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.     

Endothelial nitric oxide synthase is predominantly involved in angiotensin II modulation of renal vascular resistance and norepinephrine release

Nitric oxide (NO) is mainly generated by endothelial NO synthase (eNOS) or neuronal NOS (nNOS). Recent studies indicate that angiotensin II generates NO release, which modulates renal vascular resistance and sympathetic neurotransmission. Experiments in wild-type [eNOS(+/+) and nNOS(+/+)], eNOS-deficient [eNOS(-/-)], and nNOS-deficient [nNOS(-/-)] mice were performed to determine which NOS isoform is involved. Isolated mice kidneys were perfused with Krebs-Henseleit solution. Endogenous norepinephrine release was measured by HPLC. Angiotensin II dose dependently increased renal vascular resistance in all mice species. EC(50) and maximal pressor responses to angiotensin II were greater in eNOS(-/-) than in nNOS(-/-) and smaller in wild-type mice. The nonselective NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 0.3 mM) enhanced angiotensin II-induced pressor responses in nNOS(-/-) and wild-type mice but not in eNOS(-/-) mice. In nNOS(+/+) mice, 7-nitroindazole monosodium salt (7-NINA; 0.3 mM), a selective nNOS inhibitor, enhanced angiotensin II-induced pressor responses slightly. Angiotensin II-enhanced renal nerve stimulation induced norepinephrine release in all species. L-NAME (0.3 mM) reduced angiotensin II-mediated facilitation of norepinephrine release in nNOS(-/-) and wild-type mice but not in eNOS(-/-) mice. 7-NINA failed to modulate norepinephrine release in nNOS(+/+) mice. (4-Chlorophrnylthio)guanosine-3', 5'-cyclic monophosphate (0.1 nM) increased norepinephrine release. mRNA expression of eNOS, nNOS, and inducible NOS did not differ between mice strains. In conclusion, angiotensin II-mediated effects on renal vascular resistance and sympathetic neurotransmission are modulated by NO in mice. These effects are mediated by eNOS and nNOS, but NO derived from eNOS dominates. Only NO derived from eNOS seems to modulate angiotensin II-mediated renal norepinephrine release.     

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.     

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.     

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.     

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.     

Poster Sessions CP13: Hypoxia,Ischemia and Oxidative Stress. Changes of brain nitric oxide production in experimental cerebral ischemia

In order to study the role of nitric oxide (NO) in ischemic brain injury. Global cerebral ischemia was established in SD rats by modified Pulsinelli's method. The activities of constitutive nitric oxide synthase (cNOS), inducible NOS (iNOS), neuronal NOS (nNOS), nitrite (NO₂) and cyclic GMP in cerebral cortex, hippocampus, striatum and cerebellum at different time intervals were measured by radioimmunoassy, NADPH‐d histochemistry and fluorometry methods. The results showed that the activities of cNOS increased at 5 min in four regions and decreased in cortex, hippocampus and striatum at 60 min, in cerebellum at 15 min iNOS increased in cortex and striatum at 15 min, in hippocampus and cerebellum at 10 min, and persisted to 60 min. The expression of nNOS increased after 5 min ischemia in cortex, striatum and hippocampus, and return to normal at 30–60 min. The NO₂ and cGMP also increased after 5–15 min ischemia and returned to normal after 30–60 min ischemia. These results indicated that the NO participated in the pathogenesis of cerebral ischemia injury and different types of NOS play different role in the cerebral ischemia injuries. Selected specific NOS inhibitors to decreased the excessive production of NO at early stage may help to decrease the ischemic injury.     

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.     

Regulation of nitric oxide production in limb and ventilatory muscles during chronic exercise training

In this study, we evaluated the differential influence of chronic treadmill training (30 m/min, 15% incline, 1 h/day, 5 days/wk) on nitric oxide (NO) production and NO synthase (NOS) isoform expression as well as 3-nitrotyrosine formation (footprint of peroxynitrite) both in limb (gastrocnemius) and ventilatory (diaphragm) muscles. A group of exercise-trained rats and a control group (no training) were examined after a 4-wk experimental period. Exercise training elicited an approximate fourfold rise in gastrocnemius NOS activity and augmented protein expression of the endothelial (eNOS) and neuronal (nNOS) isoforms of NOS to approximately 480% and 240%, respectively. Qualitatively similar but quantitatively smaller elevations in NOS activity and eNOS and nNOS expression were observed in the diaphragm. No detectable inducible NOS (iNOS) protein expression was found in any of the muscle samples. Training increased the intensity of 3-nitrotyrosine only in the gastrocnemius muscle. We conclude that whole body exercise training enhances both limb and ventilatory muscle NO production and that constitutive and not iNOS isoforms are responsible for increased protein tyrosine nitration in trained limb muscles.     

Roles of nitric oxide in brain hypoxia-ischemia.

A large body of evidence has appeared over the last 6 years suggesting that nitric oxide biosynthesis is a key factor in the pathophysiological response of the brain to hypoxia-ischemia. Whilst studies on the influence of nitric oxide in this phenomenon initially offered conflicting conclusions, the use of better biochemical tools, such as selective inhibition of nitric oxide synthase (NOS) isoforms or transgenic animals, is progressively clarifying the precise role of nitric oxide in brain ischemia. Brain ischemia triggers a cascade of events, possibly mediated by excitatory amino acids, yielding the activation of the Ca2+-dependent NOS isoforms, i.e. neuronal NOS (nNOS) and endothelial NOS (eNOS). However, whereas the selective inhibition of nNOS is neuroprotective, selective inhibition of eNOS is neurotoxic. Furthermore, mainly in glial cells, delayed ischemia or reperfusion after an ischemic episode induces the expression of Ca2+-independent inducible NOS (iNOS), and its selective inhibition is neuroprotective. In conclusion, it appears that activation of nNOS or induction of iNOS mediates ischemic brain damage, possibly by mitochondrial dysfunction and energy depletion. However, there is a simultaneous compensatory response through eNOS activation within the endothelium of blood vessels, which mediates vasodilation and hence increases blood flow to the damaged brain area.     

Neuronal nitric oxide synthase contributes to the regulation of hematopoiesis

Nitric oxide (NO) signaling is important for the regulation of hematopoiesis. However, the role of individual NO synthase (NOS) isoforms is unclear. Our results indicate that the neuronal NOS isoform (nNOS) regulates hematopoiesis in vitro and in vivo. nNOS is expressed in adult bone marrow and fetal liver and is enriched in stromal cells. There is a strong correlation between expression of nNOS in a panel of stromal cell lines established from bone marrow and fetal liver and the ability of these cell lines to support hematopoietic stem cells; furthermore, NO donor can further increase this ability. The number of colonies generated in vitro from the bone marrow and spleen of nNOS-null mutants is increased relative to wild-type or inducible- or endothelial NOS knockout mice. These results describe a new role for nNOS beyond its action in the brain and muscle and suggest a model where nNOS, expressed in stromal cells, produces NO which acts as a paracrine regulator of hematopoietic stem cells.     

Neuronal nitric oxide synthase: expression in rat parietal cells

Nitric oxide synthases (NOS) are enzymes that catalyze the generation of nitric oxide (NO) from L-arginine and require nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. At least three isoforms of NOS have been identified: neuronal NOS (nNOS or NOS I), inducible NOS (iNOS or NOS II), and endothelial NOS (eNOS or NOS II). Recent studies implicate NO in the regulation of gastric acid secretion. The aim of the present study was to localize the cellular distribution and characterize the isoform of NOS present in oxyntic mucosa. Oxyntic mucosal segments from rat stomach were stained by the NADPH-diaphorase reaction and with isoform-specific NOS antibodies. The expression of NOS in isolated, highly enriched (>98%) rat parietal cells was examined by immunohistochemistry, Western blot analysis, and RT-PCR. In oxyntic mucosa, histochemical staining revealed NADPH-diaphorase and nNOS immunoreactivity in cells in the midportion of the glands, which were identified as parietal cells in hematoxylin and eosin-stained step sections. In isolated parietal cells, decisive evidence for nNOS expression was obtained by specific immunohistochemistry, Western blotting, and RT-PCR. Cloning and sequence analysis of the PCR product confirmed it to be nNOS (100% identity). Expression of nNOS in parietal cells suggests that endogenous NO, acting as an intracellular signaling molecule, may participate in the regulation of gastric acid secretion.     

Reduced neuronal nitric oxide synthase is involved in ischemia-induced hippocampal neurogenesis by up-regulating inducible nitric oxide synthase expression

Nitric oxide (NO), a free radical with signaling functions in the CNS, is implicated in some developmental processes, including neuronal survival, precursor proliferation, and differentiation. However, neuronal nitric oxide synthase (nNOS) -derived NO and inducible nitric oxide synthase (iNOS) -derived NO play opposite role in regulating neurogenesis in the dentate gyrus after cerebral ischemia. In this study, we show that focal cerebral ischemia reduced nNOS expression and enzymatic activity in the hippocampus. Ischemia-induced cell proliferation in the dentate gyrus was augmented in the null mutant mice lacking nNOS gene (nNOS−/−) and in the rats receiving 7-nitroindazole, a selective nNOS inhibitor, after stroke. Inhibition of nNOS ameliorated ischemic injury, up-regulated iNOS expression, and enzymatic activity in the ischemic hippocampus. Inhibition of nNOS increased and iNOS inhibitor decreased cAMP response element-binding protein phosphorylation in the ipsilateral hippocampus in the late stage of stroke. Moreover, the effects of 7-nitroindazole on neurogenesis after ischemia disappeared in the null mutant mice lacking iNOS gene (iNOS−/−). These results suggest that reduced nNOS is involved in ischemia-induced hippocampal neurogenesis by up-regulating iNOS expression and cAMP response element-binding protein phosphorylation.     

Vasodilator mechanisms in the coronary circulation of endothelial nitric oxide synthase-deficient mice

Previous studies have demonstrated that responses to endothelium-dependent vasodilators are absent in the aortas from mice deficient in expression of endothelial nitric oxide synthase (eNOS -/- mice), whereas responses in the cerebral microcirculation are preserved. We tested the hypothesis that in the absence of eNOS, other vasodilator pathways compensate to preserve endothelium-dependent relaxation in the coronary circulation. Diameters of isolated, pressurized coronary arteries from eNOS -/-, eNOS heterozygous (+/-), and wild-type mice (eNOS +/+ and C57BL/6J) were measured by video microscopy. ACh (an endothelium-dependent agonist) produced vasodilation in wild-type mice. This response was normal in eNOS +/- mice and was largely preserved in eNOS -/- mice. Responses to nitroprusside were also similar in arteries from eNOS +/+, eNOS +/-, and eNOS -/- mice. Dilation to ACh was inhibited by N(G)-nitro-L-arginine, an inhibitor of NOS in control and eNOS -/- mice. In contrast, trifluoromethylphenylimidazole, an inhibitor of neuronal NOS (nNOS), decreased ACh-induced dilation in arteries from eNOS-deficient mice but had no effect on responses in wild-type mice. Indomethacin, an inhibitor of cyclooxygenase, decreased vasodilation to ACh in eNOS-deficient, but not wild-type, mice. Thus, in the absence of eNOS, dilation of coronary arteries to ACh is preserved by other vasodilator mechanisms.     

Nitric oxide is proangiogenic in the retina and choroid

Nitric oxide (NO) has been shown to have proangiogenic or antiangiogenic effects depending upon the setting. In this study, we used mice with targeted deletion of one of the three isoforms of nitric oxide synthase (NOS) to investigate the effects of NO in ocular neovascularization. In transgenic mice with increased expression of vascular endothelial growth factor (VEGF) in photoreceptors, deficiency of any of the three isoforms caused a significant decrease in subretinal neovascularization, but no alteration of VEGF expression. In mice with laser-induced rupture of Bruch's membrane, deficiency of inducible NOS (iNOS) or neuronal NOS (nNOS), but not endothelial NOS (eNOS), caused a significant decrease in choroidal neovascularization. In mice with oxygen-induced ischemic retinopathy, deficiency of eNOS, but not iNOS or nNOS caused a significant decrease in retinal neovascularization and decreased expression of VEGF. These data suggest that NO contributes to both retinal and choroidal neovascularization and that different isoforms of NOS are involved in different settings and different disease processes. A broad spectrum NOS inhibitor may have therapeutic potential for treatment of both retinal and choroidal neovascularization.     

Angiotensin II response in afferent arterioles of mice lacking either the endothelial or neuronal isoform of nitric oxide synthase

The aim of the study is to evaluate the impact of nitric oxide (NO) produced by endothelial NO synthase (eNOS) and neuronal NOS (nNOS) on the angiotensin II response in afferent arterioles (Af). Dose responses were assessed for angiotensin II in microperfused Af of mice homozygous for disruption of the eNOS gene [eNOS(-/-)], or nNOS gene [nNOS(-/-)], and their wild-type controls, eNOS(+/+) and nNOS(+/+). Angiotensin II at 10(-8) and 10(-6) mol/l reduced the lumen to 69% and 68% in eNOS(+/+), and to 59% and 50% in nNOS(+/+). N(G)-nitro-L-arginine methyl ester (L-NAME) did not change basal arteriolar diameters, but augmented angiotensin II contraction, reducing diameters to 23% and 13% in eNOS(+/+), and 7% and 10% in nNOS(+/+) at 10(-8) and 10(-6) mol/l. The response to angiotensin II was enhanced in nNOS(-/-) mice (41% and 25% at 10(-8) and 10(-6) mol/l) and even more enhanced in eNOS(-/-) mice (12% and 9%) compared with nNOS(+/+) and eNOS(+/+). L-NAME led to complete constriction of Af in these groups. Media-to-lumen ratios of Af did not differ between controls and gene-deficient mice. mRNA expression of angiotensin II receptor types 1A and 1B and type 2 also did not differ. The results reveal that angiotensin II-induced release of NO from both eNOS and nNOS significantly contributes to the control of Af. Results also suggest that eNOS-derived NO is of greater importance than nNOS-derived NO in this isolated arteriolar preparation.     

Distinct roles of nitric oxide synthases and interstitial cells of Cajal in rectoanal relaxation

Nitric oxide (NO) relaxes the internal anal sphincter (IAS), but its enzymatic source(s) remains unknown; neuronal (nNOS) and endothelial (eNOS) NO synthase (NOS) isoforms could be involved. Also, interstitial cells of Cajal (ICC) may be involved in IAS relaxation. We studied the relative roles of nNOS, eNOS, and c-Kit-expressing ICC for IAS relaxation using genetic murine models. The basal IAS tone and the rectoanal inhibitory reflex (RAIR) were assessed in vivo by a purpose-built solid-state manometric probe and by using wild-type, nNOS-deficient (nNOS-/-), eNOS-deficient (eNOS-/-), and W/W(v) mice (lacking certain c-Kit-expressing ICC) with or without L-arginine or N(omega)-nitro-L-arginine methyl ester (L-NAME) treatment. Moreover, the basal tone and response to electrical field stimulation (EFS) were studied in organ bath using wild-type and mutant IAS. In vivo, the basal tone of eNOS-/- was higher and W/W(v) was lower than wild-type and nNOS-/- mice. L-arginine administered rectally, but not intravenously, decreased the basal tone in wild-type, nNOS-/-, and W/W(v) mice. However, neither L-arginine nor L-NAME affected basal tone in eNOS-/- mice. In vitro, L-arginine decreased basal tone in wild-type and nNOS-/- IAS but not in eNOS-/- or wild-type IAS without mucosa. The in vivo RAIR was intact in wild-type, eNOS-/-, and W/W(v) mice but absent in all nNOS-/- mice. EFS-induced IAS relaxation was also reduced in nNOS-/- IAS. Thus the basal IAS tone is largely controlled by eNOS in the mucosa, whereas the RAIR is controlled by nNOS. c-Kit-expressing ICC may not be essential for the RAIR.     

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.     

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.     

Mitochondrial nitric oxide synthase is not eNOS, nNOS or iNOS

Recent studies indicated that there is a distinct mitochondrial nitric oxide synthase (mtNOS) enzyme, which may be identical to the other known NOS isoforms. We investigated the possible involvement of the endothelial, the neuronal, and the inducible NOS isoforms (eNOS, nNOS, iNOS, respectively) in mitochondrial NO production. Mouse liver mitochondria were prepared by Percoll gradient purification from wild-type and NOS knockout animals. NOS activity was measured by the arginine conversion assay, NO production of live mitochondria was visualized by the fluorescent probe DAF-FM with confocal microscopy and measured with flow cytometry. Western blotting or immunoprecipitation was performed with 12 different anti-NOS antibodies. Mitochondrial NOS was purified by arginine, 2,5 ADP and calmodulin affinity columns. We observed NO production and NOS activity in mitochondria, which was not attenuated by classic NOS inhibitors. We also detected low amounts of eNOS protein in the mitochondria, however, NO production and NOS activity were intact in eNOS knockout animals. Neither nNOS nor iNOS were present in the mitochondria. Furthermore, we could not find mitochondrial targeting signals in the sequences of either NOS proteins. Taken together, the presented data do not support the hypothesis that any of the known NOS enzymes are present in the mitochondria in physiologically relevant levels.