1 Department of Veterinary Pharmacology, Graduate School of Agriculture and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657; and 2 Department of Anatomy and Cell Biology, Nagoya University Graduate School of Medicine, Tsurumai, Nagoya 466-8550, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We investigated the effect of lipopolysaccharide (LPS) on the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in muscularis resident macrophages of rat intestine in situ. When the tissue was incubated with LPS for 4 h, mRNA levels of iNOS and COX-2 were increased. The majority of iNOS and COX-2 proteins appeared to be localized to the dense network of muscularis resident macrophages immunoreactive to ED2. LPS treatment also increased the production of nitric oxide (NO), PGE2, and PGI2. The increased expression of iNOS mRNA by LPS was suppressed by indomethacin but not by NG-monomethyl-L-arginine (L-NMMA). The increased expression of COX-2 mRNA by LPS was affected neither by indomethacin nor by L-NMMA. Muscle contractility stimulated by 3 µM carbachol was significantly inhibited in the LPS-treated muscle, which was restored by treatment of the tissue with L-NMMA, aminoguanidine, indomethacin, or NS-398. Together, these findings show that LPS increases iNOS expression and stimulates NO production in muscularis resident macrophages to inhibit smooth muscle contraction. LPS-induced iNOS gene expression may be mediated by autocrine regulation of PGs through the induction of COX-2 gene expression.
inducible nitric oxide synthase; intestinal motility; nitric oxide; prostaglandin
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IT IS WELL KNOWN that lipopolysaccharide (LPS) or inflammatory cytokines such as interleukin (IL)-1 drive both cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) gene expression in macrophages, resulting in increases in the release of PGs and nitric oxide (NO) (2, 13, 18, 21, 24). Some reports indicate that there may be cross-talk between the COX-2 and iNOS genes in macrophages. For example, in the J744 macrophage cell line, PGE2, PGI2, and 8-bromoadenosine 3',5'-cyclic monophosphate suppress iNOS protein expression induced by LPS (31). Also, in LPS-stimulated J744 cells (27), low and high concentrations of PGE2 upregulate and downregulate iNOS expression, respectively. In the ANA-1 macrophage cell line, release of PGE2 induced by LPS was blocked by an iNOS inhibitor, aminoguanidine (32). In addition, in rat lung, COX-2 expression by acute hypoxia was suppressed by sodium nitroprusside and stimulated by dibutyryl cAMP (6). These reports suggest that although the cross-talk between iNOS and COX-2 exists in macrophage cells, their relationship is complex and cell type specific (see Ref. 15 for review).
Immunologically active cells, including macrophages in the intestinal mucosa, are thought to be closely related to the inflammatory bowel diseases. Recently, macrophages in the muscle layer were reported as a population distinct from mucosal macrophages in mammalian intestines (20, 25, 26). These macrophages are regularly distributed in the subserosa, at the level of the myenteric plexus, and inside the muscle layer. Although their unique distribution and great number imply an important role in the muscle layer, the function of those muscularis resident macrophages remains to be clarified and their pathological effects on the gastrointestinal tract have not yet been demonstrated. Recently, Schroeder et al. (34) and Eskandari et al. (11) reported that the macrophages induce NOS (iNOS) to release NO, which results in suppressed smooth muscle motility. These reports strongly suggest that the intestinal muscularis resident macrophage network should be an important mediator of endotoxin-induced gut dysmotility. However, the interaction between iNOS and COX-2 is not well investigated in the intestinal resident macrophages during endotoxemia.
The aim of the present study was to clarify the cross-talk between iNOS and COX-2 in the muscularis resident macrophages of rat small intestine stimulated by LPS. We found that LPS-induced iNOS gene expression might be mediated by autocrine regulation of PGs through the induction of COX-2 gene expression. The released NO plays an important role in mediating intestinal circular muscle contractility in the presence of endotoxin.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Muscle preparation and measurement of muscle tension. Male Wistar rats were stunned and killed in accordance with the use and treatment of animals outlined in the Guide to Animal Use and Care of the University of Tokyo. The ileum from each rat was then dissected into 2- to 3-cm-long segments and cut open along the mesenteric attachment, and the mucosa and submucosa were removed. The remaining muscle layers were incubated with physiological salt solution (PSS) containing (in mM) 136.9 NaCl, 5.4 KCl, 1.0 MgCl2, 23.8 NaHCO3, 1.5 CaCl2, and 5.5 glucose. PSS was aerated with 95% O2-5% CO2 to adjust pH to 7.3 at 37°C.
Muscle tension was recorded isometrically using a force-displacement transducer. Each muscle strip was attached to a holder in an organ bath (10 ml) containing PSS with a resting tension of 10 mN and equilibrated for 30 min to obtain a stable contractility induced by 72.7 mM KCl. The muscle strips were treated with PSS alone or with 100 µg/ml LPS dissolved in PSS in the organ bath for 4 h. The contraction induced by 3 µM carbachol before LPS treatment was considered as the reference response (100%).Quantitative RT-PCR analysis. Total RNA was extracted from the circular smooth muscle strips by the acid guanidinium isothiocyanate-phenol-chloroform method (7), and the concentration of RNA was adjusted to 1 µg/µl with RNase-free distilled water. Quantitative RT-PCR was performed as follows. First-strand cDNA was synthesized using random 9-mer primer and avian myeloblastosis virus (AMV) Reverse Transcriptase XL at 30°C for 10 min, 55°C for 30 min, 99°C for 5 min, and 4°C for 5 min. PCR amplification was performed by the hot starting method using Taq Gold (Perkin-Elmer, Branchburg, NJ). The oligonucleotide primers for iNOS designed from rat macrophage (37) were CTA CCT ACC TGG GGA ACA CCT GGG (forward) and GGA GGA GCT GAT GGA GTA GTA GCG G (reverse), and the suitable size of synthesized cDNA was 442 bp. The oligonucleotide primers for COX-2 designed from rat (12) were CTG TAT CCC GCC CTG CTG GTG (forward) and ACT TGC GTT GAT GGT GGC TGT CTT (reverse), and the suitable size of synthesized cDNA was 282 bp. The oligonucleotide primers for rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (14) used were TAC CAG CCG GGG GAC CAC (forward) and CGA GCT GAC AGA GTA GTA (reverse) or CTG GCA TGG CCT TCG TGT TC (forward) and CTT GCT CTC TCA GTA TCC TTG CTG GGC T (reverse). The suitable sizes of the synthesized cDNA of GAPDH were 308 bp in the former primers and 366 bp in the latter primers. After initial denaturation at 95°C for 10 min, 24-40 cycles (4-cycle interval) of amplifications at 94°C for 40 s, 55°C for 1.0 min, and 72°C for 1.5 min were performed using a thermal cycler (Takara PCR Thermal Cycler MP, Takara Biomedicals, Tokyo, Japan). PCR products in each cycle were electrophoresed on 2% agarose gel containing 0.1% ethidium bromide. The possible contamination of DNA was excluded by PCR with total RNA without the reverse transcription step. Detectable fluorescent bands were visualized by an ultraviolet transilluminator using FAS-III (Toyobo, Tokyo, Japan), and the area was measured using NIH Image software.
Immunohistochemistry. For immunohistochemistry, muscle strips were fixed with either 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4 for detecting iNOS or in Zamboni solution for detecting cyclooxygenase-1 (COX-1) and COX-2, and they were processed for whole mount preparations. Samples were incubated overnight at 4°C with anti-iNOS antibody (1:1,000; Transduction Labs, Lexington, KY), anti-rat resident macrophage antibody (9) (ED2, 1:500; Serotec, Oxford, UK), anti-COX-1 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) or anti-COX-2 antibody (1:50; Santa Cruz Biotechnology). They were then treated with an ABC kit (Vector, Burlingame, CA), and reaction products were detected by 0.03% diaminobenzidine and H2O2 in 0.05 M Tris · HCl buffer (pH 7.6). To check the specificity of the immunohistochemistry tests, tissues in which primary antibodies were omitted from the initial incubation were also prepared.
Muscle strips were also double-stained with the combination of anti-iNOS antibody and ED2 as well as with anti-COX-2 and ED2. iNOS and COX-2 or ED2 were detected with Texas red-conjugated streptavidin (1:100; Vector), or FITC-conjugated anti-mouse IgG (1:100; Sigma, St. Louis, MO), respectively. Colocalization was analyzed using a confocal laser scanning microscope (MRC-1024; Bio-Rad, Hercules, CA). iNOS and COX-2 could not be stained simultaneously because the fixing condition is different for each protein.Measurement of released PGs. Each muscle strip was attached to a holder in a organ bath (2 ml) containing PSS and equilibrated for 30 min. The muscle strips were then treated with or without 100 µg/ml LPS for 4 h at 37°C. After incubation with PSS alone or PSS + LPS for 4 h, 50 µl of the solution were removed, and the released PGE2 and PGI2 were measured using an enzyme immunoassay system (Amersham Pharmacia Biotech, Tokyo, Japan). Released PGs were calculated using the standard assay in the kit and expressed as picograms per milligram of wet weight.
Measurement of released NO. Each muscle strip was treated with or without LPS under the same conditions as those for the measurements of PGs. After incubation of the muscle strips with PSS alone or PSS + LPS for 4 h, 50 µl of the solution were sampled for measurement of released NO from the muscle strips. Total nitrite was measured as an indicator of released NO by means of colorimetric assay for determination of total nitrite using Greiss reagent (Bioxytech nitric oxide assay kit, Oxis International, Portland, OR). The released nitrite content was expressed as micromoles per milligram of wet weight.
Statistics. Numerical data are expressed as means ± SE. Differences between mean values were evaluated by Student's t-test, and, where appropriate, analysis of variance (one-way ANOVA; Bonferroni's test) was performed.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of LPS on induction of iNOS
and COX-2 mRNAs.
RT-PCR analysis was performed on RNA extracted from tissue treated with
or without LPS (100 µg/ml) for 4 h at 37°C in the organ bath.
As shown in Fig. 1, expression of the
RT-PCR product encoding GAPDH (308 or 366 bp) was identical in the
control and LPS-treated tissues. In contrast, iNOS (442 bp) and COX-2
(282 bp) mRNAs were strongly expressed in LPS-treated tissue compared with control tissue. In the quantitative RT-PCR analysis, 4-h treatment
of the tissue with LPS (100 µg/ml) significantly increased the
expression of iNOS and COX-2 mRNA levels at a constant level of the
housekeeping gene GAPDH (Fig. 1, right). As shown in Fig. 2, the mRNA level of COX-2 began to
increase at 30 min and reached a plateau at 60 min. On the other hand,
iNOS mRNA started to increase at 90 min and reached its peak at >240
min. In muscle incubated with normal PSS for 4 h, in contrast,
mRNAs of COX-2 or iNOS did not increase.
|
|
Immunohistochemistry of iNOS and
COX-2.
Because intestinal tissue, even after separation from the mucosal
layer, is composed of multiple types of cells, we performed immunohistochemistry using anti-iNOS and anti-COX-2 antibody. In the
whole-mount tissue preparation without LPS treatment, iNOS and COX-2
proteins were not detected in the muscle layer; however, in muscle
treated with LPS for 4 h, cells expressing iNOS (Fig. 3A) or COX-2 (Fig.
3B) immunoreactivities were observed among the smooth muscle
cell layer. iNOS- or COX-2-immunoreactive cells were apparently
muscularis resident macrophages but not smooth muscle cells.
|
|
Effect of LPS on NO release.
We next measured total nitrite content as an indicator of NO released
from the intestinal muscles in the absence or presence of LPS for
4 h (Fig. 5A). In the absence of LPS, the total
released nitrite content was 0.15 ± 0.15 µmol/mg wet wt
(n = 4). After incubation of muscle strips with LPS for
4 h, the total nitrite content was significantly increased, as
shown in Fig. 5A (2.16 ± 0.73 µmol/mg wet wt; P < 0.01, n = 4).
|
Effect of LPS on PG release. Because LPS induces COX-2 mRNA and protein in myenteric resident macrophages, we next examined the amount of PGs released from the tissue using an enzyme immunoassay method (Fig. 5B). In muscle not stimulated by LPS, the amounts of PGE2 and PGI2 were 1.14 ± 0.32 (n = 4) and 2.53 ± 0.62 (n = 4) pg/mg wet wt, respectively. After the incubation of muscle tissue with LPS for 4 h, released PGE2 and PGI2 were increased to 10.5 ± 2.64 (P < 0.01) and 6.22 ± 0.32 (P < 0.01) pg/mg wet wt, respectively.
Effects of indomethacin and L-NMMA on
the expression of iNOS and COX-2
mRNAs.
We next examined the effects of a COX inhibitor, indomethacin, and a
nonselective NOS inhibitor, L-NMMA, on the expression of
COX-2 and iNOS mRNAs. As shown in Fig. 6,
COX-2 mRNA levels increased by LPS were not affected by treatment of
the tissue with indomethacin (10 µM) or L-NMMA (300 µM). In contrast, the increased iNOS mRNA level induced by LPS was
decreased by treatment with indomethacin but not by L-NMMA.
|
Effects of LPS on muscle contractions. Finally, we examined the effects of LPS on smooth muscle contractility. In control tissue, 3 µM of carbachol induced contractions, the amplitude of which was almost identical to that of the contractions induced by 72.7 mM KCl (93.5 ± 2.17% of high K+-induced contractions; n = 43). Treatment of the tissue with PSS alone for 4 h slightly inhibited the 3 µM carbachol-induced contractions (72.3 ± 7.35% of carbachol-induced contraction before treatment of PSS for 4 h; n = 9). Treatment of muscle strips with a nonselective NOS inhibitor, L-NMMA (300 µM), for 4 h did not reverse this contraction (78.1 ± 11.0% of carbachol-induced contraction before treatment of PSS for 4 h; n = 10). Treatment of the tissue with 100 µg/ml LPS for 4 h inhibited the carbachol-induced contraction. Combined treatment with LPS and L-NMMA for 4 h restored the suppressed muscle force. Indomethacin (10 µM) also restored the attenuated carbachol-induced contraction to the level obtained in the muscle without LPS treatment. Treatment of muscle strips with LPS and aminoguanidine (300 µM), a selective iNOS inhibitor, or NS-398 (10 µM), a selective COX-2 inhibitor, for 4 h also suppressed the inhibitory effect of LPS. The effects of indomethacin, NS-398, L-NMMA, and aminoguanidine on LPS-induced inhibition of muscle force were not significantly different from each other.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bacterial toxins, particularly endotoxins such as LPS, can activate macrophages to induce iNOS and COX-2 (16-19, 27, 30). In this study, exposure of ileal circular smooth muscle to LPS for 4 h increased mRNA levels of both iNOS and COX-2, as shown in Fig. 1. The majority of iNOS and COX-2 proteins appeared to be restricted in ED2-positive resident macrophages, based on the results of double staining of ED2 and iNOS or COX-2 (Figs. 3 and 4). Similar results in regard to iNOS expression were obtained in our previous work (35). These results indicate that induction of iNOS gene and COX-2 gene was mainly exhibited in muscularis resident macrophages but not in intestinal smooth muscle cells. However, we cannot rule out the possibility of weak induction of these genes in smooth muscle cells, because it is difficult to compare the expression in the smooth muscle layer quantitatively. We also cannot completely rule out the contribution of a minor component of immunological cells such as mast cells, T cells, and natural killer cells within the rat intestinal muscle layer (20). In addition, a minor population of ED2-positive cells expressed neither iNOS nor COX-2, as shown in Fig. 4, indicating the possibility of heterogeneity in the muscularis resident macrophage. Further experiments are necessary to clarify this point.
Consistent with COX-2 mRNA induction and protein expression, incremental releases of PGE2 and PGI2 from the tissue were observed after LPS treatment. We also confirmed the increment of NO in the medium treated with LPS, as an indicator of NO, as shown in Fig. 5A.
It has been reported that expression of iNOS is stimulated by cAMP elevation in many cell types. In macrophages, however, the effect of increased cAMP on iNOS expression is variable (15). Therefore, we next examined the interaction between iNOS and COX-2 in muscularis resident macrophages in situ. In the presence of a nonselective COX inhibitor, indomethacin (10 µM), the induction of iNOS mRNA by LPS was completely inhibited whereas the induction of COX-2 mRNA was unaffected. On the other hand, neither iNOS mRNA nor COX-2 mRNA expression was affected by a nonselective NOS inhibitor, L-NMMA. These results suggest that expression of COX-2 may be essential for LPS-induced iNOS gene expression in muscularis resident macrophages. In the time courses of mRNA expression in iNOS and COX-2, the increment of COX-2 mRNA expression was faster than that of iNOS mRNA expression, supporting the suggestion. In agreement with these results, Milano et al. (27) reported that the expression of iNOS is stimulated by exogenously applied PGE2 in the J774 murine macrophage cell line. In addition, most recently it was reported that LPS increased cAMP level via COX-2 induction and PGE2 production, resulting in iNOS expression to produce NO in RAW264.7 macrophages (5).
Also working with a murine macrophage cell line, ANA-1, Perkins and Kniss (32) reported that NO is necessary for maintaining prolonged COX-2 gene expression. In the present study, however, the iNOS and COX-2 mRNA levels stimulated by LPS were not affected by the inhibition of NO production with L-NMMA, suggesting that, in the intestinal resident macrophage, NO does not regulate the iNOS and COX-2 genes.
We also observed that after treatment of intestinal tissue with LPS for
4 h in vitro, carbachol-induced contraction was significantly inhibited. The reduced contractility was restored by L-NMMA
(Fig. 7) without changing the iNOS mRNA
level (Fig. 6), suggesting that increased production of NO is
responsible for the reduced smooth muscle contractility. A selective
iNOS inhibitor, aminoguanidine, also suppressed the inhibitory effect
of LPS on carbachol-induced contractions. In addition, we showed the
increment of released NO in muscle treated with LPS for 4 h (Fig.
5A).
|
Interestingly, the contractility reduced by LPS was also restored by indomethacin. These results support the finding that indomethacin inhibited the LPS-induced increase in iNOS mRNA, as shown in Fig. 6. We further demonstrated that a selective COX-2 inhibitor, NS-398 (10 µM), also restored the LPS-induced inhibitory effects of muscle force (Fig. 7) and that PGE2 and PGI2 were released in LPS-treated muscles (Fig. 5B). In addition, immunohistochemical analysis indicated that the level of COX-1 protein expression did not change after LPS treatment. These results suggest that the elevated production of PGE2 and PGI2 may be attributable to COX-2 but not to COX-1.
As for the molecular mechanism by which PGs upregulate iNOS induction,
it has been reported that the transcription of iNOS genes in
macrophages is regulated mainly by the nuclear factor (NF)-B
transcription family (22, 38). The promoter of the murine
gene encoding iNOS contains two
B binding sites (23), and protein binding to the
B binding sites is necessary to confer inducibility by LPS (38). Thus PGs produced by LPS
treatment may bind to the EP3 receptor to stimulate the
adenylate cyclase/cAMP pathway in the resident macrophage. In
rat intestinal resident leukocytes, ED9- and CD14 (specific antibody
for LPS receptor)-positive leukocytes make up ~55% of total
leukocytes (5), suggesting that these ED9-positive cells,
which may be ED2-positive resident macrophages, should be target cells
of LPS in the smooth muscle layer. As is reported in cultured
macrophage cell lines (28, 29), activated cAMP-dependent
protein kinase (PKA) phosphorylates NF-
B inhibitory protein (I
B)
to induce the activation of NF-
B. A pathway that is independent of
the upregulation of NF-
B has also been reported (16,
17). Further studies are required to clarify the molecular
mechanism of iNOS expression in the muscularis resident macrophage.
However, most recently, Chen and co-workers (5)
demonstrated that LPS increases cAMP via COX-2 induction and
PGE2 production, resulting in PKA activation to induce iNOS expression and NO production via NF-
B activation in the RAW264.7 macrophage cell line. This report strongly supports our conclusions in
muscularis resident macrophages.
In the present study, we found that LPS stimulates muscularis resident macrophages to induce COX-2 and iNOS, resulting in inhibited muscle motility in rat ileum. However, in human mononuclear phagocytes, the ability to generate NO stimulated by LPS is lower than in mouse and rat macrophages, indicating the possibility that human macrophages cannot generate enough NO (8, 36). In contrast, a number of recent studies documented that the capability for iNOS activity can be induced in some human macrophage cell lines (33) or in the presence of unconventional stimuli such as infection with human immunodeficiency virus type 1 (4). These reports indicate that iNOS expression may depend on the state of macrophage differentiation, the complex of stimuli, or tissue location. In addition, LPS alone has been shown to stimulate COX-2 induction in several cell lines of human macrophages (1, 3, 10). These results suggest that LPS-stimulated muscularis resident macrophages may play a critical role in the pathogenesis of gastrointestinal dysfunction during sepsis and multisystem organ failure in humans.
In summary, LPS induces iNOS gene expression in the muscularis resident macrophage in rat small intestine and releases NO to inhibit smooth muscle contractility. The effect of LPS seems to be mediated by the upregulation of COX-2 followed by the production of PGs that stimulates iNOS expression in an autocrine manner. These findings implicate the role of iNOS and COX-2 in the muscularis resident macrophage network in the pathogenesis of gastrointestinal dysfunction during sepsis and multisystem organ failure.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Yakult Bio-Science Foundation, and Takeda Science Foundation, Japan.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: M. Hori, Dept. of Veterinary Pharmacology, Graduate School of Agriculture and Life Sciences, Univ. of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan (E-mail: ahori{at}mail.ecc.u-tokyo.ac.jp).
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.
Received 14 February 2000; accepted in final form 13 November 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Arias-Negrete, S,
Keller K,
and
Chadee K.
Proinflammatory cytokines regulate cyclooxygenase-2 mRNA expression in human macrophages.
Biochem Biophys Res Commun
208:
582-589,
1995[ISI][Medline].
2.
Barratt, GM,
Raddassi K,
Petit JF,
and
Tenu JP.
MDP and LPS act synergistically to induce arginine-dependent cytostatic activity in rat alveolar macrophages.
Int J Immunopharmacol
13:
159-165,
1991[ISI][Medline].
3.
Barrios-Rodiles, M,
and
Chadee K.
Novel regulation of cyclooxygenase-2 expression and prostaglandin E2 production by IFN- in human macrophages.
J Immunol
161:
2441-2448,
1998
4.
Bukrinsky, MI,
Nottet HS,
Schmidtmayerova H,
Dubrovsky L,
Flanagan CR,
Mullins ME,
Lipton SA,
and
Gendelman HE.
Regulation of nitric oxide synthase activity in human immunodeficiency virus type 1 (HIV-1)-infected monocytes: implications for HIV-associated neurological disease.
J Exp Med
181:
735-745,
1995[Abstract].
5.
Chen, C-C,
Chiu K-T,
Sun Y-T,
and
Chen W-C.
Role of the cyclic AMP-protein kinase A pathway in lipopolysaccharide-induced nitric oxide synthase expression in RAW264.7 macrophages. Involvement of cyclooxygenase-2.
J Biol Chem
274:
31559-31564,
1999
6.
Chida, M,
and
Voelkel NF.
Effects of acute and chronic hypoxia on rat lung cyclooxygenase.
Am J Physiol Lung Cell Mol Physiol
270:
L872-L878,
1996
7.
Chomczynski, P,
and
Sacchi N.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162:
156-159,
1987[ISI][Medline].
8.
Denis, M.
Human monocytes/macrophages: NO or no NO?
J Leukoc Biol
55:
682-684,
1994[Abstract].
9.
Dijkstra, CD,
Dopp EA,
Joling P,
and
Kraal G.
The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulations in the rat recognized by monoclonal antibodies ED1, ED2 and ED3.
Immunology
54:
589-599,
1985[ISI][Medline].
10.
Eligini, S,
Colli S,
Basso F,
Sironi L,
and
Tremoli E.
Oxidized low density lipoprotein suppresses expression of inducible cyclooxygenase in human macrophages.
Arterioscler Thromb Vasc Biol
19:
1719-1725,
1999
11.
Eskandari, MK,
Kalff JC,
Billiar TR,
Lee KKW,
and
Bauer AJ.
LPS-induced muscularis macrophage nitric oxide suppresses rat jejunal circular muscle activity.
Am J Physiol Gastrointest Liver Physiol
277:
G478-G486,
1999
12.
Feng, L,
Sun W,
Xia Y,
Tang WW,
Chanmugam P,
Soyoola E,
Wilson CB,
and
Hwang D.
Cloning two isoforms of rat cyclooxygenase: differential regulation of their expression.
Arch Biochem Biophys
307:
361-368,
1993[ISI][Medline].
13.
Forstermann, U,
Gath I,
Schwarz P,
Closs EI,
and
Kleinert H.
Isoforms of nitric oxide synthaseproperties, cellular distribution and expressional control.
Biochem Pharmacol
50:
1321-1332,
1995[ISI][Medline].
14.
Fort, P,
Marty L,
Piechaczyk M,
el Sabrouty S,
Dani C,
Jeanteur P,
and
Blanchard JM.
Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigenic family.
Nucleic Acids Res
13:
1431-1442,
1985[Abstract].
15.
Galea, E,
and
Feinstein DL.
Regulation of the expression of the inflammatory nitric oxide synthase (NOS2) by cyclic AMP.
FASEB J
13:
2125-2137,
1999
16.
Greenberg, SS,
Ouyang J,
Xhao X,
Xie J,
Wang J-F,
and
Giles TD.
Interaction of ethanol with inducible nitric oxide synthase messenger RNA and protein: direct effects on autacoids and endotoxin in vivo.
J Pharmacol Exp Ther
282:
1044-1054,
1997
17.
Greenberg, SS,
Zhao X,
Wang J-F,
Hua L,
and
Ouyang J.
cAMP and purinergic P2y receptors upregulate and enhance inducible NO synthase mRNA and protein in vivo.
Am J Physiol Lung Cell Mol Physiol
273:
L967-L979,
1997
18.
Hempel, SL,
Monick MM,
He B,
Yano T,
and
Hunninghake GW.
Synthesis of prostaglandin H synthase-2 by human alveolar macrophages in response to lipopolysaccharide is inhibited by decreased cell oxidant tone.
J Biol Chem
269:
32979-32984,
1994
19.
Hu, Y,
Fisette PL,
Denlinger LC,
Guadarrama AG,
Sommer JA,
Proctor RA,
and
Bertics PJ.
Purinergic receptor modulation of lipopolysaccharide signaling and inducible nitric-oxide synthase expression in RAW 264.7 macrophages.
J Biol Chem
273:
27170-27175,
1998
20.
Kalff, JC,
Schwarz NT,
Walgenbach KJ,
Schraut WH,
and
Bauer AJ.
Leukocytes of the intestinal muscularis: their phenotype and isolation.
J Leukoc Biol
63:
683-691,
1998[Abstract].
21.
Kerwin, JF, Jr.,
Lancaster JR, Jr.,
and
Feldman PF.
Nitric oxide: a new paradigm for second messengers.
J Med Chem
38:
4343-4362,
1995[ISI][Medline].
22.
Kleinert, H,
Euchenhofer C,
Ihrig-Biedert I,
and
Forstermann U.
In murine 3T3 fibroblasts, different second messenger pathways resulting in the induction of NO synthase II (iNOS) converge in the activation of transcription factor NF-B.
J Biol Chem
271:
6039-6044,
1996
23.
Lowenstein, CJ,
Alley EW,
Raval P,
Snowman AM,
Snyder SH,
Russell W,
and
Murphy WJ.
Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon and lipopolysaccharide.
Proc Natl Acad Sci USA
90:
9730-9734,
1993[Abstract].
24.
Maier, JAM,
Hla T,
and
Maciag T.
Cyclooxygenase is an immediate-early gene induced by interleukin-1 in human endothelial cells.
J Biol Chem
265:
10805-10808,
1990
25.
Mikkelsen, HB.
Macrophages in the external muscle layers of mammalian intestines.
Histol Histopathol
10:
719-736,
1995[ISI][Medline].
26.
Mikkelsen, HB,
Thuneberg L,
Rumessen JJ,
and
Thoball N.
Macrophage-like cells in the muscularis externa of mouse small intestine.
Anat Rec
213:
77-86,
1985[ISI][Medline].
27.
Milano, S,
Arcoleo F,
Dieli M,
D'Agostino P,
De Nucci G,
and
Cillari E.
Prostaglandin E2 regulates inducible nitric oxide synthase in the murine macrophage cell line J774.
Prostaglandins
49:
105-115,
1995[Medline].
28.
Muroi, M,
and
Suzuki T.
Role of protein kinase A in LPS-induced activation of NF-B proteins of a mouse macrophage-like cell-line, J774.
Cell Signal
5:
289-298,
1993[ISI][Medline].
29.
Novotney, M,
Chang Z-L,
Uchiyama H,
and
Suzuki T.
Protein kinase C in tumoricidal activation of mouse macrophage cell lines.
Biochemistry
30:
5597-5604,
1991[ISI][Medline].
30.
Paug, L,
and
Hoult JRS
Induction of cyclooxygenase and nitric oxide synthase in endotoxin-activated J774 macrophages is differentially regulated by indomethacin: enhanced cyclooxygenase-2 protein expression but reduction of inducible nitric oxide synthase.
Eur J Pharmacol
317:
151-155,
1996[ISI][Medline].
31.
Paug, L,
and
Hoult JRS
Repression of inducible nitric oxide synthase and cyclooxygenase-2 by prostaglandin E2 and other cyclic AMP stimulants in J774 macrophages.
Biochem Pharmacol
53:
493-500,
1997[ISI][Medline].
32.
Perkins, DJ,
and
Kniss DA.
Blockade of nitric oxide formation down-regulates cyclooxygenase-2 and decreases PGE2 biosynthesis in macrophages.
J Leukoc Biol
65:
782-799,
1999.
33.
Rockett, KA,
Brookes R,
Udalova I,
Vidal V,
Hill AVS,
and
Kwiatkowski D.
1,25-Dihydroxyvitamin D3 induces nitric oxide synthase and suppresses growth of Mycobacterium tuberculosis in human macrophage-like cell line.
Infect Immun
66:
5314-5321,
1998
34.
Schroeder, RA,
Delatorre A,
and
Kuo PC.
CD14-dependent mechanism for endotoxin-mediated nitric oxide synthesis in murine macrophages.
Am J Physiol Cell Physiol
273:
C1030-C1039,
1997
35.
Torihashi, S,
Ozaki H,
Hori M,
Kita M,
Ohota S,
and
Karaki H.
Resident macrophages activated by lipopolysaccharide suppress muscle tension and initiate inflammatory response in the gastrointestinal muscle layer.
Histochem Cell Biol
113:
73-80,
2000[ISI][Medline].
36.
Weinberg, JB,
Misukonis MA,
Shami PJ,
Mason SN,
Sauls DL,
Dittman WA,
Wood ER,
Smith GK,
McDonald B,
and
Bachus KE.
Human mononuclear phagocyte inducible nitric oxide synthase (iNOS): analysis of iNOS mRNA, iNOS protein, biopterin, and nitric oxide production by blood monocytes and peritoneal macrophages.
Blood
86:
1184-1195,
1995
37.
Wood, ER,
Berger H, Jr.,
Sherman PA,
and
Lapetina EG.
Hepatocytes and macrophages express an identical cytokine inducible nitric oxide synthase gene.
Biochem Biophys Res Commun
191:
767-774,
1993[ISI][Medline].
38.
Xie, QW,
Kashiwabara Y,
and
Nathan C.
Role of transcription factor NFB/Rel in induction of nitric oxide synthase.
J Biol Chem
269:
4705-4708,
1994