Electrogenic Ion Pump as an Energy Source for Active Amino Acid Transport in Ehrlich Cells

Heinz, Erich; Geck, Peter; Pietrzyk, C.; Pfeiffer, B.

doi:10.1007/978-3-642-66564-6_16

Cited by 7 publications

(9 citation statements)

References 6 publications

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“…(Heinz et al, 1977;Smith & Robinson, 1981b;Valdeolmillos et al, 1986), a finding consistent with the view that V,+, is not a K + diffusion potential. Our evaluation of the effects of variations in [K+I+ on the [14C]SCN distribution in control cells and in the presence of propranolol or Ba 2+ (Fig.…”

Section: Discussionsupporting

confidence: 83%

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

Smith

Robinson

1989

J. Membrain Biol.

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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.

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Section: Discussionsupporting

confidence: 83%

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

Smith

Robinson

1989

J. Membrain Biol.

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“…cal increases in the transmembrane potential could be taken into account (21,22). We have observed, however, conditions under which steeply uphill, Na+-dependent uptake of 2-aminoisobutyric acid (AIB) or its N-methyl derivative (MeAIB) continues with little if any associated uptake of Na' (13).…”

Section: Jss:207mentioning

confidence: 94%

Amino acid transport systems in animal cells: Interrelations and energization

Christensen

1977

J Supramol Struct

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After summarizing the discrimination of the several transport systems for neutral amino acids in the cell of the higher animal, I discuss here the ways in which 2 dissimilar transport systems interact, so that one tends t o run torward for net entry and the other backwards for net exodus. An evaluation of the proposals for energization shows that uphill transport continues when neither alkali-ion gradients nor ATP levels are favorable. Evidence is presented that under these conditions a major contribution is made by another mode of energization, which may depend on the fueling of an oxidoreductase in the plasma membrane. This fueling may involve the export by the mitochondrion of the reducing equivalents of NADH by one of the known shuttles, e.g., the malate-aspartate shuttle. After depletion of the energy reserves in the Ehrlich cell by treating it with dinitrophenol plus iodoacetate concentrative uptake of test amino acids is restored by pyruvate, but in poor correlation with the restoration of alkali-ion gradients and ATP levels. This restoration by pyruvate but not by glucose is highly sensitive to rotenone. A combination of phenazine methosulfate and ascorbate will also produce transport restoration, before either the alkali-ion gradients or ATP levels have begun to rise. The restoration of transport applies to a model amino acid entering by the Na+-independent system, as well as to one entering by the principal Na+-dependent system, restoration being blocked by ouabain, despite the weak effect of ouabain on the alkali-ion gradients in the Ehrlich cell. Quinacrine terminates very quickly the uptake of model amino acids, before the alkali-ion gradients have begun to fall and before the ATP level has been halved. Quinacrine is also effective in blocking restoration of uphill transport by either pyruvate or the phenazine reagent. Preliminary results show that vesicles prepared from the plasma membrane of the Ehrlich cell quickly reduce cytochrome c or ferricyanide in the presence of NADH, and that the distribution of a test amino acid between the vesicle and its environment is influenced by NADH, quinacrine, and an uncoupling agent in ways consistent with the above proposal, assuming that a majority of the vesicles are everted.Key words: amino acid transport in animal cells, energization of transport systems, discrimination of transport systems, reverse operation of transport systems, Ehrlich cell, NADH dehydrogenase, alkali-ion gradients, phenazine methosulfate, ouabain TRANSPORT SYSTEMS AND THEIR INTERACTIONThe transport systems for neutral amino acids in the Ehrlich cell are taken t o be approximately representative of those of the cells of the higher animal in general, with more and more evidence supporting that interpretation. Most conspicuous are a broad-

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“…4). This "paradoxical" C1-uptake is associated with an almost stoichiometric movement of K+ as is usually observed with K+-depleted cells if the extracellular K+ rises above 5-10 mM (12). Since under these conditions the electrical p.d.…”

mentioning

confidence: 96%

Energy sources for amino acid transport in animal cells

et al. 1977

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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),

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Electrogenic Ion Pump as an Energy Source for Active Amino Acid Transport in Ehrlich Cells

Cited by 7 publications

References 6 publications

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

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

Amino acid transport systems in animal cells: Interrelations and energization

Energy sources for amino acid transport in animal cells

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