Rh glycoprotein expression is modulated in pufferfish (Takifugu rubripes) during high environmental ammonia exposure

Abstract

Rhesus (Rh) protein involvement in ammonia transport processes in freshwater fish has received considerable attention; however, parallel investigations in seawater species are scant. We exposed pufferfish to high environmental ammonia (HEA; 1 and 5 mmol l(-1) NH(4)HCO(3)) and evaluated the patterns of ammonia excretion and gill Rh mRNA and protein expression. Gill H(+)-ATPase, NHE1, NHE2, NHE3, Na(+)/K(+)-ATPase (NKA), Na(+)/K(+)/2Cl(-) co-transporter (NKCC1) mRNA, H(+)-ATPase activity, NKA protein and activity, were also quantified. Activation of NKA by NH(4)(+) was demonstrated in vitro. The downregulation of Rhbg mRNA and simultaneous upregulations of Rhcg1, H(+)-ATPase, NHE3, NKA, NKCC1 mRNA, H(+)-ATPase activity, and NKA protein and activity levels suggested that during HEA, ammonia excretion was mediated mainly by mitochondria-rich cells (MRCs) driven by NKA with basolateral NH(4)(+) entry via NKA and/or NKCC1, and apical NH(3) extrusion via Rhcg1. Reprotonation of NH(3) by NHE3 and/or H(+)-ATPase would minimise back flux through the Rh channels. Downregulated Rhbg and Rhag mRNA observed in the gill during HEA suggests a coordinated protective response to minimise the influx of external ammonia via the pavement cells and pillar cells, respectively, while routing ammonia excretion through the MRCs. Exposure to hypercapnia (1% CO(2) in air) resulted in downregulated gill and erythrocyte Rhag mRNA. Surprisingly, Rhag, Rhbg, Rhcg1 and Rhcg2 proteins responded to both hypercapnia and HEA with changes in their apparent molecular masses. A dual NH(3)/CO(2) transport function of the pufferfish Rh proteins is therefore suggested. The results support and extend an earlier proposed model of pufferfish gill ammonia excretion that was based on immunolocalisation of the Rh proteins. Passive processes and/or Rhbg and Rhcg2 in the pavement cells may maintain basal levels of plasma ammonia but elevated levels may require active excretion via NKA and Rhcg1 in the MRCs.