Vol. 275, Issue 4, H1122-H1129, October 1998
Induction and cDNA sequence of inducible nitric oxide synthase
from canine aortic smooth muscle cells
Xiaofang
Wang,
Christopher G. A.
McGregor, and
Virginia M.
Miller
Division of Cardiothoracic Surgery, Departments of Surgery and
Physiology and Biophysics, Mayo Clinic and Foundation, Rochester,
Minnesota 55905
 |
ABSTRACT |
An inducible isoform of nitric oxide synthase
(type II, iNOS) is expressed in cardiac and vascular smooth muscle in
response to inflammatory cytokines. The dog is an important large
animal used for cardiovascular research including effects of exercise, heart failure, and allograft rejection. However, molecular probes for
iNOS developed in other mammals have not been reliable for the study of
iNOS induction in canine vascular smooth muscle. Experiments were
designed to develop a molecular probe for canine iNOS. Smooth muscle
cells were isolated from canine aortas. The cells
(passages 3-10) were incubated
for 1, 3, 6, 12, 24, 48, or 72 h in the absence and presence of
Escherichia coli lipopolysaccharide (LPS) to induce iNOS. Total RNA was isolated from the cells using standard techniques. RT-PCR with primers against conserved regions of
all known iNOS enzyme was used to clone the iNOS cDNA. RT-PCR showed a
single band only from cells treated with LPS. Cloned cDNA from cultured
canine aortic smooth muscle cells has 84% homology to human, 81% to
rat, and 81% to mouse iNOS gene. Identification of the cDNA for canine
iNOS will be useful in the study of differential, transcriptional
regulation of inducible (type II) compared with constitutive
endothelial (type III) NOS in canine studies of allograft rejection and
cardiovascular disease.
glucocorticoids; type II nitric oxide synthase
| |
INTRODUCTION |
NITRIC OXIDE participates in diverse physiological
functions, including neurotransmission, regulation of blood pressure,
and immune defense. The biosynthesis of nitric oxide is catalyzed from
L-arginine by nitric oxide
synthase (NOS). Three isoforms of NOS are known to exist that can be
classified as constitutive or inducible (20). Constitutive type I NOS,
or neural NOS (nNOS), is found in the brain, stomach, adrenal glands,
and skeletal muscle (19). cDNA for constitutive type III
NOS, or endothelial NOS (ecNOS), has been cloned from rat (2) and human
brain (19) and from bovine (15, 21, 25) and human endothelial cells (13, 17).
When stimulated appropriately, macrophages (5, 16), hepatocytes (3, 9,
23, 29), pancreatic cells (4, 7), vascular smooth muscle cells (14,
22), glial cells (8), retinal epithelial cells (11), and keratinocytes
(12) express the inducible type II NOS isoform (iNOS). However, there
is no report of iNOS in a canine cell line. The dog is an important large animal for the study of the physiology and pathophysiology of the
cardiovascular system including effects of exercise, heart failure,
vascular grafts, and allograft rejection. Differentiating the
regulation of constitutive endothelial NOS (type III) from that of the
inducible (type II) form of the enzyme has been difficult in dogs
because molecular probes for iNOS developed in other mammals have not
been reliable when used with canine tissue. Therefore, the purpose of
this study was to clone and sequence canine cDNA for iNOS.
| |
METHODS |
Preparation and Culture of Vascular Smooth Cells
Aortas obtained from adult male and female mongrel dogs (25 kg) were
stripped of adventitia and opened so that the endothelial cells could
be removed by scraping. The aortas were then cut into pieces (0.5 mm3) and placed 1 cm apart on
the wall of culture flasks (72 cm2, ~80 pieces/flask). The
pieces were incubated with Eagle's minimal essential medium (MEM; Life
Technologies, Gaithersburg, MD) supplemented with 20% fetal bovine
serum (FBS) and 1% antibiotic-antimycotic (Life Technologies). The
flasks were kept in an atmosphere of 5%
CO2 in humidified air to allow the
cells to migrate from the edge of aortic pieces. On the sixth day,
confluent cells were lifted by scraping and seeded into flasks
containing MEM plus 10% FBS and 1% antibiotic-antimycotic. Cells from
passages 3-10 were used for the
experiments. Cells stained positively for smooth muscle actin.
Measurement of Nitric Oxide
Total oxidized products of nitric oxide
(NOx) were measured in culture
medium by chemiluminescence (nitric oxide analyzer, model 270B; Sievers
Instruments, Boulder, CO) (18).
NOx was reduced to nitric oxide by
0.1 M vanadium(III) (Aldrich Chemical, Milwaukee, WI) in 3 M HCl. At
85°C, vanadium(III) reduces
NOx to NO (1). Standard curves for
sodium nitrite (50-2,000 pmol; Sigma Chemical, St. Louis, MO) and
potassium nitrate (50-2,000 pmol; Fisher Scientific, Pittsburgh,
PA) were obtained every day before the culture medium samples were
analyzed. The samples (100 µl) were injected into the reducing
solution. Output from the nitric oxide analyzer was recorded on a
Chromatopac integrator (model C-R601; Shimadzu, Kyoto, Japan). The
calculated areas of the output signal were used for both the standard
curves and the samples.
PCR Primers
The sequence of oligonucleotide primers used to amplify the mRNA for
iNOS in canine aortic smooth muscle cells was based on the sequence of
the cDNA for human iNOS and compared against conserved regions of all
known iNOS enzymes. Overlapping cDNA clones encoding canine aortic
smooth muscle cell iNOS were isolated by RT-PCR. A potential problem
with PCR amplification is the creation of mutations as a result of DNA
synthesis errors. Therefore, three complete sets of clones from
separate PCR reactions were sequenced in both forward and reverse
directions to reduce the probability of reporting synthesis errors.
Also, as a consequence of the PCR process, the sequence of the end of
each amplified fragment is identical to the sequence of the primers
used. Overlapping clones were generated to overcome the effect (Fig.
1). Sequences utilized for the experiments
are given in Table 1. The position of
inconsistent bases is given in Table 2.
View larger version (5K):
[in this window]
[in a new window]
|
Fig. 1.
Schematic showing positions (indicated by arrows) of oligonucleotide
primers (1a, 1b, 2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b, 6a, and 6b) used for
RT-PCR reactions. Potential fragments (F1-F6) amplified using
these primers are shown as horizontal bars above cDNA sequence diagram.
Nucleotide numbering (bp) is given for orientations.
|
|
RT-PCR reactions were used for each set of primers in Table 1, except
for primers 1a,
1b,
6a, and
6b. For these primers, rapid
amplification of cDNA ends (RACE) procedures were used.
3'-RACE procedure to sequence 3'-iNOS cDNA ends.
Total RNA was prepared from canine aortic smooth muscle cells cultured
with 1 µg/ml lipopolysaccharide (LPS) for 48 h and applied to a
3'-RACE procedure (Life Technologies). First, RNA was applied to
single-strand cDNA synthesis with the use of RT and the adapter primer
(5'-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTT-3', Life
Technologies). Next, the cDNA was amplified by the
gene-specific primer 6a and universal
amplification primer 6b (Life
Technologies). PCR amplification was performed as
described below except for the schedule: denaturation, annealing, and
elongation at 94, 68, and 72°C for 40 s, 1 min, and 2 min,
respectively, for 35 cycles.
5'-RACE procedure to sequence 5'-iNOS cDNA ends.
Total RNA was prepared from canine aortic smooth muscle cells cultured
with 1 µg/ml LPS for 48 h and applied to 5'-RACE procedure (Life Technologies). Total RNA (1 µg) was reverse transcribed into
first strand cDNA with the use of RT and the gene-specific primer 1b. The cDNA was purified
(GlassMAX DNA). An anchor sequence was then added to the 3' end
of the cDNA using terminal deoxymelotidyl transferase and dCTP. PCR
amplification was performed as described for the 3'-RACE
procedure by using the gene-specific primer
1b and anchor primer
1a.
Induction and Identification of Canine iNOS
Aortic smooth muscle cells were cultured in MEM with or without LPS (1 µg/ml; Sigma Chemical) in the absence or presence of dexamethasone (1 ng/ml) for up to 72 h. After this incubation, total RNA was prepared
from the cultures using RNA Stat-60 RNA isolation reagent (TEL-TEST B,
Friendswood, TX).
Total RNA was reverse transcribed into cDNA with the use of the
SuperScript preamplification system for first strand cDNA synthesis
(Life Technologies) according to the manufacturer's protocol. Briefly,
the first strand cDNA was synthesized by incubating 1 µg of total RNA
with 0.5 µg/µl of
oligo(dT)12-18 primer and 200 units of SuperScript II RT, 2.5 mM
MgCl2, 500 µM dNTP, 10 mM
dithiothreitol, 50 mM KCl, and 20 mM Tris · HCl (pH
8.4) in a final volume of 20 µl at 42°C for 50 min. The reaction
was terminated by heating to 70°C for 15 min. Two units of RNase H were added, and incubation continued for 20 min at 37°C. The cDNA was amplified by PCR in a 50-µl reaction mixture containing 2 µl of
cDNA, 2.5 units of Taq DNA polymerase
(Promega, Madison, WI), 0.5 µM of primers
2a and 2b (Table 1),
1.5 mM of MgCl2, and 200 µM dNTP
in 1× reaction buffer provided by the supplier (Promega).
Amplification was performed in a Perkin-Elmer GeneAmp PCR System 9600 (Perkin-Elmer, Norwalk, CT). Initial incubation was for 3 min at
94°C, followed by 35 cycles of 40 s at 94°C, 1 min at 62°C,
2 min at 72°C, and final extension for 10 min at 72°C. To
confirm the expression of mRNA for iNOS, expression of mRNA for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with an RT-PCR Control
Amplimer Set (Clontech Laboratories, Palo Alto, CA) was used as a
control. Ten microliters of the reaction mixture was mixed with loading
buffer, separated by electrophoresis on 2% agarose gels containing
ethidium bromide, and visualized by ultraviolet transillumination.
Northern Blot Analysis
Total RNA was isolated from canine aortic smooth muscle cells cultured
for 48 h in the absence or presence of LPS (1 µg/ml). Cells were
placed directly into RNA Stat-60. Isolated total RNA was quantified by
measuring the optical density at 260- and 280-nm wavelengths. Total RNA
(20 µg) was denatured by heating (65°C) for 10 min and separated
electrophoretically through a 1.2% agarose gel containing 2.2 M
formaldehyde. Total RNA was transferred to nylon membranes by capillary
transfer with 20× SSC (60 M NaCl, 6 M sodium citrate). After the
transfer, membranes were baked in a vacuum oven at 80°C for 2 h.
Membranes were prehybridized for 30 min at 65°C in Rapid-hyb
solution (Amersham, Amersham, UK) and hybridized with herring sperm DNA
and 32P-labeled RT-PCR product
amplified with primers 2a and
2b (Table 1) or GAPDH probe (600 bp; a
gift from Dr. Bruce Kline, Mayo Foundation, Rochester, MN) for 2.5 h at
65°C. After hybridization, membranes were washed once in 2×
SSC-0.1% SDS for 20 min at room temperature and then washed twice in
0.1× SSC-0.1% SDS for 15 min at 65°C. Membranes were dried
and exposed to X-ray film at 
70°C for 2 days.
Isolation and Sequence of cDNA Clone Encoding iNOS
The PCR products were ligated into the pGEM-T Easy Vector (Promega)
according to the manufacture's procedure, and the inserted cDNA
fragments were sequenced (Automated PCR Mayo Molecular Core).
| |
RESULTS |
Measurement of NOx
To verify product of iNOS induction,
NOx was measured in the culture
medium. NOx in the culture medium
of aortic smooth muscle cells exposed to LPS for 24 or 48 h was twice
that in culture medium alone or in medium from cells only (Fig.
2). Concentrations of
NOx in medium from cells exposed
to LPS at 24 or 48 h were comparable.
View larger version (11K):
[in this window]
[in a new window]
|
Fig. 2.
Total oxidized products of nitric oxide
(NOx) in medium from cultures of
canine aortic smooth muscle cells in absence of [control
(Con)] or after exposure to lipopolysaccharide (LPS) (1 µg/ml)
for 24 h or 48 h. NOx increased
comparably in medium after 24- and 48-h exposure of cells to LPS. Bars
represent means ± SE; n = 2 for
each group.
|
|
Time Course of Induction of NOS in Canine Aortic Smooth Muscle Cells
The RT-PCR products amplified using iNOS- and GAPDH-specific primers
showed clear bands at predicted sizes of 661 (iNOS; Fig. 3, upper
band) and 452 bp (GAPDH; Fig. 3,
lower band), respectively. These
bands were absent in the PCR-amplified products for which RNA was used
as a template or that were lacking a cDNA template (data not shown).
This indicates that the 661- and 452-bp bands originated from mRNA but
not from genomic DNA or other contamination. The iNOS mRNA was seen as
early as 3 h after cells were exposed to LPS, increased at 6, 12, 24, and 48 h, and then decreased at 72 h (Fig. 3).
View larger version (39K):
[in this window]
[in a new window]
|
Fig. 3.
Time course of induction of inducible nitric oxide synthase (iNOS) in
canine aortic smooth muscle cells. Total RNA was isolated from smooth
muscle cells treated with 1 µg/ml LPS for 0, 1, 3, 6, 12, 24, 48, or
72 h and analyzed by RT-PCR (using primers
2a and 2b) to
measure mRNA for iNOS and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) as described in
METHODS. iNOS mRNA was seen as early
as 3 h after exposure to LPS, and induction was further enhanced at 6, 12, and 24 h, reached high levels at 48 h, and then decreased at 72 h.
|
|
Effect of Dexamethasone on Expression of iNOS in Cultured Canine
Aortic Smooth Muscle Cells
The iNOS signal was absent in RNA isolated from aortic smooth muscle
cells cultured in the absence of LPS (control). A large iNOS signal was
observed in RNA isolated from LPS-treated cells. This signal was
suppressed by coincubation of the cells with LPS plus dexamethasone.
Neither LPS nor dexamethasone affected GAPDH signals (Fig.
4).
View larger version (58K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of dexamethasone on expression of iNOS in cultured canine aortic
smooth muscle cells. Total RNA was isolated and analyzed by RT-PCR to
measure iNOS and GAPDH mRNA levels as described in
METHODS. Cells were incubated either
in absence of LPS (Con), in LPS alone (1 µg/ml), or in LPS + dexamethasone (LPS/DEX; 1 µg/ml) for 48 h. Incubation of cells with
dexamethasone decreased induction of iNOS mRNA by LPS.
|
|
Northern Blot Analysis
Total RNA from canine aortic smooth muscle cells grown in the absence
(control) or presence of LPS (1 µg/ml) was resolved by
formaldehyde gel electrophoresis, transferred to a nylon membrane, and
analyzed by hybridization with the fragment 2 cDNA probe (F2, Fig.
1). A hybridization band with an estimated mRNA size of 4.4 kb under
high stringency was observed. This band was undetectable in RNA from
control cells (Fig. 5).
View larger version (74K):
[in this window]
[in a new window]
|
Fig. 5.
Northern blot analysis of canine aortic smooth muscle iNOS. Total RNA
(20 µg per lane) was isolated from canine aortic smooth muscle cells
incubated in absence (Con) or presence of LPS (1 µg/ml) for 48 h.
|
|
Amplification and Cloning of NOS cDNA From Induced Cultured Canine
Aortic Smooth Muscle Cells
No DNA fragments were detectable with the use of template RNA from
cells not treated with LPS (data not shown). Six overlapping fragments,
F1-F6 (Table 1), were produced with primers that encompass the
entire coding region of the human iNOS cDNA (Fig.
6).
View larger version (53K):
[in this window]
[in a new window]
|
Fig. 6.
Reaction products of RT-PCR were performed using total RNA isolated
from canine aortic smooth muscle cells induced with LPS as template
with indicated oligonucleotide primers (see Table 1). Ten microliters
of each fifty-microliter RT-PCR reaction mixture were analyzed.
Lane 1: primers
1a and 1b.
Lane 2: primers
2a and 2b.
Lane 3: primers
3a and 3b.
Lane 4: primers
4a and 4b.
Lane 5: primers
5a and 5b.
Lane 6: primers
6a and 6b.
|
|
DNA Sequence of Amplified Canine Aortic Smooth Muscle Cell iNOS cDNA
The sequence of the canine aortic smooth muscle cell iNOS cDNA was
determined by sequencing the PCR products. A contiguous full-length
construct of a canine aortic iNOS cDNA sequence of 4,050 bp was
compiled from sequences of the overlapping PCR products. The sequence
contains a single open reading frame beginning with an ATG methionine
codon at position 1. Comparison with other NOS sequences indicates that
the canine iNOS cDNA sequenced from explants of aorta shares 84%
homology with human iNOS, 81% with mouse iNOS, 81% with rat iNOS, and
61% with bovine ecNOS. Similar to other reported sequences for NOS (2,
3, 15), NADPH, flavin mononucleotide, and FAD binding regions are
highly conserved (Fig. 7). The
encoded protein contains 1,154 amino acids (Fig.
8). The GenBank accession number is
AF077821.
View larger version (84K):
[in this window]
[in a new window]
|
Fig. 8.
Nucleotide and deduced amino acid sequences of canine aortic smooth
muscle cell iNOS. Sequence numbering begins at 1st potential initiating
methionine codon. Deduced amino acid sequence is shown using
single-letter code.
|
|
| |
DISCUSSION |
The results of this study report the molecular cloning of a cDNA
encoding iNOS from explanted canine aortic smooth muscle cells. The
identity of this clone as iNOS is demonstrated by
1) induction of expression by LPS,
2) decrease of induction by LPS in
the presence of dexamethasone, 3)
the homology to iNOS cDNA from other species, and
4) increases in
NOx in the culture medium following induction by LPS.
LPS stimulation of iNOS in canine tissue is in agreement with other in
vitro studies using endothelial cells (24), macrophages (6),
hepatocytes (10), and rat smooth muscle cells (26). Attenuation of the
expression of iNOS by dexamethasone suggests that the regulation of
iNOS in canine tissue is similar to that of other mammalian species.
The high degree of similarity among all iNOS mRNA suggests that there
is a unique gene encoding iNOS in the mammalian genome and that
mismatches may be a consequence of species or strain differences.
However, the possibility of tissue-specific posttranslational
variations in the inducible isoform cannot be excluded.
Regulation of NOS isoforms may not be the same between different
phenotypic forms of smooth muscle cells in vivo. Smooth muscle cells in
blood vessels express a contractile phenotype with an abundance of
microfilaments. However, proliferating cells in vitro, originating and
isolated from arteries, express a synthetic phenotype with a higher
volume fraction of cell organelles for de novo protein synthesis and
thereby express growth and transcription factors (27). A problem with
identifying and localizing the various NOS isoforms in canine tissue is
the lack of a specific antibody for iNOS. The use of the cloned product
of the gene identified in this study will enable future studies to
particularly address differential regulation of NOS isoforms in vivo as
they may be expressed during allograft rejection, vascular remodeling,
and/or response to injury (28).
| |
ACKNOWLEDGEMENTS |
The authors thank Kevin Rud for performing measurements of
NOx and Dr. Norman Eberhardt for
expert comments on the manuscript.
| |
FOOTNOTES |
This work was supported by a grant from the Mayo Foundation.
Address for reprint requests: V. M. Miller, Dept. of Surgery, Mayo
Clinic and Foundation, 200 First St. S.W., Rochester, MN 55905.
Received 29 December 1997; accepted in final form 4 June 1998.
| |
REFERENCES |
1.
Braman, R. S.,
and
S. A. Hendrix.
Nanogram nitrite and nitrate determination in environmental and biological materials by vanadium (III) reduction with chemiluminescence detection.
Anal. Chem.
61:
2715-2718,
1989[Medline].
2.
Bredt, D. S.,
P. M. Hwang,
C. E. Glatt,
C. Lowenstein,
R. R. Reed,
and
S. H. Snyder.
Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase.
Nature
351:
714-718,
1991[Medline].
3.
Chartrain, N. A.,
D. A. Geller,
P. P. Koty,
N. F. Sitrin,
A. K. Nussler,
E. P. Hoffman,
T. R. Billiar,
N. I. Hutchinson,
and
J. S. Mudgett.
Molecular cloning, structure, and chromosomal localization of the human inducible nitric oxide synthase gene.
J. Biol. Chem.
269:
6765-6772,
1994[Abstract/Free Full Text].
4.
Corbett, J. A.,
M. A. Sweetland,
J. L. Wang,
J. R. Lancaster, Jr.,
and
M. L. McDaniel.
Nitric oxide mediates cytokine-induced inhibition of insulin secretion by human islets of Langerhans.
Proc. Natl. Acad. Sci. USA
90:
1731-1735,
1993[Abstract/Free Full Text].
5.
Cunha, F. Q.,
W. Y. Weiser,
J. R. David,
D. W. Moss,
S. Moncada,
and
F. Y. Liew.
Recombinant migration inhibitory factor induces nitric oxide synthase in murine macrophages.
J. Immunol.
150:
1908-1912,
1993[Abstract].
6.
Di Rosa, M.,
M. Radomski,
R. Carnuccio,
and
S. Moncada.
Glucocorticoids inhibit the induction of nitric oxide synthase in macrophages.
Biochem. Biophys. Res. Commun.
172:
1246-1252,
1990[Medline].
7.
Eizirik, D. L.,
A. Bjorklund,
and
N. Welsh.
Interleukin-1-induced expression of nitric oxide synthase in insulin-producing cells is preceded by c-fos induction and depends on gene transcription and protein synthesis.
FEBS Lett.
317:
62-66,
1993[Medline].
8.
Galea, E.,
D. L. Feinstein,
and
D. J. Reis.
Induction of calcium-independent nitric oxide synthase activity in primary rat glial cultures.
Proc. Natl. Acad. Sci. USA
89:
10945-10949,
1992[Abstract/Free Full Text].
9.
Geller, D. A.,
C. J. Lowenstein,
R. A. Shapiro,
A. K. Nussler,
M. Di Silvio,
S. C. Wang,
D. K. Nakayama,
R. L. Simmons,
S. H. Snyder,
and
T. R. Billiar.
Molecular cloning and expression of inducible nitric oxide synthase from human hepatocytes.
Proc. Natl. Acad. Sci. USA
90:
3491-3495,
1993[Abstract/Free Full Text].
10.
Geller, D. A.,
A. K. Nussler,
M. Di Silvio,
C. J. Lowenstein,
R. A. Shapiro,
S. C. Wang,
R. L. Simmons,
and
T. R. Billiar.
Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes.
Proc. Natl. Acad. Sci. USA
90:
522-526,
1993[Abstract/Free Full Text].
11.
Goreau, O.,
M. Lepoivre,
F. Becquet,
and
Y. Courtois.
Differential regulation of inducible nitric oxide synthase by fibroblast growth factors and transforming growth factor 
in bovine retinal pigmented epithelial cells: inverse correlation with cellular proliferation.
Proc. Natl. Acad. Sci. USA
90:
4276-4280,
1993[Abstract/Free Full Text].
12.
Heck, D. E.,
D. L. Laskin,
C. R. Gardner,
and
J. D. Laskin.
Epidermal growth factor suppresses nitric oxide and hydrogen peroxide production by keratinocytes.
J. Biol. Chem.
267:
21277-21280,
1992[Abstract/Free Full Text].
13.
Janssens, S. P.,
A. Shimouchi,
T. Quertermous,
D. B. Bloch,
and
K. D. Bloch.
Cloning and expression of cDNA encoding human endothelium-derived relaxing factor/nitric oxide synthase.
J. Biol. Chem.
267:
14519-14522,
1992[Abstract/Free Full Text].
14.
Koide, M.,
Y. Kawahara,
T. Tsuda,
and
M. Yokoyama.
Cytokine-induced expression of an inducible type of nitric oxide synthase gene in cultured vascular smooth muscle cells.
FEBS Lett.
318:
213-217,
1993[Medline].
15.
Lamas, S.,
P. A. Marsden,
G. K. Li,
P. Tempst,
and
T. Michel.
Endothelial nitric oxide synthase: Molecular cloning and characterization of a distinct constitutive enzyme isoform.
Proc. Natl. Acad. Sci. USA
89:
6348-6352,
1992[Abstract/Free Full Text].
16.
Lorsbach, R. B.,
M. J. Murphy,
C. J. Lowenstein,
S. H. Snyder,
and
S. W. Russell.
Expression of the nitric oxide synthase gene in mouse macrophages activated for tumor cell killing.
J. Biol. Chem.
268:
1908-1913,
1997[Abstract/Free Full Text].
17.
Marsden, P. A.,
K. T. Schappert,
H. S. Chen,
M. Flowers,
C. L. Sundell,
J. N. Wilcox,
S. Lamas,
and
T. Michel.
Molecular cloning and characterization of human endothelial nitric oxide synthase.
FEBS Lett.
307:
287-293,
1992[Medline].
18.
Menon, N. K.,
A. Wolf,
M. Zehetgruber,
and
R. J. Bing.
An improved chemiluminescence assay suggests non nitric oxide-mediated action of lysophosphatidylcholine and acetylcholine.
Proc. Soc. Exp. Biol. Med.
37:
316-319,
1989.
19.
Nakane, M.,
H. H. H. W. Schmidt,
J. S. Pollock,
U. Forstermann,
and
F. Murad.
Cloned human brain nitric oxide synthase is highly expressed in skeletal muscle.
FEBS Lett.
316:
175-180,
1993[Medline].
20.
Nathan, C.
Nitric oxide as a secretory product of mammalian cells.
FASEB J.
6:
3051-3064,
1992[Abstract].
21.
Nishida, K.,
D. G. Harrison,
J. P. Navas,
A. A. Fisher,
S. P. Dockery,
M. Uematsu,
R. M. Nerem,
R. W. Alexander,
and
T. J. Murphy.
Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase.
J. Clin. Invest.
90:
2092-2096,
1992.
22.
Nunokawa, Y.,
N. Ishida,
and
S. Tanaka.
Cloning of inducible nitric oxide synthase in rat vascular smooth muscle cells.
Biochem. Biophys. Res. Commun.
191:
89-94,
1993[Medline].
23.
Nussler, A. K.,
M. Di Silvio,
T. R. Billiar,
R. A. Hoffman,
D. A. Geller,
R. Selby,
J. Madariaga,
and
R. L. Simmons.
Stimulation of the nitric oxide synthase pathway in human hepatocytes by cytokines and endotoxin.
J. Exp. Med.
176:
261-264,
1992[Abstract/Free Full Text].
24.
Radomski, M. W.,
R. M. J. Palmer,
and
S. Moncada.
Glucocorticoids inhibit the expression of an inducible, but not the constitutive, nitric oxide synthase in vascular endothelial cells.
Proc. Natl. Acad. Sci. USA
87:
10043-10047,
1990[Abstract/Free Full Text].
25.
Sessa, W. C.,
J. K. Harrison,
C. M. Barber,
D. Zend,
M. D. Durieus,
D. D. D'Angelo,
K. R. Lynch,
and
M. J. Deach.
Molecular cloning and expression of a cDNA encoding cell nitric oxide synthase.
J. Biol. Chem.
267:
15274-15276,
1992[Abstract/Free Full Text].
26.
Sirsjo, A.,
P. Soderkvist,
T. Sundqvist,
M. Carlsson,
M. Ost,
and
A. Gidlof.
Different induction mechanisms of mRNA for inducible nitric oxide synthase in rat smooth muscle cells in culture and in aortic strips.
FEBS Lett.
338:
191-196,
1994[Medline].
27.
Thyberg, J.,
U. Hedin,
M. Sjolund,
L. Palmberg,
and
B. A. Bottger.
Regulation of differentiated properties and proliferation of arterial smooth muscle cells.
Arteriosclerosis
10:
966-990,
1990[Free Full Text].
28.
Wang, X,
D. A. Lewis,
H.-K. Kim,
H. D. Tazelaar,
Y.-S. Park,
C. G. A. McGregor,
and
V. M. Miller.
Alterations in mRNA for inducible and endothelial nitric oxide synthase and plasma nitric oxide with rejection and/or infection of allotransplanted lungs.
Transplantation
66:
1-6,
1998[Medline].
29.
Wood, E. R.,
H. Berger, Jr.,
P. A. Sherman,
and
E. G. Lapetina.
Hepatocytes and macrophages express an identical cytokine inducible nitric oxide synthase gene.
Biochem. Biophys. Res. Commun.
191:
767-774,
1993[Medline].
Am J Physiol Heart Circ Physiol 275(4):H1122-H1129
0002-9513/98 $5.00
Copyright © 1998 the American Physiological Society
Top
Abstract
Introduction
