*5.4.1. Thyroid hormones rapidly modulate synaptosomal protein phosphorylation via second messenger systems*

In other studies, L-T3 induction has also been shown to nongenomically regulate Ca2+ influx and nitric oxide synthase activity within seconds in adult rat brain [57]. Thus, THs are likely to have numerous rapid nongenomic effects on signaling mechanisms in neural tissue, including alterations in the levels of intracellular second messengers (cAMP and Ca2+) which regulate cAMP- and/or Ca2+/calmodulin (CaM)-dependent protein kinases leading to protein phosphorylation. Effects of TH on Ca2+-dependent activation of PKC and/or Ca2+/CaMdependent protein kinases are also possible, followed by direct or indirect activation of phosphorylation of the proteins. Thus further investigation demonstrated for the first time the rapid nongenomic second messenger mediated regulation of protein phosphorylation by TH in mature mammalian brain and provided additional support for the contention that TH has a unique and complex signaling function in adult brain [12].

## *5.4.1.1. Role of calcium and calmodulin on synaptosomal protein phosphorylation, in vitro*

Many nongenomic mechanisms are modulated by phosphorylation–dephosphorylation of substrate proteins. Multiple Ca2+/calmodulin (CaM)-dependent protein kinases (CaM kinases) and Ca2+/phospholipid-dependent protein kinases (PKCs) have been identified in brain. Among these, CaMPK-II is the most abundant Ca2+/CaM-stimulated protein kinase in brain. CaMPK-II is important in several neuronal functions, including neurotransmitter release and the modulation of the functional properties of ion channels and receptors. CaMPK-II is differentially expressed in different brain regions of cells, exists in both cytosolic and membrane-associated forms and is especially concentrated in the postsynaptic density and synaptic vesicles. A distinct property of CaMPK-II is that autophosphorylation of its threonine residue near the calmodulin binding domain converts it to a Ca2+ independent state. Further, it has been shown that calmodulin-dependent autophosphorylation of CaMPK-II induces a conformational changes in the region of the calmodulin binding domain that allows additional stabilizing interactions with calmodulin. This autophosphorylation may involve in extending the effects triggered by a transient calcium signal. PTU-induced mild hypothyroidism in chick brain during posthatch development has been shown to increase the level of Ca2+/CaM-stimulated phosphorylation in cytosol, but lower it in the membrane, indicating a role of thyroid hormones in distributing CaMPK-II during developmental changes [78,86].

#### 5.4.1.1.1. Effect of L-T3 on total protein phosphorylation

22 Thyroid Hormone

action of THs and its metabolites [11].

*second messenger systems* 

proteins were significantly increased followed by L-T3 and L-T4 treatment as well. Both L-T3 and L-T4 indicated bi-phasic nature of effect for each of these proteins phosphorylated. Maximum levels of phosphorylation were noticed at concentration range from 10-30 nM. However, no significant effect on protein phosphorylation was observed as an effect of rT3 on any of these proteins. This effect of rT3 clearly confirmed very structural and functional specificity of L-T3 on protein phosphorylation. Determination of time course of protein phosphorylation followed by one single *in vitro* dose of L-T3 showed it peaked rapidly between 180 seconds to 240 seconds and thereafter it decreased. This indicated a rapid

Our next interest was to see which amino acids present in these phosphoyraled proteins are targets. Hence phospho-specifc antibodies for tyrosine and serine were used in western bolt analysis. Immunoblot analysis of synaptosomal lysates incubated with L-T3 (1 nM-1 M) confirmed phosphorylation at the seryl residues of a ~112 kD protein and phosphorylation at tyrosyl residues of a distinct ~ 95 kD protein. These data support that THs have a diversity of rapid nongenomic pathways for regulation of protein phosphorylation in mature mammalian brain [11]. Especially, the α-subunit of NKA is a ~112 kD membrane protein. Indeed, inhibition of NKA is associated with the phosphorylation of its subunits by both PKA and PKC. This inhibition of NKA has been attributed to the site-specific phosphorylation of the α1-subunit of the NKA at seryl residues by PKA and PKC [61-63]. In adult rat alveolar epithelial cell L-T3 induced translocation of NKA to plasma membrane. NKA stimulation by L-T3 was assigned to L-T3-induced stimulation of PI3K/PKB pathway via the Src family of tyrosine kinases nongenomically [84]. These data suggest possible

involvement of membrane components in TH-induced protein phosphorylation.

expression, whereas PKC and tyrosine kinase did not influence it significantly [85].

*5.4.1. Thyroid hormones rapidly modulate synaptosomal protein phosphorylation via* 

In other studies, L-T3 induction has also been shown to nongenomically regulate Ca2+ influx and nitric oxide synthase activity within seconds in adult rat brain [57]. Thus, THs are likely to have numerous rapid nongenomic effects on signaling mechanisms in neural tissue, including alterations in the levels of intracellular second messengers (cAMP and Ca2+) which regulate cAMP- and/or Ca2+/calmodulin (CaM)-dependent protein kinases leading to protein phosphorylation. Effects of TH on Ca2+-dependent activation of PKC and/or Ca2+/CaMdependent protein kinases are also possible, followed by direct or indirect activation of phosphorylation of the proteins. Thus further investigation demonstrated for the first time the rapid nongenomic second messenger mediated regulation of protein phosphorylation by

Examples of nongenomic control of protein phosphorylation by L-T3 also have been reported in few other tissues. Nongenomic relationship of MAPK and MAPK-mediated protein phosphorylation at the seryl residue of nuclear TH receptor has been described in 293T cells [68]. This indicated a control of nongenomic mechanism on genomic mechanism. In developing brain, inhibition of PKA transcriptionally blocked L-T3-induced actin gene The effect of Ca2+ and calmodulin on TH-induced total protein phosphorylation and their regulation was explored. L-T3 significantly and dose-dependently (10 nM-1 M) increased total 32P- incorporation into synaptosomal proteins, *in vitro*, over the basal level of phosphorylation. Although L-T3 exerted its own independent effect on increase in overall total protein phosphorylation, specifically it established its role to be at least dependent on Ca2+ and calmodulin. Ca2+ also showed its independent influence on the basal L-T3-induced total protein phosphorylation in synaptosomal isolates. The dependency of L-T3-induced total synaptosomal protein phosphorylation was evaluated and finally confirmed using EGTA (Ca2+-ion chelator) and KN-62 (a specific blocker of CaMK-II). *In vitro* addition of 10 nM and 100 nM doses of L-T3 alone did not alter significantly the basal levels of phosphorylation. However, the 1 M dose of L-T3 significantly amplified the signal by ~1.3 fold compared to the basal level (P<0.05). Next, we wanted to determine whether Ca2+ augments protein phosphorylation in the presence of L-T3. Ca2+ (0.5 mM) were able to significantly increase the basal phosphorylation level. However, no further significant changes were noticed with additional 10 nM or 100 nM L-T3. However, 1 μM concentration of L-T3 augmented the signal significantly (P<0.05) by ~1.5-fold (0.2167 pmols/min/mg protein) as compared to the Ca2+-treated baseline (0.1475 pmols/min/mg protein), and by ~2.2-fold over the basal phosphorylation (0.097 pmols/min/mg protein). In contrast, the effects of low physiological concentrations of L-T3 were dramatically enhanced when 2 M CaM was added to the Ca2++ L-T3-treatment group. In the presence of Ca2+ and CaM, L-T3

(10 nM-1 μM) induced a dose-dependent increase in 32P- incorporation into synaptosomal protein, by 47±8 , 74±13 and 52±11 % (F = 6.77, P<0.0001) rapidly within 1 min compared with the Ca2+/CaM-treated control phosphorylation (0.189 pmols/min/mg protein) [87].

"Quo Vadis?" Deciphering the Code of Nongenomic Action of Thyroid Hormones in Mature Mammalian Brain 25

**Figure 10.** A. L-T3-stimulated phosphorylation of 63- and 53 kDa proteins are regulated by Ca2+/CaMdependent protein kinase II. (a) Representative autoradiogram of the 63- and 53 KD proteins followed by various treatment conditions as described. (b) Corresponding protein bands from silver stained gel used for normalization of the data and demonstrates comparable equal amounts of sample loading. B. The quantification of the L-T3 (10 nM)-induced phosphorylation presented as a graph of ratio of the band densities of the phosphorylated proteins in the autoradiogram (a) and the corresponding protein in the silver stained gel (b) at different treatment conditions as indicated. \* represents the level of significance of P<0.05 compared to the corresponding basal level (control). The data presented are normalized results (mean ± S.E.M.) for an indicated protein band (Ref. Sarkar 2008 Life Sciences 82: 920-

5.4.1.1.3. Inert action of Ca2+ and calmodulin on the independent effect of L-T3 on the

L-T3 also increased the phosphorylation of 23- and 38 kD proteins. The effect was independent of EGTA or KN62. L-T3 only slightly enhanced the phosphorylation of the 38 kD protein (p<0.05, F = 3.74) by ~1.2-fold in the presence of Ca2+/CaM compared to Ca2+/CaM control group. Although addition of Ca2+ decreased the level of L-T3-induced phosphorylation of 38 kD protein (P = non-significant), it was significantly increased

demonstrated differential regulation of phosphorylation status of ve different synaptosomal proteins (63-, 53-, 38-, 23-, and 16 kD) in both a Ca2+/CaM-dependent and independent manner in mature rat brain cortical synaptosomes. L-T3 increased the level of phosphorylation of all these ve proteins. Ca2+/CaM further stimulated phosphorylation of 63- and 53 kD proteins by L-T3, which were inhibited both specifically by EGTA (Ca2+ chelator) or KN62 (Ca2+/CaM kinase-II [CaMK-II] inhibitor), suggesting the role of CaMK-II. However, presence of Ca2+ significantly decreased L-T3-induced phosphorylation of 63-, 53

kD proteins (Figure 10).

927 acknowledged [12]).

phosphorylation of 38- and 23 kD proteins

Physiological concentrations L-T3 in nerve terminals are difficult to measure. Predictable levels of L-T3 within the nerve terminals range from ~10 nM to 64 nM. PTU-induced peripheral hypothyroidism in adult rats showed endogenous synaptosomal level of L-T3 is about ~126 nM. Thus L-T3 (1 μM) is well above this range, and would be considered to have a more pharmacological type of action on 32P- incorporation to synaptosomal phosphoproteins. Treatment with agents regulating Ca2+ could be a potential strategy for enhancing clinical treatment of conditions, such as certain affective disorders, which may be responsive to pharmacological doses of TH. In an earlier *in vitro* study, L-T3 doses (0.1 to 100 nM), have been shown to induce an increase in intrasynaptosomal Ca2+ levels with an optimum at 100 nM of L-T3. Although higher levels of L-T3 (1 μM) produced slight depression of intrasynaptosmal Ca2+ levels, picomolar levels of L-T3 were also shown to be able to significantly increase intrasynaptosomal Ca2+ levels *in vitro*. The synergistic effect of L-T3 and Ca2+/CaM on protein phosphorylation would likely be further amplified by the effect of L-T3 to increase Ca2+ levels intracellularly in the physiological situation. In particular, this study demonstrated that of 10 nM dose of L-T3 (brain physiological concentration) and a ten times higher dose of L-T3 (as observed to be the brain levels of L-T3 in PTU-induced hypothyroid young adult rat brain synaptosomes alone or with Ca2+ did dramatically increased L-T3-induced total protein phosphorylation. Thus the present study demonstrated that Ca2+/CaM-dependent mechanisms synergistically increase the rapid nongenomic effect of L-T3 on synaptosomal protein phosphorylation [87]. The Ca2+/CaMdependent effects could be due to an activation of an unknown CaM-dependent protein kinase(s), deactivation of protein phosphatase(s) or a combination of effects. However, it proved to be highly sensitive to L-T3 activation.

Numerous phosphoproteins are greatly influenced by PKA and PKC in a Ca2+- and/or CaMdependent way. Often Ca2+ also functions in combination with CaM or with phosphoinositides/diacylglycerol to induce additional signal transduction pathways within the synaptic network. Regulation of intracellular Ca2+, CaM and subsequent protein phosphorylation are important for brain and cognitive functions affected by various psychiatric disorders. Membrane depolarization-induced Ca2+-influx activates extracellularly regulated kinases/MAPK in a Ca2+/CaM-dependent way in PC12 cells. THs also promote MAPK-mediated serine phosphorylation of the nuclear TH receptor β-1 isoform nongenomically in 293T cells. Ca2+ and CaM also differentially regulate of THinduced neuronal protein phosphorylation [12,87].

5.4.1.1.2. L-T3-induced stimulation of phosphorylation of 63- and 53 kD proteins was regulated by Ca2+ and calmodulin

After getting an idea of L-T3-induced total protein phosphorylation within neuronal membrane it was an obvious interest to look for specific proteins phosphorylated under the influence of L-T3. *In vitro* addition of L-T3 (10 nM, brain physiologic concentrations of L-T3)

proved to be highly sensitive to L-T3 activation.

induced neuronal protein phosphorylation [12,87].

regulated by Ca2+ and calmodulin

(10 nM-1 μM) induced a dose-dependent increase in 32P- incorporation into synaptosomal protein, by 47±8 , 74±13 and 52±11 % (F = 6.77, P<0.0001) rapidly within 1 min compared with the Ca2+/CaM-treated control phosphorylation (0.189 pmols/min/mg protein) [87].

Physiological concentrations L-T3 in nerve terminals are difficult to measure. Predictable levels of L-T3 within the nerve terminals range from ~10 nM to 64 nM. PTU-induced peripheral hypothyroidism in adult rats showed endogenous synaptosomal level of L-T3 is about ~126 nM. Thus L-T3 (1 μM) is well above this range, and would be considered to have a more pharmacological type of action on 32P- incorporation to synaptosomal phosphoproteins. Treatment with agents regulating Ca2+ could be a potential strategy for enhancing clinical treatment of conditions, such as certain affective disorders, which may be responsive to pharmacological doses of TH. In an earlier *in vitro* study, L-T3 doses (0.1 to 100 nM), have been shown to induce an increase in intrasynaptosomal Ca2+ levels with an optimum at 100 nM of L-T3. Although higher levels of L-T3 (1 μM) produced slight depression of intrasynaptosmal Ca2+ levels, picomolar levels of L-T3 were also shown to be able to significantly increase intrasynaptosomal Ca2+ levels *in vitro*. The synergistic effect of L-T3 and Ca2+/CaM on protein phosphorylation would likely be further amplified by the effect of L-T3 to increase Ca2+ levels intracellularly in the physiological situation. In particular, this study demonstrated that of 10 nM dose of L-T3 (brain physiological concentration) and a ten times higher dose of L-T3 (as observed to be the brain levels of L-T3 in PTU-induced hypothyroid young adult rat brain synaptosomes alone or with Ca2+ did dramatically increased L-T3-induced total protein phosphorylation. Thus the present study demonstrated that Ca2+/CaM-dependent mechanisms synergistically increase the rapid nongenomic effect of L-T3 on synaptosomal protein phosphorylation [87]. The Ca2+/CaMdependent effects could be due to an activation of an unknown CaM-dependent protein kinase(s), deactivation of protein phosphatase(s) or a combination of effects. However, it

Numerous phosphoproteins are greatly influenced by PKA and PKC in a Ca2+- and/or CaMdependent way. Often Ca2+ also functions in combination with CaM or with phosphoinositides/diacylglycerol to induce additional signal transduction pathways within the synaptic network. Regulation of intracellular Ca2+, CaM and subsequent protein phosphorylation are important for brain and cognitive functions affected by various psychiatric disorders. Membrane depolarization-induced Ca2+-influx activates extracellularly regulated kinases/MAPK in a Ca2+/CaM-dependent way in PC12 cells. THs also promote MAPK-mediated serine phosphorylation of the nuclear TH receptor β-1 isoform nongenomically in 293T cells. Ca2+ and CaM also differentially regulate of TH-

5.4.1.1.2. L-T3-induced stimulation of phosphorylation of 63- and 53 kD proteins was

After getting an idea of L-T3-induced total protein phosphorylation within neuronal membrane it was an obvious interest to look for specific proteins phosphorylated under the influence of L-T3. *In vitro* addition of L-T3 (10 nM, brain physiologic concentrations of L-T3) demonstrated differential regulation of phosphorylation status of ve different synaptosomal proteins (63-, 53-, 38-, 23-, and 16 kD) in both a Ca2+/CaM-dependent and independent manner in mature rat brain cortical synaptosomes. L-T3 increased the level of phosphorylation of all these ve proteins. Ca2+/CaM further stimulated phosphorylation of 63- and 53 kD proteins by L-T3, which were inhibited both specifically by EGTA (Ca2+ chelator) or KN62 (Ca2+/CaM kinase-II [CaMK-II] inhibitor), suggesting the role of CaMK-II. However, presence of Ca2+ significantly decreased L-T3-induced phosphorylation of 63-, 53 kD proteins (Figure 10).

**Figure 10.** A. L-T3-stimulated phosphorylation of 63- and 53 kDa proteins are regulated by Ca2+/CaMdependent protein kinase II. (a) Representative autoradiogram of the 63- and 53 KD proteins followed by various treatment conditions as described. (b) Corresponding protein bands from silver stained gel used for normalization of the data and demonstrates comparable equal amounts of sample loading. B. The quantification of the L-T3 (10 nM)-induced phosphorylation presented as a graph of ratio of the band densities of the phosphorylated proteins in the autoradiogram (a) and the corresponding protein in the silver stained gel (b) at different treatment conditions as indicated. \* represents the level of significance of P<0.05 compared to the corresponding basal level (control). The data presented are normalized results (mean ± S.E.M.) for an indicated protein band (Ref. Sarkar 2008 Life Sciences 82: 920- 927 acknowledged [12]).

5.4.1.1.3. Inert action of Ca2+ and calmodulin on the independent effect of L-T3 on the phosphorylation of 38- and 23 kD proteins

L-T3 also increased the phosphorylation of 23- and 38 kD proteins. The effect was independent of EGTA or KN62. L-T3 only slightly enhanced the phosphorylation of the 38 kD protein (p<0.05, F = 3.74) by ~1.2-fold in the presence of Ca2+/CaM compared to Ca2+/CaM control group. Although addition of Ca2+ decreased the level of L-T3-induced phosphorylation of 38 kD protein (P = non-significant), it was significantly increased (P<0.05) in the presence of CaM compared to Ca2+ + L-T3 treatment and only L-T3 effect. However, the presence of Ca2+ or the Ca2+/CaM did not further affect the phosphorylation status of the 38 kD protein. This further suggested no involvement of Ca2+/CaM-dependent pathways mediated through CaMK-II.

"Quo Vadis?" Deciphering the Code of Nongenomic Action of Thyroid Hormones in Mature Mammalian Brain 27

abolished the L-T3-induced phosphorylation. EGTA or KN62 could not restore the effect of

Immunoblotting experiment with anti-phosphoserine antibodies also showed significant enhancement of seryl residue phosphorylation of this protein by Ca2+/CaM (Figure 13). Abolition of this effect by EGTA and KN-62 further suggested an important role of CaMK-II. This study identied the role of Ca2+/CaM in the regulation of L-T3-induced protein phosphorylation and supported a unique nongenomic mechanism of second messenger-

**Figure 12.** A. Phosphorylation of 16 kD protein by L-T3 was conquered by the dephosphorylation activity of CaM. (a) A representative autoradiogram of the 16 kD protein separated by SDS-PAGE is showing independent stimulatory action of L-T3 upon the phosphorylation of the 16 kD protein. (b). Corresponding protein bands of silver stained gel. B. The quantification of the L-T3 (10 nM)-induced phosphorylation and its dephosphorylation by CaM are presented as a graph of ratio of the band densities of the phosphorylated proteins in the autoradiogram (a) and the corresponding protein in the silver stained gel (b) at different treatment conditions as indicated. The data presented are normalized results (mean ± S.E.M.) for an indicated protein band. \* Indicates levels of significance P<0.05, compared to the basal level (control group) (Ref. Sarkar 2008 Life Sciences 82: 920-927 acknowledged [12]).

CaM-induced dephosphorylation of this protein (Figure 12).

mediated regulation of protein phosphorylation by TH in mature rat brain.

The study also described the phosphorylation status of a 23 kD protein. Phosphorylation level of 23 kD protein was highest among all the proteins. L-T3 significantly increased the phosphorylation level of 23 kD by ~2.2-fold compared to the basal level. Especially of interest, EGTA or KN62 did not show any more or less influence on the L-T3-induced increase in the phosphorylation status of the 23 kD protein suggesting lack of significant regulation by CaMK-II (Figure 11).

**Figure 11.** A. Ca2+/CaM do not modulate L-T3-stimulated phosphorylation of 23- and 38 kD proteins. (a) A representative autoradiogram of the 23- and 38 kD protein separated by SDS-PAGE showing independent stimulatory action of L-T3 upon the phosphorylation of the 23- and 38 kD proteins. B. The quantification of the L-T3-induced phosphorylation presented as a graph of ratio of the band densities of the phosphorylated proteins in the autoradiogram (a) and the corresponding protein in the silver stained gel (b) at different treatment conditions as indicated. The data presented are normalized results (mean ± S.E.M.) for an indicated protein band. \* Indicates levels of significance P<0.05 (Ref. Sarkar 2008 Life Sciences 82: 920-927 acknowledged [12]).

#### 5.4.1.1.4. Calmodulin dephosphorylated 16 kD protein following L-T3-induction

*In vitro* addition of L-T3 (10 nM) significantly increased the level of phosphorylation of 16 kD protein by ~8-fold. The L-T3-induced phosphorylation of 16 kD protein was not further activated in the presence of Ca2+. Surprisingly, L-T3-induced phosphorylation of 16 kD protein was not augmented further with Ca2+ or Ca2+/CaM; instead, the presence of CaM abolished the L-T3-induced phosphorylation. EGTA or KN62 could not restore the effect of CaM-induced dephosphorylation of this protein (Figure 12).

26 Thyroid Hormone

pathways mediated through CaMK-II.

regulation by CaMK-II (Figure 11).

Life Sciences 82: 920-927 acknowledged [12]).

(P<0.05) in the presence of CaM compared to Ca2+ + L-T3 treatment and only L-T3 effect. However, the presence of Ca2+ or the Ca2+/CaM did not further affect the phosphorylation status of the 38 kD protein. This further suggested no involvement of Ca2+/CaM-dependent

The study also described the phosphorylation status of a 23 kD protein. Phosphorylation level of 23 kD protein was highest among all the proteins. L-T3 significantly increased the phosphorylation level of 23 kD by ~2.2-fold compared to the basal level. Especially of interest, EGTA or KN62 did not show any more or less influence on the L-T3-induced increase in the phosphorylation status of the 23 kD protein suggesting lack of significant

**Figure 11.** A. Ca2+/CaM do not modulate L-T3-stimulated phosphorylation of 23- and 38 kD proteins. (a) A representative autoradiogram of the 23- and 38 kD protein separated by SDS-PAGE showing independent stimulatory action of L-T3 upon the phosphorylation of the 23- and 38 kD proteins. B. The quantification of the L-T3-induced phosphorylation presented as a graph of ratio of the band densities of the phosphorylated proteins in the autoradiogram (a) and the corresponding protein in the silver stained gel (b) at different treatment conditions as indicated. The data presented are normalized results (mean ± S.E.M.) for an indicated protein band. \* Indicates levels of significance P<0.05 (Ref. Sarkar 2008

5.4.1.1.4. Calmodulin dephosphorylated 16 kD protein following L-T3-induction

*In vitro* addition of L-T3 (10 nM) significantly increased the level of phosphorylation of 16 kD protein by ~8-fold. The L-T3-induced phosphorylation of 16 kD protein was not further activated in the presence of Ca2+. Surprisingly, L-T3-induced phosphorylation of 16 kD protein was not augmented further with Ca2+ or Ca2+/CaM; instead, the presence of CaM Immunoblotting experiment with anti-phosphoserine antibodies also showed significant enhancement of seryl residue phosphorylation of this protein by Ca2+/CaM (Figure 13). Abolition of this effect by EGTA and KN-62 further suggested an important role of CaMK-II. This study identied the role of Ca2+/CaM in the regulation of L-T3-induced protein phosphorylation and supported a unique nongenomic mechanism of second messengermediated regulation of protein phosphorylation by TH in mature rat brain.

**Figure 12.** A. Phosphorylation of 16 kD protein by L-T3 was conquered by the dephosphorylation activity of CaM. (a) A representative autoradiogram of the 16 kD protein separated by SDS-PAGE is showing independent stimulatory action of L-T3 upon the phosphorylation of the 16 kD protein. (b). Corresponding protein bands of silver stained gel. B. The quantification of the L-T3 (10 nM)-induced phosphorylation and its dephosphorylation by CaM are presented as a graph of ratio of the band densities of the phosphorylated proteins in the autoradiogram (a) and the corresponding protein in the silver stained gel (b) at different treatment conditions as indicated. The data presented are normalized results (mean ± S.E.M.) for an indicated protein band. \* Indicates levels of significance P<0.05, compared to the basal level (control group) (Ref. Sarkar 2008 Life Sciences 82: 920-927 acknowledged [12]).

"Quo Vadis?" Deciphering the Code of Nongenomic Action of Thyroid Hormones in Mature Mammalian Brain 29

unchanged. This suggests L-T3-membrane interaction was independent of the activation of the 2-ADR system. Overall these data implicate that PKA and CaMK-II both contribute for L-T3 regulated protein phosphorylation in adult mammalian brain and reveals a

**Figure 14.** L-T3 induced phosphorylation of the 51-/53- kD proteins are abolished by Protein Kinase A Inhibitor (H7). (A) One representative phosphorylation status of the 51-/53-kD protein immunoblotted with anti-phosphoserine (PS) antibodies. (B) Corresponding protein band of silver stained gel. (C) Graphical representation of the levels of phosphorylation of the 51-/53-kD protein at various treatment conditions. dbcAMP is dibutyryl cyclic AMP. The data presented are normalized results (mean ± S.E.M.) for an indicated protein band. \* Indicates levels of significance p<0.05, compared to the basal

In conclusion the recent evidence-based information regarding the nongenomic mechanism of action of THs are opening new signal transduction clues to be studied and to reveal the underlying mechanism in mature mammalian brain. The results of the study conducted will advance our knowledge of the fundamental molecular mechanism of TH action in mature CNS, likely in future will lead to more rational and effective approach to the development of novel therapeutic agents, and thus will shed insights on to the neuropshychological manifestations of adult on-set thyroid disorders in humans, particularly in relation to higher

level (control group).

**6. Conclusion** 

mental functions.

nongenomic mechanistic pathway in relation to higher mental functions.

**Figure 13.** L-T3 induced phosphorylation of the 53 kD protein is regulated by Ca2+/calmodulin protein kinase II: Serine residue phosphorylation. (A) Phosphorylation status of the 53 kD protein immunoblotted with anti-phosphoserine (PS) antibody. (B) Corresponding protein band of silver stained gel. (C) Graphical representation of the levels of phosphorylation of the 53 kD protein at various treatment conditions. The data presented are normalized results (mean ± S.E.M.) for an indicated protein band. \* Indicates levels of significance P<0.05, compared to the basal level (control group).

#### *5.4.1.2. Role of cAMP on synaptosomal protein phosphorylation, in vitro*

After searching for whether Ca2+ plays a major role as second messenger following L-T3 induced protein phosphorylation, our next step was to examine for the role of cyclic AMP (cAMP) as another second messenger upon L-T3-induction, *in vitro,* to explore furthermore the nongenomic mechanism of TH. To search for any role of cAMP-dependent protein kinase (PKA) the effects of cAMP and H7 (a specific blocker of PKA) were studied. *In vitro* addition of H7 significantly diminished the effect of L-T3-induced increase in serine phosphorylation of two closely associated proteins with 51- and 53 kD by ~14-fold and ~11 fold respectively (Figure 14). This suggested prevalence of a PKA-mediated mechanism in L-T3-induced synaptosomal protein phosphorylation. To test further whether THs exert adrenergic-like actions by binding to or modulating adrenergic receptor activities another study was performed to test this hypothesis. The idea of formation of thyronamines and its possible binding to the ADR is considered here. Effect of clonidine was studied on the L-T3 induced protein phosphorylation and on the L-T3-binding to the synaptosomal membrane receptors. Scatchard plot analysis revealed clonidine and yohimbine (2-ADR antagonist) could not alter specific L-T3 binding at the high affinity L-T3 synaptosomal membrane binding sites. L-T3 induced phosphorylation of this 51-/53 kD protein was blocked by H7, a PKA inhibitor. Activation of 2-ADR by clonidine normally decreases the levels of cAMP via inhibiting adenylate cyclase activity. Possibly in the absence of adequate cAMP levels during clonidine treatment, the phosphorylation status of the 51-/53 kD protein remained unchanged. This suggests L-T3-membrane interaction was independent of the activation of the 2-ADR system. Overall these data implicate that PKA and CaMK-II both contribute for L-T3 regulated protein phosphorylation in adult mammalian brain and reveals a nongenomic mechanistic pathway in relation to higher mental functions.

**Figure 14.** L-T3 induced phosphorylation of the 51-/53- kD proteins are abolished by Protein Kinase A Inhibitor (H7). (A) One representative phosphorylation status of the 51-/53-kD protein immunoblotted with anti-phosphoserine (PS) antibodies. (B) Corresponding protein band of silver stained gel. (C) Graphical representation of the levels of phosphorylation of the 51-/53-kD protein at various treatment conditions. dbcAMP is dibutyryl cyclic AMP. The data presented are normalized results (mean ± S.E.M.) for an indicated protein band. \* Indicates levels of significance p<0.05, compared to the basal level (control group).
