Urea Pulse Research Articles

Nitrogen pulses increase fungal pathogens in Amazonian lowland tropical rain forests

Abstract Animals represent an overlooked source of horizontal redistribution of primary production and concentration of elements in ecosystems. For example, the high nutrient concentration of excretions by animals creates a mosaic of short‐term nutrient hotspots. However, how this impacts soil microbial communities, especially fungi, and in turn plant species diversity remains little known. This study quantified the temporal dynamics of soil mineral nitrogen (N) availability and its relationship with soil fungal community and functional group composition in simulated high‐N patches in an Amazonian rain forest. We hypothesised that (H1) changes in local resource dynamics would increase the abundance of pathotrophs and reduce that of saprotrophs and that, (H2) compared with previously reported bacterial community dynamics, fungi would be more resilient after a pulse disturbance event. A single urea pulse was applied and the relationship between fungal community composition and functional groups and soil N availability were determined before and twice after the urea treatment. An increase in mineral N availability and soil pH two months after the applied urea pulse was found to be associated with significant changes in fungal community composition and the abundance of functional groups. There was a notable decrease in the relative abundance of saprotrophs, accompanied by an increase in plant pathogenic fungi. Five months after the treatment, no differences were detected either in mineral N availability and soil pH or the composition of fungal communities and functional groups between the control and urea treatment. Synthesis. By locally favouring the abundance of plant pathogens, temporally short‐lived, but frequent high‐N patches created by animal excretions could potentially be involved in maintaining spatially and temporally variable soil microbial diversity and thus contributing to high plant community diversity in tropical rain forests as predicted by the Janzen–Connell hypothesis. The tendency of soil fungal communities in this study to return to their initial composition after 5 months suggests that they are resilient to perturbation by N pulse, and more so than previously observed in bacterial communities.

Fate of a 15N-labeled Urea Pulse in Heavily Fertilized Banana Crops

Banana crops in the Caribbean are characterized by the use of high rates of nitrogen (N) fertilization which causes severe environmental damages. The aim of this study was to assess the fertilizer N use efficiency (NUE) of banana crops in the field. To do so, a field trial was carried out during the first (GS1) and the fourth (GS4) growing seasons of banana crops, and the fate of a 15N-labeled pulse applied late in the growing season (flowering stage) was determined. At harvest, NUE (average 24% 15N applied) and the total recovery of fertilizer 15N in the soil–plant system (i.e., 40% in GS1 and 62% in GS4) were low. Low NUE resulted mainly from the dilution in a large soil mineral N pool derived from earlier applications of the labeled-N fertilizer applied at flowering, combined with leaching caused by numerous high-intensity rainfall events (>20 mm d−1). Crop residues from previous cycles present at time of fertilizer application in the fourth growing season, promoted fertilizer N immobilization, which in turn favored fertilizer N recovery by decreasing N leaching. The results suggest that N fertilization after the first season could be reduced by 30% (i.e., −90 kg N ha−1) corresponding to the suppression of two applications from flowering to harvest with the current fertilizer management, as available N derived from earlier applications is sufficient to meet plant requirements.

Usefulness of different prognostic scores for AECOPD: APACHE II, BAP65, 2008, and CAPS scores

PurposeThe purpose of this study was to compare four different scores [Acute Physiology and Chronic Health Evaluation (APACHE II); elevated blood urea nitrogen, altered mental status, pulse >109/min, age >65 years (BAP65); chronic obstructive pulmonary disease (COPD) and Asthma Physiology Score (CAPS); and 2008 score) to test their predictive properties for the need of mechanical ventilation (MV) and short-term mortality in patients with acute exacerbation COPD (AECOPD).Patients and methodsThis study enrolled 100 consecutive patients with acute exacerbation COPD, over a 6-month duration, admitted to the Emergency Department in Alexandria Main University Hospitals. The four scores were calculated for each patient, and clinical data and outcome (need for MV and mortality during hospitalization or within a week after discharge) were recorded.ResultsTheir mean age was 61.1±10.7 years, and 88% were males. Duration of hospital stay was less than or equal to 20 days in 67%. Mortality rate was 4%. Overall, 40% required MV. Blood urea nitrogen, pulse, CO2, pH, altered consciousness, and white blood cell were significant predictors of mortality in univariate but not multivariate analysis. Previous MV, cyanosis, and paradoxical abdominal movement were significant predictors of need for MV. The highest area under the receiver operating characteristic curve was that of APACHE II score regarding either mortality prediction [area under the curve (AUC), 0.982; P=0.001] or need for MV (AUC, 0.959; P<0.001), followed by BAP65 score for mortality prediction (AUC, 0.967; P=0.002) and 2008 score for predicting the need for MV (AUC, 0.851; P<0.001).ConclusionAll studied scores correlated significantly with mortality, but only APACHE II and 2008 score correlated significantly with the need for MV. The highest area under the receiver operating characteristic curve was that of APACHE II score regarding either mortality or need for MV prediction. Previous need for MV was the most important predictor for the need for MV. The routine use of these practical scores in triage of patients may direct early interventions to reduce mortality rate.

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.

Pulsatile urea excretion in Gulf toadfish: the role of circulating serotonin and additional 5-HT receptor subtypes.

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% 1h 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.

PO-107 Applied Research on Heart Rate Variability in Monitoring Sports Fatigue of Boxing Athletes

Objective Based on the diagnosis of sports fatigue using physiological and biochemical indicators, to detect the changes of heart rate variability (HRV) index before and after heavy load training in boxing athletes, and observe the effect of heavy load training on cardiac autonomic nerves. The purpose of this study was to investigate the application of HRV to monitor boxing athletes’ sports fatigue.&#x0D; Methods 16 athletes from Shanghai men's boxing team were recruited. The coach organized a 4-week heavy load training, on Monday morning before and after heavy load training, to evaluate whether athletes have exercise fatigue by testing white blood cell (WBC), red blood cell (RBC), hemoglobin (Hb), blood testosterone (T), cortisol (C), testosterone/cortisol ratio (T/C), creatine kinase (CK), blood urea (BU) and morning pulse. Heart rate variability (HRV) indicators were detected simultaneously. The data were analyzed by SPSS 19.0 statistical software. Pearson correlation analysis was used to compare the correlation between HRV and physiological and biochemical indexes. The paired sample T test was used to compare the differences between the indicators, P&lt;0.05, P&lt;0.01 was statistically significant.&#x0D; Results After heavy load training, when compared with indexes before heavy load training, T and T/C ratios decreased significantly (-38%, -52.7%, p&lt;0.01), C and morning pulse increased significantly (+32.4%, +20.4%, p&lt;0.05), BU and CK had an increasing trend but no statistical significance (+16.5%, +52.7%, p&gt;0.05), while WBC, RBC and Hb showed no statistical significance (p&gt;0.05), these changes in physiological and biochemical indexes can diagnose sports fatigue of boxing athletes after heavy load training. SDNN of HRV index was significantly correlated with morning pulse (p&lt;0.05), RMSSD was significantly correlated with CK (p&lt;0.05), LF was significantly correlated with Hb (p&lt;0.05), and LF/HF was significantly correlated with T, C, T/C, morning pulse, CK (p&lt;0.05). After heavy load training, LF and LF/HF of HRV index in boxing athletes were significantly increased than that before heavy load training (1744.7±1526.3 ms2 vs. 1134.5±1003.3 ms2, 2.5±1.3 vs. 1.6±1.0, p&lt;0.05), the other HRV indexes showed no statistical significance (p&gt;0.05).&#x0D; Conclusions The LF and LF/HF changed significantly when boxing athletes appeared sports fatigue, suggesting that the sympathetic nervous system had enhanced activity and increased tension, the imbalance between Sympathetic and parasympathetic tend to predominate in sympathetic activity. LF and LF/HF are sensitive HRV indicators for monitoring sports fatigue in boxing athletes.

Of mammals and bacteria in a rainforest: Temporal dynamics of soil bacteria in response to simulated N pulse from mammalian urine

Abstract Pulse‐type perturbation through excreta by animals creates a mosaic of short‐term high nutrient‐load patches in the soil. How this affects microbial community composition and how long these impacts last are important for microbial community dynamics and nutrient cycling. Our study focused on the short‐term responses to N by bacterial communities and ‘functional groups’ associated with the N cycle in a lowland evergreen tropical rainforest. We applied a single urea pulse, equivalent to urine‐N deposition by medium‐sized mammals to simulate N enrichment and changes in soil N availability, and analysed soil bacterial communities using molecular methods, before and after urea application. Urea addition increased mineral N availability and changed bacterial community composition, from phylum to operational taxonomic unit levels, however, taxon richness and diversity were unaffected. Taxa involved in the physiologically “narrow” processes of nitrification (e.g. Nitrosospira) and denitrification (e.g. Phyllobacteriaceae, Xanthomonadaceae and Comamonadaceae) increased their relative abundance, while N2‐fixers (e.g. Rhodospirillales, and Rhizobiales) decreased after treatment. While a temporal legacy on both community composition and functional group profile was observable 58 and 159 days after treatment, at the latter date bacterial communities were already tending towards pre‐treatment composition. We suggest that pulse‐type perturbation by mammal urine that occurs on a daily basis has strong short‐term effects on patch dynamics of soil microbiota and N availability. Such a spatio‐temporally dynamic soil environment enhances overall microbial richness and diversity, and contributes to the apparent temporal resilience of community composition. A plain language summary is available for this article.

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.

A waterborne chemical cue from Gulf toadfish, Opsanus beta, prompts pulsatile urea excretion in conspecifics

The Gulf toadfish (Opsanus beta) has a fully functional ornithine urea cycle (O-UC) that allows it to excrete nitrogenous waste in the form of urea. Interestingly, urea is excreted in a pulse across the gill that lasts 1–3h and occurs once or twice a day. Both the stress hormone, cortisol, and the neurotransmitter, serotonin (5-HT) are involved in the control of pulsatile urea excretion. This and other evidence suggests that urea pulsing may be linked to toadfish social behavior. The hypothesis of the present study was that toadfish urea pulses can be triggered by waterborne chemical cues from conspecifics. Our findings indicate that exposure to seawater that held a donor conspecific for up to 48h (pre-conditioned seawater; PC-SW) induced a urea pulse within 7h in naïve conspecifics compared to a pulse latency of 20h when exposed to seawater alone. Factors such as PC-SW intensity and donor body mass influenced the pulse latency response of naïve conspecifics. Fractionation and heat treatment of PC-SW to narrow possible signal candidates revealed that the active chemical was both water-soluble and heat-stable. Fish exposed to urea, cortisol or 5-HT in seawater did not have a pulse latency that was significantly different than seawater alone; however, ammonia, perhaps in the form of NH4Cl, was found to be a factor in the pulse latency response of toadfish to PC-SW and could be one component of a multi-component cue used for chemical communication in toadfish. Further studies are needed to fully identify the chemical cue as well as determine its adaptive significance in this marine teleost fish.

Therapeutic effect evaluation on the treatment of acute upper gastrointestinal bleeding with freezing Zidi mixture lavage

Objective To observe the clinical effects of treating acute upper gastrointestinal bleeding（AUGIB） with freezing Zidi mixture lavage. Method 183 UGIB patients from the First Affiliated Hospital to Guanzhou University of TCM in August 2013 were randomly recruited into a control group with 91cases and a treatment group with 92 cases. The treatment group was treated with intragastric administration of freezing Zidi mixture, while the control group was treated with intragastric administration of cryohydrate and norepinephrine. The changes of clinical effects, blood pressure, hemoglobin, blood urea nitrogen, pulse and dark stool were observed and the bad emotions as anxiety, depression and fear were evaluated. Results ① On therapeutic effect, the total effective rate was 96.7% and 85.7% in the treatment group and the control group respectively, with statistical difference（χ^2=2.943,P〈0.05）. ② On the time of symptom disappearance：the disappearing time of vomiting, dark stools and occult blood test in the treatment group was obviously shorter than the control group after the treatment（t=6.628、14.052、21.706,P〈0.05 or 0.01）. ③ On body symptom changes： after the treatment, the systolic pressure, heart rate, hemoglobin, and blood urea nitrogen [（114.3±12.1）mmHg,（78.6±11.4）/min,（98.1±10.5）g/L,（5.9±1.1）mmol/L] in the treatment group were all obviously improved than those [（106.3±11.2）mmHg, （88.7±12.1）/min,（88.2±11.7）g/L,（6.9±1.4）mmol/L] in the control group;body temperature returned to normal in both groups, and there was no statistical difference score was either in or between groups（P〉0.05）. ④ On changes of anxiety, depression and fear：the treatment group [the（42.5±9.4）, （41.6±9.7）, （26.3±5.4）for each]was obviously improved after the treatment than the control group[the score was（51.2±10.1）,（50.4±11.5）,（30.4±6.9）for each],P〈0.05. Conclusion Freezing Zidi mixture lavage can obviously elevate systolic pressure, decrease heart rate, enhance hemoglobin, decrease blood urea nitrogen, relieve body stress reaction and other clinical symptoms. Its effects were better than intragastric administration of cryohydrate and norepinephrine in treating AUGIB. Key words: Freezing Zidi mixture; AUGIB; Intragastric administration

The toadfish serotonin 2A (5-HT2A) receptor: molecular characterization and its potential role in urea excretion

Based on early pharmacological work, the serotonin 2A (5-HT2A) 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-HT2A 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-HT2A receptor antagonist, on resting rates of pulsatile urea excretion. The full-length toadfish 5-HT2A receptor encodes a 496 amino acid sequence and shares 57–80% sequence identity to 5-HT2A receptors of other organisms, with 100% conservation among important ligand-binding residues. Toadfish 5-HT2A 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-HT2A receptor mRNA expression within the brain was highest in the hindbrain, telencephalon and midbrain/diencephalon regions. Treatment with the 5-HT2A 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-HT2A receptor in the regulation of pulsatile urea excretion in toadfish.

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

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

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

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

Cortisol-sensitive urea transport across the gill basolateral membrane of the gulf toadfish (Opsanus beta)

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 (K(m) = 0.24 mmol/l; V(max) = 6.95 micromol x mg protein(-1) x h(-1)) at low urea concentrations (<1 mmol/l). BLMV urea transport in toadfish was unaffected by in vitro treatment with ouabain, N-ethylmaleimide, or the absence of sodium, conditions that are known to inhibit sodium-coupled and proton-coupled urea transport in vertebrates. Transport kinetics were temperature sensitive with a Q(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.

Is urea pulsing in toadfish related to environmental O2 or CO2 levels?

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 alpha-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 alpha-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.

Glucocorticoid receptors are involved in the regulation of pulsatile urea excretion in toadfish

The objectives of this study were to characterize the pattern of pulsatile urea excretion in the gulf toadfish in the wake of exogenous cortisol loading and to determine the receptors involved in the regulation of this mechanism. Toadfish were fitted with indwelling arterial catheters and were infused with isosmotic NaCl for 48 h after which fish were treated with cortisol alone, cortisol + peanut oil, cortisol + RU486 (a glucocorticoid receptor antagonist) or cortisol + spironolactone (a mineralocorticoid receptor antagonist). Upon cortisol loading, fish treated with cortisol alone, cortisol + oil or cortisol + spironolactone experienced a two- to threefold reduction in pulsatile urea excretion. This reduction was due to a decrease in urea pulse size with no effect on pulse frequency compared to values measured during the control NaCl infusion period. In addition, these fish showed an increase in plasma urea concentrations upon treatment. These apparent effects of cortisol treatment were abolished in fish treated with cortisol + RU486. In contrast, these fish showed an increase in pulsatile urea excretion mediated by a twofold increase in pulse size with no change in frequency. Likewise, fish treated with cortisol + RU486 showed a significant decrease in plasma urea concentrations over the course of the experiment. The findings of this study indicate that high levels of cortisol reduce pulsatile urea excretion by decreasing pulse size. In addition, it appears that glucocorticoid receptors and not mineralocorticoid receptors are involved in the regulation of the toadfish pulsatile urea excretion mechanism.

Molecular characterization of a urea transporter in the gill of the gulf toadfish (Opsanus beta).

Urea excretion by the gulf toadfish (Opsanus beta) has been shown in previous studies to be a highly pulsatile facilitated transport, with excretion probably occurring at the gill. The present study reports the isolation of an 1800 base pair (kb) cDNA from toadfish gill with one open reading frame putatively encoding a 475-residue protein, the toadfish urea transporter (tUT). tUT, the first teleostean urea transporter cloned, has high homology with UTs (facilitated urea transporters) cloned from mammals, an amphibian and a shark, and most closely resembles the UT-A subfamily. When expressed in Xenopus laevis oocytes, tUT increased urea permeability (as measured by [(14)C]urea uptake) five- to sevenfold, and this permeability increase was abolished by phloretin, a common inhibitor of other UTs. Northern analysis using the 1.8 kb clone was performed to determine the tissue distribution and dynamics of tUT mRNA expression. Of six tissues examined (gill, liver, red blood cells, kidney, skin and intestine), only gill showed expression of tUT mRNA, with a predominant band at 1.8 kb and a minor band at 3.5 kb. During several points in the urea pulse cycle of toadfish (0, 4, 6, 12 and 18 h post-pulse), measured by excretion of [(14)C]urea into the water, gill mRNA samples were obtained. Expression of tUT mRNA was found to be largely invariant relative to expression of beta-actin mRNA over the pulse cycle. These results further confirm the gill localization of urea transport in the toadfish and suggest that tUT regulation (and the regulation of pulsatile urea excretion) is probably not at the level of mRNA control. The results are discussed in the context of the mechanisms of vasopressin-regulated UT-A in mammalian kidney and morphological data for the toadfish gill.

Urea and water permeability in the ureotelic gulf toadfish (Opsanus beta).

The permeability of toadfish gills and skin to urea and water has been measured in order to investigate the mechanisms behind the pulsatile excretion of urea previously described in this species. A perfused gill preparation was used in the gill studies and isolated pieces of skin mounted in an Ussing chamber in the skin studies. Simultaneously, urea and water permeability was measured in vivo in free swimming fish. In vivo the nonpulsing urea permeability was exceptionally low compared to other teleosts, while the tritiated water permeability was similar to that of other teleosts. The urea permeability increased 30-fold during a pulse while water permeability stayed unaffected. Compared to in vivo, tritiated water permeability was approximately 50% lower in the gills and the skin when measured directly in the isolated preparations. The urea permeability was almost identical between the three preparations. Four out of 20 perfused gill preparation showed a spontaneous urea pulse during perfusion. Several treatments were tested to elicit the pulse artificially but without success. Hormones and drugs tested were: arginine-vasotocin (AVT), 10(-10) M; adrenaline, 10(-7) M; isoprenaline, 10(-5) M; acetylcholine, 10(-7) and 10(-6) M; serotonin, 10(-7) and 10(-6) M; adenosine, 10(-6) M; cortisol, 10(-7) M; and combinations of AVT, adrenaline, and cortisol. Adrenaline and isoprenaline increased tritiated water permeability without affecting urea permeability. Gradually increasing the ammonia levels in the perfusate from 0.1 mM to 1.6 mM caused a slight increase in water permeability but a marked and progressive increase in urea permeability. No indications of an ammonia trapping mechanism in the gills were found. There was no effect of AVT (10(-10) mol l-1) in the urea permeability of the skin preparation while cortisol (10(-7) M) led to a modest increase in urea permeability. Based on a comparison between the in vivo and in vitro preparations used here, we conclude that the urea pulse in a urea-pulsing toadfish occurs through the gills and not the skin. We still do not know which internal mechanism or signal triggers the urea pulse in the toadfish.