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ISSN 0582-9879            ACTA BIOCHIMICA et BIOPHYSICA SINICA 2001, 33(5): 489-496    CN 31-1300/Q

Effect on Glucose Transport and Anion Exchange in Human Erythrocytes by Mechanical Force Factors

LI Yue-Zhou, ZHOU Han-Qing, PENG Feng, ZHANG Zhi-Hong*

( Liren Laboratory, Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai 200433, China )

Abstract The mechanical forces play an important role in both the normal physiology and the pathology of the cardiovascular system. It was observed that an increase in speed of rotary flow of the intact erythrocyte suspension resulted in increase in the rate of glucose entry across the erythrocyte membranes. The effects of osmotic stress and a membrane curvature-altering drug, chlorpromazine, on the glucose and anion transport were also shown. The decrease in the activation energy for glucose and anion transport according to Arrhenius plot for temperature dependence of membrane transport meant that thermodynamic barrier for transporter reorientation in the membranes was reduced. The data from the measurement of intrinsic fluorescence quench in ghosts indicated that the conformations of both glucose transport protein (GLUT1) and anion transport protein (band 3) in the erythrocyte membranes were affected by the mechanical force factors. After inhibition of anion transport, a modification of the response of glucose transport by the mechanical force was observed, which also indicates that there is an information linkage between GLUT1 and band 3 in the erythrocyte membranes.

Key words mechanical force; erythrocytes; glucose transport; anion exchange

Cells in body are subjected to various forms of mechanical forces, including the tangential shear stress that is due to fluid flow, the volume stress that results from osmotic pressure, and other mechanical stimuli such as tensile stress exerted by neighboring cells, the morphologic stress by drug and the internal tension of the cytoskeleton^[1,2]. It is well known that the cardiovascular system generates an appropriate pressure gradient for the delivery of blood flow to serve metabolic and other functional needs to tissues. The rheological functions of the system result from the coordinated activities of its components, especially the erythrocytes. The molecular events are beneficial for the body to cope with altered hemodynamic demands, but they may be inappropriate and detrimental under some disease states.

It has been reported that physiological shear stresses enhanced the Ca^2＋ permeability in human erythrocyte membranes^[3]. Health erythrocytes deformed by shear respond monovalent and divalent cations with increased permeability^[4]. Similarly, the erythrocytes of sickle cell anemia, which acquire distinctive elongated spicules upon deoxygenation, exhibit increased permeability to both monovalent and divalent cations^[5]. Volume-sensitive ion transport in the erythrocytes was also observed^[6]. Mechanosensitive ion channels are well known recently, especially because the three-dimensional structure of KcsA potassium channel has been determined^[7]. But, relatively little is known about the role of mechanical force in the function performance of the membrane transport proteins.

Because of the simplicity of the structure of the human erythrocyte, this physically induced response is particularly amenable to study in detail, and may afford insight into the responses of more complex mammalian cells. In this paper, we focused on the effect of mechanical force, including osmotic stress and drug-induced cell deformation, on the properties of membrane transporters, glucose transporter 1 (GLUT1) and band 3, in human erythrocyte membranes. GLUT1 is a 55 kD integral membrane glycoprotein that facilitates the passive diffusion of D-glucose across the cell membrane and contains 12 membrane-spanning helices^[8]. Band 3 is a 911-amino-acid glycoprotein that exchanges Cl^- for HCO₃^- as part of the process of CO₂ elimination. The protein comprises two domains: the 52 kD carboxyl-terminal domain embedded in the membrane which contains the anion-transporting site, and the 43 kD amino-terminal domain which contains the binding sites for the cytoskeletal proteins, hemoglobin, and glycolytic enzymes^[9]. We attempt to determine the response of both the GLUT1 and band 3 to the mechanical signal, and to examine is there any linkage between GLUT1 and band 3 during the process of signal transduction.

1 Materials and Methods

1.1 Materials

Human blood samples were obtained from the Shanghai Red Cross Blood Center and used within 2 weeks. D-glucose, phloretin, N-acetyltryptophanamide(NATA), 4,4'-diisothiocyno-stilbene-2,2'-disulfonate(DIDS), Con A and chlorpromazine were purchased from Sigma. “GOD-PAP” kit for determination of glucose was from Shanghai Kexin Biotech Institute. All other reagents were of analytical grade.

1.2 Preparation of erythrocytes and ghosts

The erythrocytes were washed three times with PBS(155 mmol/L NaCl, 5 mmol/L sodium phosphate, pH 7.4). For preparation of white-stripped ghosts, the erythrocytes at 1:40 were hemolyzed in lysis medium (5 mmol/L sodium phosphate, pH 7.4) at 4 ℃ for 30 min. When membrane fraction became a pearly white appearance after several times washing and centrifuging at 15 000 r/min, they were collected and then exposed to 10 volumes of ice-cold alkaline lysis medium (5 mmol/L sodium phosphate, pH 12.0 adjusted with dilute NaOH) for 20 s to deplete peripheral proteins. The ghosts were then washed repeatedly with PBS until the pH of the supernatant was 7.4. Aliquots of the ghosts were typically resuspended in PBS and incubated for 1 h at 37 ℃ to promote resealing, and used to measure the fluorescence under osmotic stress. The membrane protein concentration was calibrated according to Lowry's method.

1.3 Fluorescence measurements

Fluorescence measurements were performed at 20 ℃ with a Hitachi M850 spectrofluorometer equipped with a circulating water bath^[10]. The wavelength of excitation was 295 nm with a bandwidth of 3 nm and the emission was at 340 nm with a bandwidth of 9 nm to minimize the contribution of tyrosine residues. A 310 nm filter was used. Samples containing 25 mg of protein/L were constantly stirred in a 1 cm cuvette during measurements with a magnetic stirrer bar at the bottom of the cuvette. The unspecific effects of dilution, inner filter and ligands fluorescence reabsorption were corrected by a standard, NATA.

Tryptophan residues in GLUT1 can be divided into two distinct populations, i.e., “accessible” and “not accessible” to aqueous solvent, and for fluorescence quenching studies modified Stern-Volmer equation was introduced^[11],

F0/ΔF＝1/f＋1/fK_q[Q] (1)

where ΔF(＝F0-F) is fluorescence change as quencher [Q] binding, F0 and F are the unquenched and quenched fluorescence intensity, respectively. f is the fractional accessible protein fluorescence. K_q is apparent quenching constant and can be obtained from a plot of F0/ΔF vs. 1/[Q], which value equals intercept/slope of the straight line. Dissociation constant (K_d) is deduced from K_q(K_d＝1/K_q).

1.4 Sugar transport measurements

Both the zero-trans entry and infinite-cis exit were determined. For measurement of zero-trans entry, washed erythrocytes were incubated at 37 ℃ in PBS for 40 min for depletion of glucose inside the cells. 50 ml of the erythrocyte suspension (～3×10⁸ cell/ml) was rapidly mixed with 2 ml PBS containing 200 mmol/L D-glucose in 1 cm cuvette which contained a stirring bar. The time course of the change in absorbance of the cell suspension at 660 nm on a spectrometer (Model 722, Shanghai No.3 Analytical Instrument Factory) was measured at 37 ℃ with the circular water. The output electrical signal was fed to a computer. The sampling point for each measurement was 1000. The initial transport rate V₀ was determined from the recorded curve of “absorbanre vs. time” and accumulated over 5 times of measurement. For the erythrocytes treated with a series of osmolalities, the infinite-cis exit of glucose was determined by use of GOD-PAP method. The erythrocytes were incubated in the solution with a series of osmolalities, which contained 80 mmol/L D-glucose, for 40 min at 37 ℃. 50 mg of packed erythrocytes was mixed rapidly with 1 ml the solution containing a series of (0～10 mmol/L) D-glucose at 16 ℃. Efflux was terminated at required interval (0～120 s) by the rapid addition of 3 ml ice-cold stopping buffer which contained 50 mmol/L phloretin and maintained the same osmolality as the reacting solution. Then, the suspension was quickly centrifuged at 0 ℃, and the D-glucose amount in the supernatant was determined. According to the data of Solomon et al.^[12] and our observation, the total volume of soluble water inside the erythrocytes under different osmolalities can be deduced. Thus, the glucose concentration inside of the erythrocytes at any time interval was calculated from the exterior amount of glucose. The initial rate of glucose exit was obtained by regression analysis of the initially linear segment of the time course data. The maximal rate of glucose infinite-cis exit (V_max) and affinity constant (K_m) were deduced according to Harris^[13].

1.5 Mechanical force factors

The spectrometer was modified to set up a magnetic stirrer at the bottom of the photometric cell holder. The solution in cuvette was driven to rotate by a stirring bar and form a rotary flow field. The relationship between rotation speed and mechanical force acted on the cells was studied when the erythrocytes were rotated in the 1-cm cuvette. The mechanical force was increased with the rotation speed of the stirring bar in this model^[14]. The parameter of the rotation speed is w(r/s, revolutions per second). In the experiments of osmotic stress, PBS was required to diluted 1:1 with water, then added sucrose to obtain the solutions with osmolalities from 240 to 480 mOsm. The osmolalities of solution were measured by a vapor pressure osmometer, VAPRO model 5520(Wescor Inc.). Cell deformation was induced by chlorpromazine. 50 ml of the erythrocyte suspension (～3×10⁸ cell/ml) was incubated with 2 ml PBS containing a series of chlorpromazine (0～200 mmol/L) at 37 ℃ for 15 min.

1.6 Anion transport measurements

50 ml of isotonic erythrocytes (～3×10⁸ cell/ml) was rapidly mixed with 2 ml isotonic solution containing 15.5 mmol/L NaNO₂, 139.5 mmol/L NaCl, 5 mmol/L sodium phosphate, pH 7.4 in cuvette. When NO₂^- ions reacted with hemoglobin after penetrating into the erythrocytes, methemoglobin was formed, and hence the optical properties of cell suspension were changed. According to the time course of the change in absorbance of the cell suspension at 576 nm on spectrometer, the rate constant, K_t, of NO₂^- entry into the erythrocyte was obtained^[15].

2 Results

Blood flow and the associated shear stress have been shown to play an active role in the regulation of the structure and function of vascular cells. We were interested in examining the effect of mechanical force on the membrane transport proteins in the human erythrocytes. Fig.1 shows the effect of mechanical force induced by rotary flow on the properties of glucose transport across the erythrocyte membranes. As rotation speed (w), i.e., mechanical force, was increased the initial rate of glucose zero-trans entry, V₀, was increased significantly. V₀ rose from 13.2 mmol·L^-1/min to 77.1 mmol·L^-1/min after rotation speed was changed to be 40 r/s from zero. Because the concentration of glucose in the medium was 200 mmol/L, at which the binding sites of GLUT1 for glucose were occupied completely, V₀ can be regarded as the maximum transport rate approximately^[16]. Using fluorescence quenching method and according to Eq.1 the value of dissociation constant, K_d, was obtained. It was deduced from the relationship between K_d and rotation speed [Fig.1(B)] that the mechanical force would affect the conformational state of GLUT1. For estimation of unstirred layer effect, we plotted 1/V vs. S/(V_max-V) during infinite-cis exit under rotary flow (w＝10 r/s)^[10] according to Lieb and Stein, where V is the initial efflux rate of glucose and S is glucose concentration in the medium^[17]. Results from linear regression analysis showed that the P^-1, x-intercept of the extrapolated straight line, was 0.0022±0.0021. The value of P^-1 found to be not significantly different from zero means that the unstirred layer on the outer side of the erythrocyte membrane needs not be considered.

Fig.1 Effect of rotation speed, w, on the initial rate of glucose zero-trans entry, V₀, and dissociation constant, K_d

(A) Relationship between the rotation speed of the cell suspension, w, and V₀. (B) Dissociation constant, K_d, of glucose binding to GLUT1 at different rotation speed at 20 ℃. Mean values and standard deviation are indicated from three separate experiments.

There are several methods for studying glucose transport in the erythrocytes. One of the rapid and simple means is optical method based on the osmotic swelling and shrinking of the erythrocytes during the course of glucose transport. In the experiment of osmotic stress, we must always keep the erythrocytes in a condition of certain osmolality and avoid any possible volume change. So, an enzymatic method, “GOD-PAP”, was used in this research instead. The maximal rate of glucose infinite-cis exit (V_max) and affinity constant (K_m) under a series of osmotic stresses were determined at 16 ℃ (Fig.2). Both of them were increased with the enhancement of osmotic stress. V_max rose from 16.6 mmol·L^-1/min to 106.1 mmol·L^-1/min when the medium osmolality was increased from 240 mOsm to 480 mOsm. Meanwhile, K_m was increased from 4.0 mmol/L to 18.0 mmol/L. The anion transport mediated by band 3 was also responded to the osmotic stress. The rate constant of NO₂^- permeability, K_t, was increased from 7.0 mmol·L^-1/min to 81.6 mmol·L^-1/min with the osmolality enhancement from 240 mOsm to 480 mOsm [Fig.2(C)]. Fig.3 shows the relationship between medium osmolality and dissociation constant (K_d) for glucose binding to GLUT1 or DIDS bound to band 3. Resealed white stripped ghosts were treated with 20 mmol/L glucose or 10 mmol/L DIDS at 20 ℃, pH 7.4. The intrinsic tryptophan fluorescence of the ghosts was measured. According to the fluorescence quenching an apparent quenching constant was obtained, and then dissociation constant, K_d, of glucose binding to GLUT1 or DIDS binding to band 3 was deduced. It seems that the environment of tryptophan residues in GLUT1 or band 3 was changed by the osmotic stress.

Fig.2 Effect of osmolality on the glucose and anion transport

Relationships between the maximal rate of glucose infinite-cis exit, V_max, (A), affinity constant, K_m, (B), rate constant, K_t, of NO₂^- entry (C) and the medium osmolality. Mean values and standard deviation are indicated from three separate experiments.

Fig.3 Relationship between osmolality and dissociation constant (K_d) for glucose binding to GLUT1 (●) or DIDS binding to band 3 (○)

Knowledge of the temperature dependence of membrane transport makes an appraisal of the thermodynamic features of the transport system possible from Arrhenius plot of the data. The Arrhenius plots of effect of mechanical forces on the temperature dependence of glucose flux and anion transport are shown in Fig.4. The activation energy, E_a, for glucose efflux was 95.8 kJ/mol for osmolality of 240 mOsm , 78.4 kJ/mol for 300 mOsm, and 45.4 kJ/mol for 480 mOsm [Fig.4(A)]. Mechanical force also affected the temperature dependency of glucose influx. As shown in [Fig.4(B)], the slopes of the straight lines, E_a, was decreased with increase in rotation speed, w. Fig.4C shows the temperature dependence of anion transport with three different osmolalities. We found that an increase in medium osmolality resulted in decrease in Ea for anion exchange. When osmolality was increased from 240 mOsm to 300 mOsm , and finally reached to 480 mOsm, the E_a was reduced from 47.4 kJ/mol to 42.7 kJ/mol, and then to 33.8 kJ/mol, respectively.

Fig.4 Temperature dependence of glucose transport and anion exchange with various mechanical force factors

(A) For glucose efflux under three different osmolalities: 240 mOsm (□), 300 mOsm (●) and 480 mOsm (△). (B) For glucose influx under various rotation speed of the cell suspension: w＝0 r/s (□), w＝10 r/s (●) and w＝40 r/s (△). (C) For anion exchange under three different osmolalities: 240 mOsm (□), 300 mOsm (●) and 480 mOsm (△).

A wild range of amphipathic drugs can change membrane tension and curvature in the erythrocytes according to the bilayer-couple hypothesis^[18]. Chlorpromazine molecules preferentially partition into the inner leaflet of the phospholipid bilayer to bend the erythrocytes inward and form cup shape. The rates of both glucose and anion transport were affected by the shape change induced by chlorpromazine (Fig.5). V₀ of glucose influx was increased from 10.3 mmol·L^-1/min to 82.4 mmol·L^–1/min when chlorpromazine concentration was risen from 0 to 200 mmol/L. In the meanwhile, the rate constant of NO₂^- transport was reduced from 9.8 mmol·L^–1 / min to 2.2 mmol·L^–1/min.

Fig.5 Effect of chlorpromazine on the glucose entry (A) and anion transport (B)

Mean values and standard deviation are indicated from three separate experiments.

Janoshazi et al suggested that a transport protein complex, centered on band 3 and included GLUT1, was responsible for the entire transport process^[19]. We used DIDS, a specific inhibitor for anion transport, as a probe to study the interaction of GLUT1 and band 3. Treated with DIDS from 0 to 50 mol/L, the response of V₀ to the mechanical force (rotation speed, w was lowered [Fig.6(A)]. It can be inferred that the conformational changes in band 3 after binding with DIDS would be somehow transferred to GLUT1. This would alter the response behavior of GLUT1 to the mechanical force. Con A, a kind of lectin that can agglutinate human erythrocytes, binds to band 3 through oligosaccharide chain. In the experiments, Con A did not change the effect of mechanical force on the transport property of GLUT1 [Fig.6(B)]. What interested was that when we treated the erythrocytes with Con A and DIDS together, the pattern of response of V₀ to the mechanical stress was different from that of only DIDS treated, i.e., V₀ was decreased with the enhancement of mechanical force. Meanwhile, Con A did not affect the anion exchange in our experimental condition (data not shown).

Fig.6 Effect of DIDS and Con A on the rate of glucose transport at various mechanical forces

(A) Relationship between the initial rate of glucose zero-trans entry, V₀ , and the rotation speed of the cell suspension, w at a set of DIDS concentration: control(□), 2 mmol/L(■), 5 mmol/L(△), 10 mmol/L(▼), 20 mmol/L(○), 50 mmol/L(●). (B) Effect of Con A on the relationship between V₀ and w: control(■), 50 mmol/L DIDS(●), 4 mg/L Con A (△), 50 mmol/L DIDS＋4 mg/L Con A(○). Mean values and standard deviation are indicated from at least three separate experiments at 37 ℃.

3 Discussion

A variety of cell types, such as endothelial cells, osteoblasts, kidney epithelial cells, and fibroblasts have been shown to respond to fluid shear stress in vitro. Results presented here demonstrate that the important functions of erythrocyte membranes, i.e., glucose and anion transport across the membranes, do have certain response to the mechanical force.

Unlike endothelial cells in vascular system, in which the main stress to be endured is tangential shear stress, the erythrocytes are subjected to complicated hemodynamic force which was mimicked by a rotary flow field in our experiments. The deformation of the erythrocyte is very important for performance of the cell function in blood circulation. Deformation of the erythrocyte needs energy that is provided completely by the ATP. The ATP inside the erythrocyte is produced uniquely through glycolytic process, and its level closely interrelates with the rate of glucose transport across the erythrocyte membranes. We obtained that the rate of glucose influx (V₀) was increased with the enhancement of mechanical stress. In the meantime, the rate of glucose exit was decreased^[14]. This may play an important role for the erythrocytes in the high mechanical force condition because in such circumstances the cells need more ATP to make intensive cell deformation, which can be satisfied by increase in glucose level inside the cell. The temperature dependence of the glucose transport across the erythrocyte membranes showed that the Arrehnius activation energy was responded to the mechanical force. To make a comparison between Fig.1 or Fig.2 and Fig.4, it is deduced that a decreased in the activation energy for glucose transport induced by mechanical force results in an increase in the rate of glucose transport. This result is obviously reasonable. Reduction of the activation energy means the decrease in thermodynamic barrier for GLUT1 reorientation in the membrane and then substrates are much easy to transport.

The intrinsic protein fluorescence due to its endogenous tryptophan(s) is known to be sensitive to local microenvironments and has served as useful probe in studying protein tertiary structure and its dynamics. For GLUT1, quenching of the intrinsic fluorescence by ligands has been studied to describe protein dynamics induced by transporter substrates and inhibitors^[20]. GLUT1 consists of 492 amino acid residues among them four tryptophan residues are located in the hydrophobic environment and another two tryptophan residues are in the hydrophilic environment. When binding with substrates or inhibitors the fluorescence quantum yield of these exposed tryptophan residues will be decreased for their moving from a hydrophilic to a hydrophobic environment. Because the fluorescence quench came from binding of GLUT1 with their specific substrates, glucose, so we could exclude the contribution of the tryptophan residues of other membrane proteins from the value of fluorescence change (DF). On account of the lack of a suitable method to eliminate the scattering effect of the ghosts from the intrinsic fluorescence, the total amount of membrane proteins for each sample was the same in the experiment. Under this condition it was still possible to estimate the apparent quenching constant, K_q, according to Eq. 1 from plot of “1/DF vs.1/[Q]” even if we did not know the exact value of F₀.

As a consequence of the cell membrane permeability for water molecules, which follows hydrostatic and osmotic pressure gradients, mammalian cell volume constancy is challenged by any alteration in intracellular and/or extracellular osmolality. A notable example is the kidney medulla, where extracellular osmolality may range from isotonicity to more than 1 500 mOsm during water deprivation. Many evidences show that osmotic stress is a ubiquitous stimulus. An increase in osmolality leads to decrease in cell volume at constant surface area, which would facilitate the passage of the erythrocytes through narrow channels, and reduce the erythrocytes to deform in shear flow. A decrease in osmolality causes the opposite actions. Erythrocyte osmotic regulatory mechanisms are specifically important in limiting alterations of cell volume during their passage through the hypertonic kidney medulla and during HCO₃^- transport in the lung and the periphery. One disorder exacerbated by altered erythrocytes cell volume regulatory mechanisms is sickle cell anemia. Our data indicated that the change in membrane tension resulted from the osmotic stress could regulate the structure and function of GLUT1.

It was reported that the erythrocytes (normal or sickle cell anemia) deformed by shear stress have an increased permeability to monovalent and divalent cations. On the other hand, anion exchange is not affected by shear stress^[21]. In spite of modifying membrane skeletons, changing ion concentration, altering metabolism and aging condition, anion exchange did not respond to rotate speed in our experiment (unpublished observation). It is very interesting that the rate constant of NO₂^- permeability, K_t, was increased with the enhancement of osmolality [Fig.2(C)]. Meanwhile, the activation energy for NO₂^- binding to band 3 was decreased [Fig.4(C)]. Shear stress and osmotic stress are two kinds of mechanical force and different in action pattern. The force of former is tangential to the membrane and that of latter is vertical. It is obvious that responses of the membrane proteins to them are various, and this would be a reason why band 3 answers to osmotic stress but not to shear stress. Band 3 is the most abundant protein in the erythrocyte membranes and is responsible for the exchange of HCO₃^- formed from tissue CO₂ with Cl^- in the erythrocyte cytoplasm. This anion exchange occurs during the passage of the erythrocytes through the tissue capillaries, and then later in the reverse direction, as the erythrocytes pass through the lung. There was reported that conformational changes in band 3 mediated shape changes of the human erythrocytes^[22].

The erythrocyte membranes can been directly manipulated by applying amphipathic drugs known to change membrane tension and curvature^[18]. This is another way to mimic the effect of mechanical force. We applied chlorpromazine to bend membranes inward and found the expected morphologic change in the erythrocytes. It has long been known that anionic and cationic amphiphaths can induce echinocytic shape and stomatocytic (cup) shape change in the erythrocytes, respectively. However, neither of these shape changes was associated with an increase in cation flux in the erythrocytes^[23]. Our data shows that an increase in chlorpromazine concentration resulted in enhancement of glucose influx and decrease of NO₂^– permeabilitiy (Fig.6). It demonstrates that transport property of GLUT1 and band 3 was sensitive to the membrane tension induced by chlorpromazine and different from that of cations transport.

In light of the above results that the three mechanical force factors do affect the glucose transport and anion exchange in human erythrocytes, it is our further task to establish more quantitative relationship and to unveil the detailed molecular mechanism for inducing the change in conformation and function of the membrane transports by the mechanical force.

Some specific inhibitors of anion transport can modulate the transport function of GLUT1 and the information can be transmitted from GLUT1 to band 3^[15,19]. In the present study, we observed that DIDS lowered the response of the rate of glucose transport to the mechanical force. This demonstrates once again that there is signal linkage between GLUT1 and band 3 in the erythrocyte membrane. To consider the effect of Con A [Fig.6(B)], it seems that oligosaccharide chain may be involved in the transduction of mechanical signals between them.

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Received: March 20, 2001 Accepted: May 8, 2001

This work was supported by the grants from the National Natural Science Foundation of China, No.39730150 and No.30070205

*Corresponding author: Tel, 86-21-65643673; Fax, 86-21-65650149; e-mail, [email protected]