Dogmas and controversies in the handling of nitrogenous wastes: 5-HT2-like receptors are involved in triggering pulsatile urea excretion in the gulf toadfish, Opsanus beta
Division of Marine Biology and Fisheries, NIEHS Marine and Freshwater Biomedical Science Center, Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, Florida, 33149-1098, USA
* Author for correspondence (e-mail: dmcdonald{at}rsmas.miami.edu)
Accepted 23 February 2004
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Summary |
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Key words: serotonin, ketanserin, serotonin receptor, -methyl-5-HT, 8-OH-DPAT
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Introduction |
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Linked to this periodic increase in urea permeability across the gill is a
dramatic decrease in plasma cortisol levels, suggesting that circulating
cortisol may be an important regulator of pulsatile urea excretion
(Hopkins et al., 1995; Wood et
al., 1997
,
2001
). When ureotelic,
toadfish maintain plasma cortisol concentrations that are typical for
chronically (but moderately) stressed teleosts (reviewed by
Mommsen et al., 1999
).
However, 24 h preceding a natural urea pulse event, plasma cortisol
levels fall steadily and then rise rapidly thereafter (Wood et al.,
1997
,
2001
). Since plasma cortisol
levels will also decrease without the occurrence of a natural pulse, the
decline in cortisol concentrations is not believed to be the direct trigger to
pulsatile urea excretion (Wood et al.,
2001
). While the decline in cortisol may be permissive to
pulsatile excretion, a recent study has suggested that the drop in cortisol
does not have to take place in order for pulses to occur (M. D. McDonald, C.
M. Wood, M. Grosell and P. J. Walsh, unpublished data). Indeed, continuous
infusion with cortisol in an attempt to prevent a pre-pulse decline in levels
had no effect on the frequency of urea pulses, although the infusion did cause
a significant reduction in pulse size. These recent results suggest perhaps a
role for cortisol in the regulation of the number of transporters, i.e.
through the regulation of transcription as described in mammalian UT
transporters (Knepper et al.,
1975
; Naruse et al.,
1997
; Peng et al.,
2002
) or perhaps through non-genomic pathways. However, cortisol
may not directly be involved in the regulation of the activation of the
transport mechanism.
Most recently, Wood et al.
(2003) outlined the
possibility of serotonin (5-hydroxytryptamine; 5-HT) as the trigger for the
pulsatile mechanism since arterial injections of this substance result in
pulses of natural size. Serotonin is implicated in a variety of psychological
and physiological roles in mammals and is intimately associated with the
mammalian stress response, namely the regulation of the
hypothalamicpituitaryadrenal (HPA) axis, resulting in an
elevation in circulating cortisol (reviewed by
Chaouloff, 1993
;
Carrasco and Van De Kar, 2003
).
Potentially relevant to pulsatile excretion, 5-HT has also been shown to
regulate the hypothalamicpituitaryinterrenal axis (HPI), the
teleost homologue of the mammalian HPA axis
(Winberg et al., 1997
;
Overli et al., 1999
;
Höglund et al., 2002
).
Reciprocally, central 5-HT synthesis and/or release is under complex control
by glucocorticoids in mammals (reviewed by
Chaouloff, 1993
;
Carrasco and Van De Kar, 2003
).
In mammals, 5-HT1A and 5-HT2A receptors have been
attributed to mediating serotonergic activation of the HPA axis (reviewed by
Barnes and Sharp, 1999
), and
evidence suggests that a 5-HT1A-like receptor is present
(Yamaguchi and Brenner, 1997
)
and exerts the same function in teleosts
(Winberg et al., 1997
;
Höglund et al.,
2002
).
Therefore, the goal of this study was to determine the 5-HT receptors involved in the regulation of the pulsatile urea excretion mechanism of the gulf toadfish. Since circulating cortisol and urea pulse events are strongly correlated in toadfish, the hypothesis tested was that either 5-HT1A or 5-HT2 5-HT receptors mediate 5-HT-triggered urea pulse events. To test this hypothesis, toadfish were treated with agonists and antagonists specific for these types of receptors and the pattern of urea excretion was characterized. In addition, the cortisol response to these pharmacological agents was monitored.
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Materials and methods |
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Experimental protocol
As outlined by Wood et al.
(1997) and McDonald et al.
(2000
), caudal artery
catheterizations were performed on fish anaesthetized with MS-222 (0.5 g
l1; Sigma-Aldrich, St Louis, MO, USA) and wrapped with wet
towels. Intraperitoneal (i.p.) catheters (Clay-Adams PE50) filled with
toadfish saline (Walsh, 1987
)
were inserted through a small ventral incision and threaded approximately 4 cm
inside the body cavity. The wound was treated with oxytetracycline powder in
order to prevent infection and the catheter sutured securely with 3-0 silk at
the site of exit. An additional suture secured the catheter just anterior to
the arterial catheter incision and two more secured it along the arterial PE
160 sleeve. The fish were left to recover undisturbed in individual shielded
flux chambers with a PVC pipe shelter for 24 h, at which time the patency of
the arterial catheters was confirmed. The functioning arterial and i.p.
catheters were then left outside the flux chamber attached to a syringe, so as
not to disturb the fish a second time by searching for the catheters within
the chamber. The fish were left overnight (for a total of 36 h of recovery),
after which time the water flow to the flux chamber was stopped. Without
disturbing the fish, the water level quickly dropped to an exact volume mark
of 1.352.0 liters through a small hole in the flux chamber, and
vigorous aeration maintained thorough mixing and the oxygen partial pressure
(PO2) close to air saturation. A water sample
(5 ml) was taken for the measurement of initial urea and ammonia concentration
and [3H]PEG 4000 counts. Thereafter, water samples were taken every
hour until the injection of the first pharmacological agent (see below), after
which water samples were taken approximately every 30 min (unless otherwise
stated) for the remainder of the experiment.
Pharmacological experiments
Pharmacological experiments were performed in order to determine the 5-HT
receptors involved in triggering the pulsatile urea excretion mechanism of the
gulf toadfish. The pharmacological agents used in the present study included
the selective 5-HT1A receptor agonist 8-OH-DPAT
[8-hydroxy-2-(di-n-pro-pylamino) tetralin hydrobromide; Sigma-Aldrich], the
5-HT2 receptor agonist -methyl-5-HT (Sigma-Aldrich) and the
5-HT2 receptor antagonist ketanserin (ketanserin tartrate salt;
Sigma-Aldrich). Eight different experimental series were performed. In Series
1 and Series 2, fish were injected (1 µmol ml1 saline
kg1 fish) with either 8-OH-DPAT (N=6) or
-methyl-5-HT (N=27), respectively, via the arterial
catheter followed by 3 ml kg1 toadfish saline. This dose was
calculated to yield circulating concentrations of
3x106 mol l1, which is close to
circulating serotonin levels measured in toadfish
(107106 mol l1)
and identical to the dose of serotonin used on toadfish in a recent study
(Wood et al., 2003
). In a
separate experimental series (Series 3), the possibility of a non-specific
branchial permeability increase in response to
-methyl-5-HT injection
was examined by injecting a dose of 50 µCi kg1 body mass
of [3H]PEG 4000 (Perkin Elmer, Wellesley, MA, USA) via the
caudal arterial catheter followed by an additional 3 ml kg1
of saline (N=8). Toadfish were then left overnight to allow for
equilibration of the [3H]PEG 4000 throughout the extracellular
space as described by McDonald et al.
(2000
). In Series 4, the time
for
-methyl-5-HT to elicit a urea pulse was measured (N=6). In
this series, a timer was started immediately following injection of the first
toadfish and water samples were taken precisely every five minutes for the
next 50 min. At the same time, each remaining fish was injected and the
injection time noted. An estimate of the time it took for each individual fish
to pulse was then determined. In four separate experimental series (Series 5,
N=4; Series 6, N=5; Series 7, N=5; Series 8,
N=7), four different doses of the 5-HT2 receptor
antagonist ketanserin (0.01, 0.1, 1 and 10 µmol ml1
kg1) were injected via an i.p. catheter in 45%
(w/v) HBC (2-hydroxypropyl-ß-cyclodextrin, a non-toxic solubilizer;
Sigma-Aldrich) one hour before the arterial injection of
-methyl-5-HT.
Due to the necessity of a solubilizing agent, i.p. injection of ketanserin was
considered to be a more suitable method of treatment.
Analytical techniques and calculations
Urea concentrations in blood and water were measured using the diacetyl
monoxime method of Rahmatullah and Boyde
(1980), with appropriate
adjustments of reagent strength for the different urea concentration ranges in
water and blood plasma. Ammonia concentrations in the water were measured by
the indophenol blue method (Ivancic and
Degobbis, 1984
). Plasma cortisol concentrations were measured
using a commercial [125I] radioimmunoassay kit (ICN Biomedicals
Inc., Costa Mesa, CA, USA) with standards diluted to the same protein range as
toadfish plasma. For measurements of [3H]PEG 4000, water samples (2
ml) were added to 10 ml of Ecolume fluor (ICN Biomedicals Inc.) and analyzed
by scintillation counting on a TM Analytic 6895 BetaTrac counter.
The majority of urea excretion is via the gills (>90%), the
kidney of toadfish contributing only a small percentage (<10%)
(McDonald et al., 2000). In
addition, the permeability of the skin to urea is extremely low
[5.07±0.56 x107 cm s1
(N=8); Pärt et al.,
1999
]. The excretion (E; µmol kg1)
of any substance (X) was calculated from the increase in concentration of the
substance in the water [
X]w during a pulse corrected for
fish body mass (m) and calculated as:
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Statistics
Data are reported as means ± 1
S.E.M. (N=number of fish). In the
case when only two means are compared, the significance of differences between
means was evaluated using Student's unpaired two-tailed t-test
(P<0.05; Nemenyi et al.,
1977). When looking at the significance of differences between a
treatment group over time, a one-way, repeated measures analysis of variance
(ANOVA) with time as the main factor was used and followed by a Bonferroni
correction for multiple comparisons. When looking at the significance of
differences between two treatment groups over time, a two-way repeated
measures ANOVA with time and treatment group as the main factors was used and
followed by a HolmSidak test for multiple sample comparisons.
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Results |
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|
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In contrast to 8-OH-DPAT, the 5-HT2 receptor agonist
-methyl-5-HT caused a dramatic increase in the excretion of urea,
eliciting urea pulse events in >95% (N=27) of all fish injected,
with a mean pulse size of 652±102 µmol N kg1
(N=26; Fig. 2A). There
was no corresponding elevation in the excretion of ammonia or
[3H]PEG 4000, indicating that the increase in the permeability of
the gill was specific for urea (Fig.
2B,C). Remarkably,
-methyl-5-HT mediated its effect within
5 min [4.25±0.41 min (N=8)] of injection, with a pulse size
within that time frame of 517±121 µmol N kg1
(N=8; Fig. 3).
However, the constraints of the sampling protocol most likely resulted in an
overestimation of this time, as in two cases a urea pulse event was measured
from individuals after <3 min following
-methyl-5-HT injection. The
mean duration of one urea pulse was 6.75±2.63 min (N=8), with
seven of eight fish completing the pulse within 5 min (i.e. one increasing
increment of urea appearance) and one of eight taking 25 min to complete one
pulse (i.e. five consecutive increasing increments of urea appearance). Within
the first 30 min of
-methyl-5-HT injection, five of eight fish pulsed a
second time, with a mean pulse size of 468±256 µmol N
kg1 (N=5) and a mean duration of 7.0±1.2 min
(N=5; Fig. 3).
|
|
Treatment with the 5-HT2 receptor antagonist ketanserin caused a
significant, dose-dependent inhibition of pulse events elicited by
-methyl-5-HT (Fig. 4A).
Intraperitoneal injection of the vehicle (HBC) alone had no effect on the
potency or the pulse size elicited by
-methyl-5-HT (N=4), thus
values were combined with those measured from fish injected with the agonist
alone. Ketanserin not only caused a significant, dose-dependent decrease in
the size of urea pulses in those fish that did respond to
-methyl-5-HT
injection (Fig. 4B) but also
caused a dose-dependent decrease in the percentage of fish that pulsed upon
injection with
-methyl-5-HT (Fig.
4C). Urea pulse size was sensitive to inhibition by the antagonist
and was inhibited at concentrations of ketanserin (0.01 µmol
ml1 kg1) that were two orders of magnitude
less than that of
-methyl-5-HT (Fig.
4B). While pre-treatment with ketanserin at doses lower than the
agonist (0.01 or 0.1 µmol ml1 kg1
antagonist versus 1 µmol ml1
kg1 agonist) resulted in a significant reduction in urea
pulse size, the number of fish that responded to
-methyl-5-HT by
pulsing, i.e. the effectiveness of
-methyl-5-HT to elicit a urea pulse,
did not change (Fig. 4C).
However, the effectiveness of
-methyl-5-HT to elicit urea pulses was
significantly inhibited at doses of ketanserin that were greater than or equal
(1 and 10 µmol ml1 kg1) to the agonist
(Fig. 4C). Despite pulsatile
excretion of urea, plasma urea concentrations were relatively constant
throughout all experimental treatments
(Table 1).
|
Similar to fish treated with 8-OH-DPAT, fish injected with
-methyl-5-HT did experience a significant elevation of plasma cortisol
concentrations over time that was not statistically different from the
injection control, suggesting that the blood sampling protocol alone caused
the rise in cortisol (data not shown). Furthermore, pre-treatment with
ketanserin at any dose had no effect on plasma cortisol concentrations
compared with
-methyl-5-HT alone or the injection control (data not
shown).
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Discussion |
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In mammals, the 5-HT2 receptor family currently consists of
three receptor subtypes, 5-HT2A, 5-HT2B and
5-HT2C, which are similar in terms of their molecular structure,
pharmacology and signal transduction pathways (reviewed by
Barnes and Sharp, 1999). All
genes in the 5-HT2 receptor family couple positively to
phopholipase C and mobilize intracellular calcium and inositol phosphates
(Hoyer et al., 1994
). In
addition, 5-HT2 receptors have a relatively low affinity for 5-HT,
which may explain why an injection of 5-HT at the same dose as
-methyl-5-HT is less effective at eliciting urea pulses
(Wood et al., 2003
).
In mammals, -methyl-5-HT shows a selectivity between the three
different 5-HT2 receptor subtypes, with pEC50 values of
8.4, 7.3 and 6.1 corresponding to a selectivity of
5-HT2B>5-HT2C>5-HT2A
(Baxter et al., 1995
; reviewed
by Barnes and Sharp, 1999
).
Ketanserin also differentiates between the three different subtypes, with
pKi values of 8.9, 7.0 and 5.4 corresponding to a selectivity of
5-HT2A>5-HT2C>5-HT2B
(Baxter et al., 1995
; reviewed
by Barnes and Sharp, 1999
).
This indicates that ketanserin is almost 100-fold more and over 1000-fold more
selective for 5-HT2A receptors than for either 5-HT2C or
5-HT2B receptors, respectively. In addition, 5-HT2A is
almost 1000-fold more sensitive to ketanserin than to
-methyl-5-HT,
5-HT2C is approximately equal in sensitivity to the two compounds,
and 5-HT2B is 1000-fold less sensitive to ketanserin than to
-methyl-5-HT. In the present study, ketanserin first caused a
significant inhibition at a dose 100-fold lower than that of the agonist,
corresponding to an IC50 of approximately 0.0095 µmol
l1. Based on the affinities of the 5-HT2 family
of receptors for the agonist and antagonist, an antagonist IC50
that is 100 times less than the effective concentration of the agonist
suggests that 5-HT2A receptors are involved in the mediation of
pulsatile urea excretion. However, the specific involvement of the
5-HT2A receptor subtype is yet to be positively identified.
The rapid action of -methyl-5-HT to elicit a urea pulse event
suggests that the 5-HT2 receptor could be in close proximity to the
urea transporter(s) involved in pulsatile urea excretion. A recent study gives
evidence against central nervous system (CNS) activation of individual urea
transporters, as there is no effect of bilateral surgical sectioning of
cranial nerves IX (glossopharyngeal) and X (vagus) on pulse size
(Wood et al., 2003
). That
being the case, a co-localization of tUT and 5-HT2 could
potentially result in the direct activation of tUT through 5-HT2
receptor-mediated phosphorylation. Sequence analysis of tUT shows two
potential phosphorylation sites within a much longer C-terminal sequence that
is unique to tUT and has been suggested to be related to the rapid
upregulation of urea transport during a urea pulse
(Walsh et al., 2000
).
Correspondingly, ketanserin could then be inhibiting the
5-HT2-mediated phosphorylation of proximal tUTs, reducing the
number of activated transporters and subsequently causing a reduction in pulse
size. Theoretically, increasing the dose of ketanserin could result in a more
pronounced reduction in activated transporters until a threshold is achieved
when so few transporters are activated that a urea pulse can no longer be
detected.
In addition to 5-HT2 receptors and tUT being in close proximity,
it is also possible that the site of 5-HT release is close to or within the
gill. As discussed above, central mediation does not appear to be involved.
However, there are several 5-HT storage sites located outside the CNS and even
within the gill of teleost fish that could potentially be sources of 5-HT for
triggering urea pulsing in vivo. These include neuroepithelial cells
(NECs) and neurons (Dunel-Erb et al.,
1982,
1989
; Bailly et al.,
1989
,
1992
;
Jonz and Nurse, 2003
) located
within the gill itself as well as the posterior cardinal vein that runs
adjacent to the kidney (Fritsche et al.,
1993
). While the data of the present study suggest the involvement
of 5-HT2 receptors by introducing a specific concentration of
agonist and antagonist into the entire circulation, the relevant concentration
of 5-HT necessary to elicit urea pulses in vivo may be local gill
levels, which are difficult to predict.
A previous study by Wood et al.
(1997) described sharp drops
in plasma urea concentrations in association with natural pulse events, which
return to pre-pulse concentrations within 46 h. In the present study,
plasma urea concentrations show no relationship with
-methyl-5-HT-induced urea pulses as they do not change over the course
of the experiment. However, the findings of the present study describe an
activation of branchial urea excretion upon introducing
-methyl-5-HT
into the systemic circulation. It is possible that by doing so, tissue urea
transporters are also activated, thereby allowing an even more rapid
equilibration of tissue and plasma, as even under resting conditions there is
excellent equilibration of urea between the water compartments of plasma,
liver and white muscle (Wood et al.,
1997
). This might not be the case during natural pulsatile
excretion, when there could likely be a more localized, branchial release of
5-HT. The variation observed in initial plasma urea concentrations between the
different groups of fish is consistent with previous observations (reviewed by
Wood et al., 2003
).
There are extensive data lending support to an excitatory influence of
central serotonergic systems upon the HPA axis in mammals and the HPI axis in
fish (see reviews by Chaouloff,
1993; Carrasco and Van De Kar,
2003
). Depending on the dose and status of the fish, 8-OH-DPAT has
been shown to cause both increases and decreases in circulating cortisol
levels, suggesting the presence of 5-HT1A-like receptors as
mediators of HPI activity in teleosts
(Winberg et al., 1997
;
Höglund et al., 2002
). In
the present study, 8-OH-DPAT injection did not result in an increase in plasma
cortisol concentrations that was significantly different from injection
controls. Interestingly, central 5-HT1A sensitivity is under the
permissive control of glucocorticoids in mammals, and high cortisol levels
decrease the sensitivity but not the Bmax of these
receptors (Laaris et al.,
1997
; Czyrak et al.,
2002
). The high circulating cortisol levels typical of cannulated
toadfish in the present study (200400 ng ml1; Wood et
al., 1997
,
2001
; M. D. McDonald, C. M.
Wood, M. Grosell and P. J. Walsh, unpublished data) could have served to
decrease the sensitivity of the 5-HT1A receptor, making it
difficult to further stimulate the HPI axis in these fish. However, the lack
of a substantial cortisol response mediated by 5-HT1A or
5-HT2 receptor agonists could also suggest that these receptors are
not involved in the regulation of the HPI axis in toadfish. Indeed, in
amphibians, 5-HT4 receptors and not 5-HT1A or
5-HT2 receptors mediate the direct stimulatory effect on
glucocorticoid release (Idres et al.,
1991
).
Thus, 5-HT2 receptors appear to be involved in mediating the
activation of the urea pulse, but they are probably not directly responsible
for the changes in plasma cortisol observed in toadfish around the time of a
pulse. However, a strong correlation still exists between cortisol
fluctuations and urea pulses in toadfish under normal, resting conditions
(Wood et al., 1997,
2001
). While the activation of
the urea transporter mediated by 5-HT2 receptors is a relatively
fast event (<5 min), the natural pulsatile process is rather slow; cortisol
levels drop 24 h prior to a urea pulse and then rise within 2 h
thereafter (Wood et al., 1997
,
2001
). Perhaps through the
sensitization/desensitization of 5-HT2 receptors, fluctuating
plasma cortisol concentrations during the natural pulse cycle are involved in
mediating pulsatile urea excretion. In fact, cortisol-infused toadfish show a
significant 70% decrease in the size of urea pulses (M. D. McDonald, C. M.
Wood, M. Grosell and P. J. Walsh, unpublished data), lending support to the
theory that high circulating cortisol concentrations may have a desensitizing
effect on 5-HT2 receptors.
Notably, 5-HT also stimulates arginine vasopressin (AVP; antidiuretic
hormone) secretion via 5-HT2 receptors, whereas
5-HT1A receptors appear not to be involved
(Jorgensen et al., 2003). In
mammals, AVP is an important stimulator of UT-A-facilitated urea transport by
mediating acute, cAMP-dependent changes in the activity of membrane-bound UT-A
proteins as well as through a gradual recruitment of transporters to the
membrane from intracellular pools
(Grantham and Burg, 1966
;
Star et al., 1988
;
Inoue et al., 1999
; reviewed
by Smith and Rousselet, 2001
).
With respect to the toadfish, arterial injection of arginine vasotocin (AVT;
the teleost homologue of AVP) does cause a urea pulse event to occur,
suggesting the involvement of this hormone in pulsatile urea excretion
(Perry et al., 1998
;
Wood et al., 2001
). However,
these urea pulses are at most 10% the size of natural pulses, and occur only
at supraphysiological levels of AVT
(1010109 mol l1).
As mammalian AVP is also a regulator of the HPA axis, an interaction between
teleostean AVT, cortisol and 5-HT2 receptors in the regulation of
pulsatile urea excretion cannot entirely be ruled out and will be directly
investigated in the future.
Interestingly, both 5-HT and -methyl-5-HT have been shown to cause a
rapid and pronounced constriction of gill blood vessels, resulting in an
increase in branchial vascular resistance and a reduction in arterial oxygen
pressure (Sundin et al.,
1998
). It is not believed that the increase in branchial vascular
resistance associated with
-methyl-5-HT injection results in a
non-selective increase in branchial permeability, as simultaneous increases in
branchial urea, ammonia and PEG 4000 excretion would have been evident in the
present study. While the physiological significance of 5-HT on branchial
resistance and gas transfer is unknown
(Sundin et al., 1998
), it has
been suggested that 5-HT may be involved in ventilation
(Fritsche et al., 1992
),
acidbase balance (Thomas et al.,
1979
) or environmental sensory systems
(Dunel-Erb et al., 1982
;
Bailly et al., 1989
). In
theory, the factors that cause variations in any one of these systems could
also be potential cues for pulsatile urea excretion in toadfish under natural
conditions.
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Acknowledgments |
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