M. Danielle McDonald

Branchial and renal handling of urea in the gulf toadfish, Opsanus beta: the effect of exogenous urea loading

The objective of this study was to determine whether the pulsatile facilitated diffusion transport mechanism (tUT) found in the gills of the gulf toadfish (Opsanus beta) and the active secretion transporter thought to be present in its kidney could be saturated when faced with elevated plasma urea concentrations. Toadfish were infused with four consecutive exogenous urea loads at a rate of 0, 150, 300 and 600 micromol kg(-1) h(-1). Initial plasma and urine urea concentrations were 8.1+/-0.9 and 12.4+/-1.5 mmol l(-1), respectively, and steadily increased with increasing infused loads of urea to a maximum of 36.8+/-2.8 mmol l(-1) in the plasma and 39.8+/-6.5 mmol l(-1) in the urine. There was only a very weak relationship (r=0.17) between pulse size (measured as branchial excretion during pulsatile excretion of urea) and plasma urea concentration (slope=9.79 micromol-N kg(-1) per mmol-N l(-1); P<0.05) suggesting that the branchial excretion mechanism was already saturated at normal plasma urea concentrations. Urine flow rate (0.15+/-0.03 ml kg(-1) h(-1)) and glomerular filtration rate (0.025+/-0.004 ml kg(-1) h(-1)) remained constant throughout the experiment despite the increased volume load. Renal urea secretion rate maintained a strong linear relationship (r=0.84) to plasma urea levels (slope=0.391 micromol-N kg(-1) h(-1) per mmol-N l(-1); P<0.001) with no observable transport maximum, suggesting that the renal secretory transport mechanism was not saturated even at plasma urea levels well above normal, in contrast to the branchial excretion mechanism.

Do circulating plasma AVT and/or cortisol levels control pulsatile urea excretion in the gulf toadfish (Opsanus beta)?

Previous work has shown that pulsatile urea excretion at the gills of the gulf toadfish is due to periodic activation of a facilitated diffusion transport system with molecular and pharmacological similarity to the UT-A transport system of the mammalian kidney. In mammals, AVP and glucocorticoids are two important endocrine regulators of this system. The present study focused on the potential role of circulating AVT (the teleost homologue of AVP) and cortisol levels as possible triggers for urea pulses. Long-term (34-84 h) monitoring of plasma levels by repetitive sampling at 2-h intervals from chronic cannulae in individual toadfish demonstrated that circulating AVT concentrations are low (10(-12)-10(-11) M), and show no relationship to the occurrence of natural urea pulses. In contrast, plasma cortisol levels decline greatly prior to natural pulses and rise rapidly thereafter. AVT injections into the caudal artery or ventral aorta elicited pulse events, but these were extremely small (1-10%) relative to natural pulses, and occurred only at unphysiological dose levels (10(-9) M in the plasma). AVP was a partial agonist, but isotocin, insulin-like growth factor-1, and atrial natriuretic peptide were without effect at the same concentration. Artificially raising plasma cortisol levels by cortisol injection tended to reduce responsiveness to AVT. Pharmacological reduction of plasma cortisol levels by metyrapone injection elicited small pulses similar to those caused by AVT. Following such pulse events, AVT was ineffective in inducing pulses. We conclude that decreases in circulating cortisol play an important permissive role in urea pulsing, but that circulating AVT levels are not involved.

Fluoxetine treatment affects nitrogen waste excretion and osmoregulation in a marine teleost fish.

Measurable quantities of the selective serotonin reuptake inhibitor (SSRI), fluoxetine, have been found in surface waters and more recently in the tissues of fish. This highly prescribed pharmaceutical inhibits the reuptake of the monoamine, serotonin (5-HT; 5-hydroxytryptamine), causing a local amplification of 5-HT concentrations. Serotonin is involved in the regulation of many physiological processes in teleost fish including branchial nitrogen excretion and intestinal osmoregulation. Since the gill and intestine are directly exposed to the environment, environmental exposure to fluoxetine has the potential of affecting both these mechanisms. In the present study, we test the potential sensitivity of these processes to fluoxetine by implanting gulf toadfish, Opsanus beta, intraperitoneally with different concentrations of fluoxetine (0 (control), 25, 50, 75 and 100 microgg(-1)). Fluoxetine treatments of 25 and 50 microgg(-1) were sub-lethal and were used in subsequent experiments. Fish treated with both 25 and 50 microgg(-1) fluoxetine had significantly higher circulating levels of 5-HT than control fish, suggesting that any 5-HT sensitive physiological process could potentially be affected by these two fluoxetine doses. However, only fish treated with 25 microgg(-1) fluoxetine showed a significant increase in urea excretion. A similar increase was not measured in fish treated with 50 microgg(-1) fluoxetine, likely because of their high circulating levels of cortisol which inhibits urea excretion in toadfish. Intestinal fluid absorption appeared to be stimulated in fish treated with 25 microgg(-1) fluoxetine but inhibited in 50 microgg(-1) treated fish. Despite these differing responses, both doses of fluoxetine resulted in lowered plasma osmolality values, which was expected based on the stimulation of fluid absorption in the 25 microgg(-1) fluoxetine-treated fish but is surprising with the 50 microgg(-1) treated fish. In the case of the latter, the corresponding stress response invoked by this level of fluoxetine may have resulted in an additional osmoregulatory response which accounts for the lowered plasma osmolality. Our findings suggest that branchial urea excretion and intestinal osmoregulation are responsive to the SSRI, fluoxetine, and further investigation is needed to determine the sensitivity of these processes to chronic waterborne fluoxetine contamination.

The serotonin subtype 1A receptor regulates cortisol secretion in the Gulf toadfish, Opsanus beta.

It is well established that serotonin (5-HT; 5-hydroxytryptamine) plays a role in mammalian regulation of the hypothalamic-pituitary-adrenal (HPA) axis via the 5-HT receptor subtype 1A (5-HT(1A)). To date, there has not been a comprehensive investigation of the molecular, pharmacological and physiological aspects of the 5-HT(1A) receptor and its role in the activation of the hypothalamic-pituitary-interrenal (HPI) axis in teleost fish. The 5-HT(1A) receptor of the Gulf toadfish (Opsanus beta) was cloned and sequenced, showing 67.5% amino acid similarity to the human homologue. The 5-HT(1A) receptor was distributed throughout the brain, with the whole brain containing significantly higher levels of 5-HT(1A) mRNA compared to all other tissues and the midbrain/diencephalon region containing significantly higher levels of transcript than any other brain region. Substantial levels of transcript were also found in the pituitary, while very low levels were in the kidney that contains the interrenal cells. Xenopus oocytes injected with toadfish 5-HT(1A) receptor cRNA displayed significantly higher binding of [(3)H]5-HT that was abolished by the mammalian 5-HT(1A) receptor agonist, 8-OH-DPAT, indicating a conserved binding site of the toadfish 5-HT(1A) receptor and a high specificity for the agonist. Supporting this, binding of [(3)H]5-HT was not affected by the mammalian 5-HT(1B) receptor agonist, 5-nonyloxytryptamine, the 5-HT(7) receptor antagonist, SB269970, or the 5-HT(2) receptor agonist, alpha-methylserotonin. Confirming these molecular and pharmacological findings, intravenous injection of 8-OH-DPAT stimulated the HPI axis to cause a 2-fold increase in circulating levels of cortisol. The present study of the 5-HT(1A) receptor in a single teleost species illustrates the high conservation of this 5-HT receptor amongst vertebrates.

Urea transporter and glutamine synthetase regulation and localization in gulf toadfish gill

SUMMARY The goal of the present study was to investigate the role of circulating cortisol and urea in the transcriptional regulation of branchial glutamine synthetase (GS), which incorporates NH3 into glutamate to form glutamine, and the toadfish urea transporter, tUT, which is involved in urea excretion across the gill of the gulf toadfish. GS (of which there are two isoforms, LGS and GGS) and tUT mRNA expression and activity were measured in toadfish exposed to treatments that would induce variable stress responses. In addition, the role of circulating urea in tUT regulation was investigated by infusing toadfish with urea alone or in combination with intraperitoneal injection of RU486, a corticosteroid type II receptor antagonist. There was a 4.8-fold upregulation in the mRNA expression of the gill-specific GS isoform (GGS) in response to cortisol infusion and a similar upregulation in the more ubiquitous isoform (LGS). Furthermore, there was a significant 1.9-fold and 3.3-fold upregulation in the mRNA expression of the toadfish urea transporter, tUT, in response to stress through crowding or exogenous cortisol loading through infusion, respectively. In addition, tUT was found to have a urea-sensitive component to transcriptional regulation that was independent of circulating cortisol concentrations. However, the changes measured in mRNA expression of GGS, LGS and tUT did not correspond with changes in protein activity. To determine the cell type(s) involved in glutamine production and urea excretion, we attempted to localize GGS, LGS and tUT using in situ hybridization. This study is the first to show that GGS and tUT expression appear to occur in gill mitochondria-rich cells of toadfish, suggesting that these cells play a combined glutamine production and urea excretion role, which may have implications for predator avoidance.

Dogmas and controversies in the handling of nitrogenous wastes: 5-HT2-like receptors are involved in triggering pulsatile urea excretion in the gulf toadfish, Opsanus beta.

SUMMARY When injected arterially, serotonin (5-hydroxytryptamine; 5-HT) has been shown to elicit naturally sized urea pulse events in the gulf toadfish, Opsanus beta. The goal of the present study was to determine which 5-HT receptor(s) was involved in mediating this serotonergic stimulation of the pulsatile excretion mechanism. Toadfish were surgically implanted with caudal arterial catheters and intraperitoneal catheters and injected with either 8-OH-DPAT (1 μmol kg–1), a selective 5-HT1A receptor agonist, α-methyl-5-HT (1 μmol kg–1), a 5-HT2 receptor agonist, or ketanserin, a 5-HT2 receptor antagonist (0.01, 0.1, 1 and 10 μmol kg–1) plus α-methyl-5-HT. 8-OH-DPAT injection did not mediate an increase in urea excretion, ruling out the involvement of 5-HT1A receptors in pulsatile excretion. However, within 5 min,α -methyl-5-HT injection caused an increase in the excretion of urea in> 95% (N=27) of the fish injected, with an average pulse size of 652±102 μmol N kg–1 (N=26). Withα -methyl-5-HT injection there was no corresponding increase in ammonia or [3H]PEG 4000 permeability. Urea pulses elicited byα -methyl-5-HT were inhibited in a dose-dependent fashion by the 5-HT2 receptor antagonist ketanserin, which at low doses caused a significant inhibition of pulse size and at higher doses significantly inhibited the occurrence of pulsatile excretion altogether. However, neither 8-OH-DPAT nor α-methyl 5-HT injection had an effect on plasma cortisol or plasma urea concentrations. These findings suggest the involvement of a 5-HT2-like receptor in the regulation of pulsatile urea excretion.

Concentration of MgSO4 in the intestinal lumen of Opsanus beta limits osmoregulation in response to acute hypersalinity stress

Marine teleosts constantly lose water to their surrounding environment, a problem exacerbated in fish exposed to salinity higher than normal seawater. Some fish undergo hypersaline exposures in their natural environments, such as short- and long-term increases in salinity occurring in small tidal pools and other isolated basins, lakes, or entire estuaries. Regardless of the degree of hypersalinity in the ambient water, intestinal absorption of monovalent ions drives water uptake to compensate for water loss, concentrating impermeable MgSO(4) in the lumen. This study considers the potential of luminal [MgSO(4)] to limit intestinal water absorption, and therefore osmoregulation, in hypersalinity. The overall tolerance and physiological response of toadfish (Opsanus beta) to hypersalinity exposure were examined. In vivo, fish in hypersaline waters containing artificially low [MgSO(4)] displayed significantly lower osmolality in both plasma and intestinal fluids, and increased survival at 85 parts per thousand, indicating improved osmoregulatory ability than in fish exposed to hypersalinity with ionic ratios similar to naturally occurring ratios. Intestinal sac preparations revealed that in addition to the osmotic pressure difference across the epithelium, the luminal ionic composition influenced the absorption of Na(+), Cl(-), and water. Hypersalinity exposure increased urine flow rates in fish fitted with ureteral catheters regardless of ionic composition of the ambient seawater, but it had no effect on urine osmolality or pH. Overall, concentrated MgSO(4) within the intestinal lumen, rather than renal or branchial factors, is the primary limitation for osmoregulation by toadfish in hypersaline environments.

Does Pulsatile Urea Excretion Serve as a Social Signal in the Gulf Toadfish Opsanus beta

This study evaluated the hypothesis that the pulsatile excretion of urea by toadfish could serve as a social signal. In the first experiment, physiological parameters were measured in pairs of dominant and subordinate toadfish. Subordinate toadfish had elevated concentrations of circulating plasma cortisol, an effect maintained even after cannulation. In the second experiment, one fish of a pair was injected with 14C‐urea, and the occurrence of urea pulses during social encounters was documented. Social status did not influence the order of pulsing, that is, whether a dominant or subordinate fish pulsed first during a social encounter. However, in seven out of eight pairs, both toadfish pulsed within 2 h of each other, indicating some form of communication between fish. In the third and final experiment, the response of toadfish to urea (natural or synthetic) was observed. There was a tendency for toadfish to avoid synthetic urea but there was no apparent behavioural response to water containing toadfish urea. Pulsing events do not appear to play an integral role during social encounters as previously hypothesised, but the close timing of pulses in toadfish pairs suggests some transfer of information.

The regulatory role of glucocorticoid and mineralocorticoid receptors in pulsatile urea excretion of the gulf toadfish, Opsanus beta.

SUMMARY Gulf toadfish, Opsanus beta, are one among a group of unusual teleosts that excrete urea as their predominant nitrogen end product in response to stressful conditions. Under conditions of crowding or confinement, fasted toadfish excrete the majority of their nitrogen waste in large pulses of urea (>90% of total nitrogen) lasting up to 3 h. An earlier study demonstrated that cortisol has an inhibitory influence on urea pulse size. The present study tested the hypothesis that cortisol mediates changes in urea pulse size in ureotelic toadfish through the glucocorticoid receptor (GR) and not the mineralocorticoid receptor (MR). In vivo pharmacological investigations were used to manipulate the corticosteroid system in crowded toadfish, including experimentally lowering plasma cortisol levels by the injection of metyrapone, blocking cortisol receptors through exposure to either RU-486 (GR antagonist) and spironolactone (MR antagonist), or through exogenous infusion of the tetrapod mineralocorticoid aldosterone (tetrapod MR agonist). The data demonstrate that lowering the activity of cortisol, either by inhibiting its synthesis or by blocking its receptor, resulted in a two- to threefold increase in pulse size with no accompanying change in pulse frequency. Treatment with spironolactone elicited a minor (∼1.5-fold) reduction in pulse size, as did aldosterone treatment, suggesting that the anti-mineralocorticoid spironolactone has an agonistic effect in a piscine system. In summary, the evidence suggests that urea transport mechanisms in pulsing toadfish are upregulated in response to low cortisol, mediated primarily by GRs, and to a lesser extent MRs.

Evidence for transcriptional regulation of the urea transporter in the gill of the Gulf toadfish, Opsanus beta

Ureotelic Gulf toadfish (Opsanus beta) do not excrete urea continuously; instead, urea is accumulated internally until a branchial urea transport mechanism is activated to facilitate the excretion of urea in distinct pulses. This unusual pulsatile urea excretion pattern is regulated, in part, by permissive declines in circulating cortisol concentrations. The current study examined toadfish urea transporter (tUT) and glucocorticoid receptor (GR) transcript levels in toadfish gill following chronic (days) and acute (hours) changes in corticosteroid activity. Experimentally lowering circulating cortisol did not significantly alter tUT mRNA abundance but increased GR mRNA. On an acute timescale, a 6.2-fold upregulation of tUT mRNA occurred 12 to 18 h following a urea pulse event with no change in GR mRNA. In silico analysis of an isolated 1.2 kb fragment, upstream promoter region of the tUT gene, revealed 6 putative glucocorticoid response element (GRE) half sites. In vivo reporter assays of the tUT promoter fragment demonstrated relative luciferase activity was enhanced 3.4- and 9.8-fold following exposure to moderate (via a 48 h crowding stress) and high (via infusion for 48 h) cortisol. We conclude that a GRE-mediated upregulation of mRNA may be required to maintain tUT activity by offsetting post-transcriptional and/or post-translational changes that may be associated with chronically elevated plasma cortisol.