*5.2.1. In vivo and in vitro actions of L-T3 on synaptosomal Na+-K+-ATPase activity*

A dose-dependent inhibition of synaptosomal NKA activity by L-T3 both in *in vivo* [45], and in *in vitro* [46] conditions have been shown. This may be related to the differences in L-T3 status in adult rat cerebrocortical synaptosomes. L-T3 administration in a single i.p. injection showed inhibition of synaptosomal NKA specific activity maximally at 24 hours postinjection by ~ 44% compared to respective control euthyroid values. A range of L-T3 concentration (0.1 to 4.0 g/g BW, single i. p. injection) administered *in vivo* showed dosedependent inhibition of the synaptosomal NKA activity. In contrast PTU-treated hypothyroid animals showed ~ 38% increase in the NKA activity compared to the control values. This increase in NKA activity was abolished by injection of a single L-T3 injection

mammalian brain.

**cerebrocortical synaptosomes** 

and uptake across the synaptic membrane [43].

lower than found by other investigators. Some assay in brain regions was also performed in tissue homogenates instead of particular subcellular fractions. Possibly differences in the concentrations of THs could be due to a different method of severe extraction procedure

The data emerged from our study reveal the quantitative aspects of involvement of L-T3 in synaptosomes in different thyroid states, and favors its role in neuronal functions as formerly described [10,41]. A stimulation of synthesis of synapsin-1 protein (related to neurotransmission) by L-T3 in the developing brain has been reported [42]. Although, the synaptosomal L-T3 levels varied widely with different treatments, our result illustrates a unique, but unknown regulatory mechanism of the TH metabolism in the mature

**5.2. Modulation of neuronal plasma membrane Na+-K+-ATPase specific activity** 

Subsequently the idea of concentration, distribution and metabolism of THs within the mature brain generated interest to search for potential role of TH and its nongenomic interaction, if any, with neuronal plasma membrane. TH is well known for its regulation of energy metabolism in developing tissues including brain. However, adult brain has not shown this effect on energy metabolism under the influence of TH until recently. Maintenance of ionic gradients by plasma membrane Na+-K+-ATPase (NKA) is one of the important cellular events by which TH regulate energy metabolism. NKA is an ion pump responsible for maintaining Na+ and K+ ion gradients across the cellular plasma membrane in eukaryotic cells. The Na+ and K+ ion gradients are important for establishment of resting membrane potentials as well as for transport of certain molecules. NKA has special significance in maintaining membrane potentials in neurons. Inhibition of NKA has been shown to release acetylcholine [43] and norepinephrine [44] from rat cortical synaptosomes, presumably as a result of depolarizing effects of lowered K+ gradients. The level of NKA activity could therefore have consequence for the regulation of the neurotransmitter release

**as a function of specific binding of L-triiodothyronine in adult rat brain** 

*5.2.1. In vivo and in vitro actions of L-T3 on synaptosomal Na+-K+-ATPase activity* 

A dose-dependent inhibition of synaptosomal NKA activity by L-T3 both in *in vivo* [45], and in *in vitro* [46] conditions have been shown. This may be related to the differences in L-T3 status in adult rat cerebrocortical synaptosomes. L-T3 administration in a single i.p. injection showed inhibition of synaptosomal NKA specific activity maximally at 24 hours postinjection by ~ 44% compared to respective control euthyroid values. A range of L-T3 concentration (0.1 to 4.0 g/g BW, single i. p. injection) administered *in vivo* showed dosedependent inhibition of the synaptosomal NKA activity. In contrast PTU-treated hypothyroid animals showed ~ 38% increase in the NKA activity compared to the control values. This increase in NKA activity was abolished by injection of a single L-T3 injection

employed to extract brain tissue THs resulting in loss of it.

(2g/g BW) to almost close to the euthyroid levels. However, this study could not distinguish between the genomic and nongenomic effects of L-T3. TH has also been reported to inuence K+-evoked release of [3H]-GABA in adult rat cerebrocortical synaptosomes. Such evidence indicates a possible role of TH in neurotransmission in adult mammalian brain. A functional correlation between L-T3 binding and the corresponding inhibition of NKA activity under *in vitro* conditions in the synaptosomes of adult rat cerebral cortex were established [46]. To further test the hypothesis of nongenomic action of TH we investigated NKA activity in isolated synaptosomes which is devoid of nucleus to avoid the chances of nuclear activation [46]. In fact, *in vitro* addition of L-T3 (1x10-12 M to 10x10-8 M) within 10 minutes of incubation indicated a dose-dependent inhibitory response to NKA activity. Such immediate action of L-T3 added in *in vitro* in synaptosomes was concluded as rapid nongenomic action of L-T3 on synaptosomal membrane NKA [46]. Further inhibition of NKA activity was corroborated with gradual binding of [125I]-L-T3 to specific L-T3-binding sites in synaptosomes. Thus a physiologic response tied to the specific L-T3-binding in the synaptosomal membrane was demonstrated.

The presence of high affinity low capacity nuclear TH receptors in adult rat brain has been reported. Further evidence shows selective uptake of [125I]-L-T3 and rapid conversion of L-T4 to L-T3 in synaptosomal fraction of adult rat brain. Specic [125I]-L-T3 binding sites have also been demonstrated in the synaptosomes of adult rat brain [47] and chick embryo [48]. However, no functional relationship could be established due to the interaction of TH and its membrane receptor so far in adult brain.

Scatchard plot analysis demonstrated two sets of specific L-T3 binding sites: one with high affinity (Kd1: 12 pM; Bmax1: 3.73±0.07 fmols/mg protein), and the other with low affinity (Kd2: 1.4±0.05 nM; Bmax2: 349±7 fmols/mg protein). Kd represents dissociation constant. Bmax represents maximum binding capacity. Rationale between gradual L-T3 binding and the corresponding dose-dependent L-T3-induced inhibition of synaptosomal NKA was established *in vitro* [46].

The relative order of potencies of binding afnities for the synaptosomal L-T3 binding sites and relative inhibition of NKA activity in the presence of different L-T3 analogues were as follows: L-T3>L-T3-amine>L-T4=L-TRIAC>r-T3>L-T2, and L-T3>L-T3-amine>L-T4>L-TRIAC>r-T3>L-T2, respectively. The concentrations of TH analogues required to displace 50% specic binding (ED50 value) of 125I-L-T3 to its synaptosomal binding sites were 10-, 63-, 63-, 1000- and 6250 nM, respectively. This study showed the nature of inhibition of synaptosomal NKA activity as a function of L-T3 occupancy of synaptosomal receptor sites in mature rat brain [46].

This investigation demonstrates a novel action of TH in mature rat brain. This is the rst report presenting a relationship between the inhibitions of synaptosomal NKA as a functional effect of L-T3 binding to its synaptosomal receptor in the cerebral cortex of adult rat. Occupancy of specic high afnity L-T3 binding sites demonstrated a concentrationdependent inhibition of the NKA activity with a maximum of 59%. At 1x10–10 M L-T3 concentration the enzyme inhibition was ~35% and the saturation of the L-T3 binding sites 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.

"Quo Vadis?" Deciphering the Code of Nongenomic Action of Thyroid Hormones in Mature Mammalian Brain 15

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

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

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

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

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

Metabolic Research 35: 562-564 acknowledged [57])).

changes between euthyroid and hypothyroid values.

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

we further intended to explore the role of Ca2+ in L-T3-induction.

*hypothyroid brain* 

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 mental functions in adult mammalian brain.
