Increased iNOS Expression Research Articles

Enhanced expression of inducible nitric oxide synthase without vasodilator effect in chronically infected lungs.

We hypothesized that abnormal ventilation-perfusion matching in chronically infected lungs was in part due to excess nitric oxide (NO) production after upregulation of inducible NO synthase (iNOS) expression. Rats were anesthetized and inoculated intratracheally with Pseudomonas aeruginosa incorporated into agar beads (chronically infected) or with sterile agar beads (placebo inoculated) and killed 10-15 days later. Immunohistochemistry demonstrated increased expression of iNOS and reduced expression of endothelial NOS (eNOS) in chronically infected compared with placebo-inoculated or noninoculated lungs. In isolated lungs from chronically infected rats, NOS inhibition with N(omega)-nitro-L-arginine methyl ester increased the mean perfusion pressure (14.4 +/- 2.7 mmHg) significantly more than in the placebo-inoculated (4.8 +/- 1.0 mmHg) or noninoculated (5.3 +/- 0.8 mmHg) lungs (P < 0.01). Although the chronically infected lungs were more sensitive to NOS inhibition, further evidence suggested that the increased iNOS expression was not associated with enhanced iNOS activity. Selective inhibitors of iNOS did not produce an increase in vascular resistance similar to that produced by nonselective inhibitors. Accumulation of nitrate/nitrite in the perfusate of isolated lungs was unchanged by chronic infection. Thus although iNOS expression was increased in chronic pulmonary infection, iNOS activity in the intact lung was not. Nonetheless, endogenous NO production was essential to maintain normal vascular resistance in these lungs.

From rats to man: a perspective on dietary L-arginine supplementation in human renal disease.

Experimental studies have shown both therapeutic and detrimental consequences of modifying dietary L-arginine intake in renal diseases which likely reflect the complexity of L-arginine metabolism. L-Arginine intake is semi-essential and provides substrate for a number of L-arginine metabolites involved in renal pathology. Dietary L-arginine restriction has been identified as a key mediator of the beneficial effects of low protein diets on human renal fibrosis. Supplementing dietary L-arginine in renal diseases with increased iNOS expression appears to be detrimental and thus, may be harmful in immune-mediated human kidney disorders. Increasing L-arginine intake is beneficial in experimental models of hypertensive renal disease. Based upon available data, we believe additional questions must be answered experimentally, not only to prevent an adverse outcome in humans, but to enhance our chances of human trials which will result in substantially better amelioration of disease than currently available.

Frequent expression of inducible nitric oxide synthase in esophageal squamous cell carcinomas.

Nitric oxide (NO) plays important biological roles in cardiovascular, nervous and immune systems, and is synthesized by nitric oxide synthase (NOS). Intracellular NO is known to cause DNA damage as a mutagen. We examined the expression of cytokine-inducible NOS (iNOS) in human esophageal squamous cell carcinomas. Weak iNOS immunoreactivity was seen in the basal and parabasal layers of non-neoplastic esophageal stratified squamous epithelium. iNOS expression was detected in 50 (87.7%) of the 57 esophageal squamous cell carcinomas, regardless of the depth of tumor invasion, histological differentiation and lymph node status. Early-stage cancers, i.e. mucosal squamous cell carcinomas, also showed significant iNOS expression. We speculate that increased iNOS expression is associated with the carcinogenesis of human esophageal cancer.

Glycated Serum Albumin-Induced Nitric Oxide Production in Vascular Smooth Muscle Cells by Nuclear Factor κB-Dependent Transcriptional Activation of Inducible Nitric Oxide Synthase

Glycated proteins, including serum albumin, may be involved in the pathogenesis of diabetic vasculopathy. Recent evidence suggests that expression of inducible nitric oxide synthase (iNOS) in vascular smooth muscle cells (VSMC) may, in part, promote atherosclerosis by increasing local oxidative stress. We therefore investigated whether VSMC exposed to glycated human serum albumin (GHSA) produce nitric oxide (NO) by increasing iNOS expression through transcriptional activation of the iNOS gene and whether this process is dependent on nuclear factor κB (NF-κB) activation. Treatment of VSMC with GHSA causes activation of NF-κB and the iNOS promoter. Induction of NF-κB and the iNOS promoter by GHSA exhibited dose-dependent kinetics at concentrations ranging from 3 to 1000 μg/ml. GHSA alone was a weak inducer of NO production in VSMC as measured by determining nitrite levels, and interferon-γ alone was totally ineffective, whereas the combination of GHSA and interferon-γ was a strong stimulus. This synergy for NO production corresponded to Northern blot analyses of iNOS mRNA expression. Thus, GHSA may promote atherosclerosis in part by activation of NF-κB and upregulation of iNOS, thereby fostering local inflammation and oxidative stress.

Myometrial constitutive nitric oxide synthase expression is increased during human pregnancy.

Nitric oxide (NO), derived from L-arginine by the action of nitric oxide synthase (NOS), is a mediator of many diverse biological activities, including vasodilation, neurotransmission and inhibition of platelet adhesion. A role for NO in the maintenance of rat and rabbit pregnancy is supported by a variety of studies. A recent study in women demonstrated that myometrial inducible NOS (iNOS) expression was greater in the early third trimester than either the late third trimester or in the non-pregnant condition, suggesting that increased iNOS expression is involved in the maintenance of human pregnancy. Constitutive NOS (cNOS) expression was not determined. The aim of this study was to compare constitutive NOS (both eNOS and bNOS) expression in the human non-pregnant uterus, preterm pregnant uterus (25-34 weeks gestation) and term pregnant uterus (>37 weeks gestation) using immunohistochemistry and Western blotting. Preterm pregnant samples were taken from women with a variety of pathologies necessitating early delivery. We found that eNOS and bNOS protein concentrations were greater in the preterm pregnant myometrium than non-pregnant myometrium. eNOS, but not bNOS, protein concentration was lower in myometrial samples obtained at term compared with those obtained preterm. We conclude that the constitutive isoforms of NOS are also up-regulated in human pregnancy and may play a role in the maintenance of myometrial quiescence.

Nitric Oxide Is Involved in Apoptosis Induced by Thapsigargin in Rat Mesangial Cells

The purpose of this study was to examine the mechanisms of thapsigargin-induced apoptosis in rat glomerular mesangial cells and the possible involvement of nitric oxide (NO) in this process. In mesangial cell monolayers incubated for 12 h in a medium without growth factors and with 10^–6 M thapsigargin, a known specific inhibitor of endoplasmic reticulum Ca²⁺-ATPase, a high percentage of cells showed typical nuclear features of apoptosis, assessed either by staining with propidium iodide (23 vs. 9% in control conditions) or by terminal desoxynucleotidyl transferase-mediated dUTP biotin nick end labelling (TUNEL; 17 vs. 5% in control conditions). When cells were maintained in a medium containing 10% fetal calf serum (FCS) or in a free-calcium medium, the thapsigargin-induced apoptosis rate was very low. In rat mesangial cells treatment with thapsigargin decreased the expression of BCL-2 protein and bcl-2 mRNA, whereas it did not alter the levels of BAX protein or bax mRNA. When mesangial cells were incubated with thapsigargin in the absence of FCS, we detected a significant increase in nitrite production (3.78 ± 0.96 vs. 1.76 ± 0.44 μmol/well). Furthermore, the treatment with the NO synthesis inhibitor L-NAME (10^–4 M) induced a significant decrease in the number of apoptotic cells (9%), whereas incubation with the NO donor SIN-1 (10^–5 M) induced a marked increase in the rate of apoptosis (29%). Western and Northern blot analysis of macrophage-type inducible NO synthase (iNOS) demonstrated that thapsigargin treatment induces the expression of the iNOS protein and iNOS mRNA. Treatment with L-NAME prevented the thapsigargin-induced BCL-2 decrease, whereas incubation with SIN-1 potentiated the effect of thapsigargin on BCL-2. Double labelling by immunohistochemistry for iNOS and TUNEL revealed that the same cells that suffered apoptosis were positive for iNOS. In summary, our results indicate that thapsigargin is able to enhance the apoptosis rate of rat mesangial cells by a mechanism that is mediated by an increase in cytosolic free calcium. Increased iNOS expression, and hence increased NO production, seems to be involved in this effect.

Expression of inducible nitric oxide synthase in human gastric cancer.

Inducible nitric oxide synthase (iNOS) is an enzyme that catalyzes the formation of nitric oxide (NO) from L-arginine. iNOS expression and activity results in the production of high levels of NO[1]. The generation of physiological levels of NO is important for mucosal function and it also exerts a cytoprotective effect on the gastrointestinal mucosa. However, increased iNOS expression has been observed in patients with chronic inflammatory diseases of the gastrointestinal tract, such as ulcerative colitis[2,3], and gastritis[4] and it has been speculated that increased NO may induce DNA damage[5,6] and angiogenesis[7]. Nonetheless, the role of iNOS in human GI neoplasia is largely unkown. Previous studies have demonstrated increased iNOS expression in breast cancer[8,9], and increased iNOS activity and protein levels have been demonstrated in colorectal cancer[10] and adenocarcinoma of the esophagus[11]. However, to date, the role of iNOS in gastric carcinogenesis has not been elucidated.

Ciliary neurotrophic factor potentiates the beta-cell inhibitory effect of IL-1beta in rat pancreatic islets associated with increased nitric oxide synthesis and increased expression of inducible nitric oxide synthase.

Proinflammatory cytokines are implicated as effector molecules in the pathogenesis of IDDM. Interleukin-6 (IL-6) alone or in combination with IL-1beta inhibits glucose-stimulated insulin release from isolated rat pancreatic islets by unknown mechanisms. Here we investigated 1) if the effects of IL-6 are mimicked by ciliary neurotrophic factor (CNTF), another member of the IL-6 family of cytokines signaling via gp130, 2) the possible cellular mechanisms for these effects, and 3) if islet endocrine cells are a source of CNTF. CNTF (20 ng/ml) potentiated IL-1beta-mediated (5-150 pg/ml) nitric oxide (NO) synthesis from neonatal Wistar rat islets by 31-116%, inhibition of accumulated insulin release by 34-49%, and inhibition insulin response to a 2-h glucose challenge by 31-36%. CNTF potentiated IL-1beta-mediated NO synthesis from RIN-5AH cells by 83%, and IL-1beta induced islet inducible NO-synthase (iNOS) mRNA expression fourfold. IL-6 (10 ng/ml) also potentiated IL-1beta-mediated NO synthesis and inhibition of insulin release, whereas beta-nerve growth factor (NGF) (5 or 50 ng/ml) had no effect. mRNA for CNTF was expressed in rat islets and in islet cell lines. In conclusion, CNTF is constitutively expressed in pancreatic beta-cells and potentiates the beta-cell inhibitory effect of IL-1beta in association with increased iNOS expression and NO synthesis, an effect shared by IL-6 but not by beta-NGF. These findings indicate that signaling via gp130 influences islet NO synthesis associated with iNOS expression. We hypothesize that CNTF released from destroyed beta-cells during the inflammatory islet lesion leading to IDDM may potentiate IL-1beta action on the beta-cells.

B-Cell Chronic Lymphocytic Leukemia Cells Express a Functional Inducible Nitric Oxide Synthase Displaying Anti-Apoptotic Activity

AbstractThe expression of different isoforms of nitric oxide synthase (NOS) was investigated in B-cell chronic lymphocytic leukemia (B-CLL) to delineate a possible role for nitric oxide (NO) in the control of apoptosis of the tumoral cells. By reverse transcription-polymerase chain reaction (RT-PCR), all B-CLL cells were found to express spontaneously inducible NOS (iNOS) mRNA, whereas endothelial constitutive NOS (ecNOS) mRNA was undetectable. The iNOS protein was detected by immunofluorescence in the cytoplasm of permeabilized leukemic cells and identified by Western blotting, using different anti-iNOS antibodies, as a protein of 135 kD in B-CLL cytoplasmic extracts. B-CLL cell lysates also displayed basal NOS enzymatic activity, as measured by the conversion of14C-labeled L-arginine into 14C-L-citrulline. Ligation of CD23, expressed on the vast majority of B-CLL cells, resulted in increased iNOS expression and activity. The NO released exerted an anti-apoptotic effect on B-CLL cells that was counteracted by NOS inhibitors and engagement of the APO-1/Fas pathway. Therefore, the existence of a functional iNOS in B-CLL cells will provide further insights into the mechanisms that control proliferation and apoptosis in these tumor cells.© 1998 by The American Society of Hematology.

B-Cell Chronic Lymphocytic Leukemia Cells Express a Functional Inducible Nitric Oxide Synthase Displaying Anti-Apoptotic Activity

The expression of different isoforms of nitric oxide synthase (NOS) was investigated in B-cell chronic lymphocytic leukemia (B-CLL) to delineate a possible role for nitric oxide (NO) in the control of apoptosis of the tumoral cells. By reverse transcription-polymerase chain reaction (RT-PCR), all B-CLL cells were found to express spontaneously inducible NOS (iNOS) mRNA, whereas endothelial constitutive NOS (ecNOS) mRNA was undetectable. The iNOS protein was detected by immunofluorescence in the cytoplasm of permeabilized leukemic cells and identified by Western blotting, using different anti-iNOS antibodies, as a protein of 135 kD in B-CLL cytoplasmic extracts. B-CLL cell lysates also displayed basal NOS enzymatic activity, as measured by the conversion of14C-labeled L-arginine into 14C-L-citrulline. Ligation of CD23, expressed on the vast majority of B-CLL cells, resulted in increased iNOS expression and activity. The NO released exerted an anti-apoptotic effect on B-CLL cells that was counteracted by NOS inhibitors and engagement of the APO-1/Fas pathway. Therefore, the existence of a functional iNOS in B-CLL cells will provide further insights into the mechanisms that control proliferation and apoptosis in these tumor cells.© 1998 by The American Society of Hematology.

Enhanced Collagen Accumulation Following Direct Transfection of the Inducible Nitric Oxide Synthase Gene in Cutaneous Wounds

Inducible nitric oxide synthase (iNOS) is expressed during cutaneous wound repair. Mounting evidence suggests that wound nitric oxide (NO) augments collagen accumulation. We hypothesized thatin vivotransfection of wound cells with the iNOS gene would increase physiological wound NO levels and thus augment collagen accumulation. Polyvinyl alcohol sponges were instilled with a mammalian expression plasmid (pMP6) containing either the chloramphenicol acetyl transferase (CAT) reporter or murine iNOS gene driven by a CMV immediate-early promoter. Plasmid DNA was injected alone or in complex with cationic liposomes, and the sponges were placed subcutaneously in male Sprague–Dawley rats which had received a longitudinal dorsal midline incision. Animals were sacrificed at different time points post-wounding and the sponges assayed for CAT activity, transfected iNOS mRNA, total nitrate and nitrite concentration (NOX) (as an index of wound NO synthesis), and hydroxyproline content (as an index of sponge collagen accumulation). The results demonstrate that wound cells were more efficiently transfected by naked DNA than by liposome mediated transfection and that maximal expression of both iNOS and CAT occurred at 48 hrs with a rapid decline after this time point. After 7 days, iNOS transfected sponges had accumulated significantly more collagen than those transfected with CAT. We conclude that cutaneous wounds can be successfully transfected by direct injection of naked DNA and that increased iNOS expression precedes an increase in collagen synthesis.

Oleuropein, the bitter principle of olives, enhances nitric oxide production by mouse macrophages

The Mediterranean diet, rich in fresh fruits and vegetables, has been associated with a lower incidence of cardiovascular disease and cancer, partly because of its high proportion of bioactive compounds such as vitamins, flavonoids and polyphenols. The major lipid component of such diet is the drupe-derived olive oil, that can be distinguished from other seed oils for the peculiar composition of its non-triglyceride fraction. In fact, several minor components, including polyphenols, grant the oil its particular taste and aroma. Oleuropein, the most abundant among these components, has been shown to be a potent antioxidant endowed with antiinflammatory properties. We investigated the effects of oleuropein on NO release in cell culture and its activity toward nitric oxide synthase (iNOS) expression. The results show that oleuropein dose-dependently enhance nitrite production in LPS-challenged mouse macrophages. This effect was blocked by the iNOS inhibitor L-NAME, indicating increased iNOS activity. Also, Western blot analysis of cell homogenates show that oleuropein increases iNOS expression in such cells. Taken together, our data suggest that, during endotoxin challenge, oleuropein potentiates the macrophage-mediated response, resulting in higher NO production, currently believed to be beneficial for cellular and organismal protection.

O.30 Arginine-enriched total enteral nutrition increaseliver albumin gene expression and does not increase iNOS expression

liver albumin gene expression and does not increase iNOS expression J. Ren, S. Wang, Y. Zhao, J. Li and A. Li Research Institute of General Surgery, Jingling Hospital, Nanjing University, Nanjing, China. Aim: To examine the effects of arginine enriched total enteral nutrition (TEN) on liver albumin, TNF, IL-lcq IL-6R and inducible nitric oxide synthase (iNOS) gene expression. Methods: A 30% burn was applied in 40 rats after 7 days of gastrostomy. The rats received a continuous intragastric infusion of two different diets 2 hours after burn: low arginine (AL, arginine 0.01 g/kg/day) and arginine enriched (AE, arginine 0.264 g/kg/day). The two formulas were isocaloric (1 75 Kcal/kg/day) and isonitrogenous (1.25 gN/kg/day). The animals were sacrificed on day 1 post burn (PBD1) and PBD9. The mRNA of albumin, TNF, IL-l(z, IL-6R and iNOS of the liver were reverse transcribed (RT) into cDNA and the cDNA were amplifed by polymerase chain reaction (PCR). The RT-PCR products of albumin, TNF, IL-lc~, IL-6R and iNOS were determined by capillary electrophoresis (CE) and semi-quantified by the specific peak area of the CE chromatogram. Results: RT-PCR products of albumin, TNF, IL-6R and iNOS (absorbance unit). Albumin TNF IL-6R iNOS AL(PBD1) 869_*:52 1031 _+23 4320_+108 965-+41 AE(PBD1) 813_+34 984+_27 3810+_217 984_+39 AL(PBD9) 1417 -+ 43 ~ 743 _+ 29* 2570 -+ 90* 547 _+ 23* AE(PBDg) 1812_+41** 603_+32** 972-+87 **~: 522_+48* Means -+ SD, t-test: * P < 0.05 vs AL(PBD9), ** P < 0.01 vs AL(PBD9), 1: P < 0.01 vs PBDI.

In situ characterization of inflammatory responses in the rectal mucosae of patients with shigellosis.

Shigella species cause bacillary dysentery in humans by invading epithelial cells of the colonic mucosa leading to colonic epithelial cell destruction and inflammation. For further analysis of local gut inflammation, morphological changes and the potential involvement of mediators in regulatory mechanisms of cell activation and cell proliferation were studied immunohistochemically in rectal mucosal biopsies taken from patients during the acute phase of shigellosis and at convalescence. Rectal biopsies from 25 Shigella dysenteriae-1 and 10 Shigella flexneri-infected patients and from 40 controls were studied. The frequencies of proliferative cells (Ki67-positive cells), p53-immunostaining cells, and cells coexpressing Ki67 with CD3 or with p53 were analyzed. Immunostaining for the inducible nitric oxide synthase (iNOS) and the endothelial NOS was assessed. In addition, the frequencies of apoptotic cells and CD68+ cells that engulf apoptotic cells were assessed. By morphological grading, 20% of the patients had advanced inflammation (grade 3) in the acute phase; mild inflammation (grade 1) was seen in 37% of the patients at convalescence as well as in 10% of the controls. The findings in the present study suggest that in the acute phase of shigellosis inflammation is characterized by increased cell turnover in the lamina propria (LP) and the epithelium, increased iNOS expression in the surface epithelium, and apoptosis, which seems to be associated with LP macrophages. The findings also suggest that neither p53 nor iNOS are important factors for the induction of apoptosis in shigellosis. Expression of p53 may be related to early cell activation in crypt epithelium. Moreover, there is an indication of an active, low-level inflammatory process at convalescence. The results thus indicate that Shigella-induced inflammation is associated with a complex series of cellular reactions in the rectal gut mucosa which persist long after clinical symptoms have resolved.

Effects of the Murine L929 and L1210 Cell Lines on Nitric Oxide and TNF-α Production by RAW 264.7 Murine Macrophages

Co-cultures of the murine macrophagic cell line RAW 264.7 with the L929 fibrosarcoma cell line, but not with the leukemia L1210 cell line, showed enhanced NO production over control RAW 264.7 cells. This potentiating effect, which was observed in detectably mycoplasma-free conditions and required low concentrations of recombinant murine IFN-γ, was due to soluble factors released from L929 cells and not to physical contact between the two cell types. The soluble factors were able to induce TNF-α in the macrophages and to potentiate the TNF-α release induced by IFN-γ. Increased generation of NO in RAW 264.7 cells co-cultured with L929 cells was prevented by a neutralizing anti-TNF-α antibody, suggesting that TNF-α is an autocrine factor for iNOS expression in these conditions. Also the L929 cell line showed a 4- to 5-fold enhanced NO production following co-culture with RAW 264.7 cells, thus indicating that exposure of tumor cells to macrophages can lead to an increased iNOS expression in tumor cells themselves.

Transcriptional regulation of iNOS by IL-1 beta in cultured rat pulmonary artery smooth muscle cells.

Transcriptional regulation of iNOS by IL-1 beta in cultured rat pulmonary artery smooth muscle cells. Am. J. Physiol. 271 (Lung Cell. Mol. Physiol. 15): L166-L171, 1996.-Interleukin-1 beta (IL-1 beta) is the critical cytokine affecting peripheral vascular expression of inducible nitric oxide synthase (iNOS). Accordingly, we sought to determine a role for IL-1 beta in stimulating iNOS transcription in cultured rat pulmonary artery smooth muscle cells (RPASMC). Treatment of RPASMC with IL-1 beta caused a concentration-dependent increase in iNOS gene expression by Northern and Western blotting. To demonstrate IL-1 beta-mediated transcriptional activation, we used transient liposome-mediated transfection of RPASMC with promoter-luciferase constructs containing deletional mutations of the murine macrophage iNOS 5' flanking promoter region. IL-1 beta increased promoter activity approximately two- to threefold over baseline in fragments ranging from -1592 (full-length) to -242 bp. Activity was lost, however, when the promoter fragment was shorter than -242 bp. IL-1 beta-mediated increases in steady-state iNOS mRNA were sensitive to pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappa B activation. Nuclear proteins from IL-1 beta-stimulated cells demonstrated PDTC-sensitive binding to an oligonucleotide containing the sequence for the NF-kappa B binding element present in the region between -242 and -42 bp. These data document that IL-1 beta, by itself, increases iNOS expression in RPASMC by transcriptional activation, mediated in part by NF-kappa B.

Induction of Inducible Nitric-oxide Synthase by the Heterotrimeric G Protein Gα13*

While the functions of several G protein alpha subunits such as alpha(s( and alpha(q) are relatively well understood, the action of others such as alpha13 remain largely undefined. Because of recent interest in regulation of nitric-oxide synthase (NOS) by G protein-coupled signaling systems and findings that receptors for two proinflammatory substances, thrombin and thromboxane couple to alpha13, we studied the effect of alpha13 on NOS activity in a renal epithelial cell line. We found that stable overexpression of alpha13 or its GTPase-deficient mutant, alpha13Q226L, in a continuous renal epithelial cell line (MCT) increased NOS activity. The increased NOS activity was due to increased expression of the macrophage-inducible form of NOS (iNOS). iNOS protein and activity were not increased in similar cells expressing an activated alpha(s) (alpha(s)Q227L) or were minimally increased in cells expressing activated alpha(i1) (alpha-i1Q204L) and alpha(q) (alpha(q)Q209L), members of the three other G protein alpha chain families. Transient co-expression of alpha13 or alpha13Q226L increased the activity of an iNOS promoter-CAT construct demonstrating that alpha13 increases iNOS expression through transcription. Consequently, alpha13 induces iNOS through a novel mechanism that is distinct from that of other G protein alpha chains and that may mediate the actions of G protein-dependent proinflammatory agents.

Role of nitric oxide in sepsis-induced hyporeactivity in isolated rat lungs.

The aim of the present study was to test the hypothesis that pulmonary microvascular reactivity is depressed in sepsis and that inducible nitric oxide synthase (iNOS) contributes to the vascular hyporeactivity. Rats were made septic by cecal ligation and puncture. After 16 h, pulmonary vascular reactivity was evaluated by measurement of perfusion pressures while the vasculature was challenged with angiotensin II and KCl. The results showed that vascular reactivity was significantly depressed in lungs from septic rats in comparison to sham-operated controls. Pretreatment with the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) restored the depressed vasoreactivity while the nitric oxide (NO) synthase substrate L-arginine (1 mM) reversed the contraction-restoring effect of L-NAME. NO production in lungs from septic rats increased about 4-fold in comparison to sham-operated controls. iNOS protein was expressed in lung tissues, mainly the resistance vessels, from septic rats but not from sham-operated controls. Reverse transcription and polymerase chain reaction also showed a strong induction of iNOS mRNA in lung tissues from septic rats. These results suggest that increased iNOS expression and NO production may contribute to depressed pulmonary vascular reactivity in sepsis.

Nitric oxide suppression of human hematopoiesis in vitro. Contribution to inhibitory action of interferon-gamma and tumor necrosis factor-alpha.

IFN-gamma and TNF-alpha, potent inhibitors of hematopoiesis, induce nitric oxide synthase (NOS) in various cell types. When normal human bone marrow (BM) or CD34+ cells were exposed to NO, inhibition of colony formation was dose dependent and direct. NO induced apoptosis in BM progenitors, as shown by electrophoretic detection of DNA degradation and deoxynucleotidyl transferase assay. Using PCR and immunoprecipitation, we found inducible NOS (iNOS) mRNA and iNOS protein in BM after stimulation with IFN-gamma or TNF-alpha. iNOS mRNA was also detected by PCR in highly purified CD34+ cells; TNF-alpha or IFN-gamma increased iNOS expression. The presence of iNOS in CD34+ cells was confirmed in single cells by immunochemical staining. NG-Monomethyl-L-arginine (MM-Arg), an NOS inhibitor, partially reversed the effects of TNF-alpha and, to a lesser extent, IFN-gamma in methylcellulose culture of total BM and CD34+ cells, and inhibited apoptosis of BM cells induced by these cytokines. When the effects of competitive iNOS inhibition were tested on more immature progenitors, MM-Arg increased the number of long-term BM culture-initiating cells in control cultures but failed to protect these cells from the inhibitory action of IFN-gamma and TNF-alpha. Our results suggest that NO may be one mediator of cytokine-induced hematopoietic suppression.