Ammonia Excretion in Freshwater Rainbow Trout (Oncorhynchus Mykiss) and the Importance of Gill Boundary Layer Acidification: Lack of Evidence for Na+/NH4+ Exchange

Abstract

Net ammonia fluxes (JAmm) were measured in adult freshwater rainbow trout in vivo under a variety of conditions designed to inhibit unidirectional sodium uptake (JinNa; low external [NaCl], 10(-4) mol l-1 amiloride), alter transbranchial PNH3 and NH4+ gradients [24 h continuous (NH4)2SO4 infusion, or exposure to 1 mmol l-1 external total ammonia at pH 8] and prevent gill boundary layer acidification (5 mmol l-1 Hepes buffer). Inhibition of JinNa with amiloride or low external [NaCl] under normal conditions reduced JAmm by about 20 %, but did not prevent the net excretion of ammonia during exposure to high concentrations of external ammonia. Increasing the buffer capacity of the ventilatory water with Hepes buffer (pH 8) reduced JAmm by 36 % and abolished the effect of amiloride on ammonia excretion. No evidence could be found to support a directly coupled apical Na+/NH4+ exchange. We suggest that any dependence of ammonia excretion on sodium uptake is caused by alteration of transbranchial PNH3 gradients within the gill microenvironment secondary to changes in net H+ excretion. Under normal conditions (pH 8, low external ammonia) gill boundary layer acidification facilitates over one-third of the total ammonia excretion. During exposure to high concentrations of external ammonia in poorly buffered water, estimates of transbranchial PNH3 gradients from measurements of bulk water pH and total ammonia concentration (TAmm) may be grossly in error because of boundary layer acidification. Prevention of boundary layer acidification with Hepes buffer during exposure to high cocncentrations of external ammonia revealed that the local transbranchial PNH3 gradient at the gill may in fact be positive (blood to water), negating the need for an active NH4+ transport mechanism. In freshwater trout, NH3 diffusion may account for all ammonia excretion under all experimental conditions used in the present study.

Hardness does not affect the physiological responses of wild and domestic strains of diploid and triploid rainbow trout Oncorhynchus mykiss to short‐term exposure to pH 9.5

“…3(a)]. This increase is consistent with other studies (Wilkie & Wood, 1991;Wilson et al, 1994;McGeer & Eddy, 1998), but the data do not agree with the study by Yesaki & Iwama (1992), where it was shown that hard water had a protective effect on ammonia accumulation in the plasma. Liver ammonia also did not change in any condition during the exposure [Fig.…”

Mechanisms of ion transport inPotamotrygon, a stenohaline freshwater elasmobranch native to the ion-poor blackwaters of the Rio Negro

“…8). Indeed, this was larger than the marginal decreases in ammonia efflux (0-30%) normally seen in freshwater teleosts in response to amiloride blockade of Jin Na (Kirschner et al, 1973;Wright and Wood, 1985;Wilson et al, 1994;Nelson et al, 1997;Clarke and Potts, 1998;Patrick and Wood, 1999). While originally seen as evidence for Na + /NH4 + exchange, the latter observations have more recently been interpreted as an inhibition of 'diffusion trapping' of NH3 in gill boundary layer water associated with blockade of H + extrusion via Na + /H + and/or Na + channel/H + -ATPase systems (Wilson, 1996;Wilkie, 1997).…”

“…In this regard, the stingray is similar to most freshwater teleosts, in which a 60-95% blockade of Na + uptake induced by this concentration of amiloride has been widely reported (e.g. Kirschner et al, 1973;Wright and Wood, 1985;Wilson et al, 1994;Nelson et al, 1997;Clarke and Potts, 1998;Patrick and Wood, 1999). Interestingly, in two native Rio Negro characids that demonstrated the high-capacity, high-affinity, low-pHresistant-strategy of Na + transport (i.e.…”

The skin of adult rainbow trout is not a significant site of ammonia clearance from the blood

“…Exposure to HEA in live intact fish (1 mmol l −1 NH 4 + for 24 h) resulted in a 4.5‐fold increase in plasma ammonia, reaching an average concentration of 1199 ± 144 μmol l −1 (Figure 2a), similar to the response observed in other studies on adult trout (Liew et al ., 2013; Nawata et al ., 2007; Sinha et al ., 2013; Wilson et al ., 1994; Wood & Nawata, 2011). Skin tissue ammonia concentration in these same fish increased from 2322 ± 302 μmol kg −1 in control fish to 4018 ± 402 μmol kg −1 following HEA exposure (Figure 2b), demonstrating for the first time that ammonia accumulates in the skin in response to HEA.…”

“…A role for the skin in whole-body ammonia excretion is also supported by increased transcript abundance of genes encoding ammonia-transporting Rhesus (Rh) glycoproteins in this tissue in response to manipulations of ammonia status, such as high environmental ammonia (HEA; Hung et al, 2007;Nawata & Wood, 2009;Nawata et al, 2007). HEA exposure initially results in significant ammonia uptake and elevated plasma ammonia, followed by the re-establishment of outwardly directed ammonia excretion (Nawata et al, 2007;Wilson et al, 1994;Wood & Nawata, 2011;Zimmer et al, 2010). This occurs in conjunction with increased expression of Rh genes in the gills (Hung et al, 2007;Nawata et al, 2007Nawata et al, , 2010Sinha et al, 2013;Zimmer et al, 2010Zimmer et al, , 2017.…”