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Tritiated 2-deoxy-d -glucose as a probe for cell membrane permeability studies
ANALYTICAL
BIOCHEMISTRY
120,
Tritiated
8- 11 (
1982)
2-Deoxy-o-Glucose as a Probe for Cell Membrane Permeability Studies ERIKWALUMANDANDERSPETERSON
University
of Stockholm,
Unit
of Neurochemistry and Neurotoxicology. S- I72 46 Sundbyberg, Sweden
Enktipingsviigen
126,
Received May 6, 198 1 Tritiated 2-deoxy-D-glucose was taken up and phosphorylated by cultured cells of neuronal (NIE 115) glial (138 MG), muscle (L 6) and liver (BRL 123) origin. Upon perfusion the cells slowly released 2-deoxy-D-glucose 6-phosphate. The following values for rate constants, half-lives, and activation energies for the efflux were obtained: NIE 115: 0.0048 min-‘, 143 min, and 72 kJ mol-‘; 138 MG: 0.0013 min?, 547 min, and 85 kJ mol-‘; L 6 : 0.0022 min-‘, 311 mitt, and 60 kJ mol-‘; and BRL 123: 0.0013 min-‘, 528 min and 63 kJ mol-‘. When the cultures were perfused with buffer containing Triton X-100 a time- and concentration-dependent increase in the rate of efflux of 2-deoxy-D-glucose 6-phosphate was obtained. It is suggested that 2-deoxy-D-[%I]glucose can be used as a probe in studies of general cell membrane permeability changes.
The glucose analog, 2-deoxy-D-glucose (dGlc)’ is taken up into cells by the same transport system as D-glucose (1,2), and phosphorylated to 2-deoxyD-glucose 6-phosphate (dGlc-6-P) by hexokinase (1,3,4). Hexokinase is not feedback inhibited by dGlc-6-P (3). Accordingly there is no control at this level of dGlc influx into the hexose 6-phosphate pool of cells. Since dGlc-6-P is not further metabolized ( 1,4) and the cell membrane permeability is low for dGlc-6-P, it accumulates inside the cell. These properties of dGlc have been made use of, e.g., in studies of hexose transport across the cell membrane (5,6) and determinations of metabolic activities in the brain (7,8). In the present study we have investigated the possibility of using dGlc as a probe for general cell membrane permeability changes in monolayer cell cultures by following alterations in the efflux of the phosphorylated dGlc from the cells.
MATERIALS
Cultures. The mouse neuroblastoma cell line NIE 115 (9), the human glioma cell line 138 MG (IO), the rat muscle cell line L 6 (1 l), and the rat liver cell line BRL 123 (an epithelial cell line established and cloned in our laboratory) were grown under identical conditions as described before (12). Four days before experiments cells were trypsinized, deluted, and plated in 22-cm2 tissue culture dishes at a density of about lo6 cells/ dish. Incubation and perfusion procedure. Cul-
tures were incubated at 37°C for 2 h in 4 ml/dish of a phosphate-buffered saline (PBS) (13) containing 0.5 ,Ki [3H]dGlc/ml. The incubation was terminated by removal of the incubation solution and subsequent washing of the cultures with three 5-ml portions of ice-cold PBS. The cultures were then perfused at various temperatures for 1 h with PBS containing D-glucose (1 mg/ml), at a rate of 1 ml/min and fractions of the perfusate collected in scintillation vials every 5 min. The method used for continuous per-
’ Abbreviations used: dGlc, 2-deoxyD-glucose; dGlc6-P, 2-deoxy-D-glucose 6-phosphate; PBS, phosphatebuffered saline. 0003-2697/82/030008-04$02.00/O Copyright 0 1982 by Academic Press. Inc. All rights of reproduction m any form reserved.
AND METHODS
8
ALTERED
MEMBRANE
PERMEABILITY
fusion of monolayer cultures will be described in detail elsewhere (Peterson and Walum, in preparation). At the end of the experiment cells were dissolved in 1 ml of 1 M NaOH, transferred to scintillation vials together with a subsequent wash of the dishes with 1 ml of 1 M HCl. Radioactivities were determined in Unisolve I scintillation cocktail in a Packard Tri-Carb 2450 liquid scintillation counter. The counting efficiency was 20-2570. Calculations. The radioactivity remaining in the cells (value calculated by subtracting the cumulative amount of radioactivity released by the cells from the total amount of radioactivity incorporated) divided by the amount of radioactivity in the cells at the beginning of the experiment was plotted logarithmically vs time according to Kotyk and Janacek (14). The exponential curve obtained was resolved in its components, and the least-square fit of the lines calculated. Pool sizes, half-lives, and rate constants were calculated from the equations of the lines. Rate constants were then plotted vs the reciprocal of the absolute temperature and activation energies determined from the straight lines so obtained (15). Paper chromatography. The intracellular products of [3H]dGlc transport and the radioactive material in the perfusate were separated on Whatman No. 1 paper in n-butanol:acetic acid:water (2: 1: 1) according to Schultz and Culp (16). Chemicals. An aqueous solution of 2deoxyD-[ l-3H]glucose (23 Ci/mmol, 1 mCi/ ml) was purchased from the Radiochemical Centre, Amersham, England. Unisolve I is a registered trade mark of Koch-Light Laboratories Ltd, Colnbrook Buckinghamshire, England. The nonionic detergent Triton X100 was bought from Kebo-Grave, Stockholm, Sweden. RESULTS
AND
DISCUSSION
As can be seen from Table 1 [3H]dGlc uptake varied considerably between the dif-
STUDIED
WITH
TABLE UPTAKE
AND
1
PHOSPHORYLATION
GLUCOSE
IN CULTURED
Uptake”
Amount of [‘H]dClc-6-p
Cell line
(dpm/lO’ cells/2 h)
(70 of total radioactivity)
NIE II5 I38 MG L6
I164 482 64
74 92 94
I9
87
BRL 123
9
DEOXYGLUCOSE
OF 2-DEoxu-D-[‘H] CELL
LINES
Size of [‘H]dGlc-6-P pool’ (% of total radioactivity) 92 93 93 86
Nore. Cells were suspended in Ham’s FL0 medium containing 9% calf vxum and 4% fetal calf serum and plated in plastic petri dishes at a concentration of approximately lo6 cells/dish. After 4 days the cultures were incubated at 37°C for 2 h in PBS containing 0.5 &i/ml of 2-deoxyo-[‘H]glucose. Incuhatmn was interrupted by three rapid washes with 5 ml of ice-cold PBS. o Cells were dissolved in I M NaOH and the amount of radioxtivity taken up determined. Mean values of two independent experiments with duplicate dishes. b Cultures were extracted 2 X 30 min at 0°C with I ml of I M acetic acid. Extracts were neutralized with pyridine. freeze-dried, resolved in 0.25 ml water, and applied to Whatman No. I paper. After ascending chromatography in n-butanolacetic acidxatcr (2:l:l). 5 X SO-mm strips were eluted separately in 0.5 ml water and counted in Unisolve 1. Values obtained after integration of peak areas in a single chromatogram for each cell line. ’ Values obtained from the graphs in Fig. I,
ferent cell lines. In the cells 74-94% of the hexose taken up was converted to hexose 6phosphate as judged by both paper chromatography and efflux curves (Table 1). Similar figures for the phosphorylation of dGlc have been reported for cultured mouse 3T3 cells (16,17). When cell cultures, preloaded with [3H]dGlcfor2 h,wereperfusedwithD-glucose containing PBS a very rapid efflux of radioactivity from the minor (free dGlc) pool was accompanied by a slow efflux from the major (phosphorylated dGlc) pool (Fig. 1). By paper chromatography of the released material (fraction 5, i.e., 20-25 min perfusion) it was found that virtually all radioactivity released by all cell lines appeared as [3H]dGlc-6-P. No significant amounts of radioactivity were found to comigrate with standard [ 3H]dGlc. Characteristics of the dGlc-6-P release from NIE 115, 138 MG, L 6, and BRL 123 cells are given in Table 2. The very low k values, low fl/2r and high AE are compatible with the theories presented by Sokoloff
10
WALUM
AND PETERSON
- 0.8 0
- 1.00
0
20
LO
60
0
20
LO
60 0 Tlme,
20
LO
60
mtn
FIG. 1. Effects of Triton X-100 on the efflux of 2-deoxyb[‘HIglucose 6-phosphate from cultures of NIE 115 (A), 138 MC (B), L 6 (C), and BRL 123 (D) cells. Experimental conditions are given under Materials and Methods. The cultures were perfused at 20°C with PBS containing 1 mg D-glucose/ml and 0 (0), 0.015 (o), 0.022 (o), 0.030 (m), and 0.050% (A) Triton X-100. Each point represents the mean of two independent duplicate experiments.
et al. for the accumulation
of dGlc-6-P in the brain (18). Perfusion of the cell cultures in the presence of Triton X- 100, classified as producing large functional holes in plasma membranes (19), resulted in an increase in the rate of dGlc-6-P efflux. Figure 1 shows that these
TABLE CHARACTERISTICS PHOSPHATE
Cell line NIE II5 138 MG L6 BRL 123
2
OF 2-DEOXY-D-[‘H] EFFLUX
FROM
k (min?)
0.0048 0.0013 0.0022 0.0013
(l.000) (0.998) (0.997) (0.995)
GLUCOSE
CULTURED
CELL
6-
LINES
llil
bin)
AE (ld moP)
143 547 31 I 528
(I.000) (0.998) (0.997) (0.995)
72 85 60 63
(0.986) (0.995) (0.973) (0.996)
Note. Cultures grown and incubated as described in Table I wetc perfused with Dglttwsc (1 mg/ml) containing PBS at 20°C for 60 min at a rate of I ml/min. For determinations of activation energies efflux cxperiments were repeated at the additional temperatures of 25, 30, 35. and 40°C. Rate wnstants (k). half-lives (I,,~), and activation energies(U) for the efllux of 2dwxy-D-[‘HI glucose 6-phosphate were calculated BS described under Materials and Methods. Results show the mean of nine (k and II/~) 01 two (A.?) independent duplicate experiments. Correlation coefficients of the straight lines on which the determinations were based UC given within parentheses.
changes in the flux rates were time and concentration dependent. Various types of cultured cells are known to possess differences in general cell membrane properties. This was confirmed in the present work, where marked differences between the four cell lines with respect to the characteristics of [ 3H]dGlc-6-P efflux were observed (Table 2). In spite of this all tested cell lines showed a similar sensitivity to Triton X-100 (Fig. 1), which is consistent with the concept of Triton X-100 having drastic unspecific effects on plasma membranes (20). A number of methods have been developed for studies of general cell membrane permeability alterations (for review see Ref. (20)). We here describe a procedure which utilizes the efflux of tritiated dGlc-6-P from cells. This method implies some advantages: A biochemically well-characterized probe, tritiated dGlc, is used. This substance is rapidly taken up into cells (Table 1) via the Dglucose transport system, phosphorylated to a metabolically inert compound, dGlc-6-P (Table 1) which slowly penetrates out through the cell membrane (Table 2). The high levels of accumulated intracellular ra-
ALTERED
MEMBRANE
PERMEABILITY
dioactivity and the comparatively small size of the compound makes the method a sensitive one. When combined with a perfusion technique it becomes possible to study general membrane permeability alterations in detail, as done in this work for changes induced by alteration in temperature and the presence of low concentrations of Triton X100, The method has come into use in our laboratory within studies of cell membrane effects of organic solvents and heavy metals as well as for investigations of membrane changes accompanying morphological and biochemical differentiation of cultured cells. ACKNOWLEDGMENTS This work was supported by the Swedish Work Environment Foundation (Grant 78/260) and by the National Swedish Board for Technical Development (Grant 79-3852). The authors are grateful to Miss Mathilda Marcks von Wtlrtemberg and Miss Elisabeth Nyberg for excellent technical assistance.
STUDIED
11
WITH DEOXYGLUCOSE
5. Hatanaka, M., and Hanafusa, H. (1970) Virology 41,647~652.
Walum, E., and Edstrom, A. (1976) Exp. Cell Res. 100, 111-116. 7. Kennedy, C., DesRosiers, M. H., Jehle, J. W., Reivich, M., Sharpe, F., and Sokoloff, L. (1975) Sci6.
ence 187, 850-853.
Sakurada, O., Sokoloff, L., and Jacquet, Y. (1978) .Brain Rex 110, 127-139. 9. Amano, T., Richelson, E., and Nirenberg, M. 8.
(1972)
Proc. Nat.
Acad.
Sci. USA 69, 258-263.
10. Ponttn, J., and Maclntyre, E. H. (1968) Acta Pathol.
Microbial.
Stand.
74, 465-486. Acad. Sci. USA
11. Yaffe, D. (1968) Proc. Nat
61,
477-483.
12. Walum, E. (1975) Glucose Uptake into Cultured Tumour Cells from the Nervous System, Thesis, University of Gothenburg, Sweden. 13. Dulbecco, R., and Vogt, M. (1954) J. Exp. Med. 99, 167-182.
14. Kotyk, A., and Janacek, K. (1970) Cell Membrane Transport: Principles and Techniques, pp. 240244, Plenum, NY. 15. Arrhenius, S. (1915) Quantitative Laws in Biological Chemistry, Bell, London. 16. Schultz, A. R., and Gulp, L. A. (1973) Exp. Cell Res. 81, 95-103.
17. Hassell, J. A., Colby, C., and Romano, A. H. (1975)
REFERENCES 1. Smith, D. E., and Gorski, J. (1968) J. Biol. Chem. 243,4169-4174. 2. 3.
LeFevre, P. G. (1961) Phormacol. Rev. 13, 39-70. Sals, A., and Crane, R. K. (1954) J. Biol. Chem.
4.
Kipnis, D. M., and Cori, C. F. (1959) J. Biol. Chem.
210, 581-595. 234, 171-177.
J. Cell.
Physiol.
86, 37-46.
18. Sokoloff, L., Reivich, M., Kennedy, C., DesRosiers, M. H., Patlak, C. S., Pettigrew, K. D., Sakurada, O., and Shinohara, M. (1977) J. Neurochem. 28, 897-916.
19. Thelestam, M., and Mbllby, R. (1975) Infect. Zmmunity
11, 640-648.
20. Thelestam, M., and Miillby, R. (1976) Med. Biol. 54, 39-49.
BIOCHEMISTRY
120,
Tritiated
8- 11 (
1982)
2-Deoxy-o-Glucose as a Probe for Cell Membrane Permeability Studies ERIKWALUMANDANDERSPETERSON
University
of Stockholm,
Unit
of Neurochemistry and Neurotoxicology. S- I72 46 Sundbyberg, Sweden
Enktipingsviigen
126,
Received May 6, 198 1 Tritiated 2-deoxy-D-glucose was taken up and phosphorylated by cultured cells of neuronal (NIE 115) glial (138 MG), muscle (L 6) and liver (BRL 123) origin. Upon perfusion the cells slowly released 2-deoxy-D-glucose 6-phosphate. The following values for rate constants, half-lives, and activation energies for the efflux were obtained: NIE 115: 0.0048 min-‘, 143 min, and 72 kJ mol-‘; 138 MG: 0.0013 min?, 547 min, and 85 kJ mol-‘; L 6 : 0.0022 min-‘, 311 mitt, and 60 kJ mol-‘; and BRL 123: 0.0013 min-‘, 528 min and 63 kJ mol-‘. When the cultures were perfused with buffer containing Triton X-100 a time- and concentration-dependent increase in the rate of efflux of 2-deoxy-D-glucose 6-phosphate was obtained. It is suggested that 2-deoxy-D-[%I]glucose can be used as a probe in studies of general cell membrane permeability changes.
The glucose analog, 2-deoxy-D-glucose (dGlc)’ is taken up into cells by the same transport system as D-glucose (1,2), and phosphorylated to 2-deoxyD-glucose 6-phosphate (dGlc-6-P) by hexokinase (1,3,4). Hexokinase is not feedback inhibited by dGlc-6-P (3). Accordingly there is no control at this level of dGlc influx into the hexose 6-phosphate pool of cells. Since dGlc-6-P is not further metabolized ( 1,4) and the cell membrane permeability is low for dGlc-6-P, it accumulates inside the cell. These properties of dGlc have been made use of, e.g., in studies of hexose transport across the cell membrane (5,6) and determinations of metabolic activities in the brain (7,8). In the present study we have investigated the possibility of using dGlc as a probe for general cell membrane permeability changes in monolayer cell cultures by following alterations in the efflux of the phosphorylated dGlc from the cells.
MATERIALS
Cultures. The mouse neuroblastoma cell line NIE 115 (9), the human glioma cell line 138 MG (IO), the rat muscle cell line L 6 (1 l), and the rat liver cell line BRL 123 (an epithelial cell line established and cloned in our laboratory) were grown under identical conditions as described before (12). Four days before experiments cells were trypsinized, deluted, and plated in 22-cm2 tissue culture dishes at a density of about lo6 cells/ dish. Incubation and perfusion procedure. Cul-
tures were incubated at 37°C for 2 h in 4 ml/dish of a phosphate-buffered saline (PBS) (13) containing 0.5 ,Ki [3H]dGlc/ml. The incubation was terminated by removal of the incubation solution and subsequent washing of the cultures with three 5-ml portions of ice-cold PBS. The cultures were then perfused at various temperatures for 1 h with PBS containing D-glucose (1 mg/ml), at a rate of 1 ml/min and fractions of the perfusate collected in scintillation vials every 5 min. The method used for continuous per-
’ Abbreviations used: dGlc, 2-deoxyD-glucose; dGlc6-P, 2-deoxy-D-glucose 6-phosphate; PBS, phosphatebuffered saline. 0003-2697/82/030008-04$02.00/O Copyright 0 1982 by Academic Press. Inc. All rights of reproduction m any form reserved.
AND METHODS
8
ALTERED
MEMBRANE
PERMEABILITY
fusion of monolayer cultures will be described in detail elsewhere (Peterson and Walum, in preparation). At the end of the experiment cells were dissolved in 1 ml of 1 M NaOH, transferred to scintillation vials together with a subsequent wash of the dishes with 1 ml of 1 M HCl. Radioactivities were determined in Unisolve I scintillation cocktail in a Packard Tri-Carb 2450 liquid scintillation counter. The counting efficiency was 20-2570. Calculations. The radioactivity remaining in the cells (value calculated by subtracting the cumulative amount of radioactivity released by the cells from the total amount of radioactivity incorporated) divided by the amount of radioactivity in the cells at the beginning of the experiment was plotted logarithmically vs time according to Kotyk and Janacek (14). The exponential curve obtained was resolved in its components, and the least-square fit of the lines calculated. Pool sizes, half-lives, and rate constants were calculated from the equations of the lines. Rate constants were then plotted vs the reciprocal of the absolute temperature and activation energies determined from the straight lines so obtained (15). Paper chromatography. The intracellular products of [3H]dGlc transport and the radioactive material in the perfusate were separated on Whatman No. 1 paper in n-butanol:acetic acid:water (2: 1: 1) according to Schultz and Culp (16). Chemicals. An aqueous solution of 2deoxyD-[ l-3H]glucose (23 Ci/mmol, 1 mCi/ ml) was purchased from the Radiochemical Centre, Amersham, England. Unisolve I is a registered trade mark of Koch-Light Laboratories Ltd, Colnbrook Buckinghamshire, England. The nonionic detergent Triton X100 was bought from Kebo-Grave, Stockholm, Sweden. RESULTS
AND
DISCUSSION
As can be seen from Table 1 [3H]dGlc uptake varied considerably between the dif-
STUDIED
WITH
TABLE UPTAKE
AND
1
PHOSPHORYLATION
GLUCOSE
IN CULTURED
Uptake”
Amount of [‘H]dClc-6-p
Cell line
(dpm/lO’ cells/2 h)
(70 of total radioactivity)
NIE II5 I38 MG L6
I164 482 64
74 92 94
I9
87
BRL 123
9
DEOXYGLUCOSE
OF 2-DEoxu-D-[‘H] CELL
LINES
Size of [‘H]dGlc-6-P pool’ (% of total radioactivity) 92 93 93 86
Nore. Cells were suspended in Ham’s FL0 medium containing 9% calf vxum and 4% fetal calf serum and plated in plastic petri dishes at a concentration of approximately lo6 cells/dish. After 4 days the cultures were incubated at 37°C for 2 h in PBS containing 0.5 &i/ml of 2-deoxyo-[‘H]glucose. Incuhatmn was interrupted by three rapid washes with 5 ml of ice-cold PBS. o Cells were dissolved in I M NaOH and the amount of radioxtivity taken up determined. Mean values of two independent experiments with duplicate dishes. b Cultures were extracted 2 X 30 min at 0°C with I ml of I M acetic acid. Extracts were neutralized with pyridine. freeze-dried, resolved in 0.25 ml water, and applied to Whatman No. I paper. After ascending chromatography in n-butanolacetic acidxatcr (2:l:l). 5 X SO-mm strips were eluted separately in 0.5 ml water and counted in Unisolve 1. Values obtained after integration of peak areas in a single chromatogram for each cell line. ’ Values obtained from the graphs in Fig. I,
ferent cell lines. In the cells 74-94% of the hexose taken up was converted to hexose 6phosphate as judged by both paper chromatography and efflux curves (Table 1). Similar figures for the phosphorylation of dGlc have been reported for cultured mouse 3T3 cells (16,17). When cell cultures, preloaded with [3H]dGlcfor2 h,wereperfusedwithD-glucose containing PBS a very rapid efflux of radioactivity from the minor (free dGlc) pool was accompanied by a slow efflux from the major (phosphorylated dGlc) pool (Fig. 1). By paper chromatography of the released material (fraction 5, i.e., 20-25 min perfusion) it was found that virtually all radioactivity released by all cell lines appeared as [3H]dGlc-6-P. No significant amounts of radioactivity were found to comigrate with standard [ 3H]dGlc. Characteristics of the dGlc-6-P release from NIE 115, 138 MG, L 6, and BRL 123 cells are given in Table 2. The very low k values, low fl/2r and high AE are compatible with the theories presented by Sokoloff
10
WALUM
AND PETERSON
- 0.8 0
- 1.00
0
20
LO
60
0
20
LO
60 0 Tlme,
20
LO
60
mtn
FIG. 1. Effects of Triton X-100 on the efflux of 2-deoxyb[‘HIglucose 6-phosphate from cultures of NIE 115 (A), 138 MC (B), L 6 (C), and BRL 123 (D) cells. Experimental conditions are given under Materials and Methods. The cultures were perfused at 20°C with PBS containing 1 mg D-glucose/ml and 0 (0), 0.015 (o), 0.022 (o), 0.030 (m), and 0.050% (A) Triton X-100. Each point represents the mean of two independent duplicate experiments.
et al. for the accumulation
of dGlc-6-P in the brain (18). Perfusion of the cell cultures in the presence of Triton X- 100, classified as producing large functional holes in plasma membranes (19), resulted in an increase in the rate of dGlc-6-P efflux. Figure 1 shows that these
TABLE CHARACTERISTICS PHOSPHATE
Cell line NIE II5 138 MG L6 BRL 123
2
OF 2-DEOXY-D-[‘H] EFFLUX
FROM
k (min?)
0.0048 0.0013 0.0022 0.0013
(l.000) (0.998) (0.997) (0.995)
GLUCOSE
CULTURED
CELL
6-
LINES
llil
bin)
AE (ld moP)
143 547 31 I 528
(I.000) (0.998) (0.997) (0.995)
72 85 60 63
(0.986) (0.995) (0.973) (0.996)
Note. Cultures grown and incubated as described in Table I wetc perfused with Dglttwsc (1 mg/ml) containing PBS at 20°C for 60 min at a rate of I ml/min. For determinations of activation energies efflux cxperiments were repeated at the additional temperatures of 25, 30, 35. and 40°C. Rate wnstants (k). half-lives (I,,~), and activation energies(U) for the efllux of 2dwxy-D-[‘HI glucose 6-phosphate were calculated BS described under Materials and Methods. Results show the mean of nine (k and II/~) 01 two (A.?) independent duplicate experiments. Correlation coefficients of the straight lines on which the determinations were based UC given within parentheses.
changes in the flux rates were time and concentration dependent. Various types of cultured cells are known to possess differences in general cell membrane properties. This was confirmed in the present work, where marked differences between the four cell lines with respect to the characteristics of [ 3H]dGlc-6-P efflux were observed (Table 2). In spite of this all tested cell lines showed a similar sensitivity to Triton X-100 (Fig. 1), which is consistent with the concept of Triton X-100 having drastic unspecific effects on plasma membranes (20). A number of methods have been developed for studies of general cell membrane permeability alterations (for review see Ref. (20)). We here describe a procedure which utilizes the efflux of tritiated dGlc-6-P from cells. This method implies some advantages: A biochemically well-characterized probe, tritiated dGlc, is used. This substance is rapidly taken up into cells (Table 1) via the Dglucose transport system, phosphorylated to a metabolically inert compound, dGlc-6-P (Table 1) which slowly penetrates out through the cell membrane (Table 2). The high levels of accumulated intracellular ra-
ALTERED
MEMBRANE
PERMEABILITY
dioactivity and the comparatively small size of the compound makes the method a sensitive one. When combined with a perfusion technique it becomes possible to study general membrane permeability alterations in detail, as done in this work for changes induced by alteration in temperature and the presence of low concentrations of Triton X100, The method has come into use in our laboratory within studies of cell membrane effects of organic solvents and heavy metals as well as for investigations of membrane changes accompanying morphological and biochemical differentiation of cultured cells. ACKNOWLEDGMENTS This work was supported by the Swedish Work Environment Foundation (Grant 78/260) and by the National Swedish Board for Technical Development (Grant 79-3852). The authors are grateful to Miss Mathilda Marcks von Wtlrtemberg and Miss Elisabeth Nyberg for excellent technical assistance.
STUDIED
11
WITH DEOXYGLUCOSE
5. Hatanaka, M., and Hanafusa, H. (1970) Virology 41,647~652.
Walum, E., and Edstrom, A. (1976) Exp. Cell Res. 100, 111-116. 7. Kennedy, C., DesRosiers, M. H., Jehle, J. W., Reivich, M., Sharpe, F., and Sokoloff, L. (1975) Sci6.
ence 187, 850-853.
Sakurada, O., Sokoloff, L., and Jacquet, Y. (1978) .Brain Rex 110, 127-139. 9. Amano, T., Richelson, E., and Nirenberg, M. 8.
(1972)
Proc. Nat.
Acad.
Sci. USA 69, 258-263.
10. Ponttn, J., and Maclntyre, E. H. (1968) Acta Pathol.
Microbial.
Stand.
74, 465-486. Acad. Sci. USA
11. Yaffe, D. (1968) Proc. Nat
61,
477-483.
12. Walum, E. (1975) Glucose Uptake into Cultured Tumour Cells from the Nervous System, Thesis, University of Gothenburg, Sweden. 13. Dulbecco, R., and Vogt, M. (1954) J. Exp. Med. 99, 167-182.
14. Kotyk, A., and Janacek, K. (1970) Cell Membrane Transport: Principles and Techniques, pp. 240244, Plenum, NY. 15. Arrhenius, S. (1915) Quantitative Laws in Biological Chemistry, Bell, London. 16. Schultz, A. R., and Gulp, L. A. (1973) Exp. Cell Res. 81, 95-103.
17. Hassell, J. A., Colby, C., and Romano, A. H. (1975)
REFERENCES 1. Smith, D. E., and Gorski, J. (1968) J. Biol. Chem. 243,4169-4174. 2. 3.
LeFevre, P. G. (1961) Phormacol. Rev. 13, 39-70. Sals, A., and Crane, R. K. (1954) J. Biol. Chem.
4.
Kipnis, D. M., and Cori, C. F. (1959) J. Biol. Chem.
210, 581-595. 234, 171-177.
J. Cell.
Physiol.
86, 37-46.
18. Sokoloff, L., Reivich, M., Kennedy, C., DesRosiers, M. H., Patlak, C. S., Pettigrew, K. D., Sakurada, O., and Shinohara, M. (1977) J. Neurochem. 28, 897-916.
19. Thelestam, M., and Mbllby, R. (1975) Infect. Zmmunity
11, 640-648.
20. Thelestam, M., and Miillby, R. (1976) Med. Biol. 54, 39-49.