Catecholamine secretion in trout chromaffin cells experiencing nicotinic receptor desensitization is maintained by non-cholinergic neurotransmission
Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario K1N 6N5, Canada
* Author for correspondence (e-mail: sfperry{at}science.uottawa.ca)
Accepted 14 August 2003
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Summary |
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Key words: catecholamine, adrenaline, noradrenaline, VIP, PACAP, stress, nicotinic receptor, angiotensin II, muscarinic receptor, rainbow trout, Oncorhynchus mykiss
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Introduction |
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For cholinergic-evoked catecholamine secretion, the relative involvement of
nicotinic versus muscarinic receptor stimulation is species
dependent. In rainbow trout (Oncorhynchus mykiss), activation of the
nicotinic receptor is believed to be the principal direct pathway for
secretion (Montpetit and Perry,
1999). Activation of the muscarinic receptor, while able to cause
secretion at high agonist concentrations
(Julio et al., 1998
), is
mainly thought to enhance nicotinic-evoked catecholamine secretion
(Montpetit and Perry,
1999
).
It is well documented that in rats
(Khiroug et al., 2002) and in
humans (Ke et al., 1998
), the
nicotinic receptor undergoes desensitization after brief exposure to nicotinic
receptor agonists. Desensitization results in an inactive receptor that does
not allow for the passage of Na+. Thus, further release of
catecholamines via this pathway is diminished or prevented until the
nicotinic receptor is resensitized
(Reitstetter et al., 1999
;
Mahata et al., 1999
).
Recently, Lapner et al. (2000
)
developed an in vivo protocol to desensitize chromaffin cell
nicotinic receptors in rainbow trout. Interestingly, the ability to secrete
catecholamines during acute hypoxia was not impaired in those fish
experiencing nicotinic receptor desensitization
(Lapner et al., 2000
). The
continued ability to secrete catecholamines despite nicotinic receptor
desensitization was later attributed to hypoxic activation of the RAS
(Lapner and Perry, 2001
). The
results of these studies suggested that during periods of nicotinic receptor
desensitization, the importance of normally minor pathways evoking
catecholamine secretion might be substantially increased.
The neurotransmitters PACAP and VIP are potent secretagogues of adrenal
catecholamine secretion in mammals
(Lamouche and Yamaguchi, 2001;
Fukushima et al., 2001
), and in
rainbow trout (Montpetit and Perry,
2000
). It was recently demonstrated that in trout, these
neuropeptides are preferentially released (i.e. in comparison to Ach) from the
preganglionic fibres innervating the chromaffin cells under conditions of
low-frequency (1 Hz) nerve stimulation
(Montpetit and Perry, 2000
).
Because these neuropeptides (VIP and PACAP) and Ach are released from the same
nerve fibers, but at different frequencies, it is possible that the
neuropeptides might play an important role in maintaining catecholamine
secretion during periods of nicotinic receptor desensitization. Thus, the
goals of the present study were (i) to develop an in situ nicotinic
receptor desensitization model that could be used in conjunction with
electrical stimulation of the nerve fibers controlling catecholamine secretion
and (ii) to test the hypothesis that catecholamine secretion could be
sustained at such times owing to increased contribution of non-cholinergic and
muscarinic pathways.
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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. If electrical stimulation was to be administered, electrodes were sutured
to the skin on each side of the fish in the anterior region of the body
(Montpetit and Perry, 1999). A
ventral incision was made from the anus to the pectoral girdle, and the tissue
overlying the heart was removed by blunt dissection to expose the ventricle
and the bulbus arteriosus. The fish were then cannulated (PE 160 polyethylene
tubing, Clay-Adams, Maryland, USA), with the inflow cannula inserted into the
posterior cardinal vein (PCV) and the outflow cannula inserted into the
ventricle through the bulbus arteriosus. Each preparation was perfused for 20
min with modified aerated Cortland saline
(Wolf, 1963
;
Montpetit and Perry, 1999
)
using positive pressure differences to maintain constant flow (approximately
1.5 ml min1) through the PCV. The 20 min perfusion period
allowed for the stabilization of the catecholamine secretion rates prior to
the beginning of the experiment. Two samples were collected in pre-weighed
microcentrifuge tubes following the 20 min period of perfusion to assess the
catecholamine secretion rates prior to any experimental procedure. In the
control group, perfusion with saline was continued whereas in the experimental
groups, perfusion media were rapidly switched using a three-way valve.
Perfusion media were identical except for the addition of specific agonists
and/or antagonists. In both groups, perfusate samples were collected every 20
s within the first 2 min of the experiment, then at 3, 4, 5 and 10 min.
Following the 10 min sample collection period, preparations were either given
a bolus injection of an agonist or electrically stimulated using a previously
validated field stimulation technique
(Montpetit and Perry, 1999
).
Preparations were stimulated at 60 V at either high frequency (20 Hz), or low
frequency (1 Hz) for 2 min; perfusate samples were collected at 20, 60 and 100
s to determine the maximum value. Catecholamine secretion rates
post-stimulation were subsequently presented as maximal secretion rates. The
high voltage used to electrically stimulate the nerves is required owing to
the resistance imparted by the skeletal muscle; extensive prior validation
(Montpetit and Perry, 1999
)
demonstrated that this procedure results in specific activation of nerves
innervating the chromaffin tissue without non-selectively depolarizing the
chromaffin cells.
Series 1: Developing an in situ model for nicotinic receptor
desensitization
After the collection of pre-samples, the preparations were administered
unmodified control saline or saline containing hexamethonium (nicotinic
receptor antagonist; 1x103 mol l1;
Sigma) or nicotine (nicotinic receptor agonist; 1x105
mol l1; Sigma). After 10 min, all preparations were
electrically stimulated at 20 Hz.
Series 2: Catecholamine storage levels
Catecholamine storage levels were measured to assess the potential
contribution of non-chromaffin tissue to electrically evoked catecholamine
secretion Three tissues, heart, white muscle (in the vicinity of the anterior
PCV) and the anterior PCV were obtained by blunt dissection and frozen in
liquid nitrogen. Tissues were cleaned using Cortland saline. Solution (1 ml)
of 4% perchloric acid containing 2 mg ml1 EDTA and 0.5 mg
ml1 sodium bisulphite was added to each tube
(Woodward, 1982). Heart and
white muscle were processed by mortar and pestle while the PCV was sonicated.
The supernatant was diluted 100-fold with 4% perchloric acid and analysed by
HPLC.
Series 3: Assessing the mechanisms of catecholamine secretion in
preparations experiencing nicotinic receptor desensitization
This series of experiments was performed to determine if low-frequency
electrical stimulation could elicit catecholamine secretion during nicotinic
receptor desensitization, and if so, to determine if this secretion was
non-cholinergic in origin. Preparations were perfused with either unmodified
saline or saline containing nicotine (1x105 mol
l1) or with hexamethonium (1x103 mol
l1) plus atropine (1x105 mol
l1). After 10 min, all preparations were electrically
stimulated at low frequency (1 Hz).
In a separate group, preparations were either perfused with unmodified saline or saline containing nicotine (1x105 mol l1) plus VIP6-28 (VIP receptor antagonist; Peninsula Laboratories, San Carlos, CA, USA; 1x106 mol l1). After 10 min, the preparations were divided into two groups; one group was stimulated at high frequency (20 Hz) and the other at low frequency (1 Hz).
Series 4: Assessing the efficacy of cholinergic and noncholinergic
secretagogues in preparations experiencing nicotinic receptor
desensitization
Nicotinic receptors were desensitized during a 10 mininfusion period with
saline containing nicotine (see above). Following desensitization, separate
preparations received a bolus injection of either cod VIP
(1x1011 mol kg1 body mass),
homologous angiotensin II ([Asn1, Val5] Ang II;
5x107 mol kg1 body mass; Sigma) or
the muscarinic receptor agonist methylcholine (1x103
mol kg1 body mass; Sigma); control preparations received a
bolus injection of saline.
Analytical procedures
Catecholamine levels in perfusate and tissue extracts were determined on
alumina-extracted samples (200 µl) using high-pressure liquid
chromatography (HPLC) with electrochemical detection
(Woodward, 1982).
Concentrations were calculated relative to appropriate standards, using
3,4-dihydroxybenzalamine hydrobromide (DHBA) as an internal standard. The
secretion rates for adrenaline and noradrenaline were calculated then summed
to yield total catecholamine secretion rates. Owing to a high degree of
temporal variability, peak catecholamine secretion rates, generally obtained 2
or 3 min after stimulation/agonist addition, were calculated by taking the
mean of the maximal secretion rates in response to stimulation for each fish
within a given group.
Statistical analyses
The data are presented as means ± 1 standard error of the mean
(S.E.M.). All data sets were analyzed using `all pair-wise' two-way
repeated measure analysis of variance (ANOVA) followed by Bonferroni's
t-test.
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Results |
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Series 2: Catecholamine storage levels
Fig. 2 illustrates the
levels of stored catecholamines in several tissues. The catecholamine
(adrenaline plus noradrenaline) concentration in either the heart or the white
muscle was less than 1% of the concentration found in the anterior PCV.
|
Series 3: Assessing the mechanisms of catecholamine secretion in
preparations experiencing nicotinic receptor desensitization
Fig. 3 illustrates the
effect of low frequency (1 Hz) electrical stimulation on preparations treated
with saline or nicotine. The nicotine treated group displayed the
characteristic increase in catecholamine secretion in response to nicotine
followed by a transient decrease to baseline levels
(Fig. 3A). However, during
electrical stimulation at 1 Hz, both the control and nicotine-treated
(desensitized) groups showed an equivalent increase in maximum catecholamine
secretion rate (Fig. 3B). The
use of hexamethonium (nicotinic receptor antagonist) plus atropine (muscarinic
receptor antagonist) to block all cholinergic receptors prevented the
nicotine-evoked stimulation of catecholamine secretion
(Fig. 3A) but was without
effect on electrically evoked (1 Hz) secretion
(Fig. 3B).
|
Regardless of the presence or absence of the VPAC receptor antagonist VIP6-28 in the perfusate, all preparations responded similarly to nicotine addition by showing an increase in catecholamine secretion rates followed by a return to baseline levels (Fig. 4Ai,Bi). Both the control and the VIP6-28 treated preparations exhibited similar increases in catecholamine secretion rates when stimulated at 20 Hz (Fig. 4Aii). However, when stimulated at lower frequency (1 Hz), the presence of VIP6-28 significantly reduced the rate of evoked catecholamine secretion (Fig. 4Bii).
|
Series 4: Assessing the efficacy of cholinergic and noncholinergic
secretagogues in preparations experiencing nicotinic receptor
desensitization
All preparations that received nicotine exhibited a transient elevation of
catecholamine secretion rates (Fig.
5AiCi), a response that is indicative of nicotinic receptor
desensitization. However, the extent of catecholamine secretion in response to
the subsequent addition of the non-cholinergic secratagogues, cVIP
(Fig. 5Aii) and homologous Ang
II (Fig. 5Bii), was markedly
increased in preparations that had previously received nicotine. In contrast,
addition of the muscarinic receptor agonist methylcholine caused identical
increases in catecholamine secretion rates in the control and desensitized
preparations (Fig. 5Cii).
|
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Discussion |
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The nature of nicotinic receptor desensitization
In the present study, catecholamine secretion in a perfused PCV preparation
was initially stimulated by nicotine, but then declined to baseline rates
within 3 min despite the continuing presence of nicotine in the perfusion
fluid. This biphasic response pattern is indicative of nicotinic receptor
desensitization. The decline in catecholamine secretion could not be explained
by exhaustion of catecholamine stores or intracellular signaling molecules
because stimulation of muscarinic (using methylcholine) or non-cholinergic
receptors (using VIP or Ang II) was able to rapidly reactivate catecholamine
secretion. It was previously demonstrated that high-frequency (20
Hz)electrical stimulation of the trout PCV preparation evokes catecholamine
secretion via selective activation of the nicotinic receptor pathway
(Montpetit and Perry, 1999).
However, during low-frequency (1 Hz) stimulation, a significant component of
catecholamine secretion is mediated by VIP and/or PACAP via
activation of VPAC receptors (Montpetit
and Perry, 2000
). Thus, in the present study, further evidence for
nicotinic receptor desensitization was provided by the fact that catecholamine
secretion in preparations that had received nicotine was largely prevented
during high-frequency electrical stimulation. Because the addition of the
nicotinic receptor antagonist, hexamethonium, eliminated the small amount of
residual catecholamine secretion during high-frequency stimulation, it would
appear that the desensitization protocol used in this study, though effective,
was unable to totally desensitize all nicotinic receptors.
Previous studies (e.g. Montpetit and
Perry, 1999) that employed electrical stimulation to evoke
secretion assumed that the catecholamines appearing in the perfusate originate
from the chromaffin cells lining the PCV. However, owing to extensive neuronal
innervation of the heart and the likely depolarization of skeletal muscle in
the vicinity of the PCV, catecholamines could potentially also arise
via spillover from cardiac adrenergic nerves or secretion from
cardiac and/or muscle tissue. However, based on the low levels of
catecholamines stored in the heart and skeletal muscle (less than 1% of
anterior PCV), this is unlikely. The fact that catecholamine secretion during
high-frequency electrical stimulation is prevented by nicotinic receptor
blockade is further evidence that the chromaffin tissue is the predominant, if
not exclusive, site of catecholamine release in the perfused PCV
preparation.
The potential role of non-cholinergic neurotransmitters during
nicotinic receptor desensitization
Vasoactive intestinal polypeptide (VIP) and pituitary adenylyl
cyclase-activating polypeptide (PACAP) are potent secretagogues of
catecholamine release from chromaffin cells in mammals
(Fukushima et al., 2001;
Geng et al., 1997
;
Mazzocchi et al., 2002
) and
fish (Montpetit and Perry,
2000
). Furthermore, previous studies have demonstrated that VIP
and PACAP are localized in pre-ganglionic sympathetic nerve fibers in the
vicinity of chromaffin cells in rats
(Holgert et al., 1996
;
Lamouche and Yamaguchi, 2001
)
and several fish species (Reid et al.,
1995
). In the perfused rat adrenal gland, it was shown that these
endogenous neuropeptides are released into the perfusate during splanchnic
nerve stimulation (Arimura and Shioda,
1995
). On the basis of studies employing electrical stimulation of
chromaffin tissue, a model has emerged in which the non-cholinergic
neurotransmitters, VIP and PACAP, are preferentially released in mammals
(Wakade et al., 1991
) and
trout (Montpetit and Perry,
2000
) during low-frequency neuronal stimulation. An important
difference between mammals and fish, however, is that the catecholaminotropic
response to VIP/PACAP is mediated by PACAP type receptors in mammals
(Hamelink et al., 2002
) and
VPAC type receptors in trout (Montpetit
and Perry, 2000
). PACAP receptors exhibit a much greater efficacy
for PACAP binding than for VIP binding, whereas VPAC receptors exhibit similar
affinities for both PACAP and VIP. Thus, in mammalian chromaffin cells, PACAP
is about 1000x more potent than VIP (Watanebe et al., 1995;
Geng et al., 1997
;
Fukushima et al., 2001
;
Hamelink et al., 2002
) whereas
in fish, it would appear that VIP and PACAP are equally potent as
catecholamine secretagogues (Montpetit and
Perry, 2000
).
In the present study, desensitization of the nicotinic receptor or blockade of the nicotinic receptor using hexamethonium did not impair the ability of chromaffin cells to secrete catecholamines in response to low-frequency electrical stimulation. Moreover, in the presence of the VPAC receptor blocker VIP6-28, catecholamine secretion evoked by low-frequency electrical stimulation was markedly reduced in the desensitized reparations. Conversely, VIP6-28 was without affect during high-frequency stimulation. Because blockade of all cholinergic (nicotinic and muscarinic) receptors did not affect catecholamine secretion elicited by low-frequency stimulation, it is clear that neuronal release of VIP and/or PACAP and subsequent binding to chromaffin cell VPAC receptors is the principal mechanism underlying catecholamine secretion in desensitized preparations subjected to low-frequency excitation.
Upregulation of non-cholinergic pathways during nicotinic receptor
desensitization
Not only are the non-cholinergic secretagogues VIP/PACAP (this study) and
Ang II (Lapner and Perry,
2001) implicated in sustaining catecholamine secretion
capabilities during periods of nicotinic receptor desensitization, their
ability to evoke catecholamine secretion at such times is actually enhanced.
Indeed, after only 10 min of nicotinic receptor desensitization, the rate of
catecholamine secretion evoked by cVIP or homologous Ang II was approximately
doubled in preparations experiencing desensitization. The mechanism(s)
underlying this interesting phenomenon has not yet been established. However,
owing to the rapidity of the response, it is likely to be a non-genomic
mechanism that may involve modulation of existing membrane receptors or one or
more intracellular signaling pathways.
Conclusion
The results of this study have demonstrated a potential novel mechanism for
sustaining catecholamine secretion during periods of nicotinic receptor
desensitization that involves low-frequency neuronal transmission in nerve
fibres innervating the chromaffin tissue. It remains to be seen, however,
whether this strategy is actually exploited by fish to preserve the acute
humoral adrenergic stress response at times when the nicotinic receptor is in
a refractory state. In future research, it would be interesting to record from
afferent nerve fibres during acute stress to ascertain whether the frequency
of neuronal impulses is modulated according to the sensitivity state of the
nicotinic receptor.
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Acknowledgments |
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