Abstract 208: Augmented Cerebrovascular Responses to Diazoxide, an Opener of MitokAtp Channels, Following Middle Cerebral Artery (MCA) Occlusion in Rats

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Ibolya Rutkai ◽  
Somhrita Dutta ◽  
Dana Liu ◽  
Prasad V Katakam ◽  
David W Busija
Keyword(s):  
Mitochondrial Membrane ◽  
Major Effect ◽  
Mitochondrial Proteins ◽  
Mitochondrial Morphology ◽  
Western Blots ◽  
Vascular Responses ◽  
Cox Inhibitor ◽  
Mitochondrial Membrane Depolarization ◽  
Nos Inhibitor ◽  
And Function

Previous research has not examined the effects of ischemia on cerebral vascular responses to mitochondrial activation in experimental strokes caused by occlusion of the MCA (MCAO). We investigated the role and mechanisms of mitochondrial derived vasoreactivity in the MCA of male SD rats following 90 min ischemia/48 h reperfusion injury. Ischemia was induced ipsilaterally (I) and the contralateral (C) side was non-ischemic. Electron microscopy showed disrupted mitochondrial morphology on the I side. Western blots for expression of mitochondrial proteins (Mean±SEM of immunoband intensity normalized to β-actin and as C vs. I): DRP-1 (1±0.1 vs. 3.1±0.2); VDAC (0.4±0.1 vs. 0.8±0.2); and complex-V (1.3±0.3 vs.2.0±0.3) as well as the non-mitochondrial proteins: phosphorylated (ph) ph-nNOS (0.4±0.1 vs. 0.7±0.1); ph-eNOS (0.2±0.06 vs. 0.7±0.2); COX-1 (0.5±0.1 vs. 0.8±0.1); and COX-2 (0.16±0.04 vs. 0.3±0.03) were elevated on I compared with C. The I mitochondrial membrane potential was greater (165±7 %) compared with C using TMRE fluorescence. Mitochondrial membrane depolarization decreased the TMRE intensity in both groups (100 to 80±3 vs. 165±7 to 70±4). Vascular responses of the MCA were characterized using the isolated, pressurized artery technique. Vasodilation in response to Ach (12±2 vs. 3±1), BK (42±9 vs. 32±6), SNP (75±7 vs. 38±4), and vasoconstriction to serotonin (65±8 vs. 33±7), were significantly decreased in I compared to C MCAs. On the other hand, 50 μM DZ induced vasodilation was enhanced in I arteries compared with C (5±1 vs. 17±2). Diazoxide induced dilation was decreased in the presence of the NOS inhibitor L-NAME (5±1 to 1±2 vs. 17±2 to 3±2) and the non-selective COX inhibitor indomethacin (5±1 to 1±2 vs.17±2 to 6±3). Our results indicate that experimental stoke has an major effect on mitochondria via inducing mitochondrial biogenesis leading to altered morphology, protein expression and function. Furthermore, the nitric-oxide and prostanoid pathways appear to be involved in enhanced diazoxide mediated vasodilation after MCAO. We speculate that targeting mitochondria may be useful therapy for improving outcome in stroke patients.

scholarly journals Analysis of mitochondrial morphology and function with novel fixable fluorescent stains.

1996 ◽  
Vol 44 (12) ◽  
pp. 1363-1372 ◽  
Author(s):  
M Poot ◽  
Y Z Zhang ◽  
J A Krämer ◽  
K S Wells ◽  
L J Jones ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Epifluorescence Microscopy ◽  
Mitochondrial Morphology ◽  
Microtubule Network ◽  
Multiple Features ◽  
Permeabilized Cells ◽  
Dye Staining ◽  
And Function

Investigation of mitochondrial morphology and function has been hampered because photostable, mitochondrion-specific stains that are retained in fixed, permeabilized cells have not been available. We found that in live cell preparations, the CMXRos and H2-CMXRos dyes were more photostable than rhodamine 123. In addition, fluorescence and morphology of mitochondria stained with the CMXRos and CMXRos-H2 dyes were preserved even after formaldehyde fixation and acetone permeabilization. Using epifluorescence microscopy, we showed that CMXRos and H2-CMXRos dye fluorescence fully co-localized with antibodies to subunit I of cytochrome c oxidase, indicating that the dyes specifically stain mitochondria. Confocal microscopy of these mitochondria yielded colored banding patterns, suggesting that these dyes and the mitochondrial enzyme localize to different suborganellar regions. Therefore, these stains provide powerful tools for detailed analysis of mitochondrial fine structure. We also used poisons that decrease mitochondrial membrane potential and an inhibitor of respiration complex II to show by flow cytometry that the fluorescence intensity of CMXRos and H2-CMXRos dye staining responds to changes in mitochondrial membrane potential and function. Hence, CMXRos has the potential to monitor changes in mitochondrial function. In addition, CMXRos staining was used in conjunction with spectrally distinct fluorescent probes for the cell nucleus and the microtubule network to concomitantly evaluate multiple features of cell morphology.

scholarly journals Dissociation of mitochondrial HK-II elicits mitophagy and confers cardioprotection against ischemia

2019 ◽  
Vol 10 (10) ◽  
Author(s):  
Valerie P. Tan ◽  
Jeffrey M. Smith ◽  
Michelle Tu ◽  
Justin D. Yu ◽  
Eric Y. Ding ◽  
...  
Keyword(s):  
Mitochondrial Membrane ◽  
Intracellular Localization ◽  
Mitochondrial Proteins ◽  
Hexokinase Ii ◽  
Pharmacological Interventions ◽  
Mitochondrial Quality Control ◽  
Cellular Event ◽  
Mitochondrial Membrane Depolarization ◽  
Ischemic Stress

Abstract Preservation of mitochondrial integrity is critical for maintaining cellular homeostasis. Mitophagy is a mitochondria-specific type of autophagy which eliminates damaged mitochondria thereby contributing to mitochondrial quality control. Depolarization of the mitochondrial membrane potential is an established mechanism for inducing mitophagy, mediated through PINK1 stabilization and Parkin recruitment to mitochondria. Hexokinase-II (HK-II) which catalyzes the first step in glucose metabolism, also functions as a signaling molecule to regulate cell survival, and a significant fraction of cellular HK-II is associated with mitochondria (mitoHK-II). We demonstrate here that pharmacological interventions and adenoviral expression of a mitoHK-II dissociating peptide which reduce mitoHK-II levels lead to robust increases in mitochondrial Parkin and ubiquitination of mitochondrial proteins in cardiomyocytes and in a human glioblastoma cell line 1321N1, independent of mitochondrial membrane depolarization or PINK1 accumulation. MitoHK-II dissociation-induced mitophagy was demonstrated using Mito-Keima in cardiomyocytes and in 1321N1 cells. Subjecting cardiomyocytes or the in vivo heart to ischemia leads to modest dissociation of mitoHK-II. This response is potentiated by expression of the mitoHK-II dissociating peptide, which increases Parkin recruitment to mitochondria and, importantly, provides cardioprotection against ischemic stress. These results suggest that mitoHK-II dissociation is a physiologically relevant cellular event that is induced by ischemic stress, the enhancement of which protects against ischemic damage. The mechanism which underlies the effects of mitoHK-II dissociation can be attributed to the ability of Bcl2-associated athanogene 5 (BAG5), an inhibitor of Parkin, to localize to mitochondria and form a molecular complex with HK-II. Overexpression of BAG5 attenuates while knockdown of BAG5 sensitizes the effect of mitoHK-II dissociation on mitophagy. We suggest that HK-II, a glycolytic molecule, can function as a sensor for metabolic derangements at mitochondria to trigger mitophagy, and modulating the intracellular localization of HK-II could be a novel way of regulating mitophagy to prevent cell death induced by ischemic stress.

scholarly journals Heterochronic Parabiosis: Old Blood Induces Changes in Mitochondrial Structure and Function of Young Mice

2020 ◽  
Author(s):  
Jenny L Gonzalez-Armenta ◽  
Ning Li ◽  
Rae-Ling Lee ◽  
Baisong Lu ◽  
Anthony J A Molina
Keyword(s):  
Mitochondrial Respiration ◽  
Complex I ◽  
Structure And Function ◽  
Muscle Performance ◽  
Mitochondrial Morphology ◽  
Positive Effects ◽  
Mitochondrial Structure ◽  
And Function ◽  
Old Mice ◽  
Young Mice

Abstract Heterochronic parabiosis models have been utilized to demonstrate the role of blood-borne circulating factors in systemic effects of aging. In previous studies, heterochronic parabiosis has shown positive effects across multiple tissues in old mice. More recently, a study demonstrated old blood had a more profound negative effect on muscle performance and neurogenesis of young mice. In this study, we used heterochronic parabiosis to test the hypothesis that circulating factors mediate mitochondrial bioenergetic decline, a well-established biological hallmark of aging. We examined mitochondrial morphology, expression of mitochondrial complexes, and mitochondrial respiration from skeletal muscle of mice connected as heterochronic pairs, as well as young and old isochronic controls. Our results indicate that young heterochronic mice had significantly lower total mitochondrial content and on average had significantly smaller mitochondria compared to young isochronic controls. Expression of complex IV followed a similar pattern: young heterochronic mice had a trend for lower expression compared to young isochronic controls. Additionally, respirometric analyses indicate that young heterochronic mice had significantly lower complex I, complex I + II, and maximal mitochondrial respiration and a trend for lower complex II-driven respiration compared to young isochronic controls. Interestingly, we did not observe significant improvements in old heterochronic mice compared to old isochronic controls, demonstrating the profound deleterious effects of circulating factors from old mice on mitochondrial structure and function. We also found no significant differences between the young and old heterochronic mice, demonstrating that circulating factors can be a driver of age-related differences in mitochondrial structure and function.

scholarly journals The Interplay between Mitochondrial Morphology and Myomitokines in Aging Sarcopenia

2020 ◽  
Vol 22 (1) ◽  
pp. 91
Author(s):  
Vanina Romanello
Keyword(s):  
Mitochondrial Dysfunction ◽  
Poor Quality ◽  
Whole Body ◽  
Fibroblast Growth Factor 21 ◽  
Mitochondrial Morphology ◽  
Mass Force ◽  
Major Mechanism ◽  
Muscle Aging ◽  
And Function

Sarcopenia is a chronic disease characterized by the progressive loss of skeletal muscle mass, force, and function during aging. It is an emerging public problem associated with poor quality of life, disability, frailty, and high mortality. A decline in mitochondria quality control pathways constitutes a major mechanism driving aging sarcopenia, causing abnormal organelle accumulation over a lifetime. The resulting mitochondrial dysfunction in sarcopenic muscles feedbacks systemically by releasing the myomitokines fibroblast growth factor 21 (FGF21) and growth and differentiation factor 15 (GDF15), influencing the whole-body homeostasis and dictating healthy or unhealthy aging. This review describes the principal pathways controlling mitochondrial quality, many of which are potential therapeutic targets against muscle aging, and the connection between mitochondrial dysfunction and the myomitokines FGF21 and GDF15 in the pathogenesis of aging sarcopenia.

Mitochondrial Morphology and Function

Muscle ◽  
2012 ◽  
pp. 217-229
Author(s):  
Fabio Di Lisa ◽  
Luca Scorrano
Keyword(s):  
Mitochondrial Morphology ◽  
And Function

Congenital neutropenia

Hematology ◽  
2009 ◽  
Vol 2009 (1) ◽  
pp. 344-350 ◽  
Author(s):  
Christoph Klein
Keyword(s):  
Ribosomal Proteins ◽  
Mitochondrial Proteins ◽  
Phenotypic Traits ◽  
Genetic Defects ◽  
Congenital Neutropenia ◽  
Lysosomal Function ◽  
Genes Encoding ◽  
And Function ◽  
Neutrophil Differentiation ◽  
Remarkable Progress

Abstract Congenital neutropenia comprises a variety of genetically heterogeneous phenotypic traits. Molecular elucidation of the underlying genetic defects has yielded important insights into the physiology of neutrophil differentiation and function. Non-syndromic variants of congenital neutropenia are caused by mutations in ELA2, HAX1, GFI1, or WAS. Syndromic variants of congenital neutropenia may be due to mutations in genes controlling glucose metabolism (SLC37A4, G6PC3) or lysosomal function (LYST, RAB27A, ROBLD3/p14, AP3B1, VPS13B). Furthermore, defects in genes encoding ribosomal proteins (SBDS, RMRP) and mitochondrial proteins (AK2, TAZ) are associated with congenital neutropenia syndromes. Despite remarkable progress in the field, many patients with congenital neutropenia cannot yet definitively be classified by genetic terms. This review addresses diagnostic and therapeutic aspects of congenital neutropenia and covers recent molecular and pathophysiological insights of selected congenital neutropenia syndromes.

VEGF increases endothelial permeability by separate signaling pathways involving ERK-1/2 and nitric oxide

2003 ◽  
Vol 284 (1) ◽  
pp. H92-H100 ◽  
Author(s):  
Jerome W. Breslin ◽  
Peter J. Pappas ◽  
Joaquim J. Cerveira ◽  
Robert W. Hobson ◽  
Walter N. Durán
Keyword(s):  
Nitric Oxide ◽  
Signaling Pathways ◽  
Mapk Pathway ◽  
Umbilical Vein ◽  
Endothelial Permeability ◽  
Western Blots ◽  
Cell Monolayers ◽  
Dna Oligonucleotides ◽  
Dextran 70 ◽  
Nos Inhibitor

We tested the hypothesis that VEGF regulates endothelial hyperpermeability to macromolecules by activating the ERK-1/2 MAPK pathway. We also tested whether PKC and nitric oxide (NO) mediate VEGF-induced increases in permeability via the ERK-1/2 pathway. FITC-Dextran 70 flux across human umbilical vein endothelial cell monolayers served as an index of permeability, whereas Western blots assessed the phosphorylation of ERK-1/2. VEGF-induced hyperpermeability was inhibited by antisense DNA oligonucleotides directed against ERK-1/2 and by blockade of MEK and Raf-1 activities (20 μM PD-98059 and 5 μM GW-5074). These blocking agents also reduced ERK-1/2 phosphorylation. The PKC inhibitor bisindolylmaleimide I (10 μM) blocked both VEGF-induced ERK-1/2 activation and hyperpermeability. The NO synthase (NOS) inhibitor N G-nitro-l-arginine methyl ester (200 μM) and the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidiazoline-1-oxyl-3-oxide (100 μM) abolished VEGF-induced hyperpermeability but did not block ERK-1/2 phosphorylation. These observations demonstrate VEGF-induced hyperpermeability involves activation of PKC and NOS as well as Raf-1, MEK, and ERK-1/2. Furthermore, our data suggest that ERK-1/2 and NOS are elements of different signaling pathways in VEGF-induced hyperpermeability.

Mitochondrial dysfunction is an early event in high-NaCl-induced apoptosis of mIMCD3 cells

2002 ◽  
Vol 282 (6) ◽  
pp. F981-F990 ◽  
Author(s):  
Luis Michea ◽  
Christian Combs ◽  
Peter Andrews ◽  
Natalia Dmitrieva ◽  
Maurice B. Burg
Keyword(s):  
Mitochondrial Dysfunction ◽  
Collecting Duct ◽  
Early Event ◽  
Mitochondrial Morphology ◽  
Cytochrome C Release ◽  
Activity Increase ◽  
Transmission Electron ◽  
Mitochondrial Membrane Depolarization ◽  
Medullary Collecting Duct ◽  
Induced Apoptosis

Raising osmolality to 700 mosmol/kgH2O by the addition of NaCl rapidly kills most murine inner renal medullary collecting duct cells (mIMCD3), but they survive at 500 mosmol/kgH2O. At 300 and 500 mosmol/kgH2O, NADH autofluorescence is present in a mitochondria-associated, punctate perinuclear pattern. Within 45 s to 30 min at 700 mosmol/kgH2O, the autofluorescence spreads diffusely throughout the cell. This correlates with mitochondrial membrane depolarization, measured as decreased tetramethylrhodamine methyl ester perchlorate (TMRM) fluorescence. Mitochondrial dysfunction should increase the cellular ADP/ATP ratio. In agreement, this ratio increases within 1–6 h. Mitochondrial morphology (transmission electron microscopy) is unaffected, but nuclear hypercondensation becomes evident. Progressive apoptosis occurs beginning 1 h after osmolality is raised to 700, but not to 500, mosmol/kgH2O. General caspase activity and caspase-9 activity increase only after 6 h at 700 mosmol/kgH2O. The mitochondrial Bcl-2/Bax ratio decreases within 1–3 h, but no cytochrome c release is evident. The mitochondria contain little p53 at any osmolality. Adding urea to 700 mosmol/kgH2O does not change NADH or TMRM fluorescence. We conclude that extreme acute hypertonicity causes a mitochondrial dysfunction involved in the initiation of apoptosis.

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