Neurochemical Research (2) 695-706 (1977)
T E M P E R A T U R E D E P E N D E N C E OF A M I N O ACID TRANSPORT IN BRAIN SLICES M. BANAY-SCHWARTZ,K. LAJTHA,H. SERSHEN, AND A . LAJTHA Research Institute of Neurochemistry Rockland Research Institute Ward's Island, New York, New York 10035
Accepted June 8, 1977
A decrease in amino acid influx and exit in incubated slices when the temperature was lowered from 37 to 20~ was observed with all 16 amino acids examined at two concentrations (1 mM and l0/zM). The temperature dependence of cellular amino acid influx observed in slices in vitro contrasts with the absence of temperature dependence of capillary amino acid influx in the brain in vivo that we recently reported. The temperature effects in slices varied somewhat among the various amino acid transport classes. With some amino acids (Phe, Val, Tyr, Asp, Glu), especially at lower concentrations, the greater inhibition of exit than of uptake resulted in an actual increase in tissue amino acid uptake at lower temperatures in long-term experiments. The results indicate heterogeneity in the effect of temperature on the various transport classes and show a difference between capillary and cellular amino acid transport in the brain.
INTRODUCTION We found in recent studies (1) that the rate of incorporation of tyrosine into brain proteins in vivo was decreased 6% for each degree of temperature decrease in the range 37-10~ Temperature gradients and rates of turnover were similar in rat and goldfish brain. In our study of the relationship of amino acid transport to protein metabolism, we found that the uptake of low doses of amino acids by the brain in vivo from the circulation (brain uptake index) was independent of brain temperature in the range 37-20~ in rats (2). This lack of dependence on temperature of uptake in vivo was unexpected, since past work measuring uptake in
695 This journal is copyrighted by Plenum. Each article is available for $7.50 from Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 10011.
696
BANAY-SCHWARTZ ET AL.
incubated brain slices of tyrosine (3); histidine (4); and lysine, valine, and GABA in our laboratory (5) showed that uptake in vitro decreases with decreasing temperature. The present report is an extension of measurements of the activity of various amino acid transport classes at lower temperature in vitro to compare the temperature dependence of cellular transport with that of capillary transport. To estimate possible changes in cellular transport, we measured the effects of lowered temperatures at low and high amino acid concentrations. In short-time incubations, we measured uptake or exit; to estimate effects on unidirectional transport in longer incubations, we measured the concomitant activities of uptake and exit to estimate effects on steady state.
EXPERIMENTAL PROCEDURE Brain slices (0.42 mm) from adult Wistar rats or young adult Swiss mice were prepared with a Mcllwain tissue slicer and incubated as described previously (6). Slices from half a cerebrum were incubated in oxygenated HEPES-buffered medium: 119 mM NaC1, 5.0 mM KC1, 0.75 mM CaCI2, 1.2 mM MgSO4, 1.0 mM NaH2PO4, 1.0 mM N a H C Q , 10 mM glucose, and 25 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), adjusted to pH 7.35 with NaOH. After the slices were incubated for 30 min in a water bath at the temperature indicated ( - 1 4 0 mg of tissue was incubated in 5 ml of medium), labeled amino acid (0.05/zCi/5 ml) was added to the medium. The final medium concentration was either 1 mM (high) or 0.01 mM (low) amino acid, or that indicated in the legends. After the indicated time, the medium was filtered from the slices through Whatman No. 54 filter paper with suction. The tissue was frozen on dry ice, weighed, homogenized in 2 ml of 3% perchloric acid (wt/vol), and centrifuged. For measurements of radioactivity, 0.5 ml of the perchloric acid supernatant was added to 15 ml of scintillation fluid (6) and counted in an Intertechnique SL30 scintillation counter (Teledyne). The amount of added amino acid when the medium was 1 mM was greatly in excess of that contained in the tissue; therefore, any leakage of endogenous tissue amino acid into the medium would not have influenced the specific activity of the added amino acid. Under such conditions, metabolic conversions of the labeled amino acids are small and do not affect the calculated results. In experiments with 0.01 mM amino acids in the medium, if the endogenous content of the amino acids that are at high level in the tissue (Glu, Tau, Gly, GABA) became mixed with that in the medium during the experiment, resulting in a lowered specific activity in the medium, then the flux of amino acid would be higher than calculated. We have shown previously, using incubated brain slices with 0.2 mM amino acids (7), that with Val, Leu, Phe, Pro, His, Lys, and Arg, in 30 min, less than 6%, and with Ala and Asp, approximately 13%, of the label is in compounds other than the amino acid added. When slices were incubated with 1 /zM amino acids (8), less than 10% conversion occurred in 30 min with Thr, Val, Leu, Tyr, Lys, His, and Gly, but significant conversion occurred with Asp, Ala, and GABA. At low levels in the medium (1 I~M), the tissue amino acid specific activity after 30 min was about half of that in the medium (8). In initial experiments, we tested the effects of in vivo hypothermia on in vitro transport: rats were anesthetized with chloral hydrate (380 mg/kg) and submerged in an ice-water bath up to their necks for 25 min to lower brain temperature to 20~ (2). The brains were
TEMPERATURE DEPENDENCE OF AMINO ACID TRANSPORT
697
removed, sliced, and incubated at 20~ in HEPES medium. Because rates of uptake were the same for slices of in vivo-cooled brains as for slices from uncooled brains upon subsequent incubation at 20~ the in vivo cooling was discontinued. For measurements of exit, slices were preloaded with a labeled amino acid, and the exit of this labeled amino acid was measured (9). To minimize the contribution of initial exit from the extracellular fluid, longer incubation times were used for exit than for uptake measurements. Uptake values are expressed as concentrative uptake, i.e., intracellular level minus medium level. When results are expressed in this manner, if only diffusion equilibrium is reached, concentrative uptake is 0; i.e., no concentrative uptake occurs (inhibition is 100%) when uphill transport is inhibited. Since the absolute value of uptake of the various amino acids is different, exit was expressed as percent decrease for easier comparison. For expressing flux, intracellular concentrations are used; tissue levels are corrected to amino acid in the extracellular space (6,10). For measuring rates of amino acid exit from slices after the usual preincubation, the tissue was incubated for 30 rain at 37~C with labeled amino acid; the slices were then gently filtered on Hirsch funnels. A portion of the tissue was frozen, and the radioactivity was determined. Two other portions of the slices were taken; one was added to 5 ml of amino acid-free HEPES medium at 20~ and the other to the same medium at 37~ After 5-20 rain, the medium was filtered from the slices and the radioactivity was determined. A comparison was made of the exit of labeled amino acid from the slices at the two temperatures. Unlabeled amino acids were purchased from either Sigma or Calbiochem, labeled amino acids from New England Nuclear or Calatomic.
RESULTS
In the first set of experiments, slices from either mouse or rat brain were incubated for 30 min. We used both mice and rats, since the in vivo study of temperature dependence of brain uptake index was done with rats (2) and the delineation of transport classes in brain slices was done with mice (11). Uptake approaches steady state after a 30-min incubation (see also Figure 2). In contrast to initial rate of uptake, steady-state uptake is defined as maximal level in tissue, when no more net uptake occurs, i.e., when uptake and exit are equal. Steady-state uptake values (rather than initial rates) indicate changes under most in vivo conditions of hypothermia, when the brain is exposed to the lower temperature for a longer time and the established new steady state is of importance. After the 30-rain incubation, there were significant differences in the temperature dependence of uptake among the various transport classes (Table I). Decrease of uptake with decreasing temperature at high amino acid concentrations was greatest with taurine, followed by small neutral amino acids, glycine, and possibly the ASC (alanine, serine, cysteinepreferring) class (12), basic amino acids (lysine, arginine), GABA, and acidic amino acids (glutamate and aspartate). In this order, the temperature sensitivity of large neutral amino acids (L, leucine-preferring class)
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T E M P E R A T U R E D E P E N D E N C E OF A M I N O ACID TRANSPORT IN BRAIN SLICES M. BANAY-SCHWARTZ,K. LAJTHA,H. SERSHEN, AND A . LAJTHA Research Institute of Neurochemistry Rockland Research Institute Ward's Island, New York, New York 10035
Accepted June 8, 1977
A decrease in amino acid influx and exit in incubated slices when the temperature was lowered from 37 to 20~ was observed with all 16 amino acids examined at two concentrations (1 mM and l0/zM). The temperature dependence of cellular amino acid influx observed in slices in vitro contrasts with the absence of temperature dependence of capillary amino acid influx in the brain in vivo that we recently reported. The temperature effects in slices varied somewhat among the various amino acid transport classes. With some amino acids (Phe, Val, Tyr, Asp, Glu), especially at lower concentrations, the greater inhibition of exit than of uptake resulted in an actual increase in tissue amino acid uptake at lower temperatures in long-term experiments. The results indicate heterogeneity in the effect of temperature on the various transport classes and show a difference between capillary and cellular amino acid transport in the brain.
INTRODUCTION We found in recent studies (1) that the rate of incorporation of tyrosine into brain proteins in vivo was decreased 6% for each degree of temperature decrease in the range 37-10~ Temperature gradients and rates of turnover were similar in rat and goldfish brain. In our study of the relationship of amino acid transport to protein metabolism, we found that the uptake of low doses of amino acids by the brain in vivo from the circulation (brain uptake index) was independent of brain temperature in the range 37-20~ in rats (2). This lack of dependence on temperature of uptake in vivo was unexpected, since past work measuring uptake in
695 This journal is copyrighted by Plenum. Each article is available for $7.50 from Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 10011.
696
BANAY-SCHWARTZ ET AL.
incubated brain slices of tyrosine (3); histidine (4); and lysine, valine, and GABA in our laboratory (5) showed that uptake in vitro decreases with decreasing temperature. The present report is an extension of measurements of the activity of various amino acid transport classes at lower temperature in vitro to compare the temperature dependence of cellular transport with that of capillary transport. To estimate possible changes in cellular transport, we measured the effects of lowered temperatures at low and high amino acid concentrations. In short-time incubations, we measured uptake or exit; to estimate effects on unidirectional transport in longer incubations, we measured the concomitant activities of uptake and exit to estimate effects on steady state.
EXPERIMENTAL PROCEDURE Brain slices (0.42 mm) from adult Wistar rats or young adult Swiss mice were prepared with a Mcllwain tissue slicer and incubated as described previously (6). Slices from half a cerebrum were incubated in oxygenated HEPES-buffered medium: 119 mM NaC1, 5.0 mM KC1, 0.75 mM CaCI2, 1.2 mM MgSO4, 1.0 mM NaH2PO4, 1.0 mM N a H C Q , 10 mM glucose, and 25 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), adjusted to pH 7.35 with NaOH. After the slices were incubated for 30 min in a water bath at the temperature indicated ( - 1 4 0 mg of tissue was incubated in 5 ml of medium), labeled amino acid (0.05/zCi/5 ml) was added to the medium. The final medium concentration was either 1 mM (high) or 0.01 mM (low) amino acid, or that indicated in the legends. After the indicated time, the medium was filtered from the slices through Whatman No. 54 filter paper with suction. The tissue was frozen on dry ice, weighed, homogenized in 2 ml of 3% perchloric acid (wt/vol), and centrifuged. For measurements of radioactivity, 0.5 ml of the perchloric acid supernatant was added to 15 ml of scintillation fluid (6) and counted in an Intertechnique SL30 scintillation counter (Teledyne). The amount of added amino acid when the medium was 1 mM was greatly in excess of that contained in the tissue; therefore, any leakage of endogenous tissue amino acid into the medium would not have influenced the specific activity of the added amino acid. Under such conditions, metabolic conversions of the labeled amino acids are small and do not affect the calculated results. In experiments with 0.01 mM amino acids in the medium, if the endogenous content of the amino acids that are at high level in the tissue (Glu, Tau, Gly, GABA) became mixed with that in the medium during the experiment, resulting in a lowered specific activity in the medium, then the flux of amino acid would be higher than calculated. We have shown previously, using incubated brain slices with 0.2 mM amino acids (7), that with Val, Leu, Phe, Pro, His, Lys, and Arg, in 30 min, less than 6%, and with Ala and Asp, approximately 13%, of the label is in compounds other than the amino acid added. When slices were incubated with 1 /zM amino acids (8), less than 10% conversion occurred in 30 min with Thr, Val, Leu, Tyr, Lys, His, and Gly, but significant conversion occurred with Asp, Ala, and GABA. At low levels in the medium (1 I~M), the tissue amino acid specific activity after 30 min was about half of that in the medium (8). In initial experiments, we tested the effects of in vivo hypothermia on in vitro transport: rats were anesthetized with chloral hydrate (380 mg/kg) and submerged in an ice-water bath up to their necks for 25 min to lower brain temperature to 20~ (2). The brains were
TEMPERATURE DEPENDENCE OF AMINO ACID TRANSPORT
697
removed, sliced, and incubated at 20~ in HEPES medium. Because rates of uptake were the same for slices of in vivo-cooled brains as for slices from uncooled brains upon subsequent incubation at 20~ the in vivo cooling was discontinued. For measurements of exit, slices were preloaded with a labeled amino acid, and the exit of this labeled amino acid was measured (9). To minimize the contribution of initial exit from the extracellular fluid, longer incubation times were used for exit than for uptake measurements. Uptake values are expressed as concentrative uptake, i.e., intracellular level minus medium level. When results are expressed in this manner, if only diffusion equilibrium is reached, concentrative uptake is 0; i.e., no concentrative uptake occurs (inhibition is 100%) when uphill transport is inhibited. Since the absolute value of uptake of the various amino acids is different, exit was expressed as percent decrease for easier comparison. For expressing flux, intracellular concentrations are used; tissue levels are corrected to amino acid in the extracellular space (6,10). For measuring rates of amino acid exit from slices after the usual preincubation, the tissue was incubated for 30 rain at 37~C with labeled amino acid; the slices were then gently filtered on Hirsch funnels. A portion of the tissue was frozen, and the radioactivity was determined. Two other portions of the slices were taken; one was added to 5 ml of amino acid-free HEPES medium at 20~ and the other to the same medium at 37~ After 5-20 rain, the medium was filtered from the slices and the radioactivity was determined. A comparison was made of the exit of labeled amino acid from the slices at the two temperatures. Unlabeled amino acids were purchased from either Sigma or Calbiochem, labeled amino acids from New England Nuclear or Calatomic.
RESULTS
In the first set of experiments, slices from either mouse or rat brain were incubated for 30 min. We used both mice and rats, since the in vivo study of temperature dependence of brain uptake index was done with rats (2) and the delineation of transport classes in brain slices was done with mice (11). Uptake approaches steady state after a 30-min incubation (see also Figure 2). In contrast to initial rate of uptake, steady-state uptake is defined as maximal level in tissue, when no more net uptake occurs, i.e., when uptake and exit are equal. Steady-state uptake values (rather than initial rates) indicate changes under most in vivo conditions of hypothermia, when the brain is exposed to the lower temperature for a longer time and the established new steady state is of importance. After the 30-rain incubation, there were significant differences in the temperature dependence of uptake among the various transport classes (Table I). Decrease of uptake with decreasing temperature at high amino acid concentrations was greatest with taurine, followed by small neutral amino acids, glycine, and possibly the ASC (alanine, serine, cysteinepreferring) class (12), basic amino acids (lysine, arginine), GABA, and acidic amino acids (glutamate and aspartate). In this order, the temperature sensitivity of large neutral amino acids (L, leucine-preferring class)
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