Kinetics of subcellular distribution in rat intestine of 1,25-dihydroxycholecalciferol administered in vivo. Evidence for concentration within 5 min into purified nuclei.
Kinetics of subcellular distribution in rat intestine of 1,25-dihydroxycholecalciferol administered in vivo. Evidence for concentration within 5 min into purified nuclei.
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体内施用 1,25-二羟基胆钙化醇在大鼠肠道中的亚细胞分布动力学。
DOI：
10.1042/bj2080567
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发表时间：
1982
期刊：
影响因子：
--
通讯作者：
Klohs,WD
中科院分区：
文献类型：
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作者：
Bloor,JH;Dasmahapatra,A;Weiser,MM;Klohs,WD
为了更好地了解 1,25-二羟基胆钙化醇 [1,25(OH)2D3] 诱导肠道 Ca2+ 转运的初始步骤，我们研究了 1,25(OH)2D3 在大鼠肠道中的早期亚细胞定位。维生素 D 缺乏的大鼠在处死前 5 分钟至 4 小时内静脉注射 300 pmol 1,25(OH)2[3H]D3。将在 I = 90 mmol/L 的缓冲液中匀浆的细胞通过离心分级分离成粗核沉淀、纯化的细胞核、高尔基体和基底侧膜、细胞质和核后沉淀。根据生化和形态学标准建立核纯化，产率为 32 +/- 2%（平均值 +/- S.E.M.；n = 21）。尽管高尔基体重新吸收 Ca2+ 是最早报道的肠道对 1,25(OH)2D3 的反应之一，但没有检测到 1,25(OH)2D3 直接定位于高尔基体。在所有研究时间中，纯化的细胞核具有最高的比放射性，在 5 分钟时可检测到核定位，在 1 小时时可检测到峰值核摄取。细胞核对细胞质的相对特异性放射性从5分钟增加到30分钟，此时细胞质和细胞核之间似乎达到平衡。核摄取发生在从绒毛到隐窝的所有细胞中。总核结合中的 10% 对高离子强度缓冲液具有抵抗力 (I = 365 mmol/L)；在此缓冲液中 30 分钟时观察到峰值核摄取。这种紧密结合可能代表 1,25(OH)2D3 的活性部分。这些结果表明，1,25(OH)2D3 在观察到的高尔基膜效应之前定位于大鼠肠核，并表明存在高亲和力的核 1,25(OH)2D3 结合位点。
To better understand the initial steps in the induction of intestinal Ca2+ transport by 1,25-dihydroxycholecalciferol [1,25(OH)2D3], we studied the early subcellular localization of 1,25(OH)2D3 in rat intestine. Vitamin D-deficient rats received 300 pmol of 1,25(OH)2[3H]D3 intravenously at 5 min to 4h before being killed. Cells homogenized in buffer of I = 90 mmol/litre were fractionated by centrifugation into a crude nuclear pellet, purified nuclei, Golgi and basal-lateral membranes, cytosol and a post-nuclear pellet. Nuclear purification was established by biochemical and morphological criteria and gave a yield of 32 +/- 2% (mean +/- S.E.M.; n = 21). Although re-establishment of Ca2+ uptake by Golgi is one of the earliest reported intestinal responses to 1,25(OH)2D3, no direct localization of 1,25(OH)2D3 to Golgi was detected. Purified nuclei had the highest specific radioactivity at all times studied, with nuclear localization detectable at 5 min and peak nuclear uptake at 1 h. Relative specific radioactivity of nuclei to cytosol increased from 5 min to 30 min, at which time equilibrium between cytosol and nucleus appeared to be attained. Nuclear uptake occurred in all cells from villus to crypt. Of total nuclear binding 10% was resistant to high ionic strength buffer (I = 365 mmol/litre); peak nuclear uptake was observed at 30 min in this buffer. This tight binding may represent the active fraction of 1,25(OH)2D3. These results indicate that localization of 1,25(OH)2D3 to rat intestinal nuclei precedes the observed Golgi-membrane effects and suggest the existence of high-affinity nuclear 1,25(OH)2D3-binding sites.