Using 15N to determine the metabolic fate of dietary nitrogen in North Pacific spiny dogfish (Squalus acanthias suckleyi).

Marine elasmobranchs are ureosmotic, retaining large concentrations of urea to balance their internal osmotic pressure with that of the external marine environment. The synthesis of urea requires the intake of exogenous nitrogen to maintain whole-body nitrogen balance and satisfy obligatory osmoregulatory and somatic processes. We hypothesized that dietary nitrogen may be directed toward the synthesis of specific nitrogenous molecules in post-fed animals; specifically, we predicted the preferential accumulation and retention of labelled nitrogen would be directed towards the synthesis of urea necessary for osmoregulatory purposes. North Pacific spiny dogfish (Squalus acanthias suckleyi) were fed a single meal of 7 mmol l-1 15NH4Cl in a 2% ration by body mass of herring slurry via gavage. Dietary labelled nitrogen was tracked from ingestion to tissue incorporation and the subsequent synthesis of nitrogenous compounds (urea, glutamine, bulk amino acids, protein) in the intestinal spiral valve, plasma, liver and muscle. Within 20 h post-feeding, we found labelled nitrogen was incorporated into all tissues examined. The highest δ15N values were seen in the anterior region of the spiral valve at 20 h post-feeding, suggesting this region was particularly important in assimilating the dietary labelled nitrogen. In all tissues examined, enrichment of the nitrogenous compounds was sustained throughout the 168 h experimental period, highlighting the ability of these animals to retain and use dietary nitrogen for both osmoregulatory and somatic processes.

Investigating nitrogen movement in North Pacific spiny dogfish (Squalus acanthias suckleyi), with focus on UT, Rhp2, and Rhbg mRNA abundance.

For ureosmotic marine elasmobranchs, the acquisition and retention of nitrogen is critical for the synthesis of urea. To better understand whole-body nitrogen homeostasis, we investigated mechanisms of nitrogen trafficking in North Pacific spiny dogfish (Squalus acanthias suckleyi). We hypothesized that the presence of nitrogen within the spiral valve lumen would affect both the transport of nitrogen and the mRNA abundance of a urea transporter (UT) and two ammonia transport proteins (Rhp2, Rhbg) within the intestinal epithelium. The in vitro preincubation of intestinal tissues in NH4Cl, intended to simulate dietary nitrogen availability, showed that increased ammonia concentrations did not significantly stimulate the net uptake of total urea or total methylamine. We also examined the mRNA abundance of UT, Rhp2, and Rhbg in the gills, kidney, liver, and spiral valve of fasted, fed, excess urea fed, and antibiotic-treated dogfish. After fasting, hepatic UT mRNA abundance was significantly lower, and Rhp2 mRNA in the gills was significantly higher than the other treatments. Feeding significantly increased Rhp2 mRNA levels in the kidney and mid spiral valve region. Both excess urea and antibiotics significantly reduced Rhbg mRNA levels along all three spiral valve regions. The antibiotic treatment also significantly diminished UT mRNA abundance levels in the anterior and mid spiral valve, and Rhbg mRNA levels in the kidney. In our study, no single treatment had significantly greater influence on the overall transcript abundance of the three transport proteins compared to another treatment, demonstrating the dynamic nature of nitrogen balance in these ancient fish.

Nitrogen transporters along the intestinal spiral valve of cloudy catshark (Scyliorhinus torazame): Rhp2, Rhbg, UT.

As part of their osmoregulatory strategy, marine elasmobranchs retain large quantities of urea to balance the osmotic pressure of the marine environment. The main source of nitrogen used to synthesize urea comes from the digestion and absorption of food across the gastrointestinal tract. In this study we investigated possible mechanisms of nitrogen movement across the spiral valve of the cloudy catshark (Scyliorhinus torazame) through the molecular identification of two Rhesus glycoprotein ammonia transporters (Rhp2 and Rhbg) and a urea transporter (UT). We used immunohistochemistry to determine the cellular localizations of Rhp2 and UT. Within the spiral valve, Rhp2 was expressed along the apical brush border membrane, and UT was expressed along the basolateral membrane and the blood vessels. The mRNA abundance of Rhp2 was significantly higher in all regions of the spiral valve of fasted catsharks compared to fed catsharks. The mRNA abundance of UT was significantly higher in the anterior spiral valve of fasted catsharks compared to fed. The mRNA transcript of four ornithine urea cycle (OUC) enzymes were detected along the length of the spiral valve and in the renal tissue, indicating the synthesis of urea via the OUC occurs in these tissues. The presence of Rhp2, Rhbg, and UT along the length of the spiral valve highlights the importance of ammonia and urea movement across the intestinal tissues, and increases our understanding of the mechanisms involved in maintaining whole-body nitrogen homeostasis in the cloudy catshark.

A review of reductionist methods in fish gastrointestinal tract physiology.

A holistic understanding of a physiological system can be accomplished through the use of multiple methods. Our current understanding of the fish gastrointestinal tract (GIT) and its role in both nutrient handling and osmoregulation is the result of the examination of the GIT using multiple reductionist methods. This review summarizes the following methods: in vivo mass balance studies, and in vitro gut sac preparations, intestinal perfusions, and Ussing chambers. From Homer Smith's initial findings of marine fish intestinal osmoregulation in the 1930s through to today's research, we discuss the methods, their advantages and pitfalls, and ultimately how they have each contributed to our understanding of fish GIT physiology. Although in vivo studies provide substantial information on the intact animal, segment specific functions of the GIT cannot be easily elucidated. Instead, in vitro gut sac preparations, intestinal perfusions, or Ussing chamber experiments can provide considerable information on the function of a specific tissue and permit the delineation of specific transport pathways through the use of pharmacological agents; however, integrative inputs (e.g. hormonal and neuronal) are removed and only a fraction of the organ system can be studied. We conclude with two case studies, i) divalent cation transport in teleosts and ii) nitrogen handling in the elasmobranch GIT, to highlight how the use of multiple reductionist methods contributes to a greater understanding of the organ system as a whole.

The effects of digesting a urea-rich meal on North Pacific spiny dogfish (Squalus acanthias suckleyi).

Marine elasmobranchs are nitrogen-limited owing to the requirement of nitrogen for both somatic growth and urea-based osmoregulation, and due to the loss of urea across the gills and kidney as nitrogenous waste. In this study we used in vitro stomach and intestinal gut sacs to investigate the effects of consuming a urea-rich meal (700 mM within a 2% body-mass ration of food-slurry) on nitrogen movement across the gastrointestinal (GI) tract of North Pacific spiny dogfish (Squalus acanthias suckleyi). Plasma urea concentrations did not differ between fasted (359 ± 19 mM), urea-poor fed (340 ± 16 mM), and urea-rich fed (332 ± 24 mM) dogfish. Interestingly, in vitro gut sacs of urea-rich fed dogfish showed no net urea absorption from the lumen over 3 h incubation, which contrasts previously published data on urea-poor fed dogfish that absorb urea from the lumen. In addition, ammonium (NH4+) concentration within the gut sac intestinal lumen significantly increased from 0.62 to 4.35 mM over 3 h. This is likely due to a combination of tissue production and microbial urease activity in the intestine. The overall results highlight the ability of S. a. suckleyi to regulate and maintain internal nitrogen concentrations despite the addition of excess dietary urea.

Nitrogen handling in the elasmobranch gut: a role for microbial urease.

Ureotelic elasmobranchs require nitrogen for both protein growth and urea-based osmoregulation, and therefore are probably nitrogen-limited in nature. Mechanisms exist for retaining and/or scavenging nitrogen in the gills, kidney, rectal gland and gut, but as yet, the latter are not well characterized. Intestinal sac preparations of the Pacific spiny dogfish shark (Squalus acanthias suckleyi) incubated in vitro strongly reabsorbed urea from the lumen after feeding, but mucosal fluid ammonia concentrations increased with incubation time. Phloretin (0.25 mmol l-1, which blocked urea reabsorption) greatly increased the rate of ammonia accumulation in the lumen. A sensitive [14C]urea-based assay was developed to examine the potential role of microbial urease in this ammonia production. Urease activity was detected in chyme/intestinal fluid and intestinal epithelial tissue of both fed and fasted sharks. Urease was not present in gall-bladder bile. Urease activities were highly variable among animals, but generally greater in chyme than in epithelia, and greater in fed than in fasted sharks. Comparable urease activities were found in chyme and epithelia of the Pacific spotted ratfish (Hydrolagus colliei), a ureotelic holocephalan, but were much lower in ammonotelic teleosts. Urease activity in dogfish chyme was inhibited by acetohydroxamic acid (1 mmol l-1) and by boiling. Treatment of dogfish gut sac preparations with acetohydroxamic acid blocked ammonia production, changing net ammonia accumulation into net ammonia absorption. We propose that microbial urease plays an important role in nitrogen handling in the elasmobranch intestine, allowing some urea-N to be converted to ammonia, which is then reabsorbed for amino acid synthesis or reconversion to urea.