Comparative analysis of nitric oxide and SALMFamide neuropeptides as general muscle relaxants in starfish
School of Biological Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK
* Author for correspondence (e-mail: m.r.elphick{at}qmul.ac.uk)
Accepted 6 December 2002
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Summary |
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The molecular mechanisms by which NO and SALMFamides cause muscle relaxation in starfish are not known, but previous pharmacological studies on the cardiac stomach using the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazol[4,3-a]quinoxalin-1-one (ODQ) indicate that the cyclic nucleotide second messenger cGMP may mediate effects of NO. Consistent with this hypothesis, here we report that ODQ also causes partial inhibition of the relaxing effect of SNAP on tube foot and apical muscle preparations. To further investigate the involvement of cyclic nucleotides as mediators of the effects of NO and SALMFamides on starfish muscle, we have measured both cGMP and cAMP in cardiac stomach and in apical muscle after treatment with S1, S2 or SNAP. However, no significant changes in cyclic nucleotide content were observed compared with controls. Further experiments were performed on apical muscle tissue in the presence of the cyclic-nucleotide-phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), a drug that also causes cardiac stomach relaxation in starfish. Treatment with IBMX caused a 2-3-fold increase above basal levels for cGMP and cAMP, but co-treatment with IBMX and S1 or S2 or SNAP resulted in no significant further increase above the level observed with IBMX alone. We conclude from these data that the relaxing action of NO on starfish muscle may be mediated by both cGMP-dependent and cGMP-independent pathways. However, the mechanisms by which SALMFamides cause muscle relaxation in starfish remain unknown and, although our results do not rule out the involvement of cGMP or cAMP, other signalling pathways may now need to be investigated.
Key words: cardiac stomach, tube feet, apical muscle, cyclic GMP, cyclic AMP, soluble guanylyl cyclase, adenylyl cyclase, Asterias rubens, starfish, SALMFamide
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Introduction |
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The discovery that NO and the SALMFamides S1 and S2 cause relaxation of the
cardiac stomach in starfish prompted us to investigate whether these are
general muscle relaxants that also cause relaxation of other starfish
neuromuscular preparations. This issue has been addressed previously for the
SALMFamides (Elphick et al.,
1995) but we have revisited it here in combination with tests
using the NO donor SNAP. Two preparations were examined: tube feet and the
apical muscle, which is located in a midline position on the inner surface of
the aboral body wall in each of the five rays (see
Moore and Thorndyke, 1993
for
a diagram). Previously, we tested both S1 and S2 on these preparations but did
not observe relaxing effects (Elphick et
al., 1995
). However, here we have incorporated two modifications
to the test conditions, building on experience obtained with the cardiac
stomach (as discussed in Elphick and
Melarange, 2001
). Firstly, for both the tube foot and apical
muscle preparations we have used 30 mmol l-1 KCl to induce muscle
contracture prior to testing S1, S2 or SNAP. Secondly, for tube foot
preparations we have recorded under isotonic conditions (as with cardiac
stomach) whilst for apical muscle we have recorded under isometric conditions
(as previously, Elphick et al.,
1995
).
Little is known about the molecular mechanisms by which NO and SALMFamide
neuropeptides cause relaxation of muscle in starfish. Pharmacological
experiments using the soluble guanylyl cyclase (SGC) inhibitor
1H-[1,2,4]oxadiazol[4,3-a]quinoxalin-1-one (ODQ) indicate that NO exerts its
relaxing action on the cardiac stomach via a guanosine
3',5'-cyclic monophosphate (cGMP)-dependent pathway
(Elphick and Melarange, 1998;
Melarange et al., 1999
). ODQ
does not, however, cause inhibition of S2-induced relaxation of the cardiac
stomach (Melarange et al.,
1999
). Therefore, it seems likely that S1 and S2 act in parallel
with NO via a separate pathway. One candidate signal transduction
cascade that we have begun to explore for the SALMFamides is G-protein coupled
receptor-dependent activation of adenylyl cyclase leading to adenosine
3',5'-cyclic monophosphate (cAMP)-mediated relaxation. We have
found that the adenylyl cyclase activator forskolin causes relaxation of the
cardiac stomach; however, pre-treatment of the cardiac stomach with an
adenylyl cyclase inhibitor (SQ 22,536) does not affect S1- or S2-induced
relaxation (Elphick and Melarange,
2001
). To investigate further the involvement of cGMP or cAMP in
mediating the relaxing actions of NO, S1 and S2 on the cardiac stomach, here
we have employed a biochemical approach by measuring the cGMP and cAMP content
of cardiac stomach and apical muscle after treatment with SNAP, S1 or S2.
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Materials and methods |
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Pharmacology
Stock solutions (1 mmol l-1) of S1 and S2 were prepared using
distilled water and then diluted with seawater to obtain an organ bath
concentration of 10 µmol l-1. Stock solutions of all other drugs
tested were prepared in absolute ethanol prior to dilution in seawater.
However, where ethanol was used as a solvent for drugs, the final
concentration of ethanol that the tissue was exposed to did not exceed 0.03%.
Control tests with 0.03% ethanol showed no effects on muscle in any of the
preparations examined here.
Cardiac stomach preparations were dissected and linked to an isotonic
transducer (model 60-3001; Harvard, South Natick, MA, USA) in a 20 ml organ
bath containing seawater at 11°C, as described previously
(Elphick et al., 1995;
Elphick and Melarange, 2001
).
Sustained contracture of the cardiac stomach was induced and maintained by
replacing the seawater with seawater containing 30 mmol l-1 added
KCl (KCl/SW) as described previously (Elphick and Melarange,
1998
,
2001
;
Melarange et al., 1999
). SNAP,
S1 and S2 were then added to the organ bath individually at a concentration of
10 µmol l-1 in random order at 10-40 min intervals. After
maximal relaxation had been reached in each test, the organ bath was emptied
and then filled with several washes of KCl/SW. SNAP, S1 and S2 were tested
only once on each preparation, but a total of five preparations were used to
obtain mean percentage relaxation values for S1 and S2 with respect to
SNAP.
Tube foot preparations were dissected from the starfish ambulacrum as
described previously (Elphick et al.,
1995) and then linked to an isotonic transducer (as illustrated in
Fig. 1) in a 3 ml organ bath at
11°C containing seawater followed by KCl/SW. Preliminary experiments were
carried out to compare the effect of SNAP on tube foot preparations where the
external epithelium was left intact (`unstripped') and on `stripped'
preparations where the epithelium was scraped away using a scalpel blade to
expose the underlying muscle layer (see
Moore and Thorndyke, 1993
;
Newman et al., 1995a
for
photographs and diagrams of starfish tube foot histology).
N-acetylpenicillamine (NAP) was also tested in these preliminary
experiments to establish whether or not relaxing effects observed with SNAP
could be attributed specifically to its ability to release NO, as with
previous tests on the cardiac stomach
(Elphick and Melarange, 1998
).
NAP had no effect on tube feet but SNAP caused relaxation of both unstripped
and stripped preparations. However, as might be expected, the magnitude of
SNAP-induced relaxation was greater in stripped preparations than in
unstripped preparations. Therefore, stripped preparations were used for all
subsequent tests with SNAP, S1 and S2. The protocol for testing and comparing
the effects of SNAP, S1 and S2 on a total of six tube foot preparations was as
described above for the cardiac stomach. To investigate whether effects of
SNAP on tube foot preparations are mediated by SGC, the SGC inhibitor ODQ
(Tocris Cookson, Bristol, UK) was applied for 15 min at a concentration of 10
µmol l-1 prior to application of SNAP.
|
Strips of apical muscle approximately 1.5 cm in length were dissected from
the aboral body wall of starfish arms, as described in Elphick et al.
(1995), and then linked to an
isometric transducer (model 60-2997; Harvard) in a 3 ml organ bath at 11°C
containing seawater followed by KCl/SW. The effects of NAP, SNAP, S1 and S2 at
a concentration of 10 µmol l-1 were then tested on a total of
six preparations, as described above for the cardiac stomach. To investigate
whether effects of SNAP on apical muscle preparations are mediated by SGC, the
SGC inhibitor ODQ was applied for 15 min at a concentration of 10 µmol
l-1 prior to application of SNAP.
Analysis of the cGMP and cAMP content of cardiac stomach and apical
muscle with and without drug treatments
Preparations of the cardiac stomach were dissected as described previously
(Elphick et al., 1995) and then
cut into five equivalent segments reflecting its pentaradial symmetry. The
stomach segments were incubated in glass vials containing 5 ml of seawater at
11°C for 30 min, with seawater replenishment after the first 15 min.
Following this equilibration period, the five segments of each preparation
were subjected to a further 5 min incubation period at 11°C in 2 ml of
0.2% ethanol in seawater (general control) or in 2 ml of 0.2% ethanol in
seawater containing 10 µmol l-1 NAP (control for SNAP), 10
µmol l-1 SNAP, 10 µmol l-1 S1 or 10 µmol
l-1 S2. Stomach segments were then removed from the glass vials,
transferred individually to 2 ml plastic microcentrifuge tubes containing 1.5
ml of 50 mmol l-1 sodium acetate (pH 4.75) and held in boiling
water for 5 min. The stomach segments were transferred with the sodium acetate
solution into glass pestle tubes and homogenised using a glass mortar.
Homogenates were then subjected to centrifugation at 10 000 g
for 10 min in a bench-top microcentrifuge. The supernatants were removed and
stored at -20°C. Samples of supernatant were assayed for protein using a
Coomassie Plus Protein Assay kit (Pierce; Rockford, IL, USA) with bovine serum
albumin (BSA) diluted in 50 mmol l-1 sodium acetate (pH 4.75) to
establish a standard curve. Samples of supernatant were diluted 2-fold and
8-fold in radioimmunoassay buffer (50 mmol l-1 sodium acetate, pH
4.75) and assayed for cGMP or cAMP, respectively, as described below.
The methods used for assay of cyclic nucleotides in apical muscle were as described above for cardiac stomach but with the following modifications. The initial equilibration period in seawater was for 15 min in a volume of 2 ml. Drugs (SNAP, NAP, S1, S2) were tested at a concentration of 20 µmol l-1 (1 ml) for an incubation period of 5 min. Additional experiments were also performed on apical muscle using the cyclic-uncleotide-phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). To determine the effect of IBMX on basal cyclic nucleotide levels, apical muscle strips were incubated with 100 µmol l-1 IBMX for 15 min. To investigate the effects of SNAP, NAP, S1 and S2 in the presence of IBMX, apical muscle strips were first incubated with 100 µmol l-1 IBMX for 10 min, and then 20 µmol l-1 SNAP, NAP, S1 or S2 was added for a further 5 min.
cGMP and cAMP radioimmunoassays
Solutions of the cGMP and cAMP analogues succinylguanosine
3',5'-cyclic monophosphate tyrosyl methyl ester (ScGMP-TME) and
succinyladenosine 3',5'-cyclic monophosphate tyrosyl methyl ester
(ScAMP-TME), respectively, were prepared in 0.2 mol l-1 sodium
phosphate (pH 7.5). ScGMP-TME or ScAMP-TME (0.3 nmol in 10 µl) was
iodinated by addition of 11.1 MBq (3 µl) of Na[125I] (Amersham
International, Amersham, UK) followed by chloramine T (10 µl; 0.5 mg
ml-1). After vigorous mixing for 30 s, cysteine (100 µl; 43
µg ml-1) was added, followed by potassium iodide (500 µl; 0.2
mg ml-1) to stop the reaction. Labelled ScGMP-TME or ScAMP-TME were
separated from salts and unreacted iodide on a C18 Sep-Pak
cartridge (Waters; Milford, MA, USA) by eluting first with 10 ml of distilled
water and then with 3 ml of 40% isopropanol. Labelled cyclic nucleotides with
specific activity in the range of 11.1-25.9 MBq mmol l-1 were used
for radioimmunoassay.
Stock solutions (20 µmol l-1) of cGMP or cAMP were prepared in 50 mmol l-1 sodium acetate (pH 4.75) and then serial dilutions were prepared in test tubes using acetate buffer for dilution to obtain standard curves in the range of 1 fmol to 1 pmol per tube. Triplicate samples (50 µl) of standards and tissue extracts were acetylated with acetic anhydride followed by triethylamine (in a 1:2 ratio, respectively). Then, 25 µl samples of cGMP antibody or cAMP antibody (raised in rabbits and provided by Dr J. De Vente, University of Maastricht, The Netherlands) were added to each tube after dilution with acetate buffer (1:2000 for anti-cGMP; 1:8000 for anti-cAMP). Finally, 25 µl of radiolabelled cyclic nucleotide (approximately 20 000 c.p.m.) was added to each tube. Following overnight incubation (approximately 18 h) at 4°C, 25 µl of donkey anti-rabbit IgG-coated cellulose (Sac-Cel, Immunodiagnostics Ltd, Boldon, UK) was added to each tube and, after a 1 h incubation at room temperature, 1 ml of ice-cold distilled water was added to each tube. Bound and free radiolabelled cyclic nucleotide was separated by centrifugation (2070 g, 30 min), supernatant was removed and then pellets were analysed using a Wallac 1480 Wizard gamma counter. Cyclic nucleotide concentrations in tissue samples were determined using the programme RiaCalc Wiz (Wallac Oy; Turku, Finland).
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Results |
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SNAP, S1 and S2 also caused relaxation of tube foot preparations; however, in contrast to the cardiac stomach, 10 µmol l-1 SNAP was much more effective compared with 10 µmol l-1 S1 or 10 µmol l-1 S2 (Figs 3A, 4). Thus, the mean relaxation induced by S1 and S2 was only 5.5% and 13.5%, respectively, of that induced by SNAP (Fig. 4). Consistent with tests on the cardiac stomach, 10 µmol l-1 S1 was less effective than 10 µmol l-1 S2 in causing tube foot relaxation.
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SNAP, S1 and S2 also caused relaxation of apical muscle preparations; however, as with tube feet but in contrast to the cardiac stomach, 10 µmol l-1 SNAP was much more effective compared with 10 µmol l-1 S1 and 10 µmol l-1 S2 (Figs 3B, 4). Thus, the mean relaxation induced by S1 and S2 was only 11% and 32%, respectively, of that induced by SNAP (Fig. 4). Consistent with tests on the cardiac stomach and tube feet, NAP had no effect on apical muscle tone (data not shown) while 10 µmol l-1 S1 was less effective than 10 µmol l-1 S2 in causing apical muscle relaxation.
As previous studies have shown that the relaxing effect of SNAP on the cardiac stomach is inhibited (>70%) by the SGC inhibitor ODQ, we tested the effect of this compound on SNAP (10 µmol l-1)-induced relaxation of the tube foot and apical muscle preparations. ODQ (10 µmol l-1) caused partial inhibition of the effect of SNAP on both preparations, with mean responses to SNAP in the presence of ODQ of 32.5±6.02% (mean ± S.E.M., N=6) and 50.2±20.8% (N=4) of responses to SNAP without ODQ for tube foot and apical muscle preparations, respectively.
Cyclic nucleotide assays
The mean basal concentrations of cGMP and cAMP in cardiac stomach were 29.5
pmol mg-1 protein and 1032.1 pmol mg-1 protein,
respectively (N=8). However, individual measurements ranged from 6.9
pmol mg-1 protein to 64.4 pmol mg-1 protein for cGMP and
208.0 pmol mg-1 protein to 1936.9 pmol mg-1 protein for
cAMP. It was against this background variability in basal cyclic nucleotide
content that the effects of SNAP, NAP, S1 and S2 were examined
(Table 1). No significant
changes above or below the basal levels (control) of cGMP or cAMP were
observed with any of the drugs tested (t-test; P>0.05).
One possible explanation for these results may be that the muscular part of
cardiac stomach represents a relatively small, but inseparable, component of
the total tissue mass and therefore any putative changes in the cyclic
nucleotide content of cardiac stomach muscle would be small and perhaps
undetectable against a much higher and variable content contributed by all
tissue types. In this respect, tube feet offered no advantage over cardiac
stomach because the muscle layer of tube feet is separated from the external
environment by layers of epithelial, nervous and connective tissue. As
discussed above, these layers need to be stripped off to facilitate drug
access to the muscle layer and we considered it unfeasible to do this for
experiments in which many tube feet (e.g. N=40) would have to be
prepared simultaneously for different drug treatments. Therefore, we decided
to focus our analysis not on cardiac stomach or tube feet but on apical
muscle, which, as its name implies, is largely comprised of muscle tissue.
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The mean basal concentration of cGMP in apical muscle (51.5 pmol mg-1 protein; N=6) was slightly higher than in cardiac stomach (29.5 pmol mg-1 protein; N=8). Interestingly, however, the mean basal concentration of cAMP in apical muscle (40.2 pmol mg-1 protein; N=5) was much lower than in cardiac stomach (1032.1 pmol mg-1 protein; N=8). Thus, whilst in cardiac stomach the cAMP:cGMP ratio is approximately 35:1, in apical muscle the cAMP:cGMP ratio is approximately 1:1. As with cardiac stomach, however, treatment of apical muscle tissue with NAP, SNAP, S1 or S2 did not cause any significant changes above or below the basal levels (control) of cGMP or cAMP (Table 1; t-test; P>0.05). One possible explanation for these results may be that the activity of cyclic-nucleotide-phosphodiesterases in apical muscle may prevent accumulation of cGMP and/or cAMP in response to drug treatment to a level that is significantly detectable above variable basal levels. To address this possibility, we performed further experiments in which apical muscle was treated with NAP, SNAP, S1 or S2 in the presence of the cyclic-nucleotide-phosphodiesterase inhibitor IBMX. Treatment of apical muscle with IBMX alone caused a significant 2-3-fold increase in the content of cGMP (t-test; P<0.05) and cAMP (t-test; P<0.001) in apical muscle (Table 2; cf. Table 1), demonstrating the effectiveness of IBMX in inhibiting phosphodiesterase activity in this tissue. However, co-treatment with IBMX and NAP, SNAP, S1 or S2 did not result in any significant further increase in cyclic nucleotide content above the levels observed with IBMX alone (Table 2; t-test; P>0.05).
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Discussion |
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The modest relaxation of tube feet and apical muscle caused by S1 and S2 at
a concentration of 10 µmol l-1 probably explains why in a
previous study (Elphick et al.,
1995) we failed to observe relaxation of these preparations when
testing S1 and S2 at concentrations of <10 µmol l-1.
Nevertheless, the discovery that S1 and S2 can cause relaxation of muscle
preparations other than the cardiac stomach, albeit modestly, is important
because it demonstrates that SALMFamides are general muscle relaxants in
starfish. Moreover, the presence of both S1- and S2-immunoreactivity in the
innervation of tube feet (Moore and
Thorndyke, 1993
; Newman et
al., 1995a
) suggests that both peptides are released by neurons
and contribute physiologically to the control of muscle relaxation in this
organ. However, although S1- and/or S2-immunoreactivity are present in the
innervation of the cardiac stomach, tube feet and apical muscle, the
pharmacological data reported here indicate that the impact of SALMFamide
release on muscle relaxation in vivo would be much greater in the
cardiac stomach than in the tube feet or apical muscle. This may reflect
organ-specific differences in the relative abundance or the
activationrelaxation coupling efficiency of the putative SALMFamide
receptor.
Importantly, the discovery that SALMFamides act as general muscle relaxants
in starfish is consistent with previous analysis of the actions of the
holothurian SALMFamide neuropeptide GFSKLYFamide on muscle preparations from
the sea cucumber Holothuria glaberrima
(Díaz-Miranda et al.,
1992; Díaz-Miranda and
García-Arrarás, 1995
). GFSKLYFamide caused
relaxation of both the intestine and the longitudinal body wall muscle,
preparations that can be considered functionally equivalent to the starfish
cardiac stomach and apical muscle, respectively. Moreover,
GFSKLYFamide-immunoreactivity is present in the innervation of these organs
(Díaz-Miranda et al.,
1995
), indicating that the actions of GFSKLYFamide in
vitro are physiologically relevant. Thus, it appears that SALMFamides may
act as general muscle relaxants throughout the Echinodermata
(Elphick and Melarange, 2001
),
and, if SALMFamide neuropeptides are identified in species from other
echinoderm classes, it will be interesting to test this hypothesis
further.
The results of this study indicate that NO also acts as a general muscle
relaxant in starfish. Moreover, unlike the SALMFamides, the NO donor SNAP
caused substantial relaxation of all three preparations tested. The relaxing
action of SNAP on starfish tube feet is of particular interest because it is
consistent with the results of a previous study on tube feet from the sea
urchin Arbacia punctulata in which Billack et al.
(1998) report that SNAP
increased tube foot length whilst the NOS inhibitor
N
-nitro-L-arginine methyl ester (L-NAME) caused a reduction
in tube foot length compared with control preparations bathed in seawater.
Thus, release of NO by neurons in tube feet may be required to facilitate
relaxation-dependent extension of these organs as part of their stepping
action during locomotion and other behaviours in echinoderms.
The principal effector for the physiological actions of NO in mammals is
the enzyme soluble guanylyl cyclase (SGC), and pharmacological tests on
cardiac stomach, tube foot and apical muscle preparations using the SGC
inhibitor ODQ indicate that the relaxing effect of NO on starfish muscle is
mediated, at least partially, by SGC
(Elphick and Melarange, 1998;
Melarange et al., 1999
; this
study). However, here we observed no significant increase in the cGMP content
of the cardiac stomach after treatment with the NO donor SNAP. This result
does not rule out the involvement of SGC and cGMP in the relaxing effect of
SNAP on the cardiac stomach because, as discussed above, the cells relevant to
the relaxing action of SNAP in this organ (muscle cells) represent a
relatively small component of the total tissue, and, therefore, any putative
increases in the cGMP content of these cells may be small compared with the
total basal cGMP content of the cardiac stomach. In addition, it is possible
that SNAP-induced cGMP formation in cardiac stomach muscle is not detectable
due to the activity of cyclic-nucleotide-phosphodiesterases that rapidly
metabolise cGMP. Nevertheless, analysis of cGMP in the apical muscle, where
muscle cells represent the bulk of the total tissue, also showed no
significant increase after treatment with SNAP either in the absence or the
presence of the cyclic-nucleotide-phosphodiesterase inhibitor IBMX.
Collectively, these data suggest that the relaxing effect of SNAP on starfish
apical muscle may be mediated, at least in part, by a cGMP-independent
mechanism. Intriguingly, cGMP-independent relaxation of smooth muscle by SNAP
has also been reported in mammalian preparations. Janssen et al.
(2000
) obtained evidence that
the relaxing action of SNAP on tracheal smooth muscle is caused by release of
internal Ca2+ in a cGMP-independent manner, leading to activation
of Ca2+-dependent K+ channels and relaxation. It will be
interesting, therefore, to investigate whether a similar mechanism operates in
echinoderm muscle.
Potential mechanisms by which the SALMFamide neuropeptides S1 and S2 could
cause relaxation of starfish muscle have been discussed in detail previously
(Elphick and Melarange, 2001),
and here we have specifically investigated the cyclic nucleotides cGMP and
cAMP. However, as with the NO donor SNAP, no increases in either cGMP or cAMP
were observed in cardiac stomach or apical muscle after treatment with S1 or
S2. Taking into account the relatively modest relaxing effects of S1 and S2 on
apical muscle, the failure to detect any changes in cyclic nucleotide levels
in this preparation is perhaps not surprising. S1 and S2 do, however, cause
substantial relaxation of the cardiac stomach and are more effective than SNAP
when tested at the same concentration. As discussed above for SNAP, however,
the difficulty with analysis of cardiac stomach is that the muscle component
is relatively small, so any putative increases in cGMP or cAMP caused by S1 or
S2 may be insignificant against the basal levels derived from other
non-muscular tissues. Therefore, unless a tissue containing a high
concentration of cells expressing the putative SALMFamide receptor(s) can be
identified, it may be necessary to clone and express the gene encoding this
receptor(s) before it will possible to determine the signalling mechanisms of
SALMFamide neuropeptides in starfish.
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Acknowledgments |
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References |
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---|
Billack, B., Laskin, J. D., Heck, P. T., Troll, W., Gallo, M. A.
and Heck, D. E. (1998). Alterations in cholinergic signaling
modulate contraction of isolated sea urchin tube feet: potential role of
nitric oxide. Biol. Bull. Mar. Biol. Lab. Woods Hole
195,196
-197.
Díaz-Miranda, L. and García-Arrarás, J. E. (1995). Pharmacological action of the heptapeptide GFSKLYFamide in the muscle of the sea cucumber Holothuria glaberrima (Echinodermata). Comp. Biochem. Physiol. C 110,171 -176.[Medline]
Díaz-Miranda, L., Price, D. A., Greenberg, M. J., Lee, T.
D., Doble, K. E. and García-Arrarás, J. E.
(1992). Characterization of two novel neuropeptides from the sea
cucumber Holothuria glaberrima. Biol. Bull. Mar. Biol. Lab. Woods
Hole 182,241
-247.
Díaz-Miranda, L., Blanco, R. E. and García-Arrarás, J. E. (1995). Localization of the heptapeptide GFSKLYFamide in the sea cucumber Holothuria glaberrima (Echinodermata): A light and electron microscopic study. J. Comp. Neurol. 352,626 -640.[Medline]
Elphick, M. R. and Melarange, R. (1998). Nitric
oxide function in an echinoderm. Biol. Bull. Mar. Biol. Lab. Woods
Hole 194,260
-266.
Elphick, M. R. and Melarange, R. (2001). Neural
control of muscle relaxation in echinoderms. J. Exp.
Biol. 204,875
-885.
Elphick, M. R., Price, D. A., Lee, T. D. and Thorndyke, M. C. (1991). The SALMFamides: a new family of neuropeptides isolated from an echinoderm. Proc. R. Soc. Lond. B 243,121 -127.[Medline]
Elphick, M. R., Newman, S. J. and Thorndyke, M. C.
(1995). Distribution and action of SALMFamide neuropeptides in
the starfish Asterias rubens. J. Exp. Biol.
198,2519
-2525.
Janssen, L. J., Premji, M., Lu-Chao, H., Cox, G. and Keshavjee,
S. (2000). NO+ but not NO radical relaxes airway
smooth muscle via cGMP-independent release of internal Ca2+.
Am. J. Physiol. Lung Cell Mol. Physiol.
278,L899
-L905.
Martinez, A., Riveros-Moreno, V., Polak, J. M., Moncada, S. and Seesma, P. (1994). Nitric oxide (NO) synthase immunoreactivity in the starfish Marthasterias glacialis. Cell Tissue Res. 275,599 -603.
Melarange, R., Potton, D. J., Thorndyke, M. C. and Elphick, M. R. (1999). SALMFamide neuropeptides cause relaxation and eversion of the cardiac stomach in starfish. Proc. R. Soc. Lond. B 266,1785 -1789.[CrossRef]
Moncada, S., Palmer, R. M. J. and Higgs, E. A. (1991). Nitric oxide physiology, pathophysiology and pharmacology. Pharmacol. Rev. 43,109 -142.[Medline]
Moore, S. J. and Thorndyke, M. C. (1993). Immunocytochemical mapping of the novel echinoderm neuropeptide SALMFamide 1 (S1) in the starfish Asterias rubens. Cell Tissue Res. 274,605 -618.[Medline]
Newman, S. J., Elphick, M. R. and Thorndyke, M. C. (1995a). Tissue distribution of the SALMFamide neuropeptides S1 and S2 in the starfish Asterias rubens using novel monoclonal and polyclonal antibodies. I. Nervous and locomotory systems. Proc. R. Soc. Lond. B 261,139 -145.[Medline]
Newman, S. J., Elphick, M. R. and Thorndyke, M. C. (1995b). Tissue distribution of the SALMFamide neuropeptides S1 and S2 in the starfish Asterias rubens using novel monoclonal and polyclonal antibodies. II. Digestive system. Proc. R. Soc. Lond. B 261,187 -192.