Ammonia excretion in Caenorhabditis elegans: Mechanism and evidence of ammonia transport of the Rhesus protein CeRhr-1

Abstract

The soil-dwelling nematodeCaenorhabditis elegans is a bacteriovorous animal, excreting the vast majority of its nitrogenous waste as ammonia (25.3±1.2 μmol gFW?1 day?1) and very little urea (0.21± 0.004 μmolgFW?1 day?1).Although theseroundworms have beenused for decades as genetic model systems, very little is known about their strategy to eliminate the toxic waste product ammonia fromtheir bodies into theenvironment.The current studyprovidesevidence that ammonia is at least partially excreted via the hypodermis. Starvation reduced the ammonia excretion rates bymore than half, whereasmRNA expression levels of the Rhesus protein CeRhr-2, V-type H+-ATPase (subunit A) and Na+/K+-ATPase (α-subunit) decreased correspondingly.Moreover, ammonia excretion rates were enhanced in media buffered to pH 5 and decreased at pH 9.5. Inhibitor experiments, combined with enzyme activity measurements and mRNA expression analyses, further suggested that the excretion mechanism involves the participation of the V-type H+-ATPase, carbonic anhydrase, Na+/K+-ATPase, and a functionalmicrotubule network. These findings indicate that ammonia is excreted, not only by apical ammonia trapping, but also via vesicular transport and exocytosis. Exposure to 1 mmol l?1 NH4Cl caused a 10-fold increase in body ammonia and a tripling of ammonia excretion rates. Gene expression levels of CeRhr-1 and CeRhr-2, V-ATPase and Na+/K+-ATPase also increased significantly in response to 1 mmol l?1 NH4Cl. Importantly, a functional expression analysis showed, for the first time, ammonia transport capabilities for CeRhr-1 in a phylogenetically ancient invertebrate system, identifying these proteins as potential functional precursors to the vertebrate ammoniatransporting Rh-glycoproteins.