Hasler Udo, Nielsen Søren, Féraille Eric, Martin Pierre-Yves
Service de Néphrologie, Fondation pour Recherches Médicales, 64 Ave. de la Roseraie, CH-1211, Genève 4, Switzerland.
Am J Physiol Renal Physiol. 2006 Jan;290(1):F177-87. doi: 10.1152/ajprenal.00056.2005. Epub 2005 Jun 28.
Prevailing expression levels of aquaporin-2 (AQP2) mRNA play a major role in regulating AQP2 protein abundance. Here, we investigated whether AQP2 protein abundance is regulated at a posttranscriptional level as well. The expression levels of both AQP2 mRNA and protein increase in response to arginine vasopressin (AVP) in a concentration- and time-dependent manner in cultured immortalized mouse collecting duct principal cells (mpkCCD(cl4) cells). AVP washout from the medium of AVP-pretreated cells revealed that AQP2 mRNA expression progressively decreased over time, whereas AQP2 protein abundance first increased immediately after AVP washout and then gradually decreased over time. Inversely, increasing AVP concentration led to a time-dependent increase of AQP2 mRNA, whereas AQP2 protein abundance first decreased immediately after AVP supplementation and then gradually increased over time. These transient effects arose from altered V2-receptor activity because they could be abolished by SR-121463B, a specific V2-receptor antagonist. Although cycloheximide administration had no effect on transient alterations of AQP2 protein content, these effects were attenuated by administration of chloroquine, a lysosomal inhibitor, or lactacystin, a proteasomal inhibitor. Short-term inhibition of PKA activity significantly increased AQP2 protein abundance and blunted the transient alterations of AQP2 protein content induced by AVP washout and supplementation. In addition, phosphorylated AQP2 abundance increased immediately after AVP supplementation. These results indicate that in response to AVP AQP2 protein abundance in collecting duct principal cells is principally influenced by AQP2 mRNA content but is additionally regulated by PKA-dependent negative feedback acting on AQP2 protein degradation.
水通道蛋白2（AQP2）mRNA的普遍表达水平在调节AQP2蛋白丰度方面起主要作用。在此，我们还研究了AQP2蛋白丰度是否也在转录后水平受到调节。在培养的永生化小鼠集合管主细胞（mpkCCD(cl4)细胞）中，AQP2 mRNA和蛋白的表达水平均以浓度和时间依赖性方式响应精氨酸加压素（AVP）而增加。从经AVP预处理的细胞培养基中洗脱AVP后发现，AQP2 mRNA表达随时间逐渐降低，而AQP2蛋白丰度在AVP洗脱后立即首先增加，然后随时间逐渐降低。相反，增加AVP浓度导致AQP2 mRNA随时间依赖性增加，而AQP2蛋白丰度在补充AVP后立即首先降低，然后随时间逐渐增加。这些瞬时效应源于V2受体活性的改变，因为它们可被特异性V2受体拮抗剂SR-121463B消除。尽管给予环己酰亚胺对AQP2蛋白含量的瞬时改变没有影响，但这些效应可被溶酶体抑制剂氯喹或蛋白酶体抑制剂乳胞素的给药所减弱。短期抑制PKA活性显著增加AQP2蛋白丰度，并减弱由AVP洗脱和补充诱导的AQP2蛋白含量的瞬时改变。此外，补充AVP后磷酸化的AQP2丰度立即增加。这些结果表明，在集合管主细胞中，响应AVP时，AQP2蛋白丰度主要受AQP2 mRNA含量影响，但还受到对AQP2蛋白降解起作用的PKA依赖性负反馈的调节。
