Thermoregulatory role of inducible nitric oxide synthase in lipopolysaccharide-induced hypothermia

We have tested the hypothesis that nitric oxide (NO) arising from inducible nitric oxide synthase (iNOS) plays a role in hypothermia during endotoxemia by regulating vasopressin (AVP) release. Wild-type (WT) and iNOS knockout mice (KO) were intraperitoneally injected with either saline or Escherichia coli lipopolysaccharide (LPS) 10.0 mg/kg in a final volume of 0.02 mL. Body temperature was measured continuously by biotelemetry during 24 h after injection. Three hours after LPS administration, we observed a significant drop in body temperature (hypothermic response) in WT mice, which remained until the seventh hour, returning then close to the basal level. In iNOS KO mice, we found a significant fall in body temperature after the fourth hour of LPS administration; however, the hypothermic response persisted until the end of the 24 h of the experiment. The pre-treatment with beta-mercapto-beta,beta-cyclopentamethylenepropionyl(1), O-Et-Tyr2, Val4, Arg8-Vasopressin, an AVP V1 receptor antagonist (10 microg/kg) administered intraperitoneally, abolished the persistent hypothermia induced by LPS in iNOS KO mice, suggesting the regulation of iNOS under the vasopressin release in this experimental model. In conclusion, our data suggest that the iNOS isoform plays a role in LPS-induced hypothermia, apparently through the regulation of AVP release.     

Melatonin inhibits expression of the inducible NO synthase II in liver and lung and prevents endotoxemia in lipopolysaccharide-induced multiple organ dysfunction syndrome in rats.

We evaluated the role of melatonin in endotoxemia caused by lipopolysaccharide (LPS) in unanesthetized rats. The expression of inducible isoform of nitric oxide synthase (iNOS) and the increase in the oxidative stress seem to be responsible for the failure of lungs, liver, and kidneys in endotoxemia. Bacterial LPS (10 mg/kg b. w) was i.v. injected 6 h before rats were killed and melatonin (10-60 mg/kg b.w.) was i.p. injected before and/or after LPS. Endotoxemia was associated with a significant rise in the serum levels of aspartate and alanine aminotransferases, gamma-glutamyl-transferase, alkaline phosphatase, creatinine, urea, and uric acid, and hence liver and renal dysfunction. LPS also increased serum levels of cholesterol and triglycerides and reduced glucose levels. Melatonin administration counteracted these organ and metabolic alterations at doses ranging between 20 and 60 mg/kg b. w. Melatonin significantly decreased lung lipid peroxidation and counteracted the LPS-induced NO levels in lungs and liver. Our results also show an inhibition of iNOS activity in rat lungs by melatonin in a dose-dependent manner. Expression of iNOS mRNA in lungs and liver was significantly decreased by melatonin (60 mg/kg b. w., 58-65%). We conclude that melatonin inhibits NO production mainly by inhibition of iNOS expression. The inhibition of NO levels may account for the protection of the indoleamine against LPS-induced endotoxemia in rats.     

Endogenous peroxynitrite mediates mitochondrial dysfunction in rat diaphragm during endotoxemia.

It has been shown that nitric oxide (NO), synthesized by the inducible NO synthase (iNOS) expressed in the diaphragm during endotoxemia, participates in the development of muscular contractile failure. The aim of the present study was to investigate whether this deleterious action of NO was related to its effects on cellular oxidative pathways. Rats were inoculated with E. coli lipopolysaccharide (LPS) or sterile saline solution (controls) and studied at 3 and 6 h after inoculation. iNOS protein and activity could be detected in the rat diaphragm as early as 3 h after LPS, with a sustained steady-state concentration of 0.5 microM NO in the muscle associated with increased detection of hydrogen peroxide (H(2)O(2)). In vitro, the same NO concentration produced a marked increase in H(2)O(2) production by isolated control diaphragm mitochondria, thus reflecting a higher intramitochondrial concentration of nondiffusible superoxide anion (O(2)(-.)). In a similar way, whole diaphragmatic muscle and diaphragm mitochondria from endotoxemic rats showed a progressive increase in H(2)O(2) production associated with uncoupling and decreased phosphorylating capacity. Simultaneous with the maximal impairment in respiration (6 h after LPS), nitration of mitochondrial proteins (a peroxynitrite footprint) was detected and diaphragmatic force was reduced. Functional mitochondrial abnormalities, nitration of mitochondrial proteins, and the decrease in force were significantly attenuated by administration of the NOS inhibitor L-NMMA. These results show that increased and sustained NO levels lead to a consecutive formation of O(2)(-.) that reacts with NO to form peroxynitrite, which in turn impairs mitochondrial function, which probably contributes to the impairment of muscle contractility. during endotoxemia.     

NO plays a role in LPS-induced decreases in circulating IGF-I and IGFBP-3 and their gene expression in the liver

In this study, we administered aminoguanidine, a relatively selective inducible nitric oxide synthase (iNOS) inhibitor, to study the role of nitric oxide (NO) in LPS-induced decrease in IGF-I and IGFBP-3. Adult male Wistar rats were injected intraperitoneally with LPS (100 microg/kg), aminoguanidine (100 mg/kg), LPS plus aminoguanidine, or saline. Rats were injected at 1730 and 0830 the next day and killed 4 h after the last injection. LPS administration induced an increase in serum concentrations of nitrite/nitrate (P < 0.01) and a decrease in serum concentrations of growth hormone (GH; P < 0.05) and IGF-I (P < 0.01) as well as in liver IGF-I mRNA levels (P < 0.05). The LPS-induced decrease in serum concentrations of IGF-I and liver IGF-I gene expression seems to be secondary to iNOS activation, since aminoguanidine administration prevented the effect of LPS on circulating IGF-I and its gene expression in the liver. In contrast, LPS-induced decrease in serum GH was not prevented by aminoguanidine administration. LPS injection decreased IGFBP-3 circulating levels (P < 0.05) and its hepatic gene expression (P < 0.01), but endotoxin did not modify the serum IGFBP-3 proteolysis rate. Aminoguanidine administration blocked the inhibitory effect of LPS on both IGFBP-3 serum levels and its hepatic mRNA levels. When aminoguanidine was administered alone, IGFBP-3 serum levels were increased (P < 0.05), whereas its hepatic mRNA levels were decreased. This contrast can be explained by the decrease (P < 0.05) in serum proteolysis of this binding protein caused by aminoguanidine. These data suggest that iNOS plays an important role in LPS-induced decrease in circulating IGF-I and IGFBP-3 by reducing IGF-I and IGFBP-3 gene expression in the liver.     

Time-dependent expression of renal vaso-regulatory molecules in LPS-induced endotoxemia in rat

To elucidate roles of microvascular factors in the pathogenesis of renal complications during endotoxemia, that is characterized by renal vasoconstriction and systemic hypotension/generalized non-renal vasodilation, we profile the expression pattern and time-course of three key vaso-regulators, namely endothelin (ET)-1, nitric oxide (NO), and angiotensin II (Ang II). We hypothesize that disruption of the overall balance between vasodilatation and vasoconstriction in the kidney, during the early phase of sepsis, contribute to its (kidney) predisposition to acute renal failure. Adult male Wistar rats were rendered endotoxemic at different time points (1, 3, 6 and 10 h) by a single i.p. injection of lipopolysaccharide (LPS) (15 mg/kg) dissolved in saline. Control group was injected vehicle only (saline). Both systolic and diastolic blood pressures significantly decreased at different time points after LPS administration. Surprisingly, renal histopathological evaluation showed no remarkable changes in LPS-induced endotoxemia. However, overall, levels of the vaso-regulators and, where applicable, their respective receptors were upregulated: (1) plasma ET-1 increased 25-fold and peaked, as renal ET-1 mRNA, at 3 h; renal ET-1 protein and its receptors, ET type A (ET(A)) receptor (vasoconstrictive) and ET type B (ET(B)) receptor (vasodilatatory) increased in a time-dependent fashion, (2) Ang II increased by 53% compared to control, peaking at 6 h. However, while levels of Ang II type 1 (AT1) receptor increased over time after LPS injection, those of Ang II type 2 (AT2) receptor were downregulated, (3) data of NO system (NO-NOS), the key vasodilator, were the most intriguing. Whereas levels of renal NO increased time-dependently following LPS administration, with a 2240-fold increase in renal iNOS expression, levels of eNOS, were almost unchanged. In conclusion, the present study overall reveals intriguing and complex dynamics between levels of vasoconstrictors and vasodilators during the early phase of LPS-induced endotoxemia. These shifts in molecular expressions are likely triggered by compensatory mechanisms aimed at counteracting the undesirable and dominant effects of one group of vaso-regulatory moiety over the other.     

Nitric oxide-cGMP pathway is involved in endotoxin-induced contractile dysfunction in rat hearts.

The mechanisms by which endotoxemia causes cardiac depression have not been fully elucidated. The present study examined the involvement of nitric oxide (NO) in this pathology. Rats were infused with lipopolysaccharide (LPS) or saline, and the plasma and myocardial NO(2)(-) and NO(3)(-) (NOx) concentrations were measured before or 3, 6, and 24 h after treatment. The hearts were then immediately isolated and mounted in a Langendorff apparatus, and left ventricular developed pressure (LVDP) was determined before biochemical analysis of the myocardium. LPS injection effected the expression of inducible NO synthase (iNOS) in the myocardium, a marked increase in plasma and myocardial NOx levels, and a significant decline in LVDP compared with saline controls. The LPS-induced NO production and concomitant cardiac depression were most pronounced 6 h after LPS injection and were accompanied by a significant increase in myocardial cGMP content. Myocardial ATP levels were not significantly altered after LPS injection. Significant negative correlation was observed between LVDP and myocardial cGMP content, as well as between LVDP and plasma NOx levels. Aminoguanidine, an inhibitor of iNOS, significantly attenuated the LPS-induced NOx production and contractile dysfunction. Furthermore, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase, significantly decreased myocardial cGMP content and attenuated the contractile depression, although aminoguanidine or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one was not able to completely reverse myocardial dysfunction. Our data suggest that endotoxin-induced contractile dysfunction in rat hearts is associated with NO production by myocardial iNOS and a concomitant increase in myocardial cGMP.     

Inducible nitric oxide synthase mediates cytokine release: the time course in conscious and septic rats

Nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), and interleukin 1-beta (IL-1beta) are postulated to play a key pathophysiologic role during sepsis. In this study, we examined the time course of inducible NO synthase (iNOS) mRNA expression and the plasma TNF-alpha and IL-1beta in lipopolysaccharide (LPS)-treated conscious rats. The hemodynamic pattern in septic shock is more similar to clinical conditions without anesthesia. The data showed that a significant increase in iNOS mRNA levels was found in the spleen, lung, liver, with slight elevation in the heart and kidney at 3 h after LPS administration. However, iNOS mRNA levels were not elevated significantly in all tissues examined at 24 h. In the plasma, TNF-alpha and IL-1beta culminated within 1 h, and reduced gradually to baseline levels in a relatively short period (within 9 h). The results suggest that local NO production by activation of iNOS mRNA expression and cytokine release may contribute to LPS-induced organ dysfunction at various time points.     

Organ sites of lipopolysaccharide-induced nitric oxide production in the anesthetized rat.

The objective of this research was to determine the amount and timing of nitric oxide (NO, nitrogen monoxide) gas produced by the lungs, intestinal mucosa, and organ surfaces facing the peritoneal cavity after iv injection of a bacterial toxin, lipopolysaccharide (LPS). Some of the deleterious effects of LPS on organ function have been attributed to NO or strong oxidants formed locally from NO. Medical-grade air was used as an inspiratory air source (50 strokes/min x 3 ml/stroke) or was pumped through the ileal lumen or peritoneal cavity (20 strokes/min x 3 ml/stroke). The air was collected at intervals of 15-30 min for 3 h after LPS and analyzed for authentic NO gas by chemiluminescence. LPS (5 mg/kg) or saline was injected iv. Sodium nitroprusside (SNP) was injected to determine the appearance of its NO released into the perfused compartments. Blood pressure, plasma nitrate plus nitrite (NO(x)), and total plasma leukocytes were measured as other manifestations of LPS effects. NO began to increase in the pulmonary expired air 90 min after LPS and continued to increase for the remainder of the experiment. The final pulmonary post-LPS [NO] was about 20-fold greater than the [NO] before LPS. LPS had no effect on intraluminal or intraperitoneal [NO]. The saline injection had no effect on [NO] in any compartment. SNP injection increased NO entry into all three air-perfused compartments. Thus, NO from an exogenous tissue source was not prevented from being detected. Blood pressure was decreased by LPS only during the pulmonary perfusion. There were no significant effects of LPS on leukocytes or plasma NO(x). LPS decreased blood pressure and leukocytes and increased plasma NO(x) when air perfusion was not done. It was concluded that different organs can produce LPS-induced NO at markedly different rates and times. However, some aspect of the experimental technique of air perfusion could alter the effects of LPS.     

Co-induction of nitric oxide and tetrahydrobiopterin synthesis in the myocardium in vivo

Induction of the inducible isoform of nitric oxide (NO) synthase (iNOS) in the myocardium is implicated as a mechanism in the development of cardiac depression in immune activated states associated with an enhanced release of cytokines, such as septic shock. We evaluated the in vivo synthesis of NO and tetrahydrobiopterin (BH4), a cofactor of NOS, in the heart tissue using a model of LPS injection in rats (LPS: 10 mg/kg, i.v.). In control rats, iNOS activity or iNOS mRNA in the heart was negligible. Three hours after LPS administration, a marked induction of iNOS mRNA and activity was observed in the heart. A significant increase in BH4 content and GTP cyclohydrolase mRNA abundance was also observed in the heart from LPS-treated rats. Our results demonstrate induction of NO synthesis and parallel increase in BH4 concentration in the heart of rats after LPS treatment in vivo and may provide molecular evidence responsible for the increased production of BH4 which may up-regulate iNOS activity in the heart in vivo. (Mol Cell Biochem 166: 177-181, 1997)     

Modulation of cerebellar and hepatic nitric oxide synthase by exogenous arginine and endotoxin.

We have studied in mice the effect of treatment with exogenous arginine and/or LPS by monitoring serum nitrite/nitrate levels and by investigating the response of cerebellar and liver nitric oxide synthase (NOS). We measured NOS activity in cerebellar extracts while changes in iNOS mRNA were followed in the liver since direct assay of NOS activity proved unreliable with this tissue. In fact, liver and cerebellum extracts were both very active in converting arginine into a citrulline-like metabolite, but only cerebellum conversion was dependent on addition of NADPH and inhibitable by N(G)-methyl-l-arginine. Treatment with LPS, on its own, increased serum nitrite/nitrate levels at 5 and 20 h after injection, while treatment with LPS and arginine produced nitrite/nitrate levels in the serum even greater at 5 h, but significantly lower at 20 h. Liver iNOS mRNA levels were markedly increased by LPS, and this effect was significantly decreased when mice were also given exogenous arginine. A stimulatory effect of LPS was also found on NOS activity in the cerebellum, where a very small stimulation may have also been caused by arginine feeding. These findings indicate that LPS stimulates NOS expression/activity both in the cerebellum and in the liver and suggest a complex pattern of modulation of iNOS by arginine, with NO being first produced in excess and then downregulating iNOS expression.     

Interleukin-1 is not essential for expression of inducible NOS in hepatocytes induced by lipopolysaccharide in vivo

Interleukin (IL)-1 and tumor necrotic factor alpha (TNFalpha) are pivotal in the pathogenesis of endotoxemia. In spite of the in vitro finding that IL-1beta, but not TNFalpha, can induce iNOS mRNA and NO production as a single stimulus in hepatocytes in primary culture, the involvement of IL-1 in iNOS induction in the liver has been less clear in vivo. To address this, we challenged IL-1alpha/beta double-knockout (IL-1alpha/beta(-/-)) and TNFalpha(-/-) mice with lipopolysaccharide (LPS). As compared with wild-type mice, the increases in the plasma NO level measured as nitrite and nitrate and hepatic iNOS were significantly reduced in IL-1alpha/beta(-/-) and TNFalpha(-/-) mice 8 and 12h after the LPS challenge. In the wild-type mice, iNOS protein was first detected in Kupffer cells around the portal vein 2h after LPS challenge; and then it spread to hepatocytes throughout the intralobular region of the liver by 8h. Although the expression of iNOS protein was detected in Kupffer cells of both IL-1alpha/beta(-/-) and TNFalpha(-/-) mice, its level was moderate in hepatocytes of IL-1alpha/beta(-/-) mice, but negligible in those of TNFalpha(-/-) mice, 8h after LPS challenge. Concomitant with the expression of iNOS protein in the liver, Toll-like receptor 4, the signaling receptor for LPS, was expressed in hepatocytes of wild-type and IL-1alpha/beta(-/-) mice, but not of TNFalpha(-/-) mice. These results demonstrate that the expression of Toll-like receptor 4 is well correlated with that of iNOS protein in hepatocytes in vivo after LPS challenge and that IL-1 is not essential for the induction of iNOS in hepatocytes in vivo.     

S-nitrosothiol formation in blood of lipopolysaccharide-treated rats

The administration of the gram-negative bacterial cell wall component lipopolysaccharide (LPS) to experimental animals results in the dramatic up-regulation of the inducible form of nitric oxide synthase (iNOS). The resulting sustained overproduction of nitric oxide (NO) is thought to contribute to the septic shock-like state in these animals. Numerous studies have characterized the kinetics and magnitude of expression of iNOS as well as the production of NO-derived nitrite and nitrate. However, little is known regarding the ability of iNOS-derived NO to interact with physiological substrates such as thiols to yield biologically active S-nitrosothiols during endotoxemia. It has been hypothesized that these relatively stable, vaso-active compounds may serve as a storage system for NO and they may thus play an important role in the pathophysiology associated with endotoxemia. In the present study, we demonstrate that 5 h after i.p. administration of LPS in rats, circulating S-nitrosoalbumin was increased by approximately 3. 4-fold over control. S-nitrosohemoglobin was increased by approximately 25-fold over controls and by threefold over S-nitrosoalbumin. No increase in low molecular weight S-nitrosothiols (i.e., S-nitrosoglutathione and S-nitrosocysteine) could be detected under our experimental conditions. Taken together these data demonstrate that endotoxemia dramatically enhances circulating S-nitrosothiol formation.     

Up-regulation of rat adrenomedullin gene expression by endotoxin: Relation to nitric oxide synthesis

The effect of bacterial endotoxin (LPS) on adrenomedullin (AM) gene expression was investigated in cultured rat aortic vascular smooth muscle (VSM) cells and in tissues from anesthetized rats. The addition of LPS together with interferon-y to VSM cells resulted in a marked increase in the abundance of AM mRNA as well as the appearance of mRNA for the inducible isoform of nitric oxide (NO) synthase (iNOS). Intravenous injection of LPS into rats also increased AM mRNA abundance and induced iNOS mRNA in lung, heart, liver, and kidney. AM significantly enhanced NO synthesis evoked by LPS and interferon-v in cultured VSM cells. These data suggest that AM may contribute to circulatory failure during endotoxin shock, in part, by modulating NO synthesis.     

Regulation of genes for inducible nitric oxide synthase and urea cycle enzymes in rat liver in endotoxin shock

Arginine is an intermediate of the urea cycle in the liver. It is synthesized by the first four enzymes of the cycle, carbamylphosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase, and argininosuccinate lyase, and is hydrolyzed to urea and ornithine by arginase I, forming the cycle. In endotoxemia shock, inducible nitric oxide (NO) synthase (iNOS) is induced in hepatocytes and arginine is utilized for NO production. Regulation of the genes for iNOS and the urea cycle enzymes was studied using lipopolysaccharide (LPS)-treated rat livers. When rats were injected intraperitoneally with LPS, iNOS mRNA was markedly induced. Cationic amino acid transporter-2 and C/EBPbeta mRNAs were also highly increased. In contrast, mRNAs for all the urea cycle enzymes except ornithine transcarbamylase were gradually decreased and reached 16-28% of controls at 12 h. However, all these enzymes remained unchanged at protein level up to 24 h. In light of these results, we suggest that synthesis of urea cycle enzymes is downregulated and that the protein synthetic capacity is directed to synthesis of proteins required for defense against endotoxemia.     

Effects of various antioxidants on endotoxin-induced lung injury and gene expression: mRNA expressions of MnSOD, interleukin-1beta and iNOS

Antioxidants have been shown to be effective in attenuating acute lung injury. In this study, we determine the effects of various antioxidants by different mechanisms on the lipopolysaccharide (LPS)-induced changes. LPS was administered intravenously at a dose of 10 mg/kg to anesthetized rats. LPS induced a significant decrease in blood pressure (P < 0.01) and increased exhaled nitric oxide (NO) from 3.60+/-0.18 to 35.53+/-3.23 ppb (P < 0.01) during an observation period of 4 h. Plasma nitrate concentrations also increased from 0.61+/-0.06 to 1.54+/-0.22 micromol/l (P < 0.05). LPS-induced oxygen radical release from white blood cells isolated from rat peripheral blood also increased significantly (P < 0.001). After the experiment, the lung weight was obtained and lung tissues were taken for the determination of mRNA expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta) and manganese superoxide dismutase (MnSOD). Histological examination of the lungs was also performed. In the control group injected with saline solution, mRNA expressions of iNOS, IL-1beta, TNF-alpha and MnSOD were absent. Four hours after LPS administration, mRNA expressions of iNOS, IL-1beta, and MnSOD were significantly enhanced, but TNF-alpha was not discernibly expressed. LPS also caused a twofold increase in lung weight. Pathological examination revealed endothelial cell damage and interstitial edema. Various antioxidants were given 1 h after LPS administration. These agents include SOD, catalase (CAT), SOD + CAT or vitamin C (ascorbic acid). These antioxidants effectively reversed the systemic hypotension, reduced the quantity of exhaled NO and plasma nitrate concentration, and prevented acute lung injury. Administration of various antioxidants also significantly attenuated LPS-induced oxygen radical release by rat white blood cells. LPS induced mRNA expressions of MnSOD and iNOS were significantly depressed by these antioxidants. However, only SOD + CAT and vitamin C inhibited the mRNA expression of IL-1beta. These results suggest that oxygen radicals are responsible for LPS-induced lung injury. Antioxidants can attenuate the lung injury by inhibiting mRNA expressions of iNOS and IL-1beta.     

Osteopontin is induced by nitric oxide in RAW 264.7 cells

Nitric oxide (NO) produced by macrophages is thought to contribute to various pathological conditions. Osteopontin (OPN) is a phosphorylated glycoprotein produced principally by macrophages. OPN inhibits inducible nitric oxide synthase (iNOS), which generates large amounts of NO production. However, the relationship between NO and endogenous OPN in activated macrophages has not yet been elucidated. We therefore examined expression of endogenous iNOS and OPN in a murine macrophage cell line, RAW 264.7 cells, by treating the cells with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Treatment of cells with LPS and IFN-gamma resulted in an increase of iNOS mRNA to maximum at 12 h after stimulation. In contrast, OPN mRNA was induced more slowly than iNOS mRNA. Induction of both iNOS and OPN mRNA in RAW 264.7 cells was markedly suppressed by addition of the specific iNOS inhibitor S-2-aminoethyl isothiourea dihydrobromide. The NOS inhibitor NG-methyl-L-arginine also suppressed induction of OPN mRNA but hardly affected iNOS mRNA expression. The NO-releasing agent spermine-NONOate but not peroxynitrite enhanced induction of OPN mRNA. These results suggest that NO directly up-regulates the endogenous OPN in macrophages stimulated with LPS and IFN-gamma. This up-regulation of endogenous OPN may represent a negative feedback system acting to reduce iNOS expression.     

Nitric oxide released from iNOS in polymorphonuclear leukocytes makes them deformable in an autocrine manner.

The objective of this study was to determine whether endogenous nitric oxide (NO) derived from reaction catalyzed by the inducible isoform of NO synthase (iNOS: NOS II) in polymorphonuclear leukocytes (PMNs) makes the PMNs deformable. Previous studies have shown that NO increases the deformability of PMNs and decreases the sequestration of PMNs in the lungs. However, there was little information regarding the effect of PMN-derived NO on the cells' deformability. In the present study PMNs were isolated from the blood of rats 24h after ip injection of saline (control) or lipopolysaccharide (LPS), and expression of iNOS in the PMNs of the LPS group was confirmed by immunocytochemistry. PMN deformability was evaluated by measuring the pressure generated during their passage through a microfilter at a constant flow rate. The nitrite/nitrate content of the solution in which the isolated PMNs were incubated was measured by the Griess method. In the control group, no iNOS was detectable in the PMNs, and the nitrite/nitrate level in the PMN incubation solution was low. Deformability was unchanged after incubation with specific iNOS inhibitor aminoguanidine, but decreased after incubation with N-formyl-methionyl-leucyl-phenyl-alanine. In the LPS group, PMN deformability was decreased compared to that of the control group. iNOS was detectable in the PMNs, and the deformability further decreased after incubation with aminoguanidine. These results suggest that endogenous NO generated during reactions catalyzed by iNOS in PMNs makes them deformable in an autocrine manner.