Garrett J E, Tamir H, Kifor O, Simin R T, Rogers K V, Mithal A, Gagel R F, Brown E M
Department of Molecular Biology, NPS Pharmaceuticals, Salt Lake City, Utah 84108, USA.
Endocrinology. 1995 Nov;136(11):5202-11. doi: 10.1210/endo.136.11.7588259.
Calcitonin (CT) secretion by parafollicular cells of the thyroid (C cells) is regulated by small changes in the concentration of extracellular calcium ([Ca2+]e). Elevation of [Ca2+]e elicits a rise in the C cell cytoplasmic calcium concentration and stimulates CT release. The molecular entity through which C cells detect changes in [Ca2+]e and modulate hormone secretion is unknown. Recently, an extracellular calcium-sensing receptor (CaR) complementary DNA was isolated from bovine parathyroid gland. To assess whether parathyroid cells and C cells use similar mechanisms to detect changes in ambient Ca2+, rat, human, and sheep C cells were examined for expression of the parathyroid CaR or a related receptor isoform. Reverse transcription-polymerase chain reaction analysis identified CaR transcripts in rat and human thyroid gland. Northern blot analysis demonstrated CaR messenger RNA (mRNA) in rat thyroid gland, a human medullary thyroid carcinoma (MTC) isolate, and a highly enriched preparation of sheep C cells. Rat MTC 44-2 cells, a cell line responsive to changes in [Ca2+]e, express abundant levels of CaR mRNA. Human TT cells, a C cell line lacking the extracellular calcium-sensing function, have undetectable levels of CaR mRNA by Northern blot analysis. Western blot analysis, using antiserum specific to the parathyroid CaR, detected CaR protein in rMTC 44-2, but not TT cells. Immunostaining of both dispersed sheep C cells and rat thyroid gland sections identified C cell-specific expression of the CaR protein, and in situ hybridization analysis confirmed the C cell-specific expression of CaR mRNA in the intact rat thyroid. The nucleotide sequence of the coding region of the rMTC 44-2 CaR transcripts was found to encode the same CaR protein as that expressed in the parathyroid and kidney. The results demonstrate that C cells express the same extracellular calcium-sensing receptor that is found in parathyroid and kidney, and the presence of this receptor protein in C cell lines correlates with the extracellular calcium-sensing function. This CaR is likely to represent the primary molecular entity through which C cells detect changes in [Ca2+]e and control CT release, suggesting that activation of the same receptor can either stimulate or inhibit hormone secretion in different cell types.
甲状腺滤泡旁细胞（C细胞）分泌的降钙素（CT）受细胞外钙浓度（[Ca2+]e）的微小变化调节。[Ca2+]e升高会引起C细胞胞质钙浓度升高，并刺激CT释放。C细胞检测[Ca2+]e变化并调节激素分泌的分子机制尚不清楚。最近，从牛甲状旁腺中分离出一种细胞外钙敏感受体（CaR）互补DNA。为了评估甲状旁腺细胞和C细胞是否使用相似的机制来检测周围Ca2+的变化，对大鼠、人类和绵羊的C细胞进行了检测，以确定甲状旁腺CaR或相关受体异构体的表达情况。逆转录-聚合酶链反应分析在大鼠和人类甲状腺中鉴定出CaR转录本。Northern印迹分析在大鼠甲状腺、一株人类甲状腺髓样癌（MTC）分离株以及高度富集的绵羊C细胞制剂中证实了CaR信使核糖核酸（mRNA）的存在。大鼠MTC 44-2细胞是一种对[Ca2+]e变化有反应的细胞系，表达大量的CaR mRNA。人类TT细胞是一种缺乏细胞外钙传感功能的C细胞系，通过Northern印迹分析检测不到CaR mRNA水平。使用针对甲状旁腺CaR的抗血清进行的蛋白质印迹分析在rMTC 44-2细胞中检测到了CaR蛋白，但在TT细胞中未检测到。对分散的绵羊C细胞和大鼠甲状腺切片进行免疫染色，确定了CaR蛋白在C细胞中的特异性表达，原位杂交分析证实了CaR mRNA在完整大鼠甲状腺中的C细胞特异性表达。发现rMTC 44-2 CaR转录本编码区的核苷酸序列编码的CaR蛋白与甲状旁腺和肾脏中表达的相同。结果表明，C细胞表达与甲状旁腺和肾脏中相同的细胞外钙敏感受体，并且该受体蛋白在C细胞系中的存在与细胞外钙传感功能相关。这种CaR可能是C细胞检测[Ca2+]e变化并控制CT释放的主要分子实体，这表明同一受体的激活在不同细胞类型中既可以刺激也可以抑制激素分泌。
