*5.3.2. Is thyroid hormone-membrane interaction is linked to G-protein coupled receptors (GPCR)?*

#### *5.3.2.1. Association of G proteins with membrane receptors*

G proteins are GTP-binding proteins that couple activation of seven-helix receptors by neurotransmitters at the cell surface for the activation of the effector enzymes-adenylate cyclase (AC) or guanylate cyclase (GC), which synthesize the corresponding cyclic nucleotides, cAMP or cGMP respectively and regulate protein kinases., such as protein kinase A (PKA), protein kinase C (PKC) etc. Metabotropic events are often initiated at the membrane level, mediated and amplified through GPCR followed by activation of second

conditions.

T3.

*(GPCR)?* 

nM to 10 nM). This evidence indicates role of Ca2+ as a second messenger in synaptic functions. L-T3 also has been documented to increase 45Ca uptake and Ca2+-influx in adult euthyroid rat synaptosomes, and in hypothyroid mouse cortex. An enhancement of nitric oxide synthase (NOS) activity in adult rat cerebrocortical synaptosomes was shown [55]. This present study demonstrated a significant increase in Ca2+ accumulation in hypothyroid rat brain cerebrocortical synaptosomes compared to euthyroid control at below (0.1 nM) and at about brain physiologic concentrations (10 nM) of L-T3. At present clear understanding for the L-T3-induced release of intracellular calcium is not known; however possibility for L-T3-induced action in neuronal cells cannot be left out. Use of sodium azide blocked any mitochondrial accumulation of calcium. Our earlier studies have shown that 10 nM and 100 nM dose of L-T3 could saturate the specific synaptosomal L-T3-binding sites by ~69% and ~74% respectively. L-T3-mediated physiological increase in synaptosomal Ca2+ accumulation could be attributed to receptor-mediated physiological response having its maximal effect at 10 nM dose of L-T3. The differences in the observation of increased rate of Ca2+ accumulation in hypothyroid synaptosomes compared to the euthyroid values reflected an adaptive mechanism. This could be credited to homeostatic mechanism to overcome PTUinduced stress conditions persisted in the adult neuron. High intrasynaptosomal L-T3 level (~9.5-fold higher; 2.56 ng/mg synaptosomal protein 126 nM L-T3) could be one of the reasons. Although hypothyroid condition showed an appreciable decrease in both serum levels of L-T4 and L-T3 as predicted, supportive studies showed maintenance of similar levels of brain L-T3 in hypothyroid conditions through increased activity of D-II suggesting high fractional rate of L-T4 to L-T3 conversion. In brain approximately ~80% of L-T3 is produced locally from L-T4 by D-II. This data supports thyroid hormone-Ca2+-ion interaction for normal functioning of adult brain during different neuropsychological

The important functional role of Ca2+ and several calcium-dependent proteins in neuronal signal transduction are well recognized. Ca2+ has been shown to inhibit neuronal NKA activity. Ca2+-influx also lead to Ca2+-dependent activation of protein kinase C and/or Ca2+/CaM-dependent protein kinases followed by direct or indirect activation of phosphorylation of several target proteins. This indicated a rapid nongenomic action of L-

*5.3.2. Is thyroid hormone-membrane interaction is linked to G-protein coupled receptors* 

G proteins are GTP-binding proteins that couple activation of seven-helix receptors by neurotransmitters at the cell surface for the activation of the effector enzymes-adenylate cyclase (AC) or guanylate cyclase (GC), which synthesize the corresponding cyclic nucleotides, cAMP or cGMP respectively and regulate protein kinases., such as protein kinase A (PKA), protein kinase C (PKC) etc. Metabotropic events are often initiated at the membrane level, mediated and amplified through GPCR followed by activation of second

*5.3.2.1. Association of G proteins with membrane receptors* 

messenger system and subsequent substrate protein phosphorylation. Phospholipase C (PLC), another effector enzyme, generates inositol triphosphate (IP3) and diacylglycerol (DAG), the latter of which releases intracellular stores of calcium. The cAMP, cGMP, Ca2+, DAG and IP3 act as second messengers and activate protein kinases with broad substrate specificity. The kinases phosphorylate key intracellular proteins, including ion channels, enzymes, and transcription factors which modulate cellular biological processes [58,59]. Guanine nucleotides are known to have dual effects on most hormone-sensitive AC systems. This modulates activation of AC and binding of hormone to receptor. In neuronal membranes guanylate nucleotides has been shown to be required for the stimulation of AC. However, no modulation of TH binding at appropriate guanylate nucleotide concentrations has been reported. It is well established that cholera toxin enhances the activity of Gs (stimulatory G protein -subunit) by ADP-ribosylating Gs subunit and inhibiting GTPase activity associated with the protein. This increases cAMP production.

The activity of NKA is regulated by various catecholamines [45,46,60] as well as by L-T3 [45,46]. Inhibition of NKA has been demonstrated in intact cell preparations by phorbol esters, dibutyryl cAMP, and phospho-DRPP-32 (dopamine- and cAMP-regulated phosphoprotein of molecular weight 32 kD), a protein phosphatase inhibitor [61-63].

Some information focuses to effect of TH or its metabolites on noradrenergic like responses. This idea develops since TH has possibility to produce a family of biogenic amine-like neurotransmitter compounds catalyzed by aromatic amino acid decarboxylase, such as iodothyronamines. Physiologic identification of these family of TH-derived iodothyronamines have not yet been discovered until recently in rat brain and in rat and human blood. These two compounds are monoiodothyronamine and thyronamine [10]. Thinking this could be a possibility before this identification of monoiodothyronamine and thyronamine were reported we studied the effect of L-T3 on synaptosomal NKA activity using various - and -adrenergic agonists and antagonists known to regulate Gs and Gi proteins of the neuronal signal transduction system, *in vitro*.

Our studies showed that although both L-T3 and isoproterenol (-adrenergic receptor [ADR] agonist and activator of Gs-protein) similarly inhibited synaptosomal NKA activity, propranolol (-ADR antagonist) could only block the effect of isoproterenol, but not the effect of L-T3. Instead propranolol produced a dose-dependent potentiation of the inhibitory influence of L-T3 (Figure 6). The augmentation of L-T3-effect by propranolol appeared to be a type of synergistic action and it might be due to some changes in the pre-synaptic membrane properties, the mechanism of which is unclear at present. However, clonidine (2-ADR agonist, and Gi-protein activator) (Figure 7) and glutamate (acts through metabotropic glutamate receptors and activator of Gi protein) (Figure 8) attenuated L-T3 effect, suggesting its possible coupling with GPCR. Equimolar concentration of clonidine (1 nM – 100 nM) counteracted the inhibitory effect of L-T3 on the NKA activity (Figure 7). This counteraction by clonidine, 2-ADR agonist, appears to be mediated through the inhibition of adenylate cyclase activity with the activation of inhibitory G protein (Gi) followed by inhibition of cAMP synthesis and protein phosphorylation cascade mechanism. It is known that 2-adrenergic receptor agonist system act through Gi protein activation [64].

"Quo Vadis?" Deciphering the Code of Nongenomic Action of Thyroid Hormones in Mature Mammalian Brain 19

been reported to inhibit TH-induced mitogen-activated protein kinase (MAPK) phosphorylation nongenomically in 293T cells which is consistent with a cell membrane mechanism mediated via a G-protein [66]. 3-iodothyronine (T1AM), an endogenous and rapid-acting derivative of TH, is associated with Gs-protein coupled-trace amine receptor TAR1 in HEK cells. However, no modulation of TH binding at appropriate guanylate nucleotide concentrations in adult brain has been reported [67]. Determination of whether activation or inactivation of a specific type(s) of G-protein influences TH-effects on protein

phosphorylation is crucial.

Na+-K+-ATPase ( mols Pi.h-1.mg protein-1)

0

5

Control

**Figure 8.** Attenuation of L-T3-effect on synaptosomal NKA activity by glutamate, *in vitro*.

10 nM T3

A diverse nongenomic effect of TH has been observed in non-neural tissues including liver, heart, adipocytes, and blood [12,68]. Some possible nongenomic actions of THs include modulation of GABA uptake, regulation of NKA activity and increase of presynaptic Ca2+ influx. In synaptoneurosomes TH inhibits the stimulation of chloride flux by GABA [69]. L-T4 has been shown to stimulate the MAPK pathway in a variety of cultured cell lines including HeLa and CV-1 cells which lack functional nuclear TH receptors [66,70-73], consistent with a cell membrane mediated mechanism via G-proteins. L-T4 and L-T3 were found to inhibit Go-protein activities in synaptosomes from developing chick brain [48].

Direct interactions of G protein subunits with Ca2+-channels are not well documented. However, increased evidences showed receptor activated G proteins modulate activities of ion channels by membrane-confined mechanisms [74]. Isoproterenol induced phosphorylation of ventricular Ca2+-channels via PKA has been reported [75]. Gs protein also has been shown to regulate Ca2+-channels both in a cAMP-independent membraneconfined mechanism [74] and in a cAMP-dependent phosphorylation of one of the subunits of L-type Ca2+-channel [76]. Synaptosomal NKA has previously been described to be inhibited by cAMP in a dose-dependent manner suggesting a role of PKA. The activated form of this protein kinase was further phosphorylated a substrate protein which in turn depressed the total Na+-dependent phosphorylation of the synaptosomal NKA [77]. Overall,

a

100 M Glutamate

10 nM T3 + 100 M Glutamate

10

15

20

25

30

**Figure 6.** *In vitro* effect of L-T3, isoproterenol (ISO) and propranolol (P), on synaptosomal NKA activity.

**Figure 7.** *In vitro* effect of L-T3, clonidine (CLON, and yohimbine (YOH, 2-ADR antagonist) on synaptosomal NKA activity.

Thus it seems that the L-T3 action could be ascribed more to stimulate Gs protein during beta-blockade which might be directed to manage this adverse condition. The results also suggest that the L-T3-effect on the synaptosomal NKA activity was not mediated via the - ADR-dependent systems, since it was not blocked by propranolol. Based on these results it was also hypothesized that L-T3-effect would alter adenylate cyclase activity. In cultured neuroblastoma plasma membrane increased adenylate cyclase activity was noticed followed by L-T3-treatment [65]. In fact, later, increased adenylate cyclase activity was noticed in brain hypothyroid condition which increases brain L-T3 levels. This observation was correlated well with increased D-II activity to the increased brain L-T3 levels in brain hypothyroid situations [15]. Guanosine 5'-O-(3-thiotriphosphate) or pertussis toxin also has

Na+-K+-ATPase (mols Pi. h-1.mg protein-1)

Na+-K+-ATPase ( mols Pi. h-1.mg protein-1

synaptosomal NKA activity.

0

5

1 0

1 5

2 0

2 5

3 0

)

0

Control

1 nM T3

Control

1 nM T3

1 nM Clon

**Figure 7.** *In vitro* effect of L-T3, clonidine (CLON, and yohimbine (YOH, 2-ADR antagonist) on

10 nM Clon

Thus it seems that the L-T3 action could be ascribed more to stimulate Gs protein during beta-blockade which might be directed to manage this adverse condition. The results also suggest that the L-T3-effect on the synaptosomal NKA activity was not mediated via the - ADR-dependent systems, since it was not blocked by propranolol. Based on these results it was also hypothesized that L-T3-effect would alter adenylate cyclase activity. In cultured neuroblastoma plasma membrane increased adenylate cyclase activity was noticed followed by L-T3-treatment [65]. In fact, later, increased adenylate cyclase activity was noticed in brain hypothyroid condition which increases brain L-T3 levels. This observation was correlated well with increased D-II activity to the increased brain L-T3 levels in brain hypothyroid situations [15]. Guanosine 5'-O-(3-thiotriphosphate) or pertussis toxin also has

100 nM Clon

1 nM YOH

1 nM T3 + 1 nM Clon

1 nM T3 + 10 nM Clon

1 nM T3 + 100 nM Clon

1 nM T3

+ 1 nM YOH

1 nM ISO

a a

1 nM P

10 nM P

**Figure 6.** *In vitro* effect of L-T3, isoproterenol (ISO) and propranolol (P), on synaptosomal NKA activity.

0.1

M P

1 nM ISO + 1 nM P

1 nM T3

1 nM P

1 nM T3

a,b a,c

10 nM P

a

0.1 M P

1 nM T3

5

10

15

20

25

30

been reported to inhibit TH-induced mitogen-activated protein kinase (MAPK) phosphorylation nongenomically in 293T cells which is consistent with a cell membrane mechanism mediated via a G-protein [66]. 3-iodothyronine (T1AM), an endogenous and rapid-acting derivative of TH, is associated with Gs-protein coupled-trace amine receptor TAR1 in HEK cells. However, no modulation of TH binding at appropriate guanylate nucleotide concentrations in adult brain has been reported [67]. Determination of whether activation or inactivation of a specific type(s) of G-protein influences TH-effects on protein phosphorylation is crucial.

**Figure 8.** Attenuation of L-T3-effect on synaptosomal NKA activity by glutamate, *in vitro*.

A diverse nongenomic effect of TH has been observed in non-neural tissues including liver, heart, adipocytes, and blood [12,68]. Some possible nongenomic actions of THs include modulation of GABA uptake, regulation of NKA activity and increase of presynaptic Ca2+ influx. In synaptoneurosomes TH inhibits the stimulation of chloride flux by GABA [69]. L-T4 has been shown to stimulate the MAPK pathway in a variety of cultured cell lines including HeLa and CV-1 cells which lack functional nuclear TH receptors [66,70-73], consistent with a cell membrane mediated mechanism via G-proteins. L-T4 and L-T3 were found to inhibit Go-protein activities in synaptosomes from developing chick brain [48].

Direct interactions of G protein subunits with Ca2+-channels are not well documented. However, increased evidences showed receptor activated G proteins modulate activities of ion channels by membrane-confined mechanisms [74]. Isoproterenol induced phosphorylation of ventricular Ca2+-channels via PKA has been reported [75]. Gs protein also has been shown to regulate Ca2+-channels both in a cAMP-independent membraneconfined mechanism [74] and in a cAMP-dependent phosphorylation of one of the subunits of L-type Ca2+-channel [76]. Synaptosomal NKA has previously been described to be inhibited by cAMP in a dose-dependent manner suggesting a role of PKA. The activated form of this protein kinase was further phosphorylated a substrate protein which in turn depressed the total Na+-dependent phosphorylation of the synaptosomal NKA [77]. Overall,

our data indirectly support the involvement of second messenger system (cAMP and/or Ca2+) mediated through G protein activation after specific L-T3-membrane receptor interaction. The membrane NKA has been implicated in several aspects of physiologic processes including its role in neurotransmitter release [43].

"Quo Vadis?" Deciphering the Code of Nongenomic Action of Thyroid Hormones in Mature Mammalian Brain 21

While a direct effect of TH on protein kinase activity has not been formerly studied in tissues from mature brain, hypothyroidism has been linked with reduced levels of phosphorylated MAPK in the hippocampus [83]. Based on these observations, possibility of a metabotropic pathway for rapid actions of TH on protein phosphorylation in

**Figure 9.** Representative autoradiogram of SDS-PAGE separation of proteins incorporating 32P in the presence of L-T3. Lanes were loaded with synaptosomal lysates which had been preincubated at 0°C for 60 min and 37°C for 5 min with (from left): 1mM Na3VO4 (V), 1, 3, 10, 30, 100, 300, 1000, or 0 (C = control) nM L-T3 and then incubated with 20μM of [γ-32P]-ATP (3 μCi) for 1 min at 37°C. Left panel (a): Silver-stained gel for visualization of protein bands. Right panel (b): Autoradiogram of same gel showing increased incorporation of 32P in four prominent bands (α: 381 kD, β: 531 kD, γ: 631 kD, δ: 1131 kD). (c) Normalized data showing effect of *in vitro* addition of graded doses of L-T3 on the levels

(c)

Our observation demonstrated that TH induces rapid changes in synaptosomal protein phosphorylation. Incubation with L-T3 or L-T4 specifically showed significant biphasic dose-dependent effects on the phosphorylation of 381, 531, 621, and 1131 kD proteins. *In vitro* brain physiologic concentrations of TH (1-30 nM) showed significant increase in the levels of protein phosphorylation rapidly within minutes (Figure 9). In contrast, incubations with similar doses of reverse-T3 (rT3) were without significant effect, indicating specificity for L-T3 and L-T4. The protein phosphorylation statuses of these four synaptosomal

of protein phosphorylation expressed as optical density (OD)/protein (Ref. Sarkar et al. 2006

(a) (b)

*Neuroscience* 137: 125-132 acknowledged [11]).

synaptosomes from adult rat brain was investigated.
