The effects of CO2 and external buffering on ammonia excretion and Rhesus glycoprotein mRNA expression in rainbow trout

Abstract

SUMMARYRhesus (Rh) proteins were recently characterized as ammonia gas (NH 3 ) channels. Studies indicate, however, that Rh proteins also facilitate CO 2 transport in a green alga and in human erythrocytes. Previously, we reported that Rh mRNA expression in various rainbow trout tissues responded to high environmental ammonia. To determine whether or not Rh proteins may also be involved in CO 2 transport in rainbow trout, we examined the effects of a 12 h exposure to external hypercapnia (1% CO 2 in air) on Rh mRNA expression in the gill, skin and erythrocytes. External hypercapnic conditions lowered the water pH and facilitated ammonia excretion; therefore, we also studied the effects of hypercapnia and normocapnia in the presence of 10 mmol l -1 Hepesbuffered water. Hepes treatment prevented water acidification, but resulted in elevated plasma ammonia levels and reduced ammonia excretion rates. Hypercapnia exposure without buffering did not elicit changes in Rh mRNA expression in the gill or skin. However, Rhcg2 mRNA expression was downregulated in the gills and upregulated in the skin of both normocapnia-and hypercapnia-exposed fish in Hepes-buffered water. mRNA expression of a newly cloned Rhbg2 cDNA was downregulated in the skin of fish exposed to buffered water, and Rhag mRNA expression in erythrocytes was decreased with exposure to normocapnia in buffered water but not with hypercapnia exposure in either buffered or unbuffered water. With the aid of Hepes buffering, we were able to observe the effects of both CO 2 and ammonia on Rh mRNA expression. Overall, we conclude that high CO 2 did not directly elicit changes in Rh mRNA transcription levels in the gill and skin, and that the changes observed probably reflect responses to high plasma ammonia, mirroring those in trout exposed to high environmental ammonia. Therefore a dual function for gill and skin Rh proteins in CO 2 and ammonia transport is not evident from these results. Rhag expression, however, responded differentially to high CO 2 and high ammonia, suggesting a possible dual role in the erythrocytes.

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

“…An earlier study on freshwater trout did not reveal any significant changes in gill Rh transcript levels after exposure to 12h of hypercapnia (Nawata and Wood, 2008). In the present study, 24h of hypercapnia resulted in a downregulation of Rhag mRNA levels in the gill (Table2), a similar response observed with HEA exposure (Fig.3).…”

“…In the trout, Rh mRNA levels in the skin increased after branchial excretion was blocked by HEA or Hepes exposure, and after ammonia infusion Nawata and Wood, 2008). In the mangrove killifish, which volatilises ammonia through the skin, Rh mRNA levels were upregulated in the skin after HEA and also aerial exposure .…”

Aedes aegypti Rhesus glycoproteins contribute to ammonia excretion by larval anal papillae

“…This result suggests that, at the very least, both AeRh50s play an equally important role in ammonia excretion by the anal papillae. In fish, there are also multiple Rh protein homologs in the gills that function to facilitate ammonia excretion Nawata and Wood, 2008). The Rhcg isoforms (Rhcg1 and Rhcg2) are more abundant in the gills, kidney and skin, in contrast to Rhbg mRNA, which is widespread throughout the body Nawata et al, 2007).…”

Interactions between cortisol and Rhesus glycoprotein expression in ureogenic toadfish, Opsanus beta

“…These studies, carried out on rainbow trout, reported rather different patterns (Nawata and Wood, 2008;Nawata and Wood, 2009;Tsui et al, 2009). In particular, Nawata and Wood reported that cannulation stress resulted in an elevated ammonia excretion rate accompanied by upregulation of Rhbg, Rhcg1 and Rhcg2 in the gill, a trend opposite to that observed in toadfish in the present study.…”

“…Vertebrates express three main Rh-associated glycoproteins that facilitate ammonia transport: type A (Rhag), type B (Rhbg) and type C (Rhcg) (Huang and Peng, 2005;Peng and Huang, 2006 (Nakada et al, 2007), rainbow trout (Oncorhynchus mykiss) Nawata and Wood, 2008), mangrove killifish (Kryptolebias marmoratus) and zebrafish (Danio rerio) (Shih et al, 2008). Recently, we cloned four toadfish Rh genes -Rhag, Rhbg, Rhcg1 and Rhcg2 -that show considerable sequence similarity to other piscine Rh proteins (Rodela et al, 2012).…”