Use of a lipophilic cation for determination of membrane potential in neuroblastoma-glioma hybrid cell suspensions

Abstract

Neuroblastoma-glioma hybrid cells (NG108-15) in suspension accumulate the permeant lipophilic cation [3Hltetraphenylphosphonium (TPP+) against a concentration gradient. The steady-state level of TPP+ accumulation is about twice as great in physiological media of low K+ concentration (i.e., 5 mM K+/135 mM Na+) than in a medium of high K+ concentration (i.e., 121 mM K+/13.5 mM Na+ Many of the basic neurophysiological properties that underlie nervous system function have been described and analyzed in neuroblastoma-glioma hybrid cell line NG108-15, thereby establishing this line as an appropriate model system for studies of the nervous system. These properties include: (i) generation of action potentials (1); (ii) synthesis, storage, and release of the neurotransmitter acetylcholine (2, 3); (iii) synapse formation (1, 4); and (iv) presence of specific plasma membrane receptor sites for neurotransmitters and neuromodulators, such as acetylcholine, opiates, adenosine, and prostaglandins, which regulate adenylate cyclase (5)(6)(7)(8)(9). Because many of these phenomena are clearly related to alterations in the electrical potential (AI) across the plasma membrane, correlations between changes in AI and specific biochemical functions take on fundamental importance. Traditionally, measurement of AI in nervous tissue relies on techniques that require impaling selected cells with microelectrodes. However, because this technique is limited by the size of the cells and cannot be applied to populations of cells at large, it is apparent that alternative methods would be of value.This communication describes the application of a biochemical method for determining AI in cultured neuroblastoma-glioma hybrid cells. The technique involves the use of the permeant lipophilic cation tetraphenylphosphonium (TPP+) which has been used to measure AI in mitochondria (10), Ehrlich's ascites cells (11), and bacteria and bacterial membrane vesicles.$ The use of "lipophilic ions" to measure AI across biological membranes was introduced by Skulachev and Liberman (for review, see ref. 12) and their coworkers in the early 1970s, and this class of compounds has been applied to a number of prokaryotic and eukaryotic systems (10-16, ¶). The ions are constructed in such a fashion that they are sufficiently lipophilic to enter the hydrophobic core of the membrane, and they also have the propensity for charge delocalization which allows passive equilibration with AM (17). It should be noted, however, that in no case have AA determinations with lipophilic cations been validated quantitatively by direct electrophysiological measurements. The results presented here provide a strong indication that TPP+ distribution can be used to determine AI in populations of neuroblastoma-glioma hybrid cells. METHODSCells. The mouse neuroblastoma-rat glioma hybrid clone NG108-15 was used and was grown and maintained as described (18). Harvesting was accomplished by shaking the cells off the culture flask and centrifuging at 250-500 X g for 5 min at room temperat...

Regulation of initial vinblastine influx by P-glycoprotein

“…Digitonin, but not DPM, also produced a large increase in permeability to propidium iodide as determined by measuring cellular fluorescence of this agent (data not shown). Furthermore, DPM did not alter the membrane potential in KB-3-1 cells as determined by measuring the accumulation of [3H]-TPP+ at 2 h (Lichtshtein et al, 1979). These experiments provided evidence that DPM did not permeabilise cellular membranes.…”

Energetics of Sucrose Transport into Protoplasts from Developing Soybean Cotyledons

“…The same concentration of 3-0-MG did not induce any intracellular pH change, even though 3-0-MG was accumulated by the protoplasts (14,22). PCMBS, which inhibits sucrose influx (10,22,26), abolished this transient acidification of the intracellular pH.…”

Effect of Steroid Hormones and Capacitation on Membrane Potential of Human Spermatozoa

“…All the values obtained for the accumulation of TPMP' were corrected for the entrapped suspending medium in the cell pellet using ('4C)-sorbitol added to the incubation suspension [18]. Using these values in the Nernst equation, I) = -61 log R,,,,+, a value of -69 k 2 mV was obtained.…”

“…A similar methodology was used to determine intracellular volume with 3Hz0 (0.2 pCi, 250 pCilg sp act) and ('4C)-sorbitol (0.05 pCi, 285 mCi/mmol sp act) [18]. Under these conditions the external HzO volume carried down with the cells did not exceed 5 4 % of the total volume of the sample.…”