Measurement of the electrical potential difference across the membrane of the ehrlich mouse ascites tumor cell

The electrical potential difference (PD) across the membrane of the Ehrlich mouse ascites tumor cell has been measured with intracellular microelectrodes. The mean for 111 cells in control Ringer solution was ? 11.2 mV ± 0.29 (SE), interior negative. When sulfate replaced chloride in the external medium the PD fell to ? 2.8 mV if measured as soon as possible after mixing the cells with a sulfate medium, but when nitrate replaced chloride the PD fell only to ? 8.5 mV. Cells equilibrated in nitrate had the same PD as those in control Ringer. These results indicate that the PD is sensitive to changes in the external chloride concentration and that nitrate can substitute for chloride electrically. However, since the PD for chloride, based on the Nernst equation and calculated on the basis of 70% exchangeability of cell chloride, is three times greater than the measured PD, it is hypothesized that sodium contributes significantly to the membrane potential in addition to chloride. On the other hand, potassium does not influence the PD to any great extent.     

The influence of cellular amino acids and the Na+: K+ pump on the membrane potential of the Ehrlich ascites tumor cell

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. 30 mM), 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 60 mM, 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.     

Changes in membrane potential and resistance caused by transient increase of potassium conductance in the unicellular green alga Eremosphaera viridis

The electrophysiological membrane parameters of the unicellular green alga Eremosphaera viridis were determined using an improved computer-supported single-microelectrode technique. These cells developed an average membrane potential of-150 mV in the light and a specific resistance of 1 Ω m² with an external potassium concentration of 1.1 mM and pH 5.5. In the dark, many cells showed a less polarized potential of 30–40 mV and a smaller membrane resistance. At potassium concentrations in the external medium higher than 1 mM, the membrane potential strongly depends on the external potassium content apart from a small electrogenic component. At concentrations lower than 1 mM K⁺, a dependence of the membrane potential upon external potassium concentrations could not be verified. Inserting the internal ion activities in the Goldmann equation shows that, in this range, the proton conductance seems to be predominant over the potassium conductance. Transient changes in the membrane potential and in the membrane resistance were observed after switching off the light, after addition of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea or N,N′-dicyclohexylcarbodiimide, after a sudden decrease in temperature, and after current pulses. These changes resemble the action potentials (AP) found in other plant cells (Chara, Acetabularia). On average, the AP has a delay period of 5.1 s and a duration of 43.8 s showing a sudden decrease and a slower regeneration. The voltage peak during an AP followed exactly the Nernst potential of potassium over a range of external potassium concentrations from 5 μM to 0.2 M. This is true for depolarization or hyperpolarization, depending on the external K⁺-concentration. Tetraethylammonium-hydrogensulphate, a rather specific inhibitor of K⁺ channels in nervous cells, suppressed the AP. The correlation of the appearance of the AP with a short-term opening of potassium channels in the membrane of Eremosphaera is discussed.     

Electrophysiology of phagocytic membranes: III. Evidence for a calcium-dependent potassium permeability change during slow hyperpolarizations of activated macrophages

The roles of potassium and calcium in the slow hyperpolarizations of membranes of activated macrophages are investigated using standard intracellular electrical recording techniques.The amplitude of spontaneous slow hyperpolarizations decreases as a logarithmic function of the external potassium concentration in the culture medium. Similar dependence on the potassium gradient is observed when different levels of membrane potentials are imposed by constant current injection. The reversal potential for electrically evoked slow hyperpolarizations is ?90 mV. A 10-fold increase in external potassium concentration causes a 60 mV shift of the reversal potential towards zero.Divalent cation ionophores (A23187 and X537A) can induce slow hyperpolarization responses in quiescent cells or permanent hyperpolarization in spontaneously active cells. The amplitude of the ionophore-induced hyperpolarizations is reduced by an increase in external potassium concentration in a manner consistent with data on slow hyperpolarization responses in the absence of ionophore.The calcium antagonist, verapamil, depresses the slow hyperpolarization responses at the concentration of 10^?5 M.It is suggested that the development of the hyperpolarizing response is due to a calcium-dependent potassium channel. The data support the assumption that spontaneous and artificially elicited slow hyperpolarization responses share a common calcium-dependent mechanism.     

Modulation of the membrane potential of intact guinea pig aortic endothelium

The role of different ion conductances in regulation of the membrane potential (MP) of resting and agonist-stimulated intact endothelium from the guinea pig aorta was investigated. Under resting conditions, the MP measured by the patch-clamp technique varied within the range from –29 to –56 mV (the mean value of –40.8 ± 8.1 mV). Blockers of anomalous (inward) rectifier potassium channels cesium (100 µM) and barium (100 µM) exerted no effect on the MP of endothelium. Superfusion of preparations with calcium-free solution and application of 2 mM nickel depolarized the endothelium. ATP (10 µM) induced hyperpolarization of endothelium with the mean amplitude of 11.4 ± 0.6 mV. The initial phase of this hyperpolarization depended on the external potassium concentration and on the state of intracellular calcium stores, whereas the prolonged phase required the presence of external calcium. In the absence of external calcium, in 25% of recordings transient hyperpolarization was followed by depolarization, which was not observed after substitution of external NaCl for choline. It was concluded that basal activity of calcium-dependent potassium channels contributes to the regulation of the MP of resting endothelium. Stimulation with ATP led to activation of calcium-dependent potassium and nonselective cationic channels. Activation of the former channels produced the initial phase of hyperpolarization, whereas activation of the second channel type evoked the prolonged phase of hyperpolarization.Neirofiziologiya/Neurophysiology, Vol. 28, No. 6, pp. 260–266, November–December, 1996.     

Transmembrane potential and amino acid transport in rat hepatocytes in primary monolaver culture

Transmembrane potential (E_m) and alpha-aminoisobutyric acid (AIB) transport were measured in primary monolayer cultures of rat hepatocytes obtained from unoperated control rats and from rats 12 hr following partial hepatectomy. Measurements were performed 20–24 hr after plating the cells. The capacity of both kinds of cells to concentrate AIB depended upon extracellular sodium; however, the steady-state accumulation in regenerating cells was twice that of control cells. Transmembrane potentials, recorded with glass microelectrodes, were –13 ± 0.6 mV and –27 ± 1.6 mV in control and regenerating cells, respectively. Ouabain (1 mM) depolarlized regenerating cells to –18 ± 1.0 mV, but it had no effect on control cells. The initial rates of 1 mM AIB transport into control and regenerating cells were 1.2 ± 0.1 and 3.1 ± 0.1 nanomoles/mg protein × 4 min, respectively. Ouabain (1 mM) reduced the initial rate of AIB transport into regenerating cells to 2.7 ± 0.1 nanomoles/mg protein × 4 min, but it had no effect on AIB transport into control cells. Glucagon (10^?7 M) added to control cells 12 hr before measurements hyperpolarized E_m to –31 ± 1.3 mV and increased AIB transport rate to 3.1 nanomoles/mg protein × 4 min. The results suggest a relationship between increases in E_m and increases in AIB transport in rat hepatocytes. An electrogenic Na-K pump may be involved in both of these events.     

Cooperative phenomena in the membrane potential of parathyroid cells induced by divalent cations

Membrane potentials of mouse parathyroid cells were measured by means of the intracellular microelectrode method. The membrane potential in external Krebs solution containing 2.5 mM of Ca++ was -23.6 +/- 0.4 mV (mean +/- standard error of mean). The low concentration of Ca++ (1.0 mM) caused hyperpolarization of the membrane potential to -61.7 +/- 0.8 mV. The membrane potential was proportional to the logarithm of the concentration of K ion in the solution of low Ca ion. The concentration of external Na+, C1- and HPO4-- had no effect on the membrane potential. The sigmoidal transition of membrane potentials was induced by the change of Ca ion concentration in the range from 2.5 to 1.0 mM. The change of the membrane potentials in low Ca ion is originated from increase in potassium permeability of the cell membrane. The similar sigmoidal changes of the membrane potentials were observed in the solution containing 4 to 3 mM of Sr ion. The Mg and Ba ion showed smaller effect on the membrane potential. The Goldman equation was extended to divalent ions. Appling the extended membrane potential equation, ratios of the permeability coefficients were obtained as follows: PK/PCa = 0.067 for 2.5 mM Ca++, 0.33 for 1.0 mM Ca++; PK/PSr = 0.08 for 4 mM Sr++ and 0.4 for 3 mM Sr++; PK/PMg = 0.5; PK/PBa = 0.67 for all range of concentration. The Hill constants of Sr ion and Ca ion were 20; the relationship between Sr ion and Ca ion was competitive. The Hill constants of Mg and Ba ion were 1 each. The Hill constant of Ca ion was depend of the temperature; nmax = 20 at 36 degrees C, n = 9 at 27 degrees C, n = 2 at 22 degrees C. The enthalpy of Ca-binding reaction was obtained from the Van't Hoff plot as 0.58 kcal. The activation energies of the K+ permeability increase were obtained from the Arrhenius plots as 3.3 kcal and 4 kcal. The difference, 0.7 kcal, corresponds to the enthalpy change of this reaction, of which value is close to that of the Ca-binding reaction.     

Membrane potential of the unfertilized sea urchin egg

The membrane potential, specific resistance, and potassium selectivity of the unfertilized Strongylocentrotus purpuratus egg were determined by two independent methods: tracer flux and microelectrode. The potassium influx was 0.50 ± 0.2 pmole/cm²· sec, which was greater than the sodium, chloride, and calcium influxes by factors of 4, 7, and 75, respectively. By means of the constant-field equations, the flux data were used to calculate membrane potential (?70 mV) and specific resistance (420 kΩ · cm²). The effect of the external potassium concentration on the sodium influx was determined and the results closely fit the result expected if the membrane behaved as a potassium electrode. Microelectrode measurements of the potential and resistance were ?75 ± 3 mV and 380 ± kΩ · cm².     

Regulation of cytosolic sodium ion activity in frog sartorius

To explore the regulation of cytosolic sodium ion activity in the frog sartorius, we used Na(+)-selective microelectrodes to monitor intracellular sodium ion activity in situations of lowering external sodium concentration and elevating external potassium concentration. Reductions of 20%, 40%, 60% and 80% in extracellular sodium concentration produced slight but statistically insignificant changes in the membrane potential of the muscle. However, cytosolic sodium ion activity decreased significantly from 10.0 +/- 1.1 mM to 7.8 +/- 1.1 mM, 7.1 +/- 1.4 mM, 6.5 +/- 1.2 mM and 5.9 +/- 1.1 mM, respectively. In addition, elevation of the external potassium concentration from 2 mM to 12 mM, 32 mM and 62 mM caused respective stepwise depolarization of membrane potential from -87.2 +/- 1.6 mV to -62.4 +/- 3.6 mV, -45.4 +/- 3.0 mV, -27.2 +/- 1.8 mV. Under these conditions, the cytosolic sodium ion activity decreased from 10.5 +/- 1.4 mM to 7.3 +/- 1.6 mM, 6.4 +/- 1.1 mM and 5.2 +/- 0.8 mM, respectively. The results illustrate that the net sodium flux is out of cell either in the reduction of sodium chemical gradient or in the potassium depolarization across the cell membrane.     

Functional differentiation of a human ganglion cell derived neuroblastoma cell line SH-SY5Y induced by a phorbol ester (TPA)

When the human neuroblastoma cell line SH-SY5Y is exposed to 12-o-tetradecanoyl-phorbol-13-acetate (TPA) the cells grow long processes indicative of neural differentiation. Concomitantly there is an increase in the resting membrane potential from ?44 ± 2 mV found in untreated cells to ?63 ± 4 mV after induction. The TPA treated cells are depolarized when the external potassium concentration is increased to 46 mM and upon addition of veratridine. In contrast to the untreated cells depolarization in differentiated cells leads to an increase in the rate of Ca²⁺ influx. This increase in Ca²⁺ influx is blocked by the Ca²⁺ channel antagonist, verapamil, while the Na⁺ channel blocker tetrodotoxin only marginally inhibits the K⁺ depolarization-induced Ca²⁺ influx.The results suggest that the induction of morphological differentiation in this cell line is associated with the appearance of features of excitable cells.     

The influence of cellular amino acids and the Na+ : K+ pump on the membrane potential of the Ehrlich ascites tumor cell.

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.     

Effects of applying oxytocin to Helix pomatia neurons. II. Hyperpolarization

The ionic mechanisms of hyperpolarization produced by applying oxytocin (OT) were investigated at the membrane of identifiedHelix pomatia neurons. Two types of neuron were known to exist, in one of which hyperpolarization is produced by a reduction in chloride ions at the membrane and a rise in membrane permeability to potassium ions in the other. In the first of these, response to OT had a reversal potential of –40 mV and decreased when furosemide and tolbutamide were added to the external medium. In the second case, the potential of the reversal of the response to OT was –70 mV. Upon doubling of potassium ion concentration in the external solution it was shifted towards depolarization by 15 mV. It is sugested thatHelix pomatia neurons have different types of OT receptors, some of which, when activated, manifest reduced chloride permeability at the membrane (probably through the cell cyclase system) with a rise in potassium permeability at the membrane in others.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 5, pp. 659–666, September–October, 1988.     

A model of oscillation generation by molluscan neurons

A model describing slow oscillations of membrane potential in molluscan neurons is suggested. It is based on the view that the depolarization phase is due to the slow calcium current, whereas the hyperpolarization phase is due to the potassium current activated by intracellular Ca ions. It is shown that depending on values of the parameters of the model there are three possible types of electrical activity of the neurons: stable membrane hyperpolarization up to the resting potential which is between ?49 and ?53 mV; slow oscillations of membrane potential from ?30 to ?60 mV, with a period of 12–17 sec, and stable membrane depolarization to between ?40 and ?30 mV, which may lead to the onset of stable rhythmic activity of these neurons. Dependence of the amplitude of the oscillations of potential on the extracellular concentration of Ca, K, and Na ions was calculated and agrees qualitatively with the experimental data of Barker and Gainer [4].     

Inwardly rectifying K(+) channels in esophageal smooth muscle

The whole cell patch-clamp technique was used to investigate whether there were inwardly rectifying K(+) (K(ir)) channels in the longitudinal muscle of cat esophagus. Inward currents were observable on membrane hyperpolarization negative to the K(+) equilibrium potential (E(k)) in freshly isolated esophageal longitudinal muscle cells. The current-voltage relationship exhibited strong inward rectification with a reversal potential (E(rev)) of -76.5 mV. Elevation of external K(+) increased the inward current amplitude and positively shifted its E(rev) after the E(k), suggesting that potassium ions carry this current. External Ba(2+) and Cs(+) inhibited this inward current, with hyperpolarization remarkably increasing the inhibition. The IC(50) for Ba(2+) and Cs(+) at -60 mV was 2.9 and 1.6 mM, respectively. Furthermore, external Ba(2+) of 10 microM moderately depolarized the resting membrane potential of the longitudinal muscle cells by 6.3 mV while inhibiting the inward rectification. We conclude that K(ir) channels are present in the longitudinal muscle of cat esophagus, where they contribute to its resting membrane potential.     

The electrical changes accompanying fertilization and cortical vesicle secretion in the medaka egg

The electrical properties of the egg of the medaka, Oryzias latipes, were studied before, during, and after fertilization. The resting potential of the unfertilized egg averaged ?39 ± 9 mV in Yamamoto's Ringers (Y. Ringers), but 20% of the values were between ?50 and ?60 mV. Fertilization triggers a small depolarization of 4 ± 3 mV in 10% Y. Ringers with an average duration of 20 ± 10 sec. The amplitude of this depolarization is independent of [Na⁺]_o, [Ca²⁺]_o, and [Cl^?]_o, so it appears to be due to a nonspecific leak triggered by sperm-egg fusion. The depolarization is followed by a longer hyperpolarizing phase with an average amplitude of 31 ± 12 mV. Recovery from this hyperpolarization has a fast phase lasting 155 ± 18 sec, followed by a slower phase which reaches a steady average membrane potential of ?19 ± 1 mV by 9 min after fertilization. The membrane resistance falls 10-fold during the first 2 min after fertilization, from 40 (1520 kΩ-cm²) to 3 MΩ. This is largely due to an increase in the K⁺ conductance. At the peak of the hyperpolarization, the membrane potential exhibits a 28 mV/decade [K⁺]_o dependence and a 6 mV/decade [Na⁺]_o dependence. The membrane resistance slowly recovers over the next 8 min to a value about 30% larger than before fertilization. The relation of current vs voltage was linear before, during, and after fertilization and indicated a reversal potential of ?98 ± 20 mV for the hyperpolarization peak. The egg's capacitance averaged 0.04 ± 0.01 μF (0.9 μF/cm²) before fertilization and approximately doubles within 90 sec after fertilization. It then decreases over a 9-min period, reaching a value 25% smaller than before fertilization.     

Membrane Potential Measured During Potassium-Evoked Release of Noradrenaline from Rat Brain Neurons: Effects of Normorphine

A single slice of rat pons that contained the locus ceruleus (LC) or two slices of cerebellum were loaded with [3H]noradrenaline; superfusion with high (35 or 60 mM) potassium solutions evoked a release of 3H. In the presence of normorphine, the release of 3H evoked by 35 mM potassium and 60 mM potassium was reduced. In some of those experiments in which the release of 3H from the LC slice was measured, an intracellular microelectrode was used to measure membrane potential. This showed that solutions of increased potassium concentration depolarized the neurons to a potential at which inward calcium currents flowed (calcium action potentials occurred). Normorphine hyperpolarized the neurons; during this hyperpolarization the depolarization caused by 35 mM potassium did not reach the threshold for significant calcium entry. The results suggest that the inhibition by normorphine of transmitter release evoked by solutions of raised potassium concentration could result in part from the membrane hyperpolarization caused by the normorphine.     

Relationship between membrane potential and external potassium in human glioblastoma cells in tissue culture

Cells from a human glioblastoma (TC 526) maintained in tissue culture for ten years had a mean membrane potential of 27 ± 0.9 mV at an external potassium concentration [K₀] of 5.3 mM. When [K₀] was varied between 2.5 and 5.3 mM, membrane potential changes were close to those predicted by the Nernst equation. At higher [K₀], the Nernstian slope was approached only in the presence of 10^?5 M ouabain, which did not affect membrane potential at a [K₀] of 5.3 mM. An electrogenic sodium pump activated by high [K₀] could explain these findings; such a mechanism has been demonstrated in other tissues.     

The effect of altered transmembrane ion gradients on membrane potential and aggregation of human platelets in blood plasma

The resting membrane potential of platelets suspended in blood plasma buffer mixture measured using tritiated triphenylmethylphosphonium was 48 ± 13 mV. Increases in extracellular potassium concentration caused depolarization of the platelet membrane. Ouabain (1.0 μM) produced a depolarization of 13 ± 5 mV. Depolarization with elevated extracellular potassium or ouabain potentiated the aggregating effects of adenosine diphosphate even in the presence of methysergide suggesting that the effects are not mediated through ion linked serotonin transport. These observations are consistent with the suggestion that transmembrane ion gradients and membrane potential can regulate platelet sensitivity to the aggregating agent adenosine diphosphate.     

Inward rectification in rat olfactory receptor neurons.

Inwardly rectifying currents in enzymically dissociated olfactory receptor neurons of rat were studied by using patch-clamp techniques. Upon hyperpolarization to membrane potentials more negative than -100 mV, small inward-current relaxations were observed. Activation was described by a single exponential with a time constant that decreased e-fold for a 21 mV hyperpolarization. The current was not reduced by the external application of 5 mM Ba2+, but was abolished by the addition of 5 mM Cs+ to the bath solution. Increasing the external K+ concentration ([K+]o) to 25 mM dramatically enhanced the current without affecting the voltage range or the kinetics of activation. In 25 mM [K+]o, tail currents reversed at -26 mV, significantly more positive than the K+ equilibrium potential of -44 mV. These characteristics are consistent with those of a mixed Na+/K+ inward rectification that has been reported in several types of neuronal, cardiac and smooth muscle cells. The current may contribute to controlling cell excitability during the response to some odorants.     

An electrophysiological study of the non-junctional membrane of epidermal cells in Tenebrio molitor

The contribution of K and Cl to the membrane potential of the epidermal cells of the recently-ecdysed larva of the mealworm was examined. The ionic basis for the membrane potential is complex. Although increasing the external K level depolarized the cell membrane, the relationship obtained suggests that ions other than K contribute largely to the recorded membrane potential. In particular, exposing the cells to K concentrations below the normal level of 40 mM has only slight effects on membrane potential, irrespective of whether K is lowered by direct substitution with Na or under conditions in which Na and Cl levels are held constant. Increasing the external Cl levels from 4 mM to 154 mM while holding K and Na levels constant resulted in a 10 mV hyperpolarization. The slight hyperpolarizing effects of high external Cl could be mimicked by citrate, but not by acetate, the latter drastically hyperpolarizing the cell membrane at levels of K that normally maintain a reduced membrane potential. External Na has little effect on the membrane potential at normal physiological levels of K, but may depolarize the cell at low K levels. The results suggest that several inorganic ions, and possibly organic acids, participate in generating the membrane potential of the epidermal cell. The passive ionic properties of non-junctional epidermal membrane and muscle membrane appear to the similar in this insect.The electrical resistance on the non-junctional membrane is highly dependent on the external K level, and can be reduced by three orders of magnitude by increasing external K from 1 mM to 120 mM. The resistance of the junctional membrane remains constant over this range of external K concentrations.