Seminar 2 - Nitric Oxide synthesis, properties and enzymatic function PDF

Nitric Oxide synthesis, properties and enzymatic function NOSI, NOSII and NOSIII  The nitric oxide synthases  The nitric oxide synthases (NOSs) are heme- and flavin- containing enzymes;  Two serial monooxygenase reactions;  Functional dimers: a heme-containing...

Nitric Oxide synthesis, properties and enzymatic function NOSI, NOSII and NOSIII  The nitric oxide synthases  The nitric oxide synthases (NOSs) are heme- and flavin- containing enzymes;  Two serial monooxygenase reactions;  Functional dimers: a heme-containing oxygenase domain - structurally unrelated to cytochrome P450s;  Flavin-containing reductase domain - structurally resembles the P450 reductase. Electrons are transferred from NADPH into the reductase domain of one monomer , through the flavins FAD and FMN , to the heme iron of the other monomer where molecular oxygen is bound and activated; The domains are connected by a calmodulin- binding site; Calmodulin is required for electron transfer from the reductase to the oxygenase domain. NOS  Model of the dimeric structure of bovine NOSIII heme domain showing the protoporphyrin IX and tetrahydrobiopterin prosthetic groups;  BH4 - functions in electron transfer;  The substrate L-arginine (green) is in the active site of the NOSIIl;  A zinc atom is at the interface between the two monomers;  Tetra-coordinated zinc atom - important for structural integrity. The NOS reductase domain  Ribbon diagram of nNOS reductase domain derived from its crystal structure;  Similar to cytochrome P450 reductase;  Several subdomains are present:  The FMN binding domain (blue);  The FAD/NADPH binding domain (green);  The intervening connecting domain (purple).  Unlike the P450 reductase, the nNOS reductase contains a calmodulin-binding site and regulatory regions at the C-terminus (CT), in the FMN domain (AR), and in the connecting domain (SI).  NOS  The general reaction catalyzed by the NOSs is sequential monooxygenation of the amino acid L-arginine to form NO and citrulline;  The reaction cycle is initiated by the binding of calmodulin to "open the gate" between the reductase and oxygenase domains;  The electron donor is NADPH - donates two electrons to FAD – which reduces FMN;  The FMN reduces the heme iron to its ferrous form - to which oxygen can bind;  The NO syntheses  In the first step, arginine is oxidized to the stable intermediate N- hydroxy-L-arginine;  In the second step, N-hydroxy-L-arginine is oxidized to NO and citrulline;  The source of NO is the guanidino group of the substrate, arginine;  This entire process requires 2 oxygen molecules and l.5 molecules of NADPH;  Like the cytochromes P450 - is inhibited by carbon monoxide.  NO synthesis  Calmodulin is not bound to the enzyme at intracellular, basal calcium levels;  Influx of calcium - raise the calcium concentration - increasing the binding of calcium to calmodulin;  The calcium-calmodulin complex - activates NOSs;  In the absence of calmodulin binding - heme reduction occurs only very slowly and transfer between FAD and FMN is also slow;  Calmodulin binding causes a conformational change in the enzyme, potentiating electron transfer.  NO synthesis  Difference between the NOSs and P450s - substrate binding is not required by the NOSs to initiate the reaction cycle and the binding of substrate does not alter the reduction potential of the heme;  If substrate is not present - the electron transferred to the heme can activate molecular oxygen, forming the oxy-ferrous complex - decay and be released as superoxide;  If the second electron is transferred before the release occurs, the peroxy-form of oxygen is generated - dissociate as hydrogen peroxide.  If substrate is not present, but the NOS is active - potentially destructive reactive oxygen species may be formed;  NO synthesis  Tetrahydrobiopterin cofactor is required for NO synthesis by NOS, but is not required by any known cytochrome P450;  BH4 serves as an electron donor to the oxy-ferrous complex of NOS, delivering the second electron to the heme much faster than the relatively slow interdomain transfer of an electron from the NOS FMN;  In the absence of tetrahydrobiopterin - NOS will produce superoxide due to the autoxidation of the oxy-ferrous complex;  In the presence of tetrahydrobiopterin, but in the absence of substrate, NOS can also form hydrogen peroxide, due to the formation and subsequent decay of the peroxy-ferrous complex;  The formation of these reduced oxygen species has been implicated in several inflammatory disease processes - endothelial dysfunction, ischemia/reperfusion injury. NITRIC OXIDE SYNTHASE ISOFORMS  There are three major NOS isoforms:  Neuronal (NOSI or nNOS);  Inducible (NOSII or iNOS);  Endothelial (NOSIII or eNOS).  NITRIC OXIDE SYNTHASE PHYSIOLOGICAL FUNCTIONS  Many of the physiological functions of NO are mediated through activation of soluble guanylate cyclase;  Heterodimeric (alfa/beta) heme-containing protein - convenrts GTP to cGMP;  cGMP is a second messenger involved in many signal transduction cascades;  The inactive form of soluble guanylate cyclase has a penta-coordinate heme, bound by a histidine on the beta-subunit;  NO binds as the sixth ligand to form a hexa- coordinate heme and causes breakage of the heme-histidine bond - yielding the active, penta-coordinate nitrosyl complex of soluble guanylate cyclase. NOSI  Found primarily in skeletal muscle and in neurons of both the central and peripheral nervous systems;  It is a soluble enzyme, although it is localized to the membrane via protein- protein interactions with a host of regulatory and localization proteins;  It is constitutively expressed - not normally induced at the transcriptional level and is activated by the influx of calcium;  The NO produced serves as a neurotransmitter in the central and peripheral nervous systems;  In skeletal muscle, NO also serves as a mediator of contractile force.  NOSI  In the CNS - NO is produced generally by nNOS in the postsynaptic neuron;  Diffuses back across the synapse to the presynaptic neuron;  NO synthesis is regulated by the influx of calcium through receptor-dependent channels - following postsynaptic stimulation of N-- methyl-D-aspartate (NMDA) receptors by the excitatory neurotransmitter glutamate.  Guanylate cyclase is activated by NO - producing cGMP - regulates synthesis of the neurotransmitters norepinephrine and glutamate - enhancing NO production in a positive feedback manner. NOSI  NO has been implicated in:  Neural signaling;  Neurotoxicity;  Synaptic plasticity;  Learning and memory;  Perception of pain.  NO  NO is synthesized on demand and cannot be stored in lipid vesicles, as it freely diffuses across membranes;  The action of NO is not terminated by degradation or reuptake but is removed by interaction with its target, which is an intracellular second messenger rather than a membrane bound receptor.  NO in PNS  In the PNS - NO is produced by nNOS in myenteric neurons of the gastrointestinal, pulmonary, vascular and urogenital systems;  In the GI - NO is involved in gut motility and control of the pyloric sphincter;  NO mediates the main bronchodilator pathway in the human pulmonary system;  Neural control of the vascular system - important in cerebral blood flow;  In the urogenital system - NO is involved in urethra and bladder control and in penile erection - relaxation of vessels causes blood to engorge the corpus cavernosum, producing erection;  NOSI is activated in these systems by the influx of calcium through voltage dependent calcium channels.  NO in muscles  High levels of NOSI are expressed in the skeletal muscle - involved in mediation of contractile force, innervation of developing muscle and glucose uptake;  Skeletal muscle relaxation by NO also occurs through the cGMP pathway;  NOSI is targeted to the sarcolemma of the muscle due to its association with a- syntrophin , a member of the muscle dystrophin complex;  NOSI is activated by influx of calcium through voltage-dependent calcium channels, as well as from the sarcoplasmic reticulum. The therapeutic manipulation of cGMP levels for treatment of erectile dysfunction  cGMP levels - dependent on formation by guanylate cyclase and degradation by phosphodiesterases (PDEs) - hydrolyze to 5' –nucleotides.  The inhibition of PDE destruction of cyclic nucleotides would prolong the physiological response elicited by the cyclic nucleotides.  The therapeutic manipulation of cGMP levels for treatment of erectile dysfunction  Sildenafil (Viagra) - a drug prescribed for erectile dysfunction in males;  Potent inhibitor of PDE5 - specific for cGMP and is found primarily in the human corpus cavernosum and in vascular and gastrointestinal smooth muscle;  As the cGMP is degraded by PDE5, the vasodilatory signal attenuates and the erection subsides. The therapeutic manipulation of cGMP levels for treatment of erectile dysfunction  Sildenafil is highly selective for PDE5 over PDE3, a cAMP specific PDE involved in the regulation of cardiac contractility;  This is important because inhibition of PDE3 increases the incidence of cardiac arrhythmias and decreases long-term mortality in heart failure patients;  Sildenafil also inhibits - PDE6 - photoreceptors in the retina, and one of the side effects sometimes reported with Sildenafil use is a blue-green tint in the patient's vision.  The therapeutic manipulation of cGMP levels for treatment of erectile dysfunction  Because PDE5 is present in vascular smooch muscle, Sildenafil causes a mild, clinically insignificant reduction in blood pressure due to vasodilation;  Greatly exacerbates the effects of nitrates and external NO donors such as nitroglycerin - taken for angina, causing potentially fatal hypotension;  Occur regardless of the sequence and/or timing of the drugs;  Sildenafil is therefore contraindicated in patients using nitrates in any form.  Sildenafil is metabolized by both the CYP2C9 and 3A4 pathways;  Inhibition of these pathways by other drugs may increase the plasma concentration of Sildenafil, intensifying side effects.  NOSII  NOSII is found primarily in activated neutrophils and macrophages, astrocytes and hepatocytes;  Involved in the early immune response;  It is a soluble enzyme;  Induced at the transcriptional level by cytokines - IFN-gama, IL1, TNF-a and endotoxins, such as lipopolysaccharides;  Induction occurs over several hours via the inflammatory NFKB pathway.  Calmodulin is bound to NOSII under basal physiological conditions- independent of calcium influx, so the enzyme is always activated once it is synthesized;  The amount of NO synthesized by NOSII is in the nanomolar range, approximately 1000 times greater than that of either NOSI or NOSIII. NO and immune system  The NO produced by NOSII is a potent cytotoxin;  Role - destroy pathogens engulfed by neutrophils and macrophages;  NOSII controls intracellular pathogens as parasites - plasmodia (malaria), schistosoma (schisrosomiasis), leishmania (leishmaniasis), and toxoplasma (toxoplasmosis), as well as microbials - bacteria and mycobacteria (tuberculosis and leprosy), fungi, and even tumor cells.  NOSII  The cellular targets for the NO produced by NOSII are metal-containing heme proteins, such as cytochromes P450 and iron-sulfur proteins, such as aconitase and mitochondrial complexes I and II, all of which are inhibited.  NO also reacts with thiol groups and tyrosines, causing nitrosation or oxidation of proteins and with oxygen or superoxide, forming highly reactive nitrogen oxide compounds – peroxynitrite;  These reactive intermediates cause a variety of DNA alterations including strand cleavage and deamination;  Lead to cytostatic or cytotoxic effects. NO and immune system  In addition to being produced during acute inflammation, NO is generated by NOSII during chronic inflammation;  NOSII is present and active in a number of chronic inflammatory conditions including rheumatoid arthritis, Crohn disease, asthma;  It may potentially exacerbate some situations, although protective effects have also been reported. Nitric Oxide Overproduction in Septic Shock  Overproduction of NO by NOSII has been implicated in septic/cytokine-induced circulatory shock;  Septic shock is characterized by a systemic inflammatory response to microbial infection in which blood pressure drops precipitously, causing decreased tissue perfusion and oxygen delivery and, eventually, multiple organ failure;  Hypotension in these patients is often refractory to conventional vasoconstrictor drugs. Nitric Oxide Overproduction in Septic Shock  In septic shock - production of too much NO leads to massive systemic vasodilation and fatal drop in blood pressure - multiple organ failure;  Septic shock is associated with an extremely high mortality rate of 50%-70%; Nitric Oxide Overproduction in Septic Shock  NO-induced hypotension in patients occurs through the mechanism for smooth muscle relaxation - activation of guanylate cyclase;  Therapeutic intervention by NOS inhibitors has been investigated for treatment of septic shock;  Although NOS inhibition successfully and rapidly raises blood pressure and systemic vascular resistance, it also led to a progressive fall in cardiac output and exacerbated organ dysfunction, leading to increased mortality in clinical trials;  NO may also play a beneficial role in survival , due to localized vasodilation or the antiplatelet aggregation,antileukocyte adhesion, antiapoptotic or antioxidative properties of NO.  NOSIII  NOSIII is found primarily in vascular endothelial cells - line all blood vessels and cardiac myocytes;  It is localized to the membrane;  Like NOSI , it is constitutively expressed and is activated by the influx of calcium - binds to calmodulin facilitating its binding to both NOSI and NOSIII for activation;  The amount of NO synthesized in the activated state is very low, picomolar;  NO produced serves as a vasodilator of vascular smooth muscle, both ligand mediated and flow dependent;  The NO also serves antithrombotic and anti- inflammatory functions by inhibiting platelet and leukocyte adhesion and aggregation;  In addition, NO also inhibits angiotensin II, a vasoconstrictor. NO synthesis by NOSIII  Activated in response to an increase in calcium following the binding of ligands such as acetylcholine, bradykinin, histamine, insulin or following shear stress, the mechanical force of blood flow on the luminal surface of the vascular endothelium;  Increased blood flow velocity thus stimulates calcium release and increased NOSIII activity, leading to vasodilation.  NO diffuses out of the endothelial cell and into adjacent smooth muscle cells where it binds to and activates soluble guanylate cyclase - makes cGMP;  NO synthesis by NOSIII  cGMP activates protein kinase G - phosphorylates a variety of channels, including L-type calcium channels and receptors, all leading to inhibition of calcium influx into the smooth muscle cell;  Vasoconstriction in vascular smooth muscle requires the influx of calcium - binds to calmodulin - activates muscle light chain kinase (MLCK);  MLCK phosphorylates myosin light chain (MLC) - leading to cross bridge formation between the myosin heads and the actin filament - resulting in contraction. NO synthesis by NOSIII  Inhibition of calcium influx leads to decreased calmodulin stimulation of MLCK and decreased phosphorylation of MLC – diminished development of smooth muscle tension and vasodilacion;  Increased cGMP also directly causes MLC dephosphorylation by activation of MLC phosphatase;  As with NOSI, cGMP levels are regulated by a balance between guanylate cyclase activity and the phosphodiesterases;  Several therapeutic treatments are based on the administration of NO , either directly or in a precursor form.  Biological Effects of Nitroglycerin  Nitroglycerin - more correctly called glyceryl trinitrate (GTN);  Alleviate the pain of angina pectoris - dilation of the coronary blood vessels;  The mitochondrial aldehyde dehydrogenase (ALDH2) metabolizes GTN to 1,2-giycerol dinitrite and nitrite;  The nitrite is further reduced to form NO. Biological Effects of Nitroglycerin  When used over a long period of time for chronic conditions - GTN gradually loses its efficacy;  Several mechanisms have been proposed to explain patient tolerance to this drug:  Impaired biotransformation of GTN by ALDH2 - occurs on depletion of an ALDH2 reductant;  An increase in oxidative stress due to impaired mitochondrial metabolism. Therapeutic Uses of Inhaled Nitric Oxide  Systemically delivered nitric oxide causes systemic hypotension;  NO- inhaled theoretically relax the pulmonary vasculature, but any excess NO reached the bloodstream would be scavenged by oxy- hemoglobin - preventing systemic vasodilation;  Studies have shown - NO inhalation increased the systemic oxygenation of hypoxemic newborns with persistent pulmonary hypertension of the newborn (PPHN);  Inhaled NO also decreased the incidence and mortality of bronchopulmonary dysplasia (BPD) in premature infants;  In adults, chronic obstructive pulmonary disease (COPD) has been treated with a combination of supplemental oxygen and NO;  Reduced pulmonary arterial pressure and pulmonary vascular resistance and increased cardiac output in treated patients. Therapeutic Uses of Inhaled Nitric Oxide  Many patients do not respond to inhaled NO therapy;  The effects of NO are transient - requiring continuous therapy;  Although inhaled NO has no effect on systemic blood pressure, other systemic effects have been reported - platelet and leukocyte inhibition;  Result in decreased thrombosis and reduced ischemia/reperfusion injury;  It is believed that these systemic effects are mediated by blood- borne low- and high-molecular-weight thiols, which react with NO and transport it in a stable form throughout the body;  S-nitrosylated hemoglobin and serum albumin, as well as other nitrosamines, iron-nitrosyls and nitrated lipids are proposed mediators.  NOSIII  NOSIII - unique N-terminal sequence - localizing the enzyme to the membrane - targeted to plasmalemma caveolae;  The caveolae - small invaginations of the plasma membrane, characterized by the presence of proteins called caveolins;  Serve to organize and attach signaling molecules such as receptors G-proteins and NOS to plasma  eNOS localizes to plasma membrane membranes; caveolae, where it directly binds to Cav1.  At low cytoplasmic calcium concentrations -  This interaction inhibits basal eNOS caveolin-1 binds to and inhibits NOSIII, maintaining it activity and NO synthesis; in an inactive state;  Increase in Ca2+ facilitate activation of  On calcium influx - calmodulin competitively CaM - recruited to eNOS and promotes displaces the caveolin from NOSIII , resulting in dissociation of the enzyme from activation of NOS synthesis. Cav1.  Binding of CaM to free eNOS increases its enzymatic activity.  NOSIII  The cationic amino acid transporter CAT-1 - present in caveolae - involved in the uptake of L-arginine - ensuring a supply of substrate for NO synthesis;  NOSIII interacts with porin, a voltage-dependent anion/cation channel -localizes NOSIII near a source of calcium influx;  The bradykinin B2 receptor is also present - it binds and inactivates NOSIII;  On stimulation with bradykinin - this complex dissociates and NOSIII becomes activated;  If palmitoylation is inhibited - NOSIII is not found in the caveolae and synthesis of NO does not occur;  Palmitoylation is a reversible process - influenced by some agonists Palmitoylation is aand type is of essential for membrane post-translational modification localization. where a fatty acid called palmitic acid (a 16-carbon saturated fatty acid) is covalently attached to specific cysteine residues of proteins through a thioester bond.  The Role of eNOS in Endothelial Dysfunction  NO - eNOS - the vascular endothelial cells is responsible for regulation of vascular tone - inhibition of platelet and leukocyte aggregation/adhesion;  Decrease expression of pro-inflammatory genes and limit vascular smooth muscle cell proliferation;  Endothelial dysfunction - characterized by reduced NO synthesis leading to increased vasospasm, vascular inflammation and thrombosis. The Role of eNOS in Endothelial Dysfunction Concomitant with other cardiovascular risk factors, such as diabetes, hypertension, atherosclerosis, hypercholesterinemia.  The Role of eNOS in Endothelial Dysfunction  Vascular oxidative stress - increased production of radical oxygen species, superoxide, contributes to endothelial dysfunction;  Reaction of superoxide with NO - form peroxynitrite;  Powerful oxidant and nitrating agent - damage protein, DNA, lipid molecules;  Covalent nitration of tyrosine residues in proteins by reactive oxygen species is often used as a marker of oxidative stress;  Peroxynitrite - responsible for depletion of tetrahydrobiopterin, which leads to uncoupling of eNOS electron transfer, causing eNOS to produce superoxide rather than nitric oxide;  Proinflammatory and proatherosclerotic pathways are activated. The Role of eNOS in Endothelial Dysfunction  Treatment with antioxidant drugs - vitamin C or E, does not restore bioavailable NO levels, nor does it improve cardiovascular outcome;  Perhaps because the vitamins do not scavenge the superoxide before it reacts with NO;  One of the most effective class of drugs - statins, which function both by:  decreasing oxidative stress, via inhibition of NADPH oxidase activity, a prominent source of superoxide production;  increasing NO production by eNOS, via both protein expression and phosphorylation;  Angiotensin converting-enzyme (ACE) inhibitors and angiotensin II receptor agonists also effectively improve cardiovascular outcome;  These drugs decrease the formation/action of angiotensin II, a protein that activates the NADPH oxidases, thus reducing oxidative stress. Thank you for Attention! Lecturer: Salome Abechkhrishvili