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Effect of erythropoietin on the glucose transport of rat erythrocytes and bone marrow cells
BIOCHEMICAL
MEDICINE
AND
METABOLIC
BIOLOGY
38,
134-141 (1987)
Effect of Erythropoietin on the Glucose Transport of Rat Etythrocytes and Bone Marrow Cells JHARNA GHOSAL,
MUNMUN
CHAKRABORTY, TULI BISWAS, CHAYAN AND ASOKE G. DATTA
K. GANGULY,
Indian institute of Chemical Biology, 4, Raja S.C. M&lick Road, Calcutta-700032, India Received March 17, 1986, and in revised form September 10, 1986
Modern studies on the structure of membranes have revealed that chain length and extent of saturation of fatty acids of phospholipids influence membrane permeability (1). Differences in permeabilities of glycerol, erythritol, urea, and anions have been reported to be related to the membrane phospholipids (2). Erythrocyte membrane permeability increases with a rise in phosphatidylcholine content of the membrane lipids (3). These results suggest that the lipid environment of the membrane transport proteins can affect transmembrane fluxes in erythrocytes. Erythrocytes of different species which differ in lipid composition, particularly having loosely bound phospholipids, exhibited a marked difference in their permeability characteristics (4). In short, phospholipids in biological membranes have been shown to modulate the permeability behavior of the erythrocyte membrane. The role of erythropoietin (Ep) on the erythrocyte membrane is quite clear from our recent studies (5-8). We have demonstrated that Ep not only stimulates [14C]acetate incorporation into membrane lipids (6) but also alters the fatty acid composition of the erythrocyte membrane as well as the ratio of saturated to unsaturated fatty acids (7). This hormone also has an influence on the exchange of cholesterol and phospholipid between erythrocyte and plasma (8). In view of the above observations we were interested in studying the effect of this hormone on the permeability of glucose in erythrocytes. MATERIALS Albino rats, weighing about 100-120 g, were chosen for this study. Sheep plasma Step I erythropoietin was obtained from Connaught Laboratories Limited, Canada. Uniformly labeled [L4C]glucose (sp act 210 mCi/mmole) was purchased from Bhaba Atomic Research Centre, Bombay, and [‘4C]methylk-a-o-glucoside (a-MG) was prepared by the method of Bollenback (9). a-MG, 2,4-dinitrophenol (DNP), azide, ouabain, and paru-hydroxymercuribenzoate (pHMB) were purchased from Sigma Chemical Company, St. Louis, Missouri. 134 0885-4505187 $3.00 Copyright All rights
Q 1987 by Academic Press, Inc. of reproduction in any fomt reserved.
EFFECT
OF
ERYTHROPOIETIN
ON
GLUCOSE
TRANSPORT
135
METHODS Mature erythrocytes were obtained from rats of the control and experimental groups according to our previous report (6). In order to deplete the endogenous level of Ep, all the animals were kept in a fasting state during the experiment. Rats of the experimental groups were subjected to subcutaneous injection with different amounts of Ep in two successive doses on the third and fourth days of starvation. Animals were killed 48 hr after administration of Ep, and heparinized blood was collected by cardiac puncture. Plasma and buffy coat (mainly leukocytes) were separated from erythrocytes according to the method of Marks et al. (10). The packed erythrocytes were washed three times with isotonic saline (pH 7.4) and were used in subsequent incubations. Bone marrow cells were collected by flushing out the femora with heparinized saline and the cells were isolated by the method of Krantz et al. (11). The homogeneous cell suspension was filtered through cheesecloth to remove the cell aggregates, bits of bones, and connective tissues. The marrow cells were then washed three times with normal saline (pH 7.4) and were used in the experiments. Erythrocytes and bone marrow cell counts were carried out by standard hematological techniques. Determination of [‘4C]glucose and [‘4C]methyl-a-o-glucoside transport. Transport of [‘4C]glucose and [‘4C]methyl-a-u-glucoside in erythrocytes and bone marrow cells of control and experimental groups was measured by the method of Nath and Datta (12). Samples of cell suspension (0.5 ml) containing approximately equal numbers of cells (10’ cells/incubation mixture) were incubated with 0.1 ml rat serum and 0.25 pmole [‘4C]methyl-a-n-glucoside (5.61 x 10’ counts/min) or 0.25 pmole [‘4C]glucose (5.63 x 10’ counts/min) in a final volume of 5.0 ml at 37°C for 20 min. For glucose and a-MG concentration experiments, these two reagents were diluted from the above two solutions. The cells were chilled and washed three times with 5.0 ml of chilled isotonic saline (pH 7.4) and were subjected to radioactivity measurements. Radioactivities were measured in a LKB/WALLACK.1217 RACKBETA. liquid scintillation counter Model 43. RESULTS
AND DISCUSSION
This report demonstrates the effect of Ep on glucose transport by rat erythrocytes and bone marrow cells. It has already been mentioned under Methods that all the experiments were performed on starved animals. Figure 1 shows the depletion of erythropoietin levels of blood with starvation. It can further be seen from the figure that on the third day of starvation (when the first dose of erythropoietin was injected) about 75% of the circulatory erythropoietin was already depleted, rendering the animals more sensitive to exogenous erythropoietin. To assess glucose transport, radioactive o-glucose and radioactive methyl-a-n-glucoside were used and both of them are transported inside the cell by the same carrier protein (13). The greatest advantage of using this sugar analog (methyl-a-oglucoside) for the transport study is that it enters the cell but is not metabolized. Figure 2 shows the initial linear increase of glucose transport in the physiological range of glucose concentration (around 5 mM) and subsequent saturation kinetics of the erythrocytes of normal, starved, and starved plus Ep-treated rats at different
136
GHOSAL
FIG. 1. Endogenous erythropoietin given under Methods.
ET AL.
levels in rats during starvation. Experimental
procedures are
concentrations of glucose. The effect of Ep on glucose permeability does not seem to be mediated by passive transport as glucose uptake tends to attain the saturation kinetics. Moreover, the rate of uptake was temperature dependent (results not shown). The glucose transport was linear up to 20 min (Fig. 3) and showed proportionality with increasing concentration of Ep up to 3 units (Fig. 4). It is evident from Table 1 that starvation caused about 30% inhibition of transport of glucose and administration of Ep (6 units) to starved rats stimulated glucose transport by about 61% over that of the starved rats. Six units of Ep stimulated the transport process further (about 101%). However, Ep administration in normal animals produced very little change (data not shown) in glucose transport. (u-MG which competes with the transport of glucose, abolished Ep-stimulated glucose transport by erythrocytes to a significant extent. Cohen and Monod (14) showed that methyl-a-n-glucoside, a nonmetabolizable glucose derivative, is a very useful reagent in the study of glucose transport
24.0
S (mhI of Glucorr)
2. Effect of different concentration of glucose on the transport process by rat erythrocytes. 0, normal; A, starved; 0, starved + erythropoietin (6 units). Experimental procedures are given under Methods. FIG.
EFFECT
OF ERYTHROPOIETIN
ON GLUCOSE
Time
TRANSPORT
137
in min
FIG. 3. Transport of glucose by rat erythrocytes at different time periods. 0, normal; 0, starved; A, Starved + erythropoietin (6 units). Experimental procedures are given under Methods.
across the membrane. Hence, its rate of entry is an index of glucose entry into a cell and it acts as a strong competitive inhibitor of glucose transport. Table 2 shows that transport of radioactive glucose analog was stimulated by Ep administration to starved animals and n-glucose abolished the Ep-stimulated transport of methyl-a-o-glucoside. The inhibition of the transport of o-glucose by methyl-a-o-glucoside and vice versa indicates a common transport system for both o-glucose and its analog. As bone marrow cells (BMC) are more sensitive to Ep action, transport of glucose in these cells was studied next. A similar stimulatory effect of Ep was noted on the glucose and methyl-a-n-glucoside transport in BMC of starved rats. It can be seen from Fig. 5 that in BMC, like in erythrocytes there was an initial linear increase in glucose transport up to 10 mM of glucose, beyond which transport showed saturation kinetics. The transport of glucose inside bone marrow cells also showed linearity up to 20 min (Fig. 6) and up to 3 units of Ep (Fig. 4). Table 3 shows that Ep stimulated glucose transport in BMC of starved rats and a-MG abolished the stimulation. cz-MG transport by bone marrow cells was
0
2 Erythrowetin
4 (in
6 units)
FIG. 4. Effect of different amounts of erythropoietin on the transport of glucose by rat erythrocytes (0) and bone marrow cells (0). Experimental procedures are given under Methods.
138
GHOSAL
Effect of Erythropoietin
ET AL.
TABLE 1 on the Transport of [‘4C]-Glucose in Rat Erythrocyte pmole of glucose transported/lO’ cells
% Stimulation or inhibition
Normal Starved Starved + (3 units) Ep
0.357 f 0.0324 0.233 2 0.015 0.376 t 0.022
35 (-I* 61 (+I**
Starved + (6 units) Ep
0.469 f 0.052
Group
P < 0.05
101 (+)**
P < 0.01
Starved + (6 units) Ep + (w-MG
0.226 f 0.02
52 (-)***
Note. The results are the means k SD of three independent experiments. Experimental are given under Methods. a-MC was added to the incubation mixture (0.2 mg/ml). * Inhibition from normal animals. ** Stimulation/inhibition from starved animals. *** Inhibition from erythropoietin-treated animals.
procedures
also stimulated by Ep and the stimulation was abolished by glucose (Table 4), indicating that in BMC, like in erythrocytes, transport fo glucose and a-MG is mediated by a common transport system. Figure 7 shows the effect of metabolic inhibitors (2,4-DNP, pHMB, ouabain, and azide) on glucose transport in rat erythrocytes. Inhibition of Ep-stimulated glucose transport by DNP and azide suggests that the permeation process is energy dependent. It is well known that the active transport process involves coupling of pump ATPase or metabolically maintained ionic gradient and that the permeated molecule binds to a specific membrane carrier (15). The inhibitory effect of ouabain on glucose transport also suggests that the transport system is active in nature. Furthermore, inhibition by pHMB reveals the importance of sulfhydryl groups in the glucose carrier system. The exact mechanism by which Ep stimulates the glucose transport
Effect of Erythropoietin Group Normal Starved Starved + (6 units) Ep Starved + (6 units) Ep + o-glucose
TABLE 2 on the Transport of [“Cl-Methyl
a-o-Glucoside
pmole of cr-MG transported/IO’ cells 0.331 -c 0.023 0.181 f 0.018 0.449 5 0.012 P < 0.01 0.183 + 0.025
Inside Rat Erythrocytes % Stimulation or inhibition 45 (-)* 175 (+)* 60 (-)***
Note. The results are the means + SD of three independent experiments. Experimental procedures are given under Methods. o-Glucose was added to the incubation mixture (0.2 pmole/ml). * Inhibition from normal animals ** Stimulation/inhibition from starved animals. *** Inhibition from erythropoietin-treated animals.
EFFECT OF ERYTHROPOIETIN
ON GLUCOSE
139
TRANSPORT
FIG. 5. effect of different concentrations of glucose on the transport process by rat bone marrow cells. 0, normal; A, starved; 0, starved + erythropoietin (6 units). Experimental procedures are given under Methods.
0
IO Time
20 tn mtn
30
40
FIG. 6. Transport of glucose by rat bone marrow cells at different time periods. 0, normal; 0, starved; A, starved + erythropoietin (6 units). Experimental procedures are given under Methods.
Effect of Erythropoietin Group Normal Starved Starved + (6 units) Ep Starved + (6 units) Ep + (x-MG
TABLE 3 on the Transport of [‘4C]-Glucose in Rat Bone Marrow
Cells
fimoles of glucose transported/IO’ cells
% Stimulation or inhibition
0.499 + 0.02 0.351 5 0.013 0.967 t 0.029 P < 0.001 0.284 2 0.033
29 (-)* 175 (+)**
Note. The results are the Mean 2 SD of three independent experiments. Experimental are given under methods. 0.2 mg (Y-MG was added/ml of incubation mixture. * Inhibition from normal animals. ** Stimulations/Inhibitions from starved animals. *** Inhibition from erythropoietin treated animals.
71 (-j*** procedures
140
GHOSAL
Effect of Erythropoietin
TABLE 4 on the Transport of [‘4C]-Methyl a-o-Glucoside
Group Normal Starved Starved + (6 units) Ep Starved f (6 units) Ep +
ET AL.
in Rat Bone Marrow
Cell
pmoles of a-MG transported/ 10’ cells
% Stimulation or inhibition
0.451 t 0.033 0.259 +- 0.02 0.618 -c 0.022 P > 0.002 0.212 i- 0.02
43 (-)* 138 (+)** 66 (-)***
D-ghCOSC
Note. The results are the Mean t S.D. of three independent experiments. Experimental procedures are given under methods. 0.2 pmoles o-glucose was added/ml incubation mixture. * Inhibition from normal animals. ** Stimulations/inhibitions from starved animals. *** Inhibition from erythropoietin treated animals.
inside red blood cells is not yet known. However, changes in membrane lipid composition due to Ep administration (7) may be partly responsible. Ep has been found to increase membrane phosphatidylcholine content (7), which has been reported to increase membrane permeability (3). Stimulation of Na’ + K+ ATPase activity of rat erythrocyte membrane (16) may also partly explain increased glucose transport in rat erythrocytes. SUMMARY The effect of Ep on radioactive glucose and methyl-cr-o-glucoside transport by rat erythrocytes and bone marrow cells were studied. There is initial linearity followed by saturation kinetics of [‘4C]glucose transport by the erythrocytes of starved and starved plus Ep-treated rats at different concentrations of glucose. Starvation caused slight inhibition of glucose transport which increased markedly on Ep administration to starved rats. Normal animals failed to show any significant change in glucose transport after Ep treatment. Methyl-cu-p-glucoside inhibited the Ep-stimulated glucose transport significantly. 05r
FIG. 7. Effect of different inhibitors on glucose transport by rat erythrocytes. 0, control (no inhibitor); q , 2,4-DNP; a, pHMB; q , ouabain; W azide; S, starved; S + E, starved + erythropoietin (6 units). Experimental procedures are given under Methods. Results are the means f SD of three independent experiments. Each inhibitor was added to the incubation mixture separately (0.6 mmole/ml).
EFFECT OF ERYTHROPOIETIN
ON GLUCOSE
TRANSPORT
141
Ep also stimulated the transport of radioactive methyl-a-u-glucoside which was competitively inhibited in presence of o-glucose. Glucose transport in erythrocytes was found to be sensitive to metabolic inhibitors like azide and DNP. A sulfhydryl reagent and ouabain also inhibited the transport process. Ep stimulated glucose and methyl-cu-o-glucoside transport in the bone marrow cells of starved rats. The sugar analog competitively inhibited the glucose transport in bone marrow cells and vice versa. REFERENCES 1. De Gier, J., Mandersloot, J. G., and Van Deenen, L. L. M., Biochim. Biophys. Acra 150, 606 (1968). 2. Dueticke, B., and Gruber, W., J. Membrane Biol. 13, 19 (1973). 3. Gary. R., Kirk: Biochim. Biophys. Acra 464, 157 (1977). 4. De Gier, J., Mulder, I. and Van Deenen, L. L. M., Narurwissenschafren 48, 54 (1961). 5. Ghosal, J., Biswas, T., and Ghosh, D. K., IRCS Med. Sci. 8, 355 (1980). 6. Chaudhuri, T., Ghosal, J., Ghosh, D. K., and Datta. A. G., Biochem. Med. 24, 162 (1980). 7. Ghosal, J., Biswas, T., Ghosh, A., and Datta, A. G., Biochim. Med. 32, 1 (1984). 8. Biswas, T., Ghosal, J., Ganguly, C., and Datta, A. G.. Biochem. Med. Merab. Biol. 35, 120 (1986). 9. Bollenback, G. N., Methods Curbohydr. Chem. 2, 236 (1963). 10. Marks, P. A., Gelhorn, A., and Kidson, C., J. Biol. Chem. 235, 2579 (1960). II. Krantz. S. B., Gallien-Lartigm, O., and Goldwasser, E., J. Biol. Chem. 238, 4085 (1963). 12. Nath, J., and Datta, A. G., Jr., Gen. Microbial. 62, 17 (1970). 13. Kasahara, M., and Hinkle, P. C., Proc. Nor/. Acad. Sci. USA 73, 396 (1976). 14. Cohen, N. G., and Monod, J., Bacreriol. Rev. 21, 169 (1957). 15. Harrison, R., and Lunt, G. G., in “Biological Membranes: Their Structure and Function,” Chap. 2, p. 10. Thomson Litho, Great Britain, 1980. 16. Chakraborty, M.. Ghosal, J., Biswas, T.. and Datta, A. G., Biochem. Med. Merabl. Biol. 36, 231 (1986).
MEDICINE
AND
METABOLIC
BIOLOGY
38,
134-141 (1987)
Effect of Erythropoietin on the Glucose Transport of Rat Etythrocytes and Bone Marrow Cells JHARNA GHOSAL,
MUNMUN
CHAKRABORTY, TULI BISWAS, CHAYAN AND ASOKE G. DATTA
K. GANGULY,
Indian institute of Chemical Biology, 4, Raja S.C. M&lick Road, Calcutta-700032, India Received March 17, 1986, and in revised form September 10, 1986
Modern studies on the structure of membranes have revealed that chain length and extent of saturation of fatty acids of phospholipids influence membrane permeability (1). Differences in permeabilities of glycerol, erythritol, urea, and anions have been reported to be related to the membrane phospholipids (2). Erythrocyte membrane permeability increases with a rise in phosphatidylcholine content of the membrane lipids (3). These results suggest that the lipid environment of the membrane transport proteins can affect transmembrane fluxes in erythrocytes. Erythrocytes of different species which differ in lipid composition, particularly having loosely bound phospholipids, exhibited a marked difference in their permeability characteristics (4). In short, phospholipids in biological membranes have been shown to modulate the permeability behavior of the erythrocyte membrane. The role of erythropoietin (Ep) on the erythrocyte membrane is quite clear from our recent studies (5-8). We have demonstrated that Ep not only stimulates [14C]acetate incorporation into membrane lipids (6) but also alters the fatty acid composition of the erythrocyte membrane as well as the ratio of saturated to unsaturated fatty acids (7). This hormone also has an influence on the exchange of cholesterol and phospholipid between erythrocyte and plasma (8). In view of the above observations we were interested in studying the effect of this hormone on the permeability of glucose in erythrocytes. MATERIALS Albino rats, weighing about 100-120 g, were chosen for this study. Sheep plasma Step I erythropoietin was obtained from Connaught Laboratories Limited, Canada. Uniformly labeled [L4C]glucose (sp act 210 mCi/mmole) was purchased from Bhaba Atomic Research Centre, Bombay, and [‘4C]methylk-a-o-glucoside (a-MG) was prepared by the method of Bollenback (9). a-MG, 2,4-dinitrophenol (DNP), azide, ouabain, and paru-hydroxymercuribenzoate (pHMB) were purchased from Sigma Chemical Company, St. Louis, Missouri. 134 0885-4505187 $3.00 Copyright All rights
Q 1987 by Academic Press, Inc. of reproduction in any fomt reserved.
EFFECT
OF
ERYTHROPOIETIN
ON
GLUCOSE
TRANSPORT
135
METHODS Mature erythrocytes were obtained from rats of the control and experimental groups according to our previous report (6). In order to deplete the endogenous level of Ep, all the animals were kept in a fasting state during the experiment. Rats of the experimental groups were subjected to subcutaneous injection with different amounts of Ep in two successive doses on the third and fourth days of starvation. Animals were killed 48 hr after administration of Ep, and heparinized blood was collected by cardiac puncture. Plasma and buffy coat (mainly leukocytes) were separated from erythrocytes according to the method of Marks et al. (10). The packed erythrocytes were washed three times with isotonic saline (pH 7.4) and were used in subsequent incubations. Bone marrow cells were collected by flushing out the femora with heparinized saline and the cells were isolated by the method of Krantz et al. (11). The homogeneous cell suspension was filtered through cheesecloth to remove the cell aggregates, bits of bones, and connective tissues. The marrow cells were then washed three times with normal saline (pH 7.4) and were used in the experiments. Erythrocytes and bone marrow cell counts were carried out by standard hematological techniques. Determination of [‘4C]glucose and [‘4C]methyl-a-o-glucoside transport. Transport of [‘4C]glucose and [‘4C]methyl-a-u-glucoside in erythrocytes and bone marrow cells of control and experimental groups was measured by the method of Nath and Datta (12). Samples of cell suspension (0.5 ml) containing approximately equal numbers of cells (10’ cells/incubation mixture) were incubated with 0.1 ml rat serum and 0.25 pmole [‘4C]methyl-a-n-glucoside (5.61 x 10’ counts/min) or 0.25 pmole [‘4C]glucose (5.63 x 10’ counts/min) in a final volume of 5.0 ml at 37°C for 20 min. For glucose and a-MG concentration experiments, these two reagents were diluted from the above two solutions. The cells were chilled and washed three times with 5.0 ml of chilled isotonic saline (pH 7.4) and were subjected to radioactivity measurements. Radioactivities were measured in a LKB/WALLACK.1217 RACKBETA. liquid scintillation counter Model 43. RESULTS
AND DISCUSSION
This report demonstrates the effect of Ep on glucose transport by rat erythrocytes and bone marrow cells. It has already been mentioned under Methods that all the experiments were performed on starved animals. Figure 1 shows the depletion of erythropoietin levels of blood with starvation. It can further be seen from the figure that on the third day of starvation (when the first dose of erythropoietin was injected) about 75% of the circulatory erythropoietin was already depleted, rendering the animals more sensitive to exogenous erythropoietin. To assess glucose transport, radioactive o-glucose and radioactive methyl-a-n-glucoside were used and both of them are transported inside the cell by the same carrier protein (13). The greatest advantage of using this sugar analog (methyl-a-oglucoside) for the transport study is that it enters the cell but is not metabolized. Figure 2 shows the initial linear increase of glucose transport in the physiological range of glucose concentration (around 5 mM) and subsequent saturation kinetics of the erythrocytes of normal, starved, and starved plus Ep-treated rats at different
136
GHOSAL
FIG. 1. Endogenous erythropoietin given under Methods.
ET AL.
levels in rats during starvation. Experimental
procedures are
concentrations of glucose. The effect of Ep on glucose permeability does not seem to be mediated by passive transport as glucose uptake tends to attain the saturation kinetics. Moreover, the rate of uptake was temperature dependent (results not shown). The glucose transport was linear up to 20 min (Fig. 3) and showed proportionality with increasing concentration of Ep up to 3 units (Fig. 4). It is evident from Table 1 that starvation caused about 30% inhibition of transport of glucose and administration of Ep (6 units) to starved rats stimulated glucose transport by about 61% over that of the starved rats. Six units of Ep stimulated the transport process further (about 101%). However, Ep administration in normal animals produced very little change (data not shown) in glucose transport. (u-MG which competes with the transport of glucose, abolished Ep-stimulated glucose transport by erythrocytes to a significant extent. Cohen and Monod (14) showed that methyl-a-n-glucoside, a nonmetabolizable glucose derivative, is a very useful reagent in the study of glucose transport
24.0
S (mhI of Glucorr)
2. Effect of different concentration of glucose on the transport process by rat erythrocytes. 0, normal; A, starved; 0, starved + erythropoietin (6 units). Experimental procedures are given under Methods. FIG.
EFFECT
OF ERYTHROPOIETIN
ON GLUCOSE
Time
TRANSPORT
137
in min
FIG. 3. Transport of glucose by rat erythrocytes at different time periods. 0, normal; 0, starved; A, Starved + erythropoietin (6 units). Experimental procedures are given under Methods.
across the membrane. Hence, its rate of entry is an index of glucose entry into a cell and it acts as a strong competitive inhibitor of glucose transport. Table 2 shows that transport of radioactive glucose analog was stimulated by Ep administration to starved animals and n-glucose abolished the Ep-stimulated transport of methyl-a-o-glucoside. The inhibition of the transport of o-glucose by methyl-a-o-glucoside and vice versa indicates a common transport system for both o-glucose and its analog. As bone marrow cells (BMC) are more sensitive to Ep action, transport of glucose in these cells was studied next. A similar stimulatory effect of Ep was noted on the glucose and methyl-a-n-glucoside transport in BMC of starved rats. It can be seen from Fig. 5 that in BMC, like in erythrocytes there was an initial linear increase in glucose transport up to 10 mM of glucose, beyond which transport showed saturation kinetics. The transport of glucose inside bone marrow cells also showed linearity up to 20 min (Fig. 6) and up to 3 units of Ep (Fig. 4). Table 3 shows that Ep stimulated glucose transport in BMC of starved rats and a-MG abolished the stimulation. cz-MG transport by bone marrow cells was
0
2 Erythrowetin
4 (in
6 units)
FIG. 4. Effect of different amounts of erythropoietin on the transport of glucose by rat erythrocytes (0) and bone marrow cells (0). Experimental procedures are given under Methods.
138
GHOSAL
Effect of Erythropoietin
ET AL.
TABLE 1 on the Transport of [‘4C]-Glucose in Rat Erythrocyte pmole of glucose transported/lO’ cells
% Stimulation or inhibition
Normal Starved Starved + (3 units) Ep
0.357 f 0.0324 0.233 2 0.015 0.376 t 0.022
35 (-I* 61 (+I**
Starved + (6 units) Ep
0.469 f 0.052
Group
P < 0.05
101 (+)**
P < 0.01
Starved + (6 units) Ep + (w-MG
0.226 f 0.02
52 (-)***
Note. The results are the means k SD of three independent experiments. Experimental are given under Methods. a-MC was added to the incubation mixture (0.2 mg/ml). * Inhibition from normal animals. ** Stimulation/inhibition from starved animals. *** Inhibition from erythropoietin-treated animals.
procedures
also stimulated by Ep and the stimulation was abolished by glucose (Table 4), indicating that in BMC, like in erythrocytes, transport fo glucose and a-MG is mediated by a common transport system. Figure 7 shows the effect of metabolic inhibitors (2,4-DNP, pHMB, ouabain, and azide) on glucose transport in rat erythrocytes. Inhibition of Ep-stimulated glucose transport by DNP and azide suggests that the permeation process is energy dependent. It is well known that the active transport process involves coupling of pump ATPase or metabolically maintained ionic gradient and that the permeated molecule binds to a specific membrane carrier (15). The inhibitory effect of ouabain on glucose transport also suggests that the transport system is active in nature. Furthermore, inhibition by pHMB reveals the importance of sulfhydryl groups in the glucose carrier system. The exact mechanism by which Ep stimulates the glucose transport
Effect of Erythropoietin Group Normal Starved Starved + (6 units) Ep Starved + (6 units) Ep + o-glucose
TABLE 2 on the Transport of [“Cl-Methyl
a-o-Glucoside
pmole of cr-MG transported/IO’ cells 0.331 -c 0.023 0.181 f 0.018 0.449 5 0.012 P < 0.01 0.183 + 0.025
Inside Rat Erythrocytes % Stimulation or inhibition 45 (-)* 175 (+)* 60 (-)***
Note. The results are the means + SD of three independent experiments. Experimental procedures are given under Methods. o-Glucose was added to the incubation mixture (0.2 pmole/ml). * Inhibition from normal animals ** Stimulation/inhibition from starved animals. *** Inhibition from erythropoietin-treated animals.
EFFECT OF ERYTHROPOIETIN
ON GLUCOSE
139
TRANSPORT
FIG. 5. effect of different concentrations of glucose on the transport process by rat bone marrow cells. 0, normal; A, starved; 0, starved + erythropoietin (6 units). Experimental procedures are given under Methods.
0
IO Time
20 tn mtn
30
40
FIG. 6. Transport of glucose by rat bone marrow cells at different time periods. 0, normal; 0, starved; A, starved + erythropoietin (6 units). Experimental procedures are given under Methods.
Effect of Erythropoietin Group Normal Starved Starved + (6 units) Ep Starved + (6 units) Ep + (x-MG
TABLE 3 on the Transport of [‘4C]-Glucose in Rat Bone Marrow
Cells
fimoles of glucose transported/IO’ cells
% Stimulation or inhibition
0.499 + 0.02 0.351 5 0.013 0.967 t 0.029 P < 0.001 0.284 2 0.033
29 (-)* 175 (+)**
Note. The results are the Mean 2 SD of three independent experiments. Experimental are given under methods. 0.2 mg (Y-MG was added/ml of incubation mixture. * Inhibition from normal animals. ** Stimulations/Inhibitions from starved animals. *** Inhibition from erythropoietin treated animals.
71 (-j*** procedures
140
GHOSAL
Effect of Erythropoietin
TABLE 4 on the Transport of [‘4C]-Methyl a-o-Glucoside
Group Normal Starved Starved + (6 units) Ep Starved f (6 units) Ep +
ET AL.
in Rat Bone Marrow
Cell
pmoles of a-MG transported/ 10’ cells
% Stimulation or inhibition
0.451 t 0.033 0.259 +- 0.02 0.618 -c 0.022 P > 0.002 0.212 i- 0.02
43 (-)* 138 (+)** 66 (-)***
D-ghCOSC
Note. The results are the Mean t S.D. of three independent experiments. Experimental procedures are given under methods. 0.2 pmoles o-glucose was added/ml incubation mixture. * Inhibition from normal animals. ** Stimulations/inhibitions from starved animals. *** Inhibition from erythropoietin treated animals.
inside red blood cells is not yet known. However, changes in membrane lipid composition due to Ep administration (7) may be partly responsible. Ep has been found to increase membrane phosphatidylcholine content (7), which has been reported to increase membrane permeability (3). Stimulation of Na’ + K+ ATPase activity of rat erythrocyte membrane (16) may also partly explain increased glucose transport in rat erythrocytes. SUMMARY The effect of Ep on radioactive glucose and methyl-cr-o-glucoside transport by rat erythrocytes and bone marrow cells were studied. There is initial linearity followed by saturation kinetics of [‘4C]glucose transport by the erythrocytes of starved and starved plus Ep-treated rats at different concentrations of glucose. Starvation caused slight inhibition of glucose transport which increased markedly on Ep administration to starved rats. Normal animals failed to show any significant change in glucose transport after Ep treatment. Methyl-cu-p-glucoside inhibited the Ep-stimulated glucose transport significantly. 05r
FIG. 7. Effect of different inhibitors on glucose transport by rat erythrocytes. 0, control (no inhibitor); q , 2,4-DNP; a, pHMB; q , ouabain; W azide; S, starved; S + E, starved + erythropoietin (6 units). Experimental procedures are given under Methods. Results are the means f SD of three independent experiments. Each inhibitor was added to the incubation mixture separately (0.6 mmole/ml).
EFFECT OF ERYTHROPOIETIN
ON GLUCOSE
TRANSPORT
141
Ep also stimulated the transport of radioactive methyl-a-u-glucoside which was competitively inhibited in presence of o-glucose. Glucose transport in erythrocytes was found to be sensitive to metabolic inhibitors like azide and DNP. A sulfhydryl reagent and ouabain also inhibited the transport process. Ep stimulated glucose and methyl-cu-o-glucoside transport in the bone marrow cells of starved rats. The sugar analog competitively inhibited the glucose transport in bone marrow cells and vice versa. REFERENCES 1. De Gier, J., Mandersloot, J. G., and Van Deenen, L. L. M., Biochim. Biophys. Acra 150, 606 (1968). 2. Dueticke, B., and Gruber, W., J. Membrane Biol. 13, 19 (1973). 3. Gary. R., Kirk: Biochim. Biophys. Acra 464, 157 (1977). 4. De Gier, J., Mulder, I. and Van Deenen, L. L. M., Narurwissenschafren 48, 54 (1961). 5. Ghosal, J., Biswas, T., and Ghosh, D. K., IRCS Med. Sci. 8, 355 (1980). 6. Chaudhuri, T., Ghosal, J., Ghosh, D. K., and Datta. A. G., Biochem. Med. 24, 162 (1980). 7. Ghosal, J., Biswas, T., Ghosh, A., and Datta, A. G., Biochim. Med. 32, 1 (1984). 8. Biswas, T., Ghosal, J., Ganguly, C., and Datta, A. G.. Biochem. Med. Merab. Biol. 35, 120 (1986). 9. Bollenback, G. N., Methods Curbohydr. Chem. 2, 236 (1963). 10. Marks, P. A., Gelhorn, A., and Kidson, C., J. Biol. Chem. 235, 2579 (1960). II. Krantz. S. B., Gallien-Lartigm, O., and Goldwasser, E., J. Biol. Chem. 238, 4085 (1963). 12. Nath, J., and Datta, A. G., Jr., Gen. Microbial. 62, 17 (1970). 13. Kasahara, M., and Hinkle, P. C., Proc. Nor/. Acad. Sci. USA 73, 396 (1976). 14. Cohen, N. G., and Monod, J., Bacreriol. Rev. 21, 169 (1957). 15. Harrison, R., and Lunt, G. G., in “Biological Membranes: Their Structure and Function,” Chap. 2, p. 10. Thomson Litho, Great Britain, 1980. 16. Chakraborty, M.. Ghosal, J., Biswas, T.. and Datta, A. G., Biochem. Med. Merabl. Biol. 36, 231 (1986).