Zeytinoğlu F N, Brazeau P, Culkin M K
Regul Pept. 1983 May;6(2):155-62. doi: 10.1016/0167-0115(83)90008-3.
Calcitonin (CT) and neurotensin (NT) secreting cell lines (6-23,44-2) were established from a transplantable rat medullary thyroid carcinoma (rMTC). The 44-2 line was used to obtain two colony clones 44-2 B and C secreting 20-30-fold greater NT than CT. These cells were used to study the regulation of Ca2+-modulated NT secretion and to ascertain the role of K+ and NE in the rapid release of NT. Medium NT was measured by a specific RIA with the antiserum N-1-11. Secretion experiments were in replicate 35 mm dishes in Krebs-Ringer-bicarbonate buffer supplemented with 90 mg% glucose (KRBG). Ca2+ (0.5-4.0 mM) stimulated NT release in a dose-dependent manner with an ED50 of 2.0 mM. Ca2+ was required for NT release induced by the Ca2+ ionophore, ionomycin, also K+ (50 mM) and norepinephrine (NE). To determine the mode of NT release in the presence of control (1.0 mM Ca2+) or experimental conditions (Ca2+ 1.0 mM plus 10(-6) NE and/or 50 mM K+), 44-2 cells were incubated in KRBG using an experimental paradigm wherein medium was changed at 10-min intervals, and NT release was quantitated. NE stimulated release of NT by these cells and the amount of NT released with each repetitive pulse of NE remained constant throughout the experiment. K+ (50 mM) elicited a rapid release of NT in the first 0-10 min incubation and the amount of NT released into the buffer was greater than that measured with NE; however, in these experiments, the response to K+ declined progressively and reached basal values at 20-30 min. Our results show neither pulse stimulation nor continuous incubation of cells with NE affected the response of the cells to a subsequent challenge with K+. These results suggest the presence of differentially stimulated, releasable pools of NT in these cells. We conclude that these newly established 44-2 B and C cells provide a useful model to study the regulation of NT release.
从可移植的大鼠甲状腺髓样癌（rMTC）中建立了分泌降钙素（CT）和神经降压素（NT）的细胞系（6 - 23、44 - 2）。44 - 2细胞系用于获得两个克隆菌落44 - 2 B和C，它们分泌的NT比CT多20 - 30倍。这些细胞用于研究Ca2 +调节的NT分泌的调控，并确定K +和去甲肾上腺素（NE）在NT快速释放中的作用。用抗血清N - 1 - 11通过特异性放射免疫分析（RIA）测定培养基中的NT。分泌实验在补充有90 mg%葡萄糖的Krebs - Ringer - 碳酸氢盐缓冲液（KRBG）中的35 mm培养皿中进行重复实验。Ca2 +（0.5 - 4.0 mM）以剂量依赖方式刺激NT释放，半数有效浓度（ED50）为2.0 mM。Ca2 +离子载体离子霉素、K +（50 mM）和去甲肾上腺素（NE）诱导NT释放都需要Ca2 +。为了确定在对照（1.0 mM Ca2 +）或实验条件（1.0 mM Ca2 +加10(-6) NE和/或50 mM K +）下NT释放的模式，使用一种实验范式将44 - 2细胞在KRBG中孵育，其中每隔10分钟更换培养基，并对NT释放进行定量。NE刺激这些细胞释放NT，并且在整个实验过程中，每次重复给予NE脉冲时释放的NT量保持恒定。K +（50 mM）在孵育的最初0 - 10分钟内引起NT的快速释放，释放到缓冲液中的NT量大于用NE测量的量；然而，在这些实验中，对K +的反应逐渐下降，并在20 - 30分钟时达到基础值。我们的结果表明，无论是脉冲刺激还是用NE持续孵育细胞，都不会影响细胞对随后K +刺激的反应。这些结果表明这些细胞中存在受不同刺激的、可释放的NT池。我们得出结论，这些新建立的44 - 2 B和C细胞为研究NT释放的调控提供了一个有用的模型。
