First published online May 5, 2005
Journal of Experimental Biology 208, 2005-2017 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01607
Nitric oxide modulates peristaltic muscle activity associated with fluid circulation in the sea pansy Renilla koellikeri
Michel Anctil*,
Isabelle Poulain and
Claudine Pelletier
Département de sciences biologiques, Université de
Montréal, Case postale 6128, Succ. Centre-Ville, Montréal,
Québec, Canada H3C 3J7
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Fig. 1. Morphological organization of sea pansy colonies with emphasis on water
circulation. (A) Schematic diagram of the upper surface of the discoid colony
showing the distribution of water polyps (siphonozooids, shown in green) and
the direction of water flow in the channels beneath the polyps (arrows). Water
from the channels and the peduncle reaches the axial canal and moves forward
to the large exhalent siphonozooid (boxed region, shown in B) where it is
ejected. The triangle shaded in blue represents the area excised for reduced
preparation recording. Syringe symbols represent site and orientation of drug
(left) and vehicle (right) injections for behavioural experiments. The
autozooids (feeding and reproductive polyps) are not represented for better
clarity. (B) Photomicrograph of area corresponding to the boxed region in A,
from a living colony. Note the autozooid, the miniature inhalent siphonozooids
(small arrows) and the single exhalent siphonozooid (large arrow) in which the
eight septa irradiating from the mouth opening are typical of the
octocorallian body organization. Note also the masses of spicules (shown in
violet) forming the calcified skeleton of the colony.
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Fig. 8. Whole mounts showing the distribution of NADPH diaphorase staining in
autozooid polyps of R. koellikeri. (A,B) Overview at two different
focal planes of the upper part of a polyp where the base of tentacles is
heavily populated by brown endosymbiotic algae. Note that focus is on staining
of the oral disc in A, and on aboral tentacle and body column staining in B
where bundles of tentacle neurites (arrows) extend to the body column
(arrowheads). (C) Enlarged view of an oral disc region corresponding to the
rectangle in A. Note clusters of cell somata at the margin (arrowheads) and
meshwork of cell processes throughout the oral disc (arrow). (D) Enlarged view
of a tentacle area corresponding to the rectangle in B. Note neurites running
parallel to each other and cell somata (arrows) at the tentacle/body column
interface. (E) Two bands of basiectodermal stained cell somata flank a
tentacle on the aboral side. Processes from these cell bands extend towards
the endodermal layer of the tentacle (arrow) and in the septum endoderm of the
column (arrowhead). (F) Bands of stained neurites on the oral side of a
tentacle (arrows) appear to merge into the similarly stained oral disk
nerve-net. (G) Enlarged view of stained basiectodermal cell somata with an
apical process (arrowheads). Neuritic processes from these cells (between
arrows) extend towards the ectoderm/mesoglea interface. Scale bars: 200 µm
(A,B), 25 µm (C), 30 µm (D), 80 µm (E), 50 µm (F) and 15 µm
(G).
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Fig. 9. NADPH diaphorase staining and citrulline immunostaining in the polyp
anthocodium and the colonial mass. (A) Whole mount of tentacle/oral disk
region showing citrulline immunoreactivity in basiectodermal cells
(arrowheads). (B) Cross section through two tentacles showing bands of
citrulline immunoreactivity at the ectoderm/mesoglea and at the
mesoglea/endoderm boundaries (arrows). (C) Citrulline immunoreactivity
(arrows) at the interface of body wall (parietal) and septal muscle in a
whole-mount of polyp tissue. (D) Whole mount of lower polyp body column
displaying NADPH diaphorase staining in circular muscle of the body wall
(arrows) and in the muscle sheet of a flattened septum (between arrowheads).
(E) Whole mount of circular muscle sheet inside the colonial mass in which
both multipolar cells (arrowheads) and muscle feet (arrows) show citrulline
immunoreactivity. (F) Section through the colonial mass in which are embedded
the endodermal muscles of the submerged part (zooecium) of the polyps. Note
the citrulline immunostaining in the septal (radial and longitudinal) muscles
and in the circular muscle. Scale bars: 30 µm (A,C), 50 µm (B), 100
µm (D), 10 µm (E) and 40 µm (F).
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Fig. 2. Recordings of effects of NO (nitric oxide) donors on peristaltic
contractions of reduced preparations of R. koellikeri. (A) Recording
of a single preparation immersed in 40 ml of ASW and first exposed to 40 ml of
solvent (ASW), then consecutively to 40 and 400 µl of 1 mmol l-1
SIN-1 (amino-3-morpholinyl-1,2,3-oxadiazolium) chloride to obtain final bath
concentrations of 1 and 10 µmol l-1, respectively. Note the
diversity of shape of traces reflecting the contribution of different
contraction waves to peristaltic events. Note also the dose-dependent increase
in contraction amplitudes and in wave synchrony after exposure to SIN-1 and
the reversibility of this response after washing. (B) Effect of SNAP
(S-nitroso-N-acetylpenicillamine) on contractions induced by field
electrical stimulation. Stimulation trains of 6 monopolar pulses of 10 ms each
at 5 V and 2 Hz were applied as shown at arrows. Note that the induced robust
contractions were invariably followed by a sharp, transient drop of the
ongoing peristaltic waves. Note also the reversible drop in amplitude and
duration of the induced contraction in the presence of SNAP. µN,
microNewton.
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Fig. 3. Quantitative summary of effects of NO donors on peristalsis (A) and
representative effects on NO donors on the behaviour of whole colonies (B,C).
(A) Histogram showing the change, relative to controls, in the amplitude of
peristaltic contractions induced by the NO donors SIN-1
(amino-3-morpholinyl-1,2,3-oxadiazolium) and SNAP
(S-nitroso-N-acetylpenicillamine). Note that the change is largely
reversible after exposure of reduced preparations to 50 µmol l-1
SIN-1. Statistical significance: *P<0.05 or
**P<0.01 with the Mann-Whitney test. Note that 10
µmol l-1 1-(1-naphthyl)piperazine (NP) reduced the increase in
peristaltic force induced by 10 µmol l-1 SNAP, but the
difference was not significant (n.s., P>0.05). N=10 (1
µmol l-1 SIN-1), 6 (10 µmol l-1), 4 (50 µmol
l-1) and 8 (wash), 6 (SNAP) and 4 (SNAP + NP). (B) Injection of
SIN-1 in the left half of an initially relaxed colony led to a contracted
state relative to the control (right) half. Black line indicates the position
of the axial canal. (C) Injection of SNAP in the left half of an initially
contracted colony led to a relaxed state relative to the control half.
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Fig. 4. Recordings of effects of NOS inhibitors on peristaltic contractions of
reduced preparations. L-NAME (N( )-nitro-L-arginine methyl
ester) dose-dependently reduced both the amplitude of peristaltic waves and
basal tension whereas aminoguanidine, in a separate preparation, increased
peristaltic waves without affecting basal tension. Dotted line indicates
initial basal tension.
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Fig. 5. Quantitative summary of effects of NOS inhibitors on peristalsis (A) and
representative effects of NOS inhibitors on the behaviour of whole colonies
(B,C). (A) Histogram showing the actions of NOS inhibitors on peristalsis as
well as the interactions between NOS inhibitors and conventional transmitter
effects (see Results). Statistical significance:
**P<0.01 with a paired t-test. There was no
significant difference between 0.1 and 1.0 mmol l-1 L-NAME
(N()-nitro-L-arginine methyl ester) or between melatonin alone
and with aminoguanidine. N=6 (L-NAME), 3 (D-NAME), 5
(aminoguanidine), 4 (melatonin/aminoguanidine and GnRH/aminoguanidine). (B)
Injection of L-NAME in the left half of a contracted colony led to a relaxed
state relative to the control (right) half. (C) Injection of aminoguanidine in
the left half of a relaxed colony led to a contracted state relative to the
control half. Black line indicates the position of the axial canal.
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Fig. 6. Recordings of effects of the soluble guanylate cyclase inhibitor ODQ
(1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one) and of cGMP-specific
phosphodiesterase inhibitor zaprinast on peristaltic contractions of reduced
preparations. Note the enhancing effect of ODQ in contrast to the additive
depressing effects of zaprinast and db cGMP. The latter two also depressed
basal tension below initial level (dotted line).
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Fig. 7. Quantitative summary of effects of guanylate cyclase and phosphodiesterase
inhibitors [ODQ (1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one) and zaprinast,
respectively] on peristalsis (A) and their representative effects on the
behaviour of whole colonies (B,C). (A) Histogram showing the actions of ODQ
and zaprinast on peristalsis, as well as their interactions with the effects
of other signalling molecules (see Results). Statistical significance:
P>0.05 (n.s.), *P<0.05 or
**P<0.01 with the paired t-test. N=6
(ODQ/SNAP), 4 (melatonin/ODQ) and 5 (zaprinast/db cGMP/IBMX). (B) Injection of
ODQ in the left half of the colony led to a slightly contracted state and
stronger peristalsis (as illustrated by the marginal fold) relative to the
control (right) half. (C) Injection of aminoguanidine in the left half of
another colony led to a relaxed and quiescent state relative to the control
half where robust peristalsis occurs (compare the two pictures of the same
colony photographed 30 s apart). Black line indicates the position of the
axial canal.
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© The Company of Biologists Ltd 2005
