Ammonia excretion in Caenorhabditis elegans: mechanism and evidence of ammonia transport of the Rhesus protein CeRhr-1.
The soil-dwelling nematode Caenorhabditis 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 µmol gFW(-1) day(-1)). Although these roundworms have been used for decades as genetic model systems, very little is known about their strategy to eliminate the toxic waste product ammonia from their bodies into the environment. The current study provides evidence that ammonia is at least partially excreted via the hypodermis. Starvation reduced the ammonia excretion rates by more than half, whereas mRNA 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 functional microtubule 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 ammonia-transporting Rh-glycoproteins.
Faculty of Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T2N2.
Aida
A
Adlimoghaddam
Biology of Membrane Transport, IBMM, Université Libre de Bruxelles, Rue des Professeurs Jeener et Brachet 12, Gosselies 6041, Belgium.
Mélanie
M
Boeckstaens
Biology of Membrane Transport, IBMM, Université Libre de Bruxelles, Rue des Professeurs Jeener et Brachet 12, Gosselies 6041, Belgium.
Anna-Maria
AM
Marini
Faculty of Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T2N2 Faculty of Human Ecology, University of Manitoba, Winnipeg, Manitoba, Canada R3T2N2.
Jason R
JR
Treberg
Faculty of Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T2N2.
Ann-Karen C
AK
Brassinga
Faculty of Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T2N2 weihrauc@cc.umanitoba.ca.
Dirk
D
Weihrauch
D000595:Amino Acid Sequence; D000641:Ammonia / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D017173:Caenorhabditis elegans / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000378:metabolism*; D002256:Carbonic Anhydrases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D021381:Protein Transport; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism
D029742:Caenorhabditis elegans Proteins; D008565:Membrane Proteins; C000603091:Rhr-1 protein, C elegans; D000641:Ammonia; D025262:Vacuolar Proton-Translocating ATPases; D002256:Carbonic Anhydrases; D000254:Sodium-Potassium-Exchanging ATPase
Carbonic anhydrase; Na+/K+-ATPase; V-ATPase; Vesicular transport