Cytoplasmic pH modulates the activity of the plasma membrane electron transport system in Ehrlich ascites tumour cells

Summary

We report evidence for a modulation of Ehrlich cell plasma membrane redox activity by cytoplasmic pH. When Ehrlich cells were submitted to treatments leading to a slight decrease of cytoplasmic pH, there was a significant inhibition of plasma membrane ferricyanide reductase activity. However, those treatments which produced a slight alkalinization of the cytoplasm enhanced the activity of the plasma membrane redox system. Since it has been shown previously that plasma membrane redox activity controls cytoplasmic pH, it seems that plasma membrane redox activity and cytoplasmic pH mutually modulate each other.     

Characterization of plasma membrane redox activity from ehrlich cells

Ferricyanide reductase activity of plasma membranes isolated from Ehrlich ascites tumour cells was very sensitive to trypsin treatment. The decreases of activity observed after treatment with different glycosidases suggests that ferricyanide reductase is a glycoprotein. The opposite effects of phospholipase A₂ and phospholipase C on the redox activity indicate that the phospholipidic environment plays an important role in the function of ferricyanide reductase. Sodium ions at millimolar concentrations, and some divalent cations at micromolar concentrations (Ca²⁺, Mg²⁺, Sr²⁺, and Mn²⁺) behaved as stimulators of ferricyanide reductase activity.     

The possible role of redox-associated protons in growth of plant cells

The protons excreted by plant cells may arise by two different mechanisms: (1) by the action of the plasma membrane H⁺-ATPase and (2) by plasma membrane redox reactions. The exact proportion from each source is not known, but the plasma membrane H⁺-ATPase is, by far, the major contributor to proton efflux. There is still some question of whether the redox-associated protons produced by NADH oxidation on the inner side of the plasma membrane traverse the membrane in a 1 : 1 relationship with electrons generated in the redox reactions. Membrane depolarization observed in the presence of ferricyanide reduction by plasma membranes of whole cells or tissues or the lag period between ferricyanide reduction and medium acidification argue that only scalar protons may be involved. The other major argument against tight coupling between protons and electrons involves the concept of strong charge compensation. When ferricyanide is reduced to ferrocyanide on the outside of cells or tissues, an extra negative charge arises, which is compensated for by the release of H⁺ or K⁺, so that the total ratio of increased H⁺ plus K⁺ equals the electrons transferred by transmembrane electron transport. These are strong arguments against a tight coupling between electrons and protons excreted by the plasma membrane. On the other hand, there is no question that inhibitor studies provide evidence for two mechanisms of proton generation by plasma membranes. When the H⁺-ATPase activity is totally inhibited, the addition of ferricyanide induces a burst of extra proton excretion, orvice versa, when plasma membrane redox reactions are inhibited, the H⁺-ATPase can function normally. Since plasma membrane redox reactions and associated H⁺ excretion are related to growth, it is possible that in plants the ATPase-generated protons have a different function from redox-associated protons. The H⁺-ATPase-generated protons have been considered for many years to be necessary for cell wall expansion, allowing elongation to take place. A special function of the redox-generated protons may be in initiating proliferative cell growth, based on the presence of a hormone-stimulated NADH oxidase in membranes of soybean hypocotyls and stimulation of root growth by low concentrations of oxidants. Here we propose that this NADH oxidase and the redox protons released by its action control growth. The mechanism for this may be the evolution of protons into a special membrane domain, from which a signal to initiate cell proliferation may originate, independent of the action of the H⁺-ATPase-generated protons. It is also possible that both expansion and proliferative growth are controlled by redox-generated protons.     

Ascorbate free-radical reduction by glyoxysomal membranes

Glyoxysomal membranes from germinating castor bean (Ricinus communis L. cv Hale) endosperm contain an NADH dehydrogenase. This enzyme can utilize extraorganellar ascorbate free-radical as a substrate and can oxidize NADH at a rate which can support intraglyoxysomal demand for NAD⁺. NADH:ascorbate free-radical reductase was found to be membrane-associated, and the activity remained in the membrane fraction after lysis of glyoxysomes by osmotic shock, followed by pelleting of the membranes. In whole glyoxysomes, NADH:ascorbate free-radical reductase, like NADH:ferricyanide reductase and unlike NADH:cytochrome c reductase, was insensitive to trypsin and was not inactivated by Triton X-100 detergent. These results suggest that ascorbate free-radical is reduced by the same component which reduces ferricyanide in the glyoxysomal membrane redox system. NADH:ascorbate free-radical reductase comigrated with NADH:ferricyanide and cytochrome c reductases when glyoxy-somal membranes were solubilized with detergent and subjected to rate-zonal centrifugation. The results suggest that ascorbate free-radical, when reduced to ascorbate by membrane redox system, could serve as a link between glyoxysomal metabolism and other cellular activities.     

Orientation of electron transport activities in the membrane of intact glyoxysomes isolated from castor bean endosperm

Intact glyoxysomes were isolated from castor bean endosperm on isometric Percoll gradients. The matrix enzyme, malate dehydrogenase, was 80% latent in the intact glyoxysomes. NADH:ferricyanide and NADH:cytochrome c reductase activities were measured in intact and deliberately broken organelles. The latencies of these redox activities were found to be about half the malate dehydrogenase latency. Incubation of intact organelles with trypsin eliminated NADH:cytochrome c reductase activity, but did not affect NADH:ferricyanide reductase activity. NADH oxidase and transhydrogenase activities were negligible in isolated glyoxysomes. Mersalyl and Cibacron blue 3GA were potent inhibitors of NADH:cytochrome c reductase. Quinacrine, Ca²⁺ and Mg²⁺ stimulated NADH:cytochrome c reductase activity in intact glyoxysomes. The data suggest that some electron donor sites are on the matrix side and some electron acceptor sites are on the cytosolic side of the membrane.     

α-Adrenergic stimulation of trans-sarcolemma electron efflux in perfused rat heart. Possible regulation of Ca2+-channels by a sarcolemma redox system

The role of trans-sarcolemma membrane electron efflux in the α-adrenergic control of Ca²⁺ influx in perfused rat heart was examined. Electron efflux was measured by monitoring the rate of reduction of extracellular ferricyanide and compared with changes in contractility, as an indirect assessment of changes in cytoplasmic Ca²⁺ concentration. Methoxamine and phenylephrine each increased the rate of ferricyanide reduction from 80 to approx. 114 nmol/min per g wet wt. of heart, with half-maximal activation occurring at 10 μM for each agonist. Activation of the rate of ferricyanide reduction by both 10 μM methoxamine and 10 μM phenylephrine was blocked by the α-adrenergic antagonist, phenoxybenzamine, but not by the β-antagonist, propranolol. Stimulation of the rate of ferricyanide reduction by the α-agonist coincided with the increase in contractility, each reaching maximum values at approx. 80 s. Removal of the α-agonists led to parallel decreases in contractility and the rate of reduction, each returning to pre-stimulation values in approx. 400 s. In addition, the relationship between Ca²⁺ and ferricyanide reduction was examined. Perfusion of the heart with medium containing 6 mM CaCl₂ significantly increased contractility and the rate of ferricyanide reduction. Perfusion of the heart with low Ca²⁺ diminished contractility, did not affect the rate of ferricyanide reduction, but amplified the stimulatory effect of methoxamine on this rate. The increase in ferricyanide reduction by α-adrenergic agonists resulted from a change in the apparent V_max, indicative of an increase in electron efflux sites in the plasma membrane. It is concluded that α-adrenergic control of electron efflux closely parallels changes in contractility and therefore changes in the cytoplasmic concentration of Ca²⁺. The data suggest that α-agonist-mediated changes in electron efflux may lead to Ca²⁺ influx.     

PHYTOPLANKTON PLASMA MEMBRANE REDOX ACTIVITY: EFFECT OF IRON LIMITATION AND INTERACTION WITH PHOTOSYNTHESIS1

Phytoplankton plasma membrane electron transport activity was determined by monitoring the reduction of the impermeant artificial electron acceptor ferricyanide in a range of diatoms. The results revealed that constitutive plasma membrane electron transport activity of marine diatoms is high compared with chlorophytes and higher plant cells. Diatom plasma membrane electron transport activity was not significantly increased by iron limitation. This lack of induction on iron limitation indicates that diatoms have an iron acquisition strategy that is distinct from chlorophytes and the dicotyledon higher plants that exhibit marked increases in plasma membrane ferricyanide reductase activity on iron limitation. The interaction of the constitutive plasma membrane electron transport with photosynthesis was also investigated. We found that 1) ferricyanide reduction at the plasma membrane was progressively inhibited in response to increasing irradiances; 2) the presence of extracellular ferricyanide, but not the reduced couple ferrocyanide, caused a marked inhibition of carbon fixation at high irradiance; and 3) extracellular electron acceptors ferricyanide and hexachloroiridate (but not ferrocyanide) induced an immediate and reversible decrease in fluorescence yields (F_o and F_m). The extent to which extracellular electron acceptors affected CO₂ fixation, F_o, and F_m was related to the level of constitutive ferricyanide reductase activity, the species with highest ferricyanide reduction rates being most sensitive. The data suggest that consumption of electrons and/or reductant at the plasma membrane by external acceptors may compete directly with CO₂ fixation for electrons, alter cytosolic‐chloroplast redox poise, and/or induce a redox‐signaling cascade that alters photosynthetic metabolism.     

Plasma membrane redox system activity in tumour cell lines of mammary origins with different proliferation rates

Summary Plasma membrane redox systems seem to play a role in the control of cell growth. In fact, we have found that in mammary tumour cell lines the increase in the proliferation rate is accompanied by a decrease in the plasma membrane redox activity. The oxygen consumption rates, the glycolytic fluxes and other bioenergetic parameters have been studied in two cell strains of Ehrlich ascites tumour with different proliferation rates. In the more proliferative Ehrlich cell strain, the decrease in plasma membrane redox system activity is accompanied by decreased oxygen consumption and glycolytic flux and to a generally less energised status.     

Pinocytosis and locomotion in amoebae

Summary Different methods were used to demonstrate the existence of Ca⁺⁺-binding sites (Ca⁺⁺-bs) at the plasma membrane ofAmoeba proteus. In pinocytoting animals the number (indicated by the average distanced in nm) and size (average longitudinal axiss in nm) of Ca⁺⁺-bs at the cytoplasmic surface of the cell membrane were significantly increased (d=162±15;n=41 ands=93±5;n=47) in comparison to controls (d=208 ±21;n=37 ands=59±8;n=45). The ratio of P: Ca obtained by X-ray microanalysis was in the range of 1.5. The differences observed in the two experimental groups of amoebae are explained by conformational changes in the molecular structure and an increased Ca⁺⁺-permeability of the plasma membrane during induced pinocytosis.Microplasmodia of the acellular slime moldPhysarum polycephalum investigated for comparison were found to have no Ca⁺⁺-bs at the interior cell surface.     

Properties of a transplasma membrane electron transport system in HeLa cells

A transmembrane electron transport system has been studied in HeLa cells using an external impermeable oxidant, ferricyanide. Reduction of ferricyanide by HeLa cells shows biphasic kinetics with a rate up to 500 nmoles/min/g w.w. (wet weight) for the fast phase and half of this rate for the slow phase. The apparentK _m is 0.125 mM for the fast rate and 0.24 mM for the slow rate. The rate of reduction is proportional to cell concentration. Inhibition of the rate by glycolysis inhibitors indicates the reduction is dependent on glycolysis, which contributes the cytoplasmic electron donor NADH. Ferricyanide reduction is shown to take place on the outside of cells for it is affected by external pH and agents which react with the external surface. Ferricyanide reduction is accompanied by proton release from the cells. For each mole of ferricyanide reduced, 2.3 moles of protons are released. It is, therefore, concluded that a transmembrane redox system in HeLa cells is coupled to proton gradient generation across the membrane. We propose that this redox system may be an energy source for control of membrane function in HeLa cells. The promotion of cell growth by ferricyanide (0.33–0.1 mM), which can partially replace serum as a growth factor, strongly supports this hypothesis.     

Transmembrane ferricyanide reductase activity in Ehrlich ascites tumor cells

A transmembrane ferricyanide reductase activity was assayed in intact Ehrlich ascites tumor cells. Kinetic measurements gave a Km of 0.14 mM and a Vmax of 0.31 mumol/min per 10(6) cells. In short-term batch experiments, this activity was enhanced in the presence of 10 mM lactate, a source of cytosolic NADH. The transmembrane redox activity was accompanied by alkalinization of the cytosol. Both ferricyanide reduction and proton extrusion were diminished in the presence of 0.2 mM amiloride. Several cytotoxic drugs significantly inhibited the ferricyanide reductase activity at concentrations at which they show antineoplastic activity.     

Plasma Membrane Redox System in Guard Cell Protoplasts of Pea (Pisum sativum L.)

Guard cell protoplasts (GCP) from leaves of pea (Pisum sativum)were capable of reducing/oxidizing the membrane impermeableelectron carriers, ferricyanide/NADH. The redox activity ofGCP required the presence of both ferricyanide and NADH, althoughsome ferricyanide reduction occurred even in the absence ofNADH. The GCP preferred NADH to NADPH during ferricyanide reductionand the reduction was slow with DCPIP or cytochrome c. A stoichiometryof about 2 existed between moles of ferricyanide reduced andNADH oxidized by GCP. The redox activities of GCP were severaltimes greater than those of mesophyll protoplasts from pea leaves.The ferricyanide reduction or NADH oxidation by GCP was unaffectedby abscisic acid or sodium orthovanadate and fusicoccin indicatingthe non-involvement of plasma membrane ATPase in these redoxreactions.The redox activities were markedly inhibited by chloroquineor 8-hydroxyquinoline. The findings are discussed in relationto the possible regulatory role of a guard cell plasma membraneredox system in stomatal function. Key words: Plasma membrane redox system, mesophyll protoplasts, pea, guard cell protoplasts, stomatal function     

Studies on cessation of cytoplasmic streaming under K+-induced depolarization inNitella axilliformis

Internodal cells ofNitella axilliformis had a membrane potential of about−120mV and showed active cytoplasmic streaming with a rate of about 90 μm/sec in artificial pond water (APW) at 25C. When APW was replaced with 50 mM KCl solution, the membrane potential depolarized accompanying an action potential, and the cytoplasmic streaming stopped. Soon after this quick cessation, the streaming started again, but its velocity remained very low for at least 60 min. Removal of KCl from the external medium led to repolarization of the membrane and accelerated recovery of the streaming. The change in the concentration of free Ca²⁺ in the cytoplasm ([Ca²⁺]_c) was monitored by light emission from aequorin which had previously been injected into the cytoplasm. Upon application of KCl to the external medium, the light emission, i.e., [Ca²⁺]_c, quickly increased. It then decreased exponentially and reached the original low level within 100 sec. The cause of the long-lasting inhibition of cytoplasmic streaming observed even when [Ca²⁺]_c had returned to its low resting level is discussed based on the mechanism proposed for action potential-induced cessation of cytoplasmic streaming; inactivation of myosin by Ca²⁺-dependent phosphorylation or formation of cross bridge between actin filaments and myosin.     

Ascorbate is regenerated by HL-60 cells through the transplasmalemma redox system

Ascorbate was maintained in the media during a long-term culture by HL-60 cells. The chemical oxidation of ascorbate was reversed in vitro by living HL-60 cells and was related to the amount of cells added. The increase of NADH concentration by lactate addition to cells was accompanied by an increase of both ascorbate regeneration and ferricyanide reduction. Further, plasma membrane enriched fractions from HL-60 cells revealed enhancement of both ascorbate regeneration and ferricyanide reduction in the presence of NADH when previously treated with detergent. The blockage of cell surface carbohydrates by wheat germ agglutinin (WGA) and Concanavalina ensiformis (Con A) lectins significantly inhibited the regeneration of ascorbate caused by the cells. These results support the idea that ascorbate is externally regenerated by the NADH-ascorbate free radical reductase as a part of the transplasma membrane redox system.     

Comparison of the redox activities in plasma membranes from roots and shoots of etiolated bean seedlings

Summary Plasma membrane (PM) vesicles were purified in parallel from the roots and shoots of 6-day-old etiolated bean (Phaseolus vulgaris L.) seedlings, grown in water culture at 25 °C, by aqueous polymer two-phase partitioning. The purity of PM fractions was determined by measuring the activity of known marker enzymes (vanadate-sensitive Mg-ATPase, 1,3--glycan synthase, latent ID-Pase, cytochromec oxidase, and antimycin-A-insensitive cytochromec reductase). NADH-(acceptor) oxidoreductase activities were determined with the following electron acceptors: ferricyanide, cytochromec, duroquinone, monodehydroascorbate, Fe³⁺-EDTA, and oxygen. Cytochromeb content was also determined. In general, results show that redox activities are higher in the root PM than in the shoot PM which follows the glycan synthase II (PM marker) pattern. The relative activities of the distinct redox enzymes measured (activity profile) are remarkably similar in the root and shoot PM preparations. The cytochromeb content and level of ascorbate reduction were also similar in both plant organs. However, the ratio of NADH-(acceptor) oxidoreductase activity to cytochrome content was signifcantly higher in roots when compared to the shoots.Abbreviations CCO cytochromec oxidase - CCR cytochromec reductase - GSII 1,3--glycan synthase - MF microsomal fraction - N-CC-OR NADH-cytochromec oxidoreductase - N-DQ-OR NADH-duroquinone oxidoreductase - N-FC-OR NADH-ferricyanide oxidoreductase - N-FE-OR NADH-Fe³⁺-EDTA oxidoreductase - N-MDA-OR NADH-monodehydroascorbate oxidoreductase - PM plasma membrane     

Calcium mobilizing hormones activate the plasma membrane Ca2+ pump of pancreatic acinar cells

Summary ⁴⁵Ca fluxes and free-cytosolic Ca²⁺ ([Ca²⁺] _i ) measurements were used to study the effect of Ca²⁺-mobilizing hormones on plasma membrane Ca²⁺ permeability and the plasma membrane Ca²⁺ pump of pancreatic acinar cells. We showed before (Pandol, S.J., et al., 1987.J. Biol. Chem. 262:16963–16968) that hormone stimulation of pancreatic acinar cells activated a plasma membrane Ca²⁺ entry pathway, which remains activated for as long as the intracellular stores are not loaded with Ca²⁺. In the present study, we show that activation of this pathway increases the plasma membrane Ca²⁺ permeability by approximately sevenfold. Despite that, the cells reduce [Ca²⁺]i back to near resting levels. To compensate for the increased plasma membrane Ca²⁺ permeability, a plasma membrane Ca²⁺ efflux mechanism is also activated by the hormones. This mechanism is likely to be the plasma membrane Ca²⁺ pump. Activation of the plasma membrane Ca²⁺ pump by the hormones is time dependent and 1.5–2 min of cell stimulation are required for maximal Ca²⁺ pump activation. From the effect of protein kinase inhibitors on hormone-mediated activation of the pump and the effect of the phorbol ester 12-0-tetradecanoyl phorbol, 13-acetate (TPA) on plasma membrane Ca⁺ efflux, it is suggested that stimulation of protein kinase C is required for the hormone-dependent activation of the plasma membrane Ca²⁺ pump.     

Evidence for coenzyme Q function in transplasma membrane electron transport

Transplasma membrane electron transport activity has been associated with stimulation of cell growth. Coenzyme Q is present in plasma membranes and because of its lipid solubility would be a logical carrier to transport electrons across the plasma membrane. Extraction of coenzyme Q from isolated rat liver plasma membranes decreases the NADH ferricyanide reductase and added coenzyme Q10 restores the activity. Piericidin and other analogs of coenzyme Q inhibit transplasma membrane electron transport as measured by ferricyanide reduction by intact cells and NADH ferricyanide reduction by isolated plasma membranes. The inhibition by the analogs is reversed by added coenzyme Q10. Thus, coenzyme Q in plasma membrane may act as a transmembrane electron carrier for the redox system which has been shown to control cell growth.     

Modulation of the Plasma Membrane Electron Transfer System in Root Cells of Limnobium stoloniferum by External pH

The influence of ferricyanide on transmembrane electron transfer,proton secretion, membrane potential, and cytoplasmic pH ofLimnobium stoloniferum (G.F. Mey) Griseb. root cells was investigatedat different external pH HCF III reduction by the roots was accompanied by membrane depolarization,an increase in proton secretion and by alkalinization of thecytoplasm. Change of membrane potential and cytoplasmic pH aswell as transmembrane e^– transfer was more pronouncedat acid external pH. The rate of proton flux was linearly dependenton the rate of electron transfer. The slope of the relationshipwas around 1, independent of external pH The data are in agreement with the hypothesis that electrontransfer at the plasma membrane is directly coupled to protonsecretion. It is suggested that both e^– and redox-coupledH⁺ transport are activated by acid external pH Key words: Plasmalemma redox system, electron transfer, proton transport, pH, membrane potential, Limnobium stoloniferum     

Cation stimulation of the proton-translocating redox activity at the plasmalemma of Catharanthus roseus cells

Cultured Catharanthus roseus cells exhibit transmembrane ferricyanide reduction through a plasma membrane redox system which may be associated with proton translocation. Evidence shows that endogenous pyridine nucleotides serve as hydrogen donors for the reaction. The proton translocating function of the redox system is confirmed, in intact cells and isolated protoplasts, by the ability of Ca²⁺ and other cations to increase both the redox activity and the efflux of protons. The role of the cations is seen to be not a simple general charge screening phenomenon as already described. By using ionic surfactants (CP⁺, SDS^–) it was shown that the net surface charge of the membrane can interact in the activation process via a cation attraction effect. It is proposed that specific binding of cations to the plasma membrane could alter the conformation of the redox system facilitating its interaction with NADH.Abbreviations CP⁺ cetylpyridinium - EGTA ethylene glycol bis (-aminoethyl)-N,N-tetraacetic acid - FeCN potassium ferricyanide - SDS^– sodium dodecyl sulfate - SHAM salicylhydroxamic acid     

Local accumulation of membrane-associated calcium according to cell pattern formation inMicrasterias denticulata,visualized by chlorotetracycline fluorescence

Summary Developing cells ofMicrasterias denticulata Bréb. show a characteristic fluorescence of the plasma membrane (or cortical protoplasm) after treatment with chlorotetracycline (CTC), which is known to be an indicator for membrane-bound Ca²⁺. Depending on the stage of development the fluorescing sites of the young half cell are distributed in a specific pattern which corresponds to cell pattern formation. Therefore growth and thus cytomorphogenesis inMicrasterias seem to be mediated by a patterned accumulation of Ca²⁺ at the periphery of the differentiating cell. Participation of Ca²⁺ in a membrane-recognition process responsible for local vesicle incorporation is discussed.