Frigeri Antonio, Nicchia Grazia Paola, Balena Rosalba, Nico Beatrice, Svelto Maria
Department of General and Environmental Physiology and Centre of Excellence in Comparative Genomics (CEGBA), University of Bari, Bari, Italy.
FASEB J. 2004 May;18(7):905-7. doi: 10.1096/fj.03-0987fje. Epub 2004 Mar 19.
Aquaporin-4 (AQP4) is the major water channel of the neuromuscular system, but its physiological function in both perivascular astrocytes and skeletal muscle sarcolemma is unclear. The purpose of this study was to assess the following in skeletal muscle: a) the expression of all cloned water cannels; b) the functional role of AQP4 using sarcolemma vesicles purified by means of several fractionation methods, and c) the functional effect of AQP4 reduction in mdx mice, the animal model of Duchenne muscular dystrophy (DMD). Immunofluorescence and immunoblot experiments performed with affinity purified antibodies revealed that only AQP1 and AQP4 are expressed in mouse skeletal muscle: AQP1 in endothelial cells of continuous capillaries and AQP4 on the plasma membrane of muscle fiber. Plasma membrane vesicle purification was performed with a procedure extensively used to purify and characterize dystrophin-associated proteins (DAPs) from rabbit skeletal muscle. Western blot analysis showed strong co-enrichment of the analyzed DAPs and AQP4, indicating that the membrane vesicle preparation was highly enriched in sarcolemma. Stopped-flow light-scattering measurements showed high osmotic water permeability of sarcolemma vesicles (approximately 150 microm/s) compatible with the AQP-mediated pathway for water movement. Sarcolemma vesicles prepared from mdx mice revealed, in parallel with AQP4 disappearance from the plasma membrane, a strong reduction in water permeability compared with wild-type mice. Altogether, these results demonstrate high AQP4-mediated water permeability of the skeletal muscle sarcolemma. Expression of sarcolemmal AQP4 together with that of vascular AQP1 may be responsible for the fast water transfer from the blood into the muscle during intense activity. These data imply an important role for aquaporins in skeletal muscle physiology as well as an involvement of AQP4 in the molecular alterations that occur in the muscle of DMD patients.
水通道蛋白4（AQP4）是神经肌肉系统的主要水通道，但其在血管周围星形胶质细胞和骨骼肌肌膜中的生理功能尚不清楚。本研究的目的是评估骨骼肌中的以下方面：a）所有克隆的水通道的表达；b）使用通过几种分级分离方法纯化的肌膜囊泡来研究AQP4的功能作用；c）在杜兴肌营养不良症（DMD）的动物模型mdx小鼠中降低AQP4的功能效应。用亲和纯化抗体进行的免疫荧光和免疫印迹实验表明，小鼠骨骼肌中仅表达AQP1和AQP4：AQP1在连续毛细血管的内皮细胞中表达，AQP4在肌纤维的质膜上表达。采用广泛用于从兔骨骼肌中纯化和表征肌营养不良蛋白相关蛋白（DAPs）的方法进行质膜囊泡纯化。蛋白质印迹分析显示，所分析的DAPs和AQP4有很强的共富集，表明膜囊泡制剂高度富集了肌膜。停流光散射测量显示肌膜囊泡具有高渗透水通透性（约150微米/秒），这与水通过AQP介导的转运途径一致。与野生型小鼠相比，从mdx小鼠制备的肌膜囊泡显示，随着AQP4从质膜消失，水通透性大幅降低。总之，这些结果证明了骨骼肌肌膜具有高AQP4介导的水通透性。肌膜AQP4与血管AQP1的共同表达可能是剧烈活动期间血液向肌肉快速输水的原因。这些数据表明水通道蛋白在骨骼肌生理学中具有重要作用，并且AQP4参与了DMD患者肌肉中发生的分子改变。
