Antifolate transport in L1210 leukemia cells. Kinetic evidence for the non-identity of carriers for influx and efflux

The kinetics of methotrexate transport in L1210 cells are described. Data derived from the measurements of initial influx, the complete time-course of uptake, intracellular steady-state level and unidirectional efflux were found to be consistent with a simple empirical equation containing three constants. Properties of the system include the following: (1) saturability of initial influx; (2) approach to steady state during uptake is expoential; (3) the half-time for drug uptake is independent of external concentration and qual to half-time for efflux; and (4) transport is concentrative at low external concentrations, whereas the reverse is true at high external concentrations. These observations are incorporated into a kinetic model which quantitatively accounts for the data on the basis of the hypothesis that influx and efflux take place via different carriers.     

Evidence for a phosphoenolpyruvate-dependent sugar phosphotransferase in Mycoplasma strain Y

The uptake of (14)C-alpha-methyl-d-glucoside (alphaMG) by washed cells of Mycoplasma strain Y was found to be dependent on the supply of metabolic energy. Glycerol or d-mannose, but not l-lactate, would serve as an energy source. Uptake was inhibited by fluoride, iodoacetate, and arsenate, but not by 2,4-dinitrophenol. d-Glucose was inhibitory, presumably by competing for the transport system. The initial product of accumulation had the properties of a phosphate ester of alphaMG. The proportion of free alphaMG in the cells increased with time, until a steady state was reached in which uptake was balanced by the efflux of free alphaMG from the cells. Broken-cell preparations catalyzed a phosphoenolpyruvate-dependent phosphorylation of alphaMG and of d-glucose.     

Rapid changes in bidirectional K+ fluxes preceding DMSO-induced granulocytic differentiation of HL-60 human leukemic cells

When grown in medium containing 5 mM potassium and 140 mM sodium, HL-60, a human promyelocytic cell line, maintained a steady-state intracellular K+ concentration of 145 mmol/L cells and a steady-state intracellular Na+ concentration of 30 mmol/L cells. Nearly 90% of the unidirectional 42K+ influx could be inhibited by the cardiac glycoside ouabain with a Ki of 5 X 10(-8) M. This ouabain-sensitive component of influx rose as a saturating function of the extracellular K+ concentration with a K1/2 of 0.85 mM. The component of 42K+ influx resistant to ouabain inhibition was a linear function of the extracellular K+ concentration and was insensitive to inhibition by the diuretic furosemide. Unidirectional K+ efflux followed first order kinetics with a half-time of 55 min. Addition of 1.5% dimethyl sulfoxide (DMSO) to a culture of HL-60 cells allowed two population doublings followed by the cessation of growth without an impairment of cell viability. Beginning 2 to 3 days after DMSO addition, the cells underwent a dramatic reduction in volume (from 925 microns 3 to 500 microns 3) and began to take on the morphological features of mature granulocytes. Throughout this process of differentiation there was no change in the intracellular sodium or potassium concentration. However, immediately following the addition of DMSO to a culture of cells, there began an immediate, coordinated reduction in bidirectional K+ flux. The initial rate of the ouabain-sensitive component of K+ influx fell with a half-time of 11 h to a final rate, at 6 days induction, equal to one ninth that of the uninduced control, and over the same period, the rate constant for K+ efflux fell with a half-time of 14 h to a final value one fourth that of the uninduced control. The rapidity with which these flux changes occur raises the possibility that they play some role in the control of subsequent events in the process of differentiation.     

Kinetic Analyses of Calcium Movements in HeLa Cell Cultures : II. Calcium efflux

Calcium efflux was studied in monolayers of HeLa cells. The fast phase of exchange was studied in an open system by continuous washout. Its half-time was 1.58 min which is practically identical to the fast phase of calcium influx previously found to be 1.54 min. This suggests that the fast component of efflux represents calcium exchange from an extracellular compartment probably from calcium bound to the cell membrane surface. Dinitrophenol (DNP) and iodoacetate (IAA) do not inhibit calcium efflux from this compartment. The slow phase of calcium exchange was studied in a closed three compartment system. The half-time of calcium efflux measured under these conditions is almost identical to that obtained previously in studies of calcium influx: 33.0 and 37.0 min, respectively. This slow compartment is likely to be the intracellular exchangeable calcium pool. DNP and IAA inhibit calcium efflux from this compartment, lengthening the half-time from 33 min to 55.0 and 216 min, respectively. This suggests that calcium extrusion from the cell is an active process. Since calcium influx is not affected by metabolic inhibitors, the cellular calcium concentration increases as would be predicted under these conditions. Calcium efflux is also markedly depressed by lowering the temperature.     

Transport of leucine and sodium in central nervous tissue: studies on retina in vitro

Unidirectional leucine fluxes were measured in isolated rabbit retina maintained under steady state conditions in medium resembling CSF but with leucine varied from 2 to 20,000 microM. At physiological leucine concentration (11 microM), 1/2 time for outward transport was 88 s and intracellular fluid was cleared of isotopically labelled leucine at 2.3 ml/g dry wt./min; 1/2 time for inward transport was 16 s and interstitial fluid was cleared at 7.5 ml/g dry wt./min. The rate of leucine influx corresponded quite well with its rate of disappearance from the intracellular fluid, over a wide range of concentrations. Exchange diffusion was demonstrated for transport in both directions. There was competition by other amino acids, but no interaction between Na+ and leucine transport could be demonstrated. Kinetic analysis indicated the presence of more than one transport system for leucine. There was an unexpected fall in the efflux coefficient, with reduction in leucine concentration at the lower end of the concentration range, for which an explanation is proposed. Under control conditions, 1/2 time for efflux of intracellular 24Na+ was about 0.9 min. With intracellular Na+ increased 4 fold, 1/2 time for efflux was slightly reduced. Problems encountered in measuring fluxes in organized tissue are discussed.     

One-way fluxes of alpha-aminoisobutyric acid in Ehrlich ascites tumor cells. Trans effects and effects of sodium and potassium

One-way fluxes in the steady state and one-way influxes at zero intracellular concentrations were measured for alpha-aminoisobutyric acid (AIB) in Ehrlich ascites tumor cells at 32 degrees C. The one-way fulxes show trans effects in the concentration of AIB and are dependent on sodium levels. The one-way fluxes for initial influx and for the steady state were fitted with the equations derived for the frequently used two-state carrier model. Estimates of the parameters of these equations were obtained with use of nonlinear least squares. These gave relatively good fits of the flux data and the data on steady-state distribution ratios. The two-state carrier model predicted a trans inhibition of one-way influx and a trans stimulation of one-way efflux. The former phenomenon has been demonstrated for AIB transport in Ehrlich ascites cells and there is evidence, through less firm, for the latter.     

Characteristics of alpha-aminoisobutyric acid transport in rat skeletal muscles.

alpha-Aminoisobutyric acid (AIB) transport into the intracellular compartment of extensor digitorum longus and soleus muscles was measured (in vitro) after allowance for the equilibration of the amino acid in the extracellular space. The latter was determined with three markers, [14C]inulin, 60Co-EDTA and [3H]mannitol. Net transport of AIB was subsequently divided into its two components, i.e. influx and efflux. Rates of influx were measured as the intracellular accumulation of [14C]AIB after a short incubation (5 min), and efflux was measured as the release of AIB with time (up to maximum of 50 min) from muscles that had previously been preloaded with AIB. This intracellular efflux was resolved into two phases, which probably represent two separate components of exit. The influence of extracellular Na+ on the transport of this neutral amino acid (representing the A system) was investigated. Na+ depletion resulted in lower accumulations of AIB, the effects becoming more pronounced with progressive depletions of external Na+. These changes arose from an inhibition of AIB influx, concomitant with an enhancement of its efflux. In contrast, all components of tyrosine transport (representing the L system) were unaffected by lowering external Na+ concentrations. The net accumulation of AIB was also suppressed by cortisol. This inhibitory effect was, however, Na+-dependent and resulted solely from the steroid's enhancement of AIB efflux, the hormone being without effect on AIB influx.     

Rapid, futile K+ cycling and pool-size dynamics define low-affinity potassium transport in barley

Using the short-lived radiotracer 42K+, we present a comprehensive subcellular flux analysis of low-affinity K+ transport in plants. We overturn the paradigm of cytosolic K+ pool-size homeostasis and demonstrate that low-affinity K+ transport is characterized by futile cycling of K+ at the plasma membrane. Using two methods of compartmental analysis in intact seedlings of barley (Hordeum vulgare L. cv Klondike), we present data for steady-state unidirectional influx, efflux, net flux, cytosolic pool size, and exchange kinetics, and show that, with increasing external [K+] ([K+]ext), both influx and efflux increase dramatically, and that the ratio of efflux to influx exceeds 70% at [K+]ext > or = 20 mm. Increasing [K+]ext, furthermore, leads to a shortening of the half-time for cytosolic K+ exchange, to values 2 to 3 times lower than are characteristic of high-affinity transport. Cytosolic K+ concentrations are shown to vary between 40 and 200 mm, depending on [K+]ext, on nitrogen treatment (NO3- or NH4+), and on the dominant mode of transport (high- or low-affinity transport), illustrating the dynamic nature of the cytosolic K+ pool, rather than its homeostatic maintenance. Based on measurements of trans-plasma membrane electrical potential, estimates of cytosolic K+ pool size, and the magnitude of unidirectional K+ fluxes, we describe efflux as the most energetically demanding of the cellular K+ fluxes that constitute low-affinity transport.     

Ca2+ homeostasis in permeabilized human neutrophils. Characterization of Ca2+-sequestering pools and the action of inositol 1,4,5-triphosphate

The regulation of Ca2+ transport by intracellular compartments was studied in digitonin-permeabilized human neutrophils, using a Ca2+-selective electrode. When incubated in a medium containing ATP and respiratory substrates, the cells lowered within 6 min the ambient [Ca2+] to a steady state of around 0.2 microM. A vesicular ATP-dependent and vanadate-sensitive non-mitochondrial pool maintained this low [Ca2+] level. In the absence of ATP, a higher Ca2+ steady state of 0.6 microM was seen, exhibiting the characteristics of a mitochondrial Ca2+ "set point." Both pools were shown to act in concert to restore the previous ambient [Ca2+] following its elevation. Thus, the mitochondria participate with the other pool(s) in decreasing [Ca2+] to the submicromolar range whereas only the nonmitochondrial pool(s) lowers [Ca2+] to the basal level. The action of inositol 1,4,5-triphosphate (IP3) which has been inferred to mediate Ca2+ mobilization in a few cell types was studied. IP3 released (detectable within 2 s) Ca2+ accumulated in the ATP-dependent pool(s) but had no effect on the mitochondria. The response was transient and resulted in desensitization toward subsequent IP3 additions. Under experimental conditions in which the ATP-dependent Ca2+ influx was blocked, the addition of IP3 resulted in a very large Ca2+ release from nonmitochondrial pool. The results strongly suggest that IP3 is a second messenger mediating intracellular Ca2+ mobilization in human neutrophils. Furthermore, the nonmitochondrial pool appears to have independent influx and efflux pathways for Ca2+ transport, a Ca2+ ATPase (the influx component) and an IP3-sensitive efflux component activated during Ca2+ mobilization.     

Effects of metabolic deprivation on methotrexate transport in L1210 leukemia cells: Further evidence for separate influx and efflux systems with different energetic requirements

Summary Measurements of methotrexate transport in L1210 cells in the presence and absence ofd-glucose reveal that both influx and efflux are depressed in the absence ofd-glucose, whereas the steady-state accumulation of drug is enhanced. The reason for the increase in steady state is that the relative decline in efflux is greater than the decline in influx. Analysis of the concentration dependence of steady-state methotrexate accumulation ind-glucose-deprived cells indicates a linear relationship consistent with a one-carrier active transport model. Similar data in nondeprived cells is highly nonlinear and strongly supports the postulate that under physiological conditions influx and efflux of methotrexate are mediated by separate carrier systems. These results indicate that the efflux system, preferentially transporting methotrexate under normal conditions, cannot operate in the absence ofd-glucose, whereas the influx system is only partially inhibited under conditions of glucose deprivation.     

The relationship between calcium flux and beating in hyperpermeable heart muscle cells

Beating hyperpermeable myocardial fragments, suspended in fresh medium, show a Ca uptake rate which greatly exceeds the release rate. Under these conditions, Ca influx and contraction frequeny co-vary with temperature. Ca efflux, however, is not temperature responsive. In addition, influx, but not efflux, is ATP-dependent. This implies that influx, but not efflux, is a controlled process, the activity of which has the same determinants as does contraction. However, under conditions approximating the steady state, the Ca influx rate was found to decrease, while the efflux rate increased. This suggests the possibility, at least, that the source of contraction-activating Ca can vary with the prevailing conditions.     

Concentrative accumulation of choline by human erythrocytes

Influx and efflux of choline in human erythrocytes were studied using ¹⁴C-choline. When incubated at 37°C with physiological concentrations of choline erythrocytes concentrate choline; the steady-state ratio is 2.08 ± 0.23 when the external choline is 2.5 µM and falls to 0.94 ± 0.13 as the external concentration is raised to 50 µM. During the steady state the influx of choline is consistent with a carrier system with an apparent Michaelis constant of 30 x 10^-6 and a maximum flux of 1.1 µmoles per liter cells per min. For the influx into cells preequilibrated with a choline-free buffer the apparent Michaelis constant is about 6.5 x 10^-6 M and the maximum flux is 0.22 µmole per liter cells per min. At intracellular concentrations below 50 µmole per liter cells the efflux in the steady state approximates first order kinetics; however, it is not flux through a leak because it is inhibited by hemicholinium. Influx and efflux show a pronounced exchange flux phenomenon. The ability to concentrate choline is lost when external sodium is replaced by lithium or potassium. However, the uphill movement of choline is probably not coupled directly to the Na⁺ electrochemical gradient.     

Significance of the inactivation of transport in thermal death of Escherichia coli.

Cells of Escherichia coli ML308-225, harvested from the exponential phase, were heated in 50 mM potassium phosphate, and the loss in viability and inability to transport lactose, proline, and alpha-methylglucoside was compared. After cells were heated at 48 degrees C for 15 min, there was a 16% loss in viability and a similarly small reduction in the steady-state accumulation of lactose at 25 degrees C. The initial rates of lactose and proline transport were severely inhibited by heating at either 48 or 50 degrees C, but substantial recovery occurred within 5 to 7 min at 25 degrees C. Heating at 50 degrees C for 15 min caused an 86% loss in viability, but only a 53% decrease in the steady-state accumulation of lactose and only a 24% reduction in the initial rate of alpha-methylglucoside uptake. Twice as much alpha-methylglucoside was accumulated at 50 degrees C as at 25 degrees C. Although alpha-methylglucoside phosphate leaked from the cells at 50 degrees C, the concentration retained within the cells was about 500 times that externally, when only about 14% of the cells were viable. Overall, these results indicate that cells made nonviable by heating at 50 degrees C still have significant membrane integrity.     

Regulatory interaction of ATP Na+ and Cl- in the turnover cycle of the NaK2Cl cotransporter

To probe the mechanism by which intracellular ATP, Na+, and Cl- influence the activity of the NaK2Cl cotransporter, we measured bumetanide-sensitive (BS) 86Rb fluxes in the osteosarcoma cell line UMR- 106-01. Under physiological gradients of Na+, K+, and Cl-, depleting cellular ATP by incubation with deoxyglucose and antimycin A (DOG/AA) for 20 min at 37 degrees C reduced BS 86Rb uptake from 6 to 1 nmol/mg protein per min. Similar incubation with 0.5 mM ouabain to inhibit the Na+ pump had no effect on the uptake, excluding the possibility that DOG/AA inhibited the uptake by modifying the cellular Na+ and K+ gradients. Loading the cells with Na+ and depleting them of K+ by a 2-3- h incubation with ouabain or DOG/AA increased the rate of BS 86Rb uptake to approximately 12 nmol/mg protein per min. The unidirectional BS 86Rb influx into control cells was approximately 10 times faster than the unidirectional BS 86Rb efflux. On the other hand, at steady state the unidirectional BS 86Rb influx and efflux in ouabain-treated cells were similar, suggesting that most of the BS 86Rb uptake into the ouabain-treated cells is due to K+/K+ exchange. The entire BS 86Rb uptake into ouabain-treated cells was insensitive to depletion of cellular ATP. However, the influx could be converted to ATP-sensitive influx by reducing cellular Cl- and/or Na+ in ouabain-treated cells to impose conditions for net uptake of the ions. The BS 86Rb uptake in ouabain-treated cells required the presence of Na+, K+, and Cl- in the extracellular medium. Thus, loading the cells with Na+ induced rapid 86Rb (K+) influx and efflux which, unlike net uptake, were insensitive to cellular ATP. Therefore, we suggest that ATP regulates a step in the turnover cycle of the cotransporter that is required for net but not K+/K+ exchange fluxes. Depleting control cells of Cl- increased BS 86Rb uptake from medium-containing physiological Na+ and K+ concentrations from 6 to approximately 15 nmol/mg protein per min. The uptake was blocked by depletion of cellular ATP with DOG/AA and required the presence of all three ions in the external medium. Thus, intracellular Cl- appears to influence net uptake by the cotransporter. Depletion of intracellular Na+ was as effective as depletion of Cl- in stimulating BS 86Rb uptake.(ABSTRACT TRUNCATED AT 400 WORDS)     

Active transport of potassium by the Cecropia midgut; tracer kinetic theory and transport pool size.

1. Tracer influx kinetics have been analysed theoretically to determine the size of the transport pool with no assumptions regarding the transport pathway. 2. For a calculation of the size of the transport pool to be made, the following six conditions are required by the theory: tracer steady state attained, tissue steady state attained, Isc measures next flux, small magnitude and constant time-course of efflux, and correction for decay in pumping rate. 3. The size of the pool, SI, is given by the steady state influx, Finfinity, divided by the mixing-time constant, alpha. 4. Some experimental results are analysed by three different graphical methods, and it is shown that these three methods are equivalent. Specifically, alpha is equal to the reciprocal of the 75% mixing time, t75, divided by 1n 4 and is equal to the reciprocal of the lag time, X. 5. The tracer kinetic theory is applied to active potassium transport across the isolated short-circuited midgut: the transport meets the six conditions required by the theory. 6. The size of the transport pool of potassium in one midgut is calculated to be 80.5 muequiv./g wet weight under high-K steady-state conditions. A value as high as this suggests that the pool is intracellular.     

Significance of the inactivation of transport in thermal death of Escherichia coli.

Cells of Escherichia coli ML308-225, harvested from the exponential phase, were heated in 50 mM potassium phosphate, and the loss in viability and inability to transport lactose, proline, and alpha-methylglucoside was compared. After cells were heated at 48 degrees C for 15 min, there was a 16% loss in viability and a similarly small reduction in the steady-state accumulation of lactose at 25 degrees C. The initial rates of lactose and proline transport were severely inhibited by heating at either 48 or 50 degrees C, but substantial recovery occurred within 5 to 7 min at 25 degrees C. Heating at 50 degrees C for 15 min caused an 86% loss in viability, but only a 53% decrease in the steady-state accumulation of lactose and only a 24% reduction in the initial rate of alpha-methylglucoside uptake. Twice as much alpha-methylglucoside was accumulated at 50 degrees C as at 25 degrees C. Although alpha-methylglucoside phosphate leaked from the cells at 50 degrees C, the concentration retained within the cells was about 500 times that externally, when only about 14% of the cells were viable. Overall, these results indicate that cells made nonviable by heating at 50 degrees C still have significant membrane integrity.     

Transport of methotrexate in L1210 cells: Effect of ions on the rate and extent of uptake

Transport of methotrexate (MTX) in L1210 cells is highly dependent upon the ionic composition of the external medium. Half-maximal rates of MTX transport (K_t values) vary from 0.9 μm in cells suspended in potassium-Hepes buffer containing Mg²⁺ (Hepes-Mg), to 10 μm in phosphate-buffered saline (PBS). At saturating levels of substrate, however, transport rates approach the same maximum velocity (V) regardless of the buffering medium. The increased K_t value for MTX in PBS is due to the presence of the competitive inhibitors, phosphate (K_i = 0.87 mM) and Cl^? (K_i = 46 mM). Concentration gradients for MTX at the steady state are also much lower (about 20-fold) in PBS than in Hepes-Mg; the components of PBS that reduce this uptake parameter are phosphate, Cl^?, Ca²⁺, and Na⁺. Ions that decrease the influx rate or the steady-state level also produce an increase in MTX efflux. Glucose (which increases ATP levels) reduces influx rates and steady-state levels of MTX, and induces efflux in both PBS and Hepes-Mg. Conversely, the combination of azide plus iodoacetate (which reduces ATP levels) stimulates MTX uptake in PBS, but has little effect on MTX transport parameters in Hepes-Mg. The unusually high sensitivity of MTX transport to various anions is consistent with the hypothesis that this system catalyzes the exchange of external MTX for an intracellular anion, and that efflux of the anion down a concentration gradient provides the driving force for active transport of MTX.     

A confirmation of the proposed model for the hexose uptake system ofChlorella vulgaris. Anaerobic studies in the light and in the dark

Summary Energy for accumulation of sugar analogues inChlorella vulgaris can be supplied by respiration and by light. Under anaerobic conditions a quantum efficiency of close to one has been determined for light-dependent uptake of 6-deoxyglucose at 712 nm. Although the rate of uptake under anaerobic conditions in the dark is less than 15% of that in the light, the steady-state plateau of accumulation does not change when the light is turned off. This is explained by the observation that efflux of sugar is inhibited by lack of energy to the same extent as influx. This agrees with a model proposed earlier (Komor, Haass & Tanner,Biochim. Biophys. Acta 266:649, 1972). Under aerobic conditions during the steady-state plateau in the dark, the rate of influx of 6-deoxyglucose is twice that of initial influx (positive transmembrance effect). By anaerobiosis the initial influx is almost completely inhibited, whereas the steady-state influx is affected only to 50%. This points to an energyless homoexchange. However, this flux is completely inhibited by uncoupling agents. To explain these results, the existence of an energygenerating efflux has been assumed, which is affected by uncouplers. The efficiency of energy production by efflux and the re-use of this energy for influx is inversely related to the amount of energy supplied by other energy-generating processes in the cells; under anaerobic conditions in the dark, 90% of the efflux energy seems to be available for influx.     

EXCHANGE OF TAURINE IN BRAIN SLICES OF ADULT and 7-DAY-OLD RATS

Abstract— The influx or efflux of taurine in brain slices prepared from adult and 7-day-old rats was studied in Krebs-Ringer bicarbonate-glucose medium with 0,2 and 10 m m -taurine. The exchange of taurine between the slices and the medium was slow, and no steady-state concentration was reached within the experimental period of 150 min. In both experimental groups there was a net influx of taurine into the slices from 10 m m -taurine and a slight net efflux from the slices into 2 m m -taurine. The rate of influx from 10 m m -taurine was about the same in the two groups after an initial period of faster influx into the slices of adult rats. There was some rapid initial efflux into 0 and 2 m m -taurine solutions from the slices from 7-day-old rats, but with prolonged incubation these slices were better able to maintain their intracellular taurine than the slices from adult rats. The reasons and significance of the high cerebral concentration of taurine in immature brain in vivo are briefly discussed in the light of the present and earlier studies.     

Characterization of Na+/H+ exchange in a rabbit corneal epithelial cell line (SIRC)

Continuous intracellular pH (pHi) measurements were performed in SIRC rabbit corneal epithelial cells using the pH-sensitive absorbance of intracellularly trapped 5(and 6)-carboxy-4',5'-dimethylfluorescein. Steady-state pHi in nominally bicarbonate free Ringer's solution averaged 6.87 +/- 0.02 (mean +/- S.E., n = 53). After intracellular acidification induced by the NH4Cl-prepulse technique, there was a sodium-dependent pHi recovery towards the normal steady-state pHi. The initial pHi recovery rate was a saturable function of extracellular sodium concentration with an apparent Km for external sodium of about 25 mM and a Vmax of about 0.28 pH units/min. Virtually no pHi recovery was observed in the absence of extracellular sodium. Sodium removal during steady state acidified the cells by 0.36 +/- 0.05 pH units (mean +/- S.E., n = 13) within 5 min. There was a dose-dependent inhibition of pHi recovery after NH4Cl prepulse by amiloride with an IC50 of about 15 microM. Amiloride in a concentration of 1 mM almost completely abolished pHi recovery. Amiloride (1 mM) applied during steady state induced an intracellular acidification of 0.2 +/- 0.03 pH units (mean +/- S.E., n = 7) within 5 min. These findings suggest that a Na+/H+ exchange is present in SIRC rabbit corneal epithelial cells. Na+/H+ exchange seems to be the major process involved in pHi recovery in SIRC cells after an intracellular acid load. Na+/H+ exchange also plays a role in the maintenance of steady-state pHi.