Validation of the use of the lipophilic thiocyanate anion for the determination of membrane potential in Ehrlich ascites tumor cells

Abstract

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.

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

“…Because of the cell shrinkage induced by A23187, this hyperpolarization is slightly underestimated, although the effect of the volume changes is probably small. Membrane hyperpolarization in Ehrlich cells is similarly seen after addition of propranolol, which is known to activate Ca2+-dependent K + channels (Laris et al, 1976;Valdeolmillos, Garcia-Sancho & Herreros, 1986;Smith & Robinson, 1989). It was previously demonstrated that A23187 activated separate, conductive K § and C1 pathways and we find in the present report that the increase in GK and Gcl are 13 and 50 times, respectively, after addition of A23187 (see Table 3).…”

“…8). A similar depolarization is observed in Ehrlich cells when the potassium channels are inhibited by addition of Ba 2+ (Gstrein et al, 1987;Smith & Robinson, 1989).…”

“…Gstrein, Paulmichl and Lang (1987), on the other hand, find a much larger membrane potential at -57 mV, using the same technique. Indirect techniques for estimation of the cell membrane potential include the use of (i) lipid-soluble cations (dibenzyldimethylammonium), the distribution of which indicates a membrane potential of -17 to -23 mV (Cespedes & Christensen, 1974), (ii) lipophilic anion (SCN-), the distribution of which indicates a membrane potential of -19.6 mV (Smith & Robinson, 1989) and (iii) fluorescent cyanine dyes. The membrane potential of Ehrlich cells has been assessed with two fluorescent probes 3,3'-dipropylthiodicarbocyanine (DISC3-(5)) and 1,1'-dipropyloxadicarbocyanine (DiOC3-(5)), and the values obtained using these dyes were all in the range -35…”

ATP‐driven, Na+‐independent inward Cl– pumping in neuroblastoma cells

“…The membrane potentiel ( V m ) was estimated by measuring the extra‐ over intracellular distribution ratios of [ 14 C]SCN – (Catterall et al . 1976; Smith and Robinson 1989; Bettendorff and Wins 1994).…”