Biomedical subjects

S Olsnes

Publications and source records attributed to S Olsnes.

At least 109 records · Page 6Linked to original sources

PubMed1987

Cell-mediated reduction of the interfragment disulfide in nicked diphtheria toxin. A new system to study toxin entry at low pH.

When 125I-labeled nicked diphtheria toxin bound to Vero cells was exposed to pH less than 5.0, a small fraction was reduced to yield A- and B-fragments. The pH required for reduction correlates well with that required to induce intoxication, and the amount of A-fragment released was of the same order as that required to intoxicate the cells. Conditions that protect cells against intoxication, such as acidification of the cytosol, treatment with anion transport inhibitors, or treatment with anti-diphtheria toxin antibodies, prevented the reduction of the interfragment disulfide in cell-bound toxin. In vitro, thioredoxin reduced nicked diphtheria toxin only at pH 5.0 and lower, and the reduction was inhibited by anti-toxin antibodies. This indicates that a conformational change in the toxin, necessary for reduction by the thioredoxin system, is prevented by the antibodies. Reduction by glutathione and cysteine was most efficient at neutral pH and was not inhibited by anti-toxin. The results are consistent with the possibility that cell-mediated reduction of the interfragment disulfide is a measure of the entry of fragment A into the cytosol.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1987

Selective inhibition of sodium-linked and sodium-independent bicarbonate/chloride antiport in Vero cells.

The effects of compounds previously described to inhibit anion transport were tested for their ability to inhibit anion antiport in Vero cells as measured by uptake of 36Cl- by chloride self-exchange and as bicarbonate-linked uptake of 22Na+. While 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibited both processes, ethacrynic acid and probenecid selectively inhibited the uptake of 36Cl-. Low concentrations of pyridoxal phosphate and picrylsulfonic acid selectively inhibited the bicarbonate linked uptake of 22Na+, while higher concentrations of these compounds also inhibited the uptake of 36Cl-. Measurements of the internal pH indicated that ethacrynic acid inhibits Na+-independent HCO-3/Cl- exchange, while it has no measurable effect on Na+-linked bicarbonate-dependent regulation of the internal pH. Conversely, picrylsulfonic acid selectively inhibits the latter process. The results indicate that anion antiport in Vero cells occurs by two independent processes.

Animals↗

PubMed1987

Effect of intracellular pH on the rate of chloride uptake and efflux in different mammalian cell lines.

The effect of the internal pH on the rate of 36Cl- uptake and efflux was measured. While the rate of 36Cl- uptake by the antiport increased almost 10-fold over a narrow pH range in a number of cell lines, in other cells the pH-induced increase was considerably less. With increasing pH, the rate of 36Cl- efflux into chloride-free buffers increased strongly over a narrow pH range in all cell lines studied. Two groups of cell lines appeared, one group where the half-maximal increase in uptake and efflux rate was at pH 7.0-7.1 and another group where the corresponding values were 0.2-0.4 pH unit higher. The stoichiometry between chloride influx and chloride-stimulated efflux was close to 1:1 under various conditions.

Animals↗

PubMed1987

Effect of potassium depletion of Hep 2 cells on intracellular pH and on chloride uptake by anion antiport.

The effect of K+ depletion of Hep 2 cells on ion fluxes, internal pH, cell volume, and membrane potential was studied. The cells were depleted of K+ by incubation in K+-free buffer with or without a preceding exposure to hypotonic medium. Efflux of K+ in cells not exposed to hypotonic medium was inhibited by furosemide or by incubation in Na+-free medium, indicating that in this case at least part of the K+ efflux occurs by Na+/K+/Cl- cotransport. After exposure to hypotonic medium, K+ efflux was not inhibited by furosemide, whereas it was partly inhibited by 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS). Exposure to hypotonic medium induced acidification of the cytosol, apparently because of efflux of protons from intracellular acidic vesicles. When isotonicity was restored, a rebound alkalinization of the cytosol was induced, because of activation of the Na+/H+ antiporter. While hypotonic shock and a subsequent incubation in K+-free buffer rapidly depolarized the cells, depolarization occurred much more slowly when the K+ depletion was carried out by incubation in K+-free buffer alone. The cell volume was reduced in both cases. K+ depletion by either method strongly reduced the ability of the cells to accumulate 36Cl- by anion antiport, and K+-depleted cells were unable to increase the rate of 36Cl- uptake in response to alkalinization of the cytosol.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1987

Effect of reduced endocytosis induced by hypotonic shock and potassium depletion on the infection of Hep 2 cells by picornaviruses.

Potassium depletion after a brief exposure of the cells to hypotonic medium was used to inhibit endocytosis from coated pits in Hep 2 cells. After such treatment the endocytic uptake of transferrin was arrested, and electron microscopy revealed that virtually no coated pits were present at the cell surface, while smooth (uncoated) pits were abundant. Under the same conditions the cells were strongly protected against poliovirus, while the cytopathogenic effect of human rhinovirus type 2, HRV 2, was increased. The cytopathogenic effect of encephalomyocarditis (EMC) virus was only slightly affected. Potassium depletion without hypotonic shock reduced the endocytic uptake of transferrin 2-3-fold and the number of coated pits at the cell surface about 3-fold. Furthermore, the cells were not protected against poliovirus after such treatment. The data indicate that the productive uptake of poliovirus occurs by receptor-mediated endocytosis from coated pits, while the productive uptake of the other two picornaviruses may occur by another endocytic pathway. In order to efficiently arrest endocytosis from coated pits in these cells, hypotonic shock seems to be a critical component of the potassium depletion protocol.

Cell Line↗

PubMed1987

Bicarbonate/chloride antiport in Vero cells: I. Evidence for both sodium-linked and sodium-independent exchange.

The effect of bicarbonate on the ability of cells to regulate the internal pH after acid and alkali loads was studied. In the presence of Na+, the normalization of the internal pH after acid loads occurred more rapidly in the presence than in the absence of bicarbonate. DIDS (4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid) strongly inhibited the pH increase, whereas amiloride inhibited it to a lesser extent. The Na+-linked, bicarbonate-dependent pHi increase after an acid load was strongly reduced in cells depleted of Cl-. When cells were transferred to gluconate or mannitol balanced buffers containing bicarbonate, there was a rapid alkalinization of the cytosol, apparently due to influx of bicarbonate induced by chloride efflux. When the internal pH was below 7.0, the pH increase was much more rapid in the presence than in the absence of Na+, whereas at higher internal pH, there was no measurable effect of Na+. The ability of the cells to reduce the internal pH after an alkali load was increased in the presence of bicarbonate. The data indicate that both Na+-linked and Na+-independent bicarbonate/chloride exchange occur in Vero cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1987

Bicarbonate/chloride antiport in Vero cells: II. Mechanisms for bicarbonate-dependent regulation of intracellular pH.

The rates of bicarbonate-dependent uptake and efflux of 22Na+ in Vero cells were studied and compared with the uptake and efflux of 36Cl-. Both processes were strongly inhibited by DIDS. Whereas the transport of chloride increased approximately ten-fold when the internal pH was increased over a narrow range around neutrality, the uptake of Na+ was much less affected by changes in pH. The bicarbonate-linked uptake of 22Na+ was dependent on internal Cl- but not on internal Na+. At a constant external concentration of HCO3-, the amount of 22Na+ associated with the cells increased when the internal concentration of HCO3- decreased and vice versa, which is compatible with the possibility that the ion pair NaCO3- is the transported species and that the transport is symmetric across the membrane. Bicarbonate inhibited the uptake of 36Cl- both in the absence and presence of Na+. At alkaline internal pH, HCO3- stimulated the efflux of 36Cl- from preloaded cells, while at acidic internal pH both Na+ and HCO3- were required to induce 36Cl- efflux. We propose a model for how bicarbonate-dependent regulation of the internal pH may occur. This model implies the existence of two bicarbonate transport mechanisms that, under physiological conditions, transport OH(-)-equivalents in opposite directions across the plasma membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1987

Delivery of internalized ricin from endosomes to cisternal Golgi elements is a discontinuous, temperature-sensitive process.

Galactose-terminating membrane glycoproteins and glycolipids on two established human breast carcinoma cell lines were tagged at 4 degrees C with a ricin-horseradish peroxidase conjugate (Ri-HRP). The cells were then incubated for various periods of time at 37 or 18 degrees C. After fixation and diaminobenzidine cytochemistry, the compartments reached by Ri-HRP were studied by analyzing thin serial sections. In both cell types a highly pleomorphic endosomal system comprising vacuolar elements as well as smaller, sometimes branched, tubular elements (tubular endosomes) was revealed at both 37 and 18 degrees C. At 37 degrees C Ri-HRP was consistently observed in flattened cisterns of the Golgi region in 30-40% of the Golgi complexes examined after 30-60 min of incubation. However, no Ri-HRP reached such Golgi elements at 18 degrees C, even after incubation for 180 min. Moreover, at 18 degrees C the ability of ricin to inhibit protein synthesis was virtually abolished, whereas the effect of diphtheria toxin was reduced much less. Following incubation with a monovalent transferrin-HRP conjugate or with unconjugated HRP, no labeling of cisternal Golgi elements was detected. These data indicate that delivery of galactose-terminating membrane molecules from endosomes to the Golgi complex is a discontinuous, temperature-sensitive process and that this process may be required for optimal ricin A-chain translocation.

Breast Neoplasms↗

PubMed1987

Formation and activity of covalent conjugates of poliovirus and ligands binding to cell surface structures.

Disulfide-linked conjugates of poliovirus with streptavidin or concanavalin A were formed and the binding of the conjugates to mouse L cells that lack natural poliovirus receptors was studied. The conjugate with streptavidin was specifically bound to biotinylated L cells, but not to unmodified L cells. The conjugate with conA was bound to L cells in the absence of, but not in the presence of alpha-methyl mannoside. Incubation of L cells with bound conjugates did not produce virus, although the conjugates were highly infectious in HeLa cells, containing natural poliovirus receptors. This suggests that the artificially bound virus was unable to penetrate the L cells and start replication. The possibility that binding of the virus to the natural receptor is required for efficient infection is discussed.

Animals↗

PubMed1987

Acidification of the cytosol inhibits endocytosis from coated pits.

Acidification of the cytosol of a number of different cell lines strongly reduced the endocytic uptake of transferrin and epidermal growth factor. The number of transferrin binding sites at the cell surface was increased in acidified cells. Electron microscopic studies showed that the number of coated pits at the cell surface was not reduced in cells with acidified cytosol. Experiments with transferrin-horseradish peroxidase conjugates and a monoclonal anti-transferrin receptor antibody demonstrated that transferrin receptors were present in approximately 75% of the coated pits both in control cells and in cells with acidified cytosol. The data therefore indicate that the reason for the reduced endocytic uptake of transferrin at internal pH less than 6.5 is an inhibition of the pinching off of coated vesicles. In contrast, acidification of the cytosol had only little effect on the uptake of ricin and the fluid phase marker lucifer yellow. Ricin endocytosed by cells with acidified cytosol exhibited full toxic effect on the cells. Although the pathway of this uptake in acidified cells remains uncertain, some coated pits may still be involved. However, the data are also consistent with the possibility that an alternative endocytic pathway involving smooth (uncoated) pits exists.

Cell Line↗

PubMed1986

Requirement of a transmembrane pH gradient for the entry of diphtheria toxin into cells at low pH.

The effects of acidification of the cytosol and of electrical depolarization on the entry of diphtheria toxin were studied. Entry of the toxin from the cell surface was induced by low pH, and the presence of the toxin in the cytosol was monitored as toxin-induced inhibition of protein synthesis. To reduce the membrane potential the cells were incubated in a buffer containing a high concentration of potassium. The cytosol was acidified either by incubating the cells with acetic acid, by incubating them with ammonium chloride which was subsequently removed in the presence of amiloride to prevent pH regulation by the Na+/H+ exchanger, or by incubating the cells in isotonic KCl in the presence of nigericin and valinomycin. The results showed that when the cytosol was acidified by either method toxin entry was inhibited, while a reduction in the membrane potential did not strongly interfere with the entry. A pH gradient across the membrane of at least 1 pH unit was required for entry. Possibly this gradient acts as a driving force for diphtheria toxin entry.

Animals↗

PubMed1986

Interactions between diphtheria toxin entry and anion transport in Vero cells. IV. Evidence that entry of diphtheria toxin is dependent on efficient anion transport.

Entry of prebound diphtheria toxin at low pH occurred rapidly in the presence of isotonic NaCl, NaBr, NaSCN, NaI, and NaNO3, but not in the presence of Na2SO4, 2-(N-morpholino)ethanesulfonic acid neutralized with Tris, or in buffer osmotically balanced with mannitol. SCN- was the most efficient anion to facilitate entry. Uptake studies with radioactively labeled anions showed that SCN- was transported into cells 3 times faster than Cl-, while the entry of SO2-4 occurred much more slowly. The anion transport inhibitors 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and piretanide inhibited entry at low pH even in the presence of permeant anions. When cells with bound toxin were exposed to low pH in the absence of permeant anions, then briefly exposed to neutral pH and subsequently exposed to pH 4.5 in the presence of isotonic NaCl, toxin entry was induced. The data indicate that efficient anion transport at the time of exposure to low pH is required for entry of surface-bound diphtheria toxin into the cytosol. Since insertion of diphtheria toxin into the membrane occurs even in the absence of permeant anions, the results indicate that low pH is required not only for insertion of fragment B into the membrane, but also for the subsequent entry of fragment A into the cytosol.

Animals↗

PubMed1986

Interactions between diphtheria toxin entry and anion transport in vero cells. III. Effect on toxin binding and anion transport of tumor-promoting phorbol esters, vanadate, fluoride, and salicylate.

When Vero cells were incubated with TPA (12-O-tetradecanoylphorbol 13-acetate) and related tumor promoters, their ability to bind diphtheria toxin in a functional way was rapidly reduced to less than 1% of the normal value. Upon further incubation with TPA, the cells recovered their ability to bind the toxin, apparently because they became resistant to TPA. Treatment with Na3VO4 reduced the ability of the cells to bind diphtheria toxin to approximately the same extent as treatment with TPA, but the reduction required longer time to develop and it persisted upon prolonged incubation with Na3VO4. ATP depletion of the cells prevented the reduction in binding capability. Such treatment also prevented the reduction in toxin binding induced by treatment with salicylate or fluoride. Treatment with TPA, fluoride, vanadate, and salicylate altered the ability of the cells to carry out anion transport and interfered with their ability to regulate the transport. The results indicate that the binding sites for diphtheria toxin on Vero cells are modulated by TPA, Na3VO4, salicylate, and fluoride by a process which requires ATP. The possibility is discussed that the modulation consists in phosphorylation of the toxin binding sites, which may be identical with, or closely linked to, the anion antiporter in the cells.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMed1986

Interactions between diphtheria toxin entry and anion transport in Vero cells. I. Anion antiport in Vero cells.

In sodium-free buffer of low ionic strength, the uptake of chloride and sulfate in Vero cells was found to occur mainly by antiport which was very sensitive to inhibition by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. Efflux of anions from the cells appeared to energize the uptake. While the uptake of Cl- occurred over a wide pH range, that of SO4(2-) showed a clear maximum at pH 6-7. The rate of efflux of 36Cl- and 35SO4(2-) was strongly increased by the presence of permeant anions in the efflux buffer. Preincubation of the cells at slightly alkaline pH strongly increased the rate of C1- efflux into buffers nominally free of permeant anions, as well as the efflux by exchange. This increase did not occur if the cells were depleted for ATP during the preincubation. Depolarization of the cells reduced the rate of efflux into buffers without permeant anions, indicating that the efflux is at least partly due to net, electrogenic, anion transport. The efflux by antiport was not affected by manipulations of the membrane potential, indicating electroneutral exchange. The uptake and efflux were increased to the same extent with increasing temperature, the activation energies were Ea = 25 kcal/mol of Cl- and Ea = 12 kcal/mol of SO4(2-). Similar anion antiport appears to occur in L, baby hamster kidney, and HeLa S3 cells.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMed1986

Interactions between diphtheria toxin entry and anion transport in Vero cells. II. Inhibition of anion antiport by diphtheria toxin.

When cells with surface-bound diphtheria toxin were exposed to pH 4.5, the toxin became shielded against lactoperoxidase-catalyzed radioiodination, indicating that the toxin was inserted into the membrane. Cells thus treated had strongly reduced ability to take up 36Cl-, 35SO4(2-), and [14C]SCN-. The reduction of chloride uptake was strongest at neutral pH, whereas that of sulfate was strongest at acidic pH. Lineweaver-Burk plots indicated that the toxin treatment reduced the Jmax but not the Km for the anions. The toxin also inhibited the NaCl-stimulated efflux of 35SO4(2-), indicating that the toxin inhibits the antiporter. No inhibition was found when toxin-treated cells were not exposed to low pH, whereas exposure to pH 4.5 for 20 s induced close to maximal inhibition. Half-maximal inhibition was obtained after exposure to pH 5.4. The concentration of diphtheria toxin required to obtain maximal inhibition (0.3 micrograms/ml) was sufficient to ensure close to maximal toxin binding to the cells. Even in ATP-depleted cells and in the absence of permeant anions, low pH induced inhibition of anion antiport in toxin-treated Vero cells. There was no measurable inhibition of anion antiport in cells with little or no ability to bind the toxin.

Adenosine Triphosphate↗

PubMed1986

Routing of internalized ricin and ricin conjugates to the Golgi complex.

Receptor-mediated endocytosis and intracellular routing of native ricin, and of ricin conjugated to colloidal gold (Ri-Au) and to horseradish peroxidase (Ri-HRP), have been studied in cultured MCF-7 and Vero cells by electron microscopical techniques including serial section analysis. Both native ricin, as demonstrated by immunoperoxidase cytochemistry, and the ricin conjugates were internalized via a common coated pit-coated vesicle pathway to reach vacuolar and tubulo-vesicular portions of the endosomal system. In addition, native ricin and a purified monovalent fraction of Ri-HRP reached distinct Golgi cisterns, whereas Ri-Au and polyvalent Ri-HRP did not. The results delineate intracellular routing of native ricin and compare it with the routing of different ricin conjugates. Moreover, our study shows that conjugates of a particular ligand (ricin) and various probes (e.g., gold and peroxidase), may be handled differently by cells. Sorting apparently takes place in the endosomal system, allowing some but not other molecules to reach Golgi elements. This sorting seems to depend on the valency of the ricin conjugate.

Biological Transport↗

PubMed1986

pH-regulated anion antiport in nucleated mammalian cells.

The uptake of 36Cl- into cells was measured after preincubation in medium containing nigericin and KCl to allow control of the intracellular pH. When the pH was increased from pH 7.0 to pH 7.3 there was a 10-fold increase in the rate of 36Cl- uptake. The increase was half maximal at pH 7.15 in Vero and L-cells, whereas in phorbol 12-myristate 13-acetate-treated Vero cells the increase was half maximal at pH 6.9. Kinetic studies showed that in cells preincubated with nigericin and isotonic KCl, both at pH 7.0 and at pH 8.0, the Km for Cl- was 7 mM. In the two cases the Jmax was 1.7 X 10(8) Cl- ions X cell-1 X s-1 and 1.6 X 10(9) Cl- ions X cell-1 X s-1, respectively. Bicarbonate inhibited 36Cl- uptake with a Ki of 5-6 mM. Probably, the anion antiporter plays a role in the regulation of the intracellular pH.

Animals↗

PubMed1986

Ability of inhibitors of glycosylation and protein synthesis to sensitize cells to abrin, ricin, Shigella toxin, and Pseudomonas toxin.

A number of compounds that interfere with glycoprotein synthesis and transport have been tested for their ability to sensitize cells to cancerostatic protein toxins. Tunicamycin, swainsonine, cycloheximide, and puromycin sensitized Vero cells and HeLa cells to abrin and ricin, as we have found previously with monensin (K. Sandvig and S. Olsnes, J. Biol. Chem., 257: 7504-7513, 1982). Cycloheximide, but not swainsonine, sensitized Vero cells to Pseudomonas exotoxin A and Shigella toxin. The ability of ricin to intoxicate cells was much lower at 19 degrees C than at 37 degrees C and there was almost no sensitizing effect of cycloheximide and monensin at 19 degrees C. Studies by electron microscopy showed that ricin conjugated to horseradish peroxidase appeared in trans Golgi elements in Vero cells. Possibly, transport of ricin into the cytosol requires passage through the Golgi apparatus. The possibility that the sensitizing agents here described may be valuable in enhancing the action of immunotoxins is discussed.

ADP Ribose Transferases↗