Peptidergic innervation of the vasoconstrictor muscle of the abdominal aorta in Aplysia kurodai
Department of Biological Science, Graduate School of Science, Hiroshima University, Kagamiyama 1-3-1, Higashi-Hiroshima 739-8526, Japan
* Author for correspondence at present address: Laboratory of Neurobiology, Faculty of Integrated Arts and Sciences, Hiroshima University, Kagamiyama 1-7-1, Higashi-Hiroshima 739-8521, Japan (e-mail: yasfuru{at}hiroshima-u.ac.jp)
Accepted 6 September 2004
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Summary |
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Key words: cardiovascular system, peptide, mollusc, Aplysia
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Introduction |
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The blood distribution of crustaceans is regulated by neuronal as well as
neurohormonal control of the cardioarterial valves located at the base of each
artery (Kihara and Kuwasawa,
1984; Kuramoto and Ebara,
1984
; Kuramoto et al.,
1992
; F.-Tsukamoto and
Kuwasawa, 2003
). Regulation of the blood flow by the valves is
presumably a mechanism underlying the physiological modification of the blood
distribution observed in a decapode crustacean in vivo
(Airriess and McMahon, 1994
;
De Wachter and McMahon, 1996
;
McGaw and McMahon, 1996
). A
functionally similar mechanism is observed in a marine mollusc,
Aplysia. There are three major arteries in Aplysia and one
of them, the abdominal aorta, carries blood from heart to hepatopancreas and
ovotestis. The abdominal aorta has a sphincter (the vasoconstrictor muscle) at
the base of the artery (Mayeri et al.,
1974
). The activity of the vasoconstrictor muscle influences the
blood distribution because the contraction of this muscle prevents the blood
flow into the abdominal aorta and enhances the flow into the other two
arteries (Mayeri et al., 1974
;
Koch and Koester, 1982
;
Koch et al., 1984
). Activity
of the vasoconstrictor muscle is relevant to feeding
(Koch and Koester, 1982
;
Koch et al., 1984
) and
respiratory pumping (Koester et al.,
1974
; Byrne and Koester,
1978
; Kandel 1979
),
and is known to be modulated by some peptides (Alevizos et al.,
1989
,
1991
).
Recently, three different types of peptides (AMRP, enterin, NdWFamide) were
identified in Aplysia (Morishita
et al., 1997; Fujisawa et al.,
1999
; Furukawa et al.,
2001
). Immunohistochemical experiments showed that the
peptide-containing neuronal processes exist in the cardiovascular system
including the abdominal aorta (Fujisawa et
al., 1999
; Sasaki et al.,
2002b
; Morishita et al.,
2003a
). Although the immunohistochemical results imply a
regulatory role for these peptides in the abdominal aorta, physiological
actions of the peptides on the contractile activity of the vasoconstrictor
muscle are not yet established. The aim of the present study is to examine the
actions of AMRP, enterin and NdWFamide in the vasoconstrictor muscle, and to
delineate possible physiological roles of the peptides in the cardiovascular
regulation of Aplysia.
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Materials and methods |
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Immunohistochemistry
Animals were anesthetized by intra-abdominal injection of 0.33 mol
l1 MgCl2. The abdominal aorta together with the
abdominal ganglion was excised from the anesthetized animal and was bathed in
a chamber containing the artificial sea water (ASW). ASW had the following
composition (mmol l1): NaCl 460, KCl 10, CaCl2
10, MgCl2 55, Tris-HCl 10, pH 7.8. The abdominal aorta was fixed by
4% paraformaldehyde for 2024 h at 4°C or for 23 h at room
temperature. Whole mount immunocytochemistry was carried out essentially as
described previously (Fujisawa et al.,
1999). The anti-enterin and anti-AMRP antibodies were kindly
provided by F. S. Vilim. 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 (v5, Adobe
Systems, San Jose, CA, USA).
Physiological recordings
The abdominal aorta was excised from the animal with the innervation from
the pericardial nerve or the spermathecal nerve left intact. To record
contractions of the vasoconstrictor muscle, the abdominal aorta was cut open
longitudinally. One end of the vasoconstrictor muscle was pinned to the bottom
of a recording chamber (0.8 ml volume) and the other end was connected to a
force-transducer (Type 45196A, NEC San-ei Instrument Ltd, Tokyo, Japan) by a
cotton thread. The signal from the transducer was monitored on a chart
recorder (FBR-251A, TOA Electronics Ltd, Tokyo, Japan) and also 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).
The data analysis and the compilation of figures were done using Origin (v6,
Microcal Software Inc., Northampton, MA, USA). During the experiment, the
recording chamber was continuously perfused with ASW (3-4 ml
min1). All the peptides and drugs were applied by perfusing
the bath with the peptide and/or drug containing ASW. In some experiments, we
monitored the activity of the vasoconstrictor muscle by measuring the
intra-arterial pressure change as described
(Liebeswar et al., 1975).
Briefly, one end of a three-way perfusion tube was inserted into the distal
portion of aorta. The other two ends of the three-way tube were connected to a
pressure transducer (DT4812J, Nippon Becton Dickinson Company, Ltd, Tokyo,
Japan) and a peristalic pump, respectively. The abdominal artery was
continuously perfused with ASW by the pump (4 ml min1). In
this way, the contraction of the vasoconstrictor muscle was monitored as an
increase in the inner pressure of the perfusion line. In most experiments, the
contraction of the vasoconstrictor muscle was evoked by electrical
stimulations of the pericardial nerve with a suction electrode. The
pericardial nerve was stimulated by a train of the electrical pulse (1 ms,
1.03.0 V, 10 Hz) for 0.12.0 s every 20 s. In some experiments,
the contraction was induced by bath application of acetylcholine (ACh) for
2040 s.
Membrane potential of the vasoconstrictor muscle was measured using a
conventional microelectrode method as described
(Sasaki et al., 2002b). To
restrict the movement of the vasoconstrictor muscle, 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 l1
CH3COOK and 0.1 mol l1 KCl (resistance
4060 M
). The preparations were grounded directly using an
Ag/AgCl electrode. Excitatory junction potentials (EJPs) were elicited by the
pericardial nerve stimulation. The membrane potentials were amplified by the
Duo 773 electrometer (World Precision Instruments, Sarasota, FL, USA), and
were stored as described above. The results are expressed as means ±
S.E.M. All the experiments were performed at room temperature
(2023°C). To assess the statistical difference between the control
and the experimental groups, an F-test was conducted first to
determine the equality of the variances between the two groups. When the
variance was not considered to be equal, the difference between the groups was
tested by a Mann-Witney U-test. Otherwise, a Student's
t-test was used. The results were considered significant if
P<0.05.
Peptides and chemicals
ACh, 4-aminopyridine (4-AP) and hexamethonium were purchased from Sigma (St
Louis, USA) and were dissolved in ASW just before use. APSFGHSFVamide (ENpa),
GSPRFFamide, and NdWFamide were synthesized with an automated solid-phase
peptide synthesizer (PSSM8, Shimadzu, Kyoto, Japan) and purified by
reversed-phase high-performance liquid chromatography. Although enterins and
AMRPs are families of multiple related peptides, we used ENpa (enterin) and
GSPRFFamide (AMRP) throughout this study because previous studies in the
anterior aorta showed little difference in potency among different enterins or
AMRPs (Sasaki et al.,
2002a,b
).
Peptide was dissolved in distilled water to make a concentrated stock solution
(102 mol l1). The stock solution was
stored at 20°C and diluted appropriately just before use.
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Results |
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We next verified distribution of the AMRP-, enterin- and NdWFamide-immunoreactivity in the vasoconstrictor muscle. Fig. 1A shows an example of the distribution of AMRP-immunoreactivity in the vicinity of the abdominal ganglion and three main arteries. All three arteries were densely innervated by AMRP-immunoreactive fibers. In the abdominal aorta and gastroesophageal artery, all the AMRP-immunoreactive fibers originated from the pericardial nerve and the spermathecal nerve. Main immunoreactive nerve trunks on the abdominal aorta were oriented in the longitudinal direction, and many small immunoreactive fibers surrounded the vasoconstrictor muscle (Fig. 1A). No peripheral immunoreactive cell bodies were found in the abdominal aorta. The distribution pattern of the enterin- or NdWFamide-immunoreactive fibers in the abdominal aorta was almost the same to that of the AMRP-immunoreactivity (Fig. 1B). Quantitatively, however, the enterin- or NdWFamide-immuno-reactivity seemed to be less than that of AMRP. These immunohistochemical analyses suggest that AMRP, enterin and NdWFamide may play roles in the regulation of the vasoconstrictor muscle of the abdominal aorta.
Action of the peptides on the contractility of the vasoconstrictor muscle
To clarify the function of the peptides, their actions on the nerve-evoked
contraction of the vasoconstrictor muscle were examined. The stimulus trains
applied to the pericardial nerve (see Materials and methods) reliably induced
contractions of the vasoconstrictor muscle, which permitted testing the action
of the peptides unequivocally. Although the data were not presented in this
paper, similar results were obtained on the contractions elicited by the
spermathecal nerve stimulation.
Fig. 2 shows the effects of 107 mol l1 ENpa and GSPRFFamide on the nerve-evoked contractions of the vasoconstrictor muscle. The peptide was applied for 2 min because a longer application did not affect the contraction further in preliminary experiments. In our perfusion system, it took about 25 s to replace the bath solution completely (estimated by the dye perfusion). Amplitude of the contractions was reversibly inhibited by application of either peptide. Both peptides sometimes relaxed the basal tonus of the vasoconstrictor muscle slightly. To quantify the concentrationresponse relationship of the peptide action, the relative amplitude of the most depressed contraction after the peptide application was calculated and plotted against the concentration of the peptide (Fig. 2B). Threshold concentrations of both peptides were less than 109 mol l1, and their overall doseresponse relationships were almost identical. To compare the time course for the onset and the recovery of the inhibitory actions, each contraction elicited every 20 s was normalized and plotted as a function of time. The inhibitory actions of ENpa and GSPRFFamide had a similar time course up to the concentration of 107 mol l1 or less. The differences in the time course, however, became apparent at the concentration of more than 106 mol l1 (Fig. 2C). The inhibitory action of 105 mol l1 ENpa became to its peak within a minute, and started to decline even in the presence of the peptide. By a sharp contrast, the inhibitory action of GSPRFFamide grew slowly, and its maximum effect was always seen during washing out the peptide. The different time course at higher concentration of the peptides suggests that the mechanisms for the inhibitory actions of ENpa and GSPRFFamide are not identical.
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In contrast to ENpa and GSPRFFamide, NdWFamide enhanced the amplitude of the nerve-evoked contractions of the vasoconstrictor muscle (Fig. 3). The amplitude of the nerve-evoked contraction could be enhanced to more than 500% of the control contraction. NdWFamide also evoked a tonic contraction of the muscle although the amplitude of the tonic contraction was quite different among preparations. When the tonic contraction was large enough, the potentiation of the nerve-evoked contraction was masked (Fig. 3B). Although the threshold concentration for the potentiating action of NdWFamide was less than 109 mol l1 in all the tested preparations, it was difficult to evaluate the potentiating action by the amplitude of the nerve-evoked contraction in most preparations because of the apparently mixed actions of NdWFamide. In those preparations which showed relatively small tonic contractions, the potentiating action of the nerve-evoked contraction reached a plateau at more than 106 mol l1. In general, the action on the nerve-evoked contraction was long lasting compared with the action on the basal tonus and persistent after washing out the peptide (see also Fig. 12).
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Action of the peptides on the ACh-induced contraction of the vasoconstrictor muscle
In A. californica, the identified vasoconstrictor motoneurons are
suggested to be cholinergic because their actions are blocked by a cholinergic
antagonist, hexamethonium (Liebeswar et
al., 1975). The nerve-evoked contraction of the vasoconstrictor
muscle in the present preparation was also blocked reversibly by
103 mol l1 hexamethonium
(Fig. 4A; N=4),
suggesting that the excitatory innervation of the muscle in A.
kurodai is also mainly cholinergic. To determine whether the modulatory
actions of the peptides are mediated postsynaptically, we next examined
actions of the peptides on the ACh-induced contraction. ACh was applied by a
brief perfusion (<40 s) every 2025 min to avoid a desensitization of
the ACh response. In Fig. 4B,
106 mol l1 ACh was perfused for 40 s to
evoke each contraction. In the presence of 107 mol
l1 ENpa, the ACh-induced contraction was depressed to 40% of
the control (Fig. 4Bi). In the
same preparation, 107 mol l1 GSPRFFamide
did not inhibit the ACh-induced contraction but rather enhanced it slightly
(Fig. 4Bii). Effect of
GSPRFFamide was quite variable depending on the preparations, and either
modest inhibition or some potentiation of the ACh-induced contraction was
observed (see Fig. 5).
108 mol l1 NdWFamide did not affect the
ACh-induced contraction (Fig.
4Biii).
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Effects of each peptide on the nerve-evoked contraction and the ACh-induced contraction are compared in Fig. 5. ENpa always inhibited the contractions evoked by both methods although the inhibitory potencies were somewhat variable among the preparations (Fig. 5A). Mean ± S.E. of the relative contractions evoked by the nerve stimulation and the ACh application were 23.0±4.2% (N=13) and 51.7±8.5% (N=12), respectively. The different potencies of ENpa examined in the two conditions are statistically significant (P<0.05, Mann-Witney U-test), suggesting that the inhibitory action of ENpa is produced by both presynaptic and postsynaptic mechanisms. In some preparations, ENpa reduced the amplitude of the nerve-evoked contraction to 7.1±4.3% of the control although the ACh-induced contraction was rarely inhibited (93.9±1.2%, N=3).
Similarly, GSPRFFamide always decreased the nerve-evoked contraction (Fig. 5B). The mean inhibitory action (33.7±7.6% of the control, N=11) was comparable to that of ENpa. By contrast, its effect on the ACh-induced contraction was extremely variable as noted above. In the presence of GSPRFFamide, the relative contractions of seven preparations were between 70.2 and 156.9% of the control (109.6±11.9%, N=7). Such variability indicates the complexity of the AMRP action in this system. Nevertheless, the differences between the mean effects of GSPRFFamide in two conditions are statistically significant, suggesting that the inhibitory action of GSPRFFamide is mainly presynaptic.
NdWFamide enhanced the nerve-evoked contraction considerably (430.4±45.1% of the control, N=10; Fig. 5C), but had essentially no effect on the ACh-induced contraction (110.3±3.2%, N=4). These results clearly suggest that potentiation of the contractility of the vasoconstrictor muscle by NdWFamide is due to the enhancement of the excitatory transmitter release.
Action of the peptides on the resting and excitatory junction potentials of the vasoconstrictor muscle
We next examined whether the resting potential of the arterial muscle and
the excitatory junction potentials (EJPs) evoked by the nerve stimulation were
affected by the peptides. In ASW, the resting potential of the vasoconstrictor
muscle was 77.4±1.2 mV (N=37). We often observed
spontaneous EJPs but not the inhibitory junction potentials (IJPs, not shown).
Also, the nerve stimulation did not evoke IJPs in the present experiments.
Fig. 6A shows the EJP in the
vasoconstrictor muscle elicited by a single brief stimulation of the
pericardial nerve. In this preparation, a threshold stimulus intensity to
evoke an EJP was between 2.0 and 2.1 V. The threshold was different among
preparations, ranging from 1.0 to 2.2 V. In the preparation shown in
Fig. 6, the peak amplitude of
the EJP evoked by 2.3 V became twice of that evoked by 2.2 V. Further increase
in the stimulus intensity did not increase the size of the EJP. When the
intensity was increased to 2.9 V, multiple EJPs having longer latencies were
also observed in addition to the initial EJP. These results suggest that a
single vasoconstrictor muscle fiber is innervated by multiple excitatory
axons. Multiple excitatory innervation and the lack of IJPs are also described
in A. californica (Mayeri et al.,
1974). Repetitive nerve stimulations produced a summation of EJPs,
and in some cases, a marked facilitation of EJPs was observed
(Fig. 6B). EJPs were reversibly
abolished by hexamethonium (Fig.
6B; N=4), supporting the above mentioned hypothesis that
ACh is the main transmitter released from the excitatory nerve terminals
following the stimulation of the pericardial nerve.
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To quantify the effects of the peptides on EJP, stimulation intensity was adjusted before each experiment so that a single nerve stimulation evokes an EJP with a single peak. Because the nerve stimulation sometimes failed to evoke EJP and the size of a single EJP was often quite small, summated EJPs in response to several stimuli were examined to see the effects of peptides in most cases.
Fig. 7 illustrates the effect of ENpa on EJP. Summating EJPs were elicited by three consecutive stimuli of the pericardial nerve. Application of 107 mol l1 ENpa reduced the amplitude of each EJP as well as the summated EJPs. In control, the peak depolarization of the summated EJPs was 17.7±0.7 mV whereas it became 10.1±1.3 mV in the presence of 107 mol l1 ENpa (Fig. 7C, N=15). ENpa slightly hyperpolarized the resting potential of some muscle fibers (3.3±0.4 mV, 7 out of 15).
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Fig. 8 shows the effect of GSPRFFamide on EJP of the vasoconstrictor muscle. When 107 mol l1 GSPRFFamide was applied, EJP was depressed markedly. The summated EJPs of 18.6±0.6 mV was depressed to 8.4±1.2 in the presence of GSPRFFamide (Fig. 8C, N=14). Unlike ENpa, however, GSPRFFamide did not hyperpolarize the resting potential in all the tested muscle fibers (N=14).
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The effect of NdWFamide on EJP is illustrated in Fig. 9. In these experiments, EJP was elicited by a single nerve stimulation. NdWFamide enhanced the amplitude of EJP reversibly but did not affect the resting potential. In the presence of 108 mol l1 NdWFamide, the EJP (14.1±1.0 mV) was increased to 23.4±1.3 mV (Fig. 9C, N=9).
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Action of enterin and AMRP in the presence of 4-AP
In the anterior aorta of A. kurodai, enterin hyperpolarizes the
membrane potential of the arterial muscle through the activation of the
4-AP-sensitive K+ channels, and the inhibitory action on the
nerve-evoked contraction disappears in the presence of 4-AP
(Sasaki et al., 2002b). AMRP
also activates the K+ channels that are highly sensitive to 4-AP in
mechanosensory neurons of A. californica
(McDearmid et al., 2002
). To
examine a possible involvement of the 4-AP sensitive K+ channels in
the inhibitory actions of ENpa and GSPRFFamide, the actions of the peptides
were re-examined in the presence of 103 mol
l1 4-AP that is enough to block the 4-AP sensitive
K+ channels (McDearmid et al.,
2002
; Sasaki et al.,
2002b
). The interval between the stimulus trains was prolonged to
25 min because the trains applied with shorter interval increased a basal
tonus considerably in the presence of 103 mol
l1 4-AP.
An example for the effect of 4-AP on the inhibitory action of ENpa is shown
in Fig. 10A. In this series of
experiments, the peptide actions in the absence or the presence of 4-AP were
obtained in the same preparation to permit paired comparison. In the presence
of 103 mol l1 4-AP, the basal tonus of the
vasoconstrictor muscle was increased, and the nerve-evoked contraction was
enhanced vigorously as described in the anterior aorta of A. kurodai
(Sasaki et al., 2002b). In
some preparations, spontaneous phasic contractions were also observed under
this condition (Fig. 11A). The
inhibitory action of ENpa was strongly depressed in the presence of
103 mol l1 4-AP. In the absence of 4-AP,
the contraction was reduced to 30.7±7.4% of the control by
107 mol l1 ENpa
(Fig. 10Bi; N=5).
However, in the presence of 103 mol l1
4-AP, the same concentration of ENpa reduced the contraction only to
80.4±5.4% of the control.
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4-AP had similar effect on the inhibitory action of GSPRFFamide. In 107 mol l1 GSPRFFamide, the contractions in the absence and the presence of 4-AP were depressed to 16.1±5.1% and 73.4±8.5% of the control, respectively (Fig. 10Bii; N=4). These results suggest that the inhibitory actions of the two peptides on the nerve-evoked contraction are at least partly mediated by the activation of the 4-AP sensitive K+ channels.
Because ENpa inhibited the ACh-induced contraction consistently, we next examined the inhibitory action of ENpa on the ACh-induced contraction in the presence of 4-AP. In three out of four preparations, the ACh-induced contraction was markedly enhanced and prolonged in the presence of 4-AP (Fig. 11A). The amplitude of ACh-induced contraction was reduced to 43.9±12.7% of the control in the absence of 4-AP whereas it was 100.2±5.8% of the control in the presence of 4-AP (Fig. 11B, N=4). The result suggests that the inhibitory action of ENpa on the ACh-induced contraction was exclusively due to the activation of the 4-AP sensitive K+ channels.
Modulation of the intra-arterial pressure by the peptides
In previous sections, we showed that ENpa, GSPRFFamide and NdWFamide had
modulatory actions on the contractility of the vasoconstrictor muscle in the
abdominal aorta. To determine whether the modulatory actions of the peptides
on the muscle contraction affect the blood flow into the abdominal aorta
effectively, we next examined the actions of the peptides on the
intra-arterial pressure change (see Materials and methods). Following the
stimulus trains applied to the pericardial nerve, reproducible pressure
changes were evoked (Fig. 12),
indicating that the constriction of the abdominal aorta was indeed brought up
by the pericardial nerve stimulation. Bath application of ENpa or GSPRFFamide
inhibited the evoked pressure change, and the concentrationresponse
relationships of ENpa and GSPRFFamide were almost identical
(Fig. 12A,B). The threshold
concentration was less than 109 mol l1,
and the nerve-evoked pressure change was almost completely blocked at the
concentration of more than 107 mol l1.
Bath application of NdWFamide potentiated the nerve-evoked pressure change
with a threshold of <1010 mol l1
(Fig. 12C). As expected from
the result in Fig. 3, NdWFamide
also increased the basal pressure at >108 mol
l1 and the potentiation of the nerve-evoked pressure change
was more persistent than the change of the basal pressure.
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Discussion |
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The main concern of the present study was whether AMRP, enterin or
NdWFamide affects the contractile activity of the vasoconstrictor muscle. As
described previously in A. californica
(Liebeswar et al., 1975), a
main excitatory innervation of the vasoconstrictor muscle of A.
kurodai seems to be cholinergic because the nerve-evoked contraction as
well as EJP are completely blocked by a cholinergic blocker, hexamethonium.
Having confirmed the cholinergic excitatory innervation, we discuss the
feature of the actions of the peptides one by one in the following sections.
Our conclusion is summarized in Fig.
13.
|
Enterin is a family of nona/decapeptides identified in the nervous system
of Aplysia (Furukawa et al.,
2001). We previously showed that enterin is an inhibitory
regulator of the anterior aorta in A. kurodai
(Sasaki et al., 2002b
). In the
present study, enterin was found to inhibit the contractile activity of the
vasoconstrictor muscle. We suggest that enterin inhibits the contraction of
this muscle both presynaptic and postsynaptic mechanisms based on the
following observations: (1) both the nerve-evoked and the ACh-induced
contraction were depressed by enterin; (2) the potency of the inhibitory
actions on the nerve-evoked and the ACh-induced contraction was not identical
(the same potency would be expected if the inhibitory action is totally
postsynaptic); and (3) the enterin hyperpolarized the resting potential of the
muscle. The inhibitory mechanisms of enterin has been described in some detail
in the anterior aorta of Aplysia
(Sasaki et al., 2002b
). In the
anterior aorta, enterin activates the 4-AP sensitive K+ channels,
thereby hyperpolarizes the resting potential of the arterial muscle. The
activation of the 4-AP sensitive K+ channels is a main factor for
the inhibitory action of enterin on the nerve-evoked contraction of the
anterior aorta (Sasaki et al.,
2002b
). Because enterin also hyperpolarized the resting potential
of some vasoconstrictor muscles, we examined whether the inhibitory action of
enterin on this muscle was also mediated by the 4-AP sensitive K+
channels. Although the action of enterin on the ACh-induced contraction was
completely blocked by 4-AP, the inhibitory action on the nerve-evoked
contraction was not abolished by 4-AP. The result is consistent with the
hypothesis that enterin acts at both pre- and postsynaptic sites to inhibit
the nerve-evoked contraction of the vasoconstrictor muscle. A main
postsynaptic action is probably the activation of the 4-AP sensitive
K+ channels, which should inhibit the nerve-evoked depolarization
of the muscle membrane. Because the inhibition of the nerve-evoked contraction
by enterin was still observed in the presence of 4-AP, we think that enterin
may act on the presynaptic receptor to reduce the release of the excitatory
transmitter.
AMRP belongs to the Mytilus inhibitory peptide family
(Fujisawa et al., 1999) whose
members have been isolated from various molluscs
(Muneoka et al., 2000
). The
family members are shown to have inhibitory actions in many peripheral organs
and in the central neurons of different species
(Muneoka et al., 2000
). In
accord with the previous reports, AMRP inhibited the contraction of the
vasoconstrictor muscle. The inhibitory action of AMRP in the vasoconstrictor
muscle is different from that of enterin because AMRP decreases the amplitude
of EJP without affecting the resting potential of the muscle and inhibitory
action on the ACh-induced contraction was not always observed (see below).
Thus, a main inhibitory action of AMRP in the vasoconstrictor muscle seems to
be the reduction of the transmitter release from the excitatory nerve
terminals. This hypothesis is consistent with the observations in molluscan
neurons including Aplysia neurons that AMRP as well as other
Mytilus inhibitory peptides activate 4-AP sensitive K+
channels (Kiss et al., 1999
;
McDearmid et al., 2002
). The
activation of such K+ channels, in principle, suppresses
depolarization-dependent Ca2+ influx into the nerve terminals,
thereby, reducing the transmitter release. In the present study, the
inhibitory action of AMRP on the nerve-evoked contraction was indeed markedly
depressed in the presence of 4-AP. Action of AMRP on the ACh-induced
contraction was variable among the preparations. In some cases, no effect or
modest inhibition was observed. In the other cases, potentiation of the
contraction was observed. Such diverse effects are not easily explainable at
present, and more experiments are required to clarify the phenomenon.
NdWFamide is originally purified from the heart extract of A.
kurodai (Morishita et al.,
1997), and identified recently from the extract of the central
nervous systems of two other gastropod molluscs, Euhadra congenita
and Lymnaea stagnalis (Morishita
et al., 2003b
). NdWFamide-immunopositive neurons are widespread in
the central and peripheral nervous systems of Aplysia, and modulates
the contractility of a variety of peripheral organs of this animal, suggesting
that NdWFamide is ubiquitous signaling molecule in Aplysia
(Morishita et al., 2003a
).
NdWFamide evoked a contraction of the vasoconstrictor muscle and potentiated
the nerve-evoked contraction. Although NdWFamide is known to enhance the
activity of L-type Ca2+ channels in Aplysia ventricular
myocytes (Kanemaru et al.,
2002
), the upregulation of L-type Ca2+ channels by
NdWFamide is not seemed to be related to the NdWFamide-induced contraction of
the vasoconstrictor muscle because the resting potential of this muscle (well
below 70 mV) is far from the activation range of L-type Ca2+
channels. We also did not observe meaningful depolarization of the muscle
membrane following the application of 107 mol
l1 NdWFamide (data not shown). NdWFamide may, therefore,
evoke the contraction by releasing stored Ca2+ inside the muscle
without the membrane depolarization. NdWFamide-induced vasoconstriction may
restrict the blood flow into the abdominal aorta tonically, unlinked to the
activity of the vasoconstrictor motoneurons.
We previously proposed that NdWFamide may have presynaptic actions in the
arteries because NdWFamide did not evoke longitudinal contractions of the
abdominal aorta and the gastroesophageal artery in spite of their dense
NdWFamide-immunoreactivity (Morishita et
al., 2003a). In the present study, two pieces of evidence suggest
that the potentiation of the nerve-evoked contraction of the vasoconstrictor
muscle by NdWFamide is due to presynaptic modulation of the excitatory
transmitter release: (1) NdWFamide enhances EJP without affecting the resting
potential of the muscle; and (2) NdWFamide does not affect the ACh-induced
contraction. A plausible mechanism may be upregulation of the voltage-gated
Ca2+ channels in the nerve terminals by NdWFamide as is shown for
L-type Ca2+ channels in Aplysia ventricular myocytes
(Kanemaru et al., 2002
).
In the present study, three types of peptides (AMRP, enterin and NdWFamide)
were shown to affect the contractile activity of the vasoconstrictor muscle in
the abdominal aorta of Aplysia. The modulatory actions of the
peptides were strong enough to affect the intra-arterial pressure of the aorta
in the present in vitro preparation. Although the contraction of the
vasoconstrictor muscle is basically determined by the activity of the
motoneurons (Mayeri et al.,
1974; Koch et al.,
1984
; Alevizos et al.,
1989
), the potency of the muscle contraction seems to be modulated
by peptidergic innervations. The present results as well as others (Alevizos
et al., 1989
,
1991
) indicate that some
neuropeptides modulate the contractility of the vasoconstrictor muscle in the
abdominal aorta of Aplysia. Peptidergic innervations of the
vasoconstrictor muscle would enable the fine tuning of the vasoconstriction
and the blood distribution in Aplysia.
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Acknowledgments |
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References |
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