**5.3. In search for possible second messenger mediated events in synaptosomal L-T3-induced signaling**

The evidence of L-T3-synaptosomal membrane interaction in association with the inhibition of the synaptosomal membrane NKA activity led us to search for if the L-T3-induced action is mediated via activation or regulation of the second messenger cascade systems. Besides the cyclic nucleotide cyclase systems calcium (Ca2+) also plays an important role in cellular signal transmission. Ca2+-influx is a major event in neurotransmission. Keeping such visions we further intended to explore the role of Ca2+ in L-T3-induction.

14 Thyroid Hormone

was ~74%. This appears to be physiological. Further inhibition of NKA activity as found with higher concentrations of L-T3 (5x10–10 – 1x10–7 M), corresponds to the increase in the occupancy of the L-T3 binding sites (maximum of ~80%) at the low afnity binding range. However, this site was not saturated by 15.4 M L-T3 used for determining non-specic binding. Hence, it is possible that this low afnity binding is due to non-specic effects of several other proteins located in synaptosomes. The relationship between the binding of L-T3 to its synaptosomal binding sites and the concentration dependent inhibition of the enzyme activity appears to hold good only with the occupancy of high afnity sites up to 5 x 10–10 M L-T3 [46]. Synaptosomes prepared from chick embryo cortex were also reported to have two sets of L-T3 binding sites [48]. Their properties and ontogeny showed a marked difference from those of nuclear receptors. Even though NKA activity was suppressed beyond the saturating concentration of L-T3 at high afnity binding sites, this may be nonspecic and non-physiological. The relative order of binding afnities for TH analogues to the L-T3 binding sites and the inhibitory potencies for NKA activity were also correlated in the synaptosomes. L-T3-amine was used to examine its potency to inhibit specic [125I]-L-T3 binding in synaptosomes with the idea that it may be a decarboxylated product of L-T3 and may have actions like L-T3. The ED50 value for L-T3-amine was determined as 10 nM. At this dose, L-T3-amine also inhibited the synaptosomal NKA activity by ~51% compared with L-T3. This result is also in good agreement with earlier studies, in which L-T3-amine was shown to be ~71% as effective as L-T3 in stimulating Ca2+-ATPase activity at a dose of 10 nM in human RBC [49]. In earlier studies, L-T3-induced increase in NKA activity in the developing brain [50] and kidney cortex [51] of rat was reported to be due to an increase in the mRNA levels of , + and -subunits of the enzyme, while the NKA in adult was not responsive to L-T3. However, a dose-dependent inhibition and regulation of synaptosomal NKA activity in different *in vivo* situations was noticed. The immediate effect of added L-T3 on the synaptosomes appears to be nongenomic as synaptosomes do not have nuclei. This may exclude the possibility of involvement of nuclear receptors as reported earlier by us. One possible effect of L-T3 may be mediated through membrane receptors. Recently, membrane binding proteins for iodothyronines has been described in plasma membranes of most cells [52]. This protein has been designated as an integrin V3. Also a role of MCT-8, a membrane spanning protein, has been ascribed as a very active and specific transporter of THs and some of its metabolites across the membrane [25,53]. However, its action through

cytoplasmic L-T3-responsive proteins cannot be ruled out.

mental functions in adult mammalian brain.

**T3-induced signaling** 

In conclusion this study demonstrates, for the rst time, a correlation between the binding of TH to its putative receptors and inhibition of NKA activity in the synaptosomes of adult rat brain [46]. This may have implications in the involvement of thyroid hormone on important

**5.3. In search for possible second messenger mediated events in synaptosomal L-**

The evidence of L-T3-synaptosomal membrane interaction in association with the inhibition of the synaptosomal membrane NKA activity led us to search for if the L-T3-induced action
