**6. Conclusion**

28 Thyroid Hormone

**Figure 13.** L-T3 induced phosphorylation of the 53 kD protein is regulated by Ca2+/calmodulin protein


\* \*

\*

\*

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

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).

**Ca2+** - + + - + + + + **CaM** - - + - - + + + **T3 (10 nM)** - - - + + + + + **EGTA** - - - - - - + - **KN62 (M)** - - - - - - - <sup>40</sup>

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

**0**

**150000**

**300000**

**450000**

**(C)**

**Protein phosphorylation/Protein ratio**

**(Integrated band density in arbitrary units)**

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 mental functions.
