**3.2. Somatostatin**

Somatostatin (SS) was first identified in 1973 in ovine hypothalamus as a tetradecapeptide (SS-14) with a disulfure bond between 3 and 14 cysteines. Its main function was to inhibit the release of growth hormone [84]. Subsequently, a big family of functional and structural peptides related to somatostatin was discovered. Over the years several forms of somatostatin depending on the tissue and species have been described.

SS-like immunoreactivity has been found in all vertebrates as well as in some invertebrate species and in the plant kingdom. SS-producing cells occur at high densities throughout the central and peripheral nervous systems, such as the hypothalamus, pituitary, cerebellum, limbic lobe, retina, sympathetic ganglia and the vagus and sciatic nerves. SS-producing cells have also been reported in the endocrine pancreas, the gut and in small numbers in the thyroid, adrenals, submandibular glands, kidneys, prostate, and placenta [85-87].

SS has many physiological actions, it functions as a neurotransmitter in the brain with effects on cognitive, locomotor, sensory, and autonomic functions [88]. Moreover, it has inhibitory effects on the endocrine system, gut exocrine secretion, adrenal glands, kidneys, and on proliferation of lymphocytes, cartilage and bone precursor cells [86, 87, 89, 90]. Other effects consist on the inhibition of the release of growth factors (IGF1, EGF, PDGF) and cytokines (IL6, IFN-g) [91]. All of these diverse effects of SS can be explained by the inhibitory actions of the peptide on two key cellular processes: secretion and cell proliferation.

60 Thyroid Hormone

muscle, breast cancer cell lines and human primary breast cancer cells [77]. Numerous studies have demonstrated that multiple CTR mRNA isoforms are present in a number of species. In humans, two isoforms (h-CTR1 and h-CTR2) differing by a 16 amino-acid insert in the first intracellular loop have been reported [78]. Finally, studies have revealed h-CTR2 expression in normal thyroid glands, in medullary thyroid carcinoma (MTC), and in a model of human MTC cell line (TT), suggesting an autocrine regulation of CTR in C cells

A family of receptors for CGRP and CT has been demonstrated throughout the nervous systems of both man and rat. CGRP-specific binding sites have been found in the cerebellum, spinal cord and other brain regions including vessels and pia mater. In contrast to calcitonin receptors found in bone, these receptors are not linked to adenylate cyclase, however, CGRP stimulates adenylate cyclase activity by acting at the calcitonin receptor in more than one tissue. Furthermore, specific cAMP linked CGRP receptors have been identified both in the intima and media of vessels as well as the atria and ventricles of the heart [80]. Seifert et al. [81] demonstrated the presence of a novel specific receptor for CGRP in the acinar cells of the pancreas. Finally, specific CGRP binding has also been

CGRP has a double location in the thyroid, one in nerve fibers around blood vessels and follicles and the other in C cells. This may suggest a functional role on the follicular cells; however, it seems not to affect basal or TSH-stimulated thyroid hormone secretion [83]. In contrast, CT, CGRP and katacalcin, which have been demonstrated to have no effect on basal and TSH-stimulated thyroid hormone secretion when separately administered alone, inhibited TSH-stimulated thyroid hormone secretion when the three peptides were applied together [83]. Despite this, further studies about the CTRs or calcitonin like receptors (CLRs) expression in follicular cells are necessary to dilucidate the role of CT/CGRP family peptides

Somatostatin (SS) was first identified in 1973 in ovine hypothalamus as a tetradecapeptide (SS-14) with a disulfure bond between 3 and 14 cysteines. Its main function was to inhibit the release of growth hormone [84]. Subsequently, a big family of functional and structural peptides related to somatostatin was discovered. Over the years several forms of

SS-like immunoreactivity has been found in all vertebrates as well as in some invertebrate species and in the plant kingdom. SS-producing cells occur at high densities throughout the central and peripheral nervous systems, such as the hypothalamus, pituitary, cerebellum, limbic lobe, retina, sympathetic ganglia and the vagus and sciatic nerves. SS-producing cells have also been reported in the endocrine pancreas, the gut and in small numbers in the

SS has many physiological actions, it functions as a neurotransmitter in the brain with effects on cognitive, locomotor, sensory, and autonomic functions [88]. Moreover, it has

thyroid, adrenals, submandibular glands, kidneys, prostate, and placenta [85-87].

somatostatin depending on the tissue and species have been described.

[79]. Nevertheless, there is still no evidence of the CTR expression in follicular cells.

demonstrated on cloned lung and bone cancer cells [82].

in the thyroid gland function.

**3.2. Somatostatin** 

The above-listed functions of SS are mediated by a family of receptors that recognizes these ligands with different affinities [92]. Originally, five different somatostatin receptor types (SSR1-5) were identified, cloned and found able to bind not only native SS forms (SS-14 and SS-28), but also their synthetic analogues [93]. SS receptors belong to the family of G protein-coupled receptors, typically consisting of extracellular domains forming the ligand binding site, a single polypeptide chain with seven transmembrane domains and intracellular domains mediating signal transduction. Each SS receptor subtype mediates different biological actions of SS, via the activation of different intracellular systems such as inhibition of adenylate cyclase (with decrease of intracellular cyclic AMP levels), reduction of intracellular calcium levels, and stimulation of phosphotyrosine phosphatase or MAP kinase activity [94].

Within the thyroid, SS coexists with CT in a subpopulation of C cells, with a variable extension. Thus, while the frequency for somatostatin-immunoreactive (SS-IR) CC varies in rats throughout their lives, as they are scarce in the foetus, abundant at the time of birth and scarce again in adults (see Figure 6) [33, 95-97], the majority of calcitonin immunoreactive (CT-IR) CC in guinea pigs and rabbits are also SS-IR, besides positivity is seen in the parathyroid gland [33]. On the contrary, SS-IR CC are observed very occasionally in normal pig and human thyroid glands [98].

**Figure 6.** Immunohistochemical demonstration of calcitonin (A), somatostatin (B) and serotonin (C) in consecutive sections of the rat thyroid gland. Only a subpopulation of calcitonin-positive C cells is also immunopositive for somatostatin. Serotonin is exclusively located in mast cells, being C cells negative in the rat. (x400).

The effects of SS on the thyroid include the inhibition of the TSH-stimulated release of T4 and T3 [99], as well as the inhibition of TSH-stimulated radioiodine turnover accumulation in thyroid glands of euthyroid volunteers [100]. SS inhibition of basal as well as TSHstimulated adenyl cyclase activity has been demonstrated in normal and neoplastic human

thyroid tissues [101] as well as in cultures of human thyrocites [102]. Besides its effects on thyroid function, suppression of thyroid follicular cell growth has been demonstrated by the inhibition of both TSH and IGF-1 proliferative stimulation [103].

Paracrine Regulation of Thyroid-Hormone Synthesis by C Cells 63

**Figure 7.** Immunofluorescent staining for calcitonin (A) and TRH (B) in rat thyroid tissue. All

Ghrelin, is a 28 amino-acid acylated-peptide with powerful GH-releasing, orexigenic and adipogenic functions that, at hypothalamic level, regulates appetite, food-intake and energy metabolism in mammals [125, 126]. Since its initial description in 1999 [125], a variety of new functions for ghrelin have been characterized in the literature. Thus, apart from its GHreleasing and orexigenic effects, ghrelin has been reported to influence sleep and behaviour [127, 128], and the pituitary-gonadal axis at both peripheral [129] and central levels [130, 131]. The enteroendocrine cells of the stomach mucosa are the main source of circulating ghrelin [132]. Ghrelin synthesis and plasma levels rise and fall in relation to food intake, increasing with fasting and decreasing after eating [133, 134].Besides its gastric secretion, ghrelin is also expressed in many other tissues and organs [38, 135, 136] and despite the fact that molecular mechanisms have not yet been characterized, recent investigations have implicated ghrelin in many pathological conditions of heart, bone, liver, kidney and, specifically thyroid tissues [137]. The first observation regarding the presence of ghrelin in thyroid tissue was that by Kanamoto et al. in 2001 [138], describing the production of ghrelin in human medullary thyroid carcinoma tissue. Also, Gnanapavan et al. in 2002 [139] showed a very low expression of ghrelin at mRNA level and suggested to be carried-out by a very minor thyroid cell-population in normal human thyroid tissue. Those findings were concordant to those from Volante et al. [140] who failed to detect ghrelin-immunopositive cells in normal human adult thyroid gland. This last fact, being probably due to the very scarce presence of C cells in the human adult thyroid gland as compared to rats, where their percentage, in relation to follicular cells, ranges from 4.5 to 10.4% [18]. These observations were supported by the study by Raghay et al. in 2006 [38] in which ghrelin was described in the thyroid

Moreover, the ghrelin functional receptor has been demonstrated to be expressed in the neighbouring follicular cells [141], supporting, one more time the idea, increasingly found in the literature, that C cells would modulate thyroid function [38, 60, 63, 64], in this case, in a paracrine fashion, via ghrelin. Addressing this last point, up to date there are evidences

calcitonin-immunopositive C cells are also immunopositive for TRH. (x400).

**3.4. Ghrelin** 

gland to be synthesized only by C cells.

These evidences of the expression of SS in thyroid, more precisely limited to the C cells, and the actions in the follicular cells have led us to the speculation that this peptide could elicit local effects on thyroid hormone release acting locally in a paracrine fashion.
