RESUMEN
The influx of L-threonine through system ASC does not influence the membrane potential in cultured human fibroblasts although comparable fluxes of amino acids through another Na(+)-dependent agency, system A, effectively depolarize the cells. The membrane potential, however, stimulates the influx of amino acids through system ASC with a maximal effect at -50 mV. The sensitivity of amino acid influx through system ASC to the membrane potential is not constant, but rather, is dependent on intracellular and extracellular concentrations of the substrates, Na+ and amino acids, of the system. Conditions which favor the loading of the ASC carrier at the external surface reduce the sensitivity of ASC-mediated amino acid influx to the membrane potential; in contrast, the sensitivity of this amino acid influx increases under conditions which favor loading of the carrier at the internal surface. Trans-stimulation, a well-known characteristic of system ASC, also varies with the concentrations of the substrates of the system and, in fact, this characteristic is not observed when external Na+ is low. These data may be accommodated by a model in which an electrically silent mode of operation of the transporter is dominant. The influence of the membrane potential on the transport system is dependent on the extent to which a charge-translocating step in the cycling of the carrier is rate limiting (relative rate limitance).
Asunto(s)
Aminoácidos/metabolismo , Potenciales de la Membrana , Sodio/metabolismo , Adolescente , Transporte Biológico , Células Cultivadas , Electricidad , Polarización de Fluorescencia , Humanos , Cinética , Masculino , Treonina/metabolismoRESUMEN
The transport of L-threonine was studied in cultured human fibroblasts. A kinetic analysis of L-threonine transport in a range of extracellular concentrations from 0.01 to 20 mM indicated that this amino acid enters cells through both Na(+)-independent and Na(+)-dependent routes. These routes are: (1) a non-saturable, Na(+)-independent route formally indistinguishable from diffusion; (2) a saturable, Na(+)-independent route inhibitable by the analog BCH and identifiable with system L; (3) a low-affinity, Na(+)-dependent component (Km = 3 mM) which can be attributed to the activity of system A since it is adaptively enhanced by amino acid starvation and suppressed by the characterizing analog MeAIB and (4) a high-affinity, Na(+)-dependent route (Km = 0.05 mM). This latter route is identifiable with system ASC since it is insensitive to adaptive regulation, uninhibited by MeAIB, trans-stimulated by intracellular substrates of system ASC, markedly stereoselective, and relatively insensitive to changes in external pH. At an external concentration of 0.05 mM more than 90% of L-threonine transport is referrable to the activity of system ASC; in these conditions, the transport of the amino acid exhibits typical ASC-features even in the absence of inhibitors of other transport agencies, and, therefore, it can be employed as a reliable indicator of the activity of transport system ASC in cultured human fibroblasts.
Asunto(s)
Piel/metabolismo , Sodio/farmacología , Treonina/metabolismo , Aminoácidos/farmacología , Arginina/farmacología , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Glutamatos/farmacología , Ácido Glutámico , Humanos , Cinética , Masculino , Modelos BiológicosRESUMEN
The net influx of L-arginine (JARG) was employed as an indicator of the membrane potential in human fibroblasts. Cell depolarization, obtained by increasing [K+]out, decreased both JARG and the net influx of the lipid soluble cation tetraphenylphosphonium (JTPP), a probe of membrane potential. JTPP, but not JARG, was influenced by the mitochondrial potential and exhibited a component dependent on intracellular and/or extracellular binding. JARG was sensitive to changes in the membrane potential induced by Na+-dependent transport of L-proline or by the activity of Na+-K+-ATPase. In the presence of 50 microM valinomycin, JARG was markedly influenced by the distribution ratio of K+ in a range of [K+]out from 1.5 to 100 mM. In this range of [K+]out, membrane potential (Em) varied from -90 to -23 mV, and calibration of JARG vs. the membrane potential yielded a linear relationship. These results indicate the following: 1) that the net influx of TPP+ is not a reliable indicator of membrane potential in cultured human fibroblasts; 2) that in the same cells the net influx of L-arginine can be employed as an index of membrane potential; 3) that in a range of Em from -23 to -90 mV the activity of system y+ (the membrane agency devoted to L-arginine transport in cultured human fibroblasts) exhibits no saturation of potential-dependent activation of transport.
Asunto(s)
Arginina/metabolismo , Fibroblastos/fisiología , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Humanos , Cinética , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/fisiología , Compuestos Onio/metabolismo , Compuestos Organofosforados/metabolismo , Potasio/metabolismo , Prolina/metabolismo , Sodio/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Valinomicina/farmacologíaRESUMEN
The cell-to-medium distribution ratios at steady state of L-arginine (RArg) and of the lipid-soluble cation tetraphenylphosphonium (RTPP) were studied as a function of the membrane potential (Em) in adult human fibroblasts. The relationship between RArg and Em was qualitatively similar to that of RTPP and Em. Quantitatively, RArg and RTPP differed in that 1) RTPP was much greater than RArg when Em was near zero, indicating a significant binding component in the uptake of TPP+ but not of L-arginine, and 2) after a correction for binding when Em is near zero, RTPP was still greater than RArg so that RT/F . ln RTPP exceeded RT/F . ln RArg by 10-25 mV. The pattern of the redistribution of accumulated TPP+ and arginine after an alteration of Em was identical. In null-point experiments, the external [K+] for which there were no changes in cellular TPP+ or L-arginine in the presence of high valinomycin (the null points) were very similar for the two probes. Em calculated from the null-point measurements (-70(-)-80 mV) was also very similar to RT/F . ln RArg and thus smaller than RT/F.ln RTPP. It was concluded that 1) there was an additional TPP+ binding as cellular [TPP] rose in response to more negative membrane potentials, 2) the transport system for L-arginine in these cells (system y+) operates as a facilitated diffusion system driven by the membrane potential, and 3) in some circumstances, L-arginine could be employed as a probe of Em.
Asunto(s)
Arginina/metabolismo , Piel/metabolismo , Calibración , Células Cultivadas , Fibroblastos/metabolismo , Homeostasis , Humanos , Potenciales de la Membrana , Métodos , Compuestos Onio/metabolismo , Compuestos Organofosforados/metabolismo , Piel/citología , Factores de Tiempo , Valinomicina/farmacologíaAsunto(s)
Carcinoma de Ehrlich/fisiopatología , Membrana Eritrocítica/fisiología , Colorantes Fluorescentes , Potenciales de la Membrana , Microscopía/métodos , Aminoácidos/fisiología , Animales , Transporte Biológico Activo , Línea Celular , Conductividad Eléctrica , Humanos , Canales Iónicos/fisiología , Matemática , Ratones , Microscopía/instrumentación , Óptica y Fotónica/instrumentación , Potasio/fisiología , Sodio/fisiologíaRESUMEN
The distribution ratio of the lipophilic cation tetraphenylphosphonium (TPP+) has been used to estimate the electrical potential difference across the plasma membrane in cultured human fibroblasts. These cells exhibit a membrane potential markedly influenced by the diffusion potential of K+. High extracellular potassium concentrations depolarize human fibroblasts and depress the activity of transport systems A, ASC (both serving for zwitterionic amino acids), X-AG (for anionic amino acids), and y+ (for cationic amino acids). High doses (100 microM) of the K+-ionophore valinomycin hyperpolarize the cells. This condition enhances the activity of systems A, ASC and y+. Transport systems L (for neutral amino acids) and x-C (for anionic amino acids) are insensitive to changes in extracellular K+ or to valinomycin. System X-AG is inhibited by the addition of 100 microM valinomycin, but the effect of the ionophore appears to be potential-independent. These results indicate that: (a) the activity of systems L and x-C is potential-independent and (b) the activity of systems A, ASC, X-AG and y+ is sensitive to alterations of external [K+] associated to changes in membrane potential.
Asunto(s)
Aminoácidos/metabolismo , Potasio/farmacología , Ácidos Aminoisobutíricos/metabolismo , Ácido Aspártico/metabolismo , Transporte Biológico Activo , Agua Corporal , Femenino , Fibroblastos/metabolismo , Humanos , Potenciales de la Membrana/efectos de los fármacos , Compuestos Onio/metabolismo , Compuestos Organofosforados/metabolismo , Embarazo , Valinomicina/farmacologíaRESUMEN
Ehrlich ascites tumor cells lose KCl and shrink after swelling in hypotonic media and in response to the addition of 2-deoxyglucose, propranolol, or the Ca2+ ionophore, A23187, plus Ca2+ in isotonic media. All of these treatments activate cell shrinkage via a pathway with the following characteristics: (1) the KCl loss responsible for cell shrinkage does not alter the membrane potential; (2) NO3(-) does not substitute for Cl-; (3) the net KCl movements are not inhibited by quinine or DIDS; and (4) early in this study furosemide was effective in inhibiting cell shrinkage but this sensitivity was subsequently lost. This evidence suggests that the KCl loss in these cells occurs via a cotransport mechanism. In addition, hypotonic media and the other agents used here stimulate a Cl(-) - Cl(-) exchange, a net loss of K+ and a net gain of Na+ which are not responsible for cell shrinkage. The Ehrlich cell also appears to have a Ca2+-activated, quinine-sensitive K+ conductive pathway but this pathway is not part of the mechanism by which these cells regulate their volume following swelling or shrink in isotonic media in response to 2-deoxyglucose or propranolol. Shrinkage by the loss of K+ through the Ca2+ stimulated pathway appears to be limited by Cl- conductive movements; for when NO3(-), an anion demonstrated here to have a higher conductive movement than Cl-, is substituted for Cl-, the cells will shrink when the Ca2+-stimulated K+ pathway is activated.
Asunto(s)
Carcinoma de Ehrlich/fisiopatología , Desoxiazúcares/farmacología , Desoxiglucosa/farmacología , Cloruro de Potasio/metabolismo , Propranolol/farmacología , Animales , Calcimicina/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Medios de Cultivo , Líquido Intracelular/fisiología , Cinética , Potenciales de la Membrana/efectos de los fármacos , Ratones , Concentración Osmolar , Quinina/farmacologíaRESUMEN
The studies reported in this chapter indicate that the Na/K pump's contribution to Em of human red blood cells with normal cellular Na is no more than 0.1 to 0.2 mV. Estimates based on this magnitude of the electrogenic component and the pump current (taken as one-third the ouabain-sensitive Na efflux) indicate that Rm in these cells is about 1 to 2 X 10(5) ohm cm2.
Asunto(s)
Membrana Celular/fisiología , Eritrocitos/fisiología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/análogos & derivados , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Conductividad Eléctrica , Electrofisiología , Humanos , Canales Iónicos/fisiología , Potenciales de la Membrana , Ouabaína/farmacologíaRESUMEN
A unique cytoplast preparation from Ehrlich ascites tumor cells (G. V. Henius, P. C. Laris, and J. D. Woodburn (1979) Exp. Cell. Res. 121, 337-345), highly enriched in plasma membranes, was employed to characterize the high-affinity plasma membrane calcium-extrusion pump and its associated adenosine triphosphatase (ATPase). An ATP-dependent calcium-transport system which had a high affinity for free calcium (K0.5 = 0.040 +/- 0.005 microM) was identified. Two different calcium-stimulated ATPase activities were detected. One had a low (K0.5 = 136 +/- 10 microM) and the other a high (K0.5 = 0.103 +/- 0.077 microM) affinity for free calcium. The high-affinity enzyme appeared to represent the ubiquitous high-affinity plasma membrane (Ca2+ + Mg2+)-ATPase (calcium-stimulated, magnesium-dependent ATPase) seen in normal cells. Both calcium transport and the (Ca2+ + Mg2+)-ATPase were significantly stimulated by the calcium-dependent regulatory protein calmodulin, especially when endogenous activator was removed by treatment with the calcium chelator ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid. Other similarities between calcium transport and the (Ca2+ + Mg2+)-ATPase included an insensitivity to ouabain (0.5 mM), lack of activation by potassium (20 mM), and a requirement for magnesium. These similar properties suggested that the (Ca2+ + Mg2+)-ATPase represents the enzymatic basis of the high-affinity calcium pump. The calcium pump/enzyme system was inhibited by orthovanadate at comparatively high concentrations (calcium transport: K0.5 congruent to 100 microM; (Ca2+ + Mg2+)-ATPase: K0.5 greater than 100 microM). Upon Hill analysis, the tumor cell (Ca2+ + Mg2+)-ATPase failed to exhibit cooperative activation by calcium which is characteristic of the analogous enzyme in the plasma membrane of normal cells.
Asunto(s)
ATPasas Transportadoras de Calcio/metabolismo , Calcio/metabolismo , Carcinoma de Ehrlich/enzimología , Animales , Transporte Biológico Activo/efectos de los fármacos , ATPasa de Ca(2+) y Mg(2+) , Calmodulina/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Quelantes/farmacología , Cinética , Ratones , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Trifluoperazina/farmacologíaRESUMEN
Under controlled conditions, fluorescent cyanine dyes can be used to measure membrane potentials of cell suspensions. Similar changes in membrane potential can be followed both with fluorescent dyes and electrophysiological probes in response to changes in the ion composition of the medium. Recent reports that attempt to abrogate the use of the cyanine dyes in measurements of the membrane potential are misleading.
Asunto(s)
Carbocianinas , Colorantes Fluorescentes , Potenciales de la Membrana , Quinolinas , Adenosina Trifosfato/metabolismo , Animales , Células Cultivadas , Conductividad Eléctrica , Electrofisiología , Humanos , Potasio/metabolismo , Valinomicina/farmacologíaRESUMEN
Incubating Ehrlich ascites tumor cells with 10 mM glucose at room temperature resulted in the following changes. The cells shrank, reaching a minimum volume after 1 h. The decrease in cell volume was 50-90% inhibited by 1 mM furosemide. The mmol K+ and Cl-/mg dry wt decreased, and mmol Na+/mg dry wt increased over the 1 h incubation. The net loss of KCl was inhibited by 1 mM furosemide. Immediately after the addition of glucose, the influx of 86Rb sensitive to ouabain decreased, whereas the influx sensitive to furosemide increased. The total influx of 86Rb with glucose was similar to that of controls. The effluxes of 86Rb and 36Cl increased immediately after the addition of glucose. These effluxes did not increase, however, in the presence of 1 mM furosemide. Initially the ouabain-sensitive Na+ efflux was not changed with glucose, but the ouabain-insensitive Na+ efflux decreased. Furosemide (1 mM) did not influence Na+ efflux. With time the ouabain-sensitive Na+ efflux increased as cellular Na+ levels rose so that at 1 h the ouabain-sensitive Na+ efflux from glucose-treated cells was 2.5-3 times that of control cells. The potential difference across the membrane gradually became more negative by approximately 25 mV, reaching a maximum after 1 h. The hyperpolarization was reversed by 1 mM ouabain. The changes in ionic fluxes on the addition of glucose are compared with changes in ionic fluxes seen during volume regulation.
Asunto(s)
Carcinoma de Ehrlich/patología , Glucosa/farmacología , Iones , Animales , Transporte Biológico/efectos de los fármacos , Carcinoma de Ehrlich/metabolismo , Carcinoma de Ehrlich/fisiopatología , Cloro/metabolismo , Fluorescencia , Furosemida/farmacología , Potenciales de la Membrana/efectos de los fármacos , Radioisótopos , Rubidio/metabolismo , Radioisótopos de Sodio/metabolismoAsunto(s)
Membrana Celular/fisiología , Membranas Intracelulares/fisiología , Potenciales de la Membrana , Animales , Fenómenos Fisiológicos Bacterianos , Plaquetas/fisiología , Carcinoma de Ehrlich , Línea Celular , Chlorophyta/fisiología , Membrana Eritrocítica/fisiología , Eucariontes/fisiología , Leucocitos/fisiología , Macrófagos/fisiología , Mastocitos/fisiología , Organoides/fisiología , Potenciometría/métodosRESUMEN
Cells depleted of amino acids show lower rates of glycine or aminoisobutyric acid uptake than do freshly isolated cells. In the amino acid-depleted cells, addition of valinomycin stimulates amino acid influx at least to the level observed in freshly isolated cells. In cells containing high levels of cellular amino acids, valinomycin has little effect on influx of amino acids. It is concluded that the transport of amino acids in freshly isolated cells is elevated compared to depleted cells because the cells are hyperpolarized by the continuous loss of cellular amino acids during the transport assay. During this hyperpolarization by amino acid loss, transport of amino acids is not further stimulated by valinomycin at low external [K+] (10 mM +/- 5 mM). With the exception of preloading with glycine, cells preloaded with a single amino acid to a concentration greater than 20 mM show reduced rates of glycine and aminoisobutyric acid influx at early times (less than 15 min) compared to amino acid-depleted cells. The reduction of influx is transient and by 30 min, influx is greater in preloaded than in amino acid-depleted cells. Knowing that increases and decreases in the membrane potential are achieved by using varying external [K+] in the presence of valinomycin and propranolol, and using amino acid-depleted cells, it can be shown that an increased membrane potential increases the V for glycine and aminoisobutyric acid influx. A decrease in the potential difference results in a decreased V. Changes in Km also occur when the membrane potential is varied.
Asunto(s)
Aminoácidos/metabolismo , Transporte Biológico/efectos de los fármacos , Carcinoma de Ehrlich/metabolismo , Sodio/farmacología , Ácidos Aminoisobutíricos/metabolismo , Animales , Glicina/metabolismo , Cinética , Potenciales de la Membrana/efectos de los fármacos , Ratones , Potasio/farmacología , Valinomicina/farmacologíaAsunto(s)
Carcinoma de Ehrlich/metabolismo , Sodio/metabolismo , Ácidos Aminoisobutíricos/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico Activo/efectos de los fármacos , Membrana Celular/metabolismo , Cinética , Ratones , Potasio/farmacología , Espectrometría de FluorescenciaAsunto(s)
Transporte Biológico Activo , Fraccionamiento Celular/métodos , Citoplasma/metabolismo , Adenosina Trifosfato/metabolismo , Ácidos Aminoisobutíricos/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Citocalasina B/farmacología , Glucosa/metabolismo , Potasio/metabolismo , Sodio/metabolismoRESUMEN
The membrane potential of the Ehrlich ascites tumor cell was shown to be influenced by its amino acid content and the activity of the Na+ :K+ pump. The membrane potential (monitored by the fluorescent dye, 3,3'-dipropylthiodicarbocyanine iodide) varied with the size of the endogenous amino acid pool and with the concentration of accumulated 2-aminoisobutyrate. When cellular amino acid content was high, the cells were hyperpolarized; as the pool declined in size, the cells were depolarized. The hyperpolarization seen with cellular amino acid required cellular Na+ but not cellular ATP. Na+ efflux was more rapid from cells containing 2-aminoisobutyrate than from cells low in internal amino acids. These observations indicate that the hyperpolarization recorded in cells with high cellular amino acid content resulted from the electrogenic co-efflux of Na+ and amino acids. Cellular ATP levels were found to decline rapidly in the presence of the dye and hence the influence of the pump was seen only if glucose was added to the cells. When the cells contained normal Na+ (approx. 30mM), the Na+ :K+ pump was shown to have little effect on the membrane potential (the addition of ouabain had little effect on the potential). When cellular Na+ was raised to 60mM, the activity of the pump changed the membrane potential from the range -25 to -30 mV to -44 to -63 mV. This hyperpolarization required external K+ and was inhibited by ouabain.
Asunto(s)
Aminoácidos/metabolismo , Carcinoma de Ehrlich/fisiopatología , Membrana Celular/fisiología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Ácidos Aminoisobutíricos/metabolismo , Animales , Transporte Biológico , Carcinoma de Ehrlich/metabolismo , Cinética , Potenciales de la Membrana/efectos de los fármacos , Ratones , Potasio/farmacología , Sodio/farmacología , Espectrometría de FluorescenciaRESUMEN
(1) (DL)-Propranolol and Ca2+ are shown to alter the transmembrane potential difference of Ehrlich ascites tumor cells as measured by means of the cyanine dye, 3,3'-dipropyl-2,2'-thiodicarbocyanine iodide, whose fluorescent intensity changes as a function of membrane potential. (2) The changes in membrane potential elicited by these agents are dependent of the external K+ concentration in a manner which suggest that the potential changes result from a specific increase in the permeability of the plasma membrane to K+. (3) Na+-dependent amino acid transport in the presence of propranolol can be modulated by varying the external K+ concentration (K+o). The initial rate of uptake is stimulated by propranolol at low K+o and inhibited at high K+o. The change in transport rate is nearly directly proportional to the natural logarithm of [K+]o in the presence of propranolol. (4) ATP depletion of the cells by preincubation with rotenone abolishes the changes in fluorescence and amino acid uptake seen with propranolol as a function of K+o. Restoration of cellular ATP with glucose in presence of Ca2+ restores both fluorescence and amino acid transport changes which occur in response to propranolol. (5) The fluorescence changes and amino acid transport changes in response to propranolol are pH dependent, with little effect seen at pH6. (6) It is concluded that the rate of Na+-dependent amino acid uptake is a function of membrane potential and is dependent on the electrochemical potential difference for Na+.
Asunto(s)
Aminoácidos/metabolismo , Calcio/farmacología , Carcinoma de Ehrlich , Propranolol/farmacología , Adenosina Trifosfato/metabolismo , Animales , Transporte Biológico Activo/efectos de los fármacos , Carbocianinas , Carcinoma de Ehrlich/fisiopatología , Concentración de Iones de Hidrógeno , Potenciales de la Membrana/efectos de los fármacos , Ouabaína/farmacología , Potasio/farmacología , Sodio/farmacología , Valinomicina/farmacologíaRESUMEN
A transient decrease in the fluorescent intensity of the dye, 3,3t'-dipropylthiodicarbocyanine iodide was seen upon the addition of ATP to rat liver mitochondria which had been pre-treated with 2-10(-6) M rotenone and 3.3 mug oligomycin/ml. This decrease which is indicative of a hyperpolarization (internal more negative) was half maximal at 2-10(-5) M ATP was not seen with either ADP or AMP. Atractyloside, bongkrekic acid, ADP, and to a lesser extent AMP inhibited the decrease observed with ATP. The characteristics of inhibition by these compounds were similar to those observed in experiments where either transport or binding of adenine nucleotides was measured. The addition of ADP after ATP led to a transient increase in fluorescent intensity indicative of depolarization. This increase was also blocked by atractyloside and bongkrekic acid. The evidence presented supports the hypothesis that the exchange of ADP and ATP via an adenine nucleotide exchange mechanism is electrogenic.