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Efflux mechanism of phenylalanine and tryptophan in rat cerebral cortex slices
0306-4522/80:O701-1357502.00,0
Neuroscience Vol. 5, pp. 1357 to 1365 Pergamon Press Ltd 1980. Printed in Great Britain © IBRO
EFFLUX MECHANISM OF PHENYLALANINE A N D T R Y P T O P H A N IN RAT CEREBRAL CORTEX SLICES E. R. KORPI Department of Biomedical Sciences, University of Tampere, Box 607, SF-33101 Tampere 10, Finland Abstraet--Phenylalanine and tryptophan effluxes from rat cerebral cortex slices were studied in small oscillating superfusion cups. The slices superfused for 120 min gained sodium and water and lost potassium only slightly more than those only preincubated for 30 min. There was a linear correlation between the rates of spontaneous efflux of [3Hlphenylalanine and [3H]tryptophan and the phenylalanine or tryptophan contents in the slices. No signs of saturation of the exit were discernible within the amino acid concentration range of 0.1 to 18 mmol per kg slice wet weight. Extracellular histidine, phenylalanine and tryptophan stimulated the efflux of [3H]phenylalanine and I-3H]tryptophan by saturable exchange. Only histidine inhibited the efflux intracellularly, while intracellular phenylalanine and tryptophan seemed to enhance it. The results suggest that the efflux of phenylalanine and tryptophan from brain cells is carriermediated in the presence of other extracellular amino acids but that spontaneous efflux into amino acid-free medium may mainly occur via non-mediated physical diffusion.
EFFLUX mechanisms of amino acids from brain tissue are rather poorly known (see OJA & VAHVELAINEN, 1975). Aromatic amino acids as precursors of catechol and indole amines have, however, attracted more attention than most others. An involvement of carrier-mediated processes in phenylalanine and tryptophan effluxes is suggested by the enhancement of efflux by a number of other extracellular amino acids (BARBOSA, JOANNY & CORRIOL, 1970; CRNtC, HAMMERSTAD t~ CUTLER, 1973; LAAKSO, 1978; KORPI & OJA, 1979a, b). Both amino acids have been similarly affected (KORPI & OJA, 1979a, b) and the same amino acids that enhanced efflux also strongly inhibit influx (VAHVELAINEN• OJk, 1975). If the spontaneous efflux of aromatic amino acids in fact employs the same transport sites as influx, then other amino acids at high intracellular concentrations should inhibit efflux in relation to their structural similarity to phenylalanine and tryptophan molecules. CHRISTENSEN & HANDLOGTEN (1968) have indeed demonstrated inhibition of phenylalanine efflux from Ehrlich carcinoma cells by such an intracellular amino acid which in the influx process shares with phenylalanine the leucinepreferring transport system 'L'. Oddly enough, in our previous studies we have not been able to detect any such behaviour with brain slices (KORPI & OJA, 1979a, b). The intracellular amino acid concentrations attained may possibly have been too small, however, to provoke any gross effects. We have now further pursued our studies on the kinetics of spontaneous efflux of phenylalanine and tryptophan from rat cerebral cortex slices. Particular emphasis has been laid on the possible effects of high intracellular amino acid concentrations on efflux processes. A superfusion apparatus was also employed Abbreviation: HEPES, N-2-hydroxyethyl-l-piperazineN'-2-ethane sulphonic acid.
here in which tissue slices preserve their viability better than earlier. EXPERIMENTAL PROCEDURES Superfusion of cerebral cortex slices Superficial slices from the cerebral cortex (0.5 mm thick) of adult rats trimmed with a non-wetted tissue slicer of Stadie-Riggs type were preloaded for 30min at 310K under 02 in 5 ml of Krebs-Ringer-glucose solution buffered with N-2-hydroxyethyl-l-piperazine-N'-2-ethane sulphonic acid (HEPES) (KORPI & OJA, 1979b) with 4.0 mCi/1. of L-[G-3H]tryptophan or L-[2,6-aH]phenylalanine (SA 1.0 and 57.4 kCi/mol, respectively, The Radiochemical Centre, Amersham) and varying concentrations (generally 0.05 mM) of the corresponding unlabelled amino acids. The slices were separated from the preincubation medium by filtration, rinsed with l0 ml of Krebs-Ringer-HEPES solution (310 K)and transferred into 5 superfusion cups (15-40 mg of slices in each) adjusted to contain 0.25 ml of incubation solution. Superfusion was commenced immediately at a rate of 0.25 ml/min with the aid of two peristaltic pumps (Desaga 132100) installed as shown in Fig. I. The cups were shaken continuously (2 oscillations/s) and the superfusion solution inside vigorously flushed with humified oxygen gas. The superfusate was collected in 2-min fractions by a faction collector (Gilson TDC 220) for determination of radioactivity. After 30 min the superfusion solution was usually exchanged for 10 rain to the same solution supplemented with various unlabelled amino acids. From 40 to 80 min the original superfusion medium was again switched on. In the end the slices were rapidly picked out, weighed, homogenized in 5°~o(w/v) ice-cold trichloroacetic acid, centrifuged, and the radioactivity in the supernatants determined as described (KoRPI & OJA, 1979b). In the above experiments the first superfusion period up to 30min characterized the spontaneous efflux of preloaded [aH] amino acids into the amino acid-free medium. Between 30 and 40 min there occurred exchange of the labelled intracellular amino acid with the unlabelled extracellular amino acid which started to accumulate in the
1357
1358
E.R. KORVl by BLOXMAN• WARREN (1974), and histidine according to H,~KANSSON, R()NNBERG & SJOLUND (1974). Tissue and standard samples and tissue and reagent blanks were all analysed in duplicate.
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Fie. 1. A superfusion cup with its auxiliary apparatus. Superfusion medium is forced into the glass cup at a fixed rate by a pump operating at the plastic inlet tube coiled in a waterbath. The amount of medium in the cup is kept constant by an outlet pump, the pumping efficiency of which is somewhat larger than that of the inlet pump. A flow of moist, warm oxygen is directed to the drops of medium entering the cup. The central hole in the rubber stopper allows oxygen to escape from the cup. slices. From 40 min onwards both amino acids were then released together into amino acid-free medium. The halftime of the medium in the superfusion cups was 1.2 min. This was estimated from the appearance and disappearance of [3H]phenylalanine added to the second superfusion solution in the collected superfusate fractions. In some experiments the superfusion rate was doubled to 0.5 ml/min and the half-time of the medium diminished accordingly. In order to study the spontaneous efflux of amino acids at different intracellular amino acid concentrations, some slices were preloaded with 0.05 to 20.0 mM [3H]tryptophan or [3H]phenylalanine solutions for 30 to 90min, i.e. until an approximate steady state prevailed between amino acid influx and efflux, and then superfused as above for 30 min. Metabolic breakdown of [3H]tryptophan and [3H]phenylalanine in rat cerebral cortex slices has earlier been shown to be negligible during superfusions like the ones used in this work (LAAKSO, 1978; KORPI & OJA, 1979a,b). The 02 saturation of the medium samples from the superfusion cups was measured with a Corning blood gas analyzer (model 165). The partial pressure of oxygen was 49 + 7 kPa (mean + S.D., n = 8). Determination of sodium, potassium and water contents and inulin spaces Sodium, potassium and water contents of the slices were determined as described by KORPI & OJA (1979b). Inulin spaces of the slices were measured after a 30-min superfusion with 1.0 mCi/1. (27 mg/1.) [3H]inulin (SA 380 Ci/kg; The Radiochemical Centre, Amersham). The slices were briefly rinsed with 2 ml of cold (277 K) superfusion solution, extracted with 5 ~ trichloroacetic acid, and the radioactivities of the trichloroacetic acid extracts and the incubation solutions determined as above. Inulin spaces were calculated from these data as indicated by VAHVELAINEN & OJA (•972). Determination of amino acid concentrations Phenylalanine, tryptophan and histidine contents of the slices were determined from trichloroacetic acid extracts of two or three pooled slices with an Aminco-Bowman spectrophotofluorometer by the following methods: phenylalanine according to MCCAMAN & ROBINS (1962), tryptophan according to DENCKLA & DEWEY (1967) as modified
Calculations Effiux rate constants for various superfusion periods were calculated by computer as negative slopes for the regression lines of the logarithms of the remaining proportion ( x 1000 = permillage) of trichloroacetic acid soluble radioactivity in the slices vs superfusion time (see Fig. 5). Correlation coefficients for these straight lines estimated by the method of least squares always exceeded 0.996. The first rate constant (kl) corresponds to the superfusion period from 20 to 30 min. It represents the basic spontaneous effiux rate for each slice. The efflux rates for other periods (k2 for 32-38 min, k3 for 50-60min and k, for 70-80 min) were calculated as percentages of the corresponding control rate, kt, for the same slice. In this way the influences of small variations in spontaneous efflux rates between different slices were eliminated from further comparisons. The efflux rates at varying amino acid concentrations in the slices were calculated in /~mol of [3H] amino acid released in 1 s by 1 kg of incubated slices. These rates were then plotted against the [3HI amino acid concentration in the slices at the superfusion time under study (10 or 24min). Intracellular water/medium ratios (A) of [3H] amino acids at the steady state were calculated from the equation A = (C - Es)/s(1 - E - D), in which C indicates the [3H] amino acid content in mol per kg slice wet weight, s the [SH] amino acid concentration in the incubation medium (mol/1.), E the extracellular (inulin) space (l/kg slice wet weight) and D the proportion of the dry weight of the slices (kg/kg). Student's t-test was used to assess the statistical significance of all observed differences. RESULTS Stability o f slices during superfusions Cation and water contents of the slices were altered significantly (P < 0.01) already during the preloading period (Table 1). During superfusions the slices gained some sodium a n d water a n d lost potassium. The changes were slow, however, the cation and water contents of the slices changed significantly (P < 0.05) only after the 2-h superfusion. Extracellular (inulin) space likewise increased somewhat during superfusions. N o statistically significant differences in any of the measured parameters were discernible between the slices superfused for 30, 60 or 120 min. Spontaneous efflux o f phenylalanine and tryptophan The intracellular/extracellular concentration ratios of [ a H ] p h e n y l a l a n i n e and [ a H ] t r y p t o p h a n at steadystate condition (i.e. the concentrative capacities of the slices) were inversely proportional to the a m i n o acid concentrations in the preloading solution (Fig. 2). T r y p t o p h a n was more effectively concentrated (P < 0.01) than phenylalanine when the a m i n o acid concentrations in the preloading medium were low.
Amino acid efflux mechanisms
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TABLE 1. SODIUM, POTASSIUM AND WATER CONTENTS AND INULIN SPACES OF SUPERFUSED RAT CEREBRAL CORTEX SLICES
Treatment of slices Unincubated Preloaded for 30 min Preloaded for 30 min and --superfused for 30 min --superfused for 60 min --superfused for 120min
Sodium content (mmol/kg wet wt)
Potassium content (mmol/kg wet wt)
Water content (kg/kg dry wt)
Inulin space (1/kg wet wt)
60.6 + 5.7(11) 81.6 + 8.5(8)
84.0 + 6.4(11) 66.3 + 7.5(8)
4.62 + 0.21(6) 5.76 + 0.22(9)
0.43 + 0.07(7)
88.9 + 1.6(4) 88.3 + 2.6(4) 93.0 + 4.2(6)*
57.4 + 6.9(4) 59.3 + 4.9(4) 54.3 + 5.3(6)*
6.06 ___0.42(9) 6.00 + 0.36(8) 6.10 + 0.32(8)*
0.46 ___0.10(5) 0.49 ___0.13(5) 0.54 + 0.09(5)*
Rat cerebral cortex slices were preloaded with unlabelled phenylalanine or tryptophan (0.05 mM) and superfused for times indicated as described in Experimental Procedures. Cation contents were measured by atomic absorption from 2 to 3 separately superfused and then pooled slices, and water content and inulin space were estimated from single slices. The results (means + S.D.) are given per final wet or dry weight of slices. Number of experiments in brackets. Significance of the differences between superfused and only preloaded slices: * P < 0.05.
At 10raM concentrations there was no longer a significant difference, however. The ettlux rates of phenylalanine and tryptophan from these preloaded slices into amino acid-free medium were greatest during the first 2-6 min of superfusion. This fast phase rapidly attenuated. The efflux rates of both amino acids after 10- and 24-min superfusions increased apparently linearly with the amino acid content of the slices (Figs 3 and 4). Phenylalanine etflux was more than two times faster than tryptophan efflux. Doubling the pumping rate of the superfusate had no apparent effect on [3H]phenylalanine efflux. I-3H] Amino acids preloaded in the slices were released at a continuously diminishing rate into the amino acid-free medium during the 80-min superfusions when the slices were gradually depleted of amino acids (Tables 2 and 3, Figs 5 and 6). Another general rule was also established when the behaviour of individual slices upon superfusion was observed: the slower the efflux during the initial spontaneous efflux period (20-30min), the slower was it in the
same slice also during other subsequent superfusion periods.
Extracellular effects of amino acids Figures 5 and 6 show representative examples of the effects of unlabelled amino acids on the effluxes of [3H]phenylalanine and [3H]tryptophan. Amino acids added to the superfusion medium invariably caused stimulatory trans-effects on the efflux characterized by the rate constant k2 for this phase. Phenylalanine, tryptophan and histidine stimulated the efflux of [3H]phenylalanine in a concentration-dependent manner, even if the stimulation by histidine was less pronounced (Table 2). An addition of 5 mM phenylalanine and 5 mM histidine together caused no greater effect than phenylalanine (5 or 10 rnM) alone. The efflux of [3H]tryptophan was stimulated most by extracellular 10 mM tryptophan (Table 3). The stimulation percentage was even larger than the corresponding stimulation in the efflux of [3H]phenylalanine, obviously because of the slower spontaneous efflux rate of [3H]tryptophan. Otherwise the results were very similar to those with [3H]phenylalanine. Histidine was again the least potent stimulator.
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Intracellular effects of amino acids
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5 10 /'~ MEDIUM AMINO ACID CONCENTRATION mM
20
FIG. 2. Accumulation ratios of [3H]phenylalanine and [3H]tryptophan in rat cerebral cortex slices at varying amino acid concentrations in the incubation medium. The slices were incubated at 310K under 02 with labelled phenylalanihe (0) or tryptophan (O) for 30 to 90 min, i.e. until an equilibrium was reached between the intracellular and extracellular I-3H] amino acid concentrations. The accumulation ratios, calculated as described in Experimental Procedures, are given as means -b S.D.S of 4 to 8 experiments.
Figures 5 and 6 also depict the effects of the accumulated unlabelled amino acids on the efflux of phenylalanine and tryptophan during later superfusion phases when the medium was again amino acidfree and most of the extracellular amino acids had also been washed out of the slices. These cis-effects by the intracellularly accumulated amino acids were either stimulatory or inhibitory. They are characterized by the rate constants designated by k 3 (50-60 min) and k4 (70-80 min). [3H]Phenylalanine efflux between 50 to 60 min was enhanced by intracellular phenylalanine, and to a lesser extent by tryptophan (Table 2). Histidine, however, decreased the efflux. [3H]Tryptophan efflux (50-60 and 70-80 min) was also significantly enhanced by intracellular phenylalanine and diminished by histidine (Table 3). These intracellular influences were clearly related to the concentrations of unlabelled
1360
E.R. KORPI
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2 4 6 SLICE PHENYLALANINE CONTENT m m ~ / k 9
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FIG. 3. Rate of [3H]phenylalanine effiux from rat cerebral cortex slices as a function of [3H]phenylalanine content of the slices. The slices were preloaded with 4.0 mei/l L-[3H]phenylalanine (0.05-20.0 mM) for 30 to 90 rain and superfused (0.25 ml/min) for 30 rain. The specific radioactivity of [3H]phenylalanine in the preloading medium, the radioactivity ultimately remaining in the slices, outlet tubes and superfusion cups was used for calculation of the etttux rates and slice concentrations of [3H]phenyla!anine at 10 rain as shown in the larger graph. The smaller graph depicts similarly calculated [3H]phel~? lalanine efllux rates and slice concentrations after the 24-min superfusion. The regression lines through the experimental points were calculated by the method of least squares, their equations and correlation coefficients are in the Figure. phenylalanine, histidine and tryptophan taken up and retained by the slices towards the end of superfusions (Table 4). The less an accumulated amino acid was retained, the faster was the efflux of [3H] amino acids. Although histidine was retained much more efficiently t h a n phenylalanine, the levels of both these accumulated amino acids significantly exceeded the levels of preloaded [3H] amino acids (Table 4). Superfusion of the slices with 5 mM phenylalanine and 5 mM histidine together largely abolished the intracellular effects of
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both amino acids (Tables 2 and 3). The concentrations of phenylalanine and histidine in these experiments were both so high that they would have exerted a profound effect on the efflux if present in the slices alone (Table 4). The amounts of free [3H]phenylalanine and [3H]tryptophan left in the slices after superfusions also reflected the effects of other amino acids on the efflux (Tables 2 and 3). Incorporation of amino acids into proteins. Labelled
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FIG. 4. Rate of [3H]tryptophan efflux from rat cerebral cortex slices as a function of [3H]tryptophan content of the slices. The concentration range of tryptophan in the preloading medium was 0.05-10.0 mM. See the legend to Fig. 3 for other experimental details. The larger graph shows the rates after the 10-rain superfusion and the smaller graph after the 24-min superfusion.
Amino acid efflux mechanisms
1361
TABLE 2. EFFLUX AND EXCHANGE OF [3H']PHENYLALANINE IN RAT CEREBRAL CORTEX SLICES AS INFLUENCED BY VARIOUS CONCENTRATIONS OF UNLABELLED EXTRA- AND INTRACELLULAR PHENYLALANINE, TRYPTOPHAN AND HISTIDINE
Amino acid in superfusion medium from 30 to 40 min
No. of expts
kl (x 10 -3 min -1)
None (control) 10.0 mM Phe 5.0mM Phe 0.05 mM Phe 10.0mM His 5.0mM His 1.0mM His 0.05 mM His 5.0mM Phe + 5.0mM His 10.0mM Trp 0.05 mM Trp
17 9 5 4 7 6 7 6 5 8 4
16.9 + 2.6 17.2 ___1.7 16.4,-_0.7 18.3 +- 3.2 17.7 +- 1.6 18.5 + 3.0 16.9 + 3.8 18.7 +- 3.8 15.9 +- 1.5 16.4 + 1.5 17.9 _+ 3.3
Effiux rate constants k2 k3 (% of kl) (% of k0
k4 (% ofkt)
92 ___5 426 ___62t 345 + 43t 135 ___6]" 248 ___27t 242 _+_38]" 176 +_ 12]" 108 +- 5t 379 +- 48]" 432 ___49]" 123 + 8]"
78 + 13 78 ___23 89+10 85 +- 8 32 +__7"1" 37 + 7"t 55 +- l i t 78 + 9 81 +_. 13 73 + 12 77 + 10
85 135 155 92 34 39 57 81 98 102 84
___6 ___ 19tl" + 10t ___6 + 5t ___ 13t __. 9t __+4 +- 11" + 13t ___5
Per cent of radioactivity left at the end of superfusion 5.3 0.7 0.8 3.4 5.2 4.9 5.4 3.9 1.6 1.2 4.2
+ 1.9 + 0.2]" ___0.2t +- 1.8 +- 1.7 + 1.9 ___2.7 -4- 2.1 +- 0.2]" + 0.2t + 1.9
Experimental details as in Fig. 5. Means + S.D. are given, k2, k3 and k4 are expressed as percentages of k~ of the same slice. Significance of the differences between control and other groups: * P < 0.05; ]. P < 0.001. TABLE 3.
EFFLUX AND EXCHANGE OF [3H]TRYPTOPHAN IN RAT CEREBRAL CORTEX SLICES AS INFLUENCED BY VARIOUS CONCENTRATIONS OF UNLABELLED EXTRA- AND INTRACELLULAR PHENYLALANINE, TRYPTOPHAN AND HISTIDINE
Amino acid in superfusion medium from 30 to 40 rain
No. of expts
(x 10 -3 min -1)
None (control) 10.0 mM Trp 10.0 mM Phe 10.0 mM His 1.0mM His 5.0mM Phe + 5.0mM Hi's
10 8 5 7 7 7
8.95 + 1.10 9.36 ___ 1.22 9.64 + 1.63 9.82 ___ 1.87 9.61 +__0.89 9.90 ___ 1.42
kl
Effiux rate constants k2 k3 (% of kl) (% ofkx)
k4 (% of kl)
97 + 5 632 + 81t 484 ___61"f" 355 __+64t 262 ___42t 466 + 48t
83 83 130 30 61 63
91 96 199 36 63 86
___8 ___ 11 -t- 31"1" -I- 6t ___ 12t + 18
Per cent of radioactivity left at the end of superfusion
+- 13 ___9 ___31" + 4t + 4t + 17"
19.9 + 5.3 5.2 ___ 1.3"t" 3.9 ___ 1.8t 17.7 + 4.1 16.2 + 2.1 7.9 + 2.5t
Experimental details as in Fig. 6. Means _+ S.D.S are given, k2, k 3 and k4 are expressed as percentages of kl of the same slice. Significance of the differences between control and other groups: * P < 0.05; ";"P < 0.001. [fl o
phenylalanine and tryptophan molecules were also incorporated into proteins in the slices during the ex-
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SUPERFUSION TIME MIN
FIG. 5. Effects of other amino acids on the efflux of [aH]phenylalanine from rat cerebral cortex slices. Slices were preloaded for 30min with L-[3H]phenylalanine (4.0 mCi/1., 0.05 mM) and superfused for 30 min with amino acid-free medium. The medium was then changed for 10min either to the same medium (e) or to that supplemented with 10 mM phenylalanine (A), 10 mM tryptophan (A) or 10raM histidine (O). The effiux curves shown are from representative single experiments. The regression lines fitted to the lowermost curve illustrate estimation of the efflux rate constants. NSC. 5/7--L
~ ~ 40
60
80
SUPERFUSlON TIME MIN
F1G. 6. Effects of other amino acids on the efflux of [aH]tryptophan from rat cerebral cortex slices. The slices were preloaded with L-[3H]tryptophan (4.0mCi/I., 0.05 mM). Otherwise the experimental details and symbols are as in Fig. 5.
1362
E.R. KORPI
alanine at the end of some superfusions. Protein synthesis does not seem to be an important factor influencing amino acid efltux rates, however, because only a small fraction of the labelled free amino acids which had been present at the beginning of superfusions were eventually incorporated into proteins. Already after the 30-rain preloading period 1.8 ± 0.3 and 0.5 ± 0.2°~, (mean ± S.D., n = 6) of [3H]phenytalanine and [3H]tryptophan, respectively, in the slices had been incorporated into protein. Unlabelled 10raM phenylalanine and tryptophan significantly diminished the amount of label incorporated during superfusions, apparently in part owing to the enhanced efflux of the [3HI amino acids from the slices (Table 5). DISCUSSION
Superfusion of brain slices The paucity of relevant studies on amino acid efflux from brain slices results partially no doubt from methodological difficulties encountered. The present superfusion apparatus, a modification from that of Laakso (1978), appears to be more advantageous than the system we have previously used (a stationary slice in a flow of preoxygenated medium, KORPI & OJA, 1979a). The slices were well preserved in an approximate steady state with regard to their cation and water contents for at least 2 h, which time appears reasonably long enough for efflux studies. Sodium and potassium contents, swelling and inulin spaces of the slices now superfused for up to 120 min agree well
with the data reported earlier for cerebral cortex slices incubated under the best standard conditions (BAcnEt,ARD, CAMPBELL & MCILWAJN, 1962; JOANNY & HILLMAN, 1963: FRANCK, CORNETTE 8L SCHOFFENIELS, 1968). The superfusion medium in the cups could be rapidly renewed and reuptake of the label by the slices thus minimized. Since doubling of the superfusion rate did not influence effiux, the apparatus itself obviously does not in any way limit efflux rates.
Mode ~1" spontaneous efflux q] phenylalanine and tryptophan An apparent linear relationship was found between the efflux rates of phenylalanine and tryptophan and their intracellular concentrations in cerebral cortex slices. Diffusion of the label residing in the extracellular spaces after preloading cannot be responsible for this phenomenon, since the half-time for even inulin efflux from this compartment is less than 5rain (LuND-ANDERSEN, 1974), and the present actual studies were made after a much longer lag period. Several more plausible explanations can be found. Firstly, efflux could in fact occur through any nonsaturable mechanism, like simple physical diffusion through pores in plasma membranes. Secondly, efflux may be a mediated process whose true nature remains undetected. It could thus take place if diffusion of amino acid molecules out of the extracellular spaces of the slices is slow compared to their transfer across cell membranes. The heterogeneity of cellular elements and the tortuosity of the extracellular spaces in slices render determination of the rate-limiting step
TABLE 4. PHENYLALANINE, TRYPTOPHAN AND HISTn)INE CONTENTS OF SUPERFUSEI) RAT CEREBRAL CORTEX SLICES
Amino acid from 30 to 40 min in superfusion medium
Amino acid content (mmol/kg slice wet weight) at 55 min at 75 rain
Slice/medium concentration ratio
Phenylalanille None 10.0mM Phe 5.0mM Phe + 5.0mM His
0.10 ± 0.01(4J 0.63 + 0.18(3j 1.7 ± 0.1(41
None 10.0 mM Trp
0.05 ± 0.01(41 1.2 + 0.2(4}
0.05 mM His 1.0 mM His 10.0raM His 5.0mM Phe + 5.0 mr~ His
0.25 + 0.06(4) 2.4 + 0.5(4) 10.8 ± 1.7(6) 7.3 ___ 1.1(4)
0.08 ___0.01(4) 0.23 + 0.06(4) 0.87 _+ 0.16(4)
1130 270 390
0.04 ± 0.01(4) 0.45 ___0.12(4)
1t30 270
0.23 1.5 7.1 4.6
3000 540 610 550
Tryptophan
Hist ±dine +_ 0.03(4) + 0.2(4) ± 1.7(4) +__0.7(4)
The slices were preloaded for 30 min with unlabelled phenylalanine or tryptophan (0.05 mM) and superfused for 30 min with amino acid-free medium, from 30 to 40 min with the medium supplemented with unlabelled amino acids as indicated, and from 40 min onwards again with amino acid-free medium. The slices were harvested at the time periods indicated, pooling 2 or 3 separately superfused slices for each analysis. Means +_ S.D.S are given and the number of experiments in brackets. Average slice/medium concentration ratios between 55 and 75 min were calculated on the assumption that the amino acids released from the slices immediately mix with the medium in the superfusion cups.
1363
Amino acid efflux mechanisms TABLE 5. INCORPORATION OF [3H']PHENYLALANINE AND ['3H'ITRYPTOPHAN INTO PROTEINS DURINGSUPERFUSIONEXPERIMENTS
Amino acid in superfusion medium from 30 to 40 min
No. of expts
Radioactivity incorporated into protein at the end of superfusions as percentage of trichloroacetic acid soluble radioactivity Initial Final
[ 3H]Phenylalanine efflux None (control) 10rnM Phe 10 mM Trp 10 mM His
6 9 7 7
3.5 + 2.1 + 2.9 + 3.7 +
0.9 0.6* 0.7 1.3
None (control) 10 mM Trp 10mM Phe 10 mM His
I-3H]Tryptophan efflux 9 1.2 _ 0.3 6 0.6 _ 0.02* 5 0.6 + 0.1" 6 0.9 + 0.2
55 + 352 _ 268 + 72 +
18 145" 85* 19
6.1 + 11.9 + 20.3 + 5.4 +
1.5 1.5" 12.7' 0.5
Experimental design as in Figs 5 and 6. Protein in the trichloroacetic acid-insoluble pellets of slices was purified as described in detail by DUNLOP,VAN ELDEN& LAJTHA (1975), solubilized in 1 N NaOH, neutralized with HCI and measured for radioactivity as indicated in Experimental Procedures. Means + S.D.S are given. Significanceof differences from controls: * P < 0.01.
difficult, since the extrusion rate via a hypothetical high-affinity transport system may well exceed the rate of net diffusion out of the extracellular spaces (see LUND-ANDERSEN & KJELDSEN, 1976), and we are measuring only the eventual appearance of the labelled molecules in the medium. The rate of diffusion out of the extracellular spaces was not, however, a likely rate-limiting step in efflux in the present experiments, because extracellularly added unlabelled amino acids were able to produce quickly beginning and ending increments in efflux by exchange. Because efllux could not be studied reliably at very low preloading amino acid concentrations owing to dilution of the label by endogenous amino acids, mediated processes in the spontaneous efflux could possibly escape detection if they become saturated at very low intracellular amino acid concentrations. We do not see, however, any teleological reason for the existence of such a high-affinity efflux system in brain cells. The detection of saturability of the spontaneous e~ux may also be hampered by stimulation of intracellular amino acid efflux by exchange with amino acid molecules already released into the extracellular spaces of the slices, if this exchange does not operate with a one-to-one ratio. Leucine-valine exchange is such a case, as one leucine molecule taken up by the brain slice may cause the release of three to four valine molecules (BATTISTIN, PICCOLI & LAJTHA, 1972). Let us provisionally assume that the spontaneous efflux of phenylalanine and tryptophan occurs through mediated saturable mechanisms in addition to possible diffusion. The influx of phenylalanine and tryptophan into brain cortex slices has been shown to be saturable (VAHvELAINEN • OJA, 1972). The present efflux rates were smaller than thc reported influx rates
at all amino acid concentrations. This relationship makes the concentrative uptake possible. If diffusion and translation of the amino acid carriers in the cell membranes occurred at equal rates in both directions, the magnitude of the concentrative uptake would then be determined by the ratio of the transport constants of mediated influx and efflux (see OJA & VABVELAINEN, 1975). Even at concentrations that almost saturate mediated influx, phenylalanine and tryptophan are to some extent concentrated by brain slices. Accumulation ratios are then about 2 and 5 for phenylalanine and tryptophan respectively (Fig. 2; BLASBERG & LAJTHA, 1965; KIELY & SOURKES, 1972). The transport constant for influx of both amino acids are about 0.5 mM (VAHVELAINEN& OJA, 1972; 1975; KIELY & SOURKES, 1972). The estimated transport constants for emux should then be about 1 to 3 mM. We did not now observe any signs of saturation in phenylalanine and tryptophan efflux, although the highest concentrations studied were several-fold greater. This outcome is distinctly at variance with the assumption posited above. Keeping in mind all methodological reservations outlined above we must state that no positive indications of saturable mediated processes were found in the spontaneous efflux of phenylalanine and tryptophan from cerebral cortex slices. This is at variance with the observations of LEVI, BLASBERG& LAJTnA (1966), who report an auto-activation of phenylalanine and leucine efttux from mouse brain slices by some unknown mechanisms and a saturable component in the efflux of ~t-aminoisobutyric acid, lysine and D-glutamate.
Efflux in the presence of other amino acids Trans-stimulation of efflux has been proposed to
1364
E.R. KORPI
result from increased exchange of intracellular [3HI amino acids with extracellular unlabelled amino acids through common transport sites at cell membranes, since the substrate specificities of phenylalanine and tryptophan exchange and influx are identical (KORPI & OJA, 1979a, b). The present study also reveals saturation of such a stimulation by high extracellular amino acid concentrations. The data are not complete enough for a precise estimation of how large a concentration causes extracellularly the half-maximal stimulation, but a reasonable estimate for histidine in phenylalanine efflux would be about 1 mM (Table 2). This figure matches well with the estimated transport constant in influx for histidine itself (VAHVELAINEN& OJA, 1972; 1975). The present effects of intracellular histidine, phenylalanine and tryptophan on the efflux of [aH]phenylalanine and I-3H]tryptophan were quite similar to those reported earlier either by us (KORPI & OJA, 1979a, b) or others (Levl et al., 1966). The inhibition of efflux by histidine may result from competition for intracellular transport sites, but the apparent lack of homo-cis-inhibition in efflux is then more difficult to understand. A number of other explanations can be offered, remembering also the structural complexity of brain slices: (1) Histidine may by exchange processes recapture [3HI amino acid molecules already released
into the extracellular spaces and thus decrease e~ux, since it is also more effectively concentrated by the slices than for instance phenylalanine (Table 4). Phenylalanine, in turn, may be released into the extracellular spaces and from there accelerate eftiux by exchange with intracellular [aH] amino acids. This suggestion is also in accordance with the rapid exchangeability of large neutral amino acids across the blood-brain barrier in vivo (OLDENDORF, 1971; TOVH & LAJTHA, 1977). The high slice/medium concentration ratios of unlabelled histidine and phenylalanine (Table 4) do not necessarily undermine this possibility, since the efflux from the intracellular spaces to medium must mostly occur through the extracellular spaces. (2) The distribution of orientation of 'acceptor' sites of membrane carriers towards extra- and intracellular sides may be differently affected by intracellular histidine and phenylalanine. (3) The presence or fluxes of histidine and phenylalanine may have opposite, still unidentified effects on metabolic reactions supplying energy for amino acid transport. excellent technical assistance of Miss IRMA SALMINEN, Miss EUA KYR6L,~ and Mr ARIPEKKA VIIRTEL.~ is gratefully acknowledged. The author thanks Dr S. S. OJA for reviewing the manuscript. Acknowledgements--The
REFERENCES BACHELARDH. S., CAMPBELLW. J. & MCILWAIN H. (1962) The sodium and other ions of mammalian cerebral tissues, maintained and electrically stimulated in vitro. Biochem. J. 84, 225-232. BARBOSAE., JOANNYP. & CORRIOLJ. (1970) Accumulation active du tryptophane duns le cortex ~r6bral isol6 du rat. C.r. S6anc. Soc. Biol. 164, 345-350. BATTIST1NL., PICCOLIF. & LAJTHAA. (1972) Heteroexchange of amino acids in incubated slices of brain. Archs Biochem. Biophys. 151, 102-t 11. BLASBERGR. & LAJTHAA. (1965) Substrate specificity of steady-state amino acid transport in mouse brain slices. Archs Biochem. Biophys. 112, 361-377. BLOXAMD. L. & WARRENW. H. (1974) Error in the determination of tryptophan by the method of Denckla and Dewey. A revised procedure. Anal. Biochem. 60, 621-625. CHRISTENSENH. N. • HANDLOGTENM. E. (1968) Modes of mediated exodus of amino acids from the Ehrlich ascites tumor cells. J. biol. Chem. 243, 5428-5438. CRNIC D. M., HAMMERSTADJ. P, ~¢ CUTLER R. W. P. (t973) Accelerated efflux of [14C] and [3H]amino acids from superfused slices of rat brain. J, Neurochem. 20, 203-209. DENCKLAW. D. & DEWEYH. K. (1967) The determination of tryptophan in plasma, liver, and urine. J. Lab. clin. Med. 69, 160-169. DUNLOP D. S., VAN ELDEN W. & LAJTHAA. (1975) Optimal conditions for protein synthesis in incubated slices of rat brain. Brain Res. 99, 303-318. FRANCK G., CORNETTEM. & SCHOFFENIELSE. (1968) The cationic composition of incubated cerebral cortex slices. J. Neurochem. 15, 843-857. H,~KANSON R., R()NNBERGA. L. ~, SJOLUNDK. (1974) Improved fluorometric assay of histidine and peptides having NH2-terminal histidine using o-phthalaidehyde. Anal. Biochem. 59, 98-109. JOANNYP. & HILLMANH. H. (1963) Substrates and the potassium and sodium levels of guinea pig: cerebral cortex slices in vitro: Effects of application of electrical pulses, of inhibitors and of anoxia. J. Neurochem. 10, 655-664. KIELY M. ~,~ SOURKEST. L. (1972) Transport of L-tryptophan into slices of rat cerebral cortex. J. Neurochem. 19, 2863-2872. KORPI E. R. & OJA S. S. (1979a) Efltux of phenylalanine from rat cerebral cortex slices as influenced by extra- and intracellular amino acids. J. Neurochem. 32, 789-796. KORPI E. R. & OJA S. S. (1979b) Exchange and efflux of tryptophan from superfused cerebral cortex slices. Expl Brain Res. 35, 99-106. LAAKSOM.-L. (1978) Efflux of phenylalanine and tryptophan from cerebral cortex slices of adult and 7-day-old rats. Acta physiol, scand. 102, 74-83.
Amino acid effiux mechanisms
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L~vl G., Bt.ASn~G R. & LAJTHA A. (1966) Substrate specificity of cerebral amino acid exit in vitro. Archs Biochem. Biophys. 114, 339-351. LUND-ANDERS~NH. (1974) Extracellular and intracellular distribution of inulin in rat brain cortex slices. Brain Res. 65, 239-254. LUND-AND~,SENH. & IOELDS~NC. S. (1976) Uptake of glucose analogues by rat brain cortex slices: a kinetic analysis based upon a model. J. Neurochem. 27, 361-368. M c C A ~ M. W. & ROBINSE. (1962) Fluorimetric method for the determination of phenylalanine in serum. J. Lab. clin Med. 59, 885-890. OJA S. S. & V~VEt~IN~N M.-L. (1975) Transport of amino acids in brain slices. In Research Methods in Neurochemistry, Vol. 3 (eds MARKSN. & RODNIOHTR.), pp. 67-137. Plenum Press, New York. OLDENDORVW. H. (1971) Brain uptake of radiolabelled amino acids, amines, and hexoses after arterial injection. Am. J. Physiol. 221, 1629-1639. TOTH J. & LAJTHAA. (1977) Rates of exchange of free amino acids between plasma and brain in mice. Neurochem. Res. 2, 149-160. VAHVELAXNENM.-L. & OJA S. S. (1972) Kinetics of influx of phenylalanine, tyrosine, tryptophan, histidine and leucine into slices of brain cortex from adult and 7-day-old rats. Brain Res. 40, 477-488. VAHWLAINENM.-L. & OJA S. S. (1975) Kinetic analysis of phenylalanine-induced inhibition in the saturable influx of tyrosine, tryptophan, leucine and histidine into brain cortex slices from adult and 7-day-old rats. J. Neurochem. 24, 885-892. (Accepted 12 February 1980)
Neuroscience Vol. 5, pp. 1357 to 1365 Pergamon Press Ltd 1980. Printed in Great Britain © IBRO
EFFLUX MECHANISM OF PHENYLALANINE A N D T R Y P T O P H A N IN RAT CEREBRAL CORTEX SLICES E. R. KORPI Department of Biomedical Sciences, University of Tampere, Box 607, SF-33101 Tampere 10, Finland Abstraet--Phenylalanine and tryptophan effluxes from rat cerebral cortex slices were studied in small oscillating superfusion cups. The slices superfused for 120 min gained sodium and water and lost potassium only slightly more than those only preincubated for 30 min. There was a linear correlation between the rates of spontaneous efflux of [3Hlphenylalanine and [3H]tryptophan and the phenylalanine or tryptophan contents in the slices. No signs of saturation of the exit were discernible within the amino acid concentration range of 0.1 to 18 mmol per kg slice wet weight. Extracellular histidine, phenylalanine and tryptophan stimulated the efflux of [3H]phenylalanine and I-3H]tryptophan by saturable exchange. Only histidine inhibited the efflux intracellularly, while intracellular phenylalanine and tryptophan seemed to enhance it. The results suggest that the efflux of phenylalanine and tryptophan from brain cells is carriermediated in the presence of other extracellular amino acids but that spontaneous efflux into amino acid-free medium may mainly occur via non-mediated physical diffusion.
EFFLUX mechanisms of amino acids from brain tissue are rather poorly known (see OJA & VAHVELAINEN, 1975). Aromatic amino acids as precursors of catechol and indole amines have, however, attracted more attention than most others. An involvement of carrier-mediated processes in phenylalanine and tryptophan effluxes is suggested by the enhancement of efflux by a number of other extracellular amino acids (BARBOSA, JOANNY & CORRIOL, 1970; CRNtC, HAMMERSTAD t~ CUTLER, 1973; LAAKSO, 1978; KORPI & OJA, 1979a, b). Both amino acids have been similarly affected (KORPI & OJA, 1979a, b) and the same amino acids that enhanced efflux also strongly inhibit influx (VAHVELAINEN• OJk, 1975). If the spontaneous efflux of aromatic amino acids in fact employs the same transport sites as influx, then other amino acids at high intracellular concentrations should inhibit efflux in relation to their structural similarity to phenylalanine and tryptophan molecules. CHRISTENSEN & HANDLOGTEN (1968) have indeed demonstrated inhibition of phenylalanine efflux from Ehrlich carcinoma cells by such an intracellular amino acid which in the influx process shares with phenylalanine the leucinepreferring transport system 'L'. Oddly enough, in our previous studies we have not been able to detect any such behaviour with brain slices (KORPI & OJA, 1979a, b). The intracellular amino acid concentrations attained may possibly have been too small, however, to provoke any gross effects. We have now further pursued our studies on the kinetics of spontaneous efflux of phenylalanine and tryptophan from rat cerebral cortex slices. Particular emphasis has been laid on the possible effects of high intracellular amino acid concentrations on efflux processes. A superfusion apparatus was also employed Abbreviation: HEPES, N-2-hydroxyethyl-l-piperazineN'-2-ethane sulphonic acid.
here in which tissue slices preserve their viability better than earlier. EXPERIMENTAL PROCEDURES Superfusion of cerebral cortex slices Superficial slices from the cerebral cortex (0.5 mm thick) of adult rats trimmed with a non-wetted tissue slicer of Stadie-Riggs type were preloaded for 30min at 310K under 02 in 5 ml of Krebs-Ringer-glucose solution buffered with N-2-hydroxyethyl-l-piperazine-N'-2-ethane sulphonic acid (HEPES) (KORPI & OJA, 1979b) with 4.0 mCi/1. of L-[G-3H]tryptophan or L-[2,6-aH]phenylalanine (SA 1.0 and 57.4 kCi/mol, respectively, The Radiochemical Centre, Amersham) and varying concentrations (generally 0.05 mM) of the corresponding unlabelled amino acids. The slices were separated from the preincubation medium by filtration, rinsed with l0 ml of Krebs-Ringer-HEPES solution (310 K)and transferred into 5 superfusion cups (15-40 mg of slices in each) adjusted to contain 0.25 ml of incubation solution. Superfusion was commenced immediately at a rate of 0.25 ml/min with the aid of two peristaltic pumps (Desaga 132100) installed as shown in Fig. I. The cups were shaken continuously (2 oscillations/s) and the superfusion solution inside vigorously flushed with humified oxygen gas. The superfusate was collected in 2-min fractions by a faction collector (Gilson TDC 220) for determination of radioactivity. After 30 min the superfusion solution was usually exchanged for 10 rain to the same solution supplemented with various unlabelled amino acids. From 40 to 80 min the original superfusion medium was again switched on. In the end the slices were rapidly picked out, weighed, homogenized in 5°~o(w/v) ice-cold trichloroacetic acid, centrifuged, and the radioactivity in the supernatants determined as described (KoRPI & OJA, 1979b). In the above experiments the first superfusion period up to 30min characterized the spontaneous efflux of preloaded [aH] amino acids into the amino acid-free medium. Between 30 and 40 min there occurred exchange of the labelled intracellular amino acid with the unlabelled extracellular amino acid which started to accumulate in the
1357
1358
E.R. KORVl by BLOXMAN• WARREN (1974), and histidine according to H,~KANSSON, R()NNBERG & SJOLUND (1974). Tissue and standard samples and tissue and reagent blanks were all analysed in duplicate.
HII
I
I,
4-1--t ,I [3
I
Fie. 1. A superfusion cup with its auxiliary apparatus. Superfusion medium is forced into the glass cup at a fixed rate by a pump operating at the plastic inlet tube coiled in a waterbath. The amount of medium in the cup is kept constant by an outlet pump, the pumping efficiency of which is somewhat larger than that of the inlet pump. A flow of moist, warm oxygen is directed to the drops of medium entering the cup. The central hole in the rubber stopper allows oxygen to escape from the cup. slices. From 40 min onwards both amino acids were then released together into amino acid-free medium. The halftime of the medium in the superfusion cups was 1.2 min. This was estimated from the appearance and disappearance of [3H]phenylalanine added to the second superfusion solution in the collected superfusate fractions. In some experiments the superfusion rate was doubled to 0.5 ml/min and the half-time of the medium diminished accordingly. In order to study the spontaneous efflux of amino acids at different intracellular amino acid concentrations, some slices were preloaded with 0.05 to 20.0 mM [3H]tryptophan or [3H]phenylalanine solutions for 30 to 90min, i.e. until an approximate steady state prevailed between amino acid influx and efflux, and then superfused as above for 30 min. Metabolic breakdown of [3H]tryptophan and [3H]phenylalanine in rat cerebral cortex slices has earlier been shown to be negligible during superfusions like the ones used in this work (LAAKSO, 1978; KORPI & OJA, 1979a,b). The 02 saturation of the medium samples from the superfusion cups was measured with a Corning blood gas analyzer (model 165). The partial pressure of oxygen was 49 + 7 kPa (mean + S.D., n = 8). Determination of sodium, potassium and water contents and inulin spaces Sodium, potassium and water contents of the slices were determined as described by KORPI & OJA (1979b). Inulin spaces of the slices were measured after a 30-min superfusion with 1.0 mCi/1. (27 mg/1.) [3H]inulin (SA 380 Ci/kg; The Radiochemical Centre, Amersham). The slices were briefly rinsed with 2 ml of cold (277 K) superfusion solution, extracted with 5 ~ trichloroacetic acid, and the radioactivities of the trichloroacetic acid extracts and the incubation solutions determined as above. Inulin spaces were calculated from these data as indicated by VAHVELAINEN & OJA (•972). Determination of amino acid concentrations Phenylalanine, tryptophan and histidine contents of the slices were determined from trichloroacetic acid extracts of two or three pooled slices with an Aminco-Bowman spectrophotofluorometer by the following methods: phenylalanine according to MCCAMAN & ROBINS (1962), tryptophan according to DENCKLA & DEWEY (1967) as modified
Calculations Effiux rate constants for various superfusion periods were calculated by computer as negative slopes for the regression lines of the logarithms of the remaining proportion ( x 1000 = permillage) of trichloroacetic acid soluble radioactivity in the slices vs superfusion time (see Fig. 5). Correlation coefficients for these straight lines estimated by the method of least squares always exceeded 0.996. The first rate constant (kl) corresponds to the superfusion period from 20 to 30 min. It represents the basic spontaneous effiux rate for each slice. The efflux rates for other periods (k2 for 32-38 min, k3 for 50-60min and k, for 70-80 min) were calculated as percentages of the corresponding control rate, kt, for the same slice. In this way the influences of small variations in spontaneous efflux rates between different slices were eliminated from further comparisons. The efflux rates at varying amino acid concentrations in the slices were calculated in /~mol of [3H] amino acid released in 1 s by 1 kg of incubated slices. These rates were then plotted against the [3HI amino acid concentration in the slices at the superfusion time under study (10 or 24min). Intracellular water/medium ratios (A) of [3H] amino acids at the steady state were calculated from the equation A = (C - Es)/s(1 - E - D), in which C indicates the [3H] amino acid content in mol per kg slice wet weight, s the [SH] amino acid concentration in the incubation medium (mol/1.), E the extracellular (inulin) space (l/kg slice wet weight) and D the proportion of the dry weight of the slices (kg/kg). Student's t-test was used to assess the statistical significance of all observed differences. RESULTS Stability o f slices during superfusions Cation and water contents of the slices were altered significantly (P < 0.01) already during the preloading period (Table 1). During superfusions the slices gained some sodium a n d water a n d lost potassium. The changes were slow, however, the cation and water contents of the slices changed significantly (P < 0.05) only after the 2-h superfusion. Extracellular (inulin) space likewise increased somewhat during superfusions. N o statistically significant differences in any of the measured parameters were discernible between the slices superfused for 30, 60 or 120 min. Spontaneous efflux o f phenylalanine and tryptophan The intracellular/extracellular concentration ratios of [ a H ] p h e n y l a l a n i n e and [ a H ] t r y p t o p h a n at steadystate condition (i.e. the concentrative capacities of the slices) were inversely proportional to the a m i n o acid concentrations in the preloading solution (Fig. 2). T r y p t o p h a n was more effectively concentrated (P < 0.01) than phenylalanine when the a m i n o acid concentrations in the preloading medium were low.
Amino acid efflux mechanisms
1359
TABLE 1. SODIUM, POTASSIUM AND WATER CONTENTS AND INULIN SPACES OF SUPERFUSED RAT CEREBRAL CORTEX SLICES
Treatment of slices Unincubated Preloaded for 30 min Preloaded for 30 min and --superfused for 30 min --superfused for 60 min --superfused for 120min
Sodium content (mmol/kg wet wt)
Potassium content (mmol/kg wet wt)
Water content (kg/kg dry wt)
Inulin space (1/kg wet wt)
60.6 + 5.7(11) 81.6 + 8.5(8)
84.0 + 6.4(11) 66.3 + 7.5(8)
4.62 + 0.21(6) 5.76 + 0.22(9)
0.43 + 0.07(7)
88.9 + 1.6(4) 88.3 + 2.6(4) 93.0 + 4.2(6)*
57.4 + 6.9(4) 59.3 + 4.9(4) 54.3 + 5.3(6)*
6.06 ___0.42(9) 6.00 + 0.36(8) 6.10 + 0.32(8)*
0.46 ___0.10(5) 0.49 ___0.13(5) 0.54 + 0.09(5)*
Rat cerebral cortex slices were preloaded with unlabelled phenylalanine or tryptophan (0.05 mM) and superfused for times indicated as described in Experimental Procedures. Cation contents were measured by atomic absorption from 2 to 3 separately superfused and then pooled slices, and water content and inulin space were estimated from single slices. The results (means + S.D.) are given per final wet or dry weight of slices. Number of experiments in brackets. Significance of the differences between superfused and only preloaded slices: * P < 0.05.
At 10raM concentrations there was no longer a significant difference, however. The ettlux rates of phenylalanine and tryptophan from these preloaded slices into amino acid-free medium were greatest during the first 2-6 min of superfusion. This fast phase rapidly attenuated. The efflux rates of both amino acids after 10- and 24-min superfusions increased apparently linearly with the amino acid content of the slices (Figs 3 and 4). Phenylalanine etflux was more than two times faster than tryptophan efflux. Doubling the pumping rate of the superfusate had no apparent effect on [3H]phenylalanine efflux. I-3H] Amino acids preloaded in the slices were released at a continuously diminishing rate into the amino acid-free medium during the 80-min superfusions when the slices were gradually depleted of amino acids (Tables 2 and 3, Figs 5 and 6). Another general rule was also established when the behaviour of individual slices upon superfusion was observed: the slower the efflux during the initial spontaneous efflux period (20-30min), the slower was it in the
same slice also during other subsequent superfusion periods.
Extracellular effects of amino acids Figures 5 and 6 show representative examples of the effects of unlabelled amino acids on the effluxes of [3H]phenylalanine and [3H]tryptophan. Amino acids added to the superfusion medium invariably caused stimulatory trans-effects on the efflux characterized by the rate constant k2 for this phase. Phenylalanine, tryptophan and histidine stimulated the efflux of [3H]phenylalanine in a concentration-dependent manner, even if the stimulation by histidine was less pronounced (Table 2). An addition of 5 mM phenylalanine and 5 mM histidine together caused no greater effect than phenylalanine (5 or 10 rnM) alone. The efflux of [3H]tryptophan was stimulated most by extracellular 10 mM tryptophan (Table 3). The stimulation percentage was even larger than the corresponding stimulation in the efflux of [3H]phenylalanine, obviously because of the slower spontaneous efflux rate of [3H]tryptophan. Otherwise the results were very similar to those with [3H]phenylalanine. Histidine was again the least potent stimulator.
o_
Intracellular effects of amino acids
5
0 -
5 10 /'~ MEDIUM AMINO ACID CONCENTRATION mM
20
FIG. 2. Accumulation ratios of [3H]phenylalanine and [3H]tryptophan in rat cerebral cortex slices at varying amino acid concentrations in the incubation medium. The slices were incubated at 310K under 02 with labelled phenylalanihe (0) or tryptophan (O) for 30 to 90 min, i.e. until an equilibrium was reached between the intracellular and extracellular I-3H] amino acid concentrations. The accumulation ratios, calculated as described in Experimental Procedures, are given as means -b S.D.S of 4 to 8 experiments.
Figures 5 and 6 also depict the effects of the accumulated unlabelled amino acids on the efflux of phenylalanine and tryptophan during later superfusion phases when the medium was again amino acidfree and most of the extracellular amino acids had also been washed out of the slices. These cis-effects by the intracellularly accumulated amino acids were either stimulatory or inhibitory. They are characterized by the rate constants designated by k 3 (50-60 min) and k4 (70-80 min). [3H]Phenylalanine efflux between 50 to 60 min was enhanced by intracellular phenylalanine, and to a lesser extent by tryptophan (Table 2). Histidine, however, decreased the efflux. [3H]Tryptophan efflux (50-60 and 70-80 min) was also significantly enhanced by intracellular phenylalanine and diminished by histidine (Table 3). These intracellular influences were clearly related to the concentrations of unlabelled
1360
E.R. KORPI
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FIG. 3. Rate of [3H]phenylalanine effiux from rat cerebral cortex slices as a function of [3H]phenylalanine content of the slices. The slices were preloaded with 4.0 mei/l L-[3H]phenylalanine (0.05-20.0 mM) for 30 to 90 rain and superfused (0.25 ml/min) for 30 rain. The specific radioactivity of [3H]phenylalanine in the preloading medium, the radioactivity ultimately remaining in the slices, outlet tubes and superfusion cups was used for calculation of the etttux rates and slice concentrations of [3H]phenyla!anine at 10 rain as shown in the larger graph. The smaller graph depicts similarly calculated [3H]phel~? lalanine efllux rates and slice concentrations after the 24-min superfusion. The regression lines through the experimental points were calculated by the method of least squares, their equations and correlation coefficients are in the Figure. phenylalanine, histidine and tryptophan taken up and retained by the slices towards the end of superfusions (Table 4). The less an accumulated amino acid was retained, the faster was the efflux of [3H] amino acids. Although histidine was retained much more efficiently t h a n phenylalanine, the levels of both these accumulated amino acids significantly exceeded the levels of preloaded [3H] amino acids (Table 4). Superfusion of the slices with 5 mM phenylalanine and 5 mM histidine together largely abolished the intracellular effects of
~
both amino acids (Tables 2 and 3). The concentrations of phenylalanine and histidine in these experiments were both so high that they would have exerted a profound effect on the efflux if present in the slices alone (Table 4). The amounts of free [3H]phenylalanine and [3H]tryptophan left in the slices after superfusions also reflected the effects of other amino acids on the efflux (Tables 2 and 3). Incorporation of amino acids into proteins. Labelled
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FIG. 4. Rate of [3H]tryptophan efflux from rat cerebral cortex slices as a function of [3H]tryptophan content of the slices. The concentration range of tryptophan in the preloading medium was 0.05-10.0 mM. See the legend to Fig. 3 for other experimental details. The larger graph shows the rates after the 10-rain superfusion and the smaller graph after the 24-min superfusion.
Amino acid efflux mechanisms
1361
TABLE 2. EFFLUX AND EXCHANGE OF [3H']PHENYLALANINE IN RAT CEREBRAL CORTEX SLICES AS INFLUENCED BY VARIOUS CONCENTRATIONS OF UNLABELLED EXTRA- AND INTRACELLULAR PHENYLALANINE, TRYPTOPHAN AND HISTIDINE
Amino acid in superfusion medium from 30 to 40 min
No. of expts
kl (x 10 -3 min -1)
None (control) 10.0 mM Phe 5.0mM Phe 0.05 mM Phe 10.0mM His 5.0mM His 1.0mM His 0.05 mM His 5.0mM Phe + 5.0mM His 10.0mM Trp 0.05 mM Trp
17 9 5 4 7 6 7 6 5 8 4
16.9 + 2.6 17.2 ___1.7 16.4,-_0.7 18.3 +- 3.2 17.7 +- 1.6 18.5 + 3.0 16.9 + 3.8 18.7 +- 3.8 15.9 +- 1.5 16.4 + 1.5 17.9 _+ 3.3
Effiux rate constants k2 k3 (% of kl) (% of k0
k4 (% ofkt)
92 ___5 426 ___62t 345 + 43t 135 ___6]" 248 ___27t 242 _+_38]" 176 +_ 12]" 108 +- 5t 379 +- 48]" 432 ___49]" 123 + 8]"
78 + 13 78 ___23 89+10 85 +- 8 32 +__7"1" 37 + 7"t 55 +- l i t 78 + 9 81 +_. 13 73 + 12 77 + 10
85 135 155 92 34 39 57 81 98 102 84
___6 ___ 19tl" + 10t ___6 + 5t ___ 13t __. 9t __+4 +- 11" + 13t ___5
Per cent of radioactivity left at the end of superfusion 5.3 0.7 0.8 3.4 5.2 4.9 5.4 3.9 1.6 1.2 4.2
+ 1.9 + 0.2]" ___0.2t +- 1.8 +- 1.7 + 1.9 ___2.7 -4- 2.1 +- 0.2]" + 0.2t + 1.9
Experimental details as in Fig. 5. Means + S.D. are given, k2, k3 and k4 are expressed as percentages of k~ of the same slice. Significance of the differences between control and other groups: * P < 0.05; ]. P < 0.001. TABLE 3.
EFFLUX AND EXCHANGE OF [3H]TRYPTOPHAN IN RAT CEREBRAL CORTEX SLICES AS INFLUENCED BY VARIOUS CONCENTRATIONS OF UNLABELLED EXTRA- AND INTRACELLULAR PHENYLALANINE, TRYPTOPHAN AND HISTIDINE
Amino acid in superfusion medium from 30 to 40 rain
No. of expts
(x 10 -3 min -1)
None (control) 10.0 mM Trp 10.0 mM Phe 10.0 mM His 1.0mM His 5.0mM Phe + 5.0mM Hi's
10 8 5 7 7 7
8.95 + 1.10 9.36 ___ 1.22 9.64 + 1.63 9.82 ___ 1.87 9.61 +__0.89 9.90 ___ 1.42
kl
Effiux rate constants k2 k3 (% of kl) (% ofkx)
k4 (% of kl)
97 + 5 632 + 81t 484 ___61"f" 355 __+64t 262 ___42t 466 + 48t
83 83 130 30 61 63
91 96 199 36 63 86
___8 ___ 11 -t- 31"1" -I- 6t ___ 12t + 18
Per cent of radioactivity left at the end of superfusion
+- 13 ___9 ___31" + 4t + 4t + 17"
19.9 + 5.3 5.2 ___ 1.3"t" 3.9 ___ 1.8t 17.7 + 4.1 16.2 + 2.1 7.9 + 2.5t
Experimental details as in Fig. 6. Means _+ S.D.S are given, k2, k 3 and k4 are expressed as percentages of kl of the same slice. Significance of the differences between control and other groups: * P < 0.05; ";"P < 0.001. [fl o
phenylalanine and tryptophan molecules were also incorporated into proteins in the slices during the ex-
~ 2.5
k~Lg~i.g~
iiii ~ i
iiii iiiI
periments (Table 5)" Themuch a m ° ugreater n t ° f [3H]phenylalanine incorporated was even than the amount of trichloroacetic acid-soluble [3H]phenyl-
i i 2.0
1.5
~[
|
1.0
~
o k3
O~
• • •
I- z
SUPERFUSION TIME MIN
FIG. 5. Effects of other amino acids on the efflux of [aH]phenylalanine from rat cerebral cortex slices. Slices were preloaded for 30min with L-[3H]phenylalanine (4.0 mCi/1., 0.05 mM) and superfused for 30 min with amino acid-free medium. The medium was then changed for 10min either to the same medium (e) or to that supplemented with 10 mM phenylalanine (A), 10 mM tryptophan (A) or 10raM histidine (O). The effiux curves shown are from representative single experiments. The regression lines fitted to the lowermost curve illustrate estimation of the efflux rate constants. NSC. 5/7--L
~ ~ 40
60
80
SUPERFUSlON TIME MIN
F1G. 6. Effects of other amino acids on the efflux of [aH]tryptophan from rat cerebral cortex slices. The slices were preloaded with L-[3H]tryptophan (4.0mCi/I., 0.05 mM). Otherwise the experimental details and symbols are as in Fig. 5.
1362
E.R. KORPI
alanine at the end of some superfusions. Protein synthesis does not seem to be an important factor influencing amino acid efltux rates, however, because only a small fraction of the labelled free amino acids which had been present at the beginning of superfusions were eventually incorporated into proteins. Already after the 30-rain preloading period 1.8 ± 0.3 and 0.5 ± 0.2°~, (mean ± S.D., n = 6) of [3H]phenytalanine and [3H]tryptophan, respectively, in the slices had been incorporated into protein. Unlabelled 10raM phenylalanine and tryptophan significantly diminished the amount of label incorporated during superfusions, apparently in part owing to the enhanced efflux of the [3HI amino acids from the slices (Table 5). DISCUSSION
Superfusion of brain slices The paucity of relevant studies on amino acid efflux from brain slices results partially no doubt from methodological difficulties encountered. The present superfusion apparatus, a modification from that of Laakso (1978), appears to be more advantageous than the system we have previously used (a stationary slice in a flow of preoxygenated medium, KORPI & OJA, 1979a). The slices were well preserved in an approximate steady state with regard to their cation and water contents for at least 2 h, which time appears reasonably long enough for efflux studies. Sodium and potassium contents, swelling and inulin spaces of the slices now superfused for up to 120 min agree well
with the data reported earlier for cerebral cortex slices incubated under the best standard conditions (BAcnEt,ARD, CAMPBELL & MCILWAJN, 1962; JOANNY & HILLMAN, 1963: FRANCK, CORNETTE 8L SCHOFFENIELS, 1968). The superfusion medium in the cups could be rapidly renewed and reuptake of the label by the slices thus minimized. Since doubling of the superfusion rate did not influence effiux, the apparatus itself obviously does not in any way limit efflux rates.
Mode ~1" spontaneous efflux q] phenylalanine and tryptophan An apparent linear relationship was found between the efflux rates of phenylalanine and tryptophan and their intracellular concentrations in cerebral cortex slices. Diffusion of the label residing in the extracellular spaces after preloading cannot be responsible for this phenomenon, since the half-time for even inulin efflux from this compartment is less than 5rain (LuND-ANDERSEN, 1974), and the present actual studies were made after a much longer lag period. Several more plausible explanations can be found. Firstly, efflux could in fact occur through any nonsaturable mechanism, like simple physical diffusion through pores in plasma membranes. Secondly, efflux may be a mediated process whose true nature remains undetected. It could thus take place if diffusion of amino acid molecules out of the extracellular spaces of the slices is slow compared to their transfer across cell membranes. The heterogeneity of cellular elements and the tortuosity of the extracellular spaces in slices render determination of the rate-limiting step
TABLE 4. PHENYLALANINE, TRYPTOPHAN AND HISTn)INE CONTENTS OF SUPERFUSEI) RAT CEREBRAL CORTEX SLICES
Amino acid from 30 to 40 min in superfusion medium
Amino acid content (mmol/kg slice wet weight) at 55 min at 75 rain
Slice/medium concentration ratio
Phenylalanille None 10.0mM Phe 5.0mM Phe + 5.0mM His
0.10 ± 0.01(4J 0.63 + 0.18(3j 1.7 ± 0.1(41
None 10.0 mM Trp
0.05 ± 0.01(41 1.2 + 0.2(4}
0.05 mM His 1.0 mM His 10.0raM His 5.0mM Phe + 5.0 mr~ His
0.25 + 0.06(4) 2.4 + 0.5(4) 10.8 ± 1.7(6) 7.3 ___ 1.1(4)
0.08 ___0.01(4) 0.23 + 0.06(4) 0.87 _+ 0.16(4)
1130 270 390
0.04 ± 0.01(4) 0.45 ___0.12(4)
1t30 270
0.23 1.5 7.1 4.6
3000 540 610 550
Tryptophan
Hist ±dine +_ 0.03(4) + 0.2(4) ± 1.7(4) +__0.7(4)
The slices were preloaded for 30 min with unlabelled phenylalanine or tryptophan (0.05 mM) and superfused for 30 min with amino acid-free medium, from 30 to 40 min with the medium supplemented with unlabelled amino acids as indicated, and from 40 min onwards again with amino acid-free medium. The slices were harvested at the time periods indicated, pooling 2 or 3 separately superfused slices for each analysis. Means +_ S.D.S are given and the number of experiments in brackets. Average slice/medium concentration ratios between 55 and 75 min were calculated on the assumption that the amino acids released from the slices immediately mix with the medium in the superfusion cups.
1363
Amino acid efflux mechanisms TABLE 5. INCORPORATION OF [3H']PHENYLALANINE AND ['3H'ITRYPTOPHAN INTO PROTEINS DURINGSUPERFUSIONEXPERIMENTS
Amino acid in superfusion medium from 30 to 40 min
No. of expts
Radioactivity incorporated into protein at the end of superfusions as percentage of trichloroacetic acid soluble radioactivity Initial Final
[ 3H]Phenylalanine efflux None (control) 10rnM Phe 10 mM Trp 10 mM His
6 9 7 7
3.5 + 2.1 + 2.9 + 3.7 +
0.9 0.6* 0.7 1.3
None (control) 10 mM Trp 10mM Phe 10 mM His
I-3H]Tryptophan efflux 9 1.2 _ 0.3 6 0.6 _ 0.02* 5 0.6 + 0.1" 6 0.9 + 0.2
55 + 352 _ 268 + 72 +
18 145" 85* 19
6.1 + 11.9 + 20.3 + 5.4 +
1.5 1.5" 12.7' 0.5
Experimental design as in Figs 5 and 6. Protein in the trichloroacetic acid-insoluble pellets of slices was purified as described in detail by DUNLOP,VAN ELDEN& LAJTHA (1975), solubilized in 1 N NaOH, neutralized with HCI and measured for radioactivity as indicated in Experimental Procedures. Means + S.D.S are given. Significanceof differences from controls: * P < 0.01.
difficult, since the extrusion rate via a hypothetical high-affinity transport system may well exceed the rate of net diffusion out of the extracellular spaces (see LUND-ANDERSEN & KJELDSEN, 1976), and we are measuring only the eventual appearance of the labelled molecules in the medium. The rate of diffusion out of the extracellular spaces was not, however, a likely rate-limiting step in efflux in the present experiments, because extracellularly added unlabelled amino acids were able to produce quickly beginning and ending increments in efflux by exchange. Because efllux could not be studied reliably at very low preloading amino acid concentrations owing to dilution of the label by endogenous amino acids, mediated processes in the spontaneous efflux could possibly escape detection if they become saturated at very low intracellular amino acid concentrations. We do not see, however, any teleological reason for the existence of such a high-affinity efflux system in brain cells. The detection of saturability of the spontaneous e~ux may also be hampered by stimulation of intracellular amino acid efflux by exchange with amino acid molecules already released into the extracellular spaces of the slices, if this exchange does not operate with a one-to-one ratio. Leucine-valine exchange is such a case, as one leucine molecule taken up by the brain slice may cause the release of three to four valine molecules (BATTISTIN, PICCOLI & LAJTHA, 1972). Let us provisionally assume that the spontaneous efflux of phenylalanine and tryptophan occurs through mediated saturable mechanisms in addition to possible diffusion. The influx of phenylalanine and tryptophan into brain cortex slices has been shown to be saturable (VAHvELAINEN • OJA, 1972). The present efflux rates were smaller than thc reported influx rates
at all amino acid concentrations. This relationship makes the concentrative uptake possible. If diffusion and translation of the amino acid carriers in the cell membranes occurred at equal rates in both directions, the magnitude of the concentrative uptake would then be determined by the ratio of the transport constants of mediated influx and efflux (see OJA & VABVELAINEN, 1975). Even at concentrations that almost saturate mediated influx, phenylalanine and tryptophan are to some extent concentrated by brain slices. Accumulation ratios are then about 2 and 5 for phenylalanine and tryptophan respectively (Fig. 2; BLASBERG & LAJTHA, 1965; KIELY & SOURKES, 1972). The transport constant for influx of both amino acids are about 0.5 mM (VAHVELAINEN& OJA, 1972; 1975; KIELY & SOURKES, 1972). The estimated transport constants for emux should then be about 1 to 3 mM. We did not now observe any signs of saturation in phenylalanine and tryptophan efflux, although the highest concentrations studied were several-fold greater. This outcome is distinctly at variance with the assumption posited above. Keeping in mind all methodological reservations outlined above we must state that no positive indications of saturable mediated processes were found in the spontaneous efflux of phenylalanine and tryptophan from cerebral cortex slices. This is at variance with the observations of LEVI, BLASBERG& LAJTnA (1966), who report an auto-activation of phenylalanine and leucine efttux from mouse brain slices by some unknown mechanisms and a saturable component in the efflux of ~t-aminoisobutyric acid, lysine and D-glutamate.
Efflux in the presence of other amino acids Trans-stimulation of efflux has been proposed to
1364
E.R. KORPI
result from increased exchange of intracellular [3HI amino acids with extracellular unlabelled amino acids through common transport sites at cell membranes, since the substrate specificities of phenylalanine and tryptophan exchange and influx are identical (KORPI & OJA, 1979a, b). The present study also reveals saturation of such a stimulation by high extracellular amino acid concentrations. The data are not complete enough for a precise estimation of how large a concentration causes extracellularly the half-maximal stimulation, but a reasonable estimate for histidine in phenylalanine efflux would be about 1 mM (Table 2). This figure matches well with the estimated transport constant in influx for histidine itself (VAHVELAINEN& OJA, 1972; 1975). The present effects of intracellular histidine, phenylalanine and tryptophan on the efflux of [aH]phenylalanine and I-3H]tryptophan were quite similar to those reported earlier either by us (KORPI & OJA, 1979a, b) or others (Levl et al., 1966). The inhibition of efflux by histidine may result from competition for intracellular transport sites, but the apparent lack of homo-cis-inhibition in efflux is then more difficult to understand. A number of other explanations can be offered, remembering also the structural complexity of brain slices: (1) Histidine may by exchange processes recapture [3HI amino acid molecules already released
into the extracellular spaces and thus decrease e~ux, since it is also more effectively concentrated by the slices than for instance phenylalanine (Table 4). Phenylalanine, in turn, may be released into the extracellular spaces and from there accelerate eftiux by exchange with intracellular [aH] amino acids. This suggestion is also in accordance with the rapid exchangeability of large neutral amino acids across the blood-brain barrier in vivo (OLDENDORF, 1971; TOVH & LAJTHA, 1977). The high slice/medium concentration ratios of unlabelled histidine and phenylalanine (Table 4) do not necessarily undermine this possibility, since the efflux from the intracellular spaces to medium must mostly occur through the extracellular spaces. (2) The distribution of orientation of 'acceptor' sites of membrane carriers towards extra- and intracellular sides may be differently affected by intracellular histidine and phenylalanine. (3) The presence or fluxes of histidine and phenylalanine may have opposite, still unidentified effects on metabolic reactions supplying energy for amino acid transport. excellent technical assistance of Miss IRMA SALMINEN, Miss EUA KYR6L,~ and Mr ARIPEKKA VIIRTEL.~ is gratefully acknowledged. The author thanks Dr S. S. OJA for reviewing the manuscript. Acknowledgements--The
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Amino acid effiux mechanisms
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L~vl G., Bt.ASn~G R. & LAJTHA A. (1966) Substrate specificity of cerebral amino acid exit in vitro. Archs Biochem. Biophys. 114, 339-351. LUND-ANDERS~NH. (1974) Extracellular and intracellular distribution of inulin in rat brain cortex slices. Brain Res. 65, 239-254. LUND-AND~,SENH. & IOELDS~NC. S. (1976) Uptake of glucose analogues by rat brain cortex slices: a kinetic analysis based upon a model. J. Neurochem. 27, 361-368. M c C A ~ M. W. & ROBINSE. (1962) Fluorimetric method for the determination of phenylalanine in serum. J. Lab. clin Med. 59, 885-890. OJA S. S. & V~VEt~IN~N M.-L. (1975) Transport of amino acids in brain slices. In Research Methods in Neurochemistry, Vol. 3 (eds MARKSN. & RODNIOHTR.), pp. 67-137. Plenum Press, New York. OLDENDORVW. H. (1971) Brain uptake of radiolabelled amino acids, amines, and hexoses after arterial injection. Am. J. Physiol. 221, 1629-1639. TOTH J. & LAJTHAA. (1977) Rates of exchange of free amino acids between plasma and brain in mice. Neurochem. Res. 2, 149-160. VAHVELAXNENM.-L. & OJA S. S. (1972) Kinetics of influx of phenylalanine, tyrosine, tryptophan, histidine and leucine into slices of brain cortex from adult and 7-day-old rats. Brain Res. 40, 477-488. VAHWLAINENM.-L. & OJA S. S. (1975) Kinetic analysis of phenylalanine-induced inhibition in the saturable influx of tyrosine, tryptophan, leucine and histidine into brain cortex slices from adult and 7-day-old rats. J. Neurochem. 24, 885-892. (Accepted 12 February 1980)