Belleannée C, Da Silva N, Shum W W C, Marsolais M, Laprade R, Brown D, Breton S
Center for Systems Biology, Program in Membrane Biology/Nephrology Division, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.
Biol Reprod. 2009 Jan;80(1):134-43. doi: 10.1095/biolreprod.108.070797. Epub 2008 Oct 1.
Water and solute transport in the efferent ducts and epididymis are important for the establishment of the appropriate luminal environment for sperm maturation and storage. Aquaporin 9 (AQP9) is the main water channel in the epididymis, but its regulation is still poorly understood. Components of the kinin-kallikrein system (KKS), leading to the production of bradykinin (BK), are highly expressed in the lumen of the male reproductive tract. We report here that the epididymal luminal fluid contains a significant amount of BK (2 nM). RT-PCR performed on epididymal epithelial cells isolated by laser capture microdissection (LCM) showed abundant BK type 2 receptor (Bdkrb2) mRNA expression but no type 1 receptor (Bdkrb1). Double-immunofluorescence staining for BDKRB2 and the anion exchanger AE2 (a marker of efferent duct ciliated cells) or the V-ATPase E subunit, official symbol ATP6V1E1 (a marker of epididymal clear cells), showed that BDKRB2 is expressed in the apical pole of nonciliated cells (efferent ducts) and principal cells (epididymis). Triple labeling for BDKRB2, AQP9, and ATP6V1E1 showed that BDKRB2 and AQP9 colocalize in the apical stereocilia of principal cells in the cauda epididymidis. While uniform Bdkrb2 mRNA expression was detected in the efferent ducts and along the epididymal tubule, marked variations were detected at the protein level. BDKRB2 was highest in the efferent ducts and cauda epididymidis, intermediate in the distal initial segment, moderate in the corpus, and undetectable in the proximal initial segment and the caput. Functional assays on tubules isolated from the distal initial segments showed that BK significantly increased AQP9-dependent glycerol apical membrane permeability. This effect was inhibited by BAPTA-AM, demonstrating the participation of calcium in this process. This study, therefore, identifies BK as an important regulator of AQP9.
输出小管和附睾中的水和溶质转运对于建立适合精子成熟和储存的管腔环境至关重要。水通道蛋白9（AQP9）是附睾中的主要水通道，但其调节机制仍知之甚少。激肽-激肽释放酶系统（KKS）的成分可导致缓激肽（BK）的产生，在雄性生殖道管腔中高度表达。我们在此报告，附睾管腔液中含有大量BK（2 nM）。对通过激光捕获显微切割（LCM）分离的附睾上皮细胞进行的逆转录聚合酶链反应（RT-PCR）显示，有丰富的缓激肽2型受体（Bdkrb2）mRNA表达，但无1型受体（Bdkrb1）。对BDKRB2与阴离子交换蛋白AE2（输出小管纤毛细胞的标志物）或V-ATP酶E亚基（官方符号ATP6V1E1，附睾透明细胞的标志物）进行双重免疫荧光染色，结果显示BDKRB2在非纤毛细胞（输出小管）和主细胞（附睾）的顶端极表达。对BDKRB2、AQP9和ATP6V1E1进行三重标记显示，BDKRB2和AQP9在附睾尾部主细胞的顶端静纤毛中共定位。虽然在输出小管和附睾小管中检测到均匀的Bdkrb2 mRNA表达，但在蛋白质水平上检测到明显差异。BDKRB2在输出小管和附睾尾部中最高，在远端起始段居中，在体部中等，在近端起始段和头部未检测到。对从远端起始段分离的小管进行的功能分析表明，BK显著增加了AQP9依赖性甘油顶端膜通透性。该效应被BAPTA-AM抑制，表明钙参与了这一过程。因此，本研究确定BK是AQP9的重要调节因子。
