scholarly journals The membrane potential of mouse ascites-tumour cells studied with the fluorescent probe 3,3′-dipropyloxadicarbocyanine. Amplitude of the depolarization caused by amino acids

1978 ◽  
Vol 174 (3) ◽  
pp. 801-810 ◽  
Author(s):  
R D Philo ◽  
A A Eddy
Keyword(s):  
Amino Acids ◽  
Membrane Potential ◽  
Fluorescence Intensity ◽  
Maximum Amplitude ◽  
Null Point ◽  
Tumour Cells ◽  
Fluorescence Signal ◽  
Electrogenic Na Pump ◽  
Mouse Ascites ◽  
Ascites Tumour Cells

1. The magnitude of the K+ gradient across the plasma membrane, which was in equilibrium with the membrane potential (E) of the tumour cells, was determined by the “null point” procedure of Hoffman & Laris (1974) [J. Physiol. (London) 239, 519–552] in which the fluorescence of the dye serves as an indicator of changes in the magnitude of E. 2. A mixture of oligomycin, 2,4-dinitrophenol and antimycin was used to stop the mitochondria from interfering with the fluorescence signal. Transport functions at the plasmalemma were maintained under these conditions in the presence of glucose. 3. Physiological circumstances were found in which incubation with glycine or with glucose changed the “null point” value of E within the range–20mV to–100mV. The fluorescence intensity at the “null point” was an approximately linear function of E over that range. The procedure enabled E to be inferred form the fluorescence intensity in circumstances where titration to the “null point” was not feasible. 4. The rapid depolarization caused by l-methionine or glycine was shown in this way to have a maximum amplitude of about 60mV. A mathematical model of this process was devised. 5. The electrogenic Na+ pump hyperpolarized the cells up to about −80mV when the cellular and extracellular concentrations of K+ were roughly equal. 6. The observations show that the factors generating the membrane potential represent a major source of energy available for the transport of amino acids in this system.

scholarly journals The accumulation of amino acids by mouse ascites-tumour cells. Dependence on but lack of equilibrium with the sodium-ion electrochemical gradient

1981 ◽  
Vol 194 (2) ◽  
pp. 415-426 ◽  
Author(s):  
C Hacking ◽  
A A Eddy
Keyword(s):  
Amino Acids ◽  
Steady State ◽  
Amino Acid ◽  
Rate Coefficient ◽  
Tumour Cells ◽  
Cellular Respiration ◽  
Mouse Ascites ◽  
Predicted Values ◽  
Leak Pathway ◽  
Ascites Tumour Cells

1. The fluorescent dye 3,3′-dipropyloxadicarbocyanine was used to show that the tumour cells absorbed 2-aminoisobutyrate, glycine, L-leucine and L-isoleucine and certain other amino acids electrogenically. The Km values with respect to amino acid concentration ([A]o), obtained from the fluorescence assays, varied through the above series from 0.8 to 26 mM, with Vmax. fairly constant. 2. Similar Km values described the uptake of the 14C-labelled amino acids in five instances where this was measured. 3. Each amino acid lowered the membrane potential (E) by 10-20 mV when its cellular concentration ([A]i) had reached a steady value and [A]o was 10mM. In these experiments energy metabolism was maintained by glycolysis, 2,4-dinitrophenol was present and cellular respiration was inhibited. The corresponding net flow of amino acid through the Na+ symport was deduced by making use of the fact that the depolarization an amino acid initially caused was roughly proportional to the net influx of amino acid itself. 4. The steady-state depolarization was attributed to the presence of a leak pathway for the amino acid with a rate coefficient PA. As assayed in the absence of Na+, PA was about 5-fold larger for isoleucine than for glycine. 5. Direct estimates of Vmax./PA were similar to those inferred from the extent of depolarization in the steady state and [A]i. 6. A mathematical model was used to predict [A]i/[A]o in term of the measured values of [Na]o, [Na]i, E, Km and Vmax./PA. The predicted and observed values agreed fairly well when [A]o was 1 mM or 10 mM. 7. [A]i/[A]o varied from about 2.5 for 10 mM-isoleucine to 30 for 1 mM-2-aminoisobutyrate when delta microNa, expressed as a ratio, was ostensibly in the range 19-43. 8. The concentration of 2-aminoisobutyrate from a 0.1 mM solution in the presence or absence of ouabain was consistent with the model, whereas the concentration of isoleucine from a 0.1 mM solution exceeded the predicted values 2-5-fold. 9. The tumour cells concentrated 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid by a non-electrogenic mechanism, with which isoleucine may also interact.

scholarly journals Equilibrium and steady-state models of the coupling between the amino acid gradient and the sodium electrochemical gradient in mouse ascites- tumour cells

1978 ◽  
Vol 174 (3) ◽  
pp. 811-817 ◽  
Author(s):  
R D Philo ◽  
A A Eddy
Keyword(s):  
Mathematical Model ◽  
Steady State ◽  
Membrane Potential ◽  
Amino Acid ◽  
Tumour Cells ◽  
Electrochemical Gradient ◽  
Mouse Ascites ◽  
State Models ◽  
Leak Pathway ◽  
Ascites Tumour Cells

1. The tumour cells were incubated at 37 degrees C in Ringer solutions containing glucose, 1 mM-methionine, various concentrations of Na+ and K+ and, in some instances, ouabain or valinomycin to lower the membrane potential generated by the Na+ pump. After about 30 min, when the system had reached a steady state, the ratio [extracellular Na+]/[cellular Na+] varied from about 0.6 to 3.2 with the ionic conditions. The membrane potential, determined by means of the fluorescent probe 3,3′-dipropyloxadicarbocyanine, also varied systemically from about zero to–55mV.2. the ratio [cellular methionine]/[extra-cellular methionine] varied from about 1 to 35 in these circumstances. The Na+ electrochemical gradient, measured in the same units, varied from about 1 to 30. Its magnitude in 46 assays was almost directly proportional to, though slightly smaller than, the methionine gradient. 3. A mathematical model was used to define the relation between these two gradients, which were not in equilibrium, owing to the presence of a leak pathway for the amino acid. On the assumption that the values of [cellular Na+] were correct, the methionine gradient was about 1.8 times larger than this version of the gradient hypothesis predicted.

scholarly journals Ionophore-mediated coupling between ion fluxes and amino acid absorption in mouse ascites-tumour cells. Restoration of the physiological gradients of methionine by valinomycin in the absence of adenosine triphosphate

1974 ◽  
Vol 140 (3) ◽  
pp. 383-393 ◽  
Author(s):  
M. Reid ◽  
L. E. Gibb ◽  
A. A. Eddy
Keyword(s):  
Amino Acid ◽  
Sodium Pump ◽  
Tumour Cells ◽  
Ascites Tumour ◽  
Maximum Value ◽  
Methionine Concentration ◽  
Mouse Ascites ◽  
Na Ions ◽  
Physiological Gradients ◽  
Ascites Tumour Cells

1. Preparations of mouse ascites-tumour cells depleted of ATP and Na+ ions accumulated l-methionine, in the presence of cyanide and deoxyglucose, from a 1mm solution containing 80mequiv. of Na+/l and about 5mequiv. of K+/l. Valinomycin increased, from about 4 to 16, the maximum value of the ratio of the cellular to extracellular concentrations of methionine formed under these conditions without markedly affecting the distributions of Na+ and of K+. Similar observations were made with 2-aminoisobutyrate, glycine and l-leucine. Increasing the extracellular concentration of K+ progressively decreased the accumulation of methionine in the presence of valinomycin. Over the physiological range of ionic gradients, the system behaved as though the absorption of methionine with Na+ was closely coupled to the electrogenic efflux of K+ through the ionophore. The process was insensitive to ouabain and so the sodium pump was probably not involved. 2. The amount of methionine accumulated during energy metabolism was similar to the optimal accumulation in the presence of valinomycin when ATP was lacking. It was also similarly affected by increasing the methionine concentration. 3. A mixture of nigericin and tetrachlorosalicylanilide mimicked the action of valinomycin. The anilide derivative inhibited the absorption of 2-aminoisobutyrate in the presence of valinomycin, but not in its absence. 4. Gramicidin inhibited methionine absorption and caused the preparations to absorb Na+ and lose K+. 5. The observations appear to verify the principle underlying the gradient hypothesis by showing that the tumour cells can efficiently couple the electrochemical gradient of Na+ to the amino acid gradient.

scholarly journals Metabolism and sodium transfer of mouse ascites tumour cells

1958 ◽  
Vol 140 (1) ◽  
pp. 80-93 ◽  
Author(s):  
M. Maizels ◽  
Mary Remington ◽  
R. Truscoe
Keyword(s):  
Tumour Cells ◽  
Ascites Tumour ◽  
Sodium Transfer ◽  
Mouse Ascites ◽  
Ascites Tumour Cells
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