Microsoft PowerPoint - 1-7-LECTURE-Nitric oxideUgly Dr. Christiaan Leeuwenburgh
Outline
• Sources and Types of Nitric Oxide • Chemistry of Nitric Oxide • Nitric Oxide and Aging • Nitric Oxide, Endothelial Dysfunction,
and Atheriosclerosis
-Homodimer, MW = 135-150kD/monomer
-Each monomer has two domains for activity 1) reductase domain and 2) oxygenase
domain
Nitric oxide synthase isoenzymes
NOSNOS endothelial (endothelial (eNOSeNOS, NOS, NOS--III) III)
NOSNOS mitochondrial (mitochondrial (mNOSmNOS))
2. Memory Formation
3. Penile Erection
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to form ONOO-.
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Physiology of Nitric Oxide
• Endothelial nitric oxide synthase (eNOS) catalyzes the synthesis of •NO from L- arginine.
L-arginine + NADPH + O2
Endothelial Function
• The endothelium is actively involved in the control of vascular tone and homeostasis.
• In response to various stimuli endothelial cells release both vasodilating and vasoconstricting substances.
Physiology of Nitric OxidePhysiology of Nitric Oxide
NOS
+
hyperpolarizing factor (EDHF)
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to form ONOO-. This may deplete NO
N=O + O-O:- O=N-O-O:- .... ....
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• NO• reacts very rapidly with the superoxide anion (O2
•-) to form peroxynitrite (ONOO-) • Superoxide dismutase (SOD) is an endogenous
antioxidant enzyme that can scavenge O2 •-
before this reaction takes place • However, there appears to be a relative deficit
in extracellular (EC) SOD in patients with endothelial dysfunction, possibly further depleting NO•
Inactivation of •NO by Reactive Oxygen Species
• Markers of oxidative stress are elevated in in patients with ED.
• Acute and chronic treatment with vitamin C normalizes flow-dependent dilation in HF.
• Pretreatment with exogenous SOD normalizes the vascular response to Ach in the aortas of rats with HF.
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Structures of L-Arginine and its methylated derivatives
• Nitric oxide synthase generates NO from the amino acid L-arginine. Synthesis of NO leads to vasodilation essential for regulation of blood pressure.
• NG-monomethyl-L-arginine (NMMA) is a competitivecompetitive inhibitor of NO synthase that causes endothelium- dependent vasoconstriction.
• Methylated L-arginine derivatives, including NG- monomethyl-L-arginine, asymmetric dimethylarginine, an inhibitor of NO synthase, and symmetric dimethylamine, its inactive isomer, are present in human plasma in urine.
• The inhibition of NO synthase by these compounds could explain the hypertension and white blood cell dysfunction that occur in acute renal failureacute renal failure.
PhysiologyPhysiology • Receptor activation of vascular endothelium by
bradykinin bradykinin or or acetlycholineacetlycholine, and shear stressshear stress causes an uptake of calciumcalcium. L-arginine is converted to NO by NOS, thus increasing the concentration of NO in the vasculature. NO diffuses into smooth-muscle cells and activates sGC.
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Pathophysiology
Pathophysiology
• The inductioninduction of nitric oxide synthase (independent of calcium) resulting from cytokines attaching to their receptors on endothelial and smooth-muscle cells is inhibited by glucocorticoids.
• Once NOS is induced, it produces NO continuously. This results in: – sustained activation of sGC – prolonged relaxation of smooth-muscle cells – decreased responsiveness to vasoconstrictors – possibly damage to tissues
EndotoxinsEndotoxins VasocontrictorVasocontrictor agentsagents such as such as angiotensin angiotensin IIII and and tromboxane tromboxane A2A2
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Vascular Changes during Septic Shock and the Effect of NG-Monomethyl-L-
Arginine. • Exposure to lipopolysaccharide, an endotoxin, causes an
increase in both vasodilators (nitric oxide; internal internal arrowsarrows) and vasoconstrictors (sympathetic agonists, angiotensin II, thromboxane A2, vasopressin; external external arrowsarrows).
• Eventually, the level of nitric oxide becomes high enough to overpower the vasoconstrictor influences, causing the blood pressure to fall.
• A low dose of NG-monomethyl-L-arginine can bring blood pressure back to the normal level.
• The synthesis of NO is totally destroyed by a high dose of NG-monomethyl-L-arginine. End-organ damage and death can result from the influences of the unopposed vasocontrictors.
PATHOLOGICAL IMPLICATIONS OF REACTIVE NITROGEN SPECIES
1. Effects tyrosine phosphorylation (Martin et al. 1990) 2. Inhibits binding of HDL to liver plasma membrane (Chacko, 1985) 3. Peroxynitrate modification of LDL leads to recognition by the
macrophage scavenger receptor. (Graham et al., FEBS:330, 181-185, 1993.) 4. Peroxynitrate rapidly depletes antioxidants from LDL. (A. v. d. Vliet
et al. Biochem. J. 303, 295-301, 1995.)
DISEASES ASSOCIATED WITH REACTIVE NITROGEN SPECIES
1. Cancer; nitration of polycyclic aromatic structures - mutagenic - 2. Chronic Inflammation 3. Atheriosclerosis 4. Alzheimer’s disease and Aging?
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Enhanced peroxynitrite formation is associated with
vascular aging. • van der Loo B, Labugger R, Skepper JN, Bachschmid M,
Kilo J, Powell JM, Palacios-Callender M, Erusalimsky JD, Quaschning T, Malinski T, Gygi D, Ullrich V, Luscher TF.
• J Exp Med 2000 Dec 18;192(12):1731-44.
• Reduction in NO release from Aorta • Increase in iNOS and eNOS • Increase in superoxide production • Increase in Nitrotyrosine
O2 •- + NO• → ONOO- + H+⇔ ONOOH → NO2
–
- + metHb
CO2 + ONOO- → NO2 • + CO3
2GSH + H2O2→ → GSSG + 2H2O
GPx
• Leeuwenburgh C, Hansen P, Shaish A, Holloszy JO, Heinecke JW.
• Am J Physiol 1998 Feb;274(2 Pt 2):R453-61
• Markers of protein oxidation by hydroxyl radical and reactive nitrogen species in tissues of aging rats.
Nitration of Proteins Good or Bad or ..Ugly
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Effect of age on the levels of 3-nitrotyrosine in; (A) the liver of young, midaged, and old mice on an ad libitum diet or caloric restricted (cr) diet, (B) in liver of young and old rats. Ad libitum and caloric restricted mice were sacrificed at 4 months (young; n = 5) and 14 months of age (midage; n = 10, ad libitum n=5; cr n= 5) and 28-30 months (very old n= 10 ad libitum n=5; cr n= 5). Rats were sacrificed at 9 months (young; n = 5) and 24 months of age (old; n = 6).
Zainal TA, Oberley TD, Allison DB, Szweda LI, Weindruch R.
FASEB J 2000 Sep;14(12):1825-36
Caloric restriction of rhesus monkeys lowers oxidative damage in skeletal muscle.
Using a polyclonal antibody raised against NT, skeletal muscle from both a 4-year-old (Fig. 5A) and a 26-year-old (Fig. 5B) animal gave similar moderate antibody reactions.
17-year-old CR animal (Fig. 5C) was less than that observed in a normally fed animal of the same age (Fig. 5D).
Conclusion • Our immunogold light and EM in 3-nitrotyrosine
results are consistent with a report (50) describing no accumulation of nitrotyrosine adducts with advancing age. Using isotope dilution gas chromatography- mass spectroscopy, Leeuwenburgh et al. (50) observed no change in 3-nitrotyrosine levels when comparing vastus lateralis from rats aged 9 and 24 months. These authors speculated that their analysis of whole tissue homogenates yielded an average value for all proteins, thereby failing to detect a possible selective increase in the oxidation of specific proteins.
Is there evidence that Nitration alters SERCA2a isoform of the sarcoplasmic
reticulum Ca2+-ATPase in skeletal muscle • Viner RI, Ferrington DA, Williams TD,
Bigelow DJ, Schoneich C.
• Protein modification during biological aging: selective tyrosine nitration of the SERCA2a isoform of the sarcoplasmic reticulum Ca2+-ATPase in skeletal muscle.
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• Ferrini M, Magee TR, Vernet D, Rajfer J, Gonzalez-Cadavid NF.
• Aging-related expression of inducible nitric oxide synthase and markers of tissue damage in the rat penis.
• Biol Reprod 2001 Mar;64(3):974-82 • young (3-5 mo old) and old (24-30 mo old) rats • 4.1-fold increase in iNOS mRNA and Protein • 3.6-fold increase in apoptotic cells • 1.6-fold increase in nitrotyrosine
GSHGSH