GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
86, 162-169 (1992)
Alterations of Ion-Dependent ATPase Activities in the Brain of Singi Fish; Heteropneustes fossilis (Bloch), by Triiodothyronine SRABANI DE, A.K. Department
DASMAHAPATRA,AND
A. K. MEDDA
of Animal Physiology, Bose Institute, P-1112 CIT Scheme VII-M, Calcutta-700054, India Accepted July 23, 1991
The activities of Na+K+- and Mg2+ -ATPases in mitochondrial, microsomal, and cytosolic fractions of Singi fish (Heteropneustes fossilis Bloch) brain were investigated after injections of various doses (0.012, 0.025, 0.05, and 0.10 p,g/g) of triiodothyronine (T,) for 3 consecutive days. Both ATPases were found in the mitochondrial and microsomal fractions. The cytosolic fraction showed only Mg ‘+-ATPase activity. Mitochondrial Na+K+-ATPase activity increased to almost the same level in fish treated with 0.02.5,0.05, or 0.10 (*g of T,/g, while the T3 dose of 0.012 kg/g was ineffective in this respect. Microsomal Na+K+-ATPase activity increased to about the same level with all of the doses of Ts used. No detectable amount of Na+K+-ATPase was found in the brain cytosolic fraction. Mitochondrial Mg’+ATPase activity was enhanced with 0.025,0.05, and 0.10 p,g of T,/g. The last dose, however, produced a higher increase in activity than the other two doses. Surprisingly, microsomal and cytosolic Mg ‘+-ATPase activity was not increased by T, treatment. Although T, concentrations rose sharply after each T3 injection, the serum T, level in T,-injected fish was not different from that in the control as observed on the fourth day. The TX-induced rise of Na+K+- and MgZf-ATPase activities was inhibited by cycloheximide treatment. Immersion of Singi fishes in thiourea significantly reduced brain Na+K+-ATPase activity in microsomal and mitochondrial fractions but decreased Mg*+-ATPase activity only in the mitochondrial fraction. Three consecutive daily injections of T, (0.10 l&g) into the thioureatreated fishes increased their ATPase activities even beyond the control level. Our results thus indicate that T, has a controlling influence on the ATPase system in fish brain. 0 1992 Academic
Press. Inc.
Previous reports from our laboratory have provided evidence for the responsiveness of fish brain to thyroid hormone with respect to changes in protein, nucleic acid, glycogen, and lipid metabolism and activities of some specific enzymes like mitochondrial cx-glycerophosphate dehydrogenase and cytosolic malic enzyme (Ghosh and Medda, 1982, 1984; Ghosh et al., 1983; Medda and Ghosh, 1984; De et al., 1989). In higher animals, the presence of putative triiodothyronine receptors in neuronal nuclei of adult brain indicates that it may be a site of thyroid hormone action (Haider et al., 1983; Yokota et al., 1986; Gull0 et al., 1987). There are also reports that thyroid hormone status may control neurotransmitter levels (Jacoby et al., 1975; Ito et al.,
1977; Savard et al., 1983), B-adrenoceptor concentrations (Gross et al., 1980), and RNA polymerase I activity in neural nuclei (Nakamura et al., 1987) of adult rat brain. Triidothyronine (T3) stimulates leucine incorporation into TCA-precipitable material in primary cell cultures of neurons of rat brain in a dose-dependent manner (Pickard et al., 1987). The brain of nonmammalian vertebrates also possesses T, receptors in nerve cell nuclei (Haider et al., 1983; Bres and Eales, 1988). All these reports clearly indicate that both nonmammalian and mammalian adult brains undergo changes in intermediary metabolism under the influence of thyroid hormone. In our continuing investigations of the action of thyroid hormone in fish brain, we 162
0016-6480/92 $1.50 Copyright Q 1592 by Academic Press, Inc. All rights of reproduction in any form reserved.
T, ACTION
ON FISH BRAIN ATPase
attempted in the present experiments to evaluate the changes in the activities of ionspecific ATPases (Na+K+ and Mg2+ dependent) in subcellular fractions of the brain of Singi fish, Heteropneustes fossilis (Bloch), treated with different doses of T,. MATERIALS
AND METHODS
Treatment of the animals. Singi fish (H. fossilis Bloch) of 10-14 g body wt were purchased from a local supplier and acclimatized to laboratory conditions at 25 + B” for 4 days before our experiments. They were fed once daily ad iibitum with Tubifex tubifex. The animals were randomly divided into different groups and were kept in tap water in polythene trays. L-Triiodothyronine (T,, free acid, Sigma Chemical Co.) was dissolved in an alkaline 0.65% sodium chloride solution (pH 8-9). Various doses (0.012, 0.025, 0.05, and 0.10 &g/g of body wt) of T, were injected intraperitoneally for 3 consecutive days. The volume of the injected material did not exceed 35 )*I in any case. Control fish were injected with the same volume of alkaline saline only. The fish were fasted during the experimental period and kept at 25 + 1”. They were killed for biochemical analyses on Day 4. Some fish were immersed in thiourea-containing water (0.1%) for 30 days at 25” to make them hypothyroid as described previously (Ray and Medda, 1972). Corresponding control fish were maintained in simple tap water. These fish were fed during this period. After 30 days of treatment some of the fish from the thioureatreated and control groups were injected with T, (0.1 kg/g) on 3 consecutive days (Days 31, 32, and 33). Thiourea treatment was discontinued on Day 3 1. All of these fish were killed for biochemical analyses on Day 35. In another set of experiments, fish were maintained in cycloheximide-containing water (0.5 mglliter). Some of them were injected with T, (0.1 pg/g) on 3 consecutive days and were killed for analysis on the fourth day. The untreated control and TX-injected fish were maintained in tap water and sampled on the same day. The brains from different groups of animals were quickly dissected out on ice. Three brains were pooled together, weighed, and homogenized in isolation buffer (0.32 M sucrose, 10 mM Tris-HCl, 1 mM EDTA, and 5 m&f 2-mercaptoethanol, pH 7.4) at O-4” using a glass-Teflon homogenizer. The homogenate was centrifuged at 700g for 10 minutes to remove the nuclei and cell debris at o-4”. The supematant was further centrifuged at 16,000g for 20 min, and the precipitate was used as a crude mitochondrial fraction.
The supematant was saved for the isolation, of nicrosomes. The crude mitochondrial fraction was resuspended in isolation buffer and centrifuged again at 16,000g for 20 min to obtain purified mitochondria. Finally, the purified mitochondrial pellet was resnspended in 2 ml of isolation buffer and retained. for ATPase assay. The postmitochondrial supematant was further centrifuged in 105,OOOgfor 1 hr to obtain a crude dmicrosomal pellet. The pellet was resuspended in 7 ml isolation buffer and recentrifuged at 105,OOOg for t hr to obtain purified microsomes. The pellet was resuspended in 2 ml isolation buffer and nsed for ATPase assay. The first postmicrosomal supernatant was saved as the cytosolic fraction used for ATPase assay, Enzyme assay method. The assay of Na’K+ATPase activity followed the method of Xsmailand Edelman (1971) with some modifications. In brief, the final reaction mixture in 1.5 ml volume contained 50 mM Tris, 1 a EDTA, 3 ri04 MgCl,, 3 mW ATP* 100 mM NaCi, and 30 m&f KC1, pH 7.4. Ouabain, a specific inhibitor of Na”K+-ATPase activity, was used as a concentration of 1 x 10e3 M in some of p&e tubes as a control. The reaction mixture was preincubated for 10 min at 37” in a water bath. The reaction was started by the addition of ATP (c&odium salt) and was terminated by adding 1 ml of 10% TCA. The tubes were then transferred to an ice water bath and left far 15 min, after wbicb time the samples were ce~tr~foged at 1OOOgfor 5 min. The supernatant was saved and used for the determination of inorganic phosphorous (Chen et al., 1956). The protein content was determined following the method of Lowry e: nl. (1951). Na+K’-ATPase activity was calculated from the difference in phosphorous liberation in the presence and absence of ouabain and expressed as pmol of Pi x mg protein ’ x hr-‘. Iv&‘+-ATPase w mined in the presence of ouabain (I NaCl (100 rr&f) and KC1 (30 m&R) from the reaction mixture. The data were statistically analyzed by analysis of variance followed by Duncan’s multiple range test. Radioimmunoassay
(RIA)
of serum
TX. Singi
fisfi
were injected daily (intraperitoneally) for 3 days with different doses of T3 (0.012,0.025,O.Q5, and 0.1 pg/g). The blood from the control and treated fish was taken; from the cardinal vein very carefully on the fourth day after the first injection and allowed to clot to obtain the serum that was used for the determination of tbe T, level using RIA kits purchased from the Bhabha Atomic Research Centre, Bombay, India. In another set of experiments, 48 fish were injected with T, at the dose of 0. I kg/g at 0, 24, and %& hr and bled at various intervals between 1 to 96 hr. Each fish: was bled only once during the entire period of the experiment. The serum T, level was estimated by RIA as mentioned earlier.
164
DE,
DASMAHAPATRA,
RESULTS Effect ATPases
of T3 on Na+K+-
and Mg2+-
activities. The variance analyses of the data of the activities of NaiKfand Mg2 -t -ATPase in the subcellular fractions of brain of Singi fish treated with different doses of T, (0.012, 0.025, 0.05, and 0.1 pg/ g) are shown in Table 1. It was evident that T, significantly altered the activity of brain NafKf-ATPase in mitochondrial and microsomal fractions and Mg2+-ATPase in the mitochondrial fraction. No such changes in the microsomal and cytosolic 2t-ATPase activity after T, injections W were observed. Brain cytosolic fraction from normal Singi fish did not show any Na+K+-ATPase activity. Na+K+-ATPase activity in the brain mitochondrial fraction was increased in fish treated with 0.025 pg of T,/g in comparison to controls (P < 0.05). The higher doses of T, (0.05 and 0.10 l&g) caused about the same extent of increases (P < 0.05 or 0.01) in activity (Table 1). The lowest dose of T, (0.012 pg/g) was ineffective in inducing any change in mitochondrial Na + K + -ATPase activity. Microsomal Na+K+-ATPase activity also increased (P < 0.05 or 0.01) to
EFFECT
AND
MEDDA
about the same level with all of the doses of T, used in comparison to the control (Table 1). Mitochondrial Mg2+-ATPase activity was also enhanced in fish treated with 0.025 pg of T,/g (P < 0.05). Almost the same level of increase in activity was observed in fish treated with 0.05 kg of T,/g (Table 1). A clean dose-response relationship was not apparent at the lower T, doses (0.025 and 0.05 pg/g). More enhancement of Mg2+ATPase activity occurred in fish treated with 0.10 pg of T3/g in comparison to that observed in fish treated with 0.025 or 0.05 pg of T,/g (P c 0.05 or 0.01). The lower dose of 0.012 pg of T,/g was ineffective in elevating brain Mg2+-ATPase activity. The microsomal and cytosolic Mg2+-ATPase activity of Singi fish brain was not altered by T, treatment at the doses used. Effect of cycloheximide on T,-induced changes in Na+ Kt - and Mg” -ATPases activities. As already mentioned, T3 in-
creased Na+K+-ATPase activity in both mitochondrial and microsomal fractions and Mg2+-ATPase activity in the mitochondrial fraction, but when T, (0.10 pg/g) was injected for 3 consecutive days with cycloheximide simultaneously, it did not cause
TABLE 1 OF T, ON THE Na+K’AND Mg ‘+-ATPase ACTIVITIES OF DIFFERENT SUBCELLULAR THE BRAIN OF SINGI FISH, Heteropneustes fossilis (Bloch)
Name of the ATPase Na+K+-ATPase M2 + -ATPase
Subcellular fractions Mitochondria Microsome Mitochondria Microsome Cytosol
FRACTIONS
OF
Dose of T3 Wg) Control 17.91 20.73 16.06 36.85 16.82
k f k f f
0.012 1.84 2.09 2.41 4.76 3.05
21.83 27.56” 19.40 34.43 12.83
l?r 2.30 -r- 1.86 i 1.61 f 4.23 2 1.69
0.025 24.77a 27.53a 24.59” 34.49 14.11
f f i f *
0.05 Mean f SE 2.17 25.66a 1.68 29.67” 1.75 27.43ab 1.56 32.65 1.15 14.91
+ f * f f
0.10 1.71 2.22 3.67 3.62 2.31
27.07” 31.46” 36.82abcd 33.15 14.52
EMS ” f f f 2
1.71 2.78 3.14 3.10 0.96
37.37* 37.56* 55.77** 105.33 24.02
Note. SE, standard error. ATPase activity was expressed as pmol of Pi X mg protein-’ x hr-‘. EMS, error mean square obtained from ANOVA (df = 35). The animals were maintained at 25” and various doses of T3 were injected for 3 consecutive days. The fish were sacrificed on the fourth day, with the day of the first injection considered zero day. The brains from three fish were pooled in each set and the data of eight such pooled sets were expressed as means k SE. The data were statistically analyzed by ANOVA followed by Duncan’s multiple range test. P < 0.05 considered as significant. Superscripts a, b, c, and d indicate differences relative to control, 0.012, 0.025, and 0.05 pg x g -’ of Ts-injected fish, respectively. * Significance of F at 5% level. ** Significance of F at 1% level.
T, ACTION
ON
FISH
BRAIN
TABLE
ATPase
i65
2
EFFECT OF CYCLOHEXIMIDE ON T,-INDUCED CHANGES IN Na+K+AND Mgzf-ATPase MITOCHONDRIAL AND Na+K+-ATPase ACTIVITY IN MICROSOMAL FRACTIONS OF BRAIN Heteropneustes fossilis @loch) Subcellular fractions
Cycloheximide ATPases
Control
(0.1 p&
g-1)
(0.5 mg X liter-‘)
ACTIVITIES IN OF SINGI FISH,
T, f cycloheximide
EMS
Mean t SE Mitochondria
Na+K+-ATPase Mg’ + -ATPase Na+ K + -ATPase
Microsome
19.40 f 1.70 18.89 + 1.34
26.83"
26.72
34.82"
f
1.06
f
1.97
19.36b 21.94b 24.3?
29.81” f 2.87 k 2.66
+ 2.08 ? 2.20 -+ 2.32
18.92b 19.46b 26.93b
k 1.68 r 2.26 + 2.52
27.89* 39X8** 40.12**
?Gte. SE, standard error. ATPase activities were expressed as pmol of Pi X mg protein-’ x hr-‘. EMS, error mean square obtained from ANOVA (df = 28). The animals were maintained at 25” and various doses of T, were injected for 3 consecutive days. The fish were sacrificed the fourth day, with the day of the first injection considered zero day. The brains from three fishes were pooled in each set the data of eight such pooled sets were expressed as means rt SE. The data were statisticaliy analyzed by ANOVA followed Duncan’s multiple range test. P < 0.5 considered as significant. Superscripts a and b indicate differences relative to control P-a-injected fish, respectively. * Significance of F at 5% level. ** Significance of F at 1% level.
any increase in mitochondrial and microsoma1 Na+K+-ATPase activity or in mitochondrial Mg2+-ATPase activity in comarison to controls (Table 2). No significant aherations of ATPase activities were observed in fish treated with only cycloheximide. Effect of thiourea on NafKiand Mgzi-ATPase activities. Treatment of Singi fish with thiourea caused a reduction in the Na+K’-ATPase activity in both the
mitochondrial and microsomal fractions and in Mg2+ -ATPase activity in the n&ochondrial fraction in comparison to tive controls (Table 3). Injection o reated fish caused enha -ATPase activity above trol level in both the mitocho~dr~ 0.01) and microsomal (P < 0.01) fra~t~~~s~ Also, mitochondrial Mg2+-ATPase a~t~~~t~ in thiourea-treated fish was similarly ktcreased by T, treatment (P < 0.01) but mi-
TABLE EFFECT
OF THIOUREA
FRACTIONS
Subcellular fractions Mitochondria Microsome
BY BRAIN
ON Na+K”OF SINGI
ATPases Na+K+-ATPases Mg’+-ATPases Na+K’-ATPases Mg*+-ATPases
on aad by and
3
AND Mg’+- ATPase ACTIVITIES IN MITOCHONDRIAL AND MICXO~OMAL FISH, Heteropneustes fossilis (Bloch), AND SUBSEQUENT RECOVERY BY T, INJECTIONS
Control 18.69 20.89 19.99 31.16
? k C &
1.58 1.71 2.07 2.73
T3 (0.1 I% x s-‘1 26.58” 31.86” 33.68” 30.24
2 + I L
Mean 2.13 2.75 2.83 2.78
Thiourea
T3 + thiourea
* SE 12.02”b -c 1.09 24.2Y” +- 2.25 14.7gab ” 0.85 34.25” I 2.37 12.16ab k 2.11 33.94”’ I 2.32 26.14 2 1.87 29.63 i 2.45
EMS 26.68”” 33.48** 43.53”* $I.%?
IVote. SE, standard error. ATPase activity was expressed as urn01 of Pi x mg protein-’ x h-‘. EMS, error mean square obtained from ANOVA (df = 28). The animals were kept at 25”, immersed in thiourea (1 mg/mI) for 30 days, then transferred to tap water and injected with T, (0.1 pg/g) for 3 consecutive days (Days 31, 32, and 33) and sacrificed on Day 35 (frrst day of thiourea immersion was taken as zero day). The brains from three fishes were pooled in each set and the data of eight such pooled sets were expressed by means i SE. The data were statistically analyzed by ANOVA followed by Duncan’s multiple range test. P < 0.05 considered as significant. Superscripts a, b, and c indicate differences relative to control, T,, and thiourea-treated fish, respectively. ** Significance of F at 1% level.
166
DE,
DASMAHAPATRA,
AND
TABLE
MEDDA
4
MEAN VALUES OF SERUM T, LEVELS OBTAINED AFTER INJECTIONS OF DIFFERENT DOSES OF T, FOR 3 CONSECUTIVE DAYS
Serum T, (n&-4
Control
0.012
0.025
0.05
0.1
EMS
3.54 kO.5.5
4.51 e-o.51
Mean + SE 3.33 k0.29
4.20 -+0.85
4.10 20.73
56.23“
Note. The animals were kept at 2.5”and bled on the fourth day after the first T, injection. Each group consisted of six animals. SE, standard error. EMS, error mean squares, obtained from variance analysis (df = 25). u Not significant.
crosomal Mg2 + -ATPase remained unchanged after thiourea treatment as well as after T, injection in thiourea-treated fish. Serum T3 level in T,-injected Singi fish. The serum T, level of control Singi fish was 3.54 k 0.55 rig/ml (n = 6). No significant difference was observed in the blood T3 level between the control and T,-treated fish (0.012, 0.025, 0.05, and 0.10 pglg, 3 consecutive days injections) on the fourth day (Table 4). The distribution patterns of T, in Singi fish serum from 1 to 96 hr during
5
10
15
20
25
30
35
40
45
and after T, injection (0.1 pg/g, 3 consecutive days) are represented in Fig. 1. It was observed that serum T, reached the maximum level within l-2 hr postinjection (about 2.5- to 3-fold higher in comparison to the control level) and came down within 8-16 hr, tllz ranged from 3 to 7 hours, depending upon the day of treatments. DISCUSSION
Our results show that the thyroid
50 55 HOURS
60
65
70
75
80
85
90
95
100
FIG. 1. Effect of T3 injection (0.1 cl.g/g, 3 consecutive days) on the serum T, level of Singi fish, Heteropneustes fossilis (Bloch). Fish were injected at 0, 24, and 48 hr. Blood was drawn from the cardinal vein of the fish for different time periods mentioned in the abscissa. Only two fish were bled each time, and each fish was bled only once during the entire experimental period. The broken line represents the mean serum T3 level (n = 6, standard error = 0.54) obtained from control fish. The straight line represents treated data. Each point in the treated curve is the mean of two fish where individual variation is about 20%.
hor-
T, ACTION
ON
FISH
mone-induced metabolic alterations in fish brain include stimulation of ion-dependent ATPases, viz. Na+K’and Mg2+-ATPase. As already pointed out, certain enzymes, such as mitochondrial ol-glycerophosphate dehydrogenase and NADP-dependent cytosolic malic dehydrogenase, also show enhanced activities in response to T, treatment (Ghosh et al., 1983; De et al., 1989). The present experimental results indicate that NafKf-ATPase activity in brain mitochondrial and microsomal fractions significantly increases after 3 consecutive days of injections of T,. The Mg2+-ATPase activity of the mitochondrial fraction, not of microsomal or cytosolic fraction, was also enhanced. The brain cytosolic fraction showed no detectable NatKf-ATPase activity, although it contained a considerable amount of Mg2+ -ATPase. The microsomal ATPases activities were found to be higher than the mitochondrial ATPases, and the microsomal Na+K+-ATPase appears to be more responsive to T, treatment than the mitochondrial ATPase, as evidenced from the enhanced activity with the lowest dose of T, used (0.012 pg/g). This lowest dose of T, was found to be ineffective in causing any change in Na+K+-ATPase activity in the mitochondrial fraction. The changes in different ion-dependent ATPases in crude homogenate or in membrane fractions of liver, kidney, and skeletal muscle of thyroid hormone-treated mammalian and nonmammalian vertebrates have been reported by other workers (Tobin et al., 1979a,b; Guernsey and Edelman, 1983; De et aE., 1987).
The increase in the activities of iondependent ATPases is presumably due to an increased synthesis of these enzymes, which may be a reflection of the action of T, primarily at the nuclear level. This is ported by an observation that cycloheximide treatment inhibited the stimulation of Singi fish brain Na+K+and Mg2+ATPases by T,. We found that mitochondrial protein was also increased by T,, and
B&UN
ATPase
167
in contrast to this, no change in microsomal protein was detectable, although microsoma1 Na+M+-ATPase activity was enhanced by T,. T,-induced increased synthesis of the enzyme has also been indicated by increased incorporation of [3H]- or [35S],methionine in an a-subunit of ATPase of rat kidney at ter administration of T, ( and Lo, 1980). Moreover~ increas tion or decreased degradation of ATPase in T,-treated rat liver, m kidney has been suggested b binding of ]3H]ouabain in Na (Lin and Akera, 1978). The presence of a putative thyroid hormone-binding receptor and the binding of ‘251-labeled T, in the nuclear fraction of Singi fish brain have been mahapatra et a/., 1991). S with the brain nuclear re multaneous increase in a! al., 1983), malic enzyme ( and ATPases activities m clear activation by T, for the st~rn~~~t~o~ of Na+K+and Mg”+-ATPases in Singi fish brain. Similarly, there ar
crease in c&PI> activity, 0, consumption, and Nat K f -ATPase activity ~~srna~~-~e~~~ and Edelman, 1974; Oppenheimer et a!., 1978; Oppenheimer, 1983), w suggest a rmclear activation for the stimulation of Na+K+-ATPase. It has baby suge increase in the Ma+ or K’ pump units and the t Na+ across the nuclear membr T, treatment, as shown in rat 11 sey and Edelman, 1983), may be one of the causes for the T,-induced ~~ti~~at~~~ of Na+K’-ATPase in fish liver ( 1987). This mechanism may be mitochondria and microsomes o as found in the present experi regard to the T, t~mu~atio~ of Na 4 K + ATPase activity. noted that Na+K+-ATPase not be de-
168
DE,
DASMAHAPATRA,
tected yet in the nuclear membrane of Singi fish brain (unpublished observation). The T,-induced enhancement of Mg2+-ATPase activity in the mitochondrial fraction may be due to the increased transport of Mg2+ ions across the mitochondrial membrane. A difference was noted regarding the responsiveness of Mg2+-ATPase to T, between the mitochondrial and microsomal fraction; the former showed enhanced activity while the latter did not show any change in Mg2+ATPase activity in response to T, injection. That thyroid hormone is responsible for the activation of Na+K+and Mg2+ATPases in Singi fish brain has been further documented by a significant fall in the activities of these ATPases in the thioureatreated Singi fish. The Mg2+-ATPase activity in the brain microsomal fraction did not alter in thiourea-treated fish. Injections of T, (0.1 pg/g) in thiourea-treated fish raised the Na+K+and Mg2+-ATPase activities in the mitochondrial fraction and only Na+K+-ATPase activity in the microsomal fraction to a level higher than the control value. It appears that the decrease in ATPases activities in thiourea-treated fish is due to the inadequacy of the endogenous circulating thyroid hormone since we have detected in some representative experiments that reduction of serum T, and T, levels occurs in Singi fish after 15 days of immersion in thiourea (unpublished). A similar reduction in ion-dependent ATPases in hypothyroid or thyroidectomized rats has been reported by other investigators (Herd, 1978; Somjen et al., 1981; Tobin et al., 1979a). It also deserves mention that a reduction in the mitochondrial a-GPD activity occurs in the brain of thiourea-treated Singi fish and that this decrease could be restored even beyond the control level by T, treatment (Medda and Ghosh, 1984). Similar changes have also been noted in the case of malic dehydrogenase activity in Singi fish brain in a hypothyroid condition with and without T, treatment (unpublished observation).
AND
MEDDA
Thus it appears that the thyroid hormone exerts its regulatory influence on fish brain through induction of a number of enzyme systems as found in other thyroid hormone target organs. REFERENCES Bres, O., and Eales, J. G. (1988). High affinity, limited capacity triiodothyronine binding sites in nuclei from various tissues of the rainbow trout (Salvo gairdneri). Gen. Comp. Endochrinol. 69, 71-79. Chen, P. S., Toribara, T. Y., and Warner H. (1956). Microdetection of phosphorous. Anal. Chem. 28, 17561758. Dasmahapatra, A. K., De, S., and Medda, A. K. (1991). Demonstration of putative thyroid hormone receptor in the brain nuclei of Singi fish, Heteropneustes fossifis (Bloch). Gen. Comp. Endocrinol.
82, 60-68.
De, S., Dasmahapatra, A. K., Ray, A. K., and Medda, A. K. (1989). Induction of NADP dependent malic dehydrogenase activity in brain of Singi fish, (Heteropneustes fossilis Bloch) by 3,5,3’ triiodothyronine. Neurochem. Znt. 14,261266. De, S., Ray, A. K., and Medda, A. K. (1987). Nuclear activation by thyroid hormone in liver of Singi fish: Changes in different ion-specific adenosine triphosphatase activities. Horm. Metab. Res. 19, 367-370. Ghosh, R. K., Ghosh, N., De, S., Ray, A. K., and Medda, A. K. (1983). Effect of L-triiodothyronine on the mitochondrial a-glycerophosphate dehydrogenase activity, mitochondrial and total protein contents of brain of Singi fish (Heteropneustes fossilis Bloch). Neurochem. Znt. 5, 635640. Ghosh, R. K., and Medda, A. K. (1982). Effect ofthyroxine on protein and nucleic acid contents of different parts of brain of Singi fish (Heteropneustes fossilis Bloch). Endocrinology 79, 355-361. Ghosh, R. K., andMedda, A. K. (1984). Effect ofthyroxine and thiourea on cholesterol, total lipids and glycogen contents of brain of Singi fish (Heteropneustes fossilis Bloch). Neurochem. Znt. 6, 97101. Gross, G., Brodde, 0. E., and Schuemann, H. J. (1980). Decreased number of B-adrenoceptors in cerebral cortex of hypothyroid rats. Eur. J. Pharmacof.
61, 191-194.
Guernesy, D. L., and Edelman, I. S. (1983). Regulation of thermogenesis by thyroid hormones. In “Molecular Basis of Thyroid Hormone Action” (J. H. Oppenheimer and H. H. Samuels, Eds.), pp. 293-324, Academic Press, New York.
T, ACTION
ON FISH BRAIN ATPase
Gullo, D., Sinha, A. K., Bashir, A., Hubank, M., and Ekins, R. P. (1987). Differences in nuclear triiodothyronine binding in rat brain cells suggest phylogenetic specialization of neuronal functions. Endocrinology 120, 2398-2403. Haider, M. A., Dube, S., and Sarkar, P. K. (1983). Thyroid hormone receptors of developing chick brain are predominantly in the neurones. Bio&em. Biophys. Res. Commun. 112, 221-227. Herd, P. A. (1978). Thyroid hormone divalent cation interactions. Arch. Biochem. Biophys. 188, 220225. Ismail-Beigi, F., and Edelman, I. S. (1971). The mechanisms of the calorigenic action of thyroid hormone: Stimulation of Na+K+ activated adenosine triphosphatase activity. .I. Gen. Physiol. 51, 710-722. Ismail-Beigi, F., and Edelman, I. S. (1974). Time course of the effects of thyroid hormone on respiration and Na+K+-ATPase activity in rat liver. Proc. Sot. Exp. Bioi. Med. 146, 983-988. Ito, J. M., Valcana, T., and Timiras, P. S. (1977). Effect of hypo- and hyperthyroidism on regional monoamine metabolism in the adult rat brain. Neuroendocrinology 24, 55-64. Jacoby, J. H., Muller, G., and Wurtman, R. .I. (1975). Thyroid state and brain monoamine metabolism. Endocrinology 97, 1332-1335. Lin, M. II., and Akera, T. (1978). Increased (Na+K+)-ATPase concentrations in various tissues ofrats caused by thyroid hormone treatment. 3. Biol. Chem. 253, 723-726. Lo, C. S., August, T. R., Liberman, U. A., and Edelman, I. S. (1976). Dependence of renal cortical (Na+K+)-adenosine triphosphatase. .I. Biol. Chem. 251, 7834-7840. Lo, G. S., and Lo, T. N. (1980). Effect of triiodothyronine on the synthesis and degradation of the small subunit of renal cortical (Na+K+)adenosine triphosphatase. J. Biol. Chem. 255, 2131-2136. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the fohn phenol reagent. J. Biol. Chem. 193, 265275. Medda, A. K., and Ghosh, R. K. (1984). Inhibitory influence of thiourea on brain of Singi fish (Heteropneustes fossilis Bloch) and subsequent recovery by L-triiodothyronine. Neurochem. Int. 6, 527-532.
169
Nakamura, II., Yokota, T., Akamizu, T., Mori, T., and Imura, H. (1987). Triiodothyronine effects on RNA polymerase activities in isolated neuronaI and glid nuclei of the mature rat brain cortex. Metnb. Clin. Exp. 36, 931-934. Oppenheimer, .I. II. (1983). The nuclear receptor triiodothyronine complex: Relationship to thyroid hormone distribution, metabolism and biological action: In “Molecular Basis of Thyroi.d Hormone Action” (J. H. Oppenheimer and H. II. Samuels, Eds.), pp. l-34. Academic Press, New York. Oppenheimer, J. H., Coulombe, P., Schwartz, H. L., and Gutfeld, N. (1978). Nonlinear (amplifie lationship between nuclear occupancy by triiodothyronine and the appearance rate of Repatic o-glycero-phosphate dehydrogenase and malic enzyme in the rat. .I. Clin. Invest. 61, 981-997. Pickard, M. R., Sinha, A. K., Gullo, D., Pate& N., Hubank, M., and Ekins, R. P. (1987). The effect of 3,5,3’ triiodothyronine on leucine uptake and incorporation into protein in cultured neurones and subcellular fractions of rat central nervous system. Endocrinology 121, 2018-2026. Ray, A. K., and Medda, A. K. (1972). ~rod~ct~o~ of goiter in Lata fish (0phicephalus p~n~tat~s~. Ind. J. Physioi. Allied Sci. 26, 83-86. Savard, P., Merand, Y., Di Paolo, T., and Dupont, A. (1983). Effects of thyroid state on serotonin 5 hydroxyindoleacetic acid and substance P contents in discrete brain nuclei of aduit rats. ~Ve~uascience 10, 1399-1404. Somjen, D., Ismail-Beigi, F., and Edelman, I. S. (1981). Nuclear binding of T, and effects on Qo2, Na+K’-ATPase and (x-GPD in liver and kidney. Amer. J. Physiol. 240, El46154. Tobin, R. B., Baridanier, C. D., and Euklund, (1979a). Effects of thyroxine treatment on the hepatic plasma membrane ATPase activity. 9. Environ. Pathol. Toxicol. 2, 1235-1245. Tobin, R. B., Baridanier, C. I)., and Euklund, (1979b). L-Thyroxine effects upon ATPase activities of several subcellular fractions of liver of the rat and the guinea pig. J. Environ. rattan. Taxicol. 2, 1247-1266. Yokota, T., Nakamura, H., and Akamizu, T. (I986). Thyroid hormone receptors in neuronal and ghal nuclei from mature rat brain. Enndocrinology 118, 1770-1776.
AND
COMPARATIVE
ENDOCRINOLOGY
86, 162-169 (1992)
Alterations of Ion-Dependent ATPase Activities in the Brain of Singi Fish; Heteropneustes fossilis (Bloch), by Triiodothyronine SRABANI DE, A.K. Department
DASMAHAPATRA,AND
A. K. MEDDA
of Animal Physiology, Bose Institute, P-1112 CIT Scheme VII-M, Calcutta-700054, India Accepted July 23, 1991
The activities of Na+K+- and Mg2+ -ATPases in mitochondrial, microsomal, and cytosolic fractions of Singi fish (Heteropneustes fossilis Bloch) brain were investigated after injections of various doses (0.012, 0.025, 0.05, and 0.10 p,g/g) of triiodothyronine (T,) for 3 consecutive days. Both ATPases were found in the mitochondrial and microsomal fractions. The cytosolic fraction showed only Mg ‘+-ATPase activity. Mitochondrial Na+K+-ATPase activity increased to almost the same level in fish treated with 0.02.5,0.05, or 0.10 (*g of T,/g, while the T3 dose of 0.012 kg/g was ineffective in this respect. Microsomal Na+K+-ATPase activity increased to about the same level with all of the doses of Ts used. No detectable amount of Na+K+-ATPase was found in the brain cytosolic fraction. Mitochondrial Mg’+ATPase activity was enhanced with 0.025,0.05, and 0.10 p,g of T,/g. The last dose, however, produced a higher increase in activity than the other two doses. Surprisingly, microsomal and cytosolic Mg ‘+-ATPase activity was not increased by T, treatment. Although T, concentrations rose sharply after each T3 injection, the serum T, level in T,-injected fish was not different from that in the control as observed on the fourth day. The TX-induced rise of Na+K+- and MgZf-ATPase activities was inhibited by cycloheximide treatment. Immersion of Singi fishes in thiourea significantly reduced brain Na+K+-ATPase activity in microsomal and mitochondrial fractions but decreased Mg*+-ATPase activity only in the mitochondrial fraction. Three consecutive daily injections of T, (0.10 l&g) into the thioureatreated fishes increased their ATPase activities even beyond the control level. Our results thus indicate that T, has a controlling influence on the ATPase system in fish brain. 0 1992 Academic
Press. Inc.
Previous reports from our laboratory have provided evidence for the responsiveness of fish brain to thyroid hormone with respect to changes in protein, nucleic acid, glycogen, and lipid metabolism and activities of some specific enzymes like mitochondrial cx-glycerophosphate dehydrogenase and cytosolic malic enzyme (Ghosh and Medda, 1982, 1984; Ghosh et al., 1983; Medda and Ghosh, 1984; De et al., 1989). In higher animals, the presence of putative triiodothyronine receptors in neuronal nuclei of adult brain indicates that it may be a site of thyroid hormone action (Haider et al., 1983; Yokota et al., 1986; Gull0 et al., 1987). There are also reports that thyroid hormone status may control neurotransmitter levels (Jacoby et al., 1975; Ito et al.,
1977; Savard et al., 1983), B-adrenoceptor concentrations (Gross et al., 1980), and RNA polymerase I activity in neural nuclei (Nakamura et al., 1987) of adult rat brain. Triidothyronine (T3) stimulates leucine incorporation into TCA-precipitable material in primary cell cultures of neurons of rat brain in a dose-dependent manner (Pickard et al., 1987). The brain of nonmammalian vertebrates also possesses T, receptors in nerve cell nuclei (Haider et al., 1983; Bres and Eales, 1988). All these reports clearly indicate that both nonmammalian and mammalian adult brains undergo changes in intermediary metabolism under the influence of thyroid hormone. In our continuing investigations of the action of thyroid hormone in fish brain, we 162
0016-6480/92 $1.50 Copyright Q 1592 by Academic Press, Inc. All rights of reproduction in any form reserved.
T, ACTION
ON FISH BRAIN ATPase
attempted in the present experiments to evaluate the changes in the activities of ionspecific ATPases (Na+K+ and Mg2+ dependent) in subcellular fractions of the brain of Singi fish, Heteropneustes fossilis (Bloch), treated with different doses of T,. MATERIALS
AND METHODS
Treatment of the animals. Singi fish (H. fossilis Bloch) of 10-14 g body wt were purchased from a local supplier and acclimatized to laboratory conditions at 25 + B” for 4 days before our experiments. They were fed once daily ad iibitum with Tubifex tubifex. The animals were randomly divided into different groups and were kept in tap water in polythene trays. L-Triiodothyronine (T,, free acid, Sigma Chemical Co.) was dissolved in an alkaline 0.65% sodium chloride solution (pH 8-9). Various doses (0.012, 0.025, 0.05, and 0.10 &g/g of body wt) of T, were injected intraperitoneally for 3 consecutive days. The volume of the injected material did not exceed 35 )*I in any case. Control fish were injected with the same volume of alkaline saline only. The fish were fasted during the experimental period and kept at 25 + 1”. They were killed for biochemical analyses on Day 4. Some fish were immersed in thiourea-containing water (0.1%) for 30 days at 25” to make them hypothyroid as described previously (Ray and Medda, 1972). Corresponding control fish were maintained in simple tap water. These fish were fed during this period. After 30 days of treatment some of the fish from the thioureatreated and control groups were injected with T, (0.1 kg/g) on 3 consecutive days (Days 31, 32, and 33). Thiourea treatment was discontinued on Day 3 1. All of these fish were killed for biochemical analyses on Day 35. In another set of experiments, fish were maintained in cycloheximide-containing water (0.5 mglliter). Some of them were injected with T, (0.1 pg/g) on 3 consecutive days and were killed for analysis on the fourth day. The untreated control and TX-injected fish were maintained in tap water and sampled on the same day. The brains from different groups of animals were quickly dissected out on ice. Three brains were pooled together, weighed, and homogenized in isolation buffer (0.32 M sucrose, 10 mM Tris-HCl, 1 mM EDTA, and 5 m&f 2-mercaptoethanol, pH 7.4) at O-4” using a glass-Teflon homogenizer. The homogenate was centrifuged at 700g for 10 minutes to remove the nuclei and cell debris at o-4”. The supematant was further centrifuged at 16,000g for 20 min, and the precipitate was used as a crude mitochondrial fraction.
The supematant was saved for the isolation, of nicrosomes. The crude mitochondrial fraction was resuspended in isolation buffer and centrifuged again at 16,000g for 20 min to obtain purified mitochondria. Finally, the purified mitochondrial pellet was resnspended in 2 ml of isolation buffer and retained. for ATPase assay. The postmitochondrial supematant was further centrifuged in 105,OOOgfor 1 hr to obtain a crude dmicrosomal pellet. The pellet was resuspended in 7 ml isolation buffer and recentrifuged at 105,OOOg for t hr to obtain purified microsomes. The pellet was resuspended in 2 ml isolation buffer and nsed for ATPase assay. The first postmicrosomal supernatant was saved as the cytosolic fraction used for ATPase assay, Enzyme assay method. The assay of Na’K+ATPase activity followed the method of Xsmailand Edelman (1971) with some modifications. In brief, the final reaction mixture in 1.5 ml volume contained 50 mM Tris, 1 a EDTA, 3 ri04 MgCl,, 3 mW ATP* 100 mM NaCi, and 30 m&f KC1, pH 7.4. Ouabain, a specific inhibitor of Na”K+-ATPase activity, was used as a concentration of 1 x 10e3 M in some of p&e tubes as a control. The reaction mixture was preincubated for 10 min at 37” in a water bath. The reaction was started by the addition of ATP (c&odium salt) and was terminated by adding 1 ml of 10% TCA. The tubes were then transferred to an ice water bath and left far 15 min, after wbicb time the samples were ce~tr~foged at 1OOOgfor 5 min. The supernatant was saved and used for the determination of inorganic phosphorous (Chen et al., 1956). The protein content was determined following the method of Lowry e: nl. (1951). Na+K’-ATPase activity was calculated from the difference in phosphorous liberation in the presence and absence of ouabain and expressed as pmol of Pi x mg protein ’ x hr-‘. Iv&‘+-ATPase w mined in the presence of ouabain (I NaCl (100 rr&f) and KC1 (30 m&R) from the reaction mixture. The data were statistically analyzed by analysis of variance followed by Duncan’s multiple range test. Radioimmunoassay
(RIA)
of serum
TX. Singi
fisfi
were injected daily (intraperitoneally) for 3 days with different doses of T3 (0.012,0.025,O.Q5, and 0.1 pg/g). The blood from the control and treated fish was taken; from the cardinal vein very carefully on the fourth day after the first injection and allowed to clot to obtain the serum that was used for the determination of tbe T, level using RIA kits purchased from the Bhabha Atomic Research Centre, Bombay, India. In another set of experiments, 48 fish were injected with T, at the dose of 0. I kg/g at 0, 24, and %& hr and bled at various intervals between 1 to 96 hr. Each fish: was bled only once during the entire period of the experiment. The serum T, level was estimated by RIA as mentioned earlier.
164
DE,
DASMAHAPATRA,
RESULTS Effect ATPases
of T3 on Na+K+-
and Mg2+-
activities. The variance analyses of the data of the activities of NaiKfand Mg2 -t -ATPase in the subcellular fractions of brain of Singi fish treated with different doses of T, (0.012, 0.025, 0.05, and 0.1 pg/ g) are shown in Table 1. It was evident that T, significantly altered the activity of brain NafKf-ATPase in mitochondrial and microsomal fractions and Mg2+-ATPase in the mitochondrial fraction. No such changes in the microsomal and cytosolic 2t-ATPase activity after T, injections W were observed. Brain cytosolic fraction from normal Singi fish did not show any Na+K+-ATPase activity. Na+K+-ATPase activity in the brain mitochondrial fraction was increased in fish treated with 0.025 pg of T,/g in comparison to controls (P < 0.05). The higher doses of T, (0.05 and 0.10 l&g) caused about the same extent of increases (P < 0.05 or 0.01) in activity (Table 1). The lowest dose of T, (0.012 pg/g) was ineffective in inducing any change in mitochondrial Na + K + -ATPase activity. Microsomal Na+K+-ATPase activity also increased (P < 0.05 or 0.01) to
EFFECT
AND
MEDDA
about the same level with all of the doses of T, used in comparison to the control (Table 1). Mitochondrial Mg2+-ATPase activity was also enhanced in fish treated with 0.025 pg of T,/g (P < 0.05). Almost the same level of increase in activity was observed in fish treated with 0.05 kg of T,/g (Table 1). A clean dose-response relationship was not apparent at the lower T, doses (0.025 and 0.05 pg/g). More enhancement of Mg2+ATPase activity occurred in fish treated with 0.10 pg of T3/g in comparison to that observed in fish treated with 0.025 or 0.05 pg of T,/g (P c 0.05 or 0.01). The lower dose of 0.012 pg of T,/g was ineffective in elevating brain Mg2+-ATPase activity. The microsomal and cytosolic Mg2+-ATPase activity of Singi fish brain was not altered by T, treatment at the doses used. Effect of cycloheximide on T,-induced changes in Na+ Kt - and Mg” -ATPases activities. As already mentioned, T3 in-
creased Na+K+-ATPase activity in both mitochondrial and microsomal fractions and Mg2+-ATPase activity in the mitochondrial fraction, but when T, (0.10 pg/g) was injected for 3 consecutive days with cycloheximide simultaneously, it did not cause
TABLE 1 OF T, ON THE Na+K’AND Mg ‘+-ATPase ACTIVITIES OF DIFFERENT SUBCELLULAR THE BRAIN OF SINGI FISH, Heteropneustes fossilis (Bloch)
Name of the ATPase Na+K+-ATPase M2 + -ATPase
Subcellular fractions Mitochondria Microsome Mitochondria Microsome Cytosol
FRACTIONS
OF
Dose of T3 Wg) Control 17.91 20.73 16.06 36.85 16.82
k f k f f
0.012 1.84 2.09 2.41 4.76 3.05
21.83 27.56” 19.40 34.43 12.83
l?r 2.30 -r- 1.86 i 1.61 f 4.23 2 1.69
0.025 24.77a 27.53a 24.59” 34.49 14.11
f f i f *
0.05 Mean f SE 2.17 25.66a 1.68 29.67” 1.75 27.43ab 1.56 32.65 1.15 14.91
+ f * f f
0.10 1.71 2.22 3.67 3.62 2.31
27.07” 31.46” 36.82abcd 33.15 14.52
EMS ” f f f 2
1.71 2.78 3.14 3.10 0.96
37.37* 37.56* 55.77** 105.33 24.02
Note. SE, standard error. ATPase activity was expressed as pmol of Pi X mg protein-’ x hr-‘. EMS, error mean square obtained from ANOVA (df = 35). The animals were maintained at 25” and various doses of T3 were injected for 3 consecutive days. The fish were sacrificed on the fourth day, with the day of the first injection considered zero day. The brains from three fish were pooled in each set and the data of eight such pooled sets were expressed as means k SE. The data were statistically analyzed by ANOVA followed by Duncan’s multiple range test. P < 0.05 considered as significant. Superscripts a, b, c, and d indicate differences relative to control, 0.012, 0.025, and 0.05 pg x g -’ of Ts-injected fish, respectively. * Significance of F at 5% level. ** Significance of F at 1% level.
T, ACTION
ON
FISH
BRAIN
TABLE
ATPase
i65
2
EFFECT OF CYCLOHEXIMIDE ON T,-INDUCED CHANGES IN Na+K+AND Mgzf-ATPase MITOCHONDRIAL AND Na+K+-ATPase ACTIVITY IN MICROSOMAL FRACTIONS OF BRAIN Heteropneustes fossilis @loch) Subcellular fractions
Cycloheximide ATPases
Control
(0.1 p&
g-1)
(0.5 mg X liter-‘)
ACTIVITIES IN OF SINGI FISH,
T, f cycloheximide
EMS
Mean t SE Mitochondria
Na+K+-ATPase Mg’ + -ATPase Na+ K + -ATPase
Microsome
19.40 f 1.70 18.89 + 1.34
26.83"
26.72
34.82"
f
1.06
f
1.97
19.36b 21.94b 24.3?
29.81” f 2.87 k 2.66
+ 2.08 ? 2.20 -+ 2.32
18.92b 19.46b 26.93b
k 1.68 r 2.26 + 2.52
27.89* 39X8** 40.12**
?Gte. SE, standard error. ATPase activities were expressed as pmol of Pi X mg protein-’ x hr-‘. EMS, error mean square obtained from ANOVA (df = 28). The animals were maintained at 25” and various doses of T, were injected for 3 consecutive days. The fish were sacrificed the fourth day, with the day of the first injection considered zero day. The brains from three fishes were pooled in each set the data of eight such pooled sets were expressed as means rt SE. The data were statisticaliy analyzed by ANOVA followed Duncan’s multiple range test. P < 0.5 considered as significant. Superscripts a and b indicate differences relative to control P-a-injected fish, respectively. * Significance of F at 5% level. ** Significance of F at 1% level.
any increase in mitochondrial and microsoma1 Na+K+-ATPase activity or in mitochondrial Mg2+-ATPase activity in comarison to controls (Table 2). No significant aherations of ATPase activities were observed in fish treated with only cycloheximide. Effect of thiourea on NafKiand Mgzi-ATPase activities. Treatment of Singi fish with thiourea caused a reduction in the Na+K’-ATPase activity in both the
mitochondrial and microsomal fractions and in Mg2+ -ATPase activity in the n&ochondrial fraction in comparison to tive controls (Table 3). Injection o reated fish caused enha -ATPase activity above trol level in both the mitocho~dr~ 0.01) and microsomal (P < 0.01) fra~t~~~s~ Also, mitochondrial Mg2+-ATPase a~t~~~t~ in thiourea-treated fish was similarly ktcreased by T, treatment (P < 0.01) but mi-
TABLE EFFECT
OF THIOUREA
FRACTIONS
Subcellular fractions Mitochondria Microsome
BY BRAIN
ON Na+K”OF SINGI
ATPases Na+K+-ATPases Mg’+-ATPases Na+K’-ATPases Mg*+-ATPases
on aad by and
3
AND Mg’+- ATPase ACTIVITIES IN MITOCHONDRIAL AND MICXO~OMAL FISH, Heteropneustes fossilis (Bloch), AND SUBSEQUENT RECOVERY BY T, INJECTIONS
Control 18.69 20.89 19.99 31.16
? k C &
1.58 1.71 2.07 2.73
T3 (0.1 I% x s-‘1 26.58” 31.86” 33.68” 30.24
2 + I L
Mean 2.13 2.75 2.83 2.78
Thiourea
T3 + thiourea
* SE 12.02”b -c 1.09 24.2Y” +- 2.25 14.7gab ” 0.85 34.25” I 2.37 12.16ab k 2.11 33.94”’ I 2.32 26.14 2 1.87 29.63 i 2.45
EMS 26.68”” 33.48** 43.53”* $I.%?
IVote. SE, standard error. ATPase activity was expressed as urn01 of Pi x mg protein-’ x h-‘. EMS, error mean square obtained from ANOVA (df = 28). The animals were kept at 25”, immersed in thiourea (1 mg/mI) for 30 days, then transferred to tap water and injected with T, (0.1 pg/g) for 3 consecutive days (Days 31, 32, and 33) and sacrificed on Day 35 (frrst day of thiourea immersion was taken as zero day). The brains from three fishes were pooled in each set and the data of eight such pooled sets were expressed by means i SE. The data were statistically analyzed by ANOVA followed by Duncan’s multiple range test. P < 0.05 considered as significant. Superscripts a, b, and c indicate differences relative to control, T,, and thiourea-treated fish, respectively. ** Significance of F at 1% level.
166
DE,
DASMAHAPATRA,
AND
TABLE
MEDDA
4
MEAN VALUES OF SERUM T, LEVELS OBTAINED AFTER INJECTIONS OF DIFFERENT DOSES OF T, FOR 3 CONSECUTIVE DAYS
Serum T, (n&-4
Control
0.012
0.025
0.05
0.1
EMS
3.54 kO.5.5
4.51 e-o.51
Mean + SE 3.33 k0.29
4.20 -+0.85
4.10 20.73
56.23“
Note. The animals were kept at 2.5”and bled on the fourth day after the first T, injection. Each group consisted of six animals. SE, standard error. EMS, error mean squares, obtained from variance analysis (df = 25). u Not significant.
crosomal Mg2 + -ATPase remained unchanged after thiourea treatment as well as after T, injection in thiourea-treated fish. Serum T3 level in T,-injected Singi fish. The serum T, level of control Singi fish was 3.54 k 0.55 rig/ml (n = 6). No significant difference was observed in the blood T3 level between the control and T,-treated fish (0.012, 0.025, 0.05, and 0.10 pglg, 3 consecutive days injections) on the fourth day (Table 4). The distribution patterns of T, in Singi fish serum from 1 to 96 hr during
5
10
15
20
25
30
35
40
45
and after T, injection (0.1 pg/g, 3 consecutive days) are represented in Fig. 1. It was observed that serum T, reached the maximum level within l-2 hr postinjection (about 2.5- to 3-fold higher in comparison to the control level) and came down within 8-16 hr, tllz ranged from 3 to 7 hours, depending upon the day of treatments. DISCUSSION
Our results show that the thyroid
50 55 HOURS
60
65
70
75
80
85
90
95
100
FIG. 1. Effect of T3 injection (0.1 cl.g/g, 3 consecutive days) on the serum T, level of Singi fish, Heteropneustes fossilis (Bloch). Fish were injected at 0, 24, and 48 hr. Blood was drawn from the cardinal vein of the fish for different time periods mentioned in the abscissa. Only two fish were bled each time, and each fish was bled only once during the entire experimental period. The broken line represents the mean serum T3 level (n = 6, standard error = 0.54) obtained from control fish. The straight line represents treated data. Each point in the treated curve is the mean of two fish where individual variation is about 20%.
hor-
T, ACTION
ON
FISH
mone-induced metabolic alterations in fish brain include stimulation of ion-dependent ATPases, viz. Na+K’and Mg2+-ATPase. As already pointed out, certain enzymes, such as mitochondrial ol-glycerophosphate dehydrogenase and NADP-dependent cytosolic malic dehydrogenase, also show enhanced activities in response to T, treatment (Ghosh et al., 1983; De et al., 1989). The present experimental results indicate that NafKf-ATPase activity in brain mitochondrial and microsomal fractions significantly increases after 3 consecutive days of injections of T,. The Mg2+-ATPase activity of the mitochondrial fraction, not of microsomal or cytosolic fraction, was also enhanced. The brain cytosolic fraction showed no detectable NatKf-ATPase activity, although it contained a considerable amount of Mg2+ -ATPase. The microsomal ATPases activities were found to be higher than the mitochondrial ATPases, and the microsomal Na+K+-ATPase appears to be more responsive to T, treatment than the mitochondrial ATPase, as evidenced from the enhanced activity with the lowest dose of T, used (0.012 pg/g). This lowest dose of T, was found to be ineffective in causing any change in Na+K+-ATPase activity in the mitochondrial fraction. The changes in different ion-dependent ATPases in crude homogenate or in membrane fractions of liver, kidney, and skeletal muscle of thyroid hormone-treated mammalian and nonmammalian vertebrates have been reported by other workers (Tobin et al., 1979a,b; Guernsey and Edelman, 1983; De et aE., 1987).
The increase in the activities of iondependent ATPases is presumably due to an increased synthesis of these enzymes, which may be a reflection of the action of T, primarily at the nuclear level. This is ported by an observation that cycloheximide treatment inhibited the stimulation of Singi fish brain Na+K+and Mg2+ATPases by T,. We found that mitochondrial protein was also increased by T,, and
B&UN
ATPase
167
in contrast to this, no change in microsomal protein was detectable, although microsoma1 Na+M+-ATPase activity was enhanced by T,. T,-induced increased synthesis of the enzyme has also been indicated by increased incorporation of [3H]- or [35S],methionine in an a-subunit of ATPase of rat kidney at ter administration of T, ( and Lo, 1980). Moreover~ increas tion or decreased degradation of ATPase in T,-treated rat liver, m kidney has been suggested b binding of ]3H]ouabain in Na (Lin and Akera, 1978). The presence of a putative thyroid hormone-binding receptor and the binding of ‘251-labeled T, in the nuclear fraction of Singi fish brain have been mahapatra et a/., 1991). S with the brain nuclear re multaneous increase in a! al., 1983), malic enzyme ( and ATPases activities m clear activation by T, for the st~rn~~~t~o~ of Na+K+and Mg”+-ATPases in Singi fish brain. Similarly, there ar
crease in c&PI> activity, 0, consumption, and Nat K f -ATPase activity ~~srna~~-~e~~~ and Edelman, 1974; Oppenheimer et a!., 1978; Oppenheimer, 1983), w suggest a rmclear activation for the stimulation of Na+K+-ATPase. It has baby suge increase in the Ma+ or K’ pump units and the t Na+ across the nuclear membr T, treatment, as shown in rat 11 sey and Edelman, 1983), may be one of the causes for the T,-induced ~~ti~~at~~~ of Na+K’-ATPase in fish liver ( 1987). This mechanism may be mitochondria and microsomes o as found in the present experi regard to the T, t~mu~atio~ of Na 4 K + ATPase activity. noted that Na+K+-ATPase not be de-
168
DE,
DASMAHAPATRA,
tected yet in the nuclear membrane of Singi fish brain (unpublished observation). The T,-induced enhancement of Mg2+-ATPase activity in the mitochondrial fraction may be due to the increased transport of Mg2+ ions across the mitochondrial membrane. A difference was noted regarding the responsiveness of Mg2+-ATPase to T, between the mitochondrial and microsomal fraction; the former showed enhanced activity while the latter did not show any change in Mg2+ATPase activity in response to T, injection. That thyroid hormone is responsible for the activation of Na+K+and Mg2+ATPases in Singi fish brain has been further documented by a significant fall in the activities of these ATPases in the thioureatreated Singi fish. The Mg2+-ATPase activity in the brain microsomal fraction did not alter in thiourea-treated fish. Injections of T, (0.1 pg/g) in thiourea-treated fish raised the Na+K+and Mg2+-ATPase activities in the mitochondrial fraction and only Na+K+-ATPase activity in the microsomal fraction to a level higher than the control value. It appears that the decrease in ATPases activities in thiourea-treated fish is due to the inadequacy of the endogenous circulating thyroid hormone since we have detected in some representative experiments that reduction of serum T, and T, levels occurs in Singi fish after 15 days of immersion in thiourea (unpublished). A similar reduction in ion-dependent ATPases in hypothyroid or thyroidectomized rats has been reported by other investigators (Herd, 1978; Somjen et al., 1981; Tobin et al., 1979a). It also deserves mention that a reduction in the mitochondrial a-GPD activity occurs in the brain of thiourea-treated Singi fish and that this decrease could be restored even beyond the control level by T, treatment (Medda and Ghosh, 1984). Similar changes have also been noted in the case of malic dehydrogenase activity in Singi fish brain in a hypothyroid condition with and without T, treatment (unpublished observation).
AND
MEDDA
Thus it appears that the thyroid hormone exerts its regulatory influence on fish brain through induction of a number of enzyme systems as found in other thyroid hormone target organs. REFERENCES Bres, O., and Eales, J. G. (1988). High affinity, limited capacity triiodothyronine binding sites in nuclei from various tissues of the rainbow trout (Salvo gairdneri). Gen. Comp. Endochrinol. 69, 71-79. Chen, P. S., Toribara, T. Y., and Warner H. (1956). Microdetection of phosphorous. Anal. Chem. 28, 17561758. Dasmahapatra, A. K., De, S., and Medda, A. K. (1991). Demonstration of putative thyroid hormone receptor in the brain nuclei of Singi fish, Heteropneustes fossifis (Bloch). Gen. Comp. Endocrinol.
82, 60-68.
De, S., Dasmahapatra, A. K., Ray, A. K., and Medda, A. K. (1989). Induction of NADP dependent malic dehydrogenase activity in brain of Singi fish, (Heteropneustes fossilis Bloch) by 3,5,3’ triiodothyronine. Neurochem. Znt. 14,261266. De, S., Ray, A. K., and Medda, A. K. (1987). Nuclear activation by thyroid hormone in liver of Singi fish: Changes in different ion-specific adenosine triphosphatase activities. Horm. Metab. Res. 19, 367-370. Ghosh, R. K., Ghosh, N., De, S., Ray, A. K., and Medda, A. K. (1983). Effect of L-triiodothyronine on the mitochondrial a-glycerophosphate dehydrogenase activity, mitochondrial and total protein contents of brain of Singi fish (Heteropneustes fossilis Bloch). Neurochem. Znt. 5, 635640. Ghosh, R. K., and Medda, A. K. (1982). Effect ofthyroxine on protein and nucleic acid contents of different parts of brain of Singi fish (Heteropneustes fossilis Bloch). Endocrinology 79, 355-361. Ghosh, R. K., andMedda, A. K. (1984). Effect ofthyroxine and thiourea on cholesterol, total lipids and glycogen contents of brain of Singi fish (Heteropneustes fossilis Bloch). Neurochem. Znt. 6, 97101. Gross, G., Brodde, 0. E., and Schuemann, H. J. (1980). Decreased number of B-adrenoceptors in cerebral cortex of hypothyroid rats. Eur. J. Pharmacof.
61, 191-194.
Guernesy, D. L., and Edelman, I. S. (1983). Regulation of thermogenesis by thyroid hormones. In “Molecular Basis of Thyroid Hormone Action” (J. H. Oppenheimer and H. H. Samuels, Eds.), pp. 293-324, Academic Press, New York.
T, ACTION
ON FISH BRAIN ATPase
Gullo, D., Sinha, A. K., Bashir, A., Hubank, M., and Ekins, R. P. (1987). Differences in nuclear triiodothyronine binding in rat brain cells suggest phylogenetic specialization of neuronal functions. Endocrinology 120, 2398-2403. Haider, M. A., Dube, S., and Sarkar, P. K. (1983). Thyroid hormone receptors of developing chick brain are predominantly in the neurones. Bio&em. Biophys. Res. Commun. 112, 221-227. Herd, P. A. (1978). Thyroid hormone divalent cation interactions. Arch. Biochem. Biophys. 188, 220225. Ismail-Beigi, F., and Edelman, I. S. (1971). The mechanisms of the calorigenic action of thyroid hormone: Stimulation of Na+K+ activated adenosine triphosphatase activity. .I. Gen. Physiol. 51, 710-722. Ismail-Beigi, F., and Edelman, I. S. (1974). Time course of the effects of thyroid hormone on respiration and Na+K+-ATPase activity in rat liver. Proc. Sot. Exp. Bioi. Med. 146, 983-988. Ito, J. M., Valcana, T., and Timiras, P. S. (1977). Effect of hypo- and hyperthyroidism on regional monoamine metabolism in the adult rat brain. Neuroendocrinology 24, 55-64. Jacoby, J. H., Muller, G., and Wurtman, R. .I. (1975). Thyroid state and brain monoamine metabolism. Endocrinology 97, 1332-1335. Lin, M. II., and Akera, T. (1978). Increased (Na+K+)-ATPase concentrations in various tissues ofrats caused by thyroid hormone treatment. 3. Biol. Chem. 253, 723-726. Lo, C. S., August, T. R., Liberman, U. A., and Edelman, I. S. (1976). Dependence of renal cortical (Na+K+)-adenosine triphosphatase. .I. Biol. Chem. 251, 7834-7840. Lo, G. S., and Lo, T. N. (1980). Effect of triiodothyronine on the synthesis and degradation of the small subunit of renal cortical (Na+K+)adenosine triphosphatase. J. Biol. Chem. 255, 2131-2136. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the fohn phenol reagent. J. Biol. Chem. 193, 265275. Medda, A. K., and Ghosh, R. K. (1984). Inhibitory influence of thiourea on brain of Singi fish (Heteropneustes fossilis Bloch) and subsequent recovery by L-triiodothyronine. Neurochem. Int. 6, 527-532.
169
Nakamura, II., Yokota, T., Akamizu, T., Mori, T., and Imura, H. (1987). Triiodothyronine effects on RNA polymerase activities in isolated neuronaI and glid nuclei of the mature rat brain cortex. Metnb. Clin. Exp. 36, 931-934. Oppenheimer, .I. II. (1983). The nuclear receptor triiodothyronine complex: Relationship to thyroid hormone distribution, metabolism and biological action: In “Molecular Basis of Thyroi.d Hormone Action” (J. H. Oppenheimer and H. II. Samuels, Eds.), pp. l-34. Academic Press, New York. Oppenheimer, J. H., Coulombe, P., Schwartz, H. L., and Gutfeld, N. (1978). Nonlinear (amplifie lationship between nuclear occupancy by triiodothyronine and the appearance rate of Repatic o-glycero-phosphate dehydrogenase and malic enzyme in the rat. .I. Clin. Invest. 61, 981-997. Pickard, M. R., Sinha, A. K., Gullo, D., Pate& N., Hubank, M., and Ekins, R. P. (1987). The effect of 3,5,3’ triiodothyronine on leucine uptake and incorporation into protein in cultured neurones and subcellular fractions of rat central nervous system. Endocrinology 121, 2018-2026. Ray, A. K., and Medda, A. K. (1972). ~rod~ct~o~ of goiter in Lata fish (0phicephalus p~n~tat~s~. Ind. J. Physioi. Allied Sci. 26, 83-86. Savard, P., Merand, Y., Di Paolo, T., and Dupont, A. (1983). Effects of thyroid state on serotonin 5 hydroxyindoleacetic acid and substance P contents in discrete brain nuclei of aduit rats. ~Ve~uascience 10, 1399-1404. Somjen, D., Ismail-Beigi, F., and Edelman, I. S. (1981). Nuclear binding of T, and effects on Qo2, Na+K’-ATPase and (x-GPD in liver and kidney. Amer. J. Physiol. 240, El46154. Tobin, R. B., Baridanier, C. D., and Euklund, (1979a). Effects of thyroxine treatment on the hepatic plasma membrane ATPase activity. 9. Environ. Pathol. Toxicol. 2, 1235-1245. Tobin, R. B., Baridanier, C. I)., and Euklund, (1979b). L-Thyroxine effects upon ATPase activities of several subcellular fractions of liver of the rat and the guinea pig. J. Environ. rattan. Taxicol. 2, 1247-1266. Yokota, T., Nakamura, H., and Akamizu, T. (I986). Thyroid hormone receptors in neuronal and ghal nuclei from mature rat brain. Enndocrinology 118, 1770-1776.
