**3.7. Melatonin**

66 Thyroid Hormone

specific isoform [58].

*uptake inhibitors* (SSRI) [168].

hormone synthesis [58].

role of the C-cell secreted serotonin [181].

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 brain-

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

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 re-*

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

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

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

low during the day and increases to a peak during the night [167].

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

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

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

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 and protein kinase C.

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

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

HIOMT and, moreover, melatonin membrane receptors have also been found in the rat thyroid gland, specifically, on follicular-cell and C-cell membranes [209] (see Figure 8).

Paracrine Regulation of Thyroid-Hormone Synthesis by C Cells 69

In addition to those described along the present chapter, and despite the fact that there is still limited evidence in the literature, there is a small group of regulatory factors such as bombesin/GRP, CCK (cholecystokinin) and helodermin, that should be observed as

Bombesin/GRP (gastrin-releasing peptide) was first described as being expressed by neoplastic C cells from MTCs by the group of Kameya et al. In 1983 [217]. Moreover, Ahren in 1989 [57] confirmed that GRP had the capacity to stimulate both basal and TSHstimulated thyroid hormone secretion in mice. Furthermore, differences in GRP synthesis among developing thyroid C cells, normal adult C cells, and neoplastic C cells led Sunday et al [32] to hypothesize that C-cell GRP gene-expression might play a role in both normal and

In the case of CCK, its expression in C cells was described by Arias et al. in rat thyroid gland [42]. Recently, its receptor CCK2R, which is shared with gastrin, has been described to be expressed at different intensities in normal and malignant C cells by Blakër et al. [218]. No additional data has been published with regard to the putative functional role of CCK signalling in thyroid, however, there are initial encouraging therapeutic results with the use

The last factor from this "other" group is the amidated peptide helodermin. Originally isolated from *Heloderma suspectum* [221, 222], helodermin was described to be expressed by rat C cells by Grunditz et al. in 1999 [35]. Furthermore, these authors reported dose and time course experiments which showed a consistent stimulatory effect of helodermin on thyroid

Although some of these data seemed to have well-characterized new pathways for C-cell and thyrocytes interrelationships, surprisingly no further studies that illustrated new insights for this evidence have been reported so far. Therefore, to our knowledge, future studies are still

necessary to finally clarify their involvement in thyroid function and homeostasis.

**Figure 9.** Schematic diagram of the different mechanisms of regulation of the synthesis of thyroid

of CCK/Gastrin R2-binding peptides in patients with MTC [219, 220].

potential new candidates for thyroid local-modulators.

neoplastic growth processes.

hormone synthesis.

hormones by follicular cells.

**Figure 8.** Immunolocalization of AANAT in a rat C-cell line (CA77) (A), and melatonin receptor (MT1) in a follicular-cell line (PC-Cl3) (B) by immunofluorescence.

Many effects of melatonin on the thyroid gland have been described so far in the literature. In rodents, high doses of melatonin inhibit basal and TSH-stimulated mitotic activity of thyroid follicular cells *in vivo* and in primary culture [210]. Besides, melatonin has a direct inhibitory effect on T4 secretion and, also, depresses the response of the thyroid to TSH [211]. Furthermore, melatonin plays a protective role against oxidative stress in the rat thyroid gland [212-214].

As regard to the latter, reactive oxygen species (ROS) are deeply involved in cellular processes of the thyroid gland. Follicular cells are subjected to high oxidative stress since they require hydrogen peroxide (H2O2) for thyroid hormones biosynthesis and moreover, a large number of diseases associated with H2O2 accumulation in the thyroid gland have been described. For example, H2O2 participates in the Wolff-Chaikoff's effect and in hypothyroidism caused by iodine excess in the thyroid [213]. Melatonin has been suggested to be able to directly scavenge H2O2 [215]; in accordance to this last point melatonin synthesized by C cells might play a role in thyroid antioxidant defense against oxidative stress. Endogenous synthesized intrathyroidal melatonin could, thus, be a kind of local regulator that could regulate redox homeostasis and modulate thyroid function [216].
