Constitutive NOS Isoforms Research Articles

Rapid, Nitric Oxide Synthesis-Dependent Activation of MMP-9 at Pericyte Somata During Capillary Ischemia in vivo

Nitric oxide serves essential roles in normal vascular physiology, but paradoxically contributes to vascular pathology in disease. During brain ischemia, aberrant nitric oxide levels can cause cellular injury through induction of nitrosative/oxidative stress and post-translational activation of matrix-metalloproteinase-9 (MMP-9). We recently demonstrated that brain pericyte somata were associated with very early and localized MMP-9 activation along capillaries during cerebral ischemia, leading to focal blood-brain barrier disruption. Here, we tested whether this effect was dependent upon nitric oxide production. In vivo two-photon imaging was used to directly visualize MMP9 activity using a FITC-gelatin probe and leakage of intravenous dye during photothrombotically induced capillary ischemia. Results showed that the NOS inhibitor, L-NIL, at concentrations affecting both iNOS and constitutive NOS isoforms, attenuated capillary leakage at pericyte soma-specific locations and substantially reduced FITC-gelatin cleavage. We also found that combined administration of L-NIL and anisomycin, an inhibitor of protein synthesis, led to near complete elimination of FITC-gelatin cleavage and vascular leakage. These results indicate that both nitric oxide synthase and new protein synthesis are involved in the rapid activation of MMP-9 at somata of capillary pericytes during ischemia.

Characteristics of the nitric oxide system indicators in the left ventricle myocardium in SHR

The aim was to determine the morpho-functional parameters of the left ventricular myocardium NO system in the rats with essential hypertension (SHR line). Material and methods. We used a combination of modern highly informative methods, namely: research of NOS isoform profie (nNOS, iNOS eNOS) in the myocardial slices along with an assessment of their synthesis and expression of the corresponding mRNA; NO derived nitrites level determination directly in the myocardium homogenates and concentration of nitrotyrosine in blood plasma of rats. The results of the performed studies have shown that high blood pressure in the SHR was accompanied by a signifiant increase in the concentrations of all three NOS isoforms in the myocardium and increased expression of their mRNA. Higher concentration of nitrites by 18.8 % was detected in the SHR group compared with the control animals. The concentration of nitrotyrosine in blood plasma of rats with essential hypertension was also increased by 25 %. Conclusions. The predominance of IRM to constitutive isoforms of NOS with low IRM content to iNOS was noted in the myocardium of the control group rats while in SHR rats higher IRM values were marked for all NOS isoforms. The formation of hypertension is accompanied by high content of NO end metabolites and the development of systemic nitroso-oxidative stress with the increase of nitrotyrosine concentration.

Disturbance of nitric oxide synthase constitutive isoforms expression as a pathogenetic factor of complications development after tooth extraction in experimental diabetes mellitus

The role of constitutive NOS isoforms (endothelial and neuronal) in pathogenesis of complications development and repair processes disturbances in periodontium after tooth extraction in experimental diabetes mellitus is not studied completely. The aim of work – to find out the features of constitutive NOS isoforms (endothelial and neuronal) indices alterations in periodontal tissue of rats with experimental streptozotocin-induced diabetes mellitus on different terms (1 st , 7 th and 14 th day) after mandibular 1 st molar extraction. Materials and methods. Immunohistochemical study of endothelial and neuronal NOS isoforms expression indices (content, concentration, area of immunoreactive material distribution) in periodontium tissue was conducted on 120 male rats 8–10 months-old (8 experimental groups). Results. In rats with experimental diabetes mellitus (EDM) without tooth extraction content and concentration of both constitutive NOS isoforms in periodontium were lower than in control group of animals. The extraction of tooth in rats with EDM resulted in significant increase of all indices of both isoforms expression in comparison with EDM rats without tooth extraction. On the 7 th day after tooth extraction nNOS content and area of distribution was also increased by 24.3 % and 14.2 % respectively, and concentration decreased by 58.3 %. Indices of eNOS were not reliably changed in comparison with the 1 st day after tooth extraction. On the 14 th day after tooth extraction in rats with EDM, a significant decrease in all the indices of expression of constitutive NOS was observed, moreover, the concentration and area of nNOS distribution and the content, concentration and area of eNOS become significantly lower than in the corresponding control group. Conclusions. In rats with EDM in periodontal tissues the content and concentration of NOS constitutive isoforms are reduced. Extraction of the tooth in the animal with EDM leads to a moderate increase in the content and concentration of nNOS and eNOS from the 1 st to the 7 th day. On the 14 th day after the extraction, the expression indices of both isoforms decrease lower than in the corresponding control groups, that indicates a low content of endothelial and neuronal isoforms in the periodontium of rats with EDM and probably plays an important role in the pathogenesis of long-term complications development after surgery along with violation of repair processes.

Activation of α1A -adrenoceptors desensitizes the rat aorta response to phenylephrine through a neuronal NOS pathway, a mechanism lost with ageing.

A NO-mediated desensitization of vasoconstrictor responses evoked by stimulation of α1 -adrenoceptors has been reported in different vessels. We investigated the involvement of each α1 -adrenoceptor subtype and constitutive NOS isoforms and the influence of ageing and hypertension on this process. Wistar and spontaneously hypertensive rats (SHR), 16, 32, 52 and 72weeks-old, were used to evaluate the desensitization process. Expression of α1 -adrenoceptor subtypes, endothelial NOS (eNOS) and neuronal NOS (nNOS) were determined in rat aorta and left ventricle (LV). Expression levels were also evaluated in LV of a group of heart failure patients with a wide age range. Repeated application of phenylephrine decreased subsequent α1 -adrenoceptor-mediated vasoconstriction by increasing nNOS protein expression in aorta, but not in tail or mesenteric resistance arteries, where mRNA levels of nNOS were undetectable. This desensitization process disappeared in the absence of endothelium or in the presence of L-NAME (100μM), nNOS inhibitors, SMTC (1μM) and TRIM (100μM), and 5-methylurapidil (100nM, α1A -antagonist), but not BMY7378 (10nM, α1D -antagonist). The α1A /nNOS-mediated desensitization was absent in aged SHR and Wistar animals, where the expression of α1A -adrenoceptors was reduced in aorta and LV. In human LV, a negative correlation was found between age and α1A -adrenoceptor expression. The α1A -adrenoceptor subtype, through endothelial nNOS-derived NO, may act as a physiological 'brake' against the detrimental effects of excessive α1 -adrenoceptor-mediated vasoconstriction. Reduced α1A -adrenoceptor- and nNOS-mediated desensitization in aged patients could be involved in the age-dependent elevation of adrenergic activity.

The features of expression of constitutive isoforms of the nitric oxide synthase in paraventricular and supraoptic nuclei of the hypothalamus in hypertension of different origins

Aim. The purpose was to evaluate the features of the neuronal and endothelial nitric oxide synthase expression pattern in supraoptic and magnocellular part of paraventricular nuclei of the hypothalamus in different models of hypertension. Materials and methods. The study was carried out on 20 Wistar male rats and 10 SHR male rats. There were two experimental models of hypertension used: the genetically predisposed to age-related hypertension SHR and rats with endocrine-saline model of hypertension (ESM). The object was the hypothalamus of rats. The immunohystochemical assay was used to determine the expression of NOS isoforms.Results showed the imbalance of constitutive NOS isoforms in SON and PVN of hypothalamus in both of SHR and ESM. The eNOS and nNOS expression pattern is dependent on etiological factor, topographical peculiarities of the nuclei and their functions.Conclusions. We believe the imbalance of constitutive NOS isoforms take part in the hypertension development, its progression and the formation of complications.

The effects of the lithium salt of GABA on the subcellular metabolic profile of L-arginine in the prefrontal cortex and striatum of rats during chronic stress

Our previous data suggest that depression-like behavior of rats, which is induced by the 14-daylong unpredictable influence of stress factors, is accompanied by a sustained increase in the contents of L-arginine and nitric oxide (NO) and its stable metabolites, as well as stimulation of the inducible isoform of NO synthase (iNOS) and suppression of the activity of its constitutive NOS isoforms (cNOS) in the cytosol and mitochondria of the prefrontal cortex (PFC) and striatum. A single intraperitoneal injection of GABA lithium salt (at a dose of 0.9 mg/kg) on the second day of the post-stress period normalized the levels of L-arginine and active forms of nitrogen in cellular compartments from the PFC and striatum. Although this treatment did not influence the stress-induced shifts in the activity of cNOS, it prevented stimulation of the iNOS/NO system of mitochondria due to blockage of NO hyperproduction and nitrosative stress, whose development results in the disturbance of energetic processes in these brain areas.

NOS-1-derived NO is an essential triggering signal for the development of systemic inflammatory responses

Nitric oxide (NO) produced by the NO synthase type 2 (NOS-2) is known to have a prominent role in the course of the inflammatory response but less is known concerning the role of NO derived from the constitutive NOS isoforms. We have examined the role of NO derived from NOS-1 in the initiation of the systemic inflammatory response using sepsis models. Injection of LPS in rats induced an early hypotension, NOS-2 expression, increased lung myeloperoxidase activity and increased NO metabolite (NOx) levels in the skeletal muscle. Pre-treatment with 7-nitroindazol (7-NI) prevented all these changes, but its administration after LPS injection was ineffective. Septic (cecal ligation and puncture method, CLP) rats exhibited signs of organ failure, hyporesponsiveness to vasoconstrictors and 75% mortality over 3days after surgery. Pre-treatment with 7-NI prevented or significantly reduced these alterations. Injection of 7-NI after sepsis onset was without effect. Wild type mice injected with LPS exhibited increased plasma NOx, NOS-2 and COX-2 expression and 80% mortality. NOS-1−/− mice injected with LPS exhibited smaller increase in plasma NOx, no NOS-2 and COX-2 expression and reduced mortality. Injection of an NO donor in CLP rats pre-treated with 7-NI or in NOS-1−/− mice returned the mortality rate to those of CLP in rats and LPS in mice. Our results demonstrate that NOS-1-derived NO acts as a signaling element and it is essential for the initiation of systemic inflammation as demonstrated by the reduction of the inflammatory response and mortality by both pharmacological inhibition and genetic deletion of NOS-1.

Does nitric oxide modulate cardiac ryanodine receptor function? Implications for excitation-contraction coupling

Nitric oxide (NO) is a highly reactive, free radical signalling molecule that is constitutively released in cardiomyocytes by both the endothelial and neuronal isoforms of nitric oxide synthase (eNOS and nNOS, respectively). There are increasing data indicating that NO modulates various proteins involved in excitation-contraction coupling (ECC), and here we discuss the evidence that NO may modulate the function of the ryanodine receptor Ca(2+) release channel (RyR2) on the cardiac sarcoplasmic reticulum (SR). Both constitutive isoforms of NOS have been shown to co-immunoprecipitate with RyR2, suggesting that the channel may be a target protein for NO. eNOS gene deletion has been shown to abolish the increase in spontaneous Ca(2+) spark frequency in cardiomyocytes exposed to sustained stretch, whereas the effect of nNOS-derived NO on RyR2 function remains to be investigated. Single channel studies have been performed with RyR2 reconstituted in planar lipid bilayers and exposed to various NO donors and, under these conditions, NO appears to have a dose-dependent, stimulatory effect on channel open probability (P(open)). We discuss whether NO has a direct effect on RyR2 via covalent S-nitrosylation of reactive thiol residues within the protein, or whether there are downstream effects via cyclic nucleotides, phosphodiesterases, and protein kinases. Finally, we consider whether the proposed migration of nNOS from the SR to the sarcolemma in the failing heart may have consequences for the nitrosative vs. oxidative balance at the level of the RyR2, and whether this may contribute to an increased diastolic Ca(2+) leak, depleted SR Ca(2+) store, and reduced contractility in heart failure.

Neuronal Nitric Oxide Synthase Plays a Key Role in CNS Demyelination

Nitric oxide (NO) is a small, short-lived molecule released from a variety of cells that is implicated in a multitude of biological processes. In pathological conditions, overproduction of NO may lead to the generation of highly reactive species, such as peroxynitrite and stable nitrosothiols, that may cause irreversible cell damage. Accordingly, several studies have suggested that NO may be involved in the pathogenesis of various neuroinflammatory/degenerative diseases. Increased concentrations of NO in the CNS in such cases are usually attributed to an increase in the inducible isoform of NO synthase (iNOS) usually produced by inflammatory cells. However, recent reports have suggested that the constitutive isoforms of NOS, neuronal (nNOS) and endothelial (eNOS), can also play a role. Here we examined the role that the constitutive isoforms of NOS might play in the cuprizone-induced model of demyelination/remyelination. Our results demonstrate that demyelination was greatly prevented in mice lacking nNOS. Protection was associated with a dramatic increase in mature oligodendrocyte survival and a decrease in apoptosis. Moreover, nNOS-/- mice did not respond to cuprizone with the extensive recruitment of microglia/macrophages and astrocytes, which is a typical feature in wild-type mice. Although demyelinating less, nNOS-/- mice exhibited a delay in remyelination. In eNOS-/- mice, demyelination progressed to the same extent as in wild type, but they showed a slight delay in spontaneous remyelination. In conclusion, this study highlights the importance of considering the source of NO when assessing its role in neuroinflammation/degeneration and emphasizes the differing pathological effects driven by the different NOS isoforms.

NADPH‐diaphorase activity in the superficial layers of the superior colliculus of rats during aging

Neurons in the superficial layers of the superior colliculus are key elements in the visual system of rodents since they receive extensive afferent projections from retinal ganglion cells. The NADPH-diaphorase histochemical technique was used to detect differences in neuronal nitric oxide synthase (nNOS) in the superficial layers of the superior colliculus (sSC) of young adult (3 months) and aged (24 and 26 months) rats. The orientation of the dendritic processes of NADPH-diaphorase-positive neurons, cross-sectional area, and number of neurons per mm2 were analyzed. NADPH-d histochemistry revealed a high number of NADPH-d-positive cells in the stratum zonale and stratum griseum superficiale in adult and aged animals. NADPH-d-positive neurons were classified into the following morphological types: marginal, horizontal, pyriform, narrow-field vertical, wide-field vertical, and stellate. During aging, narrow field vertical and wide field vertical neurons present somatic atrophy and an increase in dendritic processes with dorsoventral orientation, whereas wide field vertical neurons show a decrease in those with lateromedial orientation. Marginal neurons undergo somatic hypertrophy at 26 months when compared with those at 3 months. The remaining types of neurons do not undergo size changes. Finally, the number of NADPH-d-positive neurons per mm2 in the various types of morphology does not significantly change with age. It is suggested to be likely that the aging process in the nitrergic neurons of the sSC does not lead to significant changes in the synthesis of NO from the constitutive NOS isoforms.

Role of inducible nitric oxide synthase in cardiac function and remodeling in mice with heart failure due to myocardial infarction

Using inducible nitric oxide (NO) synthase (iNOS) knockout mice (iNOS-/-), we tested the hypotheses that 1) lack of iNOS attenuates cardiac remodeling and dysfunction and improves cardiac reserve postmyocardial infarction (MI), an effect that is partially mediated by reduction of oxidative stress due to reduced interaction between NO and reactive oxygen species (ROS); and 2) the cardioprotection afforded by iNOS deletion is eliminated by Nomega-nitro-L-arginine methyl ester (L-NAME) due to inhibition of endothelial NOS (eNOS) and neuronal NOS (nNOS). MI was induced by ligating the left anterior descending coronary artery. Male iNOS-/- mice and wild-type controls (WT, C57BL/6J) were divided into sham MI, MI+vehicle, and MI+l-NAME (100 mg.kg(-1).day(-1) in drinking water for 8 wk). Cardiac function was evaluated by echocardiography. Left ventricular (LV) maximum rate of rise of ventricular pressure divided by pressure at the moment such maximum occurs (dP/dt/instant pressure) in response to isoproterenol (100 ng.kg(-1).min(-1) iv) was measured with a Millar catheter. Collagen deposition, myocyte cross-sectional area, and expression of nitrotyrosine and 4-hydroxy-2-nonenal (4-HNE), markers for ROS, were determined by histopathological and immunohistochemical staining. We found that the MI-induced increase in LV chamber dimension and the decrease in ejection fraction, an index of systolic function, were less severe in iNOS-/- compared with WT mice. L-NAME worsened LV remodeling and dysfunction further, and these detrimental effects were also attenuated in iNOS-/- mice, associated with better preservation of cardiac function. Lack of iNOS also reduced nitrotyrosine and 4-HNE expression after MI, indicating reduced oxidative stress. We conclude that iNOS does not seem to be a pathological mediator of heart failure; however, the lack of iNOS improves cardiac reserve post-MI, particularly when constitutive NOS isoforms are blocked. Decreased oxidative stress and other adaptive mechanisms independent of NOS may be partially responsible for such an effect, which needs to be studied further.

Formation of a protonated trihydrobiopterin radical cation in the first reaction cycle of neuronal and endothelial nitric oxide synthase detected by electron paramagnetic resonance spectroscopy.

Nitric oxide synthase (EC 1.14.13.39; NOS) converts L-arginine into NO and L-citrulline in a two-step reaction with Nomega-hydroxy-L-arginine (NOHLA) as an intermediate. The active site iron in NOS has thiolate axial heme-iron ligation as found in the related monooxygenase cytochrome P450. In NOS, tetrahydrobiopterin (BH4) is an essential cofactor for both steps, but its function is controversial. Previous optical studies of the reaction between reduced NOS with O2 at -30 degrees C suggested that BH4 may serve as an one-electron donor in the first cycle, implying formation of a trihydrobiopterin radical. We investigated the same reaction under identical conditions with electron paramagnetic resonance spectroscopy. With BH4-containing full-length neuronal NOS we obtained an organic free radical (g-value 2.0042) in the presence of Arg, and a similar radical was observed with the endothelial NOS oxygenase domain in the presence of Arg and BH4. Without substrate the radical yield was greatly (10x) diminished. Without BH4, or with NOHLA instead of Arg, no radical was observed. With 6-methyltetrahydropterin or 5-methyl-BH4 instead of BH4, radicals with somewhat different spectra were formed. On the basis of simulations we assign the signals to trihydropterin radical cations protonated at N5. This is the first study that demonstrates the formation of a protonated trihydrobiopterin radical with the constitutive isoforms of NOS, and the first time the radical was obtained without exogenous BH4. These results offer strong support for redox cycling of BH4 in the first reaction cycle of NOS catalysis (BH4 <--> BH3.H+).

Involvement of nitric oxide synthase in the physiology and pathophysiology of facial nerve function and dysfunction.

To date few reports have discussed the presence and function of nitric oxide (NO) in structures of the facial nerve. We performed nicotinamide adenine dinucleotide phosphate (NADPH-d)-diaphorase-histochemistry and immunohistochemistry on the intratemporal portion of the facial nerve, including the geniculate ganglion, of guinea pigs using specific antibodies to the three known isoforms of NO synthase and soluble guanylyl-cyclase (sGC). Normal facial nerves were compared to those treated intratympanically with bacterial lipopolysaccharides (LPS) and tumor necrosis factor-alpha (TNF-alpha). Both constitutive NOS isoforms and sGC could be detected in the bipolar ganglion cells of normal animals, while the inducible isoform (iNOS or NOS II) was not found. Endothelial NOS (NOS III) and sGC were present in blood vessels and were predominantly found in the perineurial sheath and less in the endoneurium. sGC could be detected in all fibers in a cross section of the facial nerve. LPS and TNF treatment led to the detection of iNOS in the perikaryia of the geniculate ganglion and the perineural sheath. These findings imply that NO may be involved in neurotransmission at least in the visceroafferent system. NO regulates vascular tone of nutrient blood vessels in the perineural sheath and endoneurium. The presence of sGC indicates that NO acts via its second messenger cGMP. NOS II expression may be a contributing factor to facial nerve palsy via two different mechanisms: NOS II-generated NO may lead to an overstimulation of the visceroefferent nerve fibers and motor fibers of the facial nerve. Dysregulation in facial nerve blood vessels could lead to edema and elevated pressure on the nerve within its osseous canal.

Expression of inducible nitric oxide synthase (iNOS/NOS II) in the cochlea of guinea pigs after intratympanical endotoxin-treatment

Since NO is believed to be involved in cochlear physiology, presence of the constitutive isoforms of nitric oxide synthase (NOS), and the target enzyme of NO, soluble guanylyl cyclase (sGC) in structures of the mammalian cochlea have been demonstrated. To date, no reports have been published regarding the detection of the inducible isoform (NOS II) in the cochlea. In order to show the capability of iNOS expression in cochlear tissue, a mixture of proinflammatory bacterial lipopolysaccharides (LPS) and tumor necrosis factor α (TNF-α) was injected into the tympanic cavity of guinea pigs, vs. saline-solution as control. Paraffin sections of LPS/TNF-α treated and saline-treated cochleae (6 h) were examined immunohistochemically with specific antibodies to neuronal, endothelial and inducible NOS and to sGC. Initiated expression of iNOS in the cochlea was observed in the wall of blood vessels of the spiral ligament (SL) and the modiolus, in supporting cells of the organ of Corti, in the limbus, in nerve fibers and in a part of the perikarya of the spiral ganglion after LPS/TNFα-treatment. iNOS was not detected in saline-treated control tissue. Expression of both constitutive NOS-isoforms (endothelial and neuronal NOS) and of sGC showed no significant differences in both experimental groups. Endothelial eNOS and neuronal bNOS were detected co-localized in ganglion cells, in nerve fibers, in cells of the SL and in supporting cells of the organ of Corti, but not in sensory cells. Strong labeling for bNOS became evident in the endosteum of the cochlea, while in the endothelium of blood vessels and in the epithelium of the limbus only eNOS could be labeled. sGC could be detected in SL, in supporting and sensory cells of the organ of Corti, in nerve fibers, ganglion cells, in the wall of blood vessels and in the limbus-epithelium. While small amounts of NO, generated by bNOS and eNOS, seem to support the cochlear blood flow and auditory function as well as neurotransmission, high amounts of iNOS-generated NO could have dysregulative and neurotoxic effects on the inner ear during bacterial and viral infections of the middle and inner ear.

Free Radicals in Viral Pathogenesis: Molecular Mechanisms Involving Superoxide and NO

The importance of free radical molecular species in the pathogenesis of various viral diseases has been increasingly recognized in recent years. Oxygen radicals such as superoxide (O2-) and hydroxyl radical (.OH) have been implicated as possible pathogenic molecules in viral disease pathogenesis. Much attention has been given to another simple inorganic radical [nitric oxide (NO)] in the host's defense mechanism and pathogenesis of virus infection. The NO synthesis pathway, in particular, the inducible isoform of NO synthase (iNOS), is expressed in different viral diseases via induction of proinflammatory cytokines such as interferon-gamma. iNOS produces an excessive amount of NO for a long time compared with other constitutive isoforms of NOS (i.e., neuronal NOS and endothelial NOS). Recent studies indicate that NO and O2- are produced in excess during the host's defense responses against various intruding microbes. Reactive nitrogen oxide species such as peroxynitrite (ONOO-) and NOx (NO2 and N2O3) are produced in biological systems through the reaction of NO with either O2- or O2. Among these reactive nitrogen species, ONOO- and its biological actions are of considerable interest in that ONOO- causes oxidation and nitration of amino acid residues of proteins and guanine of DNA, lipid peroxidation, and DNA cleavage. Because the ONOO- is formed via a diffusion-limited fast reaction of NO and O2-, it may be a dominant nitrogen oxide species during the host's defense reactions, when both NO and O2- are produced in excess. Thus, understanding the role of NO and oxygen radical generation in virus infections will provide insight into not only viral pathogenesis but also the host-pathogen interaction in microbial infections at a molecular level.

Identification of the NO Synthase isoforms Expressed in Human Neutrophil Granulocytes, Megakaryocytes and Platelets

Using Western blot and fluorescent immunocytochemistry, NOS III (or ecNOS) and NOS II (or iNOS), but no NOS I (or ncNOS), were identified in preparations of human platelets. Reverse-transcription polymerase chain reactions (RT-PCR) demonstrated NOS III mRNA, but no NOS II mRNA (which is short-lived) and no NOS I mRNA in platelets. Immunofluorescent staining of human bone marrow smears showed the presence of NOS III, but not NOS I in megakaryocytes. A subpopulation of megakaryocytes also expressed NOS II. In preparations of human neutrophils, immunocytochemistry demonstrated NOS I in all cells, whereas no NOS III was detected. The few NOS II positive cells were characterized as contaminating eosinophils. Similarly, in RT-PCR, transcripts for NOS I and NOS II, but not for NOS III, were identified. Thus, the constitutive NOS isoform in megakaryocytes and platelets is NOS III, whereas neutrophils express NOS I. Some megakaryocytes and eosinophils also express NOS II.

Endothelial-Type Nitric Oxide Synthase (ec-NOS) in Skeletal Muscle Fibers: Mitochondrial Relationships

Immunohistochemistry shows that skeletal muscle fibers express endothelial-type (ec)-NOS in a heterogeneous pattern distinct from that of neuronal (nc)-NOS. Analysis of adjacent serial sections reveals: 1) a strong correlation of ec-NOS expression to mitochondrial content (visualized histochemically by succinate dehydrogenase); 2) lack of correlation of ec-NOS to fiber ATPase type (in contrast to nc-NOS); and 3) constitutive co-expression of ec- and nc-NOS in some fibers. Preparations of mitochondria from diaphragm exhibited calcium-dependent NOS activity, which functioned to inhibit in vitro oxygen consumption. The results establish that normal skeletal muscle cells can express two constitutive isoforms of NOS (nc- and ec-) and suggest a functional role for ec-NOS in oxidative, mitochondria-rich fibers.