Protein kinase C-α mediates hypertonicity-stimulated increase in urea transporter phosphorylation in the inner medullary collecting duct.
Protein kinase C-α mediates hypertonicity-stimulated increase in urea transporter phosphorylation in the inner medullary collecting duct.
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DOI：
10.1152/ajprenal.00664.2011
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发表时间：
2012-05
期刊：
影响因子：
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通讯作者：
J. Klein;Christopher F. Martin;Kimilia J Kent;J. Sands
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文献类型：
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作者：
J. Klein;Christopher F. Martin;Kimilia J Kent;J. Sands
UT-A1尿素转运蛋白在浓缩尿的产生中起关键作用。加压素和高渗性均可增加大鼠终末髓内集管（IMCD）的尿素通透性。每种激动剂单独增加UT-A1磷酸化和根尖质膜积累。加压素激活PKA并磷酸化UT-A1的486和499丝氨酸。高渗性通过蛋白激酶C （PKC）和细胞内钙刺激尿素通透性。为了确定尿素通透性的高渗刺激是否由pkc介导的UT-A1磷酸化引起，大鼠imcd被代谢标记为[(32)P]。高渗性刺激UT-A1磷酸化，这种增加被PKC抑制剂预孵育阻断。imcd生物素化以评估质膜UT-A1。高渗性增加了生物素化的UT-A1，这种增加被PKC抑制剂预孵育阻断。当PKC被磷酯直接激活时，UT-A1的总磷酸化增加，但486丝氨酸的磷酸化没有增加，这表明PKC与PKA在相同的残基上没有磷酸化UT-A1。由于PKC-α是一种钙依赖性的PKC亚型，而PKC-α敲除小鼠存在尿浓缩缺陷，这表明PKC-α可能介导了对高渗性的反应。与这一假设一致，高张力增加了大鼠imcd中的磷酸化pkc -α。最后，用PKC-α敲除小鼠来确定高渗是否会在PKC-α缺失的情况下刺激UT-A1磷酸化。高渗性显著增加了野生型小鼠的UT-A1磷酸化，但在PKC-α敲除小鼠中没有。我们得出结论，PKC-α介导高渗刺激下IMCD中UT-A1磷酸化的增加。
The UT-A1 urea transporter plays a critical role in the production of concentrated urine. Both vasopressin and hypertonicity increase urea permeability in rat terminal inner medullary collecting ducts (IMCD). Each agonist independently increases UT-A1 phosphorylation and apical plasma membrane accumulation. Vasopressin activates PKA and phosphorylates UT-A1 at serines 486 and 499. Hypertonicity stimulates urea permeability through protein kinase C (PKC) and intracellular calcium. To determine whether the hypertonic stimulation of urea permeability results from a PKC-mediated phosphorylation of UT-A1, rat IMCDs were metabolically labeled with [(32)P]. Hypertonicity stimulated UT-A1 phosphorylation, and this increase was blocked by preincubation with a PKC inhibitor. IMCDs were biotinylated to assess plasma membrane UT-A1. Hypertonicity increased biotinylated UT-A1, and this increase was blocked by preincubation with a PKC inhibitor. When PKC was directly activated using a phorbol ester, total UT-A1 phosphorylation increased, but phosphorylation at serine 486 was not increased, indicating that PKC did not phosphorylate UT-A1 at the same residue as PKA. Since PKC-α is a calcium-dependent PKC isoform and PKC-α knockout mice have a urine-concentrating defect, it suggested that PKC-α may mediate the response to hypertonicity. Consistent with this hypothesis, hypertonicity increased phospho-PKC-α in rat IMCDs. Finally, PKC-α knockout mice were used to determine whether hypertonicity could stimulate UT-A1 phosphorylation in the absence of PKC-α. Hypertonicity significantly increased UT-A1 phosphorylation in wild-type mice but not in PKC-α knockout mice. We conclude that PKC-α mediates the hypertonicity-stimulated increase in UT-A1 phosphorylation in the IMCD.