**3.6. Serotonin**

64 Thyroid Hormone

thyroid hormone synthesis [142].

thryroperoxidase (TPO), Na+/I-

**3.5. CART** 

hypothalamus-pituitary-thyroid axis [144, 145].

brain after psychostimulant drugs were administered [147].

humans, only the short form is expressed (116 amino acids).

that would help to elucidate some aspects of this putative new mechanism. Thus, Volante et al. showed differences in ghrelin content in foetal and pathologic thyroid as compared to normal adult glands, [140]. Moreover, Park et al. [141] demonstrated that ghrelin, via GHSreceptor and calcium signalling, enhanced the TSH-induced proliferation of FRLT5 rat follicular cells, suggesting the thyroid function as a target for ghrelin, via GHS-receptor and protein kinase C, one of the key signal-pathways for thyroid follicular-cell function and

In regard to the implication of ghrelin in thyroid hormone synthesis, after the demonstration of the ghrelin receptor in human thyroid tissue [143], Kluge et al. [144] described a ghrelinmediated decrease in TSH levels and an increase of serum T4, probably due to a ghrelin direct stimulatory action on the thyroid gland. These results has been supported by a study from our research group which have demonstrated that, effectively, ghrelin has a direct effect on the three tissue-specific genes involved in thyroid hormone synthesis:

effect on follicular-cell activity could be responsible for the effects observed at the

Cocaine and amphetamine related transcript (CART) is an abundantly expressed and widely distributed neuropeptide that has been implicated in a number of physiologic processes. The existence of a CART peptide fragment was first reported by Spiess et al. in 1981 in extracts of ovine hypothalamus as a somatostatine-like polypeptide [146], but its functional significance was not explored further. Almost fifteen years later, Douglass et al. found the first evidence of the existence of mRNA encoding neuropeptide CART in the rat

CART gene is composed of 3 exons and 2 introns, with rats and mice having alternatively spliced variants resulting in the production of two peptides, the longest of which has 129 amino acids (lCART) while the shortest is composed by 116 amino acids (sCART) [148]. The C-terminal end of CART, consisting of 48 amino acid residues and three disulfide bonds, it is thought to constitute the biologically active part of the molecule, and several fragments, notably, rlCART 55-102 and rlCART 62-102, have been shown to be active *in vivo*. In

Distribution of CART mRNA and peptides has been demonstrated by several techniques showing a wide distribution throughout the nervous system, including sensory processing areas, central autonomic control areas, pathways involved in drug reward and reinforcement (*nucleus accumbeus*, lateral hypothalamus and projections to dopaminergic ventral tegmental area neurons), areas controlling feeding and body weight (hypothalamic nuclei), and stress related brain regions [149-152]. Furthermore, CART expressing cells have been found in the anterior pituitary, adrenal gland, islets of Langerhans, myenteric plexus of small intestine and in the ovary [153, 154]. Wierup et al. [155] reported for the first time the existence of CART IR in C cells in the porcine thyroid, in addition to porcine pancreas,

symporter (NIS) and thyroglobulin [144, 145]. This direct

Serotonin or 5-hydroxytryptamine (5-HT) is a biogenic amine synthesized by serotonergic neurons of the CNS, pineal gland and enterochromaffin cells of the gastrointestinal tract of humans and other mammals. Serotonin was isolated for the first time by Rapport et al. in 1948 [164] as a vasoconstrictor plasma agent. In fact, platelet serotonin is released to blood clots contributing to the haemostasis regulation. Serotonin synthesized by enterochromaffin cells is mainly involved in intestinal motility, whereas that synthesized in CNS acts as a neurotransmitter implicated in the regulation of mood, appetite, sleep, memory and learning. Besides, serotonin has antidepressant actions and regulates behaviour, cardiovascular function, muscle contraction, endocrine activity and body temperature [165].

Serotonin is derived from the essential amino acid L-tryptophan. The biosynthetic pathway of serotonin has two enzymatic steps: the first is catalyzed by the enzyme tryptophan-

hydroxylase (TPH), which converts the tryptophan in 5-hydroxytryptophan (5-HTP), while the second is catalyzed by an amino-acid decarboxylase which removes a carboxyl group from 5-hydroxytryptophan, forming 5-hydroxytryptamine or serotonin. TPH has been shown to exist in two forms: TPH1, found in several tissues, and TPH2, which is a brainspecific isoform [58].

Paracrine Regulation of Thyroid-Hormone Synthesis by C Cells 67

model. Nevertheless, in accordance to the literature published so far, C-cell secreted serotonin could be, at least for some animal models, considered as a local factor involved in the control of the follicular-cell activity through the hypothalamic-pituitary-thyroid axis.

Melatonin, chemically known as *N*-acetyl-5-methoxytryptamine, is an indoleamine rhythmically secreted by the pineal gland and involved in the regulation of circadian and, sometimes, seasonal rhythms [182]. Although it was originally discovered as a skin-lightening molecule acting on frog and fish melanocytes [183], melatonin is present in all vertebrates and

Pineal melatonin biosynthesis, that takes place during the night, it is activated by norepinephrine through its specific receptor located in the membranes of pinealocytes and consists of four enzymatic reactions. The first two are common to the synthesis of serotonin. Then, serotonin is converted to N-acetylserotonin (NAS) by the enzyme arylalkylamine Nacetyltransferase (AANAT) [186]. NAS is subsequently methylated by hydroxyindole-O-

Melatonin has a wide spectrum of biological activities and it is considered as a pleiotropic compound with important chronobiotic properties. This indoleamine has the capacity to resynchronize circadian rhythms [188-190] and regulate sleep-wake-cycles [191]. Besides playing an important role as a transmitter of photoperiodic information, melatonin has wellcharacterized antioxidant capacities, either, directly as a free-radical neutralizer or, indirectly by enhancing the activity or the expression of antioxidant enzymes [192, 193]. In certain aspects, related to these last activities, there is evidence in the literature that

Action mechanisms of melatonin are very varied. In this respect, this hormone can bind to specific G-protein coupled membrane receptors, of which there are two subtypes in mammals: melatonin-receptor 1a (MT1) and melatonin-receptor 1b (MT2) [195]. MT1 and MT2 possess high-affinity binding sites for melatonin and are widely distributed in CNS and in a wide-spectrum of peripheral tissues [196, 197]. Furthermore, and in addition to its abilities as a free-radical scavenger described above, melatonin has been described to interact with nuclear receptors [198, 199] as well as cytoplasmic proteins such as calmodulin

Since its discovery, melatonin had been considered an exclusive hormone of the pineal gland. However, numerous published studies have changed this view, describing many extrapineal tissues, such as retina [200], Harderian gland [201], gut [202], ovary [203, 204],

Moreover, melatonin has also been found in the rat thyroid-gland, specifically, immunopositive C cells for melatonin have been detected [45]. Furthermore, our research group has recently demonstrated endogenous melatonin biosynthesis by rat thyroid C cells through the expression of the two key melatonin biosynthetic enzymes, AANAT and

immune system [205, 206], skin [207], and testes [208], as melatonin synthesizers.

is also produced by bacteria, protozoa, plants, fungi and invertebrates [184, 185].

describes potential anti-aging and anticarcinogenic effects for melatonin [194].

methyltransferase (HIOMT) to form melatonin [187] (see Figure 8).

**3.7. Melatonin** 

and protein kinase C.

In 1960, Giarman and Freedman confirmed that the pineal gland was the richest site of serotonin in the brain [166]. This discovery suggested the pineal gland as an important site of serotonergic activity. In the pineal gland, much of this serotonin is acetylated and then methylated to yield melatonin during the night. In fact, there are day-night variations in pineal serotonin-content which is low at night, as opposite to plasmatic melatonin, which is low during the day and increases to a peak during the night [167].

Many regulatory factors that control thyroid activity at hypothalamic level have been described. Specifically, serotonin stimulates hypothalamic TRH production, leading to an increase in TSH production from the pituitary. Adequate serotonin production is necessary to maintain thyroid hormone levels. In fact, in depressed patients with low levels of serotonin, treatment with thyroid hormones has increasing effects on *selective serotonin reuptake inhibitors* (SSRI) [168].

In normal mouse and rat thyroid glands, serotonin is stored as a component of mast-cell granules (see Figure 6C) [169, 170]. Variations on mast-cell exocytosis and serotonin-content seem to be chronobiologically-linked to circadian variations of thyroid activity [169]. In rats, thyrotropin induces a gradual amine release from mast cells within the thyroid gland, where 5-HT stimulates thyroid blood flow and/or vascular permeability [170, 171]. In addition, a 5- HT-inactivating transporter (SERT), identical to that of serotonergic neurons has been found in follicular cells of several mammals [172]. Furthermore, follicular cells express specific 5- HT2 receptors through which serotonin modulates TSH response and stimulates thyroid hormone synthesis [58].

Besides mast-cell synthesis, serotonin is also expressed by thyroid C cells of different mammal species, however, as far as we know only sheep, goats, cows, bats and marmosets apparently convert endogenous 5-HTP into 5-HT [173-175]. Mice and rats belong to these many species in which endogenous 5-HT has not yet been found in adult C cells, unless the thyroid gland is pretreated with 5-HTP. C cells store serotonin in the same secretory granules as calcitonin [41, 176], where 5-HT is linked to a specific protein called serotoninbinding-protein (SBP) [41, 177-179]. Furthermore, C cells have been proposed as a serotonergic neuron study model since they exhibit some properties of serotonergic neurons, including biosynthesis and storing of serotonin and regulated release and expression of both the 5-HT autoreceptor and the 5-HT transporter [180]. Finally, C cells have been demonstrated to be stimulated by both: extracellular calcium and, in the same way to that described for mast cells, by TSH; these results support the putative regulatory role of the C-cell secreted serotonin [181].

The effects observed on thyroid function could be partly due to extrathyroidal serotonin and, on the other hand, serotonin-synthesis is not a general feature of C cells of every animal model. Nevertheless, in accordance to the literature published so far, C-cell secreted serotonin could be, at least for some animal models, considered as a local factor involved in the control of the follicular-cell activity through the hypothalamic-pituitary-thyroid axis.
