Nitric oxide and the control of catecholamine secretion in rainbow trout Oncorhynchus mykiss
Department of Biology, University of Ottawa, 10 Marie Curie, Ottawa, ON, Canada K1N 6N5
* Author for correspondence (e-mail: sfperry{at}science.uottawa.ca)
Accepted 11 April 2005
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Summary |
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Key words: adrenaline, noradrenaline, catecholamine, nitric oxide synthase, stress, chromaffin cell, rainbow trout, Oncorhynchus mykiss
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Introduction |
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The current model for catecholamine release incorporates a number of
cholinergic and non-cholinergic neurotransmitters and/or neuromodulators that
interact either directly or indirectly with the chromaffin cells to influence
secretion (Reid et al., 1998).
The primary mechanism of catecholamine secretion, as in other vertebrates, is
believed to be cholinergic and involves the interaction of acetylcholine (ACh)
with nicotinic or muscarinic receptors
(Nilsson et al., 1976
;
Guo and Wakade, 1994
).
Non-cholinergic mechanisms of catecholamine secretion in fish include
activation of the renninangiotensin system (RAS;
Bernier and Perry, 1999
),
direct action of elevated levels of adrenocorticotropic hormone (ACTH;
Reid et al., 1998
) or
serotonin (Fritsche et al.,
1993
), and neuronal release of vasoactive intestinal polypeptide
(VIP) and/or pituitary adenylyl cyclase activating polypeptide (PACAP;
Montpetit and Perry,
2000
).
Nitric oxide (NO) is a relatively short lived, highly reactive gas molecule
that was first recognized as an endothelium-derived relaxing factor (EDRF)
implicated in blood vessel dilation
(Moncada et al., 1989).
Subsequently, NO has been identified as an endogenous mediator of numerous
physiological processes ranging from vascular regulation to immunological
responses (Kuo et al., 2003
;
Mungrue et al., 2003
). NO is
produced in various tissues by the nitric oxide synthase (NOS) family of
enzymes. Of the three isoforms of NOS, neuronal NOS (nNOS) has received the
most attention as a potential modulator of catecholamine secretion. In
mammals, nNOS is present in chromaffin cells
(Schwarz et al., 1998
;
Oset-Gasque et al., 1994
) as
well as in cholinergic fibers (Bredt et
al., 1990
; Dun et al.,
1992
; Holgert et al.,
1995
), suggesting that NO may be released along with ACh
(Marley et al., 1995
). In
rainbow trout Oncorhynchus mykiss, nNOS was localized in the head
kidney tissue (Jimenez et al.,
2001
) but, unlike in mammals, it appears to be only sparsely
present in chromaffin cells (Gallo and
Civinini, 2001
). Several studies have implicated nNOS in
catecholamine regulation in mammals
(Schwarz et al., 1998
;
Vicente et al., 2002
;
Barnes et al., 2001
) whereas
fewer studies have implicated endothelial NOS (eNOS)
(Barnes et al., 2001
;
Torres et al., 1994
); there is
no evidence for a role for inducible NOS (iNOS).
To date, all previous studies investigating the role of NO on basal and
stimulus-evoked catecholamine secretion from chromaffin cells have used
mammalian systems. Results from these studies were obtained using cultured
chromaffin cells (Torres et al.,
1994; Oset-Gasque et al.,
1994
; Rodriguez-Pascual et
al., 1995
; Vicente et al.,
2002
) or perfused adrenal glands
(Marley et al., 1995
;
Nagayama et al., 1998
;
Barnes et al., 2001
). These
prior studies have relied mainly on pharmacological approaches including the
use of NO itself (Oset-Gasque et al.,
1994
), NO donors, SNP and/or SNAP
(Marley et al., 1995
;
Schwarz et al., 1998
) and/or
NOS inhibitors (Torres et al.,
1994
; Nagayama et al.,
1998
; Schwarz et al.,
1998
; Barnes et al.,
2001
; Vicente et al.,
2002
). Surprisingly, there are no published studies that have
incorporated simultaneous measurements of NO and catecholamine levels.
Previous research using rainbow trout has led to the development of a
well-characterized in situ perfusion technique in which catecholamine
secretion can be studied in whole animal preparations without major
disturbances to the chromaffin tissue
(Reid and Perry, 1995;
Montpetit and Perry, 2000
).
This, along with a field stimulation technique which allows stimulation of the
nerves that innervate the main population of chromaffin cells
(Montpetit and Perry, 1999
),
forms a model with which in vivo catecholamine secretion can be
simulated. The goal of the present study was to investigate the effects of NO
on both basal and stimulus-evoked catecholamine secretion in rainbow trout
using this model. Experiments incorporating simultaneous measurements of NO
(estimated by analysis of nitrate and nitrite levels) and catecholamine levels
were designed specifically to test the hypothesis that NO derived
predominantly from nNOS is a negative modulator of catecholamine
secretion.
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Materials and methods |
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In situ saline-perfused posterior cardinal vein preparation
The fish were killed by a sharp blow to the head, weighed and placed on
ice. To electrically stimulate the nerves innervating the chromaffin cells, a
field stimulation technique was used whereby brass electrodes were sutured to
the skin on each side of the fish immediately behind the operculum at the
level of the lateral line (Montpetit and
Perry, 1999). A ventral incision was made from the anus to the
pectoral girdle, and the tissues overlying the heart were removed by blunt
dissection to expose the ventricle and the bulbus arteriosus. An inflow
cannula (PE 160 polyethylene tubing, VWR International, Mississuaga, ON,
Canada) was inserted into the posterior cardinal vein (PCV) in the mid kidney
area (
10 cm posterior to the heart) and an outflow cannula (PE 160) was
inserted into the ventricle through the bulbus arteriosus. Prior to beginning
the experiments, the preparations were perfused for 20 min with modified
aerated Cortland saline (Wolf,
1963
; 125 mmol l1 NaCl, 2.0 mmol
l1 KCl, 2.0 mmol l1 MgSO4, 5.0
mmol l1 NaHCO3, 7.5 mmol l1
glucose, 2.0 CaCl2, and 1.25 mmol l1
KH2PO4, final pH 7.8) to allow catecholamine levels to
stabilize (Julio et al.,
1998
). Preliminary experiments established that NO levels in the
outflowing perfusate were also stable after 20 min. Perfusion was accomplished
using positive pressure differences between the surface of the saline and the
outflow cannula, resulting in a relatively constant flow (approximately 0.3 ml
min1).
Following the stabilization period, two samples were collected in
pre-weighed microcentrifuge tubes to assess basal catecholamine and NO
secretion rates prior to any experimental procedure. In the control group
perfusion with saline was continued, whereas in the experimental group,
perfusion media were switched rapidly using a three-way valve. Perfusion media
were identical except for the addition of specific antagonists, or NO donors.
In other experiments, the preparation either received a bolus injection of an
agonist via a three-way valve fitted to the infusion line or was
electrically stimulated for 2 min using a previously validated field
stimulation technique (Montpetit and
Perry, 1999). Although the stimulation voltages and frequencies
varied between experiments (see below), the pulse duration was kept constant
at 1 ms.
During the experimental procedure, the perfusate was collected continuously for 2 min intervals over a 10 minperiod. All samples were immediately centrifuged for 20 s at 7500 g and the perfusate was quickly frozen in liquid N2 and stored at 80°C until subsequent determination of catecholamine and NO levels.
Series 1: Assessing the potential for NO to modulate catecholamine secretion
Following the collection of pre-samples, the preparations were administered
unmodified control saline or saline containing the NO donor sodium
nitroprusside (SNP; 5x103 mol l1).
Samples were collected for 6 min, at which point the preparations were
electrically stimulated at 60 V at a frequency of 20 Hz.
Series 2: Catecholamine secretion and NO production during non-specific chromaffin cell depolarization
A previous study by Mendizabal et al.
(2000) showed that a
depolarizing level of KCl was able to elicit NO production and that this
production could be inhibited using a NOS inhibitor. To determine if
KCl-induced NO production could be inhibited in the present study, a cocktail
containing the NOS inhibitors 7-nitroindazole (7-NI; 104 mol
l1) and N-nitro L-arginine methyl ester
(L-NAME; 5x103 mol l1)
was used. Preparations were either perfused with saline containing the
combination of the inhibitors or with control saline. While L-NAME
could be added directly to saline, 7-NI was prepared in methanol prior to the
addition to the saline (final concentration in the perfusate was 0.2%).
Preliminary experiments showed that 0.2% methanol was without effect on basal
or stimulus-evoked catecholamine secretion. Following the collection of
pre-samples, preparations received a bolus injection of 10 mmol
l1 KCl (1 ml kg1).
Series 3: Catecholamine secretion and NO production during electrical field stimulation
In situ preparations were continuously perfused with saline for 20
min, at which point the pre-samples were collected. The preparations were then
stimulated at 30 V at either 1, 8 or 20 Hz.
To confirm the role of NOS in the generation of NO, preparations were perfused for the entire experiment with a cocktail of the NOS inhibitors, 7-NI and L-NAME as described above. Following the pre-sample collections, preparations received an electrical stimulus of 30 V and 8 Hz.
Series 4: Assessing the mechanisms of NO
Increased catecholamine degradation vs decreased catecholamine secretion
To determine the effect of NO on catecholamine degradation, noradrenaline
and adrenaline (5x107 mol l1)
prepared in 0.1 mol l1 HCl were incubated separately in
freshly prepared saline containing a range of SNP concentrations
(108103 mol l1).
0.5 ml of SNP solution was added to 0.5 ml of catecholamine solution and
incubated for 5 min in a glass test tube. Reaction was stopped by the addition
of 0.5 ml of 0.1 mol l1 perchloric acid and 0.1% cysteine
and the solutions were then placed on ice. Catecholamines were extracted and
analyzed by high pressure liquid chromatography (HPLC), while a colorimetric
assay was used to measure NO levels.
To distinguish between the effects of NO on catecholamine degradation vs cellular catecholamine secretion, per se, experiments were performed in which the cellular effects of NO, at least those known to influence catecholamine secretion, were blocked. Experimental preparations received saline containing the selective guanylyl cyclase (sGC) inhibitor, 1H-(1,2,4) oxadiazole(4,3-alpha)quinoxaline-1-one (ODQ; 105 mol l1) for the entire experiment while controls received saline. Pre-samples were collected and the preparations were stimulated at 30 V and 8 Hz.
Series 5: Assessing the potential roles of the three NOS isoforms
To assess whether iNOS could potentially contribute to NO generation during
electrical stimulation of the chromaffin cells, experiments were performed to
localize iNOS mRNA to the PCV and anterior kidney, regions known to contain
high concentrations of chromaffin cells.
Tissue collection and RNA extraction
Fish were killed by a sharp blow to the head and tissues (brain, PCV and
kidney) were collected and frozen immediately in liquid N2 and
stored at 80°C. Total RNA was extracted using Stratagene Absolute
RNA RT-PCR miniprep kit (Stratagene, Cedar Creek, TX, USA) according to the
instructions of the manufacturer. RNA concentrations were verified using
spectrophotometry (Eppendorf BioPhotometer, VWR International).
cDNA synthesis and mRNA assessment
cDNA was synthesized from 5 µg total RNA using StrataScript reverse
transcriptase (Stratagene) and random hexamer primers. iNOS mRNA levels were
assessed by real-time PCR on duplicate samples of cDNA using Brilliant®
SYBR® Green QPCR (Stratagene) and a Stratagene MX-4000 multiplex QPCR
system. PCR conditions were as instructed by the manufacturer, except scaled
down from a 50 µl to a 25 µl final reaction volume. Gene-specific
primers for rainbow trout iNOS (AJ295230) and ß-actin (AF550583) were
designed using DNAMAN (version 4.0, Lynnon Biosoft, Vaudreuil-Dorion, Quebec,
Canada) from the cDNA sequences obtained from GenBank. Relative expression of
mRNA levels was determined (using actin as a standard) using the delta-delta
Ct method (Pfaffl, 2001). For
iNOS, the forward primer 5'-GAAGTGCAGAGGTCA-3' was used with the
reverse primer 5'-GGTATTCCAGTCGTAGGCA-3' to yield a 134 bp
product. The cycle threshold (Ct) values for actin varied little between the
tissues examined (Ct=19.2±0.05 (mean ± S.E.M.;
N=45) so it is unlikely that the data were skewed by using actin
(rather than ribosomal RNA) as a standard.
To evaluate the extent of NO production attributable to iNOS activation during electrical stimulation, eNOS and nNOS induction were prevented by perfusing with Ca2+-free saline containing the Ca2+ chelator, EGTA (1 mmol l1) for 20 min prior to the collection of pre-samples; control samples were perfused with normal saline. Following the collection of the pre-samples, both groups were electrically stimulated at 30 V and 8 Hz.
Hypoxia specifically induces eNOS to produce NO
(Yamamoto et al., 2003). Thus,
experiments were performed to evaluate whether hypoxia could directly affect
NO production and if so, whether the NO produced during hypoxia could regulate
basal and stimulus-evoked catecholamine secretion. Following the collection of
the two pre-samples, fish were perfused with saline bubbled with N2
to render the saline hypoxic. The PO2 of the
hypoxic saline solution was measured using a Foxy-AL300 fiber-optic probe and
associated hardware and software (Ocean Optics, Dunedin, FL, USA). In all
cases, the PO2 of the saline was allowed to
fall to 10 mmHg prior to use. Control preparations continued to receive
normoxic saline following the collection of the pre-samples. Both groups were
perfused for 10 min following the collection of pre-samples, with samples
collected every 2 min over that time. Both groups were then electrically
stimulated at 30 V and 8 Hz.
Subsequent experiments were performed to determine if the combined presence of both NOS inhibitors could inhibit hypoxia-induced NO production from eNOS. Preparations were perfused with either 7-NI (1x104 mol l1) and L-NAME (5x103 mol l1), or regular saline. Following the collection of the pre-samples, fish were switched to hypoxic saline (PO2<10 mmHg). The same protocol was performed on the control group, except that they were not treated with inhibitors. The preparations were perfused with hypoxic saline for 10 min, with saline being collected over 2 min intervals, after which they received an electrical stimulus of 30 V and 8 Hz.
To further assess the role of eNOS, the endothelium was removed by
perfusion of the PCV with saponin as previously described
(Donoso et al., 1996;
Cortes et al., 1999
). In brief,
following establishment of perfusion flow, one group of fish received a bolus
injection of 0.1% saponin for 60 s, while two groups received a saline
injection. Following the collection of the pre-samples, a saline-injected
group and the saponin-treated group were rapidly switched to hypoxic saline
(PO2<10 mmHg, as described above), while the
remaining group continued to receive control saline. All preparations were
perfused for an additional 10 min, with collections every 2 min. The
preparations were then electrically stimulated at 30 V and 8 Hz.
Analytical procedures
Catecholamine determination
Catecholamine levels in perfusate were determined on alumina-extracted
samples (100 µl) using HPLC with electrochemical detection
(Woodward, 1982). The HPLC
incorporated a Varian ProStar 410 solvent delivery system (Varian
Chromatography Systems, Walnut Creek, CA, USA) coupled to a Princeton Applied
Research 400 electrochemical detector (EG & G Instruments, Princeton, NJ,
USA). Concentrations were calculated relative to appropriate standards, using
3,4-dihydroxybenzalamine hydrobromide (DHBA) as an internal standard.
Nitric oxide assay
Quantification of NO is problematic because of its short lifetime.
Therefore, NO production was evaluated indirectly by measuring the
concentration of nitrite and nitrate, stable metabolites of NO in biological
fluids. This method demonstrates high accuracy and reproducibility and
adequately reflects actual NO production
(Gilliam et al., 1993;
Manukhina et al., 1999
). The
NO assay was performed as described by Gilliam et al. (1994), with
modifications. In short, a stock solution of magnesium nitrate (Sigma) was
prepared in saline at a final concentration of 1 mmol l1.
The stock solution was serially diluted in 0.14 mol l1
KHPO4 to prepare standard curves. The assay procedure consisted of
adding 50 µl of standard or sample along with 15 µl of NADPH (0.8 mmol
l1 Sigma) to a 96-well plate. Next, 2.5 µl of FAD (100
µmol l1; Sigma) was added followed by 0.01 units of
nitrate reductase (from Aspergillus niger; E.C.1.6.6.2; Sigma). The
plate was sealed, placed in the dark and incubated at room temperature
(
21°C) for 45 min. 40 µl of Griess reagents I and II (Cayman
Chemicals, Ann Arbor, MI, USA) were then added and allowed to incubate for 5
min. Color development was assessed using a Spectra Max Plus 384 (Molecular
Devices, Sunnyvale, CA, USA) micro-plate reader at a wavelength of 540 nmol
l1.
Statistical analysis
The data are presented as means ± 1 standard error of the mean
(S.E.M.). All data sets were analyzed using two-way
repeated-measures analysis of variance (ANOVA). If a statistical difference
was identified, a post-hoc multiple (`all pair wise') comparison test
(Bonferroni's t-test) was applied. All statistical tests were
performed using a commercial statistical software package (SigmaStat version
2.03).
Data presentation
Owing to a high degree of temporal variability, peak catecholamine
secretion rates, generally obtained 2 or 4 min after stimulation/agonist
addition, were calculated by taking the mean of the maximal noradrenaline and
adrenaline secretion rates in response to stimulation for each fish within a
given group. For total catecholamine secretion rates, the sum of adrenaline
and noradrenaline was determined at each time point and the resultant maximum
values were used. Statistical analysis of noradrenaline, adrenaline and total
catecholamines were performed, and all showed similar trends within each
experiment. Therefore, for clarity, only the statistical analysis of total
catecholamine secretion rates are presented on the figures.
NO peak levels, generally obtained 2 or 4 min after stimulation/agonist addition, were calculated by taking the mean of the maximal NO levels in response to stimulation for each fish within a given group.
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Results |
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Perfusion with Ca2+-free saline was used as a tool to
specifically prevent the activation of iNOS and nNOS during electrical
stimulation. The control preparations responded to electrical stimulation with
an 11-fold increase in total catecholamine secretion and a fivefold
increase in NO production. Preparations perfused with Ca2+-free
saline did not exhibit an increase in catecholamine secretion
(Fig. 8A) or NO production
(Fig. 8B) in response to
electrical stimulation.
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Discussion |
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Mechanism of inhibition of catecholamine secretion by NO
This study addressed two possible mechanisms to explain the reduction of
catecholamine appearance in the perfusate in the presence of NO. The
possibilities tested were an effect of NO on reducing catecholamine stability
after their secretion from chromaffin cells and/or specific inhibition of
catecholamines secretion caused by intracellular signaling events linked to
activation of sGC. In concurrence with the study of Kolo et al.
(2004) the results of the
in vitro experiments clearly demonstrate that NO has the capability
to rapidly degrade catecholamines. It has been suggested that the underlying
explanation for the effect of NO on catecholamine degradation involves the
conversion of catecholamines by NO to their 6-nitro derivatives
(Kolo et al., 2004
).
In mammals, studies suggest that NO inhibits catecholamine secretion by
promoting a cascade of events beginning with activation of sGC and leading to
phosphorylation of Ca2+ channels and an attenuation of the inward
Ca2+ flux in response to stimulation
(Schwarz et al., 1998;
Ferrero et al., 2000
;
Hirooka et al., 2000
;
Vicente et al., 2002
). In the
present study, the sGC inhibitor, ODQ, was used to prevent Ca2+
channel phosphorylation during electrical stimulation. In the presence of ODQ,
there was a pronounced increase in stimulus-evoked catecholamine secretion,
suggesting that the activation of sGC and the downstream events are important
factors leading to the decrease in catecholamine secretion. Of the two
mechanisms leading to the NO-induced decrease in catecholamine outflow from
the perfused PCV preparation, the activation of sGC would appear to be the
primary mechanism. In the presence of ODQ, NO levels were elevated and
catecholamine secretion was significantly increased above control levels. If
the effect of NO on accelerating catecholamine degradation was the predominant
factor, one would have expected to observe a decrease in catecholamine outflow
during this experiment.
NO production is frequency dependent
The predominant mechanism causing catecholamine secretion release from
vertebrate chromaffin cells is the activation of nicotinic receptors by ACh
released from pre-ganglionic sympathetic nerve fibers
(Montpetit and Perry, 1999;
Carrasco and Van de Kar, 2003
).
There are two main pathways that could lead to the increased production of NO
and its subsequent regulation of catecholamine secretion. One involves the
interaction of ACh with the cholinergic receptors. For example, Moro et al.
(1993
) showed that cholinergic
receptor (nicotinic or muscarinic) stimulation was accompanied by an increase
in cGMP levels in the chromaffin cells. Because the interaction of ACh with
the cholinergic receptor results in an increase in intracellular
[Ca2+], Ca2+-dependent NOS enzymes could be activated,
resulting in an increased production of NO within chromaffin cells.
Another possibility involves the release of NO from the pre-ganglionic
sympathetic nerve fibers. Previous studies have demonstrated that the specific
type of neurotransmitter (e.g. ACh vs VIP) released during electrical
stimulation of these fibers is related to the action potential frequency
(Montpetit and Perry, 2000;
McNeill et al., 2003
). Because
NOS has been identified in the pre-ganglionic nerve fibers
(Bredt et al., 1990
;
Dun et al., 1993
) and NO
production during electrical stimulation is frequency dependent
(Fig. 3B), it would appear that
a similar situation may exist for NO production and release. Interestingly,
the frequency dependency of NO production was markedly different than the
frequency dependency of catecholamine secretion. Catecholamine secretion
increased linearly with increasing frequencies whereas NO production appeared
to peak at an intermediate frequency (8 Hz) and was absent entirely at the
highest frequency (20 Hz). The lack of a tight correlation between NO
production and catecholamine secretion is consistent with the view that there
are numerous mechanisms acting in concert to regulate catecholamine secretion,
all or some of which may be frequency dependent.
NOS isoforms
On the basis of previous studies on mammals, nNOS is believed to be the
main isoform regulating of catecholamine secretion
(Schwarz et al., 1998;
Vicente et al., 2002
).
However, there is also evidence implicating eNOS
(Torres et al., 1994
;
Barnes et al., 2001
). A number
of NOS inhibitors have been identified and used as experimental tools to
investigate the biological significance of NO
(Bland-Ward and Moore, 1995
).
The use of specific inhibitors in differentiating the contribution of the
different isoforms in the production of NO is proving to be difficult. In
mammals, the NOS homodimers show high homology between isoforms. In humans,
the overall amino acid sequence identity is
55%, with particularly strong
sequence conservation in regions of the proteins involved in catalysis
(Michel and Feron, 1997
). For
these reasons, the production of selective NOS inhibitors has been difficult.
Currently, most inhibitors show a lack of selectivity on isolated enzymes
(Moncada et al., 1997
;
Mayer and Andrew, 1998
). Not
surprisingly, therefore, 7-NI, a compound often used as a selective nNOS
inhibitor (Barnes et al., 2001
;
Xu et al., 2001
), was shown to
inhibit the other isoforms with equal potency
(Bland-Ward and Moore, 1995
;
Dick and Lefebvre, 1997
;
Moncada et al., 1997
). For
this reason, the use of `selective' NOS inhibitors may not be an appropriate
method to assess the involvement of the different isoforms. Therefore, in the
current study, we used several alternative approaches to assess the
contributions of the various NOS isoforms to the regulation of catecholamine
secretion.
On the basis of its mRNA expression, iNOS is apparently present in tissues
known to contain high densities of chromaffin cells (including the posterior
cardinal vein and head kidney) and thus could potentially contribute to the
regulation of catecholamine secretion. Unlike eNOS and nNOS, iNOS does not
require an elevation of intracellular [Ca2+] for its activation
because of its high binding affinity for calmodulin
(Oset-Gasque et al., 1994).
Therefore, under Ca2+-free conditions it is expected that only the
iNOS isoform would be activated during electrical stimulation. Because NO was
not elevated under Ca2+-free conditions
(Fig. 8) it would appear that
iNOS does not contribute to NO production during electrical stimulation. The
fact that catecholamines were not released in preparations perfused with
Ca2+-free saline is consistent with previous studies (Burgoyne,
1991). Based on these results, it would appear that during electrical
stimulation, the two NOS isoforms that are potentially regulating
catecholamine secretion are nNOS and/or eNOS.
In an attempt to differentiate between nNOS and eNOS, experiments were
performed without further use of inhibitors. Yamamoto et al.
(2003) demonstrated that
hypoxia treatment is able to induce NO production from eNOS in blood vessels.
Similar findings were obtained in the present study, and moreover the
production of NO during hypoxia was associated with a marked decrease in
stimulus-evoked catecholamine secretion. Several previous studies have
demonstrated that saponin treatment destroys the vascular endothelium
(Donoso et al., 1996
;
Cortes et al., 1999
) and
theoretically this would eliminate the contribution of eNOS to NO production.
Because saponin was able to prevent the increase in NO production in response
to hypoxia treatment, it suggests that the sole source of NO production during
hypoxia was via eNOS. Thus, the results of these experiments suggest
a possible role for eNOS in regulating catecholamine secretion during hypoxia.
However because saponin had no effect on stimulus-evoked NO production, it is
apparent that nNOS rather than eNOS is the principal producer of NO during
electrical stimulation of trout chromaffin cells.
Conclusion
This is the first study to assess the role of NO in the regulation of
catecholamine secretion in a non-mammalian vertebrate. Using the rainbow trout
Oncorhynchus mykiss as a model, the results demonstrate that NO,
produced during stimulation of chromaffin cells, is able to inhibit
catecholamine secretion, confirming the results of previous studies on mammals
(Oset-Gasque et al., 1994;
Torres et al., 1994
;
Schwarz et al., 1998
;
Nagayama et al., 1998
). The
results suggest that of the three NOS isoforms potentially contributing to NO
production and catecholamine regulation, nNOS is likely to be most important,
although eNOS may play an important role during hypoxia.
List of abbreviations
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Acknowledgments |
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