*5.3.1. Effect of L-T3 on synaptosomal Ca2+-influx: A comparison between euthyroid and hypothyroid brain*

Metabotropic events are often initiated at the membrane level, mediated and amplified through G-protein coupled receptors (GPCR) and/or ion channels followed by activation of second messenger system and subsequent substrate protein phosphorylation. Ca2+-influx is an important physiological function in brain, following which cascades of membrane events occur finally leading to neurosignaling. Disruption in this crucial membrane phenomenon may lead to variety of Ca2+-dependent neuropsychological disorders. Although THmediated Ca2+ entry in adult rat brain synaptosomes [54,55], and in hypothyroid mouse cerebral cortex [56] have been reported, it's synaptic functions in adult neurons in dysthyroidism is unclear. Keeping in mind the role of Ca2+ ions as a messenger in the signaling pathway the effect of L-T3 on intracellular Ca2+-influx, *in vitro*, was studied.

**Figure 5.** Effect of L-T3 on intrasynaptosomal Ca2+-concentration in euthyroid and PTU-induced hypothyroid rat cerebral cortex *in vitro* (Ref. Modified from Sarkar and Ray 2003, Hormone and Metabolic Research 35: 562-564 acknowledged [57])).

Our study demonstrates a regulation and homeostatic mechanism of Ca2+ accumulation within cerebrocortical synaptosomes of hypothyroid adult rat [57]. Application of brain physiologic concentrations of L-T3 (0.001 nM to 10 nM), *in vitro*, significantly triggered Ca2+ sequestration both in the euthyroid and hypothyroid rat brain synaptosomes in a dosedependent manner (Figure 5). Unexpectedly, PTU-induced hypothyroid synaptosomes showed significant levels of increase in Ca2+-influx compared to euthyroid controls between 0.1 nM and 10 nM doses of L-T3. However, 0.001 nM dose of L-T3 did not show significant changes between euthyroid and hypothyroid values.

Present study validates the role of Ca2+ ions under the influence of L-T3 in the synaptosomes from adult rat brain cerebral cortex. L-T3-induced dose-dependent Ca2+-entry both in euthyroid and PTU-induced hypothyroid rat brain synaptosomes at low L-T3 doses (0.001 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 conditions.

"Quo Vadis?" Deciphering the Code of Nongenomic Action of Thyroid Hormones in Mature Mammalian Brain 17

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

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

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

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].

phosphoprotein of molecular weight 32 kD), a protein phosphatase inhibitor [61-63].

activity associated with the protein. This increases cAMP production.

proteins of the neuronal signal transduction system, *in vitro*.

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-T3.
