scholarly journals Energy transduction in intact synaptosomes. Influence of plasma-membrane depolarization on the respiration and membrane potential of internal mitochondria determined in situ

1980 ◽  
Vol 186 (1) ◽  
pp. 21-33 ◽  
Author(s):  
I D Scott ◽  
D G Nicholls
Keyword(s):  
Plasma Membrane ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Significant Proportion ◽  
Membrane Potentials ◽  
Simultaneous Estimation ◽  
Anaerobic Glycolysis ◽  
Mitochondrial Potential ◽  
Plasma Membrane Potential

A method is described, based on the differential accumulation of Rb+ and methyltriphenylphosphonium, for the simultaneous estimation of the membrane potentials across the plasma membrane of isolated nerve endings (synaptosomes), and across the inner membrane of mitochondria within the synaptosomal cytoplasm. These determinations, together with measurements of respiratory rates, and ATP and phosphocreatine concentrations, are used to define the bioenergetic behaviour of isolated synaptosomes under a variety of conditions. Under control conditions, in the presence of glucose, the plasma and mitochondrial membrane potentials are respectively 45 and 148mV. Addition of a proton translocator induces a 5-fold increase in respiration, and abolishes the mitochondrial membrane potential. The addition of rotenone to inhibit respiration does not affect the plasma membrane potential, and only lowers the mitochondrial membrane potential to 128mV. Evidence is presented that ATP synthesis by anaerobic glycolysis is sufficient under these conditions to maintain ATP-dependent processes, including the reversal of the mitochondrial ATP synthetase. Addition of oligomycin under non-respiring conditions leads to a complete collapse of the mitochondrial potential. Even under control conditions the plasma membrane (Na+ + K+)-dependent ATPase is responsible for a significant proportion of the synaptosomal ATP turnover. Veratridine greatly increases respiration, and depolarizes the plasma membrane, but only slightly lowers the mitochondrial membrane potential. High K+ and ouabain also lower the plasma membrane potential without decreasing the mitochondrial membrane potential. In non-respiring synaptosomes, anaerobic glycolysis is incapable of maintaining cytosolic ATP during the increased turnover induced by veratridine, and the mitochondrial membrane potential collapses. It is concluded that the internal mitochondria must be considered in any study of synaptosomal transport.

265 TESTICULAR DEGENERATION AFFECTED PLASMA, ACROSOMAL AND MITOCHONDRIAL MEMBRANE INTEGRITY, AND DNA FRAGMENTATION IN RAM SPERMATOZOA

2015 ◽  
Vol 27 (1) ◽  
pp. 222
Author(s):  
M. Bianchi Rodrigues Alves ◽  
A. Furugen Cesar de Andrade ◽  
R. Paes de Arruda ◽  
L. Batissaco ◽  
R. Lançoni ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Potential ◽  
Dna Fragmentation ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Membrane Integrity ◽  
Mitochondrial Potential ◽  
Plasma Membrane Integrity ◽  
Scrotal Insulation ◽  
Testicular Degeneration

Testicular degeneration, an important cause of male infertility, adversely affects sperm motility and morphology. However, few studies describe effects on integrity of plasma and acrosomal membranes, mitochondrial membrane potential, and DNA fragmentation; therefore, they were evaluated in the present study. Testicular degeneration was induced in 17 White Dorper rams (scrotal insulation for 72 h). Semen was collected (artificial vagina) twice before insulation and twice thereafter (15-day intervals between post-insulation collections). Sperm motility and morphology were analysed by SCA software (Sperm Class Analyser®, MICROPTIC®, Barcelona, Spain) and differential interference contrast microscopy (DIC, model 80i, Nikon, Tokyo, Japan), respectively. Membrane integrity and potential were assessed with fluorescent probes: Hoescht 33342, propidium iodide, FITC-PSA, and JC-1 (Celeghini et al. 2010 Arq. Bras. Med. Vet. Zootec. 62, 536–543) and imaged with fluorescence microscopy (Nikon Model 80i, Nikon, Tokyo, Japan). Fragmentation of DNA was evaluated with a Halomax® kit (Halotech® DNA, Madrid, Spain). Data were analysed with Statview software (Stat View 1998, SAS Institute Inc., Cary, NC, USA). Data obtained from the periods (before × after insulation) were evaluated by analysis of variance (ANOVA) and means were compared using Tukey's test. Total motility (before: 87.53 ± 1.21%; after: 46.53 ± 4.46%) and progressive motility (before: 58.64 ± 2.00%; after: 31.33 ± 3.82%) were reduced (P < 0.01) by scrotal insulation, as were sperm major defects (before: 10.64 ± 1.65%; after: 54.30 ± 3.67%) and total defects (before: 20.50 ± 2.40%; after: 63.85 ± 3.41%; P < 0.0001). Sperm with intact plasma and acrosomal membranes and high mitochondrial potential (PIAIH) decreased (P < 0.0001) after insulation. In that regard, 53.19 ± 2.20 and 28.48 ± 3.48% of sperm were classified as PIAIH before v. after insulation, respectively. Furthermore, plasma membrane integrity, acrosome membrane integrity, and high mitochondrial potential were assessed independently. The quantity of plasma membrane integrity cells (before: 62.01 ± 2.07%; after: 33.92 ± 3.94%), acrosome membrane integrity cells (before: 57.17 ± 2.30%; after: 31.47 ± 3.77%), and high mitochondrial potential cells (before: 85.72 ± 1.42%; after: 57.28 ± 3.12%) were also reduced (P < 0.0001) after insulation. Likewise, DNA integrity decreased (P = 0.002) from 98.87 ± 0.26% before insulation to 91.88 ± 2.6% afterward. In conclusion, sperm plasma and acrosomal membrane integrity, mitochondrial membrane potential, and DNA fragmentation were adversely affected by testicular degeneration in rams induced by scrotal insulation.Research was supported by FAPESP process 2012/00040-0 and 2011/16744-3.

scholarly journals Dependence of mammalian putrescine and spermidine transport on plasma-membrane potential: identification of an amiloride binding site on the putrescine carrier

1998 ◽  
Vol 330 (3) ◽  
pp. 1283-1291 ◽  
Author(s):  
Richard POULIN ◽  
Chenqi ZHAO ◽  
Savita VERMA ◽  
René CHAREST-GAUDREAULT ◽  
Marie AUDETTE
Keyword(s):  
Plasma Membrane ◽  
Membrane Potential ◽  
Binding Site ◽  
Biochemical Properties ◽  
Mitochondrial Potential ◽  
Plasma Membrane Potential ◽  
High K ◽  
Polyamine Transport ◽  
Low K

The mechanism of mammalian polyamine transport is poorly understood. We have investigated the role of plasma-membrane potential (ΔΨpm) in putrescine and spermidine uptake in ZR-75-1 human breast cancer cells. The rate of [3H]putrescine and [3H]spermidine uptake was inversely correlated to extracellular [K+] ([K+]o) and to ΔΨpm, as determined by the accumulation of [3H]tetraphenylphosphonium bromide (TPP). Inward transport was unaffected by a selective decrease in mitochondrial potential (ΔΨmit) induced by valinomycin at low [K+]o, but was reduced by ≈ 60% by the rheogenic protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP), which rapidly (≤ 15 min) collapsed both ΔΨpm and ΔΨmit. Plasma-membrane depolarization by high [K+]o or CCCP did not enhance putrescine efflux in cells pre-loaded with [3H]putrescine, suggesting that decreased uptake caused by these agents did not result from a higher excretion rate. On the other hand, the electroneutral K+/H+ exchanger nigericin (10 μM) co-operatively depressed [3H]TPP, [3H]putrescine and [3H]spermidine uptake in the presence of ouabain. Suppression of putrescine uptake by nigericin+ouabain was Na+-dependent, suggesting that plasma-membrane repolarization by the electrogenic Na+ pump was required upon acidification induced by nigericin, due to the activation of the Na+/H+ antiporter. The sole addition of 5-N,N-hexamethylene amiloride, a potent inhibitor of the Na+/H+ antiporter, strongly inhibited putrescine uptake in a competitive fashion [Ki 4.0±0.9 (S.D.) μM], while being a weaker antagonist of spermidine uptake. The potency of a series of amiloride analogues to inhibit putrescine uptake was clearly different from that of the Na+/H+ antiporter, and resembled that noted for Na+ co-transport proteins. These data demonstrate that putrescine and spermidine influx is mainly unidirectional and strictly depends on ΔΨpm, but not ΔΨmit. This report also provides first evidence for a high-affinity amiloride-binding site on the putrescine carrier, which provides new insight into the biochemical properties of this transporter.

scholarly journals Single Layer Centrifugation Improves the Quality of Fresh Donkey Semen and Modifies the Sperm Ability to Interact with Polymorphonuclear Neutrophils

Animals ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 2128
Author(s):  
Marion Papas ◽  
Jaime Catalán ◽  
Sandra Recuero ◽  
Jane M. Morrell ◽  
Marc Yeste ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Seminal Plasma ◽  
Plasma Proteins ◽  
Mitochondrial Membrane ◽  
Single Layer ◽  
Polymorphonuclear Neutrophils ◽  
Lipid Disorder ◽  
Seminal Plasma Proteins

This study sought to determine whether single layer centrifugation (SLC) of fresh donkey semen with Equicoll has any impact on sperm quality parameters and on the modulation of endometrial reaction following semen deposition using an in vitro model. Seventeen ejaculates from five jackasses were obtained using an artificial vagina and diluted in a skim-milk extender. Samples were either selected through SLC (Equicoll) or non-treated (control). Two experiments were performed. The first one consisted of incubating selected or non-selected spermatozoa at 38 °C for 180 min. Integrity and lipid disorder of sperm plasma membrane, mitochondrial membrane potential, and intracellular levels of calcium and reactive oxygen species were evaluated at 0, 60, 120, and 180 min. In the second experiment, polymorphonuclear neutrophils (PMN) isolated from jennies blood were mixed with selected and unselected spermatozoa. Interaction between spermatozoa and PMN was evaluated after 0, 60, 120, and 180 min of co-incubation at 38 °C. SLC-selection increased the proportions of spermatozoa with an intact plasma membrane and low lipid disorder, of spermatozoa with high mitochondrial membrane potential and with high calcium levels, and of progressively motile spermatozoa. In addition, selection through SLC augmented the proportion of phagocytosed spermatozoa, which supported the modulating role of seminal plasma proteins on sperm-PMN interaction. In conclusion, SLC of fresh donkey semen increases the proportions of functionally intact and motile spermatozoa, and appears to remove the seminal plasma proteins that inhibit sperm-PMN binding.

Mitochondrial membrane potential regulation is independent of c-fos expression

1999 ◽  
Vol 77 (3) ◽  
pp. 195-203
Author(s):  
Roger A Moorehead ◽  
Gurmit Singh
Keyword(s):  
Membrane Potential ◽  
Ovarian Carcinoma ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Membrane Potentials ◽  
Rhodamine 123 ◽  
Parental Line ◽  
Rat Fibroblasts ◽  
Human Ovarian Carcinoma ◽  
Fos Expression

Tumour cells contain mitochondria with elevated membrane potentials compared with normal cells, and thus this feature provides a selective target for destroying tumour cells. To improve mitochondrial-based therapies, a better understanding of the factors involved in regulating mitochondria are required. Since v-fos overexpression has been shown to elevate mitochondrial membrane potentials in rat fibroblasts, we investigated whether the human homologue, c-fos, was also capable of regulating the mitochondrial membrane potential in cells. Rat fibroblasts transfected with the c-fos gene did not accumulate more rhodamine 123 (Rh123) nor did they retain this Rh123 for extended periods of time compared with their parental line. Moreover, there was no difference in survival following dequalinium chloride (Deca) treatment between transfectants and controls. Similarly, reduction of c-fos expression in rat fibroblasts did not significantly alter their mitochondrial membrane potential. In addition, human ovarian carcinoma cells, which overexpress the c-fos gene, did not accumulate more Rh123 nor were they hypersensitive to Deca compared with their parental line. In another human ovarian carcinoma cell line, selection of variants with lower mitochondrial membrane potential did not alter c-fos mRNA or protein levels. These data suggest that alterations in c-fos expression do not regulate the magnitude of the mitochondrial membrane potential.Key words: c-fos, mitochondria, membrane potential, rhodamine 123 (Rh123), lipophilic cations.

scholarly journals Evaluation of mitochondria in oocytes following γ-irradiation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Qiaochu Wang ◽  
Jessica M. Stringer ◽  
Jun Liu ◽  
Karla J. Hutt
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Significant Proportion ◽  
Premature Menopause ◽  
Slight Reduction ◽  
Mtdna Copy Number ◽  
Γ Irradiation ◽  
Mitochondrial Mass ◽  
Immature Oocytes

AbstractStandard cytotoxic cancer treatments, such as radiation, can damage and deplete the supply of oocytes stored within the ovary, which predisposes females to infertility and premature menopause later in life. The mechanisms by which radiation induces oocyte damage have not been completely elucidated. The objective of this study was to determine if γ-irradiation changes mitochondrial characteristics in oocytes, possibly contributing to a reduction in oocyte number and quality. Immature oocytes were collected from postnatal day (PN) 9–11 C57Bl6 mice 3, 6 and 24 hours after 0.1 Gy γ-irradiation to monitor acute mitochondrial changes. Oocytes were classified as small (>20 µm) or growing (40–60 µm). Mitochondrial membrane potential was lost in 20% and 44% of small oocytes (~20 µm) at 3 and 6 hours after γ-irradiation, respectively, consistent with the induction of apoptosis. However, mitochondrial mass, distribution and membrane potential in the surviving small oocytes were similar to the non-irradiated controls at both time points. At 24 hours after γ-irradiation, all mitochondrial parameters analysed within immature oocytes were similar to untreated controls. Mitochondrial parameters within growing oocytes were also similar to untreated controls. When mice were superovulated more than 3 weeks after γ-irradiation, there was a significant reduction in the number of mature oocytes harvested compared to controls (Control 18 ± 1 vs 0.1 Gy 4 ± 1, n = 6/16 mice, p < 0.05). There was a slight reduction in mitochondrial mass in mature oocytes after γ-irradiation, though mitochondrial localization, mtDNA copy number and ATP levels were similar between groups. In summary, this study shows that γ-irradiation of pre-pubertal mice is associated with loss of mitochondrial membrane potential in a significant proportion of small immature oocytes and a reduction in the number of mature oocytes harvested from adult mice. Furthermore, these results suggest that immature oocytes that survive γ-irradiation and develop through to ovulation contain mitochondria with normal characteristics. Whether the oocytes that survive radiation and eventually undergo meiosis can support fertility remains to be determined.

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