Department of Internal Medicine II, Technical University of Munich, D-81675 Munich, Germany
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ABSTRACT |
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The aim of this study was to
characterize neuronal nitric oxide synthase (nNOS) activity and
5'-end splice variants in rat small intestine. nNOS was
predominantly expressed in the longitudinal muscle layer, with attached
myenteric plexus (LM-MP). The biochemical properties of NOS activity in
enriched nerve terminals resemble those of nNOS isolated from the
brain. Western blot analysis of purified NOS protein with an nNOS
antibody showed a single band in the particulate fraction and three
bands in the soluble fraction. Rapid amplification of 5' cDNA
ends-PCR revealed the presence of three different 5'-end splice
variants of nNOS. Two variants encode for nNOS, which has a specific
domain for membrane association. The third variant encodes for nNOS
,
which lacks the domain for membrane association and should therefore be
soluble. nNOS is predominantly expressed in LM-MP and can be enriched
in enteric nerve terminals. We present the first evidence that three
5'-end splice variants of nNOS encoding two different proteins
are expressed in rat small intestine. These two nNOS enzymes exhibit
different subcellular locations and might be implicated in different
biological functions.
nitric oxide synthase isoforms; endothelial nitric oxide synthase; inducible nitric oxide synthase; nerve terminals; myenteric plexus
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INTRODUCTION |
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NITRIC OXIDE (NO) is a nonadrenergic, noncholinergic (NANC) neurotransmitter that has a potent inhibitory effect on the smooth muscle in various regions of the gastrointestinal tract (11, 41, 43). NO is generated from the amino acid L-arginine and may also act as a neuromodulator by facilitation or inhibition of the release of other neurotransmitters within the same nerve ending or as a neurotransmitter acting on enteric neurons or smooth muscle cells of the gastrointestinal tract (2). Also, there is some evidence (36, 40, 46) that NO might act as a second messenger within smooth muscle or interstitial cells of Cajal.
Three distinct isoforms of NO synthase (NOS) have been purified and
cloned: an inducible NOS (iNOS) (48), which is
Ca2+ independent, and an
endothelial NOS (eNOS) (35) and a neuronal NOS (nNOS) (6, 13), which
are both Ca2+ dependent. Recent
evidence (8, 15) from the mouse brain suggests that alternative
5'-end splicing of nNOS mRNA results in at least three different
NH2-terminal nNOS protein variants (nNOS, nNOS
, and nNOS
). nNOS
, which exhibits full enzymatic activity, contains a PDZ (PSD-95/Dlg/ZO-1) domain enabling a possible interaction with proteins of the postsynaptic density (PSD) zone, such
as postsynaptic density protein-95 (PSD-95) or
-syntrophin. Because of this interaction, the nNOS
form can be membrane associated (8). The nNOS
and nNOS
splice
variants lack the PDZ domain that is encoded by exon 2. This results in
a cytosolic localization of these proteins. nNOS
and nNOS
from
the mouse central nervous system showed an enzymatic activity of
~80% and ~3%, respectively, of that of nNOS
, when expressed in
mammalian cells (8). In addition, in the rat three
different splice forms with distinct 5'-untranslated first exons
(5'-untranslated region) of the nNOS
mRNA (nNOS
-a,
nNOS
-b, nNOS
-c) have been identified, which show a tissue- and
development-specific expression (31). However, these splice forms
result in a single nNOS
protein.
Recently, it has been shown (45) that nNOS expression in the rat stomach can be changed substantially under pathophysiological conditions, such as diabetic autonomic neuropathy, causing defective relaxation in response to NANC nerve stimulation.
In the present study, we quantified the distribution of nNOS mRNA expression in various layers of rat small intestine and characterized the biochemical properties of NOS in enriched enteric nerve terminals. Subsequently, we analyzed purified NOS by Western blot analysis and investigated the expression of possible 5'-end nNOS splice variants by rapid amplification of 5' cDNA ends-PCR (5'-RACE-PCR).
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MATERIALS AND METHODS |
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Preparation of rat enteric nerve terminals.
The preparation of rat enteric nerve terminals was carried out as
described previously (28). Briefly, male Wistar rats were killed by
cervical dislocation. The small intestine was suspended in ice-cold
sucrose-MOPS buffer [25 mM MOPS, pH 7.4, 10 mM
MgCl2, and 8% (wt/vol)
sucrose], stripped of mesenteric arcade and fat, and opened along
the mesenteric attachment line, and the mucosal layer was removed by
scraping. The remaining muscle layers were minced with scissors and
homogenized with a Polytron PT20 homogenizer at a setting of ~1,500
rpm for 15 s (3 times for 5 s each). The tissue homogenate was
centrifuged at 800 g for 10 min, and
the supernatant was collected [postnuclear supernatant
(PNS)] and subjected to various differential centrifugation steps
(3,500 g for 10 min, 120,000 g for 60 min, and 10,000 g for 10 min). The resulting pellet of
the final centrifugation step was the enriched nerve terminals
(P2). Protein concentrations were
determined according to the method of Bradford (4) using bovine plasma -globulin as a standard (Bio-Rad, Munich, Germany). For a single preparation, the small intestines of five rats were used. The detailed
characterization of the enriched enteric nerve terminals and the method
of [3H]saxitoxin
binding have been described previously (1, 28). Release of bombesin-,
somatostatin-, and vasoactive intestinal polypeptide (VIP)-like
immunoreactivity from enriched enteric nerve terminals was analyzed by
RIA, as reported previously (2, 28, 29).
Extraction of proteins.
Tissue from both rat brain and rat small intestinal longitudinal muscle
layer, with attached myenteric plexus (LM-MP), was cut into pieces and
homogenized with a fivefold volume of 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 (PMSF), and 12 mM
-mercaptoethanol] with a Polytron PT20 homogenizer at ~1,500 rpm for 15 s (3 times for 5 s each). The homogenates were centrifuged at 100,000 g for 60 min, and the
resulting supernatants were referred to as soluble brain and small
intestinal fractions (BRS and
SIS, respectively).
The pellets were suspended with the same buffer and subsequently
centrifuged (60,000 g for 60 min). To
obtain the particulate fraction, we homogenized the resulting pellets with buffer containing 1% Triton X-100 and centrifuged them at 100,000 g for 60 min. The supernatants
[particulate brain and small intestinal fractions
(BRP and
SIP), respectively] were
then collected for further analysis.
Enzyme purification.
NOS was isolated and purified as previously reported (42). Briefly, the
enriched nerve terminals were treated with 1% Triton X-100 and
ultrasound (3 strokes of 15 s each) to break the nerve terminals and
centrifuged at 2,500 g for 5 min. The
resulting supernatant as well as
BRS,
BRP,
SIS, and
SIP were incubated with 1 ml of
preswollen adenosine 2',5'-diphosphate-agarose with gentle agitation (30 min at 4°C), transferred to a fritted column, and washed with five column volumes of Tris buffer (10 mM Tris chloride, pH
7.6, 0.1 mM EGTA, 0.1 mM EDTA, 3 µM leupeptin, 1 µM pepstatin, 1 mM
PMSF, and 12 mM -mercaptoethanol), approximately three column volumes of Tris buffer plus 0.5 M NaCl, and again with approximately six column volumes of Tris buffer. NOS was then eluted with five column
volumes of Tris buffer plus 10 mM NADPH. As a final step, the eluate
was concentrated with Centricon-50 concentrators (15-45 min at
3,000 g; Amicon, Witten, Germany). The
enzyme activity during the purification was determined by the rate of
conversion of
L-[3H]arginine
to
L-[3H]citrulline.
NADPH diaphorase assay.
NADPH diaphorase activity was assayed as described by Hope et al. (21)
by measuring the reduction of 0.5 mM nitro blue tetrazolium with 1 mM
-NADPH in 0.9 ml of 50 mM Tris chloride, pH 8.0, containing 0.2%
Triton X-100, at 37°C.
NOS assay. NOS activity was determined as the rate of conversion of L-[3H]arginine to L-[3H]citrulline (7). Permeabilized nerve terminals (1% Triton X-100) were incubated with ~500,000 dpm of L-[3H]arginine (68 Ci/mmol) in the presence of various exogenous compounds, as specified later, in a final volume of 200 µl for 30 min at 37°C. To test the Ca2+ dependence of the NOS activity, the experiments were performed in Ca2+-free buffer containing 0.1 or 1 mM EGTA. The reaction was stopped by boiling and dilution with 1 ml distilled water containing 1 mM L-arginine and 1 mM L-citrulline. Proteins from BRS, BRP, SIS, and SIP, before and after NOS purification, were incubated in the presence of ~500,000 dpm of L-[3H]arginine, 1 mM NADPH, 4 µM FMN, 4 µM FAD, 2 mM CaCl2, 1 µM calmodulin, 0.1 µM (6R)-5,6,7,8-tetrahydro-L-biopterin (THB), and 0.1 mM dithiothreitol in a final volume of 100 µl 50 mM HEPES, pH 7.4 (15 min at 37°C). The reaction was stopped by addition of 1 ml stop buffer (20 mM HEPES, pH 5.5, and 2 mM EDTA). Separation of L-[3H]citrulline from L-[3H]arginine was carried out with Poly-Prep chromatography columns (Bio-Rad) filled with 1 g of Dowex AG50W-X8 resin (Na+ form). A sample volume of 1 ml was applied onto the columns, L-[3H]citrulline was eluted from the columns by 1.5 ml of water, and the amount of radioactivity was counted after the addition of 3 ml scintillation fluid. To determine the background activity, we boiled proteins for 6 min before the experiments to inactivate the enzyme.
Immunoblotting. Samples of the purified enzyme were separated by SDS-PAGE on 6.5% slab gels in a Bio-Rad mini-gel apparatus (30). Protein bands were visualized by Coomassie blue staining or blotted onto a polyvinylidene difluoride membrane (Bio-Rad) using a buffer composed of 50 mM Tris, 380 mM glycine, 0.05% SDS, and 20% methanol. After blocking the membrane with 5% dry milk, we probed the blots with different antibodies for nNOS [polyclonal antibody (1:500) from BioMol, Hamburg, Germany; monoclonal antibody (1 µg/ml) from Transduction Laboratories, Lexington, KY], with an antibody to eNOS (1 µg/ml, Transduction Laboratories) or an antibody to iNOS (1:250; gift from Dr. Pressley, University of Texas, Houston, TX). The incubation lasted for 2 h at room temperature. As secondary antibodies, horseradish peroxidase-linked anti-rabbit or anti-mouse IgG were used for polyclonal or monoclonal primary antibodies, respectively (enhanced chemiluminescence system, Amersham, Braunschweig, Germany), and the optical density of the bands was measured by an imaging analyzer.
The specificity of the immunoblotting using nNOS, iNOS, and eNOS antibodies at different dilutions (1:250-1:1,000) was tested with the respective positive controls obtained from Transduction Laboratories.RNA isolation. We extracted total RNA from liquid nitrogen-frozen LM-MP, circular muscle layer (CM), and mucosa of rat small intestine. Tissues were homogenized, and RNA was isolated using the guanidine isothiocynate-phenol-chloroform extraction method (Micro RNA isolation kit; Stratagene, Heidelberg, Germany) (14), followed by DNase treatment (DNase I; GIBCO-BRL, Eggenstein, Germany).
Semiquantitative RT-PCR.
We reverse transcribed 3 µg of total RNA into complementary DNA by
using 200 U SuperScript II RNase
H RT (GIBCO-BRL) and 100 ng
oligo(dT)15 (Boehringer Mannheim,
Mannheim, Germany). To determine the expression of mRNAs encoding nNOS, iNOS, and eNOS in LM-MP, CM, and the mucosa, we performed a
semiquantitative PCR using specific primers for
-actin, nNOS, iNOS,
and eNOS (Table 1). The amplified products
span one or more putative intron sites to detect a possible DNA
amplification (sequences from DNA database, European Molecular Biology
Laboratories, Heidelberg, Germany).
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5'-RACE-PCR.
Thermal 5'-RACE-PCR was performed as previously described (16) with
minor modifications. We isolated 1 µg poly(A) RNA of rat small
intestine LM-MP with the Oligotex mRNA kit (Qiagen, Hilden, Germany)
and reverse transcribed it by 400 U SuperScript II RNase
H RT (GIBCO-BRL) for 2 h at
45°C using 2 pmol of the antisense gene-specific
primer
1 (see Table
2 for all primers) complementary to exon 3 of rat nNOS RNA (5). After removal of template mRNA by RNase H
(GIBCO-BRL) treatment, excess primers were eliminated by using a PCR
purification kit (Qiagen). The 3' end of the cDNA product was
poly(A)-tailed with dATP by terminal deoxynucleotide transferase
(GIBCO-BRL). The first round of PCR amplification was performed with
gene-specific primer
2 (25 pmol), sense generic primer
Q (2 pmol), and sense generic
primer
Q1 (25 pmol)
under the following conditions: denaturation at 97°C for 5 min,
annealing at 50°C for 2 min, and extension at 72°C for 40 min. The subsequent 29 cycles were carried out using the
step-down PCR technique (19) with the following parameters:
denaturation at 94°C for 30 s, annealing temperature of the initial
9 cycles at 66°C for 40 s, with a rundown of 4°C for each
subsequent 10 cycles, and extension at 72°C for 3 min adding 5 s
each cycle. A second round of amplification was performed for 30 cycles
in a total volume of 50 µl with the antisense gene-specific
primer
3 (25 pmol) and the sense generic primer
Q2 (25 pmol) with
denaturation at 94°C for 30 s, primer annealing at 58°C for 40 s, and extension at 72°C for 3 min. To verify specific partial cDNA
amplification, we analyzed PCR products by Southern blot hybridization.
After the first and second rounds of PCR, amplification products were
separated by 1.5% agarose gel electrophoresis, denatured, and blotted
onto Hybond N+ nylon membrane
(Amersham) by capillary transfer. The filters were hybridized overnight
at 54°C with a 5' digoxigenin end-labeled oligomer specific
for exon 3 of rat nNOS (5'-AGACCTCGATGGCAAATCG-3') and
washed twice with 2× SSC (1× SSC is 0.15 M NaCl and 0.015 M
sodium citrate, pH 7.0) containing 0.1% SDS for 15 min at room temperature and once with 0.5× SSC and 0.1% SDS for 10 min at 44°C. The labeled products were detected by the DIG nucleotide acid
detection kit (Boehringer Mannheim). Finally, 20-µl aliquots of the
PCR products were fractionated on a 1.5% agarose gel, excised, and
purified, using a gel extraction kit (Qiagen). Nucleotide sequences
were deduced by cycle sequencing of both strands of the PCR products
(Medigen, Martinsried, Germany).
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Drugs.
L-[3H]arginine
(60-68 Ci/mmol) was purchased from Amersham. All other reagents
were purchased from the indicated sources.
L-arginine, L-citrulline, -NADPH,
N
-nitro-L-arginine methyl ester
(L-NAME), THB, calmodulin, FAD, FMN, Triton X-100, adenosine 2',5'-diphosphate-agarose,
leupeptin, pepstatin, PMSF, and nitro blue tetrazolium chloride were
from Fluka (Neu-Ulm, Germany). EGTA, EDTA,
-mercaptoethanol, SDS, and glycine were from Merck (Darmstadt, Germany), and Dowex AG50W-X8 resin was from Bio-Rad. Acrylamide/bisacrylamide was from
Roth (Karlsruhe, Germany), and scintillation fluid Quickszint 212 was from Zinsser Analytic (Frankfurt, Germany).
Data analysis and statistics. Data are expressed as means ± SE; n indicates the number of independent observations in various experiments from individual preparations. The activity of NADPH diaphorase or NOS at the different treatment protocols was measured in duplicate. For multiple comparisons, ANOVA followed by post hoc test with Bonferroni correction for multiple comparisons was carried out to determine statistical differences. P < 0.05 was considered significant.
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RESULTS |
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Differential expression of NOS mRNA in rat small intestine.
RT-PCR was carried out with RNA from LM-MP, CM, and the mucosa of rat
small intestine to determine the distribution of all three NOS
isoforms. RT-PCR with specific primers for nNOS, iNOS, eNOS, and
-actin as an internal standard showed single bands for each cDNA at
the expected size in proportion to the increase of the PCR cycles or
the amount of cDNA present in the reaction. A linearity in the increase
of intensity was found between 25 and 35 cycles for
-actin and
between 30 and 40 cycles for iNOS and eNOS. nNOS increased linearly
between 25 and 35 cycles in the LM-MP and between 30 and 40 cycles in
CM and the mucosa. After 35 cycles, a linear increase of the three
isoforms and
-actin was also observed in the cDNA diluted 1:100,
1:10, and 1:1 (data not shown). The mRNA quantification was
reproducible, as tube-to-tube and day-to-day variation of the ratio of
xNOS to
-actin and the mean values of the amplification products
showed no significant difference (data not shown). The quantification
of the isoforms was carried out with the 1:1 dilution and 35 cycles.
The ratio of nNOS to
-actin was ~0.785 ± 0.078 in LM-MP and
0.015 ± 0.006 in CM. Even after 40 cycles of PCR, nNOS was not
detectable in the intestinal mucosa. The ratio of iNOS to
-actin was
0.178 ± 0.059 for LM-MP, 0.043 ± 0.036 for CM, and 0.569 ± 0.160 for the mucosa. The ratio of eNOS vs.
-actin was 0.169 ± 0.045 for LM-MP, 0.192 ± 0.061 for CM, and 0.127 ± 0.046 for
the mucosa (Fig. 1). As we were interested
in the characterization of nNOS, all further studies were performed
either on isolated enteric synaptosomes or on proteins or RNA extracted
from LM-MP preparations.
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NADPH diaphorase and NOS activity in isolated nerve terminals.
Because NADPH diaphorase and NOS are present in the same protein, we
used diaphorase activity [absorbance units (AU)/mg protein] and NOS activity
(fmol · mg1
protein · min
1),
measured by the conversion of
L-[3H]arginine
to
L-[3H]citrulline
as parameters for the NOS content of isolated nerve terminals (21). The
isolation of nerve terminals led to a 5.4-fold (n = 7) enrichment of NADPH
diaphorase activity in the synaptosomal fraction (2.69 ± 0.79 AU/mg
protein) compared with PNS (0.50 ± 0.19 AU/mg protein), which was
paralleled by a 4.3-fold (n = 3) enrichment of NOS activity in P2 (4.6 ± 2.3 fmol · mg
1
protein · min
1;
PNS, 1.1 ± 0.6 fmol · mg
1
protein · min
1).
In comparison, the enrichment factor for
[3H]saxitoxin binding
or the content of bombesin-, somatostatin-, or VIP-like
immunoreactivity was 8.2, 4.0, 3.8, and 7.1, respectively (Table
3). These experiments indicate that NOS
activity can be enriched by the isolation of enteric nerve terminals.
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Isolation, purification, and Western blot analysis of NOS.
For further characterization through Western blot analysis, NOS was
isolated and purified from BRS,
BRP,
SIS, and
SIP, and P2 by adenosine
2',5'-diphosphate-affinity chromatography. The enrichment
of NOS activity vs. the crude fractions achieved by affinity
chromatography was 133-fold for
BRS (21.2 vs. 0.16 pmol · mg1
protein · min
1),
359-fold for BRP (33.5 vs. 0.09 pmol · mg
1
protein · min
1),
114-fold for SIS (22 vs. 0.18 pmol · mg
1
protein · min
1),
and 90-fold for SIP (3.1 vs. 0.03 pmol · mg
1
protein · min
1).
In P2, the further enrichment was
50-fold (197.0 vs. 3.93 fmol · mg
1
protein · min
1;
n = 3) (Fig.
3).
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Detection of different 5'-end splice variants of nNOS by
5'-RACE-PCR.
To differentiate the positive protein bands obtained with the nNOS
antibody, we investigated possible 5'-end splice variants of
nNOS. Rat small intestinal mRNA was isolated from the LM-MP, reverse
transcribed, and 5'-RACE-PCR was performed. The amplified products with
~350, 1,050, and 1,500 bp were able to hybridize with an exon
3-specific internal oligonucleotide probe (Fig.
5, A and
B). Sequencing of these RACE
products demonstrated the existence of three different nNOS
5'-end mRNA transcripts in rat small intestine (Figs.
5D and 6).
Two distinct 5'-terminal exons (exon 1a, exon 1b) located in the
5'-untranslated region of nNOS mRNA could be determined, followed
by a common exon 2, which contains the translational initiation site
with an ATG starter methionine, 378 bases downstream of the exon 1/exon
2 splice junction. Translation of these two splice variants should
result in only one nNOS protein (nNOS). Our exon 1a is almost
identical (alignment 99.3%) to the sequence of the rat exon 1a
reported by Lee et al. (31) and shows 92% alignment to the reported
alternative spliced exon 1a of mouse nNOS mRNA (8). Our exon 1b is
almost identical to the nucleotide sequence of exon 1b reported by Lee
at al. (31) and shows 88% homology with another recently reported exon
1 subtype from the mouse brain (10). In accordance with the
nomenclature for the rat by Lee et al. (31), this exon 1 splice variant
is referred to as exon 1b. The third mRNA is an exon 1a/exon 3 splice
variant that lacks exon 2, which encodes the PDZ domain of the nNOS
splice variant. The mRNA shows 92% identity with the 5'-end
nucleotide sequence of mouse brain nNOS
mRNA reported by Brenman and
co-workers (8). The translation initiation site (CTG) of this
transcript is located 20 bases upstream of the exon 1a/exon 3 splice
junction, and translation of nNOS
mRNA should generate an
alternative nNOS protein (nNOS
) that lacks the
NH2 terminus and subsequently the PDZ domain of nNOS
. The PDZ domain can interact with proteins from
the postsynaptic density zone and subsequently influence the cellular
localization of the enzyme. We could not detect mRNA of an alternative
spliced exon 1 in rat small intestine by 5'-RACE-PCR that corresponds
to nNOS
in the brain of nNOS
-knockout mice (8).
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DISCUSSION |
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We demonstrated that NOS activity, as well as enzymatic diaphorase activity, was enriched in isolated nerve terminals and the enrichment of both enzyme activities showed the same relation as the enrichment of putative enteric neurotransmitters or neuronal markers. NOS activity in the rat enteric nervous system required NADPH, Ca2+, calmodulin, THB, and FAD plus FMN for full enzymatic activity. Kostka et al. (27) also found an increase of NOS activity in isolated nerve terminals of the canine ileum by addition of NADPH, Ca2+, and calmodulin; however, they could not demonstrate a further increase by addition of THB, which caused a twofold increase compared with the values with NADPH and Ca2+ alone in our model. The activity of NOS in isolated nerve terminals was regulated crucially by Ca2+, which enables calmodulin to bind and activate the enzyme since NOS activity was almost abolished by EGTA. These functional results are in accordance with the NOS isoform detected by Western blot analysis, and these cofactors were requirements for full activity of the nNOS enzyme isolated from brain (5, 6, 37). A small residual component of NOS activity was not abolished in the presence of EGTA (0.1 or 1 mM), reflecting small amounts of Ca2+-independent NOS activity in this fraction. This finding is in agreement with that of Kostka et al. (27), who also identified an EGTA-insensitive NOS activity in canine synaptosomes. In their detailed characterization (27), this activity was unlikely to be due to a smooth muscle cell contamination and was somewhat different from iNOS activity.
To determine the expression of NOS isoforms within different intestinal
layers, we performed semiquantitative RT-PCR with RNA from the LM-MP,
CM, and mucosa of rat small intestine. It could be demonstrated that
nNOS is predominately present in the LM-MP, whereas the inducible
isoform, according to its predominant location in immune cells (20,
44), is mainly located in the mucosa and to a lesser extent in the
LM-MP. The distribution of eNOS was equal in all three layers tested,
as expected because of the presence of blood vessels in all of them.
However, even after purification of NOS activity with adenosine
2',5'-diphosphate-affinity chromatography, Western blot
analysis with specific antibodies for eNOS and iNOS could not
demonstrate the existence of these latter two isoforms in isolated
nerve terminals or in nerve/muscle homogenates. Despite the fact that
small amounts of the enzyme could be missed by protein loss during the
purification, these results indicate that eNOS and iNOS protein
expression in the LM-MP and enriched nerve terminals was below the
limit for detection of our Western blot analysis. This corresponds with
the distribution obtained by the semiquantitative RT-PCR in the
different layers as the levels of iNOS and eNOS mRNA were considerably
lower in the LM-MP fraction compared with the nNOS expression. It is
also in agreement with recent findings (3) in whole thickness
preparations of rat stomach and rectum, in which significant amounts of
iNOS were only present after upregulation with interferon-.
As recently demonstrated (8, 15), two different
NH2-terminal splice variants of
nNOS (nNOS, nNOS
) with different molecular masses (150 and 136 kDa) are present in the brain of wild-type mice. nNOS
(125 kDa) was
found as an additional splice form besides nNOS
in the brain of
nNOS
knockout mice (8). nNOS
exhibits full enzymatic activity and
is partially membrane associated via a PDZ domain. The PDZ domain in
nNOS
is a motif of ~100 amino acids that mediates an association
of nNOS
to postsynaptic density proteins (e.g., PSD-95, PSD-93,
syntrophins), which associate with the dystrophin complex (8, 9, 39).
This association can determine the cellular localization of nNOS, and
impairment of this association in skeletal muscle was shown in Duchenne
muscular dystrophy (9). nNOS
and nNOS
, which lack the PDZ domain, are subsequently soluble and show enzymatic activities of ~80% and
~3% of the full activity observed with nNOS
, respectively (8).
Through immunoblotting with specific nNOS COOH-terminal antibodies, we
demonstrated that isolated and purified NOS from all three sources,
BRS,
SIS, and
P2, displayed three different protein bands at ~155, 145, and 135 kDa. In
SIP, only the protein band at 155 kDa could be detected, corresponding to the expected molecular mass and
cellular localization of nNOS. The molecular mass of the soluble
135-kDa protein correlates with the nNOS
variant. However, definitive proof based on Western blot analysis without specific antibodies against this splice form cannot be given. Considering the results by Lowe et al. (32), who showed a specific cleavage of nNOS
by trypsin and chymotrypsin treatment, it might also be possible that proteins with lower molecular mass arise from
enzymatic cleavage of the nNOS
protein. The enzymatic cleavage at
amino acid 221 could result in an
NH2-terminal truncated nNOS fragment with 135 kDa, devoid of the functional PDZ domain. To avoid
enzymatic cleavage, several protease inhibitors also specifically directed against trypsin and chymotrypsin were used in the present study. The single protein band obtained in
SIP argues against a proteolytic
breakdown; however, on the basis of the immunoblot data this
possibility cannot be completely ruled out.
5'-RACE-PCR with LM-MP cDNA of rat small intestine resulted in three
different splice variants: exon 1a/exon 2, exon 1b/exon 2, and exon
1a/exon 3. Translation of the splice variants exon 1a/exon 2 (nNOS-a) and exon 1b/exon 2 (nNOS
-b) would result in a 155-kDa
protein corresponding to nNOS
, which is partially particulate but
also occurs in the soluble fraction. This is in accordance with the
possible membrane association of this splice form via the PDZ-binding
domain. Translation of the exon 1a/exon 3 variant, which lacks exon 2 and subsequently the PDZ domain, would result in the soluble splice
variant nNOS
(135 kDa). nNOS
is the predominant form in the brain
of wild-type mice, whereas nNOS
and nNOS
account for nNOS
activity in nNOS
knockout mice (8, 15). In the rat, both nNOS
and
nNOS
are present in the small intestine. However, in contrast to rat
brain, the relative expression of nNOS
seems to be increased in
LM-MP and predominant in isolated nerve terminals of the intestine. The
functional roles of nNOS
and nNOS
remain speculative. In
myenteric plexus of embryonic rats, nNOS
and PSD-95, a protein with
specific PDZ motifs that interacts with nNOS
, are colocalized (8).
PDZ motif-containing proteins (e.g., PSD-95) were suggested to be involved in the subcellular localization of proteins (e.g.,
N-methyl-D-aspartate receptors, K+-channel subtypes) at
sites of membrane specialization such as synaptic sites (25,
26). Thus the membrane-associated form (nNOS
) could be
involved in the regulation of receptor or ion channel function. The
role of nNOS
that is not membrane associated but is expressed
relatively high in rat small intestine, especially in enriched nerve
terminals, has yet to be determined. If a comparable distribution of
splice variants is present in mouse intestine, it would explain why
nNOS
-knockout mice, lacking only nNOS
, show no major functional
abnormalities in the small intestine (24). Whether the nNOS splice
variants are confined to neurons or can also occur in intestinal smooth
muscle (12) or interstitial cells of Cajal (33, 49) cannot be
determined from our study.
We could further demonstrate in rat small intestine that nNOS
originates from two different splice variants, which are almost identical to splice variants of the 5'-untranslated region
recently described in the rat (nNOS
-a, nNOS
-b) (31) and show
sequence homology to exon 1 splice variants from mouse brain (8, 10). Because translation of both exon 1 variants in rat small intestine should result in an identical nNOS
protein, the question about the
physiological significance of these exon 1 splice variants arises. Lee
et al. (31) demonstrated a distinct tissue and developmental-specific expression of three 5'-untranslated region exon 1 splice variants in the rat. However, in contrast to our study, Lee et al. (31) could
only demonstrate the exon 1b/exon 2 splice variant (nNOS
-b) in
embryonal tissue. Xie et al. (47) demonstrated that two distinct first
exons of nNOS of human cerebellum are under the transcriptional control
of two closely linked, but separate promoters. Transcriptional control
by separate promoters and regulation of these promoters may result in
expression of nNOS
in response to different cellular changes or
developmental stages (10, 31, 47). The differences in first exon
structure of nNOS
may also be implicated in mRNA localization,
stability, postranscriptional control, or translation efficiency (10).
The immunoblot reactive band at 145 kDa could not be further
characterized by 5'-RACE-PCR, indicating that this band is not due to
an NH2-terminal splice variant. In
particular, the molecular size of the band does not correspond to
nNOS (125 kDa), which was also not identified by 5'-RACE-PCR and
RT-PCR. Thus our results comply with the results in the mouse brain (8)
in which nNOS
was also absent in wild-type mice and was only
expressed when nNOS
was knocked out.
In summary, nNOS activity is present in enteric neurons and can be
purified along with other neuronal markers in enteric nerve terminals.
The nNOS in enteric synaptosomes exhibits immunologic and biochemical
properties identical to those of the nNOS from rat brain. Three
different splice variants of nNOS are present in the small intestine,
resulting in two distinct proteins that differ in their
NH2-terminal ending and show a
distinct subcellular distribution. From our data there is no evidence
for the expression of the nNOS form in rat small intestine.
Expression of nNOS splice variants could be changed under developmental but also physiological or pathophysiological conditions. Because of the differences in transcriptional control, enzymatic activity, and subcellular localization, these splice variants could also be involved in functional changes. Thus, in addition to transcriptional regulation of gene expression as previously shown in diabetic rats (45), alternative splicing of nNOS has to be considered in functional disorders also.
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ACKNOWLEDGEMENTS |
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We acknowledge the courtesy of Dr. Pressley, University of Texas, Houston, TX, for providing us with a polyclonal antibody for iNOS. We thank L. Kots for proofreading the manuscript.
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FOOTNOTES |
---|
This study was supported by Deutsche Forschungsgemeinschaft Sonderforschungsbereich Grant 391 C5 and KKF Grant TU Munich F71-98.
Preliminary results of this study were presented at the annual meeting of the American Gastroenterological Association in New Orleans, LA, in May 1998.
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. §1734 solely to indicate this fact.
Address for reprint requests: H.-D. Allescher, Dept. of Internal Medicine II, Technical Univ. of Munich, Ismaningerstr. 22, D-81675 Munich, Germany.
Received 11 March 1998; accepted in final form 24 June 1998.
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