Chem. Senses 28: 207-218,
2003
© Oxford University Press 2003
Olfactory Sensitivity to Catecholamines and their Metabolites in the Goldfish
1 Centro de Ciências do Mar, Universidade do Algarve, Campus de Gambelas, 8000-810 Faro 2 Departamento de Biologia, Universidade de Évora, Apartado 94, 7001 Évora Codex, Portugal
Correspondence to be sent to: Peter Hubbbard, Centro de Ciências do Mar, Universidade do Algarve, Campus de Gambelas, 8000-810 Faro, Portugal. e-mail: phubbard{at}ualg.pt
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Abstract |
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The current study assessed the olfactory sensitivity of the goldfish (Carassius auratus L.) to the catecholamines, their immediate precursors and metabolites by use of the electro-olfactogram (EOG). The olfactory system of the goldfish was found to be sensitive to both adrenaline and dopamine with thresholds of detection of 10-7.8 and 10-7.9 M respectively, but less so to noradrenaline (threshold of detection 10-6.3 M). The 3-O-methoxy metabolites (metadrenaline, normetadrenaline and 3-O-methoxytyramine) evoked larger amplitude EOGs than the non-metabolized form with lower thresholds of detection. However, the olfactory system was less sensitive to the amino acid precursors L-tyrosine and L-DOPA, and markedly less so to the
-deaminated metabolites (3,4-dihydroxyphenyl glycol,
3,4-dihydroxy mandelic acid and dihydroxyphenyacetic acid). Sensitivity to
metabolites, both -deaminated and 3-O-methoxylated, was
similar to the -deaminated forms. Cross-adaptation studies suggested
that, while there is some degree of commonality of the receptor mechanisms
with L-tyrosine and L-serine, a proportion of the
response to the catecholamines is due to distinct receptor subtypes.
Similarly, the 3-O-methoxy metabolites also had (a) separate receptor
mechanism(s), although, again, there was overlap with the adrenaline/dopamine
receptor site(s). Presence of the -adrenoreceptor antagonist prazosin
or the peripheral DA2 dopamine receptor antagonist domperidone
caused partial attenuation of the EOG responses to adrenaline and dopamine,
but had much less effect on the responses to their 3-O-methoxy
metabolites. The ß-adrenoreceptor antagonist sotalol had no such effect.
This suggests that the olfactory catecholamine receptors are structurally and
functionally distinct from systemic adreno- and dopamine receptors. The
current study raises the possibility that release of catecholamines or their
3-O-methoxy metabolites to the water may play a role in chemical
communication.
Key words: adrenaline, dopamine, electro-olfactogram (EOG), metabolites, teleost
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The olfactory system of many species of fish has been shown to be sensitive to amino acids (Hara, 1994
;
Sorensen and Caprio, 1998).
These are detected by at least four main receptor types
(Caprio and Byrd, 1984;
Friedrich and Korsching, 1997),
recognizing amino acids with neutral (subdivided into short chain and long
chain), basic or acidic side chains. Individual amino acids may be recognized
centrally by the pattern of activity evoked in the olfactory bulb in a
combinatorial manner (Friedrich and
Korsching, 1997). However, it is the presence of the -amino
group which is of prime importance in conferring olfactory potency
(Lipschitz and Michel, 1999).
Another group of biologically important compounds with an -amino group
is the catecholamines: the olfactory potency of this group in fish is
unknown.
Adrenaline, dopamine and noradrenaline have well characterized roles as
neurotransmitters, neuromodulators, paracrine agents and circulating hormones.
Given that the plasma concentrations of catecholamines (principally
adrenaline, but to a lesser extent noradrenaline) undergo massive and rapid
increases in response to various forms of stress in fish
(Wendelaar Bonga, 1997;
Reid et al., 1998),
and these in turn have very marked effects on many aspects of physiology,
especially cardiovascular effects (Fabbri
et al., 1998), it is possible that a proportion of these,
or their metabolites, may be released into the water where they may have some
communicative function. To test this hypothesis, we investigated the olfactory
sensitivity of goldfish (Carassius auratus, L., a freshwater cyprinid
inhabiting still-waters and slow-moving reaches of rivers) to the
catecholamines, their precursors (L-tyrosine and L-DOPA)
and their metabolites (both 3-O-methoxylated and -deaminated
forms; Figure 1) by use of the
electro- olfactogram (EOG).
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The half-life of circulating catecholamines is very short: they are rapidly
metabolized to inactive forms. In mammals, catecholamines are firstly
metabolized by the enzymes monoamine oxidase (MAO) and
catechol-O-methyltransferase (COMT) which convert them to the
corresponding aldehydes (e.g. DHPG, Figure
1) and 3-O-methyl metabolites (e.g. metadrenaline,
Figure 1) respectively
(Cooper et al., 1996).
The aldehydes are rapidly oxidized to the corresponding acid (e.g. DHMA,
Figure 1). As both MAO and COMT
are relatively non-specific, both enzymes can act on the products of the other
to produce 3-O-methylated and -deaminated metabolites (e.g.
HVA and VMA, Figure 1). These
are the chief metabolites excreted via the urine. Whether the same pathways
predominate in fish is much less well studied, although the limited data
available suggest that they do (Mazeaud
and Mazeaud, 1973b; Sloley
et al., 1992). For example, a trout MAO has been cloned
(Chen et al., 1994).
However, the rates and routes of release of these metabolites are not yet
known (Mazeaud and Mazeaud,
1973a).
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Experimental animals
Goldfish of both sexes (nose to fork length: 76–204 mm; weight: 8–207 g) were kept outside in 1000 l tanks under semi-natural conditions (i.e. under natural photoperiod and temperature variation) and fed once or twice a day (depending on temperature) on commercial pond-fish food (TetraPond Pond Sticks®, TetraWerke, Melle, Germany).
Recording of the EOG
The method used for recording EOGs from goldfish has been described in
detail previously (Hubbard et
al., 2002). Briefly, the goldfish were anaesthetized by
immersion in water containing 3-aminobenzoic acid ethyl ester (MS222; 80 mg/l)
and immobilized with intramuscular injection of neuromuscular blocker
(gallamine triethiodide; 1 mg/kg in 0.9% NaCl), and maintained with water
flowing over the gills (containing 40 mg/l MS222) in a padded V-clamp. The
flap of skin overlying the nostril was removed and the recording electrode
placed near the raphe, between two adjacent olfactory lamellae—a site
previously shown to give the best response to known odorants (e.g. amino
acids). The reference electrode was placed lightly on the skin of the head and
connected to earth via the headstage of the amplifier. The DC voltage signal
was amplified (gain x 1000), filtered above 50 Hz, digitized and
recorded by computer running the appropriate software. Electrodes were made
from borosilicate glass micropipettes containing 4% agar in 0.9% NaCl,
connected to solid-state electronics via an Ag/AgCl pellet.
Stimuli preparation and delivery
All odorants were bought from SigmaAldrich Chemical Co. (Madrid, Spain). All stimuli were dissolved in distilled water (10-3 M), although some catecholamines were previously dissolved in a small volume of 0.1 M HCl prior to this, and stored aliquoted at –20°C. All test solutions were made up immediately prior to use (to avoid possible oxidation of the catecholamines) in dechlorinated, charcoal-filtered tap-water; the same water used to irrigate the nostril (see below) in acid-washed glassware. The nostril of the fish was continuously irrigated via a glass tube, the opening of which was held immediately above the olfactory rosette, at a flow-rate of 6 ml/min under gravity with dechlorinated, charcoal-filtered tap-water. Odorant containing solutions were introduced into this flow via a computer-controlled three-way solenoid valve for a period of 10 s.
Experimental design
The olfactory responses of the goldfish to the catecholamines (dopamine,
adrenaline and noradrenaline) were first assessed by concentration/response
experiments. These were carried out by stimulating the olfactory epithelium
with increasing concentrations (10-9–10-4 M),
allowing at least 1 min to elapse between stimuli to ensure washout of the
stimulus from the nasal cavity, and to counteract any possible adaptation.
Once it was clear that goldfish had an acute olfactory sensitivity to dopamine
and adrenaline, the sensitivity to a range of precursors and metabolites
(Figure 1) was assessed using
the same experimental approach. Having established the most potent odorants
(dopamine, adrenaline and their 3-O-methoxy metabolites; 3-MT and
metadrenaline, respectively) within this group of compounds, the olfactory
selectivity was investigated by means of cross-adaptation experiments (e.g.
Lipschitz and Michel, 1999).
The adapting solutions (all at 10-5 M) were continuously superfused
over the olfactory epithelium for at least 1 min before the odorant in
question (in the presence 10-5 M adapting odorant) was applied as
stimulus. Ideally, the concentration of each odorant should be chosen to evoke
EOGs of similar magnitude. However, due to variability in the relative
magnitude of the responses to the amino acids (L-tyrosine and
L-DOPA) and catecholamines among different fish, this was not
possible. The ‘self-adapted control’ (SAC) consisted of the
odorant in question (at 2 x 10-5 M; the same total
concentration of odorant as in the test solutions) against a background of
10-5 M. The amplitudes of these responses were compared to the
means of controls (10-5 M of each odorant alone) run before and
after the cross-adaptation experiments. Finally, the possibility that the
olfactory responses are mediated by ‘conventional’ dopamine and/or
adreno- receptors was investigated by continually superfusing the olfactory
epithelium with a DA2 dopamine receptor antagonist (domperidone),
an -adrenoreceptor antagonist (prazosin) or a ß-adrenoreceptor
antagonist (sotalol), and comparing the responses to the catecholamines, their
3-O-methoxy metabolites and L-tyrosine (plus
L-serine at 10-5 M, and the structurally unrelated
steroid goldfish pheromone, 4-pregnene–17,20ß-diol-3-one
(17,20ß-P at 10-9 M), as controls) in the presence of these
antagonists (10-7 and 10-6 M) to those under control
conditions (absence of antagonist). Only one antagonist was used in each
experiment, and the order of treatments was randomized.
Data treatment and statistical analysis
The amplitude of the initial sharp peak of the EOG was measured in
millivolts. This was then blank-subtracted (the amplitude of EOG response to
water treated in the same way as the odorant solutions but containing no
odorant: this was generally <10% of the amplitude of the response to
10-5 M L-serine). To reduce the variation of responses
of different fish, this was then normalized to a previously run
‘standard’ of 10-5 M L-serine, blank
subtracted in the same way. These standard responses were run at regular
intervals throughout the experiment (every 10–20 min). Thresholds of
detection were calculated by linear regression on log-transformed data using
the formula log(N + 1.5) = a log C + b,
where N is the normalized response, C is the concentration,
and a and b are constants. The threshold of detection is
then the value for x where y = 0.1761 (i.e. log 1.5;
N = 0). For each odorant, only the concentrations that elicited EOG
responses significantly greater than zero were used. This revealed that the
odorants used could be divided into groups according to chemical structure,
discarding the outliers noradrenaline and normetadrenaline (see below). Thus
the data for adrenaline and dopamine were pooled (catecholamines), likewise
3-MT and metadrenaline (3-O-methoxy metabolites), DOPAC and DHMA
(-deaminated metabolites), HVA and VMA (-deaminated and
3-O-methoxy metabolites) and L-DOPA and
L-tyrosine (amino acids). The aldehydes, DHPG and MHPG, were not
included as they are short-lived intermediates and rapidly metabolized to
their respective acid forms. Linear regressions (slopes and line elevations)
were compared within and between groups of compounds by Student's
t-test (two lines) or by ANOVA followed by Tukey's test
(Zar, 1996). Pooled,
normalized data from the cross-adaptation experiments were subjected to
repeated-measures ANOVA followed by Dunnett's test (SigmaStat 2.0, Jandel
Scientific), comparing to both controls and self-adapted controls. Pooled,
normalized data from the antagonist experiments were compared by
repeated-measures ANOVA followed by Dunnett's test for each concentration of
odorant. In all cases, a P value of <0.05 was considered
significant.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Olfactory sensitivity to catecholamines
The olfactory system of the goldfish proved to be highly sensitive to
adrenaline and dopamine (Figure
2A-C
, but less so to
noradrenaline (
Figure 2D
),
evoking typical, biphasic fish EOGs with a rapid negative deflection at
stimulus onset, followed by a period of adaptation during which the EOG fell
to approximately half of the peak amplitude
(
Figure 2A
). After the end of
the stimulus period the potential returned to baseline levels within a few
seconds. The amplitude of the EOG responses was strongly concentration
dependent, increasing by a factor of approximately two for each tenfold
increase in odorant concentration. Linear regression of log-transformed data
revealed highly significant relationships [adrenaline: F(1,162) =
371.97, P < 0.001, R2 = 0.697 ± 0.080,
a = 0.086 ± 0.005, b = 0.845 ± 0.027;
dopamine: F(1,158) = 312.55, P < 0.001,
R2 = 0.664 ± 0.082, a = 0.082 ±
0.005, b = 0.822 ± 0.028; noradrenaline: F(1,39) =
105.39, P < 0.001, R2 = 0.730 ± 0.074,
a = 0.146± 0.014, b = 1.094 ± 0.072; values
± SEM). Thresholds of detection were estimated as 10-7.8 M
for adrenaline, 10-7.9 for dopamine and 10-6.3 M for
noradrenaline. No significant differences were found between the slope and
elevation of the linear regression lines calculated for adrenaline and
dopamine [Tukey's test, q(
,3) = 0.854, P > 0.05
for comparison among slopes; q(,3) = 0.105, P >
0.05 for comparison among elevations], whereas those parameters in the linear
regression for noradrenaline were significantly different from the other two
catecholamines [comparison among slopes: dopamine, q(,3) =
5.616, P < 0.001; adrenaline, q(,3) = 5.283,
P < 0.001; comparison among elevations: dopamine,
q(,3) = 4.754, P < 0.01; adrenaline,
q(,3) = 4.833, P < 0.01]. Thus the olfactory
system of the goldfish is equally sensitive to adrenaline and dopamine but
significantly less so to noradrenaline.
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Olfactory sensitivity to catecholamine metabolites
The 3-O-methoxy metabolites, metadrenaline and 3-MT, also proved
to be potent odorants to goldfish, again evoking typical fish EOGs in a
concentration-dependent manner (Figure
2A). Significant linear relationships were obtained for
metadrenaline [F(1,111) = 248.09, P < 0.001,
R2 = 0.691 ± 0.089, a = 0.099 ±
0.006, b = 0.936 ± 0.039], 3-MT [F(1,114) = 385.79,
P < 0.001, R2 = 0.772 ± 0.073,
a = 0.095 ± 0.005, b = 0.933 ± 0.030) and
normetadrenaline [F(1,41) = 141.70, P < 0.001,
R2 = 0.776± 0.059, a = 0.098±
0.008, b = 0.885 ± 0.045]. The thresholds of detection were
estimated as 10-7.7 M (metadrenaline) and 10-7.9 M
(3-MT). No significant differences were found among the slopes of the
regression lines obtained for the three metabolites [F(3,266) =
0.098, P > 0.05]. Also, no significant differences were found
between the elevations of the linear regression lines calculated for
metadrenaline and 3-MT [Tukey's test, q(,3) = 2.383,
P > 0.05 for comparison among elevations], but the elevation in
the linear regression for normetadrenaline was significantly different from
the other two metabolites [metadrenaline, q(,3) = 3.861,
P < 0.05; 3-MT, q(,3) = 5.670, P <
0.001]: the olfactory system is equally sensitive to 3-MT and metadrenaline,
but less so to normetadrenaline. The elevation of the linear regression line
obtained for the pooled 3-O-methoxy metabolite data was significantly
different from the one obtained with the catecholamines [Tukey's test,
q(,5) = 8.086, P < 0.01] but no significant
difference was found between the slopes [F(5,797) = 0.000004, P >
0.05]. Also, both the slopes and elevations of the linear regression line
obtained for noradrenaline and normetadrenaline differed significantly
(t = 3.159, d.f. = 80, P < 0.01, for comparison between
slopes; t = 3.051, d.f. = 81, P < 0.01, for comparison
between elevations). Thus, in all cases, the sensitivity is greater for the
3-O-methoxy metabolites than the unmetabolized forms.
A significant linear regression was found for the acid metabolites of
adrenaline and dopamine [DHMA: F(1,19) = 9.414, P < 0.01,
R2 = 0.331 ± 0.089, a = 0.073 ±
0.024, b = 0.649 ± 0.120; DOPAC: F(1,21) = 17.741,
P < 0.001, R2 = 0.446 ± 0.064,
a = 0.067 ± 0.016, b = 0.591 ± 0.081], with
detection thresholds estimated as 10-6.5 M for DHMA and
10-6.2 M for DOPAC. No significant differences were found among the
slopes and the elevations of the regression lines obtained for these two
compounds (t = 0.212, d.f. = 41, P > 0.05, for comparison
between slopes; t = 1.228, d.f. = 42, P > 0.05, for
comparison between elevations). Significant differences were found between the
elevations of the regression lines calculated for catecholamines and for the
-deaminated metabolites [Tukey's test, q(
,5) = 28.725,
P < 0.0001], but no significant difference was found between the
slopes [F(5,797) = 0.000004, P > 0.05]. In addition, the
linear regression obtained for the -deaminated metabolite of
noradrenaline [DHPG; F(1,19) = 13.331, P < 0.01,
R2 = 0.412 ± 0.055, a = 0.054 ±
0.015, b = 0.512 ± 0.075] was significantly different from the
one obtained for noradrenaline (t = 4.081, d.f. = 58, P <
0.01, for comparison between slopes; t = 5.905, d.f. = 59, P
< 0.001, for comparison between elevations), and indicated a lower
estimated threshold of detection (10-6.2 M). Thus,
-deamination markedly reduces olfactory potency compared to the parent
compound. The linear regressions obtained for those metabolites both
3-O-methylated and -deaminated [MHPG, F(1,19) =
76.741, P < 0.001, R2 = 0.802 ± 0.068,
a = 0.159± 0.018, b = 1.122 ± 0.092; HVA;
F(1,26) = 47.285, P < 0.001, R2 =
0.645 ± 0.063, a = 0.073 ± 0.011, b = 0.674
± 0.060; VMA, F(1,30) = 32.908, P < 0.001,
R2 = 0.523 ± 0.075, a = 0.068 ±
0.012, b = 0.635 ± 0.067] showed similar thresholds of
detection [F(3,77) = 0.650, P > 0.05] for comparison
among elevations (MHPG 10-6.0 M, HVA 10-6.8 M and VMA
10-6.8 M), but different slopes between MHPG and the other two
compounds [HVA, Tukey's test, q(75,3) = 5.517, P < 0.001;
VMA, q(75,3) = 5.978, P < 0.001]. The sensitivity is
similar, but the amplitude of response is higher for HVA. Addition of the
3-O-methoxy group does not, therefore, restore olfactory potency to
the -deaminated metabolite.
Olfactory sensitivity to precursors
It is well established that the olfactory system of fish is highly
sensitive to amino acids (e.g. Hara,
1994). The catecholamines are derived from the amino acid
L-tyrosine (Figure
1) via L-3,4-dihydroxyphenylalanine
(L-DOPA). As expected, therefore, goldfish had olfactory
sensitivity to both L-tyrosine and L-DOPA
(Figure 2E). Significant linear
relationships were obtained for L-tyrosine [F(1,118) =
202.50, P < 0.001, R2 = 0.632 ± 0.077,
a = 0.072± 0.005, b = 0.834 ± 0.031] and
L-DOPA [F(1,42) = 120.59, P < 0.001,
R2 = 0.742 ± 0.050, a = 0.074 ±
0.007, b = 0.714 ± 0.038], with thresholds of detection
estimated as 10-7.8 and 10-7.3 M for
L-tyrosine and L-DOPA respectively. These two linear
relationships were not significantly different (t = 0.117, d.f. = 70,
P > 0.05, for comparison between slopes; t = 1.155, d.f.
= 71, P > 0.05, for comparison between elevations). The elevation
of the linear regression line obtained for the catecholamines (pooled data of
adrenaline and dopamine) was significantly different from that of the amino
acids [Tukey's test, q(,5) = 20.671, P < 0.0001]
but no significant difference was found between the two slopes
[F(5,797) = 0.000004, P > 0.05): the sensitivity is
greater for the catecholamines than their amino acid precursors.
Cross-adaptation
In the presence of dopamine, metadrenaline, 3-MT and the amino acids L-tyrosine and L-serine (at 10-5 M), the amplitude of EOG responses to adrenaline (10-5 M) was significantly lower than that of controls (Figure 3A). This suggests that part of the response olfactory response to adrenaline is mediated by (a) common receptor mechanism(s) to the other odorants. However, the responses to adrenaline in the presence of L-tyrosine or L-serine were significantly larger than adrenaline self-adapted controls (SACs): the EOG amplitudes of responses to dopamine, metadrenaline and 3-MT were statistically indistinguishable from the adrenaline SACs. This suggests that a large part of the olfactory response to adrenaline is independent of L-serine and L-tyrosine receptors, but most, if not all of the olfactory receptor sites for adrenaline can also be activated by dopamine, metadrenaline and 3-MT. Similarly, the amplitude of EOG responses to dopamine were also significantly attenuated by the presence of the other odorants when compared to controls (Figure 3B), suggesting that there is (a) common receptor mechanism(s) for all these odorants. Nevertheless, responses to dopamine in the presence of L-tyrosine, L-serine and adrenaline were significantly greater than the dopamine SACs. This is indicative of dopamine being detected by a sub-set of different receptor types to L-tyrosine and L-serine. Conversely, dopamine seems to be detected by the same receptor type(s) as 3-MT and metadrenaline. The responses to metadrenaline, however, were greater than metadrenaline SACs in the presence of all odorants other than 3-MT (Figure 3C). Again, all cross-adapted responses were lower than controls. Taken together, this suggests that whilst there is a degree of commonality among the receptor sites for metadrenaline, only 3-MT is capable of competing with metadrenaline for all of these sites. This is supported by the pattern of responses to 3-MT in the presence of the other odorants (Figure 3D); although amplitudes of responses to 3-MT were reduced compared to controls, only the responses in the presence of metadrenaline were similar to 3-MT SACs. Thus it appears that there is/are (a) specific olfactory binding site(s) for 3-O-methylated catecholamines. In contrast, the olfactory responses to L-tyrosine and L-serine were somewhat less attenuated by the presence of the catecholamines. The responses to L-tyrosine (Figure 3E) were only significantly attenuated by the presence of dopamine and L-serine, suggesting a degree of overlap in the receptor mechanisms for these odorants. However, the cross-adapted responses were all larger than L-tyrosine SACs, indicative of (a) L-tyrosine specific binding site(s) at which the other odorants are without effect. In a similar manner, the responses to L-serine (Figure 3F) were only slightly (but significantly) reduced by the presence of the other odorants. The amplitudes of cross-adapted responses were, again, all significantly larger than the L-serine SACs. In a similar way to L-tyrosine, this is indicative of (a) specific olfactory receptor mechanism(s) for L-serine.
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P < 0.05, Taken together, these data suggest a degree of commonality in the receptor sites for these structurally diverse odorants, but indicate that there are specific olfactory catecholamine receptors, of which a sub-population is relatively specific for the 3-O-methoxy metabolites.
Effect of adrenergic and dopaminergic antagonists
Having established that there is a specific olfactory response to the
catecholamines in the goldfish, an attempt was made to pharmacologically
characterize the receptor type(s) responsible by use of the
-adrenoreceptor antagonist prazosin, the ß-adrenoreceptor
antagonist sotalol and the dopamine receptor antagonist domperidone. At
concentrations of 10-7 and 10-6 M, prazosin caused a
clear, concentration-dependent reduction in the amplitude of EOGs evoked by
both adrenaline (Figure 4A) and
dopamine (Figure 4B). This was
more manifest at lower concentrations of agonist (i.e. at equimolar
concentrations to prazosin) where the response was often completely
obliterated. Although a similar trend was seen in the responses to
metadrenaline (Figure 4C), this
failed to reach statistical significance. Similarly, little effect was seen in
the responses to 3-MT (Figure
4D) in the presence of prazosin. Somewhat surprisingly, prazosin
also caused a slight concentration-dependent attenuation of EOG amplitude
evoked by L-tyrosine (Figure
4E) and the structurally unrelated controls, L-serine
and the steroid pheromone 17,20ß-P
(Figure 4F). These data suggest
that prazosin is an effective antagonist at some of the receptor sites to the
catecholamines and amino acids, but provides further evidence that there is a
specific olfactory receptor mechanism for the 3-O-methoxy metabolites
of the catecholamines, at which it is relatively ineffective.
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Perhaps surprisingly, the DA2-receptor antagonist domperidone had a similar effect to prazosin. While domperidone caused a clear, concentration dependent reduction in the EOG amplitude in response to adrenaline (Figure 5A) and dopamine (Figure 5B), such an effect was much less marked in responses to metadrenaline (Figure 5C) and 3-MT (Figure 5D) and only significant at the higher concentration of antagonist (10-6 M). A similar slight effect was seen on the amplitude of responses to L-tyrosine (Figure 5E). Again, an unexpected reduction in amplitude of response to L-serine was found (Figure 5F), but the responses to the steroid odorant 17,20ß-P were unaffected.
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In contrast, the ß-adrenergic antagonist, sotalol, had little or no effect on the responses to adrenaline (Figure 6A), dopamine (Figure 6B), metadrenaline (Figure 6C), 3-MT (Figure 6D) or L-tyrosine (Figure 6E). Only a slight reduction in the amplitude of responses to 10-5 M L-serine was seen (Figure 6F), with a similar trend in the responses to 10-9 M17,20ß-P which failed to reach statistical significance.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Olfactory sensitivity to the catecholamines
To the authors' knowledge, this is the first study to address the olfactory
sensitivity of a fish to the catecholamines or their metabolites. The
olfactory system of the goldfish is clearly sensitive to both adrenaline and
dopamine with thresholds of detection around 10 nM, similar to the most potent
amino acid (L-cysteine) and bile acid (taurocholic acid) in the
zebrafish (Michel and Lubomudrov,
1995), and well within the range of sensitivity of fish in general
to amino acids (10-7–10-9 M)
(Hara 1994;
Sorensen and Caprio, 1998).
However, removal of the -carboxylic acid group (e.g. L-DOPA
to dopamine) actually increases the olfactory potency: the catecholamines are
more potent odorants (in the goldfish, at least) than their amino acid
precursors. Surprisingly, the sensitivity to noradrenaline was much less
acute, both in terms of the amplitude and threshold of the response (nearly
two orders of magnitude higher). This was the first indication that the
olfactory receptor mechanism(s) for adrenaline may not be via conventional
adrenoreceptor(s), as these tend to have similar affinities for both
adrenaline and noradrenaline (Fabbri et
al., 1998), and much lower affinity for dopamine. This
suggests that the olfactory system seems to be able to discriminate—at
the level of the receptor—between catecholamines differing only in the
ß-hydroxyl group and the methylated -amino group. In contrast,
amino acids are believed to be discriminated in a combinatorial manner by the
pattern of activity evoked in the olfactory bulb
(Friedich and Korching, 1997),
presumably integrating the input from a range of at least four types of
olfactory amino acid receptors (Caprio and
Byrd, 1984). The sensitivity to either adrenaline or dopamine
cannot be entirely explained by activation of olfactory neutral amino acid
receptor receptor(s), as cross-adaptation with L-tyrosine (neutral
amino acid with cyclic group) or L-serine (neutral amino acid with
short aliphatic residue) does not completely block the response to the
catecholamines (Figure 3).
Furthermore, the responses to adrenaline and dopamine can be partially blocked
by the -adrenoreceotor antagonist prazosin
(Figure 4) and DA2
antagonist domperidone (Figure
5), whereas these had little effect on the responses to
L-tyrosine.
Olfactory sensitivity to catecholamine metabolites
Goldfish also had a high olfactory sensitivity to the 3-O-methoxy
metabolites of the catecholamines; metadrenaline and 3-MT consistently evoked
larger responses than adrenaline and dopamine, with slightly greater
sensitivity. As the EOG is believed to be a summation of receptor neurone
generator potentials (Scott and
Scott-Johnson, 2002), this is suggestive that more olfactory
receptors are responsive to the 3-O-methoxy metabolites than their
catecholamine precursors. This could be indicative that the
3-O-methoxy metabolites are more important in nature. The
-deaminated metabolites, however, were much less effective odorants,
whether in the aldehyde (e.g. DHPG) or acid (e.g. DHMA) form. Furthermore,
3-O-methylation of any -deaminated metabolites (e.g. HVA, VMA
and MHPG) did not restore their olfactory potency to anywhere near that of the
unmetabolized forms. This strongly suggests that the -amino group is
important in ligand binding/recognition (see below).
Olfactory selectivity
From the cross-adaptation experiments
(Figure 3), it is clear that
both amino acids (L-tyrosine and L-serine) and the
catecholamines share some common olfactory binding sites. The one feature that
all these molecules have in common is the -amino group
(Figure 1). As previously
discussed, the -deaminated catecholamine metabolites have much lower
olfactory potency than their -aminated equivalents. This is in
agreement with Lipschitz and Michel
(1999) who found that removal
of the -amino group in structural analogues of L-arginine
dramatically reduced their effectiveness as odorants in the zebrafish. This
may indicate that there are relatively non-specific olfactory receptors for
compounds with an -amino group that are relatively indifferent to the
structure of the rest of the molecule. However, the cross-adaptation
experiments also suggest that there are olfactory receptors that do not
respond to either L-serine or, more importantly,
L-tyrosine which do respond to the catecholamines or their
3-O-methoxy metabolites: the presence of either amino acid cannot
completely block the response to adrenaline or dopamine, even less
metadrenaline or 3-MT. Thus goldfish must have olfactory receptors relatively
specific to the catecholamines. Surprisingly, a proportion of these receptors
seems unable to discriminate between dopamine and adrenaline (Figure
3A,B).
The olfactory catecholamine receptors must therefore be structurally and
functionally different from ‘conventional’ adrenoreceptors and
dopamine receptors, although they might share a common evolutionary origin
(Berghard and Dryer, 1998):
vertebrate olfactory receptors (Buck,
1996; McClintock,
2000), adrenoreceptors and dopamine receptors all belong to the
G-protein linked, seven transmembrane region super-family of cell-surface
receptors (e.g. Sibley and Monsma,
1992; Aantaa et al.,
1995; Johnson,
1998; Zhong and Minneman,
1999). Furthermore, a proportion of these receptors seems to be
relatively specific for the 3-O-methoxy metabolites (Figure
3C,D).
This contention is supported by the ability of the -adrenoreceptor
antagonist prazosin to competitively block the olfactory response to both
adrenaline and dopamine (Figure
4A,B),
whilst having little or no effect on the responses to metadrenaline and 3-MT
(Figure
4C,D).
Similarly, the dopamine receptor antagonist, domperidone, was also able to
attenuate the olfactory responses to adrenaline and dopamine (Figure
6A,B)
whilst having a much lesser effect on the responses to metadrenaline and 3-MT
(Figure
6C,D).
Somewhat surprisingly, both these antagonists were able to slightly reduce the
response to L-serine (Figures
4F and
6F), while prazosin was also
able to antagonize the response to L-tyrosine and the steroid
17,20ß-P. The ß-andrenoreceptor antagonist sotalol, however, had
very little effect on the olfactory responses to either the catecholamines or
the 3-O-methoxy metabolites
(Figure 5), while slightly
reducing the response to L-serine. The most conservative
explanation for these observations is that there are at least four different
receptor types for the odorants used in the current study; one that recognizes
principally L-serine, one that recognizes principally
L-tyrosine, one that recognizes dopamine and adrenaline plus (to a
lesser extent) the 3-O-methoxy metabolites and, finally, one that is
relatively specific for the 3-O-methoxy metabolites. The first could
be the receptor for neutral amino acids with aliphatic residues, the second
for neutral amino acids with cyclic residues receptor as proposed by Friedich
and Korsching (1997). This is
represented schematically in Figure
7. Prazosin and domperidone appear to principally antagonize the
catecholamine receptor(s). We strongly suspect that there are sub-populations
of receptors within these groups.
|
Possible functional significance of olfaction of catecholamines
The possible functional significance of the ability of the goldfish to
smell catecholamines was not addressed. Nevertheless, given the roles of
circulating adrenaline in fish, it might be involved in the communication of
the alarm response. Evidence exists for a ‘disturbance’ pheromone
in fish which alerts conspecifics to the presence of danger such as predators
(Wisenden et al.,
1995; Chivers and Smith,
1998; Kats and Dill,
1998; Jordão and
Volpato, 2000; Mirza and
Chivers, 2001), without the prerequisite of damage to the sender.
This response can also occur between different species. The current study
raises the possibility that goldfish might be using catecholamines or their
3-O-methoxy metabolites as chemical messengers of some sort. Whether
or not goldfish release catecholamines or their metabolites in quantities
large enough for conspecifics to detect remains to be investigated.
Summary
The present study demonstrates that goldfish have acute olfactory sensitivity to catecholamines (principally adrenaline and dopamine) and their 3-O-methoxy metabolites, metadrenaline and 3-MT. This olfactory sensitivity is likely to be mediated by at least two specific receptor types functionally distinct from both olfactory amino acid receptors and systemic/neuronal adreno- and dopamine receptors.
|
Acknowledgments |
|---|
This work was supported financially by the Fundação para a Ciência e a Tecnologia (Portugal); grant no. SFRH/BPD/1577/2000 (to P.C.H.). The authors are, as ever, grateful to João Reis (Universidade do Algarve) for excellent technical assistance.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Aantaa, R., Marjamäki, A. and Scheinin, M. (1995) Molecular pharmacology of
2-adrenoceptor subtypes. Ann. Med.,27
, 439–449.[Web of Science][Medline]Berghard, A. and Dryer, L. (1998) A novel family of ancient vertebrate odorant receptors. J. Neurobiol., 37,383 –392.[CrossRef][Web of Science][Medline]
Buck, L.B. (1996) Information coding in the vertebrate olfactory system. Annu. Rev. Neurosci.,19 , 517–544.[CrossRef][Web of Science][Medline]
Caprio, J. and Byrd, R.P. (1984)
Electrophysiological evidence for acidic, basic, and neutral amino-acid
olfactory receptor sites in the catfish. J. Gen. Physiol.84
, 403–422.
Chen, K., Wu, H.F., Grimsby, J. and Shih, J.C. (1994) Cloning of a novel monoamine oxidase cDNA from trout liver. Mol. Pharmacol., 46,1226 –1233.[Abstract]
Chivers, D.P. and Smith, R.J.F. (1998) Chemical alarm signalling in aquatic predator–prey systems: a review and prospectus. Écoscience,5 , 338–352.[Web of Science]
Cooper, J.R., Bloom, F.E. and Roth, R.H. (1996) The Biochemical Basis of Neuropharmacology, 7th edn. Oxford University Press, Oxford.
Fabbri, E., Capuzzo, A. and Moon, T.W. (1998) The role of circulating catecholamines in the regulation of fish metabolism: an overview. Comp. Biochem. Physiol., 120C,177 –172.
Friedrich, R.W. and Korsching, S.I. (1997) Combinatorial and chemotopic odorant coding in the zebrafish olfactory bulb visualized by optical imaging.Neuron , 18,737 –752.[CrossRef][Web of Science][Medline]
Hara. T.J. (1994) Olfaction and gustation in fish: an overview. Acta Physiol. Scand.,152 ,207 –217.[Web of Science][Medline]
Hubbard, P.C., Barata, E.N. and Canário, A.V.M. (2002). Possible disruption of pheromonal communication by humic acid in the goldfish, Carassius auratus.Aquat. Toxicol. , 60,169 –183.[CrossRef][Web of Science][Medline]
Johnson, M. (1998) The
ß-adrenoceptor. Am. J. Respir. Crit. Care Med.,158
, S146-S153.
Jordão, L.C. and Volpato, G.L. (2000) Chemical transfer of warning information in the non-injured fish. Behaviour, 137,681 –690.[CrossRef]
Kats, L.B. and Dill, L.M. (1998) The scent of death: chemosensory assessment of predation risk by prey animals. Écoscience, 5,361 –394.
Lipschitz, D.L. and Michel, W.C. (1999)
Physiological evidence for the discrimination of L-arginine
from structural analogues by the zebra fish olfactory system. J.
Neurophsiol., 82,3160
–3167.
McClintock, T.S. (2000) Molecular biology of olfaction. In Finger, T.E., Silver W.L. and Restrepo D. (eds),The Neurobiology of Taste and Smell , 2nd edn. Wiley-Liss, New York, pp. 179–199.
Mazeaud, M.M. and Mazeaud, F. (1973a) Excretion and catabolism of catecholamines in fish. Part I. Excretion rates. Comp. Gen. Pharmacol., 4,183 –187.[CrossRef][Medline]
Mazeaud, M.M. and Mazeaud, F. (1973b) Excretion and catabolism of catecholamines in fish. Part II. Catabolites. Comp. Gen. Pharmacol.,4 , 209–217.[CrossRef][Medline]
Michel, W.C. and Lubomudrov, L.M. (1995) Specificity and sensitivity of the olfactory organ of the zebrafish,Danio reiro. J. Comp. Physiol. A , 177,191 –199.
Mirza, R.S. and Chivers, D.P. (2001) Chemical alarm signals enhance survival of brook charr (Salvelinus fontinalis) during encounters with predatory chain pickerel (Esox niger). Ethology, 107,989 –1005.[CrossRef]
Reid, S.G., Bernier, N.J. and Perry, S.F. (1998) The adrenergic stress response in fish: control of catecholamine storage and release. Comp. Biochem. Physiol., 120C,1 –27.
Scott, J.W. and Scott-Johnson, P.E. (2002) The electroolfactogram: a review of its history and uses. Microsc. Res. Tech., 58,152 –160.[CrossRef][Web of Science][Medline]
Sibley, D.R. and Monsma, F.J., Jr (1992) Molecular biology of dopamine receptors. Trends Pharmacol. Sci., 13,61 –69.[CrossRef][Medline]
Sloley, B.D., Trudeau, V.L. and Peter, R.E. (1992) Dopamine catabolism in goldfish (Carassius auratus) brain and pituitary—lack of influence of catecholestrogens on dopamine catabolism and gonadotropin secretion. J. Exp. Zool., 263,398 –405.[CrossRef]
Sorensen, P.W. and Caprio, J. (1998) Chemoreception. In Evans, D.H. (ed.), The Physiology of Fishes, 2nd edn. CRC Press, Boca Raton, FL, pp.375 –405.
Wendelaar Bonga, S.J. (1997) The stress
response in fish. Physiol. Rev.,77
, 591–625.
Wisenden, B.D., Chivers, D.P. and Smith, R.J.F. (1995) Early warning in the predation sequence—a disturbance pheromone in Iowa darters (Etheostoma exile).J. Chem. Ecol. , 21,1469 –1480.[CrossRef]
Zar, J.H. (1996) Biostatistical Analysis, 3rd edn. Prentice Hall, Upper Saddle River.
Zhong, H. and Minnemann, K.P. (1999)
1-adrenoceptor subtypes. Eur. J. Pharmacol.,375
,261
–276.[CrossRef][Web of Science][Medline]
Accepted February 10, 2003
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