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Effects of neonatal estrogen on in vivo transport of α-aminoisobutyric acid into rat brain
ESPEHIMEKTAL
Effects
SE~RULWY
57, 817-827
(1Y77)
of Neonatal Estrogen on in Vivo Transport a-Aminoisobutyric Acid into Rat Brain
of
The effects of neonatally administered estradiol benzoate were determined on the transport of w [ l-Y] aminoisobutyric acid into amygdala, cerebellum, corpora quadrigemina, cortices (frontal, occipital, parietal, pyriform), hypothalamus, medulla, olfactory bulbs, olfactory tubercles, and pons of the rat brain. Rats were injected subcutaneously with either 500 pg estradiol benzoate or vehicle 24 and 72 h after birth, and at 5, 10, or 17 days of age they were decapitated 2, 5, 60, or 300 min after the intraperitoneal injection of w[l-‘“Claminoisobutyric acid. By 300 min, the active transport of the isotope in cstradiol benzoate-treated rats was greater in all brain regions of 5-day-old rats and in 8 of the 12 brain regions of lo-day-old rats. Estradiol benzoate did not alter active transport in 17-day-old rats at 300 min. These data suggest that injection of estradiol benzoate during the critical period of brain differentiation (directly or indirectly) affects processes regulating the active transport of LY- [ 1-“Cl aminoisobutyric acid into brain regions of neonatal rats. The effectiveness of the steroid also decreases \vith age.
INTRODUCTION Administration of estrogens to neonatal male or female rats during the critical period of brain differentiation is followed in the adult by alterations in sexual behavior and reproductive capability (11, 13, 17). Adult male rats are characterized by testicular atrophy, inhibition of spermatogenesis, and decreased male sexual behavior (13, 17). The females are anovulatory and sexual behavior is masculinized (1, 11, 13, 17). These sequelae are Abbreviation
: AIB--or-
[ 1-“C]
aminoisobutyric
acid.
1 This study was supported by the Medical Research Service of the Veterans Administration and in part by U.S. Public Health Service Grant NS11501-02 from the National Institute of Neurological and Communicative Disorders and Stroke. I thank Drs. M. Thorner and L. Vitello for their critical review of this manuscript. C. Popoff provided technical assistance. 817 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0014-4886
818
&I.
LI'UEKIA
l)r~~lal)l!~ rrlatc~tl to rstro~cll-ill(lucct1 altcratic,ns ill tic~urnl structurc&s regulating the synthesis antl,/or release of the pituitary gonntlotrol~ins (1 ). Although the esact biochemical nature of these alterations is not known, they may be associated with estrogen-induced changes in the synthesis of brain nucleic acids and/or proteins ( 19-21). Q uantitative autoradiographic studies from this laboratory have localized some of the neural structures metabolically responsive to neonatally administered 17p-estradiol benzoate of [ 3H] lysine into proteins (EB) (19-21). F or example, incorporation of specific hypothalamic nuclei (arcuate, periventricular, paraventricular, and supraoptic) was inhibited in adult male (20) and female (21) rats treated neonatally with estradiol benzoate. The Purkinje cells, but not the stellate or granule cells, of the cerebellar cortex were similarily affected ( 19). Although autoradiography identified the sites of newly synthesized protein (S), differences in the incorporation of [3H] lysine into proteins between control and estracliol benzoate-treated rats does not imply that the steroid inhibited protein synthesis per se. The steroid could have produced alterations in the transport and, therefore, availability of [3H]lysine in the brain. The purpose of this study was to determine if the administration of estradiol benzoate to neonatal rats during the critical period of brain differentiation is followed by alterations in the transport of (Y-[ 1-14C] aminoisobutyric acid (AIB) across the blood-brain barrier into specific brain regions. AIB is a small neutral, nonmetabolized amino acid that enters the brain by carrier-mediated processes (5-7, 14). The inert character of AIB avoids the problems associated with amino acids that are metabolized by the brain. MATERIALS
AND
METHODS
Three-month-old virgin Sprague-Dawley rats (Madison, Wisconsin) exposed to a 14-h light: 10-h dark schedule and ingesting Purina Rat Chow and water ad Zibitunt served as our breeding stock. Litters were reduced to eight pups within 24 h of birth and the day of delivery is called Day 1. Male or female rats were injected subcutaneously with either 500 pg estradiol benzoate or 0.1 ml sesame oil vehicle at 24 h and again at 72 h after birth. At 5, 10, and 17 days after birth, a minimum of eight control and eight estradiol-treated rats was each injected intraperitoneally with 0.25 &i AIB/g body weight (sp. act. = 9.0 mCi/mmol, New England Nuclear, Boston, Massachusetts). The rats were placed in a beaker warmed by a lamp and containing bedding from the home cage prior to decapitation at intervals of 2, 5, 60, or 300 min after the injection of AIB. The brain was rapidly removed, placed on a petri dish over crushed ice, and freed of
tllettittgt’s at~d blootl. ‘I’\vcl\~ 1)raitt rcgiotts. i.e.. attt~gtlala (\vitll ovrrl!,itl:: cortex), ccrebelhtm, corpora c~ttadrigeminn, frontal cortex. l~~potltalatntts, medulla, occipital cortex, olfactory bulbs, olfactory tubercles, parietal cortex, pans, and pyriform tortes were removed and placed in a petri dish resting on a block of dry ice. Samples of set-ml from each rat were prepared by centrifuging the clotted blood at 2°C for 30 min at 600g. Each frozen brain region was quickly weighed and transferred while still frozen into scintillation vials containing 1.0 ml solubilizer (Soluene-350, Packard Instrument Co., Inc., Downers Grove, Illinois). Duplicate samples (10 to 50 ~1) of serutu were added to the solubilizer. The tissues were solubilized overnight at room temperature and 15 ml liquid scintillation cocktail (Dimilume-30, Packard Instrument Co., Inc.) were added to each vial. Counts per minute were determined (against a blank) in a Packard Tri-Carb liquid scintillation spectronteter, Model 333s. Corrections for tissue quench were made using the esternal standardization channels ratio and least-squares methods. Data were calculated as disintegrations per minute (dpm) per milligram tissue, disintegrations per minute per microliter serum, and as tissue to serum ratios (T/S). Only SC/, of whole brain radioactivity is due to plasma AIB ; therefore, correcting for the amount of radioactivity due to blood present in each brain region was unnecessary (13). The total water content of each brain region was determined in a separate experiment for the 5, lo-, and 17-day-old control and estradiol benzoate-treated rats. Removal and handling of the brain tissues were as described above. The brain tissues were dried to constant weight and the percentage water was calculated from the difference between the weights of the frozen and dried tissues. The significance of differences between the control and estradiol benzoate-treated rats was determined by Student’s t-test; P d 0.05 was considered significant. RESULTS (Y-[l-‘“C]rllrlilloisob~~~~~,ic Acid DOSE-Rcs~onsr. To ensure that the dose of AIB selected for this study would be below saturation kinetics, T/S ratios were determined for each brain region of 5 and lo-clay-old control rats at C1 = 0.125, C, = 0.25, and Ca = 0.40 &i AIB/g body weight. Five minutes after the injection of AIB. disintegrations per minute per milligram tissue and disintegrations per minute per microliter serutn were twice as great for Cz than Cl and approximately 3.2 times higher for C3 than Cl. Significant differences were not found between T/S ratios for any brain region of the .5- or lo-day-old rats at Cl, C,, and Cs. Cl, Cz, and Cs were, therefore, below saturation kinetics and C, was selected for this study. T/S
Ilinutes 2
604.9 688.8
‘/ Each value Ir V = control
aft&
injection
of isotolx
5
f 17.3 zt 39.5
60
300
iYi.3 715.5
f. 64.6 + 31.3
255.1 260.5
f f
10.8 12.8
li4.7 153.1
f f
12.6 10.4
1128.0 1096.3
f f
25.3 30.3
1177.8 1269.5
zk 38.2 f 41.0
228.6 239.5
f f
11.4 12.1
153.7 153.0
zk 15.4 f 3.8
1115.1 1079.5
zt 39.2 f 83.5
1232.1 1146.0
f f
277.3 2io.o
f f
9.4 i.8
163.2 165.8
f f
51.2 48.1
is the mean f SE obtained from 8 to 13 rats ; El3 = treated with estradiol benzoatc.
lxr
3.8 5.3
group.
ratios rather than absolute concentrations (disinteg-rations per minute per milligram tissue) are a meas11re of AIR transport. T/S ratios greater than unity indicate the occurrence of acti1.e transport. Urak IIrtrfcr Cotltmf. There were no sex differences in the water content of specific brain regions of control or estradiol benzoate-treated 5-, lo-, and 17-day-old rats. Differences in water content were found between control and estradiol benzoate-treated rats for some brain regions at 5 and 10 days of age. However, these changes were so snlall that corrections made for these differences did not affect the significance of the data between control and estradiol benzoate-treated groups. Sex Lli~rvwlcrs. Iiflrc~s of l!sfradiol I:rwoatc on :1IU Tramport. There m-ere no sex differences in the concentration of AIlS in serum or brain regions of control or estradiol benzoate-treated 5-, lo-, and 17-day-old rats at any of the tested intervals. Therefore, data from both sexes were coinbined in the control or estradiol benzoate-treated grouljs. Scruln. The concentration of AIL: in the serum of all control and treated rats was highest 5 min after the injection of the isotope (Table 1). There were no differences betweal the concentrations of AI13 in the sermn of control and estradiol benzoate-treated rats in an)- age group. BraipL IZcgio,ls. The absolute concentration of AIB (disintegration per minute per milligrani tissue) slowly increased with time iii all brain regions of control and estradiol benzoate-treated (S-, lo-, and 17-day-old) rats at the ages tested (Tables Z--t). For example, between 2 and 300 min, the
822
M.
LITTERIA
uptake of AIB by the brain of the control and estrogen-treated rats. By 300 min, the uptake of AIB in the control groups was consistently higher for the cerebellum, olfactory bulbs, and pons of the 5- and lo-clay-old rats and for the cerebellum and pans of the 17-day-old rats. The uptake of AIB in the estradiol benzoate-treated groups at 300 min was highest for the cerebellum and olfactory bulbs of the rat at 5 and 10 days of age and for TABLE Effects
of Neonatal
Brain
Estrogen
3
on T/S Ratios of w[l-W]Aminoisobutyric Regions of the lo-Day-Old Rat”
region
T/S ratios after injection
Minutes 2 Amygdala Cb EB Cerebellum C EB Corpora quadrigemina C EB Frontal cortex C EB H~porl~alamus C EB Medulla C EB Occipital cortex C EB Olfactory bulbs C EB Olfactory tuber&s C EB Parietal cortex C EB Polls C EB Pyriform cortex C EB 0 T/S 11C = cP 5 d P 5 *P 5 11’ <
Acid
in Brain
of isotope
5
60
300
0.028 0.027
f 0.001 * 0.002
0.05 1 f 0.051 f
0.001 0.002
0.606 0.701
f f
0.039 0.045
2.176 2.192
;t 0.076 f 0.179
0.057 0.054
f f
0.003 0’002
0.092 0.088
f f
0.004 0.005
1.377 1.334
f f
0.047 0.02 I
4.222 4.411
f f
0.048 0.274
0.030 0.027
f f
0.001 0.001
0.049 0.044
f f
0.001 0.003
0.660 0.616
f 0.048 * 0.033
1.972 2.223
f f
0.056’ 0.047
0.036 0.037
+ 0.002 l 0.003
0.074 0.078
f f
0.002 0.003
0.930 0.967
3~ 0.042 f 0.026
2.712 3.114
f l
0.107“ 0.124
0.024 f 0.001 0.02 I * 0.002
0.038 0.035
f f
0.001 0.001
0.588 0.615
f 0.036 3~ 0.024
1.789 2.232
zk 0.044~ f 0.085
0.044 0.047
* 0.005 ck 0.002
0.078 0.074
f f
0.004 0.005
0.801 0.837
3~ 0.034 f 0.037
2.833 3.186
f f
0.05 1~ 0.080
0.035 0.034
f f
0.001 0.002
0.069 0.065
f f
0.002 0.003
0.808 0.971
f f
2.683 3.048
f f
O.OJle 0.05 1
0.048 0.048
f 0.002 z!z 0.003
0.082 0.078
f 0.003 f 0.004
1.102 1.102
f 0.045 z!z 0.027
3.0 I3 f 0.059~ 3.350 + 0.115
0.025 0.026
f 0.001 f 0.002
0.044 0.041
f f
0.541 0.605
f f
0.023 0.039
1.872 2.043
f f
0.065 0.165
0.036 0.038
f 0.002 zk 0.003
0.068 0.064
* 0.022 f 0.004
0.855 0.891
f f
0.047 0.025
2.629 3.020
f f
0.060@ 0.066
0.045 0.044
f f
0.003 0.003
0.072 0.068
f 0.003 f 0.006
0.826 0.911
f 0.045 f0.022
3.057 3.032
f f
0.120 0.109
0.034 0.031
* 0.002 * 0.002
0.066 0.064
i f
0.938 0.932
f 0.044 + 0.064
2.583 3.107
f 0.067’ * 0.040
ratios are expressed as the mean f standard error control; EB = treated with estradiol benzoate. 0.005. 0.025. 0.001. 0.1)2.
0.001 O.OO2
0.002 0.004
of the mean
obtained
0.033 0.019
from
8 to 13 rats per group.
NEONATAL
ESTROGEN
AND
ACTIVE
TABLE Effects
Brain
of Neonatal
region
BRAIN
4
Estrogen on T/S Ratios of a-[I-‘~C1Aminoisobutyric in Brai; Regions of the l7-Day-Old Rat” __T/S ratios Minutes .~
after
inJection -._--___
2 Amygdala Cb EB CWZ?tXllWl C EB Corpora quadrigemina C EB Frontal cortex C EB Hypothalamus C EB Medulla C EB Occipital cortex c EB Olfactory bulbs C EB Olfactory tultercles C EB ParietaI cortex c En P011s c En Pyriiorm cortex C EB n T/S * C = cP 5 d P 5 *P <
ratios control; 0.005. 0.001. 0.01.
823
TR.iNSPOKT
-
Acid
of isotope -.-.300
60 -__-
.-___
0.540 0.557
* 0.020 ;t 0.017
1.7x1 1.747
f 0.047 f 0.012
f 0.003 * 0.002
0.740 tJ.7il
f 0.046 & 0.035
2.527 2.497
* f
0.045 tJ.038
* O.otJlc f 0.002
0.4il 0.454
* 0.015 f
0.014
1.722 1.704
f 0.044 zk 0.053
tJ.tJo.1 tJ.0o.i
0.054 0.047
* 0.002* * 0.001
0.640 0.655
f f
0.014 0.039
2.197 f 2.05 1 f
0.001 0.007
0.030 0.025
f
O.OOlJ
*
0.001
0.455 0.460
f 0.016 * 0.015
1.612 l.Si4
zt 0.040 * 0.044
0.050 0.053
f 0.006 * 0.007
0.090 0.064
f 0.0086 i 0.002
0.612 0.622
zt 0.02 1 f 0.023
2.432 2.360
f f
0.033 0.031
* 0.001 f 0.002
0.049 0.052
* WJOl l 0.003
0.581 f 0.62 1 f
2.014 1.922
3~ 0.058 f 0.027
0.037 0.039
* 0.003 f 0.002
0.065 0.067
f f
0.0ll.Z 0.004
0.723 f 0.048 tJ.iYO A 0.019
2.079 f 2.tJ7O f
0.066 0.029
0.022 0.022
f f
0.039 0.030
* *
0.001~ 0.002
0.461 0.4i5
f 0.024 * 0.015
1.437 I.341
* f
0.034 0.07t.J
0.030 O.OJl
f O.tJO2 f 0.003
0.051 0.048
f f
0.004 O.tl(JZ
0.572 0.587
zk 0.028 f 0.012
2.107 I.939
zk 0.061 * tJ.095
o.o.ix 0.038
f o.lJo2 zt 0.1JO3
0.060 0.042
f f
0.OO.V 0.003
0.760 0.781
f 0.033 f 0.013
2.768
f
2.511
* U.ltl4
o.oxJ 0.028
f f
0.049 0.03x
* 0.002~ l 0.002
0.570 0.586
f f
I.913 1.819
f *
0.023 f 0.001 0.02 1 * 0.00 1
O.O,i6 0.029
f *
0.044 0.031
f *
0.003c 0.002
0.056 0.054
0.tJ.M 0.026
i 0.002~ z!z 0.002
0.041 0.039
* *
0.02 I f 0.020 *
0.002 0.003
tI.002 0.002
are expressed as the mean f standard error EB = treated with estradiol henroate.
O.tlOl~ O.(JO?
of the mean
obtained
0.022
0.048
0.011 0.032
from
O.Oib 0.050
0.047 0.061
0.063 O.OiX
0.0X9
0.046 0.024
8 to 1.3 rats per groul>.
the cerebellum and pans of the 17-day-old rats. Variations in the concentration or availability of carrier molecules for AIB in cell membranes of neurons and/or subcellular particles (3, 22) are the most probable factors (amxig others) responsible for this regional heterogeneity. ‘I’lle data in Table 2 indicate that the administration of estradiol benzuate 10 neonatal rats did not dih tlic traiisl~0rL uf AIL’, iulu brain qions oi S-(lay-uld rats 2 mill after the injection of the isotope. The uptake of AIL:
824
M.
LITTERIA
in the estradiol benzoate-treated rats was significantly reduced in the amygdala, cerebellum, medulla, and olfactory tubercles at the 5-min interval and in all brain regions 60 min after the injection of the isotope. By 60 min, AIB had been actively transported (T/S > 1) into the cerebellum and olfactory bulbs of control S-day-old rats. By 300 min, the active transport of AIB was significantly increased in all brain regions of the S-dayold rats given estrogen (Table 2). There were no significant differences in the uptake of AIB by brain regions of the lo-day-old control and estradiol benzoate-treated rats 2, 5, and 60 min after injection of the isotope (Table 3). By 60 min, AIB was actively transported into the cerebellum and olfactory bulbs of all control and estradiol benzoate-treated rats (Table 3). By 300 min, the active transport of AIB was significantly increased in 8 of the 12 brain regions from rats treated with estradiol benzoate, i.e., corpora quadrigemina, frontal cortex, hypothalamus, medulla, occipital cortex, olfactory bulbs, parietal cortex, and pyriform cortex (Table 3). Table 4 indicates that the uptake of AIB in the 17-day-old estradiol benzoate-treated rats was reduced in the cerebellum and corpora quadrigemina at the 2-min interval and in 8 of the 12 brain regions 5 min after the injection of isotope. There were no differences in the uptake of AIB by any brain region of the 17-day-old control and estradiol benzoatetreated rats at the 60- and 300-min intervals (Table 4). In contrast to the S- and lo-day-old rats (Tables 2 and 3), active transport did not occur in any brain region of the 17-day-old rats until the 300~min interval (Table 4). Tables 2-4 indicate that the effectiveness of the steroid in influencing the transport and accumulation of AIB by the brain decreases with age. DISCUSSION Currently, available evidence indicates that the transport of amino acids across the blood-brain barrier is via specific carrier-mediated transport systems presumed to be in the endothelium of the cerebral capillaries (7, 23-25,29). Saturable and stereospecific carrier-mediated transport systems have been described in viz10 for the basic, neutral, and acidic amino acids (7, 15, 23, 24). In general, the in tizlo entry of amino acids into the brain is greater in the younger animal than in the adult (28) These differences have been ascribed to the incomplete maturation of bidirectional carrier-mediated transport systems in the younger animal (7, 16, 28). Although there are many differences between the transport of amino acids into brain slices and their transport into the living brain (2, 7), the existence of separate carrier-mediated influx and efflux systems has been proposed for AIR both itl ztfro (5-7) and irr zizro (7, 23. 27). The pro-
NEONATAL
ESTROGEN
AND
ACTIVE
BRAIIi
TRAKSPORT
82.5
posed ability of the influx and eftlux systems to respond differentially to administered drugs in z&o (10, 16) can be used as a basis in attempting to explain the effects of estradiol benzoate on the accumulation of AIB in specific regions of the brain. The concentration of AIB in the brain at any given instant is a function of the activities of the opposing influx and efflux systems (10). It is, therefore, probable that estradiol benzoate selectively and/or predominantly affected a structural, enzymic, or metabolic component( s) of the influx and ef?lux systems. Thus, the significantly higher T/S ratios observed at 300 min in all brain regions of the S-day-old estradiol benzoate-treated rats (Table 2) and in S of the 12 brain regions of the IO-day-old treated rats (Table 3) could be due to either stimulation of the influx mechanism and/or inhibition of the efflux mechanism. Similarilp, the lower T/S ratios observed at 5 min for the amygdala, cerebellum, medulla, and olfactory tubercles of the .5-day-old treated rats and for all brain regions of the S-day-old treated rats at 60 min could be due to either a transient steroid-induced stimulation of the efflux system and/or inhibition of the influx mechanism. This would also explain the inhibitory effects of estradiol benzoate in some brain regions of the 17-dayold rats at the 2- and S-min intervals (Table 4). The declining ability of the steroid to influence the transport and accumulation of AIE in the brain with increasing age is probably due to maturation of the carrier-mediated transport system for the isotope. It is interesting to note that similar data have been 0l)tained from rats treated neonatally with androgen (18). The current status of knowledge rcgardin g the transport of amino acids across the blood-brain barrier does not allow us to explain the facilitating effects of neonatally administered estradiol benzoate on the active transport of AIB into specific brain regions of the S- and lo-day-old rat at 300 min. It could be tentatively assumed that estradiol benzoate increased the synthesis and/or availability of the carrier protein(s) or cofactors required 1~~ the influx system for AIB. The development antl,‘or availability of a larger number of carriers would then esplain the inability of estradiol benzoate to increase the active transport of AIB into brain regions of the 17-day-old rat. In this regard, it should be noted that the ability of insulin to stimulate the in eitro transport of AIB into bone (12) and diaphragm (9) has been attributed to the increased synthesis of carrier protein. It is unlikely that the accumulation of AI13 in the brain at concentrations greater than the concentration in serum can be attributed to diffusion and,’ or binding to cellular components. The in PGZWpassage of amino acids from the blood into brain is mediated primarily by carrier transport systems rather than by diffusion (7. 24, 29). In addition, the unsaturable component of AIR entry into brain slices could not l)e attributetl to diffusion (6). The inxl,ility to tlenlonstrate radioacti\Gt>, in subccIlular cc~mpo~ients of the rat
826
M.
LITTEKIA
brain following the injection of labeled AIB (14) indicates that subcellular binding or compartmentation of the isotope did not occur. Administration of p-estradiol valerianate to neonatal rats is followed by markedly elevated AMP cyclase activity in the brain at 7, 14, 21, and 28 days after birth (4). Cyclic adenosine monophosphate was found to increase the in ZGZJOentry of several amino acids into the brain without affecting amino acid metabolism (26). These isolated studies (4, 26) would seem to suggest the possibility of a relationship(s) between estradiol, the cyclic AMP system and the transport of amino acids across the blood-brain barrier, We recognize that the effects of neonatally administered estradiol benzoate on the transport of AIB into specific regions of the brain may not be due to a direct action of the steroid on some aspect(s) of the blood-brain barrier or transport mechanism for AIB. These effects could be secondary to estradiol benzoate-induced metabolic changes in ‘the brain. For example, the administration of P-estradiol valerianate to neonatal rats has been reported to stimulate the activity of numerous glycolytic enzymes in the brain (4). Steroid-induced alterations in the metabolic activity of the brain could then secondarily affect the activity and/or synthesis of essential carrier system components. Although the exact mechanisms are unknown, the data presented in this study definitively link the alterations in the transport of AIB into specific regions of the brain to the administration of estradiol benzoate. REFERENCES 1. ARAI, Y. 1964. Persistent-estrous and -diestrous conditions induced by early postnatal administration of estrogen in female rats. Endocritzol. Jap. 11 : 204-208. 2. BANOS, G., P. M. DANIEL, S. R. MOORHOUSE, AND 0. E. PRATT. 1973. The influx of amino acids into the brain of the rat in Go: The essential compared with the non-essential amino acids. Proc. R. Sot. Lo4 [Biol.] 183: 59-70. 3. BATTISTIN, L., AND A. LAJTHA. 1970. Regional distribution and movement of amino acids in the brain. J. Ne1w01. Sci. 10 : 313-322. 4. CAVALLOTTI, C., AND L. BISANTI. 1972. Hormonal regulation of rat brain development II. Biochemical changes induced by p-estradiol. Prog. Braitt Rcs. 38: 6983. 5. CHERAYIL, A., J. KANDEBA, AND A. LAJTIIA. 1967. Cerebral amino acid ‘transport ift Gfro. IV. The effect of inhibitors on exit from brain slices. 1. Ncztrocl~~rt. 14 : 105-115. 6. COHEN, S. R. 1973. The rate equation and activation energies for the uptake of a-aminoisobutyric acid by mouse brain slices. J. Physiol. (Londou)228 : 105113. 7. COHEN, S. R., AND A. LAJTHA. 1972. Amino acid transport. Pages 543-572 in A. IAJTHA, Ed., Handbook of Ncurochcmistry, Vol. i. Plenum Press, New York. S. I)Koz, R., AND H. I,. E;oE,VlC. 1970. Localization of protein metabolisn1 il. lleuro1ls. l’agcs Y3-108 in A. LA.ITHA, Ed., Protrirh nilcttrbo/is~r~ of the N~ITUIIU Sj’stcrrc. Plenum Press, New York.
NEOiXATAL
ESTROGEN
AND
ACTIVE
BRAIN
TRANSPORT
827
9. ELSAS, L. J., I. ALRRECHT, AND L. E. ROSENBERG. 1968. Insulin stimulation of amino acid uptake in rat diaphragm. J. Biol. Chc~rr. 243 : 1846-1853. 10. GORDON, M. W., J. A. SIX+ R. K. HANSOX, AND R. E. KUTTNER. 1962. The effects of some psychopharmacological agents and other drugs on the uptake of an amino acid by brain. J. Nc~tvochrrrz. 9 : 477-486. 11. GORSKI, R. A. 1963. Uodification of ovulatory mechanisms by postnatal administration of estrogen to the rat. A-lr~r. J. Physiol. 205 : 842-843. 12. HAIIN, T. J., S. J. IXIWK~NG, ASI) J. M. PEEAX. 1971. Insulin effect on amino acid transport in bone : Dependence on protein synthesis and Xaf. .4r11. J. Physiol. 220: 1717-1723. 13. HENDRICKS, S. E., AND A. II\. GERAM.. 1970. Effect of neonatally administered estrogen on development of male and female rats. E~docviwloy.~ 87: 435-439. 14. KCTTNER, R., J. A. Soars, AND M. W. GORDON. 1961. The uptake of a metabolically inert amino acid by brain and other organs. 1. Nc~frochcw. 6: 311-317. 15. LAJTIIA, A., S. LAHIRI, AND J. TOTH. 1963. The brain barrier system IV. Cerebral amino acid uptake in different classes. J. I%eurochem. 10: 765-773. 16. LAJTHA, r\., AND J. TOTFI. 1962. The brain barrier system III. The efflux of intracerebrally administered amino acids from the brain. J. Ncnrorhwz. 9 : 199212. 17. LEVINE, S., ASD R. MLXLINS. 1964. Estrogen administered neonatally affects adult sexual behavior in male and female rats. Scicnrc 144 : 185-187. 18. IJTTERIA, M. 1977. Effects of neonatal androgenization on the ill z&to transport of alpha-aminoisobutyric acid into specific regions of the rat brain. Bruirt Krs. 132 : 287-299. 19. LITTERIA, M., AKD M. W. THORNER. 1974. Inhibitory effect of neonatal estrogenization on the incorporation of “H-lysine in the Purkinje cells of the adult male and female rat. Brairl Krs. 80 : 152-154. 20. LITTERIA, M., AND bl. W. THORKER. 1975. Inhibitory action of neonatal estrogenization on the incorporation of ‘H-lysine into proteins of specific hypothalamic nuclei in the adult male rat. Bvaiu Kes. 90 : 175-180. 21. LITTERIA, M., AND M. W. THORNER. 1975. Inhibition in the incorporation of [3H]lysine into proteins of specific hypothalamic nuclei of the adult female rat after neonatal estrogenization. Exp. Nettvol. 49 : 592-595. 22. NA~ON, S., A~UD A. LAJTHA. 1969. The uptake of amino acids by particulate fractions from brain. BiocAiru. Biophys. Acta 173 : 518-531. 23. OLDENDORF, W. H. 1971. Brain uptake of radiolabeled amino acids, amines, and hexoses after arterial injection. =Irrl. /. Physiol. 221 : 1629-1638. 24. OLDENDORF, W. H., AP;D J. SZABO. 1976. Amino acid assignment to one of three blood-brain barrier amino acid carriers. .-lrrl. J. PIqsiol. 230: 94-98. 25. ORI.OWSKI, M., G. SESSA, AND J. P. GREEN. 1974. y-Glutamyl transpeptidase in brain capillaries: Possible site of a blood-brain barrier for amino acids. Sciclrcr 184 : 66-68. 26. PETKOV, V., P. CSLJKov, AND V. KVSHEV. 1974. The role of 3’,5’-A11P system in amino acid transport across the blood-brain barrier. .-lcttr Biol. Med. Grr. 33 : 371-383. 27. SCHAIN, R. J., AND k;. S. WATANABE. 1972. Distinct patterns of entry of two nonmetabolizable amino acids into brain and other organs of infant guinea pigs. J. Newockerrc. 19 : 2279-2288. 28. SETA, K., H. SHERSHEN, AND =\. LAJTHA. 1972. Cerebral amino acid uptake iu &lo in newborn mice. 13wilr lirs. 47 : 415-425. 29. ZWCHIN, G., H. SERSIIEN, AKD A. LAJTIIA. 1976. The eifect of hypcrosnwlal urea on the transport of amino acids into rat brain. Exp. Ncwol. 51 : 292-303.
Effects
SE~RULWY
57, 817-827
(1Y77)
of Neonatal Estrogen on in Vivo Transport a-Aminoisobutyric Acid into Rat Brain
of
The effects of neonatally administered estradiol benzoate were determined on the transport of w [ l-Y] aminoisobutyric acid into amygdala, cerebellum, corpora quadrigemina, cortices (frontal, occipital, parietal, pyriform), hypothalamus, medulla, olfactory bulbs, olfactory tubercles, and pons of the rat brain. Rats were injected subcutaneously with either 500 pg estradiol benzoate or vehicle 24 and 72 h after birth, and at 5, 10, or 17 days of age they were decapitated 2, 5, 60, or 300 min after the intraperitoneal injection of w[l-‘“Claminoisobutyric acid. By 300 min, the active transport of the isotope in cstradiol benzoate-treated rats was greater in all brain regions of 5-day-old rats and in 8 of the 12 brain regions of lo-day-old rats. Estradiol benzoate did not alter active transport in 17-day-old rats at 300 min. These data suggest that injection of estradiol benzoate during the critical period of brain differentiation (directly or indirectly) affects processes regulating the active transport of LY- [ 1-“Cl aminoisobutyric acid into brain regions of neonatal rats. The effectiveness of the steroid also decreases \vith age.
INTRODUCTION Administration of estrogens to neonatal male or female rats during the critical period of brain differentiation is followed in the adult by alterations in sexual behavior and reproductive capability (11, 13, 17). Adult male rats are characterized by testicular atrophy, inhibition of spermatogenesis, and decreased male sexual behavior (13, 17). The females are anovulatory and sexual behavior is masculinized (1, 11, 13, 17). These sequelae are Abbreviation
: AIB--or-
[ 1-“C]
aminoisobutyric
acid.
1 This study was supported by the Medical Research Service of the Veterans Administration and in part by U.S. Public Health Service Grant NS11501-02 from the National Institute of Neurological and Communicative Disorders and Stroke. I thank Drs. M. Thorner and L. Vitello for their critical review of this manuscript. C. Popoff provided technical assistance. 817 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0014-4886
818
&I.
LI'UEKIA
l)r~~lal)l!~ rrlatc~tl to rstro~cll-ill(lucct1 altcratic,ns ill tic~urnl structurc&s regulating the synthesis antl,/or release of the pituitary gonntlotrol~ins (1 ). Although the esact biochemical nature of these alterations is not known, they may be associated with estrogen-induced changes in the synthesis of brain nucleic acids and/or proteins ( 19-21). Q uantitative autoradiographic studies from this laboratory have localized some of the neural structures metabolically responsive to neonatally administered 17p-estradiol benzoate of [ 3H] lysine into proteins (EB) (19-21). F or example, incorporation of specific hypothalamic nuclei (arcuate, periventricular, paraventricular, and supraoptic) was inhibited in adult male (20) and female (21) rats treated neonatally with estradiol benzoate. The Purkinje cells, but not the stellate or granule cells, of the cerebellar cortex were similarily affected ( 19). Although autoradiography identified the sites of newly synthesized protein (S), differences in the incorporation of [3H] lysine into proteins between control and estracliol benzoate-treated rats does not imply that the steroid inhibited protein synthesis per se. The steroid could have produced alterations in the transport and, therefore, availability of [3H]lysine in the brain. The purpose of this study was to determine if the administration of estradiol benzoate to neonatal rats during the critical period of brain differentiation is followed by alterations in the transport of (Y-[ 1-14C] aminoisobutyric acid (AIB) across the blood-brain barrier into specific brain regions. AIB is a small neutral, nonmetabolized amino acid that enters the brain by carrier-mediated processes (5-7, 14). The inert character of AIB avoids the problems associated with amino acids that are metabolized by the brain. MATERIALS
AND
METHODS
Three-month-old virgin Sprague-Dawley rats (Madison, Wisconsin) exposed to a 14-h light: 10-h dark schedule and ingesting Purina Rat Chow and water ad Zibitunt served as our breeding stock. Litters were reduced to eight pups within 24 h of birth and the day of delivery is called Day 1. Male or female rats were injected subcutaneously with either 500 pg estradiol benzoate or 0.1 ml sesame oil vehicle at 24 h and again at 72 h after birth. At 5, 10, and 17 days after birth, a minimum of eight control and eight estradiol-treated rats was each injected intraperitoneally with 0.25 &i AIB/g body weight (sp. act. = 9.0 mCi/mmol, New England Nuclear, Boston, Massachusetts). The rats were placed in a beaker warmed by a lamp and containing bedding from the home cage prior to decapitation at intervals of 2, 5, 60, or 300 min after the injection of AIB. The brain was rapidly removed, placed on a petri dish over crushed ice, and freed of
tllettittgt’s at~d blootl. ‘I’\vcl\~ 1)raitt rcgiotts. i.e.. attt~gtlala (\vitll ovrrl!,itl:: cortex), ccrebelhtm, corpora c~ttadrigeminn, frontal cortex. l~~potltalatntts, medulla, occipital cortex, olfactory bulbs, olfactory tubercles, parietal cortex, pans, and pyriform tortes were removed and placed in a petri dish resting on a block of dry ice. Samples of set-ml from each rat were prepared by centrifuging the clotted blood at 2°C for 30 min at 600g. Each frozen brain region was quickly weighed and transferred while still frozen into scintillation vials containing 1.0 ml solubilizer (Soluene-350, Packard Instrument Co., Inc., Downers Grove, Illinois). Duplicate samples (10 to 50 ~1) of serutu were added to the solubilizer. The tissues were solubilized overnight at room temperature and 15 ml liquid scintillation cocktail (Dimilume-30, Packard Instrument Co., Inc.) were added to each vial. Counts per minute were determined (against a blank) in a Packard Tri-Carb liquid scintillation spectronteter, Model 333s. Corrections for tissue quench were made using the esternal standardization channels ratio and least-squares methods. Data were calculated as disintegrations per minute (dpm) per milligram tissue, disintegrations per minute per microliter serum, and as tissue to serum ratios (T/S). Only SC/, of whole brain radioactivity is due to plasma AIB ; therefore, correcting for the amount of radioactivity due to blood present in each brain region was unnecessary (13). The total water content of each brain region was determined in a separate experiment for the 5, lo-, and 17-day-old control and estradiol benzoate-treated rats. Removal and handling of the brain tissues were as described above. The brain tissues were dried to constant weight and the percentage water was calculated from the difference between the weights of the frozen and dried tissues. The significance of differences between the control and estradiol benzoate-treated rats was determined by Student’s t-test; P d 0.05 was considered significant. RESULTS (Y-[l-‘“C]rllrlilloisob~~~~~,ic Acid DOSE-Rcs~onsr. To ensure that the dose of AIB selected for this study would be below saturation kinetics, T/S ratios were determined for each brain region of 5 and lo-clay-old control rats at C1 = 0.125, C, = 0.25, and Ca = 0.40 &i AIB/g body weight. Five minutes after the injection of AIB. disintegrations per minute per milligram tissue and disintegrations per minute per microliter serutn were twice as great for Cz than Cl and approximately 3.2 times higher for C3 than Cl. Significant differences were not found between T/S ratios for any brain region of the .5- or lo-day-old rats at Cl, C,, and Cs. Cl, Cz, and Cs were, therefore, below saturation kinetics and C, was selected for this study. T/S
Ilinutes 2
604.9 688.8
‘/ Each value Ir V = control
aft&
injection
of isotolx
5
f 17.3 zt 39.5
60
300
iYi.3 715.5
f. 64.6 + 31.3
255.1 260.5
f f
10.8 12.8
li4.7 153.1
f f
12.6 10.4
1128.0 1096.3
f f
25.3 30.3
1177.8 1269.5
zk 38.2 f 41.0
228.6 239.5
f f
11.4 12.1
153.7 153.0
zk 15.4 f 3.8
1115.1 1079.5
zt 39.2 f 83.5
1232.1 1146.0
f f
277.3 2io.o
f f
9.4 i.8
163.2 165.8
f f
51.2 48.1
is the mean f SE obtained from 8 to 13 rats ; El3 = treated with estradiol benzoatc.
lxr
3.8 5.3
group.
ratios rather than absolute concentrations (disinteg-rations per minute per milligram tissue) are a meas11re of AIR transport. T/S ratios greater than unity indicate the occurrence of acti1.e transport. Urak IIrtrfcr Cotltmf. There were no sex differences in the water content of specific brain regions of control or estradiol benzoate-treated 5-, lo-, and 17-day-old rats. Differences in water content were found between control and estradiol benzoate-treated rats for some brain regions at 5 and 10 days of age. However, these changes were so snlall that corrections made for these differences did not affect the significance of the data between control and estradiol benzoate-treated groups. Sex Lli~rvwlcrs. Iiflrc~s of l!sfradiol I:rwoatc on :1IU Tramport. There m-ere no sex differences in the concentration of AIlS in serum or brain regions of control or estradiol benzoate-treated 5-, lo-, and 17-day-old rats at any of the tested intervals. Therefore, data from both sexes were coinbined in the control or estradiol benzoate-treated grouljs. Scruln. The concentration of AIL: in the serum of all control and treated rats was highest 5 min after the injection of the isotope (Table 1). There were no differences betweal the concentrations of AI13 in the sermn of control and estradiol benzoate-treated rats in an)- age group. BraipL IZcgio,ls. The absolute concentration of AIB (disintegration per minute per milligrani tissue) slowly increased with time iii all brain regions of control and estradiol benzoate-treated (S-, lo-, and 17-day-old) rats at the ages tested (Tables Z--t). For example, between 2 and 300 min, the
822
M.
LITTERIA
uptake of AIB by the brain of the control and estrogen-treated rats. By 300 min, the uptake of AIB in the control groups was consistently higher for the cerebellum, olfactory bulbs, and pons of the 5- and lo-clay-old rats and for the cerebellum and pans of the 17-day-old rats. The uptake of AIB in the estradiol benzoate-treated groups at 300 min was highest for the cerebellum and olfactory bulbs of the rat at 5 and 10 days of age and for TABLE Effects
of Neonatal
Brain
Estrogen
3
on T/S Ratios of w[l-W]Aminoisobutyric Regions of the lo-Day-Old Rat”
region
T/S ratios after injection
Minutes 2 Amygdala Cb EB Cerebellum C EB Corpora quadrigemina C EB Frontal cortex C EB H~porl~alamus C EB Medulla C EB Occipital cortex C EB Olfactory bulbs C EB Olfactory tuber&s C EB Parietal cortex C EB Polls C EB Pyriform cortex C EB 0 T/S 11C = cP 5 d P 5 *P 5 11’ <
Acid
in Brain
of isotope
5
60
300
0.028 0.027
f 0.001 * 0.002
0.05 1 f 0.051 f
0.001 0.002
0.606 0.701
f f
0.039 0.045
2.176 2.192
;t 0.076 f 0.179
0.057 0.054
f f
0.003 0’002
0.092 0.088
f f
0.004 0.005
1.377 1.334
f f
0.047 0.02 I
4.222 4.411
f f
0.048 0.274
0.030 0.027
f f
0.001 0.001
0.049 0.044
f f
0.001 0.003
0.660 0.616
f 0.048 * 0.033
1.972 2.223
f f
0.056’ 0.047
0.036 0.037
+ 0.002 l 0.003
0.074 0.078
f f
0.002 0.003
0.930 0.967
3~ 0.042 f 0.026
2.712 3.114
f l
0.107“ 0.124
0.024 f 0.001 0.02 I * 0.002
0.038 0.035
f f
0.001 0.001
0.588 0.615
f 0.036 3~ 0.024
1.789 2.232
zk 0.044~ f 0.085
0.044 0.047
* 0.005 ck 0.002
0.078 0.074
f f
0.004 0.005
0.801 0.837
3~ 0.034 f 0.037
2.833 3.186
f f
0.05 1~ 0.080
0.035 0.034
f f
0.001 0.002
0.069 0.065
f f
0.002 0.003
0.808 0.971
f f
2.683 3.048
f f
O.OJle 0.05 1
0.048 0.048
f 0.002 z!z 0.003
0.082 0.078
f 0.003 f 0.004
1.102 1.102
f 0.045 z!z 0.027
3.0 I3 f 0.059~ 3.350 + 0.115
0.025 0.026
f 0.001 f 0.002
0.044 0.041
f f
0.541 0.605
f f
0.023 0.039
1.872 2.043
f f
0.065 0.165
0.036 0.038
f 0.002 zk 0.003
0.068 0.064
* 0.022 f 0.004
0.855 0.891
f f
0.047 0.025
2.629 3.020
f f
0.060@ 0.066
0.045 0.044
f f
0.003 0.003
0.072 0.068
f 0.003 f 0.006
0.826 0.911
f 0.045 f0.022
3.057 3.032
f f
0.120 0.109
0.034 0.031
* 0.002 * 0.002
0.066 0.064
i f
0.938 0.932
f 0.044 + 0.064
2.583 3.107
f 0.067’ * 0.040
ratios are expressed as the mean f standard error control; EB = treated with estradiol benzoate. 0.005. 0.025. 0.001. 0.1)2.
0.001 O.OO2
0.002 0.004
of the mean
obtained
0.033 0.019
from
8 to 13 rats per group.
NEONATAL
ESTROGEN
AND
ACTIVE
TABLE Effects
Brain
of Neonatal
region
BRAIN
4
Estrogen on T/S Ratios of a-[I-‘~C1Aminoisobutyric in Brai; Regions of the l7-Day-Old Rat” __T/S ratios Minutes .~
after
inJection -._--___
2 Amygdala Cb EB CWZ?tXllWl C EB Corpora quadrigemina C EB Frontal cortex C EB Hypothalamus C EB Medulla C EB Occipital cortex c EB Olfactory bulbs C EB Olfactory tultercles C EB ParietaI cortex c En P011s c En Pyriiorm cortex C EB n T/S * C = cP 5 d P 5 *P <
ratios control; 0.005. 0.001. 0.01.
823
TR.iNSPOKT
-
Acid
of isotope -.-.300
60 -__-
.-___
0.540 0.557
* 0.020 ;t 0.017
1.7x1 1.747
f 0.047 f 0.012
f 0.003 * 0.002
0.740 tJ.7il
f 0.046 & 0.035
2.527 2.497
* f
0.045 tJ.038
* O.otJlc f 0.002
0.4il 0.454
* 0.015 f
0.014
1.722 1.704
f 0.044 zk 0.053
tJ.tJo.1 tJ.0o.i
0.054 0.047
* 0.002* * 0.001
0.640 0.655
f f
0.014 0.039
2.197 f 2.05 1 f
0.001 0.007
0.030 0.025
f
O.OOlJ
*
0.001
0.455 0.460
f 0.016 * 0.015
1.612 l.Si4
zt 0.040 * 0.044
0.050 0.053
f 0.006 * 0.007
0.090 0.064
f 0.0086 i 0.002
0.612 0.622
zt 0.02 1 f 0.023
2.432 2.360
f f
0.033 0.031
* 0.001 f 0.002
0.049 0.052
* WJOl l 0.003
0.581 f 0.62 1 f
2.014 1.922
3~ 0.058 f 0.027
0.037 0.039
* 0.003 f 0.002
0.065 0.067
f f
0.0ll.Z 0.004
0.723 f 0.048 tJ.iYO A 0.019
2.079 f 2.tJ7O f
0.066 0.029
0.022 0.022
f f
0.039 0.030
* *
0.001~ 0.002
0.461 0.4i5
f 0.024 * 0.015
1.437 I.341
* f
0.034 0.07t.J
0.030 O.OJl
f O.tJO2 f 0.003
0.051 0.048
f f
0.004 O.tl(JZ
0.572 0.587
zk 0.028 f 0.012
2.107 I.939
zk 0.061 * tJ.095
o.o.ix 0.038
f o.lJo2 zt 0.1JO3
0.060 0.042
f f
0.OO.V 0.003
0.760 0.781
f 0.033 f 0.013
2.768
f
2.511
* U.ltl4
o.oxJ 0.028
f f
0.049 0.03x
* 0.002~ l 0.002
0.570 0.586
f f
I.913 1.819
f *
0.023 f 0.001 0.02 1 * 0.00 1
O.O,i6 0.029
f *
0.044 0.031
f *
0.003c 0.002
0.056 0.054
0.tJ.M 0.026
i 0.002~ z!z 0.002
0.041 0.039
* *
0.02 I f 0.020 *
0.002 0.003
tI.002 0.002
are expressed as the mean f standard error EB = treated with estradiol henroate.
O.tlOl~ O.(JO?
of the mean
obtained
0.022
0.048
0.011 0.032
from
O.Oib 0.050
0.047 0.061
0.063 O.OiX
0.0X9
0.046 0.024
8 to 1.3 rats per groul>.
the cerebellum and pans of the 17-day-old rats. Variations in the concentration or availability of carrier molecules for AIB in cell membranes of neurons and/or subcellular particles (3, 22) are the most probable factors (amxig others) responsible for this regional heterogeneity. ‘I’lle data in Table 2 indicate that the administration of estradiol benzuate 10 neonatal rats did not dih tlic traiisl~0rL uf AIL’, iulu brain qions oi S-(lay-uld rats 2 mill after the injection of the isotope. The uptake of AIL:
824
M.
LITTERIA
in the estradiol benzoate-treated rats was significantly reduced in the amygdala, cerebellum, medulla, and olfactory tubercles at the 5-min interval and in all brain regions 60 min after the injection of the isotope. By 60 min, AIB had been actively transported (T/S > 1) into the cerebellum and olfactory bulbs of control S-day-old rats. By 300 min, the active transport of AIB was significantly increased in all brain regions of the S-dayold rats given estrogen (Table 2). There were no significant differences in the uptake of AIB by brain regions of the lo-day-old control and estradiol benzoate-treated rats 2, 5, and 60 min after injection of the isotope (Table 3). By 60 min, AIB was actively transported into the cerebellum and olfactory bulbs of all control and estradiol benzoate-treated rats (Table 3). By 300 min, the active transport of AIB was significantly increased in 8 of the 12 brain regions from rats treated with estradiol benzoate, i.e., corpora quadrigemina, frontal cortex, hypothalamus, medulla, occipital cortex, olfactory bulbs, parietal cortex, and pyriform cortex (Table 3). Table 4 indicates that the uptake of AIB in the 17-day-old estradiol benzoate-treated rats was reduced in the cerebellum and corpora quadrigemina at the 2-min interval and in 8 of the 12 brain regions 5 min after the injection of isotope. There were no differences in the uptake of AIB by any brain region of the 17-day-old control and estradiol benzoatetreated rats at the 60- and 300-min intervals (Table 4). In contrast to the S- and lo-day-old rats (Tables 2 and 3), active transport did not occur in any brain region of the 17-day-old rats until the 300~min interval (Table 4). Tables 2-4 indicate that the effectiveness of the steroid in influencing the transport and accumulation of AIB by the brain decreases with age. DISCUSSION Currently, available evidence indicates that the transport of amino acids across the blood-brain barrier is via specific carrier-mediated transport systems presumed to be in the endothelium of the cerebral capillaries (7, 23-25,29). Saturable and stereospecific carrier-mediated transport systems have been described in viz10 for the basic, neutral, and acidic amino acids (7, 15, 23, 24). In general, the in tizlo entry of amino acids into the brain is greater in the younger animal than in the adult (28) These differences have been ascribed to the incomplete maturation of bidirectional carrier-mediated transport systems in the younger animal (7, 16, 28). Although there are many differences between the transport of amino acids into brain slices and their transport into the living brain (2, 7), the existence of separate carrier-mediated influx and efflux systems has been proposed for AIR both itl ztfro (5-7) and irr zizro (7, 23. 27). The pro-
NEONATAL
ESTROGEN
AND
ACTIVE
BRAIIi
TRAKSPORT
82.5
posed ability of the influx and eftlux systems to respond differentially to administered drugs in z&o (10, 16) can be used as a basis in attempting to explain the effects of estradiol benzoate on the accumulation of AIB in specific regions of the brain. The concentration of AIB in the brain at any given instant is a function of the activities of the opposing influx and efflux systems (10). It is, therefore, probable that estradiol benzoate selectively and/or predominantly affected a structural, enzymic, or metabolic component( s) of the influx and ef?lux systems. Thus, the significantly higher T/S ratios observed at 300 min in all brain regions of the S-day-old estradiol benzoate-treated rats (Table 2) and in S of the 12 brain regions of the IO-day-old treated rats (Table 3) could be due to either stimulation of the influx mechanism and/or inhibition of the efflux mechanism. Similarilp, the lower T/S ratios observed at 5 min for the amygdala, cerebellum, medulla, and olfactory tubercles of the .5-day-old treated rats and for all brain regions of the S-day-old treated rats at 60 min could be due to either a transient steroid-induced stimulation of the efflux system and/or inhibition of the influx mechanism. This would also explain the inhibitory effects of estradiol benzoate in some brain regions of the 17-dayold rats at the 2- and S-min intervals (Table 4). The declining ability of the steroid to influence the transport and accumulation of AIE in the brain with increasing age is probably due to maturation of the carrier-mediated transport system for the isotope. It is interesting to note that similar data have been 0l)tained from rats treated neonatally with androgen (18). The current status of knowledge rcgardin g the transport of amino acids across the blood-brain barrier does not allow us to explain the facilitating effects of neonatally administered estradiol benzoate on the active transport of AIB into specific brain regions of the S- and lo-day-old rat at 300 min. It could be tentatively assumed that estradiol benzoate increased the synthesis and/or availability of the carrier protein(s) or cofactors required 1~~ the influx system for AIB. The development antl,‘or availability of a larger number of carriers would then esplain the inability of estradiol benzoate to increase the active transport of AIB into brain regions of the 17-day-old rat. In this regard, it should be noted that the ability of insulin to stimulate the in eitro transport of AIB into bone (12) and diaphragm (9) has been attributed to the increased synthesis of carrier protein. It is unlikely that the accumulation of AI13 in the brain at concentrations greater than the concentration in serum can be attributed to diffusion and,’ or binding to cellular components. The in PGZWpassage of amino acids from the blood into brain is mediated primarily by carrier transport systems rather than by diffusion (7. 24, 29). In addition, the unsaturable component of AIR entry into brain slices could not l)e attributetl to diffusion (6). The inxl,ility to tlenlonstrate radioacti\Gt>, in subccIlular cc~mpo~ients of the rat
826
M.
LITTEKIA
brain following the injection of labeled AIB (14) indicates that subcellular binding or compartmentation of the isotope did not occur. Administration of p-estradiol valerianate to neonatal rats is followed by markedly elevated AMP cyclase activity in the brain at 7, 14, 21, and 28 days after birth (4). Cyclic adenosine monophosphate was found to increase the in ZGZJOentry of several amino acids into the brain without affecting amino acid metabolism (26). These isolated studies (4, 26) would seem to suggest the possibility of a relationship(s) between estradiol, the cyclic AMP system and the transport of amino acids across the blood-brain barrier, We recognize that the effects of neonatally administered estradiol benzoate on the transport of AIB into specific regions of the brain may not be due to a direct action of the steroid on some aspect(s) of the blood-brain barrier or transport mechanism for AIB. These effects could be secondary to estradiol benzoate-induced metabolic changes in ‘the brain. For example, the administration of P-estradiol valerianate to neonatal rats has been reported to stimulate the activity of numerous glycolytic enzymes in the brain (4). Steroid-induced alterations in the metabolic activity of the brain could then secondarily affect the activity and/or synthesis of essential carrier system components. Although the exact mechanisms are unknown, the data presented in this study definitively link the alterations in the transport of AIB into specific regions of the brain to the administration of estradiol benzoate. REFERENCES 1. ARAI, Y. 1964. Persistent-estrous and -diestrous conditions induced by early postnatal administration of estrogen in female rats. Endocritzol. Jap. 11 : 204-208. 2. BANOS, G., P. M. DANIEL, S. R. MOORHOUSE, AND 0. E. PRATT. 1973. The influx of amino acids into the brain of the rat in Go: The essential compared with the non-essential amino acids. Proc. R. Sot. Lo4 [Biol.] 183: 59-70. 3. BATTISTIN, L., AND A. LAJTHA. 1970. Regional distribution and movement of amino acids in the brain. J. Ne1w01. Sci. 10 : 313-322. 4. CAVALLOTTI, C., AND L. BISANTI. 1972. Hormonal regulation of rat brain development II. Biochemical changes induced by p-estradiol. Prog. Braitt Rcs. 38: 6983. 5. CHERAYIL, A., J. KANDEBA, AND A. LAJTIIA. 1967. Cerebral amino acid ‘transport ift Gfro. IV. The effect of inhibitors on exit from brain slices. 1. Ncztrocl~~rt. 14 : 105-115. 6. COHEN, S. R. 1973. The rate equation and activation energies for the uptake of a-aminoisobutyric acid by mouse brain slices. J. Physiol. (Londou)228 : 105113. 7. COHEN, S. R., AND A. LAJTHA. 1972. Amino acid transport. Pages 543-572 in A. IAJTHA, Ed., Handbook of Ncurochcmistry, Vol. i. Plenum Press, New York. S. I)Koz, R., AND H. I,. E;oE,VlC. 1970. Localization of protein metabolisn1 il. lleuro1ls. l’agcs Y3-108 in A. LA.ITHA, Ed., Protrirh nilcttrbo/is~r~ of the N~ITUIIU Sj’stcrrc. Plenum Press, New York.
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827
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