Purine nucleobase transport in human erythrocytes. Reinvestigation with a novel "inhibitor-stop" assay

A novel "inhibitor-stop" method for the determination of initial rates of purine nucleobase transport in human erythrocytes has been developed, based on the addition of seven assay volumes of cold 19 mM papaverine to terminate influx. In view of our finding that the initial velocities of adenine, guanine, and hypoxanthine influx into human erythrocytes were linear for only 4-6 s at 37 degrees C, the present method has been used to reexamine the kinetics of purine nucleobase transport in these cells. Initial influx rates of all three purine nucleobases were shown to be the result of concurrent facilitated and nonfacilitated diffusion. The nonfacilitated influx rates could be estimated either from the linear concentration dependence of nucleobase influx at high concentrations of permeant or from residual influx rates which were not inhibited by the presence of co-permeants. Appropriate corrections for nonfacilitated diffusion were made to the influx rates observed at low nucleobase concentrations. Kinetic analyses indicated that adenine (Km = 13 +/- 1 microM, n = 7), guanine (Km = 37 +/- 2 microM, n = 5), and hypoxanthine (Km = 180 +/- 12 microM, n = 6) were mutually competitive substrates for transport. The Ki values obtained with each nucleobase as an inhibitor of the influx of the other nucleobases were similar to their respective Km values for influx. Furthermore, the transport of the purine nucleobases was not inhibited by nucleosides (uridine, inosine) or by inhibitors of nucleoside transport (6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, dilazep, dipyridamole). It is concluded that all three purine nucleobases share a common facilitated transport system in human erythrocytes which is functionally distinct from the nucleoside transporter.     

Acyclovir transport into human erythrocytes

The mechanism of transport of the antiviral agent acyclovir (ACV) into human erythrocytes has been investigated. Initial velocities of ACV influx were determined with an "inhibitor-stop" assay that used papaverine to inhibit ACV influx rapidly and completely. ACV influx was nonconcentrative and appeared to be rate-saturable with a Km of 260 +/- 20 microM (n = 8). However, two lines of evidence indicate that ACV permeates the erythrocyte membrane by means other than the nucleoside transport system: 1) potent inhibitors (1.0 microM) of nucleoside transport (dipyridamole, 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, and dilazep) had little (less than 8% inhibition) or no effect upon the influx of 5.0 microM ACV; and 2) a 100-fold molar excess of several purine and pyrimidine nucleosides had no inhibitory effect upon the influx of 1.0 microM ACV. However, ACV transport was inhibited competitively by adenine (Ki = 9.5 microM), guanine (Ki = 25 microM), and hypoxanthine (Ki = 180 microM). Conversely, ACV was a competitive inhibitor (Ki = 240-280 microM) of the transport of adenine (Km = 13 microM), guanine (Km = 37 microM), and hypoxanthine (Km = 180 microM). Desciclovir and ganciclovir, two compounds related structurally to ACV, were also found to be competitive inhibitors of acyclovir influx (Ki = 1.7 and 1.5 mM, respectively). These results indicate that ACV enters human erythrocytes chiefly via the same nucleobase carrier that transports adenine, guanine, and hypoxanthine.     

2',3'-Dideoxythymidine permeation of the human erythrocyte membrane by nonfacilitated diffusion

The influx of 2',3'-dideoxythymidine into human erythrocytes was characterized to gain insight into the molecular properties of 3'-azido-3'-deoxythymidine which allow this latter nucleoside analog to permeate cell membranes by nonfacilitated diffusion (J. Biol. Chem. 262, 5748-5754 (1987]. The influx of 2',3'-dideoxythymidine was (1) nonconcentrative, (2) a linear function of permeant concentration (0.05 to 12 mM), and (3) insensitive to potent inhibitors of nucleoside transport and to permeants of either the nucleoside or nucleobase transporter. It is concluded that 2',3'-dideoxythymidine, like 3'-azido-3'-deoxythymidine, permeates the human erythrocyte membrane predominantly by nonfacilitated diffusion. This unusual characteristic of these two nucleoside analogs is attributed both to their lack of a 3'-hydroxyl moiety, a structural determinant which appears to be important for transport by the nucleoside carrier, and to their relatively high partition coefficients (greater than or equal to 0.2).     

2',3'-dideoxycytidine permeation of the human erythrocyte membrane

The mechanism by which 2,3'-dideoxycytidine, an inhibitor of HIV-I infectivity, permeates the cell membrane was investigated. The influx of ddCyd into human erythrocytes was nonconcentrative. The initial velocity of both ddCyd influx and efflux was, in contrast to compounds that permeate the cell membrane via the nucleoside transporter, a linear function of nucleoside concentration in the 1 microM to 10 mM range and relatively insensitive to temperature. Furthermore, potent inhibitors of nucleoside transporter and other nucleosides were found to inhibit ddCyd influx only partially or not at all suggesting that ddCyd permeates the human erythrocyte membrane predominantly by nonfacilitated diffusion. This unusual characteristic seems to be due to the lack of 3'-hydroxyl moiety of ddCyd which appears to be an important determinant for the nucleoside carrier specificity rather than to lipid solubility itself. As far as permeation of the cell membrane is concerned ddCyd shares these properties with 2',3'-dideoxythymidine and 3'-azido-3'-deoxythymidine.     

Enantiomeric selectivity of carbovir transport.

Carbovir (9-[4 alpha-(hydroxymethyl)cyclopent-2-ene-1 alpha-yl]guanine) (CBV) is a carbocyclic analogue of 2',3'-dideoxyguanosine that exhibits potent and selective in vitro activity against human immunodeficiency virus. Antiviral activity is associated with only the (-)-enantiomer. The transport characteristics of both (-)-CBV and (+)-CBV were investigated in human erythrocytes at 37 degrees C using a papaverine-stop assay. The influx of both enantiomers appeared saturable and was inhibited greater than 90% by a combination of adenine (a low Km permeant of the nucleobase carrier) and dilazep (a potent inhibitor of nucleoside transport). The influx of (-)-CBV and (+)-CBV proceeded primarily via the nucleobase carrier with Vmax (picomoles/second/5 microliters of cells)/Km (millimolar) values of 17/0.12 and 140/1.9, respectively. To a lesser extent, the influx of (-)-CBV and (+)-CBV also occurred via the nucleoside transporter. Although both compounds exhibited a similar low affinity for this latter carrier (Km approximately 2 mM), the Vmax for (-)-CBV influx was approximately 4-fold higher than the Vmax for (+)-CBV influx. We conclude that both CBV enantiomers enter human erythrocytes by two transporters that are enantiomerically selective.     

Modulation of diphthamide synthesis by 5'-deoxy-5'-methylthioadenosine in murine lymphoma cells

The histidine derivative diphthamide occurs uniquely in eukaryotic elongation factor 2 (EF-2), and is the specific target for the diphtheria toxin mono(ADP-ribosyl)transferase. The first step in diphthamide biosynthesis may involve the transfer of aminocarboxypropyl moiety from S-adenosylmethionine to the imidazole ring of histidine in EF-2, to yield 2-(3-carboxy-3-aminopropyl)histidine and 5'-deoxy-5'-methylthioadenosine (MeSAdo). As the possible nucleoside product of the initial reaction in the diphthamide biosynthetic pathway, MeSAdo could be an inhibitor of diphthamide formation. In the present experiments, we have analyzed the effects of MeSAdo on diphthamide synthesis in a MeSAdo phosphorylase-deficient mutant murine lymphoma cell line (R1.1, clone H3). As measured by susceptibility to diphtheria toxin-induced ADP-ribosylation, MeSAdo inhibited the formation of diphthamide in EF-2. The inhibition was not due to a nonspecific effect on protein synthesis. Indeed, exogenous MeSAdo substantially protected the lymphoma cells from the lethal effects of diphtheria toxin. These results suggest that MeSAdo can specifically modulate the biosynthesis of diphthamide in EF-2 in murine malignant lymphoma cells.     

Purine and pyrimidine transport and permeation in human erythrocytes

Time courses of the uptake of radiolabeled hypoxanthine, adenine and uracil were measured by rapid kinetic techniques over substrate ranges from 0.02 to 5000 microM in suspensions of human erythrocytes at 25 or 30 degrees C. At concentrations above 25 microM, the rate of intracellular phosphoribosylation of hypoxanthine and adenine was insignificant relative to their rates of entry into the cell and time courses of transmembrane equilibration of the substrates could be measured and analyzed by integrated rate analysis. Hypoxanthine and uracil are transported by simple facilitated carriers with directional symmetry, high capacity and Michaelis-Menten constants of about 0.2 and 5 mM, respectively. Adenine is probably transported by a carrier with similar properties but no saturability was detectable up to a concentration of 5 mM. Cytosine entered the cells much more slowly than the other three nucleobases, and its entry seems not to be mediated by a carrier. The hypoxanthine transporter resembles that of one group of mammalian cell lines, which does not exhibit any overlap with the nucleoside transporter and is resistant to inhibitors of nucleoside transport. Results from studies on the effects of the nucleobases on the influx and countertransport of each other were complex and did not allow unequivocal conclusions as to the number of independent carriers involved. At concentrations below 5 microM, radiolabel from adenine and hypoxanthine accumulated intracellularly to higher than equilibrium levels. Part of this accumulation reflected metabolic trapping, especially when the medium contained 50 mM phosphate. But part was due to an apparent concentrative accumulation of free adenine and hypoxanthine up to 3-fold at medium concentrations much less than 1 microM and when cells were incubated in phosphate-free medium. This concentrative accumulation could be due to the functioning of additional high-affinity, low-capacity, active transport systems for adenine and hypoxanthine, but other factors could be responsible, such as saturable binding to intracellular components.     

Sodium-dependent nucleoside transport in mouse leukemia L1210 cells

Nucleoside permeation in L1210/AM cells is mediated by (a) equilibrative (facilitated diffusion) transporters of two types and by (b) a concentrative Na(+)-dependent transport system of low sensitivity to nitrobenzylthioinosine and dipyridamole, classical inhibitors of equilibrative nucleoside transport. In medium containing 10 microM dipyridamole and 20 microM adenosine, the equilibrative nucleoside transport systems of L1210/AM cells were substantially inhibited and the unimpaired activity of the Na(+)-dependent nucleoside transport system resulted in the cellular accumulation of free adenosine to 86 microM in 5 min, a concentration three times greater than the steady-state levels of adenosine achieved without dipyridamole. Uphill adenosine transport was not observed when extracellular Na+ was replaced by Li+, K+, Cs+, or N-methyl-D-glucammonium ions, or after treatment of the cells with nystatin, a Na+ ionophore. These findings show that concentrative nucleoside transport activity in L1210/AM cells required an inward transmembrane Na+ gradient. Treatment of cells in sodium medium with 2 mM furosemide in the absence or presence of 2 mM ouabain inhibited Na(+)-dependent adenosine transport by 50 and 75%, respectively. However, because treatment of cells with either agent in Na(+)-free medium decreased adenosine transport by only 25%, part of this inhibition may be secondary to the effects of furosemide and ouabain on the ionic content of the cells. Substitution of extracellular Cl- by SO4(-2) or SCN- had no effect on the concentrative influx of adenosine.     

Inhibition by nucleosides of glucose-transport activity in human erythrocytes.

The interaction of nucleosides with the glucose carrier of human erythrocytes was examined by studying the effect of nucleosides on reversible cytochalasin B-binding activity and glucose transport. Adenosine, inosine and thymidine were more potent inhibitors of cytochalasin B binding to human erythrocyte membranes than was D-glucose [IC50 (concentration causing 50% inhibition) values of 10, 24, 28 and 38 mM respectively]. Moreover, low concentrations of thymidine and adenosine inhibited D-glucose-sensitive cytochalasin B binding in an apparently competitive manner. Thymidine, a nucleoside not metabolized by human erythrocytes, inhibited glucose influx by intact cells with an IC50 value of 9 mM when preincubated with the erythrocytes. In contrast, thymidine was an order of magnitude less potent as an inhibitor of glucose influx when added simultaneously with the radioactive glucose. Consistent with this finding was the demonstration that glucose influx by inside-out vesicles prepared from human erythrocytes was more susceptible to thymidine inhibition than glucose influx by right-side-out vesicles. These data, together with previous suggestions that cytochalasin B binds to the glucose carrier at the inner face of the membrane, indicate that nucleosides are capable of inhibiting glucose-transport activity by interacting at the cytoplasmic surface of the glucose transporter. Nucleosides may also exhibit a low-affinity interaction at the extracellular face of the glucose transporter.     

Nucleoside transport in cultured LLC-PK1 epithelia.

The transport of nucleosides by LLC-PK1 cells, a continuous epithelial cell line derived from pig kidney, was characterised. Uridine influx was saturable (apparent Km approximately 34 microM at 22 degrees C) and inhibited by greater than 95% by nitrobenzylthioinosine (NBMPR), dilazep and a variety of purine and pyrimidine nucleosides. In contrast to other cultured animal cells, the NBMPR-sensitive nucleoside transporter in LLC-PK1 cells exhibited both a high affinity for cytidine (apparent Ki approximately 65 microM for influx) and differential 'mobility' of the carrier (the kinetic parameters of equilibrium exchange of formycin B are greater than those for formycin B influx). An additional minor component of sodium-dependent uridine influx in LLC-PK1 cells became detectable when the NBMPR-sensitive nucleoside transporter was blocked by the presence of 10 microM NBMPR. This active transport system was inhibited by adenosine, inosine and guanosine but thymidine and cytidine were without effect, inhibition properties identical to the N1 sodium-dependent nucleoside carrier in bovine renal outer cortical brush-border membrane vesicles (Williams and Jarvis (1991) Biochem. J. 274, 27-33). Late proximal tubule brush-border membrane vesicles of porcine kidney were shown to have a much reduced Na(+)-dependent uridine uptake activity compared to early proximal tubule porcine brush-border membrane vesicles. These results, together with the recent suggestion of the late proximal tubular origin of LLC-PK1 cells, suggest that in vivo nucleoside transport across the late proximal tubule cell may proceed mainly via a facilitated-diffusion process.     

Increased glucose permeability in Babesia bovis-infected erythrocytes

Glucose influx into bovine erythrocytes was found to be significantly increased upon infection with the parasite, Babesia bovis. The influx of glucose into the infected cells over 4 min was not saturable at high concentrations of glucose (240 mM), nor was it affected by established inhibitors of mammalian glucose transport, such as cytochalasin B and phloretin (0.1-100 microM). Glucose uptake into the parasitized cells was, however, inhibited by phloridzin (phloretin-2-beta-glucoside) at concentrations over the range of 10-500 microM. Further inhibition of glucose uptake by adenosine (2.5-15 mM) was found to occur in B. bovis-infected bovine erythrocytes, suggesting an interaction of adenosine with the new or altered component of glucose transport in the parasitized cells.     

Sulfate transport in human neutrophils

The mechanism by which SO4(2-) is transported across the plasma membrane of isolated human neutrophils was investigated. Unlike the situation in erythrocytes, SO4(2-) and other divalent anions are not substrates for the principal Cl-/HCO3- exchange system in these cells. At an extracellular concentration of 2 mM, total one-way 35SO4(2-) influx and efflux in steady-state cells amounted to approximately 17 mumol/liter of cell water per min. The intracellular SO4(2-) content was approximately 1 mM, approximately 25-fold higher than the passive distribution level. Internal Cl- trans stimulated 35SO4(2-) influx. Conversely, 35SO4(2-) efflux was trans stimulated by external Cl- (Km approximately 25 mM) and by external SO4(2-) (Km approximately 14 mM), implying the presence of a SO4(2-)/Cl- countertransport mechanism. The exchange is noncompetitively inhibited by 4-acetamido-4'-isothiocyanostilbene-2,2' -disulfonate (SITS) (Ki approximately 50 microM) and competitively blocked by alpha-cyano-4-hydroxycinnamate (Ki approximately 230 microM) and by ethacrynate (Ki approximately 7 microM); furosemide and probenecid also suppressed activity. The carrier exhibits broad specificity for a variety of monovalent (NO3- approximately Cl- greater than Br- greater than formate- greater than I- approximately p-aminohippurate-) and divalent WO4(2-) greater than oxalate2- greater than SO4(2-) greater than MoO4(2-) greater than SeO4(2-) greater than AsO4(2-) anions. There was little, if any, affinity for HCO3-, phosphate, or glucuronate. The influx of SO4(2-) is accompanied by an equivalent cotransport of H+, the ion pair H+ + SO4(2-) being transported together in exchange for Cl-, thereby preserving electroneutrality. These findings indicate the existence of a separate SO4(2-)/Cl- exchange carrier that is distinct from the neutrophil's Cl-/HCO3- exchanger. The SO4(2-) carrier shares several properties in common with the classical inorganic anion exchange mechanism of erythrocytes and with other SO4(2-) transport systems in renal and intestinal epithelia, Ehrlich ascites tumor cells, and astroglia.     

3'-azido-3'-deoxythymidine. An unusual nucleoside analogue that permeates the membrane of human erythrocytes and lymphocytes by nonfacilitated diffusion

The demonstrated in vitro and in vivo activity of 3'-azido-3'-deoxythymidine (N3dThd) against the infectivity and the cytopathic effect of human immunodeficiency virus has prompted an investigation of the mechanism by which this nucleoside analogue permeates the cell membrane. As with the transport of thymidine, the influx of N3dThd into human erythrocytes and lymphocytes was nonconcentrative during short incubation times (less than 5 min) which did not allow significant metabolism of this nucleoside. However, in contrast with thymidine transport, the initial velocity of N3dThd influx was strictly a linear function of nucleoside concentration (0.5-10 mM), without evidence of saturability; insensitive to micromolar concentrations of potent inhibitors of nucleoside transport (dipyridamole, 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, and dilazep); insensitive to a 1000-fold excess of other nucleosides (thymidine, uridine, 2-chloroadenosine); and relatively insensitive to temperature, with Q10 values (37-27 degrees C) of 1.4 and 2.7 for N3dThd and thymidine, respectively, determined in erythrocytes. Although the above results indicate that N3dThd permeates the cell membrane chiefly by nonfacilitated diffusion and not via the nucleoside transporter, millimolar concentrations of this nucleoside analogue were observed to inhibit both zero-trans influx of thymidine and efflux of thymidine from [3H]thymidine-loaded erythrocytes. The partition coefficients (1-octanol:0.1 M sodium phosphate, pH 7.0) of N3dThd and thymidine were determined to be 1.26 and 0.064, respectively. The unusual ability of N3dThd to diffuse across cell membranes independently of the nucleoside transport system may be attributed to the considerable lipophilicity imparted to this molecule by the replacement of the 3'-hydroxyl group of thymidine with an azido moiety.     

Characterization of Na(+)-dependent, active nucleoside transport in rat and mouse peritoneal macrophages, a mouse macrophage cell line and normal rat kidney cells

Peritoneal rat macrophages expressed solely an Na(+)-dependent, concentrative nucleoside transporter, which possesses a single Na(+)-binding site and transports purine nucleosides and uridine but not thymidine or deoxycytidine. The Michaelis-Menten constants for formycin B and Na+ were about 6 microns and 14 mM, respectively, and the estimated Na+:formycin B stoichiometry was 1:1. Rat macrophages accumulated 5 microM formycin B to a steady-state level exceeding that in the medium by about 500-fold during 60 min of incubation at 37 degrees C. Concentrative formycin B transport was resistant to inhibition by nitrobenzylthioinosine, lidoflazine, dilazep and nifedipine, but was slightly inhibited by high concentrations of dipyridamole (greater than 10 microM) and probenecid (greater than 100 microM). Mouse peritoneal macrophages and lines of mouse macrophages and normal rat kidney cells expressed Na(+)-dependent, active nucleoside transport but in addition significant Na(+)-independent, facilitated nucleoside transport. Facilitated nucleoside transport in these cells was sensitive to inhibition by nitrobenzylthioinosine, dilazep and dipyridamole. The presence of these inhibitors greatly enhanced the concentrative accumulation of formycin B by these cells by inhibiting the efflux via the facilitated transporter of the formycin B actively transported into the cells. Whereas rat macrophages lacked high-affinity nitrobenzylthioinosine-binding sites, mouse macrophages and normal rat kidney cells possessed about 10,000 such sites/cell. Rat and mouse erythrocytes, rat lymphocytes, and lines of Novikoff rat hepatoma cells, Chinese hamster ovary cells, Mus dunni cells and embryonic monkey kidney cells expressed only facilitated nucleoside transport.     

Serum has a differential effect on DNA replication in a human melanoma cell line cultured in methionine or 5'-deoxy-5'-methylthioadenosine

A human melanoma cell line called MeWo-LC1 exhibits a reduced ability to synthesize DNA when cultured in serum-supplemented medium containing 5'-deoxy-5'-methylthioadenosine (MeSAdo) in place of methionine. However, DNA replication in these cells occurs normally if the cells are cultured in serum-free medium containing transferrin, and MeSAdo in place of methionine. Although the presence of serum alters the cells' ability to respond to MeSAdo, it is not likely a consequence of any increased extracellular metabolism by MeSAdo-phosphorylase or adenosine deaminase activity, or due to the diminished uptake of the nucleoside. In the presence of methionine, MeSAdo appears to have a stronger cytostatic effect in medium containing serum than in serum-free medium supplemented with transferrin. MeWo-LC1 cells contain MeSAdo-phosphorylase activity as measured both in vivo and in vitro. The diminished replication of DNA in medium containing serum and MeSAdo is likely not due to the inhibition of polyamine synthesis by the nucleoside. These results indicate that serum (factors) can have an important influence upon the ability of MeSAdo to act as a methio-source for cells cultured in the absence of methionine.     

Effects of Ca2+-channel antagonists on nucleoside and nucleobase transport in human erythrocytes and cultured mammalian cells

Lidoflazine strongly inhibited the equilibrium exchange of uridine in human erythrocytes (Ki approximately 16 nM). Uridine zero-trans influx was similarly inhibited by lidoflazine in cultured HeLa cells (IC50 approximately to 80 nM), whereas P388 mouse leukemia and Novikoff rat hepatoma cells were three orders of magnitude more resistant (IC50 greater than 50 microM). Uridine transport was also inhibited by nifedipine, verapamil, diltiazem, prenylamine and trifluoperazine, but only at similarly high concentrations in both human erythrocytes and the cell lines. IC50 values ranged from about 10 microM for nifedipine and about 20 microM for verapamil to more than 100 microM for diltiazem, prenylamine and trifluoperazine. The concentrations required for inhibition of nucleoside transport are several orders higher than those blocking Ca2+ channels. Lidoflazine competitively inhibited the binding of nitrobenzylthioinosine to high-affinity sites in human erythrocytes, but did not inhibit the dissociation of nitrobenzylthioinosine from these sites on the transporter as is observed with dipyridamole and dilazep.     

Lactic acid secretion by human neutrophils. Evidence for an H+ + lactate- cotransport system.

The pathway by which L-lactate (Lac) crosses the plasma membrane of isolated human neutrophils was investigated. The influx of [14C]Lac from a 2 mM Lac, 145 mM Cl-, 5.6 mM glucose medium was approximately 1.5 meq/liter of cell water.min and was sensitive to the organomercurial agent mersalyl (apparent Ki approximately 20 microM), to alpha-cyano-4-hydroxycinnamate (CHC), the classical inhibitor of monocarboxylate transport in mitochondria, and to UK-5099 (apparent Ki approximately 40 microM), a more potent analogue of CHC. Transport was also strongly blocked (greater than 80%) by 1 mM of either 3,5-diiodosalicylic acid, MK-473 (an indanyloxyacetate derivative), or diphenyl-amine-2-carboxylate, and by 0.4 mM pentachlorophenol, but not by 1 mM ethacrynic acid, furosemide, or the disulfonic stilbenes SITS or H2DIDS. One-way [14C]Lac efflux from steady-state cells amounted to approximately 6 meq/liter.min and was likewise affected by the agents listed above. Influx, which was membrane potential insensitive and Na+ independent, displayed a strong pH dependence: extracellular acidification enhanced uptake while alkalinization inhibited the process (pK' approximately 5.7 at 2 mM external Lac). The rate of [14C]Lac influx was a saturable function of external Lac, the Km being approximately 7 mM. Steady-state cells exhibited an intracellular Lac content of approximately 5 mM and secreted lactic acid into the bathing medium a a rate of approximately 4 meq/liter.min. Secretion was completely suppressed by 1 mM mersalyl which inactivates the carrier, leading to an internal accumulation of Lac. That the Lac carrier truly mediates an H+ + Lac- cotransport (or formally equivalent Lac-/OH- exchange) was documented by pH-stat techniques wherein an alkalinization of poorly buffered medium could be detected upon the addition of Lac; these pH changes were sensitive to mersalyl. Thus, the Lac carrier of neutrophils possesses several features in common with other monocarboxylate transport systems in erythrocytes and epithelia.     

Ca2+-activated Na+ fluxes in human red cells. Amiloride sensitivity

The effect of Ca2+ on the ouabain- and bumetanide-resistant Na+ fluxes in intact red cells was studied at relatively constant internal Ca2+, membrane potential, and cell volume. The red cell calcium concentration was modified using the ionophore A23187. In fresh red cells, the Na+ influx and efflux (1.2 +/- 0.13 and 0.26 +/- 0.07 mmol/liter cells x h, respectively) were not affected by amiloride (1 mM). When external Ca2+ was raised from 0 to 150 microM, in the presence of A23187, both the Na+ influx and efflux were stimulated (about 3.5-fold). The Ca2+-activated Na+ efflux and influx had an apparent Km for activation by Ca2+o of about 25 microM. The Ca2+-dependent Na+ transport was inhibited 30-60% by amiloride (ID50 = 17.3 +/- 8 microM). Amiloride, however, had no effect on the Ca2+-dependent K+ influx. The amiloride-sensitive (AS) transport pathway was a linear function of the Na+o concentration in the range from 0 to 75 mM. The Ca2+i activation seems to depend on the metabolic integrity of red cells. 1) It does not take place in ATP-depleted red cells; 2) ATP-repletion of ATP-depleted red cells fully restored AS Na influx; and 3) ATP-enrichment (ATP-red cells) enhanced the AS Na influx by about 100%. The Ca2+-activated AS Na+ influx was not affected by either DIDS or trifluoperazine. The present results indicate that in human erythrocytes an increase in internal Ca2+ activates on otherwise silent AS Na+-transport system, which is dependent on the metabolic integrity of the red cells.     

Alteration of folate analogue transport inward after induced maturation of HL-60 leukemia cells. Molecular properties of the transporter in an overproducing variant and evidence for down-regulation of its synthesis in maturating cells.

Studies are described examining further the decline in folate analogue influx mediated by the one-carbon reduced-folate transport system in HL-60 cells following induction of maturation by cytodifferentiation agents. To facilitate the investigation of the underlying basis of this phenomenon, we derived a variant (HL-60/LCV) with 4-5-fold elevated influx capacity (Vmax) for folate analogues. A commensurate increase in the putative transporter for this system was documented by affinity labeling of these cells with N-hydroxysuccinimide-[3H]aminopterin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the affinity labeled plasma membrane in HL-60/LCV cells delineated a protein peak at Mr = 75,000-80,000. This was substantially greater than the analogous transporter (Mr = 45,000-47,000) we had delineated (Yang, C.-H., Sirotnak, F.M., and Mines, L.S. (1988) J. Biol. Chem. 263, 9703-9709) with the same methodology in the L1210 cell plasma membrane. In addition, the rate of translocation of the Mr = 75,000-80,000 transporter in HL-60 and HL-60/LCV cells was 2-fold lower than the rate of translocation determined for the Mr = 45,000-47,000 transporter in L1210 cells. During induced maturation of HL-60/LCV cells toward the granulocyte pathway, [3H]methotrexate (MTX) influx capacity and the amount of the affinity labeled transporter decreased rapidly in a parallel fashion. The decrease in [3H]MTX influx and in affinity labeling and in the amount of the Mr = 75,000-80,000 transporter was 5-fold following exposure to 210 mM dimethyl sulfoxide (Me2SO) for 5 days during growth in culture. Moreover, during cycloheximide treatment, the decay in [3H]MTX influx at 37 degrees C and in amount of affinity labeled transporter was the same (t1/2 = 144-155 min) for both control and Me2SO-treated HL-60/LCV cells. These results, which reveal no difference in metabolic turnover for control and Me2SO-treated cells, suggest that the decline in folate analogue influx in HL-60/LCV influx cells is a very early event in the program of differentiation and probably occurs by down-regulation of synthesis of the transporter for the one-carbon reduced-folate transport system.     

Effects of temperature on the transport of nucleosides in guinea pig erythrocytes

The initial rate of [14C]uridine transport by guinea pig erythrocytes was investigated at different temperatures. At 37, 22, and 10 degrees C the concentration dependence of uridine zero-trans influx and equilibrium exchange influx was resolved into two components; (a) a saturable component which followed simple Michaelis-Menten kinetics and which was inhibited by nitrobenzylthioinosine, and (b) a linear component of low magnitude and insensitive to nitrobenzylthioinosine inhibition. The maximum velocity, Vmax, of zero-trans uridine influx for the saturable transport system was 70-fold higher at 37 than 10 degrees C (1.24, 0.20, and 0.018 mmol/L of cells per hour at 37, 22, and 10 degrees C, respectively). Similarly, the apparent affinity, Km, for zero-trans influx decreased as the temperature was lowered (0.27, 0.066, and 0.038 mM at 37, 22, and 10 degrees C, respectively). In contrast, uridine equilibrium exchange influx was less temperature dependent (Vmax, 2.80, 0.89, and 0.14 mmol/L of cells per hour; apparent Km 0.61, 0.36, and 0.24 mM at 37, 22, and 10 degrees C, respectively). These results demonstrate that the mobility of the empty carrier is impaired to a greater extent than the mobility of the loaded carrier temperature decreased. However, the kinetic constants for zero-trans uridine influx and efflux at 37 degrees C were similar, indicating that the nucleoside transporter exhibited directional symmetry at 37 degrees C. Arrhenius plots of the maximum velocity for equilibrium exchange and zero-trans uridine influx were discontinuous above 25 degrees C, but between 20 and 5 degrees C the plots were linear (Ea = 22 and 30 kcal/mol for equilibrium exchange and zero-trans influx, respectively.