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Posts Tagged ‘mitochondrial dysfunction’


Mitochondrial Dysfunction and Cardiac Disorders

Curator: Larry H Bernstein, MD, FACP

This article is the THIRD in a four-article Series covering the topic of the Roles of the Mitochondria in Cardiovascular Diseases. They include the following;

  • Mitochondria and Cardiovascular Disease: A Tribute to Richard Bing, Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/14/chapter-5-mitochondria-and-cardiovascular-disease/

  • Mitochondrial Metabolism and Cardiac Function, Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-metabolism-and-cardiac-function/

  • Mitochondrial Dysfunction and Cardiac Disorders, Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-dysfunction-and-cardiac-disorders/

https://pharmaceuticalintelligence.com/2013/04/14/reversal-of-cardiac-mitochondrial-dysfunction/

Mitochondrial Metabolism in Impaired Cardiac Function

Mitochondrial Dysfunction and the Heart

Chronically elevated plasma free fatty acid levels in heart failure are associated with
  • decreased metabolic efficiency and cellular insulin resistance.
The mitochondrial theory of aging (MTA) and the free-radical theory of aging (FRTA) are closely related.
They were in fact proposed by the same researcher about 20 years apart. MTA adds
  • the mitochondria and its production of free radicals
  • into the concept that free-radicals damage DNA over time.
Tissue hypoxia, resulting from low cardiac output with or independent of endothelial impairment,
This dysfunctional state causes loss of mitochondrial mass. Therapies aimed at protecting mitochondrial function
  • have shown promise in patients and animal models with heart failure that will be the subject of Chapter III.

Myocardial function in hypertension

Genetic variation in vitamin D-dependent signaling
  • is associated with congestive heart failure in human subjects with hypertension.
Functional polymorphisms were selected from five candidate genes:
  1. CYP27B1,
  2. CYP24A1,
  3. VDR,
  4. REN and
  5. ACE.
Using the Marshfield Clinic Personalized Medicine Research Project,
  • 205 subjects with hypertension and congestive heart failure,
  • 206 subjects with hypertension alone and
  • 206 controls (frequency matched by age and gender) were genotyped.
In the context of hypertension, a SNP in CYP27B1 was associated with congestive heart failure
(odds ratio: 2.14 for subjects homozygous for the C allele; 95% CI: 1.05–4.39).
Genetic variation in vitamin D biosynthesis is associated with increased risk of heart failure.
RA Wilke, RU Simpson, BN Mukesh, SV Bhupathi, et al. Genetic variation in CYP27B1 is associated

Heart Failure and Coronary Circulation

There is a decrease in resting and peak stress myocardial function in chronic heart failure patients,
  • with recovery of skeletal muscle phosphocreatine following exercise induced by perhexiline treatment.
This suggested that mitochondrial deficiencies, caused by excessive free fatty acids (FFAs)
  • underlie a common cardiac and skeletal muscle myopathy in heart failure patients.
Tissue hypoxia in chronic heart failure from inadequate circulation in heart failure
  • increases the oxidative stress in lean body mass and in the heart itself.
The heterodimeric transcription factor hypoxia-inducible factor (HIF)-1
  • induces changes in the transcription of genes that encode proteins involved in the adaptation to hypoxia.
HIF-1 activity depends on levels of the HIF-1a subunit, which has a short half-life.
HIF-1a increases in rats with experimentally induced myocardial infarction together with elevated levels of
  • GLUT1 and haemoxygenase-1 in the peri-infarct region of the heart
The cardiac metabolic response to hypoxia is considered to be
  • a return to a pattern of fetal metabolism, in which
  • carbohydrates predominate as substrates for energy metabolism.
The reliance on carbohydrate energy source is thought to be a result of  the downregulation of PPARa with a decreased activity of
The sarcolemmal fatty acid transporter protein (FATP) levels are also decreased with palmitate oxidation,
  • transitioning away from fatty acid metabolism proportional to the degree of cardiac impairment.
The hypoxic changes of heart failure drives a switch toward
  1. glycolysis and glucose oxidation
  2. restriction of myocardial fatty acid uptake.
Nevertheless, late in the progression of heart failure, substrate metabolism is insufficient to support cardiac function, because
  • the hypoxic failing heart is no longer able to oxidize fats and may also be insulin resistant.
The author surmises that mitochondrial dysfunction caused by tissue hypoxia might be mediated by the
  • proapoptotic protein BCL2/adenovirus E1B 19kDa interacting protein (Bnip)3.
It  is strongly upregulated in response to hypoxia. In the isolated, perfused rat heart, Bnip3 expression was
  • induced after 1h of hypoxia, with Bnip3 integrating into the mitochondria of hypoxic ventricular myocytes.
This resulted in mitochondrial defects associated with
  1. opening of the permeability transition pore, leading to
  2. loss of inner membrane integrity and
  3. loss of mitochondrial mass.

Mitochondrial Dysfunction caused by Bnip3 Precedes Cell Death.

Experimentally induced myocardial ischemia had evidence of contractile dysfunction but preserved viability. A progressive
  • decline in circulating levels of endothelial progenitor cells was documented 3 months following instrumentation (P<0.001).
Quantitative polymerase chain reaction analysis revealed that
  • chronic myocardial ischemia produced a biphasic response in both
    • hypoxic-inducible factor 1 and
    • stromal-derived factor 1 mRNA expression.
While initially unregulated, a gradual decline was observed over time (from day 45 to 90), in
  • hypoxic-inducible factor 1 and
  • stromal-derived factor 1 mRNA expression .
On serial assessment, endothelial progenitor cell migration was progressively impaired in response to chemo-attractant gradients of:
  1. vascular endothelial growth factor (10-200 ng/mL)
  2. and stromal cell-derived factor-1 (10-100 ng/mL) .
Decreased circulating levels and migratory dysfunction of bone marrow derived endothelial progenitor cells
  • were documented in a reproducible clinically relevant model of myocardial ischemia.

Nitric Oxide (NO) in Myocardial Ischemia and Infarct

Nitric oxide (NO) is a free radical with an unpaired electron; it is an important physiologic messenger,
  • produced by nitric oxide synthases, which catalyze the reaction l-arginine to citrulline and NO.
The constitutive isoforms exists in neuronal and endothelial cells and is calcium dependent. Calcium binds to calmodulin and
  1. the calcium calmodulin complex activates the constitutive NO synthase that releases NO,
  2. relaxing smooth muscle cells through activation of guanylate cyclase and the production cGMP.
Therefore, the NO produced has a negative inotropic effect on the heart and is instrumental in the autoregulation of the coronary circulation.
The inducible form of NO synthase (iNOS), mostly produced in macrophages, is activated by cytokines and endotoxin. It eliminates intracellular pathogens,
damaging cells by inhibiting
  1. ATP production
  2. oxidative phosphorylation
  3. DNA synthesis.
In infection, lipopolysaccharide released from bacterial walls, stimulates production of iNOS. The large amount of NO produced
  • causes extensive vasodilation and hypotension.
We sought to assess whether oxidation products of
  • nitric oxide (NO), nitrite (NO2−) and nitrate (NO3−), referred to as NOx,
  • are released by the heart of patients after acute myocardial infarction (AMI) and
  • whether NOx can be determined in peripheral blood of these patients.
Previously we reported that in experimental myocardial infarction (rabbits) NOx is released mainly by inflammatory cells
  • (macrophages) in the myocardium 3 days after onset of  ischemia.
NOx is formed in heart muscle from NO; It originates through the activity of the inducible form of nitric oxide synthase (iNOS).
Eight patients with acute anterior MI and an equal number of controls were studied. Coronary venous blood was obtained by
coronary sinus catheterization; NOx concentrations in coronary sinus, in arterial and peripheral venous plasma were measured.
Left ventricular end-diastolic pressure was determined. Measurements were carried out 24, 48 and 72 h after onset of symptoms.
The type and location of coronary arterial lesions were determined by coronary angiography. Plasma NO3− was reduced to NO2−
by nitrate reductase before determination of NO2− concentration by chemiluminescence.
The results provided evidence that in patients with acute anterior MI, the myocardial production of nitrite and nitrate (NOx) was increased,
  • as well as the coronary arterial–venous difference.
Increased NOx production by the infarcted heart accounted for the increase of NOx concentration in arterial and the peripheral venous plasma.
The peak elevation of NOx occurred on days 2 and 3 after onset of the symptoms, suggesting that NOx production was at least in part the result of
  • production of NO by inflammatory cells (macrophages) in the heart.
The appearance of oxidative products of NO (NO2− and NO3−) in peripheral blood of patients with acute MI is
  • the result of their increased release from infarcted heart during the inflammatory phase of myocardial ischemia.
Further studies are needed to define the clinical value of these observations.
K Akiyama,  A Kimura, H Suzuki, Y Takeyama, …. R Bing.  Production of oxidative products of nitric oxide in infarcted human heart.  J Am Coll Cardiol. 1998;32(2):373-379.   http://dx.doi.org/10.1016/S0735-1097(98)00270-8
OPA1 Mutation and Late-Onset Cardiomyopathy
No cardiac disorders have been described in patients with OPA1 or similar mutations
  • involving the fission/fusion genes as seen in inherited maladies like Charcot–Marie–Tooth disease.
Our results indicate that, at least for OPA1, cardiac abnormalities are not completely
  • manifest until the development of blindness.

The OPA1-mutant mice survived more than 1 year and appeared healthy.

In patients with these diseases, reduced cardiac function may go undetected
secondary to reduced physical activity secondary to loss of vision.
It would be expected that patients with such mutations would have impaired cardiac reserve with
  • reduced ability to respond to high-stress disease states such as myocardial infarction and sepsis.
The OPA1-mutant mice have reduced cardiac reserve, as shown by
  • the lack of response to isoproterenol or to ischemia/reperfusion injury,
This suggests that patients with OPA1 and related inherited mitochondrial diseases
  • should be screened for abnormalities of cardiac function.
Le Chen; T Liu; A Tran; Xiyuan Lu; …AA. Knowlton. OPA1 Mutation and Late-Onset Cardiomyopathy:
Mitochondrial Dysfunction and mtDNA Instability.  http://jaha.ahajournals.org/content/1/5/e003012.full

Oxidative Stress and Mitochondria in the Failing Heart

The major problem in tissue hypoxia in the failing heart is oxidative stress. Reactive oxygen species (ROS), including
  • superoxide,
  • hydroxyl radicals and
  • hydrogen peroxide,
are generated by a number of cellular processes, including
  • mitochondrial electron transport,
  • NADPH oxidase and
  • xanthine dehydrogenase/xanthine oxidase.
The low availability of oxygen, the final receptor of mitochondrial electron transport (ET), results in
  • electron accumulation in the ET chain as the complexes become highly reduced.
A number of experimental and clinical studies have suggested that ROS generation is
  • enhanced in heart failure because of electron leak, and complexes I and II
  • are implicated as the primary sites of this loss.
Prolonged oxidative stress in cardiac failure results in damage to mitochondrial DNA.
The continued ROS generation and consequent cellular injury leads to functional decline.
Thus, mitochondria are both the sources and targets of a cycle of ROS-mediated injury in the failing heart.
Mice with a cardiac/skeletal muscle specific deficiency in the scavenger enzyme superoxide dismutase
  • developed progressive congestive heart failure
  • with defects in mitochondrial respiration.
Oxidative stress in these mice also caused specific morphological changes in cardiac mitochondria
  • characterized by decreased ATP levels,
  • impaired contractility,
  • dramatically restricted exercise capacity and
  • decreased survival.
This was in part corrected by treatment with the antioxidant superoxide dismutase mimetic, namely
  • manganese5,10,15,20-tetrakis-(4-benzoic acid)-porphyrin.
EUK-8, a superoxide dismutase and catalase mimetic improved survival and contractile parameters in a mutant mouse model
  • of pressure overload-induced oxidative stress and heart failure and in wild-type mice subjected to pressure overload.
In addition, mitochondria show
  • functional impairment and
  • morphological disorganization
in the left ventricle of Hypertrophic Cardiomyopathy (HCM)  patients without baseline systolic dysfunction.
These mitochondrial changes were associated with impaired myocardial contractile and relaxation reserves.
A strategy to protect the heart against oxidative stress could lie with
  • the modulation of mitochondrial electron transport itself.
Mild mitochondrial uncoupling may offer a potential cardioprotective effect by decreasing ROS production
  • preventing electron accumulation at complex III and
  • the Fe–S centres of complex I, and may therefore

mtDNA, Autophagy, and Heart Failure

Mitochondria are evolutionary endosymbionts derived from bacteria and contain DNA similar to bacterial DNA.
Mitochondria damaged by external haemodynamic stress are degraded by the autophagy/lysosome system in cardiomyocytes.
Mitochondrial DNA (mtDNA) that escapes from autophagy cell-autonomously leads to Toll-like receptor (TLR) 9-mediated
  • inflammatory responses in cardiomyocytes and
  • is capable of inducing myocarditis and dilated cardiomyopathy.
Cardiac-specific deletion of lysosomal deoxyribonuclease (DNase) II showed no cardiac phenotypes under baseline conditions,
but increased mortality and caused severe myocarditis and dilated cardiomyopathy 10 days after treatment with pressure overload.
Early in the pathogenesis, DNase II-deficient hearts showed
  • infiltration of inflammatory cells
  • increased messenger RNA expression of inflammatory cytokines
  • accumulation of mitochondrial DNA deposits in autolysosomes in the myocardium.
Administration of inhibitory oligodeoxynucleotides against TLR9, which is known to be activated by bacterial DNA6, or ablation of Tlr9
  • attenuated the development of cardiomyopathy in DNase II-deficient mice.
Furthermore, Tlr9 ablation
  • improved pressure overload-induced cardiac dysfunction and
  • inflammation even in mice with wild-type Dnase2a alleles.
These data provide new perspectives on the mechanism of genesis of chronic inflammation in failing hearts.
T Oka, S Hikoso, O Yamaguchi, M Taneike, T Takeda, T Tamai, et al.  Mitochondrial DNA that escapes from autophagy causes inflammation and heart failure.

Mitochondrial Dysfunction Increases Expression of Endothelin-1 and Induces Apoptosis

We developed an in vitro model of mitochondrial dysfunction using rotenone, a mitochondrial respiratory chain complex I inhibitor, and studied
  • preproendothelin-1 gene expression and apoptosis.
Rotenone greatly increased the gene expression of preproendothelin-1 in cardiomyocytes.
This result suggests that the gene expression of preproendothelin-1 is induced by the mitochondrial dysfunction.
Furthermore, treatment of cardiomyocytes with rotenone induced an elevation of caspase-3 activity, and caused a marked
  • increase in DNA laddering, an indication of apoptosis.
In conclusion, it is suggested that mitochondrial impairment in primary cultured cardiomyocytes induced by rotenone in vitro,
  • mimics some of the pathophysiological features of heart failure in vivo, and that ET-1 may have a role in myocardial dysfunction
    • with impairment of mitochondria in the failing heart.

Summary

This review focused on the evidence accumulated to the effect that mitochondria are key players in
  • the progression of congestive heart failure (CHF).
Mitochondria are the primary source of energy in the form of adenosine triphosphate that fuels the contractile apparatus,
  • essential for the mechanical activity and the Starling Effect of the heart.
We evaluate changes in mitochondrial morphology and alterations in the main components of mitochondrial energetics, such as
  • substrate utilization and
  • oxidative phosphorylation,
in the context of their contribution to the chronic energy deficit and mechanical dysfunction in HF.
REFERENCES
Zachman AL, Page JM, Prabhakar G, Guelcher SA, and Sung HJ, “Elucidation of adhesion-dependent spontaneous apoptosis in macrophages using phase separated PEG/polyurethane films.”
Acta Biomater. 2012 Nov 2.    http://dx.doi.org/pii: S1742-7061(12)00530-2. 10.1016/j.actbio.2012.10.038.    http://www.ncbi.nlm.nih.gov/pubmed/23128157

Other Related articles published on this Open Access Scientific Journal, include the following:

Perspectives on Nitric Oxide in Disease Mechanisms: The Nitric Oxide Discovery, Function, and Targeted Therapy  Opportunities, 2013, Aviral Vatsa, PhD and Larry H Bernstein, MD, FACP, Editors, Amazon e-Books (forthcoming). https://pharmaceuticalintelligence.com/biomed-e-books/perspectives-on-nitric-oxide-in-disease-mechanisms-v2/

Mitochondria: More than just the “powerhouse of the cell” Ritu Saxena, Ph.D. Consultants: Aviva Lev-Ari, PhD, RN and Pnina G. Abir-Am, PhD 7/9/2012

https://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/

Mitochondrial dynamics and cardiovascular diseases, Ritu Saxena, PhD 11/14/2012
https://pharmaceuticalintelligence.com/2012/11/14/mitochondrial-dynamics-and-cardiovascular-diseases/

Mitochondrial Damage and Repair under Oxidative Stress, Larry H Bernstein, MD, FACP 10/28/2012
https://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/

Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation, Larry H Bernstein, MD, FACP 9/26/2012

https://pharmaceuticalintelligence.com/2012/09/26/mitochondria-origin-from-oxygen-free-environment-role-in-aerobic-glycolysis-metabolic-adaptation/

Ca2+ signaling: transcriptional control, Larry H Bernstein, MD, FACP 3/6/2-13
https://pharmaceuticalintelligence.com/2013/03/06/ca2-signaling-transcriptional-control/

MIT Scientists on Proteomics: All the Proteins in the Mitochondrial Matrix identified, Aviva Lev-Ari, PhD, RN 2/3/2013
https://pharmaceuticalintelligence.com/2013/02/03/mit-scientists-on-proteomics-all-the-proteins-in-the-mitochondrial-matrix-identified/

Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function, Larry H Bernstein, MD, FACP 9/16/2012
https://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-glycolysis-with-a-concomitant-influence-on-mitochondrial-function/

Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis, Larry H Bernstein, MD, FACP 2/14/2013
https://pharmaceuticalintelligence.com/2013/02/14/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-proteolysis-and-cell-apoptosis-reconsidered/

Low Bioavailability of Nitric Oxide due to Misbalance in Cell Free Hemoglobin in Sickle Cell Disease – A Computational Model   Anamika Sarkar, PhD 11/9/2012
https://pharmaceuticalintelligence.com/2012/11/09/low-bioavailability-of-nitric-oxide-due-to-misbalance-in-cell-free-hemoglobin-in-sickle-cell-disease-a-computational-model/

The rationale and use of inhaled NO in Pulmonary Artery Hypertension and Right Sided Heart Failure, , Larry H Bernstein, MD, FACP 8/20/2012

https://pharmaceuticalintelligence.com/2012/08/20/the-rationale-and-use-of-inhaled-no-in-pulmonary-artery-hypertension-and-right-sided-heart-failure/

Clinical Trials Results for Endothelin System: Pathophysiological role in Chronic Heart Failure, Acute Coronary Syndromes and MI – Marker of Disease Severity or Genetic Determination? Aviva Lev-Ari, PhD, RN 10/19/2012

https://pharmaceuticalintelligence.com/2012/10/19/clinical-trials-results-for-endothelin-system-pathophysiological-role-in-chronic-heart-failure-acute-coronary-syndromes-and-mi-marker-of-disease-severity-or-genetic-determination/

Endothelin Receptors in Cardiovascular Diseases: The Role of eNOS Stimulation, Aviva Lev-Ari, PhD, RN 10/4/2012

https://pharmaceuticalintelligence.com/2012/10/04/endothelin-receptors-in-cardiovascular-diseases-the-role-of-enos-stimulation/

Inhibition of ET-1, ETA and ETA-ETB, Induction of NO production, stimulation of eNOS and Treatment Regime with PPAR-gamma agonists (TZD): cEPCs Endogenous Augmentation for Cardiovascular Risk Reduction – A Bibliography, Aviva Lev-Ari, PhD, RN 10/4/2012

https://pharmaceuticalintelligence.com/2012/10/04/inhibition-of-et-1-eta-and-eta-etb-induction-of-no-production-and-stimulation-of-enos-and-treatment-regime-with-ppar-gamma-agonists-tzd-cepcs-endogenous-augmentation-for-cardiovascular-risk-reduc/

Genomics & Genetics of Cardiovascular Disease Diagnoses: A Literature Survey of AHA’s Circulation Cardiovascular Genetics, 3/2010 – 3/2013, L H Bernstein, MD, FACP and Aviva Lev-Ari,PhD, RN  3/7/2013

https://pharmaceuticalintelligence.com/2013/03/07/genomics-genetics-of-cardiovascular-disease-diagnoses-a-literature-survey-of-ahas-circulation-cardiovascular-genetics-32010-32013/

Cardiovascular Disease (CVD) and the Role of agent alternatives in endothelial Nitric Oxide Synthase (eNOS) Activation and Nitric Oxide Production, Aviva Lev-Ari, PhD, RN 7/19/2012

https://pharmaceuticalintelligence.com/2012/07/19/cardiovascular-disease-cvd-and-the-role-of-agent-alternatives-in-endothelial-nitric-oxide-synthase-enos-activation-and-nitric-oxide-production/

Cardiovascular Risk Inflammatory Marker: Risk Assessment for Coronary Heart Disease and Ischemic Stroke – Atherosclerosis.

Aviva Lev-Ari, PhD, RN 10/30/2012

https://pharmaceuticalintelligence.com/2012/10/30/cardiovascular-risk-inflammatory-marker-risk-assessment-for-coronary-heart-disease-and-ischemic-stroke-atherosclerosis/

Cholesteryl Ester Transfer Protein (CETP) Inhibitor: Potential of Anacetrapib to treat Atherosclerosis and CAD, Aviva Lev-Ari, PhD, RN 4/7/2013

https://pharmaceuticalintelligence.com/2013/04/07/cholesteryl-ester-transfer-protein-cetp-inhibitor-potential-of-anacetrapib-to-treat-atherosclerosis-and-cad/

Hypertriglyceridemia concurrent Hyperlipidemia: Vertical Density Gradient Ultracentrifugation a Better Test to Prevent Undertreatment of High-Risk Cardiac Patients, Aviva Lev-Ari, PhD, RN  4/4/2013

https://pharmaceuticalintelligence.com/2013/04/04/hypertriglyceridemia-concurrent-hyperlipidemia-vertical-density-gradient-ultracentrifugation-a-better-test-to-prevent-undertreatment-of-high-risk-cardiac-patients/

Fight against Atherosclerotic Cardiovascular Disease: A Biologics not a Small Molecule – Recombinant Human lecithin-cholesterol acyltransferase (rhLCAT) attracted AstraZeneca to acquire AlphaCore, Aviva Lev-Ari, PhD, RN 4/3/2013

https://pharmaceuticalintelligence.com/2013/04/03/fight-against-atherosclerotic-cardiovascular-disease-a-biologics-not-a-small-molecule-recombinant-human-lecithin-cholesterol-acyltransferase-rhlcat-attracted-astrazeneca-to-acquire-alphacore/

High-Density Lipoprotein (HDL): An Independent Predictor of Endothelial Function & Atherosclerosis, A Modulator, An Agonist, A Biomarker for Cardiovascular Risk, Aviva Lev-Ari, PhD, RN 3/31/2013

https://pharmaceuticalintelligence.com/2013/03/31/high-density-lipoprotein-hdl-an-independent-predictor-of-endothelial-function-artherosclerosis-a-modulator-an-agonist-a-biomarker-for-cardiovascular-risk/

Peroxisome proliferator-activated receptor (PPAR-gamma) Receptors Activation: PPARγ transrepression for Angiogenesis in Cardiovascular Disease and PPARγ transactivation for Treatment of Diabetes, Aviva Lev-Ari, PhD, RN 11/13/2012

https://pharmaceuticalintelligence.com/2012/11/13/peroxisome-proliferator-activated-receptor-ppar-gamma-receptors-activation-pparγ-transrepression-for-angiogenesis-in-cardiovascular-disease-and-pparγ-transactivation-for-treatment-of-dia/

Sulfur-Deficiciency and Hyperhomocysteinemia, L H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/04/sulfur-deficiency-and-hyperhomocusteinemia/

Mitochondrial metabolism and cardiac function, L H Bernstein, MD, FACP
Cardiotoxicity and Cardiomyopathy Related to Drugs Adverse Effects, L H Bernstein, MD, FACP
Lp(a) Gene Variant Association, L H Bernstein, MD, FACP

Predicting Drug Toxicity for Acute Cardiac Events, L H Bernstein, MD, FACP

Amyloidosis with Cardiomyopathy, L H Bernstein, MD, FACP

Mitochondria and Cardiovascular Disease: A Tribute to Richard Bing, Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/14/chapter-5-mitochondria-and-cardiovascular-disease/

Mitochondrial Metabolism and Cardiac Function, Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-metabolism-and-cardiac-function/

Mitochondrial Dysfunction and Cardiac Disorders, Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/14/mitochondrial-dysfunction-and-cardiac-disorders/

Reversal of Cardiac mitochondrial dysfunction, Larry H Bernstein, MD, FACP

https://pharmaceuticalintelligence.com/2013/04/14/reversal-of-cardiac-mitochondrial-dysfunction/

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Mitochondrial Damage and Repair under Oxidative Stress

Curator: Larry H Bernstein, MD, FCAP

 


Keywords: Mitochondria, mitochondrial dysfunction, electron transport chain, mtDNA, oxidative stress, oxidation-reduction, NO, DNA repair, lipid peroxidation, thiols, ROS, RNS, sulfur,base excision repair, ferredoxin.
Summary: The mitochondrion is the energy source for aerobic activity of the cell, but it also has regulatory functions that will be discussed. The mitochondrion has been discussed in other posts at this site. It has origins from organisms that emerged from an anaerobic environment, such as the bogs and marshes, and may be related to the chloroplast. The aerobic cell was an advance in evolutionary development, but despite the energetic advantage of using oxygen, the associated toxicity of oxygen abundance required adaptive changes. Most bacteria that reduce nitrate (producing nitrite, nitrous oxide or nitrogen) are called facultative anaerobes use electron acceptors such as ferric ions, sulfate or carbon dioxide which become reduced to ferrous ions, hydrogen sulfide and methane, respectively, during the oxidation of NADH (reduced nicotinamide adenine dinucleotide is a major electron carrier in the oxidation of fuel molecules).

The underlying problem we are left with is oxidation-reduction reactions that are necessary for catabolic and synthetic reactions, and that cumulatively damage the organism associated with cancer, cardiovascular disease, neurodegerative disease, and inflammatory overload. Aerobic organisms tolerate have evolved mechanisms to repair or remove damaged molecules or to prevent or deactivate the formationof toxic species that lead to oxidative stress and disease. However, the normal balance between production of pro-oxidant species and destruction by the antioxidant defenses is upset in favor of overproduction of the toxic species, which leads to oxidative stress and disease. How this all comes together is the topic of choice.

Schematic diagram of the mitochondrial .

The transformation of energy is central to mitochondrial function. The system of energetics includes:

  • the enzymes of the Kreb’s citric acid or TCA cycle,
  • some of the enzymes involved in fatty acid catabolism (β-oxidation), and
  • the proteins needed to help regulate these systems,

central to mitochondrial physiology through the production of reducing equivalents. Reducing equivalents are also used for anabolic reactions.
Electron Transport Chain
It also houses the protein complexes involved in the electron transport component of oxidative phosphorylation and proteins involved in substrate and ion transport. The chemical energy contained in both fats and amino acids can also be converted into NADH and FADH2 through mitochondrial pathways. The major mechanism for harvesting energy from fats is β-oxidation; the major mechanism for harvesting energy from amino acids and pyruvate is the TCA cycle. Once the chemical energy has been transformed into NADH and FADH2, these compounds are fed into the mitochondrial respiratory chain.
Under physiological conditions, electrons generally enter either through complex I (NADH-mediated, examined in vitro using substrates such as glutamate/malate) or complex II (FADH2-mediated, examined in vitro using succinate).

Electrons are then sequentially passed through a series of electron carriers.

The progressive transfer of electrons (and resultant proton pumping) converts the chemical energy stored in carbohydrates, lipids, and amino acids into potential energy in the form of the proton gradient. The potential energy stored in this gradient is used to phosphorylate ADP forming ATP.
Redox-Cycling

In redox cycling the reductant is continuously regenerated, thereby providing substrate for the “auto-oxidation” reaction.

When partially oxidized compounds are enzymatically reduced, the auto-oxidative generation of superoxide and other ROS to start again. Several enzymes

  •  NADPH-cytochrome P450 reductase,
  • NADPH-cytochrome b5 reductase [EC 1.6.2.2]
  • NADPH-ubiquinone oxidoreductase [EC 1.6.5.3], and
  • xanthine oxidase [EC 1.2.3.2]),

can reduce quinones into semiquinones in a single electron process.

The semiquinone can then reduce dioxygen to superoxide during its oxidation to a quinone.

Redox cycling is thought to play a role in carcinogenesis. The naturally occurring estrogen metabolites (the catecholestrogens) have been implicated in hormone-induced cancer, possibly as a result of their redox cycling and production of ROS. It is thought that diethylstilbestrol causes the production of the mutagenic lesion 8-hydroxy-2’deoxyguanosine. It can also cause DNA strand breakage.

Another oxidative reaction that is associated with H2O2 is a significant problem for living organisms as a consequence of the reaction between hydrogen peroxide and oxidizable metals, the Fenton reaction [originally described in the oxidation of an α-hydroxy acid to an α-keto acid in the presence of hydrogen peroxide (or hypochlorite) and low levels of iron salts (Fenton (1876, 1894)).
Chemical Reactions and Biological Significance

The hydroxyl free radical is so aggressive that it will react within 5 (or so) molecular diameters from its site of production. The damage caused by it, therefore, is very site specific. Biological defenses have evolved that reduce the chance that the hydroxyl free radical will be produced to repair damage. An antioxidant would have to occur at the site of hydroxyl free radical production and be at sufficient concentration to be effective.

Some endogenous markers have been proposed as a useful measures of total “oxidative stress” e.g., 8-hydroxy-2’deoxyguanosine in urine. The ideal scavenger

  • must be non-toxic,
  • have limited or no biological activity,
  • readily reach the site of hydroxyl free radical production,
  • react rapidly with the free radical, be specific for this radical, and
  • neither the scavenger nor its product(s) should undergo further metabolism.

Unlike oxygen, nitrogen does not possess unpaired electrons and is therefore considered diamagnetic. Nitrogen does not possess available d orbitals so it is limited to a valency of 3. In the presence of oxygen, nitrogen can produce Nitric oxide which occurs physiologically with the immune system which, when activated, can produce large quantities of nitric oxide.

Nitric oxide is produced by stepwise oxidation of L-arginine catalyzed by nitric oxide synthase (NOS). Nitric oxide is formed from the guanidino nitrogen of the L-arginine in a reaction that

  • consumes five electrons and
  • requires flavin adenine dinucleotide (FAD),
  • flavin mononucleotide (FMN) tetrahydrobiopterin (BH4), and
  • iron protoporphyrin IX as cofactors.

The primary product of NOS activity may be the nitroxyl anion that is then converted to nitric oxide by electron acceptors.

NOS cDNAs show homology with the cytochrome P450 reductase family. Based on molecular genetics there appears to be at least three distinct forms of NOS:

  • A Ca2+/calmodulin-requiring constitutive enzyme (c-NOS; ncNOS or type I)
  • A calcium-independent inducible enzyme (i-NOS; type II), which is primarily involved in the mediation of the cellular immune response; and
  • A second Ca2+/calmodulin-requiring constitutive enzyme found in aortic and umbilical endothelia (ec-NOS or type III)

This has been discussed extensively in this series of posts. Recently, a mitochondrial form of the enzyme, which appears to be similar to the endothelial form, has been found in brain and liver tissue. Although the exact role of nitric oxide in the mitochondrion remains elusive, it may play a role in the regulation of cytochrome oxidase.
Nitric Oxide
Nitric oxide appears to regulate its own production through a negative feedback loop. The binding of nitric oxide to the heme prosthetic group of NOS inhibits this enzyme, and c-NOS and ec-NOS are much more sensitive to this regulation than i-NOS. It appears that in the brain, NO can regulate its own synthesis and therefore the neurotransmission process.

  • On the one hand, inhibition of ec-NOS will prevent the cytotoxicity associated with excessive nitric oxide production.
  • On the other, the insensitivity of i-NOS to nitric oxide will enable high levels of nitric oxide to be produced for cytotoxic effects.

Endogenous inhibitors of NOS (guanidino-substituted derivatives of arginine) occur in vivo as a result of post-translational modification of protein contained arginine residues by S-adenosylmethionine. The dimethylarginines (NG,NG-dimethyl-L-arginine and NG,N’G-dimethyl-L-arginine) occurs in tissue proteins, plasma, and urine of humans and they are thought to act as both regulators of NOS activity and reservoirs of arginine for the synthesis of nitric oxide.
It has been calculated that even though membrane makes up about 3% of the total tissue volume, 90% of the reaction of nitric oxide with oxygen occurs within this compartment. Thus the membrane is an important site for nitric oxide chemistry.
There are two major aspects to nitric oxide chemistry.

  • It can undergo single electron oxidation and reduction reactions producing nitrosonium and nitroxyl
  • Having a single unpaired electron in its π*2p molecular orbital it will react readily with other molecules that also have unpaired electrons, such as free radicals and transition metals.

Examples of the reaction of nitric oxide with radical species include:

  • Nitric oxide will react with oxygen to form the peroxynitrite (nitrosyldioxyl) radical (ONO2)
  • and with superoxide to form the powerful oxidizing and nitrating agent, peroxynitrite anion (ONO2-). Peroxynitrite causes damage to many important biomolecules

Importance:

  • nitrosothiols that are important in the regulation of blood pressure terminates lipid peroxidation
  • 3-nitrosotyrosine and/or 4-O-nitrosotyrosine can affect the activity of enzymes that utilize tyrosyl radicals
  • rapidly reacts with oxyhemoglobin, the primary route of its destruction in vivo
  • the reaction between nitric oxide and transition metal complexes

During the last reaction a “ligand” bond is formed (the unpaired electron of nitric oxide is partially transferred to the metal cation),

 resulting in a nitrosated (nitrosylated) complex.

For example, such complexes can be formed with free iron ions,

iron bound to heme or iron located in iron-sulfur clusters.

Ligand formation allows nitric oxide to act as a signal, activating some enzymes while inhibiting others. Thus, the binding of nitric oxide to the Fe (II)-heme of guanylate (guanalyl) cyclase [GTP-pyrophosphate lyase: cyclizing] is the signal transduction mechanism. Guanylate cyclase exists as cytosolic and membrane-bound isozymes.
Thiol-Didulfide Redox Couple

The thiol-disulfide redox couple is very important to oxidative metabolism. For example, GSH is a reducing cofactor for glutathione peroxidase, an antioxidant enzyme responsible for the destruction of hydrogen peroxide.

The importance of the antioxidant role of the thiol-disulfide redox couple:

Thiols and disulfides can readily undergo exchange reactions, forming mixed disulfides. Thiol-disulfide exchange is biologically very important. For example,

  • GSH can react with protein cystine groups and influence the correct folding of proteins.
  • GSH may also play a direct role in cellular signaling through thiol-disulfide exchange reactions with membrane bound receptor proteins
  •                        the insulin receptor complex)
  •                        transcription factors (e.g., nuclear factor κB)
  •                        and regulatory proteins in cells

Conditions that alter the redox status of the cell can have important consequences on cellular function.

The generation of ROS by redox cycling is only one possible explanation for the action of many drugs. Rifamycin not only owes its activity to ROS generation but also to its ability to block bacterial RNA synthesis as well. Quinones (and/or semiquinones) can also form adducts with nucleophiles, especially thiols. These adducts may act as toxins directly or indirectly through the inhibition of key enzymes (e.g., by reacting with essential cysteinyl residues) or the depletion of GSH.
DNA Adduct Formation

By far the most intense research in this field has been directed towards the chemistry and biology of DNA adduct formation. Attack of the free bases and nucleosides by pro-oxidants can yield a wide variety of adducts and DNA-protein cross-links. Such attack usually occurs

  • at the C-4 and C-8 position of purines and
  • C-5 and C-6 of pyrimidines.

Hydroxyl free radical-induced damage to purine bases and nucleosides can proceed through a C-8-hydroxy N-7 radical intermediate, and then either undergo oxidation with the production of an 8-hydroxy purine, or reduction, probably by cellular thiols, followed by ring opening and the formation of FAPy (formamido-pyrimidine) metabolites (hydroxyl free radical-induced damage to guanosine). Although most research has focused on 8-hydroxy-purine adducts a growing number of publications are attempting to measure the FAPy derivative.

Nitrosation of the Amines of the Nucleic Acid Bases.

Primary aromatic amines produce deaminated products, while secondary amines form N-nitroso compounds.
Formation of Peroxynitrite from Nitric Oxide.

Peroxynitrite shows complex reactivity

  • with DNA initiating DNA strand breakage, oxidation (e.g., formation of 8-hydroxyguanine, 8-OH2’dG, (5-hydroxymethyl)-uracil, and FAPyGua),
  • nitration (e.g., 8-nitroguanine), and
  • deamination of bases.

Peroxynitrite can also promote the production of lipid peroxidation related active carbonyls and cause the activation of NAD+ ADP-ribosyltransferase.

Modification of Guanine
Although all DNA bases can be oxidatively damaged, it is the modification of guanine that is the most frequent. 8OH2’dG is the most abundant DNA adduct. This can affect its hydrogen bonding between base-pairs. These base-pair substitutions are usually found clustered into areas called “hot spots”. Guanine normally binds to cytosine.

8OH2’dG, however, can form hydrogen bonds with adenine. The formation of 8OH2’dG in DNA can therefore result in a G→T transversion.

8-Hydroxyguanine was also shown to induce codon 12 activation of c-Ha-ras and K-ras in mammalian systems. G→T transversions are also the most frequent hot spot mutations found in the p53 supressor gene which is associated with human tumors.

Other mechanisms by which ROS/RNS can lead to mutations have been
proposed. Direct mechanisms include:

  • conformational changes in the DNA template that reduces the accuracy of replication by DNA polymerases
  • altered methylation of cytosine that affects gene control

Indirect mechanisms include:

  • Oxidative damage to proteins, including DNA polymerases and repair enzymes.
  • Damage to lipids causes the production of mutagenic carbonyl compounds
  • Misalignment mutagenesis (“slippery DNA”)
DNA Mismatch Repair 5

DNA Mismatch Repair 5 (Photo credit: Allen Gathman)

Repair of ROS/RNS-induced DNA Damage
The repair of damaged DNA is an ongoing and continuous process involving a
number of repair enzymes. Damaged DNA appears to be mended by two major mechanisms:

  1. base excision repair (BER) and
  2. nucleotide excision repair (NER)

Isolated DNA is found to contain low levels of damaged bases, so it appears that these repair processes are not completely effective.
Base Excision Repair

BER is first started by DNA glycosylases which recognize specific base
modifications (e.g., 8OH2’dG). For example,

  • Formamido-pyrimidine-DNA glycosylase (Fpg protein) recognizes damaged purines such as 8-oxoguanine and FAPyGua.
  • Damaged pyrimidines are recognized by endonuclease III, which acts as both a glycosylase and AP endonuclease.
  • Glycosylases cleave the N-glycosylic bond between the damaged base and the sugar

Following the glycosylase step, AP endonucleases then remove the 3′-deoxyribose moiety by cleavage of the phosphodiester bonds thereby generating a 3’-hydroxyl group that can then be extended by DNA polymerase.

The final step in mending damaged DNA is the rejoining of the free ends of DNA by a DNA ligase. It also appears that the presence of 8-oxoguanine modified bases in DNA is not only a result of ROS attack on this macromolecule. Oxidized nucleosides and nucleotides from free cellular pools can also be incorporated into DNA by polymerases and cause AT to CG base substitution mutations.

Mitochondrial DNA Repair

The mitochondrion genome encodes the various complexes of the electron transport chain, but contains no genetic information for DNA repair enzymes. These enzymes must be obtained from the nucleus. As mitochondria are continuously producing DNA damaging pro-oxidant species, effective DNA repair mechanisms must exist within the mitochondrial matrix in order for these organelles to function. Mitochondria have a short existence, and excessively damaged mitochondria will be quickly removed. Mitochondria contain many BER enzymes and are proficient at repair, but they do not appear to repair damaged DNA by NER mechanisms.

Single Strand DNA Damage and PARP Activation

Single strand DNA breakage activates NAD+ ADP-ribosyltransferase (PARP). PARP is a protein-modifying, nucleotide-polymerizing enzyme and is found at high levels in the nucleus. Activated PARP

  1. cleaves NAD+ into ADP-ribose and nicotinamide
  2. then attaches the ADP-ribose units to a variety of nuclear proteins (including histones and its own automodification domain).
  3. then polymerizes the initial ADP-ribose modification with other ADP-ribose units to form the nucleic acid-like polymer, poly (ADP) ribose.

PARP only appears to be involved with BER and not NER. In BER PARP does not appear to play a direct role but rather it probably helps by keeping the chromatin in a conformation that enables other repair enzymes to be effective. It may also provide temporary protection to DNA molecules while it is being repaired. Conflicting evidence suggests that PARP may not be an important DNA repair enzyme as cells from a PARP knockout mouse model have normal repair characteristics.

Activation of PARP can be dangerous to the cell. For each mole of ADP-ribose transferred, one mole of NAD+ is consumed, and through the regeneration of NAD+ four ATP molecules are wasted. Thus the activation of PARP can rapidly deplete a cell’s energy store and even lead to cell death. Some researchers suggest that this may be one mechanism whereby cells with excessive DNA damage are effectively removed. However, a variety of diseases may involve PARP overactivation including

  • circulatory shock,
  • CNS injury,
  • diabetes,
  • drug-induced cytotoxicity, and
  • inflammation.

The Indirect Pathway.
This (mutation) pathway does not involve oxidative damage to the protein per se. This process involves oxidative damage to the DNA molecule encoding the protein. Thus pro-oxidants can cause changes in the base sequence of the DNA molecule. If such base modification is in a coding region of DNA (exon) and not corrected, the DNA molecule may be transcribed incorrectly. Translation of the mutant mRNA can result in a mutant protein containing a wrong amino acid in its primary sequence. If this modified amino acid occurs in an essential part of the protein (e.g., the active site of an enzyme or a portion that alters folding), the function of that protein may be impaired. Fortunately, unlike modified DNA
that can pass from cell to cell during mitosis thereby continuing the production of mutant protein, damage to a protein is non-replicating and stops with its destruction.

The Direct Pathway

This (post-translational) pathway involves the action of a pro-oxidant on a protein resulting in

  • modification of amino acid residues,
  • the formation of carbonyl adducts,
  • cross-linking and
  • polypeptide chain fragmentation.

Such changes often result in altered protein conformation and/or activity. Proteins will produce a variety of carbonyl products when exposed to metal-based systems (metal/ascorbate and metal/hydrogen peroxide) in vitro. For example, histidine yields aspartate, asparagine and 2-oxoimidazoline, while proline produces glutamate, pyroglutamate, 4-hydroxyproline isomers, 2-pyrrolidone and γ-aminobutyric acid. Metal-based systems and other pro-oxidant conditions can oxidize methionine to its sulfoxide.

This portion of the presentation is endebted to THE HANDBOOK OF REDOX
BIOCHEMISTRY, Ian N. Acworth, August 2003, esa. (inacworth@esainc.com).
We shall now identify more recent work related to this presentation.

Oxygen and Oxidative Stress

The reduction of oxygen to water proceeds via one electron at a time. In the mitochondrial respiratory chain, Complex IV (cytochrome oxidase) retains all partially reduced intermediates until full reduction is achieved. Other redox centres in the electron transport chain, however, may leak electrons to oxygen, partially reducing this molecule to superoxide anion (O2_•). Even though O2_• is not a strong oxidant, it is a precursor of most other reactive oxygen species, and it also becomes involved in the propagation of oxidative chain reactions. Despite the presence of various antioxidant defences, the mitochondrion appears to be the main intracellular source of these oxidants. This review describes the main mitochondrial sources of reactive species and the antioxidant defences that evolved to prevent oxidative damage in all the mitochondrial compartments.

Reactive oxygen species (ROS) is a phrase used to describe a variety of molecules and free radicals (chemical species with one unpaired electron) derived from molecular oxygen. Molecular oxygen in the ground state is a bi-radical, containing two unpaired electrons in the outer shell (also known as a triplet state).

Since the two single electrons have the same spin, oxygen can only react with one electron at a time and therefore it is not very reactive with the two electrons in a chemical bond.

On the other hand, if one of the two unpaired electrons is excited and changes its spin, the resulting species (known as singlet oxygen) becomes a powerful oxidant as the two electrons with opposing spins can quickly react with other pairs of electrons, especially double bonds.

The formation of OH• is catalysed by reduced transition metals, which in turn may be re-reduced by O2 -•, propagating this process. In addition, O2-• may react with other radicals including nitric oxide (NO•) in a reaction controlled by the rate of diffusion of both radicals. The product, peroxynitrite, is also a very powerful oxidant. The oxidants derived from NO• have been recently called reactive nitrogen species (RNS).

‘Oxidative stress’ is an expression used to describe various deleterious processes resulting from an imbalance between the excessive formation of ROS and/or RNS and limited antioxidant defences.

  • Whilst small fluctuations in the steady-state concentration of these oxidants may actually play a role in intracellular signalling,
  • uncontrolled increases in the steady-state concentrations of these oxidants lead to free radical mediated chain reactions

which indiscriminately target

  • proteins,
  • lipids,
  • polysaccharides.

In vivo, O2-• is produced both enzymatically and nonenzymatically.

Enzymatic sources include

  • NADPH oxidases located on the cell membrane of
  • polymorphonuclear cells,
  • macrophages and
  • endothelial cells and
  • cytochrome P450-dependent oxygenases.

The proteolytic conversion of xanthine dehydrogenase to xanthine oxidase provides another enzymatic source of both O2 -• and H2O2 (and therefore constitutes a source of OH•) and has been proposed to mediate deleterious processes in vivo.

Given the highly reducing intramitochondrial environment, various respiratory components, including flavoproteins, iron–sulfur clusters and ubisemiquinone, are thermodynamically capable of transferring one electron to oxygen. Moreover, most steps in the respiratory chain involve single-electron reactions, further favouring the monovalent reduction of oxygen. On the other hand, the mitochondrion possesses various antioxidant defences designed to eliminate both O2- • and H2O2.

The rate of O2 -• formation by the respiratory chain is controlled primarily by mass action, increasing both when electron flow slows down (increasing the concentration of electron donors, R•) and when the concentration of oxygen increases (eqn (1); Turrens et al. 1982).

d[O2]/dt = k [O2] [R•].

The energy released as electrons flow through the respiratory chain is converted into a H+ gradient through the inner mitochondrial membrane (Mitchell, 1977). This gradient, in turn, dissipates through the ATP synthase complex (Complex V) and is responsible for the turning of a rotor-like protein complex required for ATP synthesis. In the absence of ADP,

  • the movement of H+ through ATP synthase ceases and
  • the H+ gradient builds up
  • causing electron flow to slow down and
  • the respiratory chain to become more reduced (State IV respiration).

Mitochondrial Antioxidant Defences

The deleterious effects resulting from the formation of ROS in the mitochondrion are, to a large extent, prevented by various antioxidant systems. Superoxide is enzymatically converted to H2O2 by a family of metalloenzymes called superoxide dismutases (SOD). Since O2-• may either reduce transition metals, which in turn can react with H2O2 producing OH• or spontaneously react with NO• to produce peroxynitrite, it is important to maintain the steady-state concentration of O2-• at the lowest possible level. Thus, although the dismutation of O2-• to H2O2 and O2 can also occur spontaneously, the role of SODs is to increase the rate of the reaction to that of a diffusion-controlled process.

The mitochondrial matrix contains a specific form of SOD, with manganese in the active site, which eliminates the O2 -• formed in the matrix or on the inner side of the inner membrane. The expression of MnSOD is further induced by agents that cause oxidative stress, including radiation and hyperoxia, in a process mediated by the oxidative activation of the nuclear transcription factor NFkB .

Turrens JF. Mitochondrial formation of reactive oxygen species. J Physiol 2003; 552(2): 335–344. DOI: 10.1113/jphysiol.2003.049478. http://www.jphysiol.org

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Reactive Oxygen Species and Control of Apoptosis

Reactive oxygen species (ROS) are products of normal metabolism and xenobiotic exposure, and depending on their concentration, ROS can be beneficial or harmful to cells and tissues.

  • At physiological low levels, ROS function as “redox messengers” in intracellular signaling and regulation, whereas
  • excess ROS induce oxidative modification of cellular macromolecules, inhibit protein function, and promote cell death.

Additionally, various redox systems, such as

  • the glutathione,
  • thioredoxin, and
  • pyridine nucleotide redox couples,
  • NADPH and antioxidant defense
  • NAD+ and the function of sirtuin proteins

participate in cell signaling and modulation of cell function, including apoptotic cell death. Cell apoptosis is initiated by extracellular and intracellular signals via two main pathways,

  1. the death receptor and
  2. the mitochondria-mediated pathways.

ROS and JNK-mediated apoptotic signaling

              GSH redox status and apoptotic signaling

Various pathologies can result from oxidative stress-induced apoptotic signaling that is consequent to

  • ROS increases and/or antioxidant decreases,
  • disruption of intracellular redox homeostasis, and
  • irreversible oxidative modifications of lipid, protein, or DNA.

We focus on several key aspects of ROS and redox mechanisms in apoptotic signaling and highlight the gaps in knowledge and potential avenues for further investigation. A full understanding of the redox control of apoptotic initiation and execution could underpin the development of therapeutic interventions targeted at oxidative stress-associated disorders.

Circu, M. L.; Aw, T. Y., Reactive oxygen species, cellular redox systems, and apoptosis, Free Radic. Biol. Med. 2010. FRB-10057; pp 14. doi:10.1016/j.freeradbiomed.2009.12.022

Assembly of Iron-sulfur (FeyS) Clusters

Iron-sulfur (FeyS) cluster-containing proteins catalyze a number of electron transfer and metabolic reactions. The components and molecular mechanisms involved in the assembly of the FeyS clusters have been identified only partially. In eukaryotes, mitochondria have been proposed to execute a crucial task in the generation of intramitochondrial and extramitochondrial FeyS proteins. Herein, we identify the essential ferredoxin Yah1p of Saccharomyces cerevisiae mitochondria as a central component of the FeyS protein biosynthesis machinery. Depletion of Yah1p by regulated gene expression resulted in a

30-fold accumulation of iron within mitochondria,

similar to what has been reported for other components involved in FeyS protein biogenesis. Yah1p was shown to be required for the assembly of FeyS proteins both inside mitochondria and in the cytosol. Apparently, at least one of the steps of FeyS cluster biogenesis within mitochondria requires reduction by ferredoxin. Our findings lend support to the idea of a primary function of mitochondria in the biosynthesis of FeyS proteins outside the organelle. To our knowledge, Yah1p is the first member of the ferredoxin family for which a function in FeyS cluster formation has been established. A similar role may be predicted for the bacterial homologs that are encoded within iron-sulfur cluster assembly (isc) operons of prokaryotes.
H Lange, A Kaut, G Kispal, and R Lill. A mitochondrial ferredoxin is essential for biogenesis of cellular iron-sulfur proteins. PNAS 2000; 97(3): 1050–1055.

DNA Charge Transport

Damaged bases in DNA are known to lead to errors in replication and transcription, compromising the integrity of the genome. The authors proposed a model where repair proteins containing redoxactive [4Fe-4S] clusters utilize DNA charge transport (CT) as a first step in finding lesions. In this model, the population of sites to search is reduced by a localization of protein in the vicinity of lesions. Here, we examine this model using single-molecule atomic force microscopy (AFM). XPD, a 5′-3′ helicase involved in nucleotide
excision repair, contains a [4Fe-4S] cluster and exhibits a DNA bound redox potential that is physiologically relevant.

In AFM studies, they observe the redistribution of XPD onto kilobase DNA strands containing a single base mismatch, which is not a specific substrate for XPD but, like a lesion, inhibits CT. They also provide evidence for DNA-mediated signaling between XPD and Endonuclease III (EndoIII), a base excision repair glycosylase that also contains a [4Fe-4S] cluster.

  • When XPD and EndoIII are mixed together, they coordinate in relocalizing onto the mismatched strand.
  • However, when a CT-deficient mutant of either repair protein is combined with the CT-proficient repair partner, no relocalization occurs.

The data presented here indicate that XPD, an archaeal protein from the NER pathway, may cooperate with other proteins that are proficient at DNA CT to localize in the vicinity of damage. XPD, a superfamily 2 DNA helicase with 5′-3′ polarity, is a component of TFIIH that is essential for repair of bulky lesions generated by exogenous sources such as UV light and chemical carcinogens. XPD contains a conserved [4Fe-4S] cluster suggested to be conformationally controlled by ATP binding and hydrolysis.

Mutations in the iron-sulfur domain of XPD can lead to diseases including TTD and XP, yet the function of the [4Fe-4S] cluster appears to be unknown.

Electrochemical studies have shown that when BER proteins MutY and EndoIII bind to DNA, their [4Fe-4S] clusters are activated toward one electron oxidation. XPD exhibits a DNA-bound midpoint potential similar to that of EndoIII and MutY when bound to DNA (approximately 80 mV vs. NHE), indicative of a possible role for the [4Fe-4S] cluster in DNA-mediated CT.

For EndoIII we have also already determined a direct correlation between the ability of proteins to redistribute in the vicinity of mismatches as measured by AFM, and the CT proficiency of the proteins measured electrochemically. Thus, we may utilize single-molecule AFM as a tool to probe the redistribution of proteins in the vicinity of base lesions and in so doing, the proficiency of the protein to carry out DNA CT.

Here we show that, like the BER protein EndoIII, XPD, involved both in transcription and NER, redistributes in the vicinity of a lesion. Importantly, this ability to relocalize is associated with the ability of XPD to carry out DNA CT. The mutant L325V is defective in its ability to carry out DNA CTand this XPD mutant also does not redistribute effectively onto the mismatched strand.

These data not only indicate a general link between the ability of a repair protein to carry out DNA CT and its ability to redistribute onto DNA strands near lesions but also provide evidence for coordinated DNA CT between different repair proteins in their search for damage in the genome. These data also provide evidence that two different repair proteins, each containing a [4Fe-4S] cluster at similar DNA bound potential, can communicate with one another through DNA-mediated CT.

Sontz PA, Mui TP, Fuss JO, Tainer JA, and Barton JK. DNA charge transport as a first step in coordinating the detection of lesions by repair proteins. PNAS 2012; 109(6):1856–1861. doi:10.1073/pnas.1120063109/-/ DCSupplemental. http://www.pnas.org/lookup/suppl/

Janus Bifron 

The signaling function of mitochondria is considered with a special emphasis on their role in the regulation of redox status of the cell, possibly determining a number of pathologies including cancer and aging. The review summarizes the transport role of mitochondria in energy supply to all cellular compartments (mitochondria as an electric cable in the cell), the role of mitochondria in plastic metabolism of the cell including synthesis of

  • heme,
  • steroids,
  • iron-sulfur clusters, and
  • reactive oxygen and nitrogen species.

Mitochondria also play an important role in the Ca2+-signaling and the regulation of apoptotic cell death. Knowledge of mechanisms responsible for apoptotic cell death is important for the strategy for prevention of unwanted degradation of postmitotic cells such as cardiomyocytes and neurons.

In accordance with P. Mitchell’s chemiosmotic concept, vectorial transmembrane transfer of electrons and protons is accompanied by generation of electrochemical difference of proton electrochemical potential on the inner mitochondrial membrane; its utilization by ATP synthase induces conformational rearrangements resulting in ATP synthesis from ADP and inorganic phosphate. Details of the mechanism responsible for ATP synthesis are given elsewhere.

Membrane potential (DY) generated across the inner mitochondrial membrane is the component of the transmembrane electrochemical potential of H+ ions (DμH+), which provides ATP synthesis together with the concentration component (DpH). Maintenance of constant membrane potential is a vitally important precondition for functioning of mitochondria and the cell. Under conditions of limited supply of the cell with oxygen (hypoxia) and inability to carry out aerobic ATP synthesis, mitochondria become ATP consumers (rather than generators) and ATP is hydrolyzed by mitochondrial ATPase, and this is accompanied by generation of membrane potential.

Redox homeostasis, i.e. the sum of redox components (including proteins, low molecular weight redox components such as NAD/NADH, flavins, coenzymes Q, oxidized and reduced substrates, etc.) is one of important preconditions for normal cell functioning.

Single-strand and double-strand DNA damage

Single-strand and double-strand DNA damage (Photo credit: Wikipedia)

Mitochondria generate such potent regulators of redox potential as

  • superoxide anion,
  • hydrogen peroxide,
  • nitric oxide,
  • peroxynitrite, etc.

They are actively involved in regulation of cell redox potential and consequently

  • control proteolysis,
  • activation of transcription,
  • changes in mitochondrial DNA (mDNA),
  • cell metabolism, and
  • cell differentiation.

Zorov DB, Isaev NK, Plotnikov EY, Zorova LD, et al. The Mitochondrion as Janus Bifrons. Biochemistry (Moscow) 2007; 72(10): 1115-1126. ISSN 0006-2979.
DOI: 10.1134/S0006297907100094

Structure of the human mitochondrial genome.

Structure of the human mitochondrial genome. (Photo credit: Wikipedia)

Gene Expression Associated with Oxidoreduction and Mitochondria
The naked mole-rat (Heterocephalus glaber) is a long-lived, cancer resistant rodent and there is a great interest in identifying the adaptations responsible for these and other of its unique traits. We employed RNA sequencing to compare liver gene expression profiles between naked mole-rats and wild-derived mice. Our results indicate that genes associated with oxidoreduction and mitochondria were expressed at higher relative levels in naked mole-rats. The largest effect is nearly

300-fold higher expression of epithelial cell adhesion molecule (Epcam), a tumour-associated protein.

Also of interest are the

  • protease inhibitor, alpha2-macroglobulin (A2m), and the
  • mitochondrial complex II subunit Sdhc,

both ageing-related genes found strongly over-expressed in the naked mole-rat.

These results hint at possible candidates for specifying species differences in ageing and cancer, and in particular suggest complex alterations in mitochondrial and oxidation reduction pathways in the naked mole-rat. Our differential gene expression analysis obviated the need for a reference naked mole-rat genome by employing a combination of Illumina/Solexa and 454 platforms for transcriptome sequencing and assembling transcriptome contigs of the non-sequenced species. Overall, our work provides new research foci and methods for studying the naked mole-rat’s fascinating characteristics.

C Yu, Y Li, A Holmes, K Szafranski, CG Faulkes, et al. RNA Sequencing Reveals Differential Expression of Mitochondrial and Oxidation reduction Genes in the Long-Lived Naked Mole-Rat When Compared to Mice. PLoS ONE 2011; 6(11): 1-9. e26729. http://www.plosone.org

The complete set of viable deletion strains in Saccharomyces cerevisiae was screened for sensitivity of mutants to five oxidants to identify cell functions involved in resistance to oxidative stress. This screen identified a unique set of mainly constitutive functions providing the first line of defense against a particular oxidant; these functions are very dependent on the nature of the oxidant. Most of these functions are distinct from those involved in repair and recovery from damage, which are generally induced in response to stress, because there was little correlation between mutant sensitivity and
the reported transcriptional response to oxidants of the relevant gene. The screen identified 456 mutants sensitive to at least one of five different types of oxidant, and these were ranked in order of sensitivity. Many genes identified were not previously known to have a role in resistance to reactive oxygen species. These encode functions including

  • protein sorting,
  • ergosterol metabolism,
  • autophagy, and
  • vacuolar acidification.

two mutants were sensitive to all oxidants examined,
12 were sensitive to at least four,

Different oxidants had very different spectra of deletants that were sensitive. These findings highlight the specificity of cellular responses to different oxidants:

  • No single oxidant is representative of general oxidative stress.
  • Mitochondrial respiratory functions were overrepresented in mutants sensitive to H2O2, and
  • vacuolar protein-sorting mutants were enriched in mutants sensitive to diamide.

Core functions required for a broad range of oxidative-stress resistance include

  • transcription,
  • protein trafficking, and
  • vacuolar function.

GW Thorpe, CS Fong, N Alic, VJ Higgins, and IW Dawes. Cells have distinct mechanisms to maintain protection against different reactive oxygen species: Oxidative-stress-response genes. PNAS 2004;101: 6564–6569. http://www.pnas.org cgi doi 10.1073 pnas.0305888101
Subcellular Thiol Redox State in Complex I Deficiency

Isolated complex I deficiency is the most common enzymatic defect of the oxidative phosphorylation (OXPHOS) system, causing a wide range of clinical phenotypes. Th authers reported before that the rates at which reactive oxygen species (ROS)-sensitive dyes are converted into their fluorescent oxidation products are markedly increased in cultured skin fibroblasts of patients with nuclear-inherited isolated complex I deficiency.

Using videoimaging microscopy we show here that these cells also display a marked increase in NAD(P)H autofluorescence. Linear regression analysis revealed a negative correlation with the residual complex I activity and a positive correlation with the oxidation rates of the ROS sensitive dyes (5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein and hydroethidine for a large cohort of 10 patient cell lines.

On the other hand, video-imaging microscopy of cells selectively expressing reduction-oxidation sensitive GFP1 in either the mitochondrial matrix or cytosol showed the absence of any detectable change in thiol redox state. In agreement with this result, neither the glutathione nor the glutathione disulfide content differed significantly between patient and healthy fibroblasts.

Finally, video-rate confocal microscopy of cells loaded with C11-BODIPY581/591 demonstrated that the extent of lipid peroxidation, which is regarded as a measure of oxidative damage, was not altered in patient fibroblasts. Our results indicate that fibroblasts of patients with isolated complex I deficiency maintain their thiol redox state despite marked increases in ROS production.

S Verkaart, WJH Koopman, J Cheek, SE van Emst-de Vries. Mitochondrial and cytosolic thiol redox state are not detectably altered in isolated human NADH:ubiquinone oxidoreductase deficiency. Biochimica et Biophysica Acta (BBA) – Molecular Basis of Disease 2007; 1772(9): 1041. DOI : 10.1016/j.bbadis.2007.05.004

  • Mitochodrial mtDNA and Cancer
  • Mitochondrial research has recently been driven by the

identification of mitochondria-associated diseases and 
the role of mitochondria in apoptosis.

Moreover, mitochondria have been implicated in the process of carcinogenesis because of their vital role in

  • energy production,
  • nuclear-cytoplasmic signal integration and
  • control of metabolic pathways.

At some point during neoplastic transformation, there is an increase in reactive oxygen species (ROS), which damage the mitochondrial genome. This accelerates the somatic mutation rate of mitochondrial DNA.

Mitochondrial characteristics

There are several biological characteristics which cast mitochondria and, in particular, the mitochondrial genome, as a biological tool for early detection and monitoring of neoplasia and its potential progression. These vital characteristics are important in cancer research, as not all neoplasias become malignant. Mitochondria are archived in the cytoplasm of the ovum and as such do not recombine.

This genome has an accelerated mutation rate, by comparison with the nucleus, and accrues somatic mutations in tumour tissue. Moreover, mitochondrial DNA (mtDNA) has a high copy number in comparison with the nuclear archive of DNA. There are potentially thousands of mitochondrial genomes per cell, which enables detection of important biomarkers, even at low levels. In addition, mtDNA can be heteroplasmic, which means that disease-associated mutations occur in a subset of the genomes.

The presence of heteroplasmy is an indication of disease and is found in many human tumours. Identification of low levels of heteroplasmy may allow unprecedented early identification and monitoring of neoplastic progression to malignancy.

Coding for just 13 enzyme complex subunits, 22 transfer RNAs and two ribosomal RNAs, the mitochondrial genome is packaged in a compact 16,569 base pair (bp) circular molecule. These products participate in the critical electron transport process of ATP production. Collectively, mitochondria generate 80 per cent of the chemical fuel which fires cellular metabolism.

As a result, nuclear investment in the mitochondria is high — that is, several thousand nuclear genes control this organelle in order to accomplish the complex interactions required to maintain a network of pathways, which coordinate energy demand and supply.

It has been proposed that these mutations may serve as an early indication of potential cancer development and may represent a means for tracking tumour progression.

Does this provide a potential utility in that these mutations may be used for the identification and monitoring of neoplasia and malignant transformation where appropriate body fluids or non-invasive tissue access is available for mtDNA recovery? Specifically discussed are:

  • prostate,
  • breast,
  • colorectal,
  • skin and
  • lung cancers

There are many important questions yet to be addressed: such as

  • the relationship between mtDNA and the actual disease;
  • are mutations causative or merely a reflection of nuclear instability?
  • And, are these processes independent events?

Alterations in the non-coding D-loop suggest genome instability;
however, as studies focus more on the coding regions of the
mitochondrial genome,

Particularly in the case of nonsynonymous mutations in the genes
contributing products to the electron transport process, metabolic
implications are evident. Moreover, mutations in mitochondrial
transfer RNAs indicate the possibility of a global mitochondrial
translational shut down.

RL Parr, GD Dakubo, RE Thayer, K McKenney, MA Birch-Machin. Mitochondrial DNA as a potential tool for early cancer detection. HUMAN GENOMICS 2006; 2(4). 252–257.
Mitochondrial DNA (mtDNA) is particularly prone to oxidation due to the lack of histones and a deficient mismatch repair system. This explains an increased mutation rate of mtDNA that results in heteroplasmy, e.g., the coexistence of the mutant and wild-type mtDNA molecules within the same mitochondrion. Hyperglycemia is a key risk factor not only for diabetes-related disease, but also for cardiovascular and all-cause mortality. One can assume an increase in the risk of cardiovascular disease by 18% for each unit (%) glycated hemoglobin HbA1c. In the Glucose Tolerance in Acute Myocardial Infarction study of patients with acute coronary syndrome, abnormal glucose tolerance was the strongest independent predictor of subsequent cardiovascular complications and death. In the Asian Pacific Study, fasting plasma glucose was shown to be an independent predictor of cardiovascular events up to a level of 5.2 mmol/L.

Glucose level fluctuations and hyperglycemia are triggers for inflammatory responses via increased mitochondrial superoxide production and endoplasmic reticulum stress. Inflammation leads to insulin resistance and β-cell dysfunction, which further aggravates hyperglycemia. The molecular pathways that integrate hyperglycemia, oxidative stress, and diabetic vascular complications have been most clearly described in the pathogenesis of endothelial dysfunction, which is considered as the first step in atherogenesis according to the response to injury hypothesis.

  • In diabetes mellitus,
  • glycotoxicity,
  • advanced oxidative stress,
  • collagen cross-linking, and
  • accumulation of lipid peroxides

in foam macrophage cells and arterial wall cells may significantly

  • decrease the mutation threshold,
  • endothelial dysfunction,
  • promoting atherosclerosis.

Alterations in mitochondrial DNA (mtDNA), known as homoplasmic and heteroplasmic mutations, may influence mitochondrial OXPHOS capacity, and in turn contribute to the magnitude of oxidative stress in micro- and macrovascular networks in diabetic patients.
The authors critically consider the impact of mtDNA mutations on the pathogenesis of cardiovascular diabetic complications.

Mutation Threshhold

Although cells may harbor mutant mtDNA, the expression of disease is dependent on the percent of alleles bearing mutations. Modeling confirms that an upper threshold level might exist for mutations beyond which the mitochondrial population collapses, with a subsequent decrease in ATP. This decrease in ATP results in the phenotypic expression of disease. It is estimated that in many patients with clinical manifestations of mitochondrial disorders, the proportion of mutant DNA exceeds 50%.

For the MELAS (mitochondrial encephalopathy, lactic acidosis and stroke-like syndrome)-causing mutation m.3243 A>G in the mitochondrial gene encoding tRNALeu, which is also associated with diabetes plus deafness, a strong correlation between the level of mutational heteroplasmy and documented disease has been found. Increased percentages of mutant mtDNA in muscle cells (up to 71%) can lead to mitochondrial myopathy. Levels of heteroplasmy of over 80% may lead to recurrent stroke and mutation levels of 95% have been associated with MELAS.

Regardless of the type of mutation or the level of heteroplasmy in affected mitochondria, unrepaired damage leads to a decrease in ATP, which in turn causes the phenotypic manifestation of disease. The manifestation of disease not only depends on the ATP level but also on the tissue affected. Various tissues have differing levels of demand on OXPHOS capacity. To evaluate a tissue threshold, Leber’s hereditary optic neuropathy can be used as a model for mitochondrial neurodegenerative disease. For neural and skeletal muscle tissues, the tissue threshold should be as high as or higher than 90% of
damaged (mutated) mtDNA. To induce mitochondrial malfunctions, the tissue threshold of the cardiac muscle is estimated to be significantly lower (approximately 64%-67%). In chronic vascular disease such as atherosclerosis, a mutation threshold in the affected vessel wall (e.g., in the postmortem aortic atherosclerotic plaques) was observed to be significantly lower. For example, for mutations m.3256 C>T, m.12315 G>A, m.15059 G>A, and m.15315 G>A, the heteroplasmy range of 18%-66% in the atherosclerotic lesions was 2-3.5-fold that in normal vascular tissue.

Mitochondrial stress and insulin resistance

  • Mitochondrial damage precedes the development of atherosclerosis and tracks the extent of the lesion in apoE-null mice, and
  • mitochondrial dysfunction caused by heterozygous deficiency of a superoxide dismutase increases atherosclerosis and vascular mitochondrial damage in the same model.

Blood vessels destined to develop atherosclerosis may be characterized by inefficient ATP production due to the uncoupling of respiration and OXPHOS. Blood vessels have regions of hypoxia, which lower the ratio of state 3 (phosphorylating) to state 4 (nonphosphorylating) respiration. Human atherosclerotic lesions have been known for decades to be deficient in essential fatty acids, a condition that causes respiratory uncoupling and atherosclerosis.

The finding by Kokaze et al.  helps to explain, at least in part, the anti-atherogenic effect of the allele m. 5178A due to its relation with the favorable lipid profile. The nucleotide change causes leucine-to-methionine substitution at codon 237 (Leu-237Met) of the NADH dehydrogenase subunit 2 located in the loop between 7th and 8th transmembrane domains of the mitochondrial protein. Given that this methionine residue is exposed at the surface of respiratory Complex I, this residue may be available as an efficient oxidant scavenger. Complex I

  • accepts electrons from NADH,
  • transfers them to ubiquinone, and
  • uses the energy released to pump protons across the mitochondrial inner membrane.

Thus, the Leu237Met replacement in the ND2 subunit might have a protective effect against oxidative damage to mitochondria.

Most fatty acid oxidation, which is promoted by peroxisome proliferator-activated receptor α (PPARα) activation, occurs in the mitochondria. Mitochondrial effects could explain why PPARα- deficient mice are protected from diet-induced insulin resistance and atherosclerosis as well as glucocorticoid induced insulin resistance and hypertension. Caloric restriction,

  • improves features of insulin resistance,
  • increases mitochondrial biogenesis and, surprisingly,
  • enhances the efficiency of ATP production.

Dysfunctional mitochondria in cultured cells can be rescued by transfer of mitochondria from adult stem cells, raising the possibility of restoration of normal bioenergetics in the vasculature to treat atherosclerosis associated with insulin resistance.
Chistiakov DA, Sobenin IA, Bobryshev YV, Orekhov AN. Mitochondrial dysfunction and mitochondrial DNA mutations in atherosclerotic complications in diabetes. World J Cardiol 2012; 4(5): 148-156. ISSN 1949-8462 (online). doi:10.4330/wjc.v4.i5.148. http://www.wjgnet.com/1949-8462/full/v4/i5/148.htm

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Nitric Oxide has a Ubiquitous Role in the Regulation of Glycolysis – with a Concomitant Influence on Mitochondrial Function

 

Reporter, Editor, and Topic Co-Leader: Larry H. Bernstein, MD, FACP, Clinical Pathologist and Biochemist

 

 

Apoptosis signaling pathways

Apoptosis signaling pathways (Photo credit: AJC1)

This discussion is a followup on a series of articles elucidating the importance of NO, eNOS, iNOS, cardiovascular and vascular endothelium effects, and therapeutic targets.

This mechanism of action and signaling actions have been introduced so that we identify endocrine, paracrine, and such effects in the normal, stressed, and dysfunctional state. The size and breadth of this vital adaptive process is now further explored.

The title is short, befitting a subtitle.  The full topic may be considered “Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function that is active in endothelium, platelets, vascular smooth muscle and neural cells and the balance has a role in chronic inflammation, asthma, hypertension, sepsis and cancer”.

Vascular endothelium

Vascular endothelium (Photo credit: Wikipedia)

Related articles

 

 

Nitric Oxide Synthase

Nitric Oxide Synthase (Photo credit: Wikipedia)

 

 

Nitric Oxide has a ubiquitous role in the regulation of glycolysis with a concomitant influence on mitochondrial function that is active in endothelium, platelets, vascular smooth muscle and neural cells and the balance has a role in chronic inflammation, asthma, hypertension, sepsis and cancer.

Uncoupling of aerobic glycolysis
Potential cytotoxic mediators of endothelial cell (EC) apoptosis include increased formation of reactive oxygen and nitrogen species (ROSRNS) during the atherosclerotic process. Nitric oxide (NO) has a biphasic action on oxidative cell killing with low concentrations protecting against cell death, whereas higher concentrations are cytotoxic. High levels of NO can be produced by inducible nitric-oxide synthase in response to cytokine stimulation, primarily from macrophages, and elevated levels of NO is injurious to endothelium.Ccytochrome c release and caspase activation are involved in NO induced apoptosis. ROS also induces mitochondrial DNA damage in ECs, and this damage is accompanied by a decrease in mitochondrial RNA (mtRNA) transcripts, mitochondrial protein synthesis, and cellular ATP levels. Mitochondria have been recognized to play a pivotal role in the signaling cascade of apoptosis leading to atherosclerosis-induced damage in endothelial cells.
The processes involved in the signaling pathways leading to apoptosis are complex but have some degree of convergence between cell types including those in the vasculature. Release of cytochrome c from mitochondria is a proapoptotic signal, which activates several downstream signaling events including formation of the apoptosome and activation of caspases. Ubiquinol cytochrome c reductase (complex III) is a site for ROS formation, and cytochrome c oxidase (complex IV) is a target for the interaction of NO in mitochondria.
The impact of the inhibition of mitochondrial protein synthesis is particularly important in NO-dependent cytotoxicity, and depends also on other factors such as glycolysis. These authors examined whether the inhibition of mitochondrial protein synthesis by chloramphenicol increases the susceptibility of endothelial cells to undergo NO-dependent apoptosis in glucose-free media. Bovine aortic endothelial cells were treated with chloramphenicol, which resulted in a decreased ratio of mitochondrial complex IV to cytochrome c and increased oxidant production in the cell. Inhibition of mitochondrial protein synthesis was associated with a greater susceptibility of the cells to apoptosis induced by NO in glucose-free medium.
Inhibition of mitochondrial protein synthesis results in increased endothelial cell susceptibility to nitric oxide-induced apoptosis. A Ramachandran, DR Moellering, E Ceaser, S Shiva, J Xu, and V Darley-Usmar. PNAS May 14, 2002: 99(10): 6643–6648 http://www.pnas.orgcgidoi10.1073pnas.102019899

Nitric oxide (NO) is a ubiquitous signaling molecule whose physiological roles mediated through the activation of the soluble guanylate cyclase are now clearly recognized. At physiological concentrations, NO also inhibits the mitochondrial enzyme cytochrome c oxidase (complex IV) in competition with oxygen, and recently we have suggested that the interplay between the two gases allows this enzyme to act as an oxygen sensor in cells. In addition, NO plays a variety of patho-physiological roles, some of which also may be the consequence of its action at a mitochondrial level. We have characterized the sequence of events that follow inhibition of complex IV by continuous exposure to NO.
The mitochondrion is a key organelle in the control of cell death. Nitric oxide (NO) inhibits complex IV in the respiratory chain and is reported to possess both proapoptotic and antiapoptotic actions. We investigated the effects of continuous inhibition of respiration by NO on mitochondrial energy status and cell viability. Serum-deprived human T cell leukemia (Jurkat) cells were exposed to NO at a concentration that caused continuous and complete (;85%) inhibition of respiration. Serum deprivation caused progressive loss of mitochondrial membrane potential (Dcm) and apoptotic cell death. In the presence of NO, Dcm was maintained compared to controls, and cells were protected from apoptosis. Similar results were obtained by using staurosporin as the apoptotic stimulus. As exposure of serum-deprived cells to NO progressed (>5 h), however, Dcm fell, correlating with the appearance of early apoptotic features and a decrease in cell viability. Glucose deprivation or iodoacetate treatment of cells in the presence of NO resulted in a collapse of Dcm, demonstrating involvement of glycolytic ATP in its maintenance. Under these conditions cell viability also was decreased. Treatment with oligomycin and or bongkrekic acid indicated that the maintenance of Dcm during exposure to NO is caused by reversal of the ATP synthase and other electrogenic pumps. Thus, blockade of complex IV by NO initiates a protective action in the mitochondrion to maintain Dcm; this results in prevention of apoptosis. It is likely that during cellular stress involving increased generation of NO this compound will trigger a similar sequence of events, depending on its concentration and duration of release. (mitochondrial membrane potential ; apoptosis ; necrosis)

The effect of nitric oxide on cell respiration: A key to understanding its role in cell survival or death. B Beltra, A Mathur, MR Duchen, JD. Erusalimsky, and S Moncada. PNAS Dec 19, 2000; 97(26):4602–14607.

Another study by this group shows that inhibition of respiration by exogenous nitric oxide (NO) in Jurkat cells leads to mitochondrial membrane hyperpolarization dependent on the utilization of glycolytic ATP by the F1Fo-ATPase and other transporters acting in reverse mode. This process also occurs in astrocytes, which are highly glycolytic cells, but not in neurons , which do not invoke glycolysis to maintain ATP concentrations. In addition, this hyperpolarization correlates with protection against apoptotic cell death. Others found an early phase of mitochondrial hyperpolarization after treatment of a variety of cells with different pro-apoptotic stimuli, which precedes the generation of free. At present, no satisfactory explanation has been proposed to explain the mechanism of hyperpolarization, the reasons why free radicals are released from the mitochondrion, or the connection of these phenomena with apoptosis.
The authors surmise that a pro-apoptotic stimulus, anti-Fas Ab, leads to release of endogenous NO from Jurkat cells in sufficient amounts to inhibit cell respiration and cause a hyperpolarization dependent on the reversal of the F1Fo-ATPase. Moreover, the reduction of the mitochondrial electron transport chain, after inhibition of cytochrome oxidase by NO, leads to generation of superoxide anion (O2). They suggest the process is a cellular defense response that may be overcome by pro-apoptotic mechanisms that occur in parallel.

Inhibition of mitochondrial respiration by endogenous nitric oxide: A critical step in Fas signaling. B Beltran, M Quintero, E Garcıa-Zaragoza, E O’Connor, JV. Esplugues, and Salvador Moncada. PNAS June 25, 2002 99(13): 8892–8897. http://www.pnas.orgcgidoi10.1073pnas.092259799

Nitric oxide has been shown to render cells resistant to oxidative stress. Mechanisms proposed for the ability of nitric oxide to protect cells against oxidative stress include reactions of nitric oxide and the induction of adaptive responses that require protein synthesis. Nitric oxide forms iron complexes preventing the formation of strong oxidants. In addition, reactions of nitric oxide with lipid and or organic radicals protect against membrane peroxidation and peroxidative chemistry-induced cell injury. Exposure to low, nonlethal doses of nitric oxide induces adaptive responses that render cells resistant to lethal concentrations of nitric oxide and or peroxides, such as, the induction of hemoxygenase-1 (HO-1) and Mn superoxide dismutase. The up-regulation of HO-1 was accompanied by an increase in ferritin to account for the release of iron from HO-1, indicating a role of both iron heme and nonheme iron for peroxide-mediated cellular injury. Further, nitric oxide, by regulating critical mitochondrial functions such as respiration, membrane potential, and release of cytochrome c, is able to trigger defense mechanisms against cell death induced by pro-apoptotic stimuli.
This study investigates the potential contribution of nitric oxide’s ability to protect cells from oxidative stress, low steady state levels of nitric oxide generated by endothelial nitric oxide synthase (eNOS) and the mechanisms of protection against H2O2. Spontaneously transformed human ECV304 cells, which normally do not express eNOS, were stably transfected with a green fluorescent-tagged eNOS cDNA. The eNOS-transfected cells were found to be resistant to injury and delayed death following a 2-h exposure to H2O2 (50–150 mM). Inhibition of nitric oxide synthesis abolished the protective effect against H2O2 exposure. The ability of nitric oxide to protect cells depended on the presence of respiring mitochondria. ECV3041 eNOS cells with diminished mitochondria respiration are injured to the same extent as non-transfected ECV304 cells, and recovery of mitochondrial respiration restores the ability of nitric oxide to protect against H2O2-induced death. Nitric oxide had a profound effect in cell metabolism, because ECV3041eNOS cells had lower steady state levels of ATP and higher utilization of glucose via the glycolytic pathway than ECV304 cells. However, the protective effect of nitric oxide against H2O2 exposure is not reproduced in ECV304 cells after treatment with azide and oligomycin suggesting that the dynamic regulation of respiration by nitric oxide represent a critical and unrecognized primary line of defense against oxidative stress.

Dynamic regulation of metabolism and respiration by endogenously produced nitric oxide protects against oxidative stress. E Paxinou, M Weisse, Q Chen, JM Souza, et al. PNAS Sept 25, 2001; 98( 20): 11575–11580. http://www.pnas.orgycgiydoiy10.1073ypnas.201293198.

Nitric oxide (NO) mediates a variety of biological effects including relaxation of blood vessels, cytotoxicity of activated macrophages, and formation of cGMP by activation of glutamate receptors of neurons. NO has also been implicated for such pathophysiological conditions as destruction of tumor cells by macrophages, rheumatoid arthritis, and focal brain ischemia. Some of these effects of NO are associated with hypoxic conditions. O2 radicals and ions that result from reactivity of NO are presumed to be involved in NO cytotoxicity. These investigators report that adaptive cellular response controlled by the transcription factor hypoxia-inducible factor 1 (HIF-1) in hypoxia is suppressed by NO. Induction of erythropoietin and glycolytic aldolase A mRNAs in hypoxically cultured Hep3B cells, a human hepatoma cell line, was completely and partially inhibited, respectively, by the addition of sodium nitroprusside (SNP), which spontaneously releases NO. A reporter plasmid carrying four hypoxia-response element sequences connected to the luciferase structural gene was constructed and transfected into Hep3B cells. Inducibly expressed luciferase activity in hypoxia was inhibited by the addition of SNP and two other structurally different NO donors, S-nitroso-Lglutathione and 3-morpholinosydnonimine, giving IC50 values of 7.8, 211, and 490 mM, respectively. Inhibition by SNP was also observed in Neuro 2A and HeLa cells, indicating that the inhibition was not cell-type-specific. The vascular endothelial growth factor promoter activity that is controlled by HIF-1 was also inhibited by SNP (IC50 5 6.6 mM). Induction generated by the addition of cobalt ion (this treatment mimics hypoxia) was also inhibited by SNP (IC50 5 2.5 mM). Increased luciferase activity expressed by cotransfection of effector plasmids for HIF-1a or HIF-1a-like factor in hypoxia was also inhibited by the NO donor. We also showed that the inhibition was performed by blocking an activation step of HIF-1a to a DNA-binding form.
Inhibition of hypoxia-inducible factor 1 activity by nitric oxide donors in hypoxia. K Sogawa, K Numayama-Tsuruta, M Ema, M Abe, et al. Proc. Natl. Acad. Sci. USA (Biochemistry) June 1998; 95:7368–7373. 1998. The National Academy of Sciences 0027-8424.98.957368-6. http:yywww.pnas.org.

The role of nitrogen metabolism in the survival of prolonged periods of waterlogging was investigated in highly flood-tolerant, nodulated Lotus japonicus plants. Alanine production revealed to be a critical hypoxic pathway. Alanine is the only amino acid whose biosynthesis is not inhibited by nitrogen deficiency resulting from RNA interference silencing of nodular leghemoglobin. The metabolic changes that were induced following waterlogging can be best explained by the activation of alanine metabolism in combination with the modular operation of a split tricarboxylic acid pathway. The sum result of this metabolic scenario is the accumulation of alanine and succinate and the production of extra ATP under hypoxia. The importance of alanine metabolism is discussed with respect to its ability to regulate the level of pyruvate, and this and all other changes are discussed in the context of current models concerning the regulation of plant metabolism.
Glycolysis and the Tricarboxylic Acid Cycle Are Linked by Alanine Aminotransferase during Hypoxia Induced by Waterlogging of Lotus japonicus[W][OA]. M Rocha, F Licausi, WL Arau´ jo, A Nunes-Nesi, et al. Plant Physiology Mar 2010; 152: 1501–1513. http://www.plantphysiol.org 2010 Amer Soc Plant Biologists

DNA damage occurs in ischemia, excitotoxicity, inflammation, and other disorders that affect the central nervous system (CNS). Extensive DNA damage triggers cell death and in the mature CNS, this occurs primarily through activation of the poly(ADP-ribose) polymerase-1 (PARP-1) cell death pathway. PARP-1 is an abundant nuclear enzyme that, when activated by DNA damage, consumes nicotinamide adenine dinucleotide (NAD)+ to form poly(ADP-ribose) on acceptor proteins. The PARP-1 activation leads to cell death. We used mouse astrocyte cultures to explore the bioenergetic effects of NAD+ depletion by PARP-1 and the role of NAD+ depletion in this cell death program. PARP-1 activation led to a rapid but incomplete depletion of astrocyte NAD+, a near-complete block in glycolysis, and eventual cell death. Repletion of intracellular NAD restored glycolytic function and prevented cell death. The addition of non-glucose substrates to the medium, pyruvate, glutamate, or glutamine, also prevented astrocyte death after PARP-1 activation.
These findings suggest a sequence of events in which NAD+ depletion is a key event linking DNA damage to metabolic impairment and cell deathm. A similar scenario has been proposed by Zong et al. (2004), based on the finding that cell types that depend on aerobic glycolysis for ATP production exhibit a particularly high sensitivity to DNA damage and PARP-1 activation. In mature brain, glucose is normally the dominant metabolic substrate due to relatively slow transport of other metabolites across the blood– brain barrier. Oncein brain, glucose may be metabolized directly by neurons and glia or may be metabolized to lactate in glia and thelactate subsequently shuttled to neurons for oxidative metabolism (Dringen et al., 1993; Pellerin and Magistretti,1994; Wender et al., 2000; Dienel and Cruz, 2004). In either case, a block in glycolytic flux produced by NAD depletion will block energy metabolism in both neurons and glia in brain. Interestingly, the lactate shuttle hypothesis raises the possibility that activation of PARP-1 selectively in astroglia might also block energy metabolism in neurons.

These studies suggest PARP-1 activation leads to rapid depletion of the cytosolic but not the mitochondrial NAD+ pool. Depletion of the cytosolic NAD+ pool renders the cells unable to utilize glucose as a metabolic substrate. Under conditions where glucose is the only available metabolic substrate, this leads to cell death. This cell death pathway is particularly germane to brain because glucose is normally the only metabolic substrate that is transported rapidly across the blood–brain barrier. © 2004 Wiley-Liss, Inc.
Key words: mitochondria; permeability transition; poly(ADP-ribose) polymerase; ischemia; peroxynitrite
NAD+as a metabolic link between DNA damage and cell death. DNA damage induced by alkylating agents, oxidative stress, or other agents causes PARP-1 activation. PARP-1 activation leads to depletion in cytosolic NAD with, initially, a relative preservation of mitochondrial NAD and mitochondrial function. The depletion in cytosolic NAD+ blocks glycolysis, and in cells in which glucose is the primary energy substrate, this in turn leads to a block in substrate flux to mitochondria. The resulting mitochondrial dysfunction leads to mitochondrial permeability transition (MPT) and subsequent downstream events culminating in cell death.
NAD+ as a Metabolic Link Between DNA Damage and Cell Death. W Ying, CC Alano, P Garnier, and RA Swanson. Journal of Neuroscience Research 2005;79:216–223
Key words: glycolysis, mitochondrial energy production, nitric oxide
Abbreviations: NO, nitric oxide; SNAP, S-nitroso-N-acetylpenicyllamine; SNP, sodium nitroprusside.
The results indicate that: 1) in porcine platelets NO is able to diminish mitochondrial energy production through the inhibition of cytochrome oxidase, 2) the inhibitory effect of NO on platelet secretion (but not aggregation) can be attributed to the reduction of mitochondrial energy production.
Nitric oxide (NO) has been increasingly recognized as an important intra- and intercellular messenger molecule with a physiological role in vascular relaxation, platelet physiology, neurotransmission and immune responses (Moncada et al., 1991; Radomski et al., 1996; Szabó, 1996; Riedel et al., 1999; Titheradge 1999). In vitro NO is a strong inhibitor of platelet adhesion and aggregation (Radomski et al., 1996; Riedel et al., 1999;nSogo et al., 2000). In the blood stream, platelets remain in contact with NO that is permanently released from the endothelial cells and from activated macrophages (Moncada et al., 1991; Riedel et al., 1999; Titheradge 1999). It has been suggested that the activated platelet itself is able to produce NO (Lantoine et al., 1995; Zhou et al., 1995; Radomski et al., 1996). The mechanism responsible for the inhibitory effect of NO on platelet responses is not entirely clear. It is believed that the main intracellular target for NO in platelets is soluble cytosolic guanylate cyclase (Waldman & Walter 1989; Schmidt et al., 1993; Wang et al., 1998). NO activates the enzyme (Schmidt et al., 1993). Thus, elevated intracellular cGMP level inhibits platelet activation. There are suggestions, however, that elevated cGMP may not be the only intracellular factor directly involved in the inhibition of platelet activation (Gordge et al., 1998; Sogo et al., 2000; Beghetti et al., 2003).
Platelets are fairly active metabolically and have a total ATP turnover rate of about 3–8 times that of resting mammalian muscle (Akkerman, 1978; Akkerman et al., 1978; Holmsen, 1981; Niu et al., 1996). Platelets contain mitochondria which enable these cells to produce energy both in the oxidative and anaerobic way (Holmsen, 1981). Under aerobic conditions, ATP is produced by aerobic glycolysis using glucose or glycogen which can account for 30–50% of total ATP production, and by oxidative metabolism using glucose and glycogen (6–11%), amino-acids (7%) or free fatty acids (20–40%) (Holmsen 1981; Guppy et al., 1990; Niu et al., 1996).
The inhibition of mitochondrial respiration by removing oxygen or by respiratory chain blockers (antimycin A, cyanide, rotenone) results in the stimulation of glycolytic flux (Guppy et al., 1990). This phenomenon is known as Pasteur effect and indicates that in platelets glycolysis and mitochondrial respiration are tightly functionally connected (Akkerman, 1978; Holmsen, 1981; Guppy et al., 1995; Niu et al., 1996). It has been reported that the activation of human platelets by high concentration of thrombin is accompanied by an acceleration of lactate production and an increase in oxygen consumption (Akkerman & Holmsen, 1981; Niu et al., 1996).
The results presented here suggest that also porcine blood platelets stimulated by collagen produce more lactate. This indicates that both glycolytic and oxidativeATP production supports platelet responses. This also indicates that blocking of energy production in platelets may decrease their responses. It is well established that platelet responses have different metabolic energy (ATP) requirements increasing in the order: aggregation< dense and alfa granule secretion < acid hydrolase secretion (Holmsen et al., 1982; Verhoeven et al., 1984; Morimoto & Ogihara, 1996).
The present results indicate that exogenously added NO (in the form of NO donors)stimulates glycolysis in intact porcine platelets. Since in platelets glycolysis and mitochondrial respiration are tightly functionally connected, this can be interpreted to mean that the stimulatory effectof NO on glycolysis in intact platelets may be produced by non-functional mitochondria.This can be really the case since NO donors are able to inhibit both mitochondrial respiration and platelet cytochrome oxidase. Interestingly, the concentrations of NO donors inhibiting mitochondrial respiration and cytochrome oxidase were similar to those stimulating glycolysis in intact platelets.
Studies performed on intact J774 cells have shown that mitochondrial complex I is inhibited only after a prolonged (6–18 h) exposure to NO and that this inhibition appears to result from S-nitrosylation of critical thiols in the enzyme complex (Clementi et al., 1998). Further studies are needed to establish whether long term exposure of platelets to NO affects Mitochondrial complexes I and II.
Comparison of the concentrations of SNP and SNAP affecting cytochrome oxidase activityand mitochondrial respiration with those reducing the platelet responses indicates that NO cannot significantly reduce platelet aggregation through the inhibition of oxidative energy production. By contrast, the concentrations of the NO donors inhibiting platelet secretion, mitochondrial respiration and cytochrome oxidase were similar. This and the fact that the platelet release reaction strongly depends on the oxidative energy production may suggest that in porcine platelets NO can affect platelet secretion through the inhibition of mitochondrial energy production at the step of cytochrome oxidase.

Taking into account that platelets may contain NO synthase and are able to produce significant amounts of NO (Berkels et al., 1997)it seems possible that nitric oxide can function in these cells as a physiological regulator of mitochondrial energy production.
Nitric oxide and platelet energy metabolism. M Tomasiak, H Stelmach, T Rusak and J Wysocka. Acta Biochimica Polonica 2004; 51(3):789–803

These authors previously investigated the bioenergetic consequences of activating J774.A1 macrophages (MФ) with interferon (IFN)γ and lipopolysaccharide (LPS) and found that there is a nitric oxide (NO)-dependent mitochondrial impairment and stabilization of hypoxia inducible factor (HIF)-1α, which synergize to activate glycolysis and generate large
quantities of ATP. We now demonstrate, using TMRM fluorescence and time-lapse confocal microscopy, that these cells maintain a high mitochondrial membrane potential (ΔΨm) despite the complete inhibition of respiration. The maintenance of high ΔΨm is due to the utilization of a significant proportion of glycolytically generated ATP as a defence mechanism against cell death. This is achieved by the reverse functioning of FoF1-ATP synthase and adenine nucleotide translocase (ANT). Treatment of activated MФ with inhibitors of either of these enzymes, but not with inhibitors of the respiratory chain complexes I to IV, led to a collapse in ΔΨm and to an immediate increase in intracellular [ATP], due to the prevention of ATP hydrolysis by the FoF1-ATP synthase. This collapse in ΔΨm was followed by translocation of Bax from cytosol to the mitochondria, release of cytochrome c into the cytosol, activation of caspase 3 and 9 and subsequent apoptotic cell death. Our results indicate that during inflammatory activation “glycolytically competent cells” such as MФ utilize significant amounts of the glycolytically-generated ATP to maintain ΔΨm and thereby prevent apoptosis.

Activated macrophages utilize glycolytic ATP to maintain mitochondrial membranepotential and prevent apoptotic cell death. A Garedew, SO Henderson, S Moncada. Cell Death and Differentiation. 2010. DOI : 10.1038/cdd.2010.27
The effects of the sodium nitroprusside (SNP), a nitric oxide (NO) donor clinically used in the treatment of hypertensive emergencies on the energy production of rat reticulocytes were investigated. Rat reticulocyte-rich red blood cell suspensions were aerobically incubated without (control) or in the presence of different concentrations of SNP (0.1, 0.25, 0.5, 1.0 mM). SNP decreased total and coupled, but increased uncoupled oxygen consumption. This was accompanied by the stimulation of glycolysis, as measured by increased glucose consumption and lactate accumulation. Levels of all glycolytic intermediates indicate stimulation of hexokinase-phosphofructo kinase (HK-PFK), glyceraldehyde 3-phosphate dehydrogenase (GAPD) and pyruvate kinase (PK) activities in the presence of SNP. Due to the decrease of coupled oxygen consumption in the presence of SNP, ATP production via oxidative phosphorylation was significantly diminished. Simultaneous increase of glycolytic ATP production was not enough to provide constant ATP production. In addition, SNP significantly decreased ATP level, which was accompanied with increased ADP and AMP levels. However, the level of total adenine nucleotides was significantly lower, which was the consequence of increased catabolism of adenine nucleotides (increased hypoxanthine level). ATP/ADP ratio and adenylate energy charge level were significantly decreased. In conclusion, SNP induced inhibition of oxidative phosphorylation, stimulation of glycolysis, but depletion of total energy production in rat reticulocytes. These alterations were accompanied with instability of energy status.

Effects of Exogenous Donor of Nitric Oxide – Sodium Nitroprusside on Energy Production of Rat Reticulocytes. SD MALETIĆ, L M DRAGIĆEVIĆ-DJOKOVIĆ, BI OGNJANOVIĆ, RV ŽIKIĆ, AŠ ŠTAJN, MB SPASIĆ.
Physiol. Res. 2004;53: 439-447.

Key points to take from this:
1. The role of NO in regulating cellular death is in many organs and central to this function is the stabilization of mitochondria through sufficient levels of NO. High levels of eNO leads to mitochondrial dysfunction that increases the dependence of ATP generated from glycolysis.
2. This is accompanied by inhibition of oxidative phosphorylation and stimulation of glycolysis, which brings the discussion to a different domain – cancer growth and Warburgh Effect.
3. This is accompanied by PPAR activation, cytoplasmic NAD+ depletion, and inhibition of glycolysis (critical in cells dependent on aerobic glycolysis), depletion of total energy production, and apoptosis.
4. Maintenance of high glycolytic generation of ATP is essential for cellular defense, but the oxygen consumption is uncoupled.
5. NO donors inhibiting mitochondrial respiration and cytochrome oxidase are similar to those stimulating glycolysis

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Author and Reporter: Ritu Saxena, Ph.D.  

Mitochondria is an important cell organelle that is associated with several key cellular functions as energy production, anabolism, calcium homeostasis and cell programmed death, and any abnormalities occurring in mitochondria would lead to alteration of normal cellular function.

Role of mitochondria in cancer has long been implicated. Post published on September 1, 2012 (https://pharmaceuticalintelligence.com/2012/09/01/mitochondria-and-cancer-an-overview/) presents a brief overview of the mechanisms by which mitochondrial defects could be associated with cancer. Different studies on various types of Cancers have tried to determine the mtDNA mutations and the mechanisms involved. An important aspect of cancerous progression is the cancer cell migration and it has been observed that mitochondrial dysfunction is involved in cancer cell migration. However, the molecular mechanism still needs to be deciphered.

A group from Taiwan recently published their findings in the Biochimica et Biophysica Acta journal stating that enhanced β5-integrin expression was involved in promoting cell migration in human gastric cancer cell line as a result of mitochondrial dysfunction.

The authors used human gastric cancer cell line, SC-M1 cells for their studies. The methodology followed was to first create mitochondrial dysfunction in the SC-M1 cells by the use of oxidative phosphorylation inhibitors: oligomycin (Complex V inhibitor) and antimycin A (Complex III inhibitor) thereby inhibiting mitochondrial function. The results indicated that impaired oxidative phosphorylation caused an increase in the intracellular Reactive Oxygen Species (ROS) that lead to an increased cell migration in SC-M1 cells.

Different types of integrin molecules have been implicated in cell migration. Hung et al extracted RNA and protein from SC-M1 cells in order to study the different types of integrins, and observed that the levels of β5-integrin were significantly upregulated in SC-M1 cells.  Simultaneously, the surface expression of the dimer- β5-integrin and αv-integrin, was studied in cancer cells with using FACS. The analysis revealed a higher surface expression of the dimer corresponding to the higher levels of the protein and RNA results of  β5-integrin expression in SC-M1 cells with mitochondrial dysfunction. Infact, a subpopulation of SC-M1 cells that showed higher migration capability (SC-M1-3rd) was observed to harbor a higher lever of β5-integrin expression, correlating β5-integrin expression with cell migration ability. The experiments supported the role of β5-integrin in cell migration in gastric cancer cells.

Finally, authors confirmed the in vitro results in the human gastric cancer samples. Immunohistochemical analysis revealed that β5-integrin was stained positive in around 73% of the cancer samples. Additionally, the higher expression levels of β5-integrin could be correlated with the invasive ability and more aggressive behavior of gastric cancer cells.

Authors stated “our study pinpoints another aspect that links the induction of intracellular ROS level in mitochondrial dysfunction gastric cancer cells with the activation of αvβ5-integrin. Taken together, the induction of β5-integrin is important to gastric cancer metastasis, especially in cancer cells that exhibit mitochondrial dysfunction.”

Thus, blockage of αvβ5-integrin function by antibodies might be tested as a potential therapy for preventing or delaying gastric cancer metastasis, especially in gastric cancers harboring mitochondrial dysfunction.

Sources:

Research article: http://www.ncbi.nlm.nih.gov/pubmed?term=22561002

Related posts: https://pharmaceuticalintelligence.com/2012/09/01/mitochondria-and-cancer-an-overview/

https://pharmaceuticalintelligence.com/2012/09/06/clinical-genetics-personalized-medicine-molecular-diagnostics-consumer-targeted-dna-consumer-genetics-conference-cgc-october-3-5-2012-seaport-hotel-boston-ma/

https://pharmaceuticalintelligence.com/2012/08/14/detecting-potential-toxicity-in-mitochondria/

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