Localisation of VIP-binding sites exhibiting properties of VPAC receptors in chromaffin cells of rainbow trout (Oncorhynchus mykiss)
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 5 March 2003
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Summary |
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Fluorescent labelling of chromaffin cells using aldehyde-induced fluorescence of catecholamines and antisera raised against dopamine ß-hydroxylase (DßH) revealed a distinct layer of chromaffin cells lining the walls of the PCV. Furthermore, specific VIP-binding sites were demonstrated on chromaffin cells using a biotinylated VIP that was previously established as being bioactive. Although multiple labelling experiments revealed that a number of DßH-positive cells were immunonegative for phenylethanolamine N-methyl transferase (PNMT; noradrenaline-containing cells versus adrenaline-containing cells, respectively), labelling of VIP-binding sites was similar to that of DßH labelling, suggesting that all chromaffin cells possess VIP-binding sites. Pharmacological assessment of the VIP-binding sites indicated that they exhibited characteristics of VPAC receptors. Specifically, the labelling of VIP-binding sites was prevented after pre-treatment of PCV tissue sections with unlabelled VIP, PACAP or the specific VPAC receptor antagonist VIP 6-28. By contrast, sections pre-treated with the PAC1 receptor blocker PACAP 6-27 displayed normal labelling of VIP-binding sites. Finally, partial cDNA clones for the trout VPAC1 and VPAC2 receptor were obtained and sequenced. Tissue distribution experiments using RT-PCR revealed the presence of VPAC1 receptor mRNA but not that of the VPAC2 receptor in the PCV tissue. The results provide direct evidence that VIP and PACAP can elicit the secretion of adrenaline from the chromaffin tissue via specific VIP-binding sites that exhibit properties of VPAC receptors. However, the selective secretion of adrenaline by VIP or PACAP cannot be explained by a lack of VIP-binding sites on the noradrenaline-containing cells.
Key words: catecholamine, adrenaline, noradrenaline, dopamine ß-hydroxylase, phenylethanolamine N-methyl transferase, chromaffin cells, VIP, vasoactive intestinal polypeptide, PACAP, pituitary adenylate cyclase-activating polypeptide, fluorescent histochemistry, stress
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Introduction |
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Catecholamines that are secreted into the circulation are stored in
separate populations of adrenaline- and noradrenaline-containing chromaffin
cells, but, unlike in mammals, the chromaffin cells of fish are not organised
into a distinct adrenal gland (reviewed by
Reid et al., 1998). In teleost
fish, chromaffin cells line the walls of the posterior cardinal vein (PCV).
Nerve fibres exhibiting immunoreactivity to VIP and PACAP have been identified
in the vicinity of the chromaffin tissue of fish, including cod (Gadus
morhua), trout (Oncorhynchus mykiss), eel (Anguilla
anguilla) and dogfish (Squalus acanthias)
(Reid et al., 1995
). Because
VIP and PACAP can evoke the secretion of adrenaline from in situ
saline-perfused PCV preparations of trout (Montpetit and Perry,
1999
,
2000
), it is conceivable that
the neuronal control of catecholamine release in fish may include VIP and/or
PACAP in addition to acetylcholine. In contrast to mammals, the effects of VIP
and PACAP appear to be mediated by receptors exhibiting properties of VPAC
receptors in rainbow trout (Montpetit and
Perry, 2000
). In mammalian and amphibian adrenal chromaffin cells,
PAC1 receptors are believed to mediate the VIP/PACAP-elicited
secretion of catecholamines. However, there is evidence that VPAC receptors
may also participate in this response. Indeed, while the adrenal medulla of
mammals exhibits a pronounced expression of PAC1 receptor mRNA,
investigations have also revealed the presence of VPAC1 and
VPAC2 receptor mRNA (Usdin et
al., 1994
; Vaudry et al.,
2000
).
In fish, the secretory profile of catecholamines during cholinergic
stimulation of chromaffin tissue features the release of both adrenaline and
noradrenaline (Nilsson et al.,
1976; Fritsche et al.,
1993
; Reid and Perry,
1995
; Al-Kharrat et al.,
1997
; Gfell et al.,
1997
; Montpetit and Perry,
1999
). By contrast, however, administration of a range of VIP and
PACAP doses selectively causes the release of adrenaline from the chromaffin
cells of all vertebrate species studied to date
(Guo and Wakade, 1994
;
Montpetit and Perry, 2000
).
These findings suggest that the variations in the secretion of adrenaline and
noradrenaline in response to acetylcholine and VIP/PACAP may reflect their
ability to specifically stimulate adrenaline- versus
noradrenaline-containing chromaffin cells. Thus, the goal of the present study
was to determine whether VIP-binding sites are present on rainbow trout
chromaffin cells and if they exhibit a differential spatial localization among
different chromaffin cell subtypes. This was achieved using a combination of
fluorescent histochemical techniques and pharmacological approaches.
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Materials and methods |
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Fluorescent histochemistry
Localization of chromaffin cells and VIP-binding sites within the PCV
tissue
Posterior cardinal veins, in the anterior region of the head kidney, were
dissected and collected in 0.1 mol l1 phosphate-buffered
saline (PBS; adjusted to pH 7.2, containing 0.5 mol l1
NaCl). The catecholamine-containing cell fraction of the PCV was identified by
detection of catecholamines using aldehyde-induced green fluorescence
(Furness et al., 1977;
Lacoste et al., 2001
). Each
PCV was cut into 5-mm pieces and incubated for 24 h at 4°C in a solution
of 4% paraformaldehyde and 0.55% glutaraldehyde (prepared in PBS). Tissues
were washed with PBS and cryoprotected by immersion in a series of PBS
solutions containing 5%, 10%, 15% and 20% sucrose (w/v) for 1 h each. Tissues
were embedded in Cryomatrix (OCT-compound; Shandon, Pittsburgh, PA, USA) and
quick-frozen in liquid N2. Cross-sections (10 µm) were prepared
using a cryostat, cooled to 17°C, and thaw-mounted on
poly-L-lysine-coated slides. Following rehydration in PBS (3x5 min),
stained cells were visualized in the PCV sections using fluorescence
microscopy (see below). Other sections were kept in the dark at
20°C until needed for immunostaining of enzymes known to be
involved in the biosynthesis of catecholamines.
Characterisation of chromaffin cells was confirmed by immunolabelling of
dopamine ß-hydroxylase (DßH) using the methods of Bernier and Perry
(1997). Following rehydration
in PBS, sections were incubated with 5% bovine serum albumin (BSA; ICN
Biomedical, Aurora, OH, USA) in PBS for 1 h. Sections were then incubated for
12 h with mouse monoclonal anti-DßH (dilution 1:500; Chemicon, Temecula,
CA, USA) and further incubated for 2 h with the Cy3-conjugated donkey
anti-mouse secondary antibody (dilution 1:400; Jackson Immunoresearch
Laboratories, West Grove, PA, USA). Sections were then examined after a final
rinse in PBS (3x5 min).
To determine whether VIP-binding sites were differentially distributed amongst the different chromaffin cell subtypes, sections were triple labelled for DßH (an enzyme found in all chromaffin cells), phenylethanolamine N-methyltransferase (PNMT; an enzyme found only in adrenaline-containing cells) and VIP-binding sites, according to the protocols described below. Following rehydration in PBS (3x5 min), sections were reduced in NaBH4 (3x5 min), to decrease autofluorescence, and immunolabelled for DßH as described above. Sections were then incubated in rabbit polyclonal anti-bovine PNMT (dilution 1:100; ProtosTech International, New York, NY, USA) and fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit secondary antibody (dilution 1:200; Jackson Immunoresearch Laboratories) using the same protocol as for DßH immunolabelling.
To visualise VIP-binding sites within the PCV, tissue sections (previously immunostained for DßH and PNMT) were incubated with a solution of biotinylated hVIP (2.58x106 mol l1 biotinyl-VIP human, porcine, rat; Peninsula Laboratories Inc., San Carlos, CA, USA) for 2 h and further incubated with Cascade blue-conjugated streptavidin (dilution 1:500; Jackson Immunoresearch Laboratories) for an additional 2 h. Stained cells were then visualised in the PCV tissues using fluorescence microscopy.
Receptor pharmacology
These experiments were performed to evaluate the pharmacological properties
of the VIP-binding sites. Sections were reduced in NaBH4 (3x5
min). VIP-binding sites (assumed to be PAC1 or VPAC receptors) were
then saturated by incubating sections with solutions containing excess VIP
receptor agonist ckVIP (105 mol l1 chicken
VIP; Peninsula Laboratories Inc.), VIP receptor antagonist hVIP 6-28
(105 mol l1 human VIP 6-28; Peninsula
Laboratories Inc.), PACAP receptor agonist hPACAP-27 (105
mol l1 human PACAP-27; Peninsula Laboratories Inc.) or the
PACAP receptor antagonist hPACAP 6-27 (105 mol
l1 human PACAP 6-27; Peninsula Laboratories Inc.) for 2 h
each prior to incubation with biotinylated VIP (described above). PACAP 6-27
and VIP 6-28 were chosen on the basis of their potent antagonism of
PAC1 (Robberecht et al.,
1991; Gaspo et al.,
1997
) and VPAC receptors
(Bodanszky et al., 1973
;
Fishbein et al., 1994
),
respectively. Excess amounts were determined from previously established
doseresponse curves for VIP- and PACAP-elicited secretion of
catecholamines (Montpetit and Perry,
2000
). To visualise binding of biotinylated VIP, sections were
then incubated with DTAF (dichlorotriazinyl aminofluorescein)-conjugated
streptavidin (dilution 1:500; Jackson Immunoresearch Laboratories) as
described earlier. In control experiments, tissue sections were incubated for
2 h with PBS prior to labelling of VIP-binding sites. Experiments were
performed on consecutive sections (eight per slide) from the PCV of six
different fish.
Control and specificity experiments
For the aldehyde-induced fluorescence of catecholamines, sections reduced
in NaBH4 (3x5 min) served as a methodological control
(Corrodi et al., 1964).
Essentially, NaBH4 reduces the aldehyde reaction with the monoamine
and eliminates the fluorescence. Heart tissue (lacking chromaffin cells)
processed for the aldehyde-induced green fluorescence of catecholamines served
as a negative control. Specificity of primary antibodies was demonstrated
previously (Reid et al., 1995
;
Lacoste et al., 2001
) and that
of secondary antibodies was confirmed by omission of the primary antibody. To
distinguish specific from non-specific binding of biotinyl-hVIP, VIP receptors
were saturated with an excess of unlabelled ckVIP (105 mol
l1) for 2 h prior to the addition of biotinyl-hVIP.
Background fluorescence was determined by replacing the primary antibodies and
fluorescent probes with PBS.
Fluorescence microscopy
All incubations were performed in a moist sealed chamber at room
temperature (approximately 20°C), consistently using 50-µl aliquots of
the various solutions. Sections were mounted using 30 µl of Vectashield
mounting medium with or without the nucleic acid dye DAPI
(4,6-diamidino-2-phenlidole; Vector Laboratories, Inc., Burlingame, CA, USA).
To preserve the slides, cover slips were sealed with nail polish and stored at
20°C. The PBS, NaBH4 and BSA solutions were freshly
prepared before use.
Tissue sections were visualised with a Zeiss Axiophot photomicroscope with appropriate filters using X10 Ph1, X16 Ph2 or X40 Ph2 Plan Neofluar objectives. Photographs were taken using Kodak MAX iso400-color 35-mm film. The negatives were developed by a local processing centre and the images were processed using a negative scanner (SprintScan 35, Polaroid) and Paint Shop Pro software (version 5.01; JASC Software Inc., Minneapolis, MN, USA).
Determination of catecholamine stores
Measurements were performed to confirm the presence of stored
catecholamines in the PCV tissue. The PCV of different fish (N=6) was
separated from the surrounding kidney tissue and both the vein and the kidney
were divided into thirds (anterior, middle and posterior for each). The
various tissues were placed into pre-weighed microcentrifuge tubes (1.5 ml),
frozen in liquid N2 and stored at 80°C until subsequent
determination of catecholamine levels. The tissues were then thawed and washed
with 1 ml of 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 mmol l1 CaCl2 and 1.25 mmol
l1 KH2PO4; final pH, 7.8). After
removing the saline by aspiration, the tubes were reweighed to determine
tissue masses. Aliquots (1 ml) of 4% perchloric acid containing 2 mg
ml1 EDTA and 0.5 mg ml1 sodium bisulphite
were added to each tube (Nilsson,
1989
), and samples were homogenised with a micro-ultrasonic cell
disrupter. The supernatant was diluted 100-fold with a 4% perchloric acid
solution containing EDTA and sodium bisulphite (as above) and analysed for
catecholamine levels (see below).
In situ saline-perfused PCV preparation
The bioactivity of the biotinylated VIP probe was tested using an in
situ saline-perfused PCV previously described by Montpetit and Perry
(2000). Briefly, the PCV and
the ventricle of the heart were catheterised (Clay-Adams PE 160 polyethylene
tubing) to serve as the inflow and outflow of the perfusion fluid,
respectively. Each preparation was perfused in the anterograde direction for
20 min with modified aerated Cortland saline
(Wolf, 1963
) to allow
stabilisation of catecholamine secretion.
After the stabilisation period, a control (pre-treatment) sample was collected in a pre-weighed microcentrifuge tube to assess basal catecholamine secretion rate. After collection of the pre-sample, a single bolus injection of biotinylated hVIP (2.58x106 mol l1; final volume 0.3 ml) was delivered through a three-way valve connected to the inflow catheter. A period of 1 min was allowed for the drug to be delivered to the chromaffin tissue before post-samples were collected in pre-weighed tubes. In total, five post-samples were collected 1 min, 2 min, 3 min, 4 min and 5 min after intervention. All samples were frozen in liquid N2 after collection and stored at 80°C until determination of catecholamine levels. Perfusate samples were reweighed before catecholamine analysis to permit an estimation of perfusion flow rates and thus allow the calculation of catecholamine secretion rates.
Determination of perfusate and tissue catecholamine levels
Perfusate and tissue adrenaline and noradrenaline levels were determined on
alumina-extracted samples (200 µl) using high-pressure liquid
chromatography (HPLC) with electrochemical detection according to Montpetit
and Perry (2000). The HPLC
consisted of a Varian Star 9012 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). The
extracted samples were passed through an Ultratechsphere ODS-C18
5-µm column (HPLC Technology Ltd, Macclesfield, UK) using Mobile Phase for
catecholamines (Chromosystems GmBh, München, Germany), and the separated
amines were integrated using the Star Chromatography software program (version
4.0; Varian Chromatography Systems, Walnut Creek, CA, USA). Concentrations
were calculated relative to appropriate standards and with
3,4-dihydroxybenzylamine hydrobromide as an internal standard in all
assays.
Cloning of VPAC1 and VPAC2 receptor partial
cDNAs
Partial cDNA clones corresponding to the third and seventh transmembrane
domains (TMDs) of the putative trout VPAC1 and VPAC2
were obtained based on PCR amplification using degenerate primers
(Chow, 1997). Briefly,
degenerate primers were designed according to conserved regions within the
third and seventh TMD of the VPAC (VPAC1 and VPAC2)
receptors of various vertebrates. Approximately 5 µg of total RNA from
trout brain was reverse-transcribed into cDNA using
oligo-(deoxythymidine)1218 primer and SuperScript II reverse
transcriptase (Gibco-BRL Life Technologies, Burlington, Ontario, Canada) in a
20-µl mixture. PCR amplification of first strand cDNA (1 µl) was
performed in 50µl reaction mixtures containing 0.2 µmol
l1 of each primer (forward and reverse), 2 mmol
l1 MgCl2, 0.2 mmol l1 of each
dNTP and 2.5 units of Taq DNA polymerase (Gibco-BRL Life Technologies) in
10x PCR buffer (supplied with the enzyme). Following a denaturation step
at 94°C for 5 min, the reaction was subjected to 34 cycles of 94°C for
40 s, 60.5°C for 40 s and 72°C for 40 s, with a final extension for 10
min at 72°C in a thermal cycler (Eppendorf Mastercycler).
For the first round of amplification, 1 µl of brain cDNA was used as template with the following primer pairs: VPAC F2 (FOR) 5'-TTCTGGCTKCTRGTGGAAGG-3' and VPAC R7 (REV) 5'-ACCACAAADCCCTGRAADGABCC-3'. PCR yielded single products of 490 bp. Validity of the PCR products was assessed on the basis of a semi-nested PCR amplification of the products obtained in the initial PCRs. For the second round, 1 µl of the first PCR reaction was used as template with the following primer pairs: VPAC F3 (FOR) 5'-GAGMGGAARTAYTTCTGGKGGTACAT-3' and the VPAC R7 (REV) primer (see above). Amplification profiles were performed as described above. Each reaction was submitted to electrophoresis on a 1.25% agarose gel stained with ethidium bromide. Gels were visualised under ultraviolet light (BioRad Chemi Doc attached to a camera) and digital images processed using commercial software (Quantity One software, version 4.1.1; BioRad).
PCR products of interest were agarose gel purified (GenElute Gel purification kit; Sigma, St Louis, MO, USA) and cloned into pCR2.1 vector (TOPO TA Cloning kit; Invitrogen, Burlington, Ontario, Canada). Plasmids from selected positive clones were purified using GeneElute Plasmid purification kit (Sigma) or Promega Wizard kit (Fisher Scientific Ltd, Nepean, Ontario, Canada) and the DNA was sequenced on both strands using Li-Cor 4200L DNA sequencer technology (Canadian Molecular Research Services, CMRSinc; Ottawa, Ontario, Canada). Of the different positive clones that were analysed, two groups could be identified after sequencing and determination of the highest nucleotide sequence identity with other known VPAC1 and VPAC2, respectively. As such, they were termed trout VPAC1 and trout VPAC2 receptor cDNA clones, respectively.
Sequence analysis
GenBank searches were performed with the standard BLAST algorithms at the
National Center for Biotechnology Information (NCBI) using the default
settings (Altschul et al.,
1997). Sequence alignments were carried out on derived protein
sequences using the default settings in CLUSTAL W with DNAMAN software (Lynnon
Biosoft, Quebec, Canada). The same software was used for sequence editing and
translation. All sequences used for BLAST searches and alignments were edited
to include only the area of the trout VPAC1 and VPAC2
receptor sequence, which was amplified in between the primer sites used to
obtain the clones.
Tissue distribution of VPAC1 and VPAC2 receptors by RT-PCR
Total RNA was isolated from various trout tissues (brain, PCV, kidney,
gill, spleen, heart, liver, intestine and blood) and reverse-transcribed as
described above. 1 µl of first strand product was then used as template for
PCR. PCR was performed using sequence-specific primers. The reaction
conditions consisted of 5 min of denaturation at 94°C, followed by 40 s at
94°C, 40 s at 62°C and 40 s extension at 72oC for 34
cycles, and a 10 min extension at 72°C on the final cycle. Furthermore,
ß-actin control RT-PCR was performed to verify the quality and integrity
of first strand cDNAs produced from the various tissues. The reaction
conditions were similar except for 40 s annealing at 55°C. Minus template
and single primer control PCRs were also performed.
Statistical analysis
The data are presented as means ± 1 S.E.M. Where appropriate, the
data were statistically analysed by a two-way repeated-measures analysis of
variance (ANOVA) followed by Dunnett's test for comparison with
pre-stimulation values. The fiducial limits of significance were set at 5%.
All statistical tests were performed using a commercial statistical software
package (SigmaStat version 2.03).
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Results |
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Multi-labelling experiments revealed that only a small number of DßH-positive cells were immunonegative for PNMT (Fig. 3). This finding indicates that, in the chromaffin tissue of trout, there are relatively few numbers of cells containing only noradrenaline. Labelling with biotinylated VIP revealed that all DßH-positive (chromaffin) cells appeared to possess VIP-binding sites. Moreover, the pattern of labelling of VIP demonstrated that VIP-binding sites were present on both PNMT-positive (adrenaline-containing) and PNMT-negative (noradrenaline-containing) chromaffin cells.
|
Control tissue sections exhibited dull non-specific autofluorescence in comparison with the bright specific fluorescence illustrated in Figs 1, 3. Excess unlabelled ckVIP eliminated the specific fluorescence obtained using the streptavidinbiotinylated VIP probe complex on chromaffin cells (see below). Incubation of sections without primary antisera did not produce any specific labelling.
To confirm that biotinylated VIP was indeed binding to VIP receptors, its bioactivity was tested using an in situ saline-perfused PCV preparation. Bolus injection of biotinylated VIP caused a significant increase in the rate of secretion of adrenaline; noradrenaline secretion was unaffected (Fig. 4). Administration of saline did not cause the release of either catecholamine.
|
Pharmacological assessment of VIP-binding sites
To assess the chromaffin cell VIP-binding sites pharmacologically, sections
of the PCV were treated with non-conjugated VIP and PACAP receptor
agonists/antagonists prior to incubation with biotinylated VIP for
visualisation of binding sites (Fig.
5). While prior treatment with the VIP receptor agonist ckVIP, the
VIP receptor antagonist VIP 6-28 and the PACAP receptor agonist hPACAP-27
eliminated the fluorescence obtained with the DTAF-conjugated
streptavidinbiotinylated VIP complex, treatment with the PACAP receptor
antagonist PACAP 6-27 had no effect on the binding of biotinylated VIP.
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Cloning and sequence analysis of the trout VPAC1 and
VPAC2 receptor partial clones
Although an apparently single 490-bp cDNA fragment was amplified following
PCR with the degenerate primers designed against the VPAC receptors, two
different clones of similar sizes were ultimately isolated. Sequence analyses
of the DNA inserts revealed that the partial sequences corresponded to
VPAC1 andVPAC2 receptors. As such, the partial cDNA
clones were subsequently termed trout VPAC1 and trout
VPAC2, respectively. A BLASTn search of the GenBank database using
the partial sequences of the trout clones produced matches corresponding to
receptors belonging to the VIP/PACAP/glucagon/secretin/parathyroid/calcitonin
receptor family. For the trout VPAC1 partial cDNA clone, the best
match was to the goldfish VPAC1 receptor (E value,
1029). At the protein level
(Fig. 6), the trout sequence
displayed highest amino acid identity (88%) with the goldfish VPAC1
receptor. For the trout VPAC2 receptor partial cDNA clone, the best
match (BLASTn search) was to the rat/mouse/human VPAC2 receptor (E
values, 104). Each of the VPAC2 receptor matches
showed approximately 60% identity with the trout VPAC2
sequence.
|
Sequence alignments of the trout sequences indicated that they are
homologous to their vertebrate counterparts. Visual inspection of the deduced
amino sequences of the trout partial clones
(Fig. 6) revealed motifs that
are common to VPAC1 and VPAC2. A signature motif for the
VPAC1 receptor was identified at a unique cysteine residue within
TMD 5, just preceding the `IIRIL' motif. A consensus phosphorylation sequence
for protein kinase C is also located in the 2nd intracellular loop between
TMDs 3 and 4 of VPAC1 receptors
(Chow 1997;
Chow et al., 1997
). The
conserved `SE-R/K' motif is also found in several other members in the same
receptor family, including the secretin, GHRH (growth hormone releasing
hormone), glucagon and PTH (parathyroid hormone) receptors. Another motif, the
`PDI I/V' found only in VPAC1, VPAC2 and PAC1
receptors between the putative TMD 5 and TMD 6
(Chow et al., 1997
), was also
present in the trout sequences. The `RLA K/R' motif immediately in front of
TMD 6 also matches the consensus found in other members of G-protein-coupled
receptors.
Tissue distribution of VPAC1 and VPAC2
receptors by RT-PCR
Sequence-specific primers were designed for each receptor. Trout
VPAC1 receptor mRNA was found in brain, liver and intestine.
Additionally, while less-prominent products were observed in the PCV, kidney,
gill, spleen and heart, no product was observed in blood
(Fig. 7). For trout
VPAC2 receptor mRNA, products were observed in the brain, spleen,
heart, liver and intestine. No products were evident in the PCV, kidney, gill
or blood (Fig. 7).
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Discussion |
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In teleost fish, catecholamines that enter the circulation are secreted
from chromaffin cells that are located within the walls of the PCV in the
region of the head kidney (Reid et al.,
1998). The distribution of chromaffin cells in a discrete layer
within the walls of the PCV supports the results of several previous
histological studies (Reid et al.,
1995
; Furimsky et al.,
1996
; Bernier and Perry,
1997
). The pattern of labelling suggests that the majority of
chromaffin cells form aggregates adjacent to the lumen of the PCV, although
individual cells can also be observed away from these groupings.
In vertebrates, a variety of studies have demonstrated that adrenaline and
noradrenaline are stored, at least in part, in separate populations of
chromaffin cells. The existence of separate noradrenaline- and
adrenaline-containing cells has been previously demonstrated in teleost fish
(Mastrolia et al., 1984;
Gallo et al., 1993
;
Kloas et al., 1994
;
Reid et al., 1995
;
Abelli et al., 1996
). In the
present study, the observation that some of the chromaffin cells were
immunopositive for DßH but not for PNMT confirmed the presence of a
sub-population of chromaffin cells containing only noradrenaline. However, the
vast majority of the chromaffin cells in the PCV were PNMT-immunoreactive.
Because PNMT catalyses the methylation of noradrenaline to adrenaline, its
presence is indicative of adrenaline-containing chromaffin cells. This finding
is consistent with previous results demonstrating that adrenaline is the more
abundant catecholamine in trout chromaffin tissue
(Reid et al., 1995
).
The concentration of catecholamines within the chromaffin tissue and the
extent of their release differ greatly within and between fish species
(Reid et al., 1998). Yet, the
fact that different secretagogues can preferentially induce the secretion of a
particular catecholamine implies an ability to specifically stimulate
subpopulations of chromaffin cells. For example, administration of cholinergic
agonists to in situ preparations causes the release of adrenaline and
noradrenaline (Nilsson et al.,
1976
; Opdyke et al.,
1983
; Al-Kharrat et al.,
1997
; Gfell et al.,
1997
; Montpetit and Perry,
1999
; Montpetit et al.,
2001
). By contrast, while the administration of angiotensin II
elicits predominantly the secretion of adrenaline in fish
(Bernier and Perry, 1997
;
Bernier et al., 1999
),
injection of natriuretic peptide in dogfish (Squalus acanthias)
causes the release of noradrenaline only
(McKendry et al., 1999
;
Montpetit et al., 2001
).
However, because the results of the present study demonstrated the presence of
VIP receptors on both types of chromaffin cells, the selectivity of VIP and
PACAP for adrenaline secretion cannot be explained by differential receptor
abundance on the chromaffin cell subtypes. Nor can the greater numbers of
PNMT-positive chromaffin cells explain the selective secretion of adrenaline.
Indeed, although the PNMT-positive cells are referred to as `adrenaline cells'
(Reid et al., 1995
,
1998
), they also contain and
secrete noradrenaline, albeit at lower levels.
VIP and PACAP exert tissue-specific effects by interacting with three
distinct receptors (Harmar et al.,
1998). Two of these receptors, VPAC1 and
VPAC2, bind VIP and PACAP with equal affinity, whereas a third
receptor, PAC1, preferentially binds PACAP. To characterise the
receptors that mediate the stimulatory effects of VIP and PACAP on
catecholamine release from trout chromaffin cells, several pharmacological
approaches were used in a previous study
(Montpetit and Perry, 2000
).
Exogenous administration of a range of doses of VIP and PACAP to in
situ perfused PCV preparations caused the release of adrenaline in a
dose-dependent manner. Evaluation of the effective dose eliciting 50% of the
maximal secretion (ED50) revealed that VIP and PACAP elicited
release of adrenaline with comparable potencies. Furthermore, neuronal
stimulation (low frequency) or adrenaline secretion in response to VIP or
PACAP was inhibited in the presence of the VPAC receptor antagonist VIP 6-28
(Montpetit and Perry, 2000
)
while being unaffected by the PAC1 receptor antagonist PACAP 6-27.
The results of the present study, employing histochemistry in conjunction with
prior application of receptor agonists and antagonists, support the findings
of the previous investigation. Indeed, labelling of VIP-binding sites was
prevented in tissues pre-treated with unlabelled VIP, VPAC receptor antagonist
and PACAP but not with PAC1 receptor antagonist. Together, these
results argue for a role of VPAC receptors regulating chromaffin cell activity
in the rainbow trout.
In the present study, different forms of VIP were used to visualise and
characterise receptors. The primary sequences of VIPs share considerable
sequence similarity among vertebrates, with only minor amino acid changes
having been reported. Indeed, the VIP amino acid sequence of fish (dogfish,
trout and cod), amphibians, reptiles and birds differs from the mammalian VIP
at only 45 positions (Nilsson,
1975; Dimaline et al.,
1987
; Thwaites et al.,
1989
; Holmgren and Jensen,
1994
; Wang and Conlon,
1995
). While the trout PACAP amino acid sequence has not yet been
ascertained, PACAPs among other vertebrates (rat, sheep, mouse, human,
amphibian, chicken and teleost fish) also share a high degree of sequence
similarity (Wong et al.,
1998
). Results indicate that species variations of VIP and PACAP
involve conservative substitutions that do not affect the biological
activities of the peptides (Lundin and
Holmgren, 1984
; Dimaline et
al., 1987
; Aldman and Holmgren,
1992
; Chow, 1997
;
Wong et al., 1998
;
Alexandre et al., 1999
;
Montpetit and Perry, 2000
;
Hoo et al., 2001
).
Whether or not trout VPAC receptors respond typically to VIP, PACAP and
receptor antagonists is not known. Recently, goldfish VPAC1 and
PAC1 receptors were cloned and functionally characterised
(Chow, 1997;
Chow et al., 1997
;
Wong et al., 1998
). Using
heterologous VIPs and PACAPs, the VPAC and PAC1 receptors responded
in an expected manner based on the pharmacological characteristics of these
receptors (Chow, 1997
;
Wong et al., 1998
). In the
present study, three partial clones exhibiting sequence properties of the
VPAC1, VPAC2 and PAC1 receptors (data shown
for VPAC receptor clones only) were obtained from trout brain. It would appear
then that, as in higher vertebrates, trout possess all three receptors.
Tissue distribution experiments using RT-PCR demonstrated the presence of VPAC1 receptors, but not VPAC2 receptors, in the PCV tissue. These preliminary results suggest that, in trout, the chromaffin cell VPAC receptors mediating adrenaline secretion are of the VPAC1 subtype. However, the results of the RT-PCR experiments cannot identify the source of cells expressing the VPAC1 mRNA. Unlike in mammals, fish chromaffin cells are not organised into a distinct gland. Indeed, the heterogeneous populations of cells within the PCV have made it difficult to isolate pure populations of chromaffin cells from the tissue. Therefore, detection of these receptors specifically on chromaffin cells could not be performed.
The inability of prior studies to clone the VPAC2 receptor in
lower vertebrates has led to the suggestion of the existence of a unique VPAC
receptor in these species (Vaudry et al.,
2000). However, recently, a VPAC2 receptor was cloned
and functionally characterised in the frog (Rana tigrina rugulosa;
Hoo et al., 2001
). By
isolating partial clones for the trout VPAC1, VPAC2 and
PAC1 receptors, we have provided direction for future
investigations into the VIP and PACAP system and the neuronal control of
catecholamine release in fish. Although it seems likely that the partial cDNA
clones reported in this paper also correspond to specific VPAC1,
VPAC2 and PAC1 receptor types, definite identification
of these receptors must await the isolation of complete gene sequences and
functional characterisation of the encoded proteins.
The results of this and an earlier study
(Montpetit and Perry, 2000)
reveal that both VIP and PACAP can potentially contribute to catecholamine
secretion from trout chromaffin cells. However, whether or not these
neuropeptides actually contribute significantly to the afferent limb
of the adrenergic stress response in vivo has yet to be determined.
Indeed, the release of these neuropeptides during sympathetic activation of
the chromaffin cell in response to acute stressors such as hypoxia, air
exposure or handling remains to be confirmed. However, we have recently shown
that cholinergic and VPAC receptor antagonists can inhibit catecholamine
secretion during electrically evoked neuronal stimulation of the chromaffin
cells in trout, thus implicating the release of acetylcholine, VIP and/or
PACAP under these conditions (Montpetit
and Perry, 2000
). Further research should now address the relative
contribution of VIP and PACAP in vivo to establish the functional
significance of these neuropeptides (and their receptors) in the control of
catecholamine secretion in fish.
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Acknowledgments |
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References |
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