Genetic Models in Applied Physiology. Differential role of nitric oxide synthase isoforms in fever of different etiologies: studies using Nos gene-deficient mice.

Male C57BL/6J mice deficient in nitric oxide synthase (NOS) genes (knockout) and control (wild-type) mice were implanted intra-abdominally with battery-operated miniature biotelemeters (model VMFH MiniMitter, Sunriver, OR) to monitor changes in body temperature. Intravenous injection of lipopolysaccharide (LPS; 50 microg/kg) was used to trigger fever in response to systemic inflammation in mice. To induce a febrile response to localized inflammation, the mice were injected subcutaneously with pure turpentine oil (30 microl/animal) into the left hindlimb. Oral administration (gavage) of N(G)-monomethyl-l-arginine (l-NMMA) for 3 days (80 mg. kg(-1). day(-1) in corn oil) before injection of pyrogens was used to inhibit all three NOSs (N(G)-monomethyl-d-arginine acetate salt and corn oil were used as control). In normal male C57BL/6J mice, l-NMMA inhibited the LPS-induced fever by approximately 60%, whereas it augmented fever by approximately 65% in mice injected with turpentine. Challenging the respective NOS knockout mice with LPS and with l-NMMA revealed that inducible NOS and neuronal NOS isoforms are responsible for the induction of fever to LPS, whereas endothelial NOS (eNOS) is not involved. In contrast, none of the NOS isoforms appeared to trigger fever to turpentine. Inhibition of eNOS, however, exacerbates fever in mice treated with l-NMMA and turpentine, indicating that eNOS participates in the antipyretic mechanism. These data support the hypothesis that nitric oxide is a regulator of fever. Its action differs, however, depending on the pyrogen used and the NOS isoform.     

Upregulation of nitric oxide synthase in mice with severe hypoxia-induced pulmonary hypertension

Background  

The importance of nitric oxide (NO) in hypoxic pulmonary hypertension has been demonstrated using nitric oxide synthase (NOS) knockout mice. In that model NO from endothelial NOS (eNOS) plays a central role in modulating pulmonary vascular tone and attenuating hypoxic pulmonary hypertension. However, the normal regulation of NOS expression in mice following hypoxia is uncertain. Because genetically engineered mice are often utilized in studies of NO, we conducted the present study to determine how hypoxia alters NOS expression in wild-type mice.     

Susceptibility to ozone-induced acute lung injury in iNOS-deficient mice

Mice deficient in inducible nitric oxide synthase (iNOS; C57Bl/6Ai-[KO]NOS2 N5) or wild-type C57Bl/6 mice were exposed to 1 part/million of ozone 8 h/night or to filtered air for three consecutive nights. Endpoints measured included lavagable total protein, macrophage inflammatory protein (MIP)-2, matrix metalloproteinase (MMP)-9, cell content, and tyrosine nitration of whole lung proteins. Ozone exposure caused acute edema and an inflammatory response in the lungs of wild-type mice, as indicated by significant increases in lavage protein content, MIP-2 and MMP-9 content, and polymorphonuclear leukocytes. The iNOS knockout mice showed significantly greater levels of lung injury by all of these criteria than did the wild-type mice. We conclude that iNOS knockout mice are more susceptible to acute lung damage induced by exposure to ozone than are wild-type C57Bl/6 mice and that protein nitration is associated with the degree of inflammation and not dependent on iNOS-derived nitric oxide.     

The role of inducible nitric oxide synthase in gamete interaction and fertilization: a comparative study on knockout mice of three NOS isoforms

Nitric oxide (NO), which is produced from l-arginine by three isoforms of NO synthase (NOS), has been implicated in reproductive functions. However, the specific role of NOS isoforms in gamete function and fertilization is not clear. Three types of NOS knockout mice were super ovulated and fertilized in vitro and in vivo. The sperm count and motility, in vivo and in vitro fertilization rate as indicated by two-cell embryos and blastocyst rate were examined. The sperm count and motility from all three knockout mice were not significantly different from that of the wild type. Inducible NOS (iNOS) knockout mice were found to have the largest number of two-cell embryos/mouse collected after fertilization in vivo (P<0.01), but the rate of blastocyst formation from two-cell embryos in vitro was similar for all three knockouts. The rate of in vitro fertilization using either iNOS-deficient sperm or oocytes, but not those deficient in the other two NOS isoforms, was significantly elevated when compared to that in the wild type (P<0.001). While all three types of NOS do not seem to play a significant role in pre-ejaculated sperm function, iNOS may play an inhibitory role in sperm and oocyte functions affecting the process of fertilization and early embryo development.     

Expression profiles of NOS isoforms in gingiva of nNOS knockout mice

Nitric oxide is a gaseous molecule associated with many distinct physiological functions, and is derived from l-arginine catalyzed by nitric oxide synthase (NOS). Nitric oxide synthase has 3 isoforms: nNOS, iNOS and eNOS. Although these NOS isoforms are believed to play an important role in gingival tissue, little information is available on their morphological dynamics. The aim of this study was to investigate the profiles of NOS isoforms in deficiency of nNOS in gingiva of mice. Twelve male (6 normal (C57BL/6) and 6 nNOS knockout) mice were used. All mice were 5-week-old, weighing approximately 20–25 g each. After sacrifice, the jaws of the mice were removed by mechanical means and specimens analyzed by histology, in situ hybridization and immunohistochemistry. Immunohistochemical observation revealed positive staining for iNOS and eNOS, especially in lamina propria. Similar results in the mRNA expression levels were shown by in situ hybridization analysis. It may suggest that iNOS and eNOS compensated nNOS deficiency in the gingiva of nNOS knockout mice.     

Lack of nitric oxide synthases increases lipoprotein immune complex deposition in the aorta and elevates plasma sphingolipid levels in lupus

Systemic lupus erythematosus (SLE) patients display impaired endothelial nitric oxide synthase (eNOS) function required for normal vasodilatation. SLE patients express increased compensatory activity of inducible nitric oxide synthase (iNOS) generating excess nitric oxide that may result in inflammation. We examined the effects of genetic deletion of NOS2 and NOS3, encoding iNOS and eNOS respectively, on accelerated vascular disease in MRL/lpr lupus mouse model. NOS2 and NOS3 knockout (KO) MRL/lpr mice had higher plasma levels of triglycerides (23% and 35%, respectively), ceramide (45% and 21%, respectively), and sphingosine 1-phosphate (S1P) (21%) compared to counterpart MRL/lpr controls. Plasma levels of the anti-inflammatory cytokine interleukin 10 (IL-10) in NOS2 and NOS3 KO MRL/lpr mice were lower (53% and 80%, respectively) than counterpart controls. Nodule-like lesions in the adventitia were detected in aortas from both NOS2 and NOS3 KO MRL/lpr mice. Immunohistochemical evaluation of the lesions revealed activated endothelial cells and lipid-laden macrophages (foam cells), elevated sphingosine kinase 1 expression, and oxidized low-density lipoprotein immune complexes (oxLDL-IC). The findings suggest that advanced vascular disease in NOS2 and NOS3 KO MRL/lpr mice maybe mediated by increased plasma triglycerides, ceramide and S1P; decreased plasma IL-10; and accumulation of oxLDL-IC in the vessel wall. The results expose possible new targets to mitigate lupus-associated complications.     

Salt sensitivity of nitric oxide generation and blood pressure in mice with targeted knockout of the insulin receptor from the renal tubule

To elucidate the role of the insulin receptor (IR) on kidney nitric oxide generation and blood pressure (BP) control, we generated mice with targeted deletion of renal tubule IR using loxP recombination driven by a Ksp-cadherin promoter. Male knockout (KO) and wild-type (WT) littermates (～4 mo old) were transitioned through three 1-wk treatments: 1) low-NaCl diet (0.085%); 2) high-NaCl diet (HS; 5%); and 3) HS diet plus 3 mM tempol, a superoxide dismutase mimetic, in the drinking water. Mice were then switched to medium-NaCl (0.5%) diet for 5 days and kidneys harvested under pentobarbital anesthesia. Twenty-four-hour urinary nitrates plus nitrites were significantly higher in the WT mice under HS (2,067 ± 280 vs. 1,550 ± 230 nmol/day in WT and KO, respectively, P < 0.05). Tempol attenuated genotype differences in urinary nitrates plus nitrites. A rise in BP with HS was observed only in KO mice and not affected by tempol (mean arterial pressure, dark period, HS, 106 ± 5 vs. 119 ± 4 mmHg, for WT and KO, respectively, P < 0.05). Renal outer medullary protein levels of nitric oxide synthase (NOS) isoforms by Western blot (NOS1-3 and phosphorylated-S1177-NOS3) revealed significantly lower band density for NOS1 (130-kDa isoform) in the KO mice. A second study, when mice were euthanized under HS conditions, confirmed significantly lower NOS1 (130 kDa) in the KO, with an even more substantial (>50%) reduction of the 160-kDa NOS1 isoform. These studies suggest that the loss of renal IR signaling impairs renal nitric oxide production. This may be important in BP control, especially in insulin-resistant states, such as the metabolic syndrome.     

The effect of embryo and maternal genotypes on prolificacy, intrauterine growth retardation and postnatal development of Nos3-knockout mice

Mice deficient for endothelial nitric oxide synthase (NOS3(-/-)) may represent a good model for studying embryo loss and intrauterine growth retardation caused by vascular deficiencies. We determined the effects of embryo genotype (homozygous vs. heterozygous descendants with paternal or maternal source of the non-functional NOS3 allele) and maternal environment (NOS3(-/-) vs. wild-type NOS3(+/+) females) on the appearance of estrus, fertility and prolificacy rates and live weight in the first week of life as well as phenotypic characteristics of offspring during the postnatal period. The results indicated that pregnancy outcomes and postnatal development of NOS3(-/-) mice seem to be related to deficiencies in fetal programming mainly determined by maternal genotype.     

Relaxation of myometrium by calcitonin gene-related peptide is independent of nitric oxide synthase activity in mouse uterus

Calcitonin gene-related peptide (CGRP) inhibits myometrial contractile activity. However, the responsiveness of the mouse myometrium to CGRP is dependent on the hormonal and gestational stage. The inhibitory effect of CGRP in the myometrium is prominent during gestation and declines at parturition. The present study was undertaken to examine if nitric oxide (NO) production by nitric oxide synthase (NOS) isoforms mediates the inhibitory action of CGRP on uterine contractions as has been suggested earlier. Transgenic mice deficient in either of the three major NOS isoforms: endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) were used. Isometric force measurements on myometrial strips obtained from NOS-deficient mice were carried out and the inhibitory capacity of CGRP was monitored. CGRP inhibited KCl-induced contractions of the myometrial strips obtained from eNOS(-/-), iNOS(-/-), and nNOS(-/-) mice with equal efficiency as in wild-type animals. Additionally, NOS protein expression in the mouse uterus during gestation and during the estrous cycle was examined by means of Western immunoblot analysis. No correlation between NOS expression and inhibitory activity of CGRP was evident. The results suggest that the inhibitory action of CGRP in the mouse uterus is independent of the activity of these NOS isoforms.     

The spinal cord expression of neuronal and inducible nitric oxide synthases and their contribution in the maintenance of neuropathic pain in mice

Background

Nitric oxide generated by neuronal (NOS1), inducible (NOS2) or endothelial (NOS3) nitric oxide synthases contributes to pain processing, but the exact role of NOS1 and NOS2 in the maintenance of chronic peripheral neuropathic pain as well as the possible compensatory changes in their expression in the spinal cord of wild type (WT) and NOS knockout (KO) mice at 21 days after total sciatic nerve ligation remains unknown.

Methodology/Principal Findings

The mechanical and thermal allodynia as well as thermal hyperalgesia induced by sciatic nerve injury was evaluated in WT, NOS1-KO and NOS2-KO mice from 1 to 21 days after surgery. The mRNA and protein levels of NOS1, NOS2 and NOS3 in the spinal cord of WT and KO mice, at 21 days after surgery, were also assessed. Sciatic nerve injury led to a neuropathic syndrome in WT mice, in contrast to the abolished mechanical allodynia and thermal hyperalgesia as well as the decreased or suppressed thermal allodynia observed in NOS1-KO and NOS2-KO animals, respectively. Sciatic nerve injury also increases the spinal cord expression of NOS1 and NOS2 isoforms, but not of NOS3, in WT and NOS1-KO mice respectively. Moreover, the presence of NOS2 is required to increase the spinal cord expression of NOS1 whereas an increased NOS1 expression might avoid the up-regulation of NOS2 in the spinal cord of nerve injured WT mice.

Conclusions/Significance

These data suggest that the increased spinal cord expression of NOS1, regulated by NOS2, might be responsible for the maintenance of chronic peripheral neuropathic pain in mice and propose these enzymes as interesting therapeutic targets for their treatment.     

Nitric oxide synthase in pulmonary hypertension: lessons from knockout mice

Nitric oxide (NO) is implicated in a wide variety of biological roles. NO is generated from three nitric oxide synthase (NOS) isoforms: neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) all of which are found in the lung. While there are no isoform-specific inhibitors of NOS, the recent development and characterization of mice deficient in each of the NOS isoforms has allowed for more comprehensive study of the importance of NO in the lung circulation. Studies in the mouse have identified the role of NO from eNOS in modulating pulmonary vascular tone and in attenuating the development of chronic hypoxic pulmonary hypertension.     

Role of the different isoforms of cyclooxygenase and nitric oxide synthase during gastric ulcer healing in cyclooxygenase-1 and -2 knockout mice

Traditional NSAIDs, selective cyclooxygenase (COX)-2 inhibitors, and inhibitors of nitric oxide synthase (NOS) impair the healing of preexisting gastric ulcers. However, the role of COX-1 (with or without impairment of COX-2) and the interaction between COX and NOS isoforms during healing are less clear. Thus we investigated healing and regulation of COX and NOS isoforms during ulcer healing in COX-1 and COX-2 deficiency and inhibition mouse models. In this study, female wild-type COX-1(-/-) and COX-2(-/-) mice with gastric ulcers induced by cryoprobe were treated intragastrically with vehicle, selective COX-1 (SC-560), COX-2 (celecoxib, rofecoxib, and valdedoxib), and unselective COX (piroxicam) inhibitors. Ulcer healing parameters, mRNA expression, and activity of COX and NOS were quantified. Gene disruption or inhibition of COX-1 did not impair ulcer healing. In contrast, COX-2 gene disruption and COX-2 inhibitors moderately impaired wound healing. More severe healing impairment was found in dual (SC-560 + rofecoxib) and unselective (piroxicam) COX inhibition and combined COX impairment (in COX-1(-/-) mice with COX-2 inhibition and COX-2(-/-) mice with COX-1 inhibition). In the ulcerated repair tissue, COX-2 mRNA in COX-1(-/-) mice, COX-1 mRNA in COX-2(-/-) mice, and, remarkably, NOS-2 and NOS-3 mRNA in COX-impaired mice were more upregulated than in wild-type mice. This study demonstrates that COX-2 is a key mediator in gastric wound healing. In contrast, COX-1 has no significant role in healing when COX-2 is unimpaired but becomes important when COX-2 is impaired. As counterregulatory mechanisms, mRNA of COX and NOS isoforms were increased during healing in COX-impaired mice.     

Differential cytokine expression in skin graft healing in inducible nitric oxide synthase knockout mice

Inducible nitric oxide synthase (iNOS) and its product, nitric oxide, have been shown to play important roles in wound biology. The present study was performed to investigate the role of iNOS in modulating the cytokine cascade during the complex process of skin graft wound healing.Fifteen iNOS-knockout mice and 15 wild-type C57BL/6J mice were subjected to autogenous 1-cm2 intrascapular full-thickness skin grafts. Three animals in each group were killed on postoperative days 3, 5, 7, 10, and 14. Specimens were then analyzed using nonisotopic in situ hybridization versus mRNA of tumor growth factor-beta1, vascular endothelial growth factor, iNOS, endothelial nitric oxide synthase (eNOS), tumor necrosis factor-alpha, and basic fibroblast growth factor, as well as positive and negative control probes. Positive cells in both grafts and wound beds were counted using a Leica microgrid. Scar thickness was measured with a Leica micrometer. Data were analyzed using the unpaired Student's t test.Expression of iNOS was 2- to 4-fold higher in knockout mice than in wild-type mice on postoperative days 5, 7, and 14. Expression of eNOS was 2- to 2.5-fold higher in knockout mice than in wild-type mice on postoperative days 5 and 7. Tumor necrosis factor-alpha expression was 2- to 7-fold higher in knockout mice than in wild-type mice on all postoperative days. In contrast, expression levels of angiogenic/fibrogenic cytokines (vascular endothelial growth factor, basis fibroblast growth factor, and tumor growth factor-beta1) were 2.5- to 4-fold higher in wild-type mice than in knockout mice. Scars were 1.5- to 2.5-fold thicker in knockout mice than in wild-type mice at all time points. All of the above results represent statistically significant differences (p < 0.05).Significantly different patterns of cytokine expression were seen in knockout and wild-type mice. Although the scar layer was thicker in knockout mice, it showed much greater infiltration with inflammatory cells. These data further delineate the modulatory effect of iNOS and nitric oxide in healing skin grafts.     

The peroxynitrite pathway in development: phenytoin and benzo[a]pyrene embryopathies in inducible nitric oxide synthase knockout mice

Nitric oxide generated by nitric oxide synthases (NOSs) can react with reactive oxygen species (ROS), forming peroxynitrite, which may contribute to the ROS-initiated macromolecular damage implicated in the embryopathic effects of both endogenous and drug-enhanced oxidative stress. Inducible NOS (iNOS) is nonconstitutive in most tissues, and its embryonic expression and developmental importance are unknown. Herein, during organogenesis (Gestational Days 9 and 10), wild-type B6129PF2 embryos in culture were highly susceptible to the ROS-initiating teratogens phenytoin and benzo[a]pyrene, whereas iNOS knockout embryos were substantially but not completely protected (p < .05), implicating iNOS in the embryopathic mechanism. However, in contrast to prostaglandin H synthase-catalyzed teratogen bioactivation and ROS formation, which occurs within the embryo, in vivo iNOS expression was limited to placental tissue. These results suggest that the diffusion of nitric oxide from placental progenitor tissue (ectoplacental cone) to embryonic target tissues contributes to the embryopathic effects of ROS-initiating teratogens in embryo culture, which may constitute a mechanism by which embryonic determinants of ROS-mediated teratogenesis can be modulated by maternal extra-embryonic processes.     

Nitric oxide-dependent penile erection in mice lacking neuronal nitric oxide synthase.

BACKGROUND: Nitric oxide (NO) has been implicated as a mediator of penile erection, because the neuronal isoform of NO synthase (NOS) is localized to the penile innervation and NOS inhibitors selectively block erections. NO can also be formed by two other NOS isoforms derived from distinct genes, inducible NOS (iNOS) and endothelial NOS (eNOS). To clarify the source of NO in penile function, we have examined mice with targeted deletion of the nNOS gene (nNOS- mice). MATERIALS AND METHODS: Mating behavior, electrophysiologically induced penile erection, isolated erectile tissue isometric tension, and eNOS localization by immunohistochemistry and Western blot were performed on nNOS- mice and wild-type controls. RESULTS: Both intact animal penile erections and isolated erectile tissue function are maintained in nNOS mice, in agreement with demonstrated normal sexual behaviors, but is stereospecifically blocked by the NOS inhibitor, L-nitroarginine methyl ester (L-NAME). eNOS is abundantly present in endothelium of penile vasculature and sinusoidal endothelium within the corpora cavemosa, with levels that are significantly higher in nNOS- mice than in wild-type controls. CONCLUSIONS: eNOS mediates NO-dependent penile erection in nNOS- animals and normal penile erection. These data clarify the role of nitric oxide in penile erection and may have implications for therapeutic agents with selective effects on NOS isoforms.     

Intrauterine growth retardation in endothelial nitric oxide synthase-deficient mice is established from early stages of pregnancy

The current study aimed to determine effects of deficiencies in nitric oxide synthase (NOS) 3 on embryo and fetal development by in vivo, noninvasive, real-time ultrasonographic assessment of phenotypic changes in Nos3-knockout pregnant mice and their wild-type counterparts. From Day 4.5 of pregnancy onwards, embryonic vesicle diameters, crown-rump lengths, and trunk diameters were obtained by serial scanning of seven adult pregnant female mice, strain B6.129P2-Nos3(tm1Unc)/J, N9 generation backcrossing with C57BL/6J mice, homozygous for the disruption of the endothelial NOS gene (group Nos3(-/-)), and 12 pregnant, wild-type C57BL/6J mice (group Nos3(+/+)). All the measurements increased in both genotypes throughout gestation. However, embryo length and width were significantly larger in Nos3(+/+) than in Nos3(-/-) mice from Day 8.5, and both longitudinal and transverse diameters of the entire gestational sacs were larger in Nos3(+/+) mice from Day 10.5. Assessment of the relative growth of embryos/fetuses and gestational annexes showed different patterns among Nos3(-/-) and Nos3(+/+) mice. Throughout pregnancy, the distance between the external limit of the gestational sac and the embryo in Nos3(+/+) mice diminished in longitudinal sections, or remained unaffected in transverse sections. In Nos3(-/-) mice, there were significant increases (P < 0.005) in the differences between embryo and gestational vesicle measurements in both longitudinal and transversal curves from Days 5.5 to 14.5, but from Day 14.5 of pregnancy onward, the changes were not significant. The results demonstrate that the processes of fetal growth retardation in the Nos3(-/-) mice are established from early pregnancy stages.     

Endothelial nitric oxide synthase decreases beta-adrenergic responsiveness via inhibition of the L-type Ca2+ current

Signaling via endothelial nitric oxide synthase (NOS3) limits the heart's response to beta-adrenergic (beta-AR) stimulation, which may be protective against arrhythmias. However, mechanistic data are limited. Therefore, we performed simultaneous measurements of action potential (AP, using patch clamp), Ca2+ transients (fluo 4), and myocyte shortening (edge detection). L-type Ca2+ current (ICa) was directly measured by the whole cell ruptured patch-clamp technique. Myocytes were isolated from wild-type (WT) and NOS3 knockout (NOS3-/-) mice. NOS3-/- myocytes exhibited a larger incidence of beta-AR (isoproterenol, 1 microM)-induced early afterdepolarizations (EADs) and spontaneous activity (defined as aftercontractions). We also examined ICa, a major trigger for EADs. NOS3-/- myocytes had a significantly larger beta-AR-stimulated increase in ICa compared with WT myocytes. In addition, NOS3-/- myocytes had a larger response to beta-AR stimulation compared with WT myocytes in Ca2+ transient amplitude, shortening amplitude, and AP duration (APD). We observed similar effects with specific NOS3 inhibition [L-N5-(1-iminoethyl)-ornithine (l-NIO), 10 microM] in WT myocytes as with NOS3 knockout. Specifically, l-NIO further increased isoproterenol-stimulated EADs and aftercontractions. l-NIO also further increased the isoproterenol-stimulated ICa, Ca2+ transient amplitude, shortening amplitude, and APD (all P < 0.05 vs isoproterenol alone). l-NIO had no effect in NOS3-/- myocytes. These results indicate that NOS3 signaling inhibits the beta-AR response by reducing ICa and protects against arrhythmias. This mechanism may play an important role in heart failure, where arrhythmias are increased and NOS3 expression is decreased.     

Relative contributions of NOS isoforms during experimental colitis: endothelial-derived NOS maintains mucosal integrity

The role of nitric oxide (NO) in inflammatory bowel diseases has traditionally focused on the inducible form of NO synthase (iNOS). However, the constitutive endothelial (eNOS) and neuronal (nNOS) isoforms may also impact on colitis, either by contributing to the inflammation or by regulating mucosal integrity in response to noxious stimuli. To date, studies examining the roles of the NOS isoforms in experimental colitis have been conflicting, and the mechanisms by which these enzymes exert their effects remain unclear. To investigate and clarify the roles of the NOS isoforms in gut inflammation, we induced trinitrobenzenesulfonic acid colitis in eNOS, nNOS, and iNOS knockout (KO) mice, assessing the course of colitis at early and late times. Both eNOS and iNOS KO mice developed a more severe colitis compared with wild-type mice. During colitis, iNOS expression dramatically increased on epithelial and lamina propria mononuclear cells, whereas eNOS expression remained localized to endothelial cells. Electron and fluorescence microscopy identified bacteria in the ulcerated colonic mucosa of eNOS KO mice, but not in wild-type, iNOS, or nNOS KO mice. Furthermore, eNOS KO mice had fewer colonic goblet cells, impaired mucin production, and exhibited increased susceptibility to an inflammatory stimulus that was subthreshold to other mice. This susceptibility was reversible, because the NO donor isosorbide dinitrate normalized goblet cell numbers and ameliorated subsequent colitis in eNOS KO mice. These results identify a protective role for both iNOS and eNOS during colitis, with eNOS deficiency resulting in impaired intestinal defense against lumenal bacteria and increased susceptibility to colitis.