Nitric oxide donors regulate nitric oxide synthase in bovine pulmonary artery endothelium

This study examined the notion that exogenous generation of nitric oxide (NO) modulates NOS gene expression and activity. Bovine pulmonary artery endothelial cells (BPAEC) were treated with the NO donors, 1 mM SNAP (S-nitroso-N-acetylpenicillamine), 0.5 mM SNP (sodium nitroprusside) or 0.2 microM NONOate (spermine NONOate) in medium 199 containing 2% FBS. Controls included untreated cells and cells exposed to 1 mM NAP (N-acetyl-D-penicillamine). NOS activity was assessed using a fibroblast-reporter cell assay; intracellular Ca2+ concentrations were assessed by Fura-2 microfluorometry; and NO release was measured by chemiluminescence. Constitutive endothelial (e) and inducible (i) NOS gene and protein expression were examined by northern and western blot analysis, respectively. Two hours exposure to either SNAP or NONOate caused a significant elevation in NO release from the endothelial cells (SNAP = 51.4 +/- 5.9; NONOate = 23.8 +/- 4.2; control = 14.5 +/- 2.8 microM); but A23187 (3 microM)-stimulated NO release was attenuated when compared to controls. Treatment with either SNAP or NONOate for 2 h also resulted in a significant increase in NOS activity in endothelial homogenates (SNAP = 23.6 +/- 2.5; NONOate= 29.8 +/- 7.7; control = 14.5 +/- 2.5fmol cGMP/microg per 10(6) cells). Exposure to SNAP and SNP, but not NONOate, for 1 h caused an increase in intracellular calcium. Between 4 and 8 h, SNAP and NONOate caused a 2- to 3-fold increase in eNOS, but not iNOS, gene (P < 0.05) and protein expression. NAP had little effect on either eNOS gene expression, activity or NO production. Our data indicate that exogenous generation of NO leads to a biphasic response in BPAEC, an early increase in intracellular Ca2+, and increases in NOS activity and NO release followed by increased expression of the eNOS gene, but not the iNOS gene. We conclude that eNOS gene expression and activity are regulated by a positive-feedback regulatory action of exogenous NO.     

Induction of inducible nitric oxide synthase by ginsenosides in cultured porcine endothelial cells

Mechanism of Nitric oxide (NO) production by ginsenosides was investigated in cultured porcine endothelial cells. Beta-nicotinamide adenine dinucleotide phosphate (beta-NADPH) staining showed that the NO production was significantly enhanced by the presence of 40 microg/ml ginsenosides with 10 microM L-arginine after 12 h incubation. NO production was suppressed by addition of 0.5 microM Nomega-Nitro-L-arginine (L-NNA), an inhibitor of NO synthases (NOSs), to the incubation medium. In addition, the immunoreactive signals of inducible NOS (iNOS) were appeared in endothelial cells after 12-h incubation of ginsenosides, whereas the signals were not observed in non-treated cells. Our findings suggest that ginsenosides can enhance NO production by induction of iNOS in addition to its direct effect on endothelial cells by increasing intracellular Ca2+ concentration.     

Interaction between NO and COX pathways in retinal cells exposed to elevated glucose and retina of diabetic rats

A nonselective inhibitor of cyclooxygenase (COX; high-dose aspirin) and a relatively selective inhibitor of inducible nitric oxide synthase (iNOS; aminoguanidine) have been found to inhibit development of diabetic retinopathy in animals, raising a possibility that NOS and COX play important roles in the development of retinopathy. In this study, the effects of hyperglycemia on retinal nitric oxide (NO) production and the COX-2 pathway, and the interrelationship of the NOS and COX-2 pathways in retina and retinal cells, were investigated using a general inhibitor of NOS [N(G)-nitro-l-arginine methyl ester (l-NAME)], specific inhibitors of iNOS [l-N(6)-(1-iminoethyl)lysine (l-NIL)] and COX-2 (NS-398), and aspirin and aminoguanidine. In vitro studies used a transformed retinal Müller (glial) cell line (rMC-1) and primary bovine retinal endothelial cells (BREC) incubated in 5 and 25 mM glucose with and without these inhibitors, and in vivo studies utilized retinas from experimentally diabetic rats (2 mo) treated or without aminoguanidine or aspirin. Retinal rMC-1 cells cultured in high glucose increased production of NO and prostaglandin E(2) (PGE(2)) and expression of iNOS and COX-2. Inhibition of NO production with l-NAME or l-NIL inhibited all of these abnormalities, as did aminoguanidine and aspirin. In contrast, inhibition of COX-2 with NS-398 blocked PGE(2) production but had no effect on NO or iNOS. In BREC, elevated glucose increased NO and PGE(2) significantly, whereas expression of iNOS and COX-2 was unchanged. Viability of rMC-1 cells or BREC in 25 mM glucose was significantly less than at 5 mM glucose, and this cell death was inhibited by l-NAME or NS-398 in both cell types and also by l-NIL in rMC-1 cells. Retinal homogenates from diabetic animals produced significantly greater than normal amounts of NO and PGE(2) and of iNOS and COX-2. Oral aminoguanidine and aspirin significantly inhibited all of these increases. The in vitro results suggest that the hyperglycemia-induced increase in NO in retinal Müller cells and endothelial cells increases production of cytotoxic prostaglandins via COX-2. iNOS seems to account for the increased production of NO in Müller cells but not in endothelial cells. We postulate that NOS and COX-2 act together to contribute to retinal cell death in diabetes and to the development of diabetic retinopathy and that inhibition of retinopathy by aminoguanidine or aspirin is due at least in part to inhibition of this NO/COX-2 axis.     

NO and NOS isoforms in the development of apoptosis in renal ischemia/reperfusion

Nitric oxide (NO) and the expression of endothelial (eNOS) and inducible (iNOS) isoforms of nitric oxide synthase (NOS) are recognized as important mediators of physiological and pathological processes of renal ischemia/reperfusion (I/R) injury, but little is known about their role in apoptosis. The ability of the eNOS/NO system to regulate the iNOS/NO system and thus promote apoptosis was assessed during experimental renal I/R. Renal caspase-3 activity and the number of TUNEL-positive cells increased with I/R, but decreased when NOS/NO systems were blocked with L-NIO (eNOS), 1400W (iNOS), and N-nitro-l-arginine methyl ester (L-NAME; a nonselective NOS inhibitor). I/R increased renal eNOS and iNOS expression as well as NO production. The NO increase was eNOS- and iNOS-dependent. Blockage of NOS/NO systems with L-NIO or L-NAME also resulted in a lower renal expression of iNOS and iNOS mRNA; in contrast, eNOS expression was not affected by iNOS-specific blockage. In conclusion, two pathways define the role of NOS/NO systems in the development of apoptosis during experimental renal I/R: a direct route, through eNOS overexpression and NO production, and an indirect route, through expression/activation of the iNOS/NO system, induced by eNOS.     

Spontaneous and receptor-controlled soluble guanylyl cyclase activity in anterior pituitary cells

Nitric oxide (NO)-dependent soluble guanylyl cyclase (sGC) is operative in mammalian cells, but its presence and the role in cGMP production in pituitary cells have been incompletely characterized. Here we show that sGC is expressed in pituitary tissue and dispersed cells, enriched lactotrophs and somatotrophs, and GH(3) immortalized cells, and that this enzyme is exclusively responsible for cGMP production in unstimulated cells. Basal sGC activity was partially dependent on voltage-gated calcium influx, and both calcium-sensitive NO synthases (NOS), neuronal and endothelial, were expressed in pituitary tissue and mixed cells, enriched lactotrophs and somatotrophs, and GH(3) cells. Calcium-independent inducible NOS was transiently expressed in cultured lactotrophs and somatotrophs after the dispersion of cells, but not in GH(3) cells and pituitary tissue. This enzyme participated in the control of basal sGC activity in cultured pituitary cells. The overexpression of inducible NOS by lipopolysaccharide + interferon-gamma further increased NO and cGMP levels, and the majority of de novo produced cGMP was rapidly released. Addition of an NO donor to perifused pituitary cells also led to a rapid cGMP release. Calcium-mobilizing agonists TRH and GnRH slightly increased basal cGMP production, but only when added in high concentrations. In contrast, adenylyl cyclase agonists GHRH and CRF induced a robust increase in cGMP production, with EC(50)s in the physiological concentration range. As in cells overexpressing inducible NOS, the stimulatory action of GHRH and CRF was preserved in cells bathed in calcium-deficient medium, but was not associated with a measurable increase in NO production. These results indicate that sGC is present in secretory anterior pituitary cells and is regulated in an NO-dependent manner through constitutively expressed neuronal and endothelial NOS and transiently expressed inducible NOS, as well as independently of NO by adenylyl cyclase coupled-receptors.     

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.     

Chronic stimulation of D1 dopamine receptors in human SK-N-MC neuroblastoma cells induces nitric-oxide synthase activation and cytotoxicity

Elevated synaptic levels of dopamine may induce striatal neurodegeneration in l-DOPA-unresponsive parkinsonism subtype of multiple system atrophy (MSA-P subtype), multiple system atrophy, and methamphetamine addiction. We examined the participation of dopamine and D1 dopamine receptors in the genesis of postsynaptic neurodegeneration. Chronic treatment of human SK-N-MC neuroblastoma cells with dopamine or H2O2 increased NO production and accelerated cytotoxicity, as indexed by enhanced nitrite levels and cell death. The antioxidant sodium metabisulfite or SCH 23390, a D1 dopamine receptor-selective antagonist, partially blocked dopamine effects but together ablated dopamine-mediated cytotoxicity, indicating the participation of both autoxidation and D1 receptor stimulation. Direct activation of D1 dopamine receptors with SKF R-38393 caused cytotoxicity, which was refractory to sodium metabisulfite. Dopamine and SKF R-38393 induced overexpression of the nitric-oxide synthase (NOS) isoforms neuronal NOS, inducible NOS (iNOS), and endothelial NOS in a protein kinase A-dependent manner. Functional studies showed that approximately 60% of total NOS activity was due to activation of iNOS. The NOS inhibitor N(G)-nitro-l-arginine methyl ester and genistein, wortmannin, or NF-kappaB SN50, inhibitors of protein tyrosine kinases phosphatidylinositol 3-kinase and NF-kappaB, respectively, reduced nitrite production by dopamine and SKF R-38393 but were less effective in attenuating H2O2-mediated effects. In rat striatal neurons, dopamine and SKF R-38393, but not H2O2, accelerated cell death through increased expression of neuronal NOS and iNOS but not endothelial NOS. These data demonstrate a novel pathway of dopamine-mediated postsynaptic oxidative stress and cell death through direct activation of NOS enzymes by D1 dopamine receptors and its associated signaling pathways.     

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.     

In Vivo Expression of Inducible Nitric Oxide Synthase in Cerebellar Neurons

Abstract: In the CNS, nitric oxide (NO) functions as both neuromodulator and neurotoxic agent. In vivo neuronal expression of NO synthase (NOS) has been attributed to constitutive NOS—both the neuronal and the endothelial types. The other class of NOS—the inducible NOS (iNOS)—is known to mediate toxic effects of NO in various tissues. In this study, we show for the first time that direct intracerebellar injection of endotoxin and cytokine (lipopolysaccharide and interferon-γ) induced in vivo neuronal expression of the iNOS gene, as demonstrated by fluorescent in situ hybridization and immunohistochemical staining analyzed by confocal laser-scanning microscopy. This raises the possibility that neuronal iNOS might contribute significantly to the vulnerability of the brain to various insults.     

Heat shock regulates the respiration of cardiac H9c2 cells through upregulation of nitric oxide synthase

Mild and nonlethal heat shock (i.e., hyperthermia) is known to protect the myocardium and cardiomyocytes against ischemic injury. In the present study, we have shown that heat shock regulates the respiration of cultured neonatal cardiomyocytes (cardiac H9c2 cells) through activation of nitric oxide synthase (NOS). The respiration of cultured cardiac H9c2 cells subjected to mild heat shock at 42 degrees C for 1 h was decreased compared with that of control. The O2 concentration at which the rate of O2 consumption is reduced to 50% was increased in heat-shocked cells, indicating a lowering of O2 affinity in the mitochondria. Western blot analyses showed a fourfold increase in the expression of heat shock protein (HSP) 90 and a twofold increase in endothelial NOS (eNOS) expression in the heat-shocked cells. Immunoblots of eNOS, inducible NOS (iNOS), and neuronal NOS (nNOS) in the immunoprecipitate of HSP90 of heat-shocked cells showed that there was a sevenfold increase in eNOS and no changes in iNOS and nNOS. Confocal microscopic analysis of cells stained with the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate showed higher levels of NO production in the heat-shocked cells than in control cells. The results indicate that heat shock-induced HSP90 forms a complex with eNOS and activates it to increase NO concentration in the cardiac H9c2 cells. The generated NO competitively binds to the complexes of the respiratory chain of the mitochondria to downregulate O2 consumption in heat-shocked cells. On the basis of these results, we conclude that myocardial protection by hyperthermia occurs at least partly by the pathway of HSP90-mediated NO production, leading to subsequent attenuation of cellular respiration.     

Phagocytosis is regulated by nitric oxide in murine microglia.

Nitric oxide (NO) is produced by inducible nitric oxide synthase (iNOS) in activated microglia and has been shown to participate in host defense mechanisms. However, the role of NO produced by constitutive nitric oxide synthase (cNOS) in microglia is poorly understood. In this report, NO was found to regulate phagocytosis in murine BV-2 microglial cells as quantified by flow cytometry. Addition of NO-generating compounds caused impaired phagocytosis as compared to untreated microglia. The addition of nitric oxide synthase (NOS) inhibitors to microglial cells resulted in potentiation of phagocytosis, suggesting that constitutive NO was participating in the regulation of phagocytosis. The inverse correlation between NO production and phagocytosis was also observed when Alzheimer's beta-amyloid peptide was added. With beta-amyloid treatment, constitutive NO production decreased while phagocytosis increased. Cell extracts prepared from untreated microglia were found to contain both neuronal and endothelial NOS isoforms, but not the inducible form. The correlation of spontaneous NO production with attenuated phagocytosis suggests that constitutive NOS enzymes participate in microglial regulation.     

Macrophage endothelial nitric-oxide synthase autoregulates cellular activation and pro-inflammatory protein expression

Expression of inducible nitric-oxide (NO) synthase (iNOS) and "high-output" production of NO by macrophages mediates many cytotoxic actions of these immune cells. However, macrophages have also been shown to express a constitutive NOS isoform, the function of which remains obscure. Herein, bone marrow-derived macrophages (BMDM?s) from wild-type and endothelial NOS (eNOS) knock-out (KO) mice have been used to assess the role of this constitutive NOS isoform in the regulation of macrophage activation. BMDM?s from eNOS KO animals exhibited reduced nuclear factor-kappaB activity, iNOS expression, and NO production after exposure to lipopolysaccharide (LPS) as compared with cells derived from wild-type mice. Soluble guanylate cyclase (sGC) was identified in BMDM?s at a mRNA and protein level, and activation of cells with LPS resulted in accumulation of cyclic GMP. Moreover, the novel non-NO-based sGC activator, BAY 41-2272, enhanced BMDM? activation in response to LPS, and the sGC inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one attenuated activation. These observations provide the first demonstration of a pathophysiological role for macrophage eNOS in regulating cellular activation and suggest that NO derived from this constitutive NOS isoform, in part via activation of sGC, is likely to play a pivotal role in the initiation of an inflammatory response.     

Regulation of the nitric oxide synthase-nitric oxide-cGMP pathway in rat mesenteric endothelial cells.

Most of the available data on the nitric oxide (NO) pathway in the vasculature is derived from studies performed with cells isolated from conduit arteries. We investigated the expression and regulation of components of the NO synthase (NOS)-NO-cGMP pathway in endothelial cells from the mesenteric vascular bed. Basally, or in response to bradykinin, cultured mesenteric endothelial cells (MEC) do not release NO and do not express endothelial NOS protein. MEC treated with cytokines, but not untreated cells, express inducible NOS (iNOS) mRNA and protein, increase nitrite release, and stimulate cGMP accumulation in reporter smooth muscle cells. Pretreatment of MEC with genistein abolished the cytokine-induced iNOS expression. On the other hand, exposure of MEC to the microtubule depolymerizing agent colchicine did not affect the cytokine-induced increase in nitrite formation and iNOS protein expression, whereas it inhibited the induction of iNOS in smooth muscle cells. Collectively, our findings demonstrate that MEC do not express endothelial NOS but respond to inflammatory stimuli by expressing iNOS, a process that is blocked by tyrosine kinase inhibition but not by microtubule depolymerization.     

Cross-talk between the calcium-sensing receptor and the epidermal growth factor receptor in Rat-1 fibroblasts

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that is activated by extracellular calcium (Cao2+). Rat-1 fibroblasts have been shown to proliferate and increase ERK activity in response to elevation of [Ca2+]o, and these responses are dependent on functional CaR expression. In this report, we examined the role of cross-talk between the CaR and the epidermal growth factor receptor (EGFR) in mediating these responses in Rat-1 cells. This report shows that AG1478, a specific inhibitor of the EGFR kinase, significantly inhibits the increase in proliferation induced by elevated Cao2+. Furthermore, we show that AG1478 acts downstream or separately from G protein subunit activation of phospholipase C. AG1478 significantly inhibits Cao2+-stimulated ERK phosphorylation and in vitro kinase activity. A similar inhibition of ERK phosphorylation was observed in response to the inhibitor AG494. In addition, treatment with inhibitors of metalloproteases involved in shedding of membrane anchored EGF family ligands substantially inhibited the increase in ERK activation in response to elevated Cao2+. This is consistent with the known expression of TGFalpha by Rat-1 cells. These results indicate that EGFR transactivation is an important component of the CaR-mediated response to increased Cao2+ in Rat-1 fibroblasts and most likely involves CaR-mediated induction of regulated proteolysis and ligand shedding.     

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.     

Effect of calcitonin gene-related peptide on nitric oxide production in osteoblasts: an experimental study

The aim of this study was to investigate the in vitro effects and regulatory mechanism of CGRP (calcitonin gene-related peptide) on NO (nitric oxide) production in osteoblasts. MOB (primary human mandibular osteoblasts) and osteoblast-like cells (MG-63) were either cultured with CGRP or co-incubated with inhibitors targeting eNOS (endothelial nitric oxide synthase), iNOS (inducible nitric oxide synthase), nNOS (neuronal nitric oxide synthase) and [Ca2+]i (intracellular Ca2+). The NO concentration in cell culture supernatants was measured during the first 24 h using the Griess test; cellular NO was marked with the fluorescent marker DAF-FM, DA (3-amino, 4-aminomethyl-2',7'-difluorescein; diacetate) and measured by fluorescence microscopy from 1 to 4 h after treatment. eNOS and iNOS mRNA expression levels were measured by quantitative RT-PCR during the first 24 h after treatment. CGRP-induced NO production in the supernatants was high between 1 to 12 h, while cellular NO was highest between 1 to 2 h after treatment and returned to basal levels by 3 h. Both in MG-63 cells and MOBs, the most effective CGRP concentration was 10 nM with a peak time of 1 h. CGRP-induced NO production decreased when eNOS activity was inhibited or when voltage-dependent L-type Ca2+ channels were blocked at 4 h. CGRP was not able to induce changes in iNOS or eNOS mRNA levels and had no effect on the cytokine-induced increase of iNOS expression. Our results suggest that CGRP transiently induces NO production in osteoblasts by elevating intracellular Ca2+ to stimulate the activity of eNOS in vitro.     

Contributions of nitric oxide synthase isozymes to exhaled nitric oxide and hypoxic pulmonary vasoconstriction in rabbit lungs

We investigated the source(s) for exhaled nitric oxide (NO) in isolated, perfused rabbits lungs by using isozyme-specific nitric oxide synthase (NOS) inhibitors and antibodies. Each inhibitor was studied under normoxia and hypoxia. Only nitro-L-arginine methyl ester (L-NAME, a nonselective NOS inhibitor) reduced exhaled NO and increased hypoxic pulmonary vasoconstriction (HPV), in contrast to 1400W, an inhibitor of inducible NOS (iNOS), and 7-nitroindazole, an inhibitor of neuronal NOS (nNOS). Acetylcholine-mediated stimulation of vascular endothelial NOS (eNOS) increased exhaled NO and could only be inhibited by L-NAME. Selective inhibition of airway and alveolar epithelial NO production by nebulized L-NAME decreased exhaled NO and increased hypoxic pulmonary artery pressure. Immunohistochemistry demonstrated extensive staining for eNOS in the epithelia, vasculature, and lymphatic tissue. There was no staining for iNOS but moderate staining for nNOS in the ciliated cells of the epithelia, lymphoid tissue, and cartilage cells. Our findings show virtually all exhaled NO in the rabbit lung is produced by eNOS, which is present throughout the airways, alveoli, and vessels. Both vascular and epithelial-derived NO modulate HPV.     

Minocycline inhibits apoptotic cell death via attenuation of TNF-alpha expression following iNOS/NO induction by lipopolysaccharide in neuron/glia co-cultures

We attempted to ascertain the neuroprotective effects and mechanisms of minocycline in inflammatory-mediated neurotoxicity using primary neuron/glia co-cultures treated with lipopolysaccharide (LPS). Neuronal cell death was induced by treatment with LPS for 48 h, and the cell damage was assessed using lactate dehydrogenase (LDH) assays and by counting microtubule-associated protein-2 (MAP-2) positive cells. Through terminal transferase deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-staining and by measuring caspase-3 activity, we found that LPS-induced neuronal cell death was mediated by apoptosis. We determined that pre-treatment with minocycline significantly inhibited LPS-induced neuronal cell death. In addition, LPS induced inducible nitric oxide synthase (iNOS) expression significantly, resulting in nitric oxide (NO) production within glial cells, but not in neurons. Both nitric oxide synthase (NOS) inhibitors (N(G)-monomethyl-L-arginine monoacetate (L-NMMA) and S-methylisothiourea sulfate (SMT)) and minocycline inhibited iNOS expression and NO release, and increased neuronal survival in neuron/glia co-cultures. Pre-treatment with minocycline significantly inhibited the rapid and extensive production of tumor necrosis factor-alpha (TNF-alpha) mediated by LPS in glial cells. We also determined that the signaling cascade of LPS-mediated iNOS induction and NO production was mediated by TNF-alpha by using neutralizing antibodies to TNF-alpha. Consequently, our results show that the neuroprotective effect of minocycline is associated with inhibition of iNOS induction and NO production in glial cells, which is mediated by the LPS-induced production of TNF-alpha.