A glucose-activated electron transfer system in the plasma membrane stimulates the H(+)-ATPase in Penicillium cyclopium.

Hyphal cells of three fungal species of the genus Penicillium reduced the nonpermeable, external electron acceptor hexabromoiridate IV (HBI IV). In Penicillium cyclopium, the rate of HBI IV reduction by hyphal cells was drastically increased by the addition of beta-glucose. The stimulation showed high specificity for this sugar and did not require its uptake and cellular metabolism. Cell wall oxidases (e.g., glucose oxidase) did not seem to be involved in the reduction of HBI IV, as no measurable H2O2 was formed from added glucose and removal of oxygen had no effect. We propose that there is a glucose-binding component outside the plasma membrane which controls transmembrane electron fluxes in response to external glucose. Reduction of HBI IV was accompanied by rapid acidification of the cellular interior (measured by confocal pH topography). Subsequently, the outer medium was acidified of the cellular interior (measured by confocal pH topography). Subsequently, the outer medium was acidified with an e-/H+ stoichiometry of > 1. In plasma membrane vesicles containing endogenous electron donors, the membrane-residing fluoroprobe Di-8-ANEPPS reported a transient depolarization of the membrane potential triggered by the external electron acceptor. Inhibitors of ATP-dependent proton pumping enhanced the extent of this depolarization, inhibited the subsequent normalization of membrane potential, and, in whole cells, reduced the amount of redox-triggered proton extrusion. From these and other findings, it is concluded that the observed trans-plasma membrane redox process activates the H(+)-ATPase via membrane depolarization and cytosolic acidification.     

Internal pH changes associated with the activity of NADPH oxidase of human neutrophils. Further evidence for the presence of an H+ conducting channel.

The internal pH (pHi) of cytoplasts, derived from human neutrophils, falls 0.05 pH units upon activation of the superoxide-generating NADPH oxidase. The decrease in pHi is absent in diphenyleneiodonium-treated cytoplasts and therefore it is likely to arise directly from the activity of the oxidase. The addition of amiloride, to diminish the Na+/H+ exchanger, enhanced the extent of the internal acidification but not the initial rate. However the electroneutral Na+/H+ exchanger cannot be a contributor to H+ efflux to compensate for charge translocated by the oxidase. In the presence of Cd ions or valinomycin, phorbol-induced acidification of the cytosol was greatly increased, suggesting an inability to translocate the cytosolic H+ generated by an electrogenic oxidase. In the presence of both Cd and valinomycin the cytoplasts retained 0.8 H+ per O2-. generated. The rate of acidification of the external medium by stimulated cytoplasts is greatly reduced in the presence of Zn and valinomycin. Our results support the view that the plasma membrane of neutrophils contains Zn2+- or Cd2+-sensitive proton-conducting channels which maintain a stable membrane potential and pHi during the activity of the electrogenic NADPH oxidase.     

Investigation of Ca2+ -induced changes of membrane potential of smooth muscle mitochondria using flow cytometric analysis

Using flow cytometric analysis and potential-sensitive fluorescent dye TMRM Ca2+ -induced changes of membrane potential of isolated smooth muscle mitochondria were studied. It was shown, that Ca2+ (100 microM) addition to the incubation medium induced mitochondrial membrane depolarization that probably could be explained by Ca2+/H+ -exchanger activation which functioning lead to membrane potential dissipation. In the case of ruthenium red (10 microM) preliminary presence in incubation medium, Ca2+ (100 microM) addition did not lead to membrane potential dissipation. Hence, membrane potential dissipation was caused by an increase of matrix Ca2+ concentration. In the presence of Mg2+ (3 mM) and ATP (3 mM), Ca2+ addition did not cause depolarization. It was supposed that in this case ATP synthase acted in the opposite direction as H+ -pump and prevented from mitochondrial membrane potential dissipation. Thus, the flow cytometry method allows to register membrane potential of isolated smooth muscle mitochondria and also to test the effectors, capable to modulate this parameter.     

Modulation of the mitochondrial cyclosporin A-sensitive permeability transition pore by the proton electrochemical gradient. Evidence that the pore can be opened by membrane depolarization.

This paper reports an investigation on the relationship between the proton electrochemical gradient (delta mu H+) and the cyclosporin A-sensitive permeability transition pore (PTP) in rat liver mitochondria. Using the SH group cross-linker phenylarsine oxide as the inducer, we show that both matrix pH and the membrane potential can modulate the process of PTP induction independently of Ca2+. We find that membrane depolarization induces the PTP per se when pHi is above 7.0, while at acidic matrix pH values PTP induction is effectively prevented. Since Ca2+ uptake leads to major modifications of the delta mu H+ (i.e. matrix alkalinization and membrane depolarization), we have explored the possibility that the Ca(2+)-induced changes of the delta mu H+ may contribute to PTP induction by Ca2+. Our data in mitochondria treated with Ca2+ plus N-ethylmaleimide and Ca2+ plus phosphate show that membrane depolarization is a powerful inducer of the PTP. Taken together, our observations indicate that the PTP can be controlled directly by the delta mu H+ both in the absence and presence of Ca2+, and suggest that a collapse of the membrane potential may be the cause rather than the consequence of PTP induction under many experimental conditions. Thus, many inducers may converge on dissipation of the membrane potential component of the delta mu H+ by a variety of mechanisms.     

Measurement of plasma membrane and mitochondrial potentials in sea urchin sperm. Changes upon activation and induction of the acrosome reaction

The relationship between the plasma membrane potential and activation of sperm motility and respiration, or induction of the acrosome reaction, was explored in sperm of the sea urchin Strongylocentrotus purpuratus. Plasma and mitochondrial membrane potentials were estimated by measuring the uptake of [14C]thiocyanate ( [14C]SCN-) and [3H]tetraphenylphosphonium ( [3H]TPP+) in intact sperm and sperm made permeant with digitonin. Mitochondrial potentials up to-185 mV were found, consistent with data for TPP+ uptake into mitochondria from other cell types. Values for TPP+ uptake corrected for mitochondrial accumulation and estimates of SCN- uptake both indicated that the plasma membrane potential was about -30 mV for actively respiring sperm in seawater and about -60 mV for quiescent sperm in Na+-free seawater. Activation of sperm motility and respiration induced by Na+ increased the intracellular pH and caused a depolarization of both the plasma membrane and mitochondrial potentials. However, membrane potential depolarization did not occur when the activation was induced by increased extracellular pH or by the peptide speract, although activation was always linked to increased intracellular pH. The acrosome reaction, on the other hand, was always associated with sperm plasma membrane potential depolarization, whether it was induced by the physiological effector from the egg surface or by several artificial triggering regimens. Thus, activation of respiration and motility is primarily controlled by increased intracellular pH (Christen, R., Schackmann, R. W., and Shapiro, B. M. (1982) J. Biol. Chem. 257, 14881-14890), whereas the acrosome reaction also requires depolarization of the plasma membrane potential.     

Generation of the electrochemical potential of Na+ by the Na+-motive NADH oxidase in inverted membrane vesicles of Vibrio alginolyticus

Inverted membrane vesicles prepared from Vibrio alginolyticus generated a membrane potential (positive inside) and accumulated Na+ by the oxidation of NADH. Generation of the membrane potential required Na+ and was inhibited by 2-heptyl-4-hydroxyquinoline N-oxide, a specific inhibitor of the Na+-dependent NADH oxidase. Collapse of the membrane potential by valinomycin stimulated the uptake of Na+. In contrast, accumulation of H+ was not detected under all the conditions tested. These results suggest that only Na+ is translocated by the Na+-dependent NADH oxidase of V. alginolyticus.     

Generation of Na+ electrochemical potential by the Na+-motive NADH oxidase and Na+/H+ antiport system of a moderately halophilic Vibrio costicola

Cells of Vibrio costicola at pH 8.5 generate both membrane potential (inside negative) and delta pH (inside acidic) in the presence of a proton conductor, carbonyl cyanide m-chlorophenylhydrazone (CCCP). The generation of CCCP-resistant membrane potential was inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide that is known to inhibit the Na+-motive NADH oxidase of Vibrio alginolyticus. NADH oxidase, but not lactate oxidase, of inverted membrane vesicles prepared from V. costicola required Na+ for a maximum activity and was inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide. By the oxidation of NADH, inverted membrane vesicles generated concentration gradients of Na+ across the membrane, whose magnitude was always larger than that of delta pH by about 50 mV. In contrast, magnitudes of delta pH and Na+ concentration gradients generated by the oxidation of lactate were similar. Na+ translocation in the presence of lactate was inhibited by CCCP but little affected by valinomycin. On the other hand, Na+ translocation in the presence of NADH was resistant to CCCP and stimulated by valinomycin. Amiloride, an inhibitor for a eucaryotic Na+/H+ antiport system, inhibited the lactate-dependent Na+ translocation but had little effect on the NADH-dependent Na+ translocation. These results indicate that a primary event of lactate oxidation is the translocation of H+, which then causes the generation of Na+ concentration gradients via the secondary Na+/H+ antiport system. We conclude that the NADH oxidase of V. costicola translocates Na+ as an immediate result of respiration, leading to the generation of Na+ electrochemical potential.     

17beta-estradiol stimulates MAPK signaling pathway in human lens epithelial cell cultures preventing collapse of mitochondrial membrane potential during acute oxidative stress

17beta-estradiol (17beta-E2) protects against H2O2-mediated depletion of intracellular ATP and lessens the degree of depolarization of mitochondrial membrane potential (DeltaPsi(m)) in cultured lens epithelial cells consequential to oxidative insult. We now report that 17beta-E2 acts as a positive regulator of the survival signal transduction pathway, MAPK which, in turn, acts to stabilize DeltaPsi(m) in effect, attenuating the extent of depolarization of mitochondrial membrane potential in the face of acute oxidative stress. The SV-40 viral transformed human cell line, HLE-B3 was treated with 17beta-E2 over a time course of 60 min and phosphorylation of ERK1/2 was analyzed by Western blot. ERK1/2 was phosphorylated within 5-15 min in the presence of 17beta-E2. Cell cultures were exposed to the MEK1/2 inhibitor, UO126, subsequent to H2O2+/-17beta-E2 treatment and the DeltaPsi(m) examined using JC-1, a potentiometric dye which serves as an indicator for the state of mitochondrial membrane potential. UO126 treatment attenuated ERK1/2 phosphorylation irrespective of whether estradiol was administered. Mitochondrial membrane depolarization resulting from H2O2 stress was substantially greater in the presence of UO126. The greater the extent of depolarization, the less effective 17beta-E2 treatment was in checking mitochondrial membrane depolarization, indicating that the relative degree of ERK phosphorylation influences mitochondrial stability with oxidative insult. The data support a positive correlation between 17beta-E2 stimulation of ERK1/2 phosphorylation and mitochondrial stabilization that would otherwise cause a complete collapse of DeltaPsi(m).     

Effect of high pH on the plasma membrane potential and conductance in Elodea densa

In leaves of Elodea densa the membrane potential measured in light equals the equilibrium potential of H+ on the morphological upper plasma membrane. The apoplastic pH on the upper side of the leaf is as high as 10.5-11.0, which indicates that alkaline pH induces an increased H+ permeability of the plasmalemma. To study this hypothesis in more detail we investigated the changes in membrane potential and conductance in response to alterations in the external pH from 7 (= control) to 9 or 11 under both light and dark conditions. Departing from the control pH 7 condition, in light and in dark the application of pH 9 resulted in a depolarization of the membrane potential to the Nernst potential of H+. In the light but not in the dark, this depolarization was followed by a repolarization to about -160 mV. The change to pH 9 induced, in light as well as in dark, an increase in membrane conductance. The application of pH 11, which caused a momentary hyper- or depolarization depending on the value at the time pH 11 was applied, brought the membrane potential to around -160 mV. The membrane conductance also increased, in comparison to its value at pH 7, as a result of the application of pH 11, irrespective of the light conditions.     

Plasma membrane potential modulates chemotactic peptide-stimulated cytosolic free Ca2+ changes in human neutrophils

The relationship between fMet-Leu-Phe-induced changes in the cytosolic free Ca2+ concentration [( Ca2+]i), plasma membrane potential depolarization, and metabolic responses was studied in human neutrophils. Receptor-activated depolarization occurred both at high and resting [Ca2+]i, but was inhibited at very low [Ca2+]i. Phorbol 12-myristate 13-acetate-induced plasma membrane depolarization, on the contrary, was independent of [Ca2+]i. The threshold fMet-Leu-Phe concentration for plasma membrane depolarization (10(-8) M) was at least 1 log unit higher than that for [Ca2+]i increases (5 X 10(-10) M) and coincident with that for NADPH oxidase activation. Nearly maximal [Ca2+]i increases were elicited by 3 X 10(-9) fMet-Leu-Phe in the absence of any significant plasma membrane potential change. This observation allowed us to investigate the effects of artificially induced plasma membrane depolarization and hyperpolarization at low fMet-Leu-Phe concentrations (10(-9) to 3 X 10(-9) M) which did not perturb plasma membrane potential. Depolarizing (gramicidin D at 10(-7) to 10(-6) M or KCl at 50 mM) and hyperpolarizing (valinomycin at 4 microM) treatments had little influence on unstimulated [Ca2+]i levels, whereas fMet-Leu-Phe-induced transients were significantly altered. Gramicidin D and KCl decreased the fMet-Leu-Phe-induced [Ca2+]i increases in Ca2+-containing or in Ca2+-free media. Valinomycin, on the contrary, increased receptor-stimulated [Ca2+]i increases, and the effect was larger in the presence of extracellular Ca2+. Valinomycin also strongly potentiated secretion. It is suggested that plasma membrane depolarization in human neutrophils is a physiological feedback mechanism inhibiting receptor-dependent [Ca2+]i changes.     

Sequential sodium-proton exchange in thrombin-induced human platelets

Thrombin stimulation of human platelets initiates a membrane depolarization attributable to a Na+ influx into, and an alkalinization of, the cytoplasm, both of which follow a similar rapid time scale and thrombin-dose dependence. These responses precede secretion of the contents of the dense granules (serotonin) and, after 1 minute, of lysosomes (beta-glucuronidase). We have evaluated these parameters in the presence of 2H2O in order to determine if the Na+ influx and H+ efflux are sequential or simultaneous. NMR evidence indicates that 2H2O equilibration in rapid, and virtually complete within the 3 min prestimulation platelet equilibration period. In response to an 0.05 U/ml addition of thrombin, the rate of depolarization is 70-80% slower in 2H2O than in H2O. The time to reach maximal depolarization is 5 to 10 seconds longer in 2H2O, the extent of depolarization 60% inhibited, and the pH change 85% inhibited. The serotonin secretion is unaltered, while the beta-glucuronidase secretion is 130-180% enhanced. Dimethylamiloride inhibits the Na+ influx and the pH change completely. These results suggest that the Na+ and H+ fluxes across the plasma membrane are interdependent but neither simultaneous nor electroneutral. Furthermore, granule secretion, previously shown by us to be independent of the existent Na+ gradient, depends on the cytoplasmic K+ and H+ concentrations.     

Membrane potential defect in hygromycin B-resistant pma1 mutants of Saccharomyces cerevisiae

The proton transport properties of hygromycin B-resistant pma1 mutants which show kinetic defects in the plasma membrane H+-ATPase were examined. It was found that net proton efflux, as measured by whole cell medium acidification in the presence of 25 mM KCl, was similar for normal and pma1 mutant cells. However, in the absence of added KCl, the extent of net proton efflux was considerably less in wild type than in pma1 mutant cells. The cellular membrane potential was implicated as an important factor in regulating net proton transport and was determined from [14C]tetraphenylphosphonium uptake studies to be considerably depolarized in the pma1 mutants. The growth of wild type cells, which is normally inhibited by hygromycin B at 200 micrograms/ml, was found to be resistant to the antibiotic by the addition of 50 mM KCl to the growth medium. These results suggest that the electrogenic behavior of proton transport by the H+-ATPase may be altered in pma1 mutants and that resistance to hygromycin B may be mediated via depolarization of the cellular membrane potential.     

Interconversion of components of the bacterial proton motive force by electrogenic potassium transport.

The influence of K+ ions on the components of the transmembrane proton motive force (delta mu H+) in intact bacteria was investigated. In K+-depleted cells of the glycolytic bacterium STreptococcus faecalis the addition of K+ ions caused a depolarization of the membrane by about 60 mV. However, since the depolarization was compensated for by an increase in the transmembrane pH gradient (delta pH), the total proton motive force remained almost constant at about 120 mV. Half-maximal changes in the potential were observed at K+ concentrations at which the cells accumulated K+ ions extensively. In EDTA-treated, K+-depleted cells of Escherichia coli K-12, the addition of K+ ions to the medium caused similar, although smaller changes in the components of delta mu H+. Experiments with various E. coli K-12 K+ transport mutants showed that for the observed potential changes the cells required either a functional TrkA or Kdp K+ transport system. These data are interpreted to mean that the inward movement of K+ ions via each of these bacterial transport systems is electrogenic. Consequently, it leads to a depolarization of the membrane, which in its turn allows the cell to pump more protons into the medium.     

Membrane potential changes during IgE-mediated histamine release from rat basophilic leukemia cells

The membrane potential of rat basophilic leukemia cells (RBL-2H3 cell line) has been determined by monitoring the distribution of the lipophilic [3H] tetraphenylphosphonium cation between the cells and the extracellular medium. By this method, the determined potential of these cells, passively sensitized with IgE, is -93 +/- 5 mV (mean +/- SEM, interior negative). Almost 40% of this membrane potential is rapidly collapsed upon the addition of the proton carrier, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP). It is suggested that the FCCP-sensitive fraction of the total membrane potential results from the accumulation of this cation by the mitochondria, which maintains a negative membrane potential. Thus, the resting plasma membrane potential of these cells equals -55 +/- 6 mV. During the process of immunological stimulation by antibodies directed against cell membrane bound IgE, the membrane potential decreases. Moreover, there is a correlation between the extent of degranulation of the cells and the depolarization. It is concluded that in common with other secretory systems, depolarization of the plasma membrane is involved in the stimulus-secretion coupling of the histamine secreting RBL cells.     

Resolution of cellular compartments involved in membrane potential changes accompanying IgE-mediated degranulation of rat basophilic leukemia cells.

The overall membrane potential of rat basophilic leukemia cells (RBL-2H3) calculated from the transmembrane distribution of the lipophilic, tritium-labelled cation tetraphenyl-phosphonium [( 3H]TPP+) was resolved into its mitochondrial and plasma membrane potential components. Using the mitochondrial uncoupler carbonylcyanide-p-trifluormethoxyphenyl hydrazone (FCCP) which collapses the mitochondrial potential, it was shown that about one third of the overall potential resulted from the mitochondrial contribution. Degranulation of the RBL cells induced by two different IgE-cross-linking agents (specific antigen and anti-IgE antibodies), was accompanied by, and well correlated with, a decrease in the overall potential. However, evaluation of the source of these observed potential changes revealed that the FCCP-insensitive fraction of the overall potential, delta psi P, (representing the plasma membrane potential), was not affected. In contrast, the FCCP-sensitive component due to the mitochondrial potential decreased when receptor cross-linking increased. Thus, the observed decrease in the overall potential is most probably a secondary event in the sequence leading from stimulus to secretion. Indeed, exposure of the RBL cells either to a high external concentration of K+ ions or to a high amount of external TPP+, both causing depolarization, failed to trigger degranulation. It is suggested that the apparent decrease in the measured overall potential is a reflection of the mitochondrial membrane depolarization. The latter is most probably caused by mitochondrial Ca2+ uptake initiated by the increase in the intracellular concentration of Ca2+ which follows cells activation.     

Effects of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSFrh) on transmembrane electrical potentials in granulocytes: relationship between enhancement of ligand-mediated depolarization and augmentation of superoxide anion (O2-) production

When human granulocytes that have been primed with recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSFrh) are activated by ligands that stimulate the respiratory burst, the amount of superoxide anion (O2-) they generate is significantly increased. We have found that the accelerated rate of O2- release occurring under these conditions is accompanied by an antecedent increase in membrane depolarization. We examined the nature of the enhancement of membrane depolarization in GM-CSFrh-primed granulocytes and investigated its relationship to the increase in O2- generation by N-formyl methionylleucylphenylalanine (fMLP)-activated granulocytes. We found that augmented depolarization could not be accounted for by a change in the resting membrane potential induced by the growth factor and was still present after either blocking passive transmembrane Na+ movement with dimethylamiloride or by increasing the membrane's permeability to K+ with valinomycin. When their ability to depolarize was virtually eliminated by dissipating the transmembrane K+ gradient, GM-CSFrh-pretreated cells continued to generate more O2- after fMLP than did control cells. These results indicate that augmentation of the granulocyte's ability to generate O2- anions, which is induced by priming with GM-CSFrh, is independent both of the resting transmembrane potential and of alterations in the extent of membrane potential change induced by stimuli such as fMLP.     

Membrane depolarization is the trigger for PI3K/Akt activation and leads to the generation of ROS

Loss of fluid shear stress (ischemia) to the lung endothelium causes endothelial plasma membrane depolarization via ATP-sensitive K(+) (K(ATP)) channel closure, initiating a signaling cascade that leads to NADPH oxidase (NOX2) activation and ROS production. Since wortmannin treatment significantly reduces ROS production with ischemia, we investigated the role of phosphoinositide 3-kinase (PI3K) in shear-associated signaling. Pulmonary microvascular endothelial cells in perfused lungs subjected to abrupt stop of flow showed membrane depolarization and ROS generation. Stop of flow in flow-adapted mouse pulmonary microvascular endothelial cells in vitro resulted in the activation of PI3K and Akt as well as ROS generation. ROS generation in the lungs in situ was almost abolished by the PI3K inhibitor wortmannin and the PKC inhibitor H7. The combination of the two (wortmannin and H7) did not have a greater effect. Activation of NOX2 was greatly diminished by wortmannin, knockout of Akt1, or dominant negative PI3K, whereas membrane depolarization was unaffected. Ischemia-induced Akt activation (phosphorylation) was not observed with K(ATP) channel-null cells, which showed minimal changes in membrane potential with ischemia. Activation of Akt was similar to wild-type cells in NOX2-null cells, which do not generate ROS with ischemia. Cromakalim, a K(ATP) channel agonist, prevented both membrane depolarization and Akt phosphorylation with ischemia. Thus, Akt1 phosphorylation follows cell membrane depolarization and precedes the activation of NOX2. These results indicate that PI3K/Akt and PKC serve as mediators between endothelial cell membrane depolarization and NOX2 assembly.     

Ion currents involved in early Nod factor response in Medicago sativa root hairs: a discontinuous single-electrode voltage-clamp study

Nod factor [NodRm-IV(Ac,S)], isolated from the bacterium Rhizobium meliloti, induces a well-known depolarization in Medicago sativa (cv Sitel) root hairs. Analysis of this membrane response using the discontinuous single-electrode voltage-clamp technique (dSEVC) shows that anion channel, K+ channel and H+-ATPase pump currents are involved in young growing root hairs. The early Nod-factor-induced depolarization is due to increase of the inward ion current and inhibition of the H+ pump. It involved an instantaneous inward anion current (IIAC) and/or a time-dependent inward K+ current (IRKC). These two ion currents are then down-regulated while the H+ pump is stimulated, allowing long-term rectification of the membrane potential (Em). Our results support the idea that the regulation of inward current plays a primary role in the Nod-factor-induced electrical response, the nature of the ions carried by these currents depending on the activated anion and/or K+ channels at the plasma membrane.     

Antigen-triggered membrane potential changes in IgE-sensitized rat basophilic leukemia cells: evidence for a repolarizing response that is important in the stimulation of cellular degranulation

We have studied Ag-induced membrane potential changes of rat basophilic leukemia cells by using the potential-sensitive dye, bis-(1,3-diethylthiobarbiturate)trimethineoxonol. A rapid membrane depolarization is triggered by a multivalent Ag, and it has a bell-shaped dose dependence that parallels the degranulation response but not the extent of cross-linking of the IgE-receptor complexes. As the temperature is reduced from 37 degrees C, this depolarization response slows and decreases in magnitude until complete inhibition is observed at 15 degrees C, similar to the temperature dependence previously observed for the Ag-stimulated rise in cytoplasmic Ca2+ and for degranulation. The results imply that a highly temperature-dependent step subsequent to Ag binding and cross-linking is necessary for the depolarization response. A partial return to the resting potential is seen to follow the depolarization response to Ag. This repolarization process is inhibited by quinidine.HCl and Ba2+ in parallel with an inhibition of the degranulation response. Repolarization is not affected by 4-aminopyridine or by the absence of K+ in the external buffer. These data suggest that the repolarization is caused by a previously uncharacterized K+ channel.     

The role of transmembrane cationic gradients in immune complex stimulation of human polymorphonuclear leukocytes

The role of monovalent cationic gradients in human polymorphonuclear leukocyte (PMNL) stimulation was investigated by monitoring immune complex-stimulated transmembrane depolarization and superoxide production, events which accompany--and have been used as indicators of --PMNL activation. Abolishing only the Na+ gradient by substitution of choline for extracellular Na+ did not affect the resting membrane potential but reduced the rate of stimulus-induced transmembrane depolarization to 50% of control. In contrast, collapsing both Na+ and K+ gradients by suspension in K+ buffer (high K-PRK) depolarized the cells and reduced the stimulus-induced rate of depolarization to 11% of control. Pretreatment of cells suspended in Na+ buffers with 5-(N,N-dimethyl)amiloride hydrochloride (DMA) or with valinomycin reduced by one-half the rate of immune complex induced membrane depolarization. Conversely, in the absence of either or of both Na+ or K+ gradients, or in the presence of valinomycin, immune complex elicited an enhanced rate of superoxide production. However, PMNL prepared via NH4Cl (NH4Cl-PMNL) instead of H2O (H2O-PMNL) lysis of residual red blood cells exhibited an absolute requirement for an intact Na+ gradient in cell stimulation. The present results thus demonstrate that: 1) both Na+ and K+ gradients participate equally in the membrane depolarization elicited by immune complex; 2) neither a Na+ or a K+ gradient is required for immune complex activation, or for activity of the respiratory burst; and 3) an artifactual requirement for an intact Na+ gradient occurs in neutrophils prepared by the NH4Cl lysis technique.