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Abstract：
The H+-ATPase-rich (HR) cells of zebrafish larvae are a sub-type of ion-transporting cell located on the yolk sac epithelium that are responsible for Na+ uptake and H+ extrusion. Current models of HR cell ion transport mechanisms in zebrafish larvae are well established, but little is known about the involvement of the various ion transport pathways in regulating intracellular acid-base status. Here, a ratiometric imaging technique was developed and validated to monitor intracellular pH (pHi) continuously in larval zebrafish HR cells in vivo Gene knockdown or CRISPR/Cas9 knockout approaches were used to evaluate the roles of the two principal apical membrane acid excretory pathways, the Na+/H+ exchanger (NHE3b; slc9a3.2) and the H+-ATPase (atpv1aa). Additionally, the role of HR cell cytosolic carbonic anhydrase (CAc) was investigated because of its presumed role in providing H+ for Na+/H+ exchange and H+-ATPase. The temporal pattern and extent of intracellular acidification during exposure of fish to 1% CO2 and the extent of post-CO2 alkalisation were altered markedly in fish experiencing knockdown/knockout of CAc, NHE3b or H+-ATPase. Although there were slight differences among the three knockdown/knockout experiments, the typical response was a greater degree of intracellular acidification during CO2 exposure and a reduced capacity to restore pHi to baseline levels post-hypercapnia. The metabolic alkalosis and subsequent acidification associated with 20 mmol l-1 NH4Cl exposure and its washout were largely unaffected by gene knockdown. Overall, the results suggest markedly different mechanisms of intracellular acid-base regulation in zebrafish HR cells depending on the nature of the acid-base disturbance.
摘要：
斑马鱼幼虫的富含H-ATPase（HR）的细胞是位于卵黄囊上皮上的离子运输细胞的亚型，负责Na摄取和H挤出。目前已经建立了斑马鱼幼虫HR细胞离子转运机制的模型,但是对各种离子转运途径在调节细胞内酸碱状态中的参与知之甚少。这里,开发并验证了比率成像技术，以连续监测体内幼虫斑马鱼HR细胞中的细胞内pH（pHi）。使用基因敲低或CRISPR/Cas9敲除方法来评估两种主要的顶膜酸排泄途径的作用，Na+/H+交换剂(NHE3b;slc9a3.2)和H+-ATP酶(atpv1aa)。此外,研究了HR细胞胞质碳酸酐酶（CAc）的作用，因为它在为Na/H交换和H-ATPase提供H中的作用。在经历CAc敲除/敲除的鱼类中，鱼类暴露于1％CO2期间细胞内酸化的时间模式和程度以及CO2后碱化的程度发生了显着改变。NHE3b或H+-ATP酶。尽管三个敲除/敲除实验之间存在细微差异，典型的反应是CO2暴露过程中细胞内酸化程度较高,高碳酸血症后pHi恢复至基线水平的能力降低.与20mmoll-1NH4Cl暴露及其清除相关的代谢性碱中毒和随后的酸化在很大程度上不受基因敲除的影响。总的来说,结果表明，根据酸碱紊乱的性质，斑马鱼HR细胞中细胞内酸碱调节的机制显着不同。
