Energy sources for amino acid transport in animal cells

Abstract

The existence of an electrogenic Naf pump in Ehrlich cells which substantially contributes t o the membrane potential, previously derived from the distribution of the lipid soluble cation tetraphenylphosphonium (TPP+), could be confirmed by an independent method based on the quenching of fluorescence of a cyanine dye derivative, after the mitochondrial respiration had been suppressed by appropriate inhibitors. The mitochondria1 membrane potential, by adding t o the overall potential as measured in this way is likely t o cause an overestimation of the membrane potential difference (p.d.). But since this error tends t o diminish with increasing pump activity, the true p.d. of the plasma membrane should easily account for the driving force to drive the active accumulation of amino acids in the absence of a n adequate Na+ concentration gradient. Accordingly, the FZ-aminoisobutyric acid (AIB) uptake rises linearly with the distribution of TPP' at constant Na' concentrations, suggesting that each responds directly t o membrane potential. There is evidence that the electrogenic (free) movement of CI-is slow, at least at normal p.d., whereas a major part of the CI-movement across the cellular membrane appears t o occur by an electrically silent CI--base exchange mechanism. By such a mode CI-, together with an almost stoichiometric amount of K+, may under certain conditions move into the cell against a high adverse electrical potential difference. This "paradoxical" movement of KfC1-contributing t o the deviation of the C1-distribution from the electrochemical equilibrium distribution, is not completely understood. It is insensitive towards ouabain but can almost specifically be inhibited by furosemide.As a likely explanation a H+-K+ exchange p u m p was previously offered, even though unequivocal evidence of such a pump is so far lacking. According t o available evidence the electrogenic movement of free CI-is t o o small, at least at normal orientation of the p.d., to significantly shunt the electrogenic pump potential so that the establishment of such a potential is plausible. T h e evidence presented is considered strong in favor of the gradient hypothesis since even in the absence of an adequate Na+ concentration gradient, the electrogenic Na+ pump will contribute sufficient extra driving force t o actively transport amino acid into the cells.Key words: amino acid transport, gradient hypothesis, electrogenic cation pump, electrolyte movements, ouabain, furosemideThe active uptake of neutral amino acids b y Ehrlich cells, as in other animal cells and tissues, is according t o many investigators secondary active transport, i.e., driven b y the electrochemical potential gradient of Nat via cotransport (gradient hypothesis),

Amino acid transport and cell volume regulation in Ehrlich ascites tumour cells.

“…If however we use the membrane potential estimated from the chloride distribution, the sodium gradients are insufficient. It has been shown previously that the distribution of Cl-, under many circumstances, is unsuitable for an estimation of the membrane potential (Heinz, Geck, Pietrzyk, Burckhardt & Pfeiffer, 1977). In Ehrlich cells this is mainly because of the anion-cation co-transport system which can lead to an accumulation of chloride in the cells (Geck, Pietrzyk, Burckhardt, Pfeiffer & Heinz, 1980;Hoffmann, Sj0holm & Simonsen, 1981).…”

Membrane potential, chloride exchange, and chloride conductance in Ehrlich mouse ascites tumour cells.

“…The values obtained in the present study (-27 + 1 0 mV (n = 74)) correspond to the previously reported values about -24 mV (Lassen et al 1971). Somewhat less negative, stable values (-12 to -14 mV) have been obtained by Smith and coworkers, employing a different technique (Smith & Levinson, 1975;Smith & Adams, 1977), whereas considerably more negative values have been reported based on indirect estimates (Heinz et al 1975(Heinz et al , 1977Burckhardt, 1977).…”

“…(12)), because the measured Vm which may be regarded as the initial value after a sudden change in external K+ concentration, was found to be reasonably constant with time and because the Nernst potentials for C1-(Ec1) and Na+ (ENa) are essentially constant in this region (see Christoffersen, 1973;Lassen, 1977;Lassen, Pape & Vestergaard-Bogind, 1978 Lassen, 1977;Lassen & Rasmussen, 1978). Although there is increasing evidence for the existence of an electrogenic Na+-K+ pump in Ehrlich ascites cells (Heinz et al 1975(Heinz et al , 1977 concentrations of K+, Na+, and Cl-, assuming equal activity coefficients in cell water and in the extracellular medium, and neglecting the available evidence ofintracellular compartmentation of Na+ and Cl- (Smith & Adams, 1977), or nuclear sequestration of these ions (Pietrzyk & Heinz, 1974 (Mills & Tupper, 1975;Tupper, 1975). This component would appear to be small, however, in cells with high intracellular K+ concentration, and suspended in ordinary Ringer solutions (see Fig.…”

“…As discussed by Heinz et al (1977) some error apparently is inherent in all the methods used for direct or indirect estimates of the membrane potential in these cells. In this study we have used the values directly recorded by micro-electrode puncture of the cells as a measure of the membrane potential.…”

The Determination of the Membrane Potential of Chlorella vulgaris. Evidence for Electrogenic Sugar Transport

“…Skulachev and coworkers have introduced special lipophilic ions for this purpose. The positively charged monovalent ion tetraphenylphosphonium, used previously for Ehrlich cells [21], was chosen for the work described here, since it was the only ion with a sufficiently high penetration rate into Chlorella cells.…”