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First published online August 31, 2007
Journal of Experimental Biology 210, 3245-3254 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.007740
Effects of elevated ecdysteroid on tissue expression of three guanylyl cyclases in the tropical land crab Gecarcinus lateralis: possible roles of neuropeptide signaling in the molting gland
1 Department of Biology, Colorado State University, Fort Collins, CO 80523,
USA
2 Bodega Marine Laboratory, University of California-Davis, Bodega Bay, CA
94923, USA
* Author for correspondence (e-mail: don{at}lamar.colostate.edu)
Accepted 5 July 2007
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ten-, four- and
twofold, respectively, whereas Gl-GC-II mRNA level was unchanged. A
single injection of 20-hydroxyecdysone into intact animals transiently lowered
Gl-GC-Iß in hepatopancreas, testis and skeletal muscle, and certain
Gl-GC-II isoforms in some of the tissues. These data suggest that YO and other
tissues can modulate responses to neuropeptides by altering GC expression.
Key words: guanylyl cyclase, molting, ecdysteroid, Crustacea, Arthropoda, Y-organ, gene expression, skeletal muscle, nervous system, digestive gland, molt-inhibiting hormone, crustacean hyperglycemic hormone, ecdysone receptor, mRNA
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Padayatti et al., 2004;
Potter, 2005;
Pyriochou and Papapetropoulos,
2005). In crustaceans, NO-sensitive GC (GC-I) and membrane
receptor GC (GC-II) are implicated in the signaling pathways for two
neuropeptide hormones, molt-inhibiting hormone (MIH) and crustacean
hyperglycemic hormone (CHH), respectively. Both hormones are produced and
released from the X-organ/sinus gland complex in the eyestalk (ES) ganglia
(Skinner, 1985). MIH inhibits
ecdysteroid synthesis and secretion in the molting glands or Y-organs (YOs),
which are a pair of epithelioid glands located in the anterior cephalothorax
(Lachaise et al., 1993;
Skinner, 1985). MIH induces a
large increase of cGMP in the YOs (Chung
and Webster, 2003; Saïdi
et al., 1994; Sedlmeier and
Fenrich, 1993). A NO synthase (NOS) is phosphorylated in activated
YOs, which suggests that a NO-sensitive GC mediates the MIH-induced increase
in cGMP (Kim et al., 2004;
Lee and Mykles, 2006). The
current thinking is that CHH binds to a GC-II membrane receptor, which
increases cGMP levels in various tissues
(Chung and Webster, 2003;
Goy, 1990;
Goy et al., 1987;
Pavloff and Goy, 1990;
Scholz et al., 1996;
Sedlmeier and Keller,
1981).
Crustacean tissues express several different GCs, including membrane
receptor GCs and both NO-sensitive and NO-insensitive soluble GCs
(Aonuma, 2002;
Goy, 1990;
Goy, 2005;
Prabhakar et al., 1997;
Scholz, 2001;
Scholz et al., 1996;
Scholz et al., 2002). cDNAs
encoding the ß subunit of a NO-sensitive GC (GC-Iß), a membrane
receptor GC (GC-II) and a NO-insensitive GC (Gl-GC-III) have been cloned
(Lee et al., 2007;
Liu et al., 2004;
Zheng et al., 2006). In G.
lateralis, isoforms of Gl-GC-Iß and Gl-GC-II appear to be generated
by alternative splicing. Two Gl-GC-Iß isoforms differ in the absence
(
32N) or presence (0N) of a 32-amino acid sequence at the N
terminus (Lee et al., 2007).
Three Gl-GC-II isoforms differ in length within the N-terminal ligand-binding
domain (+18, +9 and +0 amino acid insertions)
(Lee et al., 2007). As molting
can affect YO sensitivity to MIH and CHH
(Chung and Webster, 2003;
Nakatsuji and Sonobe, 2004;
Nakatsuji et al., 2006), the
effects of ES ablation and ecdysteroid injection on GC mRNA levels in YOs and
other tissues were determined.
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Materials and methods |
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27°C and 50% humidity and were fed raisins, carrots and lettuce twice
a week. A 12 h:12 h dark:light cycle was used. Molt stage was monitored by
regeneration of an autotomized walking leg; initiation of regenerate growth
[Regeneration index >10 (Skinner,
1985)] indicated entry into proecdysis. Only intermolt animals
with basal regenerates [Regeneration index <8
(Skinner, 1985)] were
used.
Two methods were used to increase hemolymph ecdysteroid levels in
vivo: ES ablation (ESA) and injection of intact animals with
20-hydroxyecdysone (20E). ESA removes the primary source of MIH, thus
stimulating YO ecdysteroidogenesis
(Skinner, 1985); wounds were
cauterized with a heated spatula. A 20E (Sigma-Aldrich, St Louis, MO, USA)
stock solution (10 mg ml–1) in 10% ethanol was injected into
the hemolymph through the arthrodial membrane at the base of a walking leg at
0.41 µg 20E g–1 body mass. Control animals received the
same volume of 10% ethanol. Hemolymph ecdysteroid was quantified by
radioimmunoassay (Chang and O'Connor,
1979).
Tissue expression of land crab guanylyl cyclases
Tissues were dissected from 3–5 crabs and immediately placed in
RNAlater RNA Stabilization Reagent (Qiagen, Germantown, MD, USA) and
stored at –20°C or flash frozen in liquid N2 and stored
at –80°C. Total RNA was isolated using either the RNeasy Mini column
(Qiagen) or TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the
manufacturer's instructions and treated with DNase I to remove any
contaminating genomic DNA. RNA concentration was determined by UV absorbance
at 260/280 nm and stored at –80°C. First strand cDNA was synthesized
in a reaction (20 µl) containing 1 µg total RNA, 50 mmol
l–1 Tris-HCl (pH 8.3), 75 mmol l–1 KCl, 3
mmol l–1 MgCl2, 10 mmol l–1
dithiothreitol, 0.5 mmol l–1 dNTPs, 40 units of RNaseOUT
ribonuclease inhibitor, 200–500 ng oligo(dT)12–18
primer, and 200 units SuperScript III RT (Invitrogen). The reaction mixture
was incubated for 60 min at 50°C, inactivated at 70°C for 15 min,
treated with E. coli RNase H (2 units), and stored at
–20°C.
PCR was performed in a Perkin Elmer 9600 cycler (Waltham, MA, USA) using
Takara Ex Taq HotStart polymerase (Takara, Inc., Madison, WI, USA).
Sequence-specific primers to Gl-GCIß, GC-II, Gl-III and Gl-EF2 were
synthesized by Integrated DNA Technologies, Inc.
(Table 1). Gl-EF2 (GenBank
accession no. AY552550), which is constitutively expressed, served as an
internal control (Kim et al.,
2005a; Kim et al.,
2005b). Reactions (30 µl) contained 1 µl first strand cDNA
mixture, 3 µl 10x Takara EX Taq buffer, 2 µl 250 µmol
l–1 dNTPs, 0.2 µl Takara EX Taq DNA polymerase (5 units
µl–1), and 18.8 µl PCR grade deionized water. The PCR
conditions were an initial denaturation at 96°C for 5 min, then 35 cycles
of denaturation at 96°C for 20 s, annealing at 62°C for 20 s, and
extension at 72°C for 30 s, followed by 5 min at 72°C as a final
extension. Identities of the PCR products were confirmed by sequencing a
subset of samples from specific tissues. Samples (15 µl) were separated on
2% agarose gels with a 100-bp PCR Molecular Ruler DNA size ladder (Bio-Rad,
Richmond, CA, USA) and stained with Ethidium Bromide. The gel image was
reversed and analyzed by Quantity One software (Bio-Rad). Expression data were
normalized to an internal positive control (Gl-EF2).
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Real-time reverse transcription polymerase chain reaction (RT-PCR) was used
to quantify the effect of ESA on the expression of the three GCs, ecdysone
receptor (EcR) and EF2 in the YO. RNA was purified from pooled YOs from
4–5 animals at each time point (0, 1, 3 and 7 days post-ES ablation) and
reverse transcribed as described above. Real-time PCR was performed in a
LightCycler 2.0 (Roche Applied Science, Nutley, NJ, USA) using LightCycler
FastStart DNA MasterPlus SYBR Green I (Roche Applied Science) and
sequence-specific primers to Gl-GCIß, Gl-GC-II, Gl-GC-III, Gl-EcR and
Gl-EF2 (Table 2). Reactions (20
µl) contained 2 µl first strand cDNA mixture, 4 µl 5x SYBR
Green Master Mix, 1 µl of 10 µmol l–1 forward primer
and 10 µmol l–1 reverse primer each, and 12 µl
PCR-grade water. The amount of YO cDNA from intact animals was doubled in the
PCR to compensate for the low levels of Gl-GCIß and GC-III transcripts
relative to the their levels in YOs from ES-ablated animals. The PCR
conditions were an initial denaturation at 95°C for 10 min, 42 cycles of
denaturation at 95°C for 5 s, annealing at 62°C for 5 s, and extension
at 72°C for 20 s, followed by melting curve analysis between 65°C and
95°C. Transcript copy number was calculated with the Roche LightCycler
software using standard curves of serial dilutions (10 fg to 10 ng) of each
template (Kim et al., 2005a;
Kim et al., 2005b). Transcript
levels of the GCs and EcR were normalized to EF2 copy number. As the copy
numbers differed by as much as 5 orders of magnitude between the different
transcripts, expression was related to transcript levels in YOs from intact (0
day post-ES ablated) animals so that the data could be combined in a single
graph.
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Statistical analyses
Statview 5.0.1 software (SAS Institute, Inc., Cary, NC, USA) was used for
statistical analyses. Regression analysis was used to determine the
correlations between ecdysteroid concentration and GC mRNA level. One-way
analysis of variance (ANOVA) post-hoc tests (Bonferroni–Dunn,
Tukey–Kramer and Fisher's PLSD tests) were used to determine significant
differences in GC mRNA levels in response to ES ablation and 20E
injection.
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; Kim et al.,
2005b). Terminating the PCR at 35 cycles was within the
exponential phase for generation of products. For example, the amounts of the
PCR products of the three GC-II isoforms in claw and thoracic muscles in
intact, 1 day post-ESA and 3 days post-ESA animals were quantified at 33, 35
and 37 cycles. As expected, the slopes (log products vs cycle number)
were 2 for GC-II(+18) and GC-II(+9); the means ± s.d. were
2.066±0.099 (N=30) and 1.914±0.122 (N=30),
respectively. The slope was <2 for GC-II(+0) (1.688±0.071;
N=30), indicating that the PCR conditions may underestimate the
amount of this isoform at higher mRNA levels. Similar experiments on
GC-Iß and GC-III showed an approximately twofold response per cycle
between 33 and 37 cycles (data not shown). These experiments validate this
method for quantifying GCs. In addition, the analysis enabled quantification
of the relative expression of the three Gl-GC-II isoforms.
The effects of ESA, which increased ecdysteroid levels in the hemolymph
(Table 3), were determined on
GC expression in various tissues. RT-PCR from a single set of animals
suggested that GC expression was responsive to ESA
(Fig. 1A). However, analysis of
tissues from 3 sets of animals (N=3 for each tissue at each time
period) showed no significant effect of ESA in most tissues
(Fig. 1B). The only exceptions
were the expression of the Gl-GC-II(+18) isoform in claw muscle and
Gl-GC-Iß0N and 32N isoforms and GC-III in YO. Gl-GC-II(+18)
expression increased about threefold in 3- and 7-day post-ESA animals
(Fig. 1B). Moreover,
Gl-GC-II(+18) mRNA level was significantly correlated with ecdysteroid
concentration in claw muscle, but not in heart or thoracic muscle
(Fig. 2). The expression of the
Gl-GC-Iß0N and 32N isoforms and Gl-GC-III in YO increased
in response to ESA, while the expression of the Gl-GC-II(+0) and Gl-GC-II(+9)
isoforms and Gl-EF2 remained unchanged
(Fig. 3A).
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Real-time RT-PCR was used to quantify more accurately the effect of ESA on the mRNA levels of the three GCs and EcR in the YO. The method did not distinguish the different Gl-GC-Iß and Gl-GC-II isoforms, as primers targeted to the variant sequences were unsuitable. Consequently, the data constitute the sum of all the transcripts encoding either Gl-GC-Iß or Gl-GC-II. ESA had the largest effect on Gl-GC-Iß, increasing the mRNA level 10.45-fold after 7 days post-ESA (Fig. 3B; R2=0.925; P=0.0381). Gl-GC-III and Gl-EcR mRNA levels increased 4.23-fold (R2=0.915; P=0.0435) and 2.16-fold (R2=0.988; P=0.0060), respectively (Fig. 3B). In contrast, Gl-GC-II mRNA level was not significantly affected by ESA (Fig. 3B; R2=0.288; P=0.4629). These results are consistent with those from the RT-PCR after 35 cycles (Fig. 3A).
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Morton and Simpson, 2002), and
carry out diverse physiological functions in arthropods
(Morton and Hudson, 2002). In
crustaceans, GCs are essential components of signal transduction pathways that
regulate nerve and muscle activity, molting, carbohydrate metabolism, and ion
and water balance (Fanjul-Moles,
2006; Goy, 2005;
Scholz, 2001;
Spaziani et al., 1999). Our
particular interest is the role of GCs in MIH and CHH signaling in the
crustacean YO; both neuropeptides inhibit molting by suppressing YO
ecdysteroidogenesis (Chung and Webster,
2003; Webster and Keller,
1986). Thus, entry into premolt is correlated with reduced MIH
synthesis in the eyestalk and a corresponding increase in hemolymph
ecdysteroid (Chen et al.,
2007; Lee et al.,
1998). Various environmental stressors inhibit molting, which may
be mediated by elevated CHH in the hemolymph
(Chang et al., 1998;
Chung and Webster, 2003;
Chung and Webster, 2005).
CHH increases cGMP levels in target tissues by way of a membrane receptor
GC (Chung and Webster, 2003;
Goy, 1990;
Goy et al., 1987;
Pavloff and Goy, 1990;
Scholz et al., 1996;
Sedlmeier and Keller, 1981).
Given its pleiotropic effects, one would expect the CHH receptor to be
expressed in all tissues. Crayfish, blue crab and land crab membrane receptor
GCs are widely expressed (Liu et al.,
2004; Zheng et al.,
2006) (Fig. 1).
Moreover, the Gl-GC-II(+9) is the dominant isoform in hepatopancreas and YO
(Fig. 1), both of which have
high-affinity CHH receptors (Chung and
Webster, 2003; Kummer and
Keller, 1993; Webster,
1993). The different Gl-GC-II isoforms may confer tissue-specific
responses to CHH isoforms that differ in biological activities
(Bulau et al., 2003;
Bulau et al., 2005;
Chung et al., 1999;
Chung et al., 1998;
Dircksen et al., 2001;
Gu and Chan, 1998;
Marco et al., 2003;
Serrano et al., 2003;
Soyez et al., 1994;
Yang et al., 1997;
Yasuda et al., 1994).
The identity of the MIH receptor remains controversial. YO membranes have
distinct receptors for both CHH and MIH
(Chung and Webster, 2003;
Webster, 1993). As MIH and CHH
are members of the same neuropeptide family, it seems reasonable to expect
that they would be ligands for related membrane receptors. While there is data
supporting the CHH receptor as a GC-II, the data do not support the MIH
receptor as another GC-II. Only one GC-II has been isolated in crustacean
tissues and its wide tissue distribution is not consistent with its role as a
MIH receptor (Lee et al.,
2007; Liu et al.,
2004; Zheng et al.,
2006). As MIH-binding capacity is highest in YO membranes
(Asazuma et al., 2005), a
receptor for MIH would be expected to be expressed preferentially in the YO.
This is not the case for any GC-II isoform
(Fig. 1). Moreover,
cross-linking prawn 125I-MIH with YO membrane labeled a 70 kDa
protein (Asazuma et al., 2005),
which is about one-half the estimated nonglycosylated masses of the membrane
GC cDNAs from crayfish, blue crab and land crab
(Lee et al., 2007;
Liu et al., 2004;
Zheng et al., 2006). The
actual mass of the GC-II is probably larger, due to extensive N-linked
glycosylation in the extracellular domain, which is necessary for ligand
binding and activation (Padayatti et al.,
2004). Finally, the increase (tenfold) of Gl-GC-Iß but
not Gl-GC-II in YOs of ES-ablated animals
(Fig. 3B) supports the
hypothesis that a NO-sensitive GC, and not a membrane receptor GC, mediates
MIH signaling. Although these data suggest that the MIH receptor is not a
membrane GC, the identity of the MIH receptor will not be resolved until the
protein is isolated and characterized fully.
As MIH signaling in the YO involves both cAMP and cGMP as secondary
messengers (Baghdassarian et al.,
1996; Böcking and
Sedlmeier, 1994; Chung and
Webster, 2003; Mattson and
Spaziani, 1985; Mattson and
Spaziani, 1986; Saïdi et
al., 1994; Sedlmeier and
Fenrich, 1993; Spaziani et
al., 1999; Von Gliscynski and
Sedlmeier, 1993), we propose a pathway involving a G
protein-coupled MIH receptor, adenylyl cyclase, NOS and GC-I
(Kim et al., 2004;
Lee and Mykles, 2006). YOs
express a trimeric G protein (Han and
Watson, 2005), NOS (Kim et
al., 2004) and NO-sensitive GC (Figs
1,
3). Activators of adenylyl
cyclase inhibit YO ecdysteroidogenesis
(Mattson and Spaziani, 1985;
Mattson and Spaziani, 1986;
Saïdi et al., 1994;
Sedlmeier and Fenrich, 1993).
MIH stimulates cGMP-dependent and cAMP-dependent kinases
(Baghdassarian et al., 1996;
Böcking and Sedlmeier,
1994; Spaziani et al.,
1999; Von Gliscynski and
Sedlmeier, 1993). This pathway is similar to that inhibiting
ecdysteroidogenesis in blowfly ovary, except that cAMP and cGMP are
antagonists. Stimulation of ecdsteroid secretion by cAMP is blocked by cGMP
(Maniere et al., 2003). A cGMP
analog and phosphodiesterase inhibitors (IBMX and MDBAMQ) suppress ecdysteroid
secretion by previteollogenic and vitellogenic ovaries in vitro
(Maniere et al., 2003).
Moreover, sodium nitroprusside, a NO donor, inhibits ecdysteroid secretion
(Maniere et al., 2003).
Reduced ecdysteroid secretion results from the activation of protein kinase G
(PKG), as KT 5823, a specific PKG inhibitor, increases secretion by
previteollogenic and vitellogenic ovaries
(Maniere et al., 2003). Taken
together, these data suggest an inhibitory pathway involving NOS and GC-I in
blowfly ovary. Zheng et al. (Zheng et al.,
2006) report unpublished data that sodium nitroprusside has no
effect on ecdsteroid secretion on blue crab YOs in vitro. However,
negative results are difficult to interpret without knowing if the NO donor
actually raised intracellular cGMP levels. NO is highly unstable and may have
been metabolized before reaching levels needed to activate the GC-I. Our data
from land and green crab YOs show that NO donors inhibit ecdysteroidogenesis
in vitro and will be included in a separate publication.
NO-sensitive GC has a more restricted tissue distribution than membrane
receptor GCs. As NO is rapidly metabolized, it must be produced at or near the
target tissue. Thus, it is not surprising that the tissue expression is
similar between NOS and GC-Iß. Both NOS and GC-I are highly expressed in
nervous tissues of arthropods and mammals
(Aonuma, 2002;
Collmann et al., 2004;
Mahadevan et al., 2004;
Nighorn et al., 1998;
Prabhakar et al., 1997;
Scholz et al., 1996;
Scholz et al., 2002). Lobster
tissues with significant NO-sensitive GC activity are intestine,
hepatopancreas, nerve cord, heart and gill
(Prabhakar et al., 1997). In
land crab, NOS and GC-Iß are co-expressed in ES ganglia, thoracic
ganglion, gill, ovary, YO and testis (Kim
et al., 2004) (Fig.
1).
Little is known about the functions of the `atypical' GCs. Only two GC-III
cDNAs (Gl-GC-III and Ms-GC-I from land crab and hawk moth, respectively) and
one GC-IV cDNA (Ms-GC-ß3 from hawk moth) have been characterized.
Ms-GC-I, which is expressed in the nervous system, has high NO-insensitive GC
activity (Nighorn et al.,
2001; Simpson et al.,
1999). Although it lacks a transmembrane domain, it is associated
with membranes in vivo (Nighorn
et al., 2001; Simpson et al.,
1999). Ms-GC-ß3 has a specialized function in the nervous
system of holometabolous insects; it is activated by eclosion hormone, which
triggers the sequence of behavioral movements during pupal ecdysis
(Morton and Simpson, 2002).
The tissue distribution of Gl-GC-III is similar to that of Gl-GC-Iß, as
its highest expression is in ES ganglia, gill, hepatopancreas, ovary, YO and
testis (Fig. 1). Moreover, ES
ablation upregulates Gl-GC-III in the YO
(Fig. 3). This coordinated
expression suggests that Gl-GC-III augments NO signaling in target
tissues.
The activation of ecdysteroid-responsive genes regulates development,
reproduction, growth and molting in arthropods
(Chang, 1993;
Kozlova and Thummel, 2000;
Skinner, 1985;
Truman and Riddiford, 1999).
Ecdysteroid regulates crustacean gene expression through a heterodimeric
nuclear hormone receptor consisting of ecdysone receptor (EcR) and retinoic-X
receptor (RXR) (Durica et al.,
2002; Wu et al.,
2004). Gl-GC-II(+18) mRNA increased in claw muscle in response to
ESA and was correlated with hemolymph ecdysteroid, but not in heart or
thoracic muscle (Fig. 1B,
Fig. 2). This differential
response to ES ablation is consistent with a previous study showing that ESA
stimulates expression of EcR and calpain T in claw muscle specifically
(Kim et al., 2005a). The
different sensitivities of claw and thoracic muscles may be due to differences
in the expression of RXR isoforms, which dimerize with EcR to form ecdysteroid
receptors that differ in ligand and DNA binding properties
(Kim et al., 2005b;
Wu et al., 2004).
YO activation coincides with an upregulation of the soluble GCs. ESA of
intermolt animals induced large increases in Gl-GC-Iß (tenfold) and
Gl-GC-III (fourfold) mRNAs in YOs
(Fig. 3B). There is also a
twofold increase in EcR mRNA (Fig.
3B). These data suggest that the YO responds to the absence of
neuropeptide(s) by increasing its sensitivity to hormones. The YOs of other
decapods display altered responses to ecdysteroid and neuropeptides over the
molt cycle (Chung and Webster,
2003; Dell et al.,
1999; Nakatsuji and Sonobe,
2004). YOs from premolt animals are refractory to MIH, even though
MIH receptor binding remains unchanged
(Chung and Webster, 2003;
Nakatsuji and Sonobe, 2004;
Nakatsuji et al., 2006). This
reduced sensitivity in premolt is correlated with an increased capacity to
degrade cGMP, which keeps cGMP levels low when MIH is present. YOs from
premolt crayfish have increased phosphodiesterase activity
(Nakatsuji et al., 2006). This
is correlated with reduced MIH- or CHH-induced cGMP levels in YOs from premolt
green crabs (Chung and Webster,
2003). A general conclusion one can make from these data is that
the YOs from intermolt animals are more responsive to MIH and CHH than YOs
from premolt animals. Moreover, the significance of the land crab data
(Fig. 3) is that the intermolt
YO is capable of increasing cGMP-mediated signaling in the absence of ES
neuropeptides.
As ESA removes the major site for the synthesis of MIH, CHH and other
neuropeptides, the response of tissues may be due to reduced ES factors,
increased ecdysteroid, or a combination of both. 20E was injected into intact
animals to determine the direct effects of ecdysteroid on GC expression in
hepatopancreas, testis, claw muscle and thoracic muscle. A common pattern for
those GCs showing significant changes was a reduced or low mRNA level at 4 h
post-injection followed by restored or increased levels at 8 h and 12 h
post-injection. These data suggest that high ecdsyteroid levels, which were
comparable to those at the end of the premolt period
(Skinner, 1985), can repress
expression of Gl-GC-Iß in all four tissues and certain Gl-GC-II isoforms
in certain tissues (Fig.
5).
GCs mediate neuropeptide control of various physiological processes in
arthropods. The expression of the three GC classes and NOS
(Kim et al., 2004) in
crustacean tissues other than the nervous system suggests that cGMP has
functions in addition to neuromodulation. The inhibition of YO
ecdysteroidogenesis by both MIH and CHH is associated with large increases in
intracellular cGMP (Chung and Webster,
2003; Saïdi et al.,
1994). It appears that the two neuropeptides accomplish this using
two different cGMP-mediated signaling pathways. CHH acts directly via
binding to a membrane receptor GC (Goy,
1990; Scholz et al.,
1996). MIH may stimulate GC-I by activating NOS
(Kim et al., 2004;
Lee and Mykles, 2006). The two
methods (ESA and 20E injection) used to increase hemolymph ecdysteroid levels
elicited different responses by tissues. ESA increased Gl-GC-II(+18) in claw
muscle, suggesting it has a role in molt-induced atrophy
(Mykles, 1997). ESA also
increased Gl-GC-Iß, Gl-GC-III and EcR expression in the YO, suggesting
that the tissue is compensating for low ES neuropeptide levels in the
hemolymph. Ecdysteroid (20E) at high doses generally had a transient
inhibitory effect on the expression of Gl-GC-Iß and some Gl-GC-II
isoforms, most likely resulting from the rapid clearance of 20E from the
hemolymph. These data indicate that there are interactions between ES
neuropeptides and ecdysteroids that can affect the expression of GCs in
crustacean tissues.
List of abbreviations
32N / 0N
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Acknowledgments |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Aonuma, H. (2002). Distribution of NO-induced cGMP-like immunoreactive neurones in the abdominal nervous system of the crayfish, Procambarus clarkii. Zool. Sci. 19,969 -979.[CrossRef][Medline]
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Baghdassarian, D., de Besse, N., Saidi, B., Somme, G. and Lachaise, F. (1996). Neuropeptide-induced inhibition of steroidogenesis in crab molting glands: involvement of cGMP-dependent protein kinase. Gen. Comp. Endocrinol. 104, 41-51.[CrossRef][Medline]
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Bulau, P., Meisen, I., Reichwein-Roderburg, B., Peter-Katalinic, J. and Keller, R. (2003). Two genetic variants of the crustacean hyperglycemic hormone (CHH) from the Australian crayfish, Cherax destructor: detection of chiral isoforms due to posttranslational modification. Peptides 24,1871 -1879.[CrossRef][Medline]
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