Expression of the inducible nitric oxide synthase gene in diaphragm and skeletal muscle

Nitric oxide (NO) is a pluripotent molecule that can be secreted by skeletal muscle through the activity of the neuronal constitutive isoform of NO synthase. To determine whether skeletal muscle and diaphragm might also express the macrophage-inducible form of NO synthase (iNOS) during provocative states, we examined tissue from mice at serial times after intravenous administration of Escherichia coli endotoxin. In these studies, iNOS mRNA was strongly expressed in the diaphragm and skeletal muscle of mice 4 h after intravenous endotoxin and was significantly diminished by 8 h after challenge. Induction of iNOS mRNA was followed by expression of iNOS immunoreactive protein on Western immunoblots. Increased iNOS activity was demonstrated by conversion of arginine to citrulline. Immunochemical analysis of diaphragmatic explants exposed to endotoxin in vitro revealed specific iNOS staining in myocytes, in addition to macrophages and endothelium. These results may be important in understanding the pathogenesis of respiratory pump failure during septic shock, as well as skeletal muscle injury during inflammation or metabolic stress.     

Expression of nitric oxide synthase isoforms in bone and bone cell cultures

Recent work has shown that nitric oxide (NO) acts as an important mediator of the effects of proinflammatory cytokines and mechanical strain in bone. Although several bone-derived cells have been shown to produce NO in vitro, less is known about the isoforms of NO synthase (NOS), which are expressed in bone or their cellular distribution. Here we investigated the expression, cellular localization, and regulation of NOS mRNA and protein in cultured bone-derived cells and in bone tissue sections. We failed to detect inducible NOS (iNOS) protein in normal bone using immunohistochemical techniques, even though low levels of iNOS mRNA were detected by sensitive reverse transcribed polymerase chain reaction (RT-PCR) assays in RNA extracted from whole bone samples. Cytokine stimulation of bone-derived cells and bone explant cultures caused dramatic induction of iNOS mRNA and protein in osteoblasts and bone marrow macrophages, but no evidence of iNOS expression was seen in osteoclasts by immunohistochemistry or in situ hybridization. Endothelial NOS (ecNOS) mRNA was also detected by RT-PCR in whole bone, and immunohistochemical studies showed widespread ecNOS expression in bone marrow cells and trabecular lining cells in vivo. Related studies in vitro confirmed that ecNOS was expressed in cultured osteoblasts, stromal cells, and osteoclasts. Neuronal NOS mRNA was detected by RT-PCR in whole bone, but we were unable to detect nNOS protein in bone cells in vivo or in studies of cultured bone-derived cells in vitro. In summary, our data show that mRNAs for all three NOS isoforms are expressed in bone and provide evidence for differential expression and regulation of the enzymes in different cell types. These findings confirm the likely importance of the L-arginine-NO pathway as a physiological mediator of bone cell function and demonstrate that it may be possible to exert differential effects on osteoblast and osteoclast activity in vivo by differential targeting of constitutive and inducible NOS isoforms by selective NOS inhibitors.     

Inactivation of nitric oxide synthase isoforms by diaminoguanidine and NG-amino-L-arginine

We studied the value of long video-split electroencephalographic monitoring (VSEEG) in detecting myoclonus in nearly SSPE and evaluated the natural history and outcome-affecting factors. The 32 newly diagnosed patients had VSEEG to detect myoclonus and its correlations with EEG periodic complexes. Disease progression was monitored by a special "outcome score'; the chi-square test and multi-variable statistics analysed the outcome score in relation to different variables, such as age at onset, sex, duration of symptoms at presentation, CSF measles antibody titre, type and interval between periodic complexes (EEG discharges). Myoclonus or atonia occurred in all patients and was time-related to the EEG periodic complexes; in 32% of patients, myoclonus or atonia were not clinically evident. The EEG periodic complexes were of 3 types: Type I (16 patients) periodic giant delta waves; Type II (10 patients) periodic giant delta waves intermixed with rapid spikes or fast activity; and Type III (6 patients), long spike-wave discharges interrupted by giant delta waves. Outcome score was associated with symptoms duration (P < 0.01) and EEG periodic complexes (P < 0.05). Symptom duration was inversely related to final outcome (multi-variable analysis). Long VSEEG monitoring greatly improves early diagnosis and detection of subtle atonia or segmental myoclonus. Prognostic factors were the type of EEG periodic complexes and duration of symptoms at presentation.     

Expression of differential nitric oxide synthase isoforms in human normal gastric mucosa and gastric cancer tissue

The present study investigated the expression and distribution of three isoforms of nitric oxide synthase (NOS) in different anatomical regions of the human stomach and in gastric neoplastic tissues by immunohistochemistry using specific antibodies. Intracellular localization of individual isoenzymes of NOS was detected in normal gastric mucosa. Gastric cancer tissues had a marked reduction of all three NOS isoforms expression. The expression of the endothelial NOS, neuronal NOS and inducible NOS in the tumor tissue was significantly lower than in normal gastric mucosa (P = 0.01, P = 0.02, P < 0.01, respectively). In the tumor tissue the expression of inducible NOS was significantly lower than the expression of both constitutive forms of NOS (P < 0.01). There was a tendency to higher expression of both constitutive forms of NOS in earlier stages T2 of the tumor compared to advanced T4 tumor. In contrast, the expression of inducible NOS was higher than in the advanced T4 tumor than in the earlier stages T2 of the tumor. The mapping of the expression of endothelial NOS, neuronal NOS and inducible NOS in human stomach showed higher expression of NOS isoforms in the distal third than in the proximal third of the stomach (P = 0.03, P = 0.04, P = 0.01, respectively). We conclude that there is greater expression of NOS in the stomach corpus and in antrum than in the proximal third of the normal human stomach mirroring the anatomical predilection of common pathological changes in this part of the human stomach. Furthermore, there was loss of the expression of individual isoenzymes in gastric neoplasms.     

Expression of endothelial and inducible nitric oxide synthase in human glomerulonephritis

The presence of nitric oxide (NO) in the kidney has been implicated in the pathogenesis of human glomerulonephritis. However, the exact type of glomerular cells that express NO synthase (NOS) and the NOS isoform involved in the local production of NO has not been identified in the human diseased kidney. We examined the expression of three isoforms of NOS, inducible NOS (iNOS), endothelial NOS (eNOS) and brain NOS (bNOS) in the renal tissue of patients with IgA nephropathy (IgAN, N = 10), lupus nephritis (LN, N = 5), membranous nephropathy (MN, N = 5) and minimal change nephrotic syndrome (MCNS, N = 5). Sections were immunostained and the correlation between the expression of each NOS and the degree of glomerular injury in that section was also examined. Normal portions of surgically resected kidneys served as controls. eNOS was present in glomerular endothelial cells and endothelium of cortical vessels in the control and diseased kidneys. iNOS was localized in mesangial cells, glomerular epithelial cells and infiltrating cells in the diseased glomeruli, whereas immunostaining for iNOS was hardly detected in control kidneys. In addition, the expression pattern of eNOS in each glomerulus was the reverse of that of iNOS. In IgAN and LN, the extent of staining for eNOS correlated negatively with the degree of glomerular injury, while the extent of staining for iNOS correlated positively with the degree of glomerular injury in the same tissues. bNOS was not detected in normal or nephritic glomeruli. Our results indicate the presence of a NO pathway in human diseased kidney, and suggest that NO derived from eNOS and iNOS may be involved in the progression of renal diseases and that NO derived from each NOS may play an important role in different way in human inflamed glomeruli.     

Expression of inducible nitric oxide synthase in human burn wounds

A water-soluble synthetic peptide with only nine amino acid residues, comprising the 131-139 sequence region of the cytotoxic protein alpha-sarcin (secreted by the mold Aspergillus giganteus), interacts with large unilamellar vesicles composed of acid phospholipids. It promotes lipid mixing between bilayers and leakage of vesicle aqueous contents, and it also abolishes the phospholipid phase transition. Other larger peptides containing such an amino acid sequence also produce these effects. These peptides acquire alpha-helical conformation in the presence of trifluoroethanol, but display beta-strand conformation in the presence of sodium dodecyl sulfate. The interaction of these peptides with the lipid vesicles also results in beta-structure. The obtained data are discussed in terms of the involvement of the 131-139 stretch of alpha-sarcin in its interaction with lipid membranes.     

Functional expression of three isoforms of human nitric oxide synthase in baculovirus-infected insect cells

Complementary DNAs encoding three human isoforms (neuronal, inducible, and endothelial) of nitric oxide synthase were cloned into the baculovirus expression vector pVL1392/1393. Transfection of Sf-9 insect cells with the recombinant baculovirus resulted in the expression of high levels of nitric oxide synthases. The expressed proteins of neuronal and inducible nitric oxide synthase were predominantly soluble, whereas the endothelial enzyme was for the most part, particulate. Recombinant enzymes were purified with 2',5'-ADP Sepharose affinity chromatography. The effects of reference enzymatic inhibitors (NG-methyl-L-arginine, NG-nitro-L-arginine and N-iminoethyl-L-ornithine) on recombinant expressed proteins were not significantly different from native nitric oxide synthase enzyme preparations. L-aminoguanidine was found to be much less potent in inhibiting recombinant or native human inducible nitric oxide synthase compared to the murine isoform. These findings indicate previously unappreciated interspecies differences in the action of nitric oxide synthase enzymatic inhibitors. The functional expression of human nitric oxide synthase isoforms in a heterologous expression system allowed screening of novel inhibitors. Studies indicated that S-ethylisothiourea and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine were potent novel inhibitors of human nitric oxide synthases.     

Expression of nitric oxide synthase isoforms (NOS II and NOS III) in adult rat lung in hyperoxic pulmonary hypertension

Breathing air with a high oxygen tension induces an inflammatory response and injures the microvessels of the lung. The resulting development of smooth muscle cells in these segments contributes to changes in vasoreactivity and increased pulmonary artery pressure. This in vivo study determines the temporal and spatial expression of endogenous endothelial nitric oxide synthase (NOS III) and inducible NOS (NOS II), important enzymes regulating vasoreactivity and inflammation, in the adult rat lung during the development of experimental pulmonary hypertension induced by oxidant injury. We analyzed the cellular distribution of these NOS isoforms, using specific antibodies, and assessed enzyme activity at baseline and after 1-28 days of hyperoxia (FIO2 0.87). The number of NOS III-immuno-positive endothelial cells increased early in hyperoxia and then remained high. By day 28, the relative number of these cells had increased from 40% in proximal vessels and 13-16% in distal alveolar vessels of the normal lung to 73-86% and 40-59%, respectively, in hyperoxia. Pulmonary alveolar macrophages (PAMs), normally few in number and only weakly immunopositive for NOS II or III in the normal lung, increased in number in hyperoxia and were strongly immunopositive for each isoform. These morphological data were supported by a temporal increase in total and calcium-independent NOS activity. Thus NOS expression and activity significantly increased in hyperoxia as pulmonary hypertension developed, and NOS III expression increased selectively in vascular endothelial cells, while both NOS isoforms were expressed by the PAM population. We conclude that this increase in expression of a potent vasodilator, an antiproliferative agent for smooth muscle cells, and an antioxidant molecule represents an adaptive response to protect the lung from oxidant-induced vascular and epithelial injury.     

Regulation of nitric oxide production in response to skeletal muscle activation

Nitric oxide (NO) is synthesized in normal muscle fibers by the neuronal (nNOS) and the endothelial (ecNOS) isoforms of nitric oxide synthase (NOS). NO contributes to the regulation of several processes such as excitation-contraction coupling and mitochondrial respiration. We assessed in this study whether NO production is regulated in response to an acute increase in muscle activation. Three groups of anesthetized, tracheostomized, spontaneously breathing rats were examined after an experimental period of 3 h. Group 1 served as a control (no loading), whereas groups 2 and 3 were exposed to moderate and severe inspiratory resistive loads, respectively, which elicited tracheal pressures of 30 and 70% of maximum, respectively. Ventilatory (diaphragm, intercostal, and transverse abdominis) and limb (gastrocnemius) muscles were excised at the end of the experimental period and examined for NOS activity and NOS protein expression. Neither submaximal nor maximum tracheal pressures were altered after 3 h of resistive loading. Diaphragmatic and intercostal muscle NOS activities declined significantly in response to moderate and severe loading, whereas those of transverse abdominis and gastrocnemius muscles remained unchanged. On the other hand, resistive loading had no significant effect on ventilatory and limb muscle NOS isoform expression. We propose that a contraction-induced decline in muscle NOS activity represents a compensatory mechanism through which muscle contractility and mitochondrial function are protected from the inhibitory influence of NO.     

Expression of three forms of nitric oxide synthase in peripheral nerve regeneration

Nitric oxide (NO) is a short-lived molecule with messenger and cytotoxic functions in nervous, cardiovascular, and immune systems. Nitric oxide synthase (NOS), the enzyme responsible for NO synthesis, exists in three different forms: the neuronal (nNOS), present in discrete neuronal populations; the endothelial (eNOS), present in vascular endotheliun, and the inducible isoform (iNOS), expressed in various cell types when activated, including macrophages and glial cells. In this study, we have investigated the possible involvement of NO in Wallerian degeneration and the subsequent regeneration occurring after sciatic nerve ligature, using histochemistry and immunocytochemistry for the three NOS isoforms, at different postinjury periods. Two days after lesion, the three NOS isoforms are overexpressed, reaching their greatest expression during the second week. nNOS is upregulated in dorsal root ganglion neurons, centrifugally transported and accumulated in growing axons. eNOS is overexpressed in vasa nervorum of the distal stump and around ligature, and iNOS is induced in recruited macrophages. These findings indicate that different cellular sources contribute to maintain high levels of NO at the lesion site. The parallelism between NOS inductions and well-known repair phenomena suggests that NO, acting in different ways, may exert a beneficial effect on nerve regeneration.     

Expression of inducible nitric oxide synthase in the brains of scrapie-infected mice

Thoracic trauma in the elderly population constitutes a major challenge for both thoracic and trauma surgeons as their presentation and outcomes differ from the adult population in addition to their high morbidity and mortality. One hundred and one patients, 60 years of age or older, with thoracic trauma were treated at Dicle University School of Medicine during a 6-year period. Eighty-five per cent were male and 15% were female with a mean age of 64.5 years. The cause of thoracic injury was blunt in 77.2% and penetrating in 22.8% of the patients. Sixty-two patients (61.4%) had isolated thoracic injuries. The median Injury Severity Score (ISS) was 23. The morbidity rate was 23.8%. The mortality rate was 16.8%. Seven of 10 patients (70%) who had an ISS greater than 25 died, whereas six of 24 (25%) patients with an ISS between 17 and 25, and four of 67 (5.9%) patients with an ISS less than 16 died. In the elderly the morbidity and mortality rates were higher for blunt trauma compared with penetrating trauma. For ISS greater than 25 the mortality rate was 71.4% for blunt and 66.6% for penetrating trauma. As the morbidity and mortality rate are significantly higher in the elderly patients the approach to these patients should include recognition of their high risk for morbidity and mortality, especially for those who had an ISS greater than 25.     

Expression of neural cell adhesion molecules and neurofilament protein isoforms in skeletal muscle tumors

In a series of rhabdomyosarcomas, the expression of neural cell adhesion molecules (NCAM) and neurofilament isoforms was probed in frozen sections. It was found that NCAM was widely expressed in rhabdomyosarcomas without relation to subtype or differentiation level. Neurofilament isoforms were found throughout all subtypes but were largely restricted to those neurofilament isoforms that are expressed early in neurogenesis, that is, poorly phosphorylated low- and medium-weight isoforms. It was concluded that the expression of these "neural" markers is widespread and does not signify a neural tumor.     

Expression of inducible nitric oxide synthase and formation of peroxynitrite in posttransplant obliterative bronchiolitis

BACKGROUND: Obliterative bronchiolitis is characterized histologically by inflammation, epithelial cell damage and loss, fibrosis, and eventual obliteration of airways. Production of high levels of the potential cytotoxin nitric oxide by inducible nitric oxide synthase has been implicated in several inflammatory diseases. The damaging effects of nitric oxide are mediated by peroxynitrite, are formed from nitric oxide and superoxide, and can be demonstrated by the detection of nitrotyrosine. Our previous finding of high inducible nitric oxide synthase expression in inflamed airway epithelium led us to hypothesize that release of nitric oxide in obliterative bronchiolitis mediates the characteristic epithelial damage. METHODS: Immunocytochemistry was carried out to seek expression of inducible nitric oxide synthase and nitrotyrosine in transplant samples from patients with obliterative bronchiolitis (n=10) and, as controls, unused donor lungs (n=5). RESULTS: Inducible nitric oxide synthase was strongly expressed in the damaged airway epithelium in obliterative bronchiolitis and in inflammatory cells, where its distribution was matched by that of nitrotyrosine. Normal controls showed little or no immunoreactivity for any of the antigens studied. CONCLUSIONS: Our findings suggest that nitric oxide may play a role in the pathogenesis of obliterative bronchiolitis and indicate that further work is essential to fully understand the processes and mechanisms involved.     

Expression and regulation of protein inhibitor of neuronal nitric oxide synthase in ventilatory muscles

Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus and a member of the Caulimoviridae family and closely related to viruses in the Badnavirus genus. The coat protein of RTBV is part of the large polyprotein encoded by open reading frame 3 (ORF3). ORF3 of an RTBV isolate from Malaysia was sequenced (accession no. AF076470) and compared with published sequences for the region that encodes the coat protein or proteins. Molecular mass of virion proteins was determined by mass spectrometry (matrix-assisted laser desorption/ionization-TOF) performed on purified virus particles from three RTBV isolates from Malaysia. The N- and C-terminal amino acid sequences of the coat protein were deduced from the mass spectral analysis, leading to the conclusion that purified virions contain a single coat protein of 37 kDa. The location of the coat protein domain in ORF3 was reinforced as a result of immunodetection reactions using antibodies raised against six different segments of ORF3 using Western immunoblots after SDS-PAGE and isoelectrofocusing of proteins purified from RTBV particles. These studies demonstrate that RTBV coat protein is released from the polyprotein as a single coat protein of 37 kDa.