Zebrafish (Danio rerio) Aquaporin 1a as a Multi-functional Transporter of Water, CO2, and Ammonia

Abstract

Previous in vitro studies have demonstrated that AQP1, traditionally viewed as a water channel, also facilitates the passage of CO2 and ammonia across cell membranes. This thesis summarizes the first in vivo studies confirming a physiologically-relevant role for AQP1 in acid-base balance and nitrogenous waste excretion. Zebrafish embryos were microinjected with a translation-blocking morpholino oligonucleotide targeted to the zebrafish AQP1 paralog, AQP1a. Closed-system respirometry, total CO2 analysis, tritiated water fluxes and measurement of ammonia excretion were performed on larvae at 4 days post-fertilization (dpf). Knockdown of AQP1a significantly reduced rates of water, CO2 and ammonia excretion. Use of phenylhydrazine, a haemolytic agent, provided evidence that the yolk sac epithelium AQP1a (and not erythrocyte AQP1a) is the major site of CO2 and ammonia movements. Further, the hypothesis that AQP1a and the Rh glycoprotein Rhcg1, another multi-functional gas channel, act in concert to regulate CO2 and ammonia excretion was explored. Exposure to conditions impairing ammonia excretion (such as high external ammonia (HEA) or alkaline water) modulated AQP1a protein expression in 4 dpf zebrafish larvae experiencing knockdown of Rhcg1. Chronic HEA exposure triggered a significant compensatory increase in AQP1a protein abundance in Rhcg1 morphants. Exposure of Rhcg1 morphants to pH 10 water, however, caused a significant decrease in AQP1a protein expression. Interestingly, when AQP1a mRNA and protein levels were examined in Rhcg1 morphants and vice versa, no changes were observed. Overall, zebrafish AQP1a was found to be a multi-functional transporter of water, CO2 and ammonia, though the exact relationship it holds with other such gas channels bears further exploration.