Constitutive NO synthase

Tacrolimus capacity to blockade NO pathway is weU demonstrated. NO plays a major role in the pathogenesis of cerebral hypoxia-ischemia injury mediated by glutamate/N-methyl-D-aspartate (NDMA). This injury depends on intracellular calcium influx through NDMA receptor channels, which activate calcineurin with consequent dephosphorylation of constitutive NO synthase (cNOS). Tacrohmus addition to cultured neuronal cells reduced NDMA-mediated toxicity, through the inhibition of calcineurin activation, inhibition of cNOS dephosphorylation and consequent decrease in... 

Stimulation of constitutive NO-synthase. Both S. aureus alpha-toxin and HlyA increase synthesis of NO by the constitutive NO synthase in endothelial cells (Suttorp et al., 1993). NO in turn can provoke an array of further reactions in bystander cells. 

Rogers, N. E., and Ignarro, L. J. (1992). Constitutive NO synthase from cerebellum is reversibly inhibited by NO formed from L-arginine. Biochent. Biophys. Res. Gomntun. 189, 242-249. 

Unfortunately, some of the same physiochemical characteristics make quantitation of NO difficult. Most studies in vivo and in vitro have used indirect indices of NO production such as breakdown products of NO metabolism, second messengers, or biological events in effector systems. Recent developments in probe technology and mass spectrometry have allowed the direct detection of NO in some situations. Constitutive NO synthases (cNOS) in vascular endothelium and other tissues produce small quantities of NO for continuous maintenance of vascular tone and cellular communication. Inducible NO synthases (iNOS) are induced by immunological stimuli such as interleukins and lipopolysaccharides, and synthesize relatively large amounts of NO for long periods. In general, therefore, it is much more difficult to study constitutive than inducible NO production. 

Neuronal NO synthase (nNOS) is constitutively expressed in neurons of the brain. Its activity is regulated by Ca2+ and calmodulin. Half-saturating L-arginine concentrations are around 2 pM. cDNAs encoding nNOS have been cloned from rat and human brain. The open reading frame of human nNOS consists of 4299 bp, corresponding to 1433 aa. This predicts a protein of 160 kDa, which is in accordance with the molecular mass of the purified protein. 

Inducible NO synthase (iNOS) is usually not constitutively expressed, but can be induced in macrophages by bacterial lipopolysaccharide (LPS), cytokines and other-agents. Although primarily identified in macrophages, expression of the enzyme can be stimulated in virtually any cell or tissue, provided the appropriate inducing agents have been identified (for review see [1] and [3]). 

NADH-ubiquinone reductase) and the second one was complex II (succinate-ubiquinone reductase). Chiesi and Schwaller [101] found that quercetin and tannin inhibited neuronal constitutive endothelial NO synthase. 

There are cytosolic and membrane-bound isoforms of NO synthase. Certain soluble and particulate isoforms are constitutive and other soluble isoforms are inducible. The constitutive enzyme is, by definition, present in the catalytically active form and needs only to be stimulated by an appropriate chemical species, following which there is immediate formation of NO plus L-citrulline. This form of NO synthase requires calcium, and for the most part calmodulin, for stimulation of enzymatic activity. It is likely that an increase in intracellular free calcium in the presence of calmodulin is the signal for stimulation of NO synthase, and therefore, the production of NO. This view is consistent with the general understanding that, in vascular tissue, all endothelium-dependent vaso-... 

Before discussing some of the observations concerning -NO in our laboratory, it is important to review a few of the basic principles of "NO production and mechanism of action. More detail on these topics can be found in other chapters in this book. First, "NO production can occur by one of two categories of NOS enzymes. Constitutive enzymes are expressed in various cell types, including endothelial cells, neurons, and neutrophils. Production of "NO by this type of enzyme is typically calcium/calmodulin-dependent and occurs immediately. This type of production has also been referred to as low output. Cells such as macrophages, hepatocytes, and smooth muscle cells express an inducible NO synthase. The induction of this enzyme typically results from exposure of the cells... 

Furthermore, the LPS signal transduction involves the activation of G proteins, of phospholipases C and D, the formation of diacyl-glycerol (DG) and inositol triphosphate (IP3). DG mediates the stimulation of protein kinase C (PKC) and IP3 induces an increase of cytosolic Ca++ The LPS signaling pathway also involves tyrosine kinases, constitutive nitric oxide (NO) synthase (cNOS), cGMP-dependent protein kinase, Ca channels, calmodulin and calmodulin kinase [27,28], as well as the MAP kinases [29] ERK1, ERK2 and p38 [23], The intracellular events in response to LPS are due to lipid A because they are inhibited by polymyxin B which is known to bind lipid A [27] and they are reproduced by lipids A [30,31]. 

Synthesis of NO Arginine, 02, and NADPH are substrates for cytosolic NO synthase (Figure 13.9). Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), heme, and tetrahydro- biopterin are coenzymes for the enzyme, and NO and citrulline are products of the reaction. Three NO synthases have been identified. Two are constitutive (synthesized at a constant rate regardless of physiologic demand), Ca2+-calmodulin-dependent enzymes. They are found primarily in endothelium (eNOS), and neural tissue... 

Chiesi M, Schwaller R. 1995. Inhibition of constitutive endothelial NO-synthase activity by tannin and quercetin. Biochem Pharmacol 49 495-501. 

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