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.     

Expression of nitric oxide synthase isoforms in the mouse kidney: cellular localization and influence by lipopolysaccharide and Toll-like receptor 4

We determined the cellular mRNA expression of all intrarenal nitric oxide (NO)-producing NO synthase (NOS) isoforms, endothelial NOS (eNOS) and neuronal NOS (nNOS) and inducible NOS (iNOS) in kidneys from wild-type mice (WT) and immune deficient Toll-like receptor 4 (TLR4) mutant mice, during normal physiological conditions and during a short-term (6–16 h) endotoxic condition caused by systemically administered lipopolysaccaride (LPS). Investigations were performed by means of in situ hybridization and polymerase chain reaction amplification techniques. In WT, LPS altered the expression rate of all intrarenal NOS isoforms in a differentiated but NOS-isoform coupled expression pattern, with iNOS induction, and up- and down-regulation of the otherwise constitutively expressed NOS isoforms, e.g. eNOS and nNOS and an iNOS isotype. In TLR4 mutants, LPS caused none or a lowered iNOS induction, but altered the expression rate of the constitutive NOS isoforms. It is concluded that the intrarenal spatial relation of individual NOS-isoforms and their alteration in expression provide the basis for versatile NO-mediated renal actions that may include local interactions between NOS isoforms and their individual NO-target sites, and that the NOS-isoform dependent events are regulated by TLR4 during endotoxic processes. These regulatory mechanisms are likely to participate in different pathophysiological conditions affecting NO-mediated renal functions.     

Regulation of murine airway responsiveness by endothelial nitric oxide synthase

Nitric oxide (NO) is a potent vasodilator, but it can also modulate contractile responses of the airway smooth muscle. Whether or not endothelial (e) NO synthase (NOS) contributes to the regulation of bronchial tone is unknown at present. Experiments were designed to investigate the isoforms of NOS that are expressed in murine airways and to determine whether or not the endogenous release of NO modulates bronchial tone in wild-type mice and in mice with targeted deletion of eNOS [eNOS(-/-)]. The presence of neuronal NOS (nNOS), inducible NOS (iNOS), and eNOS in murine trachea and lung parenchyma was assessed by RT-PCR, immunoblotting, and immunohistochemistry. Airway resistance was measured in conscious unrestrained mice by means of a whole body plethysmography chamber. The three isoforms of NOS were constitutively present in lungs of wild-type mice, whereas only iNOS and nNOS were present in eNOS(-/-) mice. Labeling of nNOS was localized in submucosal airway nerves but was not consistently detected, and iNOS immunoreactivity was observed in tracheal and bronchiolar epithelial cells, whereas eNOS was expressed in endothelial cells. In wild-type mice, treatment with N-nitro-L-arginine methyl ester, but not with aminoguanidine, potentiated the increase in airway resistance produced by inhalation of methacholine. eNOS(-/-) mice were hyperresponsive to inhaled methacholine and markedly less sensitive to N-nitro-L-arginine methyl ester. These results demonstrate that the three NOS isoforms are expressed constitutively in murine lung and that NO derived from eNOS plays a physiological role in controlling bronchial airway reactivity.     

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.     

Paradoxical roles of different nitric oxide synthase isoforms in colonic injury

Nitric oxide (NO) is a free radical that is largely produced by three isoforms of NO synthase (NOS): neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). NO regulates numerous processes in the gastrointestinal tract; however, the overall role that NO plays in intestinal inflammation is unclear. NO is upregulated in both ulcerative colitis and Crohn's disease as well as in animal models of colitis. There have been conflicting reports on whether NO protects or exacerbates injury in colitis or is simply a marker of inflammation. To determine whether the site, timing, and level of NO production modulate the effect on the inflammatory responses, the dextran sodium sulfate model of colitis was assessed in murine lines rendered deficient in iNOS, nNOS, eNOS, or e/nNOS by targeted gene disruption. The loss of nNOS resulted in more severe disease and increased mortality, whereas the loss of eNOS or iNOS was protective. Furthermore, concomitant loss of eNOS reversed the susceptibility found in nNOS-/- mice. Deficiencies in specific NOS isoforms led to distinctive alterations of inflammatory responses, including changes in leukocyte recruitment and alterations in colonic lymphocyte populations. The present studies indicate that NO produced by individual NOS isoforms plays different roles in modulating an inflammatory process.     

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.     

Endothelial nitric oxide synthase is protective in the initiation of caerulein-induced acute pancreatitis in mice

The effect of inhibiting nitric oxide (NO) synthase (NOS) or enhancing NO on the course of acute pancreatitis (AP) is controversial, in part because three NOS isoforms exist: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). We investigated whether inhibition or selective gene deletion of NOS isoforms modified the initiation phase of caerulein-induced AP in mice and explored whether this affected pancreatic microvascular blood flow (PMBF). We investigated the effects of nonspecific NOS inhibition with N(omega)-nitro-l-arginine (l-NNA; 10 mg/kg ip) or targeted deletion of eNOS, nNOS, or iNOS genes on the initiation phase of caerulein-induced AP in mice using in vivo and in vitro models. Western blot analysis was performed to assess eNOS phosphorylation status, an indicator of enzyme activity, and microsphere studies were used to measure PMBF. l-NNA and eNOS deletion, but not nNOS or iNOS deletion, increased pancreatic trypsin activity and serum lipase during the initiation phase of in vivo caerulein-induced AP. l-NNA and eNOS did not affect trypsin activity in caerulein-hyperstimulated isolated acini, suggesting that nonacinar events mediate the effect of NOS blockade in vivo. The initiation phase of AP in wild-type mice was associated with eNOS Thr(495) residue dephosphorylation, which accompanies eNOS activation, and a 178% increase in PMBF; these effects were absent in eNOS-deleted mice. Thus eNOS is the main isoform influencing the initiation of caerulein-induced AP. eNOS-derived NO exerts a protective effect through actions on nonacinar cell types, most likely endothelial cells, to produce greater PMBF.     

Cyclosporin-A Inhibits Constitutive Nitric Oxide Synthase Activity and Neuronal and Endothelial Nitric Oxide Synthase Expressions after Spinal Cord Injury in Rats

Nitric oxide (NO) plays a role in the pathophysiology of spinal cord injury (SCI). NO is produced by three types of nitric oxide synthase (NOS) enzymes: The constitutive Ca²⁺/calmodulin-dependent neuronal NOS (nNOS) and endothelial NOS (eNOS) isoforms, and the inducible calcium-independent isoform (iNOS). During the early stages of SCI, nNOS and eNOS produce significant amounts of NO, therefore, the regulation of their activity and expression may participate in the damage after SCI. In the present study, we used Cyclosporin-A (CsA) to further substantiate the role of Ca-dependent NOS in neural responses associated to SCI. Female Wistar rats were subjected to SCI by contusion, and killed 4 h after lesion. Results showed an increase in the activity of constitutive NOS (cNOS) after lesion, inhibited by CsA (2.5 mg/kg i.p.). Western blot assays showed an increased expression of both nNOS and eNOS after trauma, also antagonized by CsA administration.     

Nitric oxide synthase, an essential factor in peripheral nerve regeneration.

Nitric oxide (NO) exerts both, pro-apoptotic and anti-apoptotic actions and appears to be acritical factor inneuronal degenerative and regenerative processes. NO is synthesized from L-arginine by NO synthase occurring in three isoforms of (neuronal, nNOS; endothelial, eNOS; inducible, iNOS). In a mice sciatic nerve model the regenerative outcome was assessed when the endogenous NO supply was deficient by knocking out the respective NOS isoform and compared to that of wild type mice after nerve transection. In nNOS knock-out mice a delay in regeneration, preceded by slowedWallerian degeneration and a disturbed pruning of uncontrolled sprouts, was observed. This was associated with a delayed recovery of sensory and motor function. Additionally, deficiency of nNOS led after nerve cut to a substantial loss of small and medium-sized dorsal root ganglia neurons, spinal cord interneurons and, to a lesser extent, spinal cord motor neurons. A lack of iNOS resulted in a delayed Wallerian degeneration and impaired regenerative outcome without consequences for neuronal survival. A lack of eNOS was well tolerated, although a delay in nerve revascularization was observed. Thus, after peripheral nerve lesion, regular NOS activity is essential for cell survival and recovery with reference to the nNOS isoform.     

The participation of brain NO synthase in blood pressure control of adult spontaneously hypertensive rats

Increased blood pressure (BP) in genetic hypertension is usually caused by high activity of sympathetic nervous system (SNS) which is enhanced by central angiotensin II but lowered by central nitric oxide (NO). We have therefore evaluated NO synthase (NOS) activity as well as neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) protein expression in brainstem and midbrain of adult spontaneously hypertensive rats (SHR) characterized by enhanced sympathetic vasoconstriction. We also studied possible participation of brain NO in antihypertensive effects of chronic captopril treatment of adult SHR. NOS activity was increased in midbrain of SHR compared to Wistar-Kyoto (WKY) rats. This could be ascribed to enhanced iNOS expression, whereas nNOS expression was unchanged and eNOS expression was reduced in this brain region. In contrast, no significant changes of NOS activity were found in brainstem of SHR in which nNOS and iNOS expression was unchanged, but eNOS expression was increased. Chronic captopril administration lowered BP of adult SHR mainly by attenuation of sympathetic tone, whereas the reduction of angiotensin II-dependent vasoconstriction and the decrease of residual BP (amelioration of structural remodeling of resistance vessels) were less important. This treatment did not affect significantly either NOS activity or expression of any NOS isoform in the two brain regions. Our data do not support the hypothesis that altered brain NO formation contributes to sympathetic hyperactivity and high BP of adult SHR with established hypertension.     

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.     

Pulmonary NO synthase expression is attenuated in a fetal baboon model of chronic lung disease

Nitric oxide (NO), produced by NO synthase (NOS), serves multiple functions in the perinatal lung. In fetal baboons, neuronal (nNOS), endothelial (eNOS), and inducible NOS (iNOS) are all primarily expressed in proximal respiratory epithelium. In the present study, NOS expression and activity in proximal lung and minute ventilation of NO standard temperature and pressure (VeNO(STP)) were evaluated in a model of chronic lung disease (CLD) in baboons delivered at 125 days (d) of gestation (term = 185 d) and ventilated for 14 d, obtaining control lung samples from fetuses at 125 or 140 d of gestation. In contrast to the normal 73% increase in total NOS activity from 125 to 140 d of gestation, there was an 83% decline with CLD. This was related to marked diminutions in both nNOS and eNOS expression and enzymatic activity. nNOS accounted for the vast majority of enzymatic activity in all groups. The normal 3.3-fold maturational rise in iNOS protein expression was blunted in CLD, yet iNOS activity was elevated in CLD compared with at birth. The contribution of iNOS to total NOS activity was minimal in all groups. VeNO(STP) remained stable in the range of 0.5-1.0 nl x kg(-1) x min(-1) from birth to day 7 of life, and it then rose by 2.5-fold. Thus the baboon model of CLD is characterized by deficiency of the principal pulmonary isoforms, nNOS and eNOS, and enhanced iNOS activity over the first 2 wk of postnatal life. It is postulated that these alterations in NOS expression and activity may contribute to the pathogenesis of CLD.     

Expression of nitric oxide synthase isoforms is reduced in late-gestation ovine fetal brainstem

Fetal baroreflex responsiveness increases in late gestation. An important modulator of baroreflex activity is the generation of nitric oxide in the brainstem nuclei that integrate afferent and efferent reflex activity. The present study was designed to test the hypothesis that nitric oxide synthase (NOS) isoforms are expressed in the fetal brainstem and that the expression of one or more of these enzymes is reduced in late gestation. Brainstem tissue was rapidly collected from fetal sheep of known gestational ages (80, 100, 120, 130, 145 days gestation and 1 day and 1 wk postnatal). Neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) mRNA was measured using real-time PCR methodology specific for ovine NOS isoforms. The three enzymes were measured at the protein level using Western blot methodology. In tissue prepared for histology separately, the cellular pattern of immunostaining was identified in medullae from late-gestation fetal sheep. Fetal brainstem contained mRNA and protein of all three NOS isoforms, with nNOS the most abundant, followed by iNOS and eNOS, respectively. nNOS and iNOS mRNA abundances were highest at 80 days' gestation, with statistically significant decreases in abundance in more mature fetuses and postnatal animals. nNOS and eNOS protein abundance also decreased as a function of developmental age. nNOS and eNOS were expressed in neurons, iNOS was expressed in glia, and eNOS was expressed in vascular endothelial cells. We conclude that all three isoforms of NOS are constitutively expressed within the fetal brainstem, and the expression of all three forms is reduced with advancing gestation. We speculate that the reduced expression of NOS in this brain region plays a role in the increased fetal baroreflex activity in late gestation.     

Changes in the expression of NO synthase isoforms after ozone: the effects of allergen exposure

BackgroundThe functional role of nitric oxide (NO) and various nitric oxide synthase (NOS) isoforms in asthma remains unclear.ObjectiveThis study investigated the effects of ozone and ovalbumin (OVA) exposure on NOS isoforms.MethodsThe expression of inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS) in lung tissue was measured. Enhanced pause (P_enh) was measured as a marker of airway obstruction. Nitrate and nitrite in bronchoalveolar lavage (BAL) fluid were measured using a modified Griess reaction.ResultsThe nitrate concentration in BAL fluid from the OVA-sensitized/ozone-exposed/OVA-challenged group was greater than that of the OVA-sensitized/saline-challenged group. Methacholine-induced P_enh was increased in the OVA-sensitized/ozone-exposed/OVA-challenged group, with a shift in the dose-response curve to the left, compared with the OVA-sensitized/saline-challenged group. The levels of nNOS and eNOS were increased significantly in the OVA-sensitized/ozone-exposed/OVA-challenged group and the iNOS levels were reduced compared with the OVA-sensitized/saline-challenged group.ConclusionIn mice, ozone is associated with increases in lung eNOS and nNOS, and decreases in iNOS. None of these enzymes are further affected by allergens, suggesting that the NOS isoforms play different roles in airway inflammation after ozone exposure.     

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.     

Differential expression and localization of nitric oxide synthases in cirrhotic livers of bile duct-ligated rats.

Increased vascular nitric oxide (NO) production has been implicated in the pathogenesis of the hyperdynamic circulation in liver cirrhosis. This study investigated the expression of three isoforms of NO synthase (NOS) in rat cirrhotic livers. Cirrhosis was induced by chronic bile duct ligation (BDL). NOS enzyme activity was assessed by L-citrulline generation. Competitive RT-PCR was performed to detect the mRNA levels of NOS. In situ hybridization was done to localize NOS mRNA. Protein expression of NOS was evaluated by Western blotting and immunohistochemistry. The L-citrulline assay showed that constitutive NOS (cNOS) enzymatic activity was decreased, while inducible NOS (iNOS) activity was increased in BDL livers. Both endothelial NOS (eNOS) and neuronal NOS (nNOS) mRNA were detected in BDL and sham rats, but with enhanced expression in BDL rats. eNOS protein was redistributed with less expression in sinusoidal endothelial cells, but the total levels in liver were not changed. nNOS was induced in hepatocytes of BDL rats, in contrast to only a weak signal observed around some blood vessels in sham livers. Intense mRNA and protein expression of iNOS was induced in livers of BDL rats and was localized in hepatocytes, with no or a negligible amount in control livers. In conclusion, iNOS was induced in cirrhotic liver with its activity increased. In contrast, cNOS activity was impaired, regardless of unchanged eNOS protein levels and enhanced nNOS expression. These results suggest that all three types of NOS have a role in cirrhosis, but their expression and regulation are different.     

Regulation of diaphragmatic nitric oxide synthase expression during hypobaric hypoxia

Nitric oxide (NO) is normally synthesized inside skeletal muscle fibers by both endothelial (eNOS) and neuronal (nNOS) nitric oxide synthases. In this study, we evaluated the influence of hypobaric hypoxia on the expression of NOS isoforms, argininosuccinate synthetase (AS), argininosuccinate lyase (AL), and manganese superoxide dismutase (Mn SOD) in the ventilatory muscles. Rats were exposed to hypobaric hypoxia ( approximately 95 mmHg) from birth for 60 days or 9-11 mo. Age-matched control groups of rats also were examined. Sixty days of hypoxia elicited approximately two- and ninefold increases in diaphragmatic eNOS and nNOS protein expression (evaluated by immunoblotting), respectively, and about a 50% rise in diaphragmatic NOS activity. In contrast, NOS activity and the expression of these proteins declined significantly in response to 9 mo of hypoxia. Hypoxia elicited no significant alterations in AS, AL and Mn SOD protein expression. Moreover, the inducible NOS (iNOS) was not detected in normoxic and hypoxic diaphragmatic samples. We conclude that diaphragmatic NOS expression and activity undergo significant adaptations to hypobaric hypoxia and that iNOS does not participate in this response.     

Is there a role for neuronal nitric oxide synthase (nNOS) in cytokine toxicity to pancreatic beta cells?

Nitric oxide (NO), produced by the action of the inducible NO synthase, plays a crucial role in cytokine toxicity to pancreatic beta cells during type 1 diabetes development. It was the aim of this study to analyze the role of the neuronal NOS (nNOS) in proinflammatory cytokine-mediated beta cell toxicity. Expression of different isoforms of nitric oxide synthase in insulin-secreting INS1E cells and rat islets was analyzed by quantitative real-time PCR and Western blotting. The expression of nNOS in insulin-secreting INS1E cells was similar to that found in rat brain, while two other isoforms, namely the endothelial eNOS and inducible iNOS were not expressed in untreated cells. IL-1β alone or in combination with TNF-α and/or IFNγ induced iNOS but not eNOS expression. In contrast, nNOS expression was strongly decreased by the mixture of the three proinflammatory cytokines (IL-1β, TNF-α and IFNγ) both on the gene and protein level in INS1E cells and rat islet cells. The effects of cytokines on glucose-induced insulin-secretion followed the pattern of nNOS expression reduction and, on the other hand, of the iNOS induction. The data indicate that a low level of nitric oxide originating from the constitutive expression of nNOS in pancreatic beta cells is not deleterious. In particular since proinflammatory cytokines reduce this expression. This nNOS suppression can compensate for NO generation by low concentrations of IL-1β through iNOS induction. Thus, this basal nNOS expression level in pancreatic beta cells represents a protective element against cytokine toxicity.     

Developmental changes in nitric oxide synthase isoform expression and nitric oxide production in fetal baboon lung

Nitric oxide (NO), produced by NO synthase (NOS), plays a critical role in multiple processes in the lung during the perinatal period. To better understand the regulation of pulmonary NO production in the developing primate, we determined the cell specificity and developmental changes in NOS isoform expression and action in the lungs of third-trimester fetal baboons. Immunohistochemistry in lungs obtained at 175 days (d) of gestation (term = 185 d) revealed that all three NOS isoforms, neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), are primarily expressed in proximal airway epithelium. In proximal lung, there was a marked increase in total NOS enzymatic activity from 125 to 140 d gestation due to elevations in nNOS and eNOS, whereas iNOS expression and activity were minimal. Total NOS activity was constant from 140 to 175 d gestation, and during the latter stage (160-175 d gestation), a dramatic fall in nNOS and eNOS was replaced by a rise in iNOS. Studies done within 1 h of delivery at 125 or 140 d gestation revealed that the principal increase in NOS during the third trimester is associated with an elevation in exhaled NO levels, a decline in expiratory resistance, and greater pulmonary compliance. Thus, there are developmental increases in pulmonary NOS expression and NO production during the early third trimester in the primate that may enhance airway and parenchymal function in the immediate postnatal period.