Multifunctional plasma membrane redox systems

Miguel Angel Medina, Antonio del Castillo-Olivares and lgnacio Nuiiez de Castro Summary

All the biological membranes contain oxidoreduction systems actively involved in their bioenergetics. Plasma membrane redox systems seem to be ubiquitous and they have been related to several important functions, including not only their role in cell bioenergetics, but also in cell defense through the generation of reactive oxygen species, in iron uptake, in the control of cell growth and proliferation and in signal transduction. In the last few years, an increasing number of mechanistic and molecular studies have deeply widened our knowledge on the function of these plasma membrane redox systems. The aim of this review is to summarize what is currently known about the components and physiological roles of these systems. Introduction Cell membranes are not simple passive barriers delimiting compartments, but actively participate in the exchange of matter, energy and information with the cell environment and between different cell compartments inside the cell. Redox reactions are essential in the active role of cell membranes in bioenergetics. Inner mitochondrial and thylakoid membranes have the best known electron transport systems, namely respiratory and photosynthetic electron chains. However, they are not the only working membrane electron transport systems. On the contrary, it should be stressed that every bioenergetically competent cell membrane does contain redox systems(’). Such redox systems, some with more detail and others with less, have been described for the endoplasmic reticulum, the plant tonoplast, the glyoxysomal and the peroxisomal membranes and, of course, the plasma membrane. Surprisingly, the paradigm of the universal presence of redox systems at cell membranes is still not firmly established in the current biological literature, and the existence of membrane redox systems other than those of inner mitochondrialand thylacoid membranes is simply unknown by an important percentage of biologists and it is underscored by most of the rest. A plasma membrane electron transport system or plasma membrane redox system (PMRS) has been found in every living cell tested, either prokaryotic or eukaryotic, including bacteria, cyanobacteria,yeasts, algae, and all kind of plants and animal cell^(^-^). Preliminary observations can be traced back at least to the studies carried out by Voeglin e t a / . in 1925 when they examined a relation between the redox state and cancer@).In 1945, a special cytochrome system that would

Accepted 20June1997

directly drive iron uptake at the plasma membrane of plant cells was proposed(7). In 1947, Brooks reported that cell growth could be stimulated by the addition of external oxidants(*).In 1948, a PMRS was proposed as a basis for gastric secretion which would be directly coupled to proton pumping(g). Several plasma membrane redox activities were reported in the l ) . Despite all these unconnected observations, the comprehensive study of PMRS only began in the mid 1970s, thanks mainly to the pioneering effort and contribution of Crane and collaborator^(^). PMRS are not a simple curiosity, an evolutive relic; on the contrary, there is increasing experimental evidence for their direct involvement in several vital functions, as summarized in Table 1. This review is devoted to the description of PMRS and their multiple functions from a transdisciplinary viewpoint, stressing the similarities and dissimilarities among PMRS from very different organisms.

Characterization of PMRS Direct and definite evidence for the existence of PMRS was only possible to obtain when methods for isolating highly pure plasma membranes were available(5).Now it is well established that PMRS are expressed at the plasma membrane in every kind of cell. However, they remain far from being as well-defined as inner mitochondrial and thylacoid membrane electron transport systems. The main enzyme activities that have been described as components of PMRS are listed in Table 2. Their kinetic characterization has been carried out by using some of the putative natural or artificial

Table 1. Functions in which plasma membrane redox systems have been involved ~~~~~

1 Bioenergetics Modulation of membrane potential Proton extrusion and control of internal pH Energiration of solute transport Control of the cytoplasmic redox state Maintenance of high aerobic glycolytic fluxes in cells deficient or devoid of mitochondria1electron chain 2 Defense Against pathogens Stabilization of antioxidants 3 Structural Prevention of the entry of supernumerary sperm into the fertilized oocyte Cross-linking of polymers at the wall cell 4 Iron uptake

5 Control of cell growth and proliferation Control of mitogenic signals Control of elongation of plant cells Modulation of programmed cell death induced by oxidative stress

Table 2. Plasma membrane redox enzyme activities Cytochrome P450 (E C 1.6.2 4) Ferrireductase (E.C. 1.6.99.13) NADH:acceptor oxidoreductase (E C 1 6.99:) NADH:ascorbate free radical reductase (E C. 1.6.5 4 ) NADH:cytochrome b5 reductase (E C 1.6.2.2.) NADH oxidase (E.C. 1.11.1.1.) NADH:ubiquinone oxidoreductase (E.C. 1.6.5.5.) NADH:vitamine K oxidoreductase (E.C. 1.6.5:) NADPH oxidase (E.C. 1.11.1.2.) Protein disulfide isomerase (E C. 5 3.4.1 .) Thioredoxin reductase (E.C. 1.6.4.5.) This list is not exhaustive All these phenomenological enzyme activities are mentioned in the reviews cited in the text, where the original quotations can be found. ~~

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electron acceptor^(^.^,'^,^^). It is possible that some of these phenomenological enzyme activities correspond to the same PMRS, but it is now clear that there are several special and specialized PMRS; in fact, at least three different specialized PMRS have been described: superoxide generating NADPH o ~ i d a s e ( ’ ~ ,and ’ ~ ) ,two systems for iron uptake: the inducible ‘Turbo’ (in plant cells) and ferrireductase (in yeast)(l5.l6).On the other hand, all the cells - including those containing some special PMRS - do contain some kind of multifunctional PMRS. The main primary electron donors are cytoplasmic NADH and NADPH, and electron acceptors are molecular oxygen, ferric ions or ascorbate free radic a l ~ ( ~The~ cofactors ~ ~ ~ of~ the ~ PMRS ~ ~ ~are) also . varied: flavins and quinones, copper, non-heme iron and thiols. Actually, animal plasma membrane contains ubiquinone, which may play a key role as an electron shuttle(17). However, there is no direct evidence for the presence of ubiquinone in plant plasma membrane, where its role might be carried out by the quinone vitamin K(18). An interesting feature common to PMRS of different cells is their inhibition by Zn2+ ions. There are at least three con-

vergent lines of evidence: (1) Zn2+ ions at concentrations higher than 20 pM completely inhibit plasma membrane ferricyanide reductase activity of Ehrlich ascites tumor cells(1g); (2) Cakmak and Mirschner(20)obtained results suggesting that Znz+ ions directly affect the integrity of cotton plasma membranes by interfering with superoxide generation by NADPH oxidase; and (3) Zn2+ions have also been shown to inhibit selectively a H+channel associated to the NADPH oxidase of n e ~ t r o p h i l s ( ~In ~ ) .all cases, this inhibition is reversible in vitro. For further biochemical and biophysical studies of PMRS, their isolation and purification is required. However, intrinsic difficulties in isolating membrane proteins explain the scarcity of reported purifications of plasma membrane oxidoreduct a ~ e s ( ~ Further ~ - ~ ~ )characterization . of a membrane protein necessitates its functional reconstitution in artificial liposomes. Ehrlich ascites tumor cell plasma membrane ferricyanide reductase activity and the human neutrophil NADPH oxidase have been functionally reconstituted in this way(24,z5). A complementary strategy is the identification and isolation of redox genes by using recombinant DNA techniques. Much more effort must be made to achieve an adequate molecular and genetic characterization of PMRS; in fact, to date, only the components of neutrophil NADPH oxidase, NADH oxidases of several bacteria, and the ferrireductase systems of yeast have been cloned and e x p r e s ~ e d ( ’ ~ , ’ ~ , ~ ~ ) .

Bioenergetic role of PMRS The flux of electrons through PMRS causes changes by itself in the membrane potential and membrane resistance in both photosynthetic and heterotrophic ~ e l l s ( ~ 3 These ~ ) . changes may account for the modifications induced in ionic and organic solute fluxes through the plasma membrane and they stress the central roles of PMRS in bioenergetics. In all the cells tested, the functioning of PMRS is accompanied by an acidification of the medium . Thus, PMRS should be functionally connected to, or should act as, proton pump^(^-^). In plant cells, transplasma membrane electron transport could activate the H+-ATPasedepolarizing the plasma membrane. Transient acidification of the cytoplasm induced by the functioning of PMRS in some plants could also contribute to the activation of the H+-ATPase.However, it is noteworthy that H+ extrusion through the plasma membrane in plants is not solely dependent on H+-ATPase activity; Bown and Crawford(27)have shown that there is a H+ efflux stimulated by the PMRS activity which is independent of H+-ATPase. In animal cells, accumulated evidence supports a connection between PMRS and the Na+/H+a n t i p ~ r t ( ~Here, - ~ ) . proton extrusion is accompanied by an alkalynization of the cytoplasm(28),which works as a mitogenic signal for the On the other hand, neutrophil NADPH oxidase is associated with a Zn2+-sensitiveH+-channel and the activity of this channel is required for the activity of the oxidase(14). The efflux of protons across the plasma membrane results in

an electrochemical potential of protons that provides the energy required for solute transport(30).In this context, some years ago we proposed a comprehensive model for coupling between a transplasma membrane NADH dehydrogenase, the Na+/H+ antiporter, sodium-dependent organic solute transporters and the Na+/K+-ATPase at the plasma membrane in animal cells(30). An important bioenergetic role of PMRS commonly underscored is their contribution to the maintenance of appropiate NAD+/NADH and NADP+/NADPH cytoplasmic ratios(31).In fact, an increased glycolytic flux, leading to an accumulation of NADH in the cytoplasm, induces an increase of PMRS activity. Thus, it has been shown that when cells are incubated in the presence of lactate, an immediate source of cytoplasmic NADH, they maintain a PMRS activity higher than in the control situation(28). A recent paper definitively states the importance that PMRS can have to a comprehensive view of cell bioenergetpo cells (devoid of mitochondrial DNA) can i c ~ ( Vertebrate ~~). usually be grown only in media containing pyruvate and uridine, and removal of either pyruvate and/or uridine leads to a rapid cell death. Namalwa po cells were generated by prolonged treatment over 5 weeks with ethidium bromide; this procedure made an adaptative response of cells possible: there was an induced up-regulation of PMRS in these cells to maintain glycolytic fluxes higher than those usually in control Namalwa cells. When they were treated with an inhibitor of PMRS activity, they lost viability even in the presence of pyruvate and uridine. The up-regulation of PMRS in response to the loss of mitochondria1 activity could explain at least, in part - why energy deficient cells, such as those observed in disease and aging, may survive(32).

Reactive oxygen species and PMRS Generation of reactive oxygen species by PMRS A particular component of PMRS, namely a superoxide generating NADPH oxidase, is responsible for the phenomenon known as respiratory burst, described in different biological system^(^,'^,^^,^^). The best characterized one is the neutrophil NADPH ~ x i d a s e ( ~ which ~ ’ ~ ~plays ~ ~ ) a, central role in the defense against pathogens. In fact, individuals who have lost the functional enzyme or components required to activate the oxidase suffer from chronic granulomatous disease, an inherited condition in which there is an increased susceptibility to bacterial and fungal infections(14). The NADPH oxidase is a transplasma membrane heterodimeric cytochrome b, composed of a small a-subunit (p22phox)and a larger P-subunit (gp91phox),associated with two proteins located in the cytoplasma of unstimulated cells, called p47phoxand p67phox.All of the components have been purified, cloned and ~ e q u e n c e d ( ~Upon . ~ ~ ) .activation of the NADPH oxidase there is a translocation of a small fraction of cytosolic p47phoxand p67phoxto the plasma membrane. In addition, at least another three components are required for

NADPH oxidase activity: (1) Rac2, which is a cytosolic guanine nucleotide-binding protein required for oxidase activation; (2) p40phox,which enhances the activity of the purified recombinant cell-free system and bears a high degree of homology to p47phox,including two SH3 domains; (3) a H+ channel, which is essential for the activity of the oxidase, as mentioned above. Other mammalian cells, such as kidney mesangial or endothelial cells, have been shown to have a NADPH oxidase. This defense weapon is not restricted to mammals, but is also found in fishes, insects and plants(2.12).Plasma membrane-bound NADPH oxidase was suggested to generate superoxide radicals upon infection of potato tubers with incompatible races of Phytophtora i n f e ~ t a n d ~ Afterwards, ~). the oxidase burst has been shown to participate in the plant defense response to pathogens(36). A very special and specialized case is the respiratory burst oxidase of fertilization, involved in the alteration of the extracellular protein coats required to prevent the entry of supernumerary sperm(33).As was the case for other PMRS. this system is also sensitive to Zn2+ions. PMRS also generates H202, which could contribute to protein crosslinking during wall and to a modulation of the thiol-disulfide equilibrium in plant cells(38).In fact, reactive oxygen species generated by PMRS can not only act on plasma membranes but can also control the switching on of some genes through the activation of the transcription factor N F - K B @ , ~ ~ ) .

Protective role of PMRS against reactive oxygen species Many metabolic reactions and exogenous agents generate free radicals, mainly reactive oxygen species, whose levels must be strictly controlled in order to avoid serious damage to cellular structures and functions. Cells are endowed with different enzyme systems and small molecules involved in redox reactions which play a central protective role eliminating these reactive oxygen species. Cell membranes are mainly sensitive to oxidative stress damage by radical chain reactions leading to lipid peroxidation. The main mechanism of protection against reactive oxygen species at the plasma membrane is the breaking of these radical chain reactions by small molecules, namely, ubiquinone and tx-tocopherol inside the lipid bilayer and ascorbate in the interpha~e(~O). It is now well established that extracellular ascorbate is stabilized in the presence of cells, that is, the presence of cells in a medium containing ascorbate prevents its oxidation catalysed by copper ion^('^.^'). A PMRS, namely a NADHubiquinone reductase or NADH-ascorbate free radical reductase, drives electrons to ascorbate free radicals through the lipophylic antioxidant ubiquinone, in this way giving rise to ascorbate s t a b i l i z a t i ~ n (). ~ ~Furthermore, ,~~ ubiquinone would maintain a-tocopherol (a central protective agent against peroxidation of polyunsaturated membrane lipids) in its reduced state(40).Also, the a-tocopheroxyl radical may be reduced to a-tocopherol at the water-lipid

interphase by the antioxidant action of ascorbate-producing ascorbate free radicals, which could then be reduced back to ascorbate by PMRS. It is noteworthy that an increase in ascorbate stabilization can be observed in po HL-60 cells, due to increases of both ubiquinone content and NADH-ascorbate free radical reductase activity in the plasma membrane. These increases can serve to oxidize internal NADH to provide NAD+for glycolysis, as well as providing external resistance to oxidative stress(42).

Reduction of ferric ions by PMRS Ferric ions in the soil may be solubilized by all kind of chelators, including those excreted by microorganisms, such as ferrioxamine or enterochelin, or by the roots of the plants, such as mucigeneic, avenic or caffeic acids(15). In aerobic soils, iron chelates may be taken up as such by the plants; though this translocation process is slow, it can be enough for the iron requirements of grasses. On the other hand, in dicotyledonous and nongraminaceous monocotyledonous species, the reduction of ferric ion to ferrous ion is a prerequisite for uptake of iron across the root plasma mernbrane(l5). Furthermore, if a plant suffers from iron shortage, the reductive system is strongly enhanced; the induction capability of the ferric reductase system has been known since 1961. However, the role of PMRS on ferric ion reduction has been accepted only since the 19 8 0 ~ ( ~ 8The ’ ~ ) .inducible or ‘Turbo’ system, induced in response to iron stress, is different to the constitutive, ubiquitous, plasma membrane reductase present in all kind of cells. In yeasts, a ferrireductase system has been found that is also inducible and greatly increased when the cells are grown in iron-deficient conditions(16). By complementing a mutant yeast lacking this inducible system with DNA from wild-type cells, a nucleotide sequence (FRE1 ) was identified as essential for the ferrireductase activity. The product of this gene is expected to have several transmembrane domains and, interestingly, it has significant sequence homology with gp91 phox, one of the components of the neutrophil cytochrome b(43).A second gene, FREZ, responsible for a residual ferrireductase activity, has also been identified(44).Very recently, evidence for the ferrireductase system being a multicomponent PMRS has been acquired(16). In fact, the gene product of FREl has been shown to lack ferrireductase activity in vifro; it appears that an NADPH de-hydrogenase should be a second component of the ferri-reductase system, and this component should be responsible for the ferrireductase activity in vifro. Furthermore, a third component, the product of UTRl gene, would be a putative cytosolic factor, which would act synergistically with the product of FRE 1 to increase the cell ferrireductase activity. A similar gene for an iron-deficiency-induced plasma membrane reductase has been described for the fission yeast Schizosaccharomyces p ~ r n b e ( ~ ~ ) . In animals, transferrin is the predominant iron-carrying protein in serum. Iron uptake by cells from transferrin has

been proposed to be carried out by two alternative mechanisms: the first and best defined is based on the specific binding of diferric transferrin to its receptor followed by endocytosis of the complex, release of iron at acidic pH in the endosome and recycling of the protein; the second mechanism would be reduction of diferric transferrin at the cell surface and transport of ferrous ions(46).In any case, both processes could be operative simultaneously. In fact, a transmembrane electron transport system seems to be required in the endosome to channel electrons from cytoplasmic reducing agents to ferric ions, and this enzyme may be the same as that present in the plasma membrane from which the vesicle originates. There are also non-transferrin-dependent iron uptake mechanisms in some animal cells; enterocytes can only take iron previously reduced to ferrous ions by reducing compounds, such as ascorbate, in the lumen of the gut; liver and brain can also take up iron by a transferrin-independent mechanism. This is also the case in erythroleukemic K562 cells(47).

Role of PMRS in cell growth and proliferation Differentiated roles in the control of cell growth and proliferation have been reported for PMRS since the beginnings of the history of research on this subject; however, data supporting this suggestion were mostly indirect and incidental. This situation has rapidly changed: diverse lines of evidence are accumulating which show the contribution of redox reactions to control of cell growth(5).A well documented fact is that PMRS activity levels are modified in transformed cells. It has been reported that pineal and 3T3 cells transformed with SV-40 show lower PMRS activity than control non-transformed cell^(^*.^^). As a complementary example, a transient increase in PMRS activity has been shown in phorbol ester-differentiated HL-60 cells(50).On the contrary, there are cases where PMRS activity in neoplastic tissues is higher than in their corresponding normal tissues; for instance, plasma membranes from hepatoma show higher NADH oxidase activity than plasma membranes from normal liver(51).Some studies showing the relationship between oncogene overexpression and PMRS activity are particularly relevant in this context. In neuroblastoma, a positive correlation between PMRS activity and the level of expression of N-myc oncogene has been shown(52).On the other hand, transfection of Ha-ras gene into CBH 1OT1/2 mouse embryo fibroblast cells increases the rate of transplasma membrane electron transport to ferricyanide up to fivefold(53).Furthermore, in the last few years there has been an increasing amount of data pointing to a modulation of the mitogenic signal transduction pathways by PMRS and, inversely, a control of PMRS by components of the signal transduction machinery, as discussed in the next section. Several signals controling cell growth, such as cytoplasmic pH and external superoxide and peroxide, have been demonstrated to be related to PMRS. The release of protons accom-

panying the flow of electrons through PMRS can give rise to hormones, neurotransmitters and growth factors. Although significant changes in cytoplasmic pH, which in turn can attempts have been made to link the action of the plant horbehave as a mitogenic The generation of external mones gibberellins, cytokines and ethylene with PMRS superoxide or peroxide by PMRS also stimulates cell growth activity, data are fragmentary and inconclusive. On the conand is associated with activation of early growth genes trary, the relationship of auxins to PMRS is well docuthrough transcriptional control, as discussed above(12). mented(63).In animal cells, all the hormones so far tested The observation that different antitumor drugs inhibit (including glucagon, insulin, ACTH or triiodothyronine) have PMRS activity at concentrations which inhibit tumor growth is some effect on PMRS activity(3). Neurotransmitters also also consistent with the concept of growth control by affect PMRS activity: neuroexcitatory amino acids markedly PMRS(3,5.28). Very recently, it has been shown that inhibitors stimulate, whereas dopamine, adrenaline and noradrenaline of PMRS induce apoptosis, which can be inhibited by bcl-2 strongly inhibit PMRS in synaptic plasma membranes. Epiand involves calcineurin activation(55).Mild oxidative stress dermal growth factor has been shown to stimulate is a condition involved in the induction of apoptosis. Cells transplasma membrane electron transport in HL-60 cells(13). require serum to maintain growth in vitro. Serum provides The effects of other growth factors on PMRS are not so clear. growth and survival factors and its removal mimicks a mild Most of these effects have not been examined beyond the phenomenological level and more studies are needed to eluoxidative stress-inducing apoptosis through an increase in cidate the actual mechanisms involved in the action of exterceramide second messenger. Antioxidants effective at the nal signals on PMRS. plasma membrane such as ascorbate, a-tocopherol and ubiquinone can protect cells from this mild oxidative damage Cytoplasmic pH and PMRS and prevent apoptosis independently of bcl-2 content. Thus, PMRS represents a first level of protection against mild An internal signal tightly connected to PMRS is cytoplasmic oxidative damage(56,57). pH (see Fig. 1). Electron transport through a PMRS is associIn plants, elongation growth is as important as proliferaated with proton release, either vectorial, as in the case for anition. It is now well established that external oxidants stimumal cells, or non-vectorial, as occurs with plant cells. In plants, late elongation growth in shoots and a role of plant PMRS in the reduction of an external oxidant by PMRS is accompanied elongation growth has been previously d i s c u ~ s e d ( ~ ~ ~ ~ ~ by ) . acidification of both cytoplasm and extracellular medium(64). In animals, PMRS activity produces an alkalynization of cytopla~rn(3~~8). Cytoplasmic proton concentraPMRS and signal transduction tion changes appear to act as a second messenger in several cell types; thus, an increase in cytoplasmic pH seems to The involvement of PMRS in important vital functions points induce animal cell proliferation(54).Of course, cytoplasmic pH to their role as targets of different biochemical signals. It is and PMRS mutually affect each other. In plant cells, treatment expected that PMRS can be implicated in downstream secwith fusicoccin (a fungal metabolite that produces an ond messenger function by direct action on cytoplasmic increased proton extrusion, among other effects) increases components, or by modification of other membrane enzymes the rate of PMRS a~tivity(6~). In animal cells, any treatment directly involved in second messenger synthesis(59).On the leading to a slight but significant decrease of cytoplasmic pH other hand, PMRS respond to upstream modulatory external produces a significant inhibition of PMRS activity; on the conand internal signals. trary, treatments which produce a slight alkalynization of the Extracellular regulatory signals and PMRS cytoplasm enhance PMRS a ~ t i v i t y ( ~ ~ x ~ ~ ) . The first extracellular regulatory signal for plants is light. The G proteins and PMRS interconnections between PMRS and blue light are well documented(2). Blue light activates electron transport in plant plasma membranes and increases the phosphorylation state of two membrane-associated proteins(60f'1).In addition, the dephosphorylation of a redox protein has been proposed to be crucial for blue light-stimulated redox activity. On the other hand, some effects of blue light seem to involve or be mediated by PMRS. This is the case of the recently described involvement of PMRS in the photopolarization of the Fucus sp. zygote by blue light(62).The possible linkage between PMRS and red light is more controversial, although it would not be difficult to determine whether phytochrome regulates the activity of plant PMRS by using any of several phytochrome-responsive tissued2). Other external signals modulate PMRS activity, including

G proteins play key roles in many hormonal and sensory transduction processes in eukaryotes. Recently, evidence has been shown for the involvement of G proteins in the modulation of PMRS in both plant and animal cell.^(^^-^^). However, another control by GTP, different from that of G proteins, seems to occur on the NADH oxidase activity described for rat liver and soybean hypocotyl plasma membrane vesicle^(^^^^^). Araquidonic acid may play an important role as a signal transduction molecule for G protein-coupled receptors. Very recently, it has been shown that araquidonic acid activates c-jun N-terminal kinase by means of NADPH oxidase generation of superoxide, independent of eicosanoid biosynthesis(68).

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Fig. 2. Scheme of the modulation of PMRS activity by G proteins and cyclic nucleotides. PM, plasma membrane: PMRS, plasma membrane redox system; R, receptor: G, protein G: AC, adenyl cyclase.

Proliferation

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Fig. 1. Scheme of the relationship of PMRS with proton fluxes and with changes in cytoplasmic proton concentration as mitogenic signals PM, plasma membrane PMRS plasma membrane redox system, N. nucleus

Signaling involving cyclic nucleotides Currently, one of the most active areas of investigation in the study of PMRS involves protein phosphorylation/dephosphorylation. Evidence is accumulating for the involvement of different protein kinases and phosphatases in the control of PMRS activity and for the transduction of signals downstream of PMRS through pathways involving cascades of phosphorylation/dephosphorylation. Activation of certain G proteins results in an activation of adenylate cyclase and an increase in cytosolic concentrations of cyclic AMP, one of the firstly described second messengers, which is required for activation of many cellular events. Cyclic AMP-dependent protein phosphorylation has been shown to control PMRS in yeasts and animal cells(16,67.69). Although much less is known about the role of cyclic GMP as a second messenger, there is little doubt that it should play an important role in signal transduction. It seems that in Ehrlich ascites tumor cells PMRS can be activated by cyclic GMP(67).Fig. 2 summarizes these data. PMRS in signal transduction pathways involving protein kinases C and Ca2+ Several clues seem to indicate a role for certain protein kinases C in the control of PMRS. It has been hypothesized that phospholipase C activation iniciates a cascade of events by which elicitors trigger the oxidative burst in cultured plant cells(70).Active phospholipase C releases inositol tris-phosphate and diacylglycerol, giving rise to a transient increase in cytosolic Ca2+ concentrations and to activation of protein kinase C. The involvement of these signal transduction pathways in the control of Ehrlich cell PMRS has been recently

s h o ~ n ( ~ On ~,~ the ~ )other . hand, PMRS activity has been shown to increase cytosolic Ca2+concentrations and to activate protein kinase C. Fig. 3 illustrates the above described interconnections. Other data on phosphorylatioddephosphorylation and PMRS Protein phosphatases have been little studied in the context of PMRS activity control. Inhibition of phosphoprotein phosphatases produces a significant increase in Ehrlich cell PMRS activity(71). Activation of protein tyrosine phosphorylation is an early event in many signal transduction pathways. This may involve either activation of tyrosine kinases or inhibition of tyrosine-phosphate phosphatases. It has been suggested that p72Syk,the tyrosine kinase that phosphorylates the band 3 protein of erythrocytes, could be activated by PMRS(72). The activation of a tyrosine kinase is also consistent with the published activation of PMRS activity in Ha-ras transformed

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Fig. 3. Model for the interactions of PMRS with calcium ions and protein kinase C signaling pathways. PM, plasma membrane; PMRS. plasma membrane redox system, R, receptor; G . protein G: PLC. phospholipase C: PL. phospholipid. SL, sphingolipid; PKC, protein kinase C; DAG, diacyl glycerol; Ips, inositol trisphosphate; S1 P. sphingosine-1-phosphate: ER, endoplasmic reticulum.

~ells(5~ It )is. well established that neutrophil NADPH oxidase A currently emerging area of interest is the protective role activates a tyrosine k i n a ~ e ( ~on~ the ) ; other hand, the same of PMRS against oxidative stress damage. NADPH oxidase is activated by tyrosine pho~phorylation(~~). In conclusion, the multiple data presented in this review Finally, different data point to a connection of PMRS with ras confirm that the plasma membrane in all kind of cells is and MAP kinase pathways(53~70~71 ,74). involved in electron transport mechanisms implicated in a Even though the data are fragmentary and most of them variety of cellular vital functions. unconclusive, they can be hypothetically arranged as in Fig. 4.

References Prospective According to the key roles that they seem to play in cell physiology, PMRS should be expected to be accurately regulated and to be involved in more than one regulatory pathway. In fact, this is the general conclusion that can be obtained from the available data on the relationships of PMRS with signal transduction events. Much remains to be done in order to elucidate the actual involvement of different signal transduction pathways in the control of PMRS, as well as to determine with accuracy the sequence of events upstream and downstream of the PMRS. This should be an area of very active research in the near future. A deeper knowledge of the components of PMRS at the molecular level and studies on structure/function relationships are also needed. Although neutrophil NADPH oxidase and yeast ferrireductase are known in some detail and their components have been cloned, this is not the case for the PMRS constitutively expressed in all kind of cells. The actual role of PMRS in whole-cell bioenergetics and a possible modulation of proteins directly involved in the control of cell cycle by PMRS should be also further investigated. out

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Fig. 4. A speculative model for the involvement of plasma membrane redox systems in signal transduction. '+' indicates activation and '?' indicates that the detailed signal transduction pathway remains unknown. PM. plasma membrane; PMRS, plasma membrane redox system; R, receptor; G . protein G; MAP-K, MAP kinase; PPase, protein phosphatase; N, nucleus.

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Miguel Angel Medina, Antonio del Castillo-Olivares and lgnacio Nhiez de Castro are at the Laboratorio de Bioquimica y Biologra Molecular, Facultad de Ciencias, Universidad de Malaga, E-29071 Malaga, Spain ~