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1.
The uptake of nickel ions by the cyanobacterium Anabaena cylindrica was studied. Nickel transport was dependent on the membrane potential of the cells and the rate of uptake was decreased in the dark or by the addition of inhibitors, including uncouplers and electron transport inhibitors, which decreased or abolished the membrane potential of cells. The transport process obeyed hyperbolic kinetics, with a high affinity (apparent Km = 17 +/- 11 (SEM) nM) and low turnover number (maximum velocity = 22.3 +/- 5.4 (SEM) pmol h-1 mg dry wt-1 of cells or flux rate of 3.1 nmol h-1 m-2 of plasma membrane surface area). The process was also apparently specific for Ni2+, the rate being unaffected by the presence of a range of other metal ions in large excess. Equilibrium experiments showed that, over a range of nickel ion concentrations, the cells concentrated Ni2+ by a factor of 2700 +/- 240 (SEM)-fold, corresponding to a chemical diffusion potential for Ni2+ of 101 mV. It was concluded that the cells transport nickel ions by a carrier-facilitated transport process with the concentration factor for the ions being determined by the cell membrane potential according to the Nernst equation.  相似文献   

2.
The transmembrane potential of isolated chromaffin granules has been measured using the permeant ions [14C]methylamine and [35S]thiocyanate, as well as the fluorescent probe, 9-aminoacridine. At pH 7.0, the granule membrane had a Nernst proton potential of -45mV, inside negative. This potential was sensitive to the external pH, but was unaffected by K+,Na+, Ca2+, Mg2+, or other cations. The pH of zero potential was 6.25 for both methylamine and thiocyanate. Thiocyanate also had a Nernst potential of similar magnitude and sign to that of methylamine at pH 7.0, and was also sensitive to variation in external pH. Mg2+ATP was found to depolarize the granule membrane by a saturable mechanism with a K 1/2 for ATP of 40 muM. Ca2+ was only 30% as effective as Mg2+ in supporting the ATP effect. The pH optimum for this process was 6.25 and appeared to be accompanied by a marked alkalinization of the granule interior. The specificity for ATP was further tested with structural analogs of ATP and GTP. The rate of change of membrane potential in response to changes in external pH or Mg2+ATP was estimated using the fluorescent probe 9-aminoacridine. Changes came to completion in less than 1 s. This suggested that the ATP effects were not dependent on an enzymatic transformation but on an ATP-induced conformational change in the membrane. We conclude that the chromaffin granule exists in at least two proton permeability states, corresponding to the presence or absence of Mg2+ATP. These states may be related to hormone release from granules and regulation of secretion in vivo.  相似文献   

3.
Rheogenic transport in the renal proximal tubule   总被引:2,自引:2,他引:0       下载免费PDF全文
The electrophysiology of the renal Na-K ATPase was studied in isolated perfused amphibian proximal tubules during alterations in bath (serosal) potassium. Intracellular and extracellular ionic activity measurements permitted continuous evaluation of the Nernst potentials for Na+, K+, and Cl- across the basolateral membrane. The cell membrane and transepithelial potential differences and resistances were also determined. Return of K to the basal (serosal) solution after a 20-min incubation in K-free solution hyperpolarized the basolateral membrane to an electrical potential that was more negative than the Nernst potential for either Na, Cl, or K. This constitutes strong evidence that at least under stimulated conditions the Na-K ATPase located at the basolateral membrane of the renal proximal tubule mediates a rheogenic process which directly transfers net charge across the cell membrane. Interpretation of these data in terms of an electrical equivalent circuit permitted calculation of both the rheogenic current and the Na/K coupling ratio of the basolateral pump. During the period between 1 and 3 min after pump reactivation by return of bath K, the basolateral rheogenic current was directly proportional to the intracellular Na activity, and the pump stoichiometry transiently exceeded the coupling ratio of 3Na to 2K reported in other preparations.  相似文献   

4.
The synthesis of inflammation mediators produced from arachidonic acid is regulated primarily by the cellular concentration of free arachidonic acid. Since intracellular arachidonic acid is almost totally present as phospholipid esters, the concentration of intracellular arachidonic acid is primarily dependent on the balance between the release of arachidonic acid from membrane phospholipids and the uptake of arachidonic acid into membrane phospholipids. Cytosolic phospholipase A(2) is a calciumdependent enzyme that catalyzes the stimulus-coupled hydrolysis of arachidonic acid from membrane phospholipids. Following exposure of macrophages to various foreign or endogenous stimulants, cytosolic phospholipase A(2) is activated. Treatment with these compounds may also stimulate phospholipase D activity, and, in the presence of ethanol, phospholipase D catalyzes the synthesis of phosphatidylethanol. A cell-free system was used to evaluate the effect of phosphatidylethanol on cytosolic phospholipase A(2) activity. Phosphatidylethanol (0.5 microM) added to 1-stearoyl-2-[(3)H]-arachidonoyl-sn-glycero-3-phosphocholine vesicles stimulated cytosolic phospholipase A(2) activity. However, high concentrations (20-100 microM) of phosphatidylethanol inhibited cytosolic phospholipase A(2) activity. Phosphatidic acid, the normal phospholipase D product, also stimulated cytosolic phospholipase A(2) activity at 0.5 microM, but had an inhibitory effect on cytosolic phospholipase A(2) activity at concentrations of 50 and 100 microM. Ethanol (20-200 mM), the precursor of phosphatidylethanol, added directly to the assay did not alter cytosolic phospholipase A(2) activity. These results suggest that phosphatidylethanol alters the physical properties of the substrate, and at lower concentrations of anionic phospholipids the substrate is more susceptible to hydrolysis. However, at high concentrations, phosphatidylethanol either reverses the alterations in physical properties of the substrate or phosphatidylethanol may be competing as the substrate. Both interactions may result in lower cytosolic phospholipase A(2) activity.  相似文献   

5.
Chloride-dependent action potentials were elicited from embryonic skeletal muscle fibers of the chick during the last week of in ovo development. The duration of the action potentials was extremely long (greater than 8 sec). The action potentials were reversibly blocked by the stilbene derivative, SITS, a specific blocker of chloride permeability. Using patch clamp pipettes, in which the intracellular chloride concentration was controlled and with other types of ion channels blocked, the membrane potential at the peak of the action potential closely coincided with the chloride equilibrium potential calculated from the Nernst equation. These data indicate that activation of a chloride-selective conductance underlies the long duration action potential. The occurrence of the chloride-dependent action potential was found to increase during embryonic development. The percentage of fibers that displayed the action potential increased from approximately 20% at embryonic day 13 to approximately 70% at hatching. Chloride-dependent action potentials were not found in adult fibers. The voltage and time-dependent currents underlying the action potential were recorded under voltage clamp using the whole-cell version of the patch pipette technique. The reversal potential of the currents was found to shift with the chloride concentration gradient in a manner predicted by the Nernst equation, and the currents were blocked by SITS. These data indicate that chloride ions were the charge carriers. The conductance was activated by depolarization and exhibited very slow activation and deactivation kinetics.  相似文献   

6.
A critical review has been made of the literature on the use of lipophilic cations, such as triphenylmethyl phosphonium (TPMP+) as membrane potential probes in prokaryotes, uekaryote organelles in vitro, and eukaryote cells. An ideal lipophilic cation should be capable of penetrating through a biological membrane and obey the Nernst equation between a membrane bound phase and its environment. Many different forms of the Nernst equation are presented, useful in the calculation equilibrium potentials of lipophilic cations across membranes. Lipophilic cations appear to behave as valid membrane potential probes in prokaryotes and eukaryote organelles in vitro and even in vivo although some technical difficulties may be involved. On the other hand in valid forms of the Nernst equation have often been used to calculate the equilibrium potential of lipophilic cations across the plasma membranes of eukaryotic cells. In particular, the problem of intracellular compartmentation of lipophilic cations has often not been appreciated. Lipophilic cations do not appear to behave as reliable plasma membrane potential probes in eukaryotic cells. Some other avenues are discussed which might be useful in the determination of the plasma membrane potentials of small eukaryotic cells, e.g. the use of lipophilic anions as membrane potential probes.  相似文献   

7.
The resting membrane potential of isolated hepatocytes from 2- and 20-month-old rats and its changes upon activation of cells by adrenaline have been studied by the method of quantitative microfluorimetry using diethyl derivatives of polymethine probes (H-510 and D-307). The potential was estimated by the Nernst equation adapted to lipophilic cationic probes. It was shown using both probes that the transmembrane potential of hepatocytes decreases with age. The microfluorimetry data were confirmed by the results of spectrofluorimetric measurements in a cell suspension. Changes in fluorescence occurring in adrenaline-activated single cells and suspensions were unidirectional, the effect of the hormone on the cells of old animals being less pronounced. The results indicate that the potential of the plasma membrane of individual hepatocytes can be adequately estimated by microfluorimetry, which can be used in metabolic and toxicologic investigations.  相似文献   

8.
1. Pigeon erythrocytes, resealed lysed erythrocytes or liposomes derived from erythrocyte lipids were suspended in solutions containing up to 2 micrometer-3,3'-dipropyloxadicarbocyanine iodide. Gramicidin, valinomycin, nigericin or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone, or combinations of these, were used to induce electrical diffusion potentials dependent on Na+, K+ or protons. In each instance hyperpolarization of the cell membrane lowered the fluorescence of the cell suspension, a process that was completed in about 1 min. Subsequent depolarization caused an increase in fluorescence. 2. Quenching of the fluorescence of the cell suspension appeared to be due to the reversible binding of the dye to the cells. Much larger amounts of dye were bound, both to the intact and to the resealed erythrocytes, than would be expected if partitioning of the dye cation followed the Nernst equation. The dependence of the binding on the extracellular dye concentration was studied in the presence and absence of valinomycin. The results were consistent with the suggestion of Sims, Waggoner, Wang & Hoffman [(1974) Biochemistry 13, 3315-3330] that the dye was bound at both membrane surfaces and that, at low dye concentrations, hyperpolarizing the cells promoted dye binding at the inner membrane surface. 3. The applications of the technique are limited by the circumstance that the direct effect of the electric field on the uptake of the dye into the cells is amplified by a binding process that may be affected by other physiological variables.  相似文献   

9.
Valinomycin-induced potassium diffusion potential (delta psi, inside negative) in the liposomes made of phosphatidylcholine and various amounts of cholesterol was measured by uptake of 86Rb+, tetraphenylphosphonium (TPP+) or triphenylmethylphosphonium (TPMP+). In any liposome, the values of membrane potential obtained by 86Rb+ uptake (delta psi Rb) agreed well with those calculated from the imposed potassium concentration gradient using the Nernst equation, and were not affected by the presence of cholesterol. However, both delta psi TPP and delta psi TPMP showed smaller values than delta psi Rb when the cholesterol content in liposomes increased. delta psi TPMP at a stationary state was much smaller than delta psi TPP. The orientational order parameter of the lipids' bilayer with various cholesterol content was estimated from fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. The results indicated that the permeation of TPP+ or TPMP+ into liposomes containing a large amount of cholesterol is strongly restricted by the high ordering of phosphatidylcholine acyl chains.  相似文献   

10.
We have analyzed the combined utilization of highly permeant anions to induce membrane diffusion potentials and glucose uptake to probe the created potentials as a new approach to quantitative generation and estimation of membrane potential differences in vesicle studies. Rabbit jejunal brush-border membrane vesicles were used in our experiments so that membrane potential differences can be calculated from the Goldman-Hodgkin-Katz equation with the relative ion permeabilities recently reported for this preparation (Gunther, R.D., Schell, R.E. and Wright, E.M. (1984) J. Membrane Biol. 78, 119-127) or approximated by the Nernst potential for the anion. Iodide was selected as the highly permeant anion after showing its absence of effect on glucose uptake with equal concentrations of Na+ inside and outside the vesicles and the membrane potential clamped to zero with gramicidin D. Membrane potential was varied by altering the intra- and extravesicular iodide concentrations while keeping isosmolarity and isotonicity constant by chloride replacement. In these conditions, glucose uptake was sensitive and correlated to the expected membrane potentials. Moreover, a linear relationship between the log initial rate of glucose transport and membrane potential differences could be established. This linear relationship was quite insensitive to inside replacement of choline by potassium and to pH variations in the incubation medium, thus showing the reproducibility and the versatility of the method and the adequacy of glucose uptake as a probe for membrane potentials. However, no information can be gained on the stoichiometry of the Na+-glucose transporter as the slope of the straight line depends on both the charge carried by the fully loaded carrier and the point in the electric field at which the transition state of the carrier from cis to trans occurs. This new approach was compared with the more conventional one using valinomycin-induced K+-diffusion potentials and the Nernst potential for potassium as means for creating and estimating membrane potential differences. Both techniques were not equivalent, as linear relationships showing smaller slopes and sensitivity to pH were recorded with the latter. These differences are compatible with a potassium permeability in the presence of valinomycin that is lower than generally assumed, at least when compared to the permeability of the other ions present in the incubation medium.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

11.
Membrane conductance of cultured rabbit articular chondrocytes was characterized by means of the patch-clamp technique. The resting membrane potential of the articular chondrocytes was about -42 mV. The membrane potential shifted in accordance with the prediction by the Nernst equation for Cl- when intracellular and extracellular concentrations of Cl- were changed. On the other hand, change in extracellular concentration of K+ produced no shift in the membrane potential of chondrocytes. The Cl- channel blocker 4-acetamido-4'-isothiocyanatostilbene-2'2-disulfonic acid (SITS) depolarized the membrane potential. These findings suggest that the membrane potential of the chondrocytes is determined mainly by Cl- conductance. Using the cell-attached patch-clamp method, a large unitary conductance of 217 pS was observed in the articular chondrocytes. The unitary current was reversibly blocked by SITS. Therefore, the unitary current was carried by Cl-. The Cl- channel showed voltage-dependent activation and the channels exhibited long-lasting openings. Therefore, the membrane potential of rabbit cultured articular chondrocytes was mainly determined by the activities of the large-conductance and voltage-dependent Cl- channels.  相似文献   

12.
Using dialkylphospholipid (diphytanyl phosphatidylcholine) instead of the conventional diacylphospholipid (diphytanoyl phosphatidylcholine) in planar lipid bilayer membranes (BLM) led to an increase in the diffusion potential of the penetrating cation plastoquinonyl-decyl-triphenylphosphonium (SkQ1), making it close to the Nernst value, and accelerated translocation of SkQ1 across the BLM as monitored by the kinetics of a decrease in the transmembrane electric current after applying a voltage (current relaxation). The consequences of changing from an ester to an ether linkage between the head groups and the hydrocarbon chains are associated with a substantial reduction in the membrane dipole potential known to originate from dipoles of tightly bound water molecules and carbonyl groups in ester bonds. The difference in the dipole potential between BLM formed of the ester phospholipid and that of the ether phospholipid was estimated to be 100 mV. In the latter case, suppression of SkQ1-mediated proton conductivity of the BLM was also observed.  相似文献   

13.
Abstract. Accumulation of the lipophilic cation tri-phenylmethylphosphonium (TPMP+) has been used to estimate the plasmalemma potential (Φm) of Porphyra purpurea (Rhodophyta, Bangiales) and Ulva lactuca (Chlorophyta, Ulvales). Values of Φm obtained using the Nernst equation were −61 mV and −54 mV respectively; these values compare well with those obtained using glass microelectrodes. A trend of hyperpolarization of Φm in P. purpurea was observed with decreasing external salinity. This hyperpolarization was shown to be primarily due to changes in external K+ concentration. Varying external Na+ concentration was found to have little effect on Φm. The present data suggest that the membrane potential of P. purpurea is not wholly due to a K+ diffusion potential, but may have an electrogenic component.  相似文献   

14.
Membrane vesicles isolated from Azotobacter vinelandii strain O by lysis of spheroplasts in potassium of sodium phosphate buffer develop a transmembrane electric potential during respiration. The magnitude of this potential was determined by three independent methods: (i) fluorescence of 3,3'-dipropylthiodicarbocyanine and 3,3'-dihexyloxacarbocyanine; (ii) uptake of 86Rb+ in the presence of valinomycin; and (iii) uptake of [3H]triphenylmethyl phosphonium. In method (i), the relative fluorescence of these cyanine dyes in the presence of intact cells or derived vesicles is quenched during oxication of electron donors. A linear relationship between this quenching and a potassium diffusion potential was employed to calibrate the probe response. In method (ii), the steady-state concentration ratio of rubidium across the vesicle membrane during oxidation of L-malate was converted to potential by the Nernst equation. In method (iii), the steady-state concentration ratio of this lipophilic cation was likewise converted to a potential. With the exception of 3,3'-dihexyloxacarbocyanine fluorescence, these methods gave good agreement for the potential developed during L-malate oxidation by membrane vesicles. A value of 75 to 80 mV (inside negative) was obtained for vesicles prepared in potassium phosphate, and 104 mV (inside negative) was obtained for vesicles prepared in sodium phosphate. Electrogenic expulsion of hydrogen ion was observed during L-malate oxidation, and the amount of proton exodus was greater in potassium rather than the sodium-containing vesicles. This indicates the presence of a sodium-proton antiport mechanism. In addition, D-glucose uptake was observed during development of a potassium diffusion potential that was artificially imposed across the vesicle membrane. These observations suggest the presence of a glucose-proton symport mechanism in accordance with the principles of Mitchell.  相似文献   

15.
Rapid activation of phospholipase D (PLD) in response to cell stimulation was recently demonstrated in many systems, raising the hypothesis that PLD participates in transduction of extracellular signals across the plasma membrane. In the present study, we describe the identification of a neutral PLD activity in purified rat brain synaptic plasma membranes, and the in vitro conditions required to assay its catalytic activity with exogenous [3H]phosphatidylcholine as substrate. Production of [3H]phosphatidic acid, the natural lipid product of PLD and of [3H]phosphatidylethanol, catalyzed by PLD in the presence of ethanol via transphosphatidylation, were measured. The synaptic membrane PLD exhibited its highest activity at pH 7.2 and was thus defined as a neutral PLD. Enzyme activity was absolutely dependent on the presence of sodium oleate and was strongly activated by Mg2+ ions (at 1 mM). Ca2+ at concentrations up to 0.25 mM was as stimulatory as Mg2+, but at 2 mM it completely inhibited enzyme activity. Mg2+ extended the linear phase of PLD activity from 2 to 15 min, suggesting that it may stabilize the enzyme under our assay conditions. The production of [3H]phosphatidylethanol was a saturable function of ethanol concentration. Production of [3H] phosphatidic acid was inversely related to the concentration of ethanol and to the accumulation of phosphatidylethanol, indicating that the two phospholipids are indeed produced by the competing hydrolase and transferase activities of the same enzyme. beta,beta-Dimethylglutaric acid, utilized previously as a buffer in studies of rat brain PLD, inhibited enzyme activity at neutral pH but not at acidic pH. The properties of the neutral synaptic membrane PLD and its relationships with other in vitro, acid, and neutral PLD activities, as well as with the signal-dependent PLD detected in intact cells, are discussed.  相似文献   

16.
To determine if phospholipase D is present in intact adult islets, we took advantage of the fact that, in the presence of ethanol, this enzyme generates phosphatidylethanol via transphosphatidylation. Extracts of cells prelabeled with [14C]arachidonate, [14C]myristate, or [14C]stearate were analyzed via three TLC systems; the identify of phosphatidylethanol was further confirmed via incorporation of [14C]ethanol into the same phospholipid bands. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate stimulated phosphatidylethanol (to 603% of basal by 60 min) both in intact adult islets and in dispersed neonatal islet cells. A nonphorbol activator of protein kinase C (mezerein) also stimulated phospholipase D, whereas a phorbol which does not activate protein kinase C (4 alpha-phorbol-12,13-didecanoate) was virtually inactive. The effects of the active phorbol ester or of mezerein were reduced by the protein kinase C inhibitor H-7 and were virtually eliminated by prior down-regulation of that enzyme. In addition, a calcium-selective ionophore (ionomycin) or fluoroaluminate also activated the islet phospholipase D. When accumulation of phosphatidylethanol (labeled with any of three fatty acids) was induced by a preincubation in the presence of ethanol plus agonist, which then were removed, phosphatidylethanol declined by 34-47% over a subsequent 60-min incubation. Thus, while phosphatidylethanol is relatively stable metabolically, it is detectably degraded (a variable overlooked in previous studies). In the absence of ethanol, stimulated islet cells generated phosphatidic acid, although such hydrolysis was less evident than transphosphatidylation. Ethanol provision distinguished phosphatidate formed via phospholipase D (inhibition, via phosphatidylethanol formation) from that due predominantly to phospholipase C (phosphatidate not inhibited). In view of our recent findings that phosphatidic acid (or exogenous phospholipase D) has potent insulinotropic effects, this pathway could play a role in stimulus-secretion coupling; conversely, stimulation of transphosphatidylation at the expense of hydrolysis could contribute to the inhibition of secretion caused by ethanol.  相似文献   

17.
The ionic mechanism of horizontal cell potentials was investigated in the isolated retina of the axolotl Ambystoma mexicanum. The membrane potentials of both receptors and horizontal cells were recorded intracellularly while the ionic composition of the medium flowing over the receptor side of the retina was changed. The membrane potential of the horizontal cell is highly depender side of the retina was changed. The membrane potential of the horizontal cell is highly dependent on the extracellular concentration of sodium. When the external ion concentration of either chloride or potassium was changed independently of the other, there were shifts in the membrane potential of the horizontal cell which could not be explained by changes in the equilibrium potential of these ions. If the external concentrations of both potassium and chloride ions were varied so that the product of their external concentrations did not change, the shift in the membrane potential of the horizontal cell was in the direction predicted by the Nernst equation. The results are consistent with the suggestion that in the dark the receptors release a synaptic transmitter which increases primarily the sodium conductance of the horizontal cell postsynaptic membrane.  相似文献   

18.
Proteins in any solution with a pH value that differs from their isoelectric point exert both an electric Donnan effect (DE) and colloid osmotic pressure. While the former alters the distribution of ions, the latter forces water diffusion. In cells with highly Cl--permeable membranes, the resting potential is more dependent on the cytoplasmic pH value, which alters the Donnan effect of cell proteins, than on the current action of Na/K pumps. Any weak (positive or negative) electric disturbances of their resting potential are quickly corrected by chloride shifts. In many excitable cells, the spreading of action potentials is mediated through fast, voltage-gated sodium channels. Tissue cells share similar concentrations of cytoplasmic proteins and almost the same exposure to the interstitial fluid (IF) chloride concentration. The consequence is that similar intra- and extra-cellular chloride concentrations make these cells share the same Nernst value for Cl-. Further extrapolation indicates that cells with the same chloride Nernst value and high chloride permeability should have similar resting membrane potentials, more negative than -80 mV. Fast sodium channels require potassium levels >20 times higher inside the cell than around it, while the concentration of Cl- ions needs to be >20 times higher outside the cell. When osmotic forces, electroneutrality and other ions are all taken into account, the overall osmolarity needs to be near 280 to 300 mosm/L to reach the required resting potential in excitable cells. High plasma protein concentrations keep the IF chloride concentration stable, which is important in keeping the resting membrane potential similar in all chloride-permeable cells. Probable consequences of this concept for neuron excitability, erythrocyte membrane permeability and several features of circulation design are briefly discussed.  相似文献   

19.
Solutions of the Poisson-Boltzmann equation yield potential profiles and equilibrium distributions of ions on either side of a spherical shell membrane across which there exists a separation of ionic charges. For the special case in which the membrane is permeable to only one ion the total charge separation is analyzed in terms of the potential difference given by the Nernst equation. Potential profiles and ionic charge distributions are also given for situations involving a uniform distribution of fixed charges within the membrane.  相似文献   

20.
A hyperpolarized current is predicted from the Nernst equation for conditions of positive temperature derivatives with respect to time. This ion current, coupled with changes in membrane channel conductivities, is expected to contribute to a transient potential shift across the cell membrane for silent cells and to a change in firing rate for pacemaker cells.  相似文献   

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