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Uptake and utilization of [3H-]5-hydroxytryptophan by brain tissue during development
ffe~r~p~~rn~a~olo~y, 1972,11,363-372 Pergamon Press.
PrintedinGt.Britain,
UPTAKE AND UTILIZATION OF [3H-]5-HYDROXYTRYPTOPHAN BY BRAIN TISSUE DURING DEVELOPMENT* and P. LUNDBORG
C. KELLOGG
Department of Pharmacology, University of Goteborg, Gateborg, Sweden (Accepfed
23 August 1971)
Summary-The in viva utilization of tritiated S-hydrox~ryptophan was analyzed separately in the hemispheres, diencephdon, and brain stem of rats during postnatal development. A time-study of the concentration of 3H-5-hydroxytryptophan, 3H-5-hydroxytryptamine, and 3H-5-hydroxyindoieacetic acid in the respective brain regions following the S.C.injection of 3H-5-hydroxytryptophan demonstrated that the latter disappearedfrom the brain at a slower rate in rats at I and 4 days of age than in 21-day-old animals. The concentration of SH-5-hydroxytryptamine and 8H-5-hydroxyindoleacteic acid over time followed that of the precursor. Pretreating animals with an inhibitor of peripheral decarboxylase, MK-486, indicated that the capacity of the decarboxylase in peripheral organs increases markedly with age and thus alters the appearance in the brain of peripherally administered precursor.
THE ADMINISTRATIONof DL, 5-hydroxytryptophan has been used as a clinical approach in the treatment of mongoloidism (BAZELON, PAINE, Cowre, HUNT, HAUCK and ~AHANANR, 1967). Such treatment has been shown to produce an improvement in muscle tone; however, it has been reported that the effective dose level must be increased between 4 and 6 months of age. An investigation of mechanisms contributing to the decrease in the effectiveness of 5-hydroxytryptophan with age has not been reported. An increase in the capacity of peripheral L-amino acid decarboxylase, the enzyme responsible for the decarboxylation of 5hydroxytryptophan (5-HTP) to 5-hydroxytryptamine (5-HT), has been observed to occur with age in young animals (BOJANEK,BOZKOWAand KURZEPA, 1965/1966; SMITH, STACEYand YOUNG, 1962). The ontogenic development of the peripheral decarboxylase markedly affects the production of tritiated catecholamines in the brain of young rats following the peripheral administration of the tritiated precursor, L-3,4-dihydroxyphenyia~anine (KELLOGGand LUNDBORG,1971). Because little is understood of the utilization of 5-HTP in the brain during development, the present study was undertaken to analyze the uptake and utiIization of tritiated DL-SHTP by brain tissue in young animals at different stages of ontogenic development in the presence or absence of peripheral decarboxylase inhibition. This study is designed to coincide with clinical studies on the treatment of mongoloid children with 5-HTP. METHODS
Sprague-Dawley rats at one day, 4 days and 21 days postnatal age were used in -all studies. Previous reports have indicated that 5-HT in the neurones reaches a mature level by 3 weeks of age (KARKI, KUNTZMYANand BRODIE,1962). Ail animals were born in the department and the time of birth noted within 12 hr. *Data presented in part at the Third Xnternational Meeting of the International Neurochemistry Society, Budapest, Hungary, 4-9 July 1971. 363
364
C.
KELLOGG
and
P. LUNDBORG
Time-study
Animals were injected S.C.with DL [3H]-5-HTP (Amersham, specific activity 1.9 Ci/mmol) at a dose level of 10 pg/kg. A time-curve was obtained in which animals were killed at 30, 60 and 120 min following injection. The brains were removed and dissected into three regions: hemispheres (cerebral hemispheres plus corpus striatum and hippocampus), diencephalon (including the septum), and lower brain stem. The cerebellum was removed and discarded. Six animals were included per group in l- and 4-day-old rats and two animals per group in the 21-day-old animals. All tissues were homogenized in 0 ~4N PCA. The extracts were placed on DOWEX 50-X4 columns for the separation of 5-HTTP,5-HT, and 5-hydroxyindoleacetic acid (5HIAA) as outlined by ATACKand MAGNUSSON (1970) and LINDQVIST(1971). The respective eluates were counted using liquid scintillation. For statistical analysis of the time-curves, an analysis of variance determination was performed on the curves and the differences between the means tested by the Students t-test. Comparisons were made at each age across time and at individual time-points across ages (for each brain area). Inhibition of peripheral decarboxylase
Animals were pretreated with 50 mg/kg of MK-486 (/3-(3,4-dihydroxyphenyl) a-hydrazino-a-methyl propionic acid, laevo form), an inhibitor of extracerebral decarboxylase, or saline (control animals) 30 min before the administration of [3H]-5-HTP. All animals were killed 2 hr later and the tissues treated in the same manner as described above. In adult animals, the drug MK-486 does not penetrate into the brain at the dose level used in this study but is an effective inhibitor of peripheral decarboxylase (PORTER,WATSON,TITUS, TOTAROand BYER, 1962). After pretreatment with MK-486 (MK), extracerebral decarboxylase should be inhibited in the brain (i.e. brain capillary decarboxylase) as well as in the periphery. RESULTS Time-study
The disappearance of [3H]-5-HTP in the brain is described in Fig. 1. The rate of disappearance was much slower in l- and 4-day-old animals than in 21-day-old rats with the concentration of [3H]-5-HTP reaching a plateau in the younger animals from 30 to 60 min after injection, whereas the level declined markedly from the 30-min value in the 21-day-old rats. A similar pattern of disappearance was observed in all brain areas at each respective age. A decrease in the level of the time-curve occured in each brain area coincidentally with age. The disappearance of [3H]-5-HT followed a time-sequence similar to that observed for [3H]-5-HTP (Fig. 2). The magnitude of the [3H]-5-HT concentration however, was only l/l0 that of the rH]-5-HTP level, The concentration of C3H]-5-HT continued to increase at the 2-hr time-point in the younger animals but decreased from the 60-min time-point in the 21-day-old rats (with the exception of the hemispheres). The level of the [3H]-monoamine in the various brain areas appears to reach a maximum approximately 30 min after the maximum activity of the C3H]-precursor. The concentration of 13H]-5-HIAA (the major metabolite of 5-HT) in the brain regions was of a magnitude similar to ]3H]-5-HTP (Fig. 3). The time-curve for the disappearance of [3H]-5-HIAA was a function of the disappearance of [3H]-5-HTP. A decrease in the concentration of [3H]-5-HIAA occurred from the 60-min time-point in the 21”day-old animals whereas in the younger animals any decrease in concentration noted was very slight.
Utilization of [sHJ-5-HTP during development
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1. [aH]-5-HTP levels (ppmol/g) in rat brain at varying time intervals following the S.C. injection of [aH]-S-HTP (10 pg/kg) at different postnatal ages. Results are expressed as mean &S.E.M. Numbers indicate number of groups determined. The successive changes with time in the disappearance rates of [aH]-5-HTP are in general significant for each age group in each brain area (P
FIG.
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FIG. 2. rH]-S-FIT levels ~~mol/g} in rat brain at varying time intervals following the S.C. injection of [*HI-5-HTP (10 pgfkg) at different postnatal ages. Results are expressed as mean &S.E.M. Numbers indicate number of groups determined. In the l- and 4-day-old rats, the most marked increase in taH]-5-HT levels with time occurred from 30 to 60 min (P
Utilization of [3H]-5-IITP during development
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FIG. 3. [31-I]-5-WIAA levels @pmol/g) in rat brain at varying time intervals following the S.C. injection of [%I]-5-IITP (10 pg/kg) at different postnatal ages. Results are expressed as mean &S.E.M Numbers indicate number of groups determined. Statistical analysis of the timecurves indicated that in general there was no significant change with successive time intervals at any age (P > 0.1). Comparing the time-points across ages demonstrated a significant decrease in the [*II]-5-IIIAA levels from I to 4 days of age at all time-points in all brain areas (P < 0.05, P
MK-study
Pretreatment with MK induced a definite and pronounced increase in the concentration of [3H]-5-HTP in all brain regions at each age studied (Fig. 4). The greatest magnitude of response was observed in the 21-day-old rats with the concentration increasing 7-12-fold as compared to only a 2-4-fold increase in the younger animals. Little change in the effect of MK was noted from 1 to 4 days of age. An increase in the concentration of [SH]-5-HT (Fig. 5) was also observed following MK-treatment. However, the level of significance of the response varied in the different brain regions at the various ages perhaps representing a development and maturation of the 5-HT neurone. The effect of MK-treatment was si~ificant at P < 0.01 in the brain stem of
368
C. KELLOGGand P. LUNDBORG
t
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FIG. 4. [SH]-5-HTP levels (ppmol/g) in the brain of young rats pretreated with MK-486 (50 mg/kg) or saline (control) 30 min before the S.C. administration of [3H]-5-HTP (10 pg/kg). Animals were killed 2 hr after 5-HTP. Results are expressed as mean &S.E.M. Numbers indicate number of groups determined. *Denotes a significant difference between control and treated groups at P
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MK
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21
days
FIG. 5. [3H]-5-HT levels (ppmol/g) in the brain of young rats pretreated with MK-486 (50 mg/kg) or saline (control) 30 min before the S.C. administration of [3H]-5-HTP (10 pg/kg). Animals were killed 2 hr after 5-HTP. Results are expressed as mean f S.E.M. Numbers indicate number of groups determined. *Denotes a significant difference between control and treated groups at P < 0.05; o denotes significanceatP CO.01.
Utilization of [sH]-S-HTP during development
369
l-day-old rats, in the dien~phalon and brain stem of4-day-old rats, and in all three areas in the21-da~oId animals. Again the greatest magnitude of the et%& was noted in the 21-day-old rats. The final concentration of PHI-5-HT reached after MIGtreatment was similar at each age within a respective brain region. The response of PHI-5-HIAA to MK-pretreatment was variable (Fig. 6). In general the concentration of [3H]-5-HIAA decreased in the I- and 4-day-old rats. In the brain stem of the
BRAIN
HEMISPHERES
STEM
Fm,
6, [8H]-S-HIAA ievels ~~~rnol~g)in the brain of young rats pretreated with MK-486 (SOmgjkg) or saline (control) 30 min before the S.C.administration of TsEf-5-HTP (10 &kg). Aniials were killed 2 hr after 5-HTP. Results expressed as mean i S.E.M. Numbers indicate number of groups determined. *Denotes a significant difference between control and treated groups at P x0+01.
l-day-old rats an increased response was observed in 3 groups and a decrease noted in 2 groups. This division of the results may indicate that all animals utilized were not exactly l-day-old. If a transitional process is taking place in the brain stem during a time-period shortly after birth, a difference in age of half a day could contribute to the divided response observed after MK-treatment. The only definite increase in ~3H]-5-HIAA levels in the younger animals was noted in thediencephaion of 4-day-old rats. In the21-day-old animals a significant increase in [3H]-5-HIAA con~ntration was meas~ed in the dienceph~on and brain stem. No significant response occurred in the hemispheres at this age.
DISCUSSION
In the time-study the higher concentration of the [sH]-5-HTP in the brains of the young rats and the prolonged time-curve of disappearance of [aH]&HTP in these animals imply that more [SH]-5-HTP entered the brain from the periphery in the younger rats than in the Ztday-old animals during the time interval studied. The time-curves for [W&5-HT and [W&5HIAA are a function of the curve for [%H]-S-HTP and therefore have a related time-scale
370
C.KELLOGG and P.LUNDBORG
for disappearance. The high con~ntrations of ~~H]-5-HIAA in the brain at all time-points and the relatively low concentration of [3H]-5-HT indicate a rapid turnover for this exogenous monoamine at all ages. Factors that could contribute to an increased appearance of [3H]-5-HTP in the brain of the younger animals include a deficient blood-brain-barrier in these animals and/or a low capacity of decarboxylase in peripheral organs. To test the latter factor animals were pretreated with MK before the administration of PH]&HTP. If a high capacity of peripheral decarboxylation is present, inhibiting tlze enzyme in the periphery should result in a large increase in the amount of peripherally administered 5-HTP reaching the brain. The magnitude of the response in the ]3H]-5-HTP level in the brain after MK-treatment should be proportional to the capacity of decarboxylase in the periphery. In support of this hypothesis, a much greater change in the level of rH]-5-HTP was observed in the brain areas of Z-day-old animals than in the younger animals after MKtreatment suggesting a greater efficiency of peripheral decarboxylase in the oldest age group studied. Similar evidence was obtained in a previous study utilizing [3H]-DOPA (KELLOGG and LUNDBORG, 1971). If the level of [3H]-5-HTP in the brain increases following MK-treatment, a concomitant increase in the level of E3H]-5-HT could be expected. Such an increase was observed in all brain areas at each age (however the increase was not significant in the hemispheres and diencephalon at one day of age). This general increase in the [aHI-5-HT levels provides evidence that MK did not penetrate the blood-brain-barrier and inhibit neuronal decarboxylase, because in that case the [3H]-5-HT levels should have decreased after MK. However, further studies are necessary in order to verify that MK did not crossthe bloodbrain-barrier at all ages. Comparing the magnitude of increase in rH]-5-HTP in the respective brain areas at each age to the increase in rHJ-5-HT levels after MK indicates that a greater portion of the 5-HTP entering the brain was converted to 5-HT and stored as the animals matured. The change in the level of significance of the increase in [3H]-5-HT concentration in the different brain areas can be considered to demonstrate a difference in the rate of development and maturation of 5-HT neurones in the respective brain regions. These changes in development of the 5-HT neurone were not evidenced under control conditions but were revealed only after the inhibition of the peripheral decarboxylase. Continuing the line of reasoning that an increasing capacity of peripheral decarboxylase with age alters the appearance of 5-HTP in the brain, an increase in the concentration of [3H]-5-HTP in the brain should result in an increase in the level of rH]-5-HIAA. However, as the results indicate, this was not always the case. In the younger animals the general response (in 5-HIAA levels) to MK-treatment was a significant decrease in the concentration of [3HJ-5-HIAA in the brain whereas in the 21-day-old animals a definite increase was observed in the diencephalon and brain stem. The decarboxylase enzyme has been shown to be effective extraneuronally as well as intraneuronally in peripheral nerves (DAHISTR~M and JONASON, 1968). Therefore 5-HTP that enters the CNS is probably also decarboxylated in extraneuronal sites (capillaries and perhaps glia cells) as well as in the neurone. Monoamine oxidase (MAO), the enzyme responsible for the metabolism of S-HT, also appears to exist outside the neurone as well as within neuronal mitochondria (JONASON,1969).The ratio of extraneuronal to neuronal MAO in the brain is not understood. It is quite probable then that an appreciable amount of the [3H]-5-HTP in the brain
Utilization of [3H]-5-HTP during development
371
under control conditions was de~arboxylated outside the neurone and the L3H]-5-HT thus formed was rapidly metabolized outside the neurone. Therefore, in the brain regions where MK-treatment produced a decrease in the [3H]-5-HIAA level, the possibility must be considered that that level of rH]-5-HIAA in the control animals was primarily representative of extracerebral 5-HIAA (in brain capillaries) and would therefore be decreased by MKtreatment. The response of the [3H]-5-HIAA level to MK-treatment may be considered indicative of the ratio of extracerebral to cerebral decarboxylayion of 5-HTP within brain tissue. The study indicated that a greater degree of extracerebral decarboxylation could be present in the l- and4-day-old rats than in the 21-day-aid animals since, in the oldest age group, the hemisphere was the only brain area where MK-treatment did not increase the [3H]-5-HIAA level. The singular definite increase in this level in the younger animals was observed in the diencephalon of the Cday-old rats. The i3H]-5-HT measured in the brain of control animals was quite low in proportion to the concentraion of [3H]-5-HIAA. It seems likely to assume that the PH]S-HT measured was that actually stored in the neurone, since any S-HT produced outside the neurone could not be stored and would be readily metabolized. The results obtained have strongly indicated that an increase occurs in the capacity of peripheral decarboxylase (i.e. in peripheral organs) with age which can markedly affect the amount of peripherally administered monoamine precursor reaching the brain. The data also suggests that with ontogenic development there is a change in the ratio of cerebral: extracerebral decarboxylase in the brain which could alter the distribution of the precursor reaching the brain. Although previous reports have indicated that total whole brain decarboxylase activity in newborn rats is comparable to that observed in adult animals (KARKI et al., 1962), the distribution of the enzyme could change with age. Studies are currently being conducted to assist in di~erentiating cerebral and extracerabral decarboxylase during development. The apparent increase in the capacity of the peripheral decarboxylase observed in this study with age should be capable of exerting a pronounced effect on the prolonged clinical administration during ontogeny of a precursor such as 5-HTP. Acknowledgemen&-This research has been supported by the Swedish State Medical Research Council (No. B71-14P-3266-01 and B71-14X-2464-04), Expressens neonatalforskningsfond and Llkemedelsindustriforeningen. C. KELLOGG is recipient of a National Institutes of Health Fellowship, No. 1 F02 NS 31010-01 from the National Institute for Neurological Diseases and Stroke, United States Public Health Service. For a generous supply of MK-486, we are obliged to Dr. C. A. STONE,Merck, Sharp and Dohme. For skillful assistance we are much indebted to Miss LENARAWTEDT.
REFERENCES ATACK, C. V. and MAGNUSSON,T. (1970). Individual elution of noradrenaline (together with adrenaline), dopamine, 5-hydroxytryptamine and histamine from a single, strong cation exchange column, by means of mineral acid-organic solvent mixtures, J. Pharm. Pharmac. 22: 625-627. BAZEUIN, M., PAINE, R. S., COWIE,V. A., HUNT, P., HAUCK, J. C. and MAHANAND,D. (1967). Reversal of hypotonia in infants with Down’s syndrome by administration of 5-hydroxytryptophan. Lancet 1: 1130-1133. BOJAMEK,J., BOZKOWA,A. and KURZEPA, S. (1965-1966). The 5-HTP decarboxylase and MAO activities and 5-HT level in subcellular fractions of the liver during development. Biol. Neon&. 9: 203-214. DA~WTR~M, A. and JONASON,J. (1968). DOPA-decarboxylase activity in sciatic nerves of the rat after constriction. Eur. J. Phurmac. 4: 377-383. JONASON,J. (1969). Metabolism of catecholamines in the central and peripheral nervous system. Actaphysiol. stand. 75: suppl. 320. KARKI, N., KWTZMAN, R. and BRODIE,B, B. (1962). Storage, synthesis, and metabolism of monoamines in the developing brain. J. Neuroc~em, 9: 53-58,
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and P. LUNDBORG
KELLOGG,6. and L~NDBORG,P. (1971). Production of @I) ~t~holamin~ in the brain follo~g the peripheral ad~nistration of rH) DOPA during pre- and postnatal development. Brain Res. In Press. LINDQVIST,M. (1971). Quantitative estimation of S-hydroxy3-indole acetic acid and 5-hydroxyt~ptophan in the brain following isolation by means of a strong cation exchange column. Actapharmacol. toxicol. 29: 303-313. PORTER,C. C., WATSON,L. S., TITUS, D. C., TOTARO,J. A. and BYER,S. S. (1962). Inhibition of DOPA decarboxylase by the hydrazino analogue of a-methyl DOPA. Biochem. Pharmac. 11: 1067-1077. SMITH, S. E., STACEY,R. S. and YOIJNG, I. M. (1962). 5-hydroxytryptamine and 5-hydroxytryptophan decarboxylase activity in the developing nervous system of rats and guinea pigs. In: Pharmacological Analysis of Central Nervous Action (PATON,W. D. M. and LINDGREN,P., Eds.), pp. 101-105. Pergamon Press. Oxford.
PrintedinGt.Britain,
UPTAKE AND UTILIZATION OF [3H-]5-HYDROXYTRYPTOPHAN BY BRAIN TISSUE DURING DEVELOPMENT* and P. LUNDBORG
C. KELLOGG
Department of Pharmacology, University of Goteborg, Gateborg, Sweden (Accepfed
23 August 1971)
Summary-The in viva utilization of tritiated S-hydrox~ryptophan was analyzed separately in the hemispheres, diencephdon, and brain stem of rats during postnatal development. A time-study of the concentration of 3H-5-hydroxytryptophan, 3H-5-hydroxytryptamine, and 3H-5-hydroxyindoieacetic acid in the respective brain regions following the S.C.injection of 3H-5-hydroxytryptophan demonstrated that the latter disappearedfrom the brain at a slower rate in rats at I and 4 days of age than in 21-day-old animals. The concentration of SH-5-hydroxytryptamine and 8H-5-hydroxyindoleacteic acid over time followed that of the precursor. Pretreating animals with an inhibitor of peripheral decarboxylase, MK-486, indicated that the capacity of the decarboxylase in peripheral organs increases markedly with age and thus alters the appearance in the brain of peripherally administered precursor.
THE ADMINISTRATIONof DL, 5-hydroxytryptophan has been used as a clinical approach in the treatment of mongoloidism (BAZELON, PAINE, Cowre, HUNT, HAUCK and ~AHANANR, 1967). Such treatment has been shown to produce an improvement in muscle tone; however, it has been reported that the effective dose level must be increased between 4 and 6 months of age. An investigation of mechanisms contributing to the decrease in the effectiveness of 5-hydroxytryptophan with age has not been reported. An increase in the capacity of peripheral L-amino acid decarboxylase, the enzyme responsible for the decarboxylation of 5hydroxytryptophan (5-HTP) to 5-hydroxytryptamine (5-HT), has been observed to occur with age in young animals (BOJANEK,BOZKOWAand KURZEPA, 1965/1966; SMITH, STACEYand YOUNG, 1962). The ontogenic development of the peripheral decarboxylase markedly affects the production of tritiated catecholamines in the brain of young rats following the peripheral administration of the tritiated precursor, L-3,4-dihydroxyphenyia~anine (KELLOGGand LUNDBORG,1971). Because little is understood of the utilization of 5-HTP in the brain during development, the present study was undertaken to analyze the uptake and utiIization of tritiated DL-SHTP by brain tissue in young animals at different stages of ontogenic development in the presence or absence of peripheral decarboxylase inhibition. This study is designed to coincide with clinical studies on the treatment of mongoloid children with 5-HTP. METHODS
Sprague-Dawley rats at one day, 4 days and 21 days postnatal age were used in -all studies. Previous reports have indicated that 5-HT in the neurones reaches a mature level by 3 weeks of age (KARKI, KUNTZMYANand BRODIE,1962). Ail animals were born in the department and the time of birth noted within 12 hr. *Data presented in part at the Third Xnternational Meeting of the International Neurochemistry Society, Budapest, Hungary, 4-9 July 1971. 363
364
C.
KELLOGG
and
P. LUNDBORG
Time-study
Animals were injected S.C.with DL [3H]-5-HTP (Amersham, specific activity 1.9 Ci/mmol) at a dose level of 10 pg/kg. A time-curve was obtained in which animals were killed at 30, 60 and 120 min following injection. The brains were removed and dissected into three regions: hemispheres (cerebral hemispheres plus corpus striatum and hippocampus), diencephalon (including the septum), and lower brain stem. The cerebellum was removed and discarded. Six animals were included per group in l- and 4-day-old rats and two animals per group in the 21-day-old animals. All tissues were homogenized in 0 ~4N PCA. The extracts were placed on DOWEX 50-X4 columns for the separation of 5-HTTP,5-HT, and 5-hydroxyindoleacetic acid (5HIAA) as outlined by ATACKand MAGNUSSON (1970) and LINDQVIST(1971). The respective eluates were counted using liquid scintillation. For statistical analysis of the time-curves, an analysis of variance determination was performed on the curves and the differences between the means tested by the Students t-test. Comparisons were made at each age across time and at individual time-points across ages (for each brain area). Inhibition of peripheral decarboxylase
Animals were pretreated with 50 mg/kg of MK-486 (/3-(3,4-dihydroxyphenyl) a-hydrazino-a-methyl propionic acid, laevo form), an inhibitor of extracerebral decarboxylase, or saline (control animals) 30 min before the administration of [3H]-5-HTP. All animals were killed 2 hr later and the tissues treated in the same manner as described above. In adult animals, the drug MK-486 does not penetrate into the brain at the dose level used in this study but is an effective inhibitor of peripheral decarboxylase (PORTER,WATSON,TITUS, TOTAROand BYER, 1962). After pretreatment with MK-486 (MK), extracerebral decarboxylase should be inhibited in the brain (i.e. brain capillary decarboxylase) as well as in the periphery. RESULTS Time-study
The disappearance of [3H]-5-HTP in the brain is described in Fig. 1. The rate of disappearance was much slower in l- and 4-day-old animals than in 21-day-old rats with the concentration of [3H]-5-HTP reaching a plateau in the younger animals from 30 to 60 min after injection, whereas the level declined markedly from the 30-min value in the 21-day-old rats. A similar pattern of disappearance was observed in all brain areas at each respective age. A decrease in the level of the time-curve occured in each brain area coincidentally with age. The disappearance of [3H]-5-HT followed a time-sequence similar to that observed for [3H]-5-HTP (Fig. 2). The magnitude of the [3H]-5-HT concentration however, was only l/l0 that of the rH]-5-HTP level, The concentration of C3H]-5-HT continued to increase at the 2-hr time-point in the younger animals but decreased from the 60-min time-point in the 21-day-old rats (with the exception of the hemispheres). The level of the [3H]-monoamine in the various brain areas appears to reach a maximum approximately 30 min after the maximum activity of the C3H]-precursor. The concentration of 13H]-5-HIAA (the major metabolite of 5-HT) in the brain regions was of a magnitude similar to ]3H]-5-HTP (Fig. 3). The time-curve for the disappearance of [3H]-5-HIAA was a function of the disappearance of [3H]-5-HTP. A decrease in the concentration of [3H]-5-HIAA occurred from the 60-min time-point in the 21”day-old animals whereas in the younger animals any decrease in concentration noted was very slight.
Utilization of [sHJ-5-HTP during development
HEMISPHERES Ldays
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6d
OY
1 day
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&days
90
120 mm
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1
21 days
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C days
1. [aH]-5-HTP levels (ppmol/g) in rat brain at varying time intervals following the S.C. injection of [aH]-S-HTP (10 pg/kg) at different postnatal ages. Results are expressed as mean &S.E.M. Numbers indicate number of groups determined. The successive changes with time in the disappearance rates of [aH]-5-HTP are in general significant for each age group in each brain area (P
FIG.
365
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LUNDBORC
C. KELLCCGand P.
HEMISPHERES ’ L
30
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90
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p”“--’ 1Mmin
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FIG. 2. rH]-S-FIT levels ~~mol/g} in rat brain at varying time intervals following the S.C. injection of [*HI-5-HTP (10 pgfkg) at different postnatal ages. Results are expressed as mean &S.E.M. Numbers indicate number of groups determined. In the l- and 4-day-old rats, the most marked increase in taH]-5-HT levels with time occurred from 30 to 60 min (P
Utilization of [3H]-5-IITP during development
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6
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30
:6
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FIG. 3. [31-I]-5-WIAA levels @pmol/g) in rat brain at varying time intervals following the S.C. injection of [%I]-5-IITP (10 pg/kg) at different postnatal ages. Results are expressed as mean &S.E.M Numbers indicate number of groups determined. Statistical analysis of the timecurves indicated that in general there was no significant change with successive time intervals at any age (P > 0.1). Comparing the time-points across ages demonstrated a significant decrease in the [*II]-5-IIIAA levels from I to 4 days of age at all time-points in all brain areas (P < 0.05, P
MK-study
Pretreatment with MK induced a definite and pronounced increase in the concentration of [3H]-5-HTP in all brain regions at each age studied (Fig. 4). The greatest magnitude of response was observed in the 21-day-old rats with the concentration increasing 7-12-fold as compared to only a 2-4-fold increase in the younger animals. Little change in the effect of MK was noted from 1 to 4 days of age. An increase in the concentration of [SH]-5-HT (Fig. 5) was also observed following MK-treatment. However, the level of significance of the response varied in the different brain regions at the various ages perhaps representing a development and maturation of the 5-HT neurone. The effect of MK-treatment was si~ificant at P < 0.01 in the brain stem of
368
C. KELLOGGand P. LUNDBORG
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FIG. 4. [SH]-5-HTP levels (ppmol/g) in the brain of young rats pretreated with MK-486 (50 mg/kg) or saline (control) 30 min before the S.C. administration of [3H]-5-HTP (10 pg/kg). Animals were killed 2 hr after 5-HTP. Results are expressed as mean &S.E.M. Numbers indicate number of groups determined. *Denotes a significant difference between control and treated groups at P
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FIG. 5. [3H]-5-HT levels (ppmol/g) in the brain of young rats pretreated with MK-486 (50 mg/kg) or saline (control) 30 min before the S.C. administration of [3H]-5-HTP (10 pg/kg). Animals were killed 2 hr after 5-HTP. Results are expressed as mean f S.E.M. Numbers indicate number of groups determined. *Denotes a significant difference between control and treated groups at P < 0.05; o denotes significanceatP CO.01.
Utilization of [sH]-S-HTP during development
369
l-day-old rats, in the dien~phalon and brain stem of4-day-old rats, and in all three areas in the21-da~oId animals. Again the greatest magnitude of the et%& was noted in the 21-day-old rats. The final concentration of PHI-5-HT reached after MIGtreatment was similar at each age within a respective brain region. The response of PHI-5-HIAA to MK-pretreatment was variable (Fig. 6). In general the concentration of [3H]-5-HIAA decreased in the I- and 4-day-old rats. In the brain stem of the
BRAIN
HEMISPHERES
STEM
Fm,
6, [8H]-S-HIAA ievels ~~~rnol~g)in the brain of young rats pretreated with MK-486 (SOmgjkg) or saline (control) 30 min before the S.C.administration of TsEf-5-HTP (10 &kg). Aniials were killed 2 hr after 5-HTP. Results expressed as mean i S.E.M. Numbers indicate number of groups determined. *Denotes a significant difference between control and treated groups at P x0+01.
l-day-old rats an increased response was observed in 3 groups and a decrease noted in 2 groups. This division of the results may indicate that all animals utilized were not exactly l-day-old. If a transitional process is taking place in the brain stem during a time-period shortly after birth, a difference in age of half a day could contribute to the divided response observed after MK-treatment. The only definite increase in ~3H]-5-HIAA levels in the younger animals was noted in thediencephaion of 4-day-old rats. In the21-day-old animals a significant increase in [3H]-5-HIAA con~ntration was meas~ed in the dienceph~on and brain stem. No significant response occurred in the hemispheres at this age.
DISCUSSION
In the time-study the higher concentration of the [sH]-5-HTP in the brains of the young rats and the prolonged time-curve of disappearance of [aH]&HTP in these animals imply that more [SH]-5-HTP entered the brain from the periphery in the younger rats than in the Ztday-old animals during the time interval studied. The time-curves for [W&5-HT and [W&5HIAA are a function of the curve for [%H]-S-HTP and therefore have a related time-scale
370
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for disappearance. The high con~ntrations of ~~H]-5-HIAA in the brain at all time-points and the relatively low concentration of [3H]-5-HT indicate a rapid turnover for this exogenous monoamine at all ages. Factors that could contribute to an increased appearance of [3H]-5-HTP in the brain of the younger animals include a deficient blood-brain-barrier in these animals and/or a low capacity of decarboxylase in peripheral organs. To test the latter factor animals were pretreated with MK before the administration of PH]&HTP. If a high capacity of peripheral decarboxylation is present, inhibiting tlze enzyme in the periphery should result in a large increase in the amount of peripherally administered 5-HTP reaching the brain. The magnitude of the response in the ]3H]-5-HTP level in the brain after MK-treatment should be proportional to the capacity of decarboxylase in the periphery. In support of this hypothesis, a much greater change in the level of rH]-5-HTP was observed in the brain areas of Z-day-old animals than in the younger animals after MKtreatment suggesting a greater efficiency of peripheral decarboxylase in the oldest age group studied. Similar evidence was obtained in a previous study utilizing [3H]-DOPA (KELLOGG and LUNDBORG, 1971). If the level of [3H]-5-HTP in the brain increases following MK-treatment, a concomitant increase in the level of E3H]-5-HT could be expected. Such an increase was observed in all brain areas at each age (however the increase was not significant in the hemispheres and diencephalon at one day of age). This general increase in the [aHI-5-HT levels provides evidence that MK did not penetrate the blood-brain-barrier and inhibit neuronal decarboxylase, because in that case the [3H]-5-HT levels should have decreased after MK. However, further studies are necessary in order to verify that MK did not crossthe bloodbrain-barrier at all ages. Comparing the magnitude of increase in rH]-5-HTP in the respective brain areas at each age to the increase in rHJ-5-HT levels after MK indicates that a greater portion of the 5-HTP entering the brain was converted to 5-HT and stored as the animals matured. The change in the level of significance of the increase in [3H]-5-HT concentration in the different brain areas can be considered to demonstrate a difference in the rate of development and maturation of 5-HT neurones in the respective brain regions. These changes in development of the 5-HT neurone were not evidenced under control conditions but were revealed only after the inhibition of the peripheral decarboxylase. Continuing the line of reasoning that an increasing capacity of peripheral decarboxylase with age alters the appearance of 5-HTP in the brain, an increase in the concentration of [3H]-5-HTP in the brain should result in an increase in the level of rH]-5-HIAA. However, as the results indicate, this was not always the case. In the younger animals the general response (in 5-HIAA levels) to MK-treatment was a significant decrease in the concentration of [3HJ-5-HIAA in the brain whereas in the 21-day-old animals a definite increase was observed in the diencephalon and brain stem. The decarboxylase enzyme has been shown to be effective extraneuronally as well as intraneuronally in peripheral nerves (DAHISTR~M and JONASON, 1968). Therefore 5-HTP that enters the CNS is probably also decarboxylated in extraneuronal sites (capillaries and perhaps glia cells) as well as in the neurone. Monoamine oxidase (MAO), the enzyme responsible for the metabolism of S-HT, also appears to exist outside the neurone as well as within neuronal mitochondria (JONASON,1969).The ratio of extraneuronal to neuronal MAO in the brain is not understood. It is quite probable then that an appreciable amount of the [3H]-5-HTP in the brain
Utilization of [3H]-5-HTP during development
371
under control conditions was de~arboxylated outside the neurone and the L3H]-5-HT thus formed was rapidly metabolized outside the neurone. Therefore, in the brain regions where MK-treatment produced a decrease in the [3H]-5-HIAA level, the possibility must be considered that that level of rH]-5-HIAA in the control animals was primarily representative of extracerebral 5-HIAA (in brain capillaries) and would therefore be decreased by MKtreatment. The response of the [3H]-5-HIAA level to MK-treatment may be considered indicative of the ratio of extracerebral to cerebral decarboxylayion of 5-HTP within brain tissue. The study indicated that a greater degree of extracerebral decarboxylation could be present in the l- and4-day-old rats than in the 21-day-aid animals since, in the oldest age group, the hemisphere was the only brain area where MK-treatment did not increase the [3H]-5-HIAA level. The singular definite increase in this level in the younger animals was observed in the diencephalon of the Cday-old rats. The i3H]-5-HT measured in the brain of control animals was quite low in proportion to the concentraion of [3H]-5-HIAA. It seems likely to assume that the PH]S-HT measured was that actually stored in the neurone, since any S-HT produced outside the neurone could not be stored and would be readily metabolized. The results obtained have strongly indicated that an increase occurs in the capacity of peripheral decarboxylase (i.e. in peripheral organs) with age which can markedly affect the amount of peripherally administered monoamine precursor reaching the brain. The data also suggests that with ontogenic development there is a change in the ratio of cerebral: extracerebral decarboxylase in the brain which could alter the distribution of the precursor reaching the brain. Although previous reports have indicated that total whole brain decarboxylase activity in newborn rats is comparable to that observed in adult animals (KARKI et al., 1962), the distribution of the enzyme could change with age. Studies are currently being conducted to assist in di~erentiating cerebral and extracerabral decarboxylase during development. The apparent increase in the capacity of the peripheral decarboxylase observed in this study with age should be capable of exerting a pronounced effect on the prolonged clinical administration during ontogeny of a precursor such as 5-HTP. Acknowledgemen&-This research has been supported by the Swedish State Medical Research Council (No. B71-14P-3266-01 and B71-14X-2464-04), Expressens neonatalforskningsfond and Llkemedelsindustriforeningen. C. KELLOGG is recipient of a National Institutes of Health Fellowship, No. 1 F02 NS 31010-01 from the National Institute for Neurological Diseases and Stroke, United States Public Health Service. For a generous supply of MK-486, we are obliged to Dr. C. A. STONE,Merck, Sharp and Dohme. For skillful assistance we are much indebted to Miss LENARAWTEDT.
REFERENCES ATACK, C. V. and MAGNUSSON,T. (1970). Individual elution of noradrenaline (together with adrenaline), dopamine, 5-hydroxytryptamine and histamine from a single, strong cation exchange column, by means of mineral acid-organic solvent mixtures, J. Pharm. Pharmac. 22: 625-627. BAZEUIN, M., PAINE, R. S., COWIE,V. A., HUNT, P., HAUCK, J. C. and MAHANAND,D. (1967). Reversal of hypotonia in infants with Down’s syndrome by administration of 5-hydroxytryptophan. Lancet 1: 1130-1133. BOJAMEK,J., BOZKOWA,A. and KURZEPA, S. (1965-1966). The 5-HTP decarboxylase and MAO activities and 5-HT level in subcellular fractions of the liver during development. Biol. Neon&. 9: 203-214. DA~WTR~M, A. and JONASON,J. (1968). DOPA-decarboxylase activity in sciatic nerves of the rat after constriction. Eur. J. Phurmac. 4: 377-383. JONASON,J. (1969). Metabolism of catecholamines in the central and peripheral nervous system. Actaphysiol. stand. 75: suppl. 320. KARKI, N., KWTZMAN, R. and BRODIE,B, B. (1962). Storage, synthesis, and metabolism of monoamines in the developing brain. J. Neuroc~em, 9: 53-58,
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KELLOGG,6. and L~NDBORG,P. (1971). Production of @I) ~t~holamin~ in the brain follo~g the peripheral ad~nistration of rH) DOPA during pre- and postnatal development. Brain Res. In Press. LINDQVIST,M. (1971). Quantitative estimation of S-hydroxy3-indole acetic acid and 5-hydroxyt~ptophan in the brain following isolation by means of a strong cation exchange column. Actapharmacol. toxicol. 29: 303-313. PORTER,C. C., WATSON,L. S., TITUS, D. C., TOTARO,J. A. and BYER,S. S. (1962). Inhibition of DOPA decarboxylase by the hydrazino analogue of a-methyl DOPA. Biochem. Pharmac. 11: 1067-1077. SMITH, S. E., STACEY,R. S. and YOIJNG, I. M. (1962). 5-hydroxytryptamine and 5-hydroxytryptophan decarboxylase activity in the developing nervous system of rats and guinea pigs. In: Pharmacological Analysis of Central Nervous Action (PATON,W. D. M. and LINDGREN,P., Eds.), pp. 101-105. Pergamon Press. Oxford.