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

The 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.