Lin Tzung-Yi, Liao Bo-Kai, Horng Jiun-Lin, Yan Jia-Jiun, Hsiao Chung-Der, Hwang Pung-Pung
Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan.
Am J Physiol Cell Physiol. 2008 May;294(5):C1250-60. doi: 10.1152/ajpcell.00021.2008. Epub 2008 Mar 5.
H(+)-ATPase-rich (HR) cells in zebrafish gills/skin were found to carry out Na+ uptake and acid-base regulation through a mechanism similar to that which occurs in mammalian proximal tubular cells. However, the roles of carbonic anhydrases (CAs) in this mechanism in zebrafish HR cells are still unclear. The present study used a functional genomic approach to identify 20 CA isoforms in zebrafish. By screening with whole mount in situ hybridization, only zca2-like a and zca15a were found to be expressed in specific groups of cells in zebrafish gills/skin, and further analyses by triple in situ hybridization and immunocytochemistry demonstrated specific colocalizations of the two zca isoforms in HR cells. Knockdown of zca2-like a caused no change in and knockdown of zca15a caused an increase in H+ activity at the apical surface of HR cells at 24 h postfertilization (hpf). Later, at 96 hpf, both the zca2-like a and zca15a morphants showed decreased H+ activity and increased Na+ uptake, with concomitant upregulation of znhe3b and downregulation of zatp6v1a (H+-ATPase A-subunit) expressions. Acclimation to both acidic and low-Na+ fresh water caused upregulation of zca15a expression but did not change the zca2-like a mRNA level in zebrafish gills. These results provide molecular physiological evidence to support the roles of these two zCA isoforms in Na+ uptake and acid-base regulation mechanisms in zebrafish HR cells.
研究发现，斑马鱼鳃/皮肤中富含H(+)-ATP酶（HR）的细胞通过一种与哺乳动物近端肾小管细胞类似的机制进行钠离子摄取和酸碱调节。然而，碳酸酐酶（CAs）在斑马鱼HR细胞这一机制中的作用仍不清楚。本研究采用功能基因组学方法，在斑马鱼中鉴定出20种碳酸酐酶异构体。通过全胚胎原位杂交筛选，仅发现zca2-like a和zca15a在斑马鱼鳃/皮肤的特定细胞群中表达，进一步通过三重原位杂交和免疫细胞化学分析表明，这两种zca异构体在HR细胞中存在特异性共定位。敲低zca2-like a对受精后24小时（hpf）HR细胞顶端表面的H+活性没有影响，而敲低zca15a则导致H+活性增加。之后，在96 hpf时，zca2-like a和zca15a的吗啉代寡核苷酸注射胚胎均表现出H+活性降低和Na+摄取增加，同时znhe3b表达上调，zatp6v1a（H+-ATP酶A亚基）表达下调。适应酸性和低钠淡水均导致斑马鱼鳃中zca15a表达上调，但不改变zca2-like a的mRNA水平。这些结果提供了分子生理学证据，支持这两种zCA异构体在斑马鱼HR细胞的Na+摄取和酸碱调节机制中的作用。
