First published online November 19, 2004
Journal of Experimental Biology 207, 4439-4450 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01273
Peptidergic innervation of the vasoconstrictor muscle of the abdominal aorta in Aplysia kurodai
Kosei Sasaki,
Fumihiro Morishita and
Yasuo Furukawa*
Department of Biological Science, Graduate School of Science,
Hiroshima University, Kagamiyama 1-3-1, Higashi-Hiroshima 739-8526,
Japan
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Fig. 1. Innervation pattern of the vasoconstrictor muscle viewed by
immunohistochemistry. (Ai) AMRP-immunoreactivity in the vicinity of the
abdominal ganglion and three major arteries. The preparation is placed ventral
side up except for the abdominal ganglion (the ganglion is rotated 180° to
see the dorsal surface). The abdominal aorta is mainly innervated by the
branches of the pericardial and spermathecal nerves (see Aii). Scale bar is 2
mm. Inset shows a higher magnification of a part of the abdominal aorta
including the vasoconstrictor muscle. Note the extensive ramification of the
immunopositive fibers. Scale bar in the inset is 1 mm. (Aii) Diagram of the
preparation shown in Ai. AA, anterior aorta; AbA, abdominal aorta; AbG,
abdominal ganglion; GA, gastroesophageal artery; Pn, pericardial nerve; Spn,
spermathecal nerve; Vm, a region containing the vasoconstrictor muscle. (B)
Immunopositive fibers and varicosities in the vasoconstrictor muscle. Scale
bars in all figures are 100 µm. (Bi) AMRP-immunoreactivity. (Bii)
Enterin-immunoreactivity. (Biii) NdWFamide-immunoreactivity.
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Fig. 2. ENpa and GSPRFFamide depress the contraction of the vasoconstrictor muscle
evoked by the stimulation of the pericardial nerve. (A) Effects of
10–7 mol l–1 ENpa (Ai) and
10–7 mol l–1 GSPRFFamide (Aii) on the
contraction evoked by a train of the electrical pulse (1 ms, 1.6 V, 10 Hz) for
1 s. The stimulus train was applied every 20 s. Peptides were applied for 2
min as indicated by bars. Ai and Aii are from the same preparation. (B)
Concentration–response relationships of the effects of ENpa and
GSPRFFamide. Amplitude of the most depressed contraction after the peptide
application is normalized to that of the control contraction and the mean
± S.E. was plotted against the concentration of the peptides
(N=3 6). (C) Time-dependent effects of the peptide actions.
Amplitude of the muscle contraction was normalized to the control contraction
just before the peptide application and plotted against the time
(N=3). 10–5 mol l–1 of ENpa or
GSPRFFamide was applied for 2 min as indicated.
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Fig. 3. NdWFamide enhances the nerve-evoked contractions of the vasoconstrictor
muscle. (A) and (B) are from the different preparations. The contraction of
the muscle was evoked by the nerve stimuli as described in
Fig. 2. Duration of each
stimulus train in A and B were 1 s and 0.4 s, respectively. Note a tonic
contraction was also induced by NdWFamide at higher concentrations.
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Fig. 12. Actions of the peptides on the vasoconstriction of the abdominal aorta. The
vasoconstricion was monitored by an internal pressure change of the perfusion
line as described in Materials and methods. Typical examples of the peptide
actions (i) and the concentration–response relationships (ii) are shown.
(A) Action of ENpa (N=4). (B) Action of GSPRFFamide (N=3).
(C) Action of NdWFamide (N=3). The pressure change was evoked by
stimulation of the pericardial nerve using the stimulus train (1 ms, 1.6 V, 10
Hz) for 0.9 s (A,B) or 0.5 s (C).
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Fig. 4. Action of ENpa, GSPRFFamide or NdWFamide on the ACh-induced contraction of
the vasoconstrictor muscle. (A) Blockade of the nerve-evoked contraction by
10–3 mol l–1 hexamethonium. Each contraction
was evoked by the stimulus train (1 ms, 1.6 V, 10 Hz) for 0.5 s every 20 s.
(B) Effect of ENpa (Bi), GSPRFFamide (Bii) or NdWFamide (Biii) on the
ACh-induced contraction. The contraction was evoked by perfusion of
10–6 mol l–1 ACh for 40 s as indicated by
bars. Peptide was applied between arrows.
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Fig. 5. Comparison of the effects of the peptides on the nerve-evoked and the
ACh-induced contractions. (A) 10–7 mol l–1
ENpa, (B) 10–7 mol l–1 GSPRFFamide, (C)
10–8 mol l–1 NdWFamide. Upper, middle and
lower lines of the box plots show 75th percentile, median and 25th percentile,
respectively. Upper and lower bars show the largest and smallest values.
Numbers of preparations are shown in parentheses. Statistical significance was
assessed either by Mann-Witney U-test (A,C) or Student's
t-test (B).
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Fig. 6. Single vasoconstrictor muscle fiber is innervated by several excitatory
axons. (A) Relationships between stimulus intensities and EJPs. The
pericardial nerve was stimulated by a 1 ms pulse of various intensities as
indicated. Four records are superimposed. Fast vertical deflections are
stimulus artifacts. Note that the increase in stimulus intensity not only
changes the peak amplitude of EJP, but also the shape of EJP. (B) Effect of
10–3 mol l–1 hexamethonium on EJPs evoked by
the stimulus train (1 ms, 1.6 V, 10 Hz) for 0.5 s.
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Fig. 7. ENpa decreases the amplitude of EJPs. (A) Effects of 10–7
mol l–1 ENpa. EJPs were evoked by the stimulus train (1 ms,
1.6 V, 10 Hz) for 0.3 s every 20 s. Note ENpa also hyperpolarized the resting
potential of the muscle fiber. (B) Comparison of EJPs before (a) and after (b)
ENpa application. (C) Inhibitory action of ENpa on EJPs. Histogram shows mean
± S.E. of the maximum amplitude of summated EJPs before and
after ENpa application (N=15). Statistical significance was assessed
by Student's t-test.
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Fig. 8. GSPRFFamide decreases the amplitude of EJPs. (A) Effects of
10–7 mol l–1 GSPRFFamide. EJPs were evoked
by the stimulus train (1 ms, 1.6 V, 10 Hz) for 0.5 s every 20 s. (B)
Comparison of EJPs before (a) and after (b) GSPRFFamide application. (C)
Inhibitory action of GSPRFFamide on EJPs. Histogram shows mean ±
S.E. of the maximum amplitude of summated EJPs before and after
GSPRFFamide application (N=14). Statistical significance was assessed
by Student's t-test.
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Fig. 9. NdWFamide increases the amplitude of EJP. (A) Effects of
10–8 mol l–1 NdWFamide on EJP. Each EJP was
evoked by a single nerve stimulation (1 ms, 1.6 V). (B) Comparison of EJPs
before (a) and after (b) NdWFamide application. (C) Excitatory action of
NdWFamide on EJPs. Histogram shows mean ± S.E. of the
maximum amplitude of summated EJPs before and after NdWFamide application
(N=9). Statistical significance was assessed by Student's
t-test.
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Fig. 10. 4-AP depresses the inhibitory actions of ENpa and GSPRFFamide on the
nerve-evoked contraction. (A) Action of 10–7 mol
l–1 ENpa on the contraction in the absence (Ai) or presence
(Aii) of 10–3 mol l–1 4-AP. The contraction
was evoked by the stimulus train (1 ms, 1.6 V, 10 Hz) for 0.4 s. (B)
Inhibitory actions of 10–7 mol l–1 ENpa (Bi,
N=5) and 10–7 mol l–1 GSPRFFamide
(Bii, N=4) in the absence [(–)4-AP] or the presence [(+)4-AP]
of 10–3 mol l–1 4-AP. Statistical
significance was assessed by Student's t-test.
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Fig. 11. 4-AP abolishes the inhibitory action of ENpa on the ACh-induced
contraction. (A) Action of 10–7 mol l–1 ENpa
on the contraction in the absence (Ai) or presence (Aii) of
10–3 mol l–1 4-AP. 3
x10–6 mol l–1 ACh was applied for 20 s
at arrows. (B) Inhibitory action of ENpa in the absence [(–)4-AP] or the
presence [(+)4-AP] of 10–3 mol l–1 4-AP
(N=4). Statistical significance was assessed by Student's
t-test.
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Fig. 13. A model of the peptide actions on the excitatory transmission in the
vasoconstrictor muscle. AMRP acts mainly on the presynaptic receptors to
inhibit the excitatory transmitter release. Part of this inhibition is assumed
to be due to the activation of the 4-AP sensitive K+ channels.
Because 4-AP does not completely block the inhibitory action, other route to
inhibit the transmitter release seems to exist. Enterin activates both pre and
postsynaptic receptors. Activation of the postsynaptic receptors induces the
opening of the 4-AP sensitive K+ channels, and inhibits the
contraction. Because 4-AP does not completely inhibit the action of enterin,
the presynaptic inhibitory receptors are also assumed. NdWFamide also
activates both pre and postsynaptic receptors. Activation of the postsynaptic
receptors evokes the muscle contraction, and that of the presynaptic receptors
enhances the transmitter release.
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© The Company of Biologists Ltd 2004
