The enterins inhibit the contractile activity of the anterior aorta of Aplysia kurodai
1 Graduate School of Science, Department of Biological Science, Hiroshima
University, Kagamiyama 1-3-1, Higashi-Hiroshima 739-8526, Japan
2 Suntory Institute Bioorganic Research, Shimamoto, Mishima, Osaka 618-8503,
Japan
* Author for correspondence (e-mail: yasfuru{at}hiroshima-u.ac.jp)
Accepted 12 August 2002
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Summary |
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Key words: peptide, artery, smooth muscle, cardiovascular system, enterin, mollusc, Aplysia kurodai
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Introduction |
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The anterior aorta is one of the major arteries in the Aplysia
cardiovascular system, and carries hemolymph to many parts of the body
including the central nervous system (CNS), the buccal mass, the genital
organs, the opaline gland and the anterior somatic tissues
(Koester and Koch, 1987).
Previous studies on A. californica and A. kurodai have
provided evidence for regulation of the anterior aorta by several classical
transmitters, including glutamate, serotonin, acetylcholine and glycine
(Sawada et al.,
1981a
,b
,
1984a
,b
,c
).
It is, however, highly likely that the neuropeptides are also involved in
fine-tuning the function of blood vessels, as shown in other systems of
Aplysia (Weiss et al.,
1992
). In fact, several immunohistochemical studies suggest that
the nerve fibers containing different neuropeptides are distributed in the
anterior aorta (Miller et al.,
1991
; Skelton and Koester,
1992
; Giardino et al.,
1996
; Fujisawa et al.,
1999
; Morishita et al.,
2001
). Although these observations suggest numerous peptidergic
innervations of the anterior aorta, the functional roles and mechanisms of
such putative peptidergic innervations are poorly understood.
The enterins are multiple neuropeptides, recently identified in the gut and
CNS of A. californica and A. kurodai
(Furukawa et al., 2001). The
enterins inhibit gut motility of Aplysia, and also reduce
excitability of some identified Aplysia neurons that are involved in
the feeding network. Because the distribution of the enterin-immunopositive
neurons is widespread in the nervous system of Aplysia, it is
suggested that the enterins have actions in the control of several systems. In
the present study, we examined the possibility that the enterins are involved
in regulation of the arterial system of Aplysia. Immunohistochemical
results showed that the enterin-immunopositive nerve fibers are abundant in
the proximal region of the anterior aorta of Aplysia. Physiological
as well as pharmacological experiments revealed that the enterins inhibit the
contractility of the aortic muscles by at least two distinct mechanisms, and
that the multiple enterins are almost equally potent. These results suggest
that the enterins are functionally redundant multiple myoinhibitory peptides
in the arterial system of Aplysia.
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Materials and methods |
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Animals were anesthetized by intra-abdominal injection of 0.33 mol l-1 MgCl2. Approximately one third of the proximal region of the anterior aorta was dissected out from the anesthetized animal, and was kept in artificial seawater (ASW). The composition of ASW was (mmol l-1): NaCl 445, KCl 10, CaCl2, 10, MgCl2 55, Tris-HCl 10, pH 7.9. In some experiments, ASW containing either low K+ or low Cl- was used. 10% K+-ASW was made by replacing 9 mmol l-1 KCl with NaCl. 50% Cl--ASW was made by replacing 222.5 mmol l-1 NaCl with sodium methylsulfate. All the experiments were performed at room temperature (20-23°C).
Immunohistochemistry
The anterior aorta was excised from the anesthetized Aplysia, and
fixed by 4% paraformaldehyde for 24 h at 4°C or for 2-3 h at room
temperature. The fixed tissue was immunostained by using an anti-enterin
antibody (kindly provided by Dr Vilim) as described previously
(Furukawa et al., 2001). The
antibody was visualized by diluted fluorescein isothiocyanate-conjugated
rabbit anti-rat IgG (Organon Teknika, Durham, NC, USA). The preparation was
viewed with a fluorescence microscope (Nikon, Tokyo, Japan) and photographed.
The films were scanned by a film scanner (Coolscan III, Nikon), and printed
using Photoshop version 5 (Adobe Systems, San Jose, CA, USA).
Physiological recordings
To record the contraction of the anterior aorta, the proximal end of the
anterior aorta was pinned to the bottom of a chamber (0.5-1 ml volume). The
other end of the aorta was connected to a force-transducer (Type 45196A, NEC
San-ei Instrument Ltd., Tokyo, Japan). The signal from the transducer was
monitored on a chart recorder (FBR-251A, TOA Electronics Ltd, Tokyo, Japan).
In some experiments, the vulvar nerve was left attached to the anterior aorta
to permit electrical stimulation of the nerve.
The membrane potential of the muscle fibers in the aorta was measured using
a conventional microelectrode. The preparations were grounded directly using
an Ag/AgCl electrode or via a 3 mol l-1 KClagar
bridge connected to a reservoir containing 3 mol l-1 KCl and an
Ag/AgCl electrode. To restrict the movement of the anterior aorta, a small
piece of nylon mesh (approximately 100 µm between the grid) was pinned over
the aorta, and the muscle fiber was penetrated through the mesh with a sharp
microelectrode filled with a solution containing 3 mol l-1
CH3COOK and 0.1 mol l-1 KCl (resistance 40-60 M).
The membrane potentials were amplified by the Duo 773 electrometer (World
Precision Instruments, USA), and digitized using a 12-bit AD converter
(ADXM-AT10, Canopus, Kobe, Japan). The digitized data were stored on the hard
disk of a personal computer (IBM, Tokyo, Japan) for later analysis. The data
analysis and the compilation of figures were done using Origin (version 6,
Microcal Software Inc., Northampton, MA, USA). The results are expressed as
means ± S.E.M., except where indicated.
To analyze a concentrationresponse relationship of peptides, the data were fitted to an equation of the form y=r1+(r2-r1)/[1+10(logx0-x)n] by the LevenbergMarquardt algorithm using Origin. y is a response, x is a power for the concentration of the peptide (i.e. the concentration used is 10x), n is a Hill coefficient, r1 is a minimum response, r2 is a maximum response, and 10logx0 is EC50 (the concentration of peptide at which 50% response is expected). Because n values for our data were in the range 1-1.3, we fixed a value of 1 for the fittings presented in this paper. Other constraints and parameters obtained by the fittings are described in the figure legends.
Peptides and chemicals
All the peptides and drugs were applied by bath perfusion. 4-aminopyridine
(4-AP, Sigma, USA) and tetraethylammonium (TEA, Katayama, Japan) were
dissolved in ASW just before use. The enterins and an Aplysia
cardioactive peptide, NdWFamide (Asn-D-Trp-Phe-NH2;
Morishita et al., 1997), were
synthesized with an automated solid-phase peptide synthesizer (PSSM8,
Shimadzu, Kyoto, Japan) and purified by reversed-phase high performance liquid
chromatography (HPLC). The enterins used in the present study were as follows
(nomenclature is based on Furukawa et al.,
2001
): ENa, VSPKYGHNFVamide; ENe, ADLGFTHSFVamide; ENk,
APGYSHSFVamide; ENl, ELNFQHAFVamide; ENpa, APSFGHSFVamide; ENr,
DPGFNHAFVamide. ENpa is a novel enterin purified from the CNS extract of
A. kurodai (Y. Fujisawa, unpublished). Although ENpa is not coded on
the enterin precursor of A. californica
(Furukawa et al., 2001
), it was
found to be coded on the enterin precursor of A. kurodai (Y.
Furukawa, unpublished observation). In this paper, we designate APSFGHSFVamide
as ENpa because the peptide is identical to ENp of A. californica
(VPSFGHSFV amide), except for the N-terminal amino acid. The peptides were
dissolved in distilled water to make a concentrated stock solution
(10-2 mol l-1). The stock solution was diluted
appropriately just before use.
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Results |
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Action of the enterins on the quiescent anterior aorta
An isolated anterior aorta of Aplysia can show spontaneous
contractile activities and even regular beating in some cases (e.g.
Morishita et al., 1997). In
the present study, however, the isolated anterior aorta rarely showed
spontaneous contractions. When the enterin was applied to an isolated aorta by
bath perfusion, the aorta more or less relaxed, but the extent of the
relaxation was quite variable, depending on the preparations, and some
preparations did not respond at all even after the application of high
concentrations of the peptide (10-6 mol l-1).
Fig. 2 shows an example of the
enterin-induced relaxation of the anterior aorta. In this preparation,
10-8 mol l-1 ENpa induced a clear relaxation of the
basal tonus, and maximum relaxation was observed at > 10-6 mol
l-1. The enterin-induced relaxation of the aorta could be reversed
after washing out the enterin. In some preparations, however, a reduced tonus
of the aorta was never recovered after application of high concentrations of
the enterins (> 10-6 mol l-1). Because the relaxation
of the aorta by the enterins seems to be dependent on the level of basal tonus
of the aorta, we next examined the actions of the enterins on the contracted
aorta.
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Action of the enterins on the contraction of the anterior aorta
To examine the action of the enterins on the contracted aorta, we used an
Aplysia cardioactive peptide, NdWFamide
(Morishita et al., 1997), to
raise the basal tonus of the anterior aorta. Although other transmitters can
evoke contraction of the anterior aorta (Sawada et al.,
1982
,
1984a
,b
),
NdWFamide is preferable because NdWFamide evokes a tonic contraction of the
aorta with little desensitization
(Morishita et al., 2001
).
10-7 mol l-1 NdWFamide evoked a tonic contraction of the
anterior aorta, and the extent of contraction usually changed little in the
presence of NdWFamide (see Fig.
3Ai). After washing out NdWFamide, the developed tension of the
anterior aorta returned slowly to the initial level during the next 20-30 min.
When 10-7 mol l-1 ENk was applied in the presence of
NdWFamide, the contraction of the anterior aorta evoked by NdWFamide was
inhibited and the aorta showed a clear relaxation
(Fig. 3Aii), which was more
prominent at higher concentrations of ENk
(Fig. 3Aiii).
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To quantify the inhibitory action of the enterins, we normalized the minimum amplitude of the contraction in the presence of the enterin by the amplitude of the control contraction (see Fig. 3 legend). The concentrationresponse relationships obtained for six enterins (ENa, ENe, ENk, ENl, ENpa, ENr) are shown in Fig. 3B. Although the amino acid sequences of the enterins used are rather diverse (see Materials and methods), the inhibitory actions of the enterins were quite similar. A threshold concentration was close to 10-9 mol l-1, and EC50 was approximately 10-7 mol l-1 (see Fig. 3 legend). Although the curve-fittings showed different levels of maximum inhibition among the enterins (see Fig. 3 legend), the differences between relative contractions at 10-5 mol l-1 were statistically marginal but not significant (ANOVA, F5,18=2.7, P>0.05). For the experiments in Fig. 3, we applied the enterins for 3 min, although the inhibitory action was obviously not saturated during that time (see Fig. 3Aii). A longer application of the enterins produced a larger relaxation of the aorta than shown in Fig. 3, especially at the lower concentration range (data not shown). Complete recovery of the enterin-induced inhibition was only seen after a short application time, which was also necessary for the reproducible contracture by the following NdWFamide applications. Because the enterins tested had a similar potency, ENpa was used in most of the following experiments.
Action of the enterins on the phasic contraction of the anterior
aorta
We next examined the action of the enterins on the phasic contraction of
the anterior aorta, which is evoked by the electrical stimulation of the
vulvar nerve. The vulvar nerve is one of the nerve bundles arising from the
abdominal ganglion, and innervates the anterior aorta
(Sawada et al., 1981a).
Although a single electrical stimulus of the nerve can evoke a phasic
contraction of the anterior aorta, we used a repetitive stimulation (10 Hz for
1 s) to obtain a reproducible phasic contraction of the aorta.
Fig. 4A shows the effect of
10-8 mol l-1 ENpa on the phasic contraction of the
anterior aorta. In the presence of ENpa, the evoked contraction was reduced to
about 40% of the control. The phasic contraction was recovered after washing
out ENpa. The peak amplitude of the phasic contraction in the presence of ENpa
was normalized to the one obtained in the absence of ENpa, and plotted against
the concentration of ENpa (Fig.
4B). The concentrationresponse relationship showed that a
threshold for the inhibitory action is approximately 10-10 mol
l-1 and EC50 is close to 10-8 mol
l-1. In most cases, the phasic contraction was completely abolished
by 10-7 mol l-1 ENpa.
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Action of the enterins on the membrane potential of the aortic muscle
fibers
As described above, the enterins inhibit both the basal tonus and the
evoked contraction of the anterior aorta, suggesting their myoinhibitory
roles. To clarify the mechanisms of the inhibitory action, we next examined
the effect of the enterins on the membrane potential of the muscle fibers in
the anterior aorta. The resting membrane potentials of the muscle fibers were
-59.0±1.3 mV (N=38). The values are consistent with those
published by others (Sawada et al.,
1981a).
The enterins induced a hyperpolarization of the muscle fibers in a dose-dependent manner (Fig. 5A). The response turn-on was slow, and it took several minutes to reach the peak. A threshold concentration was <10-8 mol l-1, and EC50 was close to 10-7 mol l-1. A maximum hyperpolarization evoked by >10-6 moll-1 ENpa (or ENe) was more than 10 mV from the resting potential (Fig. 5B).
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Considering the resting potential level of the muscle fibers, the enterin-induced hyperpolarization can be due to the increase of either K+ conductance or Cl- conductance, or both. To determine the ionic mechanisms, we next examined the ENpa-induced hyperpolarization in K+ or Cl--deficient condition. When the anterior aorta was bathed in 10% K+-ASW, the hyperpolarizing response increased markedly (Fig. 6A). In ASW, ENpa induced the hyperpolarization of 10.7±1.0 mV, whereas it became 21.0±1.0 mV in 10% K+-ASW (N=3). The difference between the two conditions is statistically significant (t-test, P<0.01). When the anterior aorta was bathed in 50% Cl--ASW, the ENpa-induced hyperpolarization increased slightly, and became 14.6±2.6 mV compared to 12.2±3.2 mV in ASW (N=3). This small change is in the opposite direction to that expected if increased Cl- conductance is involved, and is perhaps due to a depolarized membrane potential of the muscle fibers in low Cl- solution (see Fig. 6B). These results suggest that the hyperpolarizing response of the muscle fibers by the enterins is mainly caused by an increase in K+ conductance.
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To characterize the K+ conductance activated by the enterins, we examined the effects of two conventional K+ channel blockers, 4-AP and TEA. TEA had little effect on the ENpa-induced hyperpolarization even at 10 mmoll-1 (data not shown). On the other hand, the K+ conductance was quite sensitive to 4-AP. Fig. 7 shows the blocking action of 4-AP on the ENpa-induced hyperpolarization. A threshold concentration for the blocking action was <10-7 moll-1, and the hyperpolarizing response was completely blocked by 1 mmoll-1 4-AP (Fig. 7B). The 4-AP block can be completely washed out if the concentration of 4-AP was <10-4 moll-1. These results suggest that the enterins activate the 4-AP sensitive K+ channels.
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Action of the enterins in the presence of 4-AP
The results presented above suggest that the enterins inhibit the
contraction of the muscles in the anterior aorta via the activation
of the 4-AP-sensitive K+ channels. The extra K+ currents
can hyperpolarize the membrane potential of the muscles, and keep it below the
threshold depolarization required for the muscle contraction. Because 1
mmoll-1 4-AP completely blocked the ENpa-induced hyperpolarization,
we tested the hypothesis by examining the action of the enterins in the
presence of 1 mmoll-1 4-AP. Fig.
8A shows an example of the effect of 10-6
moll-1 ENpa on the phasic contractions of the aorta in the absence
(Fig. 8Ai) or the presence of
4-AP (Fig. 8Aii) obtained in
the same preparation. In the presence of 4-AP, a basal tonus as well as the
phasic contraction of the aorta was increased. The result can be explained by
the blockade of K+ channels, which would enhance the excitability
of the muscle membrane, and/or increase the excitatory transmitter release
(Molgo et al., 1977). Although
10-6 moll-1 ENpa almost completely blocked phasic
contraction in the control (Fig.
8Ai), the contraction was not affected in the presence of 4-AP
(Fig. 8Aii). In this series of
experiments (N=4), 10-6 moll-1 ENpa reduced the
phasic contraction to 14.8±4.5% of the control in the absence of 4-AP,
compared to 93.4±6.5% in the presence of 4-AP, suggesting that
inhibition of the phasic contraction by the enterins is mostly due to
activation of the 4-AP-sensitive K+ channels. However, it is
noteworthy that ENpa also diminishes the enhanced basal contraction by 4-AP
(see Fig. 8Aii). The result
implies that the inhibitory action of the enterins is not entirely due to the
activation of the 4-AP-sensitive K+ channels.
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We next examined the effect of 4-AP on the enterin-induced inhibition of
the NdWFamide-evoked contraction (Fig.
9). In the presence of 4-AP, NdWFamide evoked oscillating
contractions superimposed on the tonic contraction, but the amplitude of the
tonic contraction was not affected so much (compare
Fig. 9Ai and Aii). Because the
contraction of the aorta via the excitatory synaptic transmission is
enhanced by 4-AP (see above), the result favors an hypothesis that NdWFamide
evokes the muscle contraction directly. The oscillating contraction of the
aorta is probably due to the enhanced excitability of the muscle membrane by
4-AP. In contrast to the nerve-evoked phasic contraction, the action of the
enterins on the NdWFamide-evoked contraction was not affected by 4-AP
(Fig. 9B). both the central and
peripheral nervous systems of Aplysia
(Furukawa et al., 2001).
Previous studies suggest that the enterins are inhibitory peptides in the gut,
and that the enterins may change the feeding motor programs. Because
enterin-positive neurons are found throughout the nervous system, the peptides
are thought to have other functions in Aplysia. In the present study,
we show that enterin-immunopositive fibers and varicosity-like structures are
abundant in the anterior aorta of Aplysia, and that the enterins
inhibit both the basal tonus and the evoked contraction of the anterior aorta.
Both the immunohistochemical and physiological results suggest that the
enterins are inhibitory neuropeptides for the contractile activity of the
aorta. Despite some structural differences between the six enterins used in
this study, their inhibitory potencies on the NdWFamide-evoked contraction
were almost identical. The results are in accord with the previous observation
that the inhibitory actions of the different enterins are similar in the gut
of Aplysia (Furukawa et al.,
2001
), supporting the previous suggestion that the enterins are
functionally redundant. A structural consideration of the present
pharmacological evidence as well as previous evidence suggests that a common
structure in the C-terminal half of the enterins (i.e.
(Y/F)XH(S/A)F(V/L)amide) is sufficient to exert the action of the enterins.
Indeed, a preliminary experiment shows that a synthetic analogue peptide,
YSHSFVamide, has an inhibitory action in the Aplysia gut (O.
Matsushima, unpublished observation).
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The anterior aorta of Aplysia is innervated by nerves arising from
the abdominal ganglion (Sawada et al.,
1981a). The vulvar nerve is one of the nerves innervating the
anterior aorta, in which axons of some identifiable motoneurons or modulatory
neurons for the anterior aorta are contained (Sawada et al.,
1981a
,
1984c
). The phasic contraction
evoked by the vulvar nerve stimulation was inhibited by the enterins. At
least, one of the mechanisms for the inhibition seems to be activation of
K+ conductance of the muscle membrane. We found that the enterins
hyperpolarize the membrane potential of the muscle fibers via the
activation of 4-AP-sensitive K+ channels. Sensitivity of the
K+ channels to 4-AP is quite high, and EC50 of 4-AP was
<10-6 mol l-1. The value is comparable to another
highly 4-AP-sensitive K+ channel described in the accessory radula
closer muscle of Aplysia (Brezina
et al., 1994
). The enterin-induced hyperpolarization of the muscle
membrane should, in principle, reduce the excitability of the muscle, and
inhibit the nerve-evoked contraction. The excitatory transmitter release may
also be reduced if the K+ channels that are activated by the
enterins are present in the presynaptic terminals. The explanation is
straightforward and in accordance with the results in
Fig. 8, in which the inhibitory
action of the enterins on the nerve-evoked contraction is almost completely
blocked by 4-AP.
However, some other results are difficult to explain completely by the
hyperpolarizing action of the enterins. A discrepancy may be noticed if the
two concentrationresponse relationships for the action of the enterins
(Figs 4 and
5) are compared. The phasic
contraction of the aorta by the vulvar nerve stimulation can be inhibited by
>10-10 mol l-1 of ENpa, and was mostly abolished by
10-7 mol l-1 ENpa. By contrast, ENpa-induced
hyperpolarization was rarely seen at 10-8 mol l-1, and
EC50 was approximately 10-7 mol l-1.
Therefore, the phasic contraction is noticeably inhibited by ENpa at levels
well below the threshold concentration to evoke a detectable hyperpolarization
of the resting membrane potential. A plausible explanation for this
discrepancy may be that the K+ channel that is activated by the
enterins may show outward rectification like the S-K+ channel of
Aplysia (Siegelbaum et al.,
1982), or may be voltage-dependent, as for the FMRFamide activated
K+ channel in Lymnaea neurons
(Kits et al., 1997
). In either
case, more K+ currents would flow during a rising phase of the
excitatory junctional potential than in the resting state, which may result in
inhibition of the nerve-evoked contraction of the aorta even though there is
little hyperpolarization of the resting potential. Strong evidence suggesting
the K+ channel-independent mechanism for the inhibitory action of
the enterins is provided by the results shown in
Fig. 9, in which the
NdWFamide-evoked contraction of the aorta was inhibited by the enterin even in
the presence of 1 mmol l-1 4-AP. Because 1 mmol l-1 4-AP
blocks the enterin-induced hyperpolarization completely
(Fig. 7), there must be a
membrane potential-independent action of the enterins. Taken together, our
working hypothesis is that the enterins activate at least two pathways leading
to the inhibition of the aortic muscle contraction: (1) indirectly by the
activation of the 4-AP-sensitive K+ channels in the muscle
membrane, and (2) direct inhibition of the contractile machinery.
Because Aplysia lacks a rigid skeleton, the blood vessels can
easily be stretched and/or twisted during a change of posture or locomotion.
The anterior aorta is one of the largest arteries of the animal, and it
encompasses approximately two thirds of the body length, from the cristae
aorta near the heart to the head ganglia. The anterior aorta, therefore, is
considered to receive a large external force during several types of animal
behavior. To accommodate to such stress, it seems reasonable that the
stiffness and the length of the aorta be under neuronal regulation. In fact,
Skelton and Koester (1992)
have shown that two identifiable neurons in pedal ganglia of Aplysia
regulate the length of the rostral anterior aorta and the right and left pedal
parapodial arteries to meet the shortening of the head and neck region during
various behaviors.
In this study, the enterins were found to relax the basal tonus of the aorta and inhibit the contraction. The action of the enterins may counterbalance the stretch posed on the blood vessels in some behavioral states to protect the vessels and maintain the blood flow. An Aplysia cardioactive peptide, NdWFamide, seems to have an opposite function because NdWFamide enhances the basal tonus of the anterior aorta. The present study also showed that the action of NdWFamide is antagonized by the action of the enterins. These two types of peptides may be physiological antagonists to regulate the basal stiffness of the blood vessels of Aplysia.
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Acknowledgments |
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References |
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