Influence of Age and Short-Term Starvation on the ATPase Activity in the Developing Rat Brain J. MOUREK F. S?ASTNY Institute of Physiology Medical Faculty Charles University Prague, Czechoslovakia Na'-K+-stimulated and Mg++dependent ATPase activities were investigated in the developing cerebral cortex, subcortical structurcs, and medulla oblongata of rats as was the effect of 24-hr lasting starvation and thirst on those enzyme activities. We found (a) a developmental increase of these ATPase activities in the developing rat brain with the maximum in the cerebral cortex and with the minimum in the medulla oblongata; (b) a decrease of the ratio of these enzyme activities, which was near unity in adult animals; (c) an increase of ATPase activities in the cerebral cortex and subcortical formations of young rats under starvation conditions followed by a decrease of the Mg++/Na+-K+-ATPase activity ratios in these structures; and (d) a decrease of these activities, especially in the cerebral cortex, and an increase of the activity ratios in adult animals under starvation conditions.
During the last 10 years much attention has been devoted to correlations between the development of Na'-K'-stimulated ATPase (adenosine triphosphate phosphohydrolase, EC 3.6.1.3), which is essential for achieving the active transport of Na' and K' in cells, and some biochemical and functional parameters of various tissues, particularly in the nervous system (Abdel-Latif, Brody, & Ramahi, 1967; Palladini, Bignanii, Venturini, Maccaghani, Correr, & Stefanelli, 1966; Samson & Quinn, 1967; Siastng, Frohlich, & Chmelik, 1969). Moreover, the inducibility of this enzyme system by glucocorticoids has been demonstrated in the developing nervous system (Ebel, Wolf, & Gunther, 1971; Siastng, 1971). In 1966 Palladini and coworkers first described a developmental increase of the Na'-K' ATPase activity concurrent with an increase in the number and structure of glial cells and the appearance of electrical activity in the chicken telencephalon. Samson and Quinn (1967) and Abdel-Latif, Smith, and Ellington (1970) carried out similar studies and found a gradual increase of Na'-K' ATPase and Mg"-ATPase activities in the developing rat brain. However, the rise of the former enzyme activity continues more slowly than that of the latter. This is true also for the nervous system of chicken embryos (Alfei & Venturini, 1972). Few data on the function and developmental changes of Mg"-dependent ATPase Reprint requests should be sent to Dr. Jindfick Mourck, Department of Physiology, Charles University, Prague 2, Albertov 5 , Czechoslovakia. Received for publication 24 February 1977 Revised for publication 20 June 1977 Developmental Psychobiology, 11(6):587-593 (1978) @ 1978 by John Wiley & Sons, Inc.
00 1 2-1 6 30/78/0011-05 8 7 $0 1.OO
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MOUREK AND STASTNY
have been published (Siege1 & Albers, 1970). Bowler and Duncan (1967) have indicated the possible influence of this enzyme on the passive permeability and on contractile proteins of the cellular membranes. The cellular localization of Mg"'dependent ATPase is different from that of Na+-K'-stimulated ATPase (Kirkpatrick, 1969). Moreover, anoxia, hypoxia, or ischemia always show a smaller effect on the Mg"-dependent ATPase activity than on the Na'-K'-stimulated ATPase (Schwartz, Mrsulja, Mrsulja, Passonneau, & Klatzo, 1976). In our previous experiments (Mourek & LoutockB, 1972) we studied the effect of ouabain, a specific inhibitor of the active transport of Na' and K' and of Na'-K'ATPase, on O2 uptake in the tissue slices isolated from different parts of the developing rat brain. We observed a developmental increase of the O2 uptake conditioned by the Na'-K'-ATPase increase in the rat cerebral cortex, but no changes in the uptake in the tissue of phylogenetically older parts of the rat brain. Also, we saw that short-term starvation and thirst during early postnatal life gave rise to a biochemical and functional retardation of the developing rat brain. The retardation was characterized by a decrease of the respiratory activity (Mourek, 1972), of free amino acids content (Mourek, Agrawal, Davis, & Himwich, 1970), and of RNA content in the rat brain (Mourek & Davis, 1970), including a decrease of some enzyme activities in plasma and brain tissue of these animals (Hrachovina & Mourek, 1976). The present work was undertaken in order to (a) describe the normal pattern of appearance of ATPase activities in different parts of tlie developing rat brain, and (b) study the effect of short-term starvation and thirst on the development of both enzyme activities in the rat brain.
Experiments were carried out on 5-, lo-, 14-, and 25-day-old rats (Rathrs norvegicus) of postnatal life (Day l-the day of birth) and on adults of both genders. Wistar rats from our own breeding colony were used. As in our previous experiments half of each standard litter, having 8 individuals in the nest, was separated from the mother and nest for 24 hr under an ambient temperature of 3OoC. Twenty-five-day-old rats and adults were deprived of food and water for the same time. After a period of 24 hr of deprivation at age 6, 11, 15, or 26 days of postnatal life the control and experimental (deprived) animals were decapitated and the heads were placed on ice. The skull and the brain membranes were opened widely and the brain was separated into cerebral cortex, subcortical structures (including thalamus and hypothalamus), and medulla oblongata. Approximately 100 mg of the individual parts were homogeniied in ice-cold .25M sucrose solution containing .005A4 ethylene diamine tetraacetic acid (EDTA-H, Serva); the pH 7.4 was adjusted with hydroxymethyl amino methane hydrochloride (Tris, Serva). Homogenates prepared under standard condition were centrifuged at 10,000 g in a Janetzki K 24 centrifuge for 10 min. The supernatant was used immediately for the ATPase assay. The total ATPase activity was measured in the medium containing 30 n1M Tris-HC1 (pH 7.4), 6 mM Mg", 20 mMK', and 100 mMNa' (as chloride salts). The Mg"-ATPase activity, which is ouabain insensitive, was determined in the same medium containing 1 mM of ouabain. The incubation media (.45 ml) were preincubated with .05 ml of
ATPASE IN THE DEVELOPING BRAIN
589
enzyme preparations for 6 m i n at 37°C. The reaction was started by adding ATP (disodium salt; Boehringer) in a final concentration of 6 mM and stopped after 10 min by addition of 1 ml of 7.5% trichloracetic acid. The solution was centrifuged at 3000 rpm for 5 min and the liberated orthophosphate was determined according to Fiske and Subbarow (1925). All ATPase assays were carried out in triplicate. The nonenzyme hydrolysis of ATP was separately estimated and released orthophosphate was subtracted from the total amount of orthophosphate released during the complete incubation. The Nat-Kt-stimulated (ouabain sensitive) and Mg"-dependent (ouabain insensitive) ATPase activity was determined in the absence or presence of ouabain and all activities were expressed as pl4 of inorganic orthophosphate (Pi)/mg protein/min. Protein was measured by the method of Lowry, Rosenbrough, Farr, and Randall (1951) and bovine serum albumine was used as a standard. The t-test for non-paired data was used for statistical evaluation of the results.
Results According to previous reports, total ATPase increases in all parts of the developing rat brain. In this study, over the period of development investigated, the increment of this enzyme activity was 249% in the cerebral cortex ( t = 10.74, p < .Ol), 228% in subcortical structures ( t = 8.85, p < .Ol), and 202% in medulla oblongata ( t = 10.19, p < .01). In all cases the increments were highly significant. The short-term starvation caused a significant increase of total ATPase activity in youngest rats in the subcortical structures (Days 6 and 11 of postnatal life) and cerebral cortex (Day 11). However, the enzyme activity decreased in adult rats (cerebral cortex; t = 3.22, p < .05) and in 26-day-old rats (subcortical structures; t = 5.05, p < .01). In the medulla oblongata the enzyme activity was unaffected by these experimental conditions in the 2 groups of youngest animals, increased in the 15-day-old and decreased in the 26-day-old and adult animals. (See Table 1.) The study of the development of Mg"-ATPase in the cerebral cortex showed an elevation of this enzyme activity, which was most rapid between 6 and 11 days and between 11 and 15 days. A similar developmental trend of the Mg^-ATPase activity was observed in the subcortical structures, but starvation was fully ineffective. The rise in the Mg"-ATPase activity of the medulla oblongata was slow but significant ( t = 5 .Y 1; p < .O1) during postnatal development. The short-term starvation induced an increase of this enzyme activity on the 15th day of postnatal life but a significant decrease of this activity on the 26th day. (See Table 2.) The Na'-K'-ATPase activity rose dramatically in the rat cerebral cortex between Days 6 and 11 and especially between Days 11 and 15 (about 53%; t = 5.65, p < .01). Starvation caused an increase of this activity on Day 11 but a diminution was observed in 26-day-old rats and adults. The developmental increment of this enzyme activity in the rat medulla oblongata was significant between Days 11 and 15 ( t = 6.82, p < .Ol) and between days 15 and 26 (t=3.10, p < .05) of postnatal life. The short-term starvation decreased the activity in adult animals, only. (See Table 3.) The results of Na'-K'-ATPase activity in the subcortical structures were quite similar to those of cerebral cortex. The activity ratio, the ratio of the Mg"-ATPase to the Na'-K'-ATPase, decreased
590
MOUREK AND STASTNY
TABLE 1. Tile Efyect of Age and Short-Term Starvation on the Total ATPusc Activitv
(z 2 S l M ) in the Various Parts of the Brain. a Cortex
Ape
- -
__
~
--
-
~
.138 t .014(5) ,174 i .005(7) t = 2.58*
.173 t .012(5) .I66 + 005(7) !,
.215 i .010(9) ,266 i .007(6) f = 3.51**
,186 * .008(9) .221 I .005(6) t = 3.04**
,215 -t .c)13(9) ,239 i .006(6) t = 1.46
,298 i .005(9) ,315 t; .011(5) f = 1.47
.241 i .005(9) .265 i .012(5) t
,252 i .006(91 ,284 i .004(5) t = 3.28**
,322 * .008(7) ,294 i .009(6) t
.360 t .004(7) ,313 t .008(6) t = 5.05**
.302 .280
i
S C S
,348 f .005(3) ,303 i .008(7) t = 3.22*
,320 i .007(3) ,286 t .009(7) t
.349 .312
f
S
C l1 S
c l5 S
A
-
Medulla Oblongata
,142 i .013(5) . I 7 3 i .006(7) t = 1.95
Ch
26
-
-
-
~
~__________
Subcortlcdl Structures
C
=
1.63
=
=
1.94
1.78
+ t
.007(7) .004(6)
=
1 =
.60
2..53*
.002(3) .008(7) t = 2.65* _____
"Activity is expressed in p.44 of inorganic orthophosphate/mg protein/min. 'C = control, S = starved, ( ) = number ofexpcriments, A = adults. * p < .05. **p < .01.
TABLE 2. The Effect of Age and Short-Term Starvation on the Mg++-Dependent ATPuse Activity 5 SKM) in the Various Parts of the Brain."
(x
Ch S
C S
Subcortical Structures
Cortex
Age
,082 i .010(5) ,093 t .004(7) I = .82
.090 i .009(5) .097 t .004(7) t = .76
. l o 2 i .010(51 ,096 i .006(7) I = .47
.I28 2 .006(9) . 1 5 6 + 011(6) / = 2 . 3 1 *
.113 i .005(9) .118 t .006(6) t = .54
.I27 -r .010(9] ,116 i 004(6) t = .h9
.I64
i .006(9) ,193 k .012(5) t
2.23*
.144 .167
i
=
i
004(9) .012(5) t = 2.15
,171 i .005(7) ,184 f .008(6) t
.195
?
.008(7)
26 S
=
1.29
.180 i .008(6) t = 1.24
.I42 .121
C A S
,188 i .003(3) . 1 8 0 i .010(7)
f =
.52
,182 i .007(3) ,172 f .007(7) t = .86
,185 i .003(3;1 ,179 t .007(7'1 t
C l5 S
C
116 + .004(9'1 166 I .004(5) t = 6.85**
"Activity is cxpressed in phi' of inorganic orthophosphate/mg protcin/min. 'C = control, S = starved, ( ) = number of experiments, A = adult. * p < .05.
**p < .01.
i f
.o05(7>1 .003(6)1 t=3.1)1*
=
.47
____
ATPASE IN THE DEVELOPING BRAIN
591
TABLE 3. The Effect of Age and Short-Term Starvation on the Na'-K 'Stimulated ATPase Activity f SEM) in the Various Parts of the Brain.a
(x
Age
Cortex
Cb S
.060 .080
t
i
.014(5) .005(7) t = 1.39
Sub cortical Structures
.048 i .010(5) .077 i .003(7) t
=
Medulla Oblongata
3.11*
.071 i .005(5) .069 i .005(7)
r = .21
.087 i .006(9) .110 i .006(6) t = 2.29*
.072 i .007(9) ,103 i .004(6) t = 3.08**
.087 i .005(9) .lo3 i .005(6) t = 2.02
C S
.133 c .004(9) .122 c .011(5) r = 1.11
.097 c .003(9) ,097 * .005(5)
.136 i .004(9) .118 t .007(5) f = 2.05
C
26 S
.150 i .007(7) .110 c .002(6) t = 4.94**
.165 t .005(7) .133 i .005(6) t = 4.09**
.159 i .006(7) .159 t .006(6) f = .16
C A S
.160 t .006(3) .123 i .005(7) t = 3.91**
.138 t .006(3) ,114 i .006(7) t = 2.05
.164 i .004(3) .133 i .005(7) t = 3.53**
C l1 S
r = 4.11
aActivity is expressed in pM of inorganic orthophosphatc/rng protein/rnin. 'C = control, S = starved, ( ) = number of experiments, A = adult.
.05. ***pp < < .01.
during the investigated developmental period. In the adult animals the value reached unity, except in the subcortical structures. Starvation caused a decrease of this ratio in 6- and 11-day-old rats but increased this value after 15 days of postnatal life.
Discussion The present study suggests that both the specific activities and total activity of ATPase increases gradually during development of the rat brain. The results are similar to those of Samson and Quinn (1967) and of Abdel-Latif et al. (1967, 1970). The comparison of Mg"-dependent ATPase and Na'-K'-stimulated ATPase activities in various parts of the rat brain revealed that the ATPase activities showed the smallest increase in the phylogenetically oldest structures and the largest in the newest structures of the brain (i.e., the cerebral cortex). Despite the gross separation of various parts of the rat brain that included functionally dissimilar structures (e.g., the subcortical structures involved the thalamus, hypothalamus, and mesencephalon) the results confirmed the biochemical heterogeneity of the developing mammalian brain. The effect of 24 hr of starvation and thirst on both enzyme activities and their activity ratios was to elevate them (after the short-term starvation) in 5- and 10-day-old rats and, in some cases, also in 14-day-old rats. Analyzing the effect of short-term starvation we can exclude an effect of the changed protein content on the increases in those activities (Mourek, 1974). However, fasting and thirst are stressors which induce the adaptive reaction characterized by an increase in the plasma level of glucocorticoids, a reaction already observed in 5-day-old rats. Glucocorticoids, which play a role
592
MOUREK AND STASTNY
in the regulation of Na'-K'-stimulated ATPase activity in the developing braln, might be responsible for the increase in ATPase activity (Siastnq, 1971). Both ATPase activities (Na'-K' and Mg+') were high in the brain of adult rats but were depressed by short-tern1 starvation and thirst. This was especially true for the activity of Na'-K'-ATPase. The decrease in the enzyme system activity may be related to the increase in the plasma concentration of Mg" induced by fasting m adult rats (Mourek & Szastnq, 1976), pointing to an alteration of membrane permeability and of Na'-K'-ATPase activity owing to its Mg" dependency. We propose that the short-term starvation was not sufficiently potent to stress the adult animals because of its similarity to conditions in nature. However, the decrement of these activities might be related to changes in metabolic substrates, for example a decrease in the plasma concentration of lipids (Hracliovina, TrojanovB, & Mourek, 1975) and to a decrease in enzyme activities synthesizing fatty acids in the rat brain (Gross & Warshaw, 1975). An alteration of phospholipid composition of cell membranes in the rat brain may be responsible for the decrease of both ATPase activities in the adult rat brain (Racker, 1974). The observed ratios of Mg"-dependent to Na'-K'-stimulated ATPase showed a developmental decrease and were near unity in adult animals. The short-term starvation influenced the ratios in young and adult animals in opposite ways. The decrease of the ratio in 5-day-old rats, induced by the starvation, was caused by an increase of the Na'-K'-ATPase activity, whereas in the adults the enzyme activity decreased and thus the activity ratio increased. The smaller changes in Mg"-ATPase activity reflect its higher resistance to acute starvation. All the observed changes in ATPase activities suggest different desrees of cell membrane maturation in various parts of the rat brain. Cell membrane rnaturation is characterized by membrane stabilization and full functional activity of ATPase molecules. The mechanism by which the Na'-K'-ATPase and Mg"-ATPase activities arc controlled is not known. We are tempted to offer the hypothesis that the intracellular ratio of Na' and K' can control the rate of synthesis and degradation of these enzyme molecules and that the enzyme activity of these molecules is probably controlled not only by the cations but also by some membrane components (e.g., by phospholipids). This hypothesis means that changes in composition of the surrounding environment can effectively control the activity of the Na'-K' pump localized in cell membranes.
References Abdel-Latif, A., Brody, J., and Rarnahi, H. (1967). Studies on sodium-potassium adenosine triphosphatase of the nerve endings and appearance of electrical activity in devcloping rat brain. J. Neurochcrn., 14:1133-1141. Abdcl-Latif, A., Smith, J. P., and Ellington, 8. P. (1970). Subcellular distribution of sodiumpotassium adenosine triphosphatase, acetylcholine and acetylcholinesterase in developins rat brain. Brain Rex, 18:441-450. Aliei, L., and Venturini, B. (1972). ATF'ase activity in the developing chick spinal cord. Brain Rcs., 43:314-316. Bowler, K., and Duncan, J. C . (1967). Evidence implicating a membrane ATPase in the control of passive permeability of excitable cells. J. Cell, Physiol., 70: 12 1-125. Ebel, H . , Wolf, J. R., and Giinther, T. (1971). Wirkung von Hormonen auf Electrolytgehalt, ATPasc und endoplasmatisches Reticulum in Rattenhirn. Z. Klin. Chem. klin. Biochern., 9x249-256.
ATPASE IN THE DEVELOPING BRAIN
593
Fiskc, C. H., and Subbarow, Y. (1925). The colorimetric determination of phosphorus. J. Biol. Chem., 66:375407. Gross, I., and Warshaw, J. B. (1975). The influence of dietary deprivation on the enzyme of fatty acid synthesis in rat brain. J. Neurochem., 25:191-192. Hrachovina, Vl., Trojanovi, M., and Mourek, J. (1974). Effect of age and fasting on the amount of pyruvic and lactic acid and total lipids in rat serum. Sbornik Ltk., 76:321-327. Hrachovina, Vl., and Mourek, J. (1976). Influence of starvation on lactic dehydrogenase activity in the serum and brain of rats of different ages. Physiol. Bohemoslov., 25:313-318. Kirkpatrick, J. B. (1969). Microtobules in brain homogenates. Science, 163:187-188. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. 3. (1951). Protein measurement with folin phenol reagent. J. Biol. Chem., 193:265-275. Mourek, J. (1972). Influence of starvation and age on aerobic metabolism in the nervous system of the rat. PhysioL Bohemoslov., 21 :473-478. Mourek, J. (1974). Effect of short-term fasting on the protein content in brain and liver of the rat during development. Sbornik Ltk., 76:97-102. Mourek, J., and Davis, J. M. (1970). Ribonucleinic acid values in the brain of rat. Relation to postnatal development and fasting. Sbornik Lkk., 72:269-275. Mourek, J., Agrawal, H. C., Davis, J. M., and Himwich, W. A. (1970). The effects of short-term starvation on amino-acid content in rat brain during ontogeny. Brain Res., 19:229-237. Mourek, J., and Loutocki, N. (1972). Effects of ouabain on the oxygen consumption by the central nervous system of the rat in the course of postnatal development. Sbornik Ltk., 74:119-124. Mourek, J., and GastnL, F. (1976). Effect of short-time fasting and hypoxia on the plasma Mg" level in the ontogenesis of the rat. Sbornik Ltk., 78:llO-116. Palladini, G., Bknami, A., Venturini, G., Maggagbani, F., Correr, S . , and Stefanelli, A. (1966). Riccerche sulla ATPai Na+-K+ dipendente nella Ontogenesi. Rend. Acc. Lincei (Ser. VIII), 40 :9 33-9 39. Racker, E. (1974). Mechanism of ATP formation in mitochondria and ion pump. In L. Ernster, R. W. Estabrook, and E. C. Slater (Eds.), Dynamics of Energy-Transducing Membranes. Amsterdam: Elsevier. Pp. 269-281. Samson, F. E., and Quinn, D. J. (1967). Na+-K+ activated ATPase in rat brain development. J. Neurochem., 14:421427. Schwartz, J. P., Mrsulja, B. B., Mrsulja, B. J., Passonneau, J. V., and Klatzo, I. (1976). Alterations of cyclic nucleotide-related enzymes and ATPase during unilateral ischemia and recirculation in gerbil cerebral cortex. J. Neurochem., 27: 101-107. Siegel, C. J., and Albers, R. W. (1970). Nucleoside triphosphate phosphohydrolases. In A. Lajtha (Ed.), Handbook of Neurochemistry. (Volume 4.). New York: Plenum Press. Pp. 13-44. ,%astnf, F. (1971). Hydrocortisone as a possible inducer of Na+-K'-ATPase in chick embryo cerebral hemispheres. Brain Res., 25:397-410. SiastnL, F., Frohlich, J., and Chmelik, V. (1969). Distribution of adenosine triphosphatase activity in the developing brain of the chick embryo. Dev. Psychobiol., 2:103-114.
During the last 10 years much attention has been devoted to correlations between the development of Na'-K'-stimulated ATPase (adenosine triphosphate phosphohydrolase, EC 3.6.1.3), which is essential for achieving the active transport of Na' and K' in cells, and some biochemical and functional parameters of various tissues, particularly in the nervous system (Abdel-Latif, Brody, & Ramahi, 1967; Palladini, Bignanii, Venturini, Maccaghani, Correr, & Stefanelli, 1966; Samson & Quinn, 1967; Siastng, Frohlich, & Chmelik, 1969). Moreover, the inducibility of this enzyme system by glucocorticoids has been demonstrated in the developing nervous system (Ebel, Wolf, & Gunther, 1971; Siastng, 1971). In 1966 Palladini and coworkers first described a developmental increase of the Na'-K' ATPase activity concurrent with an increase in the number and structure of glial cells and the appearance of electrical activity in the chicken telencephalon. Samson and Quinn (1967) and Abdel-Latif, Smith, and Ellington (1970) carried out similar studies and found a gradual increase of Na'-K' ATPase and Mg"-ATPase activities in the developing rat brain. However, the rise of the former enzyme activity continues more slowly than that of the latter. This is true also for the nervous system of chicken embryos (Alfei & Venturini, 1972). Few data on the function and developmental changes of Mg"-dependent ATPase Reprint requests should be sent to Dr. Jindfick Mourck, Department of Physiology, Charles University, Prague 2, Albertov 5 , Czechoslovakia. Received for publication 24 February 1977 Revised for publication 20 June 1977 Developmental Psychobiology, 11(6):587-593 (1978) @ 1978 by John Wiley & Sons, Inc.
00 1 2-1 6 30/78/0011-05 8 7 $0 1.OO
588
MOUREK AND STASTNY
have been published (Siege1 & Albers, 1970). Bowler and Duncan (1967) have indicated the possible influence of this enzyme on the passive permeability and on contractile proteins of the cellular membranes. The cellular localization of Mg"'dependent ATPase is different from that of Na+-K'-stimulated ATPase (Kirkpatrick, 1969). Moreover, anoxia, hypoxia, or ischemia always show a smaller effect on the Mg"-dependent ATPase activity than on the Na'-K'-stimulated ATPase (Schwartz, Mrsulja, Mrsulja, Passonneau, & Klatzo, 1976). In our previous experiments (Mourek & LoutockB, 1972) we studied the effect of ouabain, a specific inhibitor of the active transport of Na' and K' and of Na'-K'ATPase, on O2 uptake in the tissue slices isolated from different parts of the developing rat brain. We observed a developmental increase of the O2 uptake conditioned by the Na'-K'-ATPase increase in the rat cerebral cortex, but no changes in the uptake in the tissue of phylogenetically older parts of the rat brain. Also, we saw that short-term starvation and thirst during early postnatal life gave rise to a biochemical and functional retardation of the developing rat brain. The retardation was characterized by a decrease of the respiratory activity (Mourek, 1972), of free amino acids content (Mourek, Agrawal, Davis, & Himwich, 1970), and of RNA content in the rat brain (Mourek & Davis, 1970), including a decrease of some enzyme activities in plasma and brain tissue of these animals (Hrachovina & Mourek, 1976). The present work was undertaken in order to (a) describe the normal pattern of appearance of ATPase activities in different parts of tlie developing rat brain, and (b) study the effect of short-term starvation and thirst on the development of both enzyme activities in the rat brain.
Experiments were carried out on 5-, lo-, 14-, and 25-day-old rats (Rathrs norvegicus) of postnatal life (Day l-the day of birth) and on adults of both genders. Wistar rats from our own breeding colony were used. As in our previous experiments half of each standard litter, having 8 individuals in the nest, was separated from the mother and nest for 24 hr under an ambient temperature of 3OoC. Twenty-five-day-old rats and adults were deprived of food and water for the same time. After a period of 24 hr of deprivation at age 6, 11, 15, or 26 days of postnatal life the control and experimental (deprived) animals were decapitated and the heads were placed on ice. The skull and the brain membranes were opened widely and the brain was separated into cerebral cortex, subcortical structures (including thalamus and hypothalamus), and medulla oblongata. Approximately 100 mg of the individual parts were homogeniied in ice-cold .25M sucrose solution containing .005A4 ethylene diamine tetraacetic acid (EDTA-H, Serva); the pH 7.4 was adjusted with hydroxymethyl amino methane hydrochloride (Tris, Serva). Homogenates prepared under standard condition were centrifuged at 10,000 g in a Janetzki K 24 centrifuge for 10 min. The supernatant was used immediately for the ATPase assay. The total ATPase activity was measured in the medium containing 30 n1M Tris-HC1 (pH 7.4), 6 mM Mg", 20 mMK', and 100 mMNa' (as chloride salts). The Mg"-ATPase activity, which is ouabain insensitive, was determined in the same medium containing 1 mM of ouabain. The incubation media (.45 ml) were preincubated with .05 ml of
ATPASE IN THE DEVELOPING BRAIN
589
enzyme preparations for 6 m i n at 37°C. The reaction was started by adding ATP (disodium salt; Boehringer) in a final concentration of 6 mM and stopped after 10 min by addition of 1 ml of 7.5% trichloracetic acid. The solution was centrifuged at 3000 rpm for 5 min and the liberated orthophosphate was determined according to Fiske and Subbarow (1925). All ATPase assays were carried out in triplicate. The nonenzyme hydrolysis of ATP was separately estimated and released orthophosphate was subtracted from the total amount of orthophosphate released during the complete incubation. The Nat-Kt-stimulated (ouabain sensitive) and Mg"-dependent (ouabain insensitive) ATPase activity was determined in the absence or presence of ouabain and all activities were expressed as pl4 of inorganic orthophosphate (Pi)/mg protein/min. Protein was measured by the method of Lowry, Rosenbrough, Farr, and Randall (1951) and bovine serum albumine was used as a standard. The t-test for non-paired data was used for statistical evaluation of the results.
Results According to previous reports, total ATPase increases in all parts of the developing rat brain. In this study, over the period of development investigated, the increment of this enzyme activity was 249% in the cerebral cortex ( t = 10.74, p < .Ol), 228% in subcortical structures ( t = 8.85, p < .Ol), and 202% in medulla oblongata ( t = 10.19, p < .01). In all cases the increments were highly significant. The short-term starvation caused a significant increase of total ATPase activity in youngest rats in the subcortical structures (Days 6 and 11 of postnatal life) and cerebral cortex (Day 11). However, the enzyme activity decreased in adult rats (cerebral cortex; t = 3.22, p < .05) and in 26-day-old rats (subcortical structures; t = 5.05, p < .01). In the medulla oblongata the enzyme activity was unaffected by these experimental conditions in the 2 groups of youngest animals, increased in the 15-day-old and decreased in the 26-day-old and adult animals. (See Table 1.) The study of the development of Mg"-ATPase in the cerebral cortex showed an elevation of this enzyme activity, which was most rapid between 6 and 11 days and between 11 and 15 days. A similar developmental trend of the Mg^-ATPase activity was observed in the subcortical structures, but starvation was fully ineffective. The rise in the Mg"-ATPase activity of the medulla oblongata was slow but significant ( t = 5 .Y 1; p < .O1) during postnatal development. The short-term starvation induced an increase of this enzyme activity on the 15th day of postnatal life but a significant decrease of this activity on the 26th day. (See Table 2.) The Na'-K'-ATPase activity rose dramatically in the rat cerebral cortex between Days 6 and 11 and especially between Days 11 and 15 (about 53%; t = 5.65, p < .01). Starvation caused an increase of this activity on Day 11 but a diminution was observed in 26-day-old rats and adults. The developmental increment of this enzyme activity in the rat medulla oblongata was significant between Days 11 and 15 ( t = 6.82, p < .Ol) and between days 15 and 26 (t=3.10, p < .05) of postnatal life. The short-term starvation decreased the activity in adult animals, only. (See Table 3.) The results of Na'-K'-ATPase activity in the subcortical structures were quite similar to those of cerebral cortex. The activity ratio, the ratio of the Mg"-ATPase to the Na'-K'-ATPase, decreased
590
MOUREK AND STASTNY
TABLE 1. Tile Efyect of Age and Short-Term Starvation on the Total ATPusc Activitv
(z 2 S l M ) in the Various Parts of the Brain. a Cortex
Ape
- -
__
~
--
-
~
.138 t .014(5) ,174 i .005(7) t = 2.58*
.173 t .012(5) .I66 + 005(7) !,
.215 i .010(9) ,266 i .007(6) f = 3.51**
,186 * .008(9) .221 I .005(6) t = 3.04**
,215 -t .c)13(9) ,239 i .006(6) t = 1.46
,298 i .005(9) ,315 t; .011(5) f = 1.47
.241 i .005(9) .265 i .012(5) t
,252 i .006(91 ,284 i .004(5) t = 3.28**
,322 * .008(7) ,294 i .009(6) t
.360 t .004(7) ,313 t .008(6) t = 5.05**
.302 .280
i
S C S
,348 f .005(3) ,303 i .008(7) t = 3.22*
,320 i .007(3) ,286 t .009(7) t
.349 .312
f
S
C l1 S
c l5 S
A
-
Medulla Oblongata
,142 i .013(5) . I 7 3 i .006(7) t = 1.95
Ch
26
-
-
-
~
~__________
Subcortlcdl Structures
C
=
1.63
=
=
1.94
1.78
+ t
.007(7) .004(6)
=
1 =
.60
2..53*
.002(3) .008(7) t = 2.65* _____
"Activity is expressed in p.44 of inorganic orthophosphate/mg protein/min. 'C = control, S = starved, ( ) = number ofexpcriments, A = adults. * p < .05. **p < .01.
TABLE 2. The Effect of Age and Short-Term Starvation on the Mg++-Dependent ATPuse Activity 5 SKM) in the Various Parts of the Brain."
(x
Ch S
C S
Subcortical Structures
Cortex
Age
,082 i .010(5) ,093 t .004(7) I = .82
.090 i .009(5) .097 t .004(7) t = .76
. l o 2 i .010(51 ,096 i .006(7) I = .47
.I28 2 .006(9) . 1 5 6 + 011(6) / = 2 . 3 1 *
.113 i .005(9) .118 t .006(6) t = .54
.I27 -r .010(9] ,116 i 004(6) t = .h9
.I64
i .006(9) ,193 k .012(5) t
2.23*
.144 .167
i
=
i
004(9) .012(5) t = 2.15
,171 i .005(7) ,184 f .008(6) t
.195
?
.008(7)
26 S
=
1.29
.180 i .008(6) t = 1.24
.I42 .121
C A S
,188 i .003(3) . 1 8 0 i .010(7)
f =
.52
,182 i .007(3) ,172 f .007(7) t = .86
,185 i .003(3;1 ,179 t .007(7'1 t
C l5 S
C
116 + .004(9'1 166 I .004(5) t = 6.85**
"Activity is cxpressed in phi' of inorganic orthophosphate/mg protcin/min. 'C = control, S = starved, ( ) = number of experiments, A = adult. * p < .05.
**p < .01.
i f
.o05(7>1 .003(6)1 t=3.1)1*
=
.47
____
ATPASE IN THE DEVELOPING BRAIN
591
TABLE 3. The Effect of Age and Short-Term Starvation on the Na'-K 'Stimulated ATPase Activity f SEM) in the Various Parts of the Brain.a
(x
Age
Cortex
Cb S
.060 .080
t
i
.014(5) .005(7) t = 1.39
Sub cortical Structures
.048 i .010(5) .077 i .003(7) t
=
Medulla Oblongata
3.11*
.071 i .005(5) .069 i .005(7)
r = .21
.087 i .006(9) .110 i .006(6) t = 2.29*
.072 i .007(9) ,103 i .004(6) t = 3.08**
.087 i .005(9) .lo3 i .005(6) t = 2.02
C S
.133 c .004(9) .122 c .011(5) r = 1.11
.097 c .003(9) ,097 * .005(5)
.136 i .004(9) .118 t .007(5) f = 2.05
C
26 S
.150 i .007(7) .110 c .002(6) t = 4.94**
.165 t .005(7) .133 i .005(6) t = 4.09**
.159 i .006(7) .159 t .006(6) f = .16
C A S
.160 t .006(3) .123 i .005(7) t = 3.91**
.138 t .006(3) ,114 i .006(7) t = 2.05
.164 i .004(3) .133 i .005(7) t = 3.53**
C l1 S
r = 4.11
aActivity is expressed in pM of inorganic orthophosphatc/rng protein/rnin. 'C = control, S = starved, ( ) = number of experiments, A = adult.
.05. ***pp < < .01.
during the investigated developmental period. In the adult animals the value reached unity, except in the subcortical structures. Starvation caused a decrease of this ratio in 6- and 11-day-old rats but increased this value after 15 days of postnatal life.
Discussion The present study suggests that both the specific activities and total activity of ATPase increases gradually during development of the rat brain. The results are similar to those of Samson and Quinn (1967) and of Abdel-Latif et al. (1967, 1970). The comparison of Mg"-dependent ATPase and Na'-K'-stimulated ATPase activities in various parts of the rat brain revealed that the ATPase activities showed the smallest increase in the phylogenetically oldest structures and the largest in the newest structures of the brain (i.e., the cerebral cortex). Despite the gross separation of various parts of the rat brain that included functionally dissimilar structures (e.g., the subcortical structures involved the thalamus, hypothalamus, and mesencephalon) the results confirmed the biochemical heterogeneity of the developing mammalian brain. The effect of 24 hr of starvation and thirst on both enzyme activities and their activity ratios was to elevate them (after the short-term starvation) in 5- and 10-day-old rats and, in some cases, also in 14-day-old rats. Analyzing the effect of short-term starvation we can exclude an effect of the changed protein content on the increases in those activities (Mourek, 1974). However, fasting and thirst are stressors which induce the adaptive reaction characterized by an increase in the plasma level of glucocorticoids, a reaction already observed in 5-day-old rats. Glucocorticoids, which play a role
592
MOUREK AND STASTNY
in the regulation of Na'-K'-stimulated ATPase activity in the developing braln, might be responsible for the increase in ATPase activity (Siastnq, 1971). Both ATPase activities (Na'-K' and Mg+') were high in the brain of adult rats but were depressed by short-tern1 starvation and thirst. This was especially true for the activity of Na'-K'-ATPase. The decrease in the enzyme system activity may be related to the increase in the plasma concentration of Mg" induced by fasting m adult rats (Mourek & Szastnq, 1976), pointing to an alteration of membrane permeability and of Na'-K'-ATPase activity owing to its Mg" dependency. We propose that the short-term starvation was not sufficiently potent to stress the adult animals because of its similarity to conditions in nature. However, the decrement of these activities might be related to changes in metabolic substrates, for example a decrease in the plasma concentration of lipids (Hracliovina, TrojanovB, & Mourek, 1975) and to a decrease in enzyme activities synthesizing fatty acids in the rat brain (Gross & Warshaw, 1975). An alteration of phospholipid composition of cell membranes in the rat brain may be responsible for the decrease of both ATPase activities in the adult rat brain (Racker, 1974). The observed ratios of Mg"-dependent to Na'-K'-stimulated ATPase showed a developmental decrease and were near unity in adult animals. The short-term starvation influenced the ratios in young and adult animals in opposite ways. The decrease of the ratio in 5-day-old rats, induced by the starvation, was caused by an increase of the Na'-K'-ATPase activity, whereas in the adults the enzyme activity decreased and thus the activity ratio increased. The smaller changes in Mg"-ATPase activity reflect its higher resistance to acute starvation. All the observed changes in ATPase activities suggest different desrees of cell membrane maturation in various parts of the rat brain. Cell membrane rnaturation is characterized by membrane stabilization and full functional activity of ATPase molecules. The mechanism by which the Na'-K'-ATPase and Mg"-ATPase activities arc controlled is not known. We are tempted to offer the hypothesis that the intracellular ratio of Na' and K' can control the rate of synthesis and degradation of these enzyme molecules and that the enzyme activity of these molecules is probably controlled not only by the cations but also by some membrane components (e.g., by phospholipids). This hypothesis means that changes in composition of the surrounding environment can effectively control the activity of the Na'-K' pump localized in cell membranes.
References Abdel-Latif, A., Brody, J., and Rarnahi, H. (1967). Studies on sodium-potassium adenosine triphosphatase of the nerve endings and appearance of electrical activity in devcloping rat brain. J. Neurochcrn., 14:1133-1141. Abdcl-Latif, A., Smith, J. P., and Ellington, 8. P. (1970). Subcellular distribution of sodiumpotassium adenosine triphosphatase, acetylcholine and acetylcholinesterase in developins rat brain. Brain Rex, 18:441-450. Aliei, L., and Venturini, B. (1972). ATF'ase activity in the developing chick spinal cord. Brain Rcs., 43:314-316. Bowler, K., and Duncan, J. C . (1967). Evidence implicating a membrane ATPase in the control of passive permeability of excitable cells. J. Cell, Physiol., 70: 12 1-125. Ebel, H . , Wolf, J. R., and Giinther, T. (1971). Wirkung von Hormonen auf Electrolytgehalt, ATPasc und endoplasmatisches Reticulum in Rattenhirn. Z. Klin. Chem. klin. Biochern., 9x249-256.
ATPASE IN THE DEVELOPING BRAIN
593
Fiskc, C. H., and Subbarow, Y. (1925). The colorimetric determination of phosphorus. J. Biol. Chem., 66:375407. Gross, I., and Warshaw, J. B. (1975). The influence of dietary deprivation on the enzyme of fatty acid synthesis in rat brain. J. Neurochem., 25:191-192. Hrachovina, Vl., Trojanovi, M., and Mourek, J. (1974). Effect of age and fasting on the amount of pyruvic and lactic acid and total lipids in rat serum. Sbornik Ltk., 76:321-327. Hrachovina, Vl., and Mourek, J. (1976). Influence of starvation on lactic dehydrogenase activity in the serum and brain of rats of different ages. Physiol. Bohemoslov., 25:313-318. Kirkpatrick, J. B. (1969). Microtobules in brain homogenates. Science, 163:187-188. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. 3. (1951). Protein measurement with folin phenol reagent. J. Biol. Chem., 193:265-275. Mourek, J. (1972). Influence of starvation and age on aerobic metabolism in the nervous system of the rat. PhysioL Bohemoslov., 21 :473-478. Mourek, J. (1974). Effect of short-term fasting on the protein content in brain and liver of the rat during development. Sbornik Ltk., 76:97-102. Mourek, J., and Davis, J. M. (1970). Ribonucleinic acid values in the brain of rat. Relation to postnatal development and fasting. Sbornik Lkk., 72:269-275. Mourek, J., Agrawal, H. C., Davis, J. M., and Himwich, W. A. (1970). The effects of short-term starvation on amino-acid content in rat brain during ontogeny. Brain Res., 19:229-237. Mourek, J., and Loutocki, N. (1972). Effects of ouabain on the oxygen consumption by the central nervous system of the rat in the course of postnatal development. Sbornik Ltk., 74:119-124. Mourek, J., and GastnL, F. (1976). Effect of short-time fasting and hypoxia on the plasma Mg" level in the ontogenesis of the rat. Sbornik Ltk., 78:llO-116. Palladini, G., Bknami, A., Venturini, G., Maggagbani, F., Correr, S . , and Stefanelli, A. (1966). Riccerche sulla ATPai Na+-K+ dipendente nella Ontogenesi. Rend. Acc. Lincei (Ser. VIII), 40 :9 33-9 39. Racker, E. (1974). Mechanism of ATP formation in mitochondria and ion pump. In L. Ernster, R. W. Estabrook, and E. C. Slater (Eds.), Dynamics of Energy-Transducing Membranes. Amsterdam: Elsevier. Pp. 269-281. Samson, F. E., and Quinn, D. J. (1967). Na+-K+ activated ATPase in rat brain development. J. Neurochem., 14:421427. Schwartz, J. P., Mrsulja, B. B., Mrsulja, B. J., Passonneau, J. V., and Klatzo, I. (1976). Alterations of cyclic nucleotide-related enzymes and ATPase during unilateral ischemia and recirculation in gerbil cerebral cortex. J. Neurochem., 27: 101-107. Siegel, C. J., and Albers, R. W. (1970). Nucleoside triphosphate phosphohydrolases. In A. Lajtha (Ed.), Handbook of Neurochemistry. (Volume 4.). New York: Plenum Press. Pp. 13-44. ,%astnf, F. (1971). Hydrocortisone as a possible inducer of Na+-K'-ATPase in chick embryo cerebral hemispheres. Brain Res., 25:397-410. SiastnL, F., Frohlich, J., and Chmelik, V. (1969). Distribution of adenosine triphosphatase activity in the developing brain of the chick embryo. Dev. Psychobiol., 2:103-114.
