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Posts Tagged ‘cGMP-dependent kinases’


Curated/reported by : Aviral Vatsa PhD, MBBS

Based on : S Moncada et al

It was in 1980 that Furchgott & Zawadzki first described endothelium- dependent relaxation of the blood vessels by acetylcholine. Further studies in 1984 revealed that other factors such as bradykinin, histamine and 5-hydroxytryptamine release endothelium derived relaxing factor (EDRF), which can modulate vessel tone. EDRF was shown to stimulate soluble guanylate cyclase and was inhibited by haemoglobin. In 1986 it was demonstrated that superoxide (O2) anions mediated EDRF inactivation and that the inhibitors of EDRF generated superoxide (O2) anions in solution as a mean to inhibit EDRF. It was later established that all compounds that inhibit EDRF have one property in common, redox activity, which accounted for their inhibitory action on EDRF. One exception was haemoglobin, which inactivates EDRF by binding to it. In 1987 Furchgott proposed that EDRF might be nitric oxide (NO) based on a study of the transient relaxations of endothelium-denuded rings of rabbit aorta to ‘acidified’ inorganic nitrite (NO) solutions and the observations that superoxide dismutase (SOD, which removes O2) protected EDRF. Till then NO was not known to be produced in mammalian cells. In 1988 Palmer et al could detect NO production both biologically and chemically by chemiluminescence. The following year in 1989 the enzyme responsible for NO production, NO synthase, was discovered and L-arginine:NO pathway was proposed.

Roles of L-arginine:NO pathway

By 1987 it was proposed that NO is generated in tissues other than endothelium. Hibbs et al and Marletta et al proposed that NO was generated by macrophages. Moreover release of EDRF was demonstrated in cerebellar cells following activation with N-methyl-D- aspartate (NMDA ). Both noradrenergic and cholinergic responses are ‘controlled’ by the nitrergic system so that the release of NO (e.g., during electrical field stimulation) counteracts and dominates the response to the noradrenergic or cholinergic stimulus (Cellek & Moncada, 1997). Mechanism of penile erection was unveiled by the studies on nitrergic neurotransmission that led to therapeutic intervention. Selective damage of nitrergic nerves in disease states was proposed as a potent mechanism of pathophysiology. Broadly three areas of research based on three isoforms of NOS came into being;

  • cardiovascular
  • nervous
  • immunology

Identification of NG-monomethyl-L-arginine (L-NMMA) as an inhibitor of the synthesis of NO lay the basis of future research into investigating the role of NO in biological systems. In 1989 it was demonstrated that intravenous infusion of L-NMMA resulted in increase in blood pressure that was reversible by infusing L-arginine. NO was thus implicated in constantly maintaining blood vessel tone. eNOS knockout studies showed a hypertensive phenotypes in the animal models and over expression of eNOS led to lowering of the blood pressure. Furthermore, eNOS activation was attributed to phosphorylation of a specific tyrosine residue in the enzyme.

NO and Mitochondria 

https://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-glycolysis-with-a-concomitant-influence-on-mitochondrial-function/

NO reacts with some of the complexes of the respiratory chain, and inhibits mitochondrial respiration – this is a well accepted notion. Initially it was believed that the target for NO was soluble guanylate cyclase, which in vasculature would lead to elevation of cGMP that eventually results in NO mediated vasodilatation and platelet aggregation inhibition. In 1994, another potential target, cytochrome c oxidase, for inhibitory effects of NO was discovered. This was a reversible effect, in competition with oxygen concentrations. Increases in NO production were also shown to inhibit cellular respiration irreversibly by selectively inhibiting complex I . Hence in 2002 it was proposed that this might be a mechanism through which cell pathology was initiated in certain conditions. Furthermore, NO was proposed to be implicated in the activation of the grp78-dependent stress response , via modulating calcium-related interaction between mitochondria and endoplasmic reticulum . This host defence mechanism might also have role in vasculature. Further evidence was provided in 2003 to link the role of NO in mitochondrogenesis and thus indicating that NO might be involved in the regulation of the balance between glycolysis and oxidative phosphorylation in cells.

NO and Pathophysiology

Lack of NO: By 2000, NO was established as a haemostatic regulator in the vasculature. Its absence was implicated in pathological states such as hypertension and vasospasm. These pathophysiological states share a common beginning of endothelial dysfunction, which has low NO production as one of its characterstic features. This dysfunction has been observed prior to the appearance of cardiovascular disease in predisposed subjects with family history of essential hypertension and atherosclerosis. The most likely mechanism for endothelial dysfunction is that of a reduced bioavailability of NO . The mechanism of this aspect is discussed elsewhere on this site. Protection against reduction of NO bio-availability in the vasculature is a vital therapeutic target and is extensively explored. This can be achieved by the use of antioxidants and/or augmentation of eNOS expression. In 2003 statins were shown to increase the production of endothelial NO in endothelial cell cultures and in animals by the reduction of oxidative stress or by increasing the coupling of the eNOS. It was way back in 1994 that oestrogen was shown to increase both the activity and expression of eNOS. In addition, more recently in 2003, oestrogen was shown to reduce the breakdown of available NO.

Excess of NO: In 2000 it was shown that NO produced from iNOS in vasculature is involved in extensive vasodilatation in septic shock. Later it was demonstrated that inhibition of mitochondrial respiration is an important component of the NO-induced tissue damage. This inhibition of respiration, which is initially NO-dependent and reversible, becomes persistent with time as a result of oxidative stress . Such metabolic hypoxic states where in tissues cannot utilise available oxygen due to NO, could also contribute to other inflammatory and degenerative conditions. An obvious therapeutic target for reducing NO production in such conditions would be L-NMMA. L-NMM was tested in a clinical trial for septic shock in 2004. The results were however disappointing probably due to the blanket reduction in NO production from other NOS enzymes there by having deleterious effects on the treatment group. More specific inhibitors for NOS forms are being investigated for in different disease states.

In conclusion, the L-arginine: NO pathway has had a major impact in many areas of research, specially vascular biology. A lot has been understood about this pathway and its interactions, therapeutic targets are being aggressively investigated, but further investigations are required to delineate further the role of NO in human health and disease.

Further Reading

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1760731/?tool=pubmed

Nitric Oxide and Platelet Aggregation

Inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production

Nitric Oxide in bone metabolism

Nitric oxide and signalling pathways

Rationale of NO use in hypertension and heart failure

Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium

Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function

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Curator/ Author: Aviral Vatsa, PhD, MBBS

In continuation with the previous posts that dealt with short history and chemistry of nitric oxide (NO), here I will try to highlight the pathways involved in NO chemical signalling.

NO is a very small molecule, with a short half life (<5 sec). It diffuses rapidly to its surroundings and is metabolised to nitrites and nitrates. It can travel short distances, a few micrometers, before it is oxidised. Although it was previously believed that NO can only exert its effect for a very short time as other nitrogen oxides were believed to be biologically inert. Recent data suggests that other NO containing compounds such as S- or N-nitrosoproteins and iron-nitrosyl complexes can be reduced back to produce NO. These NO containing compounds can serve as storage and can reach distant tissues via blood circulation, remote from their place of origin. Hence NO can have both paracrine and ‘endocrine’ effects.

Intracellularly the oxidants present in the cytosol determine the amount of bioacitivity that NO performs. NO can travel roughly 100 microns from NOS enzymes where it is produced. NOS enzymes on the other hand are localised to specific sub-cellular areas, which have relevant proteins in the vicinity as targets for signalling.

NO signalling occurs primarily via three mechanisms (according to Martínez-Ruiz et al):

  1. Classical: This occurs via soluble guanylyl cyclase (sGC). Once NO is produced by NOS it diffuses to sGC intracellularly or even in other cells. SGC is highly sensitive for NO, even nanomolar amounts of NO activates sGC, thus making it a potent target for NO in signalling pathways. sGC in turn increases the conversion of GTP to cGMP. cGMP further mediates the regulation of contractile proteins and gene expression pathways via cGMP-activated protein kinases (PKGs). cGMPs cause confirmational changes in PKGs. Signalling by cGMP is terminated by the action of phosphodiestrases (PDEs). PDEs have become major therapeutic targets in the upcoming exciting research projects.
  2. Less classical: Within the mitochondria NO can compete with O2 and inhibit cytochrome c oxidase (CcO) enzyme. This is a reversible inhibition that depends on O2and NO concentrations and can occur at physiological levels of NO. Various studies have demonstrated that endogenously generated NO can inhibit respiration or that NOS inhibitors can increase respiration at cellular, tissue or whole animal level. Although the exact mechanism of CcO inhibition of NO is still debated, NO-CcO interaction is considered important signalling step in a variety of functions such as inhibition of mitochondrial oxidative phosphorylation, apoptosis and reactive oxygen species (ROS) generation. Interestingly, at higher concentration (~1nM) NO can cause irreversible inhibition of cellular oxidation by reversible and/or irreversible damage to the mitochondrial iron–sulfur centers,In addition to the above mentioned pathways, NO (along with AMP, ROS and O2), can also activate AMP- activated protein kinase (AMPK), an enzyme that plays a central role in regulating intracellular energy metabolism. NO can also regulate hypoxia inducible factor (HIF), an O2-dependent transcription factor that plays a key role in cell adaptation to hypoxia .
  3. Non- classical: S-nitrosylation or S-nitrosation is the covalent insertion of NO into thiol groups such as of cysteine residues of proteins. It is precise, reversible, and spatiotemporally restricted post translational modification. This chemical activity is dependent upon the reactivity between nitrosylating agent (a small molecule) and the target (protein residue). It might appear that this generic interaction results in non-specific, wide spread chemical activity with various proteins. However, three factors might determine the regulation of specificity of s-nitrosylation for signalling purposes:
  • Subcellular compartmentalisation: high concentrations of nitrosylating agents are required in the vicinity of target residues, thus making it a specific activity.
  • Site specificity: certain cysteine residues are more reactive in specific protein microenvironments than others, thus favouring their modification. As a result under physiological conditions only a specific number of cysteine residues would be modified, but under higher NO levels even the slow reacting ones would be modified. Increased impetus in research in this area to determine protein specificity to s-nitrosylation provides huge potential in discovering new therapeutic targets.
  • Denitrosylation: different rates of denitrosylation result in s-nitrosylation specificity.

Other modifications in non classical NO mechanisms include S-glutathionylation and tyrosine nitration

Peroxynitrite: It is one of the important reactive nitrogen species that has immense biological relevance. NO reacts with superoxide to form peroxynitrite. Production of peroxynitrite depletes the bioactivty of NO in physiological systems. Peroxynitrite can diffuse through membranes and react with cellular components such as mitochondrial proteins, DNA, lipids, thiols, and amino acid residues. Peroxynitrite can modify proteins such as haemoglobin, myoglobin and cytochrome c. it can alter calcium homeostasis and promote mitochondrial signalling of cell death. However, NO itself in low concentrations have protective action on mitochondrial signalling of cell death.

More details about various aspects of NO signalling can be obtained from the following references.

The post is based on the following Sources:

  1. http://www.sciencedirect.com/science/article/pii/S089158491100236Xhttp://dx.doi.org/10.1016/j.freeradbiomed.2011.04.010
  2. http://content.karger.com/produktedb/produkte.asp?doi=338150Cardiology 2012;122:55-68 (DOI: 10.1159/000338150)
  3. http://content.onlinejacc.org/article.aspx?articleid=1137266 J Am Coll Cardiol. 2006;47(3):580-581. doi:10.1016/j.jacc.2005.11.016
  4. http://goo.gl/y6oY3

 

In addition, other aspects of NO involvement in biological systems in humans are covered in the following posts on this site:

  1. Nitric Oxide and Platelet Aggregation
  2. Inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure
  3. Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production
  4. Nitric Oxide in bone metabolism

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