Exp. Brain Res. 30, 539-548 (1977)
Experimental Brain Research 9 Springer-Verl~g1977
Effect of Aromatic Acids on the Influx of Aromatic Amino Acids in Rat Brain Slices P. L/ihdesm~iki and M.-L. Hannus Department of Biochemistry, University of Oulu, SF - 90230 Oulu 23, Finland
S u m m a r y . The influx of [3H]phenylalanine, [3H]tyrosine and [3H]tryptophan into brain cells was studied using brain slices from adult rats. Each aromatic amino acid inhibited the influx of the others into the brain cells. Tryptophan inhibited non-competitively the influx ofphenylalanine, and phenylalanine similarly that of tyrosine and tryptophan. On the other hand, tyrosine inhibited competitively the influx of phenylalanine, and similarly tryptophan that of tyrosine, and tyrosine that of tryptophan. Among the aromatic organic acids tested, only phenylpyruvate and homogentisate had any inhibitory effect on the influx of the aromatic amino acids. These effects were generally competitive, non-competitive inhibition being obtained only in the inhibition of phenylalanine influx by homogentisate. The existence of only one common transport system for aromatic amino acids appears to be unlikely. K e y words: Amino acid transport - Aromatic amino acids - Phenylpyruvate - Homogentisate - Brain slices
Introduction
Aromatic amino acids interact in their influx into brain cells both in vivo (Guroff and Udenffiend, 1962; McKean et al., 1968; Lindroos and Oja, 1972) and in vitro (Neame, 1961; Barbosa et al., 1970; Vahvelainen and Oja, 1975). Their inhibitory effect is competitive only in certain cases, however (Vahvelainen and Oja, 1975), and it is thus questionable whether they share a common transport system. Aromatic amino acids also affect each others' incorporation into brain proteins (Peterson and McKean, 1969; Oja, 1972; Barra et al., 1973; L/ihdesmfiki and Oja, 1975) and tRNAs (Lghdesm~iki and Oja, 1975). Some aromatic metabolites of phenylalanine or tyrosine, which occur at higher than normal concentrations in the body in phenylketonuria (Edwards and Blau, 1972) also affect this incorporation (L/ihdesm/iki and Oja, 1975). These abnormal levels of plasma aromatic amino acids and their metabolites may influence the entry into brain cells of aromatic amino acids and the disturbance of brain amino acid levels may be related to the mental deficiency characteristic
540
P. L~ihdesm/iki and M.-L. Hannus
of phenylketonuria. In the work reported here the effect of aromatic amino acids and some other aromatic acids on the influx of the aromatic amino acids p h e n y l a l a n i n e , t y r o s i n e a n d t r y p t o p h a n i n t o b r a i n slices w a s t e s t e d , a n d a n attempt made to determine the nature of inhibition where this occurred.
Materials and Methods Radiochemically pure L-[2,3-3H]phenylalanine (15.6 Ci/mmole), L-[G-3H]tryptophan (3.8 Ci/mmole) and L-[3,5-3H]tyrosine (53 Ci/mmole) were purchased from the Radiochemical Centre, Amersham. Other chemicals were analytical grade E. Merck, Beckman or NEN products. Adult, 3-month-old, Sprague-Dawley rats were decapitated and their brains excised, briefly rinsed with ice-cold Krebs-Ringer phosphate solution and put on an ice bath at 0 ~ C. Slices 0.4 mm thick were prepared at 4 ~ C in the frontal plane from the middle parts of cerebral hemispheres with a McIlwain tissue chopper. For one incubation 80-200 mg of slices were weighed in 5 ml of cold Krebs-Ringer phosphate solution (NaC1, 120 mmol/1; KC1, 4.80 mmol/1; CaC12, 2.57 mmol/1; MgSO4, 1.21 mmol/1; KH2PO4-K2HPO4 buffer, 0.5 mol/1, pH 7.4; glucose 9.1 mmol/1). The slices were incubated in Erlenmeyer flasks at 37 ~ C with continuous agitation. Firstly the possible inhibitory influence of an excess of another aromatic amino acid or some other aromatic acid on the influx of [aH]phenylalanine, [3H]tyrosine and [3H]tryptophan into brain slices was tested using a concentration of 0.3 mmol/1 phenylalanine, tyrosine or tryptophan, and 3 mmol/1 of one of the following compounds: phenylalanine, tyrosine, tryptophan, phenylpyruvate, phenyllactate, phenylacetate, homogentisate, salicylate or benzoate. Incubation time was 20 rain. Secondly the rates of influx of [aH]phenylalanine, [3H]tyrosine and [3H]tryptophan were determined as functions of time by incubating the brain slices with 3.6 mmol/1 [3H]phenylalanine or 0.84 mmol/1 [3H]tyrosine or [3H]tryptophan (1 ~Ci/ml in each case) for varying periods of up to 60 rain. The third series of experiments tested the rate of influx as a function of medium concentrations of the above [3H]amino acids. The incubation flasks contained various amounts of the same amino acid unlabelled, and some of them 50 mmol/1 of phenylalanine, tyrosine, tryptophan, phenylpyruvate or homogentisate. These inhibitor concentrations were chosen to ensure as high a percentage inhibition as possible. Incubation time was 15 rain. The fourth series of experiments tested the rate of influx of [3H]phenylalanine, [3H]tyrosine and [3H]tryptophan in various inhibitor concentrations (5, 25 and 50 mmol/1), but otherwise the incubation conditions were identical with those in the third series of experiments. After incubation the slices were briefly rinsed on filter paper with 5 ml cold incubation medium, weighed in 3 ml of 5 % trichloroacetic acid, homogenized and centrifuged. The radioactivity of the supernatant was determined as described by Lfihdesm/iki and Oja (1972), the efficiency of counting being determined with standard samples. The number of pulses was enough to reduce the sample counting error to below 1%. The inulin space of the slices was estimated using inulin [14C]carboxylic acid (sp. act. 2.17 mCi/g, average molecular weight 5175 _+ 95, the Radiochemical Centre, Amersham). The homogeneity of the labelled inulin was checked and the low-molecular weight components (less than 2 %) eliminated by exclusion chromatography on Sephadex G-50 gel. The slices were incubated for 15 rain in the presence of 2 g/1 unlabelled inulin and 1.5 ~Ci/ml inulin [14C]carboxylic acid. The slices were filtered and homogenized as above and their radioactivity measured. The inulin space of the slices was assumed to represent their extracellular space. The amount of amino acid passing from the incubation medium into the cells of the slices was always calculated per wet weight of incubated (swollen) slices as described by Vahvelainen and Oja (1972) and Oja and Vahvelainen (1975). The amount of amino acid in the slices corresponding to the percentage volume of inulin space was subtracted from the measured values. The influx of amino acids into the slices was analyzed further on the assumption that it was mediated by two mechanisms: a saturable transport conforming to Michaelis-Menten kinetics, and non-saturable physical diffusion. The active and passive components in the influx were separated as follows (Vahvelainen and Oja, 1972; Oja and Vahvelainen, 1975). The two components of influx can be described by the equation: v-
Vm" S ~- Ka" S S~-K
m
Influx of Aromatic Amino Acids in Brain Slices
541
where v is the velocity of influx (nmol/min/g wet weight), Vm the maximal velocity of the saturable transport (nmol/min/g), Km the transport constant equivalent to the Michaelis constant (retool/I), Kd the diffusion constant (nmol/min/g/mmole), and S the amino acid concentration in the medium (retool/I). As a first step, v was plotted against S and 1/v against 1/S. With the aid of this graphical analysis the most likely values of the parameters: Vm, K~ and K d were sought. A best fit for the observed rates of influx (v) was obtained by minimizing the sums of squares by a series of sequential corrections to Vm, Km and Kd. The sums of squares at each amino acid concentration in the medium were weighted by the invariance of the observations. After successive steps of iteration, Vm, Km and Kd tended towards their best-fit values. The contribution of the active transport (Va) and passive diffusion (Vp) to the total influx (v) was then evaluated in every experiment at each amino acid concentration using the calculated Vm, Km and Ka. The parameters of active transport and their fiducial limits were finally estimated by the logarithmic method of Barber et al. (1967) which allows the variance due to Vm to be separated from any other variance. Barber et al. (1967) developed the classical Michaelis-Menten equation into the form: Km - Ko K~ ] = log 1 + 1+~Vm 2.303.(S+Ko)
log l v - l ~
where Ko is an arbitrary constant. A straight line is obtained if the right hand side of this equation is plotted against I/(S + Ko). The line's slope and intercept on the vertical axis can be used for the iterative determination of Km and Vm. The final values of Km and Vm with their confidence limits were calculated from the straight lines of the logarithmic plots9 The first equation shows that passive diffusion (vp) against S gives a straight line through the origin. Such a straight line was fitted by the method of least squares for the calculated vp values. Its slope (c) is given by
o-
N Z i = 1 vpi- Si N
Z i=l
2 Si
where vpi is an individual observation at amino acid concentration Si, and N the total number of observations. Kd with its confidence limits was calculated from the slope of the straight line. The values of diffusion constants were 24-30 nmol/min/g/mmole. The inhibitor constants (Ki) for phenylalanine, tyrosine, tryptophan, phenylpyruvate and homogentisate in the saturable transport of [3H]phenylalanine, [aH]tyrosine and [3H]tryptophan were calculated using the equation: Krn1 9 S Ki
Km2 - Kin1
where Kin1 is the transport constant of the amino acid when incubated without the inhibitor, Km2 the apparent transport constant of the same amino acid in the presence of the inhibitor, and S the concentration of the amino acid in the incubation medium. The concentrations of phenylalanine and tyrosine in the slices during incubation were monitored with an Aminco-Bowman spectro-photofluorometer, phenylalanine according to McCaman and Robins (1962), and tyrosine using the method of Waalkes and Udenfriend (1957), and tryptophan with the colorimetric method of Udenfriend and Peterson (1957). The slices were homogenized in 5 % trichloroacetic acid, centrifuged, and the amino acids determined in the supernatants. A check was kept on the possible metabolic transformations of phenylalanine and tyrosine to their other aromatic derivatives using chromatographic methods. Phenyllactic, phenylacetic, and phenylpyruvic acids, and the corresponding hydroxyphenyl acids were assayed according to Goldstein (1961) and homogentisic acid after Feldman and Bowman (1973).
542
P. L/ihdesm~ikiand M.-L. Hannus
Table 1. Inhibition by an excess of a second aromatic acid or amino acid of the influx of [3H] phenylalanine, [3H]tyrosineand [3H]tryptophaninto rat brain slices Inhibitor (3 mmol/1)
Phenylalanine %
None Phenylalanine Tyrosine Tryptophan Phenylpyruvate Phenyllactate Phenylacetate Homogentisate Salicylate Benzoate
100.0 _+13.2 --
Influx into brain slices of Tyrosine % 100.0 +10.4
Tryptophan % 100.0 _+10.3
7 9 . 8 _+ 4 . 8 b
2 1 . 8 _+ 2 . 2 a --
4 3 . 9 _+ 2 . 7 a 5 2 . 6 _+ 4 . 1 ~
6 9 . 4 +_ 6 . 7 b 7 2 . 5 _+ 7 . 0 b
4 0 . 1 _+ 7.2 a 6 5 . 1 +_ 6 . 2 b
-5 8 . 3 _+ 4 . 4 ~'
113.7 + 8.9 109.6 _+ 7.6
106.2 _+ 6.5 110.3 _+10.1
6 1 . 6 _+ 7.2 b
8 0 . 2 _+ 1 . 9 b
121.7 _+13.8 95.6 _+ 8.0
119.5 _+12.1 106.2 + 8.5
83.7 _+ 7.0 115.1 +11.1 90.3 _+ 4.8 104.4 _+ 5.9 100.8 _+ 7.7
Rat brain slices 0.4 mm thick were incubated for 20 min in Krebs-Ringer phosphate medium, pH 7.4, containing 0.3 mmol/1 [3H]phenylalanine, [3H]tyrosine or [3H]tryptophan. The inhibitor concentration was 3 mmol/1. Results (mean _+ S.E.M. of 8 to 10 experiments) are given as percentages of the corresponding control incubations without inhibitor. Significanceof difference from control: a p < 0.01; b p < 0.05 calculated from Student's t-test
Results Excess phenylalanine, tyrosine and tryptophan inhibited the influx of each of the other aromatic amino acids into rat brain slices (Table 1). Phenylpyruvate was inhibitory for the influx of phenylalanine, tyrosine and tryptophan, but homogentisate only for that of phenylalanine and tyrosine. Phenyllactate, phenylacetate, salicylate and benzoate were entirely ineffectual. After 20 min incubation only 1.8% of the phenylalanine (10 mmol/1) was hydroxylated to tyrosine, 1.2 % of the phenylalanine and 1.6 % of the tyrosine (10 mmol/1) were converted to homogentisate, and 1.0% of the phenylalanine metabolized to phenylpyruvate, but none to phenyllactate or phenylacetate. The degradation of tyrosine to the corresponding hydroxyphenyl acids could not be observed. After the incubation, 96% of the label was recovered in the original phenylalanine, and 98 % in the tyrosine. Metabolic transformation of tryptophan could not be demonstrated. The original concentrations of phenylalanine, tyrosine and tryptophan in the slices were 0.64, 0.41 and 0.06 ~mol/g, respectively. The rates of metabolic transformations of the substances studied were very low, and the effect of these metabolic interconversions on the pool of the effector substances seems to be insignificant. Considerably higher transformation rates were obtained in cell-free brain extracts (Lfihdesm~iki and Oja, 1975). Figure 1 shows the uptake of [3H]phenylalauine, [aH]tyrosine and [3H]tryptophan by brain slices as a function of incubation time. The slices took up phenylalanine and tyrosine faster than tryptophan. Influx of the label still exceeded efflux at 60 min. During the first 15 rain the radioactivity of the slices
Influx of Aromatic Amino Acids in Brain Slices
543
Fig. 1. Net influx of [3H]phenylalanine (0), [all] tyrosine (A) and [3H]tryptophan ([3) into rat brain slices as a function of incubation time. Slices were incubated for varying periods at 37 ~ C in Krebs-Ringer phosphate medium (pH 7.4) containing 9.1 mmol/1 glucose and 3.6 retool/1 [aH]phenylalanine, or 0.84 retool/1 [3H]tyrosine or [3H]tryptophan. After incubation the slices were filtered, washed and homogenized, and radioactivity determined in the supernatant. The influx into the inulin space was subtracted from the values obtained. Uptake as ~xmol/g wet weight of tissue are given (means + S. E. M.) of 5 experiments
3[~ g "2
o
N=
~.'~ o_
0.2
~_ "E 15
30 45 Incubation time (rain)
60
1500
"7=
-~
imo
E c
Experimental Brain Research 9 Springer-Verl~g1977
Effect of Aromatic Acids on the Influx of Aromatic Amino Acids in Rat Brain Slices P. L/ihdesm~iki and M.-L. Hannus Department of Biochemistry, University of Oulu, SF - 90230 Oulu 23, Finland
S u m m a r y . The influx of [3H]phenylalanine, [3H]tyrosine and [3H]tryptophan into brain cells was studied using brain slices from adult rats. Each aromatic amino acid inhibited the influx of the others into the brain cells. Tryptophan inhibited non-competitively the influx ofphenylalanine, and phenylalanine similarly that of tyrosine and tryptophan. On the other hand, tyrosine inhibited competitively the influx of phenylalanine, and similarly tryptophan that of tyrosine, and tyrosine that of tryptophan. Among the aromatic organic acids tested, only phenylpyruvate and homogentisate had any inhibitory effect on the influx of the aromatic amino acids. These effects were generally competitive, non-competitive inhibition being obtained only in the inhibition of phenylalanine influx by homogentisate. The existence of only one common transport system for aromatic amino acids appears to be unlikely. K e y words: Amino acid transport - Aromatic amino acids - Phenylpyruvate - Homogentisate - Brain slices
Introduction
Aromatic amino acids interact in their influx into brain cells both in vivo (Guroff and Udenffiend, 1962; McKean et al., 1968; Lindroos and Oja, 1972) and in vitro (Neame, 1961; Barbosa et al., 1970; Vahvelainen and Oja, 1975). Their inhibitory effect is competitive only in certain cases, however (Vahvelainen and Oja, 1975), and it is thus questionable whether they share a common transport system. Aromatic amino acids also affect each others' incorporation into brain proteins (Peterson and McKean, 1969; Oja, 1972; Barra et al., 1973; L/ihdesmfiki and Oja, 1975) and tRNAs (Lghdesm~iki and Oja, 1975). Some aromatic metabolites of phenylalanine or tyrosine, which occur at higher than normal concentrations in the body in phenylketonuria (Edwards and Blau, 1972) also affect this incorporation (L/ihdesm/iki and Oja, 1975). These abnormal levels of plasma aromatic amino acids and their metabolites may influence the entry into brain cells of aromatic amino acids and the disturbance of brain amino acid levels may be related to the mental deficiency characteristic
540
P. L~ihdesm/iki and M.-L. Hannus
of phenylketonuria. In the work reported here the effect of aromatic amino acids and some other aromatic acids on the influx of the aromatic amino acids p h e n y l a l a n i n e , t y r o s i n e a n d t r y p t o p h a n i n t o b r a i n slices w a s t e s t e d , a n d a n attempt made to determine the nature of inhibition where this occurred.
Materials and Methods Radiochemically pure L-[2,3-3H]phenylalanine (15.6 Ci/mmole), L-[G-3H]tryptophan (3.8 Ci/mmole) and L-[3,5-3H]tyrosine (53 Ci/mmole) were purchased from the Radiochemical Centre, Amersham. Other chemicals were analytical grade E. Merck, Beckman or NEN products. Adult, 3-month-old, Sprague-Dawley rats were decapitated and their brains excised, briefly rinsed with ice-cold Krebs-Ringer phosphate solution and put on an ice bath at 0 ~ C. Slices 0.4 mm thick were prepared at 4 ~ C in the frontal plane from the middle parts of cerebral hemispheres with a McIlwain tissue chopper. For one incubation 80-200 mg of slices were weighed in 5 ml of cold Krebs-Ringer phosphate solution (NaC1, 120 mmol/1; KC1, 4.80 mmol/1; CaC12, 2.57 mmol/1; MgSO4, 1.21 mmol/1; KH2PO4-K2HPO4 buffer, 0.5 mol/1, pH 7.4; glucose 9.1 mmol/1). The slices were incubated in Erlenmeyer flasks at 37 ~ C with continuous agitation. Firstly the possible inhibitory influence of an excess of another aromatic amino acid or some other aromatic acid on the influx of [aH]phenylalanine, [3H]tyrosine and [3H]tryptophan into brain slices was tested using a concentration of 0.3 mmol/1 phenylalanine, tyrosine or tryptophan, and 3 mmol/1 of one of the following compounds: phenylalanine, tyrosine, tryptophan, phenylpyruvate, phenyllactate, phenylacetate, homogentisate, salicylate or benzoate. Incubation time was 20 rain. Secondly the rates of influx of [aH]phenylalanine, [3H]tyrosine and [3H]tryptophan were determined as functions of time by incubating the brain slices with 3.6 mmol/1 [3H]phenylalanine or 0.84 mmol/1 [3H]tyrosine or [3H]tryptophan (1 ~Ci/ml in each case) for varying periods of up to 60 rain. The third series of experiments tested the rate of influx as a function of medium concentrations of the above [3H]amino acids. The incubation flasks contained various amounts of the same amino acid unlabelled, and some of them 50 mmol/1 of phenylalanine, tyrosine, tryptophan, phenylpyruvate or homogentisate. These inhibitor concentrations were chosen to ensure as high a percentage inhibition as possible. Incubation time was 15 rain. The fourth series of experiments tested the rate of influx of [3H]phenylalanine, [3H]tyrosine and [3H]tryptophan in various inhibitor concentrations (5, 25 and 50 mmol/1), but otherwise the incubation conditions were identical with those in the third series of experiments. After incubation the slices were briefly rinsed on filter paper with 5 ml cold incubation medium, weighed in 3 ml of 5 % trichloroacetic acid, homogenized and centrifuged. The radioactivity of the supernatant was determined as described by Lfihdesm/iki and Oja (1972), the efficiency of counting being determined with standard samples. The number of pulses was enough to reduce the sample counting error to below 1%. The inulin space of the slices was estimated using inulin [14C]carboxylic acid (sp. act. 2.17 mCi/g, average molecular weight 5175 _+ 95, the Radiochemical Centre, Amersham). The homogeneity of the labelled inulin was checked and the low-molecular weight components (less than 2 %) eliminated by exclusion chromatography on Sephadex G-50 gel. The slices were incubated for 15 rain in the presence of 2 g/1 unlabelled inulin and 1.5 ~Ci/ml inulin [14C]carboxylic acid. The slices were filtered and homogenized as above and their radioactivity measured. The inulin space of the slices was assumed to represent their extracellular space. The amount of amino acid passing from the incubation medium into the cells of the slices was always calculated per wet weight of incubated (swollen) slices as described by Vahvelainen and Oja (1972) and Oja and Vahvelainen (1975). The amount of amino acid in the slices corresponding to the percentage volume of inulin space was subtracted from the measured values. The influx of amino acids into the slices was analyzed further on the assumption that it was mediated by two mechanisms: a saturable transport conforming to Michaelis-Menten kinetics, and non-saturable physical diffusion. The active and passive components in the influx were separated as follows (Vahvelainen and Oja, 1972; Oja and Vahvelainen, 1975). The two components of influx can be described by the equation: v-
Vm" S ~- Ka" S S~-K
m
Influx of Aromatic Amino Acids in Brain Slices
541
where v is the velocity of influx (nmol/min/g wet weight), Vm the maximal velocity of the saturable transport (nmol/min/g), Km the transport constant equivalent to the Michaelis constant (retool/I), Kd the diffusion constant (nmol/min/g/mmole), and S the amino acid concentration in the medium (retool/I). As a first step, v was plotted against S and 1/v against 1/S. With the aid of this graphical analysis the most likely values of the parameters: Vm, K~ and K d were sought. A best fit for the observed rates of influx (v) was obtained by minimizing the sums of squares by a series of sequential corrections to Vm, Km and Kd. The sums of squares at each amino acid concentration in the medium were weighted by the invariance of the observations. After successive steps of iteration, Vm, Km and Kd tended towards their best-fit values. The contribution of the active transport (Va) and passive diffusion (Vp) to the total influx (v) was then evaluated in every experiment at each amino acid concentration using the calculated Vm, Km and Ka. The parameters of active transport and their fiducial limits were finally estimated by the logarithmic method of Barber et al. (1967) which allows the variance due to Vm to be separated from any other variance. Barber et al. (1967) developed the classical Michaelis-Menten equation into the form: Km - Ko K~ ] = log 1 + 1+~Vm 2.303.(S+Ko)
log l v - l ~
where Ko is an arbitrary constant. A straight line is obtained if the right hand side of this equation is plotted against I/(S + Ko). The line's slope and intercept on the vertical axis can be used for the iterative determination of Km and Vm. The final values of Km and Vm with their confidence limits were calculated from the straight lines of the logarithmic plots9 The first equation shows that passive diffusion (vp) against S gives a straight line through the origin. Such a straight line was fitted by the method of least squares for the calculated vp values. Its slope (c) is given by
o-
N Z i = 1 vpi- Si N
Z i=l
2 Si
where vpi is an individual observation at amino acid concentration Si, and N the total number of observations. Kd with its confidence limits was calculated from the slope of the straight line. The values of diffusion constants were 24-30 nmol/min/g/mmole. The inhibitor constants (Ki) for phenylalanine, tyrosine, tryptophan, phenylpyruvate and homogentisate in the saturable transport of [3H]phenylalanine, [aH]tyrosine and [3H]tryptophan were calculated using the equation: Krn1 9 S Ki
Km2 - Kin1
where Kin1 is the transport constant of the amino acid when incubated without the inhibitor, Km2 the apparent transport constant of the same amino acid in the presence of the inhibitor, and S the concentration of the amino acid in the incubation medium. The concentrations of phenylalanine and tyrosine in the slices during incubation were monitored with an Aminco-Bowman spectro-photofluorometer, phenylalanine according to McCaman and Robins (1962), and tyrosine using the method of Waalkes and Udenfriend (1957), and tryptophan with the colorimetric method of Udenfriend and Peterson (1957). The slices were homogenized in 5 % trichloroacetic acid, centrifuged, and the amino acids determined in the supernatants. A check was kept on the possible metabolic transformations of phenylalanine and tyrosine to their other aromatic derivatives using chromatographic methods. Phenyllactic, phenylacetic, and phenylpyruvic acids, and the corresponding hydroxyphenyl acids were assayed according to Goldstein (1961) and homogentisic acid after Feldman and Bowman (1973).
542
P. L/ihdesm~ikiand M.-L. Hannus
Table 1. Inhibition by an excess of a second aromatic acid or amino acid of the influx of [3H] phenylalanine, [3H]tyrosineand [3H]tryptophaninto rat brain slices Inhibitor (3 mmol/1)
Phenylalanine %
None Phenylalanine Tyrosine Tryptophan Phenylpyruvate Phenyllactate Phenylacetate Homogentisate Salicylate Benzoate
100.0 _+13.2 --
Influx into brain slices of Tyrosine % 100.0 +10.4
Tryptophan % 100.0 _+10.3
7 9 . 8 _+ 4 . 8 b
2 1 . 8 _+ 2 . 2 a --
4 3 . 9 _+ 2 . 7 a 5 2 . 6 _+ 4 . 1 ~
6 9 . 4 +_ 6 . 7 b 7 2 . 5 _+ 7 . 0 b
4 0 . 1 _+ 7.2 a 6 5 . 1 +_ 6 . 2 b
-5 8 . 3 _+ 4 . 4 ~'
113.7 + 8.9 109.6 _+ 7.6
106.2 _+ 6.5 110.3 _+10.1
6 1 . 6 _+ 7.2 b
8 0 . 2 _+ 1 . 9 b
121.7 _+13.8 95.6 _+ 8.0
119.5 _+12.1 106.2 + 8.5
83.7 _+ 7.0 115.1 +11.1 90.3 _+ 4.8 104.4 _+ 5.9 100.8 _+ 7.7
Rat brain slices 0.4 mm thick were incubated for 20 min in Krebs-Ringer phosphate medium, pH 7.4, containing 0.3 mmol/1 [3H]phenylalanine, [3H]tyrosine or [3H]tryptophan. The inhibitor concentration was 3 mmol/1. Results (mean _+ S.E.M. of 8 to 10 experiments) are given as percentages of the corresponding control incubations without inhibitor. Significanceof difference from control: a p < 0.01; b p < 0.05 calculated from Student's t-test
Results Excess phenylalanine, tyrosine and tryptophan inhibited the influx of each of the other aromatic amino acids into rat brain slices (Table 1). Phenylpyruvate was inhibitory for the influx of phenylalanine, tyrosine and tryptophan, but homogentisate only for that of phenylalanine and tyrosine. Phenyllactate, phenylacetate, salicylate and benzoate were entirely ineffectual. After 20 min incubation only 1.8% of the phenylalanine (10 mmol/1) was hydroxylated to tyrosine, 1.2 % of the phenylalanine and 1.6 % of the tyrosine (10 mmol/1) were converted to homogentisate, and 1.0% of the phenylalanine metabolized to phenylpyruvate, but none to phenyllactate or phenylacetate. The degradation of tyrosine to the corresponding hydroxyphenyl acids could not be observed. After the incubation, 96% of the label was recovered in the original phenylalanine, and 98 % in the tyrosine. Metabolic transformation of tryptophan could not be demonstrated. The original concentrations of phenylalanine, tyrosine and tryptophan in the slices were 0.64, 0.41 and 0.06 ~mol/g, respectively. The rates of metabolic transformations of the substances studied were very low, and the effect of these metabolic interconversions on the pool of the effector substances seems to be insignificant. Considerably higher transformation rates were obtained in cell-free brain extracts (Lfihdesm~iki and Oja, 1975). Figure 1 shows the uptake of [3H]phenylalauine, [aH]tyrosine and [3H]tryptophan by brain slices as a function of incubation time. The slices took up phenylalanine and tyrosine faster than tryptophan. Influx of the label still exceeded efflux at 60 min. During the first 15 rain the radioactivity of the slices
Influx of Aromatic Amino Acids in Brain Slices
543
Fig. 1. Net influx of [3H]phenylalanine (0), [all] tyrosine (A) and [3H]tryptophan ([3) into rat brain slices as a function of incubation time. Slices were incubated for varying periods at 37 ~ C in Krebs-Ringer phosphate medium (pH 7.4) containing 9.1 mmol/1 glucose and 3.6 retool/1 [aH]phenylalanine, or 0.84 retool/1 [3H]tyrosine or [3H]tryptophan. After incubation the slices were filtered, washed and homogenized, and radioactivity determined in the supernatant. The influx into the inulin space was subtracted from the values obtained. Uptake as ~xmol/g wet weight of tissue are given (means + S. E. M.) of 5 experiments
3[~ g "2
o
N=
~.'~ o_
0.2
~_ "E 15
30 45 Incubation time (rain)
60
1500
"7=
-~
imo
E c
