|Résumé: ||In contrast to most teleost fish that are ammoniotelic, the gulf toadfish (Opsanus beta) is both facultatively ureogenic and ureotelic. In vivo pharmacological manipulations were used to show that lowering circulating cortisol levels or blocking glucocorticoid receptors (GR) enhanced both urea excretion and urea pulse size. These findings demonstrated that changes in pulsatile urea excretion in the toadfish are mediated by the permissive action of cortisol through GRs. Measurement of urea transport across isolated basolateral gill membranes revealed a cortisol-sensitive carrier mechanism. Cortisol infusion in vivo significantly reduced urea transport capacity, suggesting that cortisol inhibits the recruitment of urea transport proteins (UT) to the basolateral membrane to ultimately decrease the size of the urea pulse in toadfish. A 1.2 kb fragment of the upstream transcription start site for the toadfish urea transporter (tUT) gene was isolated and in silico analysis revealed the presence of several putative glucocorticoid response element (GRE) half sites. Toadfish provided with this regulatory sequence in a reporter gene construct showed increased reporter gene transcription driven by cortisol. The data indicated that cortisol-mediated upregulation of tUT mRNA by GREs may be necessary to maintain tUT activity.
Four Rhesus (Rh) glycoproteins (Rhag, Rhbg, Rhcg1, Rhcg2) were isolated from toadfish; these sequences grouped with those of other vertebrates coding for membrane channels that transport ammonia. In vivo increases in circulating cortisol reduced branchial Rh glycoprotein expression and decreased ammonia excretion. These changes were accompanied by cortisol-induced increases in glutamine synthetase activity, an enzyme that captures ammonia for urea synthesis. Taken together, the data indicated that cortisol reduces the loss by branchial excretion of ammonia, instead favouring biochemical pathways that convert ammonia to urea.
This thesis confirms that nitrogen excretion in toadfish is controlled and regulated in fashions unlike those in other teleosts. The results demonstrate the importance of the GR signaling pathway in mediating changes in both urea and ammonia transport through molecular mechanisms. As a whole, the data provide a new understanding of branchial nitrogen excretion in the gulf toadfish and enhance our evolutionary perspective of the integrated biological systems involved in nitrogen excretion in fish.|