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First published online March 28, 2008
Journal of Experimental Biology 211, 1270-1280 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.012005
Changes in the control of gastric motor activity during metamorphosis in the amphibian Xenopus laevis, with special emphasis on purinergic mechanisms
Department of Zoophysiology, Göteborg University, SE 405 30 Göteborg, Sweden
* Author for correspondence (e-mail: monika.sundqvist{at}astrazeneca.com)
Accepted 7 February 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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-β-MeATP elicited
pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid
(PPADS)-sensitive contractions at all stages investigated. These results
indicate the development of an inhibitory nitrergic tonus during metamorphosis
and a 5-HT receptor involved in muscle contraction. Also, the development of
UTP receptors mediating increased tension and neural UTP receptors decreasing
contraction frequency in juveniles is indicated. An adenosine
A1-like receptor mediating relaxation and a P2X-like receptor
mediating contraction is demonstrated at all stages.
Key words: metamorphosis, development, amphibian, Xenopus laevis, 5-HT, nitric oxide, adenosine, UTP, ATP, purinergic
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Rovira et al., 1995;
Schreiber et al., 2005;
Sundqvist and Holmgren, 2004).
Previously, we have shown that the expression of neurotrophin-like receptors
is increased in the myenteric plexus during metamorphic climax
(Sundqvist and Holmgren,
2004), suggesting that the enteric nervous system also undergoes
modifications at this stage. Studies of the earliest developmental stages of
the tadpole indicate that the control of gastrointestinal motility during
these stages differs from that of adult gut
(Sundqvist and Holmgren,
2006). Thus, changes in the regulation of gut motility are likely
to take place during metamorphosis.
In adult Xenopus stomach, the transmitters pituitary adenylate
cyclase-activating polypeptide, vasoactive intestinal peptide (VIP) and nitric
oxide (NO) relax circular stomach muscle preparations
(Olsson, 2002). Furthermore,
the nitric oxide synthase (NOS) inhibitor L-NAME increases
contractions in Xenopus stomach suggesting that NO exerts an
endogenous inhibitory tonus in the preparations. In newly hatched tadpoles
from Xenopus [stage 43 according to Nieuwkoop and Faber
(Nieuwkoop and Faber, 1967)],
VIP and NO decreased gastrointestinal motility; however, tonic nitrergic
activity was absent (Sundqvist and
Holmgren, 2006). Serotonin (5-hydroxytryptamine, 5-HT), another
known modulator of gastric activity, has been shown to have a biphasic effect
in adult Xenopus stomach
(Johansson, 2003). Low
concentrations elicit excitatory responses, while high concentrations elicit
relaxation, probably indicating the presence of more than one 5-HT receptor in
the tissue. The effect of 5-HT on gastric motor activity has not been studied
in Xenopus tadpoles.
In the Xenopus intestine, changes in the regulation of motor
activity by the purinergic system occur during metamorphosis
(Sundqvist, 2007). The
purinergic system affects motor function in the gut of many vertebrate species
via purinoceptors (Burnstock,
1996; Burnstock,
2001). Responses to adenosine are mediated through metabotropic P1
receptors (A1, A2A-B, A3). Receptors that are
activated by ATP, ADP, UTP or UDP are called P2 receptors and consist of two
subfamilies, the metabotropic P2Y [P2Y1–2, P2Y4,
P2Y6, P2Y11–14; species specific: p2p3 (chicken),
p2y8 (Xenopus), tp2y (turkey)] receptors and the
ionotropic P2X (P2X1–7) receptors
(Burnstock, 2006;
von Kugelgen and Wetter,
2000). In Xenopus, the adenosine A1 receptor
(gene accession number: AJ249842) has been cloned along with a number of
receptors for ATP including P2Y1, p2y8, P2X4,
P2X7 (Bogdanov et al.,
1997; Cheng et al.,
2003; Juranka et al.,
2001; Paukert et al.,
2002) and p2y11 (gene accession number: AM040941).
In a previous study performed in our laboratory
(Sundqvist, 2007), adenosine
relaxed intestinal smooth muscle strips from Xenopus both prior to
and after metamorphosis, while ATP caused both relaxing and contracting
responses. However, prior to metamorphosis, the ATP-evoked relaxing response
appeared to be mediated by adenosine (from metabolized ATP) acting on
A1 receptors, while after metamorphosis the response to ATP was at
least partly mediated by ATP per se acting directly on a
P2Y11-like receptor. Similarly, changes in the expression pattern
or activity of the P2Y1 receptor occur in the rodent stomach and
intestine during late development. Interestingly, in longitudinal smooth
muscle of rodents, P2Y1 receptors switch from mediating contracting
responses to mediating relaxing responses around the time of weaning
(Brownhill et al., 1997;
Furukawa and Nomoto, 1989;
Giaroni et al., 2006;
Hourani, 1999;
Nicholls et al., 1990). This
may be an adaptation to the altered content of nutrients in the food, when a
more carbohydrate-containing food source replaces the lipid-rich diet of the
young pups (Giaroni et al.,
2006; Hourani,
1999).
Since our previous studies suggest changes in the control of intestinal motility during late development, the aim of this study was to investigate the changes, if any, in the control of gastric motor activity immediately around and during amphibian metamorphosis when the food intake of the animal changes from herbivorous to carnivorous and the gastrointestinal tract undergoes adaptive remodelling. We therefore studied the effect of a number of different neurotransmitters on gastric activity in stomach muscle strips prior to, during and after metamorphosis, with special emphasis on the purinergic system. The results show distinct changes in the control of gastric activity during metamorphosis including the development of a nitrergic tonus, a 5-HT receptor involved in muscle contraction and a switch in the smooth muscle response to UTP resulting in muscle contraction as well as the possible development of neural UTP receptors.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). Briefly,
adults (obtained from Nasco, Fort Atkinson, WI, USA) were injected with
chorionic gonadotropin and allowed to breed. The fertilized eggs developed
into tadpoles and were kept until metamorphosis in plastic aquariums. Tadpoles
and froglets were fed 5 days a week on suitable Xenopus diets (Blades
Biological Ltd, Edenbridge, UK). All experimental procedures were approved by
the animal ethics committee of the city of Göteborg.
Drugs
Adenosine hemisulphate salt (adenosine), adenosine 5'-triphosphate
(ATP), adenosine 5'-[
-thio]-triphosphate tetralithium salt
(ATPS), carbamoylcholine chloride (carbachol),
-β-methyleneadenosine 5'-triphosphate lithium salt
(-β-meATP), N-nitro-L-arginine methyl ester
hydrocloride (L-NAME), serotonin (5-hydroxytryptamine, 5-HT) and
uridine 5'-triphosphate (UTP) were purchased from Sigma-Aldrich Chemical
Co. (St Louis, MO, USA).
4-Amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin-3-yl)pyrido[2,3-d]pyrimidi
ne (ABT-702), N6-cyclopentyladenosine (CPA),
6-N,N-diethyl-D-β,-dibromomethylene ATP
trisodium salt (ARL67156), 1,3-dipropyl-8-cyclopentylxanthine (DPCPX),
pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and
tetrodotoxin (TTX) were obtained from Tocris Cookson Ltd (Bristol, UK). Human
neurokinin A (NKA) was obtained from Euro-Diagnostika (Malmö, Sweden) and
methysergide maleate from Sandoz (Holzkirchen, Germany).
Stock solutions of all compounds except ABT-702 and DPCPX were created by dissolving the compound in Millipore water and then stored in aliquots at –20°C. ABT-702 and DPCPX were dissolved in DMSO to stock concentrations of 10 mmol l–1. Further dilution of all compounds was made in McKenzie's amphibian Ringer solution.
Experimental procedure
The animals were anaesthetized and killed by immersion in a
NaHCO3-buffered solution of 0.05% MS 222 (3-aminobenzoic acid ethyl
ester, Sigma) and the developmental stage was determined according to
Nieuwkoop and Faber (Nieuwkoop and Faber,
1967). Prometamorphic tadpoles were taken at stages 56 and 57,
while metamorphic tadpoles were taken at stages 61–63 and juveniles were
at stage 66.
The stomach was dissected out and muscle strips from the cardiac stomach
were prepared and mounted in organ baths as previously described for
intestinal strips (Sundqvist,
2007). Briefly, ring-formed sections (
2–3 mm wide) were
cut from the stomach and the circular muscle strips were then mounted in 10 ml
organ baths containing 5 ml McKenzie's amphibian Ringer solution (NaCl 115
mmol l–1, NaHCO3 20 mmol l–1,
Hepes 5.0 mmol l–1, KCl 3.2 mmol l–1,
MgSO4 1.4 mmol l-1, CaCl2 1.3 mmol
l-1, pH 7.8, 23°C) bubbled with gas (0.3% CO2 in
air). After preliminary testing, the prometamorphic and metamorphic muscle
strips were stretched to a force of 2 mN (since higher forces caused them to
tear) while juvenile muscle strips were stretched to 5 mN to account for the
differences in muscle layer development and strength. The muscle strips were
allowed to equilibrate for 1 h before the experiments were started. The Ringer
solution was changed every 30–45 min during the experiment. The mean
force developed from the muscle strips was measured using Grass FT03 force
transducers (Astro-Med House, Slough, UK) and recorded by a Grass amplifier
coupled to a computer running a custom-made program called General Acquisition
(Labview version 6.01, National Instruments, Austin, TX, USA).
Spontaneous activity was recorded for 5–10 min after which drugs were
added in a cumulative manner to construct a concentration–response
curve. In some inhibitory concentration–response experiments, papaverine
(100 µmol l–1) was added to obtain total relaxation and
facilitate calculation of EC50 values. In the antagonist and TTX
experiments, the agonist (5-HT, ATP, adenosine or -β-meATP) was
added as a single concentration in the absence or presence of TTX or
antagonist. After the agonist response had been recorded for a minimum of 5
min, or until a maximum response was obtained, the agonist was washed out and
antagonist was administered. The antagonist was allowed to equilibrate for
20–30 min before another single concentration of the same agonist was
added. Potassium chloride (80 mmol l–1) was added at the end
of experiments to check the viability of the muscle strips.
Data analysis and statistics
The mean force elicited by the muscle strips was measured over 200 s using
a Labview-based analysis program. The mean force parameter was chosen since it
reflected most changes seen in the preparations. However, in the experiments
using UTP and TTX, the frequency was also analysed since this parameter was
affected differently to the mean force parameter. To normalize the mean force
values, the resting tension value during the control period was subtracted
from all values in an experiment. Further calculations and statistical
analyses were performed in Excel and Graphpad Prism 4.0 (GraphPad Software,
San Diego, CA, USA), respectively. EC50 values from each individual
experiment were calculated using a sigmoidal dose–response model in
GraphPad Prism 4.0, although when the plateau phase was not reached (see
respective figures) with the available concentrations, the values should only
be considered as tentative. This is indicated by using the sign for larger
than or equal to (
) in the text. The figures show the curve from the
combination of all experiments, which is why the calculated EC50
values sometimes deviate from the midpoint seen in the curve. Experiments were
analysed using repeated measures one-way analysis of variance (ANOVA) followed
by Dunnett's post hoc test, one-way ANOVA followed by Bonferroni's
post hoc test or Student's t-test for unpaired or paired
observations when appropriate, depending on the experiment type. The
statistical method used for each experiment is indicated in the figure legend.
Results are presented as means ± s.e.m., and P<0.05 is
considered statistically significant.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Carbachol
Administration of the cholinergic agonist carbachol (0.001–100
µmol l–1) resulted in a concentration-dependent increase
in mean force developed in smooth muscle from all three stages
(Fig. 1B). The potency of
carbachol was similar at all stages (EC50: 0.5±0.2 µmol
l–1 prometamorphosis, 1.4±0.3 µmol
l–1 metamorphosis and 0.9±0.6 µmol
l–1 juvenile).
NKA
The tachykinin NKA (0.0001–1 µmol l–1) elicited a
concentration-dependent increase in mean force at all stages
(Fig. 1C). The potency of NKA
was in the same range after metamorphosis as prior to or during metamorphosis
(EC50: 0.09±0.06 µmol l–1
prometamorphosis, 0.08±0.04 µmol l–1 metamorphosis
and 0.4±0.2 µmol l–1 juvenile).
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5-HT
Cumulative administration of 5-HT (0.001–10 µmol
l–1, Fig. 2A)
elicited a concentration-dependent decrease in mean force in prometamorphic
tadpoles (EC50 0.31±0.26 µmol l–1).
Tissues taken from tadpoles during metamorphosis did not respond to 5-HT.
Although not evident from Fig.
2A, which includes mean data from all experiments, tissues from
juvenile froglets sometimes responded with an increased mean force at low
concentrations of 5-HT followed by relaxation at higher concentrations, while
in other juvenile preparations only relaxation at high concentrations could be
demonstrated. The potency of 5-HT at inducing relaxations in the juvenile
stomach strips was 10-fold lower than prior to metamorphosis (EC50
4.6±2.1 µmol l–1).
The excitatory and inhibitory phases of the biphasic response of juvenile stomach strips were studied separately in another set of experiments. To determine a concentration giving only contraction for each muscle strip, low concentrations of 5-HT (between 0.01 and 1 µmol l–1, Fig. 2B,E) were administered cumulatively until an increased mean force was seen, which occurred in eight out of 14 stomach strips. Contraction evoked by 5-HT was blocked by methysergide (Fig. 2E, P<0.05). The remaining six muscle strips relaxed in response to 5-HT, an effect also blocked by methysergide (Fig. 2C,D, P<0.05). A high concentration of 5-HT (10 µmol l–1) always elicited relaxation.
Responses to ATP
ATP
Cumulative administration of ATP (0.01–1000 µmol
l–1) produced a biphasic response in muscle strips from
prometamorphic tadpoles (Fig.
3A,B), with a significant decrease in mean muscle force occurring
between 3 and 30 µmol l–1 (EC50 5.4±4.1
µmol l–1) and contraction occurring at the highest
concentrations (300–1000 µmol l–1). A single high
concentration of ATP (1000 µmol l–1) at this stage
elicited contraction in the stomach strips, demonstrating that the contraction
phase of the concentration–response curve is in fact a contraction and
is not dependent on a possible desensitization or autoinhibition of the
preceding inhibitory response. ATP did not affect muscle strips from tadpoles
in metamorphic climax in a consistent manner, although a weak relaxation at
low concentrations and a weak contraction at high concentrations could be seen
in some preparations (Fig. 3A,
P>0.05). In juvenile froglets, a decrease in mean force occurred
at 300–1000 µmol l–1 ATP (EC50
233±99 µmol l–1,
Fig. 3A,C). The potency of ATP
with regard to its relaxing effect was at least 40 times lower in juvenile
animals compared with prometamorphic animals. A single high concentration of
ATP (1000 µmol l–1) in some cases (7 of 12 strips)
elicited a dual response in juvenile stomach strips with a transient
contraction peak preceding the relaxing response
(Fig. 3D). Administration of
TTX (1 µmol l–1) did not affect the inhibitory effects of
ATP in either prometamorphic (3 µmol l–1 ATP,
N=7) or juvenile animals (1000 µmol l–1 ATP,
N=7, data not shown).
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DPCPX and ATP
To further elucidate whether the ATP-elicited relaxing response was
mediated by metabolites acting on P1 receptors, the selective adenosine
A1 receptor antagonist DPCPX (1 µmol l–1) was
administered 20 min prior to ATP in prometamorphic and juvenile
Xenopus. In prometamorphic animals, DPCPX abolished the relaxing
response to ATP (Fig. 4C). In
juvenile froglets, 1 µmol l–1 DPCPX did not affect
ATP-evoked relaxation (data not shown). At 3 µmol l–1,
DPCPX per se appeared to attenuate contractions (possibly because of
the 0.03% DMSO vehicle); however, it was not able to inhibit the ATP-induced
relaxation (Fig. 4D).
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188±76 µmol
l–1). Administration of TTX (1 µmol l–1)
did not block adenosine-dependent relaxation (1000 µmol
l–1 adenosine) in either prometamorphic or juvenile animals
(N=6 and N=7, respectively, data not shown).
CPA
The selective adenosine A1 receptor agonist CPA
(0.0001–100 µmol l–1) produced a dose-dependent
decrease in mean force developed in stomach strips from all stages
(Fig. 5B). The potency of CPA
was slightly higher before (EC50 0.6±0.3 µmol
l–1) than during and after metamorphosis (EC50
2.1±0.9 and 3.2±2.0 µmol l–1,
respectively).
DPCPX and adenosine
The selective A1 receptor antagonist DPCPX (1 µmol
l–1) blocked adenosine-induced relaxation in muscle strips
from prometamorphic tadpoles (Fig.
5C), and attenuated the adenosine-induced relaxation in juvenile
stomach strips by 50% (P<0.05). The vehicle (0.01% DMSO) had no
effect on the muscle strip preparations.
ABT-702
Administration of the adenosine kinase inhibitor ABT-702 (1 µmol
l–1) caused a significant decrease in mean force in both
prometamorphic and juvenile Xenopus
(Fig. 5D). The vehicle (0.01%
DMSO) had no effect on the muscle strip preparations.
P2 receptor-mediated responses
UTP
Administration of the P2Y receptor agonist UTP [most potent at
P2Y2, P2Y4 and P2Y6
(Abbracchio et al., 2006;
Brunschweiger and Muller,
2006)] elicited a concentration-dependent decrease in mean force
in muscle strips from prometamorphic tadpoles (EC50 7.9±3.1
µmol l–1, Fig.
6A,B). In contrast, muscle strips from metamorphic tadpoles
responded to UTP with an increase in mean force (EC50 30±13
µmol l–1, Fig.
6A,C). In juveniles, UTP elicited a decrease in phasic
contractions (Fig. 6D).
However, this was often accompanied by an increase in tension resulting in a
non-significant change in mean force (Fig.
6A).
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SS is particularly potent at
P2Y2, P2Y11 and P2X5 purinoceptors but is
also an agonist at P2Y1, P2X1–4, P2X6,
P2X1/5 and P2X2/3 purinoceptors
(Abbracchio et al., 2006;
Lambrecht, 2000).
Administration of ATPS (0.1–300 µmol l–1)
resulted in a concentration-dependent increase in mean force developed in
stomach strips from prometamorphic tadpoles (EC50
102±45 µmol l–1,
Fig. 8A) and tadpoles in
metamorphic climax (EC50 618±232 µmol
l–1). Muscle strips from juvenile froglets only responded
with a weak increase in mean force at the highest concentration (300 µmol
l–1) resulting in EC50 values too uncertain to be
reported.
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-β-MeATP
Administration of the purinergic P2X1/P2X3-selective
agonist -β-MeATP (0.01–100 µmol l–1)
elicited a concentration-dependent increase in mean force at all three stages
(EC50: 35±15 µmol l–1
prometamorphosis, 69±27 µmol l–1 metamorphosis
and 198±69 µmol l–1 juveniles,
Fig. 8B). TTX (1 µmol
l–1) failed to block the effect of -β-MeATP in
either prometamorphic tadpoles (10 µmol l–1
-β-MeATP, N=6) or juvenile froglets (30 µmol
l–1 -β-MeATP, N=7, data not shown).
Metamorphic tadpoles were not tested.
PPADS and -β-MeATP
The P2 receptor antagonist PPADS (100 µmol l–1),
effective at homomeric P2X1, P2X2, P2X3 and
P2X5 receptors, heteromeric P2X2/3 and P2X1/5
receptors and P2Y1, P2Y6 and P2Y13 receptors
(Abbracchio et al., 2006;
Jacobson and Knutsen, 2001),
inhibited -β-MeATP-induced contractions
(Fig. 8C) in both
prometamorphic tadpoles (10 µmol l–1 -β-MeATP,
P<0.05) and juvenile stomach strips (30 µmol
l–1 -β-MeATP, P<0.05). PPADS had no
effect on ATP-induced relaxation at any stage tested (N=5, data not
shown).
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Sundqvist and Holmgren,
2004).
Carbachol and NKA
Carbachol, a cholinesterase-resistant analogue of the classical
neurotransmitter acetylcholine, had a similar potency all through
metamorphosis. The larger force elicited during the juvenile stage may again
reflect an increase in smooth muscle layer thickness. The bell-shaped
concentration–response curve obtained with carbachol after metamorphosis
suggests a degree of receptor internalization or desensitization at higher
concentrations that is not seen in the earlier stages. Alternatively,
inhibitory muscarinic (M) receptors (e.g. of the M2-type) may be expressed
during this stage. The consistently excitatory effect of the tachykinin NKA
through metamorphosis, with possibly only a slightly lower potency after
metamorphosis, supports previous studies of the Xenopus gut, both in
newly hatched tadpoles (Sundqvist and
Holmgren, 2006) and in adult Xenopus
(Johansson et al., 2002). In
the present study, mammalian NKA was used, since the study by Johansson et al.
(Johansson et al., 2002)
showed that there was no difference in potency or maximal effect between
endogenous Xenopus NKA and mammalian NKA.
Nitrergic mechanisms
The NO donor sodium nitroprusside (SNP) inhibits contractions in newly
hatched tadpoles (Sundqvist and Holmgren,
2006) and relaxes stomach muscle from adult frogs
(Olsson, 2002). Neurons
containing the NO-synthesizing enzyme NOS were detected in Xenopus
gut from stage 46 (Holmberg et al.,
2001). Following this stage, VIP-induced relaxation of the
intestine was partially blocked by L-NAME, suggesting that
endogenous NOS activity can be triggered by transmitters such as VIP
(Sundqvist and Holmgren,
2006), although a constant NO tonus under basal conditions did not
appear to be present. The current experiments in the stomach indicate the
development of a nitrergic tone during metamorphosis, which could be important
in modulating the increasingly complex, adult-like spontaneous activity that
develops during metamorphosis. In the urodele amphibian the axolotl
(Ambystoma mexicanum), nitrergic neurons develop late, during the
first juvenile stages (Badawy and Reinecke,
2003).
5-Hydroxytryptaminergic mechanisms
The responses to 5-HT, with a relaxation prior to metamorphosis changing to
a biphasic effect in juvenile stomach, indicate a change in 5-HT receptor
populations during metamorphosis. Developmental changes have previously been
found in 5-HT innervation and receptor (5HT1A) expression during
the early postnatal period in rat motor neurons
(Talley et al., 1997) and in
respiratory motor control in the amphibian Rana catesbeiana during
metamorphosis (Kinkead et al.,
2002). As in Rana respiratory control and in adult
Xenopus gut (Johansson,
2003), the biphasic effect of 5-HT in the juvenile stomach is
manifested as contraction at low 5-HT concentrations followed by relaxation at
higher concentrations. The contraction could counteract a relaxation at low
concentrations, explaining the apparent decrease in potency for 5-HT-induced
relaxation in juveniles.
In mammals, several receptors are involved in gastric relaxation and
contraction in response to 5-HT (Komada
and Yano, 2007; Tamura et al.,
1996; Xue et al.,
2006). In the present study, the 5-HT1–2 and
5-HT5–7 receptor antagonist methysergide
(Komada and Yano, 2007;
Prins et al., 2001) inhibited
all 5-HT-induced changes in tonus. Even though both types of response (after
metamorphosis) were blocked, the relaxing and contracting effects could still
be mediated by different receptors given the nonselective nature of
methysergide. A previous study in our laboratory using TTX on Xenopus
gut indicated that 5-HT receptors are situated mainly on smooth muscle
(Johansson, 2003). The gastric
mucosa of amphibians, both prior to and after metamorphosis, contains numerous
enterochromaffin (EC) cells containing 5-HT
(Sundqvist and Holmgren, 2004;
Villaro et al., 2001), while
5-HT-containing neurons are reported to be absent in the amphibian (Bufo
marinus) stomach (Anderson and
Campbell, 1989). Surprisingly, muscle strips responded very weakly
to 5-HT during metamorphosis, possibly due to receptor desensitization or
internalization during a time when the EC cells, containing the major source
of 5-HT in the tissue, are being reorganized.
P1 receptor-mediated purinergic mechanisms
The response to ATP elicited in prometamorphic Xenopus stomach was
similar to responses previously described in the intestine
(Sundqvist, 2007). The
biphasic response indicates the existence of at least two receptors in the
tissue. The relaxation is probably induced by an ATP-derived metabolite, since
ATP can be rapidly metabolized by ectonucleotidases present on cell surfaces,
and blockade of ectonucleotidases using ARL67156 completely abolished the
response. That the more stable ATP analogue ATPS only produced
contraction at this stage lends further support to this hypothesis, along with
the fact that the relaxing response to ATP could be blocked by the adenosine
A1 receptor antagonist DPCPX.
Blockade of ATP breakdown revealed an ATP-induced increase in mean force, which was not seen in the ATP + DPCPX experiments. We conclude that intact ATP may cause contraction in the stomach, while a metabolite such as adenosine is responsible for relaxation prior to metamorphosis.
The blockade of adenosine-induced relaxation by an A1 receptor
antagonist, while a selective A1 receptor agonist (CPA) potently
reduced mean force suggests that the receptor mediating the adenosine-induced
relaxation is A1-like prior to metamorphosis, as was previously
shown in the intestine (Sundqvist,
2007).
The overall inconsistent response to ATP per se in the metamorphosing tadpole could be due to a rapid change in sensitivity during this stage. Some muscle strips taken in the beginning of the transition (stage 61) tended to respond similarly to prometamorphic tissues while some taken at the end (stage 63) tended to respond similarly to juvenile tissues, thereby resulting in an unchanged mean response during metamorphosis. As mentioned above, tissues collected from tadpoles during metamorphosis also responded inconsistently to 5-HT. Thus, metamorphosis appears to reflect a transition stage during the control of gastric motility.
In the juvenile stomach, ATP-induced relaxation was only seen at the highest concentrations. Adenosine is probably not the only relaxing mediator, since the relaxation was only partly blocked by ectonucleotidase inhibition. The existence of relaxing P2 receptors may be suggested, but the results could also be explained by an increase in ectonucleotidase activity over-ruling the effect of the enzyme blocker. Similarly, the A1 receptor antagonist (1 µmol l–1) failed to block the ATP-induced relaxation. However, the same concentration only blocked 50% of adenosine-induced relaxation too, possibly indicating that higher concentrations are required or that another type of receptor is present in this tissue as well, for example A3 receptors.
The decrease in tonus in both prometamorphic and juvenile animals after inhibition of adenosine kinase using ABT-702 indicates that the endogenous levels of adenosine are sufficient to elicit relaxation if inactivation of adenosine by phosphorylation is inhibited.
P2 receptor-mediated purinergic mechanisms
UTP is a potent agonist at P2Y2, P2Y4 and
P2Y6 receptors, although its metabolite UDP is the most potent
agonist known at P2Y6
(Abbracchio et al., 2006).
P2Y2, P2Y4 and P2Y6 receptors have all been
found in the stomach of different mammalian species
(Chang et al., 1995;
Communi et al., 1996;
Giaroni et al., 2002;
Moore et al., 2001;
Suarez-Huerta et al., 2001;
Van Nassauw et al., 2006). UTP
has also been reported to be an agonist at P2Y11 receptors
(White et al., 2003). The
Xenopus-specific receptor p2y8
(Bogdanov et al., 1997) also
binds UTP and may be a P2Y4 receptor orthologue
(Abbracchio et al., 2006), but
its presence in Xenopus stomach has not been investigated.
UTP had complex effects in Xenopus stomach. The response could
conceivably be mediated by UDP but, in contrast to ATP, the instantaneous
effect and the rapid decline of the response rather suggest that UTP itself is
the effector. It should be noted that ATPS is a potent agonist at
P2Y2 receptors but did not elicit comparable responses to UTP in
this study, especially not prior to metamorphosis. Further studies using
selective pharmacological tools are required to determine the receptor
subtype(s) involved in the responses to UTP.
Interestingly, a submaximal concentration of UTP induced a transient increase in basal tension in juvenile stomach. The increase in tension was not seen prior to metamorphosis, suggesting that this effect of UTP develops at metamorphosis. The immediate decline in tension after the initial peak indicates desensitization or rapid metabolism of UTP. The insensitivity to TTX of the tension increase suggests that it is non-neuronal, due to direct stimulation of smooth muscle cells, in contrast to the simultaneous TTX-sensitive decrease in frequency of phasic contractions.
That TTX was only effective in juveniles suggests that the neuronal receptors responding to UTP develop after metamorphosis. It is possible that UTP stimulates inhibitory motor- or interneurons resulting in the release of inhibitory transmitters that block phasic contractions in the muscle. Indeed, this hypothesis was supported by the fact that the UTP-induced decrease in phasic contractions was blocked by NOS inhibition. Although a tonic NOS activity is present in these preparations, this activity seems to be relatively small. Conceivably, the removal of this tonus (by L-NAME or TTX) would not on its own result in the dramatic reversal of the UTP-induced inhibition of phasic contractions.
|
). The existence of a receptor for the UTP metabolite uridine
mediating inhibitory actions has been claimed, although this receptor has yet
to be identified (Connolly and Duley,
1999). The effects of ATP, adenosine or -β-MeATP were
unaffected by TTX in this study, similarly indicating that the purinergic
receptors in Xenopus gut responding to these agents are situated
directly on the smooth muscle or on TTX-insensitive nerves.
The contractions elicited by ATP and the P2X receptor agonist
-β-MeATP at different stages, taken together with the blockade of
the -β-MeATP-induced contractions by the nonselective P2
antagonist PPADS, indicates the existence of a P2X1 and/or
P2X3 receptor in the stomach of Xenopus, as has previously
been shown in Xenopus intestine
(Sundqvist, 2007). Since the
response to -β-MeATP did not desensitize at any stage, the
possibility of a heteromeric receptor consisting of P2X2 and
P2X3 subunits could also be indicated, although the presence of
such a receptor remains to be established in Xenopus.
The present study of the stomach shows larger changes in the regulation of
motility during metamorphosis than the subtle adjustments found in the
previous study of the intestine
(Sundqvist, 2007). Both the
nutritional content, from carbohydrate to protein, and the consistency of the
food, from liquid to solid, ingested by the Xenopus frog change
during metamorphosis. But as the food is processed down the gut the
differences in mechanical properties between the food eaten at different
stages grows less pronounced, which could suggest that larger changes in the
regulation of motility could be expected in the stomach than in the intestine.
Further, one could speculate that the need to mix the food with pepsin [an
enzyme not present in tadpoles (Rovira et
al., 1995)] to start protein degradation also requires a more
complex motility and motility regulation after metamorphosis than before.
To conclude, this study indicates the development of an excitatory 5-HT
receptor, a nitrergic tonus, excitatory smooth muscle UTP receptors and
possibly inhibitory neural UTP receptors during or after metamorphosis in
Xenopus (Fig. 9).
These changes could conceivably be related to the change from liquid to solid
and from herbivorous to carnivorous food intake during this period, and
indicate that the extensive remodelling affecting the connective and muscular
tissues of the amphibian stomach during metamorphosis also affects the enteric
nervous system. The study also shows the presence of an A1-like
adenosine receptor mediating relaxation in the stomach both prior to and after
metamorphosis, as well as a P2X-like purinergic receptor mediating
contraction, similar to previous findings in Xenopus intestine
(Sundqvist, 2007).
LIST OF ABBREVIATIONS
-dibromomethylene
ATP
S-thio]-triphosphate
-β-meATP-β-methyleneadenosine 5'-triphosphate
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Acknowledgments |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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