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First published online June 29, 2006
Journal of Experimental Biology 209, 2704-2712 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02289
Metabolic and neuroendocrine effects on diurnal urea excretion in the mangrove killifish Rivulus marmoratus
Department of Integrative Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada
* Address for correspondence e-mail: patwrigh{at}uoguelph.ca
Accepted 25 April 2006
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Summary |
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Key words: nitrogen excretion, ammonia excretion, oxygen consumption, serotonin, 5-HT, ketanserin, cortisol, RU-486, killifish, Rivulus marmoratus
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Introduction |
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;
Kaushik and de Oliva-Teles,
1985; Alsop and Wood,
1997). In fed individuals, concentrations of nitrogen end products
in plasma increase within hours after a meal, followed by a washout period
typified by increased rates of excretion
(Brett and Zala, 1975;
Kaushik and Gomes, 1988;
Kaushik and de Oliva-Teles,
1985; Wicks and Randall,
2002). In contrast to most teleosts, nitrogen excretion in the
ammoniotelic Rivulus marmoratus demonstrates a most unusual pattern.
Urea (Jurea) but not ammonia excretion
(Jamm) in R. marmoratus undergoes a diurnal
rhythmicity, with the highest rates of urea excretion occurring during
mid-afternoon (Frick and Wright,
2002a; Rodela and Wright,
2006). Under the influence of circadian clocks, the phase of the
Jurea cycle was entrained fairly quickly to photic
zeitgebers and possibly feeding cues.
One of the aims of the present study was to determine if the diurnal
Jurea pattern in R. marmoratus was related to
diurnal changes in metabolism. The ammoniotelic gobiid fish Mugilogobius
abei also has a distinct daily pattern of Jurea under
light:dark cycles (Kajimura et al.,
2002). The authors postulated that the rate of urea synthesis may
vary in a similar diurnal pattern; however, rates of oxygen consumption were
not measured. An alternative possibility is that the rhythmic pattern of
Jurea in R. marmoratus is under the control of
mechanisms affecting urea permeability across cell membranes.
Urea permeability across lipid membranes is significantly lower than that
of ammonia (Collander, 1937;
Galluci et al., 1971) and
recent studies have shown the presence of urea transporters (UT) on the
branchial epithelium of many fish species
(Smith and Wright, 1999;
Walsh et al., 2000;
Walsh et al., 2001;
Mistry et al., 2001;
McDonald et al., 2002;
McDonald et al., 2004).
Pulsatile Jurea patterns in marine toadfish Opsanus
beta are attributed to periodic activation of a facilitated branchial UT,
rather than to changes in urea production pathways
(Wood et al., 1997;
Wood et al., 2003). Recent
studies have revealed that the glucocorticoid hormone, cortisol and the
monoamine, 5-hydroxytryptamine (5-HT; serotonin) may regulate pulse size and
frequency, respectively (Wood et al.,
1997; Wood et al.,
1998; Wood et al.,
2003; McDonald et al.,
2004).
Similar control mechanisms may be regulating Jurea in
R. marmoratus. Both 5-HT and cortisol are appealing candidates for
neuroendocrine mediators of diurnal urea excretion as these hormones undergo
daily rhythms under the influence of a light:dark photoperiod in other
species. For instance, circulating cortisol levels reach peak concentrations
at or near the onset of light, immediately prior to the initiation of daily
locomotor activity in several fishes
(Peter et al., 1978;
White and Fletcher, 1984;
Fivizzani et al., 1984). Daily
fluctuations in brain 5-HT levels have been documented in Anguilla
anguilla (van Veen et al.,
1982) and O. mykiss
(Zuanreiter et al., 1998).
The mangrove killifish R. marmoratus are remarkably hardy fish
that tolerate a range of environmental extremes
(Abel et al., 1987;
King et al., 1989;
Frick and Wright, 2002a;
Frick and Wright, 2002b). In
this study we hypothesized that the daily Jurea pattern in
R. marmoratus is due to diurnal changes in metabolic rate, which may
in turn alter blood to water urea gradients and ultimately, excretion rates.
Tissue concentrations of urea and ammonia, urea excretion rates, as well as
whole animal oxygen consumption rates were measured over time. A second
hypothesis, that the rate of urea excretion is influenced by neuroendocrine
messengers was also tested. We first tested the prediction that RU-486, a
cortisol receptor antagonist, will cause a dose-dependent increase in urea,
but not ammonia, excretion rates. Second, exposure to ketanserin, a
5-HT2 receptor antagonist, will cause a dose-dependent decrease in
urea excretion rates, but not ammonia excretion rates. Furthermore, we
predicted that upon exposure to RU-486 or ketanserin, the amplitude of the
diurnal Jurea pattern would be increased and decreased,
respectively, based on previous studies in the toadfish
(McDonald et al., 2004;
McDonald and Walsh, 2004).
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Materials and methods |
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). The
fish were held in an environmental chamber at 25°C on a photoperiod of 12
h:12 h light (L):dark (D). Mature fish (0.07-0.25 g) were kept in individual
translucent containers in 60 ml of 15
artificial seawater (pH 8.1),
which was changed biweekly. Artificial seawater was made from distilled water
and marine salt (Instant OceanTM, Crystal Sea, Baltimore, MA, USA). Fish
were fed Artermia salina nauplii every other day. To eliminate
effects of recent feeding on nitrogen metabolism and excretion, fish were
deprived of food 48 h prior to the initiation of an experiment.
Experimental protocol
Metabolic experiments
Four series of experiments were conducted:
Series I: measurements of tissue urea and ammonia levels over 1.5 days.
Series II: quantification of whole animal oxygen consumption rates over a 3 day period.
Series III: detailed examination of hourly oxygen consumption rates over a 6 h period.
Series IV: hourly nitrogen excretion rates in fasted R. marmoratus over a 6 h period.
Series I
In order to determine if changes in the diurnal Jurea
pattern were due to metabolic changes (i.e. changes in nitrogen production and
storage levels), tissue urea and ammonia concentrations were measured. Fish
were killed by flash freezing in liquid nitrogen every 3 h during a 1.5-day
period. Samples were stored at -80°C for up to 2 months prior to analysis
of urea and ammonia content.
Series II and III
Oxygen consumption rates were measured in R. marmoratus using
closed respirometry techniques. A single fish was placed in approximately 60
ml of brackish water in the respiratory chamber. Fish were acclimated to the
respirometry chambers 2 days prior to the start of the experiment (25°C).
Additional blank chambers (not containing a fish) were run simultaneously to
determine the rate of oxygen consumed by the oxygen probe. The background
values were subtracted from the oxygen consumption values of the fish, but
were minimal (<10.28% of average fish value). At the onset of the
measurements, the chamber was sealed for 1 h, during which changes in oxygen
levels were measured in mg l-1. During the measurement, oxygen
levels did not fall below 70% saturation in order to ensure that the values
obtained were a true representation of normoxic metabolic status in the
animal. At the end of the sampling period, the chambers were renewed with
fresh aerated water (15) for a period of 5 h to allow maximum oxygen
saturation levels before the start of the next sampling period. In Series II,
experiments involved measuring the change in water oxygen for 1 h intervals
every 5 h for 3 days. For Series III, experiments entailed measuring oxygen
consumption for every hour between 12:00 h and 18:00 h. At the end of each
series of experiments, the fish were removed from their chambers, blotted dry
and weighed. Values are presented as µmol O2 g-1
h-1.
Series IV
In order to determine correspondence between Jurea and
oxygen consumption, Jurea was measured for each 1 h
interval between 12:00 h and 18:00 h. Fish were held in individual containers
with 30 ml of 15 artificial seawater (pH 8.1, 25°C, 12 h:12 h L:D)
and water samples were collected every hour for a 1.5 day period. The water
was changed after each sampling interval to prevent accumulation of
nitrogenous wastes. Elimination of handling stress was accomplished through
the use of double-walled containers, with an inner mesh container made in the
exact same configuration as the plastic translucent holding container.
Consequently, during water changes, the inner mesh container with the fish was
removed and placed in a new outer chamber containing fresh brackish water.
Fish were acclimated to the containers 2 days prior to the start of the
experiment. Water samples were frozen at -20°C for up to 1 month and later
analyzed for urea and ammonia content.
Pharmacological experiments
Three series of experiment were conducted:
Series V: the dose effects of ketanserin and RU-486 on nitrogen excretion.
Series VI: the effects of a single application of ketanserin and RU-486 on nitrogen excretion.
Series VII: the effects of ketanserin and RU-486 on diurnal Jurea.
Series V
Pharmacological agents were used to determine if the diurnal pattern of
Jurea is controlled, in part, by either cortisol or 5-HT.
Specifically, RU-486, a cortisol receptor inhibitor, and ketanserin, a
5-HT2 receptor antagonist, were used. Initial experiments involved
testing the effect of each drug on Jurea. Concentrations
were chosen based on similar studies on other fish species
(Brustein et al., 2003;
Dasmahapatra and Lee, 1993;
Sathiyaa and Vijayan, 2003).
For ketanserin, three different treatment groups of R. marmoratus
were exposed to concentrations of 10, 30 or 50 µmol l-1.
Initially, identical concentrations of RU-486 and ketanserin were used;
however, there was a 80% or higher mortality rate associated with these
concentrations. Subsequently lower concentrations of RU-486 consisting of 1, 2
or 5 µmol l-1 were used. Both receptor antagonists required an
ethanol vehicle (0.0003% w/v), consequently control fish were exposed to
identical concentrations of the vehicle in the absence of the antagonist.
Final water concentrations of ethanol in did not exceed 0.83%. Each drug (and
ethanol control) was added to 30 ml of water (15). Initial water
samples were collected following the addition of the drugs and final water
samples were taken 6 h later, and handled as described for Series IV. Due to
the small size of the fish (<250 mg), it was not possible to measure the
plasma concentration of 5-HT and cortisol.
Series VI
Experiments involved following nitrogen excretion rates over time during
and after a 6 h exposure to 30 µmol l-1 ketanserin or 1 µmol
l-1 RU-486. Initial experiments involved measuring both
Jurea and Jamm over 6 h intervals
under control conditions for a 24 h period.
As previously described, the control treatment contained ethanol (final
concentration less than 0.83%). A single dose of either 30 µmol
l-1 ketanserin or 1 µmol l-1 RU-486 was administered
at 12:00 h. Following a 6 h exposure to the receptor antagonists (12:00
h-18:00 h), freshwater (15) with ethanol (0.83%) was replaced in each
chamber every 6 h period for the remainder of the experiment in order to
follow the recovery phase. Water samples were taken every 6 h and were treated
as described in Series IV.
Series VII
The final series of experiments involved measuring the diurnal excretion
pattern when the fish were either exposed to ketanserin or RU-486. Control
excretion rates were measured for the first 30 h and following this period the
fish was exposed to 30 µmol l-1 ketanserin or 1 µmol
l-1 RU-486 every 6 h for the next 54 h. Water samples from each
experiment were frozen at -20°C for up to 1 month and later analyzed for
urea and ammonia content.
Analytical techniques and calculation
Ammonia and urea
Extracts were prepared by grinding the intact fish to a fine powder with a
mortar and pestle in liquid nitrogen
(Wright et al., 1995). Urea
concentrations were analyzed as described previously
(Rahmatullah and Boyde, 1980)
and values were expressed as mmol N l-1. Samples were analyzed for
ammonia content using a modified published method
(Kun and Kearney, 1974) for
use with a SpectraMax 190 micoplate reader (Molecular Devices Corp.,
Sunnyvale, CA, USA). The efficiency of the assay was determined by spiking
samples with known amounts of ammonia and determining the percent recovery
(98.5±1.3% recovery rate). Values for ammonia concentration in the
tissues are expressed in mmol N l-1.
Seawater urea concentrations were measured with a colorimetric assay
(Rahmatullah and Boyde, 1980)
using an Ultrospec 3300 Pro spectrophotometer (Biochrom, Cambridge,
UK). Ammonia content of water samples were quantified by assay methods
described elsewhere (Ivancic and Degobbis,
1984). The rates of excretion (J) were calculated as
described previously (Wright and Wood,
1985).
Control experiments were carried out to determine if bacterial contamination from various sources such as the individual fish, the water supply or attached to the experimental chamber could have affected nitrogen excretion rates. Fish were placed in 30 ml of water for a 1 h interval and subsequently removed. Ammonia and urea concentrations were monitored in the water during the next 6 h. Analysis revealed that microbial contamination did not significantly affect nitrogen excretion rates from R. marmoratus (P>0.05).
Preliminary experiments were performed to determine if the hormone receptor antagonists used in this study had an effect on the colour development of either the ammonia or urea assays. There was no significant effect of either RU-486 or ketanserin on the ammonia assay, neither was the urea assay affected by RU-486. However, the colour development of the urea assay in the presence of ketanserin was slightly less intense (10%) relative to samples without ketanserin. Due to the fact that this change was relatively small and underestimated the influence of ketanserin (but did not alter the trends in the data), the values were not corrected for this effect.
Oxygen consumption
Oxygen consumption was measured by closed respirometry with eight
double-walled glass chambers connected in series to a water bath to control
the temperature inside the chambers. Changes in oxygen levels were measured
using an automatic temperature compensated dissolved oxygen sensor with
built-in thermistor and amplifier (Vernier Software and Technology, Beaverton,
OR, USA) and measurements were recorded in mg l-1. Measurements of
water oxygen levels were quantified using the LoggerPro Software (Vernier
Software and Technology). Values for oxygen consumption are expressed in
(µmol O2 min-1 g-1).
Statistical analysis
The data are presented as means ± standard error of the mean
(s.e.m.). For Series I, III and IV, a one-way analysis of variance (ANOVA) was
used to determine if tissue levels and oxygen consumption rates varied over
time (P<0.05). A Tukey test (SPSS, SPSS Inc., Chicago, IL, USA)
was used to test for differences between time intervals
(P<0.05).
For Series II, analysis of oxygen consumption values was carried out using
the single cosinor approach (Halberg et
al., 1972; Nelson et al.,
1979; Rodela and Wright,
2006). In this model a cosine function is fitted to the data by
least squares regression, defining in the process several parameters of the
circadian rhythm: its rhythm-adjusted mean value or mesor; its amplitude,
which is one half of the difference between the highest and the lowest values;
and the time at which the waveform reaches its peak value or acrophase.
Recognition of circadian activity was accomplished by testing a null
hypothesis of zero amplitude with an F-test (P<0.05).
For Series V, analysis of the dose-dependence data for both ketanserin and RU-486 was done with a one-way ANOVA (P<0.05) followed by a Dunnett's test to locate statistical differences (P<0.05). For Series VI, a paired t-test was used to test for significance differences between control and treatment values for each individual time period (P<0.05). Due to confounding effects of time and treatment on diurnal nitrogen excretion, a general linear model contrast (SAS, SPSS Inc.) was used to determine if control values during the diurnal antagonist exposure were significantly different from treatment values.
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Series II
Oxygen consumption in R. marmoratus followed a significant
circadian rhythm (F2,9=16.4, P=0.0007). The
highest rates of oxygen uptake occurred during the day between 11:30 h and
12:30 h, with an amplitude of 32% of the meson, the acrophase occurred at
13:48±00:34 h (Fig. 2).
R. marmoratus consume significantly less oxygen between 17:30 h and
18:30 h. Comparisons between the maximum and minimum rates of oxygen
consumption yield a 1.9-fold difference between the peaks and troughs.
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Series IV
When Jurea rates were examined over the same 1 h
intervals between 12:00 h and 18:00 h, the pattern was similar to the hourly
oxygen consumption measurements (Fig.
3). Jurea was significantly higher between
13:00 h and 14:00 h relative to the subsequent hourly intervals. By
17:00-18:00 h, approximately 72% less urea was excreted compared to values
obtained between the hours of 13:00 h and 14:00 h. Over the same 6 h
intervals, there was no significant difference in ammonia excretion rates
(data not shown).
Pharmacological experiments
Series V
Both receptor antagonists required the use of an ethanol vehicle to
facilitate solubility; however, ethanol concentrations in the water did not
significantly affect either urea or ammonia excretion rates (t-test,
P<0.05). Application of the ketanserin, a 5-HT2
antagonist, caused a dose-dependent inhibition of Jurea
(data not shown). A concentration of 50 µmol l-1 ketanserin
caused a 61% inhibition of Jurea. Excretion of ammonia
remained unaffected by the application of ketanserin (data not shown).
Exposure to RU-486, a cortisol receptor antagonist, also produced a
dose-dependent decrease in Jurea (data not shown).
Exposure to 5 µmol l-1 RU-486 resulted in a 65% inhibition of
Jurea (data not shown). Ammonia excretion remained
unaffected by RU-486 (data not shown).
Series VI
Under control conditions a diurnal cycle in Jurea was
present (data not shown). The highest rates of urea excretion occurred between
12:00 h and 18:00 h, and were 78% higher than rates between 00:00 h and 06:00
h. Exposure to 30 µmol l-1 of ketanserin between 12:00 h-18:00 h
resulted in a 40% inhibition of Jurea relative to control
values (Fig. 4A). During the
first 6 h of recovery (18:00 h-0:00 h), urea excretion continued to be
depressed (50%) in post-treatment fish relative to control values. In the
subsequent 6 h interval (0:00 h-6:00 h), however, Jurea
returned to control levels. Ammonia excretion rates remained unaffected by the
application of ketanserin (data not shown). RU-486 (1 µmol l-1)
exposure resulted in a 49% inhibition of Jurea rates
(Fig. 4B). Following recovery
in fresh brackish water, Jurea returned to control values
during the next 6 h interval. Ammonia excretion did not change in response to
RU-486 (data not shown).
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Series VII
A strong diurnal cycle in urea excretion was present in fish exposed to the
ethanol control prior to repeated applications of 30 µmol l-1
ketanserin (Fig. 5).
Application of ketanserin did not affect the properties (i.e. period, timing
of peaks and troughs, degree of change between peaks and troughs) of the
diurnal cycle, however, the absolute values on the rates of
Jurea were affected
(Fig. 5). Exposure to
ketanserin resulted in a 39-72% inhibition in Jurea rates.
Ammonia excretion remained constant over time (data not shown). For control
fish, Jamm constituted 72-85% of
Jnitrogen whereas in the ketanserin-treated fish,
Jamm constituted 81-95% of
Jnitrogen.
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). We
first hypothesized that the diurnal Jurea pattern in
R. marmoratus is due to metabolic changes. In particular, fluctuating
tissue urea concentrations alter the plasma-to-water urea gradient and these
variations are reflected in the pattern of Jurea.
Measurements of oxygen consumption in fasted R. marmoratus
demonstrated a diurnal pattern with the highest rates occurring during midday.
Diurnal fluctuations in urea, but not ammonia tissue concentrations paralleled
rhythms observed in oxygen uptake and urea excretion. The peak of each
variable occurred between 12:00 h and 14:00 h. Taken together, these results
suggest that urea excretion rates in R. marmoratus may be under
metabolic influence.
In R. marmoratus, urea is derived from the routine turnover of
uric acid or arginine hydrolysis, rather than the ornithine-urea cycle
(Frick and Wright, 2002b). One
possibility to explain the diurnal urea excretion pattern in fasted R.
marmoratus, is that urea is produced periodically. The highest
concentrations of urea occurred at 12:00 h (
6 mmol l-1) and
lowest levels were observed at 18:00 h (1.6 mmol l-1). This is
a profound change ( 3.8-fold) in whole body urea levels in a relatively
short period of time. Although plasma urea concentrations change by
approximately sevenfold over 2 h in gulf toadfish (O. beta) due to
their peculiar pattern of pulsatile urea excretion
(Wood et al., 1997), this is
not surprising given that the extracellular fluid volume is approximately 5%
of the mass of the fish. Whether or not similar dramatic changes occur in the
intracellular compartment in O. beta is unknown, to our knowledge.
Smaller diurnal changes in body urea ( twofold) content were observed in
the ureogenic gobiid M. abei, which also has a diurnal urea excretion
pattern (Kajimura et al.,
2002). The activity of several hepatic enzymes related to urea
synthesis in M. abei did not show any diurnal changes. Although it is
unlikely that enzymes would be rapidly up- or downregulated within a few hours
of each day, temporal changes in substrate concentrations may influence the
rates of urea synthesis and ultimately affect urea excretion rates. Hence, the
results generally support the `metabolic' hypothesis stated above, but upon
closer examination of the data, it is clear that other factors are involved in
regulating the diurnal pattern of urea excretion in R.
marmoratus.
Measurements of oxygen consumption from fasted R. marmoratus
revealed a 24 h diel pattern with approximately a twofold increase in oxygen
consumption rates during midday (12:00 h) compared to values obtained at 18:00
h. Clear diurnal patterns in oxygen consumption have been observed in other
fish species (Brett and Zala,
1975; Ghosh et al.,
1986; Ghosh et al.,
1990) but the peaks in oxygen consumption are usually indicative
of active periods. The oxygen uptake rates obtained in the present study
potentially reflect daily changes in routine and active metabolic rate
(Rao, 1968;
Sims et al., 1993). Hence, the
daily variation in urea excretion may not be due to diurnal changes in the
rates of all metabolic processes. This idea is supported by the lack of
diurnal variation in ammonia content and excretion rates.
There is indirect evidence in M. abei that diurnal fluctuations in
urea excretion and urea content are correlated with changes in the
permeability of membranes to urea
(Kajimura et al., 2002).
Hence, the diurnal changes in Jurea in R.
marmoratus may be partly explained by diurnal changes in gill urea
permeability. Our second hypothesis stated that regulation of
Jurea in R. marmoratus is influenced by 5-HT and
cortisol, that is, changes in the levels of these hormones results in a change
affecting rates of urea excretion. Indeed, ketanserin inhibited
Jurea, but not Jamm. The 5-HT
antagonist, therefore, probably did not alter protein catabolism but was
specific to Jurea mechanisms.
Of the seven different families of receptors, the 5-HT2 receptor
subtype family is the lone category of 5-HT receptors that display sensitivity
to ketanserin (Barnes and Sharp,
1999). Evidence from numerous studies on teleosts have revealed
that ketanserin blocks 5-HT-mediated locomotor activity in zebrafish
(Brustein et al., 2003) and
5-HT-mediated gonadotropin release in goldfish
(Somoza et al., 1988).
Furthermore, 5-HT-induced increases in plasma urea concentrations and
excretion rates in O. beta were abolished, in a dose-dependent
fashion, by ketanserin (McDonald and
Walsh, 2004). These findings indicate that the effects of
ketanserin specifically target 5-HT receptors in teleost fish, but whether
these physiological responses are specifically mediated by the
5-HT2 subtype in teleosts remains to be ascertained.
Repeated exposure to ketanserin for 2 consecutive days revealed that the
diurnal Jurea pattern still remained under these
conditions; however the absolute rates of excretion were significantly
diminished. In O. beta, injections of a 5-HT2 receptor
agonist elicited substantial urea pulses of comparable size and duration to
natural pulses, whereas a 5-HT2 receptor antagonist inhibited
pulsatile urea excretion implicating 5-HT2 receptors in the rapid
activation of the toadfish UT (McDonald
and Walsh, 2004). Based on the ketanserin experiments in the
present study, it is possible that 5-HT may have a role in regulating putative
urea transport proteins in R. marmoratus; however, further
experiments are required to explore this idea.
Ketanserin may be affecting other neuroendocrine cascades that ultimately
influence urea excretion pathways. In rats, brain serotonergic activity
increases corticosterone release (Fuller,
1996). In rainbow trout, 5-HT agonists elevate plasma cortisol
levels in a dose-dependent manner, suggesting that this hormone has an
important role in cortisol mobilization
(Winberg et al., 1997). It is
possible that fluctuations in 5-HT activity in the brain in R.
marmoratus affect circulating cortisol levels specifically through the
HPI axis, which in turn influences various aspects of urea metabolism and
excretion (see below).
Exposure to RU-486 resulted in an inhibition of urea, but not ammonia
excretion in R. marmoratus. As with ketanserin, the inhibition of
urea excretion following exposure to a solitary dose of RU-486 was reversible,
therefore confirming that the drug was entering the fish and influencing
Jurea pathways. Jurea rates returned
to control levels during the next 6 h period postexposure. Similar to
ketanserin, repeated application of RU-486 did not alter the timing of the
diurnal cycle; however, the absolute rates of urea excretion were
significantly depressed. Therefore, blocking cortisol appears to significantly
decrease urea excretion in R. marmoratus, in contrast to other
studies pertaining to urea transport regulation in fish and mammals by
glucocorticoids (Marsh and Knepper,
1992; Wood et al.,
1997; Wood et al.,
2001; Peng et al.,
2002; McDonald et al.,
2004).
Inhibiting cortisol receptor activity by RU-486 may alter the rate of urea
synthesis in R. marmoratus, targeting enzymes in the arginolytic
and/or uricolytic pathways. Exposure to exogenous cortisol triggers a twofold
increase in arginase activity accompanied by an increase in allantoicase
activity in the ammoniotelic sea raven, Hemipterus americanus
(Vijayan et al., 1996). An
increase in arginase activity would enhance arginine catabolism to form urea,
whereas an increase in the uricolytic enzyme, allantoicase, would enhance the
synthesis of urea from uric acid. In vivo exposure to cortisol in the
rainbow trout resulted in an increase in circulating urea concentrations
accompanied by an increase in branchial and renal excretion rates due to an
altered blood-to-water urea gradient
(McDonald and Wood, 2004).
Thus, cortisol may specifically target urea metabolism in R.
marmoratus. Furthermore, coupled with the evidence that 5-HT may also
increase cortisol mobilization in fish (see above), it is possible that these
two hormones act in tandem to increase arginolysis and uricolysis and
therefore affect plasma urea concentrations and excretion rates.
Regulation of urea synthesis and excretion by hormones such as 5-HT and
cortisol may have an adaptive significance in relation to predation risk in
R. marmoratus. Many studies have implicated the involvement of
cortisol in modifying behavioural processes and social rank in fish (e.g.
Pottinger and Pickering, 1992;
Gregory and Wood, 1999;
DiBattista et al., 2005).
Recent studies have shown that predation or threat of predation significantly
raised cortisol levels or serotonergic activity in a number of fish species
(Winberg et al., 1993;
Woodley and Peterson, 2003).
It is possible that predation risk or perceived predation risk by R.
marmoratus is greatest between 12:00 h and 14:00 h, and elevated hormone
levels, in turn, alter nitrogen excretion rates. Such a strategy may help to
evade predators. Preliminary findings from Barimo and Walsh
(Barimo and Walsh, 2005) have
shown that O. beta may be excreting pulses of urea as a form of
chemical crypsis to avoid predator detection by the grey snapper (Lutjanus
griseus). There is very little ecological information, however, on R.
marmoratus in the wild.
To conclude, there is a strong synchrony between the diurnal patterns of oxygen consumption, whole body urea content and urea excretion rates in R. marmoratus. These results support the hypothesis that the diurnal urea excretion pattern is under metabolic control, but several factors suggested a more complex regulatory process. Cortisol and 5-HT receptor antagonists inhibited the absolute rates of Jurea but did not influence the timing of the Jurea pattern. We propose that 5-HT may regulate the rate of Jurea by affecting the permeability of the gill to urea via a putative gill urea transport protein and/or indirectly altering urea synthesis mediated by other neuroendocrine agents. We further propose that changes in cortisol levels may alter the rate of urea synthesis, which in turn alters the blood-to-water urea gradient, ultimately influencing the rate of urea excretion in R. marmoratus.
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Acknowledgments |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Abel, D. C., Koenig, C. C. and Davis, W. P. (1987). Emersion in the mangrove forest fish Rivulus marmoratus: a unique response to hydrogen sulfide. Environ. Biol. Fishes 18,67 -72.
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