Robey R B, Kwon H M, Handler J S, Garcia-Perez A, Burg M B
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1991 Jun 5;266(16):10400-5.
Madin-Darby canine kidney (MDCK) cells accumulate glycinebetaine via Na(+)-dependent transport in response to hypertonic stress. When extracellular tonicity is increased by the addition of NaCl, Vmax for glycinebetaine transport increases without an associated change in Km, consistent with an increase in the number of functioning transporters. To test whether increased transport activity results from increased gene expression, we injected poly(A)+ RNA (mRNA) from MDCK cells into Xenopus oocytes and assayed for glycinebetaine uptake in ovo. RNA-induced Na(+)-dependent uptake is observed in oocytes injected with mRNA from cells exposed to high extracellular NaCl, but not in oocytes injected with either water or mRNA from cells maintained in isotonic medium. Unfractionated mRNA induces glycinebetaine uptake in ovo at a rate which is approximately 3-fold higher than in water-injected controls. Size-fractionated mRNA (median size 2.8 kilobases) induces uptake at a rate which is approximately 7-fold higher than controls. Such RNA-induced transport activity in ovo is consistent with heterologous expression of Na(+)/glucinebetaine cotransporters encoded by renal mRNA. Increased transporter mRNA in cells exposed to hypertonicity probably underlies the pattern of expression observed in ovo. This can account for the observed rise in MDCK cell glycinebetaine transport during hypertonic stress.
麦氏犬肾（MDCK）细胞在高渗应激反应中通过钠离子依赖性转运积累甘氨酸甜菜碱。当通过添加氯化钠增加细胞外张力时，甘氨酸甜菜碱转运的最大转运速率（Vmax）增加，而米氏常数（Km）没有相关变化，这与功能性转运体数量的增加一致。为了测试转运活性的增加是否源于基因表达的增加，我们将MDCK细胞的多聚腺苷酸加尾RNA（mRNA）注射到非洲爪蟾卵母细胞中，并在卵内测定甘氨酸甜菜碱的摄取情况。在注射了来自暴露于高细胞外氯化钠环境的细胞的mRNA的卵母细胞中观察到了RNA诱导的钠离子依赖性摄取，但在注射了水或来自等渗培养基中培养的细胞的mRNA的卵母细胞中未观察到。未分级的mRNA在卵内诱导甘氨酸甜菜碱摄取的速率比注射水的对照高约3倍。大小分级的mRNA（中位大小2.8千碱基）诱导摄取的速率比对照高约7倍。这种卵内RNA诱导的转运活性与肾脏mRNA编码的钠离子/甘氨酸甜菜碱共转运体的异源表达一致。暴露于高渗环境的细胞中转运体mRNA的增加可能是卵内观察到的表达模式的基础。这可以解释在高渗应激期间MDCK细胞中观察到的甘氨酸甜菜碱转运增加的现象。
