Inhibition of myogenesis by ouabain: effect on protein synthesis.

Ouabain, a specific inhibitor of the sodium- and potassium-activated adenosine triphosphatase, causes reversible inhibition of the fusion of myoblasts to form myotubes. We further examined this observation to investigate whether control of Na/K-ATPase activity may normally contribute to the regulation of myogenesis. In control cultures, fusion was preceded by a small decrease in intracellular sodium concentration, but intracellular sodium and potassium increased significantly during fusion. Levels of ouabain that produce prolonged inhibition of fusion (400 microM) virtually eliminated sodium and potassium gradients. However, lower ouabain levels (10-100 microM) also produced significant changes in intracellular potassium and/or sodium along with little apparent decrease in the eventual extent of fusion. The effect of ouabain on protein synthesis was also examined. Low levels of ouabain (<50 microM) that did not affect myogenesis also did not affect incorporation of radiolabeled amino acids, while higher concentrations produced a decline in protein synthesis that paralleled decreases in the rate of myoblast fusion. Levels of metabolic labeling were reduced 90% in cultures treated with 400 microM ouabain. Inhibition of protein synthesis would prevent membrane remodeling required for fusion and other events in myogenesis. Thus, our results do not support any specific role for the sodium- and potassium-activated adenosine triphosphatase in regulating myogenesis.

A unique subset of developmentally regulated surface proteins turns over rapidly during fusion of the L6 rat myoblast cell line.

We have previously identified several developmentally regulated surface polypeptides in the L6 rat myoblast cell line, on the basis of their susceptibility to lactoperoxidase catalyzed iodination. An analysis of the turnover rates of these polypeptides now indicates that while the bulk of the iodinated polypeptides have a half-life of 20-30 h, four low-molecular-weight polypeptides have half lives of 2-7 h. The half-lives of all of the rapid turnover class surface polypeptides were greatly increased in cultures where fusion was inhibited by chloroquine and in nonfusing variants of the L6 cell line. In contrast, inhibition of fusion by the metalloendoprotease inhibitor 1, 10-phenanthroline did not alter the turnover of any iodinatable surface proteins. We propose that some or all of the rapid turnover class of polypeptides may be surface receptors which control cell surface alterations involved in the acquisition of fusion competence or in fusion itself.

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.

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.

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.

Reduced pinosome formation in a cell-fusion-impaired mutant of mouse lymphocytic cell line L5178Y.

Accumulation and release of a fluid-phase marker ([14C]-sucrose) were studied in a subline of the mouse lymphocytic cell line L5178Y and in a polyethylene glycol-resistant, intercellular-fusion-impaired mutant of this line. The mutant was found to accumulate [14C]-sucrose at a significantly slower rate than the parent. Analysis of release of preloaded label shows that the reduced rate of accumulation is due to a correspondingly low level of internalization, i.e., pinosome formation, rather than to a reduction in delivery of label to lysosomes. Cell-to-cell fusion and pinosome formation both involve a fusion event initiated at the extracellular surface of the plasma membrane, and we propose that the coordinate reduction in both processes suggests that they are mechanistically related.

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.

Correlation of unstable multidrug cross resistance in Chinese hamster ovary cells with a homogeneously staining region on chromosome 1.

An enrichment selection method using repeated pulses of low drug concentration (1 microgram/ml) was used to isolate CHO (AK412) variants that are 20-fold more resistant to cytochalasin D (CD). CD-resistant (CydR) variants possess a unique unstable phenotype, including a longer doubling time in nonselective medium, a higher frequency of multinucleate cells in the population (probably due to a defect in cytokinesis), an altered morphology, and increased resistance or sensitivity to a number of unrelated drugs. In each of two variant lines examined cytologically, this multiple phenotype is associated with a small homogeneously staining region on chromosome 1. The homogeneously staining region is present in the CydR variants, but absent both in the CD-sensitive parent and in a CD-sensitive revertant subpopulation. Studies of CD-displaceable binding of [3H]cytochalasin B show a fourfold reduction in CD binding or uptake when whole cells of the variant line were examined. Lactoperoxidase-catalyzed iodination and metabolic labeling with [H3]fucose of cell surface proteins of the CydR variants showed multiple differences in electrophoretic band migration when compared with parental proteins.

Inactivation of phage T7 by near-ultraviolet radiation plus hydrogen peroxide: DNA-protein crosslinks prevent DNA injection.

A nonlethal concentration of H2O2 (0.05%) greatly enhances near-ultraviolet (NUV) inactivation of phage T7. Simultaneous treatment with H2O2 and NUV reduces the amount of DNA injected into the bacterial host, but not the number of phage adsorbed. Not only were recombination and gene expression of late markers reduced upon treatment of phage T7 with NUV plus H2O2, but also a gradient of recombination resulted, with markers injected first reduced to a lesser extent than those injected last. Double-strand DNA breaks were not detected; however, DNA-protein crosslinks were observed upon NUV plus H2O2 treatment of double-labeled T7. Previous studies demonstrated that single-strand DNA breaks did not account for phage death by NUV plus H2O2. It is concluded that the DNA-protein crosslinks prevent normal injection of T7 phage DNA; such crosslinks may be important lesions in NUV cellular damage.