Tresguerres Martin, Parks Scott K, Goss Greg G
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T5G 2E9, Canada.
Comp Biochem Physiol A Mol Integr Physiol. 2007 Sep;148(1):133-41. doi: 10.1016/j.cbpa.2007.03.032. Epub 2007 Apr 18.
To investigate the base secretory mechanisms in the Pacific hagfish (Eptatretus stoutii), we injected animals with NaHCO3 into the subcutaneous sinus. In the first series of experiments, hagfish were injected with 6000 micromol kg(-1) NaHCO3 (base-infused hagfish, BIH) or NaCl (controls). Blood pH increased significantly 1 h after injection in BIH (8.05+/-0.05 vs. 7.82+/-0.03 pH units), but returned to control values by t=6 h. Plasma total CO2 (TCO2) followed the same pattern. Immunolabeled sections revealed that Na+/K+-ATPase and V-H+-ATPase were usually located in the same cells. Western blotting revealed that the abundance of both proteins remained unchanged in whole gill homogenates and in a fraction enriched in cell membranes 6 h after the injections. The second experimental series was to induce long-term alkalosis by serially injecting 6000 micromol kg(-1) NaHCO3 every 6 h for 24 h. Blood pH completely recovered from the base loads within 6 h after each injection. Moreover, plasma TCO2 was not elevated 3 h after the second infusion, suggesting that HCO3(-) secreting mechanisms had been upregulated by that time. Na+/K+-ATPase and V-H+-ATPase cellular localizations did not change in the 24 h base infusion protocol. Na+/K+-ATPase abundance was similar in gill homogenates from fish from both treatments. However, Na+/K+-ATPase abundance in the membrane fraction was significantly lower in BIH, while V-H+-ATPase was greater both in whole gill and membrane fractions. Our results suggest that differential insertion of V-H+-ATPase and Na+/K+-ATPase into the basolateral membrane is involved in recovering from alkalotic stress in hagfish.
为了研究太平洋盲鳗（Eptatretus stoutii）的碱分泌机制，我们将动物皮下窦内注射碳酸氢钠。在第一组实验中，给盲鳗注射6000微摩尔/千克的碳酸氢钠（碱注入盲鳗，BIH）或氯化钠（对照组）。注射后1小时，BIH组的血液pH值显著升高（8.05±0.05对7.82±0.03 pH单位），但在t = 6小时时恢复到对照值。血浆总二氧化碳（TCO2）呈现相同模式。免疫标记切片显示，Na+/K+-ATP酶和V-H+-ATP酶通常位于同一细胞中。蛋白质印迹法显示，注射后6小时，全鳃匀浆和富含细胞膜的部分中这两种蛋白质的丰度均保持不变。第二个实验系列是通过每6小时连续注射6000微摩尔/千克的碳酸氢钠，持续24小时来诱导长期碱中毒。每次注射后6小时内，血液pH值从碱负荷中完全恢复。此外，第二次输注后3小时血浆TCO2未升高，表明此时HCO3(-)分泌机制已上调。在24小时碱注入方案中，Na+/K+-ATP酶和V-H+-ATP酶的细胞定位没有变化。两种处理的鱼鳃匀浆中Na+/K+-ATP酶丰度相似。然而，BIH组膜部分中的Na+/K+-ATP酶丰度显著较低，而V-H+-ATP酶在全鳃和膜部分中均较高。我们的结果表明，V-H+-ATP酶和Na+/K+-ATP酶向基底外侧膜的差异插入参与了盲鳗从碱中毒应激中恢复的过程。
