Intact Human Red Blood Cells Research Articles

Optimized S-nitrosohemoglobin Synthesis in Red Blood Cells to Preserve Hypoxic Vasodilation Via βCys93.

Classic physiology links tissue hypoxia to oxygen delivery through control of microvascular blood flow (autoregulation of blood flow). Hemoglobin (Hb) serves both as the source of oxygen and the mediator of microvascular blood flow through its ability to release vasodilatory S-nitrosothiol (SNO) in proportion to degree of hypoxia. β-globin Cys93Ala (βCys93Ala) mutant mice deficient in S-nitrosohemoglobin (SNO-Hb) show profound deficits in microvascular blood flow and tissue oxygenation that recapitulate microcirculatory dysfunction in multiple clinical conditions. However, the means to replete SNO in mouse red blood cells (RBCs) to restore RBC function is not known. In particular, although methods have been developed to selectively S-nitrosylate βCys93 in human Hb and intact human RBCs, conditions have not been optimized for mouse RBCs that are used experimentally. Here we show that loading SNO onto Hb in mouse RBC lysates can be achieved with high stoichiometry and β-globin selectivity. However, S-nitrosylation of Hb within intact mouse RBCs is ineffective under conditions that work well with human RBCs, and levels of metHb are prohibitively high. We developed an optimized method that loads SNO in mouse RBCs to maintain vasodilation under hypoxia and shows that loss of SNO loading in βCys93Ala mutant RBCs results in reduced vasodilation. We also demonstrate that differences in SNO/met/nitrosyl Hb stoichiometry can account for differences in RBC function among studies. RBCs loaded with quasi-physiologic amounts of SNO-Hb will produce vasodilation proportionate to hypoxia, whereas RBCs loaded with higher amounts lose allosteric regulation, thus inducing vasodilation at both high and low oxygen level. SIGNIFICANCE STATEMENT: Red blood cells from mice exhibit poor hemoglobin S-nitrosylation under conditions used for human RBCs, frustrating tests of vasodilatory activity. Using an optimized S-nitrosylation protocol, mouse RBCs exhibit hypoxic vasodilation that is significantly reduced in hemoglobin βCys93Ala mutant RBCs that cannot carry S-nitrosothiol allosterically, providing genetic validation for the role of βCys93 in oxygen delivery.

Phenol-Rich Feijoa sellowiana (Pineapple Guava) Extracts Protect Human Red Blood Cells from Mercury-Induced Cellular Toxicity.

Plant polyphenols, with broadly known antioxidant properties, represent very effective agents against environmental oxidative stressors, including mercury. This heavy metal irreversibly binds thiol groups, sequestering endogenous antioxidants, such as glutathione. Increased incidence of food-derived mercury is cause for concern, given the many severe downstream effects, ranging from kidney to cardiovascular diseases. Therefore, the possible beneficial properties of Feijoa sellowiana against mercury toxicity were tested using intact human red blood cells (RBC) incubated in the presence of HgCl2. Here, we show that phenol-rich (10–200 µg/mL) extracts from the Feijoa sellowiana fruit potently protect against mercury-induced toxicity and oxidative stress. Peel and pulp extracts are both able to counteract the oxidative stress and thiol decrease induced in RBC by mercury treatment. Nonetheless, the peel extract had a greater protective effect compared to the pulp, although to a different extent for the different markers analyzed, which is at least partially due to the greater proportion and diversity of polyphenols in the peel. Furthermore, Fejioa sellowiana extracts also prevent mercury-induced morphological changes, which are known to enhance the pro-coagulant activity of these cells. These novel findings provide biochemical bases for the pharmacological use of Fejioa sellowiana-based functional foods in preventing and combating mercury-related illnesses.

Investigations on the role of hemoglobin in sulfide metabolism by intact human red blood cells

In addition to their role as oxygen transporters, red blood cells (RBCs) contribute to cardiovascular homeostasis by regulating nitric oxide (NO) metabolism via interaction of hemoglobin (Hb) with nitrite and NO itself. RBCs were proposed to also participate in sulfide metabolism. Although Hb is known to react with sulfide, sulfide metabolism by intact RBCs has not been characterized so far. Therefore we explored the role of Hb in sulfide metabolism in intact human RBCs. We find that upon exposure of washed RBCs to sulfide, no changes in oxy/deoxyhemoglobin (oxy/deoxyHb) are observed by UV-vis and EPR spectroscopy. However, sulfide reacts with methemoglobin (metHb), forming a methemoglobin-sulfide (metHb-SH) complex. Moreover, while metHb-SH is stable in cell-free systems even in the presence of biologically relevant thiols, it gradually decomposes to produce oxyHb, inorganic polysulfides and thiosulfate in intact cells, as detected by EPR and mass spectrometry. Taken together, our results demonstrate that under physiological conditions RBCs are able to metabolize sulfide via intermediate formation of a metHb-SH complex, which subsequently decomposes to oxyHb. We speculate that decomposition of metHb-SH is preceded by an inner-sphere electron transfer, forming reduced Hb (which binds oxygen to form oxyHb) and thiyl radical (a process we here define as “reductive sulfhydration”), which upon release, gives rise to the oxidized products, thiosulfate and polysulfides. Thus, not only is metHb an efficient scavenger and regulator of sulfide in blood, intracellular sulfide itself may play a role in keeping Hb in the reduced oxygen-binding form and, therefore, be involved in RBC physiology and function.

Effects of Phenyl-1-Pyridin-2yl-Ethanone (PPY)-Based Iron Chelators on the Formation of Methemoglobin

BackgroundIron chelators are used in the treatment of iron overload related diseases and are currently receiving a major attention as potential antitumor drugs. In recent studies, the antitumor activity of thiosemicarbazones-class of iron chelator, including Di-2-pyridilketone-4,4- dimethyl-3-thiosemicarbazone (Dp44mT) has been investigated in over 20 phase I and II clinical trials [1, 2,3]. Iron chelators were also considered as anti-HIV-1 agents. However, the main obstacle to using iron chelators in vivois the deleterious side effect of methemoglobinemia induced by some iron chelators that are able to scavenge electrons from the heme-bound iron in hemoglobin. In our previous studies, we developed novel phenyl-1-pyridin-2yl-ethanone (PPY)-based iron chelators that we showed to increase IKBα expression, modulate CDK2 and CDK9 activities and inhibit HIV-1 [4].ObjectiveOur objective was to test the effect of PPYeT iron chelator for methemoglobin induction. The methemoglobin induction effect was compared with several additional iron chelators including Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and PPY analogues.MethodsFluorometric analysis was carried out in promonocytic THP-1 cells to evaluate the ability of our novel PPYeT iron chelator to reduce labile iron pool (LIP). The effect of PPYet on LIP was compared to the effect to SIH. Subsequently, spectrophotometric analysis was used to measure and quantify the production of methemoglobin in human red blood cells lysates and in isolated intact human red blood cells treated with PPYeT and various other iron chelators including DP44mT and DP4mT.ResultsPPYeT significantly reduced LIP in THP-1 cells overloaded with iron comparing to the cells treated with SIH. In RBC lysates and in intact RBC, PPYeT treatment showed notably lesser production of methemoglobin in comparison to DP44mT and DP4mT chelators. In RBC lysates, PPYeT produced about four-fold less methemoglobin than Dp44mT and ten-fold less than Dp4mT.ConclusionThe novel compound, PPYeT, shows a remarkably low ability to catalyze the formation of methemoglobin in human RBC lysates and also in intact RBCs as compared to Dp44mT. These findings indicate that PPYeT may be useful for future in vivo studies as it produces less methemoglobinemia. Further studies will evaluate the effect PPYeT as anti-cancer or anti HIV-1 inhibitor in vivo.AcknowledgmentsThis work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005, and 5G12MD007597. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIMHD or NIH.

Stability and Stoichiometry of Bilayer Phospholipid–Cholesterol Complexes: Relationship to Cellular Sterol Distribution and Homeostasis

Is cholesterol distributed among intracellular compartments by passive equilibration down its chemical gradient? If so, its distribution should reflect the relative cholesterol affinity of the constituent membrane phospholipids as well as their capacity for association with the sterol. We examined this issue by analyzing the reactivity to cholesterol oxidase of large unilamellar vesicles (LUVs) containing phospholipids and varied levels of cholesterol. The rates of cholesterol oxidation differed among the various phospholipid environments by roughly 4 orders of magnitude. Furthermore, accessibility to the enzyme increased by orders of magnitude at cholesterol thresholds that suggested cholesterol:phospholipid association ratios of 1:1, 2:3, or 1:2 (moles:moles). The accessibility of cholesterol above these thresholds was still constrained by its particular phospholipid environment. One phospholipid, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidylserine, exhibited no threshold. The analysis suggested values for the stoichiometries of the putative cholesterol-phospholipid complexes, their relative stabilities, and the fractions of bilayer cholesterol not in complexes at the threshold equivalence points. Predictably, the saturated phosphorylcholine species had the lowest apparent stoichiometric ratios and the strongest associations with cholesterol. These results are in general agreement with the equilibrium distribution of cholesterol between the various LUVs and methyl-β-cyclodextrin. In addition, the behavior of the cholesterol in intact human red blood cells matched predictions made from LUVs of the corresponding composition. These results support a passive mechanism for the intracellular distribution of cholesterol that can provide a signal for its homeostatic regulation.

Fourier transform light scattering angular spectroscopy using digital inline holography

A simple and practical method for measuring the angle-resolved light scattering (ARLS) from individual objects is reported. Employing the principle of inline holography and a Fourier transform light scattering technique, both the static and dynamic scattering patterns from individual micrometer-sized objects can be effectively and quantitatively obtained. First, the light scattering measurements were performed on individual polystyrene beads, from which the refractive index and diameter of each bead were retrieved. Also, the measurements of the static and dynamic light scattering from intact human red blood cells are demonstrated. Using the present method, an existing microscope can be directly transformed into a precise instrument for ARLS measurements.

Forced extension of delipidated red blood cell cytoskeleton with little indication of spectrin unfolding

Force-extension curves obtained on intact human red blood cells (RBC) were compared with those of delipidated RBCs to assess the contribution of cytoskeletal flexibility to the extensibility of the intact membrane skeleton. The RBCs were first delipidated by treatment with phospholipase A₂; tensile properties of the exposed cytoskeletal structures were measured using an atomic force microscope (AFM). The AFM probes were modified either with the Band 3 specific lectin, concanavalin A, (Con A) or anti-F-actin antibody, to localize the point of interaction between the probe and the cytoskeleton. Extension of the spectrin-based cytoskeleton reached up to 2-3 μm with a force less than 70 pN without showing any force peaks before the final rupture of the adhesive bonds. Our interpretation of the result is that the spectrin-based network was slack enough to allow the observed degree of extension without unfolding the tetrameric spectrin molecules. The force-extension curves obtained either on Band 3-ankyrin loci or on junction nodes of the cytoskeleton were not significantly different. Experimental results were verified by computer simulation of pulling mechanics of a network model of the RBC cytoskeleton. Our experimental results are also in agreement with the theoretical prediction of Mirijanian and Voth [2008; Proc Natl Acad Sci USA 105:1204-1208].

Elevated intracellular Ca2+ reveals a functional membrane nucleotide pool in intact human red blood cells

Elevated intracellular calcium generates rapid, profound, and irreversible changes in the nucleotide metabolism of human red blood cells (RBCs), triggered by the adenosine triphosphatase (ATPase) activity of the powerful plasma membrane calcium pump (PMCA). In the absence of glycolytic substrates, Ca2+-induced nucleotide changes are thought to be determined by the interaction between PMCA ATPase, adenylate kinase, and AMP-deaminase enzymes, but the extent to which this three-enzyme system can account for the Ca2+-induced effects has not been investigated in detail before. Such a study requires the formulation of a model incorporating the known kinetics of the three-enzyme system and a direct comparison between its predictions and precise measurements of the Ca2+-induced nucleotide changes, a precision not available from earlier studies. Using state-of-the-art high-performance liquid chromatography, we measured the changes in the RBC contents of ATP, ADP, AMP, and IMP during the first 35 min after ionophore-induced pump-saturating Ca2+ loads in the absence of glycolytic substrates. Comparison between measured and model-predicted changes revealed that for good fits it was necessary to assume mean ATPase Vmax values much higher than those ever measured by PMCA-mediated Ca2+ extrusion. These results suggest that the local nucleotide concentrations generated by ATPase activity at the inner membrane surface differed substantially from those measured in bulk cell extracts, supporting previous evidence for the existence of a submembrane microdomain with a distinct nucleotide metabolism.

Local Membrane Deformations Activate Ca2+-Dependent K+ and Anionic Currents in Intact Human Red Blood Cells

BackgroundThe mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell's diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents.Methodology/Principal FindingsThe cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K+ and Cl− currents were strictly dependent on the presence of Ca2+. The Ca2+-dependent currents were transient, with typical decay half-times of about 5–10 min, suggesting the spontaneous inactivation of a stretch-activated Ca2+ permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca2+ permeability pathway leading to increased [Ca2+]i, secondary activation of Ca2+-sensitive K+ channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents.Conclusions/SignificanceThe stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca2+-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca2+ content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia.

Human erythrocytes and molecular models of cell membranes are affected in vitro by Balbisia peduncularis (Amancay) extracts

Balbisia peduncularis, also known as “Amancay”, is a plant of the Ledocarpaceae family that can be found in the Atacama Desert in northern Chile. Infusions of the plant have long being used in traditional herbal medicine. Its chemical composition indicates the presence of flavonoids, which have antioxidant properties. Aqueous extracts from its stems were prepared to induce their interaction with human erythrocytes and their membrane models in order to elucidate whether this rare and unstudied plant produced perturbations to cell membranes. Scanning electron microscopy (SEM) of intact human red blood cells showed that the extract changed the normal erythrocytes morphology as a function of its concentration, first inducing echinocytes, and then stomatocytes and spherocytes. According to the bilayer couple hypothesis, the shape changes indicated that the flavonoids were first located in the outer monolayer of the erythrocyte membrane, and at the highest assayed concentration in both monolayers. The results obtained by fluorescence spectroscopy measurements of isolated unsealed human erythrocytes (IUM), of unilamellar vesicles (LUV) of dimyristoylphosphatidylcholine (DMPC), and by X-ray diffraction of DMPC and dimyristoylphosphatidylethanolamine (DMPE) multilayers, confirmed this conclusion. In fact, they showed that the plant aqueous extract molecules were located in both the hydrophilic polar head and in the hydrophobic acyl chain regions of the lipid bilayers. As a consequence, perturbations of the phospholipid bilayer packing arrangement were produced.

A Novel Plasmodium falciparum Microneme Protein Interacts with Host Band 3 during Red Cell Invasion.

Clinical manifestation of Plasmodium falciparum malaria is directly linked to the blood stage of the parasite life cycle. At the blood stage, circulating merozoites invade red blood cells (RBCs) through multiple receptor-ligand interactions that mediate a complex series of events in a period of approximately one minute. Certain strains of P. falciparum favor an invasion mechanism that depends on sialic acid residues on the host RBC surface, while other strains favor a sialic acid-independent mechanism to invade RBCs. Two putative non-glycosylated extracellular regions of human RBC band 3 termed, 5ABC and 6A, function as an invasion receptor binding the 42 kDa C-terminal processing product of P. falciparum merozoite surface protein-1 (MSP142) and its 19-kDa secondary processing product (MSP119) by a sialic acid-independent mechanism. Earlier, we reported screening of a P. falciparum cDNA library in a yeast two-hybrid system, which led to the identification of P. falciparum merozoite surface protein 9 (MSP9) as another parasite surface protein that interacts with the 5ABC receptor region of band 3. MSP9 formed a co-ligand complex with MSP142 and/or MSP119 in their native forms. Here, we present identification of a novel P. falciparum microneme protein from the previous cDNA library screen. Primary sequence analysis of a positive clone in the library screen revealed a cDNA fragment in the correct open reading frame of a novel P. falciparum gene of 1,221 bps, encoding a hypothetical protein of 406 amino acids. Further sequence analysis predicted it is a membrane protein transversing the lipid bilayer multiple times. Anti-peptide antibody raised against a predicted non-membrane domain recognized a single band at ~49 kDa (calculated mass, 46.9 kDa) from the P. falciparum protein extract by Western blotting. Protein co-localization studies that employed established markers for the merozoite surface, rhoptries, and micronemes by an indirect immunofluorescence assay suggested that the ~49 kDa protein was expressed in micronemes at trophozoite/schizont stages. Immunoelectron microscopy demonstrated that the 49 kDa protein is indeed localized primarily to micronemes in the merozoites. Based on our characterization, we have tentatively named this novel protein as microneme-associated membrane antigen 1 (MAMA1). Functional studies demonstrate that the MAMA1 antibody blocked RBC invasion by P. falciparum (3D7 strain) in vitro, in a concentration-dependent manner. Furthermore, MAMA1 presumably embedded in small vesicles present in the parasite protein extract bound to intact human RBCs, and the MAMA1-RBC binding was neuraminidase and trypsin resistant, but chymotrypsin sensitive. Native MAMA1 bound specifically to the 5ABC peptide of band 3 in our solution binding assay. Together, these results suggest that the novel microneme protein MAMA1 interacts with host band 3 and facilitates merozoite entry during RBC invasion.

Reduction of Arsenate to Arsenite by Human Erythrocyte Lysate and Rat Liver Cytosol – Characterization of a Glutathione- and NAD-Dependent Arsenate Reduction Linked to Glycolysis

Reduction of arsenate (AsV) to the more toxic arsenite (AsIII) is of high toxicological importance, yet in vivo relevant enzymes involved have not been identified. Purine nucleoside phosphorylase (PNP) is an efficient AsV reductase in vitro, but its role in AsV reduction is irrelevant in vivo. Intact human red blood cells (RBC) possess an AsV reductase activity that is PNP-independent, diminished by depletion of glutathione (GSH), enhanced by oxidants of erythrocytic NAD(P)H, and possibly linked to the lower part of the glycolytic pathway. In order to characterize this PNP-independent AsV reductase activity further, we examined the effects of GSH, inorganic phosphate, some inhibitors of glucose metabolism, glycolytic substrates, and pyridine, as well as adenine nucleotides on AsV reduction in lysed RBC and rat liver cytosol in the presence of BCX-1777, a PNP inhibitor. In hemolysate, GSH enhanced AsV reduction in a concentration-dependent manner, whereas phosphate inhibited it. Glycolytic substrates, especially fructose-1,6-bisphosphate and phosphoglyceric acids, improved AsV reductase activity. NAD, especially together with these substrates, strongly increased AsIII formation, whereas NADH strongly inhibited it. NADP and adenine nucleotides diminished, while 2-phosphoglycollate, which increases the breakdown of the RBC-specific compound 2,3-bisphosphoglycerate to 3-phosphoglycerate, doubled the AsV reductase activity. Although AsV reduction by the liver cytosol responded similarly to GSH, NAD, and glycolytic substrates as in the hemolysate, it was barely influenced by NADH, was diminished by 2-phosphoglycollate, and was stimulated by NADP. Collectively, hemolysate and rat liver cytosol possess a PNP-independent AsV reductase activity. This enzymatic activity requires GSH, NAD, and glycolytic substrates, and purportedly involves one or both of the two functionally linked glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. In addition, the data presented here suggest that yet another PNP-independent AsV reductase resides in the hepatic cytosol. Although this latter enzyme remains unknown, identification of the AsV reductase depending on GSH, NAD, and glycolytic substrates is presented in the following paper.

Pulmonary vascular effects of red blood cells containing S-nitrosated hemoglobin.

The role of S-nitrosated hemoglobin (SNO-Hb) in the regulation of blood flow is a central and controversial question in cardiopulmonary physiology. In the present study, we investigate whether intact human red blood cells (RBCs) synthesized to contain high SNO-Hb levels are able to export nitric oxide bioactivity and vasodilate the pulmonary circulation, and whether SNO-Hb dependent vasodilation occurs secondary to an intrinsic oxygen-linked, allosteric function of Hb. RBCs containing supraphysiological concentrations (100-1,000x normal) of SNO-Hb (SNO-RBCs) were synthesized and added to isolated, perfused rat lungs during anoxic or normoxic ventilation, and during normoxic ventilation with pulmonary hypertension induced by the thromboxane mimetic U-46619. SNO-RBCs produced dose-dependent pulmonary vasodilation compared with control RBCs during conditions of both normoxic (U-46619) and hypoxic pulmonary vasoconstriction. These effects were associated with a simultaneous, rapid, and temperature-dependent loss of SNO from Hb. Both vasodilatory effects and the rate of SNO-Hb degradation were independent of oxygen tension and Hb oxygen saturation. Furthermore, these effects were not affected by inhibition of the RBC membrane band 3 protein (anion exchanger-1), a putative membrane facilitator of NO export from RBCs. Whereas these data support observations by multiple groups that synthesized SNO-Hb can vasodilate, this effect is not under intrinsic oxygen-dependent allosteric control, nor likely to be relevant in the pulmonary circulation at normal physiological concentrations.

Bretylium, an organic quaternary amine, inhibits the Na,K-ATPase by binding to the extracellular K-site

The quaternary amine, bretylium, is a class III antiarrhythmic drug used to treat ventricular tachycardia and fibrillation. The primary mode of action for bretylium is thought to be inhibition of voltage-gated K + channels. While the Na,K-ATPase has been the pharmacological target of cardiac glycosides for over a century, recent evidence has shown that bretylium may also inhibit the Na pump. Our experimental findings support and extend these previous reports and provide definitive evidence supporting the previous suggestion that bretylium and K compete for the Na pump. We find that bretylium inhibits the Na pump in a dose-dependent manner in both Na,K-ATPase (IC 50 4.5 mM) and Rb flux experiments (IC 50 3.5 mM). Furthermore, we show that bretylium and Rb + competes for an extracellular site by measuring ouabain-sensitive 86Rb flux in intact human red blood cells; that is, there is an apparent increase in K m for Rb + in the presence of 5 mM bretylium, while V max remains unchanged. We also determined that unlike K +, bretylium does not facilitate the hydrolysis of E2-P. However, it stabilizes this conformation by reducing the ability of K + to facilitate dephosphorylation. Finally, we show that bretylium, like K +, reduces [ 3H]ouabain binding to the Na pump. Taken together, these data are consistent with bretylium binding to the extracellular facing cation site within the E2-P state of the enzyme. Moreover, these findings suggest that bretylium may serve as an effective tool for freezing the pump in an extracellularly cation-bound phosphorylated intermediate, which will aid in future structural analyses.

The secondary alcohol and aglycone metabolites of doxorubicin alter metabolism of human erythrocytes.

Anthracyclines, a class of antitumor drugs widely used for the treatment of solid and hematological malignancies, cause a cumulative dose-dependent cardiac toxicity whose biochemical basis is unclear. Recent studies of the role of the metabolites of anthracyclines, i.e., the alcohol metabolite doxorubicinol and aglycone metabolites, have suggested new hypotheses about the mechanisms of anthracycline cardiotoxicity. In the present study, human red blood cells were used as a cell model. Exposure (1 h at 37 C) of intact human red blood cells to doxorubicinol (40 M) and to aglycone derivatives of doxorubicin (40 M) induced, compared with untreated red cells: i) a ~2-fold stimulation of the pentose phosphate pathway (PPP) and ii) a marked inhibition of the red cell antioxidant enzymes, glutathione peroxidase (~20%) and superoxide dismutase (~60%). In contrast to doxorubicin-derived metabolites, doxorubicin itself induced a slighter PPP stimulation (~35%) and this metabolic event was not associated with any alteration in glutathione reductase, glutathione peroxidase, catalase or superoxide dismutase activity. Furthermore, the interaction of hemoglobin with doxorubicin and its metabolites induced a significant increase (~22%) in oxygen affinity compared with hemoglobin incubated without drugs. On the basis of the results obtained in the present study, a new hypothesis, involving doxorubicinol and aglycone metabolites, has been proposed to clarify the mechanisms responsible for the doxorubicin-induced red blood cell toxicity.

Dysprosium-bearing red cells as potential transverse relaxation agents for MRI.

The cytosol of intact human red blood cells was loaded with 28.1 +/- 3.4 mM of dysprosium DTPA-BMA using a hypoosmotic technique. When loaded cells were diluted with saline and control cells to give an average dysprosium concentration of 3.3 +/- 0.5 mM, the transverse relaxation rate constants R(*)(2) and R(2) increased. R(*)(2) increased from 7.5 +/- 0.9 sec(-1) to 356 +/- 50 sec(-1), and R(2) increased from 7.4 +/- 0.7 sec(-1) to 148 +/- 40 sec(-1). After lysing, R(*)(2) was 6.0 +/- 0.6 sec(-1) in the control and 13.4 +/- 1.5 sec(-1) in the mixture; R(2) was 6.4 +/- 1.1 sec(-1) and 9.8 +/- 2.4 sec(-1), respectively. Thus, the relaxivity effects were enhanced by sequestration of the dysprosium within intact red cells, and this effect was lost after lysis. At a circulating whole-blood concentration of 0.81 +/- 0.15 mM in rats, the liver signal intensity dropped 29.9% +/- 3.7% and kidney signal intensity dropped 19.4% +/- 8.7%. Dysprosium-loaded cells might be useful in the study of perfusion and tissue blood volume.

Nuclear Magnetic Resonance and Oxygen Affinity Study of Cesium Binding in Human Erythrocytes

We investigated the interaction of the cesium ion (Cs+) with the anionic intracellular components of human red blood cells (RBCs); the components studied included 2,3-bisphosphoglycerate (BPG), ADP, ATP, inorganic phosphate (Pi), carbonmonoxy hemoglobin (COHb), and RBC membranes. We used spin–lattice (T1) and spin–spin (T2) 133Cs NMR relaxation measurements to probe Cs+ binding, and we found that Cs+ bound more strongly to binding sites in BPG and in RBC membranes than in any other intracellular component in RBCs at physiologic concentrations. By using James–Noggle plots, we obtained Cs+ binding constants per binding site in BPG (66 ± 8 M−1), ADP (19 ± 1 M−1), ATP (25 ± 3 M−1), and RBC membranes (55 ± 2 M−1) from the observed T1 values. We also studied the effect of Cs+ on the oxygen (O2) affinity of purified Hb and of Hb in intact RBCs in the absence and in the presence of BPG. In the absence of BPG, the O2 affinity of Hb decreased upon addition of Cs+. However, in the presence of BPG, the O2 affinity of Hb increased upon addition of Cs+. The O2 affinity of Cs+-loaded human RBCs was larger than that of Cs+-free cells at the same BPG level. 31P NMR studies on the pH dependence of the interaction between BPG and Hb indicated that the presence of Cs+ resulted in a smaller fraction of BPG available to bind to the cleft of deoxyHb. Our NMR and O2 affinity data indicate that a strong binding site for Cs+ in human RBCs is BPG. A partial mechanism for Cs+ toxicity might arise from competition between Cs+ and deoxyHb for BPG, thereby increasing oxygenation of Hb in RBCs, and thus decreasing the ability of RBCs to give up oxygen in tissues. The presence of Cs+ at 12.5 mM in intact human RBCs containing BPG at normal concentrations did not, however, alter significantly the O2 affinity of Hb, thus ruling out the possibility of Cs+–BPG interactions accounting for Cs+ toxicity in this cell type.

Lipid peroxidation, antioxidant enzymes and glutathione levels in human erythrocytes exposed to colloidal iron hydroxide in vitro.

The free form of the iron ion is one of the strongest oxidizing agents in the cellular environment. The effect of iron at different concentrations (0, 1, 5, 10, 50, and 100 microM Fe3+) on the normal human red blood cell (RBC) antioxidant system was evaluated in vitro by measuring total (GSH) and oxidized (GSSG) glutathione levels, and superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px) and reductase (GSH-Rd) activities. Membrane lipid peroxidation was assessed by measuring thiobarbituric acid reactive substance (TBARS). The RBC were incubated with colloidal iron hydroxide and phosphate-buffered saline, pH 7.45, at 37 degrees C, for 60 min. For each assay, the results for the control group were: a) GSH = 3.52 +/- 0.27 microM/g Hb; b) GSSG = 0.17 +/- 0.03 microM/g Hb; c) GSH-Px = 19.60 +/- 1.96 IU/g Hb; d) GSH-Rd = 3.13 +/- 0.17 IU/g Hb; e) catalase = 394.9 +/- 22.8 IU/g Hb; f) SOD = 5981 +/- 375 IU/g Hb. The addition of 1 to 100 microM Fe3+ had no effect on the parameters analyzed. No change in TBARS levels was detected at any of the iron concentrations studied. Oxidative stress, measured by GSH kinetics over time, occurs when the RBC are incubated with colloidal iron hydroxide at concentrations higher than 10 microM of Fe3+. Overall, these results show that the intact human RBC is prone to oxidative stress when exposed to Fe3+ and that the RBC has a potent antioxidant system that can minimize the potential damage caused by acute exposure to a colloidal iron hydroxide in vitro.

Transbilayer movement of NBD-labeled phospholipids in red blood cell membranes: outward-directed transport by the multidrug resistance protein 1 (MRP1).

The outward movement (flop) of fluorescently labeled analogues of phosphatidylserine (PS) and phosphatidylcholine (PC) in human and murine red blood cells (RBC) was examined. 1-Oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl (C6-NBD) analogues of PS and PC were incorporated in the inner leaflet of the plasma membrane through the action of aminophospholipid translocase or through equilibration upon prolonged incubation, respectively. After removal of noninternalized probe, externalization of C6-NBD-PS or C6-NBD-PC from the inner to outer leaflet was monitored by continuous incubation of the cells in the presence of bovine serum albumin. Flop rates for both probes in intact human RBC were virtually identical (t1/2 approximately 1.5 h), confirming earlier findings by Bitbol et al. [Bitbol, M., et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6783-6787] and Connor et al. [Connor, J., et al. (1992) J. Biol. Chem. 267, 19412-19417]. Flop activity in resealed RBC ghosts could only be found upon coinclusion of both ATP and oxidized glutathione (GSSG). Furthermore, flop in intact cells was sensitive to verapamil (IC50 = 5-7 microM), vincristine (IC50 = 20 microM), and indomethacin (IC50 = 50 microM), suggesting the involvement of proteins conferring multidrug resistance (MDR). Experiments with RBC from knock-out mice for multidrug resistance P-glycoproteins (Mdr1a/1b-/- and Mdr2-/-) and multidrug resistance protein 1 (Mrp1-/-) revealed that Mrp1 is responsible for the observed flop of the fluorescent lipid analogues. We found no indications for outward transport of endogenous PS by any of these drug-transporting proteins as measured by a sensitive prothrombinase assay. Neither aminophospholipid translocase nor Ca2+-induced lipid scramblase activities were affected in RBC of these knock-out mice. We conclude that lipid floppase activity, as detected with lipid probes, reflects the activity of MRP1 recognizing the modified lipid analogues as xenobiotics to be expelled from the cell.

Mobility of water in biological systems studied by17O NMR via multiple-quantum filtered relaxation analysis

Multiple-quantum filtering NMR sequences were used to study the multiexponential relaxation behaviour of H217O in the presence of macromolecules. By this means, the fraction and the correlation time of water in slow motion, or ‘bound’ water, were determined, in addition to the relaxation time of bulk water in the extreme narrowing limit. Experiments were carried out on aqueous solutions of bovine serum albumin, hen egg white lysozyme and intact human red blood cells in which intracellular water properties were studied. The small fraction of bound water of less than 1% seems to correspond to ‘strongly bound’ water, whereas the behaviour of bulk water was different from that of pure water and may be due to weak (or transient) interactions with macromolecules. The experiments and the data analysis gave reproducible results which support the idea that diverse samples could be studied in this way in order to help understand their water properties. © 1997 John Wiley & Sons, Ltd.