Journal of Thyroid Research

Volume 2013 (2013), Article ID 457953, 9 pages

http://dx.doi.org/10.1155/2013/457953

Research Article

Mechanisms of L-Triiodothyronine-Induced Inhibition of Synaptosomal Na⁺-K⁺-ATPase Activity in Young Adult Rat Brain Cerebral Cortex

Pradip K. Sarkar,1,2,3 Avijit Biswas,² Arun K. Ray,³ and Joseph V. Martin²

¹Department of Basic Sciences, Parker University, 2500 Walnut Hill Lane, Dallas, TX 75229, USA

²Center for Computational & Integrative Biology, Rutgers University, 315 Penn Street, Camden, NJ 08102, USA

³Department of Molecular Medicine, Bose Institute, P-1/12, CIT, Scheme VII-M, Calcutta 700054, India

Received 29 June 2013; Revised 19 September 2013; Accepted 24 September 2013

Academic Editor: Noriyuki Koibuchi

Copyright © 2013 Pradip K. Sarkar et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

The role of thyroid hormones (TH) in the normal functioning of adult mammalian brain is unclear. Our studies have identified synaptosomal Na⁺-K⁺-ATPase as a TH-responsive physiological parameter in adult rat cerebral cortex. L-triiodothyronine (T₃) and L-thyroxine (T₄) both inhibited Na⁺-K⁺-ATPase activity (but not Mg²⁺-ATPase activity) in similar dose-dependent fashions, while other metabolites of TH were less effective. Although both T₃ and the β-adrenergic agonist isoproterenol inhibited Na⁺-K⁺-ATPase activity in cerebrocortical synaptosomes in similar ways, the β-adrenergic receptor blocker propranolol did not counteract the effect of T₃. Instead, propranolol further inhibited Na⁺-K⁺-ATPase activity in a dose-dependent manner, suggesting that the effect of T₃ on synaptosomal Na⁺-K⁺-ATPase activity was independent of β-adrenergic receptor activation. The effect of T₃ on synaptosomal Na⁺-K⁺-ATPase activity was inhibited by the -adrenergic agonist clonidine and by glutamate. Notably, both clonidine and glutamate activate -proteins of the membrane second messenger system, suggesting a potential mechanism for the inhibition of the effects of TH. In this paper, we provide support for a nongenomic mechanism of action of TH in a neuronal membrane-related energy-linked process for signal transduction in the adult condition.

1. Introduction

Thyroid hormones (TH) exert major influences on the growth and development of the mammalian brain through specific nuclear receptor-mediated gene expression. Although several different isoforms of nuclear receptors for TH have been described in adult mammalian brain, their physiological function is quite unclear [1–4]. Still, adult onset of dysthyroidism develops a number of functional, neurological and psychological manifestations in humans [5–7]. In contrast to the developing brain, most of the changes resulting from hormone variations in the adult condition are reversible with the proper adjustment of circulatory TH [5–7].

Recent evidence has demonstrated that L-triiodothyronine (T₃) is distributed, concentrated, and metabolized in the synaptosomal fraction of adult rat cerebral cortex [5, 8, 9]. Specific T₃-binding sites have also been described in cerebrocortical synaptosomes [10, 11] and a graded binding of T₃ to its synaptosomal receptor binding sites has been correlated with the corresponding inhibition of the Na⁺-K⁺-ATPase activities in adult rat brain [11]. TH rapidly alters in vitro phosphorylation of synaptosomal proteins in a dose-dependent fashion [12]. TH levels are also altered in adult rat brain in different thyroid conditions [9]. TH enhances calcium entry in adult rat brain synaptosomes [13–15], in hypothyroid mouse brain [16], and in single rat myocytes [17].

However, there is a lack of clear understanding of the mechanism(s) of action of TH in the regulation of synaptic functions in adult neurons. The present study investigates the pathways of T₃-mediated signaling from its binding to the synaptosomal membrane receptors to the subsequent activation of second messenger system components that ultimately affect the further downstream effector molecule, the Na⁺-K⁺-ATPase. In this paper, we hypothesize a nongenomic mechanism of action of TH in neuronal membrane-related energy-linked process(es) for signal transduction in adult condition. We have used - and -adrenoceptor agonists and antagonists for modulation of the activity of Gs- and Gi-proteins of the membrane adenylate cyclase system. Portions of this work have appeared elsewhere in a preliminary form [18, 19].

2. Materials and Methods

2.1. Materials

The following compounds were purchased from Sigma Chemical Company, USA: bovine serum albumin (BSA), clonidine hydrochloride (CLO), disodium-ATP, isoproterenol hydrochloride (ISO), 2-mercaptoethanol, ouabain octahydrate, prazosin hydrochloride (PRA), phenylephrine hydrochloride (PHE), propranolol hydrochloride (PROP), sodium glutamate, 3,5,3′-L-triiodothyronine (T₃), L-thyroxine (T₄), 3,3′,5′-L-triiodothyronine (reverse T₃ or r-T₃), 3,5-L-diiodothyronine (T₂), Tris-ATP, yohimbine hydrochloride (YOH), dibutyryl 3′,5′-cyclic adenosine monophosphate sodium salt (DB cAMP), and sodium orthovanadate.

2.2. Treatment of Animals

Adult male Charles Foster rats (3 months old) were housed at in 12 h dark-12 h light conditions and fed ad libitum with standard rat diet and water. The animals were sacrificed by quick decapitation and the brains were removed into ice-cold 250 mM sucrose solution. The cerebral cortices were dissected out for synaptosomal fraction preparation.

2.3. Preparation of Synaptosomes

The synaptosomes from thecerebral cortex were prepared as described previously [20]. Briefly, the cerebral cortex was homogenized (10% weight/volume) in 0.32 M sucrose and centrifuged at 1000 g for 10 minutes to remove cell debris and nuclei. The supernatant was collected and recentrifuged at 1000 g for another 10 min. The resulting pellet was discarded and the supernatant was layered over 1.2 M sucrose and centrifuged at 34,000 g for 50 min at 4°C. The fraction collected between the 0.32 M and 1.2 M sucrose layer was diluted at 1 : 1.5 with ice-cold bidistilled water, further layered on 0.8 M sucrose, and again centrifuged at 34,000 g for 30 min. The pellet thus obtained was washed and repelleted at 20,000 g for 20 min. Synaptosomal pellets were lysed by suspending in ice-cold bidistilled water to release the occluded Na⁺-K⁺-ATPase activity.

2.4. Assay of Synaptosomal Na⁺-K⁺-ATPase Activity

Synaptosomal Na⁺-K⁺-ATPase activity was assayed as ouabain-sensitive ATP hydrolysis in reaction mixtures of (i) 30 mM imidazole-HCl, 130 mM NaCl, 20 mM KCl, and 4 mM MgCl₂ and (ii) 30 mM imidazole-HCl, 4 mM MgCl₂, and 1 mM ouabain, at pH 7.4. Both the reaction media (i) and (ii) were first preincubated in vitro with or without simultaneous addition of various concentrations of thyroid hormones (T₃, T₄) and TH-analogue (T₂) (0.001 nM to 1 μM), adrenergic drugs (1 nM for ISO, PRA, PHE and YOH; 1 nM–100 nM for CLO and PROP), glutamate (100 μM), DB cAMP (1 μM–5 mM), and sodium orthovanadate (10 nM–2 mM) followed by addition of the synaptosomal lysates, each containing 20–50 μg synaptosomal protein, at 0°C for 60 minutes in dark. To get a steady-state ouabain binding, both the assay media (i) and (ii), with and without ouabain, respectively, as described above, were preincubated for 60 min at 0°C in the dark, followed by a 5-min incubation at 37°C to equilibrate the temperature. The reaction was started by adding 4 mM Tris-ATP and incubated at 37°C for 10 min. An aliquot of 100 μL of 10% sodium dodecylsulfate was added to stop the enzymatic reaction. The inorganic phosphate () formed was determined in the reaction mixture [21]. Na⁺-K⁺-ATPase activity was calculated as difference in the Pi content between media (i) and (ii) and expressed as μmols Pi/h/mg protein [22]. The ouabain-sensitive portion of the total ATPase (Na⁺-K⁺-Mg²⁺-ATPase) was determined from the Pi released in the medium (i) minus that in medium (ii). The Pi released from the reaction medium (ii) was used for determination of the synaptosomal Mg²⁺-ATPase activity. Synaptosomal Mg²⁺-ATPase activity, therefore, was assayed as the ouabain-insensitive ATP hydrolysis.

2.5. Measurement of Protein

Synaptosomal protein content was measured using bovine serum albumin as a standard [23].

2.6. Statistical Analysis

Results are expressed as the mean ± SEM of 3-4 separate experiments or as mentioned. Each experiment was made from six rats. The statistical analysis of the data was performed by Student’s t-test, considering as the significance level. The data for multiple groups were also analyzed by one-way ANOVA followed by Student Newman-Keuls post-hoc comparisons using Sigmastat software. Nonlinear regression analysis was performed using GraphPad Prism software.

3. Results

3.1. Effects of T₃ and Metabolites on Na⁺-K⁺-ATPase Activity

In vitro addition of various doses of T₃ to the synaptosomal fraction (which is devoid of cell nuclei) confirmed our previous observation [11] and showed nearly the same trend of a dose-dependent inhibition (IC₅₀ =  pM; maximal inhibition = % at 95% confidence levels) of Na⁺-K⁺-ATPase activity. No significant effect of T₃ was noticed on the Mg²⁺-ATPase specific activity (Figure 1). T₄ had a similar inhibitory effect as T₃ on Na⁺-K⁺-ATPase activity (IC₅₀ =  pM; maximal inhibition = %), while T₂ had minimal effects (Figure 2). Furthermore, the same range of doses (10⁻¹²–10⁻⁸ M) of r-T₃ did not inhibit either Na⁺-K⁺-ATPase or Mg⁺²-ATPase activities (data not shown).

Figure 1: Inhibitory effect of various doses (0.001 nM–100 nM) of T₃ on synaptosomal Na⁺-K⁺-ATPase or Mg²⁺-ATPase activity, in vitro. The data are represented as mean ± SEM of ten separate experiments, taking six animals in each group. The vertical lines denote SEM. Filled circles indicate Na⁺-K⁺-ATPase while filled triangles indicate Mg²⁺-ATPase activity.

Figure 2: Inhibitory effect of various doses (0.001 nM–10 nM) of T₄ or T₂ on synaptosomal Na⁺-K⁺-ATPase activity, in vitro. The data are represented as mean ± SEM of four separate experiments, taking six animals in each group. The vertical lines denote SEM. Filled circles indicate effects of T₄ on Na⁺-K⁺-ATPase activity while filled squares indicate effects of T₂.

3.2. Effect of T₃ and β-Adrenergic Agonists/Antagonists on Na⁺-K⁺-ATPase Activity

Equimolar doses (1 nM) of T₃ and the nonselective β-adrenergic agonist ISO were added separately in vitro, inhibited the Na⁺-K⁺-ATPase enzyme activity by 41.3% and 42.6%, respectively (Figure 3). The nonselective β-adrenergic antagonist PROP alone did not alter the enzyme activity at different doses (1 nM, 10 nM, and 100 nM). The inhibitory action of ISO (1 nM) on the Na⁺-K⁺-ATPase activity was counteracted by PROP (1 nM), whereas PROP could not block T₃-mediated inhibition of the enzyme activity. Instead PROP potentiated the T₃-mediated inhibition of the enzyme activity in a dose-dependent manner. Significant differences in the potentiation of the T₃ effect (1 nM) by PROP on Na⁺-K⁺-ATPase activity were noticed between 1 nM and 100 nM () and between 10 nM and 100 nM () doses (Figure 3).

Figure 3: Effect of T₃ on synaptosomal Na⁺-K⁺-ATPase activity and its modulation by a β-adrenergic receptor agonist (ISO) and a β-adrenergic receptor antagonist (PROP) in vitro. A half-maximally effective dose of T₃ (1 nM) was chosen from the dose-response curve for T₃ in Figure 1. The data are represented as mean ± SEM of five separate experiments taking six animals in each group. , compared to the control group. and , compared to T₃ (1 nM) + PROP (100 nM) group (one-way ANOVA followed by Newman-Keuls test). The vertical lines denote SEM.

3.3. Effects of T₃ and α-Adrenergic Agonists/Antagonists on Na⁺-K⁺-ATPase Activity

The effects of in vitro addition of 1 nM doses of PHE (selective -adrenergic receptor agonist) and PRA (-adrenergic receptor antagonist) on synaptosomal Na⁺-K⁺-ATPase activity or Mg²⁺-ATPase activity were minimal (Figure 4). Furthermore, 1 nM doses of PHE or PRA did not alter the inhibitory effect of 1 nM T₃ on Na⁺-K⁺-ATPase activity, nor did it change the Mg²⁺-ATPase activity, in vitro (Figure 4).

(a)

(b)

Figure 4: Modulation of the T₃ action on synaptosomal Na⁺-K⁺-ATPase activity by a selective -adrenergic agonist (PHE) and a selective -antagonist (PRA) in vitro. A half-maximally effective dose of T₃ (1 nM) was chosen from the dose-response curve for T₃ in Figure 1. The doses for PHE and PRA used for the in vitro experiment were 1 nM in each case. The data are represented as mean ± SEM of five separate experiments, taking six animals in each group. The vertical lines denote SEM.

Similarly, in vitro addition of CLO (-adrenergic agonist) at different doses did not elicit significant changes in the synaptosomal Na⁺-K⁺-ATPase activity (Figure 5). However, when CLO was added in the presence of an equimolar dose of T₃, the inhibitory effect of T₃ on the Na⁺-K⁺-ATPase activity was completely counteracted. The effect of T₃ on the enzyme activity remained prominent at a 100 nM dose of T₃ (100 nM T₃:  μmols Pi/h/mg protein; Control:  μmols Pi/h/mg protein) along with 1 nM CLO (100 nM T₃ + 1 nM CLO:  μmols Pi/h/mg protein); however, 1 nM CLO attenuated the effect of T₃ (100 nM) by 32% more towards the control value (data not shown graphically). The -adrenergic receptor antagonist YOH also inhibited synaptosomal Na⁺-K⁺-ATPase activity (Figure 5). Inhibition of the enzyme activity in the presence of both 1 nM T₃ and 1 nM YOH was found to be intermediate between the levels of inhibition by either compound alone, although there were no significant differences between these groups (Figure 5).

Figure 5: Modulation of the T₃ action on synaptosomal Na⁺-K⁺-ATPase activity by an -adrenergic agonist (CLO) and an -adrenergic antagonist (YOH) in vitro. The data are represented as mean ± SEM of six separate experiments taking six animals in each group. , compared to the control group (one-way ANOVA followed by Newman-Keuls test). The vertical lines denote SEM.

3.4. Effect of T₃ and Glutamate on Na⁺-K⁺-ATPase Activity

In vitro addition of 100 μM glutamate alone did not alter the synaptosomal Na⁺-K⁺-ATPase activity compared to control values, whereas, addition of 100 μM glutamate showed complete attenuation of T₃ (10 nM)-mediated inhibition of synaptosomal Na⁺-K⁺-ATPase activity in adult rat cerebral cortex (Figure 6). A higher dose of T₃ (10 nM) was chosen, in order to test the effect of glutamate against a greater inhibitory action on the Na⁺-K⁺-ATPase activity.

Figure 6: Modulatory effect of glutamate on T₃-induced inhibition of synaptosomal Na⁺-K⁺-ATPase activity in cerebral cortex, in vitro. A higher dose of T₃ (10 nM) was chosen from the T₃ dose-response curve, considering its greater inhibitory action on the Na⁺-K⁺-ATPase activity and to observe the effect of 100 μM glutamate on this T₃-induced inhibition. The data are represented as mean ± SEM of four separate experiments taking six animals in each group. , compared to the control group (one-way ANOVA followed by Newman-Keuls test).

3.5. Effect of DB cAMP and T₃ on Na⁺-K⁺-ATPase Activity

To study the effect of DB cAMP on modulation of Na⁺-K⁺-ATPase activity by T₃, first a dose response experiment with various concentrations of DB cAMP (0.001 mM to 5 mM) was performed. In vitro addition of DB cAMP showed a typical sigmoidal curve with gradual decrease in the Na⁺-K⁺-ATPase activity to a maximal inhibition at 0.2 mM (Figure 7(a)). From this standardization, we chose to use a 0.2 mM final concentration of DB cAMP for further experiments. In vitro addition of DB cAMP (0.2 mM) with and without various doses of T₃ (0.001 nM–10 nM) was examined for effects on Na⁺-K⁺-ATPase activity (Figure 7(b)). T₃-induced inhibition of synaptosomal Na⁺-K⁺-ATPase activity was further depressed in the presence of 0.2 mM DB cAMP. However, the two curves appeared to converge at the highest doses of T₃.

(a)

(b)

Figure 7: Influence of DB cAMP and T₃ on synaptosomal Na⁺-K⁺-ATPase activity, in vitro. (a) Inhibitory effect of various doses of DB cAMP on synaptosomal Na⁺-K⁺-ATPase activity, in vitro. The data are represented as mean ± SEM of four separate experiments, taking six animals in each group. The vertical bars denote SEM. (b) Interaction of the effects of of DB cAMP and T₃ on synaptosomal Na⁺-K⁺-ATPase activity, in vitro. Filled squares indicate effects of graded doses of T₃ (0.001 nM–10 nM) alone on Na⁺-K⁺-ATPase activity while filled triangles indicate effects of the 0.2 mM dose of DB cAMP with graded doses of T₃ (0.1 pM–1 μM).

3.6. Influence of Sodium Orthovanadate on Modulation of Na⁺-K⁺-ATPase Activity by T₃

The in vitro effect of sodium orthovanadate, a protein tyrosine phosphatase inhibitor, was examined in cerebrocortical synaptosomes. The cerebrocortical synaptosomes were treated with a fixed dose of T₃ (10 nM) with or without different doses of sodium -vanadate (Figure 8). A higher dose of T₃ (10 nM) was chosen from the T₃ dose-response curve, considering its greater inhibitory action on the Na⁺-K⁺-ATPase activity. T₃ caused an inhibition of Na⁺-K⁺-ATPase specific activity, and this effect was enhanced by sodium orthovanadate in a dose-dependent way. In general, the effects of sodium orthovanadate and T₃ appeared to be additive until the Na⁺-K⁺-ATPase specific activity was completely inhibited.

Figure 8: Modulation of the T₃ action on synaptosomal Na⁺-K⁺-ATPase activity by sodium orthovanadate. A higher dose of T₃ (10 nM) was chosen from the T₃ dose-response curve considering its greater inhibitory action on the Na⁺-K⁺-ATPase activity and to observe the effect of graded doses (1 nM–2 mM) of sodium orthovanadate on this T₃-induced inhibition. The data are represented as mean ± SEM of four separate experiments, taking six animals in each group. The vertical bars denote SEM. Open circles indicate mean values for a set of control incubations without T₃. Filled triangles indicate the results of a set of incubations with 10 nM T₃.

4. Discussion

The objective of the present investigation was to search for possible mechanisms for the inhibition by TH of synaptosomal Na⁺-K⁺-ATPase activity in adult rat cerebral cortex.

Initial studies examined the specificity of the effect according to the pattern of iodination of the hormone derivatives (Figures 1 and 2). In vitro inhibitory effect of T₃ on synaptosomal Na⁺-K⁺-ATPase activity supported our previous observation and showed nearly the same trend of a dose-dependent inhibition of Na⁺-K⁺-ATPase activity [11]. In addition to our earlier report, the current study showed an insignificant effect of T₃ on the synaptosomal Mg²⁺-ATPase specific activity (Figure 1). In vitro addition of T₄ also indicated similar pattern of inhibitory influence on the synaptosomal Na⁺-K⁺-ATPase activity, like the effect of T₃, with no significant changes on the Mg²⁺-ATPase activity. The effects of TH on Na⁺-K⁺-ATPase activity seemed to be specific for compounds with 2 iodine atoms on the inner ring, as T₂ and r-T₃ were without activity in the current studies. T₃ was less potent than T₄. It is consistent with reports of the relative affinities of the two compounds for a cell surface receptor, integrin known to mediate a variety of nongenomic effects of THs [24].

Binding of T₄ to integrin causes internalization of the receptor and nongenomically promotes phosphorylation of mitogen-activated protein kinase/extracellular regulated kinase 1 and 2 (MAPK/ERK1/2) in the CV-1 line of monkey fibroblasts [24]. A similar mechanism seems likely in chick chorioallantoic membrane [25]. Following the internalization of the integrin , the monomer is translocated to the nucleus, where it may transcriptionally regulate expression of protein [26]. TH causes lungs to rapidly (within hours) increase alveolar fluid clearance [27] and to express increased Na⁺-K⁺-ATPase protein by a MAPK/ERK1/2-dependent pathway [28]. Note, however, that the current finding of an immediate effect to decrease Na⁺-K⁺-ATPase activity could not be due to a mechanism involving transcriptional regulation, since the synaptosomal preparation is devoid of cell nuclei. It is also suggested that some of the effects of T₃ stimulation of the integrin could be more direct than the nuclear interaction [29].

A potential mechanism for the inhibitory effects of TH in the present study might be the regulation of phosphorylation of Na⁺-K⁺-ATPase or a modulatory molecule. It is well known that catecholamine-mediated phosphorylation of Na⁺-K⁺-ATPase inhibits enzymatic activity in Chinese hamster ovary (CHO) cells, but not through a process of internalization of the enzyme [30–32]. Intriguingly in this respect, one of the proteins found to be phosphorylated at the tyrosyl residue in synaptosomes treated for 5 s with TH had a molecular weight of 95 kD [12], matching the size of -subunit of Na⁺-K⁺-ATPase [33].

The significant inhibition of the synaptosomal Na⁺-K⁺-ATPase activity in vitro by T₃ confirmed our previous in vivo observations [22]. In order to characterize this inhibitory influence of THs on the synaptosomal membrane, we intended to study the effect of adrenergic receptor agonists and antagonists which regulate guanine nucleotide binding proteins (G-proteins) via their activating and inhibitory actions. Both T₃ and ISO (-adrenergic receptor agonist) showed an analogous but independent (parallel) inhibitory effect on the enzyme activity (Figure 3). ISO-induced inhibition of Na⁺-K⁺-ATPase activity was blocked by PROP (-adrenergic receptor blocker) indicating that the synaptosomal membrane interaction with ISO was likely a -adrenoceptor-mediated event, potentially coupled to Gs-protein. However, PROP was completely unable to block T₃-mediated inhibition of synaptosomal Na⁺-K⁺-ATPase activity. This clearly indicated that T₃-mediated inhibition of the enzyme activity was not coupled to -adrenoceptor, but rather, may have had a similar effect through another kind of receptor. The augmentation of the T₃ effect by PROP appeared to be a type of synergistic action, the mechanism of which remains unclear at present. Increased activity of adenylate cyclase caused by THs, independent of propranolol blockade, has been shown in cultured cerebral cells from embryonic mice, suggesting that the effect of T₃ was not mediated through a -adrenergic-dependent system [34]. The T₃-induced increase in sodium current in neonatal rat myocytes also could not be blocked by PROP, whereas it was antagonized by amiodarone, a nonspecific blocker of -adrenoceptor, suggesting that the effects were not mediated through -adrenergic signaling pathways [35]. However, -adrenoceptor blockade by chronic subcutaneous delivery of PROP for 14 days has been shown to downregulate levels of TH receptor TR -mRNA and -mRNA in mouse heart, which may influence the genomic effect of the hormone [36].

Next, we wanted to check for the role of an -adrenergic receptor agonist and antagonist. Agonists for the -adrenergic receptor mediate their actions through Gq protein followed by activation of phospholipase C and subsequent production of the second messengers inositol triphosphate and diacylglycerol, an activator of protein kinase C [37]. Neither PHE (selective agonist) nor PRA ( antagonist) had an influence on Na⁺-K⁺-ATPase activity. Furthermore, neither compound interacted with the effects of T₃. Mg²⁺-ATPase activity remained unaltered when treated with either of these -adrenergic drugs (agonist and antagonist) and T₃, alone or in combination (Figure 4). These results suggest that the effects of T₃ on Na⁺-K⁺-ATPase activity do not share common mechanisms with -receptors.

On the other hand, CLO, an -adrenergic receptor agonist (Figure 5), and glutamate (Figure 6), possibly acting via a metabotropic glutamate receptor (mGluR), blocked T₃-induced inhibition of Na⁺-K⁺-ATPase activity. Neither CLO nor glutamate showed any significant effect on the Na⁺-K⁺-ATPase activity in rat hippocampus and frontal cortex homogenates [38]. One possibility would be that the counteraction of the effect of T₃ on synaptosomal Na⁺-K⁺-ATPase by CLO and glutamate might be mediated through the inhibition of adenylate cyclase activity with the activation of inhibitory G-protein (Gi) followed by the inhibition of cAMP synthesis and the protein phosphorylation cascade mechanism. It is well known that -adrenergic agonists act through stimulation of Gi-protein [18, 19, 39, 40].

Association of the glutamate transporter with Na⁺-K⁺-ATPase in synaptosomes has been implicated by their correlated regulation via protein kinases [41]. Glutamate also has been reported to inhibit adenylate cyclase activity in rat hippocampal synaptosomes [39, 40, 42, 43], as well as in striatal and cerebrocortical neurons, both in intact cells and membranes [40] via metabotropic glutamate receptors (mGluRs), which are coupled to effector systems through GTP binding proteins. In fact, in the nucleus tractus solitarius of adult brain, it was shown that an antibody to the Gi inhibited the effects of mGluRs [44]. mGluR₁ and mGluR₅ subtypes are coupled to phosphatidyl inositol hydrolysis/Ca²⁺-signal transduction. mGluR₁ has also been shown to stimulate release of arachidonic acid and to increase cAMP formation. The mGluR₂, mGluR₃, mGluR₄, and mGluR₅ subtypes appear to be coupled to inhibition of cAMP synthesis, but differ in their agonist selectivity. mGluR₂ and mGluR₃ mRNAs are highly expressed in the cerebral cortex [40, 42, 43]. Activation of mGluR has been shown to counteract -adrenoceptor-mediated inhibition of afterhyperpolarization in hippocampal neurons of the CA1 area. This has been suggested to be by mGluR-mediated activation of protein kinase C, which inhibited adenylate cyclase pathways [42, 43]. The physiological functions of these mGluRs are still being clarified. Thus, T₃ action in adult rat synaptosomal membrane, ultimately to inhibit the effector molecule Na⁺-K⁺-ATPase, might be mediated through Gs stimulation. mGlu receptors may then have some regulatory roles in counteracting T₃-induced action.

Our observation showed that DB cAMP (a nonhydrolyzable form of cAMP and activator of cAMP-dependent protein kinases) had a T₃-like effect on Na⁺-K⁺-ATPase activity (Figure 7). Furthermore, the in vitro addition of increased doses of T₃ lowered the slope of the dose-response curve for DB cAMP. Such a finding might be consistent with a related mechanism for the effects of DB cAMP and T₃, and would not represent merely additive effects of two distinct mechanisms. Our previous observations suggested that the phosphorylation status of certain synaptosomal proteins could be mediated via cAMP- and/or Ca²⁺-dependent pathways [12, 45]. A differential stoichiometry of phosphorylation of the -subunit of the Na⁺-K⁺-ATPase by protein kinase A and protein kinase C has been shown to inhibit this enzymatic activity in shark rectal gland, rat renal cortex, and basolateral membrane vesicles from rat renal cortex [46].

The effect of the protein tyrosine phosphatase inhibitor sodium orthovanadate [47] appeared to be additive to the effect of T₃, implying that there could be a separate mechanism of action of the two compounds (Figure 8). Since vanadate is a blocker of tyrosine phosphatase activity, it also could be speculated that T₃-induced inhibition of Na⁺-K⁺-ATPase activity is further suppressed by synergistic action by vanadate via keeping the enzyme in its phosphorylated form, causing inhibition of its activity. A point to note here is that the -subunit is the catalytic subunit and its phosphorylation causes inhibition of this enzyme [46]. T₃ appears not to have the inhibitory effect on Na⁺-K⁺-ATPase activity by an influence on phosphatase activity.

5. Conclusion

Our results regarding T₃ action in relation to the inhibition of synaptosomal Na⁺-K⁺-ATPase are consistent with a T₃-synaptosomal membrane component binding site interaction sensitive to the activation of Gi-protein. Such a membrane binding component might interact with a Gs-protein, resulting in increased synthesis of cAMP. The membrane Na⁺-K⁺-ATPase is involved in several aspects of physiological processes. In the neuron, its inhibition is linked with neurotransmitter release [46]. Hence, the present study provides further evidence of a nongenomic membrane-related action of T₃ in the mature mammalian synaptosome. Understanding of the mechanism of action of TH in adult mammalian brain has major implications in the higher mental functions and in the regulation of several neuropsychiatric disorders developed in thyroid dysfunctions in adult humans.

Acknowledgments

Financial support was from the Council of Scientific & Industrial Research, India, and the Department of Science & Technology, Government of India, to Pradip K. Sarkar, and NSF grant IBN-0110961 to Pradip K. Sarkar and Joseph V. Martin.

References

J. H. Dussault and J. Ruel, “Thyroid hormones and brain development,” Annual Review of Physiology, vol. 49, pp. 321–334, 1987.

J. Puymirat, “Thyroid receptors in the rat brain,” Progress in Neurobiology, vol. 39, no. 3, pp. 281–294, 1992.

J. H. Oppenheimer, “Evolving concepts of thyroid hormone action,” Biochimie, vol. 81, no. 5, pp. 539–543, 1999.

J. Zhang and M. A. Lazar, “The mechanism of action of thyroid hormones,” Annual Review of Physiology, vol. 62, pp. 439–466, 2000.

M. B. Dratman and J. T. Gordon, “Thyroid hormones as neurotransmitters,” Thyroid, vol. 6, no. 6, pp. 639–647, 1996.

W. N. Henley and T. J. Koehnle, “Thyroid hormones and the treatment of depression: an examination of basic hormonal actions in the mature mammalian brain,” Synapse, vol. 27, pp. 36–44, 1997.

R. Bunevicius and A. J. Prange, “Mental improvement after replacement therapy with thyroxine plus triiodothyronine: relationship to cause of hypothyroidism,” International Journal of Neuropsychopharmacology, vol. 3, no. 2, pp. 167–174, 2000.

G. A. Mason, C. H. Walker, and A. J. Prange, “L-triiodothyronine: is this peripheral hormone a central neurotransmitter?” Neuropsychopharmacology, vol. 8, no. 3, pp. 253–258, 1993.

P. K. Sarkar and A. K. Ray, “Synaptosomal T₃ content in cerebral cortex of adult rat in different thyroidal states,” Neuropsychopharmacology, vol. 11, no. 3, pp. 151–155, 1994.

Y. Mashio, M. Inada, and K. Tanaka, “Synaptosomal T₃ binding sites in rat brain: their localization on synaptic membrane and regional distribution,” Acta Endocrinologica, vol. 104, no. 2, pp. 134–138, 1983.

P. K. Sarkar and A. K. Ray, “Specific binding of L-triiodothyronine modulates Na⁺-K⁺-ATPase activity in adult rat cerebrocortical synaptosomes,” NeuroReport, vol. 9, no. 6, pp. 1149–1152, 1998.

P. K. Sarkar, N. D. Durga, J. J. Morris, and J. V. Martin, “In vitro thyroid hormone rapidly modulates protein phosphorylation in cerebrocortical synaptosomes from adult rat brain,” Neuroscience, vol. 137, no. 1, pp. 125–132, 2006.

P. K. Sarkar and A. K. Ray, “Calcium mobilization within hypothyroid adult rat brain synaptosomes,” Hormone and Metabolic Research, vol. 35, no. 9, pp. 562–564, 2003.

G. A. Mason, C. H. Walker, and A. J. Prange Jr., “Depolarization-dependent ⁴⁵Ca uptake by synaptosomes of rat cerebral cortex is enhanced by L-triiodothyronine,” Neuropsychopharmacology, vol. 3, no. 4, pp. 291–295, 1990.

N. Chakrabarti and A. K. Ray, “Rise of intrasynaptosomal Ca²⁺ level and activation of nitric oxide synthase in adult rat cerebral cortex pretreated with 3-5-3′-L-triiodothyronine,” Neuropsychopharmacology, vol. 22, no. 1, pp. 36–41, 2000.

Z. Iqbal, H. Koenig, and J. J. Trout, “Triiodothyronine (T3) stimulates calcium influx and membrane trasport processes in nerve cells and terminals of hypothyroid mouse cortex,” Federation proceedings, vol. 43, p. 735, 2002.

R. B. Lomax, P. H. Cobbold, A. P. Allshire, K. S. R. Cuthbertson, and W. R. Robertson, “Tri-iodothyronine increases intra-cellular calcium levels in single rat myocytes,” Journal of Molecular Endocrinology, vol. 7, no. 1, pp. 77–79, 1991.

P. K. Sarkar and A. K. Ray, “Thyroid hormone and membrane-related processes in synaptosomes,” in Proceedings of the 60th Annual Meeting of Society of Biological Chemists (India), Section Endocrinology & Reproductive Biology, Abstract 147, 1991.

P. K. Sarkar, Thyroid hormone action in synaptosome of adult rat brain [Ph.D. thesis], University of Calcutta, Calcutta, India, 1995.

P. K. Sarkar and A. K. Ray, “A simple biochemical approach to differentiate synaptosomes and non-synaptic mitochondria from rat brain,” Methods and Findings in Experimental and Clinical Pharmacology, vol. 14, no. 7, pp. 493–497, 1992.

P. K. Sarkar, “A quick assay for Na⁺-K⁺-ATPase specific activity,” Zeitschrift fur Naturforschung C, vol. 57, no. 5-6, pp. 562–564, 2002.

P. K. Sarkar and A. K. Ray, “Synaptosomal action of thyroid hormone: changes in Na⁺-K⁺-ATPase activity in adult rat cerebral cortex,” Hormone and Metabolic Research, vol. 25, no. 1, pp. 1–3, 1993.

J. C. Vera, “Measurement of microgram quantities of protein by a generally applicable turbidimetric procedure,” Analytical Biochemistry, vol. 174, no. 1, pp. 187–196, 1988.

J. J. Bergh, H. Lin, L. Lansing et al., “Integrin αVβ3 contains a cell surface receptor site for thyroid hormone that is linked to activation of mitogen-activated protein kinase and induction of angiogenesis,” Endocrinology, vol. 146, no. 7, pp. 2864–2871, 2005.

F. B. Davis, S. A. Mousa, L. O'Connor et al., “Proangiogenic action of thyroid hormone is fibroblast growth factor-dependent and is initiated at the cell surface,” Circulation Research, vol. 94, no. 11, pp. 1500–1506, 2004.

A. B. Glinskii, G. V. Glinsky, H. Lin et al., “Modification of survival pathway gene expression in human breast cancer cells by tetraiodothyroacetic acid (tetrac),” Cell Cycle, vol. 8, no. 21, pp. 3554–3562, 2009.

M. Bhargava, M. R. Runyon, D. Smirnov et al., “Triiodo-L-thyronine rapidly stimulates alveolar fluid clearance in normal and hyperoxia-injured lungs,” American Journal of Respiratory and Critical Care Medicine, vol. 178, no. 5, pp. 506–512, 2008.

J. Lei, C. N. Mariash, M. Bhargava, E. V. Wattenberg, and D. H. Ingbar, “T₃ increases Na-K-ATPase activity via a MAPK/ERK1/2-dependent pathway in rat adult alveolar epithelial cells,” American Journal of Physiology—Lung Cellular and Molecular Physiology, vol. 294, no. 4, pp. L749–L754, 2008.

H. Y. Lin, H. Y. Tang, F. B. Davis et al., “Nongenomic regulation by thyroid hormone of plasma membrane ion and small molecule pumps,” Discovery Medicine, vol. 14, pp. 199–206, 2012.

X. Cheng, J. Höög, A. C. Nairn, P. Greengard, and A. Aperia, “Regulation of rat Na⁺-K⁺-ATPase activity by PKC is modulated by state of phosphorylation of Ser-943 by PKA,” American Journal of Physiology—Cell Physiology, vol. 273, no. 6, pp. C1981–C1986, 1997.

X. Cheng, G. Fisone, O. Aizman et al., “PKA-mediated phosphorylation and inhibition of Na⁺-K⁺-ATPase in response to β-adrenergic hormone,” American Journal of Physiology—Cell Physiology, vol. 273, no. 3, pp. C893–C901, 1997.

R. M. Andersson, S. X. J. Cheng, and A. Aperia, “Forskolin-induced down-regulation of Na⁺, K⁺-ATPase activity is not associated with internalization of the enzyme,” Acta Physiologica Scandinavica, vol. 164, no. 1, pp. 39–46, 1998.

W. L. Stahl and R. W. Albers, “The Na,K-ATPase of nervous tissue,” Neurochemistry International, vol. 8, no. 4, pp. 449–476, 1986.

S. G. Amur, G. Shanker, and R. A. Pieringer, “β-adrenergic stimulation of protein (arginine) methyltransferase activity in cultured cerebral cells from embryonic mice,” Journal of Neuroscience Research, vol. 16, no. 2, pp. 377–386, 1986.

C. Huang, H. M. Geller, W. L. Green, and W. Craelius, “Acute effects of thyroid hormone analogs on sodium currents in neonatal rat myocytes,” Journal of Molecular and Cellular Cardiology, vol. 31, no. 4, pp. 881–893, 1999.

S. Shahrara, C. Sylven, and V. Drvota, “Subtype specific downregulation of thyroid hormone receptor mRNA by β-adrenoreceptor blockade in the myocardium,” Biological and Pharmaceutical Bulletin, vol. 23, no. 11, pp. 1303–1306, 2000.

R. M. Graham, D. M. Perez, J. Hwa, and M. T. Piascik, “Alpha 1-adrenergic receptor subtypes. Molecular structure, function, and signaling,” Circulation Research, vol. 78, no. 5, pp. 737–749, 1996.

M. B. Conto and M. A. Venditti, “In vitro studies of the influence of glutamatergic agonists on the Na⁺, K⁺-ATPase and K⁺-p-nitrophenylphosphatase activities in the hippocampus and frontal cortex of rats,” Journal of Negative Results in BioMedicine, vol. 11, p. 12, 2012.

W. L. Stahl and W. E. Harris, “Na⁺,K⁺-ATPase: structure, function, and interactions with drugs,” Advances in Neurology, vol. 44, pp. 681–693, 1986.

A. Levitzki, “Signal transduction in hormone-dependent adenylate cyclase,” Cell Biophysics, vol. 12, pp. 133–143, 1988.

E. M. Rose, J. C. P. Koo, J. E. Antflick, S. M. Ahmed, S. Angers, and D. R. Hampson, “Glutamate transporter coupling to Na,K-ATPase,” The Journal of Neuroscience, vol. 29, no. 25, pp. 8143–8155, 2009.

M. A. Musgrave, M. A. Madigan, B. M. Bennett, and J. W. Goh, “Stimulation of postsynaptic and inhibition of presynaptic adenylyl cyclase activity by metabotropic glutamate receptor activation,” Journal of Neurochemistry, vol. 62, no. 6, pp. 2316–2324, 1994.

R. Nouranifar, R. D. Blitzer, T. Wong, and E. Landau, “Metabotropic glutamate receptors limit adenylyl cyclase-mediated effects in rat hippocampus via protein kinase C,” Neuroscience Letters, vol. 244, no. 2, pp. 101–105, 1998.

T. Endoh, “Characterization of modulatory effects of postsynaptic metabotropic glutamate receptors on calcium currents in rat nucleus tractus solitarius,” Brain Research, vol. 1024, no. 1-2, pp. 212–224, 2004.

P. K. Sarkar, “l-Triiodothyronine differentially and nongenomically regulates synaptosomal protein phosphorylation in adult rat brain cerebral cortex: role of calcium and calmodulin,” Life Sciences, vol. 82, no. 17-18, pp. 920–927, 2008.

A. M. Bertorello, A. Aperia, S. I. Walaas, A. C. Nairn, and P. Greengard, “Phosphorylation of the catalytic subunit of Na⁺,K⁺-ATPase inhibits the activity of the enzyme,” Proceedings of the National Academy of Sciences of the United States of America, vol. 88, no. 24, pp. 11359–11362, 1991.

J. A. Gordon, “Use of vanadate as protein-phosphotyrosine phosphatase inhibitor,” Methods in Enzymology, vol. 201, pp. 477–482, 1991.