Monitoring of mitochondrial membrane potential in isolated perfused rat heart

1984 ◽  
Vol 247 (4) ◽  
pp. H508-H516
Author(s):  
R. A. Kauppinen ◽  
I. E. Hassinen
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Optical Methods ◽  
Perfused Heart ◽  
Rat Hearts ◽  
Carbonyl Cyanide ◽  
Perfused Rat Hearts ◽  
Beating Rate ◽  
Isolated Perfused Rat Hearts

Optical methods were tested for measuring the membrane potential changes of mitochondria in isolated perfused rat hearts. Safranin was found to be rapidly taken up by the Langendorff-perfused heart, and after loading with the dye there was practically no washout of the stain during perfusion with Krebs-Ringer bicarbonate solution. Staining with safranin induced the appearance of an intense absorption band in the reflectance spectrum of the heart, but the absorbance spectrum changes were not useful for monitoring the mitochondrial membrane potential changes because of interference by endogenous hemoproteins. The fluorescence intensity, however, responded in a manner which indicated that its changes originated from dye attached to the mitochondria. A decrease of the fluorescence was found on energizing the mitochondria by decreasing the cellular energy consumption by arrest induced by 18 mM K+ or by decreasing the beating rate of an electrically paced heart from 5 Hz to the endogenous ventricular frequency of 1.5 Hz. In hearts arrested by Ca2+ depletion, 18 mM K+ did not affect the safranin fluorescence. This was taken to indicate that under these conditions the safranin fluorescence was not sensitive to the plasma membrane potential. The uncoupler carbonyl cyanide m-chlorophenylhydrazone induced an intense enhancement of safranin fluorescence in the perfused heart, demonstrating that the probe is sensitive to mitochondrial membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of BDM, verapamil, and cardiac work on mitochondrial membrane potential in perfused rat hearts

1995 ◽  
Vol 269 (2) ◽  
pp. H515-H523 ◽  
Author(s):  
C. Doumen ◽  
B. Wan ◽  
O. Ondrejickova
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Atp Synthase ◽  
Mitochondrial Membrane ◽  
Cardiac Work ◽  
Beta Adrenergic ◽  
Rat Hearts ◽  
Perfused Rat Hearts ◽  
Butanedione Monoxime ◽  
Adrenergic Agent

The biochemical link providing effective coordination between the mitochondrial ATP synthetic machinery and the contractile apparatus following transitions in cardiac work remains enigmatic. Studies were designed to determine whether activation of the actomyosin adenosinetriphosphatase (ATPase) is a necessary part of the signaling mechanism to the mitochondrial ATP synthase or whether a rise in cytosolic free Ca2+ is sufficient to activate the synthase. With the use of Langendorff-perfused rat hearts, cardiac work was varied via changes in perfusion pressure and by the inclusion of a beta-adrenergic agent. Furthermore, 2,3-butanedione monoxime and verapamil were used to vary independently either the activity of the actomyosin ATPase or the level of cytosolic free Ca2+. Determinations of the in vivo mitochondrial membrane potential [delta psi m; see Wan et al. Am. J. Physiol. 265 (Heart Circ. Physiol. 34): H445-H452, 1993] and its vectorial displacement during work transitions provide valuable information concerning direct activation of the ATP synthase and proton movement through the membrane domain of the synthase. Increased cardiac work in the presence of the beta-adrenergic agent resulted in a decrease in delta psi m. Addition of 2,3-butanedione monoxime decreased cardiac work but did not change delta psi m. The inclusion of verapamil resulted in similar decreases in cardiac work. However, delta psi m reversed back to a value observed under control, low-work conditions. These results in conjunction with data regarding levels of high-energy phosphates, free Mg2+, and adenosine 3',5'-cyclic monophosphate suggest a Ca(2+)-mediated increase in the activity of the ATP synthase.

ATP as a source of interstitial adenosine in the rat heart

1999 ◽  
Vol 77 (8) ◽  
pp. 579-588 ◽  
Author(s):  
Mojca Lorbar ◽  
Richard A Fenton ◽  
James G Dobson, Jr.
Keyword(s):  
Electrical Stimulation ◽  
Interstitial Fluid ◽  
Contractile Function ◽  
Stellate Ganglion ◽  
Perfused Heart ◽  
Reserpine Treatment ◽  
Rat Hearts ◽  
Perfused Rat Hearts ◽  
Stimulation Of ◽  
Isolated Perfused Rat Hearts

The contribution of neuronal ATP to interstitial adenosine levels was investigated in isolated perfused rat hearts. Ventricular surface transudates, representing interstitial fluid, were analyzed for norepinephrine, ATP, and adenosine. Exocytotic release of norepinephrine was induced by electrical stimulation of cardiac efferents emanating from the stellate ganglion. Ganglion stimulation increased contractility, interstitial norepinephrine, ATP, and adenosine. Interstitial adenosine was 11- to 27-fold higher than interstitial ATP, suggesting that the released ATP is unlikely the only source of adenosine. In the presence of AOPCP (α,β-methyleneadenosine 5'-diphosphate), an ecto-5'-nucleotidase inhibitor, the ganglion-stimulated increase in interstitial ATP and adenosine reached levels similar to those in the absence of AOPCP, also suggesting that adenosine does not derive from extracellular ATP. The perfusate Ca2+ was raised from 1 to 4 mM to determine the importance of the enhanced contractile function on the levels of norepinephrine, ATP, and adenosine. The results were increases in contractility and interstitial norepinephrine, ATP, and adenosine, which were not suppressed with atenolol, indicating a norepinephrine-independent release of ATP and adenosine. Reserpine treatment and administration of guanethidine depleted the catecholamine stores and diminished the catecholamine release, respectively. However, neither agent altered Ca2+-induced increases in ATP and adenosine. It is concluded that the amount of neuronal-derived ATP is low and most likely does not contribute significantly to interstitial levels of adenosine. Furthermore, elevations in interstitial norepinephrine, ATP, and adenosine are associated with neuronal-independent increases in contractile function.Key words: perfused heart, stellate ganglion, co-transmission, calcium, and contractility.

ATP synthase activity is required for fructose to protect cultured hepatocytes from the toxicity of cyanide

1993 ◽  
Vol 264 (3) ◽  
pp. C709-C714 ◽  
Author(s):  
J. W. Snyder ◽  
J. G. Pastorino ◽  
A. P. Thomas ◽  
J. B. Hoek ◽  
J. L. Farber
Keyword(s):  
Membrane Potential ◽  
Protective Effect ◽  
Mitochondrial Membrane Potential ◽  
Atp Synthase ◽  
Mitochondrial Membrane ◽  
Cultured Hepatocytes ◽  
Intracellular Acidosis ◽  
Mitochondrial Atp Synthase ◽  
Carbonyl Cyanide

The contributions of the loss of the mitochondrial membrane potential (MMP) and a depletion of ATP to the genesis of lethal injury were evaluated in the killing of cultured hepatocytes by cyanide (CN). The glycolytic production of ATP from fructose (Fru) maintained the MMP and prevented the killing by CN. Inhibition of the mitochondrial ATP synthase by 0.1 micrograms/ml oligomycin (Oligo) reduced ATP stores at the same rate and to the same extent as did 1 mM CN. With Oligo there was no loss of the MMP, and the hepatocytes maintained viability over the 6 h during which CN killed all of the cells. Oligo had no effect on the rate of killing by CN. However, Oligo reversed the protective effect of Fru on CN-induced killing, a result that correlated with the loss of MMP but not with the depletion of ATP. Neither Fru nor Oligo affected the intracellular acidosis achieved with CN alone. Fru also prevented toxicity of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), a result that correlated with the preservation of MMP. Oligo potentiated the toxicity of CCCP. It is concluded that a functioning mitochondrial ATP synthase is required for the production of ATP from Fru to prevent the killing of hepatocytes by CN. The extent of killing correlated closely with changes in the MMP but not with changes in the content of ATP.

TGF-β improves myocardial function and prevents apoptosis induced by anoxia–reoxygenation, through the reduction of endoplasmic reticulum stress

2016 ◽  
Vol 94 (1) ◽  
pp. 9-17 ◽  
Author(s):  
Yufeng Wang ◽  
Ligeng Zong ◽  
Xiaolei Wang
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Potential ◽  
Cell Viability ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Myocardial Function ◽  
Protective Effects ◽  
H9c2 Cells ◽  
Rat Hearts ◽  
Stress Dependent

Background: Transforming growth factor-β (TGF-β) is known for its role in ventricular remodeling, inflammatory response, cell survival, and apoptosis. However, its role in improving myocardial function in rat hearts subjected to ischemia–reperfusion (I/R) and protecting against apoptosis induced in cardiomyocytes by anoxia–reoxygenation (A/R) has not been elucidated. This study investigated the protective effects and molecular mechanisms of TGF-β on myocardial function and cardiomyocyte apoptosis. Methods and results: We used TUNEL staining, we tested cell viability, and we measured mitochondrial membrane potential and levels of mitochondrial ROS after 6 h of simulated anoxia together with various durations of simulated reoxygenation in H9c2 cells. We further observed the contractile function in rat hearts after they were subjected to 30 min global ischemia and 180 min reperfusion. Pretreatment with TGF-β markedly inhibited apoptosis in H9c2 cells, as evidenced by increased cell viability and decreased numbers of TUNEL-positive cells, maintained mitochondrial membrane potential, and diminished mitochondrial production of reactive oxygen species (ROS). These changes were associated with the inhibition of endoplasmic reticulum (ER) stress-dependent markers of apoptosis (GRP78, CHOP, caspase-12, and JNK), and the modulation of the expression of Bcl2/Bax. Furthermore, TGF-β improved I/R-induced myocardial contractile dysfunction. All of these protective effects were concentration-dependent. Conclusion: Our results show that TGF-β prevents A/R-induced apoptosis of cardiomyocytes and improves myocardial function in rat hearts injured by I/R.

scholarly journals Parkinson’s disease–associated VPS35 mutant reduces mitochondrial membrane potential and impairs PINK1/Parkin-mediated mitophagy

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Kai Yu Ma ◽  
Michiel R. Fokkens ◽  
Fulvio Reggiori ◽  
Muriel Mari ◽  
Dineke S. Verbeek
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Membrane Potential ◽  
Mitochondrial Dysfunction ◽  
Mitochondrial Membrane Potential ◽  
Ubiquitin Ligase ◽  
Mitochondrial Membrane ◽  
Mitochondrial Potential ◽  
Carbonyl Cyanide ◽  
Vacuolar Protein

Abstract Background Mitochondrial dysfunction plays a prominent role in the pathogenesis of Parkinson’s disease (PD), and several genes linked to familial PD, including PINK1 (encoding PTEN-induced putative kinase 1 [PINK1]) and PARK2 (encoding the E3 ubiquitin ligase Parkin), are directly involved in processes such as mitophagy that maintain mitochondrial health. The dominant p.D620N variant of vacuolar protein sorting 35 ortholog (VPS35) gene is also associated with familial PD but has not been functionally connected to PINK1 and PARK2. Methods To better mimic and study the patient situation, we used CRISPR-Cas9 to generate heterozygous human SH-SY5Y cells carrying the PD-associated D620N variant of VPS35. These cells were treated with a protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) to induce the PINK1/Parkin-mediated mitophagy, which was assessed using biochemical and microscopy approaches. Results Mitochondria in the VPS35-D620N cells exhibited reduced mitochondrial membrane potential and appeared to already be damaged at steady state. As a result, the mitochondria of these cells were desensitized to the CCCP-induced collapse in mitochondrial potential, as they displayed altered fragmentation and were unable to accumulate PINK1 at their surface upon this insult. Consequently, Parkin recruitment to the cell surface was inhibited and initiation of the PINK1/Parkin-dependent mitophagy was impaired. Conclusion Our findings extend the pool of evidence that the p.D620N mutation of VPS35 causes mitochondrial dysfunction and suggest a converging pathogenic mechanism among VPS35, PINK1 and Parkin in PD.

scholarly journals Effects of ionophores and metabolic inhibitors on the mitochondrial membrane potential within isolated hepatocytes as measured with the safranine method

1980 ◽  
Vol 192 (1) ◽  
pp. 183-190 ◽  
Author(s):  
K E Akerman ◽  
J O Järvisalo
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Spectral Response ◽  
Difference Spectrum ◽  
Isolated Hepatocytes ◽  
Metabolic Inhibitors ◽  
Equilibrium Potential ◽  
Carbonyl Cyanide ◽  
Subsequent Addition

A difference spectrum with a peak of absorbance at 526nm appears slowly upon addition of valinomycin or KCN in combination with oligomycin to a hepatocyte suspension in the presence of safranine. When the cells are incubated at 37 degrees C in a medium containing safranine, a slow decrease in the absorbance occurs at the wavelength pair 524-484 nm. The change in absorbance is completed within 20-30 min after additions of cells to a medium containing safranine. At this time the safranine concentration of the outer medium is considerably decreased. The safranine signal is completely reversed by valinomycin, carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone or KCN in combination with oligomycin. None of these treatments have any immediate effect on cellular ATP concentrations or the 36Cl- equilibrium potential across the plasma membrane. In the presence of iodoacetate a slow reversal of the trace can be induced upon addition of KCN, but not of oligomycin alone. Rotenone, in combination with oligomycin, does not reverse the safranine signal except when both KF and iodoacetate are present, in which case a slow reversal is seen. A subsequent addition of duroquinone brings back the signal to the same level as in the presence of rotenone alone. The results indicate that the spectral response of safranine in the presence of isolated hepatocytes is a result of a slow penetration of safranine into intracellular mitochondria, where aggregation of safranine molecules occurs as a response to the mitochondrial membrane potential.

Confocal microscopy analysis of the activity of mitochondria contained within the ‘mitochondrial cloud’ during oogenesis in Xenopus laevis

Zygote ◽  
2001 ◽  
Vol 9 (4) ◽  
pp. 347-352 ◽  
Author(s):  
Martin Wilding ◽  
Rosa Carotenuto ◽  
Vincenzo Infante ◽  
Brian Dale ◽  
Marcella Marino ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Confocal Microscopy ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
High Ratio ◽  
Steady Increase ◽  
Mitochondrial Activity ◽  
Highly Active ◽  
Carbonyl Cyanide ◽  
Mitochondrial Cloud

We have used ratiometric confocal microscopy and three fluorescence techniques to study the distribution and activity of mitochondria in frog oocytes during the early stages of oogenesis. Mitochondria in frog oocytes during oogenesis were characterised by a high ratio in the ‘mitochondrial cloud’ and peri-nuclear region and a low ratio in mitochondria freely dispersed within the cytoplasm. We tested whether the high ratio visualised by the three techniques represented mitochondrial membrane potential by perturbing the mitochondrial membrane potential. Carbonyl cyanide p-(trifluoromethyl)phenylhydrazone (FCCP) caused the immediate destruction of the membrane potential, and consequent loss of fluorescence from the membrane-potential-sensitive confocal channel. In contrast, nigericin caused an increase in membrane potential represented by a steady increase in fluorescence ratio. These data demonstrate that mitochondrial activity can be measured during oogenesis in frog oocytes, and suggest that the mitochondrial cloud and perinuclear regions are characterised by highly active mitochondria.

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