Nitric Oxide in Physiology and Pathophysiology

Nitric Oxide in Physiology and Pathophysiology
Nitric oxide (NO)

• 
  exhibits an enormous range of important functions in the organism
  

• 
  is synthesized from the substrate L-arginine by the catalyzed reaction by NO-synthases (NOS) leading to the generation of NO and L-citrulline.
  

Isoforms of NOS

• 
  the neuronal type I isoform (nNOS)
  

• 
  the inducible type II isoform (iNOS)
  

• 
  the endothelial type III isoform (eNOS)
  

• 
  a new mitochondrial isoform (mtNOS)
  

Constitutively expressed enzymes

• 
  nNOS and eNOS are constitutively expressed enzymes, that are activated by increased intracellular concentrations of Ca²⁺. Ca²⁺ binds to calmodulin and a complex of Ca²⁺/calmodulin activates nNOS or eNOS to produce NO. Physiologically low levels of NO (pM) produced by nNOS or eNOS mediate the effects namely by activation of guanylate cyclase, causing an increase of cGMP.
  

• 
  mtNOS is also a calcium-dependent and its relevance for mitochondrial bioenergetics was reported mtNOS stimulation by loading mitochondria with calcium, was found to cause mitochondrial matrix acidification and a drop in mitochondrial transmembrane potential
  

Inducible isoform

• 
  iNOS always contains tightly bound calmodulin
  
• 
  transcription of the iNOS gene is controlled
  
  • 
    positive inducers (IFNγ, TNFα, IL-1, IL-2, antigens (from both gram-negative and gram positive bacteria, tumor cells and heterologous antigens involved in transplantation immunity)
    
  • 
    inhibitory cytokines include TGF-β, IL-4, IL-10, and macrophage deactivating factor (MDF).
    

R
e^-(z NADPH)
eactions catalyzed by NO-synthase

NOS (BH4)
+BH₄ Arginin --> NO^. Arginin + O₂ NO^. + citrulin

e^-(z NADPH)


NOS
-BH₄ O₂ --> O₂^-. O₂ O₂^-.

-arginin

Inhibitors of NOS

• 
  arginin analogues – endogenous - ADMA (asymetric dimethylarginine)
  

exogenous - N-monomethylarginine, thiocitrulin, 1400W

• 
  scavengers endogenous - GSH, homocystein, hemoglobin,
  

superoxide, lipoperoxides in atheromas

exogenous - N-acetylcystein

The effects of NO

• 
  vasodilatation (EDRF), relaxation of muscles
  
• 
  neurotransmission, neuromediation
  
• 
  host defense reactions against bacteria and fungi; cytotoxicity, tumoricidal effect; contradictory roles in viral infection
  

Effects of NO

1. 
   cGMP-dependent
   

1. 
   Relaxation of smooth muscles
   

NANC Organic Nitrates





Receptor Endotel Smooth muscles


acetylcholine (M3) PLC incres. cGMP


histamin increasing Ca²⁺_i PKG

bradykinine eNOS-CaM
ADP, ATP phosphorylation of

Ca, K channels


Relaxation of

smooth muscles

2. 
   Inhibition of adhesion and agregation of platelets
   

NO produced by endothelial cells – activation of COX-PG; NO produced by platelets – negative feed-back during

activation

inhibition-TXA2
peroxynitrate(ONOO^- ) – activation of platelets

3. 
   Neurotransmission
   

glutamate-NMDA receptor on post-synaptic neuron-increasing Ca²⁺_i - activation of nNOS-CaM- production of NO-increasing cGMP- difuse to pre-synaptic neuron

CNS (long-term potentiation, depresion), PNS, Sensoric neurons

2. 
   cGMP-independent effects
   
   1. 
      Inhibition of DNA synthesis
      

ribonucleotide reductase NDP-I-->dNDP

2. 
   Inhibition of energeric metabolism of cell
   

mitochondrial cis-aconitase (Krebs pathway)

enzymes of respiratory chain (ATP synthase)

glyceraldehyde 3-phosphate dehydrogenase (glycolysis)

poly(ADP-ribose) polymerase-PARP (consumption of NAD⁺, ATP)

3. 
   Peroxynitrite
   
4. 
   Regulation of ferrum metabolism
   

IRP-1 (Iron regulatory Protein-1; cytoplasmic cis-aconitase)

decrease of Fe-increase of NO-release of Fe-S- binding to:

3´ mRNA for transferine receptor- stabilisation mRNA, increasing

translation, increasing Fe delivery

5´ mRNA for ferritine, ery ALA-S- inhibition of translation-Fe is used as

a cofactor

Nitric oxide and mitochondria

• 
  NO blocks cytochrom c oxidase by binding to its heme group and leading to
  

increased generation of superoxide in mitochondrial respiratory chain

• 
  superoxide can react with NO to yield peroxynitrite. NO or peroxynitrite inhibit respiratory chain complexes I, III, and IV, and activity of cis-aconitase, an enzyme of the tricarboxylic acid cycle, by binding to the iron-sulfur centers. Inhibition of cis-aconitase blocks metabolism of acetyl coenzyme A to carbon dioxide, an important step in generating the nicotinamide-adenine dinucleotide (NADH), which is necessary to drive oxidative phosphorylation. These effects severely impair the cell ability to maintain its pool of ATP.
  
• 
  peroxynitrite decomposes into hydroxyl radical which is the most reactive species known to cause most of cell and tissue damage in inflammation. (triggers lipid peroxidation, DNA mutations or protein modification, reveals apoptotic and cytotoxic effects in various cells. The other product of decomposition of peroxynitrite is NO₂˙ radical that causes nitration of tyrosine residues of proteins
  

Nitric oxide and virus infection

• 
  antiviral action against a ectromelia murine poxvirus, herpes simplex virus 1,
  

and VV blocked by NO at the level of DNA synthesis and it could be partially

rescued by providing deoxyribonucleosides, suggesting that NO caused

inhibition of viral ribonucleotide reductase

- stimulatory effects of NO on HIV-1 and VV replication have been observed

Nitric oxide and apoptosis

• 
  depending on its concentration, flux and the cell type
  
• 
  activates the transduction pathways leading to apoptosis. Pro-apoptotic effects are often observed when NO reacts with superoxide to produce the highly toxic peroxynitrite,
  
• 
  protects cells against spontaneous or induced apoptosis. NO inactivates caspases through oxidation and S-nitrosylation of the active cystein, stimulation of cGMP-dependent protein kinase, modulation of Bcl-2/Bax family, induction of heat shock protein Hsp 70 and interaction with ceramide pathway The redox state of the cells appears to be a crucial parameter for the determination of the ultimate effects of NO on cell multiplication and survival.
  

Nitric oxide in different diseases

Molecular aspects of pathogenesis in osteoarthritis (OA)

• 
  OA is defined as a noninflammatory arthropathy, proinflammatory cytokines such as interleukin-1 have been implicated as important mediators in the disease. In response to interleukin-1, chondrocytes upregulate the production of NO and prostaglandin E2, two factors that have been shown to induce a number of the cellular changes associated with OA
  

Osteoporosis

• 
  estrogen, statins, and essential fatty acids and their metabolites can prevent osteoporosis. They have the ability to augment constitutional (or endothelial) nitric oxide generation, which is known to be beneficial in osteoporosis
  

Inflammatory myopathies

• 
  reactive oxygen intermediates (ROI) and NO are produced in abundance in the inflammatory muscle diseases of autoimmune origin polymyositis (PM), dermatomyositis (DM), and inclusion body myositis (IBM).
  
• 
  NO released at low concentrations at target sites may even have cell-protective effects. A major mechanism of protection from apoptosis in both myocytes and inflammatory cells seems to be the upregulation of anti-apoptotic proteins like Bcl-2.
  

NOS/NO system assembly in neuromuscular junction formation

• 
  postsynaptic membrane - multi-subunit dystrophin-protein complex (DPC) and
  

its associated nitric oxide (NO)-signaling complex.

• 
  codistribution of neuronal NOS (nNOS) with known synaptic proteins, i.e.,
  

family members of the DPC, nicotinic acetylcholine receptor (AChR), NMDA-

receptor, type-1 sodium and Shaker K(+)-channel proteins, and linker proteins

(e.g., PSD- 95, 43K-rapsyn)

• 
  NO mediates agrin-induced AChR-aggregation and downstream signal
  

transduction in C2 skeletal myotubes while administration of L-arginine, the

limiting substrate for NO-biosynthesis, enhances aggregation of synapse-

specific components such as utrophin.

- early synaptic protein clustering, synaptic receptor activity and transmitter release, or downstream signaling for transcriptional control.

Changes of the brain synapses during aging

• 
  the role of nitric oxide, free radicals and apoptosis, impaired cerebral microcirculation, metabolic features of aging brain, the possible neuroprotective role of insulin-like growth factor-1 (IGF-1) and ovarian steroids, and stress and aging
  

Disorders of ammonia metabolism

• 
  urea cycle enzymopathies, Reye Syndrome, and liver failure are associated with brain edema and severe neurological impairment
  
• 
  excess blood-borne ammonia crosses the blood-brain barrier by diffusion as NH(3)
  
• 
  ammonia exerts a potent effect on glutamate (AMPA) receptor-mediated neurotransmission.
  
• 
  ammonia also inhibits high affinity transport of glutamate by an action on astrocytic glutamate transporter expression, an action which results in increased extracellular concentrations of glutamate
  
• 
  acute hyperammonemia directly activates the NMDA subclass of glutamate receptors resulting in increased intracellular Ca(2+) and increased synthesis of NO and cGMP; toxicity results in depletion of ATP in brain.
  
• 
  chronic hyperammonemia - results in a loss of NMDA receptor sites
  

Pathogenesis of portal hypertensive gastropathy

• 
  elevated portal pressure can induce changes of local hemodynamics, thus causing congestion in the upper stomach and gastric tissue damage. These changes may then activate cytokines and growth factors, such as TNF-α which are substances that activate eNOS and endothelin 1 in the portal hypertensive gastric mucosa. Overexpressed eNOS produces an excess of nitric oxide, which induces hyperdynamic circulation and peroxynitrite overproduction.
  

Pulmonary hypertension

• 
  mutations of the bone morphogenetic protein receptor 2 (BMPR2) gene, a member of the TGF-β receptor family
  
• 
  genetic predisposition might dictate the responses of pulmonary artery fibroblasts, smooth muscle cells, and endothelial cells, as well as platelets and leukocytes
  
• 
  therapy: diuretics, anticoagulants, prostaglandins, high-dose calcium-channel blockers, long-term intravenous prostacyclin infusion, inhaled of the prostacyclin analog iloprost, inhaled NO
  

Heart failure

• 
  ischemic and nonischemic heart failure, septic cardiomyopathy, cardiac allograft rejection, and myocarditis
  
• 
  regulation of myocardial contractility, distensibility, heart rate, coronary vasodilation, myocardial oxygen consumption, mitochondrial respiration, and apoptosis
  
• 
  promote left ventricular mechanical efficiency (appropriate matching between cardiac work and myocardial oxygen consumption)
  
• 
  beneficial effects are attributed to the low physiologic concentrations generated by the eNOS or nNOS
  
• 
  iNOS generates larger concentrations of NO over longer periods of time, leading to mostly detrimental effects. In addition, the recently identified beta3-adrenoceptor mediates a negative inotropic effect through coupling to endothelial nitric oxide synthase and is overexpressed in heart failure. An imbalance between beta 1 and beta2-adrenoceptor and beta3-adrenoceptor, with a prevailing influence of beta3-adrenoceptor, may play a causal role in the pathogenesis of cardiac diseases such as terminal heart failure
  
• 
  changes in the expression of eNOS or iNOS within the myocardium may alter the delicate balance between the effects of NO produced by either of these isoforms. New treatments such as selective iNOS blockade, eNOS promoting therapies, and selective beta3-adrenoceptor modulators
  

Kidney and NO

• 
  nNOS is expressed strongly in the macula densa of the kidney. Functional
  

studies show that it blunts the tubuloglomerular feedback response that

causes vasoconstriction of the renal afferent arteriole in response to sodium

chloride reabsorption at this site, and regulates renin release from the

juxtaglomerular apparatus.

- macula densa-derived NO can act via both the autocrine and probably

the paracrine routes. Nitric oxide bioactivity in the juxtaglomerular apparatus

is strongly curtailed by oxidative stress in hypertensive models

• 
  nNOS adapts renal hemodynamics and possibly renin secretion to changes in blood pressure and salt intake.
  

Sepsis and septic shock

• 
  morbidity and mortality from sepsis-related diseases have remained substantially unchanged (30 - 50%)
  

Distribution of phosphodiesterase (PDE), side-effects of sildenafile (Viagra)

Type PDE

Human – heart 1

v.saphena 1, 4, 5

a.mesenterica 1, 2, 3, 4, 5

platelets 5

corpus caverosum 1, 2, 3, 4, 5

Rabbit - aorta

platelets

Dog - coronary aa.

trabecullar tissue

Hypertension essential

experimental blood

polycythaemia

Hypotension septic shock

anemia

Arterial spasm atheromas

heart attack

acute renal failure

Coagulopathy trombosis

by-pass

DIC

Erectile dysfunction

Pulmonary hypertension newborns

ARDS

Bronchoconstriction asthma bronchiale

Megacolon congenitum

Portal hypertension portocaval shunt

hypercinetic circulation

coagulopathy

hepatal encephalopathy

Pre-eclampsia

Reperfussion damage ischemic damage of CNS

heart attack

Neurodegenerative disorders mitochondrial dysfunction

Parkinson d.

Huntington d.

Friedrich ataxia

Amyotrofic lateral sclerosis

Alzheimer d.

AIDS dementia

Inflammation local vasodilatation

bacteriaemia, sepsis

myocarditis, encephalitis, meningitis

Autoimmune disease IDDM, RA, colitis ulcerosa