Intracellular flavonoids as electron donors for extracellular ferricyanide reduction in human erythrocytes.

Reduction of extracellular ferricyanide [Fe(CN)(6)](-3) to ferrocyanide by intact cells reflects the activity of a trans-plasma membrane oxidoreductase that, in human red blood cells, utilizes ascorbic acid as an electron donor. We herein report that the flavonoids quercetin and myricetin, while inhibiting dehydroascorbic acid uptake-and thus the erythrocyte ascorbic acid content-effectively stimulate the extracellular reduction of ferricyanide. Other flavonoids such as rutin, acacetin, apigenin, and genistein do not show the same effect. The notion that quercetin or myricetin may serve as an intracellular donor for a trans-plasma membrane oxidoreductase is supported by the following lines of evidence: (i) they afford direct reduction of ferricyanide; (ii) extracellular reduction of ferricyanide was not mediated by direct effects of the flavonoids released by the cells and was abolished by the sulphydryl reagent parachloromercuribenzenesulfonic acid (pCMBS); (iii) the intracellular concentrations of quercetin or myricetin well correlate with increases in ferricyanide reduction; (iv) the intracellular concentration of the flavonoids dramatically declines after ferricyanide exposure. Taken together, the results presented in this study demonstrate that myricetin and quercetin, which accumulate in large amounts in red blood cells, act as intracellular substrates of a pCMBS-sensitive trans-plasma membrane oxidoreductase. This may represent a novel mechanism whereby these flavonoids exert beneficial effects under oxidative stress conditions.     

Effects of dinitrophenol and oligomycin on the coupling between anaerobic metabolism and anaerobic sodium transport by the isolated turtle bladder

Dinitrophenol (1 x 10^-5 M) has been found to inhibit anaerobic sodium transport by the isolated urinary bladder of the fresh water turtle. Concurrently, anaerobic glycolysis was stimulated markedly. However, tissue ATP levels diminished only modestly, remaining at approximately 75% of values observed under anaerobic conditions without DNP. The utilization of glucose (from endogenous glycogen) corresponded closely to that predicted from the molar quantities of lactate formed. Thus the glycolytic pathway was completed in the presence of DNP and if ATP were synthesized normally during glycolysis, synthesis should have been increased. On the other hand, the decrease in Na transport should have decreased ATP utilization. Oligomycin did not block sodium transport either aerobically or anaerobically, but ATP concentrations did decrease. When anaerobic glycolysis was blocked by iodoacetate, pyruvate did not sustain sodium transport thus suggesting that no electron acceptors were available in the system. Two explanations are entertained for the anaerobic effect of DNP: (a) Stimulation by DNP of plasma membrane as well as mitochondrial ATPase activity; (b) inhibition of a high energy intermediate derived from glycolytic ATP or from glycolysis per se. The arguments relevant to each possibility are presented in the text. Although definitive resolution is not possible, we believe that the data favor the hypothesis that there was a high energy intermediate in the anaerobic system and that this intermediate, rather than ATP, served as the immediate source of energy for the sodium pump.     

Phosphorylation and dephosphorylation reactions of the red beet plasma membrane ATPase studied in the transient state

The reaction mechanism of the solubilized red beet (Beta vulgaris L.) plasma membrane ATPase was studied with a rapid quenching apparatus. Using a dual-labeled substrate ([γ-³²P]ATP and [5′,8-³H]ATP), the presteady-state time course of phosphoenzyme formation, phosphate liberation and ADP liberation was examined. The time course for both phosphoenzyme formation and ADP liberation showed a rapid, initial rise while the timecourse for phosphate liberation showed an initial lag. This indicated that ADP was released with formation of the phosphoenzyme while phosphate was released with phosphoenzyme breakdown. Phosphoenzyme formation was Mg²⁺-dependent and preincubation of the enzyme with free ATP followed by the addition of Mg²⁺ increased the rate of phosphoenzyme formation 2.3-fold. This implied that phosphoenzyme formation could result from a slow reaction of ATP binding followed by a more rapid reaction of phosphate group transfer. Phosphoenzyme formation was accelerated as the pH was decreased, and the relationship between pH and the apparent first-order rate constants for phosphoenzyme formation suggested the role of a histidyl residue in this process. Transient kinetics of phosphoenzyme breakdown confirmed the presence of two phosphoenzyme forms, and the discharge of the ADP-sensitive form by ADP correlated with ATP synthesis. Potassium chloride increased the rate of phosphoenzyme turnover and shifted the steady-state distribution of phosphoenzyme forms. From these results, a minimal catalytic mechanism is proposed for the red beet plasma membrane ATPase, and rate constants for several reaction steps are estimated.     

The dynamics of heat production in erythrocytes of the scorpion fish (<Emphasis Type="Italic">Scorpaena porcus</Emphasis> Linnaeus, 1758) in vitro

Temperature measurements in a plastic tube isolated from external influences containing an erythrocyte suspension of the scorpion fish (Scorpaena porcus Linnaeus, 1758) showed that these red blood cells are able to generate heat. Heat release in the cell suspension was expressed by a linear temperature increase in the tube during the entire experiment. Addition of extracellular ATP (1 mg mL^–1) caused the effect of a thermal shift: a sharp temperature rise in the cell suspension for 30–60 s. We believe that the heat release was caused by hydrolysis of extracellular ATP by membrane ecto-ATPase. Inhibition of ecto-ATPase activity through the addition of EDTA (1 mM) to the erythrocyte suspension led to complete blockage of heat release; the effect of the thermal shift ceased. We assume that thermal properties of red blood cells play an important role in blood hemodynamics, especially in providing the “non-Newtonian” properties of blood. The thermal phenomena observed in suspensions of fish erythrocytes open new scientific directions in exploring the capabilities of multifunctional extracellular ATP.     

Light-Induced Polar pH Changes in Leaves of Elodea canadensis: II. Effects of Ferricyanide: Evidence for Modulation by the Redox State of the Cytoplasm

The effect of an extracellular electron acceptor, ferricyanide, on the light-induced polar leaf pH changes of the submerged angiosperm Elodea canadensis in light and in darkness was determined. The rate of transmembrane ferricyanide reduction was stimulated by increased light intensity and was inhibited by inorganic carbon, indicating that changes in the redox state of the chloroplast were reflected at the plasma membrane. The addition of ferricyanide inhibited the light-induced polar leaf pH reaction. This effect could be balanced by increasing the light intensity. In the dark, the acidification induced by ferricyanide was not influenced by diethylstilbestrol at concentrations that completely inhibited the polar leaf pH changes. This indicates that the ferricyanide-induced H⁺ extrusion and the H⁺ transport during the polar reaction were mediated by different mechanisms.     

Structural changes ofNoetia ponderosa red blood cell membranes during cell volume regulation in reduced salinities: A freeze fracture study

Summary Changes in molluscan blood cell membrane structure coincided with changes in membrane amino acid permeability during cell volume regulation. Blood cells were freeze fractured after the free amino acid permeability of their membranes had been altered by modifying the extracellular Ca²⁺ and intracellular ATP levels and the membrane particles examined for changes in size, number/area and distribution. Test substances that altered the divalent cation or ATP levels also altered membrane particle densities, but not size or distribution, of freeze fractured blood cells. Those test substances (Ca²⁺-free seawater, DNP, low temperature) that inhibited volume regulation and the FAA efflux caused decreased membrane particle density, while those test substances (Co²⁺, Mn²⁺) that potentiated volume regulation and the FAA efflux increased the number of membrane particles/unit area. These changes in membrane particle density appear to result from the changes in surface area due to the treatment effects on cell volume, so that the number of membrane particles per cell remained constant. Therefore, altered membrane FAA permeability is associated with altered membrane particle density, but the effect of this structural alteration on membrane permeability is not clear.Abbreviations FAA free amino acid - DMSO dimethylsulfoxide - DNP dinitrophenol - ASW artificial seawater     

The effect of ferricyanide with iodoacetate in calcium-free solution on passive cation permeability in human red blood cells: comparison with the Gardos-effect and with the influence of PCMBS on passive cation permeability

Freshly prepared human red blood cells incubated with 5 mM ferricyanide, 0.2 mM iodoacetate and 2 mM adenosine in the presence of 5 mM EGTA demonstrate comparable increases in Na+ and K+ permeability (ferricyanide effect). This effect is unrelated to the Ca2+-activated K+ channel (Gardos effect) since influx of Ca2+ from outside the cell is excluded. Also this effect is different from the non-specific Na+ and K+ permeability change elicited by PCMBS. These differences become obvious by using various reagents. For example, A23187 and quinidine exert opposite effects in Gardos and ferricyanide experiments, where A23187 and atebrin react oppositely in the latter and in PCMBS experiments. The ferricyanide effect described here does not involve formation of nonspecific channels. The change in Na+ permeability separately from K+ permeability under certain circumstances suggests a more specific effect.     

Effects of metabolic inhibitors on the release of glutamate from the retina

—The superfused, isolated retina of the chicken was used to investigate the mechanisms responsible for the increase in retinal transparency and the release of glutamate associated with stimuli known to elicit spreading depression (SD). We sought to distinguish between (1) mechanisms involving glutamate-induced increase in Na⁺ permeability and consequent uptake of extracellular material into the intracellular compartment and (2) mechanisms involving interference with operation of the Na⁺ pump that would result in a similar uptake of extracellular materials. Tetrodotoxin (which inhibits inward movements of Na⁺) depressed the transparency increase caused by stimulation with glutamate but not that elicited by application of KCl. Ouabain (which inhibits the Na⁺ pump) caused a marked increase in tissue transparency. The application of inhibitors of the aerobic metabolism, such as DNP or cyanide, or deprivation of O₂ had no effect on the retinal transparency; results suggesting that the energy for the Na⁺ pump could be supplied by glycolysis. Indeed iodoacetate (which inhibits glycolysis) caused a marked change in transparency. Furthermore we found evidence for a compound in the superfusion fluid supplemented with iodoacetate that may be a reaction product of glutamate and iodoacetate. In some preparations superfusion with glucose-free solutions caused a slowly developing increase in transparency and release of glutamate; in others the increase in transparency was more sudden and there was a larger release of glutamate. Seemingly, interference with the tissue metabolism can cause an uptake of extracellular material either by arrest of the Na⁺ pump or by the release of glutamate, depending on the conditions of the experiment.     

Relation of membrane vesicles to volume and Na+ transport in smooth muscle: effect of metabolic and transport inhibition on fresh tissues.

We tested the hypothesis that membrane vesicles of smooth muscle function as organelles controlling cell volume through a mechanochemical mechanism not involving Na+-K+ dependent membrane ATPase. Pieces of rat myometrium were incubated under various conditions at 25 degrees C, and then were analyzed after various times for Na+, K+, ATP and water contents or were prepared and examined in the electron microscope. Metabolic inhibition with iodoacetate (IAA) + dinitrophenol (DNP) rapidly depleted ATP, then decreased membrane vesicle number and increased vesicle size. Thereafter K+ loss, Na+ gain and water gain occurred. Slower depletion of ATP by treatment of tissues with IAA or ethacrynic acid produced similar, but delayed effects. Treatment with DNP alone, DNP in glucose-free Krebs-Ringer or glucose-free solution bubbled with N2 partly depleted the tissues of ATP but did not markedly affect the membrane vesicles or tissue water content. Ouabain affected neither ATP contents of tissues nor the numberof membrane vesicles, but produced large intracellular vesicles. The membrane vesicles were suggested to be sites of a mechanochemical volume control system.     

Hemolysis and ATP Release from Human and Rat Erythrocytes under Conditions of Hypoxia: A Comparative Study

Red blood cells are involved not only in transportation of oxygen and carbon dioxide but also in autoregulation of vascular tone by ATP release in hypoxic conditions. Molecular mechanisms of the ATP release from red blood cells in response to a decrease in partial oxygen pressure still remain to be elucidated. In this work we have studied effects of hypoxia on red blood cell hemolysis in humans and rats and compared the effects of inhibitors of ecto-ATPase and pannexin on the release of ATP and hemoglobin from rat erythrocytes. The 20-min hypoxia at 37°C increased hemolysis of red blood cells in humans and rats 1.5- and 2.5-fold, respectively. In rat erythrocytes a significant increase in hypoxia-induced extracellular ATP level was found only in the presence of ecto-ATPase inhibitor ARL 67156. In these conditions we observed a positive correlation (R² = 0.5003) between the increase in free hemoglobin concentration and the ATP release. Neither carbenoxolon nor probenecid, the inhibitors of low-selectivity pannexin channels, altered the hypoxia-induced ATP release from rat erythrocytes. The obtained results indicate a key role of hemolysis in the ATP release from red blood cells.     

Depolarization of Cell Membrane Potential during Trans-Plasma Membrane Electron Transfer to Extracellular Electron Acceptors in Iron-Deficient Roots of Phaseolus vulgaris L

Transfer of electrons from the cytosol of bean (Phaseolus vulgaris L.) root cells to extracellular acceptors such as ferricyanide and Fe^IIIEDTA causes a rapid depolarization of the membrane potential. This effect is most pronounced (30-40 millivolts) with root cells of Fe-deficient plants, which have an increased capacity to reduce extracellular ferric salts. Ferrocyanide has no effect. In the state of ferricyanide reduction, H⁺ (1H⁺/2 electrons) and K⁺ ions are excreted. The reduction of extracellular ferric salts by roots of Fe-deficient bean plants is driven by cellular NADPH (Sijmons, van den Briel, Bienfait 1984 Plant Physiol 75: 219-221). From this and from the membrane potential depolarization, we conclude that trans-plasma membrane electron transfer from NADPH is the primary process in the reduction of extracellular ferric salts.     

Peroxidative membrane damage in human erythrocytes induced by a concerted action of iodoacetate, vanadate and ferricyanide

Human erythrocytes incubated without substrate in the presence of iodoacetate (0.2 mM), vanadate (0.5 mM) and ferricyanide (5 mM) form aqueous membrane leaks of equivalent radii of 0.5-0.8 nm leading to complete colloid-osmotic lysis within 180 min. All three components are indispensable for the effect. Inosine but not glucose markedly enhances the rate of hemolysis. These effects are due to oxidative damage, as indicated by concomitant destruction of polyunsaturated fatty acids and suppression of both effects by radical scavengers. Hemoglobin is not oxidized under these conditions. GSH and membrane SH levels remain almost normal, and no crosslinking or irreversible aggregation of membrane proteins is observed. In the absence of O2 no membrane damage can be observed. It is proposed that radical formation originates from reduction of O2 by NADPH, analogous to processes described in microsomal membranes. NADH seems not to be involved, since leak formation occurs in spite of the blockage of NADH formation by iodoacetate. Vanadate and ferricyanide are probably required to amplify the peroxidative reaction sufficiently to overcome the cellular antioxidative capacity.     

All or none cell responses of Ca2+-dependent K channels elicited by calcium or lead in human red cells can be explained by heterogeneity of agonist distribution

Summary We have studied the all or none cell response of Ca²⁺-dependent K⁺ channels to added Ca in human red cells depleted of ATP by incubation with iodoacetate and inosine. A procedure was used which allows separation and differential analysis of responding and nonresponding cells. Responding (H for heavy) cells incubated in medium containing 5mM K lose KCl and water and increase their density to the point of sinking on diethylphthalate (specific gravity=1.12) on centrifugation. Nonresponding (L for light) cells do not lose KCl at all. There is no intermediate behavior. Increasing the Ca concentration in the medium increases the fraction of cells which become H. No differences in the sensitivity to Ca²⁺ of the individual K⁺ channels were detected in inside-out vesicles prepared either from H or from L cells. The Ca content of H cells was higher than that of L cells. Cells depleted of ATP by incubation with iodoacetate and inosine sustain pump-leak Ca fluxes of about 15 mol/liter cells per hour. ATP seems to be resynthesized in these cells at the expense of cell 2,3-diphosphoglycerate stores at a rate of about 150 mol/liter cells per hour. Inhibition of 2,3-diphosphoglycerate phosphatase by tetrathionate increased 6–8 times the measured rate of uptake of external⁴⁵Ca. This was accompanied by an increase in the fraction of H cells. All or none cell responses of Ca²⁺-dependent K channels have also been evidenced in intact human red cells on addition of Pb. They have the same characteristics as those in responding and nonresponding cells. The detailed study of the kinetics of Pb-induced shrinkage of red cells suspended in medium containing 5mM K showed that changes of Pb concentration changed not only the fraction of H cells but also the rate of shrinkage of responding cells. H cells generated by Pb treatment contained significantly more lead than L cells. The above results suggest that the two all or none cell responses studied here can be explained by heterogeneity of agonist distribution among cells. Since pump-leak fluxes exist in both cases, differences of agonist distribution could be generated by heterogeneity of pumping among cells. This interpretation turns interest from K channels to Ca pumps to explain the heterogeneous behavior of red cells in response to a uniform stimulus.     

Control of NADH ferricyanide reductase activity in the human erythrocyte by somatotrophin and insulin

Reduction of external ferricyanide by the human erythrocyte is significantly stimulated by insulin and somatotrophin at concentrations above physiological levels. Basal (in absence of hormones) and hormone-stimulated activities are attenuated in the presence of glycolytic inhibitors iodoacetate and vanadate indicating the requirement of glycolytic substrates for the reduction process and for the activation of cellular metabolism in response to the hormones. Sulfhydryl reagents like N-ethylmaleimide also attenuate the basal and hormone-stimulated activities and this effect was rationalized on the basis of action at SH sites which trigger responses to hormones. Stimulation of ferricyanide reduction by insulin and somatotrophin may be also the result of Na⁺/H⁺ antiport activation which may be prevented by amiloride. This suggests that Na⁺/H⁺ antiport is part of the membrane transduction system for insulin and somatotrophin in the human erythrocyte. These observations are a contribution to the study of plasma membrane oxidoreductase systems involved in physiological and metabolic functions of the cell.     

Use of P NMR to Assess Effects of DNP on ATP Levels in Vivo in Barley Roots

Previous work has shown that undissociated 2,4-dinitrophenol (DNP) both increases the permeability of roots to ions and alters the membrane lipids of barley roots. Anionic DNP is the main entrant form but has no effect on permeability or on the membrane lipids. The amount of anionic DNP taken up by the roots is sufficient, that were it in free solution in the cytoplasm, the DNP would uncouple oxidative phosphorylation, and thereby inhibit ATP synthesis. The present work was undertaken to assess whether DNP alters ATP levels when it is taken up by barley roots. ³¹P nuclear magnetic resonance spectra were used to monitor, in vivo, levels of ATP, cytoplasmic phosphate, vacuolar phosphate, and other phosphate compounds in barley roots in the presence of 10 micromolar DNP at pH 5 and pH 7. The spectra indicate that no change in the level of ATP or the cytoplasmic pH occurred in the roots in the presence of DNP for as long as 20 hours. Thus, the effects of undissociated DNP are effects directly on the root membranes and do not involve inhibition of ATP synthesis. Furthermore, the results explain why anionic DNP has no effect on ion uptake and accumulation.     

The effect of inorganic phosphate on sodium fluxes in dog red blood cells

The effect of extracellular inorganic phosphate on Na⁺ movements in dog red blood cells has been studied. As the phosphate concentration is increased from 0 to 30 mM, Na⁺ efflux increases by 2- to 3-fold and Na⁺ influx increases approximately 2-fold. This enhancement of Na⁺ fluxes by phosphate can be prevented by the addition of iodoacetate (1 mM), an inhibitor of glycolysis, or 4-acetamido-4′-iso-thiocyantostilbene-2,2′-disulfonic acid (0.01 mM), which blocks anion transport, to the medium. The increases in Na⁺ movements are not caused by changes in cell volumes. These results suggest that phosphate must enter the cell to enhance Na⁺ fluxes and that the mechanism of action may be via a stimulatory effect on glycolysis.     

Studies on Foliar Penetration: IV. MECHANISMS CONTROLLING THE RATE OF PENETRATION OF 2,4-DICHLOROPHENOXYACETIC ACID (2,4-D) INTO LEAVES OF PHASEOLUS VULGARIS

The effects of a wide range of metabolic inhibitors on the penetrationof 2,4-dichlorophenoxyacetic acid (2,4-D) into the leaf disksof Phaseolus vulgaris have been studied. While recognizing thelack of specificity of most inhibitors, compounds were chosenwhich are known to affect respiration, phosphorylation, photosynthesis,membrane permeability, protein synthesis, and the binding capacityof membrane systems. They were: fluoride, azide, arsenite, iodoacetate,arsenate, 2,4-dinitrophenol (DNP), 3-(3,4-dichlorophenyl), -I,I-dimethylurea (DCMU), phenylmercuric chloride, octenylsuccinicacid, decenylsuccinic acid, dimethyl sulphoxide, actinomycin-D,chloramphenicol, streptomycin, 5-fluorouracil, cycloheximide,and cetyltrimethylammoniumbromide (CTAB).At sub-toxic levelsall compounds had little or no influence on penetration in darknesssave for iodoacetate and decenylsuccinic acid, which causedsome enhanced entry at 10^-4M and 10^-3M respectively, and CTABwhich promoted penetration at concentrations known tolower thesurface tension of water.The much greater rate of penetrationof 2,4-D into disks exposed to bright light (16 000 lx) is unaffectedby fluoride, azide, DNP, octenylsuccinic acid, decenylsuccinicacid, dimethyl sulphoxide, or actinomycin-D. It is, however,progressively inhibited by increasing concentrations of arsenite,iodoacetate, arsenate, streptomycin, and 5-fluorouracil. Chloramphenicol,cycloheximide, and CTAB lower the rate of penetration at intermediateconcentrations but at high concentrations the affect is reversed.The most active inhibitors of light-induced penetration areDCMU and phenylmercuric chloride, compounds which block theproduction of ATP.These results are discussed in relation tomechanisms of transport, in particular the structureand stabilityof barriers likely to impede penetration.     

Passive membrane permeability to small molecules and ions in transformed mammalian cells: probable role of surface phosphorylation

Addition of ATP to medium surrounding intact, transformed 3T3 cells causes the formation of aqueous channels in the plasma membrane. This effect of extracellular ATP is sharply dependent on the pH and temperature of the incubation medium, and is inhibited by low levels of La3+ or ruthenium red; inhibition is also obtained with concentrations of Mg2+ ions that exceed a ratio of Mg/ATP of one. The effect of ATP on membrane channel formation is unaffected by chelators of metal ions or by prior modification of the cell surface with various surface-active enzymes or sulfhydryl reagents. Under conditions which favor aqueous channel formation, incubation of intact 3T6 cells with ATP (gamma-32P) leads to phosphorylation of two membrane components with apparent molecular weight of 40,000 (40K) and 110,000 (110K) daltons; the 110K component which is unaffected by trypsin under normal conditions is rendered trypsin-sensitive by the phosphorylation reaction, probably as a result of a conformational change. Conditions which inhibit aqueous channel formation also inhibit phosphorylation of the 110K protein and decrease the labeling of the 40K component. These results indicate the probable role of cell surface phosphorylation, involving one or both of these components, in the formation of aqueous channels in transformed 3T3 cells. Aqueous channel formation by extracellular ATP is not associated with gross unfolding of the cell surface as revealed by lactoperoxidase-catalyzed iodination of the 3T6 cell surface.     

Effect of extracellular Ca2+, K+ and OH− on erythrocyte membrane potential as monitored by the fluorescent probe 3,3′-dipropylthiodicarbocyanine

Changes in fluorescence intensity of thiodicarbocyanine, DiS-C₃(5), were correlated with direct microelectrode potential measurements in red blood cells from Amphiuma means and applied qualitatively to evaluate the effects of extracellular Ca²⁺, K⁺ and pH on the membrane potential of human red cells. Increasing extracellular [Ca²⁺] from 1.8 to 15 mM causes a K⁺-dependent hyperpolarization and decrease in fluorescence intensity in Amphiuma red cells. Both the hyperpolarization and fluorescence change disappear when the temperature is raised from 17 to 37°C. No change in fluorescence intensity is observed in human red cells with comparable increase in extracellular Ca²⁺ in the temperature range 5–37°C. Increasing the extracellular pH, however, causes human red cells to respond to an increase in extracellular Ca²⁺ with a significant but temporary loss in fluorescence intensity. This effect is blocked by EGTA, quinine or by increasing extracellular [K⁺], indicating that at elevated extracellular pH, human erythrocytes respond to an increase in extracellular Ca²⁺ with an opening of K⁺ channels and associated hyperpolarization of the plasma membrane.     

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.