Nitric oxide mediates seasonal muscle potentiation in clam gills
1 Department of Biological Sciences, University of Southern Maine, Portland,
Maine, 04104, USA
2 The Whitney Laboratory, The University of Florida, 9505 Ocean Shore
Boulevard, St Augustine, Florida, 32086, USA
* Author for correspondence (e-mail: gainey{at}usm.maine.edu)
Accepted 27 June 2003
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Summary |
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Key words: clam, Mercenaria mercenaria, gill, muscle, nitric oxide (NO), potentiation, season, signaling pathway
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Introduction |
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In addition to being eulamellibranch, the gills of Mercenaria are
also plicate (Kellogg, 1892).
That is, adjacent filaments are connected to each other by interfilament
tissue junctions, while the ascending and descending filaments within each
demibranch are connected at intervals by interlamellar tissue junctions (i.e.
the septa), an arrangement that produces the plicae and the water tubes
(Fig. 1). Water flow though the
gill is determined by both the rate of beat of the lateral cilia and the
geometry of the gill, which includes the spacing of the filaments, the shape
of the plicae, and the diameter of the water tubes. These geometric parameters
are controlled by the contractile state of the gill musculature, which
consists of two distinct domains: the longitudinal/dorso-ventral muscles, and
the water tube muscles. In particular, contraction of the longitudinal muscles
reduces the length of the gill and narrows the spacing between adjacent
filaments, whereas contraction of the water tube muscles constricts the
diameter of the water tubes and changes the shape of the plicae
(Gainey et al., 2003
).
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We recently demonstrated a complex network of dopaminergic and serotonergic
fibers associated with the musculature in the gill and proposed, on
pharmacological evidence, that 5HT is the excitatory and ACh the inhibitory
transmitter to those muscles (Gainey et
al., 2003). During that investigation, we discovered,
quite by accident, that if a gill was exposed twice to the same concentration
of 5HT (with a wash between doses), then the second contraction was typically
larger than the first. We hypothesized that this potentiation might be
mediated by NO because, although this molecule is best known as a gaseous,
inter-neuronal signaling agent, it is also synthesized in muscle, where it
modulates contractility.
In vertebrates, NO modulates the activity of the three classes of muscle:
cardiac, skeletal and smooth, but its effects in these muscles are complex and
variable (for reviews on cardiac muscle, see
Brady et al., 1993;
Hare and Stammler, 1999
;
Ohba and Kawata, 1999
;
Petroff et al., 2001
;
Satoh and Naoki, 2001
;
Reading and Barclay, 2002
; for
skeletal muscle, see Marechal and Gailly,
1999
; Stamler and Meissner,
2001
; for smooth muscle, see
Buchwalow et al., 2002
). Nitric
oxide synthase (NOS) has also been demonstrated in all classes of molluscs
except Monoplacophora and Aplacophora (reviewed by
Moroz and Gillette, 1995
;
Untch et al., 1999
), and NO
relaxes vascular muscle in the cuttlefish Sepia officinalis
(Schipp and Gebaure, 1999
).
More to the point, in Mercenaria, NOS has been localized using the
NADPH-diaphorase technique to such nonneuronal tissue as the gill muscles and
the cilia of the gut (Untch et al.,
1999
). Moreover, a small fragment of a gene encoding NOS (
129
bp) has been cloned from Mercenaria; the amino acid sequence is 71%
similar to the calmodulin binding site of human neuronal NOS (L. L. Moroz and
B. Untch, University of Florida, personal communication).
The mechanisms whereby NO exerts its effects within cells fall into two
broad categories: S-nitrosylation of effector proteins, and stimulation of
soluble guanylate cyclase (sGC) leading to an increase in the concentration of
cGMP (see reviews by Denninger and
Marletta, 1999; Jaffrey et
al., 2001
). Within invertebrate neurons and muscles, however,
signaling appears to be confined to the cGMP pathway (Aplysia
californica neurons, Koh and Jacklet,
1999
; Asterias rubens muscle,
Elphick and Melarange, 2001
;
Idotea baltica muscle, Erxleben
and Hermann, 2001
; Sepia officinalis muscle,
Schipp and Gebauer, 1999
).
To test our hypothesis about the 5HT-induced potentiation of gill
musculature, we: (1) studied the time course and seasonal occurrence of this
potentiation; (2) investigated the pharmacology of agents that act at various
stages in the NO/cGMP/PK-G signaling pathway; (3) determined the
immunohistochemical distribution of NOS and sGC. Preliminary results of this
study were presented to the Society for Integrative and Comparative Biology
and the Society for Neuroscience (Gainey
et al., 1999b; Greenberg et
al., 2000
).
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Materials and methods |
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Gill preparation and apparatus
Gills were dissected away from the body wall, separated into demibranchs,
and the main trunks of the branchial nerves were removed. Muscular
contractions were recorded as changes in the length of the anterior-posterior
axis of the isolated demibranchs.
Contractions of the branchial muscles were recorded in either of two ways.
(1) Isolated demibranchs were suspended in organ baths and attached with
thread to isometric force transducers [Grass FT03 (Grass Instruments Division,
Astro-Med, Inc., West Warwick, RI, USA) and UFI 1030 (UFI, Morro Bay, CA,
USA)] equipped with springs; the resulting contractions were therefore
semi-isotonic. The transducers were interfaced to DA 100 amplifiers and an
MP100 analog-to-digital converter (Biopac, Santa Barbara, CA, USA). (2) In one
set of experiments, ultratrasonic crystal transceivers (Sonometrics, London,
Ontario, Canada) were tied to the ends of demibranchs with thread. One end of
the demibranch was pinned to a piece of rubber band that was glued with rubber
cement to the bottom of a plastic Petri dish (4.7 cm diameter); the Petri
dishes were placed on a cooling plate to maintain temperature. Under these
conditions, the muscles were unrestrained and contracted against virtually no
external load; thus, a single demibranch could be used for an entire
dose-response experiment. The isotonic contractions were measured with a
digital ultrasonic measurement system (TRX series 8; Sonometrics). In both
cases, the magnitude of the contractions was measured with AcqKnowledge
version 3.5 (Biopac Systems). All experiments were carried out at 10°C in
aerated artificial seawater (ASW; Welsh et
al., 1968).
Contraction ratios
Each of a clam's four demibranchs was isolated, suspended in an organ bath
and attached to a force transducer. After 15 min of relaxation, each of the
demibranchs was exposed to 2x10-5 mol l-1 5HT.
After the resulting contractions had leveled off, the baths were flushed and a
variable amount of time, dependent upon the specific experiment, elapsed
before reapplying the same dose of 5HT. The experimental results are expressed
as the ratio between the second and first contractions from the same
demibranch and are thus internal contraction ratios
(Fig. 2).
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In certain experiments, designed to examine the pharmacology of potentiation, it was necessary to pretreat a demibranch with an agent before exposure to 5HT. In this experimental design, one of a pair of inner or outer demibranchs was exposed to an agent (the treated demibranch) while the control was untreated. After a predetermined time, both demibranchs of the pair were exposed to only one dose of 5HT. An external contraction ratio was constructed by dividing the response of the treated demibranch (as a percentage of its initial length) by the response of the control demibranch (again as a percentage of its initial length, Fig. 2).
The internal contraction ratios of untreated controls were analyzed using a Kolmogorov-Smirnov one-sample test, which revealed that these data were not normally distributed (P<0.001, two-tailed, N=139). Both internal and external contraction ratios were therefore normalized by a logarithmic transformation. The normality of this transformation was checked as above (Kolmogorov-Smirnov; P=0.614). The ln-transformed ratios were tested against a mean of 0 (since ln1=0) with a one-sample t-test. This is mathematically equivalent to a paired t-test because the contractions used to construct the ratios were either from the same demibranch or the same clam in the case of external contraction ratios.
Although the statistical tests were performed on the ln-transformed data, for clarity in the Results, data in tables and figures are presented untransformed. Depending upon the specific experiment, the P-values reported for these tests are either two-tailed or one-tailed probabilities and are noted as such in the results; P-values <0.05 were considered significant.
Exposure times to modulatory agents
The appropriate times for exposure to various agents were determined from
the literature and by trial and error at an initial concentration
10-5 mol l-1. The specific times are noted in the
Results.
Gill anatomy
Isolated demibranchs were allowed to relax overnight at 5°C in isotonic
MgCl2 in ASW (7.6% MgCl2 in distilled water added to an
equal volume of ASW). The relaxed demibranchs were then pinned to a small
Petri dish, the bottom of which was coated with Sylgard. Demibranchs were
fixed overnight in 4% paraformaldehyde in phosphate-buffered saline (0.01 mol
l-1 sodium phosphate, 530 mmol l-1 NaCl, pH 7.3; PBS).
The fixative was prepared as described in Gainey et al.
(1999a). Because mammalian
antibodies were used for the immunohistochemistry, subsequent rinses and
solutions were made in mammalian PBS (mPBS; 0.1 mol l-1 sodium
phosphate, 140 mmol l-1 NaCl, pH 7.3).
After fixation and a 15 min rinse in mPBS, 12 µm cryostat sections were prepared. The demibranchs were placed in a solution of 30% sucrose/mPBS overnight at 5°C. Pieces of demibranch were then placed in Tissue Tek OCT, frozen and sectioned. Sections were placed on gel-coated slides and stored at -20°C until used.
After fixation and three 15 min rinses in mPBS, 100 µm vibratome sections were prepared. Pieces of demibranch were placed in a plastic mold and covered with 12% Type A pigskin gelatin in mPBS, which had been heated to 50°C. The tissue was sectioned with the vibratome after the gelatin had cooled. The sections were placed on gel-coated slides and heated briefly at 50°C to melt the excess gelatin.
Both cryostat and vibratome sections were processed at 5°C and all of
the steps are described in detail in
Gainey et al.
(2003).
Muscles were visualized with phalloidin conjugated to the fluorescent probe Alexa Fluor 488 (Molecular Probes, Eugene, OR, USA) at a concentration of 1 unit/100 µl of mPBS. The phalloidin was added to the tissues at the same time as the secondary antibody.
The primary antibody to NOS was polyclonal, raised in rabbit, and affinity purified (Universal NOS antibody, product number N-217, Sigma-Aldrich, St Louis, MO, USA). The antibody was raised to the synthetic peptide DQKRYHEDIFG, which comprises amino acid residues 1113-1122 of mouse NOS, with an added N-terminal aspartyl residue. Since the antigenic undecapeptide was conjugated to keyhole limpet hemocyanin (KLH) through this N-terminal aspartic acid, and since heterodont clams do not contain hemocyanin, the 5-6 amino acid residues at the free C-terminal of the peptide determined the immunoreactivity of the antibody. When we used BLAST to search in Mollusca for protein sequences with alignments similar to that of the antigenic peptide, the top two results were NOS sequences from Aplysia californica (accession number AAK83069) and Lymnaea stagnalis (accession number O61039). Both proteins include an 11-residue sequence with the C-terminal sequence -HEDIFG, in the same general location as those in the mouse. Since this hexapeptide is conserved in mouse and two gastropods, and since it is almost certainly the epitope for the universal NOS antibody, we conclude that, in clam gills, the antibody is also detecting NOS. This conclusion was supported by our observation that clam gills subjected to the NADPH-diaphorase technique showed staining comparable to that seen in gills processed with the antibody (L. L. Moroz and B. Untch, unpublished observations). A 1/100 dilution of the antibody with mPBS was used for the images in Fig. 10.
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The primary antibody to soluble guanylate cyclase (sGC) was a rabbit
polyclonal raised to two antigenic peptides: EQARAQDGLKKRLGKLKAT (human
1 residues 414-432) and EDFYEDLDRFEENGTQDSR (rat
ß1 residues 188-207); the peptides were both conjugated to KLH
(product number 160890; Cayman Chemical, Ann Arbor, MI, USA). A BLAST search
revealed that the antigenic
peptide is similar to
subunit sGC
sequences in both Drosophila and Manduca; there are no
sequence data of sGC in molluscs. A 1/100 dilution of the antibody with mPBS
was used for the images in Fig.
10.
The secondary antibody in both instances was a goat anti-rabbit (IgG) conjugated to Alexa Fluor 594 (Molecular Probes); the antibody was used at a 1/200 dilution in mPBS.
Negative controls were made by omitting the primary antibodies from one slide in each series of preparations.
Fluorescent images were made with a Nikon Eclipse TE200 or a Leica DMLB microscope equipped with a Spot RT digital color camera (Diagnostic Instruments, St Sterling Heights, MI, USA). Images were prepared for publication with Adobe Photoshop.
Dose-dependent effects
All contractions, measured in mm, were expressed as a percentage of the
initial length of each demibranch. Regression lines were fitted with a
logistic function of the form:
response=/1+exp[ß0+ß1xlog(agonist)],
where
is the asymptotic value of the maximal contraction, and
ß0 and ß1 are intercept and slope parameters,
respectively. Initially, all three parameters were estimated using non-linear
regression (Systat, v. 9 and 10); later
was fixed in the model,
reducing the error estimates of the remaining parameters. The concentrations
of agonist giving half-maximal responses (EC50) were estimated
according to the following formula:
EC50=10(-ß0/ß1).
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Results |
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Duration
In our initial experiments, leading to the discovery of potentiation, the
period between the end of the first dose of 5HT (flushing of the organ baths)
and the addition of the second dose was 1 h. To study whether the potentiation
was time dependent, we varied this period between 15 min and 24 h
(Fig. 4). The mean internal
contraction ratios, as a measure of potentiation, ranged from a high of
2.45±0.69 (±S.E.M.; N=8) at 2 h to a low of
2.00±0.45 (N=15) at 24 h; but ANOVA revealed that there were
no significant differences amongst any of the times
(F5,178=0.28; P=0.92). These data were collected
in the winter.
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Seasonal effects
From data collected over 5 years, monthly mean internal contraction ratios
were calculated, and ranged from a high of 2.94±0.65 (mean ±
S.E.M.; N=23) in February to a low of 0.88±0.12
(N=30) in October. Moreover, when these monthly means were plotted, a
clear seasonal component to potentiation was revealed
(Fig. 5). From November through
June, the period of potentiation, all of the mean internal contraction ratios
were equal to each other (F7,226=1.845; P=0.08)
and significantly greater than 1 (one-tailed P<0.001). From July
through October, the season of no potentiation, the mean internal contraction
ratios were again equal to each other (F3,177=2.36;
P=0.06), but they were not different from 1 (two-tailed,
P=0.07).
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Geographic effects
Because the clams used in our experiments all came from the northeastern
USA, we wondered if geographical origin affected potentiation. To answer this
question, we compared the internal contraction ratios of northern clams to
those of Florida clams; comparisons were made in July and August, when
potentiation was absent, and in March, when potentiation was present. In
neither case, was there a statistical difference between northern and Florida
clams (Table 1).
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Acclimation temperature and geography
In the previous experiments, the northern clams were maintained at 10°C
while the Florida clams were kept at 20°C, so we tested whether the
acclimation temperature affected the internal contraction ratios. In March, we
acclimated clams from Maine and Florida to temperatures of both 10°C and
20°C for 1 week and compared the mean internal contraction ratios using a
two-way ANOVA. Neither geographic region nor acclimation temperature was
significant (F1,47=0.1, 0.17; P=0.76, 0.68,
respectively; Table 2).
Moreover, we acclimated clams from Massachusetts to temperatures of 10°C
and 20°C for 1 week in September and compared the internal contraction
ratios, and again there was no significant difference (two-tailed
P=0.11; Table 2).
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Nitric oxide mediates potentiation
To test if potentiation was mediated by nitric oxide (NO), we performed the
following experiments and immunohistochemical observations. (1) We inhibited
nitric oxide synthase (NOS) with L-NAME
(nitro-L-arginine methyl ester); (2) pretreated gills with the NO
donor DEANO (diethylamine/nitric oxide complex); and (3) prepared gill
sections were exposed to the universal NOS antibody.
Inhibition of NOS
In May, when potentiation was present, we exposed demibranchs to the NOS
inhibitor L-NAME for 15 min before the second exposure to 5HT.
Potentiation was abolished at L-NAME concentrations of
10-6, 10-5 and 10-4 mol l-1, and
none of these mean internal contraction ratios were significantly different
from 1 (Fig. 6). At lower
concentrations of L-NAME (10-8, 10-7 mol
l-1), the mean internal contraction ratios were not significantly
different from those of control clams (two-tailed P=0.14 at
10-8 mol l-1; P=0.46 at 10-7 mol
l-1; internal contraction ratio of controls=2.72±0.44, mean
± S.E.M.; N=15). As an additional control, gills
were exposed to the inactive enantiomer D-NAME, at 10-4
mol l-1; the internal contraction ratio was 2.04±0.32 (mean
± S.E.M., N=5), which was not significantly
different from that of untreated controls (two-tailed P=0.48) (not
shown).
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In the previous experiments, potentiation was studied at 2x10-5 mol l-1 5HT. But is the magnitude of potentiation dependent upon the concentration of 5HT? To answer this question, we used paired demibranchs from the same clams: one demibranch was exposed to 10-5 mol l-1 L-NAME to block potentiation, then both demibranchs were exposed to increasing concentrations of 5HT. The responses were measured with ultrasound, which minimized fatigue, thus a single demibranch could be used for an entire dose-response curve. The control demibranchs would show the effects of both 5HT and NO, because they were exposed to multiple, increasing doses of 5HT; while the demibranchs pre-treated with L-NAME would show only the effects of 5HT. At 5HT concentrations below 5x10-6 mol l-1, the responses of both treatment and control demibranchs were indistinguishable. At concentrations of 5x10-6 mol l-1 5HT and above, the data and the regression lines diverge. The data and the curve for the demibranchs treated with L-NAME lie below those of the control demibranchs, and the statistically significant difference between the control and treatment lines is the contribution of NO to the effects of 5HT (F1,52=100; P<0.001; Fig. 7). Thus, the amount of potentiation is dependent upon the dose of 5HT.
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The two previous experiments suggest that the potentiation of the second response to 5HT is mediated by NO. But does NO also contribute to the initial response to 5HT? In January, when potentiation was present, we pretreated one demibranch of each pair with L-NAME for 15 min, and then exposed both demibranchs to 2x10-5 mol l-1 5HT. The resulting external contraction ratios decreased markedly (Fig. 8); indeed, for 5HT doses from 10-9 to 10-4 mol l-1 the pooled external contraction ratio=0.73±0.05 (mean ± S.E.M.; N=27) was significantly less than 1 (one-tailed P<0.001), and these values were equal to each other (F5,19=0.27; P=0.92). The external contraction ratio at 10-10 mol l-1L-NAME was 0.96±0.10 (mean ± S.E.M.; N=3), which was not significantly different from 1 (two-tailed P=0.64). As a control, gills were pretreated with 10-5 mol l-1 D-NAME, and the resulting external contraction ratios were not significantly different from 1 (1.11±0.23, mean ± S.E.M.; N=6; two-tailed P=0.87). Finally, we repeated the experiment in August, when potentiation was absent, with 10-5 mol l-1L-NAME, and there was no significant effect upon the external contraction ratio (1.10±0.19, mean ± S.E.M.; N=10; two-tailed P=0.77; Fig. 8).
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Effect of an NO donor
To confirm the results of the experiments with L-NAME, we
pretreated one of each pair of inner and outer demibranchs for 5 min with the
NO donor DEANO. To retard the oxidation and degassing of NO, all of the
demibranchs were suspended in degassed non-aerated seawater. Then we exposed
all of the demibranchs to 2x10-5 mol l-1 5HT;
thus, external contraction ratios were calculated in this experiment. Nitric
oxide, released by DEANO, increased the external contraction ratios in a
dose-dependent manner (Fig. 9). The threshold of the response was at 10-13 mol l-1. At
concentrations of DEANO greater than 10-10 mol l-1, all
of the external contraction ratios were significantly greater than 1.
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The experiments shown in Fig. 9 were performed in the late fall, during the season of potentiation. In July, when potentiation was absent, we repeated these experiments with 10-5 mol l-1 DEANO, a concentration that had potentiated contractions in the fall. The external contraction ratio (mean ± S.E.M.) was 1.05±0.085; N=15), which was not significantly different from a mean of 1 (two-tailed P=0.99); in other words, DEANO had no effect on 5HT induced contractions (Fig. 9, open circle).
NOS immunohistochemistry
Our pharmacological data indicated that NOS should be found in the gill. We
examined thick sections (100 µm) of gills removed from clams in November
when potentiation is present, and found immunoreactive (ir) NOS to be
concentrated in the longitudinal muscles, the muscles of the blood vessel and
septa, and the epithelium of the gill filaments
(Fig. 10A,B). Little ir-NOS
was seen in the water tube muscles. However, in July and August, when
potentiation is absent, thin and thick sections of gills showed NOS
concentrated only at the base of the gill filaments and in varicose fibers
adjacent to the gill muscle (Fig.
10C,D). The enzyme was also unevenly distributed within the
horizontal blood vessels and subfilamentar tissues
(Fig. 10C). Moreover, the thin
section (Fig. 10D) was made
from Florida clams in July, 2001, while the thick section
(Fig. 10C) was made from
Massachusetts clams in August, 2002. Negative controls, lacking the primary
antibody, had low levels of background fluorescence comparable to the tips of
the filaments in Fig. 10C.
Signal transduction
Because NO exerts at least some of its effects by stimulating soluble
guanylate cyclase (sGC) and thereby increasing the concentration of cGMP, we
tested the effects of the membrane permeable cGMP analog db-cGMP (dibutyryl
cyclic GMP) on branchial muscle contraction. The experimental design was that
used in the experiments with DEANO: one of each pair of demibranchs was
pretreated with db-cGMP for 15 min then both demibranchs were then exposed to
2x10-5 mol l-1 5HT, and external contraction
ratios calculated. db-cGMP increased the external contraction ratios in a
dose-dependent manner, and the threshold was between 10-9 and
10-8 mol l-1 (Fig.
11). These experiments were performed in November and December,
during the season of potentiation.
|
To test further the hypothesis that NO augments branchial muscle contraction by stimulating sGC, we exposed gills to increasing concentrations of the sGC inhibitor ODQ (oxadiasoloquinoxalin) for 60 min between the first and second contractions. ODQ inhibited potentiation in a dose-dependent manner (Fig. 12). The internal contraction ratio at 10-4 mol l-1 ODQ was significantly lower than that of untreated controls (one-sample P=0.019), but the ratio was still significantly greater than 1(one-sample P<0.001).
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Because we could not completely abolish potentiation with ODQ in the previous experiment, we wondered if some of the effects of NO were direct via S-nitrosylation of effector proteins. Therefore, we pretreated one of each pair of demibranchs with 10-6 mol l-1 DEANO (a concentration sufficient to elicit potentiation) and with increasing concentrations of ODQ (at the time we performed this experiment, we were not aware that NO potentiated the initial contraction, hence we added the DEANO). Then we exposed the treated and control gills to 2x10-5 mol l-1 5HT. External contraction ratios were calculated as usual. ODQ inhibited the potentiation induced by DEANO (Fig. 13). The mean external contraction ratios at 10-8, 10-7 and 10-6 mol l-1 ODQ were equal to each other (F2,12=0.137; P=0.87] and significantly greater than 1 (one-tailed P=0.004). In contrast, the mean external contraction ratios at 10-5 and 5x10-5 mol l-1 ODQ were not significantly different than 1 (two-tailed P=0.63 and 0.93, respectively). In summary, we could completely inhibit NO-induced potentiation by inhibiting sGC.
|
To test whether cGMP affects potentiation directly, i.e. by activating a cGMP-gated channel or by stimulating a protein kinase G (PK-G), we treated gills with the PK-G inhibitor Rp-8-CPT-cGMPS (10-5 mol l-1; Rp-8-[(4-chlorophenyl)thio]guanosine 3',5'-cyclic monophosphothioate) for 5 min between the first and second contractions. The mean internal contraction ratio for treated demibranchs was 1.06±0.15 (±S.E.M.; N=12), which was significantly less than the control contraction ratio of 2.01±0.51 (mean ± S.E.M.; N=9; one-tailed P=0.02) (not shown).
sGC immunohistochemistry
Thick sections of demibranchs from clams in April showed ir-sGC
concentrated in the gill musculature and the gill filament epithelium
(Fig. 10E). In contrast,
demibranchs taken from clams in August, when potentiation was absent, had no
sGC in the main body of the longitudinal muscle, but the enzyme was still
present in the gill filament epithelium and in branches of the longitudinal
muscles running into the center of each filament
(Fig. 10F).
Seasonal 5HT dose-response data
Because potentiation and the distributions of NOS and sGC vary seasonally,
we expected that a 5HT dose-response curve produced with demibranchs from
summer clams would lie to the right of a curve produced with winter clams.
That is, in the summer (the season of no potentiation), muscles would be less
sensitive to 5HT. We therefore constructed a dose-response curve to 5HT during
July and August and compared it to one previously, and fortuitously, produced
during the winter (Gainey et al.,
2003). Unexpectedly, the dose-response curve of summer gills lies
to the left of that from winter gills, and the regression lines are
significantly different (F1,103=22; P<0.001;
Fig. 14). The EC50
for the line of summer is 3.8x10-5 mol l-1 (95%
CI=1.8x10-5 to 5.8x10-5 mol l-1)
whereas, that for the winter is 1.1x10-4 mol l-1
(95% CI=4.0x10-5 to 1.8x10-4 mol
l-1).
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Finally, using the data set for seasonal potentiation (Fig. 5), we plotted, for each month, the mean response of the demibranchs to their first exposure to 2x10-5 mol l-1 5HT. The mean response was measured as a fraction of the initial length of the demibranch. One-way ANOVA revealed that the mean monthly contractions were significantly different (F11,338=11.29; P<0.001, Fig. 15). The responses are lowest in February and rise incrementally to their maximum in July. March, however, appears to be an outlier, because the mean response in this month is as large as that in June. But close inspection of the data from March suggests that the magnitude of these responses is not a sampling artifact. In particular, the data for this month includes the responses of both northern clams (% contraction=4.99; mean ± S.E.M.=0.63; N=30) and Florida clams (% contraction=5.09±0.69, mean ± S.E.M.; N=24) and they are clearly similar.
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Discussion |
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Potentiation
Time course
The potentiation reported here is particularly striking because it was
maintained for up to 24 h in isolated gills, and the magnitudes of
potentiation observed between 15 min and 24 h were equal. Similar
potentiations, induced by prior stimulation or related to the contraction
history of the muscle, have been observed in a variety of vertebrate muscles,
including skeletal (Rosenthal,
1969), cardiac (Hare and
Stamler, 1999
) and airway smooth muscle
(Gunst et al., 1993
;
Meiss, 1997
). Long-term
potentiation of the buccal muscles of the gastropod mollusc Aplysia
has also been extensively studied (Cropper et al.,
1987a
,b
,
1990
). But in most of these
cases, potentiation only lasts from seconds to minutes (see previous
references and Decostre et al.,
2000
; Pilarski and Brechue,
2002
), which is much shorter than the potentiation of the gill
muscle seen in the present work.
Seasonality, geography, and acclimation temperature
The seasonality of potentiation is another of its notable aspects, and we
supposed that it would vary with the source of the experimental animals or the
temperature of their acclimation. However, neither the origin of the clams
(New England versus Florida) nor their acclimation temperature had
any effect upon potentiation.
Because potentiation was absent from July through October, we expected that
the sensitivity of branchial muscles to 5HT would also be lowest in the late
summer to early fall. In fact, although the size of the responses to single
doses of 5HT (2x10-5 mol l-1) are strongly
seasonal, the largest occur in the summer (compare Figs
4 and
13); and therefore, 5HT
dose-response curves constructed in the summer lie to the left of those
produced in the winter (Fig.
12). In contrast to the gill muscle, the heart of
Mercenaria is least sensitive to 5HT (by about an order of magnitude)
from late June through August (Greenberg,
1960). Finally, we previously reported that the lateral cilia of
Mercenaria gills are less sensitive to inhibition by dopamine from
April to June (Gainey et al.,
1999a
). Thus, although pharmacological seasonality is common, its
timing seems to vary from tissue to tissue.
The reproductive cycle of Mercenaria has been studied extensively
with respect to temperature and geography. Thus spawning varies with water
temperature, and there is a latitudinal gradient, with Florida clams spawning
in March (and a second time in the fall) and New England clams spawning once
in August. But when Massachusetts and South Carolina clams were crossed, a
genetic component to the spawning time was revealed. In addition, the time of
spawning could only be altered by a cyclic change in temperature, and this
procedure is effective only at certain times of the year
(Knaub and Eversole, 1988; for
an extensive review, see Eversole,
2001
). Finally, potentiation and the other neuropharmacological
changes may be part of an endogenous cycle. An amazing example of such a cycle
was found in the pulmonate Otala lactea. Gainer
(1972
) found that snails in
diapause, under constant laboratory conditions, would spontaneously emerge in
April, crawl about for several days, and then return to a dormant state until
the next April. These complex phenomena indicate that there may well be
geographical or physiological differences in the seasonality of potentiation
and sensitivity that our experiments were unable to resolve.
Nitric oxide mediates potentiation
Our experiments provide convincing evidence that potentiation is mediated
by NO. First, the NO donor DEANO had, by itself, no effect on the gill muscle;
i.e. the effects of NO were only apparent after the muscle was stimulated with
5HT. Therefore, NO has no direct effect upon muscle contraction. Second, when
we pretreated gills with the NOS inhibitor L-NAME, the response to
a first dose of 5HT was inhibited. Therefore, the initial exposure to 5HT
generates NO, which then potentiates the initial contraction. Finally, if
L-NAME was applied before the second dose of 5HT, the resulting
contraction was not potentiated. Therefore, NO must be produced during both
the first and the second contractions if the second one is to be
potentiated.
Signal transduction
Our results indicate that the effects of NO-mediated potentiation are
via the stimulation of sGC because potentiation is inhibited by the
SGC inhibitor ODQ in a dose-dependent manner. Additionally, the cGMP analog
dibutyryl cGMP mimicked the effects of potentiation. In some systems, e.g.
vertebrate photoreceptors, cGMP interacts directly with membrane ion channels,
leading to a change in ionic conductance, while in other systems the effects
of cGMP are elicited through stimulation of a PK-G, leading to phosphorylation
of target proteins (see references cited in the Introduction). That the PK-G
inhibitor RP-8-CPT-cGMPS inhibited potentiation implies that potentiation is
via the latter pathway, i.e. stimulation of a PK-G.
Seasonality of NO mediation and signal transduction
The expression of immunoreactive NOS is seasonal, and the pattern of
seasonality is identical to that of potentiation. Thus, in November, when
potentiation occurs, NOS was clearly present within the gill musculature; but
in July and August, during the off-season, NOS was not expressed in the
muscle, but appeared at the base of the gill filaments adjacent to the muscle.
The distance between the muscle and the base of the filaments is about 25
µm, so NO can certainly diffuse into the muscle. Moreover, when we
pretreated gills with the NO generator DEANO in July, there was no
potentiation. Finally, these observations are consistent with our observation
that the expression of immunoreactive sGC also varies seasonally; this enzyme
is not detectable in the muscle during the summer.
The mechanism of potentiation
Although we understand the basic signaling mechanism involved in
potentiation (Fig. 16), we do
not totally understand its underlying causes. Clearly NO modulates force
production in the gill musculature, but it is not clear how the initial
exposure to 5HT potentiates subsequent exposures to that transmitter. Two sets
of our experiments suggest that increased activity of both NOS and sGC occurs
following the initial exposure to 5HT. In the experiments with the NOS
inhibitor L-NAME, potentiation was inhibited by 10-9 mol
l-1L-NAME during the initial exposure to 5HT. In
contrast, a higher concentration of L-NAME (10-6 mol
l-1) was needed to inhibit potentiation during the second exposure
to 5HT (compare Figs 8 and
6, respectively). These
experiments imply that there is increased NOS activity during the second
exposure to 5HT. In the second set of analogous experiments with the sGC
inhibitor ODQ, potentiation was inhibited by 10-5 mol
l-1 ODQ during the initial exposure to 5HT. In contrast, we could
not completely inhibit potentiation with 10-4 mol l-1
ODQ during the second exposure to 5HT (compare Figs
13 and
12, respectively). Again,
there is an implication of increased sGC activity during the second exposure
to 5HT. These data do not preclude changes in the activity of PK-G,
phsophodiesterases or protein phosphatases.
|
We speculate that increased concentrations of cGMP, resulting from
increases in the activity of NOS and sGC, lead to an increased influx of
calcium through phosphorylated membrane channels in response to the second
dose of 5HT, resulting in potentiation. There are several studies in
Lymnaea showing that the effects of 5HT are mediated by
cGMP-dependent phosphorylation of calcium channels. Moreover, in
Helix, modulation of calcium currents in neuron F1 is via
regulation of a phosphorylation/dephosphorylation cycle of calcium channels
(for a summary of data for both Lymnaea and Helix, see
Kits and Mansvelder, 1996).
Although we know the seasonal patterns of expression of both NOS and sGC, we
have not yet measured changes in the activity of any of the relevant
enzymes.
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Acknowledgments |
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References |
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Brady, A. J. B., Warren, J. B., Poole-Wilson, P. A., Williams, T. J. and Harding, S. E. (1993). Nitric oxide attenuates cardiac myocyte contraction. Am. J. Physiol. 265,H176 -H182.[Medline]
Buchwalow, I. B., Podzuweit, T., Bocker, W., Samoilova, V. E.,
Thomas, S., Wellner, M., Baba, H. A., Robenek, H., Schnekenburger, J. and
Lerch, M. M. (2002). Vascular smooth muscle and nitric oxide
synthase. FASEB J. 16,500
-508.
Cropper, E. C., Lloyd, P. E., Reed, W., Tenebaum, R., Kupfermann, I. and Weiss, K. R. (1987a). Multiple neuropeptides in cholinergic motor neurons of Aplysia: evidence for modulation intrinsic to the motor circuit. Proc. Natl. Acad. Sci. USA 84,3486 -3490.[Abstract]
Cropper, E. C., Tenebaum, R., Kolks, M. A. G., Kupfermann, I. and Weiss, K. R. (1987b). Myomodulin: a bioactive neuropeptide present in an identified cholinergic buccal motor neuron of Aplysia. Proc. Natl. Acad. Sci. USA 84,5483 -5486.[Abstract]
Cropper, E. C., Price, D., Tenebaum, R., Kupfermann, I. and Weiss, K. R. (1990). Release of peptide cotransmitters from a cholinergic motor neuron under physiological conditions. Proc. Natl. Acad. Sci. USA 87,933 -937.[Abstract]
Decostre, V., Gillis, J. M. and Gailly, P. (2000). Effect of adrenaline on the post-tetanic potentiation in mouse skeletal muscle. J. Muscle Res. Cell Motil. 21,247 -254.[CrossRef][Medline]
Denninger, J. W. and Marletta, M. A. (1999). Guanylate cyclase and the NO/cGMP signaling pathway. Biochem. Biophys. Acta 1411,334 -350.[Medline]
Elphick, M. R. and Melarange, R. (2001). Neural
control of muscle relaxation in echinoderms. J. Exp.
Biol. 204,875
-885.
Erxleben, C. and Hermann, A. (2001). Nitric oxide augments voltage-activated calcium currents of crsutacea (Idotea baltica) skeletal muscle. Neurosci. Lett. 300,133 -136.[CrossRef][Medline]
Eversole, A. G. (2001). Reproduction in Mercenaria mercenaria. In Biology of The Hard Clam (ed. J. N. Kraeuter and M. Castagna), pp.221 -260. Amsterdam, The Netherlands: Elsevier Science.
Gainer, H. (1972). Effects of experimentally induced diapause on the electrophysiology and protein synthesis patterns of identified molluscan neurons. Brain Res. 39,387 -402.[CrossRef][Medline]
Gainey, L. F., Jr, Vining, K. J., Doble, K. E., Waldo, J. M.,
Candelario-Martinez, A. and Greenberg, M. J. (1999a). An
endogenous SCP-related peptide modulates ciliary beating in the gills of a
venerid clam Mercenaria mercenaria. Biol.
Bull. 197,159
-173.
Gainey, L. F., Jr, Pirone, R. T. and Greenberg, M. J. (1999b). Nitric oxide potentiates gill muscle contraction in Mercenaria mercenaria. Amer. Zool. 39, 71A.
Gainey, L. F., Jr, Walton, J. C. and Greenberg, M. J.
(2003). Branchial musculature of a venerid clam: pharmacology,
distribution, and innervation. Biol. Bull.
204, 81-95.
Greenberg, M. J. (1960). The responses of the Venus heart to catechol amines and high concentrations of 5-hydroxytryptamine. Brit. J. Pharmacol. 15,365 -374.
Greenberg, M. J., Moroz, L. L., Untch, B., Meleshkevitch, E. A. and Gainey, L. F., Jr (2000). An analog of the mammalian airway: transmitter interactions regulate the functions of the clam gill. Soc. Neurosci. Abstr. 26, 1166.
Gunst, S. J., Wu, M.-F. and Smith, D. D. (1993). Contraction history modulates isotonic shortening velocity in smooth muscle. Am. J. Physiol. 265,C467 -C476.[Medline]
Hare, J. M. and Stamler, J. S. (1999). NOS: modulator, not mediator of cardiac performance. Nature Medicine 5,273 -274.[CrossRef][Medline]
Jaffrey, S. R., Erdjument-Bromage, H., Ferris, C. D., Tempst, P. and Snyder, S. (2001). Protein S-nitrosylation: a physiological signal for neuronal nitric oxide. Nature Cell Biol. 3,193 -197.[CrossRef][Medline]
Kellogg, J. L. (1892). A contribution to our knowledge of the morphology of lamellibranchiate molluscs. Bull. US Fish. Comm. 10,389 -436.
Kits, K. S. and Mansvelder, D. (1996). Voltage gated calcium channels in molluscs: classification, Ca2+ dependent inactivation, modulation and functional roles. Invert. Neurosci. 2,9 -34.[Medline]
Knaub, R. S. and Eversole, A. G. (1988). Reproduction of different stocks of Mercenaria mercenaria. J. Shellfish Res. 7,371 -376.
Koh, H. and Jacklet, J. W. (1999). Nitric oxide
stimulates cGMP production and mimics synaptic responses in metacerebral
neurons of Aplysia. J. Neurosci.
19,3818
-3826.
Marechal, G. and Gailly, P. (1999). Effects of nitric oxide on the contraction of skeletal muscle. Cell. Mol. Life Sci. 55,1088 -1120.[CrossRef][Medline]
Meiss, R. A. (1997). Mechanics of smooth muscle contraction. In Cellular Aspects of Smooth Muscle Function (ed. C. Y. Kao and M. E. Carsten), pp.169 -208. Cambridge, UK: Cambridge University Press.
Moroz, L. and Gillette, R. (1995). From Polyplacophora to Cephalopoda: Comparative analysis of nitric oxide signalling in Mollusca. Acta Biol. Hungarica 46,169 -182.[Medline]
Ohba, M. and Kawata, H. (1999). Biphasic nature of inotropic action of nitric oxide donor NOC7 in guinea-pig ventricular trabeculae. Jap. J. Physiol. 49,389 -394.[Medline]
Petroff, M. G. V., Kim, S. H., Pepe, S., Dessy, C., Marban, E., Ballingand, J.-L., Sollott, S. J. (2001). Endogenous nitric oxide mechanisms mediate the stretch dependence of Ca 2+ release in cardiomyocetes. Nature Cell Biol. 3, 867-873.[CrossRef][Medline]
Pilarski, J. Q. and Brechue, W. F. (2002). Preservation of force generation during repetitive contractions is due to postactivation potentiation (PAP). FASEB J. 16, A770.
Reading, S. A. and Barclay, J. K. (2002). The inotropic effect of nitric oxide on mammalian papillary muscle is dependent on the level of beta 1-adrenergic stimulation. Can. J. Physiol. Pharmacol. 80,569 -577.[CrossRef][Medline]
Rosenthal, J. (1969). Post tetanic potentiation at the neuromuscular junction of the frog. J. Physiol. 203,121 -133.[Medline]
Satoh, S. and Naoki, M. (2001). Intracellular mechanisms of cGMP-mediated regulation of myocardial contraction. Basic Res. Cardiol. 96,652 -658.[CrossRef][Medline]
Schipp, R. and Gebauer, M. (1999). Nitric oxide: a vasodilatatory mediator in the cephalic aorta of Sepia officinalis (L.) (Cephalopoda). Invert. Neurosci. 4, 9-15.[CrossRef][Medline]
Stamler, J. S. and Meissner, G. (2001).
Physiology of nitric oxide in skeletal muscle. Physiol.
Rev. 81,209
-237.
Untch, B., Greenberg, M. J. and Moroz, L. (1999). Nitric oxide producing cells in the CNS and peripheral tissues of Merceneria merceneria (Bivalvia): A histochemical study. Amer. Zool. 39,41A .
Welsh, J. H., Smith, R. I. and Kammer, A. E. (1968). Laboratory Exercises in Invertebrate Physiology. 3rd edition. Minneapolis: Burgess.