228
Brain Research, 552 (1991) 228-231 © 199l Elsevier Science Publishers B.V. (1006-8993/91/$03.5(I ADONIS 000689939116705R
BRES 16705
Blocking effect of intraperitoneal injection of phenylalanine on high-threshold calcium currents in rat hippocampal neurones A.E. Martynyuk, S. Savina and G.G. Skibo Bogomoletz Institute of Physiology, Ukrainian Academy of Sciences, Kiev (U. S. S. R.) (Accepted 15 January 1991)
Key words: Hippocampal neuron; Calcium current; Phenylalanine
Calcium currents were recorded in cultured (5-7 days) hippocampal neurones isolated from one-day-old rats. The animals obtained intraperitoneal injections of L-phenylalanine which induces in the brain biochemical changes characteristic of phenylketonuria. It has been found that the amplitude of the low-threshold calcium current in L-phenylalanine-affected neurones was not appreciably changed compared with that in neurones from control (non-injected) animals. However, the amplitude of the high-threshold calcium current was essentially decreased. Its relative amplitude at Vt = +20 mV became 40 + 30% as contrasted to 416 + 130% in neurones from control animals (the amplitude of the calcium currents at Vt = -10 mV taken as 100%). The decrease remained during the whole time of culturing. Addition of L-tyrosine to the cultivation medium (50 gM) restored the high-voltage calcium current, its relative amplitude reaching 280 + 57%. The data are discussed in conjunction with the previously obtained results about antagonistic modulatory action of tyrosine and phenylalanine on the functioning of high-threshold calcium channels and possible mechanisms of brain dysfunction during phenylketonuria.
INTRODUCTION It is well k n o w n that sustained elevation of L-phenylalanine in blood plasma evoked by decreased activity of phenylalanine 4-hydroxylase in the liver during congenital disease phenylketonuria provokes severe damage of brain functions in humans. The mechanisms which determine brain dysfunction during this disease are not quite understood. In our previous paper 6 it has been shown that intracellular introduction of L-phenylalanine exerts a down-regulatory effect on the activity of highthreshold calcium channels in PC12 pheochromocytoma cells. O n the contrary, introduction of L-tyrosine supported the activity of corresponding channels. The obtained data prompted us to investigate the effects of these amino acids on brain n e u r o n e s in conditions which imitate biochemical changes characteristic of phenylketonuria. MATERIALS AND METHODS Experiments were performed on cultured hippocampal neurones obtained from one-day-old rats. Experimental and control animals were pretreated according to the technique described earlier ~'8. They were placed in a thermostat for 30 min at a temperature of 30 + 2 °C. Then the experimental animals obtained intraperitoneal injections of L-phenylalanine dissolved in 0.42% NaCI solution (1 g/kg b.w.). The control animals were injected with the same volume
of 0.9% NaCI solution. After the injections the animals were returned to the thermostat for 160 min before killing. Five groups of control and experimental animals were used. The dissociated tissue culture from the hippocampus was prepared according to Banker and Cowan2. Whole-cell current recordings were performed using glass micropipettes with a resistance between 2 and 3 MD from cells which had been in culture for 5-7 days. Control measurements were made on cells obtained in the same way from animals injected with an equivalent amount of 0.9% NaCI. The pipette solution contained (in mM): CsCI 100, HEPES 20, EGTA 5, ATP 2, MgCI2 3, cAMP 0.01, TrisOH 30, GTP (sodium salt) 1 (pH 7.3). The composition of the extraceUular solution was (in mM): NaCI 100, CaC12 20, MgCI2 1.5, HEPES 5, glucose 15, T r x 0.003 (pH 7.3). All recordings were made at room temperature (21-23 °C).
RESULTS
Calcium currents in control neurones In all cells from control animals inward calcium currents (/ca) could be evoked by m e m b r a n e depolarization from holding potential Vh = - 9 0 mV. The currents appeared already at testing potentials (Vt) between - 6 0 and - 5 0 mV. Ica reached its maximal value at Vt = +20 mV and diminished to zero at Vt between +50 and +60 mV (Fig. 1). D u r i n g prolonged depolarizing potential shifts (200 ms) lca declined (inactivated). The inactivation had a complicated time course and could be separated into fast and slow c o m p o n e n t s (time constants
Correspondence: A.E. Martynyuk, Bogomoletz Institute of Physiology, Bogolometz street 4, 252601 GSP, Kiev 24, U.S.S.R.
229 A
B -60 -40 -20 0
A 20 rnV
- 30 -
+o I
/I
/
8 -60
1
0
-40
-20
0
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~
200
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10
f 33pA J
°/o 60 pA I 100m$
lea ~o
°/o 150
100m5
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Fig. 1. Calcium inward currents in cultured hippocampal neurons from newborn rats. A: original current records obtained at indicated testing potentials; holding potential -90 mV. B: averaged currentvoltage characteristic normalized to current amplitude at Vt -- -10 mV; vertical bars indicate S.D. (n = 7). Vt = -10 mV was chosen for normalization in this and the following figures because it corresponds to maximal amplitude of the low-threshold current. The dotted line shows the current-voltage characteristic recorded in one neurone at holding potential -50 mV.
5 - 2 0 ms and several h u n d r e d s of milliseconds). The fast inactivating c o m p o n e n t of the total current was maximal at V t b e t w e e n - 2 0 and - 1 0 mV, reaching 31 + 13% of the t o t a l / c a ( m e a n + S . D . , n -- 12). If the holding potential was shifted to - 5 0 mV, the low-threshold c o m p o n e n t of the current d i s a p p e a r e d and the high-threshold component of I c , b e c a m e p r e d o m i n a n t (Fig. 1B). This component d e m o n s t r a t e d high sensitivity to intracellular perfusion; it b e c a m e ' w a s h e d - o u r in about 20 min, whereas the fast inactivating c o m p o n e n t r e m a i n e d quite stable (see Fig. 2). The described effects of varying holding potential and intracellular perfusion clearly indicate the presence of two p o p u l a t i o n s of calcium channels in h i p p o c a m p a l n e u r o n e s of n e w b o r n animals: the low-threshold meta-
Fig. 3. Calcium currents in hippocampal neurones from phenylalanine-injected animals. A: original current records, B: averaged current voltage characteristic (mean -+ S.D., n = 6) constructed in the same way as in Fig. 1. Holding potential -90 mV. bolic-independent (T-type) and high-threshold metabolic-dependent ones. Probably, the second p o p u l a t i o n is not h o m o g e n e o u s and contains inactivating and noninactivating channels (N- and L-types according to Fox et al. 4 and Takahashi et al.9). Their expression varied in different neurones, and their separation was not very reliable. T h e r e f o r e we identified only the low- and high-threshold c o m p o n e n t s of the calcium current, as it has also been done by Yaari et al. 10 in their investigations of cultured h i p p o c a m p a l n e u r o n e s from rat embryos.
Calcium currents in neurones from phenylalanine-injected animals. The calcium currents in n e u r o n e s from phenylalanineinjected animals could be activated by testing depolarizations similar to those in control n e u r o n e s ( - 6 0 to - 5 0 A
E5 -60
-40
-20
0
2 0 mV
i
-lO
10C Ica A
°/o 5C
10'
~
.
19' c.._Ea l
3O 10
lOOms
2O
~
2'o Fig. 2. Changes of calcium currents during perfusion of a hippocampal neurone by saline solution. Ordinate - relative amplitude of the fast inactivating (A) and slowly inactivating (O) components of the current normalized to their amplitude at the beginning of perfusion; abscissa - time after beginning of perfusion. The inset demonstrates current records obtained at indicated perfusion times. Holding potential -60 mV.
40 pA J lO0rns
°/o
40C
Fig. 4. Calcium currents in hippocampal ncurones from phenylalanine-injected animals, cultured in solution containing 50 /~M L-tyrosine. A: original current records; B: averaged current-voltage characteristic (mean + S.D., n = 7) constructed in the same way as in Fig. 1. Holding potential -90 mV.
230
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previous experiments, cells were cultured for 5-7 days. The results of calcium current measurements in such cells are presented in Fig. 4. A substantial restoration of the high-threshold calcium current after culturing the cells in tyrosine-containing medium was observed. A characteristic feature of the restored high-threshold current was the presence of definite inactivation which was well developed at V~ = +20 mV and had a time constant of about 100 ms. This may indicate that the restored current was generated mainly by N-type calcium channels. Fig. 5 summarizes data about the calcium currents amplitudes measured at Vt -- +20 mV in control neurones, neurones from phenylalanine-injected animals and neurones cultured in the presence of 50 /~M t.tyrosine.
I~° l °/°
|
4OO
300
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DISCUSSION 100
1
2
3
Fig. 5. Mean amplitudes (+ S.D.) of the high-voltage component of the calcium current (Vt = +20 mV) normalized to the current amplitude at gt = -10 mV in neurones from control animals (1), neurones from phenylalanine-injected animals cultured in standard (2) and u-tyrosine containing (3) media. mV). However, the maximum of current-voltage characteristics was shifted by 20-30 mV to a more negative potential region. These Vt corresponded to the maximal contribution of the inactivating component to the total Ica in control cells (-20 to -10 mV, Fig. 3). Another difference of the modified /Ca was the depression or even complete absence of the high-voltage current component, as can be seen from records presented in Fig. 3A. In 12 cells the mean relative amplitude of the latter was only 32 + 14% at Vt -- -10 mV. The data obtained definitely indicate that in phenylalanine-pretreated animals a depression of the activity of high-threshold calcium channels takes place. On the contrary, the activity of the low-threshold (T-type) channels remains practically unchanged. The most probable reason for the detected downregulation of high-threshold calcium channels could be the shift in the tyrosine-phenylalanine concentration balance towards the latter. We made an attempt to restore the function of high-threshold calcium channels by addition of L-tyrosine to the culture medium (taking into account that aromatic amino acids penetrate quite easily through the cellular membrane, cf. ref. 11). L-Tyrosine was added in a concentration of 50 kLM immediately after the beginning of culturing. As in
The data presented are in agreement with the results obtained previously on PC12 pheochromocytoma cells: in rat hippocampal neurones the activity of the highthreshold calcium channels is also up- and down-modulated correspondingly by tyrosine and phenylalanine. An additional feature of such modulation not observed in model experiments on clonal cells becomes obvious in this case: the depressing effect of phenylalanine is extremely long-lasting, it manifests itself for many days after the injection of the amino acid in the newborn animal (at least for 5-7 days of cell culturing). Obviously, some still unknown stable changes occur in highthreshold calcium channels after exposure to phenylalanine which can be removed later on by excessive tyrosine. A possible site of action of aromatic acids on calcium channels could be the microtubular apparatus, especially in dendrites. We did not perform special morphometric investigations, but visually an intense growth of neurites was observed during cell culturing in the tyrosinecontaining solution. It has been suggested by several authors that high-threshold inactivating calcium channels are expressed mainly in the dendritic membrane 7'1°. Probably, tyrosination of the a-tubulin in the microtubuli 3 creates favourable conditions for the functioning of the high-threshold calcium channels. The replacement of tyrosine by phenylalanine in the COOH-terminal of a-tubulin (such replacement did take place in case of experimental phenylketonuria shown by Rodriguez and Borisy, ref. 8) removes such favourable conditions. The behavioural changes which occur in rats after phenylalanine treatment include inability for learning (cf. ref. 5). Some authors suggested that calcium inward currents generated mainly by high-threshold calcium channels might play an important role in long-lasting
231 changes of the effectiveness of synaptic transmission which m a y form the mechanisms for plasticity and learning. The p r o f o u n d changes seen in the activity of these channels m a y substantially disturb learning and play an i m p o r t a n t role in the genesis of brain dysfuncREFERENCES 1 Aoki, K. and Siegel, EL., Hyperphenylalaninemia: disaggregation of brain polyribosomes in young rats, Science, 168 (1970) 129-130. 2 Banker, G.A. and Cowan, W.M., Rat hippocampal neurons in dispersed cell culture, Brain Research, 126 (1977) 397-425. 3 Barra, H.S., Arce, C.A., Rodriguez, J.A. and Caputto, R., Some common properties of the protein that incorporates tyrosine as a single unit into microtubule protein, Biochem. Biophys. Res. Commun., 60 (1974) 1384-1390. 4 Fox, A.P., Nowycky, M.C. and Tsien, R.W., Kinetic and pharmacological properties distinguishing three types of calcium currents in chick sensory neurons, J. Physiol., 394 (1987) 149-172. 5 Kohsaka, S. and Tsukada, Y., Neurochemical correlates of discriminative learning disabilities in experimental phenylketonuric rats and in postnataUy undernourished rats. In Y. Tsukada and B.W. Agranoff (Eds.), Neurobiological Basis of Learning and Memory, Wiley, New York, 1980.
tions during p h e n y l k e t o n u r i a .
Acknowledgements. The authors are grateful to Prof. P. Kostyuk for the discussion of the results and the critical remarks during preparation of the manuscript. 6 Kostyuk, P.G., Martynyuk, A.E. and Pogorelaya, N.Ch., Effects of intracellular administration of L-tyrosine and Lphenylalanine on voltage-operated calcium conductance in PC12 pheochromocytoma cells, Brain Research, in press.. 7 Krnjevic, K. and Leblond, J., Changes in membrane currents of hippocampal neurons evoked by brief anoxia, J. Neurophysiol., 62 (1989) 15-30. 8 Rodriguez, J.A. and Borisy, G.G., Experimental phenylketonuria: replacement of carboxyl terminal tyrosine by phenylalanine in infant rat brain tubulin, Science, 206 (1979) 463-465. 9 Takahashi, K., Wakamori, M. and Akaike, N., Hippocampal CA1 pyramidal cells of rats have four voltage-dependent calcium conductances, Neurosci. Lett., 104 (1989) 229-234. 10 Yaari, Y., Hamon, B. and Lux, H.D., Development of two types of calcium channels in cultured mammalian hippocampal neurons, Science, 235 (1987) 680-682. 11 Young, S.N., The significance of tryptophan, phenylalanine, tyrosine and their metabolites in the nervous system. In A. Lajtha (Ed.), Handbook of Neurochemistry, Plenum Press, New York, Vol. 3 (1983) 559-581.
Brain Research, 552 (1991) 228-231 © 199l Elsevier Science Publishers B.V. (1006-8993/91/$03.5(I ADONIS 000689939116705R
BRES 16705
Blocking effect of intraperitoneal injection of phenylalanine on high-threshold calcium currents in rat hippocampal neurones A.E. Martynyuk, S. Savina and G.G. Skibo Bogomoletz Institute of Physiology, Ukrainian Academy of Sciences, Kiev (U. S. S. R.) (Accepted 15 January 1991)
Key words: Hippocampal neuron; Calcium current; Phenylalanine
Calcium currents were recorded in cultured (5-7 days) hippocampal neurones isolated from one-day-old rats. The animals obtained intraperitoneal injections of L-phenylalanine which induces in the brain biochemical changes characteristic of phenylketonuria. It has been found that the amplitude of the low-threshold calcium current in L-phenylalanine-affected neurones was not appreciably changed compared with that in neurones from control (non-injected) animals. However, the amplitude of the high-threshold calcium current was essentially decreased. Its relative amplitude at Vt = +20 mV became 40 + 30% as contrasted to 416 + 130% in neurones from control animals (the amplitude of the calcium currents at Vt = -10 mV taken as 100%). The decrease remained during the whole time of culturing. Addition of L-tyrosine to the cultivation medium (50 gM) restored the high-voltage calcium current, its relative amplitude reaching 280 + 57%. The data are discussed in conjunction with the previously obtained results about antagonistic modulatory action of tyrosine and phenylalanine on the functioning of high-threshold calcium channels and possible mechanisms of brain dysfunction during phenylketonuria.
INTRODUCTION It is well k n o w n that sustained elevation of L-phenylalanine in blood plasma evoked by decreased activity of phenylalanine 4-hydroxylase in the liver during congenital disease phenylketonuria provokes severe damage of brain functions in humans. The mechanisms which determine brain dysfunction during this disease are not quite understood. In our previous paper 6 it has been shown that intracellular introduction of L-phenylalanine exerts a down-regulatory effect on the activity of highthreshold calcium channels in PC12 pheochromocytoma cells. O n the contrary, introduction of L-tyrosine supported the activity of corresponding channels. The obtained data prompted us to investigate the effects of these amino acids on brain n e u r o n e s in conditions which imitate biochemical changes characteristic of phenylketonuria. MATERIALS AND METHODS Experiments were performed on cultured hippocampal neurones obtained from one-day-old rats. Experimental and control animals were pretreated according to the technique described earlier ~'8. They were placed in a thermostat for 30 min at a temperature of 30 + 2 °C. Then the experimental animals obtained intraperitoneal injections of L-phenylalanine dissolved in 0.42% NaCI solution (1 g/kg b.w.). The control animals were injected with the same volume
of 0.9% NaCI solution. After the injections the animals were returned to the thermostat for 160 min before killing. Five groups of control and experimental animals were used. The dissociated tissue culture from the hippocampus was prepared according to Banker and Cowan2. Whole-cell current recordings were performed using glass micropipettes with a resistance between 2 and 3 MD from cells which had been in culture for 5-7 days. Control measurements were made on cells obtained in the same way from animals injected with an equivalent amount of 0.9% NaCI. The pipette solution contained (in mM): CsCI 100, HEPES 20, EGTA 5, ATP 2, MgCI2 3, cAMP 0.01, TrisOH 30, GTP (sodium salt) 1 (pH 7.3). The composition of the extraceUular solution was (in mM): NaCI 100, CaC12 20, MgCI2 1.5, HEPES 5, glucose 15, T r x 0.003 (pH 7.3). All recordings were made at room temperature (21-23 °C).
RESULTS
Calcium currents in control neurones In all cells from control animals inward calcium currents (/ca) could be evoked by m e m b r a n e depolarization from holding potential Vh = - 9 0 mV. The currents appeared already at testing potentials (Vt) between - 6 0 and - 5 0 mV. Ica reached its maximal value at Vt = +20 mV and diminished to zero at Vt between +50 and +60 mV (Fig. 1). D u r i n g prolonged depolarizing potential shifts (200 ms) lca declined (inactivated). The inactivation had a complicated time course and could be separated into fast and slow c o m p o n e n t s (time constants
Correspondence: A.E. Martynyuk, Bogomoletz Institute of Physiology, Bogolometz street 4, 252601 GSP, Kiev 24, U.S.S.R.
229 A
B -60 -40 -20 0
A 20 rnV
- 30 -
+o I
/I
/
8 -60
1
0
-40
-20
0
20mY
~
200
++3oo.
10
f 33pA J
°/o 60 pA I 100m$
lea ~o
°/o 150
100m5
5OO
Fig. 1. Calcium inward currents in cultured hippocampal neurons from newborn rats. A: original current records obtained at indicated testing potentials; holding potential -90 mV. B: averaged currentvoltage characteristic normalized to current amplitude at Vt -- -10 mV; vertical bars indicate S.D. (n = 7). Vt = -10 mV was chosen for normalization in this and the following figures because it corresponds to maximal amplitude of the low-threshold current. The dotted line shows the current-voltage characteristic recorded in one neurone at holding potential -50 mV.
5 - 2 0 ms and several h u n d r e d s of milliseconds). The fast inactivating c o m p o n e n t of the total current was maximal at V t b e t w e e n - 2 0 and - 1 0 mV, reaching 31 + 13% of the t o t a l / c a ( m e a n + S . D . , n -- 12). If the holding potential was shifted to - 5 0 mV, the low-threshold c o m p o n e n t of the current d i s a p p e a r e d and the high-threshold component of I c , b e c a m e p r e d o m i n a n t (Fig. 1B). This component d e m o n s t r a t e d high sensitivity to intracellular perfusion; it b e c a m e ' w a s h e d - o u r in about 20 min, whereas the fast inactivating c o m p o n e n t r e m a i n e d quite stable (see Fig. 2). The described effects of varying holding potential and intracellular perfusion clearly indicate the presence of two p o p u l a t i o n s of calcium channels in h i p p o c a m p a l n e u r o n e s of n e w b o r n animals: the low-threshold meta-
Fig. 3. Calcium currents in hippocampal neurones from phenylalanine-injected animals. A: original current records, B: averaged current voltage characteristic (mean -+ S.D., n = 6) constructed in the same way as in Fig. 1. Holding potential -90 mV. bolic-independent (T-type) and high-threshold metabolic-dependent ones. Probably, the second p o p u l a t i o n is not h o m o g e n e o u s and contains inactivating and noninactivating channels (N- and L-types according to Fox et al. 4 and Takahashi et al.9). Their expression varied in different neurones, and their separation was not very reliable. T h e r e f o r e we identified only the low- and high-threshold c o m p o n e n t s of the calcium current, as it has also been done by Yaari et al. 10 in their investigations of cultured h i p p o c a m p a l n e u r o n e s from rat embryos.
Calcium currents in neurones from phenylalanine-injected animals. The calcium currents in n e u r o n e s from phenylalanineinjected animals could be activated by testing depolarizations similar to those in control n e u r o n e s ( - 6 0 to - 5 0 A
E5 -60
-40
-20
0
2 0 mV
i
-lO
10C Ica A
°/o 5C
10'
~
.
19' c.._Ea l
3O 10
lOOms
2O
~
2'o Fig. 2. Changes of calcium currents during perfusion of a hippocampal neurone by saline solution. Ordinate - relative amplitude of the fast inactivating (A) and slowly inactivating (O) components of the current normalized to their amplitude at the beginning of perfusion; abscissa - time after beginning of perfusion. The inset demonstrates current records obtained at indicated perfusion times. Holding potential -60 mV.
40 pA J lO0rns
°/o
40C
Fig. 4. Calcium currents in hippocampal ncurones from phenylalanine-injected animals, cultured in solution containing 50 /~M L-tyrosine. A: original current records; B: averaged current-voltage characteristic (mean + S.D., n = 7) constructed in the same way as in Fig. 1. Holding potential -90 mV.
230
6oo[
previous experiments, cells were cultured for 5-7 days. The results of calcium current measurements in such cells are presented in Fig. 4. A substantial restoration of the high-threshold calcium current after culturing the cells in tyrosine-containing medium was observed. A characteristic feature of the restored high-threshold current was the presence of definite inactivation which was well developed at V~ = +20 mV and had a time constant of about 100 ms. This may indicate that the restored current was generated mainly by N-type calcium channels. Fig. 5 summarizes data about the calcium currents amplitudes measured at Vt -- +20 mV in control neurones, neurones from phenylalanine-injected animals and neurones cultured in the presence of 50 /~M t.tyrosine.
I~° l °/°
|
4OO
300
2OO
DISCUSSION 100
1
2
3
Fig. 5. Mean amplitudes (+ S.D.) of the high-voltage component of the calcium current (Vt = +20 mV) normalized to the current amplitude at gt = -10 mV in neurones from control animals (1), neurones from phenylalanine-injected animals cultured in standard (2) and u-tyrosine containing (3) media. mV). However, the maximum of current-voltage characteristics was shifted by 20-30 mV to a more negative potential region. These Vt corresponded to the maximal contribution of the inactivating component to the total Ica in control cells (-20 to -10 mV, Fig. 3). Another difference of the modified /Ca was the depression or even complete absence of the high-voltage current component, as can be seen from records presented in Fig. 3A. In 12 cells the mean relative amplitude of the latter was only 32 + 14% at Vt -- -10 mV. The data obtained definitely indicate that in phenylalanine-pretreated animals a depression of the activity of high-threshold calcium channels takes place. On the contrary, the activity of the low-threshold (T-type) channels remains practically unchanged. The most probable reason for the detected downregulation of high-threshold calcium channels could be the shift in the tyrosine-phenylalanine concentration balance towards the latter. We made an attempt to restore the function of high-threshold calcium channels by addition of L-tyrosine to the culture medium (taking into account that aromatic amino acids penetrate quite easily through the cellular membrane, cf. ref. 11). L-Tyrosine was added in a concentration of 50 kLM immediately after the beginning of culturing. As in
The data presented are in agreement with the results obtained previously on PC12 pheochromocytoma cells: in rat hippocampal neurones the activity of the highthreshold calcium channels is also up- and down-modulated correspondingly by tyrosine and phenylalanine. An additional feature of such modulation not observed in model experiments on clonal cells becomes obvious in this case: the depressing effect of phenylalanine is extremely long-lasting, it manifests itself for many days after the injection of the amino acid in the newborn animal (at least for 5-7 days of cell culturing). Obviously, some still unknown stable changes occur in highthreshold calcium channels after exposure to phenylalanine which can be removed later on by excessive tyrosine. A possible site of action of aromatic acids on calcium channels could be the microtubular apparatus, especially in dendrites. We did not perform special morphometric investigations, but visually an intense growth of neurites was observed during cell culturing in the tyrosinecontaining solution. It has been suggested by several authors that high-threshold inactivating calcium channels are expressed mainly in the dendritic membrane 7'1°. Probably, tyrosination of the a-tubulin in the microtubuli 3 creates favourable conditions for the functioning of the high-threshold calcium channels. The replacement of tyrosine by phenylalanine in the COOH-terminal of a-tubulin (such replacement did take place in case of experimental phenylketonuria shown by Rodriguez and Borisy, ref. 8) removes such favourable conditions. The behavioural changes which occur in rats after phenylalanine treatment include inability for learning (cf. ref. 5). Some authors suggested that calcium inward currents generated mainly by high-threshold calcium channels might play an important role in long-lasting
231 changes of the effectiveness of synaptic transmission which m a y form the mechanisms for plasticity and learning. The p r o f o u n d changes seen in the activity of these channels m a y substantially disturb learning and play an i m p o r t a n t role in the genesis of brain dysfuncREFERENCES 1 Aoki, K. and Siegel, EL., Hyperphenylalaninemia: disaggregation of brain polyribosomes in young rats, Science, 168 (1970) 129-130. 2 Banker, G.A. and Cowan, W.M., Rat hippocampal neurons in dispersed cell culture, Brain Research, 126 (1977) 397-425. 3 Barra, H.S., Arce, C.A., Rodriguez, J.A. and Caputto, R., Some common properties of the protein that incorporates tyrosine as a single unit into microtubule protein, Biochem. Biophys. Res. Commun., 60 (1974) 1384-1390. 4 Fox, A.P., Nowycky, M.C. and Tsien, R.W., Kinetic and pharmacological properties distinguishing three types of calcium currents in chick sensory neurons, J. Physiol., 394 (1987) 149-172. 5 Kohsaka, S. and Tsukada, Y., Neurochemical correlates of discriminative learning disabilities in experimental phenylketonuric rats and in postnataUy undernourished rats. In Y. Tsukada and B.W. Agranoff (Eds.), Neurobiological Basis of Learning and Memory, Wiley, New York, 1980.
tions during p h e n y l k e t o n u r i a .
Acknowledgements. The authors are grateful to Prof. P. Kostyuk for the discussion of the results and the critical remarks during preparation of the manuscript. 6 Kostyuk, P.G., Martynyuk, A.E. and Pogorelaya, N.Ch., Effects of intracellular administration of L-tyrosine and Lphenylalanine on voltage-operated calcium conductance in PC12 pheochromocytoma cells, Brain Research, in press.. 7 Krnjevic, K. and Leblond, J., Changes in membrane currents of hippocampal neurons evoked by brief anoxia, J. Neurophysiol., 62 (1989) 15-30. 8 Rodriguez, J.A. and Borisy, G.G., Experimental phenylketonuria: replacement of carboxyl terminal tyrosine by phenylalanine in infant rat brain tubulin, Science, 206 (1979) 463-465. 9 Takahashi, K., Wakamori, M. and Akaike, N., Hippocampal CA1 pyramidal cells of rats have four voltage-dependent calcium conductances, Neurosci. Lett., 104 (1989) 229-234. 10 Yaari, Y., Hamon, B. and Lux, H.D., Development of two types of calcium channels in cultured mammalian hippocampal neurons, Science, 235 (1987) 680-682. 11 Young, S.N., The significance of tryptophan, phenylalanine, tyrosine and their metabolites in the nervous system. In A. Lajtha (Ed.), Handbook of Neurochemistry, Plenum Press, New York, Vol. 3 (1983) 559-581.
