Abnormal regulation of Mg2+ transport via Na/Mg exchanger in sickle erythrocytes
http://www.100md.com
《血液学杂志》
the Department of Laboratory Medicine, Children's Hospital Boston, Boston, MA
Department of Pathology, Harvard Medical School, Boston, MA
Division of Endocrinology, Diabetes and Hypertension, Brigham and Women's Hospital, Boston, MA
Department of Medicine, Harvard Medical School, Boston, MA.
Abstract
Erythrocyte magnesium (Mg2+) deficiency has been demonstrated in sickle cell disease to contribute to erythrocyte dehydration, K loss, and thus sickling. No studies have assessed the functional properties of the Na/Mg exchanger in sickle cell disease. Using Mg2+-loaded erythrocytes, we measured Mg2+ efflux induced by extracellular Na+. We estimated that the Na/Mg exchanger had higher maximal velocity, higher affinity for Na+, and lower cooperativity for Mg2+ in sickle than in normal erythrocytes. The activity of the exchanger was markedly decreased by hypotonic and hypertonic conditions in normal erythrocytes but not in sickle erythrocytes. Studies of density-separated erythrocytes showed that the activity of the exchanger decreased as the mean cellular hemoglobin concentration increased in normal but not in sickle erythrocytes. Inhibition of protein kinase C (PKC) activity by calphostin C and chelerythrine increased the activity of the exchanger in normal but not in sickle erythrocytes. Inhibition of serine/threonine phosphatases did not affect the activity of the exchanger in either normal or sickle erythrocytes. Altogether, these data indicate that the Na/Mg exchanger is abnormally regulated in sickle erythrocytes. Therefore, Mg2+ depletion in sickle erythrocytes might be mediated by an up-regulated Na/Mg exchanger, possibly by dephosphorylation of the transporter or a closely associated regulator. (Blood. 2005;105:382-386)
Introduction
Magnesium plays a central role in a variety of cellular metabolic functions. Cytosolic Mg2+ levels are maintained within narrow limits, representing less than 10% of total cellular magnesium. However, little is known about cellular Mg2+ transporters, their molecular identities, or regulatory mechanisms in human cells. A decrease in intracellular magnesium has been observed in sickle erythrocytes.1,2 Hemoglobin S polymerization may play a role in promoting Mg2+ loss, as shown by Ortiz et al.2 One of the erythrocyte regulatory pathways of cellular magnesium is the Na/Mg exchanger.3 We and others have previously shown elevated Na/Mg exchanger activity in sickle erythrocytes compared with normal erythrocytes.4,5 However, little is known about the kinetic parameters of the exchanger, potential physiologic modulators of its activity, or signaling mechanisms in sickle erythrocytes. Furthermore, its molecular characteristics are unknown.
One of the hallmarks of sickle cell disease is dehydration of the erythrocyte mediated by potassium loss.6-8 It has been previously shown that magnesium supplementation could significantly alter the dehydration state of the sickle erythrocytes possibly via a decrease in the activity of the K/Cl cotransporter.5,9 In addition, a preliminary study showed that the activity of the Na/Mg exchanger in sickle erythrocytes was decreased after 6 months of dietary magnesium supplementation. However, the mechanism(s) inducing this change in the exchanger's activity was not identified. Therefore, the current study was designed to characterize the mechanism(s) for cellular Mg2+ regulation via the Na/Mg exchanger in sickle erythrocytes (Hb SS) in comparison to normal erythrocytes (Hb AA).
Patients, materials, and methods
Drugs and chemicals
A23187 ionophore was purchased from Calbiochem (La Jolla, CA). Bovine serum albumin (BSA) was purchased from Roche (Indianapolis, IN). All other reagents were purchased from Sigma Chemical (St Louis, MO).
Preparation of erythrocytes
Blood from patients with sickle cell disease was collected after informed consent. Blood was collected into tubes containing heparin, passed through cotton to decrease the number of white cells, and centrifuged in a Sorvall RC 28S (Kendro Laboratory, Asheville, NC) at 1188 g for 4 minutes at 4°C. The supernatant was removed by aspiration and erythrocytes were washed 4 times with ice-cold Mg2+-free choline washing solution (Mg2+-free CWS) containing the following (mM): 150 choline Cl; 10 Tris MOPS (3-[N-Morpholino]propanesulphonic acid), pH 7.4 (4°C); and 20 sucrose. A 0.5 (50%) cell suspension was made in CWS, and manual hematocrit and mean cellular volume (MCV) were measured. Aliquots of this suspension were diluted with 0.0002 (0.02%) acationox in double-distilled water to allow measurement of intracellular Na+, K+, and Mg2+ by atomic absorption spectrophotometry (Perkin Elmer 800, Wellesley, MA).
Measurements of Mg2+ fluxes via Na/Mg exchanger
Erythrocytes were loaded with Mg2+ at 0.1 (10%) hematocrit for 30 minutes at 37°C in the presence of 6 μM A23187 [GenBank] . The Mg2+-loading solution contained the following (mM): 140-122 KCl; 0-18 MgCl2; 10 glucose; and 10 Tris MOPS, pH 7.4 (37°C). To avoid Gardos channel activation due to traces of Ca2+ during the Mg2+-loading period, 25 nM charybdotoxin was added to all loading solutions. The ionophore was removed by 4 consecutive washes using the same loading solution with 0.01 (0.1%) BSA at 37°C. The cells were further washed 4 times with Mg2+-free CWS (310 mOsm) at 4°C to remove extracellular Mg2+. A 50% cell suspension was made in Mg2+-free CWS. Sodium, potassium, and magnesium cellular content were determined as described in "Preparation of erythrocytes." Flux started when 0.4 mL of 0.5 (50%) cell suspension was added to either 8 mL NaCl or choline Cl flux media containing the following (mM): 140 NaCl or 140 choline Cl; 10 glucose; 10 Tris MOPS, pH 7.4 (37°C); 0.1 ouabain; and 0.01 bumetanide. Triplicate samples were taken at 5 and 45 minutes and centrifuged at 4°C. Mg2+ efflux was shown in preliminary experiments to be linear between 5, 15, 30, and 50 minutes at 37°C (data not shown). Supernatants were removed carefully and quickly transferred to 4-mL plastic tubes. Total Mg2+ concentration of the supernatants was determined by atomic absorption. Mg2+ efflux was calculated from the slope of the linear regression of total Mg2+ supernatant content versus time (mM cell x h). The Na/Mg exchanger was estimated from the difference between Mg2+ efflux in the NaCl and choline Cl flux media, and values were corrected by MCV and expressed as mmol/1013 cell x h. We found that lowering the media osmolarity below 200 mOsm caused an increase in cell lysis, and therefore we did not decrease the osmolarity below 225 mOsm (Figure 5) for the evaluation of the osmotic effect on the exchanger activity.
Measurement of the affinity constant for cellular Mg2+
To measure the kinetic parameter of the internal Mg2+ site of the exchanger, cells were loaded at different concentrations of Mg2+ (0.15-18 mM) using A23187 [GenBank] as described in "Measurements of Mg2+ fluxes via Na/Mg exchange." Since the cells change in volume as they are loaded with Mg2+, we adjusted the K+ and the sucrose levels to maintain a relatively constant hemoglobin concentration of the loaded cells at all Mg2+ concentrations. To estimate the ionized intracellular Mg, we used the equation given by Raftos et al.10 In those experiments we measured the proton concentration ratio (Table 1) across the plasma membrane in normal and sickle Mg2+-loaded erythrocytes. After loading, the cells were spun down, the supernatant was collected, and the pH and extracellular Mg2+ concentration were measured. The cell pellet was hemolyzed to estimate the intracellular pH and total Mg2+ (Table 1). Aliquots were also taken to measure the MCV, cell Na+, and cell K+ (Table 1). To avoid cell K+ and volume changes due to Gardos channel activation, 25 nM charybdotoxin was added during the loading process. Charybdotoxin does not have any significant effect on the Na/Mg exchanger activity (data not shown). Furthermore, all fluxes were corrected to account for changes in MCV observed in loaded cells.
Density-separated erythrocytes
Freshly isolated erythrocytes were suspended in normal saline and layered over a stractan gradient. Larex (arabinogalactan) was prepared as indicated by the manufacturer (Larex, White Bear Lake, MN). Larex stock was diluted to prepare varying densities between 1.077 and 1.119 g/mL. Approximately 1 mL of each density dilution was carefully layered, from the densest to the lightest, to reach a combined volume of 10 mL. Then, 0.5 mL of 30% erythrocytes was layered on top and the entire mixture centrifuged at 42 000 g for 45 minutes at 10°C. This method yields 5 clearly visible layers of density-separated erythrocytes. The layers were separated and washed with isotonic medium and kept on ice for further processing. Due to the small number of cells recovered from the washing steps, the first 2 layers (1 + 2, lighter population) and the last 2 layers (4 + 5, denser population) were combined to yield a total of 3 fractions. An aliquot of the 50% suspension was used for total cell blood count and MCV determinations for each fraction as indicated in the figure legends.
Results
Maximal velocity of the Na/Mg exchanger in Hb AA and Hb SS erythrocytes
It has been shown that Mg2+ efflux stimulated by external Na+ is increased in sickle erythrocytes.4,5 To study this effect, we measured the activity of the Na/Mg exchanger in Hb AA and Hb SS erythrocytes under conditions similar to those described in "Patients, materials, and methods." In Hb AA erythrocytes, we found that the activity of the Na/Mg exchanger in Mg2+-loaded cells was 0.71 ± 0.07 mmol/1013 cell x h (Figure 1A). In Hb SS erythrocytes, the exchanger activity was significantly and markedly increased to 3.00 ± 0.32 mmol/1013 cell x h (n = 39; P < .0001). Studies were carried out on density-separated normal and sickle erythrocytes. Figure 1B shows the activity of the exchanger in different red cell fractions obtained from healthy subjects. Under these experimental conditions, the velocity of the exchanger decreased significantly as the mean cellular hemoglobin concentration (MCHC) increased. Thus, as the cell becomes denser, the activity of the exchanger is significantly reduced. However, in density-separated sickle cells, the activity of the exchanger did not change as a function of cell density (Figure 1B). Since the reticulocyte content in sickle erythrocyte fractions decreased from 13% to 7% in the second fraction with no corresponding changes in flux activity, reticulocyte content per se does not appear to be a major determinant of the activity of the exchanger.
Affinity constant for internal Mg2+ in normal and sickle erythrocytes
We hypothesized that the increase in Na/Mg exchanger activity could be mediated by abnormalities in the affinity constant for intracellular Mg2+ in sickle erythrocytes. To test our hypothesis, we measured the activity of the exchanger in the presence of different concentrations of cellular Mg2+ in Hb AA and Hb SS erythrocytes (Figure 2). Total cell Mg2+ and ionized cell Mg were calculated from the total extracellular Mg2+ and the proton ratio (r2) as described in "Patients, materials, and methods" (Table 1) and by Raftos et al.10 As described previously, the proton ratio decreased as the intracellular Mg2+ concentration was increased. The MCV was increased in Mg2+-loaded cells, with a correlated decrease in the Na+ and K+ total concentrations. We found that loading with 18 mM extracellular Mg2+, the maximal velocity of the exchanger changes from 0.767 ± 0.25 mmol/1013 cell x h in normal erythrocytes to 3.77 ± 1.33 mmol/1013 cell x h in sickle erythrocytes (P < .005; n = 3; Figure 2). Kinetic analysis indicates that the affinity constant (K0.5) for intracellular Mg2+ followed a sigmoid pattern, with a K0.5 of 2.65 ± 0.4 mM cell water for normal erythrocytes, and a hyperbolic pattern, with Michaelis constant (Km) at 2.57 ± 0.56 mM cell water in sickle erythrocytes. Therefore, these results suggest that the elevated exchanger activity of sickle erythrocytes is not due to abnormalities in the internal Mg2+ affinity site.
Affinity for external Na+ in normal and sickle erythrocytes
Analysis of the external Na+ site of the exchanger was performed by measuring the activity of the exchanger in Mg2+-loaded cells at different concentrations of external Na+ (Figure 3). The activity of the exchanger followed a sigmoid pattern in normal erythrocytes, with a maximal velocity of 0.93 ± 0.22 mmol/1013 cell x h and a K0.5 of 64.2 ± 4.1 mM. In sickle erythrocytes, kinetic analysis of the external site showed a significant increase in the maximal velocity to 3.15 ± 1.1 mmol/1013 cell x h (n = 9; P < .001), with a change in pattern to hyperbolic with a decrease in the Km to 50.9 ± 2.1 mM (n = 9; P < .023). The change in the kinetic pattern from sigmoidal to hyperbolic in sickle erythrocytes suggests a loss of Na ion cooperativity in the external binding site.
Effect of osmolarity on the Na/Mg exchanger
It was previously shown that when the media osmolarity was lowered to 200 mOsm, the Na/Mg exchanger activity of human erythrocytes increased.3 We found that the activity of the exchanger markedly decreased when osmolarity was lowered from 300 mOsm to 225 mOsm in normal erythrocytes (Figure 4). Hypertonic (400-575 mOsm) conditions also caused a decrease in the activity of the exchanger in normal erythrocytes. However, in sickle erythrocytes, no inhibition of the exchanger activity by hypotonicity was observed. Indeed, no significant changes in Mg2+ flux were observed in sickle erythrocytes with changes in media tonicity (Figure 4). These data indicate that regulation of the exchanger by cell volume is altered in sickle erythrocytes and the transporter appears to be abnormally up-regulated. Therefore, our results imply an abnormality of the signal pathway of the regulation of cellular Mg2+ by cell volume in sickle erythrocytes.
Effect of phosphorylation/dephosphorylation events on the activity of the Na/Mg exchanger
In order to study the signaling pathways controlling Na/Mg exchanger activation, we evaluated the effect of different inhibitors of phosphorylation and dephosphorylation in erythrocytes. Figure 5 shows the effect of 2 different protein kinase C (PKC) inhibitors, calphostin C (CC) and chelerythrine (CH), on exchanger activity. Calphostin C is known to inhibit PKC by binding to the diacylglycerol site (active site) of the PKC enzyme, abolishing its activity with very high specificity.11 Chelerythrine causes PKC inhibition by blocking the substrate-binding site of the enzyme.12 We previously showed that calphostin C decreases the activity of PKC in mouse erythrocytes.13 Here we found that inhibition of PKC activity induced a significant increase in exchanger activity in normal erythrocytes (from 0.62 ± 0.18 to 0.91 ± 0.11 with CH and to 0.80 ± 0.12 with CC). Inhibition of the PKC induces a reduction of phosphorylation activity, suggesting that phosphorylation events down-regulate the exchanger's activity. To test whether inhibition of phosphatases plays a role in the activation of the exchanger under normal (baseline) conditions, we measured the activity of the exchanger in the presence of 100 nM okadaic acid, an inhibitor of serine-threonine protein phosphatases. We found that okadaic acid did not affect Na/Mg exchanger activity in normal erythrocytes (Figure 5A). Okadaic acid is known to inhibit protein phosphatase 2A at low concentrations, but at 100 nM this agent interferes with other phosphatases as well.14 The use of calyculin A, a potent inhibitor of protein phosphatase 1, produced results similar to those obtained with okadaic acid (Figure 5A).
In sickle erythrocytes, we found that PKC inhibitors did not modify the activity of the exchanger (from 4.26 ± 0.08 to 4.21 ± 0.13 with CH and 4.13 ± .009 with CC) as seen under similar conditions in normal erythrocytes (Figure 5B). We also evaluated the effect of phosphatase inhibitors in sickle erythrocytes (Figure 5B). We found that neither okadaic acid nor calyculin A altered the activity of the exchanger in sickle erythrocytes. These data suggest that the signaling pathway for Na/Mg regulation is abnormal in sickle erythrocytes and strongly support the hypothesis that the Na/Mg exchanger is "locked" in an up-regulated mode in these cells.
Discussion
Reduced erythrocyte magnesium content has been associated with the pathophysiology of sickle cell disease (SCD), and magnesium supplementation has been proposed as a therapeutic approach to ameliorate painful crisis in SCD.5,15-20 However, the mechanisms underlying these effects are not fully understood. In erythrocytes, regulation of intracellular Mg2+ content is mediated by cytoplasmic magnesium buffers such as adenosine triphosphate (ATP) and 2,3-diphosphoglycerate10 as well as by Na+-dependent and -independent Mg ion transport mechanisms.3 One of these transport mechanisms is the plasma membrane Na/Mg exchanger. Flatman21 has previously described the dependency of the exchanger's activation upon extracellular Na+ and Mg2+. We measured activation kinetics of the exchanger by extracellular Na+ and found an increased Vmax and affinity in sickle red cells that was associated with a decrease in cooperativity of the Na ions compared with normal erythrocytes. The loss of cooperativity of the exchanger observed in SCD supports the notion of abnormal regulation of the exchanger in sickle erythrocytes. In turn, these kinetic alterations may in part explain the low total cellular Mg2+ levels observed in the denser fraction of sickle erythrocytes.2 Although the difference in affinity for extracellular Na+ might not be of major physiologic importance for the regulation of the activity of the exchanger in circulating red cells, it might be of interest in tissues and red cells exposed to dramatic changes in Na+ concentration such as in the kidney.
Normal erythrocytes show decreasing Mg2+ levels with aging,22 and younger erythrocytes contain higher Mg2+ levels than older cells. The activity of the Na/Mg exchanger also decreases with increased cell density in normal erythrocytes (Figure 1B). Perhaps the cell volume reduction associated with aging decreased the activity of the exchanger, as seen in hypertonic stimulation experiments (Figure 4) in normal erythrocytes. Neither cell shrinkage nor cell swelling had any significant effect on the activity of the exchanger in sickle erythrocytes, suggesting abnormal regulation. However, we cannot rule out the possible contribution of the large difference in the average ages of normal and sickle erythrocytes to the observed differences in Mg2+ fluxes.
The changes in Na/Mg exchanger activity with varying MCHCs (Figure 1B) cannot be explained solely by the presence of reticulocytes. A 46% decrease in reticulocyte content in sickle fraction number 2 (Figure 1B) did not significantly affect the magnitude of the Mg2+ flux, suggesting that reticulocyte content per se is not a major determinant of the total exchanger's activity. Nevertheless, we cannot rule out the contribution of reticulocyte number to the dramatic change observed from the first fraction to the second fraction in normal red cells (Figure 1B). Further experiments will be required to assess the contribution of the reticulocyte fraction to the total exchanger activity.
We hypothesize that the persistent elevation of Na/Mg exchanger activity at all density levels may play a role in generating the reduced total intracellular Mg2+ seen in dense sickle erythrocytes.2 Our data support the notion of a constant exchanger activity at different densities, whereby the cell is constantly losing Mg2+. Since Mg2+ is known to be a modulator of K/Cl activity in normal and sickle erythrocytes,9,14 the high activity of this exchanger might lead to a decrease in intracellular Mg2+ that is responsible for the high activity of the K/Cl cotransporter in these cells.9,23-25
The role of PKC in the regulation of cellular Mg2+ has been investigated in rat vascular smooth muscle cells, where PKC inhibition leads to increased Mg2+ levels.26 Zheng et al26 also reported that this increase was mediated by intracellular release of stored Mg2+, since PKC inhibitors induced the same Mg2+ response in the absence of extracellular Mg2+. Differential activation of PKC has been previously reported in sickle cells. PKC activity is significantly elevated in sickle erythrocytes when compared with normal cells, and only PKC seems to translocate to the plasma membrane when it is activated by phorbol 12-myristate 13-acetate.27 In normal red cells, we show that the exchanger is activated by PKC inhibitors and is unaffected by phosphatase inhibitors, suggesting the involvement of PKC in the regulation of the exchanger's activity. In contrast, neither PKC nor phosphatase inhibitors significantly affected the activity of the exchanger in sickle erythrocytes (Figure 5B). Therefore, we speculate that the PKC-sensitive pathway of Na+-dependent exchanger activity modulation is abnormally regulated or interrupted in sickle erythrocytes. Furthermore, the absence of any significant effect of PKC inhibitors on the sickle erythrocyte Na/Mg exchanger also suggests that the elevated PKC activity observed by Fathallah et al27 plays no role in the regulation of the Na/Mg exchanger in sickle erythrocytes. Further studies will be needed to elucidate this point.
Because of the amiloride sensitivity of the Na+-dependent Mg2+ flux, it has been suggested that the Na/Mg exchanger and the Na/H exchanger might be mediated by the same system. Our results argue against this hypothesis, in that (1) in normal erythrocytes, the external Na+ dependency of the Na/H exchanger follows a hyperbolic pattern and not the sigmoid pattern as seen in Na/Mg exchanger; (2) the Na/Mg exchanger is not sensitive to okadaic acid, as is the case for Na/H exchanger activity in human erythrocytes28; (3) PKC inhibitors decrease the activity of Na/H exchanger29 and increase the activity of Na/Mg exchanger in normal erythrocytes; and (4) hyperosmolarity does not increase the activity of the exchanger, as occurs with the Na/H exchanger in normal erythrocytes.30
In summary, our results suggest that the Na/Mg exchanger plays an important role in the pathophysiology of SCD. The initial characterization of the Na/Mg exchanger in sickle erythrocytes presented here provides a platform for further studies on the pathophysiology of Mg2+ transporters in SCD. Furthermore, these studies open new pathways for investigation of K+ transport, water loss, and the consequent cellular dehydration in sickle erythrocytes.
Acknowledgements
We would like to thank Dr Carolyn Bigelow, Dr Rathi Iyer, Carole Ward, Cindy Kendig, and Aileen Anderson from the University of Mississippi Medical Center (Jackson, MS) for generously providing blood samples from patients with sickle cell disease. We also wish to thank Ms Michelle Langlois and Mr Lin-Chie Pong for their technical support.
Footnotes
Prepublished online as Blood First Edition Paper, September 7, 2004; DOI 10.1182/blood-2003-11-3755.
Supported by National Institutes of Health (NIH) grants HL67699 (A.R.), DK50422 (C.B.), and DK02817 and DK064841 (J.R.R.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
References
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Ortiz OE, Lew VL, Bookchin RM. Deoxygenation permeabilizes sickle cell anaemia red cells to magnesium and reverses its gradient in the dense cells. J Physiol. 1990;427: 211-226.
Feray JC, Garay R. An Na+-stimulated Mg2+-transport system in human red blood cells. Biochim Biophys Acta. 1986;856: 76-84.
Feray JC, Beuzard Y, Garay RP. Na/Mg exchange in sickle cells: 22a. Proceeding of the V International Magnesium Symposium. John Libbey & Co., Ltd; 1988.
De Franceschi L, Bachir D, Galacteros F, et al. Oral magnesium pidolate: effects of long-term administration in patients with sickle cell disease. Br J Haematol. 2000;108: 284-289.
Brugnara C, Bunn HF, Tosteson DC. Ion content and transport and the regulation of volume in sickle cells. Ann N Y Acad Sci. 1989;565: 96-103.
Glader BE, Nathan DG. Cation permeability alterations during sickling: relationship to cation composition and cellular hydration of irreversibly sickled cells. Blood. 1978;51: 983-989.
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De Franceschi L, Villa-Moruzzi E, Fumagalli L, et al. K-Cl cotransport modulation by intracellular Mg in erythrocytes from mice bred for low and high Mg levels. Am J Physiol Cell Physiol. 2001;281: C1385-C1395.
Raftos JE, Lew VL, Flatman PW. Refinement and evaluation of a model of Mg2+ buffering in human red cells. Eur J Biochem. 1999;263: 635-645.
Bruns RF, Miller FD, Merriman RL, et al. Inhibition of protein kinase C by calphostin C is light-dependent. Biochem Biophys Res Commun. 1991;176: 288-293.
Herbert JM, Augereau JM, Gleye J, Maffrand JP. Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun. 1990;172: 993-999.
Rivera A, Rotter MA, Brugnara C. Endothelins activate Ca(2+)-gated K(+) channels via endothelin B receptors in CD-1 mouse erythrocytes. Am J Physiol. 1999;277: C746-C754.
Bize I, Guvenc B, Buchbinder G, Brugnara C. Stimulation of human erythrocyte K-Cl cotransport and protein phosphatase type 2A by n-ethylmaleimide: role of intracellular Mg++. J Membr Biol. 2000;177: 159-168.
De Franceschi L, Bachir D, Galacteros F, et al. Oral magnesium supplements reduce erythrocyte dehydration in patients with sickle cell disease. J Clin Invest. 1997;100: 1847-1852.
De Franceschi L, Brugnara C, Beuzard Y. Dietary magnesium supplementation ameliorates anemia in a mouse model of beta-thalassemia. Blood. 1997;90: 1283-1290.
De Franceschi L, Beuzard Y, Jouault H, Brugnara C. Modulation of erythrocyte potassium chloride cotransport, potassium content, and density by dietary magnesium intake in transgenic SAD mouse. Blood. 1996;88: 2738-2744.
De Franceschi L, Cappellini MD, Graziadei G, et al. The effect of dietary magnesium supplementation on the cellular abnormalities of erythrocytes in patients with beta thalassemia intermedia. Haematologica. 1998;83: 118-125.
De Franceschi L, Brugnara C, Rouyer-Fessard P, Jouault H, Beuzard Y. Formation of dense erythrocytes in SAD mice exposed to chronic hypoxia: evaluation of different therapeutic regimens and of a combination of oral clotrimazole and magnesium therapies. Blood. 1999;94: 4307-4313.
Bookchin RM, Tiffert JT, Davies SC, Vichinsky E, Lew VL. Sickle Cell Disease and Thalassemias: New Trends in Therapy. Vol 234. London, United Kingdom: John Libbey and Co, Ltd; 1995.
Flatman PW. The control of red cell magnesium. Magnes Res. 1988;1: 5-11.
Watson WS, Lyon TD, Hilditch TE. Red cell magnesium as a function of cell age. Metabolism. 1980;29: 397-399.
Brugnara C, Tosteson DC. Cell volume, K transport, and cell density in human erythrocytes. Am J Physiol. 1987;252: C269-C276.
Canessa M, Fabry ME, Nagel RL. Deoxygenation inhibits the volume-stimulated, Cl(-)-dependent K+ efflux in SS and young AA cells: a cytosolic Mg2+ modulation. Blood. 1987;70: 1861-1866.
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Zheng T, Li W, Altura BT, Altura BM. Use of protein kinase C inhibitors results in rapid [Mg(2+)](i) mobilization in primary cultured rat aortic smooth muscle cells: are certain kinase C isoforms natural homeostatic regulators of cystolic free Mg(2+). Eur J Pharmacol. 2001;413: R1-R3.
Fathallah H, Sauvage M, Romero JR, Canessa M, Giraud F. Effects of PKC alpha activation on Ca2+ pump and K(Ca) channel in deoxygenated sickle cells. Am J Physiol. 1997;273: C1206-C1214.
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Department of Pathology, Harvard Medical School, Boston, MA
Division of Endocrinology, Diabetes and Hypertension, Brigham and Women's Hospital, Boston, MA
Department of Medicine, Harvard Medical School, Boston, MA.
Abstract
Erythrocyte magnesium (Mg2+) deficiency has been demonstrated in sickle cell disease to contribute to erythrocyte dehydration, K loss, and thus sickling. No studies have assessed the functional properties of the Na/Mg exchanger in sickle cell disease. Using Mg2+-loaded erythrocytes, we measured Mg2+ efflux induced by extracellular Na+. We estimated that the Na/Mg exchanger had higher maximal velocity, higher affinity for Na+, and lower cooperativity for Mg2+ in sickle than in normal erythrocytes. The activity of the exchanger was markedly decreased by hypotonic and hypertonic conditions in normal erythrocytes but not in sickle erythrocytes. Studies of density-separated erythrocytes showed that the activity of the exchanger decreased as the mean cellular hemoglobin concentration increased in normal but not in sickle erythrocytes. Inhibition of protein kinase C (PKC) activity by calphostin C and chelerythrine increased the activity of the exchanger in normal but not in sickle erythrocytes. Inhibition of serine/threonine phosphatases did not affect the activity of the exchanger in either normal or sickle erythrocytes. Altogether, these data indicate that the Na/Mg exchanger is abnormally regulated in sickle erythrocytes. Therefore, Mg2+ depletion in sickle erythrocytes might be mediated by an up-regulated Na/Mg exchanger, possibly by dephosphorylation of the transporter or a closely associated regulator. (Blood. 2005;105:382-386)
Introduction
Magnesium plays a central role in a variety of cellular metabolic functions. Cytosolic Mg2+ levels are maintained within narrow limits, representing less than 10% of total cellular magnesium. However, little is known about cellular Mg2+ transporters, their molecular identities, or regulatory mechanisms in human cells. A decrease in intracellular magnesium has been observed in sickle erythrocytes.1,2 Hemoglobin S polymerization may play a role in promoting Mg2+ loss, as shown by Ortiz et al.2 One of the erythrocyte regulatory pathways of cellular magnesium is the Na/Mg exchanger.3 We and others have previously shown elevated Na/Mg exchanger activity in sickle erythrocytes compared with normal erythrocytes.4,5 However, little is known about the kinetic parameters of the exchanger, potential physiologic modulators of its activity, or signaling mechanisms in sickle erythrocytes. Furthermore, its molecular characteristics are unknown.
One of the hallmarks of sickle cell disease is dehydration of the erythrocyte mediated by potassium loss.6-8 It has been previously shown that magnesium supplementation could significantly alter the dehydration state of the sickle erythrocytes possibly via a decrease in the activity of the K/Cl cotransporter.5,9 In addition, a preliminary study showed that the activity of the Na/Mg exchanger in sickle erythrocytes was decreased after 6 months of dietary magnesium supplementation. However, the mechanism(s) inducing this change in the exchanger's activity was not identified. Therefore, the current study was designed to characterize the mechanism(s) for cellular Mg2+ regulation via the Na/Mg exchanger in sickle erythrocytes (Hb SS) in comparison to normal erythrocytes (Hb AA).
Patients, materials, and methods
Drugs and chemicals
A23187 ionophore was purchased from Calbiochem (La Jolla, CA). Bovine serum albumin (BSA) was purchased from Roche (Indianapolis, IN). All other reagents were purchased from Sigma Chemical (St Louis, MO).
Preparation of erythrocytes
Blood from patients with sickle cell disease was collected after informed consent. Blood was collected into tubes containing heparin, passed through cotton to decrease the number of white cells, and centrifuged in a Sorvall RC 28S (Kendro Laboratory, Asheville, NC) at 1188 g for 4 minutes at 4°C. The supernatant was removed by aspiration and erythrocytes were washed 4 times with ice-cold Mg2+-free choline washing solution (Mg2+-free CWS) containing the following (mM): 150 choline Cl; 10 Tris MOPS (3-[N-Morpholino]propanesulphonic acid), pH 7.4 (4°C); and 20 sucrose. A 0.5 (50%) cell suspension was made in CWS, and manual hematocrit and mean cellular volume (MCV) were measured. Aliquots of this suspension were diluted with 0.0002 (0.02%) acationox in double-distilled water to allow measurement of intracellular Na+, K+, and Mg2+ by atomic absorption spectrophotometry (Perkin Elmer 800, Wellesley, MA).
Measurements of Mg2+ fluxes via Na/Mg exchanger
Erythrocytes were loaded with Mg2+ at 0.1 (10%) hematocrit for 30 minutes at 37°C in the presence of 6 μM A23187 [GenBank] . The Mg2+-loading solution contained the following (mM): 140-122 KCl; 0-18 MgCl2; 10 glucose; and 10 Tris MOPS, pH 7.4 (37°C). To avoid Gardos channel activation due to traces of Ca2+ during the Mg2+-loading period, 25 nM charybdotoxin was added to all loading solutions. The ionophore was removed by 4 consecutive washes using the same loading solution with 0.01 (0.1%) BSA at 37°C. The cells were further washed 4 times with Mg2+-free CWS (310 mOsm) at 4°C to remove extracellular Mg2+. A 50% cell suspension was made in Mg2+-free CWS. Sodium, potassium, and magnesium cellular content were determined as described in "Preparation of erythrocytes." Flux started when 0.4 mL of 0.5 (50%) cell suspension was added to either 8 mL NaCl or choline Cl flux media containing the following (mM): 140 NaCl or 140 choline Cl; 10 glucose; 10 Tris MOPS, pH 7.4 (37°C); 0.1 ouabain; and 0.01 bumetanide. Triplicate samples were taken at 5 and 45 minutes and centrifuged at 4°C. Mg2+ efflux was shown in preliminary experiments to be linear between 5, 15, 30, and 50 minutes at 37°C (data not shown). Supernatants were removed carefully and quickly transferred to 4-mL plastic tubes. Total Mg2+ concentration of the supernatants was determined by atomic absorption. Mg2+ efflux was calculated from the slope of the linear regression of total Mg2+ supernatant content versus time (mM cell x h). The Na/Mg exchanger was estimated from the difference between Mg2+ efflux in the NaCl and choline Cl flux media, and values were corrected by MCV and expressed as mmol/1013 cell x h. We found that lowering the media osmolarity below 200 mOsm caused an increase in cell lysis, and therefore we did not decrease the osmolarity below 225 mOsm (Figure 5) for the evaluation of the osmotic effect on the exchanger activity.
Measurement of the affinity constant for cellular Mg2+
To measure the kinetic parameter of the internal Mg2+ site of the exchanger, cells were loaded at different concentrations of Mg2+ (0.15-18 mM) using A23187 [GenBank] as described in "Measurements of Mg2+ fluxes via Na/Mg exchange." Since the cells change in volume as they are loaded with Mg2+, we adjusted the K+ and the sucrose levels to maintain a relatively constant hemoglobin concentration of the loaded cells at all Mg2+ concentrations. To estimate the ionized intracellular Mg, we used the equation given by Raftos et al.10 In those experiments we measured the proton concentration ratio (Table 1) across the plasma membrane in normal and sickle Mg2+-loaded erythrocytes. After loading, the cells were spun down, the supernatant was collected, and the pH and extracellular Mg2+ concentration were measured. The cell pellet was hemolyzed to estimate the intracellular pH and total Mg2+ (Table 1). Aliquots were also taken to measure the MCV, cell Na+, and cell K+ (Table 1). To avoid cell K+ and volume changes due to Gardos channel activation, 25 nM charybdotoxin was added during the loading process. Charybdotoxin does not have any significant effect on the Na/Mg exchanger activity (data not shown). Furthermore, all fluxes were corrected to account for changes in MCV observed in loaded cells.
Density-separated erythrocytes
Freshly isolated erythrocytes were suspended in normal saline and layered over a stractan gradient. Larex (arabinogalactan) was prepared as indicated by the manufacturer (Larex, White Bear Lake, MN). Larex stock was diluted to prepare varying densities between 1.077 and 1.119 g/mL. Approximately 1 mL of each density dilution was carefully layered, from the densest to the lightest, to reach a combined volume of 10 mL. Then, 0.5 mL of 30% erythrocytes was layered on top and the entire mixture centrifuged at 42 000 g for 45 minutes at 10°C. This method yields 5 clearly visible layers of density-separated erythrocytes. The layers were separated and washed with isotonic medium and kept on ice for further processing. Due to the small number of cells recovered from the washing steps, the first 2 layers (1 + 2, lighter population) and the last 2 layers (4 + 5, denser population) were combined to yield a total of 3 fractions. An aliquot of the 50% suspension was used for total cell blood count and MCV determinations for each fraction as indicated in the figure legends.
Results
Maximal velocity of the Na/Mg exchanger in Hb AA and Hb SS erythrocytes
It has been shown that Mg2+ efflux stimulated by external Na+ is increased in sickle erythrocytes.4,5 To study this effect, we measured the activity of the Na/Mg exchanger in Hb AA and Hb SS erythrocytes under conditions similar to those described in "Patients, materials, and methods." In Hb AA erythrocytes, we found that the activity of the Na/Mg exchanger in Mg2+-loaded cells was 0.71 ± 0.07 mmol/1013 cell x h (Figure 1A). In Hb SS erythrocytes, the exchanger activity was significantly and markedly increased to 3.00 ± 0.32 mmol/1013 cell x h (n = 39; P < .0001). Studies were carried out on density-separated normal and sickle erythrocytes. Figure 1B shows the activity of the exchanger in different red cell fractions obtained from healthy subjects. Under these experimental conditions, the velocity of the exchanger decreased significantly as the mean cellular hemoglobin concentration (MCHC) increased. Thus, as the cell becomes denser, the activity of the exchanger is significantly reduced. However, in density-separated sickle cells, the activity of the exchanger did not change as a function of cell density (Figure 1B). Since the reticulocyte content in sickle erythrocyte fractions decreased from 13% to 7% in the second fraction with no corresponding changes in flux activity, reticulocyte content per se does not appear to be a major determinant of the activity of the exchanger.
Affinity constant for internal Mg2+ in normal and sickle erythrocytes
We hypothesized that the increase in Na/Mg exchanger activity could be mediated by abnormalities in the affinity constant for intracellular Mg2+ in sickle erythrocytes. To test our hypothesis, we measured the activity of the exchanger in the presence of different concentrations of cellular Mg2+ in Hb AA and Hb SS erythrocytes (Figure 2). Total cell Mg2+ and ionized cell Mg were calculated from the total extracellular Mg2+ and the proton ratio (r2) as described in "Patients, materials, and methods" (Table 1) and by Raftos et al.10 As described previously, the proton ratio decreased as the intracellular Mg2+ concentration was increased. The MCV was increased in Mg2+-loaded cells, with a correlated decrease in the Na+ and K+ total concentrations. We found that loading with 18 mM extracellular Mg2+, the maximal velocity of the exchanger changes from 0.767 ± 0.25 mmol/1013 cell x h in normal erythrocytes to 3.77 ± 1.33 mmol/1013 cell x h in sickle erythrocytes (P < .005; n = 3; Figure 2). Kinetic analysis indicates that the affinity constant (K0.5) for intracellular Mg2+ followed a sigmoid pattern, with a K0.5 of 2.65 ± 0.4 mM cell water for normal erythrocytes, and a hyperbolic pattern, with Michaelis constant (Km) at 2.57 ± 0.56 mM cell water in sickle erythrocytes. Therefore, these results suggest that the elevated exchanger activity of sickle erythrocytes is not due to abnormalities in the internal Mg2+ affinity site.
Affinity for external Na+ in normal and sickle erythrocytes
Analysis of the external Na+ site of the exchanger was performed by measuring the activity of the exchanger in Mg2+-loaded cells at different concentrations of external Na+ (Figure 3). The activity of the exchanger followed a sigmoid pattern in normal erythrocytes, with a maximal velocity of 0.93 ± 0.22 mmol/1013 cell x h and a K0.5 of 64.2 ± 4.1 mM. In sickle erythrocytes, kinetic analysis of the external site showed a significant increase in the maximal velocity to 3.15 ± 1.1 mmol/1013 cell x h (n = 9; P < .001), with a change in pattern to hyperbolic with a decrease in the Km to 50.9 ± 2.1 mM (n = 9; P < .023). The change in the kinetic pattern from sigmoidal to hyperbolic in sickle erythrocytes suggests a loss of Na ion cooperativity in the external binding site.
Effect of osmolarity on the Na/Mg exchanger
It was previously shown that when the media osmolarity was lowered to 200 mOsm, the Na/Mg exchanger activity of human erythrocytes increased.3 We found that the activity of the exchanger markedly decreased when osmolarity was lowered from 300 mOsm to 225 mOsm in normal erythrocytes (Figure 4). Hypertonic (400-575 mOsm) conditions also caused a decrease in the activity of the exchanger in normal erythrocytes. However, in sickle erythrocytes, no inhibition of the exchanger activity by hypotonicity was observed. Indeed, no significant changes in Mg2+ flux were observed in sickle erythrocytes with changes in media tonicity (Figure 4). These data indicate that regulation of the exchanger by cell volume is altered in sickle erythrocytes and the transporter appears to be abnormally up-regulated. Therefore, our results imply an abnormality of the signal pathway of the regulation of cellular Mg2+ by cell volume in sickle erythrocytes.
Effect of phosphorylation/dephosphorylation events on the activity of the Na/Mg exchanger
In order to study the signaling pathways controlling Na/Mg exchanger activation, we evaluated the effect of different inhibitors of phosphorylation and dephosphorylation in erythrocytes. Figure 5 shows the effect of 2 different protein kinase C (PKC) inhibitors, calphostin C (CC) and chelerythrine (CH), on exchanger activity. Calphostin C is known to inhibit PKC by binding to the diacylglycerol site (active site) of the PKC enzyme, abolishing its activity with very high specificity.11 Chelerythrine causes PKC inhibition by blocking the substrate-binding site of the enzyme.12 We previously showed that calphostin C decreases the activity of PKC in mouse erythrocytes.13 Here we found that inhibition of PKC activity induced a significant increase in exchanger activity in normal erythrocytes (from 0.62 ± 0.18 to 0.91 ± 0.11 with CH and to 0.80 ± 0.12 with CC). Inhibition of the PKC induces a reduction of phosphorylation activity, suggesting that phosphorylation events down-regulate the exchanger's activity. To test whether inhibition of phosphatases plays a role in the activation of the exchanger under normal (baseline) conditions, we measured the activity of the exchanger in the presence of 100 nM okadaic acid, an inhibitor of serine-threonine protein phosphatases. We found that okadaic acid did not affect Na/Mg exchanger activity in normal erythrocytes (Figure 5A). Okadaic acid is known to inhibit protein phosphatase 2A at low concentrations, but at 100 nM this agent interferes with other phosphatases as well.14 The use of calyculin A, a potent inhibitor of protein phosphatase 1, produced results similar to those obtained with okadaic acid (Figure 5A).
In sickle erythrocytes, we found that PKC inhibitors did not modify the activity of the exchanger (from 4.26 ± 0.08 to 4.21 ± 0.13 with CH and 4.13 ± .009 with CC) as seen under similar conditions in normal erythrocytes (Figure 5B). We also evaluated the effect of phosphatase inhibitors in sickle erythrocytes (Figure 5B). We found that neither okadaic acid nor calyculin A altered the activity of the exchanger in sickle erythrocytes. These data suggest that the signaling pathway for Na/Mg regulation is abnormal in sickle erythrocytes and strongly support the hypothesis that the Na/Mg exchanger is "locked" in an up-regulated mode in these cells.
Discussion
Reduced erythrocyte magnesium content has been associated with the pathophysiology of sickle cell disease (SCD), and magnesium supplementation has been proposed as a therapeutic approach to ameliorate painful crisis in SCD.5,15-20 However, the mechanisms underlying these effects are not fully understood. In erythrocytes, regulation of intracellular Mg2+ content is mediated by cytoplasmic magnesium buffers such as adenosine triphosphate (ATP) and 2,3-diphosphoglycerate10 as well as by Na+-dependent and -independent Mg ion transport mechanisms.3 One of these transport mechanisms is the plasma membrane Na/Mg exchanger. Flatman21 has previously described the dependency of the exchanger's activation upon extracellular Na+ and Mg2+. We measured activation kinetics of the exchanger by extracellular Na+ and found an increased Vmax and affinity in sickle red cells that was associated with a decrease in cooperativity of the Na ions compared with normal erythrocytes. The loss of cooperativity of the exchanger observed in SCD supports the notion of abnormal regulation of the exchanger in sickle erythrocytes. In turn, these kinetic alterations may in part explain the low total cellular Mg2+ levels observed in the denser fraction of sickle erythrocytes.2 Although the difference in affinity for extracellular Na+ might not be of major physiologic importance for the regulation of the activity of the exchanger in circulating red cells, it might be of interest in tissues and red cells exposed to dramatic changes in Na+ concentration such as in the kidney.
Normal erythrocytes show decreasing Mg2+ levels with aging,22 and younger erythrocytes contain higher Mg2+ levels than older cells. The activity of the Na/Mg exchanger also decreases with increased cell density in normal erythrocytes (Figure 1B). Perhaps the cell volume reduction associated with aging decreased the activity of the exchanger, as seen in hypertonic stimulation experiments (Figure 4) in normal erythrocytes. Neither cell shrinkage nor cell swelling had any significant effect on the activity of the exchanger in sickle erythrocytes, suggesting abnormal regulation. However, we cannot rule out the possible contribution of the large difference in the average ages of normal and sickle erythrocytes to the observed differences in Mg2+ fluxes.
The changes in Na/Mg exchanger activity with varying MCHCs (Figure 1B) cannot be explained solely by the presence of reticulocytes. A 46% decrease in reticulocyte content in sickle fraction number 2 (Figure 1B) did not significantly affect the magnitude of the Mg2+ flux, suggesting that reticulocyte content per se is not a major determinant of the total exchanger's activity. Nevertheless, we cannot rule out the contribution of reticulocyte number to the dramatic change observed from the first fraction to the second fraction in normal red cells (Figure 1B). Further experiments will be required to assess the contribution of the reticulocyte fraction to the total exchanger activity.
We hypothesize that the persistent elevation of Na/Mg exchanger activity at all density levels may play a role in generating the reduced total intracellular Mg2+ seen in dense sickle erythrocytes.2 Our data support the notion of a constant exchanger activity at different densities, whereby the cell is constantly losing Mg2+. Since Mg2+ is known to be a modulator of K/Cl activity in normal and sickle erythrocytes,9,14 the high activity of this exchanger might lead to a decrease in intracellular Mg2+ that is responsible for the high activity of the K/Cl cotransporter in these cells.9,23-25
The role of PKC in the regulation of cellular Mg2+ has been investigated in rat vascular smooth muscle cells, where PKC inhibition leads to increased Mg2+ levels.26 Zheng et al26 also reported that this increase was mediated by intracellular release of stored Mg2+, since PKC inhibitors induced the same Mg2+ response in the absence of extracellular Mg2+. Differential activation of PKC has been previously reported in sickle cells. PKC activity is significantly elevated in sickle erythrocytes when compared with normal cells, and only PKC seems to translocate to the plasma membrane when it is activated by phorbol 12-myristate 13-acetate.27 In normal red cells, we show that the exchanger is activated by PKC inhibitors and is unaffected by phosphatase inhibitors, suggesting the involvement of PKC in the regulation of the exchanger's activity. In contrast, neither PKC nor phosphatase inhibitors significantly affected the activity of the exchanger in sickle erythrocytes (Figure 5B). Therefore, we speculate that the PKC-sensitive pathway of Na+-dependent exchanger activity modulation is abnormally regulated or interrupted in sickle erythrocytes. Furthermore, the absence of any significant effect of PKC inhibitors on the sickle erythrocyte Na/Mg exchanger also suggests that the elevated PKC activity observed by Fathallah et al27 plays no role in the regulation of the Na/Mg exchanger in sickle erythrocytes. Further studies will be needed to elucidate this point.
Because of the amiloride sensitivity of the Na+-dependent Mg2+ flux, it has been suggested that the Na/Mg exchanger and the Na/H exchanger might be mediated by the same system. Our results argue against this hypothesis, in that (1) in normal erythrocytes, the external Na+ dependency of the Na/H exchanger follows a hyperbolic pattern and not the sigmoid pattern as seen in Na/Mg exchanger; (2) the Na/Mg exchanger is not sensitive to okadaic acid, as is the case for Na/H exchanger activity in human erythrocytes28; (3) PKC inhibitors decrease the activity of Na/H exchanger29 and increase the activity of Na/Mg exchanger in normal erythrocytes; and (4) hyperosmolarity does not increase the activity of the exchanger, as occurs with the Na/H exchanger in normal erythrocytes.30
In summary, our results suggest that the Na/Mg exchanger plays an important role in the pathophysiology of SCD. The initial characterization of the Na/Mg exchanger in sickle erythrocytes presented here provides a platform for further studies on the pathophysiology of Mg2+ transporters in SCD. Furthermore, these studies open new pathways for investigation of K+ transport, water loss, and the consequent cellular dehydration in sickle erythrocytes.
Acknowledgements
We would like to thank Dr Carolyn Bigelow, Dr Rathi Iyer, Carole Ward, Cindy Kendig, and Aileen Anderson from the University of Mississippi Medical Center (Jackson, MS) for generously providing blood samples from patients with sickle cell disease. We also wish to thank Ms Michelle Langlois and Mr Lin-Chie Pong for their technical support.
Footnotes
Prepublished online as Blood First Edition Paper, September 7, 2004; DOI 10.1182/blood-2003-11-3755.
Supported by National Institutes of Health (NIH) grants HL67699 (A.R.), DK50422 (C.B.), and DK02817 and DK064841 (J.R.R.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
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