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Unlike most teleosts, gulf toadfish have the capacity to switch from ammoniotely to ureotely as the predominate means of nitrogen excretion during periods of stress. The switch to ureotely is a result of increased glutamine synthetase (GS) mRNA expression/enzyme activity in the liver and muscle, which is initiated by cortisol. Cortisol typically affects gene expression through the action of cortisol-activated transcription factors, such as glucocorticoid receptors, which bind to glucocorticoid response elements (GRE) in the upstream regulatory region of genes. The purpose of the present study was to identify the GRE responsible for increased GS gene expression during crowding/ confinement in gulf toadfish using an in vivo luciferase reporter assay. Upstream promoter regions for both the ubiquitous and gill GS isoforms were amplified by PCR. Additionally, an intron was amplified from the ubiquitous GS isoform that suggested the possibility of two discreet transcripts for the mitochondrial and cytoplasmic proteins. When tested via in vivo reporter assays, both the cytoplasmic and mitochondrial ubiquitous GS promoters showed increased luciferase activity during crowding vs. noncrowded controls; the gill GS promoter showed no effects in response to crowding. In silico analysis of the mitochondrial and cytoplasmic ubiquitous GS promoter constructs showed an overlapping section of 565 bp containing two potential GREs. Mutation of either site alone had no effect on luciferase activity vs. wild-type controls. However, when both sites were mutated a significant decrease in luciferase activity was observed. We conclude that two functional GREs combine to confer cortisol-inducible GS expression in the liver of gulf toadfish. ammonia; urea; nitrogen metabolism; cortisol; ureotely; ornithine-urea cycle doi: 10.1152/ajpregu.00267.2009

Recent evidence suggests that carbonic anhydrase (CA) IX in humans is under the regulatory control of hypoxia-inducible factor and is over-expressed in certain cancers. However, little is known of its presence in nonmammalian vertebrates or its physiological function in any vertebrate. The objective of this study was to examine and characterize the presence, distribution, induction by hypoxia, and physiological function of CA IX in the zebrafish. Zebrafish CA IX was highly expressed in the eye, brain, and gastrointestinal tract and showed increased expression in the eye, brain, and muscle in response to hypoxia (water [Po.sub.2] = 24 mmHg). The hypothesis that increased CA IX expression during hypoxia would act to attenuate intracellular acidosis was then examined. Muscle intracellular pH (pHi) decreased after 4 h of hypoxic exposure (from 7.15 [+ or -] 0.02 to 7.06 [+ or -] 0.01 pH units) and did not recover by 24 h. Manipulation of extracellular CA activity via intraperitoneal injection of either bovine CA or the selective extracellular CA inhibitor F3500 revealed that although increased CA activity could fully restore [pH.sub.i], removal of extracellular activity did not result in further acidosis. An exercise-induced acidosis was also attenuated in fish treated with bovine CA; however, the increased extracellular CA expression resulting from hypoxia had no affect. These data suggest that although extracellular CA can potentially minimize the impact of hypoxia on muscle [pH.sub.i], the actual level of extracellular CA activity is likely insufficient to achieve this goal, even when enhanced by hypoxia-induced increases in CA IX expression. acid-base; brain; eye