4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and Carcinoma--Ehrlich-Tumor

The effects of changes of membrane potential on amino acid transport through systems A, ASC and L was investigated in the Ehrlich cell and the human erythrocyte. Changes of membrane potential were produced by incubating cells whose K+ permeability had been increased, either by valinomycin or by activation of Ca2+-dependent K+ channels, in medium containing different K+ concentrations. The changes in membrane potential were followed by measuring the distribution ratio reached by lipophilic indicators. Transport through Na+-dependent system A was sensitive to the membrane potential, the rate of amino acid uptake increasing 2.2-3.1-times for each 60 mV-hyperpolarization. The Na+-dependent system ASC was insensitive to membrane potential. The Na+-independent system L was not directly affected by membrane potential, but the steady-state accumulation of system L substrates was increased by hyperpolarization.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amino Acids; Amino Acids, Cyclic; Animals; Biological Transport, Active; Carcinoma, Ehrlich Tumor; Erythrocyte Membrane; Humans; In Vitro Techniques; Kinetics; Membrane Potentials; Mice; Onium Compounds; Potassium; Propranolol; Sodium; Trityl Compounds

In Ehrlich ascites tumor cells 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS) inhibits the chloride exchange both reversibly and irreversibly. The reversible inhibition is practically instantaneous and of a competitive nature with Ki about 2 microM at zero chloride concentration. This is succeeded by a slow irreversible binding of DIDS to the transporter, with a chloride dependence suggesting binding to the same site as for reversible DIDS binding/inhibition. To identify the membrane protein involved in anion exchange, cells were labeled with 3H-DIDS. Incubation of cells for 10 min with 25 microM DIDS at pH 8.2 leads to more than 95% inhibition of the DIDS-sensitive chloride exchange flux when the chloride concentration is low (15 mM). This condition was used for the 3H-DIDS-labeling experiments. After incubation the cells were disrupted, the membranes isolated and solubilized, and the proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The distribution of the 3H-activity in the gel showed only one major peak, which could be related to protein with a mol wt of about 30,000 Daltons. The number of transport sites was estimated at about 400,000 per cell, and from the DIDS-sensitive chloride flux under steady-state conditions we calculate a turnover number of 340 ions per sec per site.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anion Transport Proteins; Binding Sites; Carcinoma, Ehrlich Tumor; Carrier Proteins; Chlorides; Female; Ion Exchange; Kinetics; Mice; Molecular Weight; Neoplasm Proteins; Stilbenes

The Ehrlich tumor cell possesses an anion-cation cotransport system which operates as a bidirectional exchanger during the physiological steady state. This cotransport system, like that associated with the volume regulatory mechanism (i.e. coupled net uptake of Cl- + Na+ and/or K+), is Cl- -selective and furosemide-sensitive, suggesting the same mechanism operating in two different modes. Since Na+ has an important function in the volume regulatory response, its role in steady-state cotransport was investigated. In the absence of Na+, ouabain-insensitive K+ and DIDS-insensitive Cl- transport (KCl cotransport) are low and equivalent to that found in 150 mM Na+ medium containing furosemide. Increasing the [Na+] results in parallel increases in K+ and Cl- transport. The maximum rate of each (18 to 20 meq/(kg dry wt) . min) is reached at about 20 mM Na+ and is maintained up to 55 mM. Thus, over the range 1 to 55 mM Na+ the stoichiometry of KCl cotransport is 1:1. In contrast to K+ and Cl-, furosemide-sensitive Na+ transport is undetectable until the [Na+] exceeds 50 mM. From 50 to 150 mM Na+, it progressively rises to 7 meq/(kg dry wt) . min, while K+ and Cl- transport decrease to 9 and 16 meq/(kg dry wt) . min, respectively. Thus, at 150 mM Na+ the stoichiometric relationship between Cl-, Na+ and K+ is 2:1:1. These results are consistent with the proposal that the Cl- -dependent cation cotransport system when operating during the steady state mediates the exchange of KCl for KCl or NaCl for NaCl; the relative proportion of each determined by the extracellular [Na+].

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Biological Transport, Active; Carcinoma, Ehrlich Tumor; Chlorides; Furosemide; Ion Channels; Kinetics; Male; Mice; Mice, Inbred ICR; Ouabain; Potassium; Sodium

Cells resuspended in hypotonic medium initially swell as nearly perfect osmometers, but later recover their volume with an associated KCl loss. This regulatory volume decrease (RVD) is unaffected when nitrate is substituted for Cl- or if bumetanide or 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) is added. It is inhibited by quinine, Ba2+, low pH, anticalmodulin drugs, and depletion of intracellular Ca2+. It is accelerated by the Ca2+ ionophore A23187, or by a sudden increase in external Ca2+ and at high pH. A net KCl loss is also seen after addition of ionophore A23187 in isotonic medium. Similarities are demonstrated between the KCl loss seen after addition of A23187 and the KCl loss seen during RVD. It is proposed that separate conductive K+ and Cl- channels are activated during RVD by release of Ca2+ from internal stores, and that the effect is mediated by calmodulin. After restoration of tonicity the cells shrink initially, but recover their volume with an associated KCl uptake. This regulatory volume increase (RVI) is inhibited when NO3- is substituted for Cl-, and is also inhibited by furosemide or bumetanide, but it is unaffected by DIDS. The unidirectional Cl-flux ratio is compatible with either a coupled uptake of Na+ and Cl-, or an uptake via a K+/Na+/2Cl- cotransport system. No K+ uptake was found, however, in ouabain-poisoned cells where a bumetanide-sensitive uptake of Na+ and Cl- in nearly equimolar amounts was demonstrated. Therefore, it is proposed that the primary process during RVI is an activation of an otherwise quiescent Na+/Cl- cotransport system with subsequent replacement of Na+ by K+ via the Na+/K+ pump. There is a marked increase in the rate of pump activity in the absence of a detectable increase in intracellular Na+ concentration.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Biological Transport; Bumetanide; Calcimycin; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Chlorides; Extracellular Space; Furosemide; In Vitro Techniques; Ion Channels; Isotonic Solutions; Mice; Osmotic Fragility; Potassium; Sodium

Previous studies have shown that mediated Cl- transport which occurs by at least two processes (Cl- -dependent cation cotransport and Cl- self-exchange) becomes progressively inhibited when extracellular Cl- exceeds about 60 mM (Hoffmann et al., 1979). To account for this type of kinetic behavior, that is, self-inhibition, an anion transport system possessing two sites, a high affinity transport site and a lower affinity modifier site is suggested (Dalmark, 1976). In the present experiments we have attempted to determine which of the mediated transport pathways is susceptible to self-inhibition by studying the dependence of the steady state Cl- flux on the extracellular Cl- concentration and how DIDS, an inhibitor of Cl- self-exchange, and H + affect this relationship. Addition of DIDS to Ehrlich cells results in inhibition of Cl- transport at every Cl- concentration tested (40-150 mM). Moreover, the Cl- flux/Cl- concentration relationship no longer exhibits self-inhibition, suggesting that this phenomenon is a characteristic of the Cl- self-exchanger rather than of the Cl- -dependent cation cotransport system. Lowering the extracellular pH (pHo) from 7.35 to 5.30 stimulates Cl- transport by a process that saturates with respect to [H +]. Half-maximal stimulation occurs at pHo 6.34. A comparison of the kinetic parameters, Ks and Jmax, calculated from the ascending limb of the Cl- flux/Cl- concentration curve at pHo 7.30 to those at pHo 5.50 show that the values for Ks are almost identical (23.6 mM and 21.3 mM, respectively), while the values for Jmax [22.2 mEq/Kg dry wt) X min] differ by only 15%. This finding along with the observation that DIDS completely blocks H + stimulation of Cl- transport is compatible with the suggestion that H + interact at the modifer site of the Cl- self-exchanger and thereby prevents self-inhibition.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Biological Transport; Carcinoma, Ehrlich Tumor; Chlorides; Culture Media; Feedback; Gluconates; Hydrogen-Ion Concentration; Kinetics; Mice

Ehrlich ascites cells were preincubated in hypotonic medium with subsequent restoration of tonicity. After the initial osmotic shrinkage the cells recovered their volume within 5 min with an associated KCl uptake. The volume recovery was inhibited when NO-3 was substituted for Cl-, and when Na+ was replaced by K+, or by choline (at 5 mM external K+). The volume recovery was strongly inhibited by furosemide and bumetanide, but essentially unaffected by DIDS. The net uptake of Cl- was much larger than the value predicted from the conductive Cl- permeability. The undirectional 36Cl flux, which was insensitive to bumetanide under steady-state conditions, was substantially increased during regulatory volume increase, and showed a large bumetanide-sensitive component. During volume recovery the Cl- flux ratio (influx/efflux) for the bumetanide-sensitive component was estimated at 1.85, compatible with a coupled uptake of Na+ and Cl-, or with an uptake via a K+,Na+,2Cl- cotransport system. The latter possibility is unlikely, however, because a net uptake of KCl was found even at low external K+, and because no K+ uptake was found in ouabain-poisoned cells. In the presence of ouabain a bumetanide-sensitive uptake during volume recovery of Na+ and Cl- in nearly equimolar amounts was demonstrated. It is proposed that the primary process during the regulatory volume increase is an activation of an otherwise quiescent, bumetanide-sensitive Na+,Cl- cotransport system with subsequent replacement of Na+ by K+ via the Na+/K+ pump, stimulated by the Na+ influx through the Na+,Cl- cotransport system.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Biological Transport; Bumetanide; Carcinoma, Ehrlich Tumor; Chlorides; Female; Furosemide; Hypotonic Solutions; In Vitro Techniques; Mice; Nitrates; Osmosis; Ouabain; Potassium Chloride; Sodium; Sodium-Potassium-Exchanging ATPase; Trifluoperazine; Water-Electrolyte Balance

A major aim of this investigation was to determine whether, in steady-state ascites cells, Cl- transport can be partitioned into a furosemide-sensitive cotransport with K+ and a separate 4,4'-isothiocyanostilbene-2,2'-disulfonic acid (DIDS) sensitive self-exchange. Both Cl- and K+ fluxes were studied. The furosemide- and Cl- sensitive K+ fluxes were equivalent, both in normal ionic media and when the external K+ concentration, [K+]o, was varied from 4 to 30 mM. The stoichiometry of the furosemide-sensitive Cl- and K+ fluxes was 2 Cl-:1 K+ at 0.1 and 0.5 mM drug levels but increased to 3 Cl-:1 K+ at 1.0 mM furosemide. DIDS at 0.1 mM had no effect on the K+ exchange rate but inhibited Cl- exchange by 39% +/- 2 (S.E.). The effects of DIDS and 0.5 mM furosemide on Cl- transport were additive but 1.0 mM furosemide and DIDS had overlapping inhibitory actions. Thus furosemide acts on components of K+ and Cl- transport which are linked to each other, but the drug also inhibits an additional DIDS-sensitive Cl- pathway, when present at higher concentrations. The dependence of the furosemide-sensitive K+ and Cl- transport on [K+]o was also studied; both fluxes fell as the [K+]o increased. The latter results recall those in an earlier study by Hempling (Hempling, H.G. (1962) J. Cell. Comp. Physiol. 60, 181-198).

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Biological Transport; Carcinoma, Ehrlich Tumor; Cells, Cultured; Chlorides; Furosemide; Kinetics; Mice; Potassium

DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) can interact covalently with membrane sites, resulting in an inhibition of anion exchange. This inhibition varies from reversible to irreversible, depending on the length of times DIDS interacts with the membranes. During the reversible phase, a kinetic analysis of the nature of its inhibitory effect on C1 self-exchange can be performed. The effect of variations in the chloride concentration on the inhibitory potency of DIDS is consistent with the concept that C1 and DIDS compete for the transport site of the anion exchange system in Ehrlich cells. The value of Ki for DIDS inhibition is approx. 0.5 microM. Since the reversible binding may be specific to the anion recognition site in the transport system, it is likely that the subsequent covalent reaction (which is very slow in the Ehrlich cell) involves a nucleophilic group in the membrane close to the transport site. In this case DIDS can be used as a covalent label for the transport protein. We have synthesized a 3-H labelled DIDS with a high specific activity (0.62 X 10(8) cpm/mumol). Using the labelled DIDS, it has been possible to demonstrate that in low C1 medium, only one membrane protein seems to be labelled, and that the number of binding sites per cell is 7 X 10(7). This value is very close to the known density of binding sites in the red blood cell.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anion Transport Proteins; Biological Transport, Active; Carcinoma, Ehrlich Tumor; Carrier Proteins; Cell Membrane; Chlorides; Kinetics; Membrane Proteins; Mice

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anions; Binding Sites; Biological Transport; Carcinoma, Ehrlich Tumor; Cell Membrane; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Humans; In Vitro Techniques; Kinetics; Male; Membrane Proteins; Mice; Mice, Inbred ICR; Protein Binding; Stilbenes; Sulfates

Ehrlich ascites tumor cells spontaneously release cell surface material (glycocalyx) into isotonic saline medium. Exposure of these cells to tritium-labeled 4,4'-diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid (3H2DIDS) at 4 degrees C leads to preferential labeling of the cell surface coat. We have combined studies of the kinetics of 3H2DIDS-label release, the effects of enzymatic treatment, and cell electrophoretic mobility to characterize the 3H2DIDS-labeled components of the cell surface. Approximately 73% of the cell-associated radioactivity is spontaneously released from the cells after 5 h at 23 degrees C. The kinetics of release is consistent with the first-order loss of two fractions; a slow (tau 1/2 = 360 min) component representing 33% of the radioactivity and a fast (tau 1/2 = 20 min) component representing 26%. The remaining 14% of the labile binding may reflect mechanically induced surface release. Trypsin (1 microgram/ml) also removes approximately 73% of the labeled material within 30 min and converts the kinetics of release to that of a single component (tau 1/2 = 5.5 min). The specific activity (SA) of material released by trypsin immediately after labeling is 83% of the SA of the material spontaneously lost in 1 h. However, trypsinization following a 2-h period of spontaneous release yields material of reduced (43%) SA. Neither 3H2DIDS labeling nor the initial spontaneous loss of labeled material alters cell electrophoretic mobility. However, extended spontaneous release is accompanied by a significant decrease in surface charge density. Trypsinization immediately following labeling or after spontaneous release (2 h) reduces mobility by 32%. We have tentatively identified the slowly released compartment as contributing to cell surface negativity.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Carcinoma, Ehrlich Tumor; Cell Membrane; Electrophoresis, Polyacrylamide Gel; Kinetics; Male; Membrane Proteins; Mice; Mice, Inbred Strains; Molecular Weight; Trypsin