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PubMed1992

Modulation of calcium fluxes in Jurkat T cells by myristic acid. Inhibition is independent of membrane potential and intracellular pH.

Treatment of T lymphocytes with mitogenic antibodies against the T-cell receptor/CD3 complex induces within seconds a rise in the concentration of intracellular free Ca2+. We recently reported that free myristic acid, but not its methyl ester, inhibits both the anti-CD3-induced Ca2+ influx across the cell membrane and the Ca2+ release from intracellular stores in Jurkat T cells. Here we show that myristic acid induced a rapid hyperpolarization of the cell membrane potential and a decrease in intracellular pH in Jurkat cells. Lauric acid and palmitic acid caused minor hyperpolarization, whereas other saturated non-esterified fatty acids tested were without effect. Hyperpolarization of the membrane potential in Jurkat cells with valinomycin did not, however, inhibit the anti-CD3-induced Ca2+ signal, and the blocking effect on the Ca2+ signal in myristic acid-treated Jurkat cells was not reversed after normalization of the cell membrane potential by treatment with gramicidin. The inhibitory effect of myristic acid on the Ca2+ fluxes thus cannot be explained by changes in membrane potential. We also present evidence that the blocking effect of myristic acid on the receptor-operated Ca2+ flux is not due to the myristic acid-induced decrease in intracellular pH. Moreover, we demonstrate that myristic acid does not prevent the release of Ca2+ triggered by inositol 1,4,5-trisphosphate from intracellular pools in permeabilized cells. Our findings indicate that myristic acid blocks anti-CD3-induced Ca2+ traffic in Jurkat cells by interfering with the regulation of Ca2+ mobilization, apparently by blocking an early step in signal transduction from the T-cell-antigen receptor/CD3 complex.

Animals↗

PubMedDate not supplied

[Cyclic nucleotide levels, adenylate cyclase activity and membrane potential of the gastrocnemius muscle fibers following denervation in adult and old rats].

The denervation-induced changes of cyclic nucleotide concentrations adenylate cyclase activity and resting membrane potential were studied in gastrocnemius muscle of adult and old rats. Under conditions of denervation alterations in resting membrane potential and in adrenaline-stimulated adenylate cyclase activity had a biphasic character in the muscle studied: resting membrane potential was increased and the adenylate cyclase activity was decreased following 30 min denervation, while the opposite effect was observed after 2 weeks denervation. The denervation did not alter cGMP concentration. In old animals more pronounced changes of cAMP level were found in denervated gastrocnemius muscle.

Adenylyl Cyclases↗

PubMed1990

[Effect of acetylcholine on membrane potential in toad dorsal root ganglion neurons and its ionic basis].

Intracellular recordings were made to investigate the effect of acetylcholine (ACh) on membrane potential of neurons in isolated toad dorsal root ganglion (DRG). In the 73 neurons examined, 67 were of type A, and the remaining 6 of type C cell. The resting membrane potential of these two types of cells was -67.5 +/- 1.3 mV (mean +/- SE). During the application of ACh (4 x 10(-4)-6 x 10(-4) mol/L) four types of the membrane potential changes were observed: (1) hyperpolarization of 9.1 +/- 3.0 mV (mean +/- SE, n = 23); (2) depolarization of 12.9 +/- 2.2 mV (mean +/- SE, n = 20); (3) biphasic response, i.e., hyperpolarization of 8.0 +/- 2.4 mV (mean +/- SE) followed by depolarization of 10.9 +/- 3.1 mV (mean +/- SE, n = 24); (4) no change (n = 6). The hyperpolarization induced by ACh could be blocked by atropine (1.3 x 10(-5) mol/L, n = 23), and ACh-induced depolarization could be abolished by coperfusion of d-tubocurarine (1.4 x 10(-5) mol/L) and hexamethonium (1.4 x 10(-5) mol/L, n = 18). During ACh hyperpolarization the membrane conductance was increased by 13.8% and the reversal potential was about -96 mV (n = 3). TEA (20 mmol/L) enhanced ACh depolarization amplitude by 48.2 +/- 3.2% (mean +/- SE, n = 6), and depressed ACh hyperpolarization by 79.4 +/- 4.3% (mean +/- SE, n = 8), MnCl2 (4 mmol/L) decreased the ACh depolarization and hyperpolarization by 54.2 +/- 7.2% (mean +/- SE, n = 5) and by 69.2 +/- 6.4% (mean +/- SE, n = 14) respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗

PubMed1984

Oscillatory hyperpolarizations and resting membrane potentials of mouse fibroblast and macrophage cell lines.

L cells (a mouse fibroblast cell line) and macrophages have been reported to exhibit slow oscillatory hyperpolarizations and relatively low membrane potentials, when measured with glass micro-electrodes. This paper describes the role of micro-electrode-induced leakage in these oscillations for L cells and a mouse macrophage cell line (P388D1). Both L cells and macrophages showed fast negative-going peak-shaped potential transients upon micro-electrode entry. This shows that the micro-electrode introduces a leakage conductance across the membrane. The peak values of these fast transients were less negative for L cells (-17 mV) than for macrophages (-39 mV), although their sustained resting membrane potentials were about equal (-13 mV). This indicates that the pre-impaled membrane potential of macrophages is more negative than that of L cells. Ionophoretic injection of Ca2+ into the P388D1 macrophages showed the existence of a Ca2+ -dependent hyperpolarizing conductance presumed to be involved in the oscillatory hyperpolarizations of L cells and macrophages. Cells increased in size by X-ray irradiation to reduce membrane input resistances were still found to be susceptible to micro-electrode-induced leakage. Impalement transients upon entry of a second electrode during a hyperpolarization evoked by a first electrode, were often step-shaped instead of peak-shaped due to the high membrane conductance associated with hyperpolarization. Since peak-shaped impalement transients were always seen with the first impalement both in oscillating and non-oscillating cells, oscillatory hyperpolarizations cannot be regarded as spontaneously occurring in the unperturbed cells but are induced by micro-electrode penetration. Since the hyperpolarizing response can be evoked by ionophoretic injection of Ca2+, and oscillatory as well as single hyperpolarizing responses are absent in a Ca2+ -free medium, it is concluded that the Ca2+ needed intracellularly to activate the hyperpolarizing responses enters the cell via the leakage pathway introduced by the measuring electrode.

Animals↗

PubMed1986

Mechanisms of 1,25(OH)2D3-induced rapid changes of membrane potential in proximal tubule: role of Ca2+-dependent K+ channels.

Eleven different secosteroids or steroids (10(-10) to 10(-8) M) were acutely and reversibly introduced in solutions delivered to the lumen of single proximal tubules of the amphibian Necturus kidney while recording basolateral cell membrane potential Vm. Seven of these molecules (1,25(OH)2D3, 25(OH)D3, 24,25(OH)2D3, 5,6-trans-25(OH)D3, 19-diol-cholesterol, estradiol and testosterone) resulted in changes of Vm (delta Vm) occurring in a few seconds, the largest delta Vm being observed with 1,25(OH)2D3, +6.5 +/- 0.75 mV (n = 19); these seven (seco)steroids, but not the four inactive sterols (vitamin D3, cholesterol, 1 alpha D3 and aldosterone) possess a hydroxyl group on at least one carbon of the C17 to C25 lateral chain of the sterol ring. The delta Vm effect was present in Na+-free or Cl-free media, but it was abolished in HCO3-free media. Depolarization of cell membrane potential by addition of glucose, 11 mM, in luminal perfusion fluid abolished the 1,25(OH)2D3-evoked delta Vm effect, suggesting dependence of the latter on the absolute value of membrane potential. Barium, a blocking agent of K+ conductances, suppressed the 1,25(OH)2D3-evoked delta Vm effect, even when the proper effects of barium of cell membrane potential were canceled by current clamp. Pretreatment with quinine, a putative blocker of Ca2+-dependent K+ channels also abolished the 1,25(OH)2D3-evoked depolarization. Such observations are consistent with the presence of Ca2+-dependent K+ channels at the apical cell membrane of the proximal tubule, these channels being inactivated by 1,25(OH)2D3 and probably by other (seco)steroids.

Animals↗

PubMed1969

The mechanism of oscillatory activity at low membrane potentials in cardiac Purkinje fibres.

1. The mechanism of oscillations at low membrane potentials in Purkinje fibres has been investigated using voltage clamp experiments.2. The oscillations are generated by time-dependent variations in an outward current component, i(x1), that is activated over the voltage range -40 to 10 mV. During normal activity, this current is responsible for initiating full repolarization to the resting potential (Noble & Tsien, 1969b) so that the oscillations represent a failure of the normal repolarization process, probably as a consequence of a small change in background (leakage) current.3. These oscillations are distinct from the normal pacemaker activity of Purkinje fibres which is generated by a separate time-dependent current, i(K2) (Noble & Tsien, 1968). i(K2) shows no time-dependence when the membrane potential variations are entirely positive to -65 mV and cannot, therefore, be involved in the oscillatory activity apart from contributing a background outward current.4. The amplitude and frequency of the oscillations are very sensitive to applied currents less than 1 muA/cm(2). Larger currents abolish the oscillatory activity.5. The mechanism of the oscillations is discussed in relation to the possible mechanisms underlying the natural pacemaker activity of the sino-atrial (SA) node.

Electric Stimulation↗

PubMed1988

ATP-dependent calcium transport in rat parotid basolateral membrane vesicles is modulated by membrane potential.

The ATP-dependent Ca2+ transport activity (T. Takuma, B.L. Kuyatt and B.J. Baum, Biochem. J. 227:239-245, 1985) exhibited by inverted basolateral membrane vesicles isolated from rat parotid gland was further characterized. The activity was dependent on Mg2+. Phosphate (5 mM), but not oxalate (5 mM), increased maximum Ca2+ accumulation by 50%. Half-maximal Ca2+ transport was achieved at approximately 70 nM Ca2+ in EGTA-buffered medium while maximal activity required greater than 1 microM Ca2+ (Vmax = 54 nmol/mg protein/min). Optimal rates of Ca2+ transport were obtained in the presence of KCl, while in a KCl-free medium (mannitol or sucrose) approximately 40% of the total activity was achieved, which could not be stimulated by FCCP. The initial rate of Ca2+ transport could be significantly altered by preimposed membrane potentials generated by K+ gradients in the presence of valinomycin. Compared to the transport rate in the absence of membrane potential, a negative (interior) potential stimulated uptake by approximately 30%, while a positive (interior) potential inhibited uptake. Initial rates of Ca2+ uptake could also be altered by imposing pH gradients, in the absence of KCl. When compared to the initial rate of Ca2+ transport in the absence of a pH gradient, pHi = 7.5/pHo = 7.5; the activity was approximately 60% higher in the presence of an outwardly directed pH gradient, pHi = 7.5/pHo = 8.5; while it was approximately 80% lower when an inwardly directed pH gradient was imposed, pHi = 7.5/pHo = 6.2. The data show that the ATP-dependent Ca2+ transport in BLMV can be modulated by the membrane potential, suggesting therefore that there is a transfer of charge into the vesicle during Ca2+ uptake, which could be compensated by other ion movements.

Animals↗

PubMed1975

Sensitivity of calcium efflux from squid axons to changes in membrane potential.

Squid giant axons were internally dialyzed with a medium free of metabolic substrates but containing 45Ca buffered with EGTA to concentrations of free Ca++ in the range 0.01-230 muM. At (Ca)i of 1.0 muM OR GREATER, Ca efflux was in the range of 1-3 pmol/cm2 s, was dependent on (Na)o and (Ca)o, and was sensitive to membrane potential. At lower (Ca)i, the sensitivity of Ca efflux to membrane potential was greater. Hyperpolarization of the membrane increased, and depolarization decreased Ca efflux over the range of potentials studied (-20 to -100 mV). The maximum sensitivity of Ca efflux to membrane potential was of the order of an e-fold increase in Ca efflux for a 25-mV increase in Em; this sensitivity of Ca efflux to membrane potential was lost if (Na)o was removed and was greatly reduced when (Ca)i was increased to 230 muM.

Acetates↗

PubMed1985

Membrane potentials of individual cells of isolated gastric glands of rabbit.

Individual glands of rabbit gastric mucosa were prepared for measurements of cell membrane potentials. In the first experiments a collagenase isolation technique was used which produced gland fragments that were fixed on agarose. In later experiments a microdissection technique was used which allowed whole glands to be isolated that were held in suction pipettes. Individual parietal or chief cells could be recognized and impaled with microelectrodes, however, the yield of reliable recordings was small and the distinction from artifacts sometimes difficult. In acceptable recordings the membrane potentials of both cell types varied between around -20 and -35 mV or exceptionally -50 mV in both preparations, with mean values being around -26 mV. The significance of the recordings was tested by ion substitution experiments. Substitution of all chloride by sulfate increased the membrane potential to values ranging up to -60 and -80 mV that are commonly observed in other cells.

Animals↗

PubMed1979

[Changes in the vitro membrane potential of cancer cells induced by contact with cytotoxic lymphoid cells].

Plasma membrane potentials have been measured in a cancer cell line at different stages of growth. A chamber has been especially built for pH control of the culture medium during several hours. Individual values spread widely from 14 to 44 mV but low measurements are less frequent after 3 days of culture when the growth rate is at a peak. The same cancer line is grafted serially in syngeneic C 57 bl mice were it induces an immunological sensitization of the lymphnode cells. The effect of such cytotoxic lymphocytes on the membrane potentials of the target tumour cell has been studied after a contact (15 to 90 minutes) too short for any morphological changes to be detectable. There is a decrease in the mean values and a very significant rise in percentage of measurements below 20 mV. No such changes are observed in control experiments where naïve lymphocytes are used. These results are discussed in relation to the hypothesis of an increased permeability of the plasma membrane to ions as an early event in cell mediated cytotoxicity.

Animals↗

PubMed1977

A study of the primary effect of the uncoupler carbonyl cyanide m-chlorophenylhydrazone on membrane potential and conductance in Riccia fluitans.

1. In the presence of 10(-5) to 10 (-8) M carbonyl cyanide m-chlorophenylhydrazone (CCCP) the membrane potential of thallus cells of the aquatic liverwort Riccia fluitans responds to changes of the external pH between 5.5 and 8.3. This occurs in the light and dark, and also if respiration is abolished by addition of 10(-4) M KCN and 10 (-5) M salicyl-hydroxamic acid. 2. The ATP-level of the thallus is reduced, independently of the external pH, by greter than or equal to 10 (-6) M CCCP to 30--40% of the control level of about 1.1 nmol ATP per mg dryweight within 5 min. 3. Upon addition of 10 (-4) M CCCP at 20 degrees C the ATP-level declines exponentially with a half time of about 20--30 s, whereas the membrane potential declines exponentially with a half time of about 2--3 s. 4. At pH 7.2 the electrical membrane conductance yields a sigmoid curve as a function of the logarithm of the CCCP concentration between 10 (-8) and 3 - 10 (-6) M. On the other hand, at 3 - 10(-7) M M CCCP the gm (electrical slope conductance, muS - cm-2 = 10 (-6)-omega-1-cm-2) versus pH-curve displays an optimum between pH 6.5 and 7.5. 5. We conclude that CCCP acts upon membrane potential and conductance in Riccia predominantly by inducing a passive proton permeability of the cell membrane, i.e. CCCP raises the permeability ratio, PH/PK, more than 100-fold above its control level of about 10.

Adenosine Triphosphate↗

PubMed1999

Membrane potential as a modulator of the free intracellular Ca2+ concentration in agonist-activated endothelial cells.

We have used combined patch clamp and fura-2 fluorescence to elucidate the role of membrane potential in the regulation of the cytosolic Ca2+ concentration ([Ca2+]i) in a human umbilical vein derived endothelial cell-line, EA.hy926 (EA cells) stimulated with vasoactive agonists, such as ATP, histamine and bradykinin. This stimulation caused hyperpolarization and sustained Ca2+ plateau in nonclamped cells. Clamping agonist-stimulated cells at negative potentials enhanced the amplitude of this plateau, whereas it was smaller at more depolarized potentials, indicating that Ca2+ influx follows its driving force. Depolarization of the membrane by increasing extracellular K+ or by applying charybdotoxin, a blocker of big conductance Ca2+-dependent K+ channels during agonist stimulation diminished the plateau rise in [Ca2+]i. It is concluded that the membrane potential is an efficient regulator of Ca2+ influx during the plateau phase of agonist-mediated Ca2+ signals. In addition, the modulating effects on Ca2+ signals should be interpreted with caution if the membrane potential of the cells is not controlled.

Adenosine Triphosphate↗

PubMed1989

Thallium-201 uptake relates to membrane potential and potassium permeability in human glioma cells.

The mechanism for 201Tl+ uptake was studied in cultured human glioma cell lines. Ouabain (1 mM) decreased the uptake at steady-state to 60%, but the rate of uptake was faster in the presence of ouabain. Addition of non-radioactive Tl+ (to a K+-free medium) decreased the uptake, but much less than expected for a system limited by the number of transport sites. Changes in K+ concentration of incubation medium affected the 201Tl+ uptake as predicted by the electrochemical equilibrium (Nernst equation). Using the uptake in isotonic KCl as a reference for membrane potential (0 mV), the calculated membrane potential was -75 mV in a medium with 3.0 mM K+. The Tl+-flux constants and the membrane permeabilities for Tl+ and K+ were calculated from the rate of uptake and from wash-out experiments. This is a new method for membrane potential and permeability studies in cell populations. The mechanism for 201Tl+ uptake is relevant for the clinical interpretation of 201Tl+ scintigraphy.

Humans↗

PubMed1997

Photometric assessment of volume changes coupled with membrane potential in valinomycin-incorporated red blood cells.

Employing photometric methods, we have attempted to derive a possible quantitative relationship between volume change and membrane potential for valinomycin-incorporated red blood cells. The cells, collected from a human, rats or bullfrogs, were suspended in test solutions composed of a mixture of NaCl and KCl in varying proportions. The osmolality of the suspending medium was appropriately fixed at different values. After the addition of valinomycin to the suspension, changes in optical density (turbidity) at 620 nm and in fluorescence from a voltage-sensitive permeant dye (diS-C3-[5]) were measured in different concentrations of external potassium ions. The changes in optical density and fluorescence intensity were converted into relative cell volume and membrane potential changes in the test cells, respectively. Cell volume increased with depolarization of the membrane. We derived an empirical equation for the volume change versus membrane potential relation curves obtained experimentally, and have also shown that the observed volume change may be plausibly represented by a hyperbolic function of transmembrane potential involving the medium osmolality as an important parameter.

Animals↗

PubMed1984

Intracellular chloride activity and membrane potential in stripped frog skin (Rana temporaria).

The regulation of cell chloride activity in frog skin was investigated using double barrelled Cl--microelectrodes to measure cell membrane potentials and chloride activity in the isolated frog epidermis. Experiments were done under short-circuit conditions, impaling cells from the serosal side. The basic electrophysiological parameters of the isolated skin were similar to those reported in the literature for whole preparations. Intracellular chloride activity was on average 21.9 mM and membrane potential was about 57 mV, implying that chloride was distributed away from its electrochemical equilibrium (i.e., concentrated inside the cells). Chloride activity decreased after removal of either Cl- or Na+ from the serosal bathing solution, with no change in membrane potential. The chloride permeability of the serosal membrane was calculated to be 2.6 X 10(-6) cm X s-1 which represents about 1/4 of the total conductance of the serosal membrane. We suggest that an electrically silent sodium-dependent uphill transport of chloride is present at the basolateral membrane of the frog skin, which accounts for the non-passive distribution of chloride.

Animals↗

PubMed1990

Oscillating and triggering properties of T cell membrane potential.

Using the patch-clamp technique, membrane potential (MP) behaviour under different electrogenic stimuli for both resting and Con A-activated T lymphocytes was studied. The voltage-gated K+ channels stabilize MP at the level of their threshold of activation (-44 +/- 5 mV, n = 27). Upon depolarization of the cell membrane by external current pulse (in the current-clamp mode), MP can oscillate with a high amplitude of 20-50 mV. The amplitude depends on the number of K+ channels per cell and the external current value. After current pulse termination, MP did not return to its initial value, but continued to oscillate. However, the cells first activated by Con A did not display this triggering effect. Thus, the properties of T cell membrane revealed appear to be associated with the functional state of the cell.

Concanavalin A↗

PubMed1988

Membrane potential can be determined in individual cells from the nernstian distribution of cationic dyes.

The distribution of a selection of cationic fluorescent dyes can be used to measure the membrane potential of individual cells with a microfluorometer. The essential attributes of these dyes include membrane permeability, low membrane binding, spectral properties which are insensitive to environment, and, of course, strong fluorescence. A series of dyes were screened on HeLa cells for their ability to meet these criteria and several commercially available dyes were found to be satisfactory. In addition, two new dyes were synthesized for this work by esterification of tetramethyl rhodamine. The analysis of the measured fluorescent intensities requires correction for fluorescence collected from outside the plane of focus of the cell and for nonpotentiometric binding of the dye. The measurements and analysis were performed on three different cell types for which there exists a body of literature on membrane potential; the potentials determined in this work were always within the range of literature values. The rhodamine esters are nontoxic, highly fluorescent dyes which do not form aggregates or display binding-dependent changes in fluorescence efficiency. Thus, their reversible accumulation is quantitatively related to the contrast between intracellular and extracellular fluorescence and allows membrane potentials in individual cells to be continuously monitored.

Animals↗

PubMed1993

Effect of membrane potential on K-Cl transport in human erythrocytes.

We examined whether swelling-activated K-Cl cotransport is electrogenic in human erythrocytes. Baseline membrane potential, measured by the change in fluorescence of the carbocyanine dye diS-C3-5, was not different in hypotonically swollen (-7.6 mV) or isosmotically swollen cells (-9.5 mV). We used hemisodium, a new highly selective Na ionophore, in varying concentrations, in the presence of a fixed outwardly directed Na gradient (intracellular Na, 75 mM; external Na, 1 mM) to vary membrane potential over a wide range despite identical K and Cl concentrations. The membrane potential varied between -8 and -90 mV. K influx increased slightly with hyperpolarization in swollen and nonswollen cells. However, the difference between the two fluxes, swelling-activated K influx, a measure of K-Cl cotransport, was unaffected by voltage changes, as was swelling-activated K efflux. We conclude that K-Cl cotransport in human erythrocytes is electroneutral and by inference has a 1:1 stoichiometry.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗