Nitrogen excretion in developing zebrafish (Danio rerio): a role for Rh proteins and urea transporters.

Injection of antisense oligonucleotide morpholinos to elicit selective gene knockdown of ammonia (Rhag, Rhbg, and Rhcg1) or urea transporters (UT) was used as a tool to assess the relative importance of each transporter to nitrogen excretion in developing zebrafish (Danio rerio). Knockdown of UT caused urea excretion to decrease by approximately 90%, whereas each of the Rh protein knockdowns resulted in an approximately 50% reduction in ammonia excretion. Contrary to what has been hypothesized previously for adult fish, each of the Rh proteins appeared to have a similar effect on total ammonia excretion, and thus all are required to facilitate normal ammonia excretion in the zebrafish larva. As demonstrated in other teleosts, zebrafish embryos utilized urea to a much greater extent than adults and were effectively ureotelic until hatching. At that point, ammonia excretion rapidly increased and appeared to be triggered by a large increase in the mRNA expression of Rhag, Rhbg, and Rhcg1. Unlike the situation in the adult pufferfish (35), the various transporters are not specifically localized to the gills of the developing zebrafish, but each protein has a unique expression pattern along the skin, gills, and yolk sac. This disparate pattern of expression would appear to preclude interaction between the Rh proteins in zebrafish embryos. However, this may be a developmental feature of the delayed maturation of the gills, because as the embryos matured, expression of the transporters in and around the gills increased.

Induction of four glutamine synthetase genes in brain of rainbow trout in response to elevated environmental ammonia.

The key strategy for coping with elevated brain ammonia levels in vertebrates is the synthesis of glutamine from ammonia and glutamate, catalyzed by glutamine synthetase (GSase). We hypothesized that all four GSase isoforms (Onmy-GS01-GS04) are expressed in the brain of the ammonia-intolerant rainbow trout Oncorhynchus mykiss and that cerebral GSase is induced during ammonia stress. We measured GSase activity and the mRNA expression of Onmy-GS01-GS04 in fore-, mid- and hindbrain and liver, as well as ammonia concentrations in plasma, liver and brain of fish exposed to 9 or 48 h of 0 (control) or 670 micromol l(-1) NH(4)Cl (75% of the 96 h-LC(50) value). The mRNA of all four GSase isoforms were detected in brain (not liver). After 9 h of NH(4)Cl exposure, brain, liver and plasma ammonia content were elevated by two- to fourfold over control values. Midbrain, hindbrain and liver GSase activities were 1.3- to 1.5-fold higher in ammonia-exposed fish relative to control fish. Onmy-GS01-GS04 mRNA levels in brain (not liver) of ammonia-exposed fish (9 h) were significantly elevated by two- to fourfold over control values. After 48 h of the NH(4)Cl treatment, ammonia content and GSase activity, but not mRNA levels, in all tissues examined remained elevated compared to control fish. Taken together, these findings indicate that all four GSase isoforms are constitutively expressed in trout brain and are inducible under high external ammonia conditions. Moreover, elevation of GSase activities in fore-, mid- and hindbrain in response to environmental ammonia underlines the importance of brain GSase in the ammonia-stress response.

Long term hypoxia suppresses reproductive capacity in the estuarine fish, Fundulus grandis.

Human nutrient input has significantly altered dissolved oxygen (DO) cycles in coastal waters such that summertime hypoxia (DO <2 mg/L) and anoxia of bottom water are common worldwide. Prolonged hypoxia usually reduces metabolic rate in fish and potentially reduces reproduction, particularly in a spring and summer spawning species such as the Gulf killifish, Fundulus grandis. To evaluate the effects of long term hypoxia on reproduction, Gulf killifish were subjected to either normoxia (6.68+/-2.1 mg/L DO) or hypoxia (1.34+/-0.45 mg/L DO) for one month. Fecundity, growth, gonadosomatic index (GSI), circulating sex steroids (testosterone, T; 11-ketotestosterone, 11KT; and estradiol-17beta, E2), and egg yolk protein (vitellogenin, VTG) were measured. Hypoxia significantly reduced growth and reproduction. E2 was 50% lower in females and 11KT was 50% lower in males, although the precursor hormone T was unchanged in either sex after hypoxic exposure. Hypoxia-exposed females produced significantly fewer eggs and initiated spawning later than control fish. Plasma VTG concentration was unchanged, suggesting that hypoxia may delay VTG uptake by oocytes. Long term laboratory exposure clearly suppressed reproductive capacity in Gulf killifish. Wild populations experience cyclic hypoxia which could have equivalent effects if daily hypoxic periods are long and frequent - a potential consequence of anthropogenic nutrient enrichment in marsh systems.

Dogmas and controversies in the handling of nitrogenous wastes: osmoregulation during early embryonic development in the marine little skate Raja erinacea; response to changes in external salinity.

Marine elasmobranchs retain relatively high levels of urea to counterbalance the osmotic strength of seawater. Oviparous species, such as the little skate Raja erinacea, release encapsulated embryos that hatch after about 9 months on the seafloor. To study the ureosmotic capability of skate embryos, we measured a variety of possible osmolytes and ornithine-urea cycle (OUC) enzyme activities in little skate embryos, and determined their physiological response to dilute seawater (75% SW) exposure relative to controls (100% SW). The urea:trimethylamine oxide (TMAO) + other osmolytes ratio was 2.3-2.7:1. At the earliest stage of development investigated (4 months), there were significant levels of the key OUC enzyme, carbamoyl phosphate synthetase III, as well as ornithine transcarbamoylase, arginase and glutamine synthetase, providing evidence for a functional OUC. Embryos (4 and 8 months) survived and recovered from exposure to 5 days of 75% SW. There was a significant increase in the rate of urea excretion (five- to tenfold), no change in OUC enzyme activities, and significant decreases in the tissue content of urea, TMAO and other osmolytes in embryos exposed to 75% SW compared to 100% SW. Taken together, the data indicate that little skate embryos synthesize and retain urea, as well as a suite of other osmolytes, in order to regulate osmotic balance with the external environment. Interestingly, these ureosmotic mechanisms are in place as early as 4 months, around the time at which the egg capsule opens and the embryo is in more direct contact with the external environment.

Ammonia detoxification and localization of urea cycle enzyme activity in embryos of the rainbow trout (Oncorhynchus mykiss) in relation to early tolerance to high environmental ammonia levels.

The present study investigated the role of ammonia as a trigger for hatching, mechanisms of ammonia detoxification and the localization of urea cycle enzymes in the early life stages of freshwater rainbow trout (Oncorhynchus mykiss). The key urea cycle enzyme carbamoyl phosphate synthetase III was found exclusively in the embryonic body (non-hepatic tissues); related enzymes were distributed between the liver and embryonic body. 'Eyed-up' trout embryos were exposed either acutely (2h) to 10 mmol l(-1) NH(4)Cl or chronically (4 days) to 0.2 mmol l(-1) NH(4)Cl. Time to hatching was not affected by either acute or chronic NH(4)Cl exposure. Urea levels, but not ammonia levels in the embryonic tissues, were significantly higher than in controls after both acute and chronic NH(4)Cl exposure, whereas there were no significant changes in urea cycle enzyme activities. Total amino acid levels in the embryonic tissues were unaltered by chronic ammonia exposure, but levels of most individual amino acids and total amino acid levels in the yolk were significantly lower (by 34-58%) than in non-exposed controls. The data indicate that trout embryos have an efficient system to prevent ammonia accumulation in embryonic tissue, by conversion of ammonia to urea in embryonic tissues and through elevation of ammonia levels in the yolk.

Comparison of sympathetic nerve responses to neck and forearm isometric exercise.

PURPOSE: Although the autonomic and cardiovascular responses to arm and leg exercise have been studied, the sympathetic adjustments to exercise of the neck have not. The purpose of the present study was twofold: 1) to determine sympathetic and cardiovascular responses to isometric contractions of the neck extensors and 2) to compare sympathetic and cardiovascular responses to isometric exercise of the neck and forearm. METHODS: Muscle sympathetic nerve activity (MSNA), mean arterial pressure (MAP), and heart rate were measured in nine healthy subjects while performing isometric neck extension (INE) and isometric handgrip (IHG) in the prone position. After a 3-min baseline period, subjects performed three intensities of INE for 2.5 min each: 1) unloaded (supporting head alone), 2) 10% maximal voluntary contraction (MVC), and 3) 30% MVC, then subjects performed two intensities (10% and 30% MVC) of IHG for 2.5 min. RESULTS: Supporting the head by itself did not significantly change any of the variables. During [NE, MAP significantly increased by 10 +/- 2 and 31 +/- 4 mm Hg and MSNA increased by 67 +/- 46 and 168 +/- 36 units/30 s for 10% and 30% MVC, respectively. IHG and INE evoked similar responses at 10% MVC, but IHG elicited higher peak MAP and MSNA at 30% MVC (37 +/- 7 mm Hg (P < 0.05) and 300 +/- 48 units/30 s (P < 0.01) for IHG, respectively). CONCLUSIONS: The data indicate that INE can elicit marked increases in MSNA and cardiovascular responses but that it evokes lower peak responses as compared to IHG. We speculate that possible differences in muscle fiber type composition, muscle mass, and/or muscle architecture of the neck and forearm are responsible for these differences in peak responses.

Sympathetic nerve activity during natural stimulation of horizontal semicircular canals in humans.

We have shown that static head-down neck flexion elicits increases in muscle (MSNA) but not skin sympathetic nerve activity (SSNA) in humans. These findings suggest that stimulation of the otolith organs causes differential sympathetic outflow to vascular beds. The purpose of the present study was to determine whether yaw head rotation (YHR), which stimulates the horizontal semicircular canals, elicits sympathetic nerve responses. To test this question, we recorded MSNA (n = 33) and SSNA (n = 25) before and during 3 min of sinusoidal YHR performed at 0.1, 0.6, and 1.0 Hz. At all frequencies, YHR elicited no significant changes in heart rate and mean arterial pressure. Likewise, YHR did not significantly change either MSNA or SSNA at all frequencies. Our results indicate that stimulation of the horizontal semicircular canals by YHR does not alter SNA to either muscle or skin. Moreover, these results provide evidence to support the concept that the otolith organs but not the horizontal semicircular canals participate in the regulation of SNA in humans.