Department of Biology, Georgia Southern University, Statesboro, GA, USA; Mount Desert Island Biological Laboratory, Salisbury Cove, ME, USA.
Department of Biology, Georgia Southern University, Statesboro, GA, USA; Mount Desert Island Biological Laboratory, Salisbury Cove, ME, USA.
Comp Biochem Physiol B Biochem Mol Biol. 2022 Feb-Mar;258:110702. doi: 10.1016/j.cbpb.2021.110702. Epub 2021 Nov 29.
Complementary DNAs (cDNAs) for two aquaporin water channel genes (AQP3 and AQP15) were amplified cloned and sequenced to initiate this study. Northern blot analysis was carried out to confirm the mRNA sizes of these AQP genes with AQP3 mRNA bands exhibiting sizes of 1.2 and 1.6 k bases and AQP15 had a mRNA band of 2.1 k bases. Northern blot analysis was also performed on kidney and esophagus total RNA samples from fish acclimated to 75%, 100% or 120% seawater (SW). The level of AQP15 mRNA expression was shown to significantly decrease following salinity acclimation from 100 to 120% SW. An opposite but non-significantly different trend was observed for AQP3 mRNA levels. Full length cDNAs were then used to generate AQP3 and AQP15 mRNAs for microinjection into Xenopus oocytes. Both AQP3- and AQP15- microinjected oocytes exhibited significantly elevated apparent water permeability compared to control oocytes at neutral pH. The apparent water permeability was mercury-inhibitable, significantly so in the case of AQP3. AQP3 microinjected oocytes showed pH sensitivity in their apparent water permeability, showing a lack of permeability at acidic pH values. The Carboxyl-terminal derived amino acid sequences of AQP3 and AQP15 were used to generate rabbit affinity-purified polyclonal antibodies. Western blots with the antibodies showed a band of 31.3 kDa for AQP3 in the kidney, with minor bands at 26, 24 and 21 kDa. For AQP15 a band of 26 kDa was seen in gill and kidney. Fainter bands at 28 and 24 kDa were also seen in the kidney. There was also some higher molecular weight banding. None of the bands were seen when the antibodies were pre- blocked with their peptide antigens. Immunohistochemical localization studies were also performed in the gill and spiral valve intestine. In the gill, AQP15 antibody staining was seen sporadically in the membranes of surface epithelial cells of the secondary lamellae. Tyramide amplification of signals was employed in the spiral valve intestine. Tyramide-amplified AQP3 antibody staining was observed in the basal membrane of the invaginated epithelial cell layer of secondary intestinal folds in luminal surface of either the side wall of the spiral valve intestine or in internal valve tissue 'flaps'. For the AQP15 antibody, tyramide-amplified staining was instead found on the apical and to a lesser extent the lateral membranes of the same invaginated epithelial cell layer. The localization of AQP3 and AQP15 in the spiral valve intestine suggests that a trans-cellular water absorption pathway may exist in this tissue.
为了开展这项研究，我们扩增并克隆了两种水通道 Aquaporin (AQP) 基因（AQP3 和 AQP15）的互补 DNA (cDNA)，并对其进行了测序。通过 Northern blot 分析，我们确认了这些 AQP 基因的 mRNA 大小，AQP3 mRNA 条带的大小为 1.2 和 1.6 kb，AQP15 的 mRNA 条带大小为 2.1 kb。我们还对适应于 75%、100%或 120%海水 (SW) 的鱼类肾脏和食管总 RNA 样本进行了 Northern blot 分析。结果表明，AQP15 mRNA 的表达水平在从 100%SW 适应到 120%SW 后显著降低。AQP3 mRNA 水平则表现出相反但无统计学差异的趋势。然后，我们使用全长 cDNA 生成 AQP3 和 AQP15 mRNA，用于微注射到非洲爪蟾卵母细胞中。与对照卵母细胞相比，在中性 pH 值下，AQP3-和 AQP15-微注射卵母细胞的表观水通透性显著升高。水通透性可被汞抑制，AQP3 的抑制作用非常显著。AQP3 微注射卵母细胞在其表观水通透性方面表现出 pH 敏感性，在酸性 pH 值下缺乏通透性。AQP3 和 AQP15 的羧基末端衍生氨基酸序列被用于生成兔亲和纯化的多克隆抗体。用抗体进行的 Western blot 分析显示，肾脏中 AQP3 的条带大小为 31.3 kDa，有 26、24 和 21 kDa 的小条带。在鳃和肾脏中，AQP15 抗体显示出 26 kDa 的条带。在肾脏中还观察到 28 和 24 kDa 的较弱条带。还有一些高分子量的条带。当抗体用其肽抗原预先阻断时，没有观察到任何条带。我们还在鳃和螺旋瓣肠中进行了免疫组织化学定位研究。在鳃中，AQP15 抗体染色在二级鳃片表面上皮细胞的膜中散在出现。在螺旋瓣肠中，我们采用了酪胺放大信号。在螺旋瓣肠侧壁或内部瓣组织“瓣”的腔面的次级肠褶内陷上皮细胞层的基底膜中观察到 AQP3 抗体的酪胺放大染色。对于 AQP15 抗体，在相同内陷上皮细胞层的顶膜和侧膜上观察到酪胺放大染色，程度较轻。AQP3 和 AQP15 在螺旋瓣肠中的定位表明，该组织中可能存在跨细胞水吸收途径。
