Urea transporter and glutamine synthetase regulation and localization in gulf toadfish gill

Abstract

SUMMARYThe goal of the present study was to investigate the role of circulating cortisol and urea in the transcriptional regulation of branchial glutamine synthetase (GS), which incorporates NH 3 into glutamate to form glutamine, and the toadfish urea transporter, tUT, which is involved in urea excretion across the gill of the gulf toadfish. GS (of which there are two isoforms, LGS and GGS) and tUT mRNA expression and activity were measured in toadfish exposed to treatments that would induce variable stress responses. In addition, the role of circulating urea in tUT regulation was investigated by infusing toadfish with urea alone or in combination with intraperitoneal injection of RU486, a corticosteroid type II receptor antagonist. There was a 4.8-fold upregulation in the mRNA expression of the gill-specific GS isoform (GGS) in response to cortisol infusion and a similar upregulation in the more ubiquitous isoform (LGS). Furthermore, there was a significant 1.9-fold and 3.3-fold upregulation in the mRNA expression of the toadfish urea transporter, tUT, in response to stress through crowding or exogenous cortisol loading through infusion, respectively. In addition, tUT was found to have a urea-sensitive component to transcriptional regulation that was independent of circulating cortisol concentrations. However, the changes measured in mRNA expression of GGS, LGS and tUT did not correspond with changes in protein activity. To determine the cell type(s) involved in glutamine production and urea excretion, we attempted to localize GGS, LGS and tUT using in situ hybridization. This study is the first to show that GGS and tUT expression appear to occur in gill mitochondria-rich cells of toadfish, suggesting that these cells play a combined glutamine production and urea excretion role, which may have implications for predator avoidance.

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

“…Furthermore, branchial GS activity remains unchanged following an acute (24-48h) crowding and confinement stress Wood et al, 2003;Esbaugh and Walsh, 2009;Rodela et al, 2012). Increased branchial activity has only been reported following chronic exposure (~1week) to crowded conditions (McDonald et al, 2009). Collectively, these data suggest that additional mechanisms may play an integral role in modulating ammonia excretion in toadfish.…”

“…The transition to ureotelism is characterized by increases in liver and muscle glutamine synthetase (GS) activity (Walsh et al, 1994;Walsh and Milligan, 1995;Wood et al, 1995;Esbaugh and Walsh, 2009). GS converts ammonia into glutamine for use in the piscine ornithine-urea cycle (OUC), and induction of this enzyme results in an increase in urea production and accumulation in the plasma (Walsh et al, 1994;Hopkins et al, 1995;Walsh and Milligan, 1995;McDonald et al, 2009). However, the transition to ureotelism does not involve a significant increase in urea excretion, but rather a substantial reduction in ammonia excretion (Walsh et al, 1994;Walsh and Milligan, 1995).…”

“…Although the hepatic GS likely accounts for the bulk of ammonia entry as glutamine into the OUC, the glutamine produced by the gill-specific GS is thought to minimize ammonia leakage across the branchial epithelium of ureotelic toadfish and likely contributes only a small fraction to urea production in the OUC Walsh, 1997;Walsh et al, 2003;Esbaugh and Walsh, 2009;McDonald et al, 2009). A recent study by McDonald and colleagues demonstrated that GS expression has a limited distribution in the gills, being localized only in the mitochondrion-rich cells (MRCs) (McDonald et al, 2009), which constitute a very small proportion of the total gill cell population (Perry and Walsh, 1989). Furthermore, branchial GS activity remains unchanged following an acute (24-48h) crowding and confinement stress Wood et al, 2003;Esbaugh and Walsh, 2009;Rodela et al, 2012).…”

“…In particular, the rise in cortisol induces transcription of the hepatic GS that results in enhanced enzyme activity (Kong et al, 2000;Esbaugh and Walsh, 2009), an effect eliminated by treatment with the cortisol-synthesis inhibitor metyrapone . Elevated cortisol also causes an upregulation of toadfish urea transporter (tUT) transcript levels (McDonald et al, 2009;Rodela et al, 2011), but inhibits tUT function McDonald et al, 2009), as indicated by a reduction in urea excretion. These observations suggest that cortisol coordinates both transcriptional regulation of tUT and post-translational regulatory events to facilitate the efflux of urea in the pulsatile manner that is unique to the gulf toadfish (Wood et al, 2003;McDonald et al, 2009;Rodela et al, 2011).…”

“…Elevated cortisol also causes an upregulation of toadfish urea transporter (tUT) transcript levels (McDonald et al, 2009;Rodela et al, 2011), but inhibits tUT function McDonald et al, 2009), as indicated by a reduction in urea excretion. These observations suggest that cortisol coordinates both transcriptional regulation of tUT and post-translational regulatory events to facilitate the efflux of urea in the pulsatile manner that is unique to the gulf toadfish (Wood et al, 2003;McDonald et al, 2009;Rodela et al, 2011). Based on the strong involvement of cortisol in urea metabolism and excretion, we speculate that glucocorticoids may regulate Rh expression in toadfish to coordinate simultaneous changes in ammonia and urea excretion during the transition to ureotely.…”