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Myoinositol uptake by four cultured mammalian cell lines
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 246, No. 2, May 1, pp. 801-807, 1986
Myoinositol MARK
Uptake by Four Cultured Mammalian Cell Lines’
A. YOREK,2 JOYCE A. DUNLAP,
AND
BARRY
H. GINSBERG
Veterans Administration Medical Center, Iowa City, Iowa 52240, and the Department of Inter-d Medicine and the Diabetes Erulocrirwm Research Center, University of Iowa, Iozva City, Iowa 522&Z Received August 26,1985
The uptake of myo-[2-3H]inositol by mouse neuroblastoma, human Y’79 retinoblastoma, human HL60, and bovine pulmonary artery endothelial cells occurs by a saturable, Na+dependent and partially energy-dependent mechanism. Inositol uptake by all four cell lines occurred by both a high- and low-affinity system. The kinetic parameters for the high-affinity uptake systems were similar for all four cell lines. These data suggest that all four of these diverse cell lines have similar inositol transport systems and probably rely on extracellular inositol for anabolic processes. 0 1986 Academic Press, Inc.
Myoinositol is an important component for the synthesis of the inositol phospholipids in both neural and nonneural mammalian cells (1). Myoinositol exists in mammalian circulation at a concentration ranging from 25 to 100 pmol/liter (2-9) and is taken up and utilized by cells for phospholipid synthesis. Although myoinositol can be synthesized by the intestinal flora (10) and also in certain tissues (11,12) the most recent evidence suggests that it is an essential component of the diet (1). Myoinositol combines with CDP-diacylglycerol to form phosphatidylinositol which can be phosphorylated to form phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-diphosphate (1). The inositol phospholipids have been suggested to have an important role in many cellular functions including the motor nerve conduction velocity in peripheral nerves (13, 14), sodium-potassium adenosine triphosphatase activity (8), a fatty acid source for prostaglandin synthesis (15, 16), and neurotransmitter (1’7) and insulin release (18). Many of these cellular events occur follow1 This paper is dedicated to the memory of Dr. Edward C. Heath. ‘To whom correspondence and reprint requests should be addressed. 801
ing the activation of the “phosphoinositide cycle” which results in the release of possibly two second messengers, inositol1,4,5triphosphate and diacylglycerol (19, 20). These two compounds have been shown to stimulate Ca2+ mobilization and protein kinase C activity, respectively (19-23). It is assumed that intracellular inositol is acquired by a specific transport system. In addition, an abnormal uptake process may be responsible for some cellular disorders. For example, diabetic neuropathy has been partly attributed to a decrease in the intracellular myoinositol levels in peripheral nerve which results in a decrease in the inositol phospholipid content (24,25). The diabetic circulatory conditions have been suggested to be responsible for this abnormality apparently causing a reduction in inositol uptake (25, 26). In some cases increasing the myoinositol levels in the diet which results in an increase in inositol uptake has been shown to restore normal nerve activity (22, 27). Segal et aZ. (26) in the only published report examining inositol transport in a cultured cell line has shown that rat schwann cells take up inositol by a saturable, sodium-dependent mechanism with a K:, of about 30 PM. Since inositol seems to play an important role in neural membrane function and we have 0003-9861/86 $3.00 Copyright All rights
0 1986 by Academic Press. Inc. of reproduction in any form reserved.
802
YOREK,
DUNLAP,
shown abnormalities in inositol transport in cultured neural cells during periods of prolonged hyperglycemia (28), we felt it was important to characterize the inositol transport system in two cultured neural cell lines and compare it to cultured nonneural cells. In this study we describe the inositol transport properties of human Y79 retinoblastoma and HL 60 cells, mouse neuroblastoma, and bovine pulmonary artery endothelial cells. MATERIALS
AND
METHODS
Tissue culture Human Y79 retinoblastoma cells (29) which grow in suspension as small clusters of 6-10 cells and human HL 60 cells which also grow in suspension culture were propagated in RPM1 1640 medium containing 10% heat-inactivated fetal bovine serum (Dutchland, Denver, Pa.), 100 units/ml penicillin, 100 pg/ml streptomycin, 294 pg/ml glutamine, and 10 mM Hepes.* Bovine pulmonary artery endothelial cells were cultured in Ml99 medium containing 20% heat-inactivated fetal bovine serum, BME vitamins and amino acids, 294 rg/ml glutamine, and 2.2 mg/ml NaHCOa. Mouse neuroblastoma cells were cultured in Hams FlO medium containing 15% horse serum, 2.5% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 106 pg/ml streptomycin, and 294 pg/ml glutamine. All the cells were grown in 150-cm2 tissue culture flasks in an incubator maintained at 37”C, with 5% CO2 in humidified air as the gas phase. Prior to experimental use, the endothelial and neuroblastoma cells were transferred to Costar six-well plates and grown to confluency. The Y79 and HL 60 cells were isolated by centrifugation, washed and resuspended in 10 mM Hepes-buffered salt solution containing 128 mM NaCl, 5.2 mM KCl, 2.1 mM CaC&, 2.9 mM MgSO,, and 5 mM glucose, pH 7.4. This buffer was used in the uptake studies for all four of the cells lines. Inositol uptake. Following a wash, 2.5 ml buffer at 37°C containing 0.1 to 500 PM myoinositol and myo[2-aH]inositol as tracer was added to each well of a six-well plate containing either endothelial or neuroblastoma cells. Corresponding incubations at 0°C were carried out to determine nonspecific uptake. After the incubation the cells were washed three times with ice-cold buffer and the cells collected in a total volume of 1.5 ml. The cells suspension was sonicated to obtain a uniform mixture and samples were taken to determine cell protein content and total uptake. Triplicate 0.25-ml aliquots of the cell suspension were
3 Abbreviations used: Hepes, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid; NCS, N-chlorosuccinimide.
AND
GINSBERG
taken, 5 ml of Budget-Solve scintillation solution (Research Products International, Elk Grove Village, Ill.) was added and the radioactivity measured with a liquid scintillation counter. The Y79 and HL 60 cells were resuspended in 1.0 ml buffer containing the myo-[23H]inositol solution. Following the uptake period, three 200-~1 aliquots of the suspension were transferred to ice-cold l&ml Sarstedt microfuge tubes containing 0.8 ml buffer layered over 0.3 ml of a nbutyl phthalate/corn oil mixture (11/4 v/v) (30). The tubes were immediately centrifuged in an Eppendorf microfuge (Brinkman Instruments, Westbury, N.Y.) for 30 s to pellet the cells. After the aqueous and oil layers were removed by aspiration, the bottom of the tube containing the sedimented cells was cut off and added to a microscintillation vial containing 0.3 ml of NCS tissue solubilizer (Amersham/Searle, Arlington Heights, Ill.). The cell pellet was then dissolved by incubating the contents of the scintillation vial at 45°C for 1 h. Afterward 5 ml of Neutralizer scintillation solution (Research Products International) was added and the radioactivity determined. Quenching was monitored with a =Ra external standard. Doto an&sti. Values were calculated per milligram of cell protein. The protein content of the cells was determined by a modification of the Lowry method (31). Kinetic analysis of the myoinositol uptake data was done with an IBM personal computer using a weighted least-squares fit of the experimental points to a double reciprocal plot (32). RESULTS
Inositol Uptake Inositol uptake by the cells was measured from 0 to 10 min using 5 pmol/liter myo-[2-3H]inositol. The incubations were conducted at 3’7 and 0°C to determine the specific uptake by correcting for uptake at 0°C. The neuroblastoma and HL 60 cells took up similar amounts of myo-[23H]inositol, whereas, the Y79 retinoblastoma cells took up about 50% less (Fig. 1). The pulmonary artery endothelial cells took up the least amount of myo-[23H]inositol under these conditions and was about 10% of the total uptake observed in the neuroblastoma and HL 60 cells. Myo[2-3H]inositol uptake at 0°C accounted for less than 10% of the total uptake in the neuroblastoma, HL 60, and pulmonary artery endothelial cells, whereas, nonspecific uptake accounted for 30% of the total uptake by Y79 cells after 10 min of incubation. The retinoblastoma, HL 60, and endothelial
INOSITOL
803
TRANSPORT
60 40
a a
0
2
4
6
6
0
2
4
6
6
0
2
4
6
6
10
0
2
4
6
6
Time(
min)
10
10
10
FIG. 1. Inositol uptake by four individual cell lines. Myo-[2-3H]inositol uptake (5 pmol/liter) was analyzed from 0 to 10 min in four cell lines; (a) human Y79 retinoblastoma, (b) mouse neuroblastoma, (c) human HL 60, and (d) bovine pulmonary artery endothelial. Each data point is the mean -C SEM for three separate determinations. Specific uptake was determined by substracting the uptake which occurred at 0°C from the total uptake at 37°C.
cells demonstrated a linear uptake of myo[2-3H]inositol for a minimum of 10 min. Whereas, the myo-[2-3H]inositol uptake by the neuroblastoma cells was linear for at least 5 min. Therefore, subsequent transport studies to determine the kinetic parameters of inositol uptake were conducted for 5 min. Total myo-[2-3H]inositol uptake by the neuroblastoma cells approached saturation after 20 min of incubation, whereas the other three cell lines approached saturation after 30 min of incubation (data not shown). To determine if inositol uptake is dependent on extracellular Naf, incubations were conducted in normal buffer and buffer in which the NaCl was isosmotically replaced with sucrose, CsCl or LiCl. Incubations were also conducted in the presence of 1 mM ouabain. The data in Table I show that myo-[2-3H]inositol uptake by all four of the cell lines was dependent upon the extracellular presence of Na+. The exchange of NaCl for sucrose, CsCl or LiCl resulted in
a significant reduction in myo-[2-3H]inositol uptake. The presence of 1 mM ouabain also caused a reduction in inositol uptake, however, not to as a large an extent as the other three conditions. To determine if inositol uptake is dependent upon metabolic energy the cells were preincubated for 10 min with several metabolic inhibitors then analyzed for myo-[23H]inositol uptake (Table II). Strychnine 50 PM and dinitrophenol(1 mM) were the most effective in significantly decreasing inositol uptake in all four cell lines, whereas, iodoacetamide and potassium ferricyanide were less effective and only significantly reduced inositol uptake in the pulmonary artery endothelial cells. We wanted to determine if the kinetic parameters for inositol uptake by the four cell lines were different. Shown in Fig. 2 are the Eadie-Hofstee plots of myo-[23H]inositol up take by all four cell lines. When the concentration dependence of myo-[2-3H]inositol uptake is presented in
804
YOREK,
DUNLAP,
AND
TABLE
GINSBERG
I
THE EFFECT OF SODIUM REPLACEMENT AND OUABAIN
ON INOSITOL
UPTAKE
Ceil line Addition or modification of buffer
Mouse neuroblastoma
Human Y79 retinoblastoma Inositol
None Ouabain (1 mM) Sodium replaced by Sucrose CSCI LiCl
160.0 f 12.7 118.2 + 7.7’ 45.0 + 39.6 f 36.1 f
1.9” 6.4@ 4.5’
Human HL 60
uptake (pmol/mg
Bovine pulmonary artery endothelial
protein)
65.1 ?z 3.0 53.3 f 2.1”
92.0 +- 10.0 71.1 + 6.2
8.5 + 0.7 4.8 f 0.1
33.4 + 5.5’ 28.9 + 1.0’ 25.0 f 1.0”
20.3 + 20.5 + 20.1 f
2.4 + 1.1 2.0 k 0.7 1.9 + 0.7
1.4“ 1.6” 1.1”
Note. The cells were preincubated for 10 min in the described buffer conditions. The 5-min incubations were started by the addition of 25 pmol/liter my~2-3Hjinositol. Myo-[2-‘Hlinositol transport was linear under these conditions and the uptake was corrected for uptake which occurred at 0°C. The data are given as the means + SEM for three separate cell preparations. a P < 0.05.
this form, two graphically distinguishable components are observed. This suggests that inositol is taken up by both high- and low-affinity transport systems by each of the four cell lines examined. The high-affinity uptake system predominates at low concentrations of inositol (0.1 to 25 pmol/
liter), whereas, the low-affinity system predominates in the inositol concentration range of 50-500 PmolAiter. Kinetic parameters for these uptake systems were calculated from four separate experiments and are presented in Table III. The kinetic parameters for the high affinity uptake
TABLE THE EFFECT OF METABOLIC
II
INHIBITORS
ON INOSITOL
UPTAKE
Cell line Addition or modification of buffer
Mouse neuroblastoma
Human Y79 retinoblastoma Inositol
None Dinitrophenol(1 mM) Iodoacetamide (1 mM) Potassium ferricyanide (1 mM) Strvchnine (50 UM)
Human HL 60
uptake (pmol/mg
Bovine pulmonary artery endothelial
protein)
160.0 + 3.0 102.8 + 10.3” 151.6 2 15.6
65.1 + 3.0 50.3 + 3.3” 60.3 f 3.9
92.0 + 10.0 69.0 + 5.2” 74.4 Ik 3.9
8.5 f 0.7 7.9 + 0.1 4.0 + 0.3
133.6 + 13.7 118.4 + 5.5”
55.6 + 6.0 50.6 + 4.1”
74.7 + 22 57.4 + 3.5”
5.4 + 0.3 5.3 + 0.4
Note. The cells were preincubated for 10 min in the described buffer conditions. The 5-min incubations were Myo-[2-%X] inositol transport was linear under these started by the addition of 15 pmol/liter myo[2-‘H]inositol. conditions and the uptake was corrected for uptake which occurred at 0°C. The data are given as the means ? SEM for three separate cell preparations. a P < 0.05.
INOSITOL 160
100 (a)
(b)
160
60 i
120
60
60
.
40.
l .
40 ll..JL 0.0
20
. .
0
0
V
l
0.6
1.2
1.6
2.4
125
0.0
0.4
0.6
i.2
1.6
a2 (cl
Cd)
166 24 75 16 SO 6
26 0 L:: 0.0
0.5
1.0
1.6
2.0
0 0.0
A0.16
:0.62
%&0.46
v/s
FIG. 2. Eadie-Hofstee plots of inositol uptake. Myo[2-‘Hlinositol uptake was measured after a 5-min incubation, over a concentration range of 0.1-506 rmol/ liter in four individual cell lines: (a) human Y’79 retinoblastoma, (b) mouse neuroblastoma, (c) human HL 60 and (d) bovine pulmonary artery endothelial. The unit of (V) is pmol min-’ mg protein-‘, and S is PM. Each point is the average of four separate determinations. A least-squares fit was employed to plot the two apparent uptake components.
system are about the same for all four of the cell lines. The bovine pulmonary artery endothelial cells and mouse neuroblastoma had the lowest KL and vl,,,. However, there is only a difference of a factor of two between the four cell lines. In addition, the Kk for low-affinity uptake is also similar for the four cell lines. Although the Vl,,, for low-affinity uptake is higher in human Y79 retinoblastoma cells than in the other three cell lines. DISCUSSION
Inositol uptake was investigated in four cell lines: mouse neuroblastoma cells which are derived from peripheral nervous tissue and have retained many neuronal characteristics (33); human Y79 retinoblastoma cells which are derived from a tumor of retinal origin which have also retained many neural characteristics consistent
TRANSPORT
805
with its retinal origin (34-37); human HL 60, a widely used cell line which upon differentiation converts into a granulocyte (38); and bovine pulmonary artery which is a commonly used endothelial cell (39). The data indicate that inositol uptake occurs by a Na+-dependent and a partially energy-dependent mechanism. Based on the kinetic parameters of inositol uptake by the four cell lines these data suggest that circulating inositol, which exists at a concentration near or above the K:, for the high-affinity uptake system of the cells (29), is likely the major source of inositol for the cells. The kinetic parameters for the high-affinity uptake system of inositol is remarkably similar in all four of the cell lines considering the origin and species differences. Furthermore, the high-affinity Kk for inositol uptake by these cells is similar to isolated rat schwann cells, 30 PM (26). Combined these results suggest that the inositol transporter may be similar in many mammalian cells in respect to the capability to take up inositol. It has been shown that high-glucose levels in the medium decrease myoinositol uptake by rat schwann cells (26) and endoneural fasciles (25, 40). It has also been suggested that the high-glucose levels resulting from diabetes may be responsible for a decrease in inositol uptake by peripheral nerves (9). This effect is reversed by supplemented myoinositol(2,27). Thus far the effect of diabetic circulatory conditions on inositol uptake has only been examined in peripheral nerves or preparations derived from peripheral nerves and recently in isolated glomeruli (41). In addition, we have shown that the myoinositol transport is decreased by about 30% in neuroblastoma cells when the cells are cultured in the presence of 30 mM glucose or 1 mM sorbitol (28). However, if inositol transport properties are similar in most cells as suggested by these data then the diabetic circulatory conditions, specifically the high-glucose or sorbitol levels, may affect inositol metabolism in cells from many origins. This could be particularily pertinent to cells of retinal and vascular origin where diabetes can result in blindness and atherosclerosis.
YOREK,
DUNLAP, TABLE
AND
GINSBERG
III
KINETIC PARAMETERS OF INOSITOL UPTAKE Cell line Kinetic parameters High affinity KS vmax Low affinity Kit
Vtnax
Mouse neuroblastoma
12.4 + 3.8 14.1 + 2.7 433.0 + 28.4 215.0 + 10.0
Human Y79 retinoblastoma
27.6 + 49.3 +
3.9 5.4
632.1 21 63.2 533.6 + 106.3
Bovine pulmonary artery endothelial
Human HL60
40.9 + 66.5 k
2.2 2.1
462.9 + 190.6 169.0 f 20.3
15.9 + 8.2 f
1.1 0.2
420.8 + 150.4 90.1 + 28.9
Note Inositol uptake was conducted using a concentration range of 0.1-500 pmol/liter myo[2-%I]inositol and an incubation period of 5 min. Kk and V”‘,, values were calculated from the intercepts of double-reciprocal plots fitted by a weighted least-squares fit of the data points. The kinetic parameters are the means f SEM are PM and pmol/min/mg protein, respectively. of four separate determinations. The units for K& and I”’
ACKNOWLEDGMENT This work was supported by a Diabetes Endocrinology Research Center Grant (AM 25295) from the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, NIH.
REFERENCES 1. HOLUB, B. J. (1982) in Advances in Nutritional Research (Draper, H. H., ed), Vol. 4, pp 107141, Plenum, New York. 2. GREENE, D. A.,DJ%JEsus. P. V., JR., AND WINEGRAD, A. I. (1975) J. Clia &est55,.1326-1336. 3. CLEMENTS, R. S., JR., AND REYNERTSON, R. (1977) Diabetes 26,215~221. 4. PALMANO, K. P., WHITING, P. H., AND HAWTHORNE, J. N. (1977) Biochem J. 167,229-235. 5. CLEMENTS, R. S., JR., AND STOCKARD, C. R. (1980) Lkbetes 29,227-235. 6. RANCOUR, T. P., AND WELLS, W. W. (1980) Arch.
Biochem Biqhys. 202,150-159. 7. SERVO, C. (1980) Actu EndocrinolS4,133-137. 8. GREENE, D. A., AND LA’ITIMER, S. A. (1983) J. Clin Invest 72,1058-1063. 9. GILLON, K. R. W., HAWTHORNE, J. N., AND TOMLINSON, D. R. (1983) Diabetdogia 25,365-371. 10. WOOLLEY, D. W. (1942) J. Exp. Meal 75,227-281. 11. HALLIDAY, J. W., AND ANDERSON, L. (1955) J. Bid Chem 217,797-803. 12. EISENBERG, F., JR., AND BOLDEN, A. H. (1963) Biochem. Biophya RQS.Commua 12,72-80. 13. GREENE, D. A., LEWIS, R. A., LATTIMER, S. A., AND BROWN, M. J. (1982) Diabetes 31,573-578.
14. NATARAJAN, V., DYCK, P. J., AND SCHMID, H. H. 0. (1981) J. Neurochem 36.413-419. 15. MAUCO, G., DANGELMAIER, C. A., AND SMITH, J. B. (1984) Biochem J. 224,933-940. 16. SUGPURA, T., AND WAKU, K. (1984) B&&em Bie
phys Actu 796,190-198. 17. HARRIS, R. A., SCHMIDT, J., HITZEI~ANN, B. A., AND HITZEMANN, R. J. (1981) sciace212,1290-1291. 18. TANIGAWA, K., KUZUYA, H., AND IMURA, H. (1984)
Harm. Mekxb. Res. 16,458-460. 19. MAJERUS, P. W., NEUFELD, E. J., AND WILSON, D. B. (1984) CeU 37,701-703. 20. MARX, J. L. (1985) Science 228,312-313. 21. ADUNYAH, S. E., AND DEAN, W. L. (1985) B&hem Biophys. Res. Common 128,1274-1280. 22. EXTON, J. H. (1985) J. Clin Invest 75,1753-1757. 23. BIDEN, T. J., PRENTIKI, M., IRVINE, R. F., BERRIDGE, M. J., AND WOLLHEIM, C. B. (1984) Biochem J. 223.467-473. 24. JOSEPH, S. K., THOMAS, A. P., WILLIAMS, R. J., IRVINE, R. F., AND WILLIAMSON, J. R. (1984) J. Bill. Chem 259,3077-3081. 25. GILLON, K. R. W., AND HAWTHORNE, J. N. (1983) B&hem J. 210,775-781. 26. SEGAL, S., HWANG, S. M., STERN, J., AND PLEASURE, D. (1984) B&hem Biophgs. Res. Commun 120, 486-492. 27. MAYER, J. H., AND TOMLINSON, D. R. (1983) Diubetobgia 25.433-438. 28. YOREK, M. A., AND GINSBERG, B. H. (1985) J. Neuros& 11,775. 29. REID, T. W., ALBERT, D. M., RABSON, A. S., RusSELL, P., CREFT, J., CHU. E. W., TRALKA, T. S.,
INOSITOL
30.
31. 32.
33. 34. 35.
AND WILCOX, J. G. (1974) J. Nati Cancer Inst. 53,347-360. KADUCE, T. L., AWAD, A. B., FONTENELLE, L. J., AND SPECTOR, A. A. (1977) J. Biol Chem. 252, 6624-6630. LEES, M. B., AND PAXMAN, S. (1972) And Bkxhem. 47.184-192. C&LAND, W. W. (1967) in Advances in Enzymology (Nord, F. F., ed.), Vol. 29, pp. l-32, Wiley, New York. HAMPRECHT, B. (1977) Int. Rev. Cytol 49.99-170. YOREK, M. A., HYMAN, B. T., AND SPECTOR, A. A. (1983) J. Neuroc~ 40,70-78. YOREK, M. A., AND SPECTOR, A. A. (1983) J. Neurochem 41,809-815.
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TRANSPORT
36. YOREK, M. A., STROM, D. K., AND SPECTOR, A. A. (1984) J. Neurochem. 42,254-261. 37. KYRISIS, A. P., TSOKOS, M., TRICHE, T. J., AND CHADER, G. J. (1984) Nature (London) 307,471473. 38. BURNS, C. P., WEISHMAN, I. R., AND SPECTOR, A. A. (1977) Cancer Res. 37.1323-1327. 39. BAR, R. S., AND BOES, M. (1984) Biochem Res. Ccnnmun 124,203~209. 40. GREENE, D. A., AND LA~IMER, Invest. 70.1009-1018.
Biophp.
S. A. (1982) J. Clin.
41. ULBRECHT, J. S., BENSEN, J. T., AND GREENE, D. A. (1985) Diabetes34(Suppl. 13A).
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 246, No. 2, May 1, pp. 801-807, 1986
Myoinositol MARK
Uptake by Four Cultured Mammalian Cell Lines’
A. YOREK,2 JOYCE A. DUNLAP,
AND
BARRY
H. GINSBERG
Veterans Administration Medical Center, Iowa City, Iowa 52240, and the Department of Inter-d Medicine and the Diabetes Erulocrirwm Research Center, University of Iowa, Iozva City, Iowa 522&Z Received August 26,1985
The uptake of myo-[2-3H]inositol by mouse neuroblastoma, human Y’79 retinoblastoma, human HL60, and bovine pulmonary artery endothelial cells occurs by a saturable, Na+dependent and partially energy-dependent mechanism. Inositol uptake by all four cell lines occurred by both a high- and low-affinity system. The kinetic parameters for the high-affinity uptake systems were similar for all four cell lines. These data suggest that all four of these diverse cell lines have similar inositol transport systems and probably rely on extracellular inositol for anabolic processes. 0 1986 Academic Press, Inc.
Myoinositol is an important component for the synthesis of the inositol phospholipids in both neural and nonneural mammalian cells (1). Myoinositol exists in mammalian circulation at a concentration ranging from 25 to 100 pmol/liter (2-9) and is taken up and utilized by cells for phospholipid synthesis. Although myoinositol can be synthesized by the intestinal flora (10) and also in certain tissues (11,12) the most recent evidence suggests that it is an essential component of the diet (1). Myoinositol combines with CDP-diacylglycerol to form phosphatidylinositol which can be phosphorylated to form phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-diphosphate (1). The inositol phospholipids have been suggested to have an important role in many cellular functions including the motor nerve conduction velocity in peripheral nerves (13, 14), sodium-potassium adenosine triphosphatase activity (8), a fatty acid source for prostaglandin synthesis (15, 16), and neurotransmitter (1’7) and insulin release (18). Many of these cellular events occur follow1 This paper is dedicated to the memory of Dr. Edward C. Heath. ‘To whom correspondence and reprint requests should be addressed. 801
ing the activation of the “phosphoinositide cycle” which results in the release of possibly two second messengers, inositol1,4,5triphosphate and diacylglycerol (19, 20). These two compounds have been shown to stimulate Ca2+ mobilization and protein kinase C activity, respectively (19-23). It is assumed that intracellular inositol is acquired by a specific transport system. In addition, an abnormal uptake process may be responsible for some cellular disorders. For example, diabetic neuropathy has been partly attributed to a decrease in the intracellular myoinositol levels in peripheral nerve which results in a decrease in the inositol phospholipid content (24,25). The diabetic circulatory conditions have been suggested to be responsible for this abnormality apparently causing a reduction in inositol uptake (25, 26). In some cases increasing the myoinositol levels in the diet which results in an increase in inositol uptake has been shown to restore normal nerve activity (22, 27). Segal et aZ. (26) in the only published report examining inositol transport in a cultured cell line has shown that rat schwann cells take up inositol by a saturable, sodium-dependent mechanism with a K:, of about 30 PM. Since inositol seems to play an important role in neural membrane function and we have 0003-9861/86 $3.00 Copyright All rights
0 1986 by Academic Press. Inc. of reproduction in any form reserved.
802
YOREK,
DUNLAP,
shown abnormalities in inositol transport in cultured neural cells during periods of prolonged hyperglycemia (28), we felt it was important to characterize the inositol transport system in two cultured neural cell lines and compare it to cultured nonneural cells. In this study we describe the inositol transport properties of human Y79 retinoblastoma and HL 60 cells, mouse neuroblastoma, and bovine pulmonary artery endothelial cells. MATERIALS
AND
METHODS
Tissue culture Human Y79 retinoblastoma cells (29) which grow in suspension as small clusters of 6-10 cells and human HL 60 cells which also grow in suspension culture were propagated in RPM1 1640 medium containing 10% heat-inactivated fetal bovine serum (Dutchland, Denver, Pa.), 100 units/ml penicillin, 100 pg/ml streptomycin, 294 pg/ml glutamine, and 10 mM Hepes.* Bovine pulmonary artery endothelial cells were cultured in Ml99 medium containing 20% heat-inactivated fetal bovine serum, BME vitamins and amino acids, 294 rg/ml glutamine, and 2.2 mg/ml NaHCOa. Mouse neuroblastoma cells were cultured in Hams FlO medium containing 15% horse serum, 2.5% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 106 pg/ml streptomycin, and 294 pg/ml glutamine. All the cells were grown in 150-cm2 tissue culture flasks in an incubator maintained at 37”C, with 5% CO2 in humidified air as the gas phase. Prior to experimental use, the endothelial and neuroblastoma cells were transferred to Costar six-well plates and grown to confluency. The Y79 and HL 60 cells were isolated by centrifugation, washed and resuspended in 10 mM Hepes-buffered salt solution containing 128 mM NaCl, 5.2 mM KCl, 2.1 mM CaC&, 2.9 mM MgSO,, and 5 mM glucose, pH 7.4. This buffer was used in the uptake studies for all four of the cells lines. Inositol uptake. Following a wash, 2.5 ml buffer at 37°C containing 0.1 to 500 PM myoinositol and myo[2-aH]inositol as tracer was added to each well of a six-well plate containing either endothelial or neuroblastoma cells. Corresponding incubations at 0°C were carried out to determine nonspecific uptake. After the incubation the cells were washed three times with ice-cold buffer and the cells collected in a total volume of 1.5 ml. The cells suspension was sonicated to obtain a uniform mixture and samples were taken to determine cell protein content and total uptake. Triplicate 0.25-ml aliquots of the cell suspension were
3 Abbreviations used: Hepes, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid; NCS, N-chlorosuccinimide.
AND
GINSBERG
taken, 5 ml of Budget-Solve scintillation solution (Research Products International, Elk Grove Village, Ill.) was added and the radioactivity measured with a liquid scintillation counter. The Y79 and HL 60 cells were resuspended in 1.0 ml buffer containing the myo-[23H]inositol solution. Following the uptake period, three 200-~1 aliquots of the suspension were transferred to ice-cold l&ml Sarstedt microfuge tubes containing 0.8 ml buffer layered over 0.3 ml of a nbutyl phthalate/corn oil mixture (11/4 v/v) (30). The tubes were immediately centrifuged in an Eppendorf microfuge (Brinkman Instruments, Westbury, N.Y.) for 30 s to pellet the cells. After the aqueous and oil layers were removed by aspiration, the bottom of the tube containing the sedimented cells was cut off and added to a microscintillation vial containing 0.3 ml of NCS tissue solubilizer (Amersham/Searle, Arlington Heights, Ill.). The cell pellet was then dissolved by incubating the contents of the scintillation vial at 45°C for 1 h. Afterward 5 ml of Neutralizer scintillation solution (Research Products International) was added and the radioactivity determined. Quenching was monitored with a =Ra external standard. Doto an&sti. Values were calculated per milligram of cell protein. The protein content of the cells was determined by a modification of the Lowry method (31). Kinetic analysis of the myoinositol uptake data was done with an IBM personal computer using a weighted least-squares fit of the experimental points to a double reciprocal plot (32). RESULTS
Inositol Uptake Inositol uptake by the cells was measured from 0 to 10 min using 5 pmol/liter myo-[2-3H]inositol. The incubations were conducted at 3’7 and 0°C to determine the specific uptake by correcting for uptake at 0°C. The neuroblastoma and HL 60 cells took up similar amounts of myo-[23H]inositol, whereas, the Y79 retinoblastoma cells took up about 50% less (Fig. 1). The pulmonary artery endothelial cells took up the least amount of myo-[23H]inositol under these conditions and was about 10% of the total uptake observed in the neuroblastoma and HL 60 cells. Myo[2-3H]inositol uptake at 0°C accounted for less than 10% of the total uptake in the neuroblastoma, HL 60, and pulmonary artery endothelial cells, whereas, nonspecific uptake accounted for 30% of the total uptake by Y79 cells after 10 min of incubation. The retinoblastoma, HL 60, and endothelial
INOSITOL
803
TRANSPORT
60 40
a a
0
2
4
6
6
0
2
4
6
6
0
2
4
6
6
10
0
2
4
6
6
Time(
min)
10
10
10
FIG. 1. Inositol uptake by four individual cell lines. Myo-[2-3H]inositol uptake (5 pmol/liter) was analyzed from 0 to 10 min in four cell lines; (a) human Y79 retinoblastoma, (b) mouse neuroblastoma, (c) human HL 60, and (d) bovine pulmonary artery endothelial. Each data point is the mean -C SEM for three separate determinations. Specific uptake was determined by substracting the uptake which occurred at 0°C from the total uptake at 37°C.
cells demonstrated a linear uptake of myo[2-3H]inositol for a minimum of 10 min. Whereas, the myo-[2-3H]inositol uptake by the neuroblastoma cells was linear for at least 5 min. Therefore, subsequent transport studies to determine the kinetic parameters of inositol uptake were conducted for 5 min. Total myo-[2-3H]inositol uptake by the neuroblastoma cells approached saturation after 20 min of incubation, whereas the other three cell lines approached saturation after 30 min of incubation (data not shown). To determine if inositol uptake is dependent on extracellular Naf, incubations were conducted in normal buffer and buffer in which the NaCl was isosmotically replaced with sucrose, CsCl or LiCl. Incubations were also conducted in the presence of 1 mM ouabain. The data in Table I show that myo-[2-3H]inositol uptake by all four of the cell lines was dependent upon the extracellular presence of Na+. The exchange of NaCl for sucrose, CsCl or LiCl resulted in
a significant reduction in myo-[2-3H]inositol uptake. The presence of 1 mM ouabain also caused a reduction in inositol uptake, however, not to as a large an extent as the other three conditions. To determine if inositol uptake is dependent upon metabolic energy the cells were preincubated for 10 min with several metabolic inhibitors then analyzed for myo-[23H]inositol uptake (Table II). Strychnine 50 PM and dinitrophenol(1 mM) were the most effective in significantly decreasing inositol uptake in all four cell lines, whereas, iodoacetamide and potassium ferricyanide were less effective and only significantly reduced inositol uptake in the pulmonary artery endothelial cells. We wanted to determine if the kinetic parameters for inositol uptake by the four cell lines were different. Shown in Fig. 2 are the Eadie-Hofstee plots of myo-[23H]inositol up take by all four cell lines. When the concentration dependence of myo-[2-3H]inositol uptake is presented in
804
YOREK,
DUNLAP,
AND
TABLE
GINSBERG
I
THE EFFECT OF SODIUM REPLACEMENT AND OUABAIN
ON INOSITOL
UPTAKE
Ceil line Addition or modification of buffer
Mouse neuroblastoma
Human Y79 retinoblastoma Inositol
None Ouabain (1 mM) Sodium replaced by Sucrose CSCI LiCl
160.0 f 12.7 118.2 + 7.7’ 45.0 + 39.6 f 36.1 f
1.9” 6.4@ 4.5’
Human HL 60
uptake (pmol/mg
Bovine pulmonary artery endothelial
protein)
65.1 ?z 3.0 53.3 f 2.1”
92.0 +- 10.0 71.1 + 6.2
8.5 + 0.7 4.8 f 0.1
33.4 + 5.5’ 28.9 + 1.0’ 25.0 f 1.0”
20.3 + 20.5 + 20.1 f
2.4 + 1.1 2.0 k 0.7 1.9 + 0.7
1.4“ 1.6” 1.1”
Note. The cells were preincubated for 10 min in the described buffer conditions. The 5-min incubations were started by the addition of 25 pmol/liter my~2-3Hjinositol. Myo-[2-‘Hlinositol transport was linear under these conditions and the uptake was corrected for uptake which occurred at 0°C. The data are given as the means + SEM for three separate cell preparations. a P < 0.05.
this form, two graphically distinguishable components are observed. This suggests that inositol is taken up by both high- and low-affinity transport systems by each of the four cell lines examined. The high-affinity uptake system predominates at low concentrations of inositol (0.1 to 25 pmol/
liter), whereas, the low-affinity system predominates in the inositol concentration range of 50-500 PmolAiter. Kinetic parameters for these uptake systems were calculated from four separate experiments and are presented in Table III. The kinetic parameters for the high affinity uptake
TABLE THE EFFECT OF METABOLIC
II
INHIBITORS
ON INOSITOL
UPTAKE
Cell line Addition or modification of buffer
Mouse neuroblastoma
Human Y79 retinoblastoma Inositol
None Dinitrophenol(1 mM) Iodoacetamide (1 mM) Potassium ferricyanide (1 mM) Strvchnine (50 UM)
Human HL 60
uptake (pmol/mg
Bovine pulmonary artery endothelial
protein)
160.0 + 3.0 102.8 + 10.3” 151.6 2 15.6
65.1 + 3.0 50.3 + 3.3” 60.3 f 3.9
92.0 + 10.0 69.0 + 5.2” 74.4 Ik 3.9
8.5 f 0.7 7.9 + 0.1 4.0 + 0.3
133.6 + 13.7 118.4 + 5.5”
55.6 + 6.0 50.6 + 4.1”
74.7 + 22 57.4 + 3.5”
5.4 + 0.3 5.3 + 0.4
Note. The cells were preincubated for 10 min in the described buffer conditions. The 5-min incubations were Myo-[2-%X] inositol transport was linear under these started by the addition of 15 pmol/liter myo[2-‘H]inositol. conditions and the uptake was corrected for uptake which occurred at 0°C. The data are given as the means ? SEM for three separate cell preparations. a P < 0.05.
INOSITOL 160
100 (a)
(b)
160
60 i
120
60
60
.
40.
l .
40 ll..JL 0.0
20
. .
0
0
V
l
0.6
1.2
1.6
2.4
125
0.0
0.4
0.6
i.2
1.6
a2 (cl
Cd)
166 24 75 16 SO 6
26 0 L:: 0.0
0.5
1.0
1.6
2.0
0 0.0
A0.16
:0.62
%&0.46
v/s
FIG. 2. Eadie-Hofstee plots of inositol uptake. Myo[2-‘Hlinositol uptake was measured after a 5-min incubation, over a concentration range of 0.1-506 rmol/ liter in four individual cell lines: (a) human Y’79 retinoblastoma, (b) mouse neuroblastoma, (c) human HL 60 and (d) bovine pulmonary artery endothelial. The unit of (V) is pmol min-’ mg protein-‘, and S is PM. Each point is the average of four separate determinations. A least-squares fit was employed to plot the two apparent uptake components.
system are about the same for all four of the cell lines. The bovine pulmonary artery endothelial cells and mouse neuroblastoma had the lowest KL and vl,,,. However, there is only a difference of a factor of two between the four cell lines. In addition, the Kk for low-affinity uptake is also similar for the four cell lines. Although the Vl,,, for low-affinity uptake is higher in human Y79 retinoblastoma cells than in the other three cell lines. DISCUSSION
Inositol uptake was investigated in four cell lines: mouse neuroblastoma cells which are derived from peripheral nervous tissue and have retained many neuronal characteristics (33); human Y79 retinoblastoma cells which are derived from a tumor of retinal origin which have also retained many neural characteristics consistent
TRANSPORT
805
with its retinal origin (34-37); human HL 60, a widely used cell line which upon differentiation converts into a granulocyte (38); and bovine pulmonary artery which is a commonly used endothelial cell (39). The data indicate that inositol uptake occurs by a Na+-dependent and a partially energy-dependent mechanism. Based on the kinetic parameters of inositol uptake by the four cell lines these data suggest that circulating inositol, which exists at a concentration near or above the K:, for the high-affinity uptake system of the cells (29), is likely the major source of inositol for the cells. The kinetic parameters for the high-affinity uptake system of inositol is remarkably similar in all four of the cell lines considering the origin and species differences. Furthermore, the high-affinity Kk for inositol uptake by these cells is similar to isolated rat schwann cells, 30 PM (26). Combined these results suggest that the inositol transporter may be similar in many mammalian cells in respect to the capability to take up inositol. It has been shown that high-glucose levels in the medium decrease myoinositol uptake by rat schwann cells (26) and endoneural fasciles (25, 40). It has also been suggested that the high-glucose levels resulting from diabetes may be responsible for a decrease in inositol uptake by peripheral nerves (9). This effect is reversed by supplemented myoinositol(2,27). Thus far the effect of diabetic circulatory conditions on inositol uptake has only been examined in peripheral nerves or preparations derived from peripheral nerves and recently in isolated glomeruli (41). In addition, we have shown that the myoinositol transport is decreased by about 30% in neuroblastoma cells when the cells are cultured in the presence of 30 mM glucose or 1 mM sorbitol (28). However, if inositol transport properties are similar in most cells as suggested by these data then the diabetic circulatory conditions, specifically the high-glucose or sorbitol levels, may affect inositol metabolism in cells from many origins. This could be particularily pertinent to cells of retinal and vascular origin where diabetes can result in blindness and atherosclerosis.
YOREK,
DUNLAP, TABLE
AND
GINSBERG
III
KINETIC PARAMETERS OF INOSITOL UPTAKE Cell line Kinetic parameters High affinity KS vmax Low affinity Kit
Vtnax
Mouse neuroblastoma
12.4 + 3.8 14.1 + 2.7 433.0 + 28.4 215.0 + 10.0
Human Y79 retinoblastoma
27.6 + 49.3 +
3.9 5.4
632.1 21 63.2 533.6 + 106.3
Bovine pulmonary artery endothelial
Human HL60
40.9 + 66.5 k
2.2 2.1
462.9 + 190.6 169.0 f 20.3
15.9 + 8.2 f
1.1 0.2
420.8 + 150.4 90.1 + 28.9
Note Inositol uptake was conducted using a concentration range of 0.1-500 pmol/liter myo[2-%I]inositol and an incubation period of 5 min. Kk and V”‘,, values were calculated from the intercepts of double-reciprocal plots fitted by a weighted least-squares fit of the data points. The kinetic parameters are the means f SEM are PM and pmol/min/mg protein, respectively. of four separate determinations. The units for K& and I”’
ACKNOWLEDGMENT This work was supported by a Diabetes Endocrinology Research Center Grant (AM 25295) from the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, NIH.
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INOSITOL
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41. ULBRECHT, J. S., BENSEN, J. T., AND GREENE, D. A. (1985) Diabetes34(Suppl. 13A).