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High-pressure-induced hemolysis is characterized by release of membrane vesicles from human erythrocytes
Trends in High Pressure Bioscience and Biotechnology R. Hayashi (editor) 9 2002 Elsevier Science B.V. All rights reserved.
167
H i g h - p r e s s u r e - i n d u c e d h e m o l y s i s is c h a r a c t e r i z e d by release of m e m b r a n e vesicles f r o m h u m a n e r y t h r o c y t e s T. Yamaguchi and S. Terada Department of Chemistry, Faculty of Science, Fukuoka University, Jonan-ku, Fukuoka 8140180, Japan Upon exposure of human erythrocytes to high pressures (0.1-200 MPa), hemolysis and vesiculation start to occur at about 140 MPa. At 200 MPa, the value of hemolysis is about 45%, and the diameter of released vesicles is about 460 nm. Furthermore, when transmembrane proteins are cross-linked with cytoskeletal ones using diamide, high-pressureinduced hemolysis is greatly suppressed. In this case, the diameter of vesicles is about 230 nm. These results suggest that high-pressure-induced hemolysis may be associated with the size of released vesicles. 1. I N T R O D U C T I O N The biological membranes are mainly composed of proteins and phospholipids. The association between these components may be controlled by hydrophobic, ionic, and van der Waals interactions [1]. The membrane structure of human erythrocytes has been studied as a prototype of such biological membranes. The stability and deformability of the erythrocyte membrane are controlled by the interactions between transmembrane proteins and cytoskeletal ones [2]. The transmembrane proteins such as band 3 and glycophorin C associate with linking proteins such as ankyrin and protein 4.1 [2]. These linking proteins attach the cytoskeleton, which consists of spectrin, protein 4.1, and actin, to the red cell plasma membrane. Thus, membrane protein-protein interactions play an important role in the structure and function of the erythrocyte membrane. The default in these interactions causes the destruction of the membrane, i.e., hemolysis. Therefore, it is expected that the study of thc hemolysis provides the useful information on the interactions among membrane componcnts. It is well known that pressure affects the hydrophobic, ionic, and van der Waals interactions. Thus, it seems likely that the membrane structure of erythrocytes is affected by pressure. In fact, high pressure induces the hemolysis [3]. In this paper, we describe that high-pressure-induced hemolysis is associated with the size of released vesicles. 2. MATERIALS A N D METHODS 2 . 1 . Materials Diazinedicarboxylic acid bis-(N,N'-dimethylamide) (diamide) was purchased from Sigma. All other chemicals were of reagent grade. 2 . 2 . Chemical modification of erythrocytes Human blood was obtained from the Fukuoka Red Cross Blood Center. The blood was centrifuged at 750 g for 10 rain at 4~ The plasma and buffy coat were carefully removed.
168
The erythrocytes were washed three times with phosphate buffered saline (PBS; 10 mM sodium phosphate, 150 mM NaCI, pH 7.4). The erythrocytes in PBS (at 20% hematocrit) were treated with diamide (0.5 mM) at 100 MPa for 30 min at 37~ The erythrocytes were washed three times in PBS and used for the experiments of hemolysis and vesiculation. 2 . 3 . Hemolysis The hemolysis of the erythrocytes under high pressure was measured as follows. The erythrocytes suspended at 0.3% hematocrit in PBS were put into a syringe-type cell with a piston. The sample cell was placed in a pressure bomb made of stainless steel. The pressure was generated by means of a hand-type pump and monitored with a Heise pressure gauge. Thc mixture of ligroin and kerosine (v/v 1:1) was used as the pressure transmitting fluid. The erythrocyte suspension was compressed at a rate of 20 MPa/min, incubated at 37~ for 30 min at various pressures (110-200 MPa). Then, the samples were decompressed up to atmospheric pressure at a rate of 40 MPa/min. The suspension was centrifuged at 750 g for 10 min at 35~ Hemoglobin release into the supernatant was measured at 542 nm. One hundred percent of hemolysis was performed by adding Triton X-100 (0.02%) into the suspension.
2.4. Scanning electron microscopy Erythrocyte suspensions (5 ml at 0.3% hematocrit) were subjected to a pressure of 200 MPa for 30 min at 37~ and centrifuged for 10 min at 750 g. The pellets were washed once with 10 volumes of PBS and then suspended in 3 ml of the same buffer containing 0.5 % bovine serum albumin. Glutaraldehyde was added to a final concentration of 1% and the suspension was incubated at 22~ for 2 h. The cells were washed twice with 40 volumes of the same buffer without bovine serum albumin. Fixed erythrocytes were dehydrated with solutions of increasing acetone concentration (50, 80, 90, 95, and 100% acetone) and then suspended in 2 ml of amyl acetate. The samples were dried with a Hitachi critical point dryer (model HCP-1) and then coated with Pt-Pd using an Eiko sputtering outfit (model IB-3). A Hitachi S-430 scanning electron microscope was used. 2 . 5 . L i g h t scattering The sizes of membrane vesicles were measured at 250C by using a submicron particle sizer (model 370, NICOMP, Calif. USA) with laser wavelength at 488 nm.
3. R E S U L T S
3.1. High-pressure-induced hemolysis When human erythrocytes were subjected to various pressures (0.1-200 MPa) for 30 min at 37~ the hemolysis began to occur at a pressure of about 140 MPa. At higher pressures, the degree of hemolysis increased (Fig. 1). For instance, the value of hemolysis at 200 MPa was about 45%. To cross-link membrane proteins, the erythrocytes were treated with 0.5 mM diamide at 100 MPa. When diamide-treated erythrocytes were exposed to a pressure of 200 MPa, the value of hemolysis was about 3 %.
3.2. High-pressure-induced vesiculation To examine the shape of high-pressure-treated erythrocytes, the erythrocytes were exposed to a pressure of 200 MPa for 30 min at 37~ decompressed, and fixed with glutaraldehyde. The scanning electron micrography of 200 MPa-treated erythrocytes shows the membrane vesicles formed on the surface of red cells (Fig. 2). We can observe various size of membrane vesicles on the cell surface. These vesicles may be released into the medium from the membrane surface. If so, the released vesicles are readily separated from mother cells by centrifugation. The membrane vesicles in the supernatant are detected by measuring the
169
50
5o v
"o
4o
4O 30
tD
20
t-
O
10
0
o 110
140
160
Pressure
180
~
200
(MPa)
Figure 1. Hemolysis and vesiculation as a function of pressure in human erythrocytes activity of acetylcholinesterase. As with hemolysis, the activity was also observed above 140 MPa and increased with increasing pressure (Fig. 1).
3.3. Size of high-pressure-induced vesicles The activity of acetylcholinesterase suggests the release of membrane vesicles from the membrane surface of pressure-treated erythrocytes. So, we attempted to examine the size of vesicles by using a light scattering technique. Using this method, it was found that the diameter of membrane vesicles released from 200 MPa-treated erythrocytes is about 480 nm. On the other hand, when the diamide-treated erythrocytes, in which tmnsmembrane proteins are cross-linked with cytoskeletal ones, were exposed to a pressure of 200 MPa, the diameter of released vesicles was about 230 rim. 4. D I S C U S S I O N When human erythrocytes are exposed to high pressures, hemolysis starts to occur at pressures of 130 --140 MPa. In 200 MPa-treated cells, spectrin molecules are partially detached from the membrane [3]. Spectrin is a rod-like heterodimer molecule comprising two large subunits, ct (240 kDa) and 15 (220 kDa) [4]. The spectrin dimers form tetramers upon head-to-head association. In general, oligomeric proteins dissociate under high pressure. Therefore, it seems likely that the association of spectrin molecules and the interactions of spectrin with ankyrin are perturbed by a pressure of 200 MPa. Thus, the cytoskeletal network is destroyed by high pressure. When 200 MPa-treated erythrocytes are incubated at 37"C and atmospheric pressure, hemoglobin release from the membrane is suppressed [3]. Upon the shift from 37"C to 0*C, however, hemoglobin within the membrane is released again [3]. This indicates that the size of the membrane holes induced by pressure is dependent on temperature, i.e., resealed membranes reopen at 0*C [3].
170
Fig. 2 Scanning electron micrograph of 200 MPa-treated erythrocytes. The vesiculation of human erythrocytes is performed by various methods. For instance, membrane vesicles induced by ATP depletion [5], Ca2+-loading [6], or incubation with dimyristoylphosphatidylcholine (DMPC) liposomes [7] are spectrin-free and band 3-rich. On the other hand, membrane vesicles containing cytoskeletal proteins such as spectrin and actin in addition to band 3 are released upon the exposure of erythrocytes to high pressure (200 MPa) [8]. The average size of such vesicles is about 480 nm in diameter. In addition, the membrane protein composition of the released vesicles is modulated by cross-linking of membrane proteins in erythrocytes. In diamide-treated erythrocytes, protein 4.1-rich vesicles are released by high pressure [9]. In this case, the average size of released vesicles is about 230 nm and pressure-induced hemolysis is greatly suppressed (about few percents). These results suggest that high-pressure-induced hemolysis is associated with the size of released membrane vesicles. REFERENCES
1. C. Tanford, The Hydrophobic Effect; Formation of Micelles and Biological Membranes,Weley, New York, 1973. 2. V. Bennett, Biochim. Biophys. Acta 988 (1989) 107. 3. T. Yamaguchi, H. Kawamura, E. Kimoto,and M. Tanaka, J. Biochem., 106 (1989) 1080. 4. E. Ungewickell and W. Gratzer, Eur. J. Biochem., 88 (1978) 379. 5. H.U. Lutz, S.-C. Liu, and J. Palek, J. Cell Biol., 73 (1977) 548. 6. D. Allan, M.M. Billah, J.B. Finean, and R.H. Michell, Nature 261 (1976) 58. 7. P. Ott, M.J. Hope, A.J. Verkleij, B. Roelofsen, U. Brodbeck, and .L.L.M.Van Deenen, 641(1981) 79. 8 T. Yamaguchi, T. Kajikawa, and E. Kimoto, J. Biochem., 110 (1991) 355. 9. T.Yamaguchi, T. Saeki, and E. Kimoto, J. Biochem., 1147 (1993) 1.
167
H i g h - p r e s s u r e - i n d u c e d h e m o l y s i s is c h a r a c t e r i z e d by release of m e m b r a n e vesicles f r o m h u m a n e r y t h r o c y t e s T. Yamaguchi and S. Terada Department of Chemistry, Faculty of Science, Fukuoka University, Jonan-ku, Fukuoka 8140180, Japan Upon exposure of human erythrocytes to high pressures (0.1-200 MPa), hemolysis and vesiculation start to occur at about 140 MPa. At 200 MPa, the value of hemolysis is about 45%, and the diameter of released vesicles is about 460 nm. Furthermore, when transmembrane proteins are cross-linked with cytoskeletal ones using diamide, high-pressureinduced hemolysis is greatly suppressed. In this case, the diameter of vesicles is about 230 nm. These results suggest that high-pressure-induced hemolysis may be associated with the size of released vesicles. 1. I N T R O D U C T I O N The biological membranes are mainly composed of proteins and phospholipids. The association between these components may be controlled by hydrophobic, ionic, and van der Waals interactions [1]. The membrane structure of human erythrocytes has been studied as a prototype of such biological membranes. The stability and deformability of the erythrocyte membrane are controlled by the interactions between transmembrane proteins and cytoskeletal ones [2]. The transmembrane proteins such as band 3 and glycophorin C associate with linking proteins such as ankyrin and protein 4.1 [2]. These linking proteins attach the cytoskeleton, which consists of spectrin, protein 4.1, and actin, to the red cell plasma membrane. Thus, membrane protein-protein interactions play an important role in the structure and function of the erythrocyte membrane. The default in these interactions causes the destruction of the membrane, i.e., hemolysis. Therefore, it is expected that the study of thc hemolysis provides the useful information on the interactions among membrane componcnts. It is well known that pressure affects the hydrophobic, ionic, and van der Waals interactions. Thus, it seems likely that the membrane structure of erythrocytes is affected by pressure. In fact, high pressure induces the hemolysis [3]. In this paper, we describe that high-pressure-induced hemolysis is associated with the size of released vesicles. 2. MATERIALS A N D METHODS 2 . 1 . Materials Diazinedicarboxylic acid bis-(N,N'-dimethylamide) (diamide) was purchased from Sigma. All other chemicals were of reagent grade. 2 . 2 . Chemical modification of erythrocytes Human blood was obtained from the Fukuoka Red Cross Blood Center. The blood was centrifuged at 750 g for 10 rain at 4~ The plasma and buffy coat were carefully removed.
168
The erythrocytes were washed three times with phosphate buffered saline (PBS; 10 mM sodium phosphate, 150 mM NaCI, pH 7.4). The erythrocytes in PBS (at 20% hematocrit) were treated with diamide (0.5 mM) at 100 MPa for 30 min at 37~ The erythrocytes were washed three times in PBS and used for the experiments of hemolysis and vesiculation. 2 . 3 . Hemolysis The hemolysis of the erythrocytes under high pressure was measured as follows. The erythrocytes suspended at 0.3% hematocrit in PBS were put into a syringe-type cell with a piston. The sample cell was placed in a pressure bomb made of stainless steel. The pressure was generated by means of a hand-type pump and monitored with a Heise pressure gauge. Thc mixture of ligroin and kerosine (v/v 1:1) was used as the pressure transmitting fluid. The erythrocyte suspension was compressed at a rate of 20 MPa/min, incubated at 37~ for 30 min at various pressures (110-200 MPa). Then, the samples were decompressed up to atmospheric pressure at a rate of 40 MPa/min. The suspension was centrifuged at 750 g for 10 min at 35~ Hemoglobin release into the supernatant was measured at 542 nm. One hundred percent of hemolysis was performed by adding Triton X-100 (0.02%) into the suspension.
2.4. Scanning electron microscopy Erythrocyte suspensions (5 ml at 0.3% hematocrit) were subjected to a pressure of 200 MPa for 30 min at 37~ and centrifuged for 10 min at 750 g. The pellets were washed once with 10 volumes of PBS and then suspended in 3 ml of the same buffer containing 0.5 % bovine serum albumin. Glutaraldehyde was added to a final concentration of 1% and the suspension was incubated at 22~ for 2 h. The cells were washed twice with 40 volumes of the same buffer without bovine serum albumin. Fixed erythrocytes were dehydrated with solutions of increasing acetone concentration (50, 80, 90, 95, and 100% acetone) and then suspended in 2 ml of amyl acetate. The samples were dried with a Hitachi critical point dryer (model HCP-1) and then coated with Pt-Pd using an Eiko sputtering outfit (model IB-3). A Hitachi S-430 scanning electron microscope was used. 2 . 5 . L i g h t scattering The sizes of membrane vesicles were measured at 250C by using a submicron particle sizer (model 370, NICOMP, Calif. USA) with laser wavelength at 488 nm.
3. R E S U L T S
3.1. High-pressure-induced hemolysis When human erythrocytes were subjected to various pressures (0.1-200 MPa) for 30 min at 37~ the hemolysis began to occur at a pressure of about 140 MPa. At higher pressures, the degree of hemolysis increased (Fig. 1). For instance, the value of hemolysis at 200 MPa was about 45%. To cross-link membrane proteins, the erythrocytes were treated with 0.5 mM diamide at 100 MPa. When diamide-treated erythrocytes were exposed to a pressure of 200 MPa, the value of hemolysis was about 3 %.
3.2. High-pressure-induced vesiculation To examine the shape of high-pressure-treated erythrocytes, the erythrocytes were exposed to a pressure of 200 MPa for 30 min at 37~ decompressed, and fixed with glutaraldehyde. The scanning electron micrography of 200 MPa-treated erythrocytes shows the membrane vesicles formed on the surface of red cells (Fig. 2). We can observe various size of membrane vesicles on the cell surface. These vesicles may be released into the medium from the membrane surface. If so, the released vesicles are readily separated from mother cells by centrifugation. The membrane vesicles in the supernatant are detected by measuring the
169
50
5o v
"o
4o
4O 30
tD
20
t-
O
10
0
o 110
140
160
Pressure
180
~
200
(MPa)
Figure 1. Hemolysis and vesiculation as a function of pressure in human erythrocytes activity of acetylcholinesterase. As with hemolysis, the activity was also observed above 140 MPa and increased with increasing pressure (Fig. 1).
3.3. Size of high-pressure-induced vesicles The activity of acetylcholinesterase suggests the release of membrane vesicles from the membrane surface of pressure-treated erythrocytes. So, we attempted to examine the size of vesicles by using a light scattering technique. Using this method, it was found that the diameter of membrane vesicles released from 200 MPa-treated erythrocytes is about 480 nm. On the other hand, when the diamide-treated erythrocytes, in which tmnsmembrane proteins are cross-linked with cytoskeletal ones, were exposed to a pressure of 200 MPa, the diameter of released vesicles was about 230 rim. 4. D I S C U S S I O N When human erythrocytes are exposed to high pressures, hemolysis starts to occur at pressures of 130 --140 MPa. In 200 MPa-treated cells, spectrin molecules are partially detached from the membrane [3]. Spectrin is a rod-like heterodimer molecule comprising two large subunits, ct (240 kDa) and 15 (220 kDa) [4]. The spectrin dimers form tetramers upon head-to-head association. In general, oligomeric proteins dissociate under high pressure. Therefore, it seems likely that the association of spectrin molecules and the interactions of spectrin with ankyrin are perturbed by a pressure of 200 MPa. Thus, the cytoskeletal network is destroyed by high pressure. When 200 MPa-treated erythrocytes are incubated at 37"C and atmospheric pressure, hemoglobin release from the membrane is suppressed [3]. Upon the shift from 37"C to 0*C, however, hemoglobin within the membrane is released again [3]. This indicates that the size of the membrane holes induced by pressure is dependent on temperature, i.e., resealed membranes reopen at 0*C [3].
170
Fig. 2 Scanning electron micrograph of 200 MPa-treated erythrocytes. The vesiculation of human erythrocytes is performed by various methods. For instance, membrane vesicles induced by ATP depletion [5], Ca2+-loading [6], or incubation with dimyristoylphosphatidylcholine (DMPC) liposomes [7] are spectrin-free and band 3-rich. On the other hand, membrane vesicles containing cytoskeletal proteins such as spectrin and actin in addition to band 3 are released upon the exposure of erythrocytes to high pressure (200 MPa) [8]. The average size of such vesicles is about 480 nm in diameter. In addition, the membrane protein composition of the released vesicles is modulated by cross-linking of membrane proteins in erythrocytes. In diamide-treated erythrocytes, protein 4.1-rich vesicles are released by high pressure [9]. In this case, the average size of released vesicles is about 230 nm and pressure-induced hemolysis is greatly suppressed (about few percents). These results suggest that high-pressure-induced hemolysis is associated with the size of released membrane vesicles. REFERENCES
1. C. Tanford, The Hydrophobic Effect; Formation of Micelles and Biological Membranes,Weley, New York, 1973. 2. V. Bennett, Biochim. Biophys. Acta 988 (1989) 107. 3. T. Yamaguchi, H. Kawamura, E. Kimoto,and M. Tanaka, J. Biochem., 106 (1989) 1080. 4. E. Ungewickell and W. Gratzer, Eur. J. Biochem., 88 (1978) 379. 5. H.U. Lutz, S.-C. Liu, and J. Palek, J. Cell Biol., 73 (1977) 548. 6. D. Allan, M.M. Billah, J.B. Finean, and R.H. Michell, Nature 261 (1976) 58. 7. P. Ott, M.J. Hope, A.J. Verkleij, B. Roelofsen, U. Brodbeck, and .L.L.M.Van Deenen, 641(1981) 79. 8 T. Yamaguchi, T. Kajikawa, and E. Kimoto, J. Biochem., 110 (1991) 355. 9. T.Yamaguchi, T. Saeki, and E. Kimoto, J. Biochem., 1147 (1993) 1.