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The effects of palmitate on human erythrocyte membrane potential and osmotic stability.

To evaluate the mechanism(s) of erythrocyte stabilization in hypotonic medium by free fatty acids, we have studied the effect of palmitic acid and its derivatives on human erythrocyte membrane electrochemical properties, osmotic stability and fluidity. Palmitic acid increased the membrane potential to the highest extent as compared with its derivatives. The membrane potential was equal to -11.0 +/- 1.7 mV in the absence and -23.3 +/- 3.2 mV in the presence of 400 mumol 1(-1) of palmitic acid, -15.3 +/- 2.2 mV in the presence of 400 mumol 1(-1) of methyl ester of palmitic acid and -14.2 +/- 1.8 mV in the presence of 400 mumol 1(-1) of lauric aldehyde. We propose that free fatty acids, containing the charged carboxylic end group, in contrast to the corresponding esters or aldehydes, can act as ionophores. At the same time, the osmotic stabilization of erythrocytes by palmitic acid and its derivatives was higher in sodium-containing as compared with potassium-containing media and did not correlate with the effect of these compounds on the membrane potential. We suggest that protective potassium leakage does not play a main role in erythrocyte osmotic stabilization by free fatty acids. Possibly this stabilization is due to the structural rearrangement of the whole membrane.

Erythrocyte Membrane↗

Mutual interaction of ion uptake and membrane potential.

The concentration dependence of cation uptake by the cell may be considerably complicated when this uptake is accompanied by a depolarization of the cell membrane. In case of carrier-mediated transport deviations from Michaelis-Menten kinetics may come to the fore comparable to those found in a dual mechanism of cation uptake or when substrate inhibition is involved. This remains true when only the maximum rate of uptake and not the Km is dependent upon the membrane potential. We have proven this by means of computer simulation of cation transport mediated by a non-mobile carrier. Under restricted conditions still apparent Michaelis-Menten kinetics may be found despite the fact that the membrane potential varies with increasing substrate cation concentration. But even then there are still differences with 'normal' transport kinetics. A non-competitive inhibitor does not only affect the maximum rate of uptake but also the apparent Km. Depolarization of the cells by a cation which passes the cell membrane by means of diffusion, affects the uptake of the substrate cation almost in the same way as a non-competitive inhibitor does and causes both a decrease in the maximum rate of uptake and an increase in Km. In the case of competitive inhibition the apparent affinity of the inhibitor for the carrier depends upon the rate of transfer of this inhibitor through the cell membrane. The mutual influence of cation uptake and membrane potential is dealt with for uniport of either monovalent or divalent cations and for cotransport of monovalent cation with protons, as well. Possible effects of the surface potential are accounted for.

Cations↗

Membrane potential of squid axons: effect of internal and external ion concentration.

Transmembrane resting potentials of squid axons were measured with variation of intracellular salt concentrations. It was found that the dependence of resting potential on the extracellular K-ion concentration was practically unaffected by the change of ionic strength in the intracellular phase, and the relationship of membrane potential and the logarithm of extracellular K-ion concentration was a straight line with a slope of about 50 mV/10-fold extracellular K-ion concentration. Also, the degree of change in resting potential due to the change of intracellular K-ion concentration was not affected appreciably by the extracellular solution with various K-ion concentrations, and the membrane potentials were insensitive over a wide range of intracellular K-ion concentrations. It was also indicated that the observed transmembrane potentials are not explained by a conventional membrane potential theory.

Animals↗

The membrane potential modulates the ATP-dependent Ca2+ pump of cardiac sarcolemma.

The effect of membrane potential on the activity of the ATP-dependent Ca2+ pump of isolated canine ventricular sarcolemmal vesicles was investigated. The membrane potential was controlled by the intravesicular and extravesicular concentration of K+, and the initial rates of Ca2+ uptake both in the presence and the absence of valinomycin were determined. The rate of Ca2+ uptake was stimulated by a inside-negative potential induced in the presence of valinomycin. The valinomycin-dependent stimulation was enhanced by the addition of K+ channel blocker, tetraethylammonium ion or Ba2+. The electrogenicity of cardiac sarcolemmal ATP-dependent Ca2+ pump is suggested from the increase of Ca2+ uptake by negative potential induced by valinomycin.

Adenosine Triphosphate↗

Spontaneous changes in mitochondrial membrane potential in cultured neurons.

Using the mitochondrial membrane potential (DeltaPsi(m))-sensitive fluorescent dyes 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1) and tetramethylrhodamine methyl ester (TMRM), we have observed spontaneous changes in the DeltaPsi(m) of cultured forebrain neurons. These fluctuations in DeltaPsi(m) appear to represent partial, transient depolarizations of individual mitochondria. The frequency of these DeltaPsi(m) fluctuations can be significantly lowered by exposure to a photo-induced oxidant burden, an ATP synthase inhibitor, or a glutamate-induced sodium load, without changing overall JC-1 fluorescence intensity. These spontaneous fluctuations in JC-1 signal were not inhibited by altering plasma membrane activity with tetrodotoxin or MK-801 or by blocking the mitochondrial permeability transition pore (PTP) with cyclosporin A. Neurons loaded with TMRM showed similar, low-amplitude, spontaneous fluctuations in DeltaPsi(m). We hypothesize that these DeltaPsi(m) fluctuations are dependent on the proper functioning of the mitochondria and reflect mitochondria alternating between the active and inactive states of oxidative phosphorylation.

Animals↗

The internal pH and membrane potential of the insulin-secretory granule.

The membrane potential (DeltaPsi) and the pH gradient (DeltapH) across the membrane of the insulin-secretory granule were determined in studies in vitro from the uptake of the permeant anion thio[(14)C]cyanate or the permeant base [(14)C]methylamine. Freshly prepared granules incubated in iso-osmotic medium containing sucrose and low concentrations of buffer salts exhibited an acidic internal pH and a DeltaPsi positive inside. Addition of MgATP(2-) under these conditions did not alter the DeltapH, but produced a marked increase in the DeltaPsi. Conversely, when a permeant anion was also included, ATP produced a marked increase in the DeltapH and a lesser increment in the DeltaPsi. NH(4) (+) salts reduced the DeltapH across granule membranes. In the presence of ATP this effect was accompanied by a reciprocal increase in the DeltaPsi. A similar reciprocity was evident when nigericin was added together with K(+) or on decreasing the medium pH, suggesting that these gradients were linked by a common electrogenic process. The effects of ATP were reversed by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the combination of valinomycin, nigericin and K(+), and by the Mg(2+)-dependent ATPase inhibitor tributyltin. Uptakes of (14)C-labelled tracer molecules were also markedly reduced by cryogenic disruption of the granule membrane or hypo-osmotic incubation conditions. These results were readily interpreted within a chemiosmotic hypothesis, which proposed that the insulin granules possess an inwardly-directed electrogenic proton-translocating Mg(2+)-dependent ATPase with the additional postulate that the membrane has a low proton permeability. The intragranular pH was estimated as being between 5 and 6 in vivo. Such a value corresponds to optimal conditions for the crystallization of zinc-insulin hexamers. Several other functions related to chemiosmotic processes within insulin granules, however, may be envisaged.

Adenoma, Islet Cell↗

Membrane potentials and resistances of giant mitochondria. Metabolic dependence and the effects of valinomycin.

The membrane potentials and resistances of giant mitochondria from mice fed cuprizone have been studied. They were found to correspond approx. 10-20 mV, positive inside, and 2 M omega, respectively. These properties were found to be independent of the metabolic state. The microelectrodes were in the inner mitochondrial space since (a) the potentials in the presence of valinomycin depended on the K+ concentration of the medium and magnitude of the K+ diffusion potentials was consistent with the presence of a high internal concentration of K+, (b) almost identical results were obtained with mitochondria from which the external membrane had been removed and the cristae were evaginated, and (c) punch-through experiments, in which the microelectrodes were advanced until they emerged through the other side of the mitochondria, showed an identical membrane potential both in the presence and in the absence of valinomycin. The potentials were stable under a variety of conditions and showed no sign of decay of membrane leakiness. Detailed evidence that the impaled mitochondria are metabolically viable will be presented in a separate publication.

Animals↗

Ability to light-induced conductance change of arthropod visual cell membrane, indirectly depending on membrane potential, during depolarization by external potassium or ouabain.

Light responses (ReP) and pre-stimulus membrane potential (PMP) and conductance of photoreceptors of Astacus leptodactylus and Limulus polyphemus (lateral eye) were recorded and changes were observed when the photoreceptor was depolarized by the action of external ouabain of high potassium concentration application. 1 mM/1 ouabain application causes a transient increase of PMP and ReP in Limulus, followed by a decrease which is faster for the ReP (half time time 34 min) than for the PMP (half time 80 min). Irreversible loss of excitability occurs when the PMP is still ca. 40% of the reference value. In both preparations high external potassium concentration leads to total depolarization (beyond zero line to + 10-+ 20 mV) of the PMP and after a time lag of 10 min also to a loss of excitability (intracellular recording). In extracellular recordings (Astacus) the excitability remains at a low level of 15%. The effects are reversible and are similar whether no or 10% external sodium is present. In all experiments the light-induced changes of membrane conductance are about parallel to those of the light response. The fact that the ability of the photosensoric membrane to undergo light-induced conductance changes is membrane potential-dependent is discussed, leading to the explanation that dipolar membrane constituents such as channel forming molecules (probably not rhodopsin) have to be ordered by membrane potential to keep the membrane functional for the photosensoric action.

Animals↗

Membrane potential of Plasmodium-infected erythrocytes.

The membrane potential (Em) of normal and Plasmodium chabaudi-infected rat erythrocytes was determined from the transmembrane distributions of the lipophilic anion, thiocyanate (SCN), and cation, triphenylmethylphosphonium (TPMP). The SCN- and TPMP-measured Em of normal erythrocytes are -6.5 +/- 3 mV and -10 +/- 4 mV, respectively. The TPMP-measured Em of infected cells depended on parasite developmental stage; "late" stages (schizonts and gametocytes) were characterized by a Em = -35 mV "early stages (ring and copurifying noninfected) by a low Em (-16 mV). The SCN-determined Em of infected cells was -7 mV regardless of parasite stage. Studies with different metabolic inhibitors including antimycin A, a proton ionophore (carbonylcyanide m-chlorophenylhydrazone [CCCP] ), and a H+ -ATPase inhibitor (N,N'-dicyclohexylcarbodiimide, [DCCD] ) indicate that SCN monitors the Em across the erythrocyte membrane of infected and normal cells whereas TPMP accumulation reflects the Em across the plasma membranes of both erythrocyte and parasite. These inhibitor studies also implicated proton fluxes in Em-generation of parasitized cells. Experiments with weak acids and bases to measure intracellular pH further support this proposal. Methylamine distribution and direct pH measurement after saponin lysis of erythrocyte membranes demonstrated an acidic pH for the erythrocyte matrix of infected cells. The transmembrane distributions of weak acids (acetate and 5,5-dimethyloxazolidine-2,4-dione) indicated a DCCD-sensitive alkaline compartment. The combined results suggest that the intraerythrocyte parasite Em and delta pH are in part the consequence of an electrogenic proton pump localized to the parasite plasma membrane.

Animals↗

The effects of membrane potential and lanthanides on the conformation of the Ca2+-transport ATPase in sarcoplasmic reticulum.

The effects of Ca2+, lanthanide ions (Gd3+, La3+ and Pr3+) and membrane potential on the fluorescence of tryptophan and covalently bound fluorescein were analysed in native and fluorescein isothiocyanate (FITC)-labelled sarcoplasmic reticulum vesicles. The binding of Ca2+ and lanthanides to the Ca2+-ATPase increases the fluorescence intensity of tryptophan and decreases the fluorescence intensity of FITC; the dependence of these effects on cation concentration is consistent with the involvement of the high-affinity Ca2+-binding sites of the Ca2+-ATPase in the cation-induced fluorescence changes. The fluorescence of FITC-labelled sarcoplasmic reticulum vesicles is also influenced by membrane potential changes induced by ion substitution. Inside positive potential increases, while inside negative potential decreases, the fluorescence of bound FITC. Smaller potential-dependent changes in tryptophan fluorescence were also observed. The effects of Ca2+, lanthanides and membrane potential on the fluorescence of tryptophan and FITC are discussed in terms of the two major conformations of the Ca2+-ATPase (E1 and E2), that are assumed to alternate during Ca2+ transport. The observations support the suggestion [Dux, Taylor, Ting-Beall & Martonosi (1985) J. Biol. Chem. 260, 11730-11743] that the vanadate-induced crystals of Ca2+-ATPase represent the E2, while the Ca2+ and lanthanide-induced crystals the E1, conformation of the enzyme.

Animals↗

Direct measurement of the membrane potential of Ehrlich ascites tumor cells: lack of effect of valinomycin and ouabain.

The membrane potential of Ehrlich ascites tumor cells and the effects of valinomycin and ouabain upon it have been determined. The membrane potential in control cells was 12.0 mV, inside negative. Neither valinomycin nor ouabain alone affected this value. However, valinomycin and ouabain in combination resulted in a slight hyperpolarization of the membrane. Concomitant determinations of cellular Na+, K+ and Cl- showed that valinomycin induced net losses of K+ and Cl- and a net gain in Na+ when compared to ouabain-inhibited cells. K+ permeability was increased by approximately 30% in the presence of valinomycin. In addition, valinomycin caused a rapid depletion of cellular ATP. Inhibition of Na/K transport by ouabain was without sparing effect on the rate of ATP depletion. Possible mechanisms for the electroneutral increase in K+ permeability induced by valinomycin are discussed.

Adenosine Triphosphate↗

Effects of polyene macrolides on the membrane potential of resting and activated human leukocytes.

Membrane potential changes in human polymorphonuclear leukocytes have been studied by monitoring the fluorescence signal of a carbocyanine probe in the presence of calcium ionophore A23187, chemotactic tripeptide f-met-leu-phe, and polyene macrolides. Reduction of the signals triggered by the ionophore and the tripeptide, inhibition of the respiratory burst, and changes in the Na+ influx into the cell seem to be the dominant effect of the polyenes. Polyene-induced depolarization does not correlate with the inhibition of the respiratory burst.

Amphotericin B↗

Membrane potential changes in activated cells: connection with cytosolic calcium oscillator.

The activation of many cells by hormones, growth factors or neurotransmitters can lead to a periodic variation of the cell membrane potential. A theoretical model that describes this behaviour on the basis of the cytosolic calcium oscillator is developed. The main assumption of this model stressed that the plasma membrane potential is mainly a K+ diffusion potential. It is shown that the oscillations of the intracellular calcium concentration can induce solely the membrane potential oscillations, without any further interaction mechanisms between fluxes through the ionic channels from the plasma membrane. A one-pool model was used to account for Ca2+ release from the intracellular stores. The model and the numerical calculations are based on the theory of nonlinear dynamical systems.

Animals↗

[Measurement of human erythrocyte membrane potential with microelectrode].

The measurement of erythrocyte membrane potential (Ery-MP) in 189 healthy subjects using a microelectrode showed that the Ery-MP in 96 healthy subjects was -5.80 +/- 0.31 mV, and that in 93 healthy female subjects was -6.30 +/- 0.34 mV. Plasma lipid peroxide (P-LPO), erythrocyte lipid peroxide (Ery-LPO), plasma superoxide dismutase activity (P-SODA) and erythrocyte superoxide dismutase activity (Ery-SODA) were measured at the same time. After linear regression and correlation analysis, it was found that the Ery-MP value of healthy adults decreased as the P-LPO and Ery-LPO values increased, displaying a linear negative correlation, Ery-MP and Ery-SODA values formed a linear positive correlation.

Adolescent↗

Effect of nicotine and acetylcholine on crustacean muscle membrane potential.

We have investigated the effect of nicotine and acetylcholine on the resting membrane potential of the crayfish extensor muscle in order to determine whether crustacean muscle can be activated by cholinergic compounds. Intracellular recordings from individual deep extensor abdominal muscle cells were made using standard glass microelectrode techniques. The resting membrane potential was measured before and after treatment with glutamate, nicotine, and acetylcholine. Glutamate, which is a known activator of crayfish muscle, was used to determine whether the muscle cell preparation was viable and capable of responding to any of the test substances. Our results confirm that application of glutamate is associated with a depolarization of the muscle membrane. However, muscle cells showed no depolarization after treatment with nicotine (50 microM) or acetylcholine (66 microM). These results argue against the notion that increases in muscle tension may be responsible for the increased receptor organ discharge observed in the presence of nicotine. Rather, it supports the hypothesis that nicotine is acting directly on the mechanoreceptor membrane to change its sensitivity.

Acetylcholine↗

Regulation of Ca2+ efflux in rat liver mitochondria. Role of membrane potential.

The paper analyzes the relationship between membrane potential (delta psi), steady state pCao (-log [Ca2+] in the outer aqueous phase) and rate of ruthenium-red-induced Ca2+ efflux in liver mitochondria. Energized liver mitochondria maintain a pCao of about 6.0 in the presence of 1.5 mM Mg2+ and 0.5 mM Pi. A slight depression of delta psi results in net Ca2+ uptake leading to an increased steady state pCao. On the other hand, a more marked depression of delta psi results in net Ca2+ efflux, leading to a decreased steady-state pCao. These results reflect a biphasic relationship between delta psi and pCao, in that pCao increases with the increase of delta psi up to a value of about 130 mV, whereas a further increase of delta psi above 130 mV results in a decrease of pCao. The phenomenon of Ca2+ uptake following a depression of delta psi is independent of the tool used to affect delta psi whether by inward K+ current via valinomycin, or by inward H+ current through protonophores or through F1-ATP synthase, or by restriction of e- flow. The pathway for Ca2+ efflux is considerably activated by stretching of the inner membrane in hypotonic media. This activation is accompanied by a decreased pCao at steady state and by an increased rate of ruthenium-red-induced Ca2+ efflux. By restricting the rate of e- flow in hypotonically treated mitochondria, a marked dependence of the rate of ruthenium-red-induced Ca2+ efflux on the value of delta psi is observed, in that the rate of Ca2+ efflux increases with the value of delta psi. The pCao is linearly related to the rate of Ca2+ efflux. Activation of oxidative phosphorylation via addition of hexokinase + glucose to ATP-supplemented mitochondria, is followed by a phase of Ca2+ uptake, which is reversed by atractyloside. These findings support the view that Ca2+ efflux in steady state mitochondria occurs through an independent, delta psi-controlled pathway and that changes of delta psi during oxidative phosphorylation can effectively modulate mitochondrial Ca2+ distribution by inhibiting or activating the delta psi-controlled Ca2+ efflux pathway.

Animals↗

[Changes in the membrane potential of rat peritoneal macrophages under the extracellular action of ATP].

In rat peritoneal macrophages the patch-clamp technique in whole-cell configuration was used to study modifications of membrane potential as a result of extracellular application of micromolar ATP concentration. The registered changes in membrane potential appeared to be of different form and polarization depending on the presence of Ca2+ in extracellular solution and on the concentration of calcium buffer inside the cell. A mechanism of membrane potential changes and their possible connection with the generation of a Ca2+ signal in the cell are discussed.

Adenosine Triphosphate↗

Depolarization of the membrane potential by beta-lactams as a signal to induce autolysis.

The effect of beta-lactam antibiotics that are known to inhibit cell wall biosynthesis and induce cell wall autolysis on the electrophysiological state of the plasma membrane in Streptomyces griseus was studied. Addition of various beta-lactam antibiotics induced a dose- and growth-stage-dependent depolarization of the membrane potential of Streptomyces griseus. The hydrolyzed biologically inactive derivative penicilloic acid had no depolarizing effect on the membrane potential. The ionophore gramicidin D, while depolarizing the membrane potential, also induced a dose-dependent increase in cell wall lysis. These observations suggest that alteration of the transmembrane potential could be an important signal in triggering cell wall autolysis of S. griseus.

Anti-Bacterial Agents↗