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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nitric oxide, a radical generated by the enzyme nitric oxide synthase (iNOS), may be an important mediator of beta-cell damage in early insulin-dependent diabetes mellitus. We have investigated the molecular regulation of iNOS in insulin-producing RINm5F cells. The data obtained suggest that iNOS is maximally induced in these cells by a 6-h exposure to IL-1 beta or TNF-alpha + IFN-gamma, but not by endotoxin. iNOS mRNA degradation is rapid and it is not affected by IL-1 beta. Interestingly, NO seems to induce a negative feedback on iNOS expression, probably by decreasing iNOS transcription.
Mol Cell Endocrinol 1994 Dec
PMID:Studies on the molecular regulation of the inducible form of nitric oxide synthase (iNOS) in insulin-producing cells. 753 33

Endotoxin induces an enzyme that synthesizes nitric oxide (NO) from L-arginine (NO synthase) in vascular smooth muscle cells, resulting in nonendothelial NO release. In this study, we measured the NO release and its intracellular action on the Ca(2+)-activated K+ channel (KCa channel) in cultured smooth muscle cells of porcine coronary artery using a newly-developed porphyrinic-based microsensor and the patch-clamp technique. In smooth muscle cells pretreated with endotoxin, extracellular application of 10(-4) M L-arginine increased NO release, which induced rapid and prolonged activation of the KCa channel. This activation was only partially blocked by application of 10(-5) M 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-oxyl 3-oxide, which neutralizes NO. NO formation and activation of the KCa channel were suppressed by pretreatment with 10(-3) M NG-methyl-L-arginine or 10(-3) M N omega-nitro-L-arginine methyl ester, each of which is a specific antagonist of the L-arginine-NO pathway. One micromolar methylene blue, a blocker of guanylate cyclase, inhibited L-arginine-induced activation of the KCa channel. The effect of nitroprusside in opening the KCa channel was transient, although it induced production of larger amounts of NO in the bath. These results suggest that the endotoxin-induced and L-arginine pathway generates NO and directly modulates the KCa channel intracellularly in an autocrine manner.
J Mol Cell Cardiol 1994 Nov
PMID:Endotoxin-induced nonendothelial nitric oxide activates the Ca(2+)-activated K+ channel in cultured vascular smooth muscle cells. 753 31

Nitric oxide is a signaling molecule involved in events crucial to neuronal cell function, such as neurotransmitter release, gene transcription, and neurotoxicity, i.e., a number of processes in which a key role appears to be played by increases in intracellular Ca2+ concentration. In the neurosecretory/neuronal cell line PC-12, we have investigated the role of nitric oxide in the modulation of Ca2+ release from intracellular stores elicited by activation of three different receptors coupled to phosphatidyl-inositol-4,5-bisphosphate hydrolysis, i.e., the purinergic P2U, muscarinic M3, and bradykinin B2 receptors. The results obtained show that nitric oxide donors have an inhibitory effect on agonist-evoked Ca2+ release. This effect is not due to nitric oxide-induced modifications of Ca2+ storage, because the total releasable Ca2+ pool, measured as the radioactivity released by thapsigargin and ionomycin in cells loaded at equilibrium with 45Ca2+, was unchanged. In contrast, nitric oxide donors decreased agonist-evoked inositol-1,4,5-trisphosphate generation and total inositol phosphate accumulation. Similarly, nitric oxide inhibited total inositol phosphate accumulation stimulated by either aluminium fluoride or Ca2+. All of these effects were mimicked by the cGMP analogue 8-bromo-cGMP. When cells were incubated with nitric oxide synthase inhibitors, the results observed were opposite those produced by nitric oxide donors. All of the effects of nitric oxide were abolished when cells were treated with the cGMP-dependent protein kinase I inhibitor KT5823. Furthermore, KT5823 mimicked the effects of nitric oxide synthase inhibitors. We conclude that nitric oxide and Ca2+ signaling pathways are interconnected in PC-12 cells. Modulation of inositol phosphate generation and Ca2+ release by nitric oxide appears to be exerted primarily at the level of phospholipase C functioning and to be mediated by the activation of cGMP-dependent protein kinase I.
Mol Pharmacol 1995 Mar
PMID:Nitric oxide modulation of agonist-evoked intracellular Ca2+ release in neurosecretory PC-12 cells: inhibition of phospholipase C activity via cyclic GMP-dependent protein kinase I. 753 79

Nitric oxide synthesized by the endothelial isoform of nitric oxide synthase (ecNOS) is importantly involved in the homeostatic control of blood pressure and platelet aggregation. The different members of the nitric oxide synthase protein family have several biochemical features in common but serve distinct physiological functions and are the products of distinct genes. The ecNOS is further distinguished by its subcellular distribution in the endothelial cell membrane, and the enzyme undergoes several post-translational modifications, including myristoylation, palmitoylation, and phosphorylation. Overall, however, the ecNOS has remained less well characterized because of the challenges involved in isolating sufficient quantities of this membrane-associated protein from native or cultured endothelial cells. In this report, we describe the purification and characterization of ecNOS expressed in a heterologous system in recombinant baculovirus-infected insect Sf9 cells. Recombinant ecNOS is targeted to the Sf9 cell membrane and comprises approximately 10% of the total cellular protein, allowing purification to homogeneity in a single-step procedure to yield a stable protein that retains the essential features of the native enzyme. Using biosynthetic labeling and immunoprecipitation, we show that recombinant ecNOS is myristoylated, palmitoylated, and phosphorylated when expressed in insect Sf9 cells. The interpretation of structural and enzymological studies of recombinant ecNOS will be facilitated by the apparent fidelity of its biosynthesis and post-translational modification in insect Sf9 cells.
Mol Pharmacol 1995 Apr
PMID:Recombinant endothelial nitric oxide synthase: post-translational modifications in a baculovirus expression system. 753 85

We have identified two novel potent and selective inhibitors of inducible nitric oxide synthase, S-ethylisothiourea and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine. Ki values of 14.7 nM for S-ethylisothiourea and 4.2 nM for 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine were obtained with partially purified preparations of inducible nitric oxide synthase. These compounds demonstrate about 1000-fold greater potency than prototypical inhibitors, and the inhibitions are 10-40-fold more selective for murine inducible nitric oxide synthase, compared with the rat neuronal and bovine endothelial isoforms of nitric oxide synthase. These compounds also potently inhibit the nitric oxide synthase activity in intact J774 mouse macrophages. The inhibition is competitive with the substrate L-arginine and reversible in both enzymatic and intact cell assays. These potent and selective inhibitors of inducible nitric oxide synthase may have potential therapeutic applications in the treatment of inflammatory and autoimmune diseases.
Mol Pharmacol 1995 Apr
PMID:Novel potent and selective inhibitors of inducible nitric oxide synthase. 753 89

Vitamin E, a lipophilic antioxidant, has effectively inhibited the activation of cytokine-induced nuclear factor kB (NFkB). Since NFkB plays a critical role in the induction of an isoform of nitric oxide synthase (iNOS) gene by lipopolysaccharide (LPS), we investigated the effect of a vitamin E derivative, pentamethyl-hydroxychromane (PMC), which is an extremely potent inhibitor of NFkB activation, on the induction of nitric oxide (NO) synthesis and iNOS mRNA by LPS. PMC inhibited the LPS-stimulated induction of NO production in a concentration-dependent fashion in cultured J774 macrophages and rat vascular smooth muscle cells without evidence of cytotoxicity. However, the addition of PMC to J774 macrophages after the induction of iNOS did not inhibit NO production. Treatment of J774 macrophages with LPS resulted in a significant expression of iNOS mRNA, which was profoundly reduced by PMC. Data suggest that PMC inhibits the induction of iNOS by preventing iNOS gene expression through inhibition of NFkB activation.
Biochem Mol Biol Int 1995 Jan
PMID:Pentamethyl-hydroxychromane, vitamin E derivative, inhibits induction of nitric oxide synthase by bacterial lipopolysaccharide. 753 70

A major determinant of survival in patients with advanced viral or bacterial infection, or following severe trauma or burns complicated by multiple organ failure, is the combination of clinical signs termed the systemic inflammatory response syndrome (SIRS). SIRS is characterized by hypotension, tachypnea, hypo- or hyperthermia and leukocytosis as well as other clinical signs and symptoms, including a depression in myocardial contractile function. Heart failure complicating systemic sepsis or other causes of SIRS is usually not accompanied by coronary artery ischemia due to hypotension, myocardial necrosis, or marked cardiac interstitial inflammatory infiltrates, and thus the cause of cardiac contractile dysfunction in this syndrome has remained unclear. However, recent evidence has implicated an endogenous nitric oxide (NO) signalling pathway within cardiac myocytes and other cellular constituents of cardiac muscle, including the microvascular endothelium, as a possible contributor to the pathogenesis of heart failure in this syndrome. Cardiac myocytes are now known to express both constitutive NO synthase (cNOS) and inducible NO synthase (iNOS) activities. Activation of cNOS appears to modulate cardiac myocyte responsiveness to muscarinic cholinergic and beta-adrenergic receptor stimulation. Induction of iNOS by soluble inflammatory mediators, including cytokines, causes a marked depression in myocyte contractile responsiveness to beta-adrenergic agonists. Thus, inappropriate activation of cNOS or excessive or prolonged induction of iNOS in the myocardium may contribute to cardiac dysfunction complicating SIRS.
J Mol Cell Cardiol 1995 Jan
PMID:Myocardial contractile dysfunction in the systemic inflammatory response syndrome: role of a cytokine-inducible nitric oxide synthase in cardiac myocytes. 753 82

We investigated the effect of intratracheal (i.t.) lipopolysaccharide (LPS) on alveolar macrophage release of nitric oxide. Mice received i.t. LPS at doses ranging from 1 to 100 micrograms/100 g body weight and were killed at serial intervals for bronchoalveolar lavage. Control mice received i.t. phosphate-buffered saline. We found that after i.t. LPS, there was an early (1 to 3 days) influx of neutrophils followed by a later (5 to 7 days) influx of macrophages into the lungs. Alveolar macrophages lavaged from mice given i.t. LPS did not spontaneously release nitric oxide (measured as nitrite), but the capacity of these cells to release nitric oxide in vitro in response to interferon-gamma (IFN-gamma) or LPS was markedly upregulated. Alveolar macrophages lavaged from mice given i.t. LPS but not i.t. phosphate-buffered saline also expressed mRNA for inducible nitric oxide synthase as measured by semiquantitative reverse-transcription polymerase chain reaction. To investigate possible mechanisms for cellular priming for increased nitric oxide release after i.t. LPS, mice were depleted of CD4+ lymphocytes with an anti-CD4 antibody. Alveolar macrophages from CD4-depleted mice given i.t. LPS released significantly less nitric oxide in vitro in comparison to macrophages from nondepleted mice. Additional mice were treated with neutralizing doses of anti-tumor necrosis factor or anti-IFN-gamma antibody before i.t. LPS. Pretreatment with each cytokine antibody decreased but did not eliminate macrophage priming for nitric oxide release after i.t. LPS. We conclude that intratracheal LPS induces mRNA for nitric oxide synthase in alveolar macrophages, priming the cells for increased release of nitric oxide in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1995 Jul
PMID:Regulation of nitric oxide release by macrophages after intratracheal lipopolysaccharide. 754 Dec 22

Prolonged hypoxia in the adult rat causes a decline in endothelium-derived nitric oxide (NO) production in the pulmonary circulation. To evaluate whether this is related to a decrease in endothelial NO synthase (NOS-III) expression, we determined the effects of hypobaric hypoxia (7 or 21 days) on NOS-III gene expression in adult rat lung. Neuronal NOS (NOS-I) expression was also examined; NOS-I has been immunohistochemically localized to rat bronchiolar epithelium. NOS-III and NOS-I mRNA abundance were assessed in reverse transcription-polymerase chain reaction assays and the proteins were evaluated by immunoblot analysis. After 7 and 21 days of hypoxia, there were increases in the steady-state levels of both NOS-III and NOS-I mRNA, rising 2.7- to 3.0-fold and 2.5- to 2.8-fold, respectively. These findings were confirmed by Northern analyses. In parallel, NOS-III and NOS-I protein abundance were also increased with hypoxia by 3.0- to 3.5-fold and 2.4- to 3.0-fold, respectively. NOS activity detected by [3H]arginine to [3H]citrulline conversion rose 109%. Thus, prolonged in vivo hypoxia causes enhancement of NOS-III and NOS-I gene expression in adult rat lung, indicating that the pulmonary expression of these genes is modulated in vivo. The increase in NOS-III expression does not explain the declines in pulmonary endothelial NO production previously observed following prolonged hypoxia in this model. Alternatively, the fall in NO production may be related to diminished NOS co-factor availability.
Am J Respir Cell Mol Biol 1995 Aug
PMID:Prolonged in vivo hypoxia enhances nitric oxide synthase type I and type III gene expression in adult rat lung. 754 96

We compared inhibitory nonadrenergic noncholinergic (i-NANC) neural relaxations, evoked by electrical field stimulation (EFS), at three levels (main [MA], proximal [PA], and distal [DA] airways) of isolated human airways and correlated these with nitric oxide synthase-immunoreactive (NOS-IR) nerves, using antiserum raised to rat cerebellar NOS. Maximal relaxations to papaverine (100 microM) were reduced in PA and DA (MA: 1,712 +/- 219 mg, n = 12; DA: 862 +/- 69 mg, n = 5, P < 0.05 versus MA); hence, subsequent relaxations were expressed as a percentage of the papaverine maximum. EFS elicited frequency-dependent relaxations that were largest in MA and reduced in PA and DA, especially at high stimulation frequencies (10 Hz EFS: MA: 51.6 +/- 3.7%, n = 12; PA: 30.5 +/- 6.0%, n = 6, P < 0.01 versus MA; DA: 17.8 +/- 3.6%, n = 5, P < 0.001 versus MA). The NOS inhibitor L-NG-nitroarginine methyl ester (L-NAME) (100 microM) and tetrodotoxin (3 microM) significantly inhibited i-NANC responses at all frequencies, leaving an L-NAME-resistant non-neural relaxation at frequencies > 5 Hz which was reduced in PA and DA. Cumulative concentration-response studies to sodium nitroprusside (1 nM to 0.1 mM) and the NO donor 3-morpholinosydnonimine (1 nM to 1 mM) were not significantly different in PA and DA, suggesting impaired relaxation is not caused by impaired guanylyl cyclase activity. Total nerve density, shown by protein gene product 9.5 staining, was not significantly different in PA and DA; however, NOS-IR nerve density was reduced in PA and DA (NOS-IR [intercepts/mm2]: MA: 705 +/- 98, n = 6; DA: 284 +/- 32, n = 6, P < 0.01 versus MA). These studies demonstrate that i NANC neural relaxations are reduced in DA, apparently due to a decrease in the density of nitrergic innervation.
Am J Respir Cell Mol Biol 1995 Aug
PMID:Distribution of human i-NANC bronchodilator and nitric oxide-immunoreactive nerves. 754 97


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