Membrane potential, anion and cation conductances in Ehrlich ascites tumor cell

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Abstract
Summary The fluorescence intensity of the dye 1,1′-dipropyloxadicarbocyanine (DiOC3-(5)) has been measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potential (V m ) under different ionic conditions, after treatment with cation ionophores and after hypotonic cell swelling. Calibration is performed with gramicidin in Na+-free K+/choline+ media, i.e., standard medium in which NaCl is replaced by KCl and cholineCl and where the sum of potassium and choline is kept constant at 155mm. Calibration by the valinomycin “null point” procedure described by Lariset al. (Laris, P.C., Pershadsingh, A., Johnstone, R.M., 1976,Biochim. Biophys. Acta 436:475–488) is shown to be valid only in the presence of the Cl−-channel blocker indacrinone (MK196). Distribution of the lipophilic anion SCN− as an indirect estimation of the membrane potential is found not to be applicable for the fast changes inV m reported in this paper. Incubation with DiOC3-(5) for 5 min is demenstrated to reduce the Cl− permeability by 26±5% and the NO −3 permeability by 15±2%, while no significant effect of the probe could be demonstrated on the K+ permeability. Values forV m , corrected for the inhibitory effect of the dye on the anion conductance, are estimated at −61±1 mV in isotonic standard NaCl medium, −78±3 mV in isotonic Na+-free choline medium and −46±1 mV in isotonic NaNO3 medium. The cell membrane is depolarized by addition of the K+ channel inhibitor quinine and it is hyperpolarized when the cells are suspended in Na+-free choline medium, indicating thatV m is generated partly by potassium and partly by sodium diffusion. Ehrlich cells have previously been shown to be more permeable to nitrate than to chloride. Substituting NO −3 for all cellular and extracellular Cl− leads to a depolarization of the membrane, demonstrating thatV m is also generated by the anions and that anions are above equilibrium. Taking the previously demonstrated single-file behavior of the K+ channels into consideration, the membrane conductances in Ehrlich cells are estimated at 10.4 μS/cm2 for K+, 3.0 μS/cm2 for Na+, 0.6 μS/cm2 for Cl− and 8.7 μS/cm2 for NO −3 . Addition of the Ca2+-ionophore A23187 results in net loss of KCl and a hyperpolarization of the membrane, indicating that the K+ permeability exceeds the Cl− permeability also after the addition of A23187. The K+ and Cl− conductances in A23187-treated Ehrlich cells are estimated at 134 and 30 μS/cm2, respectively. The membrane potential is depolarized in hypotonically swollen cells, confirming that the increase in the Cl− permeability following hypotonic exposure exceeds the concommitant increase in the K+ permeability. In control experiments where the membrane potentialV m =E K =E Cl =E Na , it is demonstrated that cell volume changes has no significant effect on the fluorescence signal, apparently because of a large intracellular buffering capacity. The increase in the Cl− conductances is 68-fold when cells are transferred to a medium with half the osmolarity of the standard medium, as estimated from the net Cl− efflux and the change inV m . The concommitant increase in the K+ conductance, as estimated from the net K+ efflux, is only twofold.