Hexose transport in preimplantation rabbit blastocysts

Transtrophectodermal 3-0-methyl glucose (3-0MG) transport in the rabbit blastocyst at Days 6 and 7 post coitum was investigated to understand better how the trophectoderm can regulate inner cell mass growth by controlling substrate availability. 3-0MG rapidly traversed the trophectoderm and displayed saturation kinetics (Km = 4.3 +/- 0.5 mM, Vmax = 79 +/- 3.8 nmol.cm-2). The flux of 3-0MG was inhibited nearly 95% by 10(-4) M-phloretin, and only 15% by 10(-4) M-phlorizin. Furthermore, 3-0MG influx was inhibited by cytochalasin B (5 microM) and was unaffected by removal of sodium. The transport system had a high specificity for 2-deoxy-D-glucose and glucose, and a very low specificity for fructose and 4-alpha-methyl glucoside. Western blots probed with a polyclonal antibody to the human erythrocyte glucose transport protein and also with a polyclonal antibody to the C-terminus of the glucose transport protein of the rat brain revealed a broad band with a molecular weight of 55,000. Using immuno-gold labelling techniques, Na(+)-independent glucose transporters were localized to both the apical and basolateral borders of the trophectodermal cell. These results suggest that the mechanism in the trophectoderm responsible for transport of glucose is similar to other sodium-independent glucose transport systems. In addition, 3-0MG influx was unaffected by short-term incubation with progesterone, the progesterone antagonist mifepristone (RU-486), PGF-2 alpha, PGE-2, insulin, or cAMP. Day-7 p.c. embryos also transported hexoses by a similar system because the influx rate and the phlorizin/phloretin sensitivity were the same as in the Day-6 p.c. embryo.     

The action of inhibitors of sugar transport phlorizin, phloretin and cytochalasin B in model systems

Phlorizin, phloretin and cytochalasin B are known to be specific sugar transport inhibitors. A study was made of their effects on the carbohydrate-protein interaction in solution as a model system for examining the initial steps of sugar membrane transport. Glycogen precipitation by concanavalin A is inhibited only by alpha-methylmannoside, whereas both phlorizin and phloretin inhibit interactions between hexokinase and glucose, and between glucose-6-phosphate dehydrogenase and glucose-6-phosphate. Cytochalasin B was found to exert no effect on both the concanavalin A--glycogen interaction and the enzyme reactions investigated. The data obtained in the model system examination may suggest that the sites of glucose and cytochalasin binding are, respectively, spatially uncoupled.     

Sugar transport in the LLC-PK1 renal epithelial cell line: Similarity to mammalian kidney and the influence of cell density

Cells of confluent cultures of the established pig renal epithelial line, LLC-PK₁, accumulate α-methyl-D-glucoside against a concentration gradient. This transport system is strongly inhibited by phlorizin and 6-deoxy-D-glucose, moderately inhibited by phloretin, and only weakly inhibited by 3-0-methyl-D-glucose, paralleling the situation in mammalian kidney. The time course for the uptake of α-methyl-D-glucoside and for the carrier-mediated but passive uptake of 3-0-methyl-D-glucose are identical to those seen in mammalian kidney. Subconfluent cultures of LLC-PK₁ cells are unable to accumulate α-methyl-D-glucoside, and their transport of this glucose analog is less sensitive to phlorizin inhibition than is the transport system in confluent cultures. Transmission electron micrographs show that cells from subconfluent cultures lack the microvillous surface seen in cells from confluent cultures. Cell density is thus a factor in the occurrence of structural and functional differentiated properties related to transport in these cells.     

Reaction of the glucose carrier of erythrocytes with sodium tetrathionate: Evidence for inward-facing and outward-facing carrier conformations

Summary Sodium tetrathionate reacts with the glucose carrier of human erythrocytes at a rate which is greatly altered in the presence of competitive inhibitors of glucose transport. Inhibitors bound to the carrier on the outer surface of the membrane, either at the substrate site (maltose) or at the external inhibition site (phloretin and phlorizin), more than double the reaction rate. Inhibitors bound at the internal inhibition site (cytochalasin B and androstenedione), protect the system against tetrathionate. After treatment with tetrathionate, the maximum transport rate falls to less than one-third, and the properties of the binding sites are modified in unexpected ways. The affinity of externally bound inhibitors rises: phloretin is bound up to seven times more strongly and phlorizin and maltose twice as strongly. The affinity of cytochalasin B, bound at the internal inhibition site, falls to half while that of androstenedione is little changed. The affinity of external glucose falls slightly. Androstenedione prevents both the fall in transport activity and the increase in phloretin affinity produced by tetrathionate. An inhibitor of anion transport has no effect on the reaction. The observations support the following conclusions: (1) Tetrathionate produces its effects on the glucose transport system by reacting with the carrier on the outer surface of the membrane. (2) The carrier assumes distinct inward-facing and outward-facing conformations, and tetrathionate reacts with only the outward-facing form. (3) The thiol group with which tetrathionate is presumed to react is not present in either the substrate site or the internal or external inhibitor site. (4) In binding asymmetrically to the carrier, a reversible inhibitor shifts the carrier partition between inner and outer forms and thereby raises or lowers the rate of tetrathionate reaction with the system. (5) Reaction with tetrathionate converts the carrier to an altered state in which the conformation at all three binding sites is changed and the rate of carrier reorientation is reduced.     

Common characteristics for Na+-dependent sugar transport in Caco-2 cells and human fetal colon

Summary The recent demonstration that the human colon adenocarcinoma cell line Caco-2 was susceptible to spontaneous enterocytic differentiation led us to consider the question as to whether Caco-2 cells would exhibit sodium-coupled transport of sugars. This problem was investigated using isotopic tracer flux measurements of the nonmetabolizable sugar analog -methylglucoside (AMG). AMG accumulation in confluent monolayers was inhibited to the same extent by sodium replacement, 200 m phlorizin, 1mm phloretin, and 25mm d-glucose, but was not inhibited further in the presence of both phlorizin and phloretin. Kinetic studies were compatible with the presence of both a simple diffusive process and a single, Na⁺-dependent, phlorizin-and phloretin-sensitive AMG transport system. These results also ruled out any interaction between AMG and a Na⁺-independent, phloretin-sensitive, facilitated diffusion pathway. The brush-border membrane localization of the Na⁺-dependent system was inferred from the observations that its functional differentiation was synchronous with the development of brush-border membrane enzyme activities and that phlorizin and phloretin addition 1 hr after initiating sugar transport produced immediate inhibition of AMG uptake as compared to ouabain. Finally, it was shown that brush-border membrane vesicles isolated from the human fetal colonic mucosa do possess a Na⁺-dependent transport pathway(s) ford-glucose which was inhibited by AMG and both phlorizin and phloretin. Caco-2 cells thus appear as a valuable cell culture model to study the mechanisms involved in the differentiation and regulation of intestinal transport functions.     

Identification of proximal tubular transport functions in the established kidney cell line, OK

OK cells, derived from an American opossum kidney, were analyzed for proximal tubular transport functions. In monolayers, L-glutamate, L-proline, L-alanine, and alpha-methyl-glucopyranoside (alpha-methyl D-glucoside) were accumulated through Na+-dependent and Na+-independent transport pathways. D-Glucose and inorganic sulfate were accumulated equally well in the presence or absence of Na+. Influx of inorganic phosphate was only observed in the presence of Na+. Na+/alpha-methyl D-glucoside uptake was preferentially inhibited by phlorizin and D-glucose uptake by cytochalasin B. An amiloride-sensitive Na+-transport was also identified. In isolated apical vesicles (enriched 8-fold in gamma-glutamyltransferase), L-glutamate, L-proline, L-alanine, alpha-methyl D-glucoside and inorganic phosphate transport were stimulated by an inwardly directed Na+-gradient as compared to an inwardly directed K+-gradient. L-Glutamate transport required additionally intravesicular K+. D-Glucose transport was similar in the presence of a Na+- and a K+-gradient. Na+/alpha-methyl D-glucoside uptake was inhibited by phlorizin whereas cytochalasin B had no effect on Na+/D-glucose transport. An amiloride-sensitive Na+/H+ exchange mechanism was also found in the apical vesicle preparation. It is concluded that the apical membrane of OK cells contains Na+-coupled transport systems for amino acids, hexoses, protons and inorganic phosphate. D-Glucose appears a poor substrate for the Na+/hexose transport system.     

Reconstitution of the glucose transporter from bovine heart

Reconstitution of the glucose transporter from heart should be useful as an assay in its purification and in the study of its regulation. We have prepared plasma membranes from bovine heart which display D-glucose reversible binding of cytochalasin B (33 pmol sites/mg protein; Kd = 0.2 muM). The membrane proteins were reconstituted into liposomes by the freeze-thaw procedure. Reconstituted liposomes showed D-glucose transport activity which was stereospecific, saturable and inhibited by cytochalasin B, phloretin, and mercuric chloride. Compared to membrane proteins reconstituted directly, proteins obtained by dispersal of the membranes with low concentrations of cholate or by cholate solubilization showed 1.2- or 2.3-fold higher specific activities for reconstituted transport, respectively. SDS-polyacrylamide gel electrophoresis followed by electrophoretic protein transfer and labeling with antisera prepared against the human erythrocyte transporter identified a single band of about 45 kDa in membranes from both dog and bovine hearts, a size similar to that reported for a number of other glucose transporters in various animals and tissues.     

Facilitated transport of 3-O-methyl-D-glucose in human polymorphonuclear leukocytes

Transport of the nonmetabolizable hexose analogue 3-O-methyl-D-glucose (30MG) was measured in human polymorphonuclear leukocytes at 37 degrees C, pH 7.4. 3OMG at very low concentration (0.05 mM) equilibrated with the intracellular water with a rate constant of about 0.08 s-1. Transport of 3OMG in the presence of 20 microM cytochalasin B and transport of L-glucose were insignificant. Countertransport of 14C-labelled 3OMG was demonstrated. Exchange of 3OMG between the extracellular and intracellular water showed saturation with a Km of about 4 mM. Thus, the transport of 3OMG is mediated almost exclusively by facilitated diffusion.     

Inhibition of glucose transport into brain by phlorizin, phloretin and glucose analogues.

An indicator dilution technique with 22Na+ as the intravascular marker was used to measure unidirectional transport of D-[6-3H]glucose from blood into the isolated, perfused dog brain. 18 compounds which are structurally related to glucose were tested for their ability to inhibit glucose transport. The data suggest that no single hydroxyl group is absolutely required for glucose transport, but rather that glucose binding to the carrier probably occurs through hydrogen bonding at several sites (hydroxyls on carbons 1, 3, 4 and 6). In addition, alpha-D-glucose has higher affinity for the carrier than does beta-D-glucose. A separate series of experiments demonstrated that phlorizin and phloretin are competitive inhibitors of glucose transport into brain; however, phloretin is partially competitive and inhibits at lower concentrations than does phlorizin. Inhibition by phlorizin and phloretin is mutually competitive, indicating that these compounds compete for binding to the glucose carrier. Comparison with the results reported in the literature for similar studies using the human erythrocyte demonstrates a fundamental similarity between glucose transport systems in the blood-brain barrier and erythrocyte.     

Inhibition of glucose transport into brain by phlorizin, phloretin and glucose analogues

An indicator dilution technique with ²²Na⁺ as the intravascular marker was used to measure unidirectional transport of d-[6-³H]glucose from blood into the isolated, perfused dog brain. 18 compounds which are structurally related to glucose were tested for their ability to inhibit glucose transport. The data suggest that no single hydroxyl group is absolutely required for glucose transport, but rather that glucose binding to the carrier probably occurs through hydrogen bonding at several sites (hydroxyls on carbons 1, 3, 4 and 6). In addition, α-d-glucose has higher affinity for the carrier than does β-d-glucose.A separate series of experiments demonstrated that phlorizin and phloretin are competitive inhibitors of glucose transport into brain; however, phloretin is partially competitive and inhibits at lower concentrations than does phlorizin. Inhibition by phlorizin and phloretin is mutually competitive, indicating that these compounds compete for binding to the glucose carrier. Comparison with the results reported in the literature for similar studies using the human erythrocyte demonstrates a fundamental similarity between glucose transport systems in the blood-brain barrier and erythrocyte.     

Hexose transport in human myoblasts.

The present investigation reports on the hexose transport properties of human myoblasts isolated from normal subjects and from patients with Duchenne muscular dystrophy (DMD). Similar to rat myoblast L6, normal human myoblasts possess a high- (HAHT) and a low- (LAHT) affinity hexose transport system. The non-metabolizable hexose analogue, 2-deoxyglucose, is preferentially taken up by HAHT. The transport of this analogue is the rate-limiting step in the uptake process. This human myoblast HAHT is also similar to that of the rat myoblast in its substrate specificity and in response to the energy uncouplers, cytochalasin B and phloretin. The human myoblast LAHT resembles that of rat myoblast in its insensitivity to energy uncouplers, and in its transport affinity and capacity for 3-O-methyl-D-glucose. Although DMD myoblasts resemble their normal counterpart in their ability to differentiate, they differ significantly in their hexose transport properties. In addition to HAHT and LAHT present in normal human myoblast, DMD myoblasts contain a super-high-affinity hexose transport system (SHAHT). SHAHT can be detected only at very low substrate concentrations. It differs from HAHT not only in its much higher transport affinity, but also in its response to the traditional hexose transport inhibitors. For example, SHAHT can be activated by cytochalasin B and phlorizin, whereas it is more sensitive to inhibition by phloretin. Unlike HAHT, energy uncouplers are found to be ineffective in inhibiting SHAHT. It should be mentioned that SHAHT cannot be detected in myoblasts isolated from patients with other types of myopathy. The present study serves to demonstrate that more than one hexose transport system is operating in human skeletal muscle cells, as found in other cell types.     

Transport of 1,5-anhydro-D-glucitol into human polymorphonuclear leukocytes.

1,5-Anhydro-D-glucitol (AG) is one of the main polyols and its structure resembles glucose. It has been proposed that decreased serum AG concentrations in diabetic patients are a novel indicator of diabetic metabolic derangement. However, the pathway of AG metabolism still remains to be clarified. In this study we investigated the transport of AG into human polymorphonuclear leukocytes (PMNLs) isolated from healthy volunteers and found that 0.1 mM 3-O-methy-D-glucose (3OMG) was equilibrated with a half saturation time of 10 s, while the uptake rate of AG was much slower. The concentration dependence of AG uptake revealed that the AG transport velocity reached a plateau, with a Km of about 50 mM and Vmax of about 25 nmol/min/10(7) cells. Transport of 14C-labeled 3OMG was inhibited by unlabeled D-glucose or AG in a dose-dependent manner. The mean inhibition constant (Ki) for D-glucose and for AG were 1.06 and 4.93 mM, respectively. Cytochalasin B (20 microM) inhibited 3OMG transport by 90% but AG transport by only 50%. S/V for 14C-labeled AG transport plotted against the concentration of unlabeled 3OMG showed a non-linear and biphasic pattern. These results suggest that AG influx into PMNLs is mediated not only by the cytochalasin B-sensitive glucose transport system but also via another facilitated transport system.     

Phlorizin-receptor interactions in fat cell plasma membranes

The rate but not the extent of phlorizin binding to purified fat cell plasma membranes was temperature dependent and this binding was a saturable process. A Scatchard plot revealed a population of sites which exhibited a dissociation constant of about 0.35 mM and a maximum binding capacity of about 8 nmoles/mg membrane protein. Under the conditions of these experiments neither glucose, phloretin, nor cytochalasin B inhibited [³H]phlorizin binding. These data demonstrate the presence in fat cell plasma membrane of specific receptors for phlorizin which may mediate the inhibitory effects of this agent on hexose trasport.     

Modulation of glucose uptake in animal cells. Studies using plasma membrane vesicles isolated from nontransformed and simian virus 40-transformed mouse fibroblast cultures.

Plasma membrane vesicles isolated from nontransformed and Simian virus 40-transformed mouse fibroblast cultures catalyzed carrier-mediated D-glucose transport without detectable metabolic conversion to glucose 6-phosphate. Glucose transport activity was stereospecific, temperature-dependent, sensitive to inactivation by p-chloromercuriphenylsulfonate, and accompanied plasma membrane material during subcellular fractionation. D-Glucose efflux from vesicles was inhibited by phloretin, an inhibitor of glucose uptake in intact cells. Cytochalasin B, a potent inhibitor of glucose uptake when tested with the intact cells used for vesicle isolation did not inhibit glucose transport in vesicles despite the presence of high affinity cytochalasin binding sites in isolated membranes. The enhanced glucose uptake observed in intact cells after viral transformation was not expressed in vesicles: no significant differences in glucose transport specific activity could be detected in vesicle preparations from nontransformed and transformed mouse fibroblast cultures. These findings indicate that cellular components distinct from glucose carriers can mediate changes in glucose uptake in mouse fibroblast cultures in at least two cases: sensitivity to inhibition by cytochalasin B and the enhanced cellular sugar uptake observed after viral transformation.     

Evidence that forskolin binds to the glucose transporter of human erythrocytes

Binding of [4-3H]cytochalasin B and [12-3H]forskolin to human erythrocyte membranes was measured by a centrifugation method. Glucose-displaceable binding of cytochalasin B was saturable, with KD = 0.11 microM, and maximum binding approximately 550 pmol/mg of protein. Forskolin inhibited the glucose-displaceable binding of cytochalasin B in an apparently competitive manner, with K1 = 3 microM. Glucose-displaceable binding of [12-3H]forskolin was also saturable, with KD = 2.6 microM and maximum binding approximately equal to 400 pmol/mg of protein. The following compounds inhibited binding of [12-3H]forskolin and [4-3H]cytochalasin B equivalently, with relative potencies parallel to their reported affinities for the glucose transport system: cytochalasins A and D, dihydrocytochalasin B, L-rhamnose, L-glucose, D-galactose, D-mannose, D-glucose, 2-deoxy-D-glucose, 3-O-methyl-D-glucose, phloretin, and phlorizin. A water-soluble derivative of forskolin, 7-hemisuccinyl-7-desacetylforskolin, displaced equivalent amounts of [4-3H]cytochalasin B or [12-3H]forskolin. Rabbit erythrocyte membranes, which are deficient in glucose transporter, did not bind either [4-3H]cytochalasin B or [12-3H]forskolin in a glucose-displaceable manner. These results indicate that forskolin, in concentrations routinely employed for stimulation of adenylate cyclase, binds to the glucose transporter. Endogenous ligands with similar specificities could be important modulators of cellular metabolism.     

Differences in neutral amino acid and glucose transport between brush border and basolateral plasma membrane of intestinal epithelial cells.

A comparison of L-valine and D-glucose transport was carried out with vesicles of plasma membrane isolated either from the luminal (brush border) or from the contra-luminal (basolateral) region of small intestinal epithelial cells. The existence of transport systems for both non-electrolytes was demonstrated by stereospecificity and saturability of uptake, as well as tracer coupling. Transport of L-valine and D-glucose differs markedly in the two types of plasma membrane with respect to stimulation by Na+. The presence of Na+ stimulated initial L-valine and D-glucose uptake in brush border, but not in basolateral membrane. Moreover, an electro-chemical Na+ gradient, oriented with the lower potential on the inside, supported accumulation of the non-electrolytes above medium concentration only in the brush border membrane. L-Valine and D-glucose transport also were saturated at lower concentrations in brush border (10-20 mM) than in basolateral plasma membranes (30-50 mM). A third difference between the two membranes was found in the effectiveness of known inhibitors of D-glucose transport. In brush border membranes phlorizin was more potent than phloretin and 2', 3', 4'-trihydroxy-4-methoxy chalcone and cytochalasin B did not inhibit at all. In contrast, with the basolateral plasma membranes the order of potency was changed to phloretin = 2',3',4'-trihydroxy-4-methoxy chalcone greater than cytochalasin B greater than phlorizin. These results indicate the presence of different types of transport systems for monosaccharides and neutral amino acids in the luminal and contra-luminal region of the plasma membrane. Active transepithelial transport can be explained on the basis of the different properties of the non-electrolyte transport systems in the two cellular regions and an electro-chemical Na+ gradient that is dependent on cellular metabolism.     

UPTAKE OF 3-O-METHYL-d-GLUCOSE INTO CULTURED HUMAN GLIOMA CELLS

Human glioma cells (138 MG) were found to take up 3-O-methyl-d -glucose (3-OMG) by a saturable low affinity transport system with a K_m of 20 mm and a V_max of 500 nmol/mg protein/min. About 20 per cent of the total uptake was due to passive diffusion. d -Glucose was a competitive inhibitor with a K_i of 10 mm . Follow-up experiments indicated that the same transport mechanism is involved in the uptake of n-glucose and 3-OMG. Phloretin (0·02 mm ) and cytochalasin B (0·002 mm ) strongly inhibited the uptake of 3-OMG, whereas phlorizin (0·02 mm ), ouabain (0·1 mm ), NaCN (0·5 mm ) and iodoacetic acid (1·0 mm ) had no effect. The data suggest that 3-OMG and d -glucose enter 138 MG cells mainly by a Na⁺-independent passive carrier-mediated transport system. Serum-deprivation doubled the population doubling time (T_d) without affecting the total uptake of 3-OMG. An increase in the non-specific (diffusional) uptake was balanced by a decrease in the specific (carrier-medíated) uptake. After addition of dibutyryl cyclic AMP (dbcAMP, 0·25 mm ) the cells attained a morphology characteristic of differentiated glia cells. T_d was maintained unchanged. The non-specific uptake of 3-OMG was not affected in cells grown in serum-containing medium plus dbcAMP, whereas the specific uptake increased by 40 per cent and there-fore also the total uptake. Similar, but more pronounced, changes were observed if serum-deprived cells were treated with dbcAMP.     

Stimulation of the glucose transport system in isolated mouse pancreatic acini by cholecystokinin and analogues.

Cholecystokinin and analogues increased the uptake of 2-deoxy-D-glucose and 3-O-methylglucose into isolated mouse pancreatic acini. This uptake was mediated by a facilitated glucose transport system that was saturable, stereospecific, and was inhibited by both phloretin and cytochalasin B. In agreement with previous studies of acinar function, caerulein was more potent and pentagastrin less potent than cholecystokinin in increasing sugar transport. The cholinergic analogue carbachol mimicked the effect of caerulein; atropine completely abolished the effects of carbachol but was without influence on the effects of the polypeptide hormones. In contrast, secretion, as well as dibutyryl cyclic AMP and dibutyryl cyclic GMP, had no effect on 2-deoxy-D-glucose uptake. Two lines of evidence suggested that hormonal stimulation of this sugar transport system was related to mobilization of cellular Ca2+. First, depletion of cellular Ca2+ by incubation of acini with ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) reduced the effect of caerulein. Second, the Ca2+ ionophore A23187 mimicked the effects of caerulein on 2-deoxy-D-glucose uptake when Ca2+ was present in the medium.     

Transport of 1,5-anhydro-D-glucitol across plasma membranes in rat hepatoma cells

The transport of 1,5-anhydro-D-glucitol (AG) across plasma membranes was investigated in rat hepatoma cells, Reuber H-35. The AG uptake by the cells showed a concentration gradient dependency: the uptake was saturated within 40 s, which was less than one-third of the saturation time for 2-deoxy-D-glucose (DG) uptake. Furthermore, the Km value of the transport system for AG was higher than 100 mM. Though AG has a pyranoid structure resembling that of glucose, AG did not compete for cellular uptake with DG, D-glucose or 3-O-methyl-D-glucose, which are taken into cells through the glucose transporters. Conversely, the DG transport was not inhibited by AG at concentrations up to 50 mM. AG transport was hardly inhibited by 10 microM cytochalasin B, which strongly inhibits glucose transporters. In contrast, the AG transport was inhibited by 100 microM phloretin much more strongly than the DG transport when cells were preincubated with the inhibitor; the inhibition constant was 28.0 microM. The AG transport was not inhibited by 100 microM phloridzin, while the DG uptake was slightly inhibited by phloridzin. On the basis of these observations we propose that the AG uptake into rat hepatoma cells is mediated by a carrier distinct from glucose transporters.     

Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium

Primary cultures of rabbit-kidney epithelial cells derived from purified proximal tubules were maintained without fibroblast overgrowth in a hormone-supplemented serum-free medium (Medium RK-1). A hormone- deletion study indicated that the primary cultures derived from purified rabbit proximal tubules required all of the three supplements in Medium RK-1 (insulin, transferrin, and hydrocortisone) for optimal growth but did not grow in response to EGF and T3. In contrast, the epithelial cells in primary cultures derived from an unpurified preparation of rabbit kidney tubules and glomeruli grew in response to EGF and T3, as well as insulin, transferrin, and hydrocortisone. These observations suggest that kidney epithelial cells derived from different segments of the nephron grow differently in response to hormones and growth factors. Differentiated functions of the primary cultures derived from proximal tubules were examined. Multicellular domes were observed, indicative of transepithelial solute transport by the monolayers. The proximal tubule cultures also accumulated alpha- methylglucoside (alpha-MG) against a concentration gradient. However, little or no alpha-MG accumulation was observed in the absence of Na+. Metabolic inhibitor studies also indicated that alpha-MG uptake by the primaries is an energy-dependent process, and depends upon the activity of the Na+/K+ ATPase. Phlorizin at 0.1 mM significantly inhibited 1 mM alpha-MG uptake whereas 0.1 mM phloretin did not have a significant inhibitory effect. Similar observations have been made concerning the Na+-dependent sugar-transport system located on the lumenal side of the proximal tubule, whereas the Na+-independent sugar transporter on the peritubular side is more sensitive to inhibition by phloretin than phlorizin. The cultures also exhibited PTH-sensitive cyclic AMP synthesis and brush-border enzymes typical of proximal cells. However, the activities of the enzymes leucine aminopeptidase, alkaline phosphatase, and gamma-glutamyl-transpeptidase were lower in the cultures than in purified proximal-tubule preparations from which they are derived.