Characterization of the high-affinity uptake of fructose-1,6-bisphosphate by cardiac myocytes

Previously, we reported that fructose-1,6-bisphosphate (FBP) was taken up by rat cardiac myocytes by two processes: a component that was saturable at micromolar levels and a nonsaturable component that dominated at millimolar levels. Here, we continued to characterize the saturable high-affinity component, with the aim of identifying the physiological substrate and role for this activity. ATP, ADP, and AMP inhibited the uptake of FBP with apparent affinities of 0.2-0.5 mM. Fumarate and succinate were very weak inhibitors. Several phosphorylated sugars (ribulose-1,5-phosphate, fructose-1-phosphate, ribose-5-phosphate, and inositol-2-phosphate) inhibited FBP uptake with apparent affinities of 40-500 μM. As in our previous study, no tested compound appeared to bind as well as FBP. The data suggest that the best ligands have two phosphoryl groups separated by at least 8 ?. The rates of FBP uptake were measured from 3° to 37°. The calculated activation energy was 15-50 kJ/mol, similar to other membrane transport processes. Uptake of FBP was tested in several types of cells other than cardiac myocytes, and compared to the uptake of 2-deoxyglucose and L: -glucose. While FBP uptake in excess of that of L: -glucose was observed in some cells, in no case was the uptake as high as in cardiac myocytes. The physiological substrate and role for the high-affinity FBP uptake activity remain unknown.     

Saturable and nonsaturable process of sugar uptake: effect of oncogenic transformation in transport and uptake of nutrients.

Uptake of sugars into cells by a saturable process increased enormously during and after transformation, and uptake by a nonsaturable process increased significantly but less remarkably compared to controls. The drastic change of uptake rates, observed at around 5 x 10(-3) M sugar during and after transformation, emphasizes the significant observation that transition of the sugar uptake system from a saturable to a nonsaturable process occurs near the physiological concentration of D-glucose normally seen in animal blood. At concentrations below higher than 5 x 10(-3) M, where a saturable process is barely involved, nonsaturable uptakes of D-glucose, D-mannose, D-galactose, 2-deoxy-D-glucose and 3-O-methyl-D-glucose proceed tens to hundreds fold faster than the rate of simple diffusion of L-glucose. These findings suggest that nonsaturable uptake of the sugars known to be substrates for the saturable transport carrier system may not be a physical process or simple diffusion, as observed for L-glucose uptake. Rather, the nonsaturable uptake might be part of the total physiological process which, along with the saturable process, is controlled by a membrane-coordination mechanism. A plausible mechanism is discussed in which negative cooperativity of nutrient uptake, such as that found in bacteria, is involved.     

An Hg-sensitive channel mediates the diffusional component of glucose transport in olive cells

In several organisms solute transport is mediated by the simultaneous operation of saturable and non-saturable (diffusion-like) uptake, but often the nature of the diffusive component remains elusive. The present work investigates the nature of the diffusive glucose transport in Olea europaea cell cultures. In this system, glucose uptake is mediated by a glucose-repressible, H(+) -dependent active saturable transport system that is superimposed on a diffusional component. The latter represents the major mode of uptake when high external glucose concentrations are provided. In glucose-sufficient cells, initial velocities of D- and L-[U-(14)C]glucose uptake were equal and obeyed linear concentration dependence up to 100 mM sugar. In sugar starved cells, where glucose transport is mediated by the saturable system, countertransport of the sugar pairs 3-O-methyl-D-glucose/D-[U-(14)C]glucose and 3-O-methyl-D-glucose/3-O-methyl-D-[U-(14)C]glucose was demonstrated. This countertransport was completely absent in glucose-sufficient cells, indicating that linear glucose uptake is not mediated by a typical sugar permease. The endocytic inhibitors wortmannin-A and NH(4)Cl inhibited neither the linear component of D- and L-glucose uptake nor the absorption of the nonmetabolizable glucose analog 3-O-methyl-D-[U-(14)C]glucose, thus excluding the involvement of endocytic mediated glucose uptake. Furthermore, the formation of endocytic vesicles assessed with the marker FM1-43 proceeded at a very slow rate. Activation energies for glucose transport in glucose sufficient cells and plasma membrane vesicles were 7 and 4 kcal mol(-1), respectively, lower than the value estimated for diffusion of glucose through the lipid bilayer of phosphatidylethanolamine liposomes (12 kcal mol(-1)). Mercury chloride inhibited both the linear component of sugar uptake in sugar sufficient cells and plasma membrane vesicles, and the incorporation of the fluorescent glucose analog 2-NBDG, suggesting protein-mediated transport. Diffusive uptake of glucose was inhibited by a drop in cytosolic pH and stimulated by the protein kinase inhibitor staurosporine. The data demonstrate that the low-affinity, high-capacity, diffusional component of glucose uptake occurs through a channel-like structure whose transport capacity may be regulated by intracellular protonation and phosphorylation/dephosphorylation.     

Effect of pH on IAA Uptake by Maize Root Segments

The uptake of [5-³H]indoleacetic acid (IAA) by Zea mays L. root segments involves nonsaturable and saturable processes. The pH optimum of the saturable component was found to be 5.0. The proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone inhibited at 100 micromolar the saturable component of IAA uptake but had no effect on non-saturable uptake. This indicates that the saturable component of IAA uptake is dependent on the proton gradient across the plasmalemma. The high level of proton extrusion in the elongation zone of the root will stimulate nonsaturable and saturable uptake of IAA in that zone.     

Saturable binding sites for vesicular stomatitis virus on the surface of Vero cells.

The binding of vesicular stomatitis virus (VSV) to Vero monkey cells was studied by using virus metabolically labeled with [35S]methionine. Under conditions where viral uptake did not occur (4 degrees C), apparent binding equilibrium was achieved within 12 h at a level representing 12% of the input virus. Two distinct forms of virus-cell interaction were found. At low concentrations of VSV, corresponding to multiplicities used for tissue culture studies, saturable binding was the major form of interaction. Saturation was complete at approximately 4,000 VSV virions per cell. At higher virus concentrations, nonsaturable binding prevailed. Trypsin treatment of Vero cells did not decrease the binding of VSV to the saturable binding sites. Internalization of VSV at 37 degrees C also displayed a saturable component which was directly comparable to that observed for binding. VSV binding to high-affinity, saturable sites on the plasma membrane may represent a receptor-mediated route of viral uptake.     

Studies on avian erythrocyte metabolism. XVII. Kinetics and transport properties of myo-inositol in chicken reticulocytes

The uptake of myo-inositol was determined in a reticulocyte-enriched fraction prepared from chicken blood and compared with uptake in mature erythrocytes. While reticulocytes accumulated inositol at levels more than threefold that of the plasma concentration, erythrocyte levels were only slightly higher than that of the plasma concentration. The rate of uptake in reticulocytes was approximately 66 mumol/ml rbc/h compared to 5 mumol/ml rbc/h in mature erythrocytes when measured at an inositol medium concentration of 250 microM. The kinetic analysis of inositol influx by reticulocytes reveals a two component system: saturable and nonsaturable. The saturable component, which has a Km for inositol of approximately 222 microM, is Na-dependent. This Na-dependent saturable component, which presumably reflects active transport of inositol, accounts for 30-35% of the transport process. The saturable component is completely inhibited by amiloride but to a lesser extent by ouabain and bumetanide. Moreover, in the course of reticulocyte maturation, the saturable component is lost concomitantly with the completion of the synthesis of myo-inositol pentakisphosphate and the drastic decrease in the membrane permeability to inositol. In addition, phloretin and cytochalasin B, which bind to hexose carriers and inhibit hexose sugar transport, also inhibited inositol transport. The uptake of inositol was not affected by excesses of 3-O-methylglucose (100 mM) or by physiological concentrations of D-glucose. Thus, the transport mechanism of myo-inositol appears distinct from that of D-glucose.     

Saturable uptake of indol-3yl-acetic Acid by maize roots

The uptake of 5-[³H]indol-3yl-acetic acid (IAA^*) by segments of Zea mays L. roots was measured in the presence of nonradioactive indol-3yl-acetic acid (IAA°) at different concentrations. IAA uptake was found to have a nonsaturable component and a saturable part with (at pH 5.0) an apparent K_m of 0.285 micromolar and apparent V_max 55.0 picomoles per gram fresh mass per minute. These results are consistent with those which might be expected for a saturable carrier capable of regulating IAA levels. High performance liquid chromatography analyses showed that very little metabolism of IAA^* took place during 4 minute uptake experiments. Whereas nonsaturable uptake was similar for all 2 millimeter long segments prepared within the 2 to 10 millimeter region, saturable uptake was greatest for the 2 to 4 millimeter region. High levels of uptake by stelar (as compared with cortical) segments are partly attributable to the saturable carrier, and also to a high level of uptake by nonsaturable processes. The carrier may play an essential role in controlling IAA levels in maize roots, especially the accumulation of IAA in the apical region. The increase in saturable uptake toward the root tip may also contribute to the acropetal polarity of auxin transport.     

Metabolism of long-chain polyunsaturated fatty acids in isolated cardiac myocytes

The uptake and integrated intracellular metabolism of (n - 6) and (n - 3) polyunsaturated fatty acids was studied in isolated rat cardiac myocytes and in the perfused heart. Labeled linolenic acid (18:3(n - 3)) uptake and its subsequent metabolism into carbon dioxide as well as acylation into lipids was nonsaturable over a substrate range of 0.02 to 0.4 mM. [1-14C]Linoleic acid (18:2(n - 6)), dihomo-gamma-linolenic acid (20:3(n - 6)) and arachidonic acid (20:4(n - 6)) were transported into myocytes at rates similar to those for linolenic acid. Conversely both [1-14C]-gamma-linolenic acid (18:3(n - 6)) and eicosapentaenoic acid (20:5(n - 3)) were taken up at a slower rate. Oxidation of 18:3(n - 6) was 4-5-fold greater when compared with C18-C20 polyunsaturated fatty acids. When myocytes were incubated with labeled 18:2(n - 6), 18:3(n - 6), 18:3(n - 3), 20:4(n - 6) or 20:5(n - 3), it was not possible to detect any desaturation or chain-elongation products. Identical results were obtained when hearts were perfused with 1-14C-labeled linoleic acid.     

Cadmium uptake kinetics in human erythrocytes

Cross-membrane transport of cadmium in human erythrocytes was studied using 109Cd+(+) and liquid scintillation counting. Uptake rates were determined by depletion of radioactivity in the incubation medium and the amount of hemolyzate radioactivity taken up by the erythrocytes. Both saturable and nonsaturable components for cadmium transport were observed. The mean maximum uptake rate (Jmax) of the saturable component was 4.9 X 10(-6) mol/L/h. The transport constant (Kt) was estimated at 6.9 X 10(-5) mol/L. The diffusion constant (Kd) of the non-saturable component was 1.4 X 10(-2)/h. Both Jmax and Kt of cadmium generally decreased when Zn+(+) was present, with a biphasic response in the presence of Cu+(+). Kd of cadmium increased as Zn+(+) or Cu+(+) levels were increased. It is suggested that cadmium may penetrate human red cells via cation transport sites owing to its behavior as an analog of one or more nutrient species.     

Factors involved in the uptake of corticosterone by rat liver cells

Isolated rat liver cells take up corticosterone rapidly; the initial rates increase with increasing temperature. A plot of the initial rates against the concentration of corticosterone indicated the presence of saturable and nonsaturable uptake systems. The Eadie-Hofstee plot showed the presence of two saturable and one nonsaturable uptake components. The apparent Kt values of the saturable systems were 64 +/- 40 nM (n = 3) and 1085 +/- 313 nM (n = 12). The nonsaturable system, probably diffusion, contributed 12% to the total uptake between 15 and 72 nM corticosterone, the physiological concentration of the free corticosterone in rat serum. Metabolic inhibitors did not influence the uptake of corticosterone. N-Ethylmaleimide, 1-fluoro-2,4-dinitrobenzene and sodium ethyl mercurithiosalicylate (1 mM each) decreased the uptake by 40%. Iodoacetate did not have any influence. Treatment of cells with phospholipase A inhibited the uptake 35--45%. In the presence of cortisone, cortisol, dexamethasone, aldosterone, testosterone, estradiol-17beta and estrone (2 muM each) the uptake decreased 30--50%. The presence of serum proteins in the external medium inhibits the uptake of corticosterone. These results suggest that corticosterone is transported into the cell and is accumulated. Only the free hormone is available for uptake which in turn may be regulated by protein and lipid components in the plasma membrane of the liver cell.     

Ca+2 transport across intestinal brush border membranes of the cichlid teleost Oreochromis mossambicus

Summary Brush border membranes were isolated from tilapia (Oreochromis mossambicus) intestine by the use of magnesium precipitation and differential centrifugation. The membrane preparation was enriched 17-fold in alkaline phosphatase. The membranes were 99% right-side-out oriented as indicated by the unmasking of latent glyceraldehyde-3-phosphate dehydrogenase and acetylcholine esterase activity by detergent treatment. The transport of Ca⁺² in brush border membrane vesicles was analyzed. A saturable and a nonsaturable component in the uptake of Ca⁺² was resolved. The saturable component is characterized by a K _m much lower than the Ca⁺² concentrations predicted to occur in the intestinal lumen. The nonsaturable component displays a Ca⁺² permeability too high to be explained by simple diffusion. We discuss the role of the saturable component as the rate-limiting step in transmembrane Ca⁺² movement, and suggest that the nonsaturable component reflects a transport mechanism operating well below its level of saturation.The authors wish to thank Tom Spanings for his superb organization of fish husbandry, and Maarten de Jong (Dept. of Physiology, Faculty of Medicine, University of Nijmegen) for making the automated stopped-flow apparatus available to us.     

A radiolabel and electron microscopy study of the interaction of liposomes with synaptosomal membranes

The quantitative and qualitative interaction of liposomes with synaptosomes isolated from rat brain was examined using radiolabeled phospholipids and electron microscopy. Liposomes were prepared by sonication and detergent dialysis. Binding (adsorption) of radiolabeled phospholipid to synaptosomes was saturable when liposomes were in the liquid-crystalline state, were electrically neutral (egg-phosphatidylcholine), or carried increasing fractions (10:2 and 10:4 molar ratio) of negatively charged phosphatidic acid. Analysis using the Langmuir isotherm equation indicated a biphasic adsorption behavior. Adsorption increased with increasing temperature (4 degrees C and 37 degrees C). Binding was nonsaturable when liposomes were positively charged with stearylamine or composed of dimyristoylphosphatidylcholine and phosphatidylinositol (10:2 molar ratio). Due to the latter composition's solid state at 4 degrees C, temperature dependency was inverse. Electron micrographs revealed disc-shaped areas of adsorption that were free of integral membrane particles which appeared to form a condensed layer surrounding the areas of liposome adsorption. Following interaction with stearylamine-containing liposomes the vesicular structure of synaptosomes appeared largely destroyed. It is concluded that both liposome surface charge and membrane fluidity determine the extent of interaction with biological membranes.     

Calcium accumulation and release by the sarcoplasmic reticulum of digitonin-lysed adult mammalian ventricular cardiomyocytes.

We have developed a model for characterizing calcium handling by the intact cardiac sarcoplasmic reticulum (SR) that yields data consistent with both mathematical simulations of in situ SR Ca2+ uptake and deduced behavior of the Ca2(+)-induced Ca2+ efflux channels in mechanically skinned single cardiac cells. In Na(+)-based media (37 degrees C, pH 7.2, 50 mM Pi, 10 mM MgATP, pMg 3.3, 10 mM phosphocreatine), SR 45Ca2+ uptake by digitonin-lysed rat myocytes as a function of free [Ca2+] peaked at pCa 6.2, declined until pCa 5.6 and increased again at lower pCa. When Ca2(+)-induced Ca2+ efflux was inhibited with 30 microM ruthenium red and 10 mM procaine, uptake was saturable with a Vmax of 160 +/- 5 nmol.min-1.mg-1, K0.5 of 500 nM free [Ca2+] and slope factor of 1.6. In K(+)-based media, maximum Pi- and oxalate-supported uptake increased to 220 and 260 nmol.min-1.mg-1, respectively. Without phosphocreatine, 45Ca2+ uptake declined under all conditions; this was correlated with a decrease in ATP/ADP. Vmax for 45Ca2+ uptake was increased 20% in hyperthyroid myocytes but depressed 30% in myocytes from heart failure-prone rats. In canine myocytes, Vmax was the same as in normal rat cells, but K0.5 was 830 nM. Without efflux inhibitors, ryanodine caused a concentration-dependent decline in net Pi-supported 45Ca2+ uptake at pCa 6.3 (K0.5 = 1 microM), while 10 microM ryanodine depressed uptake at all pCa between 7.2 and 5.6. Ruthenium red/procaine fully reversed this effect.     

Adipocyte differentiation of 3T3-L1 cells involves augmented expression of a 43-kDa plasma membrane fatty acid-binding protein.

A previously described 43-kDa plasma membrane fatty acid-binding protein (FABPPM) was not observed by immunohistochemical methods in proliferating 3T3-L1 fibroblasts. However, it was detectable in plasma membranes by the second day of confluent growth, prior to accumulation of visible lipid droplets, and was strongly expressed in 8-day differentiated adipocytes. These observations were confirmed by extraction of plasma membrane proteins and subsequent immunoblotting. Kinetics of initial [3H]oleate uptake by both fibroblasts and adipocytes consisted of the sum of a saturable and a non-saturable component. During differentiation the saturable component increased progressively. Vmax increased from 3 to 25 to 110 pmol.s-1.mg cell protein-1 between the fibroblast, the 4-day, and 8 day adipocyte stages; Km was 24 nM in fibroblasts and approximately 55 nM in both 4- and 8-day differentiated adipocytes. By contrast, the rate constant for nonsaturable oleate influx decreased progressively from 0.026 to 0.010 ml.s-1.mg protein-1 between the fibroblast and 8 day adipocyte stages. In 8-day adipocytes saturable oleate uptake was inhibited by up to 55% by antibodies against rat liver FABPPM; these antibodies had no effect on uptake of 2-deoxyglucose or the medium chain fatty acid octanoate. They also had no effect on oleate uptake by fibroblasts. These studies support the hypothesis that FABPPM is a component of a saturable transport mechanism for long chain fatty acids.     

Interaction of immunoglobulin-coupled liposomes with rat liver macrophages in vitro

The interaction between liposomes coated with covalently linked rabbit immunoglobulin (RbIg-liposomes), and rat liver macrophages (Kupffer cells) in monolayer culture was studied biochemically with radioactive tracers and morphologically by electron microscopy. The attachment of immunoglobulin (Ig) to liposomes caused a five-fold increase in liposome uptake by the Kupffer cells at 37 degrees C, in comparison with uncoated liposomes. The uptake was linear with time for at least 4 h and linear with liposome concentration up to a lipid concentration of 0.2 mM. At 4 degrees C uptake, probably representing cell surface-bound liposomes, was reduced to a level of approx. 20% of the 37 degrees C values. Involvement of the Fc receptor in the uptake process was indicated by the reduction of RbIg-liposome uptake by more than 75% as a result of preincubating the cells with heat-aggregated human or rabbit Ig at concentrations (less than 2 mg/ml) at which bovine serum albumin (BSA) had virtually no effect on uptake. At high concentrations (10-35 mg/ml), however, albumin also reduced liposome uptake significantly (20-30%), which suggests an interaction of the RbIg-liposomes with the Kupffer cells that is partially non-specific. RbIg-liposome uptake was dependent on the amount of RbIg coupled to the liposomes. Maximal uptake values were reached at about 200 micrograms RbIg/mumol liposomal lipid. Electron microscopic observations on cells incubated with horseradish peroxidase-containing RbIg-liposomes demonstrated massive accumulation of peroxidase reaction product in intracellular vacuoles, showing that the uptake observed by label association represents true internalization.     

Nucleoside transport in mammalian cell membranes. III. Kinetic and chemical modification studies of cytosine-arabinoside and uridine transport in hamster cells in culture

Transport of the nucleoside analog cytosine-arabinoside (CAR) in transformed hamster cells in culture has been studied in conditions of minimal metabolic conversion. Uptake (zero-trans in) properties at 20 degrees C over a limited range of CAR concentrations were characterized by a Km of 350 micrometer and a maximal velocity (V) of 780 micrometer.min-1 (V/Km = 2.28 min-1). Equilibrium exhcange at 20 degrees C over a wider range of concentrations was best described by a saturable component with a Km of 500 micrometer and a v of 1230 micrometer.min-1 (V/Km = 2.26 min-1) and either a saturable component of high Km or a nonsaturable component of k = 0.3 min-1. For the saturable component, the v/Km values were similar in both procedures. CAR transport was inhibited by various metabolizable nucleosides. Uptake of some of these nucleosides was inhibited by CAR. CAR transport and uridine uptake were inhibited in a reversible but partially competitive fashion by high affinity probes like S-(p-nitrobenzyl-6-mercaptoinosine (NBMI) (Ki less than 0.5 nM) and in an irreversible fashion by SH reagents such as N-ethylmaleiimide (NEM). The organomercurial p-hydroxymercuribenzene sulfonate (pMBS) markedly stimulated transport of these nucleosides, but also markedly potentiated the inhibitory effects of either NBMI or NEM. The effects are interpreted either in terms of models which invoke allosteric properties or in terms of two transport systems which display distinct chemical susceptibilities to externally added probes.     

Permeabilization of phospholipid bilayer membranes induced by gas-liquid flow in an airlift bubble column

The permeability of 5(6)-carboxyfluorescein (CF) through the phospholipid bilayer membranes was measured by using the system in which the CF-containing phospholipid vesicles (liposomes) were suspended in the gas-liquid flow in an external loop airlift bubble column. The airlift was operated at various superficial gas velocities UG up to 2.4 cm/s at 25 and 40 degrees C. The CF-containing liposomes composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) had the nominal diameters of 50, 100, and 200 nm. The 50- and 100-nm liposomes were stable at 40 degrees C for 5 h even at a high UG value of 2.4 cm/s based on the observed turbidity of the liposome suspension in the airlift. On the other hand, the 200-nm liposomes were stable at a low UG value of 1.4 cm/s, although a progressive decrease in size of the liposomes was implied at the high UG value of 2.4 cm/s. The permeability coefficient PCF of CF through the lipid membrane of the 100-nm liposomes was significantly increased with increasing UG at a high temperature of 40 degrees C, while at a low temperature of 25 degrees C the PCF value was little dependent on UG. As a typical result on the above liposomes, the PCF value (9.2 x 10(-11) cm/s) at 40 degrees C and UG = 2.4 cm/s in the airlift was more than 15 times larger than that at 25 degrees C in the static liquid corresponding to UG = 0. In addition, the dependence of the PCF value on UG at 40 degrees C became more significant with increasing the size of liposomes suspended. The results obtained revealed that the permeability of the liposome membranes could be regulated by suspending the liposomes in the gas-liquid flow in the airlift without modulating the membrane composition of liposomes.     

Uptake of thyroid hormones (L-T3 and L-T4) by isolated rat adipocytes

3,5,3'-triiodo-L-thyronine is taken up by isolated rat adipocytes under physiological conditions by a saturable sigmoidal process, while L-thyroxine uptake follows Michaelian kinetics. Comparative studies performed with intact adipocytes and derived liposomes suggest that thyroid hormones are taken up into cells via carrier-mediated transport.     

Factors involved in the uptake of corticosterone by rat liver cells

Isolated rat liver cells take up corticosterone rapidly; the initial rates increase with increasing temperature. A plot of the initial rates against the concentration of corticosterone indicated the presence of saturable and nonsaturable uptake systems. The Eadie-Hofstee plot showed the presence of two saturable and one nonsaturable uptake components. The apparent K_t values of the saturable systems were 64 ± 40 nM (n =3) and 1085 ± 313 nM (n =12). The nonsaturable system, probably diffusion, contributed 12% to the total uptake between 15 and 72 nM corticosterone, the physiological concentration of the free corticosterone in rat serum. Metabolic inhibitors did not influence the uptake of corticosterone. N-Ethylmaleimide, 1-fluor-2,4-dinitrobenzene and sodium ethyl mercurithiosalicylate (1 mM each) decreased the uptake by 40%. Iodoacetate did not have any influence. Treatment of cells with phospholipase A inhibited the uptake 35–45%. In the presence of cortisone, cortisol, dexamethasone, aldosteron, testosterone, estradiol-17β and estrone (2 μM each) the uptake decreased 30–50%. The presence of serum proteins in the external medium inhibits the uptake of corticosterone. These results suggest that corticosterone is transported into the cell and is accumulated. Only the free hormone is available for uptake which in turn may be regulated by protein and lipid components in the plasma membrane of the liver cell.     

Temperature sensitivity and substrate specificity of two distinct Na+-activated D-glucose transport systems in guinea pig jejunal brush border membrane vesicles

D-Glucose transport was studied with isolated brush border membrane vesicles from guinea pig jejunum. Saturation curves were carried out at either 25 or 35 degrees C in buffers containing Na+, Li+, K+ (100 mM chloride salt), or sorbitol (200 mM). Uncorrected uptake rates were fitted by nonlinear regression analysis to an equation involving one diffusional and two saturable terms. In the presence of Na+ at 35 degrees C, two saturable systems (Km = 0.4 and 24 mM, respectively) were evident, as well as a diffusion component quantitatively identical with that measured with L-glucose in separate experiments. In contrast, at 25 degrees C only one saturable system was apparent (Km = 1.2 mM): the second exhibited diffusion-like kinetics. In the presence of Na+ at 35 degrees C, D-glucose uptake was fully inhibited by both D-glucose and D-galactose, whereas alpha-methylglucoside gave kinetics of partial inhibition. We conclude that in the presence of Na+ there are at least two distinct D-glucose transport systems: 1) System I, a low temperature-sensitive system, fully inhibited by D-glucose, D-galactose, and alpha-methylglucoside; we identify it as the "classical" D-glucose/Na+ cotransport system, insensitive to inhibition by cytochalasin B and obligatorily dependent on Na+; and 2) System II, a high temperature-sensitive system where D-glucose and D-galactose inhibit but alpha-methylglucoside is inert. Its cation specificity is unclear but it appears to be sensitive to cytochalasin B inhibition. When Li+ or K+ substituted for Na+, only one transport system was apparent. The Li+-activated transport was: independent of the incubation temperature; inhibited by D-glucose and D-galactose but not by alpha-methylglucoside, 2-deoxy-D-glucose, D-mannose, and D-xylose; and sensitive to cytochalasin B inhibition. The exact nature of the system (or systems) involved in D-glucose transport in the absence of sodium remains to be established.