Neonatal hypothyroidism alters the kinetic properties of Na+, K+-ATPase in synaptic plasma membranes from rat brain

Brain Research Bulletin 70 (2006) 55–61

Neonatal hypothyroidism alters the kinetic properties of Na+, K+-ATPase in synaptic plasma membranes from rat brain Framroze R. Billimoria a , Bharat N. Dave b , Surendra S. Katyare c,∗ a

Department of Biochemistry, Terna Medical College, Nerul, Navi Mumbai 400706, India Department of Biochemistry, Seth G.S. Medical College and K.E.M. Hospital, Parel, Mumbai 400012, India c Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat 390002, India b

Received 23 August 2005; received in revised form 24 February 2006; accepted 6 March 2006 Available online 3 April 2006

Abstract Neonatal hypothyroidism was induced in rat pups by injecting 131 I within two days of birth and the effects on kinetic properties of Na+ , K+ ATPase from synaptic plasma membranes were examined. Neonatal hypothyroidism resulted in a generalized decrease in Vmax with ATP, Na+ , K+ and Mg2+ together with an increase in the Km for ATP, appearance of a low affinity component for Na+ and allosteric characteristic for the Mg2+ -dependent activity at high Mg2+ concentrations. Binding pattern for Na+ and Mg2+ changed. Our results suggest that impairment of Na+ , K+ -ATPase activity together with altered kinetic properties could be one of the underlying biochemical mechanism leading to central nervous system (CNS) dysfunctions as a consequence of thyroid hormone deprivation during critical stages of brain development. © 2006 Elsevier Inc. All rights reserved. Keywords: Neonatal hypothyroidism; Synaptic plasma membrane; Na+ , K+ -ATPase; Rat; Brain

1. Introduction The role of thyroid hormone(s) in the development of the brain has been recognized for over a century [35] Deprivation of thyroid hormones in early developmental stages results in structural and functional deficits in the central nervous system (CNS), and deficiency during critical period of development can affect cognitive functions such as attention, learning and memory [13,16,36,37]. The effects on these neurological functions are irreversible [14]. Treatment of developing rats with propylthiouracil (PTU) was shown to result in functional impairment in synaptic transmission and plasticity in the neonatal rat hippocampus and in the dentate gyrus of the adult hippocampus [13,31]. Neonatal hypothyroidism also impairs structural maturation of the brain and results in diminished metabolic and electrical activities [11]. Neuronal hypoplasia with reduced axon count, dentritic branching, synaptic spike and interneuron connection has been reported [22]. Also, the oligodentrocytes

∗

Corresponding author. Tel.: +91 265 2795594; fax: +91 265 2795569. E-mail addresses: sskatyare [email protected], sskatyare [email protected] (S.S. Katyare). 0361-9230/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2006.03.005

decrease in number and consequently myelin content drops [22]. More recently it has been shown that thyroid hormones regulate the synthesis of several crucial proteins in the brain which include 7.2 kb dynamin III transcript [1,22], the extra-cellular signal regulated kinases (ERK-1 and ERK-2) which are important in late long-term potential, synapsin I and synaptotagmin I [22,36,37]. The latter two proteins function in the control of neurotransmitter release [36]. In the hypothyroid rats neuronal microtubule content and organizations are altered, mitochondrial number decreases and 3,5,3 -triiodothyronine (T3 ) receptors in nuclei and cytoplasm are altered [36]. It has also been suggested that T3 may regulate the levels of neurotrophins to promote the development of cerebellum [23]. Although the effects of thyroid deficiency on the neuronal outgrowth of process formation of axon and dendrites and the impairment of the synthesis of several crucial proteins has been documented as cited above, the basic biochemical defects underlying dysfunctions relating to memory, cognition, neurotransmission and plasticity are not yet clearly understood. The synaptic transmission is regulated by neurotransmitters and is dependent on action potential [14]. The action potential in turn is regulated by the synaptic plasma membrane Na+ , K+ -ATPase [14,27]. It is therefore possible that the observed

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memory and cognitive dysfunctions may be traced to possible functional alterations in the activity of the synaptic plasma membrane Na+ , K+ -ATPase. To evaluate this possibility, we examined the effect(s) of neonatal hypothyroidism on kinetic attributes of synaptic plasma membrane Na+ , K+ -ATPase in the rat model. The results indicate that the kinetic properties of the Na+ , K+ -ATPase were significantly altered as a result of neonatal hypothyroidism. 2. Materials and methods 2.1. Chemicals Vanadium free ATP and Tris were purchased from Sigma Chemical Co., USA. Imidazole was from Koch-Light and Co., U.K. All other chemicals were of analytical-reagent grade and were purchased locally. 131 I was obtained from Isotope Group, Bhabha Atomic Research Centre, Mumbai.

2.2. Animals and treatment with 131 I Female rats of Wistar strain (250 gm) were mated and within two days of birth the pups were injected with 150 ␮Ci of 131 I [28]. These pups showed significant growth retardation and at the end of the experimental period their body weight decreased by 30–35% compared to the controls. The pups in the control group did not receive any treatment. The animals were killed between 30 and 35 days of birth and their brains were quickly removed and placed in beakers containing chilled (0–4 ◦ C) 0.32 M sucrose for isolation of synaptosomal membranes. Tissues from three to four animals from the same litter were pooled to obtain a single preparation. Only the male animals were used in the studies.

2.3. Isolation of synaptosomal membranes

Table 1 Effect of neonatal hypothyroidism on ATPase activities in synaptic membranes from rat brain Enzyme

Na+ , K+ , Mg2+ -ATPase Na+ , K+ -ATPase Mg2+ -ATPase

Activity( ␮mol Pi/h/mg protein) Control

Hypothyroid

40.8 ± 1.43 28.4 ± 1.84 12.4 ± 0.91

22.5 ± 1.40a 6.1 ± 0.51a 14.4 ± 0.90

Change (%)

−44.9 −78.5 +16.1

The experimental details are as described in the text. The results are given as mean ± S.E.M. of six independent observations. a p < 0.001 compared with control. 37 ◦ C for 15 min. At the end of the incubation period, the reaction was terminated by adding 0.1 ml of 10% (w/v) sodium dodecyl sulfate (SDS) solution [30]. Na+ , K+ -ATPase activity was determined in a medium containing NaCl and KCl from which Mg2+ was omitted. Mg2+ -ATPase activity was determined in the presence of 5 mM MgCl2 in the medium from which NaCl and KCl were omitted (eg. see Table 1). ATP solutions were prepared fresh prior to use by neutralizing ATP to pH 7.4 with Tris base.

2.5. Determination of kinetic properties ATP: the medium employed for concentration dependent changes in enzyme activity with respect to ATP concentration was the same as that used for assay of Na+ , K+ , Mg2+ -ATPase activity as detailed above; concentration of ATP was varied from 0.1 to 10 mM. NaCl: the assay medium was the same as employed above for the determination of Na+ , K+ , Mg2+ -ATPase, in which the concentration of NaCl was varied from 0 to 100 mM. KCl: the medium was the same as above; concentration of KCl was varied from 0 to 20 mM. MgCl2 : the medium employed for assay of Na+ , K+ , Mg2+ -ATPase activity described above was used in which concentration of MgCl2 was varied from 0 to 5 mM. Determination of inorganic phosphate released was according to the method of Fiske and Subba Row [12]. The data for substrate kinetics were analyzed by the Lineweaver–Burk, Eadie–Hofstee and Eisenthal and Cornish–Bowden methods for the determination of Km and Vmax [10]. The values of Km and Vmax obtained by the three methods were in close agreement and were averaged. For sake of brevity only the typical Eadie–Hofstee plots are shown (Fig. 1). Hill plot analysis was carried out to find out the extent of binding of the individual substrates, viz. ATP, Na+ , K+ and Mg2+ over the concentration ranges used, by employing the equation:

The synaptic plasma membranes were isolated essentially by employing the procedure described previously [30]. Briefly, the tissue was washed repeatedly with the isolation mediun (0.32 M sucrose) to free it from adhering blood and homogenized using a Potter-Elvehjem type glass-Teflon homogenizer to obtain 20% (w/v) homogenate. The homogenate was centrifuged at 900 × g for 10 min to sediment nuclei and cell debris. The pellet (P1 ) was discarded and the clear supernatant (S1 ) was subjected to a further centrifugation at 12,000 × g for 10 min to obtain a second pellet (P2 ) and supernatant (S2 ). The pellet (P2 ) which consisted of mitochondria, synaptosomes and myelin was washed once by resuspending in 0.32 M sucrose and re-sedimenting, and lysed by suspending in 6.0 ml of 5 mM Tris–HCl buffer, pH 8.1. After incubating for 30 min at 0 ◦ C, 12.0 ml of 48% (w/w) sucrose was added to the lysate and the contents were mixed thoroughly and transferred to a 50 ml tube of Beckman L2 65B SW 25.2 rotor. The lysate was then carefully over layered with 15 ml of 28.5% (w/w) sucrose followed by 15 ml of 10% (w/w) sucrose, care being taken not to disturb the gradient and avoiding the intermixing of sucrose solution layers. The tubes were then centrifuged in a Beckman L2 65B ultracentrifuge using a SW 25.2 rotor at 60,000 × g for 90 min. At the end of the centrifugation period, the synaptic membrane fraction was removed with a syringe employing needle of broad gauge (No. 18) taking care to avoid contamination from myelin, transferred to a 12 ml tube of a Ti 50 rotor and sedimented by centrifugation at 105,000 × g for 60 min. Finally, the pellet was suspended in 0.32 M sucrose to give synaptic membrane protein concentration of about 0.3–0.4 mg/ml. The suspension was stored frozen at −25 ◦ C for assay of the ATPase activities within a week of preparation.

where v is the velocity in the presence of a given concentration of the substrate [S], V is the maximum velocity and k is a constant. The Hill coefficient (n) represents the number of substrate molecules bound. The plot of log[v/V − v] versus log[S] is expected to be a straight line, the slope of which gives the Hill coefficient, n [10]. All the kinetics data were computer analyzed using Sigma plot Version 6.1 [8,26]. Protein estimation was according to the method of Lowry et al. [21] with bovine serum albumin fraction V used as the standard.

2.4. Assay of ATPase activities

3. Results

Na+ , K+ , Mg2+ -ATPase activity was determined in a medium (total volume:1.0 ml) containing 50 mM imidazole-HCl buffer pH 7.4, 120 mM NaCl, 10 mM KCl, 5 mM MgCl2 and 4 mM ATP [30]. The reaction was started by adding 30–50 ␮g of synaptosomal membrane proteins and was carried out at

The effects of neonatal hypothyroidism on ATPase activity in synaptic membranes are summarized in Table 1. As is evident, the total and the Na+ , K+ -dependent ATPase activities decreased

log

v = n log[S] − log k V −v

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Fig. 1. Typical Eadie–Hofstee plots showing dependence of the synaptic membrane Na+ , K+ -ATPase activity on ATP, Na+ , K+ and Mg2+ . The enzyme activity v on abscissa is plotted versus v/[S] on ordinate.

by 42 and 79% in the hypothyroid group. The Mg2+ -dependent ATPase activity was not affected. The observed decreases (Table 1) could have been due to altered kinetic properties of the enzyme and/or, compositional changes in the enzyme subunits or the compositional changes in the membrane itself. In the present studies we ascertained the former possibility by determining the dependence of the Na+ , K+ , Mg2+ -ATPase activity on ATP, Na+ , K+ and Mg2+ . The typical Eadie–Hofstee plots for ATP, Na+ , K+ and Mg2+ are shown in Fig. 1. It can be noted that in the control group for ATP and Na+ the enzyme displayed patterns in which a single kinetic component was detected. As against this, for K+ and Mg2+ dependent activities two kinetically distinguishable com-

ponents were discerned. As detailed above, the Km and Vmax values were obtained by three methods employing computer analysis and the values were averaged. These values are given in Table 2. As can be noted, in the control group the Km values for ATP and Na+ were 0.05 and 0.83 mM, respectively and the Vmax values were in the range of 37–45 U (Table 2). For K+ the Km for component I was 0.11 mM whereas that for component II was about 12 times higher (1.31 mM); for component II the Vmax value was almost double. In case of Mg2+ the two Km values were 0.22 and 1.37 mM and the Vmax values were 8.3 and 22.7 U (Table 2). Hypothyroidism resulted in increased Km for ATP and the lowering of Vmax . For Na+ a high affinity component with Km of 0.19 mM appeared. Nevertheless, the Vmax values were always

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Table 2 Effect of neonatal hypothyroidism on kinetic properties of rat brain synaptic membrane Na+ , K+ -ATPase Parameter

Animals

Component I

Component II

Km (mM)

V (␮mol Pi/h/mg protein)

Km (mM)

V (␮mol Pi/h/mg protein)

ATP

Control Hypothyroid

0.05 ± 0.003 0.11 ± 0.007a

45.2 ± 3.23 28.3 ± 2.70a

– –

Na+

Control Hypothyroid

– 0.19 ± 0.012

– 18.6 ± 0.97

0.83 ± 0.060 0.99 ± 0.008

36.7 ± 3.03 26.4 ± 2.48b

K+

Control Hypothyroid

0.11 ± 0.006 0.13 ± 0.007

28.1 ± 1.81 19.7 ± 0.89c

1.31 ± 0.096 0.75 ± 0.039a

52.4 ± 4.26 35.1 ± 1.97d

Mg2+

Control Hypothyroid

0.22 ± 0.011 0.15 ± 0.005a

8.3 ± 0.59 3.3 ± 0.16a

1.37 ± 0.077

– –

22.7 ± 1.53 Allosteric

Experimental details are as given in the text. The Km and Vmax values were calculated by three different methods as described in text and averaged. The results are given as mean ± S.E.M. of six independent observations. a p < 0.001 compared with control. b p < 0.05 compared with control. c p < 0.002 compared with control. d p < 0.01 compared with control.

significantly low (Table 2). In the case of K+ , hypothyroidism resulted in lowering of Km for component II and significant decrease in the Vmax of both the components. For Mg2+ the substrate saturation kinetics followed a normal pattern up to the concentration of 0.33 mM beyond which an allosteric pattern was evident (Fig. 1). We then examined the affinity of the enzyme for binding ATP, Na+ , K+ and Mg2+ by Hill plot analysis. The typical Hill plots are shown in Fig. 2. As can be noted (Fig. 2 and Table 3) in normal euthyroid animals the enzyme bound one ATP and two Na+ over the entire concentration range employed. One K+ and one Mg2+ were bound at lower concentrations and two each of K+ and Mg2+ were bound beyond 1.7 and 0.4 mM concentrations, respectively of the two cations. In the hypothyroid animals the capacity to bind Na+ had decreased in low concentration range. However, beyond 2.5 mM concentration the enzyme bound two Na+ . The enzyme also displayed increased affinity for binding K+ and Mg2+ .

4. Discussion The present studies were initiated to examine the effects of neonatal hypothyroidism on the Na+ , K+ , Mg2+ -ATPase activity of the synaptic plasma membranes. The enzyme comprises a mixture of units consisting of isoforms containing different ␣ and ␤ subunits [5] and the different isoforms of the ␣ subunits show differential sensitivity to ouabain [32–34]. In view of this observation we decided to determine the composite picture of the kinetic properties of the enzyme as it is present in the membrane. Also, we determined the Mg2+ -ATPase activity (Table 1) in a medium devoid of Na+ and K+ which eliminated the problem of differential inhibition by ouabain of Na+ , K+ component of the enzyme activity [32–34]. It is clear from the data presented that neonatal hypothyroidism severely impaired the Na+ , K+ -ATPase activity in synaptic membrane. However, apparently the Mg2+ -ATPase activity was not affected. An overall decease in the Vmax with ATP,

Table 3 Hill coefficients for different components of synaptic membrane ATPases Component

Animals

Hill coefficient

Transitition point concentration (mM)

n1

n2

ATP

Control Hypothyroid

0.84 ± 0.069 1.06 ± 0.080

– –

– –

Na+

Control Hypothyroid

1.38 ± 0.113 0.47 ± 0.040a

– 1.53 ± 0.110a

– 2.50 ± 0.160

K+

Control Hypothyroid

0.46 ± 0.024 0.65 ± 0.040

1.67 ± 0.091 2.59 ± 0.140a

1.69 ± 0.062 1.82 ± 0.056

Mg2+

Control Hypothyroid

0.65 ± 0.023 1.11 ± 0.056a

2.08 ± 0.120 2.50 ± 0.108b

0.43 ± 0.019 0.31 ± 0.013

Experimental details are as given in the text. The Hill coefficients n1 and n2 were calculated from the corresponding Hill plots. The results are given as mean ± S.E.M. of six independent observations. a p < 0.001 compared with control. b p < 0.05 compared with control.

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Fig. 2. Typical Hill plots showing dependence of the synaptic membrane Na+ , K+ -ATPase activity on ATP, Na+ , K+ and Mg2+ . Log[v/Vmax − v] on abscissa is plotted versus log[S] on ordinate.

Na+ , K+ and Mg2+ was a generalized feature of hypothyroidism (Table 2). Also, under these conditions a high affinity component became apparent for Na+ . Whereas, two kinetic components for Mg2+ were noted in the control group, in the hypothyroid group Mg2+ -ATPase displayed allosteric characteristics at higher Mg2+ concentrations. The increased Km for ATP together with appearance of a high affinity component for Na+ and allosteric behavior of Mg2+ -ATPase at high concentration suggest that subtle regulatory changes occurred in the hypothyroid state, together with diminished rate of the enzyme activity with all the substrates. The appearance of a high affinity component for Na+ may represent a compensatory mechanism for maintaining intracellular Na+ homeostasis. The reported values of Km for Na+ , K+ and ATP are in the range of 9–28 mM, 2–6 mM and 0.07–0.46 mM, respectively [5].

Our values for Km for ATP in the control group (Table 2) are close to the reported lowest value [5]. However our values for Km for Na+ are significantly lower compared to those reported in the literature [5]. In this connection it may be pointed out that the intracellular concentration of Na+ is in the range of 10–20 mM while that of K+ is around 120 mM [17,34]. It is obvious that for the cation exchanging activity of the Na+ , K+ -ATPase of plasma membrane, the concentrations of intracellular Na+ and extra-cellular K+ would only be of importance. Viewed in this context, the reported Km for Na+ in the range of 9–28 mM [5] would seen to be abnormally high especially since the intracellular concentration of Na+ is in the range of 10–20 mM. By similar consideration the extra-cellular concentration of K+ rather than the intracellular concentration would be of importance for activation of the enzyme. The two catalytic components showing

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Km values in the range of 0.1–1 mM, respectively which we note here (Fig. 1, Table 2) would be quite compatible with efficient translocation of K+ with the reported extra-cellular K+ concentration which is in the range of 10–20 mM [17,34]. Thus, the reported Km ’s in the range of 2–5 mM [5] for K+ would once again seem to be significantly higher for efficient functioning of enzyme. The reasons for these discrepancies [5] are not clear at this stage. However, it is possible that the previously reported values were based on studies using whole cell extracts or enzyme mutated for ouabain sensitivity [5]. In the present studies we carried out measurement with purified synaptic membranes and therefore we believe that our values are more close to the normal physiological Km ’s for ATP and the three cations, viz. Na+ , K+ and Mg2+ for the enzyme as it exists in the membrane environment in situ. Our studies also indicate that besides affecting the Km and Vmax , even the binding of ATP, Na+ , K+ and Mg2+ was also differentially affected by hypothyroidism (Table 3). Regulation of Na+ , K+ -ATPase is a complex process controlled by several factors. These include the subunit composition, stoichiometry of the subunits and interaction of the enzyme with membrane proteins [5,7,20]. As is well documented, the enzyme comprises ␣, ␤ and ␥ subunits [5,7,20]. The ␣ is the catalytic subunit while ␤ functions as the regulatory subunit [5,7,20]. Four isoforms of ␣ (␣1–4) and three isoforms of ␤ (␤1–3) subunits have been reported [5]. The ␥ subunit ‘FIXIT’ plays a crucial role in the anchoring of the enzyme in the membrane [7]. As cited in the literature, the ␣3 is the major subunits in the brain although presence of ␣1 and ␣2 in cell subtypes has been reported [27]. Interestingly, ␣2 seems to be the major subunit in the neuronal cells [5]. Likewise, ␤1 seems to be the major subunit in the CNS although ␤2 is also present in specific cell types [5]. Subcellular localization for ␣ and ␤ subunit isoforms is not clear at this stage [5]. The different isoforms of ␣ and ␤ subunits seem to be regulated differentially in the tissue specific manner by the thyroid hormones [9,15,25]. Schmitt and Mc Donough reported that during postnatal development T3 regulates the ␣+ isoform of the enzyme between the 15th and 25th postnatal days but not in the adults [29]. Nomura et al. [24] reported that the number of ouabain binding sites decreased in the cerebral cortex and the cerebellum of hypothyroid rats and significantly increased in response to T3 treatment. Chaudhury et al. [6] reported that in the developing rat brain the ␣3 mRNA is expressed as a major component and that the expression is severely reduced in hypothyroidism. Treatment with 200 ␮g T3 /100 g body weight stimulated expression of all isoforms of ␣ subunits [6]. Interestingly, in the skeletal muscles, thyroid hormones seem to regulate the ␣2 isoform [2]. Thyroid hormone sensitivity of the isoforms of the Na+ , K+ -ATPase subunits in the glial cell has also been reported [3]. Besides, as referred to above the thyroid hormones also regulate synthesis of several membrane and cytoskeleton proteins [1,22,36]. In view of this it may be suggested that a dual mechanism i.e. altered membrane milieu and/or altered subunit composition may be responsible for the observed changes in the kinetic properties. Additionally, it is very well recognized that the thyroid hormones also regulate the lipid/phospholipid

biosynthesis and thereby the membrane lipid/phospholipid composition [4,38]. Dependence of membrane ATPases on specific phospholipid classes is well recognized [27]. Thus, altered membrane lipid/phospholipid milieu could be another regulatory factor. Besides, the efficiency of the enzyme can get further impaired due to increased Km for ATP and decreased ATP synthesis in the hypothyroid brain [18,19,38]. In the light of the above, it would be interesting to evaluate by direct experiments using isoform specific antibodies, if compositional changes are responsible for the observed changes in the kinetic properties that we report here. In conclusion our results suggest that hypothyroidism induced impairment in the function of Na+ , K+ -ATPase can disturb the cellular ionic homeostasis. This in turn could result in impaired nerve transmission and cognitive functions. References [1] A.M. Arnold, G.W. Anderson, B. McIver, N.L. Eberhardt, A novel dynamin III isoforms is up-regulated in the central nervous system in hypothyroidism, Int. J. Dev. Neurosci. 21 (2003) 267–275. [2] K.K. Azuma, C.B. Hensley, M.J. Tang, A.A. McDonough, Thyroid hormone specifically regulates skeletal muscle Na(+)-K(+)-ATPase alpha 2and beta 2-isoforms, Am. J. Physiol. 265 (1993) C680–C687. [3] B. Banerjee, S. Chaudhury, Thyroidal regulation of different isoforms of NaKATPase in glial cells of developing rat brain, Life Sci. 69 (2001) 2409–2417. [4] C.S. Bangur, J.L. Howland, S.S. Katyare, Thyroid hormone treatment alters phospholipid composition and membrane fluidity in rat brain mitochondria, Biochem. J. 305 (1995) 29–32. [5] G. Blanco, R. Mercer. Isozymes of the Na+ K+ -ATPase: heterogeneity in structure, diversity in function. Am. J. Physiol. 275 (Renal Physiol 44) (1998) F633–F650. [6] S. Chaudhury, M. Bajpai, S. Bhattacharya, Differential effects of hypothyroidism on Na- K-ATPase mRNA alpha isoforms in the developing rat brain, Mol. Neurosci. 7 (1996) 229–234. [7] H. Cornelius, Y.A. Mahammoud, Functional modulation of the sodium pump: the regulatory protein “Fixit”, News Physiol. Sci. 18 (2003) 119–124. [8] K.R. Dave, A.R. Syal, S.S. Katyare, Tissue cholinesterases. A comparative study of their kinetic properties, Z. Naturforsch. 55c (2000) 100–108. [9] V. Desai-Yajnik, J. Zeng, K. Omori, J. Herman, T. Morimoto, The effect of thyroid hormone treatment on the gene expression and enzyme activity of rat liver sodium-potassium dependent adenosine triphosphatase, Endocrinology 136 (1995) 629–639. [10] M. Dixon, C. Webb, Enzymes, Longman, London, 1979, pp. 47–206. [11] T. Esaki, H. Suzuki, M. Cook, K. Shimoji, S.Y. Chang, L. Sokoloff, J. Nunez, Functional activation of cerebral metabolism in mice with mutated thyroid hormone nuclear receptors, Endocrinology 144 (2003) 4117–4122. [12] C.H. Fiske, Y. Subba Row, Colorimetric determination of phosphorus, J. Biol. Chem. 66 (1925) 375–381. [13] M.E. Gilbert, C. Paczkowski, Propylthiouracil (PTU)-induced hypothyroidism in the developing rat impairs synaptic transmission and plasticity in the dentate gyrus of the adult hippocampus, Brain Res. Dev. Brain Res. 145 (2003) 19–29. [14] M.E. Gilbert, Alterations in synaptic transmission and plasticity in area CA 1 of adult hippocampus following developmental hypothyroidism, Brain Res. Dev. Brain Res. 148 (2004) 11–18. [15] C.B. Hensley, M.M. Bersohn, J.S. Sarma, B.N. Singh, M.A. Mc Donough, Amiodarone decreases Na, K-ATPase alpha 2 and beta 2 expression specifically in cardiac ventricle, J. Mol. Cell. Cardiol. 26 (1994) 417–424.

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