Dopamine stimulates snail albumen gland glycoprotein secretion through the activation of a D1-like receptor
Department of Biology, Faculty of Pure and Applied Sciences, York University, Toronto, Ontario, Canada M3J 1P3
* Author for correspondence (e-mail: saber{at}yorku.ca)
Accepted 22 April 2004
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Summary |
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Key words: dopamine, dopamine receptor, albumen gland, perivitelline fluid, glycoprotein, secretion, cAMP, snail, exocrine
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Introduction |
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Catecholaminergic axons have been shown to innervate the reproductive
organs of various molluscs (Hartwig et
al., 1980; Smith et al.,
1998
; Croll et al.,
1999
; Croll, 2001
;
Kiehn et al., 2001
), and
histochemical analyses have revealed that catecholaminergic cell bodies and
axon processes are concentrated in the region of the exocrine albumen gland
(AG) of the freshwater snails Bulinus truncatus
(Brisson and Collin, 1980
;
Brisson, 1983
) and Helisoma
duryi (Kiehn et al.,
2001
). The AG is a female accessory reproductive gland that
secretes a viscous substance known as the perivitelline fluid (PVF) around the
individual eggs as they enter the carrefour, the area where the main duct of
the AG empties. The PVF consists mainly of glycoproteins and galactogen (a
highly branched galactose polymer), which provide the main source of nutrients
to the developing embryos (Duncan,
1975
; Geraerts and Joosse,
1984
). Therefore, the timely release (secretion) of PVF is a key
regulatory process governing egg production in freshwater snails.
Although the neuroendocrine control of polysaccharide (galactogen)
synthesis in the AG has been studied extensively in freshwater pulmonate
molluscs (de Jong-Brink et al.,
1982; Wijdenes et al.,
1983
; Miksys and Saleuddin,
1985
,
1988
;
Mukai et al., 2001b
), little
is known with respect to the regulation of PVF release. We have identified the
major protein produced by the AG of the freshwater snail H. duryi as
a 288 kDa glycoprotein, which is composed of several 66 kDa subunits
(Morishita et al., 1998
). The
gene encoding for the 66 kDa glycoprotein subunit has recently been cloned
(Mukai et al., 2004
) and the
corresponding protein was given the name Helisoma duryi albumen gland
protein (HdAGP). The release of HdAGP is known to be stimulated by a novel
brain peptide (Morishita et al.,
1998
) through a cAMP signalling pathway
(Mukai et al., 2001a
).
The secretion of HdAGP coincides with the arrival of the eggs at the
carrefour, and the existence of a control mechanism over AG secretory activity
has been postulated (Mukai et al.,
2004). Kiehn et al.
(2001
) showed that the AG and
carrefour region of H. duryi is innervated by dopaminergic nerve
fibres. Moreover, dopamine has also been shown to induce the secretion of
total protein from isolated AGs of H. duryi
(Mukai, 1998
;
Saleuddin et al., 2000
) and
Biomphalaria glabrata
(Santhanagopalan and Yoshino,
2000
). Subsequent studies by Boyle and Yoshino
(2002
) showed that B.
glabrata AG dopamine levels increased during the initial stage of egg
mass production (the period during which the AG is secreting PVF), whereas
protein levels in the AG decreased during the latter stages of egg mass
production. Collectively, these results suggest that the secretion of protein
by the AG of freshwater pulmonate snails is regulated by dopamine and, by
inference, specific dopamine receptors in the AG.
Dopamine receptors were originally classified into two categories, D1 and
D2, based on their ability when activated to either stimulate or inhibit,
respectively, adenylate cyclase activity (reviewed by
Civelli et al., 1993). The
genes for five distinct dopamine receptor subtypes (D1, D2, D3, D4, D5) have
been cloned in mammals and placed into one of the two dopamine receptor groups
based on their gene structure and pharmacology
(O'Dowd, 1993
). The dopamine
D1-like receptors, which stimulate cAMP formation, comprise the D1 and D5
subtypes, whereas the dopamine D2-like receptors, comprising the D2, D3 and D4
subtypes, either inhibit or have no effect on adenylate cyclase
(Missale et al., 1998
). Using
a number of dopamine receptor agonists and antagonists, Saleuddin et al.
(2000
) suggested that a
dopamine D1-like receptor mediates total protein secretion by AGs of H.
duryi. Here, we extend our previous study and examine the intracellular
signalling pathway activated after AGs were treated with dopamine. Addition of
exogenous dopamine to AG explants stimulates secretion of HdAGP via
the activation of the cAMP signalling pathway. A number of D1-selective and
D2-selective agonists and antagonists were used to obtain a pharmacological
profile of the AG dopamine receptor. It is concluded that the AG dopamine
receptor regulating protein secretion is distinct but functionally similar to
vertebrate D1-like receptors and mediates secretion of HdAGP through an
elevation in glandular cAMP, possibly through a protein kinase A
(PKA)-independent pathway.
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Materials and methods |
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Chemicals
Dopamine (3-hydroxytyramine), serotonin, acetylcholine,
-aminobutyric acid, norepinephrine, histamine, octopamine, glutamate,
forskolin (7ß-acetoxy-8,13-epoxy-1
, 6ß,
9
-trihydroxylabd-14-en-11-one), IBMX (3-isobutyl-1-methylxanthine),
apomorphine hydrochloride, bromocriptine (2-bromo-
-ergocryptine
methanesulfonate salt), (R)(+)-SCH23390 hydrochloride, (R)(±)-SKF-38393
hydrochloride, R(+)-SKF81297 hydrobromide, (±)-SKF83566 hydrochloride,
dihydrexidine hydrochloride
{(±)-trans-10,11-dihydroxy-5,6,6a,7,8,12b-hexahydrobenzo[a]phenanthridine},
6,7-ADTN [(±)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronapthalene],
haloperidol
{4-(4-[4-chlorophenyl]-4-hydroxy-1-piperidinyl)-1-(4-fluorophenyl)-1-butanone},
()-butaclamol hydrochloride, cis-flupenthixol, chlorpromazine,
eticlopride, Rp-cAMP and H-89 were purchased from Sigma-Aldrich Canada,
Oakville, Ontario, Canada.
Bioassay
Albumen glands were dissected free from surrounding tissue under
Helisoma saline (51.3 mmol l-1 NaCl, 1.7 mmol
l-1 KCl, 4.1 mmol l-1 CaCl2, 1.5 mmol
l-1 MgCl2, 5.0 mmol l-1 Hepes, 1 mmol
l-1 glucose, pH 7.4, 120 mOsm H2O), cut into halves and
then washed in several changes of saline. One half served as a control gland
while the other was treated with test compound. Each AG piece
(0.51.5 mg) was placed in a separate well of a 96-well culture
plate (Becton-Dickinson and Co., Lincoln Park, NJ, USA) containing 100 µl
of saline. The saline surrounding the AG was removed and replaced with another
100 µl of fresh saline every 20 min. The collected saline was placed in 1.7
ml polypropylene microtubes (Brinkmann Instruments Inc., Westbury, NY, USA)
and centrifuged at 2000 g for 1 min. An 80 µl sample was
removed and added directly to a 1.5 ml polystyrene cuvette containing 420
µl of Triton X-100 (0.0095% in water). Total protein was determined by
adding 125 µl Bio-Rad Protein Dye Reagent Concentrate (Bio-Rad Laboratories
Canada Ltd, Mississauga, Ontario, Canada) and measuring the absorbance at 595
nm with a Zeiss PM2DL spectrophotometer (Carl Zeiss, Oberkochen, Germany).
Basal HdAGP secretion was measured for the first 60 min. A stock solution of
dopamine (10 mmol l-1) was prepared in deoxygenated-demineralized
water and diluted in saline to a final concentration of 10 µmol
l-1 immediately before use. All the test compounds were dissolved
in Helisoma saline (with or without dopamine) to their final
concentrations and applied to the AGs at 60 min. The test agents were removed
at 80 min and replaced with normal saline for another 60 min. Dopamine
receptor antagonists (D1-selective or D2-selective) or PKA inhibitors (Rp-cAMP
and H-89) were first preincubated with AGs at 40 min, then removed and
replaced with antagonist plus dopamine at 60 min. The amount of HdAGP secreted
between 40 and 60 min (control) was compared with the amount secreted between
60 and 80 min (treated).
Electrophoresis
To qualitatively determine the proteins secreted by the AG in
vitro after dopamine stimulation, the saline surrounding the AG was
collected and analyzed by sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE). The collected saline was first evaporated to
dryness using a Savant SVC 100 Speed-Vac (Instruments Inc., Farmingdale, NY,
USA), then resuspended in SDS-PAGE sample buffer and separated on a 9%
mini-gel apparatus (Bio-Rad) according to Laemmli
(1970). Following
electrophoresis, the gel was stained overnight with 0.2% Coomassie Brilliant
Blue R-250 in 50% methanol/10% acetic acid. The gel was destained the next day
and dried onto Whatman 3MM filter paper using a slab gel dryer (Hoefer
Scientific Instruments, San Francisco, CA, USA).
cAMP determinations
Albumen glands were dissected under snail saline, then quartered and rinsed
in normal saline. Prior to treatment, each AG piece was preincubated for 20
min with 1 mmol l-1 IBMX in saline to allow the phosphodiesterase
inhibitor to penetrate the AG cells. All test compounds were dissolved in IBMX
saline and applied to the AG for 10 min unless indicated otherwise. For the
time course experiment, one AG piece served as a control, the second piece was
treated with dopamine (10 µmol l-1), the third piece was treated
with forskolin (10 µmol l-1) and the fourth piece was treated
with both dopamine and forskolin. At the end of each time point, the AGs were
immediately plunged into liquid nitrogen, then stored in 1.7 ml microtubes at
80°C. To extract cAMP, AGs were homogenized in 3% ice-cold
perchloric acid using a motor-driven Teflon pestle and then centrifuged at 10
000 g (10 min at 4°C). The precipitates were kept for
subsequent protein determinations and resuspended in 0.1 mol l-1
NaOH (60°C for 1 h) prior to use. The acidic supernatant was transferred
to a new tube and neutralized to pH 6 with 2.6 mol 1-1 potassium
bicarbonate. The resultant potassium perchlorate precipitate was discarded and
the recovered supernatant was evaporated to dryness. The dried residue was
resuspended in cAMP assay buffer and the concentration of AG cAMP was measured
with a commercial [3H]cAMP assay kit (Diagnostic Products Corp., Los Angeles,
CA, USA). For the pharmacological characterization of the AG dopamine
receptor, AGs were quartered: one AG piece served as a control; the second
piece was treated with 10 µmol l-1 dopamine (positive control);
the third piece was treated with dopamine receptor agonist or antagonist (10
µmol l-1); and the fourth piece was treated with dopamine plus
agonist or antagonist. The AGs were extracted for cAMP as described above.
Data were expressed as pmol cAMP mg-1 AG protein.
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Results |
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The specificity of the AG response to dopamine was tested by applying other
known neurotransmitters found in molluscan nervous tissue to the AG and then
measuring in vitro secretion of HdAGP. Serotonin, acetylcholine,
-aminobutyric acid (GABA), norepinephrine, histamine, octopamine and
L-glutamate were tested at 0.1, 1 and 10 µmol l-1.
With the exception of dopamine, none of the other neurotransmitters were
capable of inducing the secretion of HdAGP from the AG
(Table 1). A significant
stimulation of HdAGP secretion was induced at a concentration of 1 and 10
µmol l-1 dopamine.
|
The effect of dopamine on cAMP production in the AG
Since the existence of a dopamine D1-like receptor on the AG was indicated
by previous protein secretion bioassays
(Saleuddin et al., 2000) and
since forskolin (an adenylate cyclase activator) is known to be a potent
stimulator of HdAGP secretion (Morishita
et al., 1998
), we tested for the effect of dopamine on
intracellular cAMP production. The time course of AG cAMP production was
measured over a 20 min period after application of 10 µmol l-1
dopamine, 10 µmol l-1 forskolin or both of these compounds
together. Both dopamine and forskolin caused a significant increase in cAMP
production by the AG between 5 and 20 min after their application.
Furthermore, dopamine-stimulated cAMP levels were augmented in the presence of
forskolin and increased linearly for at least 20 min
(Fig. 2). Subsequent cAMP
determinations were performed using a 10 min treatment unless otherwise
indicated. The addition of dopamine (Fig.
3A) or forskolin (Fig.
3B) to AGs increased cAMP production in a dose-dependent manner.
Significant elevation of cAMP levels were observed at 10 µmol
l-1 and 100 µmol l-1 for both compounds; however,
forskolin was a more potent activator of cAMP production than dopamine.
|
|
Effects of dopamine receptor agonists on AG cAMP production
To assess the effect of various dopamine receptor agonists on AG cAMP
production, D1-selective agonists (dihydrexidine, SKF81297 and 6,7-ADTN) and
D2-selective agonists (bromocriptine and apomorphine) were tested. Treatment
of AGs with dopamine (10 µmol l-1) increased AG cAMP levels
36-fold above basal AG cAMP levels
(Fig. 4). The D1-selective
agonists dihydrexidine (Fig.
4A) and 6,7-ADTN (Fig.
4B) stimulated AG cAMP production significantly (3-fold and
5-fold, respectively), whereas SKF81297 had a modest (2-fold) non-significant
stimulatory effect on AG cAMP production
(Fig. 4C). No augmentation of
cAMP production was detected when both dopamine and the D1-selective agonists
were added to the AGs. By contrast, neither of the two D2-selective agonists
apomorphine (Fig. 5A) nor
bromocriptine (Fig. 5B) had a
statistically significant effect on basal cAMP production. Bromocriptine had
no effect on dopamine-stimulated cAMP production
(Fig. 5B) but apomorphine
unexpectedly caused a significant inhibition of dopamine-stimulated cAMP
production (Fig. 5A).
|
|
Effects of dopamine receptor antagonists on dopamine-stimulated cAMP levels in the AG
Various D1-selective antagonists were tested for their ability to inhibit
dopamine-stimulated cAMP production. At the concentrations (10 µmol
l-1) tested in this study, the benzazepines SCH23390
(Fig. 6A) and SKF83566
(Fig. 6B) suppressed
dopamine-stimulated cAMP production by 62% and 48%, respectively. The compound
flupenthixol had a slight inhibitory effect (22%) on dopamine-stimulated cAMP
production but it was not statistically significant. None of the D1-selective
antagonists affected basal cAMP levels significantly. The D2-selective
antagonists chlorpromazine (Fig.
7A), eticlopride (Fig.
7B) or haloperidol (Fig.
7C) and the mixed D1/D2 antagonist butaclamol
(Fig. 7D) had no effect on
either basal or dopamine-stimulated cAMP production.
|
|
Effects of D1 and D2 agonists and D1 antagonists on HdAGP secretion
To confirm our previous results on the presence of a dopamine D1-like
receptor, which mediates protein secretion, in the AG of H. duryi, we
extended the number of D1-like receptor agonists and antagonists used. The
D1-selective agonists SKF81297 and dihyrexidine were tested for their ability
to induce protein secretion from the AGs whereas the D1-selective antagonists
SKF83566 and flupenthixol were tested for their ability to inhibit
dopamine-induced protein secretion. Both SKF81297
(Fig. 8A) and dihyrexidine
(Fig. 8B) were capable of
inducing HdAGP secretion by AG explants, although dihydrexidine was the more
potent stimulator. A concentration of 10 µmol l-1 dihyrexidine
stimulated HdAGP secretion 5-fold above control levels whereas 100 µmol
l-1 of SKF81297 was required to increase protein secretion
3.9-fold. By contrast, the D2-selective agonists apomorphine and bromocriptine
had no effect on protein secretion (Fig.
9). Previous studies showed that SCH23390 (100 µmol
l-1), a D1-selective antagonist, was a potent inhibitor of
dopamine-stimulated protein secretion
(Saleuddin et al., 2000). In
the present study, protein secretion was also inhibited by two D1-selective
antagonists, SKF83566 (50 µmol l-1) and flupenthixol (10 µmol
l-1). Both antagonists inhibited dopamine-stimulated HdAGP
secretion by 50% (Fig. 10A)
and 37% (Fig. 10B),
respectively.
|
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|
Effect of PKA antagonists on HdAGP secretion
The involvement of PKA in mediating AG protein secretion was determined
using the PKA antagonists Rp-cAMP (1 mmol l-1) and H-89 (10 µmol
l-1). Both Rp-cAMP (Fig.
11A) and H-89 (Fig.
11B) did not significantly inhibit dopamine-stimulated protein
secretion and neither inhibitor affected basal protein secretion.
|
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Discussion |
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Despite the widespread occurrence of dopamine in the peripheral tissues of
molluscs, little information is available about the pharmacological properties
of peripheral dopamine receptors, particularly in reproductive organs. In the
salivary duct muscle of Helix pomatia, dopamine-induced contraction
was inhibited by the D1-selective antagonists flupenthixol and fluphenazine,
whereas the D1-selective agonist SKF38393 mimicked the effect of dopamine
(Kiss et al., 2003). In the AG
of B. glabrata, the D2-selective antagonist chlorpromazine inhibited
dopamine-induced protein secretion
(Santhanagopalan and Yoshino,
2000
) but it was without effect in H. duryi, as shown in
an earlier report (Saleuddin et al.,
2000
) and in the present study. Previous studies have indicated
the presence of a D1-like receptor that mediated AG protein secretion in
H. duryi (Saleuddin et al.,
2000
). Since D1-like receptors are known to stimulate adenylate
cyclase activity (Missale et al.,
1998
) and since forskolin is a potent stimulator of protein
secretion (Morishita et al.,
1998
), we tested for the effect of dopamine on AG cAMP production.
Addition of dopamine or forskolin to AG explants increased cAMP production in
a time- and concentration-dependent manner. In the presence of forskolin,
dopamine-stimulated cAMP production was enhanced because forskolin bypasses
the receptor and directly activates the catalytic subunit of adenylate cyclase
(Insel and Ostrom, 2003
).
Using a number of different dopamine receptor agonists and antagonists it
was possible to obtain a pharmacological characterization for the dopamine
receptor in the AG of H. duryi. The D1-selective benzazepine agonist
SKF81297 showed a slight stimulatory effect on AG cAMP production at a
concentration of 10 µmol l-1, indicating that it is a weak
agonist at the AG dopamine receptor. This result is consistent with its effect
on the secretion of HdAGP, where it was only half as potent as dopamine. In
vertebrates, SKF81297 is considered to be a partial D1 agonist, i.e. it
modestly activates adenylate cyclase when compared with dopamine
(Andersen and Jansen, 1990).
Saleuddin et al. (2000
) found
that SKF38393, another vertebrate D1-selective agonist, also had little effect
on AG protein secretion, suggesting that benzazepines are relatively weak
agonists on the AG dopamine receptor. In locust salivary glands, SKF38393 was
also inactive in stimulating cAMP production
(Ali and Orchard, 1994
) and
caused a weak hyperpolarization of the acinar cells as compared with dopamine
(Keating and Orchard, 2001
).
In vertebrates, benzazepine analogues are effective dopamine receptor
antagonists but display only limited effectiveness as cAMP agonists
(Andersen and Jansen, 1990
).
Therefore, the H. duryi AG dopamine receptor is functionally similar
to vertebrate D1-like receptors since its activation leads to cAMP formation
but its structure is probably different because of its specificity to various
dopamine receptor agonists.
The dopamine receptor agonist that was most effective in elevating AG cAMP
levels was 6,7-ADTN. In vertebrates, this tetraline compound does not
discriminate between D1-like and D2-like receptors. The activation
characteristics of the AG dopamine receptor resembled that of a cloned D1-like
receptor in Drosophila. This primordial Drosophila dopamine
receptor also displayed poor affinity for benzazepines and was activated by
the tetraline 6,7-ADTN (Sugamori et al.,
1995). In the CNS of Lymnaea stagnalis, 6,7-ADTN mimicked
the effect of dopamine at synapses between right pedal ganglion 1 (RPeD1) and
its follower cells; however, this neuronal dopamine receptor was
pharmacologically identified as a D2-like receptor
(Magoski et al., 1995
). The
presence of a D2-like receptor that mediates dopamine-induced
hyperpolarization in the growth hormone-producing light green cells of L.
stagnalis has also been shown (de
Vlieger et al., 1986
; Werkman
et al., 1987
). Together, these results imply the existence of
different dopamine receptor subtypes in molluscan neural and peripheral
tissues. The other D1-selective agonist used in the present study was
dihydrexidine. Dihydrexidine is a phenanthridine analogue that is considered
to be a full agonist at vertebrate D1-like receptors
(Brewster et al., 1990
;
Mottola et al., 1992
);
however, it was only modestly effective in elevating AG cAMP levels in H.
duryi. Although dihyrexidine was not as effective as 6,7-ADTN in
stimulating AG cAMP production, it was a more potent agonist in inducing HdAGP
secretion when compared with 6,7-ADTN
(Saleuddin et al., 2000
).
Dihydrexidine-stimulated HdAGP secretion was dose dependent and its activity
was comparable with equivalent concentrations of dopamine. Finally, no
additive or synergistic effects on AG cAMP production were observed with any
of the agonists used in this study, indicating that these compounds probably
act on a single receptor system.
The D2-selective agonists apomorphine and bromocriptine had no effect on AG protein secretion or basal cAMP levels. However, apomorphine exhibited an unusual effect by attenuating dopamine-stimulated cAMP production. The reason for this is unclear but it is possible that apomorphine occupies the same binding site on the receptor as dopamine, thereby reducing the latter's overall effect on AG cAMP formation.
The effect of various dopamine receptor antagonists was tested on AGs for
their ability to inhibit dopamine-stimulated AG cAMP production and
dopamine-induced HdAGP secretion. In the present study, the most effective
inhibitor of dopamine-stimulated cAMP production was the benzazepine SCH23390,
followed by SKF83566. In support of these findings, SCH23390 was also the most
effective antagonist inhibiting dopamine-induced protein secretion in the AG
of H. duryi (Saleuddin et al.,
2000). Flupenthixol at a concentration of 10 µmol
l-1 suppressed dopamine-stimulated AG cAMP production only
marginally but significantly inhibited dopamine-induced protein secretion. In
the salivary duct muscle of the snail H. pomatia, SCH23390 was
inactive in suppressing dopamine-induced contractions whereas flupenthixol was
shown to be the most potent antagonist
(Kiss et al., 2003
). This
indicates that the peripheral dopamine receptor in the salivary duct muscle of
H. pomatia displays characteristics of a D1-like receptor and that
its pharmacological properties are distinct from the D1-like receptor in the
AG of H. duryi.
The AG dopamine receptor might represent a member of a novel class of
dopamine receptors that is substantially different from those characterized in
vertebrates and even other invertebrates. In support of this notion, the
dopamine receptor in the corpus allatum of the tobacco hornworm, Manduca
sexta, exhibits a pharmacological profile distinct from other insect
dopamine receptors and has both D1- and D2-like properties
(Granger et al., 2000). In the
nematode Caenorhabditis elegans, a novel dopamine D1-like receptor
that did not bind [3H]SCH23390 was recently cloned
(Suo et al., 2002
). In
addition, the D1-like receptor from C. elegans also contained introns
in the coding region, a feature not present in mammalian or previously
characterized invertebrate D1-like receptors, providing further evidence that
some invertebrate dopamine receptors represent a structurally distinct group.
However, definitive characterization of the H. duryi AG dopamine
receptor can only be achieved by molecular cloning and functional expression
in a heterologous system.
To investigate intracellular signalling events further downstream of
adenylate cyclase and cAMP, we tested for the involvement of PKA. In the vast
majority of cAMP-mediated signalling, the primary effector regulated by cAMP
is PKA. Cyclic AMP binds to the regulatory subunits of PKA, which in turn
releases the catalytic subunits of PKA
(Francis and Corbin, 1996).
The activated catalytic subunits can then phosphorylate specific intracellular
target proteins and mediate the appropriate physiological response. The PKA
inhibitors Rp-cAMP and H-89 suppress PKA-mediated signalling in a number of
invertebrates, including leeches (Ali et
al., 1998
), crustaceans
(Locatelli et al., 2002
),
insects (Hazel et al., 2003
)
and molluscs (Malagoli et al.,
2000
). Our results demonstrate that dopamine-mediated secretion of
HdAGP might be a PKA-independent process because inhibitors of both the
regulatory (Rp-cAMP) and catalytic (H-89) subunits of PKA failed to inhibit
protein secretion. PKA-independent signalling has recently become a recognized
phenomenon due to the identification of novel cAMP-binding proteins, such as
guanine exchange factors and cyclic nucleotide-gated channels (reviewed by
Dremier et al., 2003
;
Kopperud et al., 2003
), and
the establishment of `cross-talk' among other signalling pathways
(Cooper et al., 1995
;
Houslay and Milligan, 1997
;
Schwartz, 2001
). However, it
is possible that these PKA inhibitors were not able to fully penetrate into
the glandular cells to exert their effects. Alternative approaches such as
microinjection of PKA inhibitors into dissociated glandular cells combined
with electrophysiological measurements are required to rule out this
possibility.
In addition to dopamine, a putative neuropeptide from the CNS of H.
duryi also appears to be involved in regulating HdAGP secretion through
the cAMP signalling system (Morishita et
al., 1998; Mukai et al., 2001). Whether these two molecules are
co-released or perhaps affect each other's release is currently unknown.
Identifying specific interactions between these two molecules can only be done
after the H. duryi brain peptide is sequenced. The intracellular
target of cAMP action in the AG of H. duryi is not known but it is
possible that cAMP interacts with other intracellular signalling pathways such
as Ca2+. In support of this, we have recently shown that, in
addition to cAMP, the influx of Ca2+ into the AG cells is an
important regulator of protein secretion
(Kiehn et al., 2004
).
Structural elucidation of the brain peptide as well as the identification of
downstream targets of cAMP and Ca2+ is in progress.
In summary, we have identified a specific receptor to the neurotransmitter dopamine in the AG of H. duryi that participates in HdAGP secretion. Based on the pharmacological profile obtained using dopamine receptor agonists and antagonists, it is concluded that the AG dopamine receptor displays functional characteristics of a vertebrate D1-like receptor because of the following: dopamine stimulates cAMP formation and protein secretion in a dose-dependent fashion; D1-selective agonists stimulate basal cAMP formation and induce protein secretion in isolated AGs; D2-selective agonists had no effect on cAMP production or protein secretion; D1-selective antagonists suppress dopamine-stimulated cAMP production and dopamine-induced protein secretion; and D2-selective antagonists had no inhibitory effect on dopamine-stimulated cAMP production. However, the AG dopamine receptor of H. duryi also displays some unique pharmacological characteristics in that its activity is stimulated by the non-selective agonist 6,7-ADTN and is attenuated by the D2-selective agonist apomorphine. Finally, dopamine-induced protein secretion might occur through a PKA-independent mechanism involving Ca2+ or, perhaps, some unidentified protein that is regulated by cAMP.
Symbols and abbreviations
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Acknowledgments |
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References |
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