Assessing intracellular pH regulation in H + -ATPase-rich ionocytes in zebrafish larvae using in vivo ratiometric imaging

• Published in: Journal of experimental biology Vol. 223; no. Pt 5
• Main Authors: Yew, H M, Zimmer, A M, Perry, S F
• Format: Journal Article
• Language: English
• Published: England 12.03.2020
• Subjects: Animals; Hydrogen-Ion Concentration; Ion Transport; Larva - growth & development; Larva - physiology; Proton-Translocating ATPases - metabolism; Zebrafish - growth & development; Zebrafish - physiology; Ammonium chloride; Sodium–hydrogen exchanger (NHE); Hypercapnia; Morpholino knockdown; Carbonic anhydrase; Ionic regulation; Danio rerio; Acid–base balance
• ISSN: 0022-0949; 1477-9145
• DOI: 10.1242/jeb.212928

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 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; ) and the H -ATPase ( ). 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% CO and the extent of post-CO 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 CO exposure and a reduced capacity to restore pHi to baseline levels post-hypercapnia. The metabolic alkalosis and subsequent acidification associated with 20 mmol l NH Cl 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.