Regulation of sugar transport in cultured diploid human skin fibroblasts

The regulation of hexose transport under glucose-starvation conditions was studied in cultured human skin fibroblasts. Glucose starvation enhanced the transport of 2-DG and 3-O-methyl-D-glucose (3-OMG) but not of L-glucose. Glucose-starvation enhanced transport was inhibited by cytochalasin B (10 μM). The starvation-induced change in 2-DG transport was due to an increase in the V_max of both the high and low affinity transport sites (2.8- and 2.4-fold, respectively) with no effect on their K_ms. The presence of 5.55 mM galactose, fructose, or L-glucose in the medium resulted in transport increases similar to those seen in glucose-starved cells, while the presence of 5.55 mM glucose, mannose, or 3-OMG repressed 2-DG transport. Glucose-starvation enhancement of 2-DG transport was blocked by cycloheximide (20 μg/ml) but not by actinomycin D (0.03 μg/ml) or α-amanitin (3.5 μM). Readdition of glucose (5.55 mM) for six hours to glucose-starved cells led to a rapid decrease in hexose transport that could be blocked by cycloheximide but not actinomycin D. Although readdition of 3-OMG to glucose-starved cells had little effect on reversing the transport increases, glucose plus 3-OMG were more effective than glucose alone. Serum containing cultures (10% v/v) of glucose-fed or glucose-starved cells exhibited rapid decreases in 2-DG transport when exposed to glucose-containing serum-free medium. These decreases were prevented by employing glucose-free, serum-free medium. The data indicate that hexose transport regulation in cultured human fibrob asts involves protein synthesis of hexose carriers balanced by interactions of glucose with a regulatory protein(s) and glucose metabolism as they affect the regulation and/or turnover of the carrier molecules.     

Hexose transport regulation in cultured hamster cells

Hamster (nil) cells maintained overnight in culture medium containing cycloheximide and either glucose or fructose exhibit strikingly different rates of hexose transport and metabolism (i.e., uptake). Pretreatment of cultures with sulfhydryl reagents makes it possible to determine initial transport rates for a physiological sugar such as galactose which is a catabolite in hamster cells. Using galactose transport as a model, hexose uptake enhancements can now be shown to be due almost entirely to increase in the rate of the transport step. The transport regulation can best be accounted for by a model comprised of 2 antagonizing mechanisms. This model involves turnover of transport carriers as well as inhibitory units (“regulators”). The experimental as well as the theoretical model may also apply to the well-known uptake enhancements observed in oncogenically transformed cells.     

Selective high metabolic lability of uridine, guanosine and cytosine triphosphates in response to glucose deprivation and refeeding of untransformed and polyoma virus-transformed hamster fibroblasts.

Sugar deprivation of hamster fibroblasts (NIL) affected the steady state levels (pool sizes) of cellular acid soluble nucleotides in the folloing fashion: the pools of UTP, GTP and CTP decreased to a much greater extent than the cellular ATP pools, with the UTP pools undergoing the most dramatic reduction. Sugar deprivation of polyoma-transformed NIL cells (PyNIL) yielded even sharper decreases in the nucleoside triphosphate pools with relative changes similar to those of the untransformed cells. Inhibition of protein synthesis by cycloheximide, initiated at the onset of (and continued during) sugar deprivation, prevented the reduction in pool sizes and yielded values slightly higher than those observed for pool sizes in cells cultured in sugar-supplemented medium.Refeeding glucose to sugar-depleted hamster fibroblasts led to rapid increases (within 1 hour) in the UTP and CTP pools to levels well above the pool sizes observed in cells which were continuously cultured (16 hours) in sugar supplemented medium. Feeding NIL or PyNIL cells with fructose instead of glucose as the only hexose source did not appreciably affect any of the ribonucleoside triphosphate pool sizes. Measurements of hexose uptake by NIL and PyNIL cells under a variety of conditions suggest that hexose transport is not regulated by the total cellular pools of ATP or any of the other ribonucleoside triphosphates.     

Hexose transport after glucose refeeding of glucose-starved human fibroblasts: 1. The effects of tunicamycin and cycloheximide. 2. Insulin binding and action

Hexose transport in glucose-starved human fibroblasts was readily reversed by glucose refeeding. This hexose transport reversal was not inhibited by tunicamycin (1.5 microgram/ml) but was blocked by cycloheximide (20 micrograms/ml). The ability of insulin (100 mU/ml) to stimulate hexose transport was returned by glucose refeeding and this was not affected by tunicamycin. Cycloheximide which blocked the glucose refeeding effect on hexose transport, decreased the ability of insulin to stimulate hexose transport. Specific 125I-insulin binding was increased by glucose refeeding of glucose-starved cells and this change in binding was inhibited by tunicamycin and cycloheximide. Thus, it appears that under the conditions employed in human fibroblasts, the ability of insulin to stimulate hexose transport is differentially regulated more by factors affecting basal hexose transport than by those affecting changes in insulin binding.     

Dissociation by cytochalasin B of movement, DNA synthesis and transport in 3T3 cells.

Cytochalasin B was used as a tool to study the inter-relationships between cell movement, the reinitiated DNA synthesis and the enhanced transport of specific small molecules stimulated by serum in quiescent 3T3 cells. Cytochalasin at concentrations of less than 1 mug/ml inhibits serum-stimulated movement within the monolayer and migration into a wound. Even at ten times this concentration there is little effect on the increase in DNA in the culture, indicating that movement away from neighboring cells is not required for the initiation of DNA synthesis. While DNA synthesis is not inhibited by concentrations of cytochalasin up to 10 mug/ml, the increased thymidine transport which is associated with the onset of the S phase of the cell cycle is inhibited and DNA synthesis cannot be measured by the labelling of nuclei with radioactive thymidine. Cytochalasin has a differential effect on the early transport changes produced by serum addition. Glucose transport is inhibited by low concentrations of the drug (less than 1 mug/ml) while the enhanced uptake of phosphate and uridine is unaffected by a 10-fold increase in concentration. Although the doses of cytochalasin required for 50% inhibition of hexose uptake and of cell movement are the same, no causal relationship between sugar transport and locomotion can be demonstrated. Cytochalasin affects membrane functions in at least two different ways. The drug inhibits the uptake of glucose directly but affects only the S-phase associated increase in thymidine transport.     

Regulation of hexose transport in L8 myocytes by glucose: possible sites of interaction

Previous work demonstrated that glucose controls its own transport rate in rat skeletal muscle: exposure to high glucose levels down-regulates muscle hexose transport, while glucose withdrawal results in elevated transport rates (J. Biol. Chem. 261:16827-16833, 1986). The present study investigates the mechanism of this autoregulatory system. Preincubation of L8 myocytes at 16 mM glucose reduced subsequent 2-deoxy-D-glucose (dGlc) uptake by 40% within 3 h. Cycloheximide (1 microM) mimicked the action of glucose; the effects of glucose and cycloheximide were not additive. At 50 microM, cycloheximide prevented the modulations of glucose transport induced by exposure of muscle cells to high or low glucose concentrations. Inhibition of glycosylation with tunicamycin A1 reduced the basal dGlc uptake, but did not prevent its up-regulation following glucose withdrawal. Inhibition of RNA synthesis by actinomycin D prevented the down-regulatory effect of glucose. These results indicate that continuous protein synthesis and protein glycosylation are required for the maintenance of the steady-state dGlc uptake. We suggest that glucose exerts its autoregulatory effect on hexose transport by modifying the incorporation of active glucose transporters into the plasma membrane rather than changing their rate of degradation. It is hypothesized that this effect is mediated by a non-glycosylated protein involved in the translocation or activation of glucose transporters.     

Dissociation of insulin-stimulated glucose transport from the translocation of glucose carriers in rat adipose cells

Cycloheximide, a potent inhibitor of protein synthesis, has been used to examine the relationship between recruitment of hexose carriers and the activation of glucose transport by insulin in rat adipocytes. Adipocytes were preincubated +/- cycloheximide for 90 min then +/- insulin for a further 30 min. We measured 3-O-methylglucose uptake in intact cells and in isolated plasma membrane vesicles. The concentration of glucose transporters in plasma membranes and low density microsomes was measured using a cytochalasin B binding assay. Cycloheximide had no affect on basal or insulin-stimulated 3-O-methylglucose uptake in intact cells or in plasma membrane vesicles. However, the number of glucose carriers in plasma membranes prepared from cells incubated with cycloheximide and insulin was markedly reduced compared to that from cells incubated with insulin alone (14 and 34 pmol/mg protein, respectively). Incubation of cells with cycloheximide alone did not change the concentration of glucose carriers in either plasma membranes or in low density microsomes compared to control cells. When isolated membranes were analyzed with an antiserum prepared against human erythrocyte glucose transporter, decreased cross-reactivity was observed in plasma membranes prepared from cycloheximide/insulin-treated cells compared to those from insulin cells. The present findings indicate that incubation of adipocytes with cycloheximide greatly reduces the number of hexose carriers in the plasma membrane of insulin-stimulated cells. Despite this reduction, insulin is still able to maximally stimulate glucose uptake. Thus, these data suggest an apparent dissociation between insulin stimulation of glucose transport activity and the recruitment of glucose carriers by the hormone.     

“Carrier activation” and glucose transport in Chinese hamster fibroblasts

Incubation of chinese hamster fibroblasts in glucose free medium, resulted in a 4 to 8 fold increase in the rate of D-glucose uptake and in a 3 to 4 fold increase in the uptake rate of glucose analogs (D-glucosamine, 2-Deoxy-D-glucose, 3-O-Methylglucose). In contrast to what is known for chick embryo fibroblasts, this increased hexose uptake activity is not blocked by cycloheximide in chinese hamster cells. The stimulation of synthesis of the Glucose Regulated Protein, GRP 95 which preceeds by 4 hours the stimulation of GRP 75 cannot account for the increase in hexose uptake-activity. Kinetic data have shown that the activation of glucose uptake activity following sugar starvation resulted only in a V_max increase; K_m for glucose remained constant at 0.6–0.7 mM. However, only the “activated” form of glucose uptake (glucose starvation) was very sensitive to N-ethylmaleimide. A mechanism of hexose “carrier activation” by glucose or a close metabolite is discussed.     

Effect of glucocorticoids on the glucose transport system of isolated fat cells.

Glucocorticoids inhibit glucose utilization by fat cells. The possibility that this effect results from altered glucose transport was investigated using an oil-centrifugation technique which allows a rapid (within 45 s) estimation of glucose or 3-O-methylglucose uptake by isolated fat cells. At high concentration (greater than 25 muM), dexamethasone inhibited glucose uptake within 1 min of its addition to fat cells. Efflux of 3-O-methylglucose was also impaired by 0.1 mM dexamethasone. However, diminished glucose uptake was not a specific effect of glucocorticoids; high concentrations (0.1 mM) of 17beta-estradiol, progesterone, and deoxycorticosterone produced a similar response in adipocytes. At a more physiologic steroid concentration (0.1 muM), glucocorticoids inhibited glucose uptake in a time-dependent manner (maximum effect in 1 to 2 hours). This effect was specific for glucocorticoids since, under these conditions, glucose uptake was not changed by the non-glucocorticoid steroids. Lineweaver-Burk analysis showed that 0.1 muM dexamethasone treatment produced a decrease in Vmax for glucose uptake but did not change the Ku. Hexokinase activity and ATP levels were not altered by this treatment, suggesting that processes involved in glucose phosphorylation were not affected. Dexamethasone treatment also caused a reduction in uptake of 3-O-methylglucose when assayed using a low sugar concentration (0.1 mM). At a high concentration (10 mM), uptake of the methyl sugar was only slightly less than normal in treated cells. Stimulation by insulin markedly enhanced uptake of glucose and 3-O-methylglucose by both treated and untreated cells. At a low hexose concentration (0.1 mM) and in the presence of insulin, sugar uptake by dexamethasone-treated cells was slightly less than control cells. Stimulation by insulin did however completely overcome the alteration in hexose uptake when larger concentrations of sugars (greater than 5 mM) were used. There was no detectable change in total protein synthesis during incubation of fat cells with dexamethasone. However, actinomycin C blocked the inhibitory effect of dexamethasone on glucose uptake. Cycloheximide, which caused a small inhibition in glucose uptake, prevented the full expression of the inhibitory effect of dexamethasone on glucose transport. These results indicate that dexamethasone alters the facilitated transport of glucose and, secondly, suggest that synthesis of RNA and protein is needed for glucocorticoid action.     

Effects of valinomycin on hexose transport and cellular ATP pools in mouse fibroblasts

The K+ ionophore valinomycin at concentrations of 1 X 10(-8) M and over, stimulated 2-deoxy-D-glucose (2DG) and 3-O-methylglucose (3OMG) uptake in Swiss 3T3 fibroblasts. The rate-limiting step of 2DG uptake was transport rather than phosphorylation, in the control or valinomycin-treated cells. Kinetic analysis showed that valinomycin increased the Vmax for 2DG uptake without change of the Km. The valinomycin-stimulated 2DG uptake was insensitive to 10 micrograms/ml cycloheximide, and extracellular K+ concentrations between 0.1 and 50 mM. On the other hand, valinomycin at the concentration of 1 X 10(-8) M and over, induced a rapid decrease in cellular ATP content, followed by stimulation of 2DG uptake and recovery of the ATP content. A similar relationship between the reduction of cellular ATP content and the subsequent stimulation of 2DG uptake was observed when the cells were treated not only with 2,4-dinitrophenol and iodoacetic acid, but also with other monovalent cation ionophores or inhibitors of oxidative phosphorylation. These results suggest that valinomycin may posttranslationally stimulate hexose transport by increasing the number of functional carriers of hexose or changing their mobility, and the rapid decrease in cellular ATP pools by valinomycin may be a trigger of the stimulation of the hexose transport in Swiss 3T3 fibroblasts.     

Expression of kinase-dependent glucose uptake in Saccharomyces cerevisiae

There are both low- and high-affinity mechanisms for uptake of glucose in Saccharomyces cerevisiae; high-affinity uptake somehow depends on the presence of hexose kinases (L. F. Bisson and D. G. Fraenkel, Proc. Natl. Acad. Sci. U.S.A. 80:1730-1734, 1983; L. F. Bisson and D. G. Fraenkel, J. Bacteriol. 155:995-1000, 1983). We report here on the effect of culture conditions on the level of high-affinity uptake. The high-affinity component was low during growth in high concentrations of glucose (100 mM), increased as glucose was exhausted from the medium, and decreased again during prolonged incubation in the stationary phase. The higher level of uptake was found in growth on low concentrations of glucose (0.5 mM) and in growth on normal concentrations of galactose, lactate plus glycerol, or ethanol. These results suggest that some component of high-affinity uptake is repressible by glucose. A shift from medium with 100 mM glucose to medium with 5 mM glucose resulted in up to a 10-fold increase in the level of high-affinity uptake within 90 min; the increase did not occur in the presence of cycloheximide or 2,4-dinitrophenol or in buffer alone with low glucose, suggesting that protein synthesis or energy metabolism (or both) was required. Reimposition of the high glucose concentration caused loss of high-affinity uptake, a process not prevented by cycloheximide. The use of hexokinase single-gene mutants showed that the derepression of high-affinity uptake was not clearly correlated with changes in levels of the kinases themselves. These results place the phenomenon of high- and low-affinity uptake in a physiological context, in that high-affinity uptake seems to be expressed best in conditions where it might be needed. Apparent similarities between glucose uptake in yeast and animal cells are noted.     

Glucose as a regulator of insulin-sensitive hexose uptake in 3T3 adipocytes

In the present study we examined the role of glucose in the regulation of its own transport activity in the cultured 3T3 fat cell. A regulatory control of glucose became apparent after these cells were cultured in the absence of glucose. Glucose deprivation of the cells was accompanied by a specific time and protein synthesis-dependent increase in dGlc (2-deoxyglucose) uptake (up to 5-fold), which was due to an increase in the apparent Vmax of the transport system. Concomitantly, the stimulatory effect of insulin on hexose uptake almost completely disappeared. Addition of glucose to the glucose-deprived cells rapidly reversed the deprivation effects. Cycloheximide experiments revealed that the glucose deprivation-induced increase in hexose uptake required protein synthesis as well as a protein synthesis-independent response to glucose deprivation that retarded the turnover of hexose transport activity. Taken together, these data indicate that glucose deprivation is accompanied by retardation of the rate of degradation, internalization, or inactivation of hexose transporters while the increase in dGlc uptake requires at least the continuation of protein synthesis-dependent de novo synthesis, insertion, or activation of hexose transporters. Hexose competitively taken up with dGlc, including the nonmetabolizable glucose analogue 3-O-methylglucose, could replace glucose in the process of prevention and reversal of the deprivation effects, indicating that competitive transport but not the metabolism of hexose is a prerequisite for the regulatory effect of glucose on the activity of its own transport system. In conclusion, our results indicate that in cultured 3T3 fat cells glucose itself is involved in the regulation of the activity of its own transport system by influencing the rate of degradation, internalization, or inactivation of hexose transporters by a protein synthesis-independent mechanism.     

Regulation of Biotin Transport in Saccharomyces cerevisiae

The metabolic control of biotin transport in Saccharomyces cerevisiae was investigated. Nonproliferating cells harvested from cultures grown in excess biotin (25 ng/ml) took up small amounts of biotin, whereas cells grown in biotin-sufficient medium (0.25 ng/ml) accumulated large amounts of the vitamin. Transport was inhibited maximally in cells grown in medium containing 9 ng (or more) of biotin per ml. When avidin was added to biotin-excess cultures, the cells developed the ability to take up large amounts of biotin. Boiled avidin was without effect, as was treatment of cells with avidin in buffer. Avidin did not relieve transport inhibition when added to biotin-excess cultures treated with cycloheximide, suggesting that protein synthesis was required for cells to develop the capacity to take up biotin after removal of extracellular vitamin by avidin. Cycloheximide did not inhibit the activity of the preformed transport system in biotin-sufficient cells. The presence of high intracellular free biotin pools did not inhibit the activity of the transport system. The characteristics of transport in biotin-excess cells (absence of temperature or pH dependence, no stimulation by glucose, absence of iodoacetate inhibition, independence of uptake on cell concentration, and nonsaturation kinetics) indicated that biotin entered these cells by diffusion. The results suggest that the synthesis of the biotin transport system in S. cerevisiae may be repressed during growth in medium containing high concentrations of biotin.     

Effect of radiation on the regulation of sodium-dependent glucose transport in LLC-PK1 epithelial cell line: possible model for gene expression

Low concentrations of glucose induce cultured kidney epithelial cells (LLC-PK1) to produce hexose transport proteins. We have investigated the effects of ionizing radiation on this induction process in plateau-phase cultures. The induced production of hexose transporters, requiring approximately 6 to 9 days for complete expression, can be inhibited by irradiation during the first 4 days. After the fourth postinduction day, radiation sensitivity decreases with almost no radiation effect on the induction of hexose transport apparent by the sixth day of the induction period. The D0 value associated with the induction block is approximately 25 Gy, a value which is considerably greater than that necessary to inhibit cell replication. Hexose transport, itself resistant to ionizing radiation at doses in excess of 100 Gy, is sensitive to cycloheximide throughout the induction period. The sensitivity to cycloheximide decreases during the last 2 days of the induction period, approximately 1 day after the reduction in radiosensitivity. Based on these properties hexose transport may be a convenient model for the study of radiation effects upon gene expression in this cell line.     

Glucose and insulin co-regulate the glucose transport system in primary cultured adipocytes. A new mechanism of insulin resistance

We have previously shown in primary cultured rat adipocytes that insulin acts at receptor and multiple postreceptor sites to decrease insulin's subsequent ability to stimulate glucose transport. To examine whether D-glucose can regulate glucose transport activity and whether it has a role in insulin-induced insulin resistance, we cultured cells for 24 h in the absence and presence of various glucose and insulin concentrations. After washing cells and allowing the glucose transport system to deactivate, we measured basal and maximally insulin-stimulated 2-deoxyglucose uptake rates (37 degrees C) and cell surface insulin binding (16 degrees C). Alone, incubation with D-glucose had no effect on basal or maximal glucose transport activity, and incubation with insulin, in the absence of glucose, decreased maximal (but not basal) glucose transport rates only 18% at the highest preincubation concentration (50 ng/ml). However, in combination, D-glucose (1-20 mM) markedly enhanced the long-term ability of insulin (1-50 ng/ml) to decrease glucose transport rates in a dose-responsive manner. For example, at 50 ng/ml preincubation insulin concentration, the maximal glucose transport rate fell from 18 to 63%, and the basal uptake rate fell by 89%, as the preincubation D-glucose level was increased from 0 to 20 mM. Moreover, D-glucose more effectively promoted decreases in basal glucose uptake (Ki = 2.2 +/- 0.4 mM) compared with maximal transport rates (Ki = 4.1 +/- 0.4 mM) at all preincubation insulin concentrations (1-50 ng/ml). Similar results were obtained when initial rates of 3-O-methylglucose uptake were used to measure glucose transport. D-glucose, in contrast, did not influence insulin-induced receptor loss. In other studies, D-mannose and D-glucosamine could substitute for D-glucose to promote the insulin-induced changes in glucose transport, but other substrates such as L-glucose, L-arabinase, D-fructose, pyruvate, and maltose were without effect. Also, non-metabolized substrates which competitively inhibit D-glucose uptake (3-O-methylglucose, cytochalasin B) blocked the D-glucose plus insulin effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of hexose transport in BALB/c 3T3 preadipose cells: effects of glucose concentration and 12-O-tetradecanoylphorbol-13-acetate

Like many cell types in culture, both undifferentiated and differentiated BALB/c 3T3 preadipose cells respond to glucose deprivation with an increased uptake of 2-deoxy-D-glucose (deoxyglucose) and 3-O-methyl-D-glucose (methylglucose). Glucose readdition to glucose-deprived cultures resulted in a prompt fall in uptake activity; in undifferentiated cells, a half-maximally effective concentration of glucose was approximately 0.5 mM, while 0.1 mM was ineffective. Several hexoses differed in their efficacy of "deactivating" methylglucose transport in glucose-deprived cells; it appeared that a particular hexose must be metabolized beyond the 6-phosphate form to deactivate the transport system. Previous studies have shown that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates hexose transport in undifferentiated and differentiated BALB/c 3T3 cells. In this study, it was found that TPA (and insulin in differentiated cells) prevented the glucose-induced deactivation of transport activity. Glucose-induced deactivation of transport activity was also prevented by cycloheximide or actinomycin D addition concomitantly with glucose. In glucose-starved cells, agents such as TPA and insulin appear to override a cellular control mechanism sensitive to the external concentration of glucose, so that elevated levels of transport activity are maintained under environmental conditions (i.e., a return to physiological glucose concentrations) that normally induce a fall in transport activity.     

Active transport of myo-inositol in rat pancreatic islets.

myo-Inositol transport by isolated pancreatic islets was measured with a dual isotope technique. Uptake was saturable with a half-maximal response at approx. 75 microM. With 50 microM-inositol, uptake was linear for at least 2 h during which time the free intracellular concentration rose to double that of the incubation medium. Inositol transport is therefore active and probably energized by electrogenic co-transport of Na+ down its concentration gradient as uptake was inhibited by ouabain, Na+ removal or depolarizing K+ concentrations. Inositol transport was abolished by cytochalasin B which binds to hexose carriers, but not by carbamoylcholine or Li+ which respectively stimulate or inhibit phosphoinositide turnover. Uptake of inositol was not affected by 3-O-methylglucose or L-glucose (both 100 mM) nor by physiological concentrations of D-glucose. The results suggest that most intracellular inositol in pancreatic islets would be derived from the extracellular medium. Since the transport mechanism is distinct from that of glucose, inositol uptake would not be inhibited during periods of hyperglycaemia.     

Transport by the (Na+,K+) ATPase: modulation by differentiation inducers and inhibition of protein synthesis in the MDCK kidney epithelial cell line

MDCK kidney epithelial cell cultures exposed to the differentiation inducer hexamethylene bisacetamide (HMBA) for 24 hours exhibited a 50% decrease in transport activity per (Na+,K+)-ATPase molecule (turnover number) but an unchanged number of pump sites (Kennedy and Lever, 1984). Inhibition of protein synthesis by either 10 microM cycloheximide or 2 microM emetine blocked the inhibitory effects of HMBA on Na+/K+ pump efficiency assessed by measurements of [3H]-ouabain binding to intact cells, (Na+,K+) ATPase activity of detergent-activated cell extracts, and ouabain-sensitive Rb+ uptake. In the absence of inducer treatment, inhibition of protein synthesis increased Na+/K+ pump turnover number by twofold while maintaining Na+/K+ pump activity per cell at a constant level. Intracellular Na+ levels were decreased after cycloheximide treatment; therefore, pump stimulation was not due to substrate effects. Furthermore, cycloheximide effects of Rb+ uptake could be dissociated from effects on tight junctions. These observations suggest that the transport activity of the (Na+,K+) ATPase is tightly regulated by factors dependent on protein synthesis.