1 Department of Internal Medicine II, Technical University of Munich, 81675 Munich, and 2 Department of Anatomy I, University of Erlangen, 91054 Erlangen, Germany
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ABSTRACT |
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5' mRNA variants of
neuronal nitric oxide synthase (nNOS) are generated either by
alternative promoter usage resulting in different mRNAs that encode for
the same protein (nNOS) or alternative splicing encoding
NH2-terminally truncated proteins (nNOS
/
) that lack
the PDZ/GLGF domain for protein-protein interaction of nNOS
. We
studied the expression of 5' nNOS mRNA forms and nNOS-interacting
proteins (postsynaptic density protein-95; PSD-95) in the rat
gastrointestinal tract and analyzed the more distinct localization of
nNOS protein variants in the duodenum by immunohistochemistry with
COOH- and NH2-terminal nNOS antibodies. 5' nNOS mRNA
variants showed a site-specific expression along the gastrointestinal
tract with presence of all forms (nNOS
-a, -b, -c; nNOS
) in the
muscle layer of esophagus, stomach, duodenum, longitudinal muscle layer of jejunum/ileum, proximal colon, and rectum. In contrast, a lack of
nNOS
-a and nNOS
mRNA was observed in pylorus, circular muscle layer of jejunum/ileum, and cecum. Expression of nNOS
and nNOS
cDNAs revealed proteins of ~155 kDa and 135/125 kDa, respectively. Immunohistochemistry showed a differential distribution of COOH- and
NH2-terminal nNOS immunoreactivity in distinct layers of
rat duodenum, suggesting a cell-specific expression and distinct
compartmentalization of nNOS proteins. Observed distribution of 5' nNOS
mRNA variants and proteins argue for a complex control of nNOS
expression by usage of separate promoters, cell- and site-specific
splicing mechanisms, and translational initiation. These mechanisms
could be involved in gastrointestinal motor diseases and may explain the phenotype of nNOS
knockout mice with gastric stasis and pyloric stenosis, due to a total loss of nNOS in the pyloric sphincter region.
alternative promoters; alternative splicing; transcriptional and posttranscriptional control; postsynaptic density; pyloric sphincter.
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INTRODUCTION |
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NITRIC OXIDE (NO) is an important nonadrenergic, noncholinergic mediator within the enteric nervous system (4). It is generated by enzymatic NADPH-dependent electron transfer during the conversion of L-citrulline to L-arginine by NO synthase (NOS). In addition to its action as neurotransmitter and neuromodulator (1, 4, 14, 25, 27), NO might also act as a messenger within smooth muscle or interstitial cells of Cajal (19, 24). Neuronal NOS (nNOS) (2) is the predominant isoform of NOS in the enteric nervous system (5, 12, 20, 38) besides the other constitutive and calcium-dependent endothelial NOS and the calcium-independent inducible NOS (iNOS).
It has been demonstrated recently in various tissues, such as the central nervous system, skeletal muscle, testis, kidney, spleen, adrenal gland, heart, embryonic tissue, and in the rat and human gastrointestinal tract that different 5' mRNA variants of nNOS are expressed (3, 9, 12, 15, 21, 26, 32, 34, 37). These variants differ in the first untranslated exon and in exon deletions or insertions within the translated region and thus in the protein structure.
Differences in first exons of the 5' untranslated region (UTR) are due
to alternative promoter usage (26, 37), whereas exon
deletions/insertions are generated by alternative splicing (9,
12, 26, 34). In rat small intestine, three different 5' mRNA
splice variants of nNOS have been described (12). Two (nNOS-a, nNOS
-b) differ in their first untranslated exon (exon 1a
and exon 1b) but encode for the same protein nNOS
. A third variant
(exon 1a/exon 3) lacks exon 2 and encodes for nNOS
.
Lee et al. (15) found another first exon called exon 1c in different rat tissues and demonstrated a tissue and development-specific expression of nNOS exon 1a, 1b, and 1c. In addition, one kidney-specific first exon (K1) and two alternatively spliced kidney-specific second exons (K2a and K2b) have been described (21).
nNOS mutant mice, generated by targeted disruption of the nNOS gene by
homologous recombination, led to a gastrointestinal phenotype closely
resembling, but not identical to, hypertrophic pyloric stenosis with
delayed gastric emptying of solids and fluids (10, 18).
Interestingly, no other gastrointestinal abnormalities could be
observed in these animals. However, this genetic model in which exon 2 and, subsequently, full-length nNOS, was disrupted maintained nNOS
expression due to alternative splicing, resulting in the
NH2-terminally truncated proteins nNOS
(135 kDa) with ~80% catalytic activity of nNOS
and nNOS
(125 kDa) lacking
functional nNOS enzymatic activity (3). Full-length
nNOS
but not the truncated forms (nNOS
and nNOS
) contain an
NH2-terminal PDZ/GLGF motif encoded by exon 2, enabling
protein-protein interactions with various proteins such as
-syntrophin or proteins of the postsynaptic density (PSD) like
PSD-95 or PSD-93 (3, 28). PSD-95 and PSD-93 can anchor
nNOS
to N-methyl-D-aspartate (NMDA) receptor
subunits and subsequently to the cell membrane (6). Because differences in the NH2-terminal protein structure
affect functionally important parts and determines the cellular
localization of nNOS, different functional roles can be attributed to
membranebound nNOS
, cytosolic nNOS
, and the inactive form
nNOS
. Therefore, it is of physiological and functional interest to
determine the site and organ-specific expression of nNOS variants and
the presence of putative interaction partners.
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MATERIALS AND METHODS |
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Tissue preparation.
Adult male Wistar rats with an individual body weight of ~200 g were
killed by cervical dislocation. The entire gastrointestinal tract was
removed and divided into the following areas: upper and lower
esophagus; gastric fundus/corpus and antrum; pylorus; small intestine;
proximal, medial, and distal colon; and rectum. Each area of gut was
cleaned in ice-cold PBS, attached mesenterial fat was removed, and the
muscle layers including the attached myenteric plexus were separated
from the mucosa by scraping. In small intestine, the longitudinal
muscle layer with attached myentric plexus (LMMP) was additionally
peeled away from the circular muscle layer (CM). Tissues were cut into
small pieces, immediately frozen in liquid nitrogen, and stored at
80°C until use.
RNA isolation. Total RNA was extracted from muscle layer preparations of liquid nitrogen-frozen rat gastrointestinal tissues. Specimens were homogenized with a Polytron homogenizer (Kinematica), and RNA was isolated using the guanidine isothiocynate/phenol/chloroform extraction method (Peq Lab, Schwalbach, Germany), followed by DNase I treatment (1 U/µg RNA, 15 min, 25°C; GIBCO BRL, Eggenstein, Germany).
RT-PCR and Southern blot analysis.
RT-PCR and Southern blot analysis were performed as described
previously (12). Five micrograms of total RNA were reverse transcribed at 42°C for 1 h into complementary DNA using 200 units SuperScript II RNase H reverse transcriptase (GIBCO
BRL) in the presence of 100-ng random hexamer primers (Roche, Mannheim,
Germany) or 25 pmol of the gene-specific antisense primer nNOS
P1/ex5-AS (for all primers see Table 1) complementary to exon 5 of rat nNOS (2). As negative
controls, isolated RNA amplified without reverse transcriptase or
random hexamer primers were used. Site-specific expression of nNOS mRNA splice variants and PSD-95 mRNA were investigated by PCR using isoform-specific sense primers for rat nNOS exon 1a, exon 1b, and exon
1c combined with a common antisense strand primer for nNOS exon 5 (P2/ex5-AS) or sense and antisense gene-specific primers for PSD-95.
One microliter of the RT-reaction mix was subjected to 35 or 30 PCR
cycles in a Biometra UNO I thermal cycler using 2.5 units of
Taq polymerase (Sigma, Deisenhofen, Germany) with the
following conditions. After a "hot start" with an initial denaturation at 95°C for 3 min, each PCR cycle involved denaturation at 94°C for 30 s, annealing at 58°C for 45 s, and
extension at 72°C for 90 s. The last cycle was followed by an
extension step at 72°C for 7 min. If the first round of PCR yielded
no visible PCR product in the ethidium bromide-stained gel, a second
round of nested PCR was performed for 20 cycles with annealing at
58°C. PCR products were size fractionated by 1.5% agarose gel
electrophoresis, visualized by ethidium bromide staining, denaturated,
and blotted onto Hybond N+ nylon membranes
(Amersham-Pharmacia, Freiburg, Germany) by capillary transfer. Blots
were hybridized overnight at 46°C with [
-33P] ATP 5'
end-labeled internal probes specific for nNOS exon 3 or PSD-95 (see
Table 1 for all hybridization probes). Membranes were washed twice with
2× standard saline citrate (SSC) containing 0.1% SDS for 15 min at
room temperature and once with 0.5× SSC containing 0.1% SDS for 10 min at 44°C. Labeled products were detected by autoradiography.
Finally, 20-µl aliquots of the PCR products were size fractionated on
a 1.5% agarose gel, excised, and purified, using a gel extraction kit
(Qiagen, Hilden, Germany) and cloned into the TOPO PCRII plasmid
(Invitrogen, Groningen, Netherlands) as described by the manufacturer.
Nucleotide sequences were deduced by cycle sequencing of the purified
plasmids (Qiagen Mini Prep Kit) with T7 sequencing primer (GATC,
Konstanz, Germany). Sequences were analyzed by BLASTn
homology search.
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Heterologous eucaryotic expression of nNOS and nNOS
cDNA in
COS-7 cells.
Eucaryotic expression vectors containing either full-length
nNOS
or nNOS
cDNAs were constructed as follows. For nNOS
, a 5,057-bp fragment of rat nNOS cDNA, kindly provided by Dr. S. Snyder, Johns Hopkins Medical School, Baltimore, MD
(2) that contained 348 bp of the 5' UTR was subcloned into
the mammalian expression vector pcDNA3 (Invitrogen) resulting in
nNOS
-pcDNA3. For nNOS
, the 1,558-bp
Xho I/Nar I 5' end fragment of nNOS
-pcDNA3 was
replaced with a 792 bp Xho I/Nar I fragment
representing the nNOS exon 1a/exon 3 sequence. This fragment was
constructed by RT-PCR, using random hexamer-primed total RNA from LMMP
of rat ileum as template. Thirty PCR cycles with annealing at 56°C
for 45 s, extension at 72°C for 90 s, and denaturation at
94°C for 30 s were performed using a proofreading polymerase
(Pwo, Roche) and the sense and antisense strand primers nNOS exon 1a
and nNOS ex6-AS, respectively (see Table 1). The PCR product
was subsequently cloned into PCRII plasmid, sequenced with T7 and M13
reverse primers (GATC) and subcloned in to the Xho
I/Nar I site of nNOS
-pcDNA3, resulting in nNOS
-pcDNA3.
Protein expression and Western blot analysis.
Liquid nitrogen frozen specimens from the mucosa and the longitudinal
and circular muscle layer (LM/CM) with attached nerve plexus of rat
duodenum were homogenized, and proteins were extracted as described
before (12, 26). COS-7 cells were lysed 48 h after
transfection using 1× lysis buffer (50 mM Tris, pH 7.6, 0.1 mM EDTA,
0.1 mM EGTA, 3 µM leupeptin, 1 µM pepstatin, 1 mM phenylmethylsulfonyl fluoride, 12 mM -mercaptoethanol, 1% Triton X-100) and centrifuged at 21,000 g for 20 min. The total
protein concentration was measured with protein assay kit II (Bio-Rad, Munich, Germany). Proteins were separated by 7.5% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad). Blots
were probed with anti-rat nNOS antibodies [nNOS COOH-terminal monoclonal antibody (nNOS-C), amino acids 1095-1289 of nNOS
, Transduction Laboratories, Heidelberg, Germany; nNOS
NH2-terminal polyclonal antibody (nNOS-N), amino acids
38-57 of nNOS
NOS1 (K-20), Santa Cruz Biotechnologies,
Heidelberg, Germany] as described previously (12, 26).
Signal detection of the immunoreactive bands was facilitated by
enhanced chemiluminescence (Amersham-Pharmacia).
Immunohistochemistry.
Detection of nNOS immunoreactivity in rat duodenum was performed as
described previously (11, 23). In brief, rats were perfusion fixed with Zamboni's solution and segments of the duodenum were excised. After cryoprotection in 20% phosphate-buffered sucrose, 12-µm-thick cryostat sections were mounted on
poly-L-lysine-coated slides and air dried (1 h). Following
a 5-min rinse in Tris-buffered saline (TBS), sections were incubated in
TBS containing 1% BSA, 5% normal goat serum, and 0.5% Triton X-100.
All antibodies were diluted in TBS containing 1% BSA and 0.5% Triton
X-100. Immunological detection of nNOS was performed by incubating
sections overnight with the COOH-terminal antibody nNOS-C and the
NH2-terminal antibody nNOS-N (directed against an exon 2 encoded domain of nNOS) in a 1:100 dilution. Binding of the antibodies
was visualized with goat anti-mouse IgG (nNOS-C) tagged with Alexa 488 (MoBiTec; Molecular Probes, Göttingen, Germany) or goat
anti-rabbit IgG (nNOS-N) tagged with indocarbocyanin (Cy3; Dianova,
Hamburg, Germany) diluted 1:800 and 1:200, respectively. Colocalization
of COOH- and NH2-terminal nNOS immunoreactivity was
investigated by a sequential double immunostaining protocol as
described previously (11, 23). Sections were coincubated
sequentially with antibody nNOS-C and nNOS-N. Binding of the antibodies
was facilitated using goat anti-mouse IgG antibody tagged with Alexa
488 and goat anti-rabbit IgG antibody labeled with Cy3 in a dilution of
1:800 and 1:200, respectively. Controls included trials with omission
of the primary antibodies, replacing it by buffer or normal rabbit
serum (23). Preabsorption of the primary antibodies was
done with the respective antigens for 3 h at room temperature. For
nNOS-N antibody, a blocking peptide (amino acids 38-57 of human
nNOS) from Santa Cruz Biotechnologies [NOS1 (K20) P] was used with
a concentration of 10 µg/ml. For nNOS-C antibody, expressed and
purified (12) full-length nNOS
proteins were used in a
concentration of 5 µg/ml. Sections were analyzed by confocal laser
scanning microscopy (Bio-Rad MRC 1000 attached to a Nikon Diaphot 300).
Fluorochromes were excited with 488 and 568 nm lines, respectively, by
a Krypton-Argon laser. Single optical sections were taken with a 20×
objective lens (0.75 numerical aperture) and various zoom factors. When
controls and "full" incubations were compared, special care was
taken to keep the pinhole and gain of the photomultiplier constant. Two
channel scans were coded green and red, and merged images were
documented using the software package Corel PhotoPaint.
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RESULTS |
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Site-specific expression of 5' splice variants of rat nNOS in the
gastrointestinal tract.
RT-PCR experiments with equal amounts of total RNA and Southern blot
hybridization showed a distinct and site-specific expression of 5' nNOS
mRNA variants along the rat gastrointestinal tract, with expression of
all four different forms (nNOS-a, nNOS
-b, nNOS
-c, nNOS
; see
Fig. 1 for exon structure of 5' nNOS mRNA variants) in the upper and lower esophagus, gastric
fundus/corpus/antrum, duodenum, LMMP of small intestine, proximal
colon, and rectum (Fig. 2, Table
2).
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Expression of PSD-95, a nNOS PDZ/GLGF interacting protein, in the
gastrointestinal tract.
We investigated the expression of the nNOS interacting PSD-95 along
the rat gastrointestinal tract. By using RT-PCR and Southern blot
hybridization, we could demonstrate the presence of mRNA for PSD-95 as
a possible target for a membrane association of nNOS
at all
investigated localizations (Fig. 3).
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Heterologous eucaryotic expression of nNOS and nNOS
cDNA.
Expression of nNOS
and nNOS
cDNA in COS-7 cells revealed
immunoreactive bands at 155 kDa for nNOS
and 135 and 125 kDa for nNOS
cDNA with a specific COOH-terminal monoclonal antibody
(nNOS-C). A specific NH2-terminal antibody (nNOS-N)
directed against an exon 2 encoded domain of nNOS revealed just a
single band at 155 kDa for nNOS
cDNA, whereas no bands were detected
for nNOS
cDNA lacking exon 2 (Fig. 4).
Thus the two antibodies distinguish between full-length nNOS
(155 kDa; reactive with nNOS-C and nNOS-N) and the NH2-terminal
truncated variants nNOS
(135 kDa) and nNOS
(125 kDa) (both nNOS-C
positive and both nNOS-N negative). nNOS
and nNOS
proteins are
generated by translation of the same cDNA (nNOS
-cDNA) using two
different start codons in exon 1a (CUG) and exon 5 (AUG) (see Fig. 1).
Atypical translation initiation codons have been described in various
genes, including mouse nNOS
mRNA, where a CUG start codon within
exon 1a similar to rat nNOS
is used (3). This start
codon and the surrounding sequence is highly conserved between mouse
and rat with a homology of 100% between nt 395 and 431 of mouse
(European Molecular Biology Laboratories accession number U50718) and
nt 406 to 442 of rat nNOS exon 1a (European Molecular Biology
Laboratories accession no. AF008912).
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Localization of COOH- and NH2-terminal nNOS
immunoreactivity in the rat duodenum.
To investigate a possible differential distribution of the different
NH2-terminal nNOS proteins (nNOS/
/
) in the rat
gastrointestinal tract, we used confocal laser scanning microscopy and
double staining with nNOS antibodies directed against the COOH-terminal
end (nNOS-C detecting nNOS
/
/
; see Fig. 4) and the
NH2-terminal end (nNOS-N detecting only nNOS
).
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Western blot analysis of rat duodenum.
NH2-terminal nNOS immunoreactivity of rat duodenum, which
may represent novel COOH-terminal extended or deleted nNOS variants, was further characterized by Western blot analysis of tissue
homogenates from the mucosa and the LM/CM of rat duodenum. The assay
using the NH2-terminal nNOS antibody nNOS-N revealed a
single band with a molecular weight of ~85 kDa in the mucosa and
three bands with molecular weights of ~155, ~85, and ~30 kDa in
LM/CM (Fig. 6), suggesting the possible
presence of nNOS protein variants with differing COOH-terminal ends.
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DISCUSSION |
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We could demonstrate a distinct and site-specific expression of
four different 5' nNOS splice variants along the rat gastrointestinal tract. Three variants differ in their untranslated first exon (exon 1a,
exon 1b, and exon 1c) resulting in nNOS-a, nNOS
-b, and nNOS
-c
mRNA (Fig. 1). We have previously described exon 1a and exon 1b
expression in the LMMP of rat small intestine (12), whereas the third variant (exon 1c) was found by Lee et al.
(15) in rat kidney, skeletal muscle, and embryonic tissue
but not in the intestine, using RNase protection assays. However, we
identified exon 1c in all investigated nerve-muscle layer preparations
of the rat gastrointestinal tract, indicating that nNOS
-c mRNA
expression was below the detection limit of the RNase protection assay
when using RNA isolated from whole intestinal tissue preparations
including the mucosa (15). Expression of nNOS
-a has
been described in various rat tissues, such as brain, kidney, skeletal
muscle, intestine, embryonic tissue, adrenal gland, and heart
(12, 15, 21). Here we demonstrate a differential
distribution of nNOS
-a mRNA in the rat gastrointestinal tract with
expression in the proximal and distal esophagus, gastric
fundus/corpus/antrum, duodenum, LMMP of small intestine, proximal
colon, and rectum, and an altered or lacking expression in the CM of
jejunum/ileum, the pyloric sphincter, and cecum, whereas in the middle
and distal colon, expression patterns varied. In contrast, nNOS
-b
and nNOS
-c mRNAs were present at all investigated localizations.
Untranslated first exons 1a, 1b, and 1c are spliced to a common second
exon containing the AUG starter methionine for initiation of
translation (Fig. 1). Because translation of these nNOS mRNA variants
results in identical full-length nNOS nNOS proteins, the question
about their physiological significance arises. There are several
reasons for alternative first exon utilization. Described variants are
generated most likely by usage of separate promoters as demonstrated
for the human nNOS gene (26, 37). Thus nNOS gene
expression can be regulated by activation or suppression of alternative
promoters in a cell- or site-specific way, as shown in the present
study, for the rat gastrointestinal tract by the characterization of a
regional differential distribution of alternative first exon variants.
Such a differential transcriptional control of separate promoters has
been shown for the human nNOS gene for the transcription factor Oct-2
(7) and a tissue and developmental-specific expression of
rat nNOS variants has been described by Lee et al. (15)
and Oberbäumler et al. (21). In the gastrointestinal tract, the abundance of nNOS mRNA and protein expression decreases from
the proximal to the distal colon (30), during development in the submucous plexus of the small intestine (39),
during aging in the colon (31), and in animal models of
diabetic gastropathy (29, 36). In addition, the expression
level of nNOS mRNA in the CM is significantly lower compared with LMMP
(12) and nNOS mRNA expression is regulated by protein
kinase C-dependent pathways (20). These findings
demonstrate a tightly regulated nNOS gene expression and are in
agreement with our observations of a regional distinct distribution of
nNOS variants in the rat gastrointestinal tract.
In humans, more than nine distinct first exons of nNOS have been described (26, 32, 34, 37), whereas in the rat, only four alternative exon 1 variants are known (12, 15, 21). The structure of the 5' mRNA end of rat nNOS has been extensively studied by several groups in different tissues, like brain, kidney, heart, intestine, and embryo (15), cerebellum, kidney, and skeletal muscle (21), and small intestine (12), using different approaches including 5'RACE-PCR. However, due to the more expansive number of 5' mRNA variants of nNOS in man, it remains possible that additional forms are present in the rat. In the human gastrointestinal tract, we identified three alternative first exons of nNOS called exon 15'1, 15'2 and 15'3 [corresponding to exon 1g, 1f, and 1c of a recent nNOS nomenclature (34), respectively] by 5'RACE-PCR as the predominant forms (26). This is in accordance with the presence of three alternative first exons in the rat gastrointestinal tract called exon 1a, 1b, and 1c. The sequence of rat exon 1a is homologous to human exon 15'2 (exon 1f), rat exon 1b matches human exon 15'3 (exon 1c), and rat exon 1c shows sequence similarities to human exon 1b, whereas no homologue for human exon 15'1 (exon 1g) has been found in rats. Therefore additional 5' variants of rat nNOS mRNA may be present, and further studies in each of the different functional regions of the rat gastrointestinal tract using 5' RACE-PCR have to be done to clarify this issue.
In addition to the variability in the 5' UTR, posttranscriptional
control of nNOS gene expression by cis acting elements and trans-acting splicing factors can generate
NH2-terminally truncated nNOS proteins (3, 12, 26,
34). Cassette exon deletion by splicing of exon 1a to
exon 3 results in the formation of nNOS mRNA, with a loss of the
genuine translational initiation site located at exon 2 (Fig. 1). As an
alternative, a noncanonical initiation region within exon 1a (CUG) 20 bp upstream of the exon 1a/exon 3 splice junction (Fig. 1) that is
homologous to the mouse nNOS
CUG translation start site
(3) could be used, resulting in an
NH2-terminally truncated 135-kDa nNOS
protein (lacking amino acids 1-236 of full-length nNOS
), similar to nNOS
of
nNOS
knockout mice (3). By heterologous eucaryotic
expression of cloned nNOS
cDNA, isolated from rat small intestine,
we could demonstrate that a 135-kDa nNOS immunoreactive protein can be detected with a COOH-terminal, but not with an
NH2-terminal, nNOS antibody, directed against an exon 2 encoded domain. In addition, a second immunoreactive band at 125 kDa
was obtained after incubation with the COOH-terminal antibody but not
with the NH2-terminal antibody. This protein has an
identical molecular weight with nNOS
of nNOS
knockout mice
(3) and is most likely generated by an internal
consensus translational initiation codon (AUG) within exon 5 of rat
nNOS (Fig. 1). In contrast to nNOS
with a catalytic activity of
~80% of nNOS
, recombinant nNOS
of nNOS
knockout mice has
been shown to lack functional nNOS catalytic activity (3).
Thus nNOS
may function as a dominant-negative nNOS variant that
could regulate the catalytic activity of nNOS
and nNOS
by
interisoformal dimerization (22, 35). Our results demonstrate that both the nNOS
and nNOS
protein can be generated by translation of nNOS
mRNA, and therefore posttranscriptional and
translational mechanisms can regulate the expression of fully active
soluble nNOS
or the potential inhibitor nNOS
from the same mRNA.
nNOS mRNA forms are expressed in a site-specific way in the rat
gastrointestinal tract, because we could demonstrate that the CM of
small intestine and the pyloric sphincter region lack nNOS mRNA.
Just two of seven pyloric preparations showed low levels of nNOS
,
which are most likely due to a contamination with tissue from the
duodenum or antrum. In the other five preparations, nNOS
was also
undetectable after a second round of nested PCR.
Thus nNOS seems to be the predominant mRNA form in enteric nerves of
the CM and specialized sphincter regions. This could be due to
different functional roles of nNOS
, nNOS
, and nNOS
, which may
result from different subcellular localizations (3, 12).
Full-length nNOS
contains a NH2-terminal
PDZ/GLGF-domain, a motif of ~100 amino acids, which can mediate an
association to other PDZ-containing proteins (3, 28), such
as PSD-95 or PSD-93. PDZ-PDZ interactions enable the targeting of
nNOS
to the PSD (3) and therefore determine its
subcellular localization and function. As an example, PSD-95 can anchor
nNOS
to the 2B subunit of the NMDA receptor (6) or
K+ channel subtypes at synaptic sites (13).
We could demonstrate that mRNA of PSD-95 as well as nNOS is present
at all investigated regions, enabling a subcellular targeting of
nNOS
to the PSD in the rat gastrointestinal tract.
NH2-terminally truncated proteins nNOS
and nNOS
lack
the PDZ/GLGF motif for protein-protein interaction and therefore a
possible membrane association (3). Thus
Ca2+-dependent enzymatic activity of nNOS
can be
regulated by activation or inactivation of receptors (e.g., the NMDA
receptor) that increase or decrease intracellular Ca2+
concentrations (6). In turn, NO generated by nNOS
can
also regulate the function of these receptors (17), and
therefore NO may be able to determine the enzymatic activity of NOS.
These regulatory mechanisms located at the postsynaptic density
could play an important role in relaxation of the pyloric sphincter and
circular smooth muscles. They express nNOS
but lack nNOS
, indicating a possible signaling pathway via activation of NMDA receptors or other proteins and ion channels of the PSD. Therefore, membrane-associated nNOS
seems to be responsible for pyloric sphincter relaxation (10, 18), and lack of nNOS
in
nNOS
knockout mice results in gastric stasis with delayed gastric
emptying of solids and liquids (10, 18). Furthermore,
diabetic rats and mice with defects in gastric emptying and pyloric
nonadrenergic, noncholinergic relaxation, show a profound
reduction in nNOS protein and mRNA levels before neural degeneration in
the pyloric sphincter (29, 36) but not in the central
nervous system (36). Interestingly, nNOS expression and
nonadrenergic, noncholinergic relaxation are restored to normal levels
in the pyloric sphincter of diabetic mice by insulin treatment,
indicating that transcriptional and posttranscriptional mechanisms of
nNOS gene expression are involved in diabetic gastroparesis
(36). Recently, glucoresponsive neurons have been
identified within the enteric nervous system (16). Therefore, glucose or insulin-responsive signaling pathways may regulate nNOS gene expression specifically in the gastrointestinal tract by transcriptional control of distinct alternative nNOS promoters.
To obtain morphological evidence for a differential distribution of the
different nNOS proteins, we used immunohistochemistry with confocal
laser scanning microscopy. Because no specific antibodies for the
NH2-terminally truncated nNOS variants are available, we
used an antibody directed against the NH2 terminus
(detecting nNOS) and the COOH terminus (detecting nNOS
, -
, and
-
) of nNOS. In parts of the intestine (duodenum) where all known
nNOS mRNA variants are present, we could demonstrate morphological evidence for a differential localization of COOH- and
NH2-terminal nNOS immunoreactivity by doublestaining
analysis using the COOH- and NH2-terminal nNOS antibodies.
There was a good but not identical colocalization of COOH- and
NH2-terminal nNOS immunoreactivity in the myenteric plexus
and the nerve fibers running to the LM and CM, suggesting that the
majority of nNOS protein expressed at these localizations is nNOS
.
In addition, there seems to be some subcellular areas, especially in
nerve fibers, where no colocalization can be detected, suggesting that
the different nNOS proteins could be localized in different cell
compartments. Interestingly, there was additional staining with the
NH2-terminal antibody, but not the COOH-terminal antibody,
in the submucosal plexus and in nerve fibers running to the
mucosa. This suggests that submucosal neurons do not contain
nNOS
/
but also do not contain full-length nNOS
. Because this
staining could be specifically blocked by preabsorption of the antibody
with the respective immunogen, it does reflect nNOS immunoreactivity.
This staining indicates the existence of new nNOS variants containing
the NH2-terminal region of nNOS
encoded by exon 2, but
not the typical COOH-terminal end, representing COOH-terminally
truncated or extended nNOS protein variants. Using tissue homogenates
from the mucosa and the LM/CM with attached nerve plexus of the
duodenum, we further characterized these nNOS variants by Western blot
analysis. The assay revealed a single band with a molecular weight of
~85 kDa in the mucosa and three bands at ~155, ~85, and ~30 kDa
in the muscle layer using the NH2-terminal nNOS-N antibody.
This observation supports our immunohistochemical data and argues for
the possible existence of additional, yet unknown COOH-terminal nNOS
variants. Whether these variants are due to posttranscriptional,
translational, or posttranslational processing cannot be answered from
this study and has to be further investigated.
Diversity of nNOS mRNA in different tissues and developmental stages is a major characteristic of nNOS gene expression (8, 33). Here we report, in addition, a site-specific expression of nNOS mRNA forms and a differential localization of COOH- and NH2-terminal nNOS proteins in the rat gastrointestinal tract. This argues for a complex and tightly regulated gene expression of the so-called constitutive nNOS by site-specific transcriptional, posttranscriptional, and translational control, resulting in different nNOS proteins that may play a pivotal role in the motility of sphincter and nonsphincteric regions of the gastrointestinal tract.
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ACKNOWLEDGEMENTS |
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We thank Dr. S. Snyder for providing rat nNOS cDNA used in this work.
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FOOTNOTES |
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This study was supported by Deutsche Forschungsgemeinschaft Sonderforschungsbereich 391 C5 and KKF TU Munich F71-98.
Preliminary results of this study were presented at the annual meeting of the American Gastroenterological Association in Orlando, FL, 1999.
Address for reprint requests and other correspondence: D. Saur, Dept. of Internal Medicine II, Technical Univ. of Munich, Ismaningerstr. 22, 81675 Munich, Germany (E-mail: Dieter.Saur{at}lrz.tu-muenchen.de).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpgi.00226.2001
Received 31 May 2001; accepted in final form 1 November 2001.
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