Tamir H, Liu K P, Hsiung S C, Adlersberg M, Nunez E A, Gershon M D
Division of Neuroscience, New York State Psychiatric Institute, New York 10032.
J Neurosci. 1990 Nov;10(11):3743-53. doi: 10.1523/JNEUROSCI.10-11-03743.1990.
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.
甲状腺的滤泡旁（PF）细胞是神经嵴衍生物。这些细胞即使在成年动物中仍具有可塑性，并且在体外暴露于神经生长因子（NGF）时可被诱导表现出神经特性。一种源自PF细胞的人类细胞系——甲状腺髓样癌（MTC），先前已被证明能合成并储存5-羟色胺（5-HT）、一种血清素结合蛋白（SBP）以及几种神经肽；此外，当在营养匮乏的培养基中生长时，MTC细胞表现出神经特性。本研究的目的是利用MTC细胞作为神经相关的模型系统来研究介导5-HT分泌的相关因素。电子显微镜免疫细胞化学显示，MTC细胞的分泌囊泡共同储存免疫反应性5-HT与SBP和降钙素。环磷酸腺苷（cAMP）衍生物N6-2'-O-二丁酰腺苷3',5'-环磷酸（二丁酰-cAMP；1.0 mM）增加了MTC细胞中5-HT的浓度，并使由L-色氨酸合成5-HT的速率几乎增加了一倍。二丁酰-cAMP还显著增加了5-HT的分泌。放线菌酮（20微克/毫升）和茴香霉素（20微摩尔）抑制了二丁酰-cAMP诱导的5-HT释放增加，表明二丁酰-cAMP的这种作用需要蛋白质合成。在磷酸二酯酶抑制剂3-异丁基-1-甲基黄嘌呤（1.0 mM）存在的情况下，霍乱毒素（1.0微克/毫升）和福斯可林（0.05 mM）均增加了5-HT的生物合成和分泌。人们试图鉴定一种刺激cAMP介导的5-HT分泌的配体。促甲状腺激素（TSH：50 mU/毫升）和细胞外钙浓度升高（[Ca2+]e为7.0 mM），这两者均作为PF细胞的促分泌剂，刺激了5-HT的分泌。TSH的作用是依赖Ca2+的。用抗TSH受体的单克隆抗体进行免疫细胞化学证实这些受体存在于MTC细胞上。TSH和细胞外钙浓度升高均未提高cAMP水平。然而，Fura-2荧光测量表明，TSH和细胞外钙浓度升高均增加了胞质钙（[Ca2+]i），细胞外钾浓度升高（[K+]e）时也是如此。得出的结论是，多种信号转导机制可在MTC细胞中触发胞吐作用。cAMP或细胞内钙浓度升高均可刺激分泌；然而，尚未鉴定出一种能增加cAMP的促分泌剂。
