Angiotensin II directly stimulates activity and alters the phosphorylation of Na-K-ATPase in rat proximal tubule with a rapid time course.
Angiotensin II directly stimulates activity and alters the phosphorylation of Na-K-ATPase in rat proximal tubule with a rapid time course.
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DOI：
10.1152/ajprenal.00065.2004
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发表时间：
2004-05
期刊：
影响因子：
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通讯作者：
D. Yingst;Katherine J. Massey;N. Rossi;M. Mohanty;R. Mattingly
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作者：
D. Yingst;Katherine J. Massey;N. Rossi;M. Mohanty;R. Mattingly
我们提供的证据表明，大鼠近端小管中的na - k - atp酶被ANG II直接激活的速度比以前观察到的要快得多。具体来说，我们表明，暴露于0.1 nM和1 nM的ANG II 2分钟，可以减缓细胞内钠的积累速度，以响应在gramicidin d存在下添加的细胞外钠的增加。从这些数据中，我们表明ANG II在细胞内钠的限速浓度下直接刺激na - k - atp酶活性。在相同的条件下，将近端小管暴露于ANG II中，会改变na - k - atp酶α亚基的胰蛋白酶消化产生的多种磷酸肽中32P的掺入量。na - k - atp酶通过瓦巴因亲和柱从全细胞裂解物中分离，然后通过SDS-PAGE分离到其单个亚基。Na- k -ATP酶以E2构象与色谱柱结合，并通过Na+ATP将其构象改变为E1来洗脱。与对照细胞分离的Na-K-ATPase相比，经ANG II处理的细胞分离的Na-K-ATPase更容易从瓦阿因亲和柱上洗脱，这表明ANG II降低了Na-K-ATPase对瓦阿因的亲和。因此，ANG II在2分钟或更短的时间内迅速刺激na - k - atp酶的活性，其机制可能涉及na - k - atp酶的磷酸化和构象的改变。我们认为na - k - atp酶的快速直接激活的生理作用是在钠重吸收过程中对细胞内钠的更大控制。
We present evidence that Na-K-ATPase in the rat proximal tubule is directly activated by ANG II much faster than previously observed. Specifically, we show that a 2-min exposure to 0.1 and 1 nM ANG II slowed the rate of intracellular sodium accumulation in response to an increase in extracellular sodium added in the presence of gramicidin D. From these data, we show that ANG II directly stimulates Na-K-ATPase activity at rate-limiting concentrations of intracellular sodium. Under these same conditions, exposing proximal tubules to ANG II altered the amount of 32P incorporated into multiple phosphopeptides generated from a tryptic digest of the alpha-subunit of Na-K-ATPase. Na-K-ATPase was isolated from whole cell lysates by means of a ouabain-affinity column and then separated into its individual subunits by SDS-PAGE. Na-K-ATPase bound to the column in its E2 conformation and was eluted by altering its conformation to E1 using Na+ATP. Na-K-ATPase isolated from cells treated with ANG II eluted more easily from the ouabain-affinity column than Na-K-ATPase isolated from control cells, suggesting that ANG II decreased the affinity of Na-K-ATPase for ouabain. Thus ANG II rapidly stimulated the activity of Na-K-ATPase in 2 min or less by a mechanism that could involve changes in phosphorylation and conformation of Na-K-ATPase. We suggest that the physiological role for rapid direct activation of Na-K-ATPase is greater control of intracellular sodium during sodium reabsorption.