- Email: [email protected]
Early increase of amino acid transport in stimulated lymphocytes
Amino acid transport in lymphocytes chelating derivates, m a y be useful in studying pinocytosis. I n addition, considering that E D T A can enter the cell possibly as a n actively chelating form, it might be p r u d e n t , i n some cases, to re-examine the practice of i n t r o d u c ing E D T A into culture media. The authors gratefully acknowledge the technical assistance of Mrs Jane Lundin and Mrs Sarah Reynolds. This study was aided by a grant from the American Medical Association Education and Research Foundation.
REFERENCES 1. Cohn, Z A & Benson, B, J exptl med 121 (1965) 153. 2. Cohn, Z A & Ehrenreich, B A, J exptl reed 129 (1969) 201. 3. Cohn, Z A & Parks, E, J exptl med 125 (1967) 213. 4. Foreman, H, Vier, M & Magee, M, J biol chem 203 (1953) 1045. 5. Schwartz, S L, Johnson, C B & Doolan, P D, Mol pharmacol 6 (1970) 54. 6. Schwartz, S L, Johnson, C B, Hayes, J R & Doolan, P D, Biochem pharmacol 16 (1967) 2413. 7. Schwartz, S L, Hayes, J R, Ide, R S, Johnson, C B & Doolan, P D, Biochem pharmaco115 (1966) 377. Received October 27, 1970 Revised version received March 1, 1971
Early increase of amino acid transport in stimulated lymphocytes K. J. VAN DEN BERG and I. BETEL, Radiobiological Institute TNO, Rijswijk Z. H., The Netherlands A large variety of substances has been f o u n d to stimulate small lymphocytes into the form a t i o n of blast cells (reviewed by Ling [5]). A m o n g the non-specific stimulants, which do n o t require prior sensitization of cells for their action, those best studied are phytoh a e m a g g l u t i n i n ( P H A ) a b e a n extract of Phaseolus vulgar&, c o n c a n a v a l i n A (con A) f r o m the j a c k b e a n Canavalia ensiformis, a n d pokeweed mitogen ( P W M ) from Phytolacca americana. However, little is k n o w n a b o u t their p r i m a r y site of action. There are indications that they m a y have a n effect o n the
257
properties of the cell m e m b r a n e [1, 4, 10, 11]~ I n lymphocytes, a m i n o acids are a c c u m u l a t e d by a n active carrier-mediated t r a n s p o r t system of the cell m e m b r a n e [3, 14]. Since a n interaction of the stimulants with the m e m b r a n e m a y alter the supply of a m i n o acids [1], we have studied the t r a n s p o r t of the n o n - m e t a bolizable a m i n o acid 2 - a m i n o i s o b u t y r i c acid (AIB) in rat lymphocytes stimulated b y P H A , con A a n d P W M a n d in a m i x e d culture of rat lymphocytes (MLC).
Materials and Methods 14c-1,2- Aminoisobutyric acid (spec. act. 53.8 mCi[ mM1) and 8H-thymidine (spec. act. 2 Ci/mM1) were obtained from The Radiochemical Centre, Amersham~ Phytohaemagglutinin, purified Phytohaemagglutinin (p-PHA), concanavalin A and pokeweed mitogen were purchased from Burroughs Wellcome, London; Calbiochem, Los Angeles, and the Grand Island Biological Company, New York, respectively. Male WAG/Rij and BN rats 10 weeks of age were used as donors of lymphocytes. A cell suspension was prepared by gently forcing smaU pieces of lymph nodes through a nylon gauze filter, in Hanks Eagles medium containing 15 % fresh rat serum. The final concentration was 2-4 • 106 cells/ml. A MLC was set up by mixing 6 x l0 s WAG/Rij lymphocytes with the same number of BN cells in a total volume of 4 ml; cell interaction in these incubations was. promoted by centrifuging the suspension 5 rain at 500 rpm. WAG/Rij lymphocytes were stimulated by incubation with PHA, p-PHA, PWM and con A in complete medium at 37~ Amino acid transport was determined after washing: the cells to remove medium and free stimulant. 1-1.5 • 107 cells were resuspended in 1 ml of Hanks salt solution and incubated with 0.1 mM 14C-AIB for 1 h at 37~ In this period a steady state is achieved. After washing twice, the cells were lysed with hot distilled water and the radioactivity of the supernatant was measured. Thymidine incorporation was estimated in the presence of complete medium and stimulants. After 4 h of incubation of 4 • 106 cells with 2.5 /~Ci 3Hthymidine at 37~ the radioactivity was determined in the cell pellet obtained after precipitation with cold TCA. The thymidine incorporation in these cells reaches a maximum after 45 h of incubation with the optimal concentration of stimulant. The same concentrations were used in the AIB uptake experiments.
Results and Discussion Fig. l shows that cells i n c u b a t e d for o n l y 5 m i n in the presence of P H A or con A have
Exptl Cell Res 66
258
K . J . van den Berg et al.
20.
15"
10"
control
PHA
p-PHA
PWM
con A
Fig. 1. Ordinate: • 103 cpm 14C-AIB transport by stimulated rat lymphocytes. Lymphocytes stimulated in complete medium for 5 min (grey bars) and 60 rain (open bars), were washed and resuspended in 1 ml of Hanks salt solution containing 0.1 m M 14C-AIB. After 1 h of incubation at 37~ the radioactivity in the supernatant of the lysed cell pellet was measured. The concentrations of stimulants were (per ml of incubation medium): 5 /~1 of P H A solution and 20/~1 of P W M solution (the content of the vials was dissolved in 5 ml of Hanks Eagles medium): 40/zg of concanavalin A and 1 /zg of purified PHA. Each bar represents the mean of duplicate cultures.
a greatly increased uptake of AIB (in Student's t test, p <0.005 for PHA and p < 0.05 for con A), which is even more pronounced after 1 h of incubation with the stimulant (p<0.005 for PHA, p<0.01 for con A). Cells incubated with PWM show only a slight increase (p < 0.05). The thymidine incorporation by stimulated rat lymphocytes is shown in fig. 2. In these
experiments the stimulant remained present during the whole incubation period. The stimulation of thymidine incorporation shows a striking similarity with the stimulation of AIB uptake. The results obtained in the rat MLC are shown in fig. 3. A significant stimulation of AIB uptake was noted already after 8 h (p < 0.005) and was more pronounced after 16 h (p<0.001). Measurable thymi-
20
15
lo
1
5-
control
PHA
p-PHA
PWM
con A
Fig. 2. Ordinate: +__l0 -5 dpm/culture. 8H-Thymidine incorporation by stimulated rat lymphocytes. Lymphocytes were stimulated for 45 h and 3H-thymidine incorporation was estimated after a pulse label with 2.5 /zCi 8H-thymidine for 4 h. The concentrations of stimulants were the same as described in fig. 1. Each bar represents the mean of duplicate cultures. Exptl Cell Res 66
Amino acid transport in lymphocytes 8-
8h
i 16h
Fig. 3. Ordinate: x 10 .8 cpm. 14C-AIB transport in a mixed culture of rat lymphocytes. After the indicated time of incubation, the cells were washed and AIB transport was measured as described in fig. 1. Open bars: MLC; grey bars: control. Each bar represents the mean of triplicate cultures.
dine uptake in these cultures does not start before 24 h. These experiments show that increased amino acid uptake in rat lymphocytes, stimulated specifically (MLC) as well as nonspecifically (PHA, PWM, con A), is a very early effect. Our experiments do not exclude the loss of some AIB during the washing procedure and the radioactivity in the cell lYsate may not represent the actual intracellular amino acid concentration. It seems likely that the increased steady state level of AIB is due to an increased uptake rather than to a decreased efflux. Such an increased rate of uptake of AIB has been recently reported by Mendelsohn et al. [7]. These experiments suggest that blast formation of lymphocytes, induced by mitogenic stimulation, is preceded by an alteration in the properties of the cellular membrane.
259
Although the causal relation between the early increase of amino acid transport and subsequent blast formation remains to be proven, the similarity between the patterns of increased amino acid transport and thymidine incorporation may be more than a coincidence. Nowell has already proposed that stimulants may facilitate the entrance of blastogenic factors by affecting the lymphocyte membrane [9]. Our experiments and also other evidence [6, 13] suggest that amino acids play a role in blastogenesis. The possibility that amino acids may influence the metabolism of RNA [2, 8, 12] and protein in stimulated lymphocytes will be the subject of further investigations.
REFERENCES 1. Betuel, H, Richard, G B, Colobert, L & Revol, L, Nature 225 (1970) 459. 2. Byfield, J E & Scherbaum, O H, Exptl cell res 49 (1967) 202. 3. Helmreich, E & Kipins, D M, J biol chem 237 (1962) 2 582. 4. Kornfeld, R & Kornfeld, S, J biol chem 245 (1970) 2 536. 5. Ling, N R, Lymphocyte stimulation. North-Holland, Amsterdam (1968). 6. McElwain, T J & Hayward, S K, Lancet 1 (1969) 527. 7. Mendelsohn, J, Skinner, S A & Kornfeld, S, J clin invest 49 (1970) 65a. 8. Munro, H N, Fed proc 29 (1970) 1 490. 9. Nowell, P C, Cancer res 20 (1960) 462. 10. Quastel, M R, Dow, D & Kaplan, J G, J cell biol 43 (1970) 109 a. 11. Smith, J W, Steiner, A L & Parker, C W, Fed proc 29 (1970) 369 Abstr. 12. Smulson, M E & Thomas, J, J biol chem 244 (1969) 5 309. 13. Waithe, W I, Weiner, M & Hirschhorn, K, Fed proc 29 (1970) 370 Abstr. 14. Yunis, A A, Arimura, G K & Kipnis, D M, J lab clin med 62 (1963) 465. Received November 13, 1970 Revised version received February 18, 1971
Exptl Cell Res 66
REFERENCES 1. Cohn, Z A & Benson, B, J exptl med 121 (1965) 153. 2. Cohn, Z A & Ehrenreich, B A, J exptl reed 129 (1969) 201. 3. Cohn, Z A & Parks, E, J exptl med 125 (1967) 213. 4. Foreman, H, Vier, M & Magee, M, J biol chem 203 (1953) 1045. 5. Schwartz, S L, Johnson, C B & Doolan, P D, Mol pharmacol 6 (1970) 54. 6. Schwartz, S L, Johnson, C B, Hayes, J R & Doolan, P D, Biochem pharmacol 16 (1967) 2413. 7. Schwartz, S L, Hayes, J R, Ide, R S, Johnson, C B & Doolan, P D, Biochem pharmaco115 (1966) 377. Received October 27, 1970 Revised version received March 1, 1971
Early increase of amino acid transport in stimulated lymphocytes K. J. VAN DEN BERG and I. BETEL, Radiobiological Institute TNO, Rijswijk Z. H., The Netherlands A large variety of substances has been f o u n d to stimulate small lymphocytes into the form a t i o n of blast cells (reviewed by Ling [5]). A m o n g the non-specific stimulants, which do n o t require prior sensitization of cells for their action, those best studied are phytoh a e m a g g l u t i n i n ( P H A ) a b e a n extract of Phaseolus vulgar&, c o n c a n a v a l i n A (con A) f r o m the j a c k b e a n Canavalia ensiformis, a n d pokeweed mitogen ( P W M ) from Phytolacca americana. However, little is k n o w n a b o u t their p r i m a r y site of action. There are indications that they m a y have a n effect o n the
257
properties of the cell m e m b r a n e [1, 4, 10, 11]~ I n lymphocytes, a m i n o acids are a c c u m u l a t e d by a n active carrier-mediated t r a n s p o r t system of the cell m e m b r a n e [3, 14]. Since a n interaction of the stimulants with the m e m b r a n e m a y alter the supply of a m i n o acids [1], we have studied the t r a n s p o r t of the n o n - m e t a bolizable a m i n o acid 2 - a m i n o i s o b u t y r i c acid (AIB) in rat lymphocytes stimulated b y P H A , con A a n d P W M a n d in a m i x e d culture of rat lymphocytes (MLC).
Materials and Methods 14c-1,2- Aminoisobutyric acid (spec. act. 53.8 mCi[ mM1) and 8H-thymidine (spec. act. 2 Ci/mM1) were obtained from The Radiochemical Centre, Amersham~ Phytohaemagglutinin, purified Phytohaemagglutinin (p-PHA), concanavalin A and pokeweed mitogen were purchased from Burroughs Wellcome, London; Calbiochem, Los Angeles, and the Grand Island Biological Company, New York, respectively. Male WAG/Rij and BN rats 10 weeks of age were used as donors of lymphocytes. A cell suspension was prepared by gently forcing smaU pieces of lymph nodes through a nylon gauze filter, in Hanks Eagles medium containing 15 % fresh rat serum. The final concentration was 2-4 • 106 cells/ml. A MLC was set up by mixing 6 x l0 s WAG/Rij lymphocytes with the same number of BN cells in a total volume of 4 ml; cell interaction in these incubations was. promoted by centrifuging the suspension 5 rain at 500 rpm. WAG/Rij lymphocytes were stimulated by incubation with PHA, p-PHA, PWM and con A in complete medium at 37~ Amino acid transport was determined after washing: the cells to remove medium and free stimulant. 1-1.5 • 107 cells were resuspended in 1 ml of Hanks salt solution and incubated with 0.1 mM 14C-AIB for 1 h at 37~ In this period a steady state is achieved. After washing twice, the cells were lysed with hot distilled water and the radioactivity of the supernatant was measured. Thymidine incorporation was estimated in the presence of complete medium and stimulants. After 4 h of incubation of 4 • 106 cells with 2.5 /~Ci 3Hthymidine at 37~ the radioactivity was determined in the cell pellet obtained after precipitation with cold TCA. The thymidine incorporation in these cells reaches a maximum after 45 h of incubation with the optimal concentration of stimulant. The same concentrations were used in the AIB uptake experiments.
Results and Discussion Fig. l shows that cells i n c u b a t e d for o n l y 5 m i n in the presence of P H A or con A have
Exptl Cell Res 66
258
K . J . van den Berg et al.
20.
15"
10"
control
PHA
p-PHA
PWM
con A
Fig. 1. Ordinate: • 103 cpm 14C-AIB transport by stimulated rat lymphocytes. Lymphocytes stimulated in complete medium for 5 min (grey bars) and 60 rain (open bars), were washed and resuspended in 1 ml of Hanks salt solution containing 0.1 m M 14C-AIB. After 1 h of incubation at 37~ the radioactivity in the supernatant of the lysed cell pellet was measured. The concentrations of stimulants were (per ml of incubation medium): 5 /~1 of P H A solution and 20/~1 of P W M solution (the content of the vials was dissolved in 5 ml of Hanks Eagles medium): 40/zg of concanavalin A and 1 /zg of purified PHA. Each bar represents the mean of duplicate cultures.
a greatly increased uptake of AIB (in Student's t test, p <0.005 for PHA and p < 0.05 for con A), which is even more pronounced after 1 h of incubation with the stimulant (p<0.005 for PHA, p<0.01 for con A). Cells incubated with PWM show only a slight increase (p < 0.05). The thymidine incorporation by stimulated rat lymphocytes is shown in fig. 2. In these
experiments the stimulant remained present during the whole incubation period. The stimulation of thymidine incorporation shows a striking similarity with the stimulation of AIB uptake. The results obtained in the rat MLC are shown in fig. 3. A significant stimulation of AIB uptake was noted already after 8 h (p < 0.005) and was more pronounced after 16 h (p<0.001). Measurable thymi-
20
15
lo
1
5-
control
PHA
p-PHA
PWM
con A
Fig. 2. Ordinate: +__l0 -5 dpm/culture. 8H-Thymidine incorporation by stimulated rat lymphocytes. Lymphocytes were stimulated for 45 h and 3H-thymidine incorporation was estimated after a pulse label with 2.5 /zCi 8H-thymidine for 4 h. The concentrations of stimulants were the same as described in fig. 1. Each bar represents the mean of duplicate cultures. Exptl Cell Res 66
Amino acid transport in lymphocytes 8-
8h
i 16h
Fig. 3. Ordinate: x 10 .8 cpm. 14C-AIB transport in a mixed culture of rat lymphocytes. After the indicated time of incubation, the cells were washed and AIB transport was measured as described in fig. 1. Open bars: MLC; grey bars: control. Each bar represents the mean of triplicate cultures.
dine uptake in these cultures does not start before 24 h. These experiments show that increased amino acid uptake in rat lymphocytes, stimulated specifically (MLC) as well as nonspecifically (PHA, PWM, con A), is a very early effect. Our experiments do not exclude the loss of some AIB during the washing procedure and the radioactivity in the cell lYsate may not represent the actual intracellular amino acid concentration. It seems likely that the increased steady state level of AIB is due to an increased uptake rather than to a decreased efflux. Such an increased rate of uptake of AIB has been recently reported by Mendelsohn et al. [7]. These experiments suggest that blast formation of lymphocytes, induced by mitogenic stimulation, is preceded by an alteration in the properties of the cellular membrane.
259
Although the causal relation between the early increase of amino acid transport and subsequent blast formation remains to be proven, the similarity between the patterns of increased amino acid transport and thymidine incorporation may be more than a coincidence. Nowell has already proposed that stimulants may facilitate the entrance of blastogenic factors by affecting the lymphocyte membrane [9]. Our experiments and also other evidence [6, 13] suggest that amino acids play a role in blastogenesis. The possibility that amino acids may influence the metabolism of RNA [2, 8, 12] and protein in stimulated lymphocytes will be the subject of further investigations.
REFERENCES 1. Betuel, H, Richard, G B, Colobert, L & Revol, L, Nature 225 (1970) 459. 2. Byfield, J E & Scherbaum, O H, Exptl cell res 49 (1967) 202. 3. Helmreich, E & Kipins, D M, J biol chem 237 (1962) 2 582. 4. Kornfeld, R & Kornfeld, S, J biol chem 245 (1970) 2 536. 5. Ling, N R, Lymphocyte stimulation. North-Holland, Amsterdam (1968). 6. McElwain, T J & Hayward, S K, Lancet 1 (1969) 527. 7. Mendelsohn, J, Skinner, S A & Kornfeld, S, J clin invest 49 (1970) 65a. 8. Munro, H N, Fed proc 29 (1970) 1 490. 9. Nowell, P C, Cancer res 20 (1960) 462. 10. Quastel, M R, Dow, D & Kaplan, J G, J cell biol 43 (1970) 109 a. 11. Smith, J W, Steiner, A L & Parker, C W, Fed proc 29 (1970) 369 Abstr. 12. Smulson, M E & Thomas, J, J biol chem 244 (1969) 5 309. 13. Waithe, W I, Weiner, M & Hirschhorn, K, Fed proc 29 (1970) 370 Abstr. 14. Yunis, A A, Arimura, G K & Kipnis, D M, J lab clin med 62 (1963) 465. Received November 13, 1970 Revised version received February 18, 1971
Exptl Cell Res 66