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

Abstract

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.

Keywords: Carbonic anhydrase; Na+/K+-ATPase; V-ATPase; Vesicular transport.

Fig. 1.

Ammonia and urea excretion rates of C. elegans under fed and starved (24 h) conditions. (A) Ammonia excretion. (B) Urea excretion. Data represent means±s.e.m. and were analyzed employing an unpaired, two-tailed Student's t-test (N=4–5); *P≤0.05.

Fig. 2.

Changes of mRNA expression levels of CeRhr-1, CeRhr-2, V-ATPase (subunit A) and Na⁺/K⁺-ATPase (α-subunit) in C. elegans under fed and starved (24 h) conditions. CeRhr-1, N=4–5; CeRhr-2, N=5; V-ATPase (subunit A), N=4–5; Na⁺/K⁺-ATPase (α-subunit), N=4–5. Absolute mRNA expression levels of fed animals were set to 1 while values measured under starved conditions are given as ‘fold change’ of the respective control. *Significant differences between treatments (P≤0.05). Data represent means±s.e.m. and were analyzed using an unpaired, two-tailed Student's t-test prior to calculation for fold change values.

Fig. 3.

Ammonia excretion rates of C. elegans in media adjusted and buffered to various pH regimes. Control conditions are unbuffered control medium (pH=7). Data represent means±s.e.m. For statistical analysis, a Kruskal–Wallis test was applied with post hoc Mann–Whitney pairwise comparisons (N=4–6). Significant differences are indicated by different letters.

Fig. 4.

Effects of different inhibitors on ammonia excretion rates in C. elegans. Control values for each treatment were set to 1, with values measured under the influence of the inhibitors are given as ‘fold change’ of the respective control. The concentrations of the inhibitors were: concanamycin C, 5 µmol l⁻¹ (N=5–6); acetazolamide, 5 mmol l⁻¹ (N=5–6); ouabain, 5 mmol l⁻¹ (N=6); colchicine, 2 mmol l⁻¹ (N=5–6). Data represent means±s.e.m. and were analyzed employing an unpaired, two-tailed Student's t-test prior to calculation for fold change values. *P≤0.05.

Fig. 5.

Specific activity of the Na⁺/K⁺-ATPase in C. elegans exposed to control conditions or acclimated for 48 h to 1 mmol l⁻¹ NH₄Cl (HEA). Either 10 mmol l⁻¹ KCl or 10 mmol l⁻¹ NH₄Cl was used as substrate in the assay. Control worms, N=5–6; worms acclimated for 48 h to 1 mmol l⁻¹ NH₄Cl (HEA), N=5–6. *Significant differences between control worms and HEA acclimated worms (P≤0.05). Data represent means±s.e.m. and were analyzed employing an unpaired, two-tailed Student's t-test.

Fig. 6.

Growth tests of S. cerevisiae yeast cells on solid minimal medium. Medium contained 3 mmol l⁻¹ (NH₄)₂SO₄ or 6.8 mmol l⁻¹ glutamate (positive growth control) as the sole nitrogen source. Wild-type cells (23344c) were transformed with the empty p426 vector (-) and, triple-mepΔ cells (31019b), deprived of the three endogenous Mep proteins, were transformed with the empty p426 vector (-) or, with a multi-copy plasmid (p426) bearing the HsRhCG, or CeRhr1 genes. Cells were incubated for 4 days at 29°C.

Fig. 7.

Ammonia excretion rates of C. elegans acclimated for 2 days to various NH₄Cl concentrations. Excretion was measured in the corresponding NH₄Cl concentration (N=4–5 for each condition). Control conditions were unbuffered control medium (pH=7, no NH₄Cl added). Data represent means±s.e.m. For statistical analysis, a Kruskal–Wallis test was applied with post hoc Mann–Whitney pairwise comparisons. Significant differences are indicated by different letters.

Fig. 8.

Ammonia and urea body content in C. elegans. (A) Ammonia body concentration and (B) urea body concentration in control worms (N=5–6) and worms acclimated for 2 days to 1 mmol l⁻¹ NH₄Cl (N=6). Data represent means±s.e.m. and were analyzed with an unpaired, two-tailed Student's t-test. *P≤0.05.

Fig. 9.

Changes in mRNA expression levels of CeRhr-1, CeRhr-2, V-ATPase (subunit A) and Na⁺/K⁺-ATPase (α-subunit) in C. elegans. CeRhr-1 (N=3–5), CeRhr-2 (N=4–5), V-ATPase (subunit A) (N=4–5) and Na⁺/K⁺-ATPase (α-subunit) (N=4–5) in control C. elegans and worms acclimated for 2 days to 1 mmol l⁻¹ NH₄Cl (HEA). Data represent means±s.e.m. and were analyzed with an unpaired, two-tailed Student's t-test prior to calculation of fold change values. *P≤0.05.