Basham J C, Chabrerie A, Kempson S A
Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5120, USA.
Kidney Int. 2001 Jun;59(6):2182-91. doi: 10.1046/j.1523-1755.2001.00733.x.
Epithelial cells in the renal inner medulla accumulate osmolytes such as betaine to maintain normal cell volume during prolonged extracellular hypertonic stress. Betaine accumulation is the result of activation of transcription of the BGT1 transporter gene followed by increased betaine transport.
We studied the possible role of microtubules in this adaptive mechanism using renal cells in culture. RESULTS.: In cultured renal cell lines [Madin-Darby canine kidney (MDCK) and mouse inner medullary collecting duct (mIMCD-3)], up-regulation of BGT1 activity was maximal after 24 to 30 hours in growth medium made hypertonic (510 mOsm/kg) by the addition of sucrose or NaCl. Up-regulation was reversed within 24 to 36 hours after returning cells to isotonic medium. Both cycloheximide (20 micromol/L) and nocodazole (20 micromol/L) blocked the hypertonic up-regulation of BGT1. Nocodazole was partially effective even when added 16 to 20 hours after the switch to hypertonic medium. Recovery from nocodazole action was rapid, and there was full activation of BGT1 transport within three to six hours after nocodazole removal, suggesting rapid trafficking to the cell surface once microtubules repolymerized. Hypertonic activation of BGT1 transport was detected in an isolated membrane fraction and was blocked by cycloheximide but not by nocodazole. Confocal microscopy confirmed the increased abundance of BGT1 proteins in the plasma membrane of hypertonic cells and showed that BGT1 remained intracellular during nocodazole treatment.
Hypertonic activation of BGT1 in renal cells requires de novo protein synthesis and microtubule-dependent trafficking of additional transporters to the cell surface. The apparent resistance of membrane BGT1 to nocodazole blockade is likely due to the presence in the membrane fraction of an increased intracellular pool of active BGT1 transporters.
肾内髓质中的上皮细胞积累渗透压溶质，如甜菜碱，以在长期细胞外高渗应激期间维持正常细胞体积。甜菜碱的积累是BGT1转运蛋白基因转录激活，随后甜菜碱转运增加的结果。
我们使用培养的肾细胞研究微管在这种适应性机制中的可能作用。结果：在培养的肾细胞系[麦迪逊-达比犬肾（MDCK）和小鼠内髓集合管（mIMCD-3）]中，通过添加蔗糖或氯化钠使生长培养基变为高渗（510 mOsm/kg）后，24至30小时内BGT1活性的上调达到最大值。将细胞恢复到等渗培养基后，上调在24至36小时内逆转。放线菌酮（20 μmol/L）和诺考达唑（20 μmol/L）均阻断了BGT1的高渗上调。即使在切换到高渗培养基后16至20小时添加，诺考达唑也有部分效果。从诺考达唑作用中恢复很快，去除诺考达唑后三至六小时内BGT1转运完全激活，表明微管重新聚合后迅速转运到细胞表面。在分离的膜组分中检测到BGT1转运的高渗激活，并且被放线菌酮阻断，但未被诺考达唑阻断。共聚焦显微镜证实高渗细胞的质膜中BGT1蛋白丰度增加，并表明在诺考达唑处理期间BGT1保留在细胞内。
肾细胞中BGT1的高渗激活需要从头合成蛋白质以及微管依赖的额外转运蛋白向细胞表面的运输。膜BGT1对诺考达唑阻断的明显抗性可能是由于膜组分中存在增加的活性BGT1转运蛋白细胞内池。
