Self-inhibition of chloride transport in Ehrlich ascites tumor cells

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.     

Regulation of taurine transport in Ehrlich ascites tumor cells

Summary Taurine influx is inhibited and taurine efflux accelerated when the cell membrane of Ehrlich ascites tumor cells is depolarized. Taurine influx is inhibited at acid pH partly due to the concomitant depolarization of the cell membrane partly due to a reduced availability of negatively charged free carrier. These results are in agreement with a 2Na, 1Cl, 1taurine cotransport system which is sensitive to the membrane potential due to a negatively charged empty carrier. Taurine efflux from Ehrlich cells is stimulated by addition of LTD4 and by swelling in hypotonic medium. Cell swelling in hypotonic medium is known to result in stimulation of the leukotriene synthesis and depolarization of the cell membrane. The taurine efflux, activated by cell swelling, is dramatically reduced when the phospholipase A₂ is inhibited indirectly by addition of the anti-calmodulin drug pimozide, or directly by addition of RO 31-4639. The inhibition is in both cases lifted by addition of LTD₄. The swelling-induced taurine efflux is also inhibited by addition of the 5-lipoxygenase inhibitors ETH 615-139 and NDGA. It is concluded that the swelling-induced activation of the taurine leak pathway involves a release of arachidonic acid from the membrane phospholipids and an increased oxidation of arachidonic acid into leukotrienes via the 5-lipoxygenase pathway. LTD₄ seems to act as a second messenger for the swelling induced activation of the taurine leak pathway either directly or indirectly via its activation of the Cl^– channels, i.e., via a depolarization of the cell membrane.     

The transport of chloride in Ehrlich ascites tumor cells.

The steady state transport and distribution of chloride between the intracellular and extracellular phases was investigated when the extracellular chloride concentration was varied by isosmotic replacement with nitrate, bromide and acetate. The results of these experiments show that chloride transport, measured by uptake of 36Cl, is sensitive to the replacement anion. In the presence of nitrate, chloride transport is a linear function of the extracellular chloride concentration. The relationship between chloride transport and extracellular chloride in the presence of bromide is concave upward which suggests that this anion inhibits chloride movement. However, when acetate replaces chloride, the relationship between chloride transport and extracellular chloride is concave downward. The chloride distribution ratio of cells incubated in 145-155mM chloride medium is 0.386 and is not effected by the replacement of chloride with nitrate, bromide or acetate. These findings are consistent with the assertion that chloride transport is composed of two parallel pathways, a diffusional plus a saturating, mediated component. Of the total chloride flux (9.1 mmoles Cl-/kg dry weight per minute) measured in chloride medium (145-155 mM Cl-), the mediated component represents 40% and the diffusional component 60%.     

Calcium transport and distribution in Ehrlich mouse ascites tumor cells

Data from isotopic uptake experiments were used to measure calcium fluxes and compartment sizes in ascites tumor cells. The data were analyzed with two kinetic models, A and B. In 80% of the experiments model A, which is based on one exchangeable calcium compartment, was rejected in favor of Model B, which predicts two exchangeable compartments. A statistical evaluation of the model's performance, when fit to the experimental data was used to select between the two models. The results show that calcium was distributed between three cellular compartments in the ratio, non-exchangeable (88%): rapidly exchanging (7%): slowly exchanging (5%). The undirectional fluxes suggested that calcium transport could be described as a series system with the temporal sequence: environment ? rapidly exchanging ? slowly exchanging.     

Evidence for monovalent phosphate transport in Ehrlich ascites tumor cells

In an effort to determine whether the Na+-dependent Pi transport system of Ehrlich ascites tumor cells exhibits specificity for H2PO4- or HPO4(-2), Pi fluxes were determined by measuring 32Pi-Pi self-exchange. Three experimental approaches were employed. First, the effect of pH on steady-state Pi transport at 0.5 and 5 mM was studied. Second, the relationship between Pi transport and Pi concentration (0.25-9.2 mM) at pH 5.6 and 7.9 was determined. Third, the dependence of Pi transport on [H2PO4-] (0.05-4.2 mM) at constant [HPO4(-2)] (0.5 mM), and the converse, [HPO4(-2)] (0.06-4.5 mM) at constant [H2PO4-] (0.5 mM), was evaluated. Ks (apparent half-saturation constant) and Jmax (maximal transport rate) were calculated by two methods: weighted linear regression (WLR) and a nonparametric procedure. The dependence of Pi flux on pH indicates that optimum transport occurs at pH 6.9. Pi transport decreases as pH is reduced when extracellular Pi is either 0.5 or 5 mM. However, at pH 7.9, Pi flux is reduced only in 0.5 mM Pi. At pH 5.6, H2PO4- comprises 93% of the total Pi present, and the calculated Ks is 0.055 +/- 0.026 mM (WLR). This is the same as the Ks determined from the initial phase of the flux vs. [H2PO4-] relationship (0.056 +/- 0.020 mM). However, at pH 7.9 (where 94% of Pi is HPO4(-2)), the measured Ks is 0.58 +/- 0.11 mM (WLR), which is ten times higher than at pH 5.6. This value is also five times greater than the Ks calculated from the flux vs. [HPO4(-20)] curve (0.106 +/- 0.16 mM). Kinetic parameters calculated by the nonparametric method, though somewhat different, gave similar relative results. Taken together, these results support two conclusions: (1) H2PO4- is the substrate for the Na+-dependent Pi transport system of the Ehrlich cell, and (2) H+ can inhibit Pi transport.     

Quinine inhibits multiple Na+ and K+ transport mechanisms in Ehrlich ascites tumor cells

The interaction of quinine with K+ and Na+ transport mechanisms has been investigated in Ehrlich ascites tumor cells. Quinine affects both Ca2+-dependent K+ channel and total K+ influx. Activation of Ca+-dependent K+ channels by propranolol is abolished by quinine (1 mM). In addition, quinine inhibits the ouabain-sensitive component of K+ influx with an apparent Ki of 0.32 +/- 0.02 mM and the furosemide-sensitive component with a Ki of 0.24 +/- 0.01 mM. Furthermore, a significant fraction (52%) of Na+ influx is inhibited by quinine. The same component is sensitive to amiloride, suggesting that it represents Na+/H+ antiport. Concomitant with the inhibition of K+ and Na+ transport, quinine stimulates ATP hydrolysis by 57%. The results suggest that quinine exerts broad, nonspecific effects on cellular mechanisms which serve to regulate cation transport in Ehrlich cells.     

Ca2+-dependent K+ transport in the Ehrlich ascites tumor cell

The possible presence and properties of the Ca2+-dependent K+ channel have been investigated in the Ehrlich ascites tumor cell. The treatment with ionophore A23187 + CA2+, propranolol or the electron donor system ascorbate-phenazine methosulphate, all of which activate that transport system in the human erythrocyte, produces in the Ehrlich cell a net loss of K+ (balanced by the uptake of Na+) and a stimulation of both the influx and the efflux of 86Rb. These effects were antagonized by quinine, a known inhibitor of the Ca2+-dependent K+ channel in other cell systems, and by the addition of EGTA to the incubation medium. Ouabain did not have an inhibitory effect. These results suggests that the Ehrlich cell possesses a Ca2+-dependent K+ channel whose characteristics are similar to those described in other cell systems.     

Phosphate transport in Ehrlich ascites tumor cells and the effect of arsenate

The effect of extracellular Pi and arsenate on Pi-transport in Ehrlich ascites tumor cells has been studied. Pi-transport can be described by Michaelis-Menten kinetics; the maximal flux equal to 44 mmoles (kg cell water)^?1 hour^?1 and K_m equal to 3.3 × 10^?4 M . Arsenate is a competitive inhibitor of Pi-transport with an inhibition constant (K_i) equal to 2.41 × 10^?3 M . The data support the hypothesis that cellular Pi is regulated by the cell membrane through the mediation of a carrier system.     

Phosphate transport in Ehrlich ascites tumor cells: inhibition by H+

The effect of changes in extracellular pH (pHo) and intracellular pH (pHi) on Na+-dependent and Na+-independent inorganic phosphate (Pi) transport in Ehrlich cells was investigated. In the presence of Na+, acutely reducing pHo from 7.30 to 5.50 results first in a transient (approximately 7 min) stimulation of Pi transport. The enhanced rate of transport is a saturable function of the extracellular [H+]; the Ks equals 2.3 X 10(-6) M (pHo 6.68). However, Pi transport is progressively inhibited as pHi falls below 6.50. The effect of pHi on Pi transport measured at various intracellular [Na+] suggests that inhibition develops as a consequence of H+ interaction with an intracellular Na+ site(s) on the Na+-dependent carrier. At pHo 7.4, about 15% of the steady state Pi flux persists in the absence of Na+. However, when pHo is reduced, transport is stimulated to the same extent and with the same time course and kinetic characteristics as in the presence of Na+. Thus, H+ stimulated Pi transport does not require Na+, raising the possibility that the Na+-independent component is mediated by the anion (Cl-) exchanger.     

Mannose toxicity in Ehrlich ascites tumor cells

Mannosephosphate isomerase (MPI) showed a higher activity than hexokinase (HKM) in its ability to phosphorylate mannose in the spleen, thymus, brain, liver, striated muscles, kidneys, and testes from BALB/c mice. This led to a HKM/MPI ratio of less than 1 in all the organs and tissues mentioned. In contrast, Ehrlich ascites tumor cells obtained from the peritoneum of BALB/c mice had low MPI activity (half of the HKM activity and, therefore, a ratio of 2). Mannose, which is nontoxic to nontumor cells at a concentration of 0.1 M, induced marked in vitro mortality of the tumor cells. Incubation of Ehrlich ascites tumor cells with mannose resulted in a high accumulation of mannose-6-phosphate and a marked depletion of ATP which did not appear when the cells were incubated with glucose. These facts may explain the selective mortality caused by mannose in the tumor cells studied.     

Phosphate concentration and transport in Ehrlich ascites tumor cells: Effect of sodium

The effects of extracellular P_i and Na⁺ on cellular P_i concentration and transport were studied. Steady-state P_i exchange flux was measured by ³²P uptake in the presence and absence of Na⁺. Model experiments were also conducted to assess the possibility that hydrolysis of organic phosphate esters contributes to the chemically measured intracellular P_i concentration of Ehrlich ascites tumor cells. The results of these experiments indicate that hydroloysis of labile organic phosphate esters does not contribute to the measured intracellular pool of P_i. The P_i transport system exhibits an apparent K_s of 0.115 mM P_i and a maximal flux of 1.73 mmole min^?1 (kg dry wt)^?1. When incubated in a phosphate-buffered choline chloride medium (5 mM P_i) the intracellular P_i and the P_i influx fall by 65 and 88%, respectively. At 5 mM extracellular P_i, the Na⁺-dependent component of P_i transport fits Michaelis-Menten kinetics with the maximal flux equal to 2.46 mmole min^?1 (kg dry wt)^?1 and an apparent K_s of 35.4 mM Na⁺. In addition, a Na⁺-independent component of P_i transport, comprising about 12% of the total P_i flux, was identified. The data support the hypothesis that a P_i transport system, dependent on Na⁺, plays a principal role in the maintenance of intracellular P_i concentration.