the renal Na+/glucose cotransport system

Na+/D-glucose symport is a secondary active glucose transport mechanism expressed only in kidney proximal tubule and in small intestine. A monoclonal antibody that recognized the Na+/glucose symporter of pig renal brush border membranes also recognized a 75-kD protein in apical membranes isolated from highly differentiated LLC-PK1 cultures, an epithelial cell line of pig renal proximal tubule origin. The 75-kD antigen was enriched from solubilized LLC-PK1 apical membranes by means of high-pressure liquid chromatography. The symporter antigen became apparent on the apical membrane surface after the development of a confluent monolayer in correlation with the expression of transport activity. Long-term treatment of cultures with the differentiation inducer hexamethylene bisacetamide was accompanied by a dramatically increased expression of the symporter antigen as detected quantitatively by Western blot analysis and qualitatively by immunofluorescence staining. The number of symporter-positive cells was dramatically increased after inducer treatment as predicted for differentiation-regulated expression. These results identify a 75-kD protein as a component of a developmentally regulated renal Na+/glucose symporter expressed in cell culture.     

Expression of the thyroid sodium/iodide symporter in Xenopus laevis oocytes

Poly(A+) RNA isolated from FRTL-5 cells (a continuous line of cultured and fully functional rat thyroid cells (Ambesi-Impiombato, F. S., Parks, L. A. M., and Coons, H. G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 3455-3459] was injected into Xenopus laevis oocytes, and the expression of the Na+/I- symporter in the plasma membrane was assayed by measuring the Na+-dependent ClO4--sensitive uptake of 125I. Expression of the Na+/I- symporter was detected as a 7-fold average increase in transport over background, 5-6 days after injection. Poly(A+) RNA was subsequently fractionated by sucrose gradient centrifugation, and fractions were assayed for their ability to induce I- transport activity. The poly(A+) RNA encoding the Na+/I- symporter was found in a fraction containing messages of 2.8-4.0 kilobases in length.     

Transport energetics of the Na+ pump in Aplysia californica gut

Basolateral membranes of Aplysia californica foregut epithelia contain an ATP-dependent Na+ transporter (Na+ pump). Increased activity of the Na+ pump, coupled to luminal Na+/AIB symporter activity and basolateral membrane depolarization, changed the Na+ transport energetics across the basolateral membrane to a greater extent than the change in Na+ transport energetics across the luminal membrane.     

Genes involved in control of galactose uptake in Lactobacillus brevis and reconstitution of the regulatory system in Bacillus subtilis

The heterofermentative lactic acid bacterium Lactobacillus brevis transports galactose and the nonmetabolizable galactose analogue thiomethyl-beta-galactoside (TMG) by a permease-catalyzed sugar:H(+) symport mechanism. Addition of glucose to L. brevis cells loaded with [(14)C]TMG promotes efflux and prevents accumulation of the galactoside, probably by converting the proton symporter into a uniporter. Such a process manifests itself physiologically in phenomena termed inducer expulsion and exclusion. Previous evidence suggested a direct allosteric mechanism whereby the phosphocarrier protein, HPr, phosphorylated at serine-46 [HPr(Ser-P)], binds to the galactose:H(+) symporter to uncouple sugar transport from proton symport. To elucidate the molecular mechanism of inducer control in L. brevis, we have cloned the genes encoding the HPr(Ser) kinase, HPr, enzyme I, and the galactose:H(+) symporter. The sequences of these genes were determined, and the relevant phylogenetic trees are presented. Mutant HPr derivatives in which the regulatory serine was changed to either alanine or aspartate were constructed. The cloned galP gene was integrated into the chromosome of Bacillus subtilis, and synthesis of the mutant HPr proteins in this organism was shown to promote regulation of GalP, as expected for a direct allosteric mechanism. We have thus reconstituted inducer control in an organism that does not otherwise exhibit this phenomenon. These results are consistent with the conclusion that inducer exclusion and expulsion in L. brevis operates via a multicomponent signal transduction mechanism wherein the presence of glycolytic intermediates such as fructose 1,6-bisphosphate (the intracellular effector), derived from exogenous glucose (the extracellular effector), activates HPr(Ser) kinase (the sensor) to phosphorylate HPr on Ser-46 (the messenger), which binds to the galactose:H(+) symporter (the target), resulting in uncoupling of sugar transport from proton symport (the response). This cascade allows bacteria to quickly respond to changes in external sugar concentrations. Understanding the molecular mechanism of inducer control advances our knowledge of the link between metabolic and transport processes in bacteria.     

Baculovirus-mediated expression of the Na+/glucose cotransporter in Sf9 cells.

We have used baculovirus (AcNPV) to express the Na+/glucose cotransporter protein in cultured Sf9 cells. We constructed a baculovirus transfer vector containing the cDNA for the rabbit intestinal Na+/glucose cotransporter (SGLT1) under the control of the polyhedrin gene promoter. Recombinant baculovirus was obtained by cotransfection of SF9 cells with wild-type AcNPV DNA and the transfer vector. Recombinant virus was identified by Southern blotting and then purified. Recombinant infected Sf9 cells expressed a protein which was recognized by anti-peptide antibodies raised to sequences of the cloned Na+/glucose cotransporter. This protein migrated with a molecular mass of 55 kD by SDS-PAGE, similar to the in vitro translation product of SGLT1. An identical protein was metabolically labeled with [35S]methionine. Cells which synthesized the transport protein showed Na(+)-dependent alpha MeGlc transport. Micromolar phlorizin inhibited transport. Uninfected and wild-type virus infected Sf9 cells did not have Na(+)-dependent glucose transport. All transport protein migrated at 45% sucrose (w/w) by density gradient sedimentation, suggesting that the expressed transporter is membrane associated. We conclude that we have functionally expressed the rabbit intestinal Na+/glucose cotransporter in Sf9 cells. The transporter is not heavily glycosylated, and this is consistent with previous work showing that glycosylation is not necessary for function. We are poised to purify and characterize this protein from a structure-function perspective.     

Characterization of a glucose transport system in Vibrio parahaemolyticus.

Cells of a glucose-PTS (phosphoenolpyruvate:carbohydrate phosphotransferase system)-negative mutant of Vibrio parahaemolyticus transport D-glucose in the presence of Na+. Maximum stimulation of D-glucose transport was observed at 40 mM NaCl, and Na+ could be replaced partially with Li+. Addition of D-glucose to the cell suspension under anaerobic conditions elicited Na+ uptake. Thus, we conclude that glucose is transported by a Na+/glucose symport mechanism. Calculated Vmax and Km values for the Na(+)-dependent D-glucose transport were 15 nmol/min/mg of protein and 0.57 mM, respectively, when NaCl was added at 40 mM. Na+ lowered the Km value without affecting the Vmax value. D-Glucose was the best substrate for this transport system, followed by galactose, alpha-D-fucose, and methyl-alpha-glucoside, judging from the inhibition pattern of the glucose transport. D-Glucose itself partly repressed the transport system when cells were grown in its presence.     

Monoclonal antibodies that bind the renal Na+/glucose symport system. 1. Identification

Phlorizin is a specific, high-affinity ligand that binds the active site of the Na+/glucose symporter by a Na+-dependent mechanism but is not itself transported across the membrane. We have isolated a panel of monoclonal antibodies that influence high-affinity, Na+-dependent phlorizin binding to pig renal brush border membranes. Antibodies were derived after immunization of mice either with highly purified renal brush border membranes or with apical membranes purified from LLC-PK1, a cell line of pig renal proximal tubule origin. Antibody 11A3D6, an IgG2b, reproducibly stimulated Na+-dependent phlorizin binding whereas antibody 18H10B12, an IgM, strongly inhibited specific binding. These effects were maximal after 30-min incubation and exhibited saturation at increased antibody concentrations. Antibodies did not affect Na+-dependent sugar uptake in vesicles but significantly prevented transport inhibition by bound phlorizin. Antibodies recognized a 75-kDa antigen identified by Western blot analysis of brush border membranes, and a 75-kDa membrane protein could be immunoprecipitated by 18H10B12. These properties, taken together with results in the following paper [Wu, J.-S.R., & Lever, J.E. (1987) Biochemistry (following paper in this issue)], provide compelling evidence that the 75-kDa antigen recognized by these antibodies is a component of the renal Na+/glucose symporter.     

Inhibition of the Na+/I- symporter by harmaline and 3-amino-1-methyl-5H-pyrido(4,3-b)indole acetate in thyroid cells and membrane vesicles

Novel inhibitors of the Na+/I- symporter were identified using rat-thyroid-derived FRTL-5 cells and sealed vesicles from calf thyroid as model systems. Na(+)-dependent 125I- uptake was inhibited by the hallucinogenic drug harmaline and by a chemically related convulsive agent, 3-amino-1-methyl- 5H-pyrido(4,3-b)indole acetate (TRP-P-2). TRP-P-2 (Ki = 0.25 mM) was tenfold more effective as an inhibitor than harmaline (Ki = 4.0 mM). Inhibition by TRP-P-2 was competitive with respect to Na+ and was fully reversible. Although TRP-P-2 is a relatively low-affinity inhibitor, its affinity for the Na+ site of the Na+/I- symporter is over 100 times higher than that of Na+ (Km = 50 mM). 45Ca(2+)-efflux rates in calf thyroid membrane vesicles were not affected by TRP-P-2, indicating that membrane integrity is not disrupted by the drug. These findings show that TRP-P-2 may be a potentially useful tool for the identification and characterization of the Na+/I- symporter.     

Purification and reconstitution of a 75-kilodalton protein identified as a component of the renal Na+/glucose symporter

A 75-kilodalton (kDa) protein was purified from solubilized renal brush border membranes by using high-pressure liquid chromatography (HPLC) and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Functional and immunological properties identified the 75-kDa protein as a component of the Na+/glucose symport system. The purified protein was specifically recognized by a monoclonal antibody that functionally interacts with the Na+/glucose symporter. Na+-dependent phlorizin binding activity was associated with fractions containing the 75-kDa protein during HPLC fractionation on the anion exchanger Mono-Q and was greatly increased after reconstitution into egg yolk phosphatidylcholine vesicles. The final purified preparation contained glucosamine and a blocked N-terminus.     

Sodium-induced conformational changes in the glucose transporter of intestinal brush borders

Using brush-border membranes prepared from rabbit small intestine by Ca2+ precipitation and KSCN treatment, we have studied the kinetics and conformational changes of the glucose carrier. Na+ behaves as a competitive activator of glucose transport under zero-trans conditions. Phenyl isothiocyanate and fluorescein isothiocyanate (FITC) inhibit Na+-dependent transport in an irreversible but substrate-protectable manner. Vesicles pretreated with phenyl isothiocyanate in the presence of substrates were then selectively labeled at the glucose carrier with FITC. Competition experiments with Na+ and phlorizin or glucose indicated that FITC binds to the glucose site on the carrier. Carrier-bound FITC displays a saturable quenching of fluorescence in the presence of Na+. The K0.5 of the Na+-specific quench is 25 mM, which is similar to the apparent Km for Na+ activation of glucose transport. Two tyrosine group-specific reagents, N-acetylimidazole and tetranitromethane, inhibit glucose uptake and fluorescent quenching in a Na+-protectable fashion. FITC labeled a 75-kilodalton peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a substrate-sensitive manner. We conclude that Na+ binds to the glucose symporter of intestinal brush borders, a 75-kilodalton peptide, and this induces a rapid conformation change in the transporter which increases its affinity for D-glucose.     

Expression and characterization of the intestinal Na+/glucose cotransporter in COS-7 cells

Cells derived from the simian kidney, COS-7 cells, were transfected with a eucaryotic expression vector (pEUK-C1) containing the clone for the rabbit intestinal Na+/glucose cotransporter. Expression was monitored after transfection with lipofectin by measuring the initial rate of alpha-methylglucopyranoside (MeGlc) uptake. Cells transfected with vector containing the cDNA for the Na+/glucose cotransporter expressed Na(+)-dependent MeGlc transport. Neither control cells nor cells transfected with vector lacking cloned cDNA expressed the cotransporter. Na(+)-dependent MeGlc uptake into transfected cells was saturable (Km 150 microM), phlorizin-sensitive (Ki 11 microM), and inhibited by sugar analogs (D-glucose greater than MeGlc greater than D-galactose greater than 3-O-methyl-D-glucoside greater than D-allose much greater than L-glucose). Europium was able to mimic Na+ in driving MeGIC uptake. Finally, tunicamycin, an inhibitor of asparagine-linked glycosylation, inhibited the expression of Na(+)-dependent MeGlc transport 80%. We conclude that the rabbit intestinal Na+/glucose cotransporter expressed in COS-7 cell exhibits very similar kinetic properties to that in the native brush border and to that expressed in Xenopus oocytes. In addition, N-linked glycosylation appears to be important for functional expression of this membrane protein.     

Insulin-stimulated glucose transport in rat adipose cells. Modulation of transporter intrinsic activity by isoproterenol and adenosine

The mechanism of modulation of insulin-stimulated glucose transport activity in isolated rat adipose cells by lipolytic and antilipolytic agents has been examined. We have measured glucose transport activity in intact cells with 3-O-methylglucose and in plasma membranes with D-glucose, and the concentration of glucose transporters in plasma membranes using a cytochalasin B binding assay. In intact cells, isoproterenol reduced insulin-stimulated transport activity by 60%. This effect was lost after cooling and washing the cells with homogenization buffer, and neither the concentration of glucose transporters nor transport activity in the plasma membranes differed from control. However, treatment of cells with KCN prior to homogenization preserved the isoproterenol effect through the fractionation procedure. Plasma membranes from these cells contained an unchanged number of transporters (31 +/- 7, mean +/- S.E., versus 31 +/- 4 pmol/mg of protein in controls) but transported glucose at a reduced rate (19 +/- 6 versus 48 +/- 9 pmol/mg of protein/s). Conversely, incubation of intact cells in the presence of adenosine stimulated plasma membrane glucose transport activity compared to that in the absence of adenosine (44 +/- 6 versus 36 +/- 6 pmol/mg of protein/s). Kinetic studies of isoproterenol-inhibited glucose transport in plasma membranes revealed a 60% decrease in Vmax (2900 +/- 350 versus 7200 +/- 1000 pmol/mg of protein/s) and a small increase in Km (15.1 +/- 1 versus 13.0 +/- 0.6 mM). These data indicate that modifications of glucose transport activity produced by lipolytic and antilipolytic agents in intact adipose cells can be fully retained in plasma membranes isolated under appropriate conditions. Furthermore, the effects of these agents occur through a modification of the glucose transporter intrinsic activity.     

The mechanism of insulin stimulation of (Na+,K+)-ATPase transport activity in muscle

Since the mechanism underlying the insulin stimulation of (Na+,K+)-ATPase transport activity observed in multiple tissues has remained undetermined, we have examined (Na+,K+)-ATPase transport activity (ouabain-sensitive 86Rb+ uptake) and Na+/H+ exchange transport (amiloride-sensitive 22Na+ influx) in differentiated BC3H-1 cultured myocytes as a model of insulin action in muscle. The active uptake of 86Rb+ was sensitive to physiological insulin concentrations (1 nM), yielding a maximum increase of 60% without any change in 86Rb+ permeability. In order to determine the mechanism of insulin stimulation of (Na+,K+)-ATPase activity, we demonstrated that insulin also stimulates passive 22Na+ influx by Na+/H+ exchange transport (maximal 200% increase) and an 80% increase in intracellular Na+ concentration with an identical time course and dose-response curve as insulin-stimulated (Na+,K+)-ATPase transport activity. Incubation of the cells with high [Na+] (195 mM) significantly potentiated insulin stimulation of ouabain-inhibitable 86Rb+ uptake. The ionophore monensin, which also promotes passive Na+ entry into BC3H-1 cells, mimics the insulin stimulation of ouabain-inhibitable 86Rb+ uptake. In contrast, incubation with amiloride or low [Na+] (10 mM), both of which inhibit Na+/H+ exchange transport, abolished the insulin stimulation of (Na+,K+)-ATPase transport activity. Furthermore, each of these insulin-stimulated transport activities displayed a similar sensitivity to amiloride. These results indicate that insulin stimulates a large increase in Na+/H+ exchange transport and that the resulting Na+ influx increases the intracellular Na+ concentration, thus activating the internal Na+ transport sites of the (Na+,K+)-ATPase. This Na+ influx is, therefore, the mediator of the insulin-induced stimulation of membrane (Na+,K+)-ATPase transport activity classically observed in muscle.     

Utilization and transport of glucose in Olea Europaea cell suspensions

Cell suspensions of Olea europaea var. Galega Vulgar grown in batch culture with 0.5% (w/v) glucose were able to transport D-[(14)C]glucose according to Michaelis-Menten kinetics associated with a first-order kinetics. The monosaccharide carrier exhibited high affinity (K(m) approximately 50 micro M) and was able to transport D-glucose, D-fructose, D-galactose, D-xylose, 2-deoxy-D-glucose and 3-O-methyl-D-glucose, but not D-arabinose, D-mannitol or L-glucose. D-[(14)C]glucose uptake was associated with proton uptake, which also followed Michaelis-Menten kinetics. The transport of 3-O-methyl-D-glucose was accumulative (40-fold, at pH 5.0) and the protonophore carbonyl cyanide m-chlorophenylhydrazone strongly inhibited sugar accumulation. The results were consistent with the involvement of a monosaccharide: proton symporter with a stoichiometry of 1 : 1. When cells were grown with 3% (w/v) glucose, the uptake of D-[(14)C]glucose followed first-order kinetics and monosaccharide:proton symporter activity was not detected. The value obtained for the permeability coefficient of hexoses in O. europaea cells supported the hypothesis that the first-order kinetics observed in 0.5% and 3% sugar-grown cells was produced exclusively by passive diffusion of the sugar. The results indicate that in O. europaea cells sugar levels have a regulatory effect on sugar transport, because the activity for monosaccharide transport was repressed by high sugar concentrations.     

Insulin action on cardiac glucose transport. Studies on the role of the Na+/K+ pump

Isolated muscle cells from adult rat heart have been used to study the relationship between myocardial glucose transport and the activity of the Na+/K+ pump. 86Rb+-uptake by cardiac cells was found to be linear up to 2 min with a steady-state reached by 40-60 min, and was used to monitor the activity of the Na+/K+ pump. Ouabain (10(-3) mol/l) inhibited the steady-state uptake of 86Rb+ by more than 90%. Both, the ouabain-sensitive and ouabain-insensitive 86Rb+-uptake by cardiac cells were found to be unaffected by insulin treatment under conditions where a significant stimulation of 3-O-methylglucose transport occurred. 86Rb+-uptake was markedly reduced by the presence of calcium and/or magnesium, but remained unresponsive towards insulin treatment. Inhibition of the Na+/K+ pump activity by ouabain and a concomitant shift in the intracellular Na+ :K+ ratio did not affect basal or insulin stimulated rates of 3-O-methylglucose transport in cardiac myocytes. The data argue against a functional relationship between the myocardial Na+/K+ pump and the glucose transport system.     

Functional reconstitution of SdcS, a Na+-coupled dicarboxylate carrier protein from Staphylococcus aureus

In Staphylococcus aureus, the transport of dicarboxylates is mediated in part by the Na+-linked carrier protein SdcS. This transporter is a member of the divalent-anion/Na+ symporter (DASS) family, a group that includes the mammalian Na+/dicarboxylate cotransporters NaDC1 and NaDC3. In earlier work, we cloned and expressed SdcS in Escherichia coli and found it to have transport properties similar to those of its eukaryotic counterparts (J. A. Hall and A. M. Pajor, J. Bacteriol. 187:5189-5194, 2005). Here, we report the partial purification and subsequent reconstitution of functional SdcS into liposomes. These proteoliposomes exhibited succinate counterflow activity, as well as Na+ electrochemical-gradient-driven transport. Examination of substrate specificity indicated that the minimal requirement necessary for transport was a four-carbon terminal dicarboxylate backbone and that productive substrate-transporter interaction was sensitive to substitutions at the substrate C-2 and C-3 positions. Further analysis established that SdcS facilitates an electroneutral symport reaction having a 2:1 cation/dicarboxylate ratio. This study represents the first characterization of a reconstituted Na+-coupled DASS family member, thus providing an effective method to evaluate functional, as well as structural, aspects of DASS transporters in a system free of the complexities and constraints associated with native membrane environments.     

Increased Na/H antiporter and Na/3HCO3 symporter activities in chronic hyperfiltration. A model of cell hypertrophy

The effect of chronic hyperfiltration, a model of cell hypertrophy, on H/HCO3 transporters was examined in the in vivo microperfused rat proximal tubule. Hyperfiltration was induced by uninephrectomy with subsequent increased dietary protein. After 2 wk the hyperfiltration group had a higher glomerular filtration rate (2.21 +/- 0.13 vs. 1.48 +/- 0.12 ml/min), associated with increased kidney weight (1.71 +/- 0.05 vs. 1.23 +/- 0.04 g). HCO3 absorptive rate measured in tubules perfused with an ultrafiltrate-like solution (25 mM HCO3) was higher in the hyperfiltration group (183 +/- 17 vs. 109 +/- 16 pmol/mm per min). The activities of the apical membrane Na/H antiporter and basolateral membrane Na/3HCO3 symporter were assayed using the measurement of cell pH [(2'7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein] in the doubly microperfused tubule in the absence of contact with native fluids. After 2 wk of hyperfiltration Na/H antiporter activity, assayed as the effect of luminal Na removal on cell pH, was increased 114%. Basolateral membrane Na/3HCO3 symporter activity, assayed as the effect of a decrease in peritubular [HCO3] (25 to 5 mM) or in peritubular [Na] (147 to 25 mM) in the absence of luminal and peritubular chloride, was increased 77 and 113%, respectively, in the hyperfiltration group. Steady-state cell pH, measured with physiologic, ultrafiltrate-like luminal and peritubular perfusates, was significantly higher in the hyperfiltration group (7.27 +/- 0.02 vs. 7.14 +/- 0.03). In similar studies, performed 24 h after uninephrectomy and protein feeding, kidney weight was increased 10%, Na/H antiporter activity 39%, and Na/3HCO3 symporter activity 46%. At this time cell pH was not different between the two groups. The results demonstrate that chronic hyperfiltration is associated with parallel increases in Na/H antiporter and Na/3HCO3 symporter activities. If a decrease in cell pH is the signal that triggers these adaptations, it occurs early, and the adaptations can be maintained in the absence of sustained cell acidification.     

Intestinal glucose and galactose transport in the cultured gilthead bream (Sparus aurata)

1. Electrical parameters and transepithelial glucose and galactose transport were determined in vitro across anterior and posterior intestine of the culture fish Sparus aurata. 2. Electrical potential difference (PD) and short-circuit current (Isc) were serosa-positive in anterior intestine, while they were serosa-negative or near zero in posterior intestine. 3. Tissue conductance (Gt) was higher in posterior than in anterior intestine. In both parts it was decreased when the Na ion was omitted in mucosal and serosal reservoirs. 4. Addition of glucose or galactose to the mucosal side of intestine caused an increase in PD and Isc in posterior intestine but did not significantly change PD and Isc in anterior intestine. 5. Isotopic flux of glucose and galactose measurements in short-circuit conditions showed a net active glucose and galactose absorption in posterior intestine, while in anterior intestine active transport of glucose or galactose was not observed. 6. The net transport of glucose and galactose in posterior intestine was decreased to zero in the absence of Na in mucosal and serosal reservoirs or in the presence of ouabain (1 mM) in serosal solution.     

Alterations in jejunal transport and (Na+-K+)-ATPase in an experimental model of hypoxia in rats

Hypoxia was induced by exposing rats to an atmosphere of 93% N2, 7% O2 for 4-48 hr. The animals became hypoxic as indicated by a decreased blood PaO2 (mean +/- SEM: 48 +/- 10 mm Hg). Hypoxia was accompanied by metabolic acidosis (pH 7.22 +/- 0.02) and decreased serum bicarbonate levels (9.0 +/- 4.0 meq/liter). Hypoxic rats also showed evidence of tissue hypoxia; liver tryptophan oxygenase levels were increased to 21 +/- 2 nmole/min/mg protein. In the hypoxic animals there was decreased jejunal mucosal (Na+-K+)-ATPase activity and an inhibition of active intestinal transport of sodium, glucose, 3-O-methylglucose, galactose, tyrosine, phenylalanine, and glycine as determined by in vivo perfusion studies. Jejunal fructose transport, which has a large passive component, was unaffected by hypoxia. The electrolyte, carbohydrate, and amino acid transport alterations produced by hypoxia were seen in the absence of an effect on jejunal cell number, DNA synthesis, or cell turnover. There was also no evidence of histological or ultrastructural damage. Furthermore, studies with a luminal macromolecular tracer, horseradish peroxidase, indicated that the jejunal lumen-to-blood barrier to macromolecules was also unaltered in these hypoxic animals. In vitro local oxygenation of the jejunum, by bubbling of 95% O2:5% CO2, markedly improved sodium and glucose (but not 3-O-methylglucose) absorption in hypoxic rats and control rats. The (Na+-K+)-ATPase activity of the jejunal mucosa of hypoxic rats was significantly enhanced by the local bubbling of 95% O2:5% CO2. Overall, our data indicate that during relatively mild conditions of hypoxia there is an inhibition of jejunal (Na+-K+)-ATPase activity and related transport processes that is prevented by in situ oxygenation.