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Plasma serotonin (5-hydroxytryptamine, 5-HT) homeostasis is maintained through the combined processes of uptake (via the 5-HT transporter SERT, and others), degradation (via monoamine oxidase, MAO) and excretion. Previous studies have shown that inhibiting SERT, which would inhibit 5-HT uptake and degradation, attenuates parts of the cardiovascular hypoxia reflex in gulf toadfish (Opsanus beta), suggesting that these 5-HT clearance processes may be important during hypoxia exposure. Therefore, the goal of this experiment was to determine the effects of mild hypoxia on 5-HT uptake and degradation in the peripheral tissues of toadfish. We hypothesized that 5-HT uptake and degradation would be upregulated during hypoxia, resulting in lower plasma 5-HT, with uptake occurring in the gill, heart, liver and kidney. Fish were exposed to normoxia (97.6% O2 saturation, 155.6 Torr) or 2 min, 40 min or 24 h mild hypoxia (50% O2 saturation, ∼80 Torr), then injected with radiolabeled [3H]5-HT before blood, urine, bile and tissues were sampled. Plasma 5-HT levels were reduced by 40% after 40 min of hypoxia exposure and persisted through 24 h. 5-HT uptake by the gill was upregulated following 2 min of hypoxia exposure, and degradation in the gill was upregulated at 40 min and 24 h. Interestingly, there was no change in 5-HT uptake by the heart and degradation in the heart decreased by 58% within 2 min of hypoxia exposure and by 85% at 24 h. These results suggest that 5-HT clearance is upregulated during hypoxia and is likely driven, in part, by mechanisms within the gill and not the heart.

The neurochemical serotonin (5-HT) is involved in stimulating pulsatile urea excretion in Gulf toadfish (Opsanus beta) through the 5-HT2A receptor; however, it is not known if (1) the 5-HT signal originates from circulation or if (2) additional 5-HT receptor subtypes are involved. The first objective was to test whether 5-HT may be acting as a hormone in the control of pulsatile urea excretion by measuring potential fluctuations in circulating 5-HT corresponding with a urea pulse, which would suggest circulating 5-HT may be involved with urea pulse activation. We found that plasma 5-HT significantly decreased by 38% 1 h after pulse detection when branchial urea excretion was significantly elevated and then returned to baseline. This suggests that 5-HT is removed from the circulation, possibly through clearance or excretion, and may be involved in the termination of pulsatile urea excretion. There appeared to be no pulsatile release of 5-HT from peripheral tissues to trigger a urea pulse. The second objective was to determine if additional 5-HT receptor subtypes, such as an additional 5-HT2 receptor (5-HT2C receptor) or the 5-HT receptors that are linked to cAMP (5-HT4/6/7 receptors), played a role in the stimulation of urea excretion. Intravenous injection of 5-HT2C, 5-HT4, 5-HT6, and 5-HT7 receptor agonists did not result in a urea pulse, suggesting that these receptors, and thus cAMP, are not involved in stimulating urea excretion. The involvement of circulating 5-HT and the 5-HT2A receptor in the regulation of pulsatile urea excretion may provide insight into its adaptive significance.

Gulf toadfish (Opsanus beta) are exceptionally capable of switching from excreting ammonia as their primary nitrogenous waste to excreting predominantly urea in distinct pulses across the gill. Previous studies suggest that these urea pulses may be used for intraspecific chemical communication. To determine whether pulsatile urea excretion communicates reproductive status, toadfish were sexed using ultrasound and delivered conspecific-conditioned seawater (CC-SW) that previously housed a conspecific of the opposite sex, a conspecific chemical alarm cue (avoidance control), or a prey cue (attraction control). Swim behavior, attraction to or avoidance of the cues, and changes in the pattern of pulsatile urea excretion were monitored during and after delivery. Gulf toadfish did not spend more time in zones that were delivered CC-SW or prey cue. However, male toadfish spent significantly more time swimming after the delivery of female cues than control seawater (SW). In contrast, toadfish did not appear to have an immediate avoidance response to the conspecific alarm cue. Additionally, significantly more toadfish pulsed within 7 h of CC-SW and prey cue delivery compared to control SW, and pulse frequency was 1.6 times greater in response to CC-SW than control SW. These results, in combination with increased urea production and excretion the during breeding season, suggest that toadfish may use pulsatile urea excretion to communicate with conspecifics when exposed to chemosensory cues from the opposite sex.

The neurotransmitter serotonin (5-hyroxytryptamine, 5-HT) is involved in a variety of peripheral processes. Arguably most notable is its role as a circulating vasoconstrictor in the plasma of vertebrates. Plasma 5-HT is maintained at constant levels under normal conditions through the processes of cellular uptake, degradation, and excretion, known collectively as clearance. However, the degree to which each individual component of clearance contributes to this whole animal response remains poorly understood. The goal of this experiment was to determine the extent to which transporter-mediated uptake and intracellular degradation contribute to 5-HT clearance in the model teleost Gulf toadfish (Opsanus beta). Fish that were treated with the 5-HT transport inhibitors fluoxetine, buproprion, and decynium-22 had 1.47-fold higher plasma 5-HT concentrations and a 40% decrease in clearance rate compared to control fish. In contrast, fish treated with the MAO inhibitor clorgyline had a 1.54-fold increase in plasma 5-HT with no change in clearance rate. The results show that transporter-mediated 5-HT uptake plays an important role in controlling circulating 5-HT and whole body 5-HT homeostasis.

Gulf toadfish (Opsanus beta) can switch from continuously excreting ammonia as their primary nitrogenous waste to excreting predominantly urea in distinct pulses. Previous studies have shown that the neurotransmitter serotonin (5-HT) is involved in controlling this process, but it is unknown if 5-HT availability is under central nervous control or if the 5-HT signal originates from a peripheral source. Following up on a previous study, cranial nerves IX (glossopharyngeal) and X (vagus) were sectioned to further characterize their role in controlling pulsatile urea excretion and 5-HT release within the gill. In contrast to an earlier study, nerve sectioning did not result in a change in urea pulse frequency. Total urea excretion, average pulse size, total nitrogen excretion, and percent ureotely were reduced the first day post-surgery in nerve-sectioned fish but recovered by 72 h post-surgery. Nerve sectioning also had no effect on toadfish urea transporter (tUT), 5-HT transporter (SERT), or 5-HT2A receptor mRNA expression or 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) abundance in the gill, all of which were found consistently across the three gill arches except 5-HIAA, which was undetectable in the first gill arch. Our findings indicate that the central nervous system does not directly control pulsatile urea excretion or local changes in gill 5-HT and 5-HIAA abundance.

Gulf toadfish (Opsanus beta) can excrete the majority of their nitrogenous waste as urea in distinct pulses across their gill. Urea pulses are controlled by cortisol and serotonin (5-HT) and are believed to contain chemical signals that may communicate reproductive and/or social status. The objectives of this study were to determine if reproductive hormones are involved in controlling pulsatile urea excretion, and if toadfish respond to prostaglandins as a chemical signal. Specifically, 11-ketotestosterone (11-KT), estradiol (E2), and the teleost pheromone prostaglandin E2 (PGE2) were investigated. Castration during breeding season did not affect pulsatile urea excretion but serial injections of 11-KT outside of breeding season did result in a 48% reduction in urea pulse size in fish of both sexes. Injections of E2 and PGE2, on the other hand, did not alter urea excretion patterns. Toadfish also did not pulse urea in response to waterborne exposure of PGE2 suggesting that this compound does not serve as a toadfish pheromone alone. Toadfish have significantly higher plasma 5-HT during breeding season compared to the months following breeding season. Future research should focus on the composition of the chemical signal in toadfish and the potential importance of seasonal changes in plasma 5-HT in toadfish pulsatile urea excretion and teleost reproduction in general.

Freshwater- and seawater-acclimated Fundulus heteroclitus were exposed to acute hypoxia (10% air saturation, 3 h), followed by normoxic recovery (3 h). In both salinities, ventilation increased and heart rate fell in the classic manner, while Ṁ(O(2)) initially declined by ∼50%, with partial restoration by 3 h of hypoxia, and no O(2) debt repayment during recovery. Gill paracellular permeability (measured with [(14)C] PEG-4000) was 1.4-fold higher in seawater, and declined by 50% during hypoxia with post-exposure overshoot to 188%. A similar pattern with smaller changes occurred in freshwater. Drinking rate (also measured with [(14)C] PEG-4000) was 8-fold higher in seawater fish, but declined by ∼90% during hypoxia in both groups, with post-exposure overshoots to ∼270%. Gill diffusive water flux (measured with (3)H(2)O) was 1.9-fold higher in freshwater fish, and exhibited a ∼35% decrease during hypoxia, which persisted throughout recovery, but was unchanged during hypoxia in seawater fish. Nevertheless, freshwater killifish gained mass while seawater fish lost mass during hypoxia, and these changes were not corrected during normoxic recovery. We conclude that this hypoxia-tolerant teleost beneficially reduces gill water permeability in a salinity-dependent fashion during hypoxia, despite attempting to simultaneously improve Ṁ(O(2)), but nevertheless incurs a net water balance penalty in both freshwater and seawater.

In mammals, circulating serotonin [5-hydroxytryptamine (5-HT)] is sequestered by platelets via the 5-HT transporter (SERT) to prevent unintended signaling by this potent signaling molecule. Teleost fish appear to lack a similar circulating storage pool, although the diverse effects of 5-HT in teleosts likely necessitate an alternative method of tight regulation, such as uptake by peripheral tissues. Here, a 5-HT radiotracer was used to explore the 5-HT uptake capacity of peripheral tissues in the Gulf toadfish, Opsanus beta, and to elucidate the primary excretion routes of 5-HT and its metabolites. Pharmacological inhibition of SERT and other transporters enabled assessment of the SERT dependence of peripheral 5-HT uptake and excretion. The results indicated a rapid and substantial uptake of 5-HT by the heart atrium, heart ventricle, and gill that was at least partly SERT dependent. The results also supported the presence of a partial blood-brain barrier that prevented rapid changes in brain 5-HT content despite fluctuating plasma 5-HT concentrations. The renal pathway appeared to be the dominant excretory route for 5-HT and its metabolites over shorter time frames (up to ~30 min), but hepatic excretion was substantial over several hours. SERT inhibition ultimately reduced the excretion of 5-HT and its metabolites by urinary, biliary, and/or intestinal pathways. In addition, branchial excretion of 5-HT and its metabolites could not be ruled out. In summary, this study reveals that the toadfish heart and gill play active roles in regulating circulating 5-HT and yields important insights into the control of peripheral 5-HT in this teleost fish.

The serotonin transporter (SERT) functions in the uptake of the neurotransmitter serotonin (5-HT) from the extracellular milieu and is the molecular target of the selective serotonin re-uptake inhibitors (SSRIs), a common group of anti-depressants. The current study comprehensively assesses the sequence, tissue distribution, transport kinetics and physiological function of a teleost SERT. The 2022 bp toadfish SERT sequence encodes a protein of 673 amino acids, which shows 83% similarity to zebrafish SERT and groups with SERT of other teleosts in phylogenetic analysis. SERT mRNA is ubiquitous in tissues and is expressed at high levels in the heart and, within the brain, in the cerebellum. SERT cRNA expressed in Xenopus laevis oocytes demonstrates a K m value of 2.08±0.45 μmol l –1 , similar to previously reported K m values for zebrafish and human SERT. Acute systemic blockade of SERT by intraperitoneal administration of the SSRI fluoxetine (FLX) produces a dose-dependent increase in plasma 5-HT, indicating effective inhibition of 5-HT uptake from the circulation. As teleosts lack platelets, which are important 5-HT sequestration sites in mammals, the FLX-induced increase in plasma 5-HT suggests that toadfish tissues may normally be responsible for maintaining low 5-HT concentrations in the bloodstream.

Stimulation of the serotonin 1A (5-HT1A) receptor subtype by 5-HT has been shown to result in an elevation in plasma corticosteroid levels in both mammals and several species of teleost fish, including the Gulf toadfish (Opsanus beta); however, in the case of teleost fish, it is not clearly known at which level of the hypothalamic-pituitary-interrenal axis the 5-HT1A receptor is stimulated. Additionally, previous investigations have revealed that chronic elevations of plasma cortisol mediate changes in brain 5-HT1A receptor mRNA and protein levels via the glucocorticoid receptor (GR); thus, we hypothesized that the function of centrally activated 5-HT1A receptors is reduced or abolished as a result of chronically elevated plasma cortisol levels and that this response is GR mediated. Our results are the first to demonstrate that intravenous injection of the 5-HT1A receptor agonist, 8-OH-DPAT, stimulates a significant increase in corticotropin-releasing factor (CRF) precursor mRNA expression in the hypothalamic region and the release of adrenocorticotropic hormone (ACTH) from the pituitary of teleost fish compared to saline-injected controls. We also provide evidence that cortisol, acting via GRs, attenuates the 5-HT1A receptor-mediated secretion of both CRF and ACTH.

In both mammals and teleost fish, serotonin stimulates cortisol secretion via the 5-HT1A receptor. Additionally, a negative feedback loop exists in mammals whereby increased circulating levels of cortisol inhibit 5-HT1A receptor activity. To investigate the possibility of such a feedback mechanism in teleosts, plasma cortisol levels and signaling in Gulf toadfish (Opsanus beta) were manipulated and the role of cortisol in the control of 5-HT1A evaluated. Despite a significant 4-fold increase in plasma [cortisol], crowded toadfish expressed similar amounts of 5-HT1A mRNA transcript as uncrowded toadfish; whereas, cortisol-implanted fish possessed 41.8% less 5-HT1A mRNA transcript compared to vehicle-implanted controls. This cortisol effect appeared to be reversed in RU486-injected fish, which blocks glucocorticoid receptors, as these fish expressed nearly twice as much 5-HT1A receptor transcript as the vehicle-injected fish despite significantly elevated cortisol levels. The binding affinity for the 5-HT1A receptor in the brain did not vary between any groups; however, maximum binding was significantly higher in uncrowded toadfish compared to crowded, and the same significant difference was observed between the maximum binding of vehicle and cortisol-implanted fish. The opposite trend was seen in RU486-injected and vehicle-injected fish, with RU486-injected fish having significantly higher maximal binding compared to vehicle-injected controls. Injection with the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin revealed an inhibition of cortisol secretion that was independent of 5-HT1A transcript and protein binding. These results suggest that cortisol plays a role in regulating the 5-HT1A receptor via GR-mediated pathways; however, further study is necessary to elucidate how and where this inhibition is mediated.

The goal of this study was to determine the relationship between cortisol and the toadfish serotonin 2A (5-HT2A) receptor, which is believed to be responsible for the activation of the toadfish urea transporter, tUT. We hypothesize that elevations in cortisol would play a role in the regulation of the 5-HT2A receptor at the level of mRNA expression, ligand binding, and/or function. To test this idea, cortisol levels were manipulated by either crowding or through treatment with the cortisol synthesis blocker, metyrapone. Crowded fish had significantly higher circulating cortisol levels compared to uncrowded fish and cortisol levels in metyrapone-treated fish were significantly lower than saline-treated controls. No significant difference was measured in gill 5-HT2A mRNA expression levels between uncrowded and crowded, control- or metyrapone-treated fish. Furthermore, no significant difference was measured in [3H]-5-HT binding kinetics or in the competitive binding of the 5-HT2 agonist, α-methyl 5-HT, to isolated gill basolateral membranes of uncrowded or crowded toadfish. However, the binding maximum (Bmax) of the 5-HT2A receptor antagonist, [3H]-ketanserin, was significantly different between all four groups of fish (metyrapone > control > crowded > uncrowded). Furthermore, metyrapone-treated fish excreted approximately twofold more urea compared to controls when injected with α-methyl 5-HT, a 5-HT2 receptor agonist shown to stimulate urea excretion. Our results suggest that cortisol may have differential effects on 5-HT receptor binding, which could have potential implications on the control of pulsatile urea excretion in toadfish. J. Exp. Zool. 319A:237–248, 2013. © 2013 © 2013 Wiley Periodicals, Inc.

Yellowtail snapper (Ocyurus chrysurus) is an important part of the reef fish assemblage in the western, tropical Atlantic and is caught by both recreational and commercial fisheries in south Florida and the Bahamas. It is estimated that 80% of snapper caught within southeastern Florida waters are discarded due to minimum size restrictions. Neglecting to include information on delayed mortality of undersized fish has the potential for fishery managers to overestimate the abundance of smaller size classes and introduce bias into stock assessments. This study examines associations between reflex impairment, traditional physiological parameters and post-release mortality of undersized yellowtail snapper. Laboratory experiments exposed yellowtail snapper to a gradient, simulating capture conditions. Blood draws were obtained from a sub-sample of fish. There was a significant relationship between delayed mortality and the proportion of reflex impairment for both individual fish and groups of fish (P < 0.001 and P = 0.03). Within the sub-sample of blood-sampled fish, base excess and pH were significantly correlated to reflex impairment. Delayed mortality was significantly correlated to pH, base excess and lactate concentration. Results suggest that discarded, undersized yellowtail with more than 29% of their reflexes impaired will not survive.

Pharmaceuticals and personal care products (PPCPs) are found in measureable quantities within the aquatic environment. Selective serotonin reuptake inhibitor (SSRI) antidepressants are one class of pharmaceutical compound that has received a lot of attention. Consistent with most PPCPs, the pharmacokinetics and physiological impacts of SSRI treatment have been well-studied in small mammals and humans and this, combined with the evolutionary conservation of the serotonergic system across vertebrates, allows for the read-across of known SSRI effects in mammals to potential SSRI impacts on aquatic organisms. Using an Adverse Outcome Pathway (AOP) framework, this review examines the similarities and differences between the mammalian and teleost fish SSRI target, the serotonin transporter (SERT; SLC6A4), and the downstream impacts of elevated extracellular serotonin (5-HT; 5-hydroxytryptamine), the consequence of SERT inhibition, on organ systems and physiological processes within teleost fish. This review also intends to reveal potentially understudied endpoints for SSRI toxicity based on what is known to be controlled by 5-HT in fish.

Not long ago, urea was believed to freely diffuse across plasma membranes. The discovery of specialized proteins to facilitate the movement of urea across the fish gill, similar to those found in mammalian kidney, was exciting, and at the same time, perplexing; especially considering the fact that, aside from elasmobranchs, most fish do not produce urea as their primary nitrogenous waste. Increasingly, it has become apparent that many fish do indeed produce at least a small amount of urea through various processes and continued work on branchial urea transporters in teleost and elasmobranch fishes has led to recent advances in the regulation of these mechanisms. The following review outlines the substantial progress that has been made towards understanding environmental and developmental impacts on fish gill urea transport. This review also outlines the work that has been done regarding endocrine and neural control of urea excretion, most of which has been collected from only a handful of teleost fish. It is evident that more research is needed to establish the endocrine and neural control of urea excretion in fish, including fish representative of more ancient lineages (hagfish and lamprey), and elasmobranch fish.

SUMMARY In their native environment, gulf toadfish excrete equal quantities of ammonia and urea. However, upon exposure to stressful conditions in the laboratory (i.e. crowding, confinement or air exposure), toadfish decrease branchial ammonia excretion and become ureotelic. The objective of this study was to determine the influences of cortisol and ammonia on ammonia excretion relative to expression of Rhesus (Rh) glycoproteins and the ammonia-fixing enzyme, glutamine synthetase (GS). In vivo infusions and/or injections were used to manipulate corticosteroid activity and plasma ammonia concentrations in ureotelic toadfish. Metyrapone treatment to lower circulating cortisol levels resulted in a 3.5-fold elevation of ammonia excretion rates, enhanced mRNA expression of two of the toadfish Rh isoforms (Rhcg1 and Rhcg2), and decreased branchial and hepatic GS activity. Correspondingly, cortisol infusion decreased ammonia excretion 2.5-fold, a change that was accompanied by reduced branchial expression of all toadfish Rh isoforms (Rhag, Rhbg, Rhcg1 and Rhcg2) and a twofold increase in hepatic GS activity. In contrast, maintenance of high circulating ammonia levels by ammonia infusion enhanced ammonia excretion and Rh expression (Rhag, Rhbg and Rhcg2). Toadfish treated with cortisol showed an attenuated response to ammonia infusion with no change in Rh mRNA expression or GS activity. In summary, the evidence suggests that ammonia excretion in toadfish is modulated by cortisol-induced changes in both Rh glycoprotein expression and GS activity.

SUMMARY Models of branchial transport in teleosts have been reshaped by the recent discovery of Rhesus (Rh) glycoproteins, a family of proteins that facilitate the movement of NH3 across cell membranes. This study examines the effects of crowding and feeding on ammonia excretion in gulf toadfish (Opsanus beta) within the context of Rh glycoproteins and the ammonia-fixing enzyme, glutamine synthetase (GS). Four Rh isoforms (Rhag, Rhbg, Rhcg1 and Rhcg2) were isolated from toadfish. Tissue distributions showed higher levels of mRNA expression in the gills and liver, moderate levels in the intestine and lower levels in the stomach. Crowding significantly lowered branchial Rh expression and ammonia excretion rates in fasted toadfish. A comparison of Rh expression in the digestive tract revealed relatively low levels of Rhcg1 and Rhcg2 in the stomach and high mRNA abundance of Rhbg, Rhcg1 and Rhcg2 in the intestine of fasted, crowded toadfish. We speculate that these trends may reduce secretion and enhance absorption, respectively, to minimize the amount of ammonia that is lost through gastrointestinal routes. By contrast, these patterns of expression were modified in response to an exogenous ammonia load via feeding. Post-prandial ammonia excretion rates were elevated twofold, paralleled by similar increases in branchial Rhcg1 mRNA, gastric Rhcg1 mRNA and mRNA of all intestinal Rh isoforms. These changes were interpreted as an attempt to increase post-prandial ammonia excretion rates into the environment owing to a gradient created by elevated circulating ammonia concentrations and acidification of the digestive tract. Overall, we provide evidence that toadfish modulate both the expression of Rh isoforms and urea synthesis pathways to tightly control and regulate nitrogen excretion.

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.

SUMMARY The 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.

Although the number of fish species that have been studied for both hypoxia/anoxia tolerance and ammonia tolerance are few, there appears to be a correlation between the ability to survive these two insults. After establishing this correlation with examples from the literature, and after examining the role Peter Lutz played in catalyzing this convergent interest in two variables, this article explores potential mechanisms underpinning this correlation. We draw especially on the larger body of information for two human diseases with the same effected organ (brain), namely stroke and hepatic encephalopathy. While several dissimilarities exist between the responses of vertebrates to anoxia and hyperammonemia, one consistent observation in both conditions is an overactivation of NMDA receptors or glutamate neurotoxicity. We propose a glutamate excitotoxicity hypothesis to explain the correlation between ammonia and hypoxia resistance in fish. Furthermore, we suggest several experimental paths to test this hypothesis.

The neurochemical, serotonin (5-hydroxytryptamine; 5-HT) is involved in the regulation of toadfish pulsatile urea excretion as well as the teleost hypoxia response. Thus, the goal of this study was to determine whether environmental conditions that activate branchial chemoreceptors also trigger pulsatile urea excretion in toadfish, since environmental dissolved oxygen levels in a typical toadfish habitat show significant diel fluctuations, often reaching hypoxic conditions at dawn. Toadfish were fitted with arterial, venous and/or buccal catheters and were exposed to various environmental conditions, and/or injected with the O 2 chemoreceptor agonist NaCN or the 5-HT 2 receptor agonist α-methyl-5HT. Arterial PO 2 , as well as ammonia and urea excretion were monitored. Natural fluctuations in arterial PO 2 levels in toadfish did not correlate with the occurrence of a urea pulse. Chronic exposure (24 h) of toadfish to hyperoxia was without effect on nitrogen excretion, however, exposure to hypoxia caused a significant reduction in the frequency of urea pulses, and exposure to hypercapnia resulted in a reduction in the percentage of nitrogen waste excreted as urea. Of toadfish exposed acutely to hypoxia, 20% pulsed within 1 h, whereas none pulsed after normoxic or hypercapnic treatments. Furthermore, 20% of fish injected intravenously with NaCN pulsed within 1 h of injection, but no fish pulsed after injection of NaCN into the buccal cavity. To test whether environmental conditions affected 5-HT 2 receptors, toadfish were injected with α-methyl-5HT, which elicits urea pulses in toadfish. No significant differences in pulse size occurred among the various environmental treatments. Our findings suggest that neither the environmental conditions of hypoxia, hyperoxia or hypercapnia, nor direct branchial chemoreceptor activation by NaCN play a major role in the regulation of pulsatile urea excretion in toadfish.

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.

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 micro mol kg(-1)), a selective 5-HT(1A) receptor agonist, alpha-methyl-5-HT (1 micro mol kg(-1)), a 5-HT(2) receptor agonist, or ketanserin, a 5-HT(2) receptor antagonist (0.01, 0.1, 1 and 10 micro mol kg(-1)) plus alpha-methyl-5-HT. 8-OH-DPAT injection did not mediate an increase in urea excretion, ruling out the involvement of 5-HT(1A) receptors in pulsatile excretion. However, within 5 min, alpha-methyl-5-HT injection caused an increase in the excretion of urea in95% (N=27) of the fish injected, with an average pulse size of 652+/-102 micro mol N kg(-1) (N=26). With alpha-methyl-5-HT injection there was no corresponding increase in ammonia or [(3)H]PEG 4000 permeability. Urea pulses elicited by alpha-methyl-5-HT were inhibited in a dose-dependent fashion by the 5-HT(2) 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 alpha-methyl 5-HT injection had an effect on plasma cortisol or plasma urea concentrations. These findings suggest the involvement of a 5-HT(2)-like receptor in the regulation of pulsatile urea excretion.

The transport physiology of the urinary bladder of both the freshwater rainbow trout (Oncorhychus mykiss) and the marine gulf toadfish (Opsanus beta) was characterized with respect to urea, and the suitability of the urinary bladder as a model for renal urea handling was investigated. Through the use of the in vitro urinary bladder sac preparation urea handling was characterized under control conditions and in the presence of pharmacological agents traditionally used to characterize urea transport such as urea analogues (thiourea, acetamide), urea transport blockers (phloretin, amiloride), and hormonal stimulation (arginine vasotocin; AVT). Na+-dependence and temperature sensitivity were also investigated. Under control conditions, the in vitro trout bladder behaved as in vivo, demonstrating significant net reabsorption of Na+, Cl–, water, glucose, and urea. Bladder urea reabsorption was not affected by pharmacological agents and, in contrast to renal urea reabsorption, was not correlated to Na+. However, the trout bladder showed a threefold greater urea permeability compared to artificial lipid bilayers, a prolonged phase transition with a lowered Ea between 5°C and 14°C, and differential handling of urea and analogues, all suggesting the presence of a urea transport mechanism. The in vitro toadfish bladder did not behave as in vivo, showing significant net reabsorption of Na+ but not of Cl–, urea, or water. As in the trout bladder, pharmacological agents were ineffective. The toadfish bladder showed no differential transport of urea and analogues, consistent with a low permeability storage organ and intermittent urination. Our results, therefore, suggest the possibility of a urea transport mechanism in the urinary bladder of the rainbow trout but not the gulf toadfish. While the bladders may not be suitable models for renal urea handling, the habit of intermittent urination by ureotelic tetrapods and toadfish seems to have selected for a low permeability storage function in the urinary bladder. J. Exp. Zool. 292:604–617, 2002. © 2002 Wiley-Liss, Inc.

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.

Based on early pharmacological work, the serotonin 2A (5-HT 2A ) receptor subtype is believed to be involved in the regulation of toadfish pulsatile urea excretion. The goal of the following study was to characterize the toadfish 5-HT 2A receptor at a molecular level, to determine the tissues in which this receptor is predominantly expressed and to further investigate the pharmacological specificity of toadfish pulsatile urea excretion by examining the effect of ketanserin, a 5-HT 2A receptor antagonist, on resting rates of pulsatile urea excretion. The full-length toadfish 5-HT 2A receptor encodes a 496 amino acid sequence and shares 57–80% sequence identity to 5-HT 2A receptors of other organisms, with 100% conservation among important ligand-binding residues. Toadfish 5-HT 2A receptor mRNA expression was highest in the swim bladder and gonad, followed by the whole brain. All other tissues tested (esophagus, stomach, anterior intestine, posterior intestine, rectum, liver, kidney, heart, muscle and gill) had mRNA expression levels that were significantly less than whole brain. Toadfish 5-HT 2A receptor mRNA expression within the brain was highest in the hindbrain, telencephalon and midbrain/diencephalon regions. Treatment with the 5-HT 2A receptor antagonist, ketanserin, resulted in a significant decrease in the pulsatile component of spontaneous urea excretion due to a reduction in urea pulse size with no significant change in pulse frequency. These results lend further support for the 5-HT 2A receptor in the regulation of pulsatile urea excretion in toadfish.

Nitrogen excretion by the gulf toadfish (Opsanus beta) is of interest because of its high proportion of urea excretion compared with that of other teleosts. To better understand the factors influencing the timing of nitrogen excretion, the ratio of excreted urea∶ammonia, and the effector molecules regulating these processes, gulf toadfish were subjected to a series of experiments that moved them progressively from internal laboratory to outdoor mesocosm settings while assessing their behavior, nitrogen excretion patterns, levels of plasma hormones/effectors, and other parameters. In confined flux chambers in both laboratory and outdoor settings, toadfish nitrogen excretion was largely observed as urea pulses, with no apparent diel patterns to the pulses. Unrestrained toadfish in mesocosms exhibited distinctly nocturnal behavior, remaining exclusively in shelters during the day but taking several forays out into the mesocosm at night. In contrast to nitrogen excretion patterns in chambers, urea and ammonia were coexcreted in mesocosms and ratios for urea∶ammonia were very close to 1∶1 for both fed and fasted toadfish. The majority of measured excretion (and corresponding declines in plasma urea levels) occurred during two distinct periods of pulsing during daylight hours (0600-1000 and 1600-1800 hours). The declines in plasma urea associated with excretion were preceded by/coincided with declines in plasma cortisol. No day/night or hourly patterns in plasma serotonin (5-hydroxytryptamine [5-HT]) were observed, but there was a strong positive correlation among all samples between plasma urea and 5-HT. There was also a negative correlation between plasma cortisol and 5-HT. As expected for a nocturnally active species, plasma melatonin was significantly lower in daylight hours. A variety of enzyme activities (glutamine synthetase, glutaminase) and mRNA levels (glutamine synthetase, urea transporter, and Rhesus proteins) showed no significant variation over a diel cycle. Unlike prior laboratory studies, our results show that gulf toadfish in a natural setting have a distinctly diurnal pattern of nitrogen excretion and that ammonia and urea are coexcreted. The decline in plasma cortisol associated with urea pulses noted in prior laboratory studies was not as evident in the natural setting.

The acute cardiovascular and respiratory responses of the gulf toadfish, Opsanus beta, to acute hypoxia or exposure to the O(2) chemoreceptor stimulant, sodium cyanide (NaCN) were characterized and the role of serotonin type 2 (5-HT(2)) receptors in mediating these responses was investigated. Toadfish responded to hypoxia or NaCN exposure with a decrease in heart rate (fH) and an increase in breathing amplitude (V(AMP)) but no change in breathing frequency (fR). The bradycardia appeared to be mediated to some extent by 5-HT(2) receptors, as methysergide, a non-selective 5-HT(1/2) receptor antagonist, and ketanserin, a 5-HT(2) receptor antagonist, attenuated the response. Injection of alpha-methyl-5-HT, a 5-HT(2) agonist, also resulted in bradycardia that was inhibited by ketanserin, lending further support for 5-HT(2) receptor involvement, possibly 5-HT(2A) or 5-HT(2C), in the regulation of fH. External NaCN exposure resulted in a significant decrease in caudal arterial blood pressure (P(CA)) that was attenuated by methysergide. In contrast, injection with alpha-methyl-5-HT resulted in a substantial increase in P(CA) that was not affected by ketanserin, suggesting the possible involvement of 5-HT(2B) or 5-HT(2C) receptors. These data are the first to suggest a unique distribution of 5-HT(2B/2C) receptors may be involved in mediating vasoconstriction of the systemic vasculature of toadfish. These data also provide mechanistic support for why pulsatile urea excretion, believed to be regulated by 5-HT via the toadfish 5-HT(2A) receptor, is not triggered by hypoxia or external chemoreceptor activation.

SUMMARYGulf 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.

Gulf toadfish ( Opsanus beta) use a unique pulsatile urea excretion mechanism that allows urea to be voided in large pulses via the periodic insertion or activation of a branchial urea transporter. The precise cellular and subcellular location of the facilitated diffusion mechanism(s) remains unclear. An in vitro basolateral membrane vesicle (BLMV) preparation was used to test the hypothesis that urea movement across the gill basolateral membrane occurs through a cortisol-sensitive carrier-mediated mechanism. Toadfish BLMVs demonstrated two components of urea uptake: a linear element at high external urea concentrations, and a phloretin-sensitive saturable constituent ( Km= 0.24 mmol/l; Vmax= 6.95 μmol·mg protein−1·h−1) at low urea concentrations (10> 2, further suggestive of carrier-mediated processes. Our data provide evidence that a basolateral urea facilitated transporter accelerates the movement of urea between the plasma and gills to enable the pulsatile excretion of urea. Furthermore, in vivo infusion of cortisol caused a significant 4.3-fold reduction in BLMV urea transport capacity in lab-crowded fish, suggesting that cortisol inhibits the recruitment of urea transporters to the basolateral membrane, which may ultimately affect the size of the urea pulse event in gulf toadfish.

SUMMARYThe 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.

The goal of this study was to investigate the role of MgSO 4 in aglomerular kidney function, independent of changes in NaCl. The renal handling of MgSO 4 was manipulated by intravenous infusion of an isoosmotic solution containing 80 mmol/L MgSO 4 or through exposure to an environment that was reduced in MgSO 4 concentration by 90%. Intravenous infusion resulted in a transient increase in circulating Mg 2+ and SO 2- 4 levels; however, the concentration of both divalent ions in the urine remained elevated throughout the entire infusion period. Infusion also resulted in a transient increase in urine flow rate and apparent glomerular filtration rate, measured using the glomerular filtration rate marker, [ 3 H] PEG 4000. Exposure to MgSO 4 -depleted conditions resulted in a significant decrease in plasma and urine concentrations of Mg 2+ and in the urine concentrations of SO 2- 4 ; correspondingly, urine flow rate was significantly depressed. The urinary excretion of both Mg 2+ and SO 2- 4 demonstrated nonlinear saturation kinetics. The urinary excretion of Mg 2+ was significantly correlated with plasma Mg 2+ concentration (r = 0.75, P = 0.04) and yielded a Michealis constant (K m ) of 1.67±1.43 mmol/L; P = 0.26 and a maximal velocity (V max ) of 117.4±47.0 μmol/kg/ hr; P=0.046. The urinary excretion of SO 2- 4 was significantly correlated with plasma SO 2- 4 concentration (r = 0.94, P

The gulf toadfish, Opsanus beta, is a marine teleost fish with an aglomerular kidney that is highly specialized to conserve water. Despite this adaptation, toadfish have the ability to survive when in dilute hypoosmotic seawater environments. The objectives of this study were to determine the joint role of the kidney and intestine in maintaining osmotic and ionic balance and to investigate whether toadfish take advantage of their urea production ability and use urea as an osmolyte. Toadfish were gradually acclimated to different salinities (0.5, 2.5, 5, 10, 15, 22, 33, 50 and 70 ppt (1.5%, 7.5%, 15%, 30%, 45%, 67%, 100%, 151% and 212% seawater)) and muscle tissue, urine, blood and intestinal fluids were analyzed for ion and in some cases urea concentration. The renal and intestinal ionoregulatory processes of toadfish responded to changes in salinity and when gradually acclimated, toadfish maintain a relatively constant plasma osmolality at environmental salinities of 5 to 50 ppt. However, at salinities lower (2.5 ppt) or higher (70 ppt) than this range, a significant deviation from resting plasma and urine osmolality as well as changes in muscle water content was measured, suggesting osmoregulatory difficulties at these salinities. The renal system compensates for dilute seawater by reducing Na+ reabsorption by the bladder, which allowed excess water to be excreted. In the case of hypersalinity, Na+ reabsorption was increased, which resulted in a conservation of water and the concentration of Mg2+, Cl-, SO(4)2- and urea. A similar pattern was observed within the gastrointestinal system. Notably, Mg2+, HCO3- and SO4(2-) were the dominant ions in the intestinal fluid under control and hypersaline conditions due to the absorption of Na+, Cl- and water. When exposed to dilute seawater conditions, the absorption of Na+ was greatly reduced which likely increased water elimination. As a result of decreased environmental levels and a reduction in drinking rate, Mg2+ and SO4(2-) in intestinal fluids under hypoosmotic conditions were greatly reduced. While urea did play a minor role in renal osmoregulation, toadfish appear to preferentially regulate Na+ and to some extend Cl- in urine and intestinal fluids.

Opsanus beta expresses a full complement of ornithine-urea cycle (OUC) enzymes and is facultatively ureotelic, reducing ammonia-N excretion and maintaining urea-N excretion under conditions of crowding/confinement. The switch to ureotelism is keyed by a modest rise in cortisol associated with a substantial increase in cytosolic glutamine synthetase for trapping of ammonia-N and an upregulation of the capacity of the mitochondrial OUC to use glutamine-N. The entire day's urea-N production is excreted in 1 or 2 short-lasting pulses, which occur exclusively through the gills. The pulse event is not triggered by an internal urea-N threshold, is not due to pulsatile urea-N production, but reflects pulsatile activation of a specific branchial excretion mechanism that rapidly clears urea-N from the body fluids. A bidirectional facilitated diffusion transporter, with pharmacological similarity to the UT-A type transporters of the mammalian kidney, is activated in the gills, associated with an increased trafficking of dense-cored vesicles in the pavement cells. An 1814 kB cDNA ('tUT') coding for a 475-amino acid protein with approximately 62% homology to mammalian UT-A's has been cloned and facilitates phloretin-sensitive urea transport when expressed in Xenopus oocytes. tUT occurs only in gill tissue, but tUT mRNA levels do not change over the pulse cycle, suggesting that tUT regulation occurs at a level beyond mRNA. Circulating cortisol levels consistently decline prior to a pulse event and rise thereafter. When cortisol is experimentally clamped at high levels, natural pulse events are suppressed in size but not in frequency, an effect mediated through glucocorticoid receptors. The cortisol decline appears to be permissive, rather than the actual trigger of the pulse event. Fluctuations in circulating AVT levels do not correlate with pulses; and injections of AVT (at supraphysiological levels) elicit only minute urea-N pulses. However, circulating 5-hydroxytryptamine (5-HT) levels fluctuate considerably and physiological doses of 5-HT cause large urea-N pulse events. When the efferent cranial nerves to the gills are sectioned, natural urea pulse events persist, suggesting that direct motor output from the CNS to the gill is not the proximate control.

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); P0.05) 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); P0.001) 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.

The possible presence of urea transport mechanisms in the gill and kidney of the freshwater rainbow trout (Oncorhynchus mykiss) was investigated in vivo by comparing the branchial and renal handling of analogues acetamide and thiourea with the handling of urea. Trout were fitted with indwelling dorsal aortic catheters and urinary catheters and injected with an isosmotic dose of [(14)C]-labeled urea analogue (acetamide or thiourea) calculated to bring plasma analogue concentrations close to plasma urea concentrations. Urea and analogue concentrations were significantly greater in the urine than in the plasma. Branchial clearance rate of acetamide was only 48% of urea clearance, whereas the clearance of thiourea was only 22%, a pattern that was also observed in branchial uptake of these substances and was similar to our previous observations in toadfish and midshipmen. The renal secretion clearance rates of urea and acetamide were similar, and on average, both substances were secreted on a net basis, although reabsorption did occur in some cases. In contrast, thiourea was neither reabsorbed nor secreted by the kidney tubule. The secretion clearance rates of both acetamide and urea were well correlated with the secretion clearance rates of Na(+), Cl(-), and water, whereas there was no relationship between thiourea and these substances. The pattern of acetamide, thiourea, and urea handling by the gill of the trout is similar to that found in the gills of the midshipman and the gulf toadfish and strongly suggests the presence of a UT-type facilitated diffusion urea transport mechanism. The pattern of differential handling in the kidney is unlike that in the gill and also unlike that in the kidney of the midshipman and the gulf toadfish, suggesting a different mechanism. In addition, renal urea secretion occurs against a concentration gradient, suggesting the involvement of an active transport mechanism.

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