Mild hypoxia exposure impacts peripheral serotonin uptake and degradation in Gulf toadfish (Opsanus beta).

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.

Is serotonin uptake by peripheral tissues sensitive to hypoxia exposure?

In the Gulf toadfish (Opsanus beta), the serotonin (5-HT) transporter (SERT) is highly expressed in the heart, and the heart and gill both demonstrate the capacity for SERT-mediated uptake of 5-HT from the circulation. Because 5-HT is a potent vasoconstrictor in fish, we hypothesized that hypoxia exposure may increase 5-HT uptake by these tissues-and increase excretion of 5-HT-to prevent branchial vasoconstriction that would hamper gas exchange. Spot sampling of blood, bile, and urine revealed that fish exposed to chronic hypoxia (1.83 ± 0.12 mg·L-1 O2 for 24-26 h) had 41% lower plasma 5-HT in the ventral aorta (immediately following the heart) than in the hepatic vein (immediately before the heart), suggesting enhanced cardiac 5-HT uptake during hypoxia. 5-HT concentrations in the bile were greater than those in the urine, but there were no effects of acute (1.31 ± 0.06 mg·L-1 O2 for 25 min) or chronic hypoxia on 5-HT levels in these fluids. In 5-HT radiotracer experiments, the presence of tracer in the bile decreased upon hypoxia exposure, but, surprisingly, neither acute nor chronic hypoxia-induced changes in [3H]5-HT uptake in the heart, gill, or other tissues. Given the likely impact of the hypoxia exposure on metabolic rate, future studies should examine the effects of a milder hypoxia exposure on 5-HT uptake into these tissues and the role of 5-HT degradation.

The role of uptake and degradation in the regulation of peripheral serotonin dynamics in Gulf toadfish, Opsanus beta.

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.

The osmorespiratory compromise in the euryhaline killifish: water regulation during hypoxia.

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 MO2 initially declined by ∼50%, with partial restoration by 3 h of hypoxia, and no O2 debt repayment during recovery. Gill paracellular permeability (measured with [14C] 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 [14C] 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 3H2O) 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 MO2 , but nevertheless incurs a net water balance penalty in both freshwater and seawater.

Do reproductive hormones control Gulf toadfish pulsatile urea excretion?

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.

The serotonin transporter and nonselective transporters are involved in peripheral serotonin uptake in the Gulf toadfish, Opsanus beta.

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.

Molecular and functional characterization of the Gulf toadfish serotonin transporter SLC6A4.

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 Km value of 2.08±0.45 µmol l-1, similar to previously reported Km 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.

Extrinsic nerves are not involved in branchial 5-HT dynamics or pulsatile urea excretion in Gulf toadfish, Opsanus beta.

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 72h 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) gill neuroepithelial cells in response to hypoxia exposure.

Neuroepithelial cells (NECs) within the fish gill contain the monoamine neurochemical serotonin (5-HT), sense changes in the partial pressure of oxygen (PO2) in the surrounding water and blood, and initiate the cardiovascular and ventilatory responses to hypoxia. The distribution of neuroepithelial cells (NECs) within the gill is known for some fish species but not for the Gulf toadfish, Opsanus beta, a fish that has always been considered hypoxia tolerant. Furthermore, whether NEC size, number, or distribution changes after chronic exposure to hypoxia, has never been tested. We hypothesize that toadfish NECs will respond to hypoxia with an increase in NEC size, number, and a change in distribution. Juvenile toadfish (N = 24) were exposed to either normoxia (21.4 ± 0.0 kPa), mild hypoxia (10.2 ± 0.3 kPa), or severe hypoxia (3.1 ± 0.2 kPa) for 7 days and NEC size, number, and distribution for each O2 regime were measured. Under normoxic conditions, juvenile toadfish have similar NEC size, number, and distribution as other fish species with NECs along their filaments but not throughout the lamellae. The distribution of NECs did not change with hypoxia exposure. Mild hypoxia exposure had no effect on NEC size or number, but fish exposed to severe hypoxia had a higher NEC density (# per mm filament) compared to mild hypoxia-exposed fish. Fish exposed to severe hypoxia also had longer gill filament lengths that could not be explained by body weight. These results point to signs of phenotypic plasticity in these juvenile, lab-bred fish with no previous exposure to hypoxia and a strategy to deal with hypoxia exposure that differs in toadfish compared to other fish.

Immunohistochemical localization of urea and ammonia transporters in two confamilial fish species, the ureotelic gulf toadfish (Opsanus beta) and the ammoniotelic plainfin midshipman (Porichthys notatus).

This study aims to illustrate potential transport mechanisms behind the divergent approaches to nitrogen excretion seen in the ureotelic toadfish (Opsanus beta) and the ammoniotelic plainfin midshipman (Porichthys notatus). Specifically, we wish to confirm the expression of a urea transporter (UT), which is found in the gill of the toadfish and which is responsible for the unique "pulsing" nature of urea excretion and to localize the transporter within specific gill cells and at specific cellular locations. Additionally, the localization of ammonia transporters (Rhesus glycoproteins; Rhs) within the gill of both the toadfish and midshipman was explored. Toadfish UT (tUT) was found within Na(+)-K(+)-ATPase (NKA)-enriched cells, i.e., ionocytes (probably mitochondria-rich cells), especially along the basolateral membrane and potentially on the apical membrane. In contrast, midshipman UT (pnUT) immunoreactivity did not colocalize with NKA immunoreactivity and was not found along the filaments but instead within the lamellae. The cellular location of Rh proteins was also dissimilar between the two fish species. In toadfish gills, the Rh isoform Rhcg1 was expressed in both NKA-reactive cells and non-reactive cells, whereas Rhbg and Rhcg2 were only expressed in the latter. In contrast, Rhbg, Rhcg1 and Rhcg2 were expressed in both NKA-reactive and non-reactive cells of midshipman gills. In an additional transport epithelium, namely the intestine, the expression of both UTs and Rhs was similar between the two species, with only subtle differences being observed.

Pronounced in vivo hemoglobin polymerization in red blood cells of Gulf toadfish: a general role for hemoglobin aggregation in vertebrate hemoparasite defense?

Two human hemoglobin (Hb) variants, Hb C and Hb S, are known to protect against Plasmodium falciparum malaria and have evolved repeatedly in malaria endemic areas. Both aggregate to insoluble crystals (Hb C) or polymers (Hb S) under certain physiological conditions, impair parasite growth, and may facilitate retention of infected red blood cells (RBCs) in the spleen. Given the profound effects of parasites on host evolution in general, and that RBC Hb concentration is often close to its solubility limit throughout vertebrates, similar mechanisms may operate in nonhuman vertebrates. Here we show exercise-induced, profound in vivo Hb polymerization in RBCs of the Gulf toadfish. Hb aggregation was closely associated with the extent of plasma acidosis, fully reversible, and without any signs of hemolysis or anemia. Our literature analysis suggests that aggregation prone Hbs may be relatively old, evolved multiple times in nonhuman vertebrates, show enhanced aggregation during hemoparasite infections, and can be uncovered in vivo by splenectomy. We discuss the working hypothesis that widespread Hb aggregation within several vertebrate groups may be the result of ongoing or past selection pressure against RBC parasites. Further comparative studies of these evolutionary old systems may provide valuable insights into hemoparasite susceptibility and reservoir potential of livestock and companion animals but also into human malaria and sickle cell disease.

Cortisol-mediated downregulation of the serotonin 1A receptor subtype in the Gulf toadfish, Opsanus beta.

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.

A multi-targeted investigation of Deepwater Horizon crude oil exposure impacts on the marine teleost stress axis.

The toxicity of the polycyclic aromatic hydrocarbons (PAHs) in Deepwater Horizon (DWH) oil is well-established, but a knowledge gap exists regarding how this combination of PAHs affects the vertebrate stress axis. We hypothesized that (1) marine vertebrates exposed to DWH PAHs experience stress axis impairment, and co-exposure to an additional chronic stressor may exacerbate these effects, (2) serotonin (5-hydroxytryptamine; 5-HT) may act as a secondary cortisol secretagogue in DWH PAH-exposed fish to compensate for impairment, and (3) the mechanism of stress axis impairment may involve downregulation of cyclic adenosine monophosphate (cAMP; as proxy for melanocortin 2 receptor (MC2R) functionality), total cholesterol, and/or mRNA expression of CYP1A and steroidogenic proteins StAR, P450scc, and 11ß-h at the level of the kidney. We found that in vivo plasma cortisol and plasma adrenocorticotropic hormone (ACTH) concentrations in Gulf toadfish exposed to an environmentally relevant DWH PAH concentration (ΣPAH50= 4.6 ± 1.6 µg/L) for 7 days were not significantly different from controls, whether fish were chronically stressed or not. However, the rate of cortisol secretion by isolated kidneys after acute stimulation with ACTH was significantly lower in PAH-exposed toadfish compared to clean seawater (SW) controls. 5-HT does not appear to be acting as a secondary cortisol secretagogue, rather, PAH-exposed + stressed toadfish exhibited significantly lower plasma 5-HT concentrations than clean SW + stressed fish as well as a reduced sensitivity to 5-HT at the level of the kidney. There was a tendency for kidney cAMP concentrations to be lower in PAH-exposed fish (p = 0.069); however, mRNA expression of steroidogenic proteins between control and PAH-exposed toadfish were not significantly different and a significant elevation in total cholesterol concentration in PAH-exposed toadfish compared to controls was measured. Future work is needed to establish whether the slower cortisol secretion rate by isolated kidneys of PAH-exposed fish is detrimental, to determine the potential role of other secretagogues in compensating for the impaired kidney interrenal cell function, and to determine whether there is a reduction in MC2R mRNA expression or an impairment in the function of steroidogenic proteins.

Interactions between cortisol and Rhesus glycoprotein expression in ureogenic toadfish, Opsanus beta.

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.

Revisiting the effects of crowding and feeding in the gulf toadfish, Opsanus beta: the role of Rhesus glycoproteins in nitrogen metabolism and excretion.

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 NH(3) 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.

Elevated cortisol inhibits adrenocorticotropic hormone- and serotonin-stimulated cortisol secretion from the interrenal cells of the Gulf toadfish (Opsanus beta).

Stimulation of the toadfish 5-HT(1A) receptor by serotonin (5-hydroxytryptamine; 5-HT) or 8-OH-DPAT, a 5-HT(1A) receptor agonist, results in a significant elevation in plasma cortisol. Conversely, chronic elevation of plasma cortisol has been shown to decrease brain 5-HT(1A) receptor mRNA and protein levels via the glucocorticoid receptor (GR); however, there appears to be a disconnect between brain levels of the receptor and cortisol release. We hypothesized that elevated plasma cortisol would inhibit both adrenocorticotropic hormone (ACTH)- and 5-HT-stimulated cortisol release from the interrenal cells of Gulf toadfish, that ACTH sensitivity would not be GR-mediated and 5-HT-stimulated cortisol release would not be via the 5-HT(1A) receptor. To test these hypotheses, interrenal cells from uncrowded, crowded, vehicle-, and cortisol-implanted toadfish were incubated with either ACTH, 5-HT or 5-HT receptor agonists, and cortisol secretion was measured. Incubation with ACTH or 5-HT resulted in a stimulation of cortisol secretion in uncrowded toadfish. Cortisol secretion in response to ACTH was not affected in crowded fish; however, interrenal cells from cortisol-implanted toadfish secreted significantly less cortisol than controls, a response that was not reversed upon treatment with the GR antagonist RU486. 5-HT-stimulated cortisol release was significantly lower from both crowded and cortisol-implanted toadfish interrenal cells compared to controls. Incubation with either a 5-HT(4) or a 5-HT(2) receptor agonist significantly stimulated cortisol secretion; however, incubation with 8-OH-DPAT did not, suggesting that the 5-HT(1A) receptor is not a mediator of cortisol release at the level of the interrenal cells. Combined, these results explain in part the disconnect between brain 5-HT(1A) levels and cortisol secretion.

5-Hydroxytryptamine initiates pulsatile urea excretion from perfused gills of the gulf toadfish (Opsanus beta).

When stressed, toadfish become ureotelic and excrete almost all of their nitrogenous waste in 1-3 daily pulses of urea-N across the gills. Intravascular injections of 5-hydroxytyptamine (5-HT; serotonin) and analogues also elicit marked excretory pulses of urea-N from toadfish in vivo, suggesting that 5-HT release is the proximate trigger for spontaneous pulses. However it is unclear whether 5-HT is acting on the gills directly or elsewhere to cause the effect indirectly. A perfused whole gill preparation which maintained normal pressure relationships and stable vascular resistance was employed to address this question. Bolus injections into the ventral aortic perfusate of either 5-HT (1, 10 µmol kg(-1)) or the specific 5-HT(2) receptor agonist α-methyl 5-HT (1, 10 µmol kg(-1)) elicited rapid urea-N pulses from perfused toadfish gills. The effective doses, the post-injection delays (5.5 ± 1.3 min, range=2-22), the percent occurrences (57-85%), and the magnitude of the induced urea-N pulses (615.4 ± 131.3 µmol-N kg(-1), range 66.0-2634.0), were all similar to those previously reported when these agents were injected in vivo. Bolus injections of 5-HT and α-methyl 5-HT also elicited a biphasic response in ventral aortic pressure, reflecting an initial rapid short-lived vasodilation and a subsequent longer-lasting vasoconstriction. These events were similar to those which have been recorded to occur at a greater frequency during spontaneous urea-N pulsing in vivo. Neither the urea-N pulsing nor the cardiovascular responses to 5-HT were inhibited by the 5-HT(2A) receptor subtype blocker, ketanserin (pre-injection with 10 µmol kg(-1) plus 33 µmol L(-1) in the perfusate). Overall, these results provide strong support for the idea that the proximate stimulus for natural urea pulsing in vivo is 5-HT mobilization, acting directly in the gills.

The toadfish serotonin 2A (5-HT(2A)) receptor: molecular characterization and its potential role in urea excretion.

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.

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.

Exposure and Recovery of the Gulf Toadfish (Opsanus beta) to Weathered Deepwater Horizon Slick Oil: Impacts on Liver and Blood Endpoints.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants that can be responsible for a variety of deleterious effects on organisms. These adverse outcomes are relatively well studied, but at concentrations rarely found in the environment. Among the documented effects of sublethal acute PAH exposure are reductions in osmoregulatory capacity and immune function, and changes in the function of critical metabolic organs such as the liver. Gulf toadfish (Opsanus beta) were exposed to control seawater (0.006 µg tPAH50 /L) or water accommodated fractions of Deepwater Horizon spill oil diluted to 3 flow-through exposure regimes (0.009, 0.059, and 2.82 µg tPAH50 /L) for 7 d, with a recovery period of equal duration. We hypothesized that these chronic exposures would induce the aryl hydrocarbon receptor (AhR)-mediated pathways and result in significant impacts on markers of osmoregulatory, immune, and metabolic function. We further hypothesized that measurable reversal of these impacts would be observed during the recovery period. Our results indicate that activation of cytochrome P 450 (CYP)1A1 was achieved during exposure and reversed during the recovery phase. The only significant deviations from controls measured were a reduction in plasma glucose in fish exposed to medium and high levels of PAH after 7 d of exposure and a reduction in plasma osmolality fish exposed to high levels of PAHs after 7 d of recovery, when CYP1A1 messenger (m)RNA levels had returned to control levels. Our study illustrates a disconnect between the activation of CYP1A1 in response to environmentally realistic PAHs concentrations and several physiological endpoints and supports the idea that the AhR might not be associated with mediating osmoregulatory, immune, and metabolic changes in Gulf toadfish. Environ Toxicol Chem 2021;40:1075-1086. © 2020 SETAC.