Department of Biology, University of Ottawa, Ottawa, Ontario, Canada.
PLoS One. 2013 Aug 14;8(8):e70764. doi: 10.1371/journal.pone.0070764. eCollection 2013.
Teleost fish living in freshwater are challenged by passive water influx; however the molecular mechanisms regulating water influx in fish are not well understood. The potential involvement of aquaporins (AQP) and epithelial tight junction proteins in the regulation of transcellular and paracellular water movement was investigated in larval zebrafish (Danio rerio). We observed that the half-time for saturation of water influx (K(u)) was 4.3±0.9 min, and reached equilibrium at approximately 30 min. These findings suggest a high turnover rate of water between the fish and the environment. Water influx was reduced by the putative AQP inhibitor phloretin (100 or 500 μM). Immunohistochemistry and confocal microscopy revealed that AQP1a1 protein was expressed in cells on the yolk sac epithelium. A substantial number of these AQP1a1-positive cells were identified as ionocytes, either H⁺-ATPase-rich cells or Na⁺/K⁺-ATPase-rich cells. AQP1a1 appeared to be expressed predominantly on the basolateral membranes of ionocytes, suggesting its potential involvement in regulating ionocyte volume and/or water flux into the circulation. Additionally, translational gene knockdown of AQP1a1 protein reduced water influx by approximately 30%, further indicating a role for AQP1a1 in facilitating transcellular water uptake. On the other hand, incubation with the Ca²⁺-chelator EDTA or knockdown of the epithelial tight junction protein claudin-b significantly increased water influx. These findings indicate that the epithelial tight junctions normally act to restrict paracellular water influx. Together, the results of the present study provide direct in vivo evidence that water movement can occur through transcellular routes (via AQP); the paracellular routes may become significant when the paracellular permeability is increased.
生活在淡水中的硬骨鱼类受到被动水流入的挑战；然而，鱼类中水流入的分子机制还没有被很好地理解。本研究旨在探讨水通道蛋白（AQP）和上皮紧密连接蛋白是否参与了斑马鱼幼鱼（Danio rerio）细胞内和细胞间水运动的调节。我们观察到水流入的半饱和时间（K(u)）为 4.3±0.9 min，并在大约 30 min 时达到平衡。这些发现表明鱼类与环境之间的水交换率很高。水流入被假定的 AQP 抑制剂根皮苷（100 或 500 μM）所减少。免疫组织化学和共聚焦显微镜显示 AQP1a1 蛋白在卵黄囊上皮细胞中表达。大量 AQP1a1 阳性细胞被鉴定为离子细胞，包括 H⁺-ATPase 丰富细胞或 Na⁺/K⁺-ATPase 丰富细胞。AQP1a1 似乎主要表达在离子细胞的基底外侧膜上，这表明它可能参与调节离子细胞的体积和/或水流入循环。此外，AQP1a1 蛋白的翻译基因敲低使水流入减少了约 30%，进一步表明 AQP1a1 在促进细胞间水吸收中发挥作用。另一方面，用 Ca²⁺螯合剂 EDTA 孵育或上皮紧密连接蛋白 claudin-b 的基因敲低显著增加了水流入。这些发现表明上皮紧密连接通常起到限制细胞间水流入的作用。综上所述，本研究的结果提供了直接的体内证据，表明水可以通过细胞内途径（通过 AQP）进行移动；当细胞旁通透性增加时，细胞旁途径可能变得重要。
