- Email: [email protected]
Inhibition of dipeptide transport in mouse brain slices
323
Journal of the neurological Sciences
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
Inhibition of Dipeptide Transport in Mouse Brain Slices T. Y A M A G U C H I , M. Y A M A G U C H I A N D A. L A J T H A New York State Research Institute jbr Neurochemistry and Drug Addiction, Ward's Island, New York, N.Y. (U.S.A.)
(Received 19 April, 1969)
INTRODUCTION
In recent years an increasing number of peptides have been isolated from the brain (for a recent review see PISANO 1969), and it is becoming clear that peptides play an important role in cerebral function. Among peptides, fl-alaninylhistidine and methylhistidine (carnosine and anserine), and their 7-aminobutyric analogs (homocarnosine and homoanserine), have been found in nervous tissue (ABRAHAM et al. 1962 ; NAKAJIMAet al. 1967; TSUNOOet al. 1963). In rat brain slices carnosine but not homocarnosine was taken up in a concentrated manner, and carnosine uptake was similar in oxygen and glucose requirement to histidine uptake (ABRAHAMet al. 1962). Since recent work indicated that transport processes play an important role in influencing the level and the distribution of amino acids in the brain (LAJXHA 1968), it was of interest to investigate whether transport processes specific for peptides are also present in the brain. In this paper observations on the properties of the uptake of carnosine and anserine by brain slices are reported. We found that heat treatment of brain slices can selectively inactivate the transport of specific amino acids (YAMAOUCm et al. 1970), and therefore peptide uptake was compared in normal and heat-treated brain slices. There seem to be mechanisms in the brain involved in the transport of peptides with a specificity somewhat different from those of amino acid transport.
Presented at a symposium on The Blood-Brain Barrier, held 11 September, 1968, in New York, N.Y. (U.S.A.). This investigation was supported in part by Grant NB-04360 from the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service. T. YAMAGUCHI:present address: Department of Biochemistry, Tokyo Medical College, Tokyo (Japan). M. YAMAGUCHI: present address: Department of Neurology and Psychiatry, Showa University School of Medicine, Tokyo (Japan). J. neurol. Sci., 1970, 10:323-329
324
T. YAMAGUCI|I, M. YAMAGUCHI, A. [AJTHA
MATERIALS AND METHODS
Young adult Swiss mice, 6-8 weeks old, were decapitated, and the brain of each was quickly removed; the cerebral cortex was separated from the midbrain, and each hemisphere, weighing about 100 mg, was sliced by a Mc[lwain tissue slicer lo 0.42 mm thickness and put into a flask containing Krebs-Ringer medium with Tris phosphate buffer at pH 7.4, as previously described (LAJTFIAet aL 1963). The slices were shaken in 2 ml of medium at either normal (37°C) or elevated (47°C) temperature for 30 rain and cooled in the ice box; 2.5 ml medium containing glucose was added, bringing the final glucose concentration to 10 mmotes; then Oz was bubbled through the solution for 1 min. The flasks were further preincubated for 10 rain, 0.5 ml medium containing 1 #mole [14C] peptide (0.01#C) was added, and incubation proceeded at 3TC. At the end of the incubation, the flask contents were placed on filter paper on a Buchner funnel, extra medium surrounding the slices was drawn off by suction for" 30 sec. and the slices were picked up by a spatula and placed in dry ice. The frozen tissues were weighed, homogenized in 2 ml of 3 ~ perchloric acid, and centrifuged, The precipitates were discarded, and an aliquot of the supernatant fluid was counted in a Packard Tricarb scintillation counter. L-[1-t4C]anserine-fl-alanyl and t,-[l-l:tEt carnosine-fl-alanyl with specific activities of 11.4 mc/mM, and non-labeled peptid~s and amino acids were obtained from Calbiochem. Inc. Paper chromatography was done on ethanol extracts of tissues (70 ~;'ov,:vt. and o l the medium after the incubation period, in butanol-acetic acid-HzO(10:3:5) for 29 'a in an ascending unidirectional system. Spots were developed by ninhydrin (only |\~" standard) and counted for radioactivity by liquid scintillation. The incubation medium showed no evidence of hydrolysis of the peptides even after 6 h incubation, whereas in tissue extracts 8.8':',~ of anserine, but no carnosine, was hydrolyzed.
RESULTS
Uptake o/'anserine and carnosine
Dipeptide uptake was a slower process than amino acid uptake; and the accumulation still increased after 6 h incubation, especially with anserine, although it was not very great (Fig. 1). The uptake of anserine was faster and somewhat greater than that of carnosine. The relatively slow uptake of peptides is in contrast to that of amino acids, where in most cases final equilibrium of uptake in brain slices is reached in 60-120 min (BLASBERGAND LAJTHA 1965). Histidine uptake in brain reached equilibrium much sooner than did carnosine uptake (ABRAHAMet al. 1962). In our laboratory (YAMAGUCHIe t al. 1970) heat treatment of brain slices (incubation at 47°C for 30 min, for example) decreased the uptake of amino acid as determined by subsequent incubation at 37°C. The effect was not identical with all amino acids --under suitable conditions heat treatment almost completely abolished glutamate uptake but inhibited lysine uptake only 40°J~. The uptake of carnosine and anserine was almost completely destroyed by such heat treatment (47°C for 30 min before incubation) although a slight uptake was observed after longer incubation. d. neurol. Sci., 1990, t 0 : 3 2 3 - 3 2 9
INHIBITION OF DIPEPTIDE TRANSPORT IN MOUSE BRAIN SLICES
325
4
o a:
ca
3
z
F-
60
180
.360
INCUBATION
TIME
(MIN)
Fig. 1. T i m e - c o u r s e o f a c c u m u l a t i o n o f p e p t i d e s i n m o u s e b r a i n slices. A n s e r i n e : ©, c o n t r o l ; @, h e a t - t r e a t e d ; c a r n o s i n e : A , c o n t r o l , A , h e a t - t r e a t e d . T h e i n c u b a t i o n m e d i u m c o n t a i n e d 0.2 m M [~4C]peptides. V a l u e s , a v e r a g e s o f 8 e x p e r i m e n t s , are g i v e n as t h e r a t i o o f c o n c e n t r a t i o n o f t i s s u e w a t e r t o m e d i a a t t h e e n d o f the i n c u b a t i o n .
TABLE 1 INHIBITION _
OF
PEPTIDE
UPTAKE
BY
RELATED
_
COMPOUNDS m
lnhibitm"
30 min
(2 raM)
tissue/'medium
Percent of control 37°C
heated
_
180 rain
Per cent ~[control
tissue:'medium
37 C
heated
2.89i0.07 1.63-+0.12 . . 2.20-+0.17 3.14-+0.29 2.50-+0.23 2.11-+0.14 1.75±0.13 3.50_+0.74 3.06-+0.22 2.69-+0.10 3.31_+0.22
100.0 56.4
36.3 36.7
76.1 108.7 86.5 73.0 60.6 121.1 105.9 93.1 114.5
37.4 42.6 42.2 43.9 42.9 37.4 42.6 42.9 45.3
100.0
26.5
62.6 59.8 95.8 70.1 46.9 112.8 141.9 117.0 94.4 98.9
26.5 25.4 26.3 26.5 26.3 29.6 30.4 25.1 26.8 26.8
Carnosine 0.2 m M .... Anserine Carnosine Glycylglycine Glycylleucine Glycylglycylg[ycine fl-Alanine c-Histidine l-Methyl-histidine GABA DL-Leucine l.-Lysine
1.48i0.22 0.85-+0.11 . .
100.0 57.6 .
--1.56~0.17 1.32-i- 0.21 2.11~0.16
-105.4 77.3 128.1 ----
---
51.4 45.9 .
. ---41.9 46.6 42.6 ----
.
Anserine 0.2 m M Anserine Carnosine Glycylglycine Glycylleucine Glycylglycylglycine I]-Alanine L-Histidine 1- M e t h y l - h i s t i d i n e GABA DL-Leucine L-Lysine
2.20-+0.57 . 1.20-+0.18 ---1.21 ± 0 . 3 0 2.33-+0.52 2.37~0.52 ----
.
100.0 . 54.5 --55.0 105.9 107.7 ----
33.2 . 30.5 ---28.6 29.5 30.5 ----
3.58-+0.20 . . 2.24±0.35 2.14-+0.22 3.43-r:0.18 2.51-+0.23 1.685_0.09 4.04~0.25 5.08::[_0.84 4.19~:0.18 3.38--0.14 3.54-+0.34
M o u s e b r a i n slices p r e p a r e d as d e s c r i b e d in the m e t h o d s s e c t i o n w e r e t r e a t e d at 37°C or 4 7 ° C ( h e a t e d ) for 30 m i n a n d t h e n i n c u b a t e d w i t h 0.2 m M [ 1 4 C ] p e p t i d e for 30 o r 180 rain. I n h i b i t o r s w h e n p r e s e n t w e r e 2 raM. V a l u e s s h o w n a r e c o n c e n t r a t i o n r a t i o s , t i s s u e w a t e r p e r m e d i u m a t t h e e n d o f the i n c u b a t i o n -+ S.D., a n d t i s s u e m e d i u m r a t i o s as p e r c e n t o f c o n t r o l . A v e r a g e s o f 6 e x p e r i m e n t s are given.
J. nearol. Sci., 1970, 1 0 : 3 2 3 - 3 2 9
326
T. YAMAGUCHI, M. YAMAGUCHI, A. LAJTttA
Competition with dipeptides and amino acid analogs An investigation of specificity of amino acid uptake of brain slices revealed several transport classes (BLASBERGAND LAJTHA 1966). Measurement of the effect of analogs revealed some difference between carnosine and anserine uptake. For shorter and longer term experiments, 30 and 180 min incubations, respectively, and an analog to peptide ratio of 1:10 were selected. Under such experimental conditions anserine uptake was inhibited by carnosine, by glycylglycine, and to a smaller extent by glycylglycylglycine. Uptake was also inhibited by/3-alanine, but no effect was seen with L-methylhistidine, a component of anserine (Table l). Carnosine uptake was inhibited by anserine and slightly inhibited by glycylglycine and glycylglycylglycine./~-Alanine did not inhibit uptake in short-term experiments. but inhibition was observed upon longer incubation. Histidine inhibited carnosine transport, whereas it had no effect on anserine transport. /3-Alanine was the only amino acid that had an affinity to both dipeptide carriers. Heat treatment greatly decreased anserine and carnosine uptake with tissue-to-medium ratios below unity at 30 min and close to unity at 180 min. Most active transport ofpeptides was gone, and the analogs tried had no effect on the movement of peptides in heat-treated slices (Table 2). TABLE 2 SODIUM-DEPENDENCE
OF P E P T I D E U P T A K E I N B R A I N S L I C E S
Anserine / 2 8 r a m Na 12.SmM Na
Tissue per medium
30rain 180 rain
1.28~-0.18 2.29 ~0.35
Percent of control
30 rain 180 rain
100 100
Tissue per medium
30min 180 rain
0.63:~0.10 0.97 t~0.07
Percent of control
30 rain 180 rain
0.76--0,11 1.02 :~-0.07 59.4 44.5
('arnosine 1 2 8 m M Na
1 2 . 8 m M Na
1.1220,07 1.76+0.13
0.77~0.07 1.1 t ~:0.07
100
68.8
100
63.1
Heat treated
49.2 42.4
0.70-i: 0.09 1.04~0.08 54.7 45.4
0.65±0.04 0.963:~0.05 58.0 54.5
0.72i0.06 1.13-c 0.13 64.3 64.2
Brain slices were incubated in the presence of 0.2 mM[~4C] peptides in high (128 m M ) o r low (12.8 m M ) Na-containing Krebs-Ringer media. To maintain osmolarity when NaCI was lowered, it was replaced by choline chloride of equal concentration. Averages of 5 experiments z~ S.D. are shown.
Na+ion requirement for dipeptide transport Amino acid uptake in brain slices has an absolute requirement for Na ions. The absence of Na + inhibits uptake completely; lowering Na + lowers uptake, but not to the same degree in each case (MARGOLIS AND LAJTI-IA 1968). When the Na ~ concentration was reduced to 12.8 m M from 128 mM, with choline chloride replacing Na + to maintain osmolarity, the uptake of anserine and carnosine was decreased, with tissueto-medium ratios below unity at 30 min and close to unity at 180 rain. After heat J. neurol. Sci., 1970, 10:323-329
327
INHIBITION OF D1PEPTIDE TRANSPORT IN MOUSE BRAIN SLICES
treatment at 47°C for 30 min, no active transport was observed, and no difference due to the presence of high or low concentration of Na + in the medium (Table 2).
Influence of metabolic inhibitors The concentrative uptake of dipeptides was strongly inhibited by metabolic inhibitors such as iodoacctic acid, 2,4-dinitrophenol, and sodium cyanide (Table 3); inhibitors of protein synthesis like puromycin or cycloheximide did not show any inhibitory effect. As with the uptake of amino acids, cyanide was a stronger inhibitor than dinitrophenol. No change was observed in the heat-treated slices.
TABLE 3 EFFECT OF METABOLIC INH1BITORS ON PEPTIDE T R A N S P O R T
30 min Inhibitor
(Conc. mM)
tissue/mediu m
per cent of control 37°C
-IAA DNP NaCN NEM Puromycin Cycloheximide
IAA DNP NaCN NEM Puromycin Cycloheximide
180 min tissue/medium
heated
1 0.02 1 0. I 0.02 0.004
1.55±0.03 0.56± 0.66±0.06 0.63 ± 0.02 1.78±0.08 1.78+0.02 1.54±0.03
Carnosine 100.0 41.3 36.1 43.2 42.6 39.4 40.6 36.8 114.8 41.3 114.8 38.7 99.4 42.6
I 0.02 l 0.1 0.02 0.004
2.10±0.30 0.60-k0.05 0.5410.02 0.51~+0.02 2.13±0.15 2.11±0.07 2.10±0.05
A nserine 100.0 27.1 28.0 33.2 25.2 27.1 23.8 22.4 99.5 26.6 98.6 26.6 98.1 28.5
per cent o f control 37°C
heated
2.845_0.24 1.05±0.08 1.294~0.11 0.99~0.08 2.88 ~ 0.38 3.12~-0.10 3.1%k0.06
100.0 37.0 45.4 34.8 101.4 109.8 112.3
41.9 37.7 32.7 25.7 41.5 36.3 39.8
4.135_0.13 0.98~0.12 1.20i0.14 0.91~0.10 3.72~0.36 4.17~0.29 4.37~: 0.44
100.0 23.7 29.1 22.0 90.1 101.0 105.8
24.7 24.7 22.8 18.9 26.2 24.2 23.0
Brain slices were treated as described in the legends to Table 1, except that in the 10 min pre-incubation medium the inhibitor was present. Averages of 5 experiments ± S . D . are given. The concentration of carnosine and anserine was 0.2 m M i n the medium.
DISCUSSION
Anserine is taken up by brain slices against a concentration gradient--as shown previously with carnosine (A~RAHAM et al. 1962). The 2 peptides behave similarly in most aspects in that their accumulation is considerably slower than that of the aminoacids but continues to increase for a much longer time. The greater inhibition of peptide uptake by peptides than by amino acids indicates the existence of a specific transport system at least for the two peptides studied (ABRAHAM et al. 1964). The somewhat different effects of histidine and fl-alanine (Table 1) and the greater uptake of carnosine than homocarnosine (ABRAHAM et al. 1962) would indicate further possible differentiation in the transport of these two related peptides. It is of further J. neurol. Sci., 1970, 10:323-329
328
r . YAMAGU( HI, M. YAMAGUCHI, A. LAJTHA
interest that methylhistidme (a component of anserme) and ;,-aminobutyric acid (a component of homocarnosine and homoanserine) had no inhibitory effects under our experimental conditions. The sensitivity of peptide transport was equal to or greater than that of amino acid transport in brain slices with the inhibitors tried (heal treatment, lowered Na, and metabolic inhibitors). It is clear from the present and from previous results that the uptake of carnosine and anserine is similar to amino acid uptake in brain slices in its energy-dependence (stimulated by glucose and oxygen, inhibited by cyanide) and in its sodium-dependence. There seem to be important differences, however. The accumulation of the peptides tried (carnosine, anserine, and homocarnosine) increases for a much longer time than that of any amino acid tried--in fact it continues lbr so long (welt over 6 h) that it is not clear whether equilibrium was reached, or the slices were damaged, after the prolonged incubation. Equilibrium is reached much faster by the component amino acids, histidine, and :,-aminobutyric acid (A~RAHAM et al. 1962: BLASm-:R(; AND LAJTHA 1965). Peptide transport also seems to be more heat-sensitive than amino acid transport. Studies of specificity of cerebral amino acid movement indicated several transport classes in the brain (NEaME 1968, LAJTHAe t al. 1967), among them one l'or/7-alanine and ~,-aminobutyric acid, which was different from the classes that included the other amino acids. The fact that :,-aminobutyric acid, which is very strongly accumulated by brain slices, has no strong inhibitory effect on the uptake of peptides; the differences between the accumulation of homocarnosine and carnosine: and the differences in the effect of analogs on carnosine and anserine would all indicate further specificity for peptide transport. The existence of specific transport mechanisms lbr peptides in the brain indicates the significant role these compounds play in the nervous system--whether originating endogenously or exogenously---and it also indicates a specific regulation of peptide distribution in the various cerebral components. ACKNOWLEDGEMENI S We are indebted to Professor S. Tsunoo for many helpful discussions and to Mr. A. Mazeika for excellent technical assistance.
SUMMARY
Uptake of anserine (fl-alanyl-l-methylhistidine) and carnosine (fi-alanyt-histidine) by mouse brain slices was studied. Some properties of fl-alanyl peptide transport were compared with those of amino acid transport with regard to heat sensitivity, competition with analogs, and Na ion and energy requirements. Accumulation of peptides proceeded for a longer period than that of amino acids, and equilibrium was not reached by 6 h. The concentrative uptake of peptides was at least as sensitive as that of amino acid transport in that it was strongly inhibited by lowered Na + and dinitrophenol, and was J. ,eurol. Sci.. 1970, 10:323-329
INHIBITION OF DIPEPTIDE TRANSPORT IN MOUSE BRAIN SLICES
329
a b o l i s h e d b y c y a n i d e o r i o d o a c e t a t e o r b y t r e a t m e n t o f t h e slices a t 4 7 ° C f o r 30 m i n . I n h i b i t i o n b y a n a l o g s i n d i c a t e d t h e p r e s e n c e o f specific p r o c e s s e s f o r p e p t i d e t r a n s p o r t in t h e b r a i n .
REFERENCES ABRAHAM, D., J. J. PISANOAND S. UDENFRIEND(1962) The distribution of homocarnosine in mammals, Arch. Biochem. Biophys., 99 : 210-213. BLASBERG,R. AND A. LAJTHA (1965) Substrate specificity of steady-state amino acid transport in mouse brain slices, Arch. Biochem. Biophys., 112: 361-377. BLASBERG, R. AND A. LAJTHA (1966) Heterogeneity of the mediated transport systems of amino acid uptake in brain, Brain Res., l : 86-104. LAJTHA, A. (1968) Transport as control mechanism of cerebral metabolite levels. In: A. LAJTHA AND D. H. FORD (Eds.), Brain Barrier Systems (Progress in Brain Research, Vol. 29), Elsevier, Amsterdam, pp. 201-218. LAJTHA, A., G. LEVI AND R. BLASBERG (1967) Specificity of cerebral amino acid transport. In: I. KLATZO AND E. SEITELBERGER(Eds.), Brain Edema, Springer, New York, pp. 339-353. LAJTHA, A., S. LAHIRI AND J. TOTH (1963) The brain barrier system, Part 4 (Cerebral amino acid uptake in different classes), J. Neurochem., 10: 765-773. MARGOLIS, R. AND A. LAJTHA 0968) Ion dependence of amino acid uptake in brain slices, Bioehim. Biophys. Acta, 163: 374-385. NAKAJIMA,T., F. WOLFGRAMAND W. G. CLARK (1967) The isolation of homoanserine from bovine brain, J. Neurochem., 14:1107-1112. NEAME, K. D. (1968) A comparison of the transport system for amino acids in the brain, intestine, kidney and tumour. In: A. LAJTHA AND D. H. FORD (Eds.), Brain Barrier Systems (Progress in Brain Research, Vol. 29) Elsevier, Amsterdam, pp. 185-196. PISANO, J. J. (1969) Peptides. In: A. LAJTHA (Ed.), Handbook of Neurochemistry, Vol. 1, Plenum, New York, N.Y., pp. 53-74. TSUNOO, S., K. HORmAKA ANn M. KAWASUMI(1963) Concerning the free amino acids in chicken brain. The isolation and identification of histidine and anserine, J. Bioehem. (Tokyo), 54: 355-362. YAMAGUCHI, T., M. YAMAGUCHI AND m. LAJTHA(1970) The effect of heat treatment on the uptake of amino acids by brain slices, Biochim. Biophys. Acta, In press.
J. neurol. Sci., 1970, 10:323-329
Journal of the neurological Sciences
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
Inhibition of Dipeptide Transport in Mouse Brain Slices T. Y A M A G U C H I , M. Y A M A G U C H I A N D A. L A J T H A New York State Research Institute jbr Neurochemistry and Drug Addiction, Ward's Island, New York, N.Y. (U.S.A.)
(Received 19 April, 1969)
INTRODUCTION
In recent years an increasing number of peptides have been isolated from the brain (for a recent review see PISANO 1969), and it is becoming clear that peptides play an important role in cerebral function. Among peptides, fl-alaninylhistidine and methylhistidine (carnosine and anserine), and their 7-aminobutyric analogs (homocarnosine and homoanserine), have been found in nervous tissue (ABRAHAM et al. 1962 ; NAKAJIMAet al. 1967; TSUNOOet al. 1963). In rat brain slices carnosine but not homocarnosine was taken up in a concentrated manner, and carnosine uptake was similar in oxygen and glucose requirement to histidine uptake (ABRAHAMet al. 1962). Since recent work indicated that transport processes play an important role in influencing the level and the distribution of amino acids in the brain (LAJXHA 1968), it was of interest to investigate whether transport processes specific for peptides are also present in the brain. In this paper observations on the properties of the uptake of carnosine and anserine by brain slices are reported. We found that heat treatment of brain slices can selectively inactivate the transport of specific amino acids (YAMAOUCm et al. 1970), and therefore peptide uptake was compared in normal and heat-treated brain slices. There seem to be mechanisms in the brain involved in the transport of peptides with a specificity somewhat different from those of amino acid transport.
Presented at a symposium on The Blood-Brain Barrier, held 11 September, 1968, in New York, N.Y. (U.S.A.). This investigation was supported in part by Grant NB-04360 from the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service. T. YAMAGUCHI:present address: Department of Biochemistry, Tokyo Medical College, Tokyo (Japan). M. YAMAGUCHI: present address: Department of Neurology and Psychiatry, Showa University School of Medicine, Tokyo (Japan). J. neurol. Sci., 1970, 10:323-329
324
T. YAMAGUCI|I, M. YAMAGUCHI, A. [AJTHA
MATERIALS AND METHODS
Young adult Swiss mice, 6-8 weeks old, were decapitated, and the brain of each was quickly removed; the cerebral cortex was separated from the midbrain, and each hemisphere, weighing about 100 mg, was sliced by a Mc[lwain tissue slicer lo 0.42 mm thickness and put into a flask containing Krebs-Ringer medium with Tris phosphate buffer at pH 7.4, as previously described (LAJTFIAet aL 1963). The slices were shaken in 2 ml of medium at either normal (37°C) or elevated (47°C) temperature for 30 rain and cooled in the ice box; 2.5 ml medium containing glucose was added, bringing the final glucose concentration to 10 mmotes; then Oz was bubbled through the solution for 1 min. The flasks were further preincubated for 10 rain, 0.5 ml medium containing 1 #mole [14C] peptide (0.01#C) was added, and incubation proceeded at 3TC. At the end of the incubation, the flask contents were placed on filter paper on a Buchner funnel, extra medium surrounding the slices was drawn off by suction for" 30 sec. and the slices were picked up by a spatula and placed in dry ice. The frozen tissues were weighed, homogenized in 2 ml of 3 ~ perchloric acid, and centrifuged, The precipitates were discarded, and an aliquot of the supernatant fluid was counted in a Packard Tricarb scintillation counter. L-[1-t4C]anserine-fl-alanyl and t,-[l-l:tEt carnosine-fl-alanyl with specific activities of 11.4 mc/mM, and non-labeled peptid~s and amino acids were obtained from Calbiochem. Inc. Paper chromatography was done on ethanol extracts of tissues (70 ~;'ov,:vt. and o l the medium after the incubation period, in butanol-acetic acid-HzO(10:3:5) for 29 'a in an ascending unidirectional system. Spots were developed by ninhydrin (only |\~" standard) and counted for radioactivity by liquid scintillation. The incubation medium showed no evidence of hydrolysis of the peptides even after 6 h incubation, whereas in tissue extracts 8.8':',~ of anserine, but no carnosine, was hydrolyzed.
RESULTS
Uptake o/'anserine and carnosine
Dipeptide uptake was a slower process than amino acid uptake; and the accumulation still increased after 6 h incubation, especially with anserine, although it was not very great (Fig. 1). The uptake of anserine was faster and somewhat greater than that of carnosine. The relatively slow uptake of peptides is in contrast to that of amino acids, where in most cases final equilibrium of uptake in brain slices is reached in 60-120 min (BLASBERGAND LAJTHA 1965). Histidine uptake in brain reached equilibrium much sooner than did carnosine uptake (ABRAHAMet al. 1962). In our laboratory (YAMAGUCHIe t al. 1970) heat treatment of brain slices (incubation at 47°C for 30 min, for example) decreased the uptake of amino acid as determined by subsequent incubation at 37°C. The effect was not identical with all amino acids --under suitable conditions heat treatment almost completely abolished glutamate uptake but inhibited lysine uptake only 40°J~. The uptake of carnosine and anserine was almost completely destroyed by such heat treatment (47°C for 30 min before incubation) although a slight uptake was observed after longer incubation. d. neurol. Sci., 1990, t 0 : 3 2 3 - 3 2 9
INHIBITION OF DIPEPTIDE TRANSPORT IN MOUSE BRAIN SLICES
325
4
o a:
ca
3
z
F-
60
180
.360
INCUBATION
TIME
(MIN)
Fig. 1. T i m e - c o u r s e o f a c c u m u l a t i o n o f p e p t i d e s i n m o u s e b r a i n slices. A n s e r i n e : ©, c o n t r o l ; @, h e a t - t r e a t e d ; c a r n o s i n e : A , c o n t r o l , A , h e a t - t r e a t e d . T h e i n c u b a t i o n m e d i u m c o n t a i n e d 0.2 m M [~4C]peptides. V a l u e s , a v e r a g e s o f 8 e x p e r i m e n t s , are g i v e n as t h e r a t i o o f c o n c e n t r a t i o n o f t i s s u e w a t e r t o m e d i a a t t h e e n d o f the i n c u b a t i o n .
TABLE 1 INHIBITION _
OF
PEPTIDE
UPTAKE
BY
RELATED
_
COMPOUNDS m
lnhibitm"
30 min
(2 raM)
tissue/'medium
Percent of control 37°C
heated
_
180 rain
Per cent ~[control
tissue:'medium
37 C
heated
2.89i0.07 1.63-+0.12 . . 2.20-+0.17 3.14-+0.29 2.50-+0.23 2.11-+0.14 1.75±0.13 3.50_+0.74 3.06-+0.22 2.69-+0.10 3.31_+0.22
100.0 56.4
36.3 36.7
76.1 108.7 86.5 73.0 60.6 121.1 105.9 93.1 114.5
37.4 42.6 42.2 43.9 42.9 37.4 42.6 42.9 45.3
100.0
26.5
62.6 59.8 95.8 70.1 46.9 112.8 141.9 117.0 94.4 98.9
26.5 25.4 26.3 26.5 26.3 29.6 30.4 25.1 26.8 26.8
Carnosine 0.2 m M .... Anserine Carnosine Glycylglycine Glycylleucine Glycylglycylg[ycine fl-Alanine c-Histidine l-Methyl-histidine GABA DL-Leucine l.-Lysine
1.48i0.22 0.85-+0.11 . .
100.0 57.6 .
--1.56~0.17 1.32-i- 0.21 2.11~0.16
-105.4 77.3 128.1 ----
---
51.4 45.9 .
. ---41.9 46.6 42.6 ----
.
Anserine 0.2 m M Anserine Carnosine Glycylglycine Glycylleucine Glycylglycylglycine I]-Alanine L-Histidine 1- M e t h y l - h i s t i d i n e GABA DL-Leucine L-Lysine
2.20-+0.57 . 1.20-+0.18 ---1.21 ± 0 . 3 0 2.33-+0.52 2.37~0.52 ----
.
100.0 . 54.5 --55.0 105.9 107.7 ----
33.2 . 30.5 ---28.6 29.5 30.5 ----
3.58-+0.20 . . 2.24±0.35 2.14-+0.22 3.43-r:0.18 2.51-+0.23 1.685_0.09 4.04~0.25 5.08::[_0.84 4.19~:0.18 3.38--0.14 3.54-+0.34
M o u s e b r a i n slices p r e p a r e d as d e s c r i b e d in the m e t h o d s s e c t i o n w e r e t r e a t e d at 37°C or 4 7 ° C ( h e a t e d ) for 30 m i n a n d t h e n i n c u b a t e d w i t h 0.2 m M [ 1 4 C ] p e p t i d e for 30 o r 180 rain. I n h i b i t o r s w h e n p r e s e n t w e r e 2 raM. V a l u e s s h o w n a r e c o n c e n t r a t i o n r a t i o s , t i s s u e w a t e r p e r m e d i u m a t t h e e n d o f the i n c u b a t i o n -+ S.D., a n d t i s s u e m e d i u m r a t i o s as p e r c e n t o f c o n t r o l . A v e r a g e s o f 6 e x p e r i m e n t s are given.
J. nearol. Sci., 1970, 1 0 : 3 2 3 - 3 2 9
326
T. YAMAGUCHI, M. YAMAGUCHI, A. LAJTttA
Competition with dipeptides and amino acid analogs An investigation of specificity of amino acid uptake of brain slices revealed several transport classes (BLASBERGAND LAJTHA 1966). Measurement of the effect of analogs revealed some difference between carnosine and anserine uptake. For shorter and longer term experiments, 30 and 180 min incubations, respectively, and an analog to peptide ratio of 1:10 were selected. Under such experimental conditions anserine uptake was inhibited by carnosine, by glycylglycine, and to a smaller extent by glycylglycylglycine. Uptake was also inhibited by/3-alanine, but no effect was seen with L-methylhistidine, a component of anserine (Table l). Carnosine uptake was inhibited by anserine and slightly inhibited by glycylglycine and glycylglycylglycine./~-Alanine did not inhibit uptake in short-term experiments. but inhibition was observed upon longer incubation. Histidine inhibited carnosine transport, whereas it had no effect on anserine transport. /3-Alanine was the only amino acid that had an affinity to both dipeptide carriers. Heat treatment greatly decreased anserine and carnosine uptake with tissue-to-medium ratios below unity at 30 min and close to unity at 180 min. Most active transport ofpeptides was gone, and the analogs tried had no effect on the movement of peptides in heat-treated slices (Table 2). TABLE 2 SODIUM-DEPENDENCE
OF P E P T I D E U P T A K E I N B R A I N S L I C E S
Anserine / 2 8 r a m Na 12.SmM Na
Tissue per medium
30rain 180 rain
1.28~-0.18 2.29 ~0.35
Percent of control
30 rain 180 rain
100 100
Tissue per medium
30min 180 rain
0.63:~0.10 0.97 t~0.07
Percent of control
30 rain 180 rain
0.76--0,11 1.02 :~-0.07 59.4 44.5
('arnosine 1 2 8 m M Na
1 2 . 8 m M Na
1.1220,07 1.76+0.13
0.77~0.07 1.1 t ~:0.07
100
68.8
100
63.1
Heat treated
49.2 42.4
0.70-i: 0.09 1.04~0.08 54.7 45.4
0.65±0.04 0.963:~0.05 58.0 54.5
0.72i0.06 1.13-c 0.13 64.3 64.2
Brain slices were incubated in the presence of 0.2 mM[~4C] peptides in high (128 m M ) o r low (12.8 m M ) Na-containing Krebs-Ringer media. To maintain osmolarity when NaCI was lowered, it was replaced by choline chloride of equal concentration. Averages of 5 experiments z~ S.D. are shown.
Na+ion requirement for dipeptide transport Amino acid uptake in brain slices has an absolute requirement for Na ions. The absence of Na + inhibits uptake completely; lowering Na + lowers uptake, but not to the same degree in each case (MARGOLIS AND LAJTI-IA 1968). When the Na ~ concentration was reduced to 12.8 m M from 128 mM, with choline chloride replacing Na + to maintain osmolarity, the uptake of anserine and carnosine was decreased, with tissueto-medium ratios below unity at 30 min and close to unity at 180 rain. After heat J. neurol. Sci., 1970, 10:323-329
327
INHIBITION OF D1PEPTIDE TRANSPORT IN MOUSE BRAIN SLICES
treatment at 47°C for 30 min, no active transport was observed, and no difference due to the presence of high or low concentration of Na + in the medium (Table 2).
Influence of metabolic inhibitors The concentrative uptake of dipeptides was strongly inhibited by metabolic inhibitors such as iodoacctic acid, 2,4-dinitrophenol, and sodium cyanide (Table 3); inhibitors of protein synthesis like puromycin or cycloheximide did not show any inhibitory effect. As with the uptake of amino acids, cyanide was a stronger inhibitor than dinitrophenol. No change was observed in the heat-treated slices.
TABLE 3 EFFECT OF METABOLIC INH1BITORS ON PEPTIDE T R A N S P O R T
30 min Inhibitor
(Conc. mM)
tissue/mediu m
per cent of control 37°C
-IAA DNP NaCN NEM Puromycin Cycloheximide
IAA DNP NaCN NEM Puromycin Cycloheximide
180 min tissue/medium
heated
1 0.02 1 0. I 0.02 0.004
1.55±0.03 0.56± 0.66±0.06 0.63 ± 0.02 1.78±0.08 1.78+0.02 1.54±0.03
Carnosine 100.0 41.3 36.1 43.2 42.6 39.4 40.6 36.8 114.8 41.3 114.8 38.7 99.4 42.6
I 0.02 l 0.1 0.02 0.004
2.10±0.30 0.60-k0.05 0.5410.02 0.51~+0.02 2.13±0.15 2.11±0.07 2.10±0.05
A nserine 100.0 27.1 28.0 33.2 25.2 27.1 23.8 22.4 99.5 26.6 98.6 26.6 98.1 28.5
per cent o f control 37°C
heated
2.845_0.24 1.05±0.08 1.294~0.11 0.99~0.08 2.88 ~ 0.38 3.12~-0.10 3.1%k0.06
100.0 37.0 45.4 34.8 101.4 109.8 112.3
41.9 37.7 32.7 25.7 41.5 36.3 39.8
4.135_0.13 0.98~0.12 1.20i0.14 0.91~0.10 3.72~0.36 4.17~0.29 4.37~: 0.44
100.0 23.7 29.1 22.0 90.1 101.0 105.8
24.7 24.7 22.8 18.9 26.2 24.2 23.0
Brain slices were treated as described in the legends to Table 1, except that in the 10 min pre-incubation medium the inhibitor was present. Averages of 5 experiments ± S . D . are given. The concentration of carnosine and anserine was 0.2 m M i n the medium.
DISCUSSION
Anserine is taken up by brain slices against a concentration gradient--as shown previously with carnosine (A~RAHAM et al. 1962). The 2 peptides behave similarly in most aspects in that their accumulation is considerably slower than that of the aminoacids but continues to increase for a much longer time. The greater inhibition of peptide uptake by peptides than by amino acids indicates the existence of a specific transport system at least for the two peptides studied (ABRAHAM et al. 1964). The somewhat different effects of histidine and fl-alanine (Table 1) and the greater uptake of carnosine than homocarnosine (ABRAHAM et al. 1962) would indicate further possible differentiation in the transport of these two related peptides. It is of further J. neurol. Sci., 1970, 10:323-329
328
r . YAMAGU( HI, M. YAMAGUCHI, A. LAJTHA
interest that methylhistidme (a component of anserme) and ;,-aminobutyric acid (a component of homocarnosine and homoanserine) had no inhibitory effects under our experimental conditions. The sensitivity of peptide transport was equal to or greater than that of amino acid transport in brain slices with the inhibitors tried (heal treatment, lowered Na, and metabolic inhibitors). It is clear from the present and from previous results that the uptake of carnosine and anserine is similar to amino acid uptake in brain slices in its energy-dependence (stimulated by glucose and oxygen, inhibited by cyanide) and in its sodium-dependence. There seem to be important differences, however. The accumulation of the peptides tried (carnosine, anserine, and homocarnosine) increases for a much longer time than that of any amino acid tried--in fact it continues lbr so long (welt over 6 h) that it is not clear whether equilibrium was reached, or the slices were damaged, after the prolonged incubation. Equilibrium is reached much faster by the component amino acids, histidine, and :,-aminobutyric acid (A~RAHAM et al. 1962: BLASm-:R(; AND LAJTHA 1965). Peptide transport also seems to be more heat-sensitive than amino acid transport. Studies of specificity of cerebral amino acid movement indicated several transport classes in the brain (NEaME 1968, LAJTHAe t al. 1967), among them one l'or/7-alanine and ~,-aminobutyric acid, which was different from the classes that included the other amino acids. The fact that :,-aminobutyric acid, which is very strongly accumulated by brain slices, has no strong inhibitory effect on the uptake of peptides; the differences between the accumulation of homocarnosine and carnosine: and the differences in the effect of analogs on carnosine and anserine would all indicate further specificity for peptide transport. The existence of specific transport mechanisms lbr peptides in the brain indicates the significant role these compounds play in the nervous system--whether originating endogenously or exogenously---and it also indicates a specific regulation of peptide distribution in the various cerebral components. ACKNOWLEDGEMENI S We are indebted to Professor S. Tsunoo for many helpful discussions and to Mr. A. Mazeika for excellent technical assistance.
SUMMARY
Uptake of anserine (fl-alanyl-l-methylhistidine) and carnosine (fi-alanyt-histidine) by mouse brain slices was studied. Some properties of fl-alanyl peptide transport were compared with those of amino acid transport with regard to heat sensitivity, competition with analogs, and Na ion and energy requirements. Accumulation of peptides proceeded for a longer period than that of amino acids, and equilibrium was not reached by 6 h. The concentrative uptake of peptides was at least as sensitive as that of amino acid transport in that it was strongly inhibited by lowered Na + and dinitrophenol, and was J. ,eurol. Sci.. 1970, 10:323-329
INHIBITION OF DIPEPTIDE TRANSPORT IN MOUSE BRAIN SLICES
329
a b o l i s h e d b y c y a n i d e o r i o d o a c e t a t e o r b y t r e a t m e n t o f t h e slices a t 4 7 ° C f o r 30 m i n . I n h i b i t i o n b y a n a l o g s i n d i c a t e d t h e p r e s e n c e o f specific p r o c e s s e s f o r p e p t i d e t r a n s p o r t in t h e b r a i n .
REFERENCES ABRAHAM, D., J. J. PISANOAND S. UDENFRIEND(1962) The distribution of homocarnosine in mammals, Arch. Biochem. Biophys., 99 : 210-213. BLASBERG,R. AND A. LAJTHA (1965) Substrate specificity of steady-state amino acid transport in mouse brain slices, Arch. Biochem. Biophys., 112: 361-377. BLASBERG, R. AND A. LAJTHA (1966) Heterogeneity of the mediated transport systems of amino acid uptake in brain, Brain Res., l : 86-104. LAJTHA, A. (1968) Transport as control mechanism of cerebral metabolite levels. In: A. LAJTHA AND D. H. FORD (Eds.), Brain Barrier Systems (Progress in Brain Research, Vol. 29), Elsevier, Amsterdam, pp. 201-218. LAJTHA, A., G. LEVI AND R. BLASBERG (1967) Specificity of cerebral amino acid transport. In: I. KLATZO AND E. SEITELBERGER(Eds.), Brain Edema, Springer, New York, pp. 339-353. LAJTHA, A., S. LAHIRI AND J. TOTH (1963) The brain barrier system, Part 4 (Cerebral amino acid uptake in different classes), J. Neurochem., 10: 765-773. MARGOLIS, R. AND A. LAJTHA 0968) Ion dependence of amino acid uptake in brain slices, Bioehim. Biophys. Acta, 163: 374-385. NAKAJIMA,T., F. WOLFGRAMAND W. G. CLARK (1967) The isolation of homoanserine from bovine brain, J. Neurochem., 14:1107-1112. NEAME, K. D. (1968) A comparison of the transport system for amino acids in the brain, intestine, kidney and tumour. In: A. LAJTHA AND D. H. FORD (Eds.), Brain Barrier Systems (Progress in Brain Research, Vol. 29) Elsevier, Amsterdam, pp. 185-196. PISANO, J. J. (1969) Peptides. In: A. LAJTHA (Ed.), Handbook of Neurochemistry, Vol. 1, Plenum, New York, N.Y., pp. 53-74. TSUNOO, S., K. HORmAKA ANn M. KAWASUMI(1963) Concerning the free amino acids in chicken brain. The isolation and identification of histidine and anserine, J. Bioehem. (Tokyo), 54: 355-362. YAMAGUCHI, T., M. YAMAGUCHI AND m. LAJTHA(1970) The effect of heat treatment on the uptake of amino acids by brain slices, Biochim. Biophys. Acta, In press.
J. neurol. Sci., 1970, 10:323-329