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J F Ash

Publications and source records attributed to J F Ash.

At least 19 recordsLinked to original sources
PubMed1998

Inducible expression of the GLT-1 glutamate transporter in a CHO cell line selected for low endogenous glutamate uptake.

Inducible expression of the mammalian glial cell glutamate transporter GLT-1 has been established in a CHO cell line selected for low endogenous Na+-dependent glutamate uptake by [3H]aspartate suicide selection. Culturing the cells in doxycycline-containing medium, to activate GLT-1 expression via the Tet-On system, increased uptake of the GLT-1 substrate D-aspartate 280-fold, and increased cell size. Applying glutamate to whole-cell clamped, doxycycline-treated cells evoked a transporter-mediated current with characteristics appropriate for GLT-1. This cell line provides a useful tool for further examination of the electrical, biochemical and pharmacological properties of GLT-1, the most abundant glutamate transporter in the brain.

ATP-Binding Cassette Transporters↗
PubMed1998

The Na+-dependent glutamate and aspartate transporter supports glutathione maintenance and survival of CHO-K1 cells.

Glutathione synthesis, a vital cellular process, depends on L-cystine uptake by the amino acid transporter, System x-C. Here we show that a second transporter, System X-AG, is required for normal System x-C activity and glutathione maintenance by employing somatic cell mutants of CHO-K1. Uptake by System x-C in two X-AG-null mutants is significantly lower than that of CHO-K1, either under control conditions or after prolonged treatment with an electrophile. In addition, levels of glutathione in control and treated mutant cells are less than half those of wild-type CHO-K1 or of a pseudorevertant. The significance of this reduction was tested by chemical challenge: mutants are twofold more sensitive than wild type to reactive oxygen species generated by phenylbenzoquinone and to damage produced by the anticancer drug, cisplatin. These results suggest that System X-AG provides a significant portion of the glutamate used to energize the uptake of cystine required for the synthesis of glutathione.

ATP-Binding Cassette Transporters↗
PubMed1997

Rapid swelling of a CHO-K1 aspartate/glutamate transport mutant in hypo-osmotic medium.

Two Chinese hamster ovary cell (CHO-K1) mutants selected for defective glutamate transport via system X-AG are also highly permeable to small neutral molecules. Light microscopy demonstrated that exposure of one of these mutants, Ed-A1, to hypo-osmotic medium led to extremely rapid swelling, presumably due to increased water flux. When placed in 20% saline, Ed-A1 cells swelled to three times their original volume within 15 sec, a sixfold larger increase than parental CHO-K1. In spite of this rapid volume increase, mutant and wild-type cells remained viable for 20 min in dilute saline. A regulatory volume decrease in Ed-A1, and the continual swelling of CHO-K1, resulted in the two cells achieving equal size after 5 min in 20% saline. The time course of these volume changes permitted analysis of large numbers of cells by a hydrodynamic technique, steric field flow fractionation (FFF). Steric FFF demonstrated the expected inhibition of osmotic swelling of human erythrocytes by the mercurial, p-chloromercuribenzenesulfonic acid (PCMBS). However, PCMBS increased the apparent swelling rate of Ed-A1 and CHO-K1, suggesting that an aquaporin-like molecule is not responsible for any significant fraction of the water fluxes into either line. PCMBS also strongly inhibited aspartate transport by system X-AG. By taking advantage of their different swelling rates in hypotonic medium, steric FFF can separate mixtures of CHO-K1 and Ed-A1.

4-Chloromercuribenzenesulfonate↗
PubMed1996

New mutations and phenotypes associated with glutamate and aspartate transport in Chinese hamster ovary (CHO-K1) cells.

Two new Chinese hamster ovary cell (CHO-K1) mutants lacking amino acid transport System X-AG activity were isolated by [3H]aspartate suicide selection. These null mutants, Dd-B6 and Dd-B7, were analyzed by somatic cell hybridization, along with previously described partial-function mutants, Ed-A1 and Ed-B8. With respect to System X-AG activity, all four mutations fell into a single complementation group. By quantitative assay, the mutations in Ed-A1 and Ed-B8 behaved as simple recessives in fusions with wild type cells, while those in Dd-B6 and Dd-B7 were codominant. We have discovered that Ed-A1 and Ed-B8 are highly permeable to small neutral molecules. This high permeability phenotype was dominant to wild-type. Northern, Southern, and Western analyses indicated that System X-AG in CHO is not closely related to any of the three well characterized glutamate transporters represented by GLT-1, EAACI or GLAST.

Amino Acid Transport System X-AG↗
PubMed1995

Novel regulations of glutamate and aspartate uptake by HeLa cells.

Pathways of L-glutamate and L-aspartate import by HeLa S3 cells were investigated before and after the cells were depleted of internal amino acids by starvation. Two new regulations of transport were observed in starved cells. Aspartate entered nonstarved cells by two routes, one non-saturable and one, an apparent analog of saturable system X-AG, that was sodium-dependent and competitively inhibited by glutamate. Starvation for one hour in saline increased the efficiency of saturable aspartate import, increasing Vmax and decreasing Km, an effect not previously reported for system X-AG. Glutamate uptake by nonstarved cells appeared to occur through system X-AG; through an analog of system X-C, which was sodium-independent, cystine- and quisqualate-inhibitable; as well as through one or more nonsaturable pathways. Starvation in saline for one hour resulted in the appearance of a new low-affinity saturable glutamate uptake system. This new system was sodium-dependent but not inhibited by aspartate.

Aspartic Acid↗
PubMed1993

Numerical analysis reveals complexities of glutamate transport.

The uptake of radiolabeled glutamate into cultured human (HeLa S3) and hamster (CHO-K1) cells was analyzed according to modified Michaelis-Menten models fit by the Marquardt least-squares method. Kinetic parameters not previously reported for these cells were obtained. Some rarely used features available with this fitting method proved to be extremely helpful. Most importantly, a goodness-of-fit measure revealed a significant alteration of glutamate uptake in HeLa cells that was induced by starvation. This apparent regulation, unexpected for glutamate transport, might have been missed if the fit had been judged by eye or by the magnitude of parameter standard deviations. Techniques for analyzing parameter distributions, improving experimental design and performing tests of significance are also described.

Animals↗
PubMed1993

Selection of a lysine-resistant CHO-K1 mutant with reduced amino acid transport through multiple systems.

High levels of L-lysine were used to select for resistant variants of Chinese hamster ovary (CHO-K1) cells. Surviving colonies were screened for altered lysine transport and two with reduced uptake were picked. Clone CH-Kr, derived from the more severely affected colony, was analyzed in detail. In starved cells the Vmax of lysine uptake in CH-Kr was half that of CHO while Km was unaltered. The intracellular pool of lysine, a substrate of cationic amino acid transport system y+, was significantly lower in CH-Kr. However, transport and pools of other amino acids, which are not substrates of y+, were also reduced in CH-Kr, as was the internal sodium concentration, while hexose import was increased. It appears that the mutation in CH-Kr is pleiotropic, affecting some general aspects of amino acid transport.

Amino Acids↗
PubMed1993

Selection of Chinese hamster ovary cells (CHO-K1) with reduced glutamate and aspartate uptake.

Transport of L-[3H]glutamate into Chinese hamster ovary cells (CHO-K1) was characterized and the results used to design a tritium suicide selection for cells with transport defects. Replicas of surviving colonies on polyester cloth disks were screened by autofluorography for reduced uptake and two mutant clones, Ed-A1 and Ed-B8, were obtained. Uptake of glutamate through a sodium-dependent system in both mutants was characterized by significant reductions in Vmax and increases in Km compared to parental cells, but their response to removal of extracellular sodium differed, suggesting distinct mutations in the two lines. The Vmax of aspartate uptake through this system was reduced in both mutants, to one-ninth in the case of Ed-B8. Glutamate uptake through a sodium-independent system was not altered in either mutant. Surprisingly, acid-soluble intracellular pools of several amino acids were higher in both mutants.

Animals↗
PubMed1989

Steady-state physiological variations across a graded series of Na,K-ATPase-amplified cells.

Measurements of internal ion concentrations, amino acid pools, and membrane potential were made across a series of HeLa subclones which are amplified for the genes for the sodium- and potassium-activated ATPase (Na,K-ATPase). These subclones expressed heterogeneous levels of ouabain-binding sites, allowing us to construct a graded amplification series. While [K+]i levels did not vary systematically across the series studied, [Na+]i ranged from 9 to 20 mM as a function of Na,K-ATPase expression. Steady-state accumulation of tetraphenylphosphonium in low versus high potassium was used to measure membrane potential. Values for [Na+]i and the membrane potential were used to calculate the sodium electrochemical potential, which was also found to be a function of Na,K-ATPase expression. Measurements of acid-soluble amino acid pools in cell lysates demonstrated that amino acids which are substrates for sodium-dependent transport systems, or which can potentially exchange through system L for a substrate of a sodium-dependent system, varied as a function of the sodium electrochemical potential. This confirmed our prediction of increased amino acid pool sizes in Na,K-ATPase-amplified lines based on observations of elevated flux through the sodium-independent system L. Finally, we measured lactate production and glycolytic potential in a subset of clones and found that both were reduced in subclones with elevated Na,K-ATPase.

Biological Transport↗
PubMed1987

Graded amplification of the Na,K-ATPase across a subclonal series: effects on membrane physiology.

We have generated a series of clonally related cell lines which differ in the level of amplified expression of the Na,K-ATPase. These lines, originally derived from the ouabain resistant HeLa variant C+, expressed different numbers of binding sites for the Na,K-ATPase inhibitor ouabain, ranging from 2.9 X 10(6)/cell to 11.8 X 10(6)/cell. Amplification of the genes for both subunits of the enzyme was also seen but was not strictly correlated with level of expression. The influxes of histidine and tetraphenylphosphonium were measured across a series, including HeLa S3 and revertants, expressing from 0.74 X 10(6) to 10.5 X 10(6) ouabain-binding sites per cell. Tetraphenylphosphonium influx rate, presumed to be a function of membrane potential, varied linearly with ouabain binding site number, while histidine influx varied with the log of ouabain binding site number. Our results suggest that membrane potential increases in a simple fashion across our series of amplified lines. However, histidine influx was unaffected by treatments which cause membrane depolarization and a decrease in tetraphenylphosphonium influx rate. We propose that increasing histidine influx rates across our amplified series reflects exchange acceleration of L system transport due to increased intracellular pools of L system reactive amino acids. The Na,K-ATPase is ultimately responsible for most active transport across the plasma membrane. The consistent, graded physiological alterations seen across this series of closely related lines, chosen for graded enzyme expression, demonstrate the value of this novel genetic approach to the study of the energization of membrane transport.

Cell Line↗
PubMed1986

Alterations in amino acid transport in Na,K-ATPase amplified HeLa cells.

Amino acid transport was studied in C1 cells which contain amplified levels of sodium- and potassium-activated adenosine triphosphatase (Na,K-ATPase), in C4 cells which are ouabain-sensitive revertants, and in parental HeLa S3. Sodium-dependent uptake of aminoisobutyric acid and alanine was increased 2-fold in the amplified C1 cells. After a 6 h amino acid starvation period, the rate of sodium-dependent uptake of methylaminoisobutyric acid was 70-90% greater for C1 than for C4 and HeLa. This uptake was inhibitable by ouabain and the apparent Km values for high affinity uptake were similar in all three lines. Overall, neutral amino acid uptake through Systems A, ASC, and L was 2-fold higher in the Na,K-ATPase amplified C1 cells relative to C4 or HeLa. The induction of System A uptake of methylaminoisobutyric acid after starvation was more rapid in both the amplified C1 cells and the revertant C4 when compared to HeLa, which suggests that the selection for amplification of the Na,K-ATPase produced membrane alterations affecting the adaptive regulation of System A.

Alanine↗
PubMed1986

Stable gene amplification and overexpression of sodium- and potassium-activated ATPase in HeLa cells.

Cell lines stably resistant to ouabain were isolated from an unstably resistant HeLa line after growth in nonselective medium. Stable resistant lines bound ouabain at levels 10-fold higher than did HeLa cells and at similar levels to those bound by the unstable C+ line previously described (J. F. Ash, R. M. Fineman, T. Kalka, M. Morgan, and B. Wire, J. Cell Biol. 99: 971-983). Expression and synthesis of the Na+, K+ -ATPase alpha chain showed a similar amplification over that for HeLa cells by Western blots and [35S]methionine pulse-labeling. In addition, a glycoprotein labeled with [3H]fucose and comigrating with the Na+, K+ -ATPase beta chain was eight- to ninefold amplified in stably resistant lines. Dot blots with a cDNA clone specific for Na+, K+ -ATPase alpha chain gene sequences confirmed the amplification of this gene. Karyotyping suggested that the amplification is associated with an expanded, abnormal banded region on the long (q) arm of one chromosome 17.

Drug Resistance↗
PubMed1986

Amplification of DNA sequences coding for the Na,K-ATPase alpha-subunit in ouabain-resistant C+ cells.

We have studied the mechanism of cellular resistance to cardiac glycosides in C+ cells. C+ cells were resistant to ouabain and overproduced plasma membrane-bound Na,K-ATPase relative to parental HeLa cells. Overexpression of Na,K-ATPase in C+ cells correlated with increased ATPase mRNA levels and amplification (approximately 100 times) of the ATPase gene. Growth of C+ cells in ouabain-free medium resulted in a marked decline in ATPase mRNA and DNA levels. However, when cells were reexposed to ouabain, they proliferated and ATPase mRNA and DNA sequences were reamplified. Restriction analysis of C+ and other human DNA samples revealed the occurrence of rearrangements in the region of the Na,K-ATPase gene in C+ cells. Furthermore, C+ cells expressed an ATPase mRNA species not found in HeLa cells. These results suggest that amplification of the gene coding for Na,K-ATPase results in overproduction of Na,K-ATPase polypeptides. Amplification of the ATPase gene or the expression of new ATPase mRNA sequences or both may also be responsible for acquisition of the ouabain-resistant phenotype.

Base Sequence↗
PubMed1984

Amplification of sodium- and potassium-activated adenosinetriphosphatase in HeLa cells by ouabain step selection.

A multistep selection for ouabain resistance was used to isolate a clone of HeLa S3 cells that overproduces the plasma membrane sodium, potassium activated adenosinetriphosphatase (Na+,K+-ATPase). Measurements of specific [3H]ouabain-binding to the resistant clone, C+, and parental HeLa cells indicated that C+ cells contain 8-10 X 10(6) ouabain binding sites per cell compared with 8 X 10(5) per HeLa cell. Plasma membranes isolated from C+ cells by a vesiculation procedure and analyzed for ouabain-dependent incorporation of [32P]phosphate into a 100,000-mol-wt peptide demonstrated a ten- to twelvefold increase in Na+,K+-ATPase catalytic subunit. The affinity of the enzyme for ouabain on the C+ cells was reduced and the time for half maximal ouabain binding was increased compared with the values for the parental cells. The population doubling time for cultures of C+ cells grown in dishes was increased and C+ cells were unable to grow in suspension. Growth of C+ cells in ouabain-free medium resulted in revertant cells, C-, with biochemical and growth properties identical with HeLa. Karyotype analysis revealed that the ouabain-resistant phenotype of the C+ cells was associated with the presence of minute chromosomes which are absent in HeLa and C- cells. This suggests that a gene amplification event is responsible for the Na+,K+-ATPase increase in C+ cells.

Cell Membrane↗
PubMed1980

The antibody-induced clustering and endocytosis of HLA antigens on cultured human fibroblasts.

It has previously been shown by immunofluorescence experiments that the cross-linking of HLA antigens into patches (by antibody reagents directed to human beta 2--microglobulin) on the surfaces of cultured human fibroblasts leads to the lining up of the patches over the actomyosin-containing stress fibers lying immediately under the surface membrane. These experiments have now been extended to the resolution of the electron microscope by the use of ferritin-conjugated antibody. The results show that a substantial part of the HLA surface clusters that form by 5 min after the addition of the antibody reagents is found in small uncoated surface invaginations which are subsequently endocytosed and ultimately fuse with lysosomal bodies. At no stage in this process is there any indication that coated pits or coated vesicles participate. These and other results suggest, therefore, that there are at least two distinct mechanisms for the ligand-induced endocytosis and lysosomal processing of membrane components, one involving coated pits and the other the noncoated invaginations described in this paper. Transmembrane associations of clusters with intracellular actomyosin-containing structures may have a role in the endocytosis of these noncoated invaginations.

Antibodies↗