Nitric Oxide Functions
NO is a potent signaling molecule, a key determinant of endothelial function, metabolic and vascular health, also affecting the nervous and immune systems. Protective effects occur at pico- to nanomolar NO concentrations. At higher concentrations, NO and its derivatives become cytotoxic.
Mitochondria
NO effects on mitochondria have considerable implications for cell physiology and cell death. Mitochondria are primary cellular targets for NO.
mtNOS is linked to mitochondria at several sites of the mitochondrial electron transport chain (ETC), most notably at Complex I (NADH dehydrogenase) [
11] and Complex IV (cytochrome c oxidase, CcOX) [
12].
mtNOS is highly activated by activation of the ETC and Complex I, which serves as its source of electrons to produce NO. Conversely, inactivation of Complex I terminates normal mtNOS activity [
11].
Metabolism
mtNOS-derived NO effectively controls mitochondrial respiration, O2 consumption, transmembrane proton gradient and potential and adenosine triphosphate (ATP) synthesis [
12].
Acutely, NO
reduces mitochondrial oxidative metabolism [
13]:
(1) Physiologic NO levels acutely and reversibly bind to and inhibit several ETC complexes, the most sensitive target being Complex IV [
12]. The result is a transient NO-induced reduction of mitochondrial respiration with partial mitochondrial membrane depolarization [
14]. Since mtNOS derives its electrons from Complex I, there is reciprocal regulation between mtNOS and the mitochondrial ETC [
11].
(2) Very high NO levels, generated upon inflammatory iNOS induction, compete with O2, engendering NO-dependent hypoxia (‘nitroxia’) [
15]. Nitroxia promotes the generation of high levels of reactive oxygen species (ROS)/reactive nitrogen species (RNS) [
12]. NO/RNS can then shut down mitochondrial respiration at multiple sites by irreversibly inhibiting ETC complexes at the expense of ATP production, with cytotoxic effect [
16].
Chronically, NO
increases cellular oxidative metabolism [
13]:
(1) NO-guanylate cyclase signaling increases mitochondrial biogenesis in diverse cell types. NO increases sirtuin-1 expression [
17], and, with 5′-AMP-activated protein kinase (AMPK)-α1, synergistically upregulates peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, a master regulator of mitochondriogenesis [
13].
ATP formation via mitochondrial oxidative phosphorylation increases in association with the NO/cGMP-stimulated increase in mitochondrial content [
13] in a variety of tissues.
(2) NO modulates mitochondrial content and total-body energy balance in response to physiological stimuli, such as exercise or cold exposure, functioning as a unifying molecular switch to trigger the entire mitochondriogenic process [
13].
Reactive Oxygen Species
Mitochondria are the main intracellular source of ROS. Normal oxidative phosphorylation continually produces low ROS/RNS levels, as several ETC redox centers leak electrons to partially reduce O2 to the superoxide anion [
18]. Between 0.4 and 4% of O2 consumed is converted to superoxide.
The mitochondrial membrane potential is the principal parameter regulating ROS production [
18]. Since physiologic NO lowers this potential, NO reduces ROS production [
12]. However, disturbed mtNOS function, excessive or deficient NO and dysregulation of NO signaling pathways increase ROS/RNS production while lowering antioxidant levels [
19].
Efficient Mitochondria
Any increase in energy demand is matched by a coordinated rise in oxidative metabolism, which increases mitochondrial membrane potential and thus ROS generation.
It is paradoxical that NO/cGMP signaling decreases oxidative metabolism in any single mitochondrion while increasing cellular mitochondrial function. However, in the process, NO/cGMP renders mitochondria ‘efficient’, with an organized ETC that generates sufficient ATP, while lowering oxygen consumption, mitochondrial potential and ROS production.
The result is of major benefit. Exercise training increases energy demand but also stimulates NO, since NO couples demand with cellular and total-body energy generation [
20]. Instead of the expected ROS increase, oxidative stress is reduced due to ‘efficient’ mitochondria, forestalling ROS-induced cellular aging by protecting the integrity of mitochondria, telomeres or the endoplasmic reticulum.
Mitochondrial Calcium
Mitochondrial energy homeostasis responds to changes in mitochondrial Ca2+. Key mitochondrial enzymes, as in the tricarboxylic acid cycle, are upregulated by higher intramitochondrial Ca2+, enhancing the provision of reducing equivalents to the ETC and increasing mitochondrial potential and ATP generation [
16].
Cytoplasmic Ca2+ signals correspond to higher energy demand from secretory, contractile or other work. Thus, a primary function for mitochondrial Ca2+ uptake appears to be the Ca2+-dependent coordination of mitochondrial energy production with cellular energy consumption.
Ca2+ uptake into mitochondria is partly driven by the mitochondrial membrane potential. Excessive mitochondrial Ca2+ accumulation is implicated in disease.
Lowering the mitochondrial potential limits mitochondrial Ca2+. Such conditions not only lower mitochondrial metabolic activity but also protect against deleterious Ca2+ overload [
16].
NO/cGMP reduces the mitochondrial potential, thereby decreasing mitochondrial Ca2+[
14]. In fact, NO provides negative feedback on mitochondrial Ca2+ uptake: whereas higher mitochondrial Ca2+ activates mtNOS, increasing NO inhibits respiration, lowering the mitochondrial potential and further limiting Ca2+ uptake [
14].
Cell Protection
Ischemic preconditioning provides powerful cardioprotection against myocardial ischemia-reperfusion injury. Physiologic NO levels are involved in cytoprotective effects of early and late preconditioning. Not only eNOS-, but also exogenous nitrate-donor-derived NO can effect endothelial and myocardial cytoprotection [
21].
NO/cGMP may protect against mitochondrial permeability transition and apoptosis induced by manifold insults. Through its interaction with ETC components, such as CcOX, NO affects low-level ROS generation and other mitochondrial defense mechanisms, thereby triggering adaptive cell survival signaling [
15,
21].
Cell Death
High NO concentrations are cytotoxic:
(1) Excessive NO and RNS, such as peroxynitrite, may cause tyrosine nitration of mitochondrial components and play a key role in apoptosis [
19].
(2) NO-derived ROS/RNS signaling, mitochondrial permeability transition or DNA damage may activate mitochondrial pathways to apoptosis or necrosis.
(3) The irreversible inhibition of mitochondrial respiration at multiple sites by excessive NO can inhibit apoptosis and induce necrosis via energy depletion. The ensuing profound mitochondrial failure contributes to the insidious, progressive and fatal end-organ failure of sepsis, associated with signs of accelerated and refractory anaerobic metabolism [
22].
Skeletal Muscle
NO signaling in skeletal muscle is implicated in the control of multiple functions, including
• muscle metabolism,
• excitation-contraction coupling and contractility,
• immune function,
• cell growth and
• neurotransmission.
Metabolically active skeletal muscle is the most abundant tissue, constituting approximately 40% of normal-weight body mass, rendering it a critical factor in total-body metabolism [
23]. Skeletal muscle NOS thus plays a pivotal role in total-body glucose and lipid homeostasis.
Glucose
Higher skeletal muscle NOS expression and activity improve insulin action via NO/cGMP/cGK signaling [
23].
Insulin sensitivity is enhanced
• indirectly as NO increases
– skeletal muscle microvascular perfusion, delivering nutrients and insulin to target tissues [
23],
– antioxidant and anti-inflammatory actions,
– the synthesis of insulin-sensitizing adiponectin;
• directly as NO/cGK blocks the inhibitory interaction of the small GTPase Rho/Rho kinase with insulin receptor substrate (IRS)-1 [
24].
In contrast, excessive proinflammatory iNOS/NO induction impairs myocyte insulin sensitivity via prooxidant pathways.
Glucose uptake and myocyte intracellular energy stores are also stimulated by NO/cGMP/cGK signaling and NOS-derived ROS via mechanisms that are distinct from, but additive to, contraction-, insulin-, AMPK- or p38 MAPK-dependent glucose uptake pathways [
23,
25].
NO stimulates glucose oxidation in skeletal and cardiac muscle, liver and adipose tissue via cGMP-dependent mechanisms.
Fatty Acids
Increased physiologic NO/cGMP signaling enhances fatty acid catabolism [
13]. It accelerates adipocyte lipolysis while stimulating fatty acid oxidation in skeletal and cardiac muscle via AMPK activation and PGC-1α expression [
26].
Oxygen Consumption
NO reduces myocyte energy demand [
23] by
• reducing contractility. NO reduces myofilament Ca2+ sensitivity through nitrosation of target proteins, depressing submaximal and isometric skeletal muscle force, shortening contraction velocity and accelerating relaxation;
• downregulating metabolism. NO lowers glycolysis. It reduces mitochondrial respiration, the breakdown of creatine phosphate and the transfer of high-energy phosphates.
Contractile Dysfunction
Cardiac pump failure is a life-threatening response to severe inflammation in myocarditis, heart transplant rejection, sepsis or trauma. Excessive myocardial iNOS/NO/cGMP/cGK induction has a profound negative inotropic effect [
27] as it
• inhibits aerobic enzymes, including CcOX,
• depresses cAMP levels, thereby reducing Ca2+ influx through L-type Ca2+ channels and
• phosphorylates troponin I, lowering myofilament Ca2+ sensitivity.
Myocyte Loss
Human diseases, ranging from heart failure to cancer, induce skeletal muscle catabolism via proinflammatory induction of excessive iNOS/NO, which impairs myocyte differentiation and is associated with myocyte apoptosis. There is also a significant association between iNOS abundance, cardiomyocyte apoptosis and cardiomyopathy.
Vasculature
Vascular NO is produced by endothelial cells.
Vasodilation
NO is the most potent endogenous vasodilator, predominantly of conduit vessels rather than the microvasculature.
NO/cGMP/cGK signaling accomplishes vasodilation through
• the autocrine increase of NO and BH4 within the endothelium [
9],
• the paracrine relaxation of subjacent vascular smooth muscle cells (VSMCs) by
(1) lowering cytoplasmic Ca2+ concentrations and
(2) reducing myofibrillar Ca2+ sensitivity [
9,
24].
NO mediates flow-mediated vasodilation and opposes vasoconstrictor effects. It counteracts vascular stiffness and lowers blood pressure. NO is a critical modulator of blood flow, vascular tone and blood pressure [
28].
Vascular Repair and Angiogenesis
The endothelium is continuously exposed to mechanical, chemical or ischemic insults. At the site of injuries, bone marrow-derived endothelial stem and progenitor cells (EPCs) participate in repair processes, normalizing endothelial function. NO protects the functional ability of EPCs to participate in vascular repair and angiogenesis [
29].
Inhibition of Platelet Activation
NO inhibits platelet activation, aggregation and adhesion to the endothelium via cGMP-dependent [
9] and -independent mechanisms.
Oxidative Stress
Physiologic NO levels reduce oxidative stress. NO inhibits superoxide production by inactivating NADH/NADPH oxidase. NO increases the endogenous antioxidant potential by inducing endothelial superoxide dismutase (SOD), extracellular SOD in VSMCs, myocardial SOD, mitochondrial S-nitrosoglutathione synthesis [
11] and thioredoxin activity [
30], thus thwarting oxidative NO inactivation. NO inhibits low-density lipoprotein (LDL) oxidation.
In contrast, induction of high levels of NO/iNOS is highly prooxidant. NO, reacting with superoxide, generates the oxidant anion peroxynitrite (ONOO–):
NO + O2–· →ONOO–
Peroxynitrite engenders lipid peroxidation and nitrosation of amino acid residues, disrupting cell membranes, cell signaling and cell survival [
30]. Peroxynitrite also has proinflammatory effects.
Anti-Inflammatory and Antiatherogenic Activities
Physiologic NO levels are anti-inflammatory. By preventing proinflammatory cytokine activation, NO protects blood vessels from endogenous injury, interfering with early and later stages of conduit vessel atherogenesis [
28]. NO
• delays endothelial cell senescence and senescence-related proinflammatory signaling,
• reduces endothelial cell apoptosis,
• inhibits the transcription of nuclear factor-ĸB,
• inhibits redox-sensitive, cytokine-induced vascular cell adhesion molecule-1, intracellular adhesion molecule-1 and monocyte chemoattractant protein-1, preventing leukocyte adhesion to the endothelium,
• decreases endothelial permeability, reducing the influx of oxidized lipoproteins into the vascular wall,
• interferes with leukocyte migration into the vascular wall by decreasing the expression of factors, including the surface adhesion molecules CD11/CD18 and P-selectin,
• powerfully inhibits inflammatory cell activation and monocyte activity,
• blocks VSMC migration,
• thwarts VSMC proliferation,
• inhibits the synthesis and secretion of extracellular matrix proteinases, which degrade extracellular matrix proteins,
• increases the expression of tissue inhibitor of matrix metalloproteinases,
• inhibits transforming growth factor-β/Smad-regulated gene transactivation [
31,
32].
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