Cross bonding and stiffening of the red cell membrane

Cross bonding and stiffening of the human red cell membrane was studied using treatments with SH, amino, and carboxyl reagents, oxidizing and denaturing treatments and acidification. Membrane cross bonding was initiated when, after red cell treatment, opposite areas of the cytoplasmic face of the red cell membrane were brought into contact by cell shrinking. Membrane cross bonding was detected by light microscopy when this contact persisted upon swelling the cells in a hypotonic medium. Membrane stiffening was recorded as a decrease in elongation of red cells in the shear field of a viscous dextran solution. No correlation was found between membrane cross bonding and membrane stiffening. The results are explained by the existence of two modifications of spectrin, type I causing solely membrane stiffening, type II causing membrane cross bonding as well as membrane stiffening. The amino and carboxyl reagents caused only type I modification. The other treatments caused both types of modification although with varying proportions. The results support the previously suggested mechanism of membrane cross bonding which involves a rearrangement of spectrin similar to denaturation by heat or urea, a decrease in associations within the membrane skeletal network, and a lateral aggregation of membrane proteins. These changes are proposed to occur by the type II modification. The data further substantiate the membrane stiffening effect of inter- and intra-molecular cross linking of spectrin which is identified with the type I modification. Finally, hypotheses are presented concerning the mechanism of membrane stiffening due to type II modifications of spectrin.     

Transcellular cross bonding of the red blood cell membrane

When red blood cells are osmotically shrunk, opposing regions of the inner membrane surface touch each other in the dimple area. In normal red cells such a mechanical contact is undone by reswelling the cells. When the cells are treated with the SH reagents diamide or N-ethylmaleimide, or simply heated to temperatures between 42 and 48 degrees C such a mechanical contact can be made permanent by a process termed 'membrane cross bonding'. Cross bonding also occurred when the cells were treated before mechanical contact was established. The bridge between the two cross-bonded membrane regions may be assumed to be formed by membrane skeletal material. Membrane bridges become visible microscopically when the cells are swollen. These bridges are strong enough to resist the membrane tensions occurring at osmotic lysis. Bridged red cells can be a useful tool in rheology, since they are deformable but cannot adapt to shear flows by membrane tank treading.     

Membrane protein organization in ATP-depleted and irreversibly sickled red cells

It has been proposed that the spectrin-actin submembrane network participates in control of red cell shape and deformability. We have examined ATP- and calcium-dependent changes in organization of spectrin in the membrane employing cross-linking of the nearest membrane protein neighbors by spontaneous or catalyzed (CuSO₄, O-phenanthroline) intermolecular disulfide couplings and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Cross-linking of fresh red cells resulted in the formation of spectrin and actin dimers and tetramers. ATP-depleted red cells differed from fresh cells in the presence of an additional reducible polymer of MW > 1 × 10⁶ selectively enriched in spectrin. This polymer formed spontaneously when red cells were depleted of ATP under aerobic conditions. After anaerobic ATP depletion, the polymer formed in ghosts after cross-linking by catalytic oxidation. Polymerization was prevented by maintenance of ATP and coincided with an ATP-dependent discocyte-echinocyte transformation. This suggests that, in ATP-depleted red cells, spectrin is rearranged to establish closer contacts, and that this may contribute to the discocyte-echinocyte transformation. The introduction of greater than 0.5 mM Ca⁺⁺ into ghosts by inclusion in hemolysis buffer or into fresh red cells (but not ATP-depleted red cells) by treatment with ionophore A23187 spontaneously produced a nonreducible polymer which others have attributed to transamidative cross-linking of spectrin, band 3, and other proteins. Spontaneous formation of both polymer types (reducible in aerobically ATP-depleted red cells and nonreducible in fresh, Ca⁺⁺ enriched red cells) resulted in stabilization (“autocatalytic fixation”) of spheroechinocytic shape. Irreversibly sickled cells, which have increased calcium and decreased ATP, and exhibit a permanent membrane deformation, failed to form any of the above polymers. This suggests that in contrast to normal cells depleted of ATP in vitro, fixation of ISC shape in vivo is not related to Ca- and ATP-dependent membrane protein polymerization. However, ISCs had an increased propensity to form the reducible, spectrin-rich polymer during a subsequent metabolic depletion in vitro. This was associated with transformation of ISCs into spheroechinocytes. Similar echinocytic ISCs were found to constitute 5–10% of the densest fractions of freshly separated ISCs. ISCs then exhibit sphero-echniocyte transformation, both in vitro and in vivo. We propose that this is due to spectrin reorganization that presumably results from the progressively increasing calcium and decreasing ATP of ISCs. These data provide evidence of altered spectrin organization in membranes of ATP-depleted, calcium-enriched red cells in vitro and in vivo.     

Formation of disulfide bonds between glutathione and membrane sh groups in human erythrocytes

In erythrocytes treated with the SH-oxidizing agent, diamide, mixed disulfide bonds between membrane proteins and GSH are formed involving 20% of the membrane SH groups. To study the distribution of these mixed disulfides over the membrane protein fractions, intracellular GSH was labelled biosynthetically with [2-³H]glycine prior to diamide treatment of the cells and the radioactivity of defined membrane peptide fractions determined. Mixed disulfides preferentially occur in the extrinsic protein, spectrin (six SH groups), in addition to the formation of peptide disulfides. Intrinsic proteins are much less reactive: only one SH group of the major intrinsic protein (band 3) reacts with GSH, which accounts for previously observed impossibility to dimerize band 3 via disulfide bonds in intact cells. The labelling method described offers a promising strategy to label and map exposed endofacial SH groups of membrane proteins with a physiological, impermeable marker, GSH.In ghosts treated with diamide and GSH the number of mixed disulfides formed is greater than in erythrocytes. Polymerization of spectrin via intermolecular disulfide bridges is suppressed, while intramolecular disulfides are still formed, providing a means for the analysis of spectrin structure.The diamide-induced mixed membrane-GSH disulfides are readily reduced by GSH. This suggests, that GSH may also be able to reduce mixed disulfides formed in the erythrocyte membrane under oxidative stress in vivo. The reversible formation of mixed disulfides may serve to protect sensitive membrane structures against irreversible oxidative damage.     

Direct involvement of spectrin thiols in maintaining erythrocyte membrane thermal stability and spectrin dimer self-association

Human erythrocytes vesiculate upon exposure to temperatures of 49 degrees C and above. Pretreatment of the cells with the thiol-alkylating agent N-ethylmaleimide (NEM) lowers the temperature needed to produce the same effect. Concomitant with the cells' heat susceptibility, skeletal mechanical instability and an increase in spectrin dissociation have been reported (Smith and Palek (1983) Blood 62, 1190). In the present study, similar results were achieved by preincubation of the cells with diamide, which could be reversed by reduction with dithiothreitol. Another oxidative agent, sodium tetrathionate, could only induce the temperature susceptibility, with little effect on spectrin dissociation. Incubation of spectrin solutions with NEM or diamide caused decreased association of spectrin dimers and increased dissociation of spectrin tetramers. Estimation of membrane and spectrin thiols in the treated cells showed that NEM was effective while blocking less than 20% of the thiols. Diamide and tetrathionate blocked more than 50% of the thiols, but were less effective than NEM. It is suggested that some very defined population of thiols is essential for spectrin self-association and for membrane thermal stability. They are more available to NEM than to diamide and less so to tetrathionate. Other thiols participate in maintaining the membrane thermal stability only.     

The interaction of adenine nucleotides with the red cell membrane: A calorimetric study

The interaction of two adenine nucleotides with the red cell membrane was investigated using highly sensitive differential scanning calorimetry. It was found that ADP and AMP-PNP (an ATP analogue) preferentially modify the A transition, which has been shown to involve the unfolding of a portion of spectrin, an erythrocyte membrane protein complex. The interaction of ADP with spectrin was shown to be reversible and facilitated by the usual cofactor, Mg²⁺. The ADP-induced modification, however, is only observed for membrane associated spectrin; ADP has no effect on extracted spectrin. The results presented are consistent with an ADP-induced conformational change in the spectrin complex which leads to a change in the spectrin-membrane interaction. ADP, but not AMP-PNP, is shown to modify an additional calorimetric transition (B₂) associated with a structural change in the transmembrane protein band 3. This behavior is characteristic of inhibitors of anion transport in the red cell. ADP is also found to be an inhibitor of anion transport in red cells.     

Functional Links between Membrane Transport and the Spectrin Cytoskeleton

Membrane transporters precisely regulate which molecules cross the plasma membrane and when they can cross. In many cases it is also important to regulate where substances can cross the plasma membrane. Consequently, cells have evolved mechanisms to confine and stabilize membrane transport proteins within specific subdomains of the plasma membrane. A number of different transporters (including ion pumps, channels and exchangers) are known to physically associate with the spectrin cytoskeleton, a submembrane complex of spectrin and ankyrin. These proteins form a protein scaffold that assembles within discrete subdomains of the plasma membrane in polarized cells. Recent genetic studies in humans and model organisms have provided the opportunity to test the hypothesis that the spectrin cytoskeleton has a direct role in restricting transporters to specialized domains. Remarkably, genetic defects in spectrin and ankyrin can produce effects on cell physiology that are comparable to knockouts of the transporters themselves.     

Involvement of ATP-dependent aminophospholipid translocation in maintaining phospholipid asymmetry in diamide-treated human erythrocytes

Crosslinking of membrane skeletal proteins such as spectrin by oxidation of their SH-groups can be provoked by treatment of intact erythrocytes with diamide. Shortly after exposure of human erythrocytes to diamide and despite the transverse destabilization of the lipid bilayer that was observed in these cells (Franck, P.F.H., Op den Kamp, J.A.F., Roelofsen, B. and Van Deenen, L.L.M. (1986) Biochim. Biophys. Acta 857, 127-130), no abnormalities could be detected regarding the asymmetric distribution of the phospholipids when probed by either the prothrombinase assay or brief exposure of the cells to a modified phospholipase A2 with enhanced membrane penetrating capacity. This asymmetry appeared to undergo dramatic changes however, when the ATP content of the cytosol had decreased to less than 10% of its original level during prolonged incubation of the treated cells. These observations indicate that the initial maintenance of phospholipid asymmetry in diamide-treated erythrocytes can be solely ascribed to the action of the ATP-dependent aminophospholipid translocase. This view is supported by experiments involving radiolabeled phospholipids of which trace amounts had been inserted into the outer membrane leaflet of diamide-treated red cells and which still showed a preferential translocation of both aminophospholipids in favour of the inner monolayer, be it that the efficiency of the translocase was found to be impaired when compared to control cells.     

Effect of the level of ATP and of the state of spectrin on osmotic properties of bovine erythrocytes

The effect of the intracellular level of ATP and of the state of spectrin on the critical cell volume of bovine erythrocyte was studied. The state of spectrin was changed by thermal denaturation, which for the bovine red cell took place at similar temperature as for the human erythrocyte. The increase of the ATP level and the spectrin denaturation increased the critical cell volume, while metabolic starvation decreased it. The changes of the ATP level did not influence the critical volume after the denaturation of spectrin. The results suggest that the ATP-dependent effect on the critical cell volume was caused by an alteration of the membrane extensibility due to the change of the membrane skeleton-lipid bilayer interaction(s).     

Red Blood Cell Shape and Fluctuations: Cytoskeleton Confinement and ATP Activity

We review recent theoretical work that analyzes experimental measurements of the shape and fluctuations of red blood cells. Particular emphasis is placed on the role of the cytoskeleton and cell elasticity and we contrast the situation of elastic cells with that of fluid-filled vesicles. In red blood cells (RBCs), the cytoskeleton consists of a two-dimensional network of spectrin proteins. Our analysis of the wave vector and frequency dependence of the fluctuation spectrum of RBCs indicates that the spectrin network acts as a confining potential that reduces the fluctuations of the lipid bilayer membrane. However, since the cytoskeleton is only sparsely connected to the bilayer, one cannot regard the composite cytoskeleton membrane as a polymerized object with a shear modulus. The sensitivity of RBC fluctuations and shapes to ATP concentration may reflect the transient defects induced in the cytoskeleton network by ATP.     

The covalent modification of spectrin in red cell membranes by the lipid peroxidation product 4-hydroxy-2-nonenal

Spectrin strengthens the red cell membrane through its direct association with membrane lipids and through protein-protein interactions. Spectrin loss reduces the membrane stability and results in various types of hereditary spherocytosis. However, less is known about acquired spectrin damage. Here, we showed that α- and β-spectrin in human red cells are the primary targets of the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) by immunoblotting and mass spectrometry analyses. The level of HNE adducts in spectrin (particularly α-spectrin) and several other membrane proteins was increased following the HNE treatment of red cell membrane ghosts prepared in the absence of MgATP. In contrast, ghost preparation in the presence of MgATP reduced HNE adduct formation, with preferential β-spectrin modification and increased cross-linking of the HNE-modified spectrins. Exposure of intact red cells to HNE resulted in selective HNE-spectrin adduct formation with a similar preponderance of HNE-β-spectrin modifications. These findings indicate that HNE adduction occurs preferentially in spectrin at the interface between the skeletal proteins and lipid bilayer in red cells and suggest that HNE-spectrin adduct aggregation results in the extrusion of damaged spectrin and membrane lipids under physiological and disease conditions.     

ATP-dependent Mechanism Protects Spectrin against Glycation in Human Erythrocytes

Human erythrocytes are continuously exposed to glucose, which reacts with the amino terminus of the β-chain of hemoglobin (Hb) to form glycated Hb, HbA1c, levels of which increase with the age of the circulating cell. In contrast to extensive insights into glycation of hemoglobin, little is known about glycation of erythrocyte membrane proteins. In the present study, we explored the conditions under which glucose and ribose can glycate spectrin, both on the intact membrane and in solution and the functional consequences of spectrin glycation. Although purified spectrin could be readily glycated, membrane-associated spectrin could be glycated only after ATP depletion and consequent translocation of phosphatidylserine (PS) from the inner to the outer lipid monolayer. Glycation of membrane-associated spectrin led to a marked decrease in membrane deformability. We further observed that only PS-binding spectrin repeats are glycated. We infer that the absence of glycation in situ is the consequence of the interaction of the target lysine and arginine residues with PS and thus is inaccessible for glycation. The reduced membrane deformability after glycation in the absence of ATP is likely the result of the inability of the glycated spectrin repeats to undergo the obligatory unfolding as a consequence of interhelix cross-links. We thus postulate that through the use of an ATP-driven phospholipid translocase (flippase), erythrocytes have evolved a protective mechanism against spectrin glycation and thus maintain their optimal membrane function during their long circulatory life span.     

Anion transport in dog, cat, and human red cells. Effects of varying cell volume and Donnan ratio

Membrane potential and the rate constants for anion self-exchange in dog, cat, and human red blood cells have been shown to vary with cell volume. For dog and cat red cells, the outward rate constants for SO4 and Cl increase while the inward rate constant for SO4 decreases as cells swell or shrink. These changes coincide with the membrane potential becoming more negative as a result of changes in cell volume. Human red cells exhibit a similar change in the rate constants for SO4 and Cl efflux in response to cell swelling, but shrunken cells exhibit a decreased rate constant for SO4 efflux and a more positive membrane potential. Hyperpolarization of shrunken dog and cat red cells is due to a volume-dependent rate constant for SO4 efflux and a more positive membrane potential. Hyperpolarization of shrunken dog and cat red cells is due to a volume-dependent increase in PNa. If this increase in PNa is prevented by ATP depletion or if the outward Na gradient is removed, the response to shrinking is identical to human red cells. These results suggest that the volume dependence of anion permeability may be secondary to changes in the anion equilibrium ratio which in red cells is reflected by the membrane potential. When the membrane potential and cell volume of human red cells were varied independently by a method involving pretreatment with nystatin, it was found that the rate of anion transport (for SO4 and Cl) does not vary with cell volume but rather with membrane potential (anion equilibrium ratio); that is, the rate constant for anion efflux is decreased and that for influx is increased as the membrane potential becomes more positive (internal anion concentration increases) while the opposite is true with membrane hyperpolarization (a fall in internal anion concentration).     

Phosphorylation of membrane proteins from Plasmodium berghei-infected red cells.

Membrane from Plasmodium berghei-infected mouse red cells has a different pattern of phosphorylation by (γ-³²P)ATP from normal membrane. A phosphorylated membrane protein of apparent molecular weight 42,000, absent in membrane from normal cells, can be detected in membrane from infected cells. The new phosphorylated protein can be extracted by 0.1 mM EDTA but not by triton X-100, indicating that it may be red cell actin.     

Interactions of Plasmodium falciparum erythrocyte membrane protein 3 with the red blood cell membrane skeleton

Plasmodium falciparum parasites express and traffick numerous proteins into the red blood cell (RBC), where some associate specifically with the membrane skeleton. Importantly, these interactions underlie the major alterations to the modified structural and functional properties of the parasite-infected RBC. P. falciparum Erythrocyte Membrane Protein 3 (PfEMP3) is one such parasite protein that is found in association with the membrane skeleton. Using recombinant PfEMP3 proteins in vitro, we have identified the region of PfEMP3 that binds to the RBC membrane skeleton, specifically to spectrin and actin. Kinetic studies revealed that residues 38-97 of PfEMP3 bound to purified spectrin with moderately high affinity (K(D(kin))=8.5 x 10(-8) M). Subsequent deletion mapping analysis further defined the binding domain to a 14-residue sequence (IFEIRLKRSLAQVL; K(D(kin))=3.8 x 10(-7) M). Interestingly, this same domain also bound to F-actin in a specific and saturable manner. These interactions are of physiological relevance as evidenced by the binding of this region to the membrane skeleton of inside-out RBCs and when introduced into resealed RBCs. Identification of a 14-residue region of PfEMP3 that binds to both spectrin and actin provides insight into the potential function of PfEMP3 in P. falciparum-infected RBCs.     

Is an intact cytoskeleton required for red cell urea and water transport?

In order to determine the membrane protein(s) responsible for urea and water transport across the human red cell membrane, we planned to reconstitute purified membrane proteins into phosphatidylcholine vesicles. In preparatory experiments, we reconstituted a mixture of all of the red cell integral membrane proteins into phosphatidylcholine vesicles, but found that p-chloromercuribenzenesulfonate (pCMBS), which normally inhibits osmotic water permeability by approximately 90%, has no effect on this preparation. The preparation was also unable to transport urea at the high rates found in red cells, though glucose transport was normal. White ghosts, washed free of hemoglobin and resealed, also did not preserve normal urea and pCMBS-inhibitable water transport. One-step ghosts, prepared in Hepes buffer in a single-step procedure, without washing, retained normal urea and pCMBS-inhibitable water transport. Perturbations of the cytoskeleton in one-step ghosts, by removal of tropomyosin, or by severing the ankyrin link which binds band 3 to spectrin, caused the loss of urea and pCMBS-inhibitable water transport. These experiments suggest that an unperturbed cytoskeleton may be required for normal urea and pCMBS-inhibitable water transport. They also show that the pCMBS inhibition of water transport is dissociable from the water transport process and suggest a linkage between the pCMBS water transport inhibition site and the urea transport protein.     

Remodeling the shape of the skeleton in the intact red cell.

The role of the membrane skeleton in determining the shape of the human red cell was probed by weakening it in situ with urea, a membrane-permeable perturbant of spectrin. Urea by itself did not alter the biconcave disk shape of the red cell; however, above threshold conditions (1.5 M, 37 degrees C, 10 min), it caused an 18% reduction in the membrane elastic shear modulus. It also potentiated the spiculation of cells by lysophosphatidylcholine. These findings suggest that the contour of the resting cell is not normally dependent on the elasticity of or tension in the membrane skeleton. Rather, the elasticity of the skeleton stabilizes membranes against deformation. Urea treatment also caused the projections induced both by micropipette aspiration and by lysophosphatidylcholine to become irreversible. Furthermore, urea converted the axisymmetric conical spicules induced by lysophosphatidylcholine into irregular, curved and knobby spicules; i.e., echinocytosis became acanthocytosis. Unlike controls, the ghosts and membrane skeletons obtained from urea-generated acanthocytes were imprinted with spicules. These data suggest that perturbing interprotein associations with urea in situ allowed the skeleton to evolve plastically to accommodate the contours imposed upon it by the overlying membrane.     

Interaction of calmodulin with the red cell and its membrane skeleton and with spectrin

The binding of calmodulin to red cell membrane cytoskeletons and to purified spectrin from red cells and bovine brain spectrin (fodrin) has been examined. Under physiological solvent conditions binding can be measured by ultracentrifugal pelleting assays. The membrane cytoskeletons contained a single class of binding sites, with a concentration similar to that of spectrin dimers and an association constant of 1.5 X 10(5) M-1. Binding is calcium dependent and is suppressed by the calmodulin inhibitor trifluoperazine. The binding showed a marked dependence on ionic strength, with a maximum at 0.05 M, and a steep dependence on pH, with a maximum at pH 6.5. It was unaffected by 5 mM magnesium. An azidocalmodulin derivative, under the conditions of our experiments, did not label the spectrin-containing complex, although it could be used to demonstrate binding to fodrin. Binding of calmodulin to spectrin tetramers and fodrin in solution could be demonstrated by a pelleting assay after addition of F-actin. Calculations (which are necessarily rough) suggest that at the free calcium concentration prevailing in a normal red cell about 1 in 20 of the calmodulin binding sites in spectrin will be occupied; this proportion will rise rapidly with increasing intracellular calcium. To determine whether inhibition of calmodulin binding to red cell proteins disturbs the control of cell shape, as has been suggested, calcium ions were removed from the cell by addition of an ionophore and of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to the external medium. This did not affect the discoid shape. Trifluoperazine still induced stomatocytosis, exactly as in untreated cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

The human erythrocyte proteome: analysis by ion trap mass spectrometry

This report describes an analysis of the red blood cell proteome by ion trap tandem mass spectrometry in line with liquid chromatography. Mature red blood cells lack all internal cell structures and consist of cytoplasm within a plasma membrane envelope. To maximize outcome, total red blood cell protein was divided into two fractions of membrane-associated proteins and cytoplasmic proteins. Both fractions were divided into subfractions, and proteins were identified in each fraction separately through tryptic digestion. Membrane protein digests were collected from externally exposed proteins, internally exposed proteins, "spectrin extract" mainly consisting of membrane skeleton proteins, and membrane proteins minus spectrin extract. Cytoplasmic proteins were divided into 21 fractions based on molecular mass by size exclusion chromatography. The tryptic peptides were separated by reverse-phase high-performance liquid chromatography and identified by ion trap tandem mass spectrometry. A total of 181 unique protein sequences were identified: 91 in the membrane fractions and 91 in the cytoplasmic fractions. Glyceraldehyde-3-phosphate dehydrogenase was identified with high sequence coverage in both membrane and cytoplasmic fractions. Identified proteins include membrane skeletal proteins, metabolic enzymes, transporters and channel proteins, adhesion proteins, hemoglobins, cellular defense proteins, proteins of the ubiquitin-proteasome system, G-proteins of the Ras family, kinases, chaperone proteins, proteases, translation initiation factors, and others. In addition to the known proteins, there were 43 proteins whose identification was not determined.     

Effect of Ca2+, peroxides, SH reagents, phosphate and aging on the permeability of mitochondrial membranes

The mechanism by which a number of agents such as hydroperoxides, inorganic phosphate, azodicarboxylic acid bis(dimethylamide) (diamide), 2-methyl-1,4-naphthoquinone (menadione) and aging, induce Ca2+ release from rat liver mitochondria has been analyzed by following Ca2+ fluxes in parallel with K+ fluxes, matrix swelling and triphenylmethylphosphonium fluxes (as an index of transmembrane potential). Addition of hydroperoxides causes a cycle of Ca2+ efflux and reuptake and an almost parallel cycle of delta psi depression. The hydroperoxide-induced delta psi depression is biphasic. The first phase is rapid and insensitive to ATP and is presumably due to activation of the transhydrogenase reaction during the metabolization of the hydroperoxides. The second phase is slow and markedly inhibited by ATP and presumably linked to the activation of a Ca2+-dependent reaction. The slow phase of delta psi depression is paralleled by matrix K+ release and mitochondrial swelling. Nupercaine and ATP reduce or abolish also K+ release and swelling. Inorganic phosphate, diamide, menadione or aging also cause a process of Ca2+ efflux which is paralleled by a slow delta psi depression, K+ release and swelling. All these processes are reduced or abolished by Nupercaine and ATP. The slow delta psi depression following addition of hydroperoxide and diamide is largely reversible at low Ca2+ concentration but tends to become irreversible at high Ca2+ concentration. The delta psi depression increases with the increase of hydroperoxide, diamide and menadione concentration, but is irreversible only in the latter case. Addition of ruthenium red before the hydroperoxides reduces the extent of the slow but not of the rapid phase of delta psi depression. Addition of ruthenium red after the hydroperoxides results in a slow increase of delta psi. Such an effect differs from the rapid increase of delta psi due to ruthenium-red-induced inhibition of Ca2+ cycling in A23187-supplemented mitochondria. Metabolization of hydroperoxides and diamide is accompanied by a cycle of reversible pyridine nucleotide oxidation. Above certain hydroperoxide and diamide concentrations the pyridine nucleotide oxidation becomes irreversible. Addition of menadione results always in an irreversible nucleotide oxidation. The kinetic correlation between Ca2+ efflux and delta psi decline suggests that hydroperoxides, diamide, menadione, inorganic phosphate and aging cause, in the presence of Ca2+, an increase of the permeability for protons of the inner mitochondrial membrane. This is followed by Ca2+ efflux through a pathway which is not the H+/Ca2+ exchange.