Nitric Oxide Part A: Sources and Detection of NO; NO Synthase

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NO possesses several physiological properties that make it a potent cardioprotective-signaling molecule, as follows [ 43 ]: First, NO is a potent vasodilator in the ischemic myocardium which enables an essential perfusion of injured tissue. Second, NO reversibly inhibits mitochondrial respiration during early reperfusion.

Nitric Oxide Synthase Enzymes Part 1

It is known that restoration of oxygen at reperfusion leads to a lethal burst of reactive oxygen species ROS generation. An important source of ROS is the mitochondria. In mitochondria, electrons from intermediary metabolism move down the electron transport chain ETC and transferred to oxygen at complex IV [ 46 ]. When oxygenation is normal, complex I activity is high because a cysteine residue on its ND3 subunit is protected from modification.

5 Ways to Increase Nitric Oxide Naturally

During ischemia without oxygen , electrons accumulate along the ETC [ 46 ]. Reperfusion leads to a burst of ROS production from multiple sites which can attack proteins, lipids, and DNA, as well as lethal activation of the mitochondrial permeability transition pore [ 46 ]. NO inhibits mitochondrial complex I by S-nitrosation or S-nitrosylation of cysteines, which subsequently prevents damage during IR injury [ 47 ]. Reversible S-nitrosation of complex I slows the reactivation of mitochondria during the crucial first minutes of the reperfusion, thereby decreasing ROS production, oxidative damage, and tissue necrosis [ 48 ].

Third, NO is a potent inhibitor of neutrophil adherence to the vascular endothelium which is a significant event initiating further leukocyte activation and superoxide radical production [ 43 , 49 , 50 ]. Fourth, NO prevents platelet aggregation [ 51 ], and this effect attenuates capillary plugging together with the anti-neutrophil actions of NO [ 52 ]. Finally, NO inhibits apoptosis either directly or indirectly by inhibiting caspaselike activation via a cGMP-dependent mechanism [ 43 , 53 ] and by direct inhibition of caspaselike activity through protein S-nitrosylation [ 43 , 54 ].

In summary, the release of low concentrations of NO by constitutive NOS played a role in the regulation of coronary blood flow, inhibition of platelet aggregation, adherence to the endothelium, and possibly modulation of myocardial oxygen consumption. But, excessive generation of NO is detrimental to cardiovascular function as exemplified in septic shock where burst generation of iNOS-derived NO causes hypotension, cardiodepression, and vascular hyporeactivity [ 55 ].


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The detrimental effect of excess NO is attributed to the action on mitochondria. NO inhibits the mitochondrial respiratory chain, resulting in inhibition of ATP production, increased oxidant production, and increased susceptibility to cell death [ 56 ]. Inhibition of mitochondrial respiration by NO and its derivatives stimulates production of reactive oxygen and nitrogen species by mitochondria [ 56 ], which contribute to cell death in excess. In conclusion, NO can preserve blood flow in the ischemic tissues and reduce platelet aggregation and neutrophil-endothelium interaction following IR.

Besides, low concentrations of NO improve cardiomyocyte function. On the contrary, higher NO concentrations diminish cardiomyocyte function, mediate inflammatory processes following IR, worsen mitochondrial respiration, and even induce cardiomyocyte death. Therefore, it seems that NO can mediate both protective and detrimental myocardial effects which are crucially dependent upon the experimental conditions. However, the role of their product, NO, in the process of IR is still not well defined mainly because of the difficulty in measuring NO concentration in the body tissue. In the next section, we summarize real-time NO dynamics in the myocardium during myocardial IR.

And applications of electrochemical NO sensor for the evaluation of cardioprotective effects of therapeutic treatments such as hypothermia, drug administration, and ischemic preconditioning are summarized. In general, hypothermia is thought to reduce the metabolic needs of cells, specifically perhaps by reducing the oxygen demand in the hypothermic tissues [ 57 ]. Besides, in isolated heart perfusion system, hearts were placed in ice-cold buffer as quickly as possible to avoid any detrimental effects of hypoxia. Therefore, myocardial hypothermia might be a useful technique to limit ischemic damage during infarction or as adjunctive therapy during minimally invasive cardiac surgery [ 58 ].

Lee et al. They attempted to clarify the role of endogenous NO release by comparing intact and cardioprotected hearts, in which cardioprotection was conferred by hypothermic treatment of the hearts. As a result, they inferred that hypothermic treatment of the heart would promote endogenous NO production in the ischemic myocardium. It might be a helpful therapeutic strategy for protecting the myocardium from IR injury [ 4 ].

Representative real-time measurement of NO in intact A and hypothermic B groups during myocardial ischemia-reperfusion of Langendorff-perfused rat hearts. Reproduced with permission from Lee et al. Because oxygen plays a critical role in the pathophysiology of myocardial injury during subsequent reperfusion, as well as ischemia, the accurate measurement of myocardial oxygen tension is crucial for the assessment of myocardial viability by IR injury.


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And they analyzed differences in oxygen tension recovery in the post-ischemic myocardium depending on ischemic time to investigate the correlation between recovery parameters for oxygen tension and the severity of IR injury. These results show that the maximum and restoration values of p O 2 in the post-ischemic myocardium were closely related to the infarct size [ 59 ]. In summary, they demonstrated that the degree of reoxygenation in the post-ischemic myocardium was an important index of IR injury and myocardial viability, utilizing a sol-gel-derived electrochemical oxygen microsensor and recovery parameters.

Reproduced from Lee et al. Ischemic preconditioning is an adaptive response of briefly ischemic tissues that serves to protect against subsequent prolonged ischemic insults and reperfusion injury [ 60 ]. In particular, remote ischemic preconditioning RIPC is a novel method where ischemia followed by reperfusion of one organ is believed to protect remote organs either by the release of biochemical messengers into circulation or by the activation of nerve pathways, resulting in the release of messengers that have a protective effect [ 60 — 62 ].

This preserves the target tissue without trauma to major vessels or direct stress to the target organ [ 63 ]. Although some studies have demonstrated that endothelial NO is one of the major contributors to the candidate mechanism of RIPC [ 60 , 64 ], the mechanism of RIPC-induced cardioprotection has not yet been fully elucidated. By comparing control and RIPC-treated hearts, we attempted to clarify the correlation between NO release in the ischemic period and O 2 restoration in the myocardium after reperfusion. Schematic diagrams of A experimental design and B experimental setup of an isolated heart perfusion system and a real-time monitoring system for nitric oxide and oxygen tension dynamics during myocardial ischemia-reperfusion of the rat.

Reproduced from Kang et al. As a result, the concentration change of NO in the RIPC group was different from those in the control group during the ischemic period.

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In the control group, the NO level initially declined but then gradually inclined during the ischemic episode. In contrast, the NO level in the RIPC group rapidly increased after the onset of ischemia and continued to rise throughout the entire ischemic period [ 65 ]. In summary, the endogenous production of NO during the ischemic period appears to be correlated with the restoration of NO and p O 2 in the post-ischemic myocardium after early reperfusion.

Additionally, RIPC would promote endogenous NO release against ischemic stimuli and subsequently facilitate reoxygenation in post-ischemic myocardia after reperfusion [ 65 ]. Representative real-time and simultaneous measurement of nitric oxide and oxygen tension in A control and B remote ischemic preconditioning RIPC groups during myocardial ischemia-reperfusion in Langendorff-perfused rat hearts. The correlation between ischemia-evoked nitric oxide concentration and reoxygenation parameters of the post-ischemic myocardium in two groups. Reproduced from Ref. Acta , 74 Similar to NO, PGE1 has cardioprotective effects during IR [ 67 , 68 ], as well as vasodilator effects on the systemic and pulmonary circulation [ 69 ].

Fang et al.

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And this effect might depend, at least in part, on the upregulation of NO expression [ 66 ]. On the other hand, Huk et al. They investigated the correlation between endogenous NO and PGE1 in the ischemic episode, as well as oxygen recovery in the post-ischemic myocardium [ 67 ]. In addition, after the onset of reperfusion, NO level inclined to a maximum of They suggest that the cardioprotective effect of PGE1 might be attributed to a reduction in excessive NO production during early reperfusion.

B — Chem. NO plays important roles in the cardiovascular system by mediating various physiological and pathophysiological processes. From the real-time measurement of endogenous NO dynamics in the myocardium, we summarize as follows: 1 NO concentration was definitely decreased after myocardial ischemia; 2 there was endogenous NO formation as a protective response against ischemia during the ischemic episode, but it was not enough to restore pre-ischemic NO level; 3 the promotion of endogenous formation and inhibition of the time-course alteration of NO during an ischemic episode might be helpful as a therapeutic strategy for protecting the myocardium from ischemic injury; and 4 the reduction of excessive NO production in early reperfusion period might also be helpful as a therapeutic strategy to protect the myocardium from IR injury.

And NO permselective microsensors have good sensitivity and specificity for detecting biologically released NO dynamics in vivo and can be applied in real-time monitoring of NO dynamics in various organs.

www.sibteplokomplekt.ru/includes/grinder-prilozhenie/4757-minsk-zhenshina.php Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3. Help us write another book on this subject and reach those readers. Login to your personal dashboard for more detailed statistics on your publications. Edited by Seyed Soheil Saeedi Saravi. Edited by Angel Catala. We are IntechOpen, the world's leading publisher of Open Access books. The purpose of these studies is to elucidate signal transduction mechanisms of the cannabinoid-mediated nNOS activation resulting in NO production in neuronal cells.

The reagents were purchased from Sigma Chemical Company St. CP 2-[ 1 S ,2 R ,5 S hydroxy 3-hydroxypropyl cyclohexyl] 2-methyloctanyl phenol and rimonabant N - piperidin-lyl 4-chlorophenyl 2,4-dichlorophenyl methyl- H -pyrazolecarboxamide were provided by the National Institute on Drug Abuse drug supply program. The nNOS rabbit polyclonal antibody catalog no. Goat anti-rabbit IgG horseradish peroxidase catalog no. G and goat anti-mouse IgG horseradish peroxidase catalog no. NO production was measured using previously described methods McCollum et al.

Autofluorescence was minimized with 0. The inverted cover slips were mounted onto glass slides using ProLong Antifade, and images from the slides were digitalized using identical exposure time and brightness settings for all conditions using a Nikon Eclipse E fluorescence microscope Nikon Instruments, Melville, NY, USA. Quantitation of fluorescence was performed using Image Pro Plus 4. PCR products were detected from agarose gels 1. The protein concentrations were determined using the Coomassie dye binding method Bradford Immunoreactive bands were detected by enhanced chemiluminescence and exposure to Hyperfilm at various time intervals to obtain optimal signals.

The cover slips were mounted in an Attofluor Cell Chamber catalog no. A, Molecular Probes. For every experiment, the effects of cannabinoid agonists were compared to the dose-dependent response to bradykinin. The data were analyzed, and graphs were prepared using Prism 4. N18TG2 cells were treated with the indicated concentrations of cannabinoid agonists squares CP, inverted triangles WIN, triangles methanandamide.

Blots are representative of at least three independent experiments. The CB 1 receptor and nNOS protein have been identified in close proximity in brain areas suggestive of functional interactions that may be of importance in a variety of physiological states in which NO could serve either a paracrine or autocrine function.

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In striatal medium spiny neurons, CB 1 receptors coexist on at least one third of the nNOS-expressing neurons Azad et al. In the hypothalamic preoptic area and arcuate nucleus, NOS and CB 1 receptors interact in thermoregulation Rawls et al. Maccarrone and colleagues demonstrated that CB 1 receptor-stimulated NO production is involved in the anandamide translocation process Maccarrone et al. These studies support the role of CB 1 receptor-stimulated NO production in various physiological processes. In contrast, cannabinoid-stimulated NO production has not been universally observed in other neuronal preparations.

In our studies, cannabinoid agonists would be expected to promote a pattern of phosphorylation of nNOS that would be quite different from that of a neuron undergoing an excitotoxic reaction in response to NMDA Rameau et al. We determined that cannabinoid agonists stimulate a robust and prolonged translocation of NO-sensitive guanylyl cyclase from cytosol to the cell membranes Jones et al. The data obtained in our studies provide insights regarding cellular regulation of nNOS by CB 1 receptor stimulation.

Post-translational phosphorylation of nNOS, which could incorporate as many as six to seven phosphates per monomer Nakane et al. An alternative explanation for the observation of two nNOS bands on Western blots is the existence of splice variants. Because the antibody that we used would have recognized both forms, our data for cytosol and membrane-associated nNOS do not support the existence of a splice variant exhibiting these properties. A nNOS isoform in which exons 9 and 10 are deleted and therefore is missing the C-terminal portion of the dihydrofolate reductase domain is a lower molecular weight dysfunctional isoform that might serve a role in a cellular function other than NO synthesis from l- arginine Iwasaki et al.

It should be noted that other alternative splicing of exons 1 and 2 can affect the untranslated region, leading to potentially altered regulation of mRNA stability and translation Boissel et al. Studies with N18TG2 cells do not provide convincing evidence from conventional PCR analyses that would argue for the presence of alternative splice variants Lloyd and Norford, unpublished observations. In summary, we have found an autocrine regulation of the CB 1 receptor-nNOS-NO-sensitive guanylyl cyclase in a model neuronal cell line.

Our findings help to clarify the mechanism by which cannabinoids promote NO production in an autocrine or paracrine signaling in biological responses mediated by the CB 1 receptor. These studies were in partial fulfillment of the M. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author s and source are credited.

Skip to main content Skip to sections. Advertisement Hide. Download PDF. Open Access. First Online: 14 April Introduction Nitric oxide NO is a highly reactive compound that can serve as a beneficial physiologic messenger or as a toxin in disease processes in various tissues Schmidt and Walter The low background fluorescence indicates that the cellular production of NO does not occur constitutively in these cells.

Over the min period of NO accumulation, all three cannabinoid receptor agonists produced maximal NO-DAF-FM fluorescence at 10 nM concentrations, indicating that the cells were extremely sensitive to agonist stimulation Fig. Open image in new window. Other inhibitors, including 1-aminohydroxyguanidine para -toluenesulfonate, 7-nitroindazole, S -methylisothiourea sulfate, and l- thiocitrulline a constitutive NOS inhibitor also attenuated NO-DAF-FM fluorescence in response to CB 1 agonists Carney and Norford, data not shown.

Because of the relatively poor selectivity exhibited by NOS inhibitors, the question of which NOS type was responsible for the NO production was addressed using protein and gene expression studies. As shown in Fig. The two bands may be representative of the expression of nNOS splice variants Boissel et al. The decrement in nNOS protein was observed in both the cytosolic fraction as well as the membranes. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author s and source are credited.

Biochem J — Eur J Neurosci — Nitric Oxide — Biol Chem — Cell Mol Life Sci — Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem —

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