Multiple signal transduction mechanisms leading to the secretion of 5- hydroxytryptamine by MTC cells, a neurectodermally derived cell line

Abstract

Parafollicular (PF) cells of the thyroid gland are neural crest derivatives. These cells remain plastic even in adult animals and can be induced to exhibit neural properties when exposed to NGF in vitro. A human cell line derived from PF cells, medullary thyroid carcinoma (MTC), has previously been shown to synthesize and store 5-HT, a serotonin-binding protein (SBP), and several neuropeptides; moreover, when grown in impoverished media, MTC cells display neural properties. The purpose of the current study was to utilize MTC cells as a neurally relevant model system to investigate factors involved in mediating 5-HT secretion. Electron microscopic immunocytochemistry revealed that secretory vesicles of MTC cells costore immunoreactive 5-HT with SBP and calcitonin. The cAMP derivative, N6-2'-O-dibutyryl-adenosine 3',5'-cyclic monophosphate (dibutyryl-cAMP; 1.0 mM) increased the concentration of 5-HT in MTC cells and almost doubled the rate of synthesis of 5-HT from L-tryptophan. Dibutyryl-cAMP also significantly increased the secretion of 5-HT. Cycloheximide (20 micrograms/ml) and anisomycin (20 microM) inhibited the dibutyryl-cAMP-induced increase of 5-HT release, suggesting that this action of dibutyryl-cAMP requires protein synthesis. Cholera toxin (1.0 microgram/ml) and forskolin (0.05 mM) in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (1.0 mM) both increased 5-HT biosynthesis and secretion. Attempts were made to identify a ligand that stimulates cAMP-mediated secretion of 5-HT. Both thyroid-stimulating hormone (TSH: 50 mU/ml) and elevated [Ca2+]e (7.0 mM), each of which acts as a secretogogue for PF cells, stimulated the secretion of 5-HT. The effect of TSH was Ca2(+)-dependent. Immunocytochemistry with monoclonal antibodies to the TSH receptor confirmed that these receptors are present on MTC cells. Neither TSH nor elevated [Ca2+]e elevated cAMP levels. Measurements of Fura-2 fluorescence, however, indicated that both TSH and elevated [Ca2+]e increased cytosolic calcium ([Ca2+]i), as did elevation of [K+]e. It is concluded that exocytosis can be triggered in MTC cells by multiple signal transduction mechanisms. Either cAMP or elevated [Ca2+]i can stimulate secretion; however, a secretogogue that increases cAMP has yet to be identified.

Ca2+-Evoked Serotonin Secretion by Parafollicular Cells: Roles in Signal Transduction of Phosphatidylinositol 3′-kinase, and the γ and ζ Isoforms of Protein Kinase C

“…The pellet was solubilized in the above buffer containing Triton X-100 (1.0%). Proteins (100 g) were separated by 8.5% SDS-PAGE (Tamir et al, 1990). After separation, the proteins were electroblotted onto nitrocellulose membranes, and protein bands were visualized by staining the blots for 2 min in Ponceau solution (0.02% in 0.3% trichloroacetic acid).…”

“…The CaR is G-protein-coupled (Herbert and Brown, 1995;Ruat et al, 1996) and has been demonstrated to be linked to Gi and Gq (Tamir et al, 1996). After the activation of the CaR by 1[Ca 2ϩ ] e , PF cells depolarize (as a result of an increase in a nonselective plasma membrane cation conductance) (McGehee et al, 1997), secretory vesicles acidify (Tamir et al, 1994b), and 5-HT is secreted (Tamir et al, 1990(Tamir et al, , 1994bMcGehee et al, 1997).…”

“…PKC, however, appears to play an important role in the secretory process. Phorbol esters stimulate 5-HT secretion, and secretion in response to phorbol esters is abolished by phorbol ester downregulation of PKC (Tamir et al, 1990;McGehee et al, 1997). Nevertheless, despite the ability of phorbol ester downregulation of PKC to block phorbol ester-induced secretion, downregulation only slightly inhibits 5-HT secretion when it is evoked by 1[Ca 2ϩ ] e .…”

Paracrine Regulation of Thyroid-Hormone Synthesis by C Cells

“…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][178][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].…”

Development of the parafollicular cells in recurrent goiter

“…Another group showed that increased levels of TRH release calcitonin in normal PF cells as well as in MTC, similar to the effect of pentagastrin (12). Further, TSH leads to a discharge of serotonin in PF cells and MTC (4,6,13,14). On the other hand, serotonin receptors have been detected on the FRTL-5 follicular cell line.…”