Unveiling the nongenomic actions of thyroid hormones in adult mammalian brain: The legacy of Mary B. Dratman.

A comprehensive review was conducted to compile the contributions of Mary B. Dratman and studies by other researchers in the field of nongenomic actions of thyroid hormones in adult mammalian brain. Dratman and her collaborators authored roughly half of the papers in this area. It has been almost fifty years since Dratman introduced the novel concept of thyroid hormones as neurotransmitters for the first time. The characterization of unique brain-region specific accumulation of thyroid hormones within the nerve terminals in adult mammals was a remarkable contribution by Dratman. It suggested a neurotransmitter- or neuromodulator-like role of thyroid hormone and/or its derivative, 3-iodothyronamine within adrenergic systems in adult mammalian brain. Several studies by other researchers using synaptosomes as a model system, have contributed to the concept of direct nongenomic actions of thyroid hormones at synaptic regions by establishing that thyroid hormones or their derivatives can bind to synaptosomal membranes, alter membrane functions including enzymatic activities and ion transport, elicit Ca2+/NO-dependent signaling pathways and induce substrate-protein phosphorylation. Such findings can help to explain the physiological and pathophysiological roles of thyroid hormone in psychobehavioral control in adult mammalian brain. However, the exact mode of nongenomic actions of thyroid hormones at nerve terminals in adult mammalian brain awaits further study.

Autochthonous Gut Bacteria in Two Indian Air-breathing Fish, Climbing Perch (Anabas testudineus) and Walking Catfish (Clarias batrachus): Mode of Association, Identification and Enzyme Producing Ability.

Scanning electron microscopy (SEM) was used to define the location of epithelium-associated bacteria in the gastrointestinal (GI) tract of two Indian air-breathing fish, the climbing perch (Anabas testudineus) and walking catfish (Clarias batrachus). The SEM examination revealed substantial numbers of rod shaped bacterial cells associated with the microvillus brush borders of enterocytes in proximal (PI) and distal regions (DI) of the GI tract of both the fish species. Ten (two each from the PI and DI of climbing perch and three each from the PI and DI of walking catfish) isolated bacterial strains were evaluated for extracellular protease, amylase and cellulase production quantitatively. All the bacterial strains exhibited high cellulolytic activity compared to amylolytic and proteolytic activites. Only two strains, CBH6 and CBH7, isolated from the DI of walking catfish exhibited high proteolytic activity. Maximum cellulase activity was exhibited by the strain, CBF2, isolated from the PI of climbing perch. Six most promising enzyme-producing adherent bacterial strains were identified by 16S rDNA gene sequence analysis. The strain ATH1 (isolated from climbing perch) showed high similarity fo Bacillus amyloliquefaciens whereas, the remaining five strains (isolated from walking catfish) were most closely related to Bacillus licheniformis.

Mechanisms of L-triiodothyronine-induced inhibition of synaptosomal na(+)-k(+)-ATPase activity in young adult rat brain cerebral cortex.

The role of thyroid hormones (TH) in the normal functioning of adult mammalian brain is unclear. Our studies have identified synaptosomal Na(+)-K(+)-ATPase as a TH-responsive physiological parameter in adult rat cerebral cortex. L-triiodothyronine (T3) and L-thyroxine (T4) both inhibited Na(+)-K(+)-ATPase activity (but not Mg(2+)-ATPase activity) in similar dose-dependent fashions, while other metabolites of TH were less effective. Although both T3 and the ß -adrenergic agonist isoproterenol inhibited Na(+)-K(+)-ATPase activity in cerebrocortical synaptosomes in similar ways, the ß -adrenergic receptor blocker propranolol did not counteract the effect of T3. Instead, propranolol further inhibited Na(+)-K(+)-ATPase activity in a dose-dependent manner, suggesting that the effect of T3 on synaptosomal Na(+)-K(+)-ATPase activity was independent of ß -adrenergic receptor activation. The effect of T3 on synaptosomal Na(+)-K(+)-ATPase activity was inhibited by the α2-adrenergic agonist clonidine and by glutamate. Notably, both clonidine and glutamate activate Gi-proteins of the membrane second messenger system, suggesting a potential mechanism for the inhibition of the effects of TH. In this paper, we provide support for a nongenomic mechanism of action of TH in a neuronal membrane-related energy-linked process for signal transduction in the adult condition.

Membrane phospholipid augments cytochrome P4501a enzymatic activity by modulating structural conformation during detoxification of xenobiotics.

Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites. Accumulated body of evidence indicates that phospholipids play a crucial role in determining the enzymatic activity of cytochrome P450 in the microenvironment by modulating its structure during detoxification; however, the structure-function relationship of cytochrome P4501A, a family of enzymes responsible for degrading lipophilic aromatic hydrocarbons, is still not well defined. Inducibility of cytochrome P4501A in cultured catfish hepatocytes in response to carbofuran, a widely used pesticide around the world, was studied earlier in our laboratory. In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver. We examined the role of phospholipid on the of cytochrome P4501A-marker enzyme which is known as ethoxyresorufin-O-deethylase (EROD) in the context of structure and function. We purified the carbofuran-induced cytochrome P4501A protein from catfish liver. Subsequently, we examined the enzymatic activity of purified P4501A protein in the presence of phospholipid, and studied how the structure of purified protein was influenced in the phospholipid environment. Membrane phospholipid appeared to accelerate the enzymatic activity of EROD by changing its structural conformation and thus controlling the detoxification of xenobiotics. Our study revealed the missing link of how the cytochrome P450 restores its enzymatic activity by changing its structural conformation in the phospholipid microenvironment.

Targeting RET to induce medullary thyroid cancer cell apoptosis: an antagonistic interplay between PI3K/Akt and p38MAPK/caspase-8 pathways.

Mutations in REarranged during Transfection (RET) receptor tyrosine, followed by the oncogenic activation of RET kinase is responsible for the development of medullary thyroid carcinoma (MTC) that responds poorly to conventional chemotherapy. Targeting RET, therefore, might be useful in tailoring surveillance of MTC patients. Here we showed that theaflavins, the bioactive components of black tea, successfully induced apoptosis in human MTC cell line, TT, by inversely modulating two molecular pathways: (i) stalling PI3K/Akt/Bad pathway that resulted in mitochondrial transmembrane potential (MTP) loss, cytochrome-c release and activation of the executioner caspases-9 and -3, and (ii) upholding p38MAPK/caspase-8/caspase-3 pathway via inhibition of Ras/Raf/ERK. Over-expression of either constitutively active myristoylated-Akt-cDNA (Myr-Akt-cDNA) or dominant-negative-caspase-8-cDNA (Dn-caspase-8-cDNA) partially blocked theaflavin-induced apoptosis, while co-transfection of Myr-Akt-cDNA and Dn-caspase-8-cDNA completely eradicated the effect of theaflavins thereby negating the possibility of existence of other pathways. A search for the upstream signaling revealed that theaflavin-induced disruption of lipid raft caused interference in anchorage of RET in lipid raft that in turn stalled phosphorylation of Ras and PI3Kinase. In such anti-survival cellular micro-environment, pro-apoptotic signals were triggered to culminate into programmed death of MTC cell. These findings not only unveil a hitherto unexplained mechanism underlying theaflavin-induced MTC death, but also validate RET as a promising and potential target for MTC therapy.

Regulation of cytochrome P4501A by protein kinase C: the role of heat shock protein70.

Carbofuran is a pesticide, which is used throughout the world as a nematicide and an acaricide. This pesticide integrates into living organisms through aquatic ecosystem. In earlier report, we had demonstrated that cytochrome P4501A was induced in cultured catfish hepatocytes in response to carbofuran, which might be responsible for the detoxification of this pesticide. As the underlying signaling mechanism associated with induction and regulation of cytochrome P4501A has not yet been well defined, we therefore in the present study have investigated to identify the regulatory network of cytochrome P4501A in catfish liver or cultured hepatocytes by targeting several key signaling molecules such as phosphatidyl inositol (PI) or protein kinase C (PKC), which are critical molecules for many important pathways. PKC and heat shock protein70 (HSP70) have been shown to be induced in response to carbofuran in catfish hepatocytes. Results also indicate that induction of CYP1A is modulated by HSP70 and PKC in fish hepatocytes. Thus our data shed light on the regulation of EROD activity, which has been used as a bio-monitoring tool for measuring aquatic pollution.

Thyroid hormone homeostasis in brain: possible involvement of adrenergic phenomenon in adult rat.

BACKGROUND: Imbalance in thyroid hormone concentrations has been linked with profound neurobehavioral alterations in the adult. Peripheral hypothyroidism is associated with a phenomenon of central thyroid hormone homeostasis in adult rat. This central homeostasis mechanism could be maintained by adrenergic interplay due to close physiological association between sympathetic nervous system activity and thyroid hormones. The central homeostasis is characterized by increased cerebrocortical synaptosomal T(3) content, deiodinase type II (DII) activity, and cAMP content. METHODS: We injected specific alpha- and beta-adrenergic receptor (AR) agonists and antagonists along with an anti-thyroid drug to find out any AR-mediated action on central homeostasis. RESULTS: The alpha(2)-AR agonist did not alter the onset of central homeostasis, but prolonged its duration. Similar prolongation was observed with alpha(2)-AR antagonist and beta-AR agonist, but these compounds amplified the normal anti-thyroid drug-induced rise in cerebrocortical T(3) content on the day of onset of central homeostasis. Injections of the beta-AR antagonist did not cause any perturbations. All these observations have been supported by parallel changes in cerebrocortical DII activity, cAMP and [Ca(2+)](i) content. CONCLUSION: There emerges a close correlation between cerebral T(3) content, DII activity, cAMP and [Ca(2+)](i) content that are regulated by the AR system. Thus, thyroid hormone homeostasis in the adult mammalian brain is maintained primarily by the beta-adrenergic pathway along with an unexpected pharmacological involvement of the alpha-ARs.

Maintenance of homeostasis for thyroid hormone in the adult rat brain: possible involvement of a nuclear-mediated phenomenon.

During adult-onset peripheral hypothyroidism, the brain maintains normal levels of thyroid hormone for some time through a mechanism of 'central homeostasis'. Although onset, duration, and termination of such a homeostatic phenomenon have been recently evaluated in rat models, the mechanism behind remains unknown. During our investigation to understand the mechanism further, we injected the protein synthesis blockers actinomycin D and cycloheximide along with propylthiouracil to adult male rats during the days of onset (day 2) and termination (day 20) of the homeostatic mechanism. We evaluated synaptosomal T(3) level and neuronal Na(+)-K(+)-ATPase and acetylcholinesterase activities along with deiodinase II activity and cyclic adenosine monophosphate level in the cerebral cortex. The results indicated prevalence of unchanged or lower levels of synaptosomal T(3) on the 2nd and on the 20th day, respectively. Such a condition has been parallely supported by reflections in cerebrocortical deiodinase II activity and cyclic adenosine monophosphate levels. The activities of cerebrocortical synaptosomal Na(+)-K(+)-ATPase and acetylcholinesterase, which are the two important physiological parameters for neuronal function, have been found to be supportive of the involvement of a neuronal protein-mediated factor in the 'on' and 'off' reactions in central homeostasis during peripheral hypothyroidism. The results of our study indicate that the expression of 'central thyroid hormone homeostasis' is a genomic nuclear-mediated mechanism.

Variations in hepatic estradiol-17beta receptor concentrations during the annual reproductive cycle of diploid and triploid female catfish, Heteropneustes fossilis (Bloch).

Concentrations of hepatic estradiol-17beta (E2) receptors (ER) in cytosolic and nuclear fractions were evaluated in diploid and triploid female catfish Heteropneustes fossilis (Bloch) during four different reproductive periods of a complete reproductive cycle. Basal level of ER concentration was noted in the resting period of both diploids and triploids. Receptor level gradually elevated through the preparatory period and reached a peak in the pre-spawning period in both diploids and triploids. However, ER concentrations were overall reduced in triploid to that of diploid females. In a single point assay, in diploids, ER concentration showed about a 3-fold rise (p<0.001) in the cytosolic and a 4-fold rise (p<0.001) in the nuclear extracts from resting to the pre-spawning period. In triploids, only a 2-fold rise was observed both in cytosolic (p<0.01) and nuclear (p<0.05) ER concentration during the same span. Finally, a sudden fall of receptor level was observed in the spawning period in both the ploidy groups with a lower concentration in the triploids. The K(d) value did not differ between the females of diploids (cytosolic 1.12+/-0.21 nM and nuclear 6.9+/-0.9 nM) and triploids (cytosolic--1.13+/-0.17 nM, nuclear--6.8+/-2 nM). However, B(max) of the diploid showed about double the value than triploid females both in the cytosolic (diploid--367.4+/-33.24 pmol/mg protein, triploid--187.3+/-13.20 pmol/mg protein, p<0.001) and nuclear extracts (diploid--946+/-66 pmol/mg DNA, triploid-558+/-98 pmol/mg DNA, p<0.01) of liver. Lower E2 binding capacity and lower amount of E2 receptors of triploid catfish liver with a stunted vitellogenic status could be one of the major causes for reduced gonadal development and sterility in female triploids.

Estradiol-17beta: tracing its metabolic significance in female fatbody of fifth instar larvae of silkworm, Bombyx mori L (race: Nistari).

In recent years, various vertebrate peptide and steroid hormones have been identified in invertebrates, estradiol-17beta (E2) being a major one. We have specifically shown NADP-malate dehydrogenase (NADP-MDH) activity in fifth instar larval fatbody of female silkworm, Bombyx mori, as an E2 responsive parameter. Interestingly enough, estradiol-induced increase in the enzyme activity could be counteracted by simultaneous application of specific E2-inhibitor, ICI-182780. Further, a nice correlation was obtained among the E2 titre, specific *E2 binding and expression of NADP-MDH activity in fatbody during different days of normal fifth instar larval development. Though the nature of the binding sites is quite similar to known steroid receptors of vertebrate, the reported absence of estrogen receptor gene in some insects poses a question. A recent finding regarding the presence of an estrogen-related receptor ortholog in fruit fly may provide some answers. The specific effects elicited by estradiol in the female fatbody of this insect support its possibility of having an important metabolic function. This role played by E2, whether hormonal or not, is yet to be identified.

Maintenance of brain thyroid hormone level during peripheral hypothyroid condition in adult rat.

Thyroid hormones are essential for normal functioning of adult mammalian brain. The present investigation deals with the understanding of the time course of thyroid hormone homeostasis in adult rat brain. Animals were rendered hypothyroid by PTU injections (2 mg/100 g bw) for 30 consecutive days. Serum and synaptosomal T3/T4 content, synaptosomal AChE and Na+-K+-ATPase activities were determined on alternate days. While serum T4 level initially increased on the second day compared to control, serum T3 declined in a triphasic pattern; the first phase lasting from the second day to the 6th day, the second phase ended on the 14th day and last phase continued till the 30th day. Cerebro-cortical synaptosomal T3 level increased on the 2nd day from the control, attained a peak on the 4th day, remained stable until the 18th day, and abruptly declined on the 20th day. Synaptosomal T4 content remained negligible or undetected throughout. Synaptosomal membrane Na+-K+-ATPase and AChE activity exhibited an inverse relationship during the experimental regime, being much more prominent on the 2nd, 18th and 20th day coinciding with the variations in brain T3 level. Thus, the study identifies the onset of central homeostasis between the first and second day, its continuation for about 16-18 days and its termination between the 18th and 20th day.

Thyroid hormone profile during annual reproductive cycle of diploid and triploid catfish, Heteropneustes fossilis (Bloch).

Triploid fishes generally show sterility along with retarded gonadal development and aneuploid gametes. In teleosts, thyroid hormones influence seasonal adaptations and annual events such as reproduction. In addition, thyroid hormone deposition in matured ova is important for reproductive success as the role of thyroid hormones in early development and metamorphosis is well established. The present study deals with measurements of free and total thyroxine (T4) and triiodothyronine (T3) in the plasma of triploid and diploid catfish Heteropneustes fossilis (Bloch) in a complete reproductive cycle. Accumulations of total T4 and T3 within the oocytes have also been measured during the spawning period from fishes of both ploidy groups. No difference of plasma free hormones was noticed between the diploids and triploids of both the sexes in any period of reproductive cycle, although, seasonal variations were noted in both the groups. A significant decrease in the total thyroid hormone levels was noticed in plasma of the diploids in the spawning period compared to triploid fish. During the same period, accumulation of THs was significantly higher in the oocytes of diploids than that of the triploids. Thyroid gland structure also revealed a higher state of activity in the female diploids than the triploids during spawning period. Lower activity of thyroid tissue, higher levels of THs in plasma, and lower accumulation of maternally derived hormones in the oocytes of triploid females during spawning period may be associated with sterility of triploids.

Putative L-triiodothyronine receptors in the liver nuclei of mature tropical toad, Bufo melanostictus.

Thyroid hormones exert a major role in growth and differentiation of almost all types of tissues in animals, particularly in amphibian metamorphosis, through its specific nuclear receptor activation followed by gene expression. However, its function in mature tropical amphibians is less studied. The present study revealed the existence of a single class of specific nuclear receptor(s) in the liver nuclei of mature tropical toad, Bufo melanostictus, with a dissociation constant of (3.7 +/- 0.9) x 10(-10) molar and maximum binding capacity of 0.074 +/- 0.013 pmol/mg DNA. The percentage of relative binding affinities for the specific nuclear L-T3 binding site in the liver nuclei of toad were L-triiodothyronine (L-T3) > triiodothyroacetic acid (TRIAC) > L-thyroxine (L-T4) = tetraiodothyroacetic acid (TETRAC) > 3,3',5'-triiodothyronine (r-T3) > Diiodothyrtonine (L-T2) (100 > 75 > 19.4 = 19.4 > 3.7 > 0.39) and the relative ED50 values (in nanomolar) were 0.33 < 0.44 < 1.7 = 1.7 < 9 < 83.

Stimulation of AChE activity in relation to changes in electronmicroscopic structure of adult rat cerebrocortical synaptosomes pretreated with 3-5-3'-triiodo-L-thyronine.

Triiodothyronine (T3) stimulated AChE activity in depolarization-induced intact synaptosomes (isolated from adult rat cerebral cortex) suspended in calcium-supplemented choline chloride buffer in a time-dependent manner maximally 45-60 s after T3 administration and in a dose-dependent manner with an optimum at 10-100 nM. T3 (100 nM) had no such effects on AChE activity in synaptosomes at non-depolarized conditions. There was no direct effect of T3 on AChE activity of lysed synaptosomal suspension in the physiological range (nM) of T3. The experiments suggest that T3 might have a role in the calcium-dependent release/co-release of acetylcholine from intact synaptosomes concomitant with the acceleration of choline uptake mechanisms that has been reported to accompany elevation of AChE activity. Additionally, electron microscopic structures showed condensation of the cytosolic content with increase in electron density, formation of intrasynaptosomal coarse vesicles and appearance of vesicular fusion like structures (meandering) at the periphery in depolarization-induced T3-treated (60 s) intact synaptosomes, indicating the occurrence of the release of neurotransmitters. The present investigation indicates a definite role of T3 on Ca2+-dependent cholinergic neurotransmission.

The role of thyroid hormone on phenylhydrazine hydrochloride mediated inhibitory effects on blood acetylcholinesterase: an in vivo and in vitro study.

A novel phenomenon of protective counteraction by thyroid hormone has been demonstrated in phenylhydrazine hydrochloride (PHH) induced insult on blood acetylcholinesterase (AChE, EC 3.1.1.7) activity, in both, in vivo and in vitro conditions. Injection of PHH (20 microg/g) to juvenile male rats for three consecutive days caused a 48% decrease (p < 0.001) in the total blood AChE activity on the third day (i.e. 24 h after injections for three consecutive days) in comparison to the control animals. Simultaneous injections of thyroxine (T4) 1 or 2 microg/g with PHH (20 microg/g) showed a recovery in AChE activity by 27% (p < 0.02) and 55% (p < 0.001), respectively, in comparison to the only PHH-injected animals. T4 at 1, 2 and 4 microg/g doses showed unchanged levels in comparison to the untreated controls. In our in vitro system, incubations of the RBCs in PHH (2 mM) containing medium also showed an inhibition of 44% (p < 0.001) of the RBC membrane AChE activity in comparison to the control conditions. A recovery of 23-81% of the enzyme activity was observed after simultaneous use of T4 (1 nM-100 nM) or T3 (0.1 nM-100 nM), or triiodothyroacetic acid (TRIAC) (100 nM) with PHH (2 mM) in a dose-dependent manner with a potency profile of T3 > T4 > TRIAC. Incubation of RBCs only with T4, T3, or TRIAC at 0.1-100 nM concentration did not cause any alteration in the membrane AChE activity in comparison to control conditions. Thus, thyroid hormone distinctly demonstrated a counteraction or protective nature of action on the PHH-induced inhibition of total blood and RBC membrane AChE activity.

Gonadotropin releasing hormone (GnRH) neurones of triploid catfish, Heteropneustes fossilis (Bloch): an immunocytochemical study.

Gonadotropin-releasing hormone (GnRH), a regulator of gonadal maturation in vertebrates, is primarily secreted by neurosecretory cells of the pre-optic area (POA) in the forebrain of teleosts. GnRH-immunoreactive (GnRH-ir) cells of this area demonstrate positive correlation in number and size of soma with gonadal maturity and directly innervate the pituitary in most teleosts. Gonadal development in triploid fish remains impaired due to genetic sterility. The gonadal immaturity in triploid fish may be due to low levels of gonadotropin and sex steroids during the vitellogenic phase of reproductive cycle. However, the nature of GnRH-ir cells in triploid fish is not yet known. Triploid catfish (H. fossilis) showed significant decrease (P<0.001) in size and number of immunoreactive-GnRH cells of POA and low immunoreactivity in pituitary in comparison to their diploid full-sibs during the late pre-spawning phase of ovarian cycle. This study suggests that low activity of GnRH-cells in triploid may be due to lack of positive feedback stimulation by sex steroids and/or reduced responsiveness of sensory cells to environmental cues required for gonadal maturation in teleosts.

Testosterone triggers the brain-pituitary-gonad axis of juvenile female catfish (Heteropneustes fossilis Bloch) for precocious ovarian maturation.

The brain-pituitary-gonad axis of precociously matured females (PMFs) of Indian catfish (Heteropneustes fossilis), produced by testosterone treatment during juvenile stages, was analyzed by studies on immunoreactive gonadotropin-releasing hormone (ir-GnRH) secreting cells of the preoptic area of brain, plasma levels of gonadotropin (GtH-II), testosterone (T), and estradiol-17 beta (E(2)). GnRH cells of PMFs were large and strongly immunoreactive in comparison to control females. PMFs showed higher plasma levels of GtH-II, T, and E(2) than did control females. The ovaries of PMFs contained ripe ova, whereas control females had ova at maturing stages. This study suggests testosterone-mediated activation of the brain-pituitary-ovarian axis for precocious maturation in juvenile catfish.

Action of estradiol-17beta on the synthetic activity of the silk gland in Bombyx mori L.

The effects of estradiol-17beta (E(2)) were studied on several metabolic parameters in the silk gland of Bombyx mori L. race Nistari. Topical application of different doses (0.05-4.0&mgr;g/g body weight) of E(2) on the first and second day of the fifth instar larvae showed a dose dependent effect when studied on the fifth day. A significant increase in silk gland weight and fibroin content was observed between the doses 0.05 and 0.1, and 0.1 and 1.0&mgr;g/g of E(2). A similar pattern of dose-dependent rise in DNA and RNA content of posterior silk gland (PSG) was observed with the doses of E(2) when the contents were expressed per pair of PSG. Higher doses of E(2) (2.0 or 4.0&mgr;g/g) demonstrated relatively less increase, unchanged level or a decrease in the above parameters in comparison to the control values. The glutamate-pyruvate transaminase of PSG showed a significant increase from 0.1 to 2.0&mgr;g/g of E(2) doses in comparison to the control value. Simultaneous injection of ICI-182780 (1.0&mgr;g/g), a very pure and specific antiestrogenic compound, with E(2) (1.0&mgr;g/g) caused a significant counteraction of E(2)-induced increase in silk gland activity, which was reflected in DNA and RNA content of PSG, wet weight and fibroin content of silk gland, and on glutamate-pyruvate transaminase activity. Cycloheximide (0.5&mgr;g/g), a protein synthesis blocker, caused a significant inhibition of the E(2) (1.0&mgr;g/g)-induced silk gland activity when treated along with estradiol. From this study it appears that estradiol has a specific effect on silk gland function and that it may act in a nuclear mediated way.