Charles Levinson

Direct measurement of the membrane potential of ehrlich ascites tumor cells lack of effect of valinomycin and ouabain

SummaryThe membrane potential of Ehrlich ascites tumor cells and the effects of valinomycin and ouabain upon it have been determined. The membrane potential in control cells was 12.0 mV, inside negative. Neither valinomycin nor ouabain alone affected this value. However, valinomycin and ouabain in combination resulted in a slight hyperpolarization of the membrane. Concomitant determinations of cellular Na+ K+ and Cl− showed that valinomycin induced net losses of K+ and Cl− and a net gain in Na+ when compared to ouabain-inhibited cells. K+ permeability was increased by approximately 30% in the presence of valinomycin. In addition, valinomycin caused a rapid depletion of cellular ATP. Inhibition of Na/K transport by ouabain was without sparing effect on the rate of ATP depletion. Possible mechanisms for the electroneutral increase in K+ permeability induced by valinomycin are discussed.

Regulatory volume increase in Ehrlich ascites tumor cells

Ehrlich ascites tumor cells, shrunken as a result of KCl-depletion and Na+ loading, re-establish normal ionic concentrations by the combined activity of the Na+/K+ pump and the (2Cl- + K+ + Na+) cotransport system. Restoration of cell volume, however, correlates only with the increase in intracellular Cl-. This along with the finding that the equilibrium volume is linearly related to the steady state [Cl-] suggests that the extent to which cell volume increases is determined by Cl- transport. Net Cl- uptake, which is mediated almost exclusively by the cotransport system, is ultimately responsible for establishing the steady-state intracellular Cl- concentration. Transport mediated by this pathway ceases when the sum of the chemical potentials for Na+, K+ and Cl- approaches zero and corresponds with the establishment of a steady state for Cl-. These findings suggest that Cl- plays a key role in the regulation of net cotransport activity and thereby cell volume.

H+ transport and the regulation of intracellular pH in Ehrlich ascites tumor cells

SummaryThe intracellular pH (pHi) of Ehrlich ascites tumor cells, both in the steady state and under conditions of acid loading or recovery from acid loading, was investigated by measuring the transmembrane flux of H+ equivalents and correlating this with changes in the distribution ratio of dimethyloxazolidine-2,4-dione (DMO). The pHi of cells placed in an acidic medium (pHo below 7.15) decreases and reaches a steady-state value that is more alkaline than the outside. For example when pHo is acutely reduced to 5.5, pHi falls exponentially from 7.20 ± 0.06 to 6.29 ± 0.04 with a halftime of 5.92 ± 1.37 min, suggesting a rapid influx of H+. The unidirectional influx of H+ exhibits saturation kinetics with respect to extracellular [H+]; the maximal flux is 15.8 ± 0.05 mmol/(kg dry wt · min) andKm is 0.74 ± 0.09 × 10−6m.Steady-state cells with pHi above 6.8 continuously extrude H+ by a process that is not dependent on ATP but is inhibited by anaerobiosis. Acid-loaded cells (pHi 6.3) when returned to pHo 7.3 medium respond by transporting H+, resulting in a rapid rise in pHi. The halftime for this process is 1.09 ± 0.22 min. The H+ efflux measured under similar conditions increases as the intracellular acid load increases. An ATP-independent as well as an ATP-dependent efflux contributes to the restoration of pHi to its steady-state value.

INABILITY OF EHRLICH ASCITES TUMOR CELLS TO VOLUME REGULATE FOLLOWING A HYPEROSMOTIC CHALLENGE

SummaryEhrlich cells shrink when the osmolality of the suspending medium is increased and behave, at least initially, as osmometers. Subsequent behavior depends on the nature of the hyperosmotic solute but in no case did the cells exhibit regulatory volume increase. With hyperosmotic NaCl an osmometric response was found and the resultant volume maintained relatively constant. Continuous shrinkage was observed, however, with sucrose-induced hyperosmolality. In both cases increasing osmolality from 300 to 500 mOsm initiated significant changes in cellular electrolyte content, as well as intracellular pH. This was brought about by activation of the Na+/H+ exchanger, the Na/K pump, the Na++K++2Cl− cotransporter and by loss of K+ via a Ba-sensitive pathway. The cotransporter in response to elevated [Cl−]i (∼100mm) and/or the increase in the outwardly directed gradient of chemical potential for Na+, K+ and Cl−, mediated net loss of ions which accounted for cell shrinkage in the sucrose-containing medium. In hyperosmotic NaCl, however, the net Cl− flux was almost zero suggesting minimal net cotransport activity.We conclude that volume stability following cell shrinkage depends on the transmembrane gradient of chemical potential for [Na++K++Cl−], as well as the ratio of intra- to extracellular [Cl−]. Both factors appear to influence the activity of the cotransport pathway.

Steady-state distribution of phosphate across the membrane of the Ehrlich ascites tumor cell.

Abstract 1. 1. The steady-state concentrations of chloride, mono- and divalent phosphate ions in the cellular and extracellular phases of Ehrlich ascites tumor cells were determined. 2. 2. The distribution ratios of these ions were: [Cl − ] cell − [Cl − ] ex. = 0.365 , [HPO 4 2− ] cell 1 2 [HPO 4 2− ] ex. 1 2 = 0.385 and [H 2 PO 4 − ] cell [H 2 PO 4 − ] ex. = 0.627 . 3. 3. These results indicate that the divalent ion is passively distributed across the tumor cell membrane, while the monovalent ion is not.

Regulatory volume decrease in Ehrlich ascites tumor cells is not mediated by a rise in intracellular calcium

Ehrlich ascites tumor cells suspended in hyposmotic solution initially swell and then shrink back towards normal volume, a process known as regulatory volume decrease (RVD). RVD is characterized by a specific loss of KCl, although the mechanism for this is currently unknown. The hypothesis that a rise in intracellular calcium ([Ca2+]i) activates calcium-sensitive ion conductances to initiate RVD was investigated. The results indicate that in the Ehrlich cell no rise in [Ca2+]i occurs when the extracellular osmolality is reduced from 300 mosM to 180 mosM. These findings were substantiated by the lack of sensitivity of RVD to the Ca(2+)-sensitive K+ channel blockers charybdotoxin (CTX) and nifedipine. In contrast, the ionophore ionomycin induced a cell shrinkage that was sensitive to CTX and nifedipine indicating that a rise in [Ca2+]i could play a role in cell volume reduction but that this occurred by a mechanism different from that observed in RVD. The conclusion from these experiments is that Ca2+ does not act as a second messenger for RVD in the Ehrlich cell.

Sidium-dependent ion cotransport in steady-state Ehrlich ascites tumor cells

SummaryThe Ehrlich tumor cell possesses and 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 150mm 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 20mm Na+ and is maintained up to 55mm. Thus, over the range 1 to 55mm 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 50mm. From 50 to 150mm 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 150mm 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+].

Interaction of tritium-labeled H2DIDS (4,4′-diisothiocyano-1,2,diphenyl ethane-2,2′disulfonic acid) with the ehrlich mouse ascites tumor cell

SummaryThe experiments reported in this paper were undertaken to explore the interaction of tritiated H2DIDS (4,4′-diisothiocyano-1,2,diphenyl ethane-2,2′-disulfonic acid) with Ehrlich ascites tumor cells. Addition of (3H)H2DIDS to tumor cell suspension at 21°C, pH 7.3, resulted in: (i) rapid reversible binding which increased with time and (ii) inhibition of sulfate transport. Tightly bound H2DIDS, i.e., reagent not removed by cell washing, also increased with time. Binding of 0.02 nmol H2DIDS/mg dry mass or less did not affect sulfate transport, but, at greater than 0.02 nmol and up to 0.15 nmol the relationship between tight binding and inhibition of transport is linear. The fact that H2DIDS could bind to the cell and yet not affect anion transport suggests that binding sites exist unrelated to those concerned with the regulation of anion permeability. Support for this is the observation that H2DIDS is spontaneously released from cells even after extensive washings by a temperature-sensitive process. The most important source of released H2DIDS is the cell surface coat which labels rapidly (within 1 min) and is then spontaneously released into the medium. A second source is derived from H2DIDS that slowly entered the cells. Consequently, at least four modes of interaction exist between H2DIDS and ascites tumor cells. These include both reversible and irreversible binding to membrane components which regulate anion permeability, irreversible binding to cell surface proteins or glycocalyx, and finally incorporation of H2DIDS into the intracellular phase.

Volume regulatory activity of the Ehrlich ascites tumor cell and its relationship to ion transport.

SummaryThe volume regulatory response of the Ehrlich ascites tumor was studied in KCl-depleted, Na+-enriched cells. Subsequent incubation in K+-containing NaCl medium results in the reaccumulation of K+, Cl−, water and the extrusion of Na+. The establishment of the physiological steady state is due primarily to the activity of 2 transport systems. One is the Na/K pump (KM for K0+=3.5mm;Jmax=30.1 mEq/kg dry min), which in these experiments was coupled 1K+/1 Na+. The second is the Cl−-dependent (Na++K+) cotransport system (KM for K0+=6.8mm;Jmax=20.8 mEq/kg dry min) which mediates, in addition to net ion uptake in the ratio of 1K+∶1Na+∶2Cl−, the exchange of Ki+ for K0+. The net passive driving force on the cotransport system is initially inwardly directed but does not decrease to zero at the steady state. This raises the possibility of the involvement of an additional source of energy. Although cell volume increases concomitant with net ion uptake, this change does not appear to be a major factor regulating the activity of the cotransport system.

Calcium exchange in Ehrlich mouse ascites tumor cells.

Abstract 1. 1. The mean Ca2+ content of Ehrlich mouse ascites tumor cells was 0.131 μmole per 107 cells. 2. 2. Ca2+ addition to the cellular environment was unnecessary for the maintenance of high internal K+ content and low internal Na+ content. The Ca2+ content of the cell was also independent of the external Ca2+ concentration. 3. 3. Low concentrations of HClO4 (1–10%) extracted 30% of the cellular Ca2+ while the remaining 70% was tenaciously bound to the HClO4-insoluble residue. 4. 4. The cell Ca2+ which was extractable with low concentrations of HClO4 exchanged 3 times faster than the Ca2+ which remained bound. 5. 5. The results are interpreted as evidence for Ca2+ compartmentation within the tumor cell.