A crustacean nitric oxide synthase expressed in nerve ganglia, Y-organ, gill and gonad of the tropical land crab, Gecarcinus lateralis
Department of Biology, Colorado State University, Fort Collins, CO 80523, USA
* Author for correspondence (e-mail: don{at}lamar.colostate.edu)
Accepted 27 May 2004
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Summary |
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Key words: Crustacea, Arthropoda, nitric oxide synthase, Y-organ, gonad, ovary, gill, eyestalk ganglion, molting gland, nervous tissue, tissue distribution, cDNA cloning, DNA sequence, amino acid sequence, gene expression, mRNA
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Introduction |
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In mammals, there are three NOS genes: neuronal NOS
(nNOS), endothelial NOS (eNOS) and inducible NOS
(iNOS) (Bogdan, 2001;
Mungrue et al., 2003
;
Nathan and Xie, 1994
).
Although their expression and biological roles vary, they share a common
structural organization (Ghosh and
Salerno, 2003
; Kone et al.,
2003
; Torreilles,
2001
). The native enzyme is a homodimer of 130160-kDa
subunits (Torreilles, 2001
).
The N-terminal oxygenase domain contains the binding motif for a P450-like
cysteine thiolate-ligate heme and tetrahydrobiopterin (H4). The C-terminal
reductase domain contains the binding motifs for FAD, FMN and NADPH. These two
domains are linked by a calmodulin (CaM) binding motif. nNOS and eNOS are
constitutively expressed and their enzymatic activities are regulated by the
intracellular Ca2+ concentration through binding of Ca2+
to CaM (Roman et al., 2002
).
They contain a 4050 amino acid sequence linked to the FMN binding motif
that acts as an autoinhibitory loop, blocking electron transfer from FMN to
the heme in the absence of Ca2+/CaM
(Craig et al., 2002
;
Ghosh and Salerno, 2003
;
Nishida and de Montellano,
2001
; Salerno et al.,
1997
). By contrast, iNOS lacks the autoinhibitory loop and binds
CaM with high affinity at low Ca2+ levels; its activity is
regulated predominantly at the transcriptional level
(Chen and Wu, 2003
;
Nathan and Xie, 1994
).
Insect NOSs have the highest sequence identity with mammalian nNOS and
share the same organization in the oxygenase, CaM-binding and reductase
domains (Davies, 2000;
Torreilles, 2001
). Insect NOS
requires NADPH, Ca2+ and CaM for enzymatic activity. It is
expressed in a variety of adult and embryonic tissues, including abdominal
nerve cord, optic lobes, fat body, antenna, hemocytes, midgut and Malpighian
tubule (Broderick et al.,
2003
; Gibbs and Truman,
1998
; Imamura et al.,
2002
; Luckhart et al.,
1998
; Nighorn et al.,
1998
). Isoforms of NOS are generated by alternative splicing. The
Drosophila NOS gene contains at least four alternative promoters
(Stasiv et al., 2001
). Some
truncated alternative splicing variants of the Drosophila NOS lacking
the reductase domain may act as dominant negative regulators, as heterodimers
would lack enzyme activity (Stasiv et al.,
2001
).
In crustaceans, NO/cGMP signaling plays a role in neuronal development and
neuron, skeletal muscle and cardiac muscle regulation
(Aonuma et al., 2000; Aonuma
and Newland, 2001
,
2002
;
Erxleben and Hermann, 2001
;
Hermann and Erxleben, 2001
;
Johansson and Mellon, 1998
;
Mahadevan et al., 2004
;
Scholz, 1999
,
2001
; Scholz et al.,
1998
,
2001
). NOS is expressed in
neurons of the cerebral, stomatogastric, eyestalk, abdominal terminal and
cardiac ganglia (Christie et al.,
2003
; Johansson and Carlberg,
1994
; Johansson and Mellon,
1998
; Lee et al.,
2000
; Scholz et al.,
2002
; Schuppe et al.,
2001a
,b
,
2002
;
Talavera et al., 1995
;
Zou et al., 2002
). The NO/cGMP
signaling pathway is required for the dynamic assembly of the neuronal circuit
that drives rhythmic movement in crabs
(Scholz, 2001
;
Scholz et al., 2002
), alters
ion channel properties of skeletal muscle
(Erxleben and Hermann, 2001
;
Hermann and Erxleben, 2001
)
and decreases heartbeat amplitude and frequency
(Mahadevan et al., 2004
). The
biochemical properties of crustacean NOS are similar to those of mammalian
nNOS and insect NOS, as it also requires NADPH, Ca2+ and CaM for
activity (Johansson and Carlberg,
1994
; Lee et al.,
2000
; Scholz et al.,
2002
; Zou et al.,
2002
).
The NO/cGMP signaling pathway may be involved in regulating molting in
crustaceans. Molt inhibiting hormone (MIH), a neuropeptide synthesized in a
neurosecretory center (X-organ/sinus gland complex) in the eyestalk acts as a
negative regulator of ecdysteroidogenesis in the Y-organ
(Lachaise et al., 1993;
Skinner, 1985
). The signal
transduction pathway is poorly understood. Cyclic nucleotides mediate MIH
inhibition of Y-organ ecdysteroidogenesis (Spaziani et al.,
1999
,
2001
). There are species
differences in the relative importance of cAMP and cGMP, although both cyclic
nucleotides probably play a role (Lachaise
et al., 1993
; Sedlmeier and
Fenrich, 1993
; Spaziani et
al., 1999
). MIH induces an increase in cAMP and cGMP, with
subsequent activation of protein kinases in Y-organs in vitro
(Baghdassarian et al., 1996
;
Böcking and Sedlmeier,
1994
; Saïdi et al.,
1994
; Sedlmeier and Fenrich,
1993
; Von Gliscynski and
Sedlmeier, 1993
).
Given the wide expression of NOS in insect and mammalian tissues, we hypothesized that crustacean NOS is expressed in non-neuronal tissues and functions in regulating a variety of physiological processes. A cDNA encoding a full-length crustacean NOS (Gl-NOS) was cloned from land crab (Gecarcinus lateralis) Y-organ and thoracic ganglion mRNA using RT-PCR and 3' and 5' RACE PCR. The tissue expression of NOS mRNA was determined with RT-PCR. Immunohistochemistry was used to localize NOS protein in Y-organ, gill and ovary. The results show that NOS is more widely distributed than was previously supposed and suggest that NO is involved in regulating diverse functions, including MIH signaling in the Y-organ.
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Materials and methods |
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Cloning of Gl-NOS cDNA
A partial NOS cDNA was initially obtained by nested RT-PCR using degenerate
primers directed to highly conserved sequences in a wide variety of
NOS genes in the GenBank database
(http://www.ncbi.nlm.nih.gov),
including those from six insect species and three human forms (nNOS, eNOS,
iNOS) and aligned using the ClustalW program
(http://www.ebi.ac.uk/clustalw/index.html).
Two sets of degenerate primers were designed to anneal to DNA sequences
encoding F(S/N)GWYM, VF(H/F)QEM or TFGNG(E/D)PP
(Fig. 1, broken lines with
solid arrowheads): NOS F1, 5'-TT(C/T) (A/T)(G/C/A)(A/G/T/C) GG(A/G/T/C)
TGG TA(C/T) ATG-3'; NOS F2, 5'-GT(A/G/T/C) TT(C/T) (C/T)(A/T)(C/T)
CA(G/A) GA(G/A) ATG-3'; NOS R1, 5'-GG(A/G/T/C) GG(A/G/T/C)
TC(A/G/T/C) CC(G/A) TT(A/G/T/C) CC-3'; R2, 5'-G(A/G/T/C)
TC(A/G/T/C) CC(G/A) TT(A/G/T/C) CC(G/A) AA(A/G/T/C) G-3'. All the
primers were synthesized and purified by Integrated DNA Technologies, Inc.
(Des Moines, IA, USA).
|
Total RNA was isolated from thoracic ganglia and Y-organ using RNeasy Protect mini kit (Qiagen, Inc., Valencia, CA, USA). About 20 mg of tissue and 600 µl of RTL reagent were used for each spin column unit. Total RNA (100 µg) was used for isolating mRNA with an Oligotex mRNA isolation kit (Qiagen). cDNA was synthesized according to the manufacturer's protocol using the Superscript II RNase H-reverse transcriptase first-strand synthesis system (Invitrogen, Inc., Carlsbad, CA, USA). Briefly, 12 µl of a mixture containing 1 µl oligo (dT)1218 (500 µg ml1), 100 ng RNA and 1 µl of 10 mmol l1 dNTPs was heated to 65°C for 5 min and chilled on ice for 1 min. 4 µl of 5x First-Strand Buffer, 2 µl of 0.1 mol l1 dithiothreitol (DTT) and 1 µl RNaseOUT, recombinant ribonuclease inhibitor (40 units µl1) were added. The mixture was incubated at 42°C for 2 min. The reaction was initiated by the addition of 1 µl (200 units) SuperScript II at 42°C for 50 min. The reaction was inactivated by heating at 70°C for 15 min. PCRs were performed using an ABI 9600 thermal cycler (Perkin-Elmer, Inc., Wellesley, MA, USA). The first-round PCR contained 3 µl of cDNA, 3 µl of 10x Takara EX Taq buffer, 2 µl of 250 µmol l1 dNTPs, 1 µl of forward degenerate primer (NOS F1), 1 µl of reverse degenerate primer (NOS R1), 0.2 µl of Takara EX Taq DNA polymerase (5 units µl1) and 18.8 µl of PCR-grade deionized water. Initial denaturation (95°C for 5 min) was followed by 35 amplifying cycles (95°C for 30 s, 53°C for 30 s and 72°C for 1 min) and final extension at 72°C for 7 min. For the second PCR, 0.1 µl of the first PCR reaction and the nested degenerate NOS F2 and NOS R2 primers were used. Other reaction components and PCR conditions were the same as those in the first reaction.
PCR products were separated by 2% agarose gel electrophoresis and stained with ethidium bromide. The PCR products were purified from gel slices using QIAquick Gel Extraction mini kit (Qiagen), ligated into PCR2.1 vector with the TOPO TA Cloning kit (Invitrogen) and transformed into One Shot TOP 10 E. coli strain (Invitrogen). Plasmids were purified using Qiagen spin mini prep kit and sequenced using T7 and M13 reverse vector primers (Davis Sequencing, Davis, CA, USA).
A semi-nested RT-PCR strategy was used to obtain more of the ORF 3' to the initial nested RT-PCR product. The reactions used two sequence-specific forward primers (cNOS F5, 5'-CAAGTCAGAGATGTACGCCAAGAAG-3', and cNOS F6, 5'-TCTTCGGTCACACCTTCAATGCTC-3') and a degenerate reverse primer (NOS R3, 5'-RAADATRTCYTCRTGRTANC-3') to a highly conserved sequence in the reductase domain (Fig. 1, broken lines with open arrowheads). First-round PCR used cNOS F5 and NOS R3 primers and the original cDNA synthesized from thoracic ganglia and Y-organ mRNA (see above). Second-round PCR used cNOS F6 and NOS R3 primers and 0.1 µl of the first-round PCR. The PCR conditions were the same as described above, except that the extension time for the amplification cycles was 2 min instead of 1 min. PCR products were separated on 1% agarose gels, purified, cloned and sequenced as described above.
RACE (rapid amplification of cDNA ends) of mRNA was used to obtain full-length sequences. Poly(A+) RNA was isolated from total RNA using Oligotex mRNA kit (Qiagen). For the 3' sequence, the RACE System (Invitrogen) was used. Briefly, first-strand cDNA synthesis reactions contained 200 ng poly(A+) RNA and adaptor primer (5'-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3'). First-round PCR on the cDNA (20 ng) included a universal amplification primer (5'-CUACUACUACUAGGCCACGCGTCGACTAGTAC-3') and gene-specific forward primer, cNOS F1 (5'-CACTATGGCTGAGTGTGTCTACCAGAAG-3'), under the following conditions: denaturation at 96°C for 5 min, 35 amplification cycles (96°C for 30 s, 60°C for 30 s and 72°C for 2 min) and final extension at 72°C for 10 min. Nested PCR (30 µl total volume) was conducted with a gene-specific primer, cNOS F2 (5'-AGCTGAGGTCCATTGTGCAGGAGCATG-3'), and an abridged universal amplification primer (5'-GGCCACGCGTCGACTAGTAC-3') under the same conditions as the first-round PCR. PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide.
The SMARTTM RACE cDNA amplification kit (BD Biosciences, Inc., San Jose, CA, USA) was used to obtain the 5' sequence. The first-strand cDNA synthesis reaction contained 3 µl poly(A+) RNA (100 ng), 1 µl 5' CDS primer [10 mmol l1, 5'-(T)25N-1N-3'] and 1 µl SMART II A oligo (10 mmol l1, 5'-AAGCAGTGGTATCAACGCAGAGTACGCGGG-3') and was incubated at 68°C for 2 min. After cooling the reaction on ice for 2 min, 2 µl of 5x First-Strand buffer [250 mmol l1 Tris-HCl (pH 8.3), 375 mmol l1 KCl and 30 mmol l1 MgCl2], 1 µl DTT (20 mmol l1), 1 µl dNTPs (10 mmol l1) and 1 µl PowerScript Reverse Transcriptase were added. The reaction was covered with 20 µl paraffin oil and incubated at 42°C for 1.5 h in an ABI 9600 DNA thermal cycler (Perkin-Elmer). The reaction mixture was diluted 10-fold with autoclaved distilled water and was used for first-round PCR with 10x universal primer A mix (0.4 mmol l1 5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3' and 2 mmol l1 5'-CTAATACGACTCACTATAGGGC-3') and gene-specific reverse primer, cNOS R1 (5'-CGAAGTCCTCCCCATTCTCAGGAG-3'), under the following conditions: denaturation at 96°C for 5 min, 35 amplification cycles (96°C for 30 s, 66°C for 15 s and 72°C for 3 min) and final extension at 72°C for 10 min. Second-round PCR was conducted using nested gene-specific primer cNOS R2 (5'-AGCTTACTTGTGAACTTGACGGCTCTG-3') and nested universal primer A (10 mmol l1, 5'-AAGCAGTGGTATCAACGCAGAGT-3'). The PCR conditions were the same as those used for first-round PCR. PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide. Purified products were sequenced to confirm identities.
Gl-NOS expression by RT-PCR
Integument, thoracic ganglia, testis, ovary, heart, digestive gland, gill,
claw muscle, eyestalk neural ganglia and Y-organ were dissected from 35
crabs and immediately placed in RNAlater RNA stabilization reagent (Qiagen).
Tissues were stored at 20°C until RNA extractions could be
performed. Total RNA was isolated from pooled tissues using either the RNeasy
mini or midi kit according to the manufacturer's instructions (Qiagen). RNA
concentration was determined by UV absorbance at 260 nm and stored at
80°C. About 1 µg of total RNA was used for the reverse
transcription reaction. RNA was first treated with DNase I to degrade any
contaminating genomic DNA. First-strand cDNA was synthesized in a 20 µl
reaction volume containing 50 mmol l1 Tris-HCl, 75 mmol
l1 KCl, 3 mmol l1 MgCl2, 10
mmol l1 DTT, 0.5 mmol l1 of each dNTP, 40
units of RNaseOUT ribonuclease inhibitor, 1 ng oligo dT primer and 200 units
of Moloney murine leukemia virus reverse transcriptase (Invitrogen). The
reaction was incubated for 50 min at 37°C, heat-inactivated and stored at
20°C.
The quality of the cDNA was first verified by performing PCR with land crab elongation factor 2 (EF2; GenBank accession #AY552550) primers (cEF F1, 5'-TTCTATGCCTTTGGCCGTGTCTTCTC-3'; cEF R1, 5'-TGATGGTGCCCGTCTTAACCAGATAC-3'). The PCR conditions were an initial denaturation at 95°C for 2 min, 35 amplification cycles (denaturation at 94°C for 30 s, annealing at 61°C for 30 s and extension at 72°C for 30 s) and 2 min at 72°C as a final extension.
NOS PCR was then performed in a 20-µl reaction mixture as described above using 2 µl of the first-strand cDNA and an NOS gene-specific primer pair (cNOS EXF, 5'-CAACTTGAGAAGGAATAAAAGGGGAGGATG-3'; cNOS R31, 5'-CTGCTGAAGCTGCTGCCTCTGTCTTGAG-3'), each at a final concentration of 0.2 µmol l1. The PCR conditions were an initial denaturation at 95°C for 2 min, 35 amplification cycles (denaturation at 96°C for 20 s, annealing at 62°C for 20 s and extension at 72°C for 90 s) and 4 min at 72°C as a final extension. This primary PCR reaction was then used as template with a nested NOS primer pair (cNOS F1, 5'-GTACAAGCAGGAGGACGGGAG-3'; cNOS R5, 5'-AGCTTACTTGTGAACTTGACGGCTCTG-3'), each at a final concentration of 0.2 µmol l1 as described above. The primary reaction was diluted 1:10 000 in water, and 2 µl was used in the reaction. The PCR conditions were an initial denaturation at 95°C for 2 min, 35 amplification cycles (denaturation at 96°C for 20 s, annealing at 62°C for 20 s and extension at 72°C for 50 s) and 4 min at 72°C as a final extension. All PCR reactions were analyzed by separating some or all of the 20 µl reaction volume on 12% agarose gels with a 100 bp PCR Molecular Ruler DNA size ladder (Bio-Rad, Inc., Hercules, CA, USA).
Immunohistochemistry
Tissues used for immunohistochemistry were dissected and fixed for
2448 h in Bouin's fixative or Histochoice MB fixative (Amresco, Inc.,
Solon, OH, USA) containing 0.15 mol l1 NaCl. The tissue was
then dehydrated through a graded ethanol series, cleared and embedded in
paraffin. Sections (9 µm) were placed on clean glass slides and
allowed to dry at 40°C for 24 h. Sections were then deparaffinized in
xylene and rehydrated through an ethanol series into a phosphate-buffered
saline (137 mmol l1 NaCl, 2.7 mmol l1 KCl,
4.3 mmol l1 Na2HPO4, 1.4 mmol
l1 KH2PO4; pH 7) with 15 mmol
l1 glycine (PBSG). Sections were incubated at room
temperature in 1% bovine serum albumin (BSA) and normal goat serum in PBSG for
45 min and then with primary antibody (rabbit anti-NOS antibody; Affinity
BioReagents, Inc., Golden, CO, USA; 1:10002000 dilution in PBSG) at
4°C overnight. The NOS antibody was a universal antibody (generated
against a portion of murine nNOS/iNOS) that crossreacts with crustacean NOS
(Christie et al., 2003
; Scholz
et al., 1998
,
2002
). After four rinses (5
min each) in PBSG, sections were incubated with either a goat anti-rabbit or
mouse anti-rabbit biotin-conjugated antibody (Pierce, Inc., Rockford, IL, USA;
diluted 1:10001500) at room temperature for 2 h, rinsed four times in
PBSG and incubated with an avidin/alkaline phosphatase reagent (Vector
Laboratories, Inc., Burlingame, CA, USA). Sections were rinsed four times in
PBSG and incubated with BCIP/NBT (Gibco/Invitrogen) or Vector Red (Vector
Laboratories, Inc.) substrate until sufficient color development was attained.
Control incubations were also done on adjacent tissue sections and included
either omitting the primary antibody or substituting a non-immune rabbit serum
for the primary antibody.
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Results |
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The Gl-NOS cDNA encoded a protein containing 1199 amino acids with an estimated mass of 135 624 Da (Fig. 1). The Gl-NOS amino sequence was aligned with NOS sequences from five insects, a mollusk and three human types (Fig. 2). The N-terminal region varied among different NOS genes, but the oxygenase domain in Gl-NOS was 70% identical to the Drosophila NOS, 68% identical to Aplysia NOS and 66% identical to human nNOS. In the oxygenase domain, the heme-binding motif was well conserved, including the cysteine residue that acts as an axial ligand. The motifs for binding tetrahydrobiopterin (H4) cofactor and CaM were also well-conserved. The reductase domain contained all conserved binding motifs for FMN, FAD and NADPH. Interestingly, the Gl-NOS had single amino acid substitutions in all three motifs for binding FAD that differed from other NOS cDNAs (Fig. 2). The domain organization of Drosophila NOS and Gl-NOS is compared in Fig. 3 to show the high amount of similarity between the two sequences.
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The phylogenetic relationships of various NOS sequences were determined using sequence alignments of the oxygenase domains (Fig. 4; residues #54455 in Gl-NOS). Insect NOS sequences clustered according to major taxonomic groups: Lepidoptera (M. sexta and B. mori), Diptera (A. stephensi and D. melanogaster) and Hemiptera (R. prolixus). Molluskan NOS (A. californica) and vertebrate NOS formed distinct groups. Within the vertebrates, the inducible NOS (iNOS) and noninducible NOS (nNOS, eNOS) were divided. Since few NOS genes have been obtained from lower invertebrates (e.g. nematode), Gl-NOS could not be grouped with any other NOS, although overall sequence comparison showed that Gl-NOS was most closely related to Drosophila NOS.
|
Gl-NOS was expressed in both neuronal and non-neuronal tissues. Initial RT-PCR using cNOS EXF and R31 primers generated a 2110-bp product amplified from RNA isolated from testis, gill, ovary, eyestalk neural ganglia and Y-organ (Fig. 5A, lanes c, fi). RT-PCR with an elongation factor 2 (EF2) primer pair served as a positive control (Fig. 5C). Nested PCR on the first-round PCR product using cNOS F1 and R5 primers generated a product of the expected size (795 bp), which confirmed the identity of the initial product as the NOS sequence (Fig. 5B). Gl-NOS mRNA varied in Y-organ, thoracic ganglion and gill; in some preparations, no PCR product was detected in these tissues. Although no PCR product was obtained from the thoracic ganglion mRNA preparation in Fig. 5 (lane b), other thoracic ganglion mRNA preparations yielded an NOS PCR product (data not shown). By contrast, the NOS mRNA was present at consistently high levels in all RNA preparations from eyestalk ganglia, ovary and testis.
|
Immunohistochemistry
Immunohistochemistry was used to confirm that the NOS protein is present in
Y-organs and other non-neuronal tissues. A pair of Y-organs is located
adjacent to the branchial chamber on one side and a hemolymph sinus and
connective tissue on the other. A thin cuticle separates the Y-organ from the
branchial chamber. A universal anti-NOS antibody reacted with the nuclei and
cytoplasm of all Y-organ cells (Figs
6A,
7A). Some nuclei in the
connective tissue (Fig. 6A,C,D)
were stained, as well as the tendinous cells, which anchor connective tissue
to the cuticle lining the branchial chamber
(Fig. 6D).
|
|
In gill, the NOS protein was localized in the epithelium (Fig. 7B). Staining was more intense in the epithelium lining the central axis between the gill lamellae (Fig. 7B, arrows) and in the pillar cells (Fig. 7B, arrowheads). In the ovary, the NOS protein was confined to the perinuclear cytoplasm of oocytes (Fig. 7D). Control sections without primary antibody showed a low amount of staining in oocytes, indicating some non-specific binding of the biotinylated secondary antibody and/or the avidin/alkaline phosphatase reagent (Fig. 7C).
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Discussion |
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NOS genes are regulated at the transcriptional, translational and
post-translational levels (Hall et al.,
1994; Kone, 2001
;
Wang et al., 1999
). Numerous
isoforms of human nNOS are produced by alternative promoters and alternative
splicing and differ in tissue expression patterns
(Kone, 2001
;
Wang et al., 1999
). Multiple
isoforms of Drosophila NOS (dNOS) result from alternative mRNA
splicing; the truncated isoforms lack NOS activity and those that have
sequences required for dimerization in the vicinity of the CaM-binding domain
act as dominant negative regulators
(Regulski and Tully, 1995
;
Stasiv et al., 2001
). The dNOS
isoforms are differentially expressed during Drosophila development
(Stasiv et al., 2001
). In the
mosquito Anopheles stephensi, 1822 NOS alternative transcripts
are expressed (Luckhart and Li,
2001
). The number of alternative transcripts expressed in land
crab has not been determined. Our preliminary results indicate that a
truncated isoform was expressed in the Y-organ. In this cDNA, alternative
splicing introduced a stop codon in the reductase domain, resulting in a
polypeptide of 720 amino acids in length. This isoform may act as a dominant
negative regulator, as it retains the dimerization sequence but lacks a
complete reductase domain. It was not characterized further, because it was
expressed at low levels in the Y-organ and thoracic ganglion.
In insects, the NO/cGMP signaling pathway is involved in such diverse
functions as phototransduction, olfaction, neuronal development, ecdysis, food
search behavior and epithelial fluid transport
(Davies, 2000;
Morton and Hudson, 2002
).
Until recently, NOS had only been reported in the nervous system of
crustaceans, in which it is involved in regulating neuronal activity
(Aonuma et al., 2000
;
Aonuma and Newland, 2002
;
Johansson and Carlberg, 1994
;
Johansson and Mellon, 1998
;
Lee et al., 2000
; Scholz et
al., 1998
,
2001
,
2002
; Schuppe et al.,
2001a
,b
,
2002
;
Talavera et al., 1995
;
Zou et al., 2002
). In lobster,
NO produced by the cardiac muscle reduces bursting activity of the cardiac
gangion, which decreases heartbeat amplitude and frequency
(Mahadevan et al., 2004
). In
addition to nervous tissue, we have shown that the land crab Y-organ, gill,
testis and ovary express NOS by RT-PCR
(Fig. 5) and
immunohistochemistry (Figs 6,
7). However, we did not detect
NOS mRNA in land crab heart, even after a second round of PCR with nested
primers (Fig. 5B). Although
this is the first report of NOS in these tissues from a crustacean,
non-neuronal NOS has been reported in insect Malpighian tubules, imaginal
discs and salivary glands (Davies et al.,
1997
; Kuzin et al.,
1996
,
2000
;
Ribeiro and Nussenzveig, 1993
;
Stasiv et al., 2001
).
The presence of NOS in crustacean non-neuronal tissues suggests that NO
signaling is involved in physiological processes in addition to
neuromodulation. NOS was not detected in skeletal muscle
(Fig. 5), although NO and cGMP
increase inward Ca2+ current and both early and delayed outward
K+ currents in skeletal muscle of a marine isopod
(Erxleben and Hermann, 2001;
Hermann and Erxleben, 2001
).
The functions of NOS in Y-organ, ovary, testis and gill are not known. In
gonadal tissue, NOS may regulate gametogenesis and/or steroidogenesis. In
blowfly, for example, steroid synthesis is inhibited by NO and cGMP. However,
crustacean ovary accumulates ecdysteroid, but there is little evidence that it
is a significant site for ecdysteroid synthesis
(Gunamalai et al., 2003
;
Spaziani et al., 1997
;
Subramoniam, 2000
;
Suzuki et al., 1996
;
Warrier et al., 2001
). The
localization of NOS in connective tissue and epithelia in Y-organ and gill
suggests that it functions in an immune response to pathogens. In insects,
hemocytes express an NOS that is activated upon bacterial infection
(Weiske and Wiesner, 1999
).
Plasmodium infection in mosquitoes leads to a rapid induction of NOS
activity (Luckhart et al.,
1998
). The pillar cells, which extend across each lamella and
anchor in the cuticle, restrict distension caused by hemolymph pressure
(Copeland, 1968
;
Taylor and Taylor, 1992
). The
localization of NOS in tendinous and pillar cells suggests that NOS is
involved in the regulation of blood pressure, as it is in vertebrates.
The presence of NOS in the Y-organ suggests that NO regulates
ecdysteroidogenesis via the activation of a soluble NO-sensitive
GC-I. A hypothetical pathway is presented in
Fig. 8, which is consistent
with the available data. Increased cAMP, cGMP and Ca2+ levels
inhibit ecdysteroid synthesis in crustacean Y-organ (Spaziani et al.,
1999,
2001
). We propose that MIH
binds to a G protein-coupled receptor, leading to activation of NOS by the
combined effects of dephosphorylation by calcineurin and binding of CaM. The
regulation of NOS by phosphorylation/dephosphorylation is complex. Several
protein kinases phosphorylate NOS in different regions
(Bredt et al., 1992
), which
may have different effects on enzyme activity
(Kone, 2001
;
Nakane et al., 1991
). For
example, CaM kinase II phosphorylates nNOS at Ser847, which inhibits enzyme
activity by reducing its binding affinity for CaM
(Hayashi et al., 1999
;
Komeima et al., 2000
). The NO
activates a GC-I, resulting in an increase in cGMP. This is similar to the
signaling mechanism that stimulates fluid secretion in Drosophila
Malpighian tubules by the decapeptide cardioacceleratory peptide 2b
(Davies, 2000
; Davies et al.,
1995
,
1997
;
Dow et al., 1994
;
Kean et al., 2002
;
MacPherson et al., 2001
;
Rosay et al., 1997
) and
inhibits steroidogenesis in blowfly ovary
(Maniere et al., 2003
). We
have recently cloned a cDNA encoding a GC-I that is expressed in land crab
Y-organ (H.-W.K. and D.L.M., data not shown). Studies are now in progress to
determine the role of NOS and GC-I in the MIH signaling pathway.
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Acknowledgments |
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References |
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