Effects of crowding on ornithine–urea cycle enzyme mRNA expression and activity in gulf toadfish (Opsanus beta)

Abstract

SUMMARYThe gulf toadfish (Opsanus beta) is a facultatively ureotelic fish that excretes primarily urea under conditions of crowding or confinement. To examine the relationship between ammonia production, urea production and the ornithine-urea cycle (O-UC) enzyme activity and mRNA expression, we subjected toadfish to two-day and seven-day crowding regimes. Plasma cortisol levels were measured and liver tissue was assayed for ammonia and urea concentrations. Liver glutamine synthetase (GS), carbamoyl phosphate synthetase III (CPS), ornithine carbamoyl transferase (OCT) and arginase (ARG) activities were also measured. Quantitative PCR was utilized to determine liver GS, CPS, OCT, ARG, argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) mRNA expression. Hepatic ammonia concentrations decreased with increased duration of crowding whereas liver urea and circulating cortisol levels increased. An elevation in enzyme activity with increased duration of crowding was observed for all four O-UC enzymes examined. By contrast, mRNA expression was variable for the O-UC enzymes and only CPS and ASS had mRNA expression levels that were elevated in crowded fish. These results suggest that the activities of O-UC enzymes are better predictors for urea production than O-UC enzyme mRNA expression levels.

Diel Patterns of Nitrogen Excretion, Plasma Constituents, and Behavior in the Gulf Toadfish (Opsanus beta) in Laboratory versus Outdoor Mesocosm Settings

“…This pro-65% ‫ע‬ 8.5% portion is considerably lower that the ∼90% ureotely reported above and in laboratory trials with individuals stressed by confinement (Wood et al 2003), but it was similar to the proportion observed in toadfish in their natural habitat in Florida Bay in that ∼50% of waste N appeared to be excreted as urea N ( Barimo et al 2004Barimo et al , 2007. Given the much lower levels of cortisol (and presumably stress) in mesocosm-held toadfish at low densities (Laberge et al 2009;McDonald et al 2009), it is tempting to speculate that low cortisol levels are necessary to allow the 1 : 1 excretion ratio of ammonia N to urea N (see below). Because of the similarity of urea N to ammonia N ratios in mesocosm and field measurements, the mesocosm measurement system appears to be more reflective of toadfish in their natural habitat than either the laboratory or the outdoor flux chamber measurement systems.…”

“…Overall, the plasma cortisol measurements in the Series IV mesocosm experiments were higher than those observed in freshly collected toadfish from the field and at the lower densities typical of mesocosm experiments similar to those in Series III (Laberge et al 2009;McDonald et al 2009), but they were at the lower end of the range for cannulated laboratory fish in confined conditions . While plasma cortisol levels should not be viewed as a simple proxy for stress levels, these values indicate that there is at least some level of stress-related disturbance at the densities used in Series IV of this study, even in toadfish that, while fasted, are well nourished and excreting nitrogen at a relatively high rate.…”

“…On the basis of these observations, there is reason to suspect that there is a periodicity to gulf toadfish nitrogen excretion that has not yet been discovered as a result of the largely laboratory-based experimental approaches used to date. Plasma cortisol levels in toadfish in the field or held in mesocosm tanks that simulate the animals' natural habitat (Laberge et al 2009;McDonald et al 2009) are significantly lower than those in toadfish under confined conditions in the laboratory. Given the importance of cortisol in the control of nitrogen excretion in toadfish, these observations support a potentially distinct difference in the pattern of nitrogen waste excretion that is reflective of the holding conditions of these fish.…”

Metabolic and molecular responses in Nile tilapia,Oreochromis niloticusduring short and prolonged hypoxia

“…Similar association was described for liver LDH-B mRNA and the protein it codes in gulf killifish exposed to~13.5% air saturation for 4-48 h (Crawford et al 1999;Everett et al 2012) and considered to be generally true for many other metabolic genes ). This good correlation may be best explained by transcriptional regulation where mRNA is a good predictor of specific enzyme activity (Haas et al 2005) for a number of reasons including mRNA stability that is similar to the in vivo protein half-life (Laberge et al 2009). The second pattern implies poor association, where increases in mRNA levels were recorded with no equivalent increase in enzyme activities, as in the case of liver PK and muscle LDH after short-term exposure.…”

The Effect of Stress on Gill Basolateral Membrane Binding Kinetics of 5‐HT2 Receptor Ligands: Potential Implications for Urea Excretion Mechanisms

“…Glucocorticoids, specifically the stress hormone, cortisol, have been shown to play a key role in the regulation of this pulsatile process (Wood et al, ; McDonald et al, ; Rodela et al, ). While the chronic elevation of cortisol in response to a stressor initiates the production of urea (Walsh et al, ; Hopkins et al, ; Laberge et al, ) as well as an upregulation in tUT mRNA expression (McDonald et al, ) and initial membrane recruitment (Rodela et al, ), cortisol also inhibits pulsatile urea excretion as the activation or insertion of tUT into the gill membrane appears to only occur when cortisol levels are periodically reduced (Wood et al, ; McDonald et al, ; Rodela et al, ). The drop in cortisol alone does not trigger the urea pulse since a natural drop in cortisol can be observed without a corresponding pulse occurring (Wood et al, ).…”