Nitric oxide production in the ventilatory muscles in response to acute resistive loading

The effect of muscle activation on muscle nitric oxide (NO) production remains controversial. Whereas NO release increases in in vitro activated muscles and in vivo limb muscles, diaphragmatic NO synthase (NOS) activity declines after 3 h of inspiratory resistive loading (IRL). We tested in this study the hypotheses that acute IRL decreases diaphragmatic NO derivatives levels and reduces protein expression of neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) NO synthases, as well as 3-nitrotyrosine formation. Anesthetized, tracheostomized, spontaneously breathing adult rats were subjected to IRL (50% of the maximum inspiratory pressure) for 1, 3, or 6 h. Quietly breathing rats served as controls. After 3 h of IRL, muscle eNOS and nNOS protein levels rose by 80 and 60% of control values, respectively. Whereas eNOS expression did not change any further, nNOS expression reached 550% of control values after 6 h of IRL. Strong iNOS protein expression was detected in the diaphragms after 6 h of IRL. Total NO derivatives levels in the diaphragm declined during IRL as a result of reduction in nitrate, nitrite, and nitrosothiols. Diaphragmatic protein tyrosine nitration decreased in response to IRL, and this reduction was mainly due to reduced tyrosine nitration of enolase and aldolase. We conclude that diaphragmatic NO derivatives levels decline in response to IRL and that the rise in diaphragmatic NOS protein expression may be a compensatory response designed to counterbalance the decline in NOS activity.     

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.     

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.     

Nitric oxide produced via neuronal NOS may impair vasodilatation in septic rat skeletal muscle

Impaired vascular responsiveness in sepsis may lead to maldistribution of blood flow in organs. We hypothesized that increased production of nitric oxide (NO) via inducible nitric oxide synthase (iNOS) mediates the impaired dilation to ACh in sepsis. Using a 24-h cecal ligation and perforation (CLP) model of sepsis, we measured changes in arteriolar diameter and in red blood cell velocity (V(RBC)) in a capillary fed by the arteriole, following application of ACh to terminal arterioles of rat hindlimb muscle. Sepsis attenuated both ACh-stimulated dilation and V(RBC) increase. In control rats, arteriolar pretreatment with the NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside reduced diameter and V(RBC) responses to a level that mimicked sepsis. In septic rats, arteriolar pretreatment with the "selective" iNOS blockers aminoguanidine (AG) or S-methylisothiourea sulfate (SMT) restored the responses to the control level. The putative neuronal NOS (nNOS) inhibitor 7-nitroindazole also restored the response toward control. At 24-h post-CLP, muscles showed no reduction of endothelial NOS (eNOS), elevation of nNOS, and, surprisingly, no induction of iNOS protein; calcium-dependent constitutive NOS (eNOS+nNOS) enzyme activity was increased whereas calcium-independent iNOS activity was negligible. We conclude that 1) AG and SMT inhibit nNOS activity in septic skeletal muscle, 2) NO could impair vasodilative responses in control and septic rats, and 3) the source of increased endogenous NO in septic muscle is likely upregulated nNOS rather than iNOS. Thus agents released from the blood vessel milieu (e.g., NO produced by skeletal muscle nNOS) could affect vascular responsiveness.     

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.     

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.     

Role of neuronal and endothelial nitric oxide synthase in nitric oxide generation in the brain following cerebral ischemia.

Nitric oxide (NO) plays an important role in the pathogenesis of neuronal injury during cerebral ischemia. The endothelial and neuronal isoforms of nitric oxide synthase (eNOS, nNOS) generate NO, but NO generation from these two isoforms can have opposing roles in the process of ischemic injury. While increased NO production from nNOS in neurons can cause neuronal injury, endothelial NO production from eNOS can decrease ischemic injury by inducing vasodilation. However, the relative magnitude and time course of NO generation from each isoform during cerebral ischemia has not been previously determined. Therefore, electron paramagnetic resonance spectroscopy was applied to directly detect NO in the brain of mice in the basal state and following global cerebral ischemia induced by cardiac arrest. The relative amount of NO derived from eNOS and nNOS was accessed using transgenic eNOS(-/-) or nNOS(-/-) mice and matched wild-type control mice. NO was trapped using Fe(II)-diethyldithiocarbamate. In wild-type mice, only small NO signals were seen prior to ischemia, but after 10 to 20 min of ischemia the signals increased more than 4-fold. This NO generation was inhibited more than 70% by NOS inhibition. In either nNOS(-/-) or eNOS(-/-) mice before ischemia, NO generation was decreased about 50% compared to that in wild-type mice. Following the onset of ischemia a rapid increase in NO occurred in nNOS(-/-) mice peaking after only 10 min. The production of NO in the eNOS(-/-) mice paralleled that in the wild type with a progressive increase over 20 min, suggesting progressive accumulation of NO from nNOS following the onset of ischemia. NOS activity measurements demonstrated that eNOS(-/-) and nNOS(-/-) brains had 90% and < 10%, respectively, of the activity measured in wild type. Thus, while eNOS contributes only a fraction of total brain NOS activity, during the early minutes of cerebral ischemia prominent NO generation from this isoform occurs, confirming its importance in modulating the process of ischemic injury.     

Modulation by NO of acetylcholine release in the ileum of wild-type and NOS gene knockout mice

Nitric oxide (NO) inhibits the release of acetylcholine and cholinergic contractions in the small intestine of several species, but no information is available about the mouse ileum. This study examines the effects of NO on the electrically evoked release of [3H]acetylcholine and smooth muscle contraction in myenteric plexus-longitudinal muscle preparations of wild-type mice and of neuronal NO synthase (nNOS) and endothelial NOS (eNOS) knockout mice. The NOS inhibitor N(G)-nitro-L-arginine (L-NNA) and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) concentration dependently increased the evoked [3H]acetylcholine release and cholinergic contractions in preparations from wild-type mice and from eNOS knockout mice. Effects of L-NNA were specifically antagonized by L-arginine. In contrast, L-NNA and ODQ did not modify the release and contractions in preparations from nNOS knockout mice. The NO donor S-nitroso-N-acetyl-DL-penicillamine inhibited the electrically evoked release of [3H]acetylcholine and longitudinal muscle contractions in a quantitatively similar manner in wild-type preparations as well as in nNOS and eNOS knockout preparations. We conclude that endogenous NO released by electrical field stimulation tonically inhibits the release of acetylcholine. Furthermore, data suggest that nNOS and not eNOS is the enzymatic source of NO-mediating inhibition of cholinergic neurotransmission in mouse ileum.     

Neuronal and Endothelial Nitric Oxide Synthases in the Paraventricular Nucleus Modulate Sympathetic Overdrive in Insulin-Resistant Rats

A central mechanism participates in sympathetic overdrive during insulin resistance (IR). Nitric oxide synthase (NOS) and nitric oxide (NO) modulate sympathetic nerve activity (SNA) in the paraventricular nucleus (PVN), which influences the autonomic regulation of cardiovascular responses. The aim of this study was to explore whether the NO system in the PVN is involved in the modulation of SNA in fructose-induced IR rats. Control rats received ordinary drinking water, whereas IR rats received 12.5% fructose-containing drinking water for 12 wks to induce IR. Basal SNA was assessed based on the changes in renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) in response to chemicals administered to the PVN. We found an increased plasma norepinephrine level but significantly reduced NO content and neuronal NOS (nNOS) and endothelial NOS (eNOS) protein expression levels in the PVN of IR rats compared to Control rats. No difference in inducible NOS (iNOS) protein expression was observed between the two groups. In anesthetized rats, the microinjection of sodium nitroprusside (SNP), an NO donor, or Nω-nitro-L-arginine methyl ester (L-NAME), a non-selective inhibitor of NOS, into the PVN significantly decreased and increased basal SNA, respectively, in both normal and IR rats, but these responses to SNP and L-NAME in IR rats were smaller than those in normal rats. The administration of selective inhibitors of nNOS or eNOS, but not iNOS, to the PVN significantly increased basal SNA in both groups, but these responses were also smaller in IR rats. Moreover, IR rats exhibited reduced nNOS and eNOS activity in the PVN. In conclusion, these data indicate that the decreased protein expression and activity levels of nNOS and eNOS in the PVN lead to a reduction in the NO content in the PVN, thereby contributing to a subsequent enhancement in sympathoexcitation during IR.     

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.     

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.     

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.     

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.     

Locomotor performance and muscle metabolic capacities: impact of temperature and energetic status

In aquatic ectotherms, muscle metabolic capacities are strongly influenced by exogenous factors, principally temperature and food availability. Seasonal changes in temperature lead many organisms to modify their metabolic machinery so as to maintain capacity even in "slower" cold habitats. Modifications of mitochondrial capacities are central in this response. The increases in protein-specific oxidative capacities of mitochondria during cold acclimation of temperate fishes do not occur during the evolutionary adaptation to cold in Antarctic species. Instead, Antarctic fishes tend to increase the proportion of fibre volume devoted to mitochondria, perhaps to facilitate intracellular distribution of oxygen and metabolites. Variation in energetic status can drastically modify muscle metabolic status, with glycolytic muscle changing more than oxidative muscle. This in turn impacts swimming performance. A decrease in the condition of cod leads endurance at speeds above Ucrit to drop by 70%. Sprint swimming is less affected, perhaps as it does not exhaust glycolytic muscle. We used interindividual variation in muscle metabolic capacities to identify correlates of swimming performance in stickleback and cod. Activities of cytochrome c oxidase in glycolytic muscle are a correlate of sprint swimming in stickleback (Gasterosteus aculeatus) and cod (Gadus morhua), whereas lactate dehydrogenase activities in glycolytic muscle are a correlate of cod endurance swimming. In scallops, gonadal maturation leads to virtually complete mobilisation of glycogen from muscle. This does not reduce the capacity of the scallops, Chlamys islandica and Euvola ziczac, to mount escape responses, but significantly slows their recuperation from exhaustive exercise. Muscle metabolic capacities fall in parallel with glycogen mobilisation. In the compromise between muscles' dual roles as a motor and a macromolecular reserve, a significant loss in locomotory ability occurs during gametogenesis and spawning. Reproductive fitness takes the upper hand over maintenance of performance.     

Autoinhibition of endothelial nitric oxide synthase (eNOS) in gut smooth muscle by nitric oxide

Nitric oxide in the gut is produced by nNOS in enteric neurons and by eNOS in smooth muscle cells. The eNOS in smooth muscle is activated by vasoactive intestinal peptide (VIP) released from enteric neurons. In the present study, we examined the effect of nitric oxide on VIP-induced eNOS activation in smooth muscle cells isolated from human intestine and rabbit stomach. NOS activity was measured as formation of the 1:1 co-product, l-citrulline from l-arginine. VIP caused an increase in l-citrulline production that was inhibited by NO in a concentration dependent manner (IC(50)~25 microM; maximal inhibition 72% at 100 microM NO). Basal l-citrulline production, however, was unaffected by NO. The effect was not mediated by cGMP/PKG since the PKG inhibitor KT5823 had no effect on eNOS autoinhibition. The autoinhibition was selective for NO since the co-product l-citrulline had no effect on VIP-induced NOS activation. Similar effects were obtained in rabbit gastric and human intestinal smooth muscle cells. The results suggest that NO produced in smooth muscle cells as a result of the activation of eNOS by VIP exerts an autoinhibitory restraint on eNOS thereby regulating the balance of the VIP/cAMP/PKA and NO/cGMP/PKG pathways that regulate the relaxation of gut smooth muscle.     

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.     

Possible involvement of neuronal nitric oxide synthase enzyme in early-phase isoflurane-induced hypotension in rats

This study was conducted to demonstrate the involvement of nitric oxide synthase (NOS) in the early-phase isoflurane-induced hypotension and to ascertain whether this NOS is neuronal NOS (nNOS) or endothelial NOS (eNOS). Mean arterial pressures (MAPs) were directly measured from the femoral arteries of urethane-anesthetized rats. Isoflurane-induced changes in MAP were monitored in rats following pretreatment with vehicle or one of the following NOS inhibitors: L-N^G-monomethyl-L-arginine (L-NMMA), which is non-selective; L-N^G-nitro arginine (L-NOARG), which is more selective for nNOS and eNOS; and 7-nitroindazole (7-NI), which is selective for nNOS. Exposure to 2% isoflurane in oxygen produced a triphasic reduction in MAP, including an early phase in which mean arterial pressure (MAP) fell by 25-30% during the initial 2½ min. This early hypotensive response, but not subsequent phases, was abolished by i.v. pretreatment with either L-NMMA or L-NOARG. The early-phase hypotension was also significantly attenuated by i.p. pretreatment with 7-NI; however, the blockade was not as complete as with L-NMMA or L-NOARG. Cerebella and aorta were removed from vehicle- and 7-NI pretreated rats and assayed for NOS activity by determining the conversion of [¹⁴C]L-arginine to [¹⁴C]L-citrulline. The 7-NI pretreatment significantly reduced NOS activity in the cerebellum but not the aorta. These findings indicate that the early-phase isoflurane-induced hypotension may involve nNOS as well as eNOS. The nNOS may participate in regulation of isoflurane-induced neuronal release of endogenous opioid peptide, which produces a vasodilation that is dependent on NO derived from an action of eNOS.     

Chimeras of nitric-oxide synthase types I and III establish fundamental correlates between heme reduction, heme-NO complex formation, and catalytic activity

Neuronal nitric-oxide synthase (nNOS or NOS I) and endothelial NOS (eNOS or NOS III) differ widely in their reductase and nitric oxide (NO) synthesis activities, electron transfer rates, and propensities to form a heme-NO complex during catalysis. We generated chimeras by swapping eNOS and nNOS oxygenase domains to understand the basis for these differences and to identify structural elements that determine their catalytic behaviors. Swapping oxygenase domains did not alter domain-specific catalytic functions (cytochrome c reduction or H(2)O(2)-supported N(omega)-hydroxy-l-arginine oxidation) but markedly affected steady-state NO synthesis and NADPH oxidation compared with native eNOS and nNOS. Stopped-flow analysis showed that reductase domains either maintained (nNOS) or slightly exceeded (eNOS) their native rates of heme reduction in each chimera. Heme reduction rates were found to correlate with the initial rates of NADPH oxidation and heme-NO complex formation, with the percentage of heme-NO complex attained during the steady state, and with NO synthesis activity. Oxygenase domain identity influenced these parameters to a lesser degree. We conclude: 1) Heme reduction rates in nNOS and eNOS are controlled primarily by their reductase domains and are almost independent of oxygenase domain identity. 2) Heme reduction rate is the dominant parameter controlling the kinetics and extent of heme-NO complex formation in both eNOS and nNOS, and thus it determines to what degree heme-NO complex formation influences their steady-state NO synthesis, whereas oxygenase domains provide minor but important influences. 3) General principles that relate heme reduction rate, heme-NO complex formation, and NO synthesis are not specific for nNOS but apply to eNOS as well.     

Comparison of neuronal and endothelial isoforms of nitric oxide synthase in stably transfected HEK 293 cells

The neuronal and endothelial isoforms of nitric oxide (NO) synthase (nNOS and eNOS, respectively) both catalyze the production of NO but are regulated differently. Stably transfected HEK 293 cell lines containing nNOS, eNOS, and a soluble mutant of eNOS were therefore established to compare their activity in a common cellular environment. NOS activity was determined by measuring L-[3H]citrulline production in homogenates and intact cells, the conversion of oxyhemoglobin to methemoglobin, and the production of cGMP. The results indicate that nNOS is more active than eNOS, both in unstimulated as well as calcium-stimulated cells. Under basal conditions, the soluble mutant of eNOS appeared to be slightly more active than wild-type eNOS in terms of NO and cGMP formation, suggesting that membrane association may be crucial for inhibition of basal NO release but is not required for stimulation by Ca2+-mobilizing agents. The maximal activity of soluble guanylate cyclase was significantly reduced by transfection with wild-type eNOS due to downregulation of mRNA expression. These results demonstrate that nNOS and eNOS behave differently even in an identical cellular environment.