No abstract

No abstract

The effects of valinomycin (25 pM) on the membrane potential and on initial, passive Na+ and K + movements have been determined in Ehrlich ascites tumor cells. The membrane potential of steady-state cells in a physiologic environment was -23.2 mV. Addition of valinomycin induced a small, significant hyperpolarization (V, = -29.6 mV) when averaged over the population tested. However, analyses of the response of individual cells to valinomycin showed two different potential effects: (1) the majority of cells hyperpolarized after treatment; but (2) a significant fraction depolarized when exposed to valinomycin. The V, of steady-state cells incubated in saline with K+ at concentrations of 21 mM or 75 mM was -21.4 mV and -22.0 mV, respectively. Addition of valinomycin to these cells was without effect on V,, thus establishing the "null point" responses. Only for cells incubated in saline with a K + of 75 mM was there agreement between V, and K + equilibrium potential (V,). Determinations of cellular Na+ and K+ showed that valinomycin induced net losses of K + and gains of Na+ by cells incubated in either physiologic saline or saline with a K+ concentration of 21 mM. However, the cellular K + of cells incubated in saline with a K + concentration of 75 mM was unaltered by valinomycin. There was a two-to threefold increase in K+ permeability of the cell membrane in the presence of valinomycin. These results are consistent with the existence of two null points in the membrane-potential response to valinomycin: One is established when the membrane potential corresponds to V,; the second occurs when the effects of valinomycin on K + loss from the cell are exactly offset by its inhibition of active Na+ + K + transport.

The effects of altered external sodium and potassium concen- Elevation of external K+ (3-68 mM) at constant Na+ (92 mM) inhibits JE, but is without effect on JR,. The coupling ratio declines from 1.01 * 0.14 to 0.07 * 0.05, a 14-fold alteration. Reduction of external Na+ (154-25 mM) at constant K+ (6 mM) depresses JRa, but is without effect on Jk. The coupling ratio increases from 0.63 * 0.04 at 154 mM Na+ to 4.5 2.04 at 25 mM Na+. The results of this investigation are consistent with the independent regulation of active cation fluxes by the transported species. Kinetic analysis of the data indicates that elevation of external sodium stimulates active sodium efflux by interacting at "modifier sites" at the outer cell surface. Similarly, external potassium inhibits active potassium influx by interaction at separate modifier sites.

The utility of the lipophilic anion thiocyanate (SCN-) as a probe for the indirect estimation of the cell membrane potential (Vm) in Ehrlich ascites tumor cells has been evaluated by comparison to direct electrophysiological measurements. SCN accumulation is consistent with first-order uptake into a single, kinetically-identifiable cellular compartment, achieving steady-state distribution in 20-30 min at 22 degrees C. The steady state distribution ratio ([SCN-]e/[SCN-]e) in physiological saline is 0.44 +/- 0.02. Treatment of the cells with propranolol (0.13 mM), an activator of Ca2+ dependent K+ channels, reduces the steady-state distribution ratio to 0.19 +/- 0.02. Conversely, treatment with BaCl2 (10 mM), an antagonist of the pathway, increases the SCN- distribution ratio to 0.62 +/- 0.01. The equilibrium potentials (VSCN) calculated under these conditions are virtually identical to direct electrophysiological measurements of the Vm made under the same conditions. The effect of varying extracellular [K+] ([K+]e) in the presence of constant [Na+]e = 100 mM has also been tested. In control cells, elevation of [K+]e from 6 to 60 mM reduces VSCN from -20.6 +/- 1.0 to -13.2 +/- 1.2 mV. Again, microelectrode measurements give excellent quantitative agreement. Propranolol increases the sensitivity of the cells to varying [K+]e, so that a 10-fold elevation reduces VSCN by approximately 31 mV. BaCl2 greatly reduces this response: a 10-fold elevation in [K+]e yielding only a 4-mV reduction in VSCN. It is concluded that the membrane potential of Ehrlich cells can be estimated accurately from SCN- distribution measurements.