Erythrocyte Membrane Vesicles Research Articles

An Erythrocyte Membrane-Derived Nanosystem for Efficient Reversal of Endothelial Injury in Sepsis.

Sepsis is caused by a disordered host immune in response to infection and endothelial cells perform a crucial role in boosting immunity reaction in the pathophysiology of sepsis and septic organ failure. The aim of this study is to construct a novel erythrocyte membrane-derived nanosystems to reverse endothelial damage in sepsis. Herein, an innovative nanometer calcium metal-organic framework(Ca-MOF) is generated for the first time by using chelidonic acid as a ligand and calcium chloride as an ion donor for anti-inflammation. Then, zoliflodacin is loaded into Ca-MOF (CMZ) to sterilize and nanoscale erythrocyte membrane vesicles are prepared by modification with a γ3 peptide on the surface (γ3-RM) for precise targeting. Finally, γ3-RM camouflages the nanocore CMZ, to form novel erythrocyte membrane-camouflaged nanoparticle γ3-RCMZ. The superior performance of novel nanosystem results from its suitable biocompatibility, nontoxicity, specific targeting, and anti-inflammatory and bactericidal effects. Its anti-inflammatory mechanism mainly involves inhibiting the Caspase1-nuclear factor kappa-B (Caspase1-NF-κB) pathway and oxidative stress reduction to alleviate endothelial damage. Moreover, the findings have revealed for the first time that the bactericidal drug zoliflodacin also has anti-inflammatory effects in vivo and in vitro. Therefore, the novel nanosystem (γ3-RCMZ) provides a new nanotherapy strategy for sepsis treatment.

Biomimetic liposomes hybrid with erythrocyte membrane modulate dendritic cells to ameliorate systemic lupus erythematosus.

Dendritic cells (DCs)-based immunotherapy has shown immense promise in systemic lupus erythematosus (SLE) treatment. However, existing carrier strategies such as polymers, liposomes, and polypeptides, are difficult to achieve active targeting to DCs due to their intricate interaction with biological systems. Since DCs represent a class of phagocytes responsible for the removal of senescent or damaged erythrocytes, we hypothesize that hybrid vesicles containing erythrocytes membrane components could be presented to be potent drug carriers to target DCs specifically. Herein, inspired by the cell membrane fusion technique, we develop hybrid biomimetic liposomes (R-Lipo) by fusing natural erythrocyte membrane vesicles and artificial liposomes for DCs-targeted SLE therapy. The resultant R-Lipo exhibited excellent biocompatibility and was shown to be effectively internalized by DCs both in vitro and in vivo. Using an immunosuppressant, mycophenolic acid (MPA), as the model drug, MPA-loaded R-Lipo powerfully suppressed DCs maturation and efficiently controlled the duration of lupus nephritis without apparent side effects. Our findings provide a safe, effective, and easy-to-prepare biomimetic vesicle platform for the treatment of SLE and other DC-associated diseases.

Celastrol-loaded biomimetic nanodrug ameliorates APAP-induced liver injury through modulating macrophage polarization.

Drug-induced liver injury (DILI) is a major concern in clinical treatment as well as postmarketing surveillance, showing an urgent requirement for the development of protective medications. Celastrol (Cel), a highly active natural product extracted from the roots of Tripterygium wilfordii, has a potential liver protective activity due to its antioxidant and anti-inflammatory effects. However, the further application of Cel to DILI remains a challenge because of its short half-life, low solubility, and toxic side effects. Herein, we developed a Cel-loaded biomimetic nanodrug based on erythrocyte membrane vesicles (EMV) for protecting the liver from acetaminophen (APAP)-induced liver injury. The Cel-loaded EMV (C-EMV) with lower cytotoxicity had a well-sustained release effect and exhibited excellent ability for liver accumulation under physiological and pathological conditions. By suppressing the inflammatory response of pro-inflammatory macrophage M1 polarization while stimulating anti-inflammatory macrophage M2 polarization, C-EMV could significantly alleviate the primary pathological manifestations related to liver injury, including aberrant elevation of biochemical indicators, histopathological alterations, neutrophil infiltration as well as hepatocyte DNA fragmentation. The macrophage depletion experiment further demonstrated that the protective effect of C-EMV on APAP-induced liver injury appeared to be dependent on hepatic macrophages. Therefore, C-EMV as a biomimetic nanodrug exhibits great potential for attenuating the progress of DILI, providing a new approach to protecting the liver from DILI as well as other liver inflammatory diseases through a targeted nanodelivery system. KEY MESSAGES: EMV biomimetic nanocarrier has good monodispersity and sustained-release property. EMV biomimetic nanocarrier displays excellent liver-targeting capability under physiological and pathological conditions. C-EMV biomimetic nanodrug with lower cytotoxicity regulates macrophage polarization in vitro and in vivo. C-EMV biomimetic nanodrug can significantly alleviate APAP-induced liver injury. The protective effect of C-EMV on APAP-induced liver injury is dependent on hepatic macrophages.

Cyanine Dyes for Photo-Thermal Therapy: A Comparison of Synthetic Liposomes and Natural Erythrocyte-Based Carriers

Cyanine fluorescent dyes are attractive diagnostic or therapeutic agents due to their excellent optical properties. However, in free form, their use in biological applications is limited due to the short circulation time, instability, and toxicity. Therefore, their encapsulation into nano-carriers might help overcome the above-mentioned issues. In addition to indocyanine green (ICG), which is clinically approved and therefore the most widely used fluorescent dye, we tested the structurally similar and cheaper alternative called IR-820. Both dyes were encapsulated into liposomes. However, due to the synthetic origin of liposomes, they can induce an immunogenic response. To address this challenge, we proposed to use erythrocyte membrane vesicles (EMVs) as “new era” nano-carriers for cyanine dyes. The optical properties of both dyes were investigated in different biological relevant media. Then, the temperature stability and photo-stability of dyes in free form and encapsulated into liposomes and EMVs were evaluated. Nano-carriers efficiently protected dyes from thermal degradation, as well as from photo-induced degradation. Finally, a hemotoxicity study revealed that EMVs seem less hemotoxic dye carriers than clinically approved liposomes. Herein, we showed that EMVs exhibit great potential as nano-carriers for dyes with improved stability and hemocompatibility without losing excellent optical properties.

Magneto-Erythrocyte Membrane Vesicles’ Superior T2 MRI Contrast Agents to Magneto-Liposomes

Despite their high potential, most of the clinically approved iron oxide (IO)-based contrast agents for magnetic resonance imaging (MRI) have been withdrawn from the market either due to safety issues or lack of sales. To address this challenge, erythrocyte membranes have been used to prepare IO-based T2 contrast agents with superior MRI properties and higher safety margin. A simple formulation procedure has been proposed, and the nanostructures’ morphology and physicochemical properties have been evaluated. We compared their performance in terms of contrast ability in MRI to the more clinically established magneto-liposomes and non-encapsulated nanoparticles (NPs). The encapsulation of 5-nm iron oxide nanoparticles (IO NPs) in the liposomes and erythrocyte membrane vesicles (EMVs) led to a significant improvement in their r2 relaxivity. r2 values increased to r2 = 188 ± 2 mM−1s−1 for magneto-liposomes and r2 = 269 ± 3 mM−1s−1 for magneto-erythrocyte membranes, compared to “free” IO NPs with (r2 = 12 ± 1 mM−1 s−1), measured at a 9.4 T MRI scanner. The superiority of magneto-erythrocyte membranes in terms of MRI contrast efficacy is clearly shown on T2-weighted MR images. Our study revealed the hemocompatibility of the developed contrast agents in the MRI-relevant concentration range.

Erythrocyte membrane vesicles coated biomimetic and targeted doxorubicin nanocarrier: Development, characterization and in vitro studies

Ovarian cancer is the most common cause of death within gynecologic cancers and doxorubicin is an anthracycline agent, is widely used in many cancer types. Folate receptor enables of accumulation folate linked nanoparticles into tumor cells and magnetic targeting also provides localized addressing. The aim of this work is to develop a magnetic and biomolecularly targeted biomimetic doxorubicin nanocarrier system for ovarian cancer therapy. Firstly, highly dispersed starch and PEG diacid coated magnetic nanoparticles were synthesized with an easy and fabricable method and 91.45 ± 1.07 μg doxorubicin was adsorbed on per mg magnetic nanoparticles. The structure was verified with FTIR, TGA, VSM, TEM and zetasizer. Then, folate linked erythrocyte membrane vesicles were prepared from ghost cells and doxorubicin loaded magnetic nanoparticles were encapsulated into vesicles through extrusion due to enabling avoidance from immun clearance. The final nanocarrier system was nearly spherical in shape and had a hydrodynamic size of 157.4 ± 33.2 nm. Drug release profile was determined as in a controlled manner and IC50 value against SKOV3 for 72 h was calculated as 0.74 ± 0.32 μg/mL which was lower than that of free doxorubicin. In conclusion, it was suggested that biomimetic doxorubicin nanocarrier system have many advantages for targeted ovarian cancer therapy.

Black phosphrous quantum dot (BPQD)-loaded erythrocyte membrane vesicles for phothothermal therapy in breast cancer

Objective To investigate the method of preparing black phosphrous quantum dot (BPQD)-loaded erythrocyte membrane nanovesicles (BPQD-EMNVs), and to study its efficiency in photothermal therapy for breast cancer. Methods Fresh red blood cells (RBCs) of healthy mice were extracted to prepare erythrocyte membrane, and BPQD-EMNVs were prepared by sonication method. The morphology of BPQD-EMNVs was observed by a transmission electron microscopy. The particle size distribution was measured by a nano-particle size and Zeta potential meter. The encapsulation efficiency of BPQD was determined by inductively coupled plasma emission spectrometry. The uptake rate of BPQD-EMNVs was observed by a laser scanning confocal microscope. 4T1 tumor-bearing Balb/c mice were randomly divided into PBS, EMNVs, BPQD and BPQD-EMNVs groups, and the tumor sites were irradiated with 808 nm near-infrared light for 10 min after 4 hours of tail vein injection. The growth of the tumors was continually observed. Results The prepared BPQD-EMNVs have a regular spherical shape with an average particle diameter of 228 nm and an encapsulation efficiency of about 47%. Cellular uptake in vitro experiments showed that BPQD-EMNVs were rapidly taken up by 4T1 tumor cells. The results of animal in vivo experiments showed that BPQD-EMNVs had the highest enrichment after 4 h of injection at the tumor site, and BPQD-EMNVs could effectively kill tumor tissues after 10 min of 808 nm near-infrared light irradiation. Conclusions The BPQD-EMNVs are easy to prepare, and the prepared nanovesicles have good biocompatibility and photothermiotherapy effect, which is expected to be a promising method for breast cancer therapy. Key words: Erythrocyte membrane; Breast cancer; Black phosphorous quantum dot; Nanovesicles; Photothermal therapy

In vitro and in vivo evaluation of folate receptor-targeted a novel magnetic drug delivery system for ovarian cancer therapy

Doxorubicin is widely used anticancer drug; however, use of doxorubicin is limited. Under externally applied magnetic field, magnetic agents can help to transport drug directly to tumor. Folate receptor is overexpressed in ovarian carcinomas. In this study, we aimed to develop magnetically responsive and folate receptor-targeted biomimetic drug delivery system for ovarian cancer therapy. Doxorubicin-loaded and glucose/gluconic acid-coated magnetic nanoparticles were synthesized and erythrocyte membrane vesicles were used for coating of nanoparticles. Folate ligand was anchored to surface so as to target receptor. Hydrodynamic size of nanocarrier was found as 91.2 ± 20.8 nm. The results showed that delivery system has controlled drug release profile and biocompatible features. In folate-free medium, folate receptor-targeted nanocarrier showed 10.33-fold lower IC50 values for A2780 cells and 3.93-fold lower for OVCAR3 cells compared to non-targeted nanoparticles and demonstrated more cytotoxicity against ovarian cancer cells. Moreover, magnetically and folate receptor-targeted doxorubicin delivery system was significantly more effective for therapy of xenografted nude mice than free doxorubicin based on tumor shrinkages and biochemical parameters. In conclusion, it can be suggested that folate ligand-attached and biomimetically designed magnetic drug delivery system have advantages and potential for targeted ovarian cancer therapy.

Light-activatable Chlorin e6 (Ce6)-imbedded erythrocyte membrane vesicles camouflaged Prussian blue nanoparticles for synergistic photothermal and photodynamic therapies of cancer.

Multiple therapeutic modalities, such as photodynamic (PDT) and photothermal (PTT) therapies, have been jointly applied to produce a synergistic effect for tumor eradication based on the hyperthermia and generation of reactive oxygen species (ROS) mediated by photoactive agents. Effective delivery of highly efficient photosensitizers and photothermal agents is the key for combination of PDT/PTT. Herein, we propose a strategy to functionalize Prussian blue (PB) nanoparticles (NPs) with Chlorin e6 (Ce6)-imbedded erythrocyte membrane vesicles. This nanoplatform can address the major issues of these two capable photoactive agents, such as limited biocompatibility, lack of functional chemical groups, and poor bioavailability due to rapid blood clearance or self-aggregation. Specifically, PB NPs were packaged within Ce6-imbedded erythrocyte membrane vesicles, named as PB@RBC/Ce6 NPs, to take advantage of both biological functions of natural erythrocyte membranes and the unique physicochemical properties of synthetic nanoagents. Compared to bare PB NPs or free Ce6, PB@RBC/Ce6 NPs exhibited considerably enhanced cellular uptake and accumulation in tumoral tissues. Moreover, the PB@RBC/Ce6 NP-mediated PDT/PTT combination therapies produced a notable effect in boosting the necrosis and late apoptosis of tumor cells in vitro, and further showed a synergistic therapeutic effect against an orthotopic tumor model in vivo.

Modulation of multidrug resistance-associated proteins function in erythrocytes in glycerol-induced acute renal failure rats.

Evaluation of the function of multidrug resistance-associated proteins (MRPs) expressed in erythrocytes and screening of endogenous MRPs modulator(s) in glycerol-induced acute renal failure (ARF) rats. Concentrations of 2,4-dinitrophenyl-S-glutathione (DNP-SG), a substrate for MRPs, in erythrocytes after administration of 1-chloro-2,4-dintrobenzene (CDNB), a precursor of DNP-SG, were determined in control and ARF rats. The screening of endogenous MRPs modulator(s) was performed using washed erythrocytes and inside-out erythrocyte membrane vesicles (IOVs) in vitro. Accumulation of DNP-SG in erythrocytes was observed in ARF rats. Uraemic plasma components exhibited a greater inhibitory effect on DNP-SG uptake by IOVs than control plasma components and increased the DNP-SG accumulation significantly in washed erythrocytes. Several protein-bound uraemic toxins at clinically observed concentrations and bilirubin significantly inhibited DNP-SG uptake by IOVs. In washed erythrocytes, bilirubin (10 μm) and l-kynurenine (100 μm), a precursor of kynurenic acid being MRPs inhibitor, increased DNP-SG accumulation significantly. Glycerol-induced ARF rats contain various MRPs inhibitors in plasma, and membrane-permeable MRP substrates/inhibitors including their precursors inhibit the MRPs function in erythrocytes cooperatively.

【原著】 膜小胞および培養細胞を用いた生体膜輸送研究のための新規反応停止液の開発

There are certain transporters that function effectively even at ice–cold temperatures, such as facilitative glucose transporter (GLUT) 1 and equilibrative nucleoside transporter (ENT) 1 in human erythrocyte membranes. In order to evaluate the characteristics of these transporters precisely, inhibition of substrate efflux during stopping and washing processes is essential. In this study, we attempted to develop a novel stop solution that can be used universally for membrane transport studies. As candidate compounds for preparing a novel stop solution, protein denaturants such as urea, 2–mercaptoethanol, formaldehyde, and glutaraldehyde were used. D–Glucose and uridine were used as substrates for GLUT1 and ENT1, respectively. [3H]D–Glucose taken up by rightside–out erythrocyte membrane vesicles (ROVs) was almost completely effluxed during washing with ice–cold stop solution without phloretin. Protein denaturants inhibited the efflux of these substrates, and increased the remaining amount of substrates in ROVs. Especially, the combination of urea/formaldehyde or 2–mercaptoethanol/glutaraldehyde was effective as a stop solution for both GLUT1– and ENT1–mediated efflux. In addition, these stop solutions were applicable for the transport study in cultured HepG2 cells. Our results indicate that these novel stop solutions can be used universally for membrane transport studies with isolated membrane vesicles and culture cells.

Microscopic evaluation of vesicles shed by rat erythrocytes at elevated temperatures

It is hypothesized that the elevation of the temperature of the blood during heat stress may cause an increase of the shedding of erythrocyte membrane vesicles. Therefore, the increase of vesicle numbers following heat stress may be indicative of and proportional to the level heat stress. In order to test this hypothesis, erythrocytes and the vesicles shed by erythrocytes were collected from rat blood and analyzed after the elevation of body temperature by exposure to external heat. The images of erythrocytes and vesicles were analyzed by a custom light microscopy system with spatial resolution of better than 90nm. The samples were observed in an aqueous environment and required no freezing, dehydration, staining, shadowing, marking or any other manipulation. The elevation of temperature from 36.7±0.3 to 40.3±0.4°C resulted in significant increase of the concentration of vesicles in blood. At a temperature of 37°C, mean vesicle concentrations and diameters found in rat blood were (1.4±0.2)×106 vesicles/μL and 0.436±0.03μm, respectively. The concentration of free vesicles increased after exposure to heat to (3.8±0.3)×106 vesicles/μL. It was estimated that 80% of all vesicles found in rat blood are smaller than 0.45μm. The increase in the number of vesicle associated with elevated temperatures may be indicative of the heat stress level and serve as diagnostic test of erythrocyte stability and heat resistance.

Transport Characteristics of Ribavirin in Human Erythrocyte Membrane Vesicles

Transport Characteristics of Ribavirin in Human Erythrocyte Membrane Vesicles

Characterization of the ATP-dependent Sphingosine 1-Phosphate Transporter in Rat Erythrocytes

Sphingosine 1-phosphate (S1P) is a bioactive lipid signal transmitter present in blood. Blood plasma S1P is supplied from erythrocytes and plays an important role in lymphocyte egress from lymphoid organs. However, the S1P export mechanism from erythrocytes to blood plasma is not well defined. To elucidate the mechanism of S1P export from erythrocytes, we performed the enzymatic characterization of S1P transporter in rat erythrocytes. Rat erythrocytes constitutively released S1P without any stimulus. The S1P release was reduced by an ABCA1 transporter inhibitor, glyburide, but not by a multidrug resistance-associated protein inhibitor, MK571, or a multidrug resistance protein inhibitor, cyclosporine A. Furthermore, we measured S1P transport activity using rat erythrocyte inside-out membrane vesicles (IOVs). Although the effective S1P transport into IOVs was observed in the presence of ATP, this activity was also supported by dATP and adenosine 5'-(beta,gamma-imido)triphosphate. The rate of S1P transport increased depending on S1P concentration, with an apparent K(m) value of 21 microm. Two phosphorylated sphingolipids, dihydrosphingosine 1-phosphate and ceramide 1-phosphate, did not inhibit S1P transport. Similar to the intact erythrocytes, the uptake of S1P into IOVs was inhibited by glyburide and vanadate but not by the other ABC transporter inhibitors. These results suggest that S1P is exported from the erythrocytes by a novel ATP-dependent transporter.

Flow cytometric monitoring of multidrug drug resistance protein 1 (MRP1/ABCC1) -mediated transport of 2′,7′-bis-(3-carboxypropyl)-5-(and-6)- carboxyfluorescein (BCPCF) into human erythrocyte membrane inside-out vesicles

The presence of human multidrug resistance protein 1 (MRP1/ABCC1) in the human erythrocyte membrane is well established. In the present study, flow cytometric monitoring is introduced to identify MRP1 as the main transporter of 2',7'-bis-(3-carboxypropyl)-5-(and-6)-carboxyfluorescein (BCPCF) in the erythrocyte membrane and to facilitate inhibition and kinetic studies of MRP1-mediated transport. The ATP-dependent transport of BCPCF into human erythrocyte inside-out vesicles and, for comparison, into MRP1-expressing Sf9 cell membrane inside-out vesicles were studied. The MRP1-specific monoclonal antibody, QCRL-3 and the MRP1 inhibitor, MK-571 strongly decreased the uptake of BCPCF into both erythrocyte and MRP1-expressing Sf9 cell membrane inside-out vesicles. The inhibition profiles of cyclosporin A, verapamil, benzbromarone, and probenecid in erythrocyte membrane vesicles were typical for MRP1-mediated transport. Furthermore, kinetic constants K(m) and V(max) of BCPCF transport into erythrocyte membrane inside-out vesicles were determined in the absence and in the presence of selected inhibitors (MK-571, cyclosporin A, benzbromarone and verapamil). The presented results identified MRP1 as the major transporter of BCPCF in the human erythrocyte membrane and showed for the first time that the active transport of fluorescent substrate into inside-out vesicles can be monitored by flow cytometry.

CGMP (guanosine 3′,5′-cyclic monophosphate) transport across human erythrocyte membranes

Human erythrocytes produce cGMP that can be eliminated by phosphodiesterases or active efflux transporters. The efflux can be studied under controlled conditions as ATP-dependent uptake into inside-out membrane vesicles. However, widely differing values for the transport rates have been reported. We have here examined factors that influence the uptake rates measured and thus may explain these discrepancies. Both the ionic composition of the buffer used during uptake and the mode of vesicle preparation were found to affect the observed transport rates. Furthermore it was apparent that different blood donors expressed on their erythrocytes different amounts of both MRP4 and MRP5, transporters that have been putatively linked to cGMP efflux across erythrocyte membranes. These differences in expression were reflected in differences in rates of cGMP uptake into inside-out erythrocyte membrane vesicles. Calculations based on the transport rates observed using vesicles suggest that efflux may be the principal means for eliminating cGMP from human erythrocytes.

Nicotinamide is not a substrate of the facilitative hexose transporter GLUT1.

It has been proposed that GLUT1, a membrane protein that transports hexoses and the oxidized form of vitamin C, dehydroascorbic acid, is also a transporter of nicotinamide (Sofue, M., Yoshimura, Y., Nishida, M., and Kawada, J. (1992) Biochem. J. 288, 669-674). To ascertain this, we studied the transport of 2-deoxy-D-glucose, 3-O-methyl-D-glucose, and nicotinamide in human erythrocytes and right-side-out and inside-out erythrocyte membrane vesicles. The transport of nicotinamide was saturable, with a K(M) for influx and efflux of 6.1 and 6.2 mM, respectively. We found that transport of the hexoses was not competed by nicotinamide in both the erythrocytes and the erythrocyte vesicles. Likewise, the transport of nicotinamide was not affected by hexoses or by inhibitors of glucose transport such as cytochalasin B, genistein, and myricetin. On the other hand, nicotinamide blocked the binding of cytochalasin B to human erythrocyte membranes but did so in a noncompetitive manner. Using GLUT1-transfected CHO cells, we demonstrated that increased expression of GLUT1 was paralleled by a corresponding increase in hexose transport but that there were no changes in nicotinamide transport. Moreover, nicotinamide failed to affect the transport of hexoses in both control and GLUT1-transfected CHO cells. Therefore, our results indicates that GLUT1 does not transport nicotinamide, and we propose instead the existence of other systems for the translocation of nicotinamide across cell membranes.

Effect of Band 3 Subunit Equilibrium on the Kinetics and Affinity of Ankyrin Binding to Erythrocyte Membrane Vesicles

The membrane-spanning protein, band 3, anchors the spectrin-based membrane skeleton to the lipid bilayer via the bridging protein, ankyrin. To understand how band 3 subunit stoichiometry influences this membrane-skeletal junction, we have induced changes in the band 3 association equilibrium and assayed the kinetics and equilibrium properties of ankyrin binding. We observe that band 3 oligomers convert slowly to dimers and ultimately monomers following removal of ankyrin. Addition of excess ankyrin back to these membranes enriched in dissociated band 3 then shifts band 3 almost entirely to tetramers, confirming that the tetrameric form of band 3 constitutes the preferred oligomeric state of ankyrin binding. 4, 4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) labeling of band 3, which is shown to shift most of the band 3 population to dimers, eliminates the majority of ankyrin-binding sites on the membrane and greatly reduces retention of band 3 in detergent-extracted membrane skeletons. Furthermore, DIDS- modified membranes lack all low affinity ankyrin-binding sites and roughly half of all high affinity sites. Since labeled membranes lack the rapid kinetic phase of ankyrin binding and exhibit only half of the normal amplitude of the slow kinetic phase, it can be concluded that the rapid phase of ankyrin association involves low affinity sites and the slow phase involves high affinity sites. A model accounting for these data and most previous data on ankyrin-band 3 interactions is provided.

Biochemical Analysis of Potential Sites for Protein 4.1-mediated Anchoring of the Spectrin-Actin Skeleton to the Erythrocyte Membrane

Erythrocyte protein 4.1 has been hypothesized to link the spectrin-actin junctional complex directly to the cytoplasmic domain of glycophorin C, but this bridging function has never been directly demonstrated. Because an alternative protein-mediated bridge between the junctional complex and the cytoplasmic domain of band 3 is also plausible, we have undertaken to characterize the membrane sites to which protein 4.1 can anchor the spectrin and actin skeleton. We demonstrate that proteolytic removal of the cytoplasmic domain of band 3 has minimal effect on the ability of protein 4.1 to promote 125I-labeled spectrin and actin binding to KI-stripped erythrocyte membrane vesicles. We also show that quantitative blockade of all band 3 sites with either monoclonal or polyclonal antibodies to band 3 is equally ineffective in preventing protein 4.1-mediated association of spectrin and actin with the membrane. In contrast, obstruction of protein 4.1 binding to its docking site on the cytoplasmic pole of glycophorin C is demonstrated to reduce the same protein 4.1 bridging function by approximately 85%. We conclude from these data that (i) glycophorin C contributes the primary anchoring site of the protein 4.1-mediated bridge to the spectrin-actin skeleton; (ii) band 3 is incapable of serving the same function; and (iii) additional minor protein 4.1 bridging sites may exist on the human erythrocyte membrane.

Direct measurement of nitrite transport across erythrocyte membrane vesicles using the fluorescent probe, 6-methoxy-N-(3-sulfopropyl) quinolinium.

Nitrite was shown to quench the fluorescence of 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) almost twofold more than chloride. SPQ loaded inside vesicles prepared from asolectin and isolated erythrocyte ghosts allowed for the direct measurement of nitrite movement across these membranes. Movement of nitrite across asolectin occurred by diffusion as HNO2 in a pH-dependent manner. By contrast, erythrocyte ghosts had very low diffusion rates for nitrous acid. Erythrocyte ghosts preloaded with 50 mM nitrite to quench SPQ fluorescence were utilized to study heteroexchange with externally added anions. SPQ fluorescence increases (becomes unquenched) with added bicarbonate and nitrate, indicating that nitrite is moving out of the preloaded vesicles. The pH optimum for this exchange was approximately 7.6 and exchange was inhibited by N-ethylmaleimide (NEM) and dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). These data indicate that nitrite moves across erythrocyte plasma membranes as NO2- by a heteroexchange mechanism with other monovalent anions.