scholarly journals The Effect of A2E on the Uptake and Release of Calcium in the Lysosomes and Mitochondria of Human RPE Cells Exposed to Blue Light

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Mao-Mei Luo ◽  
Lin Chen ◽  
Shu Wang ◽  
Chun Zeng ◽  
De-Zhi Li ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Blue Light ◽  
Fluorescence Intensity ◽  
Mitochondrial Membrane ◽  
Light Exposure ◽  
Final Concentration ◽  
Rpe Cells ◽  
Uptake And Release ◽  
Human Rpe Cells

We aimed to explore the effect of N-retinylidene-N-retinylethanolamine (A2E) on the uptake and release of calcium in lysosomes and mitochondria by establishing a model of human retinal pigment epithelial (RPE) cell injury induced by exposure to blue light. Primary human RPE cells were cultured from passages 4 to 6 and exposed to blue light at an intensity of 2000 ± 500 lux for 6 hours. After blue light exposure, the culture was maintained for 24 hours. A2E at a final concentration of 25 μM was added to the culture 2 hours before light exposure, and nifedipine at a final concentration of 10−4 M was added 1 hour before light exposure. The levels of Ca2+ in the cytosol (CaTM/2AM), mitochondria (Rhod/2AM), and lysosomes (LysoTracker Red and Fluo-3/AM) were determined. In order to measure the calcium levels in the different organelles, RPE were imaged using a laser scanning confocal microscope. Moreover, changes in the mitochondrial membrane potential were detected by flow cytometry analysis of JC-1-stained cells. The obtained results revealed that blue light illumination increased the calcium fluorescence intensity in the cytoplasm, mitochondria, and lysosomes of human RPE cells when compared with the control cells ( P < 0.05 ). After A2E treatment, the fluorescence intensity of the calcium in the cytoplasm was further increased ( P < 0.05 ), while that in the mitochondria and lysosomes decreased ( P < 0.05 ). In addition, we observed that nifedipine reduced the fluorescence intensity of calcium in the RPE cells. Our results also showed that the mitochondrial membrane potential in the RPE treated with blue light and A2E was lower than that in the control, blue light, and A2E-treated cells ( P < 0.05 ). Blue light increased calcium levels in the cytoplasm, lysosomes, and mitochondria of RPE cells. A2E damages the lysosomal and mitochondrial membranes, resulting in calcium release into the cytoplasm. Finally, our results demonstrated that both blue light and A2E treatments reduced mitochondrial membrane potential, increasing cytosolic Ca2+ levels, which can contribute to the activation of RPE death.

scholarly journals Thioflavin T indicates membrane potential in mammalian cells and can affect it in a blue light dependent manner.

2021 ◽  
Author(s):  
Emily Skates ◽  
Hadrien Delattre ◽  
Zoe Schofield ◽  
Munehiro s Asally ◽  
orkun s soyer
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Blue Light ◽  
Mammalian Cells ◽  
Mitochondrial Membrane ◽  
Light Exposure ◽  
Thioflavin T ◽  
Dependent Manner ◽  
Tht Fluorescence ◽  
Low Concentrations

The fluorescent benzothiazole Thioflavin T (ThT) has a high binding affinity to protein aggregates and is used as a marker for the study of this process, most commonly in the context of neurodegenerative disease research and diagnosis. Recently, this same dye was shown to indicate membrane potential in bacteria due to its cationic nature. This finding prompted a question whether ThT fluorescence is linked to the membrane potential in mammalian cells, which would be important for appropriate utilisation of ThT in research and diagnosis. Here, we show that ThT localises into the mitochondria of HeLa cells in a membrane-potential dependent manner. Specifically, ThT colocalised in cells with a well-established mitochondrial membrane-potential indicator Tetramethylrhodamine methyl ester (TMRM) and gave similar temporal responses as TMRM to treatment with a protonophore, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP). Additionally, we found that presence of ThT together with exposure to blue light (λ=405 nm) exposure, but neither factor alone, caused depolarisation of mitochondrial membrane potential. This depolarisation effect was recapitulated by a mathematical model implementing the potential-dependent distribution of ThT and its light-dependent binding in mitochondria. These results show that ThT can act as a membrane potential dye in mammalian cells, when used at low concentrations and with low blue-light exposure, while it causes dissipation of the mitochondrial membrane potential at higher concentrations and in the presence of blue light excitation. This conclusion motivates a re-evaluation of ThT use at micromolar range in live-cell analyses, while indicating that this dye can enable future studies on the potential connections between membrane potential dynamics and protein aggregation.

scholarly journals Near infrared light exposure is associated with increased mitochondrial membrane potential in retinal pigmented epithelial cells

2020 ◽  
Vol 19 (10) ◽  
pp. 1455-1459
Author(s):  
Catherine Rono ◽  
Tiffany R Oliver
Keyword(s):  
Membrane Potential ◽  
Epithelial Cells ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Near Infrared ◽  
Light Exposure ◽  
Infrared Light ◽  
Near Infrared Light ◽  
Retinal Pigmented Epithelial Cells

The goal of this study was to characterize the effect of near-infrared light exposure on mitochondrial membrane potential, in vitro.

Abstract 335: Exercise Protects the Heart by Preserving Mitochondrial Membrane Potential During Early Reperfusion

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Rick J Alleman ◽  
Hetal D Patel ◽  
Fatiha Moukdar ◽  
David A Brown
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Fluorescence Intensity ◽  
Mitochondrial Membrane ◽  
Ischemia Reperfusion ◽  
Redox Homeostasis ◽  
Respiratory Control ◽  
Treadmill Running ◽  
Early Reperfusion

Exercise evokes adaptations intrinsic to the myocardium that protect against ventricular arrhythmia, yet the underlying mechanisms are not completely understood. We have previously shown that the transition to arrhythmia occurs concomitant with a collapse in mitochondrial membrane potential (ΔΨm). As our previous studies indicated that exercise preserves intracellular redox homeostasis, which directly influences mitochondrial energetics, we hypothesized that rats exposed to exercise (Ex, 10 d of treadmill running) would be protected against reperfusion arrhythmia via better maintenance of ΔΨm. To fully understand the temporal relationship between ΔΨm and cardiac electrical activity, two-photon microscopy images (using the fluorescent probe TMRM) and volume-conducted electrocardiogram were simultaneously recorded. Langendorff-perfused hearts underwent 40/30 min of ischemia/reperfusion. Exercise lowered the incidence of arrhythmia, with 3 of 8 Ex hearts experiencing tachycardia or fibrillation compared to 7 of 8 sedentary (Sed) hearts. Ex prevented the collapse of ΔΨm during the first 10 min of reperfusion (74±6.4% v 57±1.5% of baseline fluorescence intensity; P<0.05). To gain a more comprehensive understanding of energetics throughout the heterogeneous mitochondrial population, we then measured mean TMRM fluorescence intensity in isolated ventricular mitochondria harvested after reperfusion using flow cytometry (n=100,000 events per group). Interestingly, mean fluorescence intensity for ΔΨm was similar in Ex and Sed mitochondria (278±33 v 309 ±44 AU, respectively). Mitochondrial respiratory control ratios were also similar in Ex and Sed (9.03±0.70 v 9.00±0.92, respectively). Taken together, the isolated mitochondrial assessment did not reflect what was observed in vivo. This suggests that either intracellular factors influenced in vivo mitochondrial energetics, or our isolated mitochondria may have been enriched with predominantly healthy mitochondria. Our studies demonstrate for the first time that exercise prevents electrical dysfunction following an ischemic insult through better preservation of mitochondrial energetics, and that this preservation is only observed in the intact organ.

scholarly journals Inhibiting Mitochondrial Cytochrome c Oxidase Downregulates Gene Transcription After Traumatic Brain Injury in Drosophila

2021 ◽  
Vol 12 ◽  
Author(s):  
Ekta J. Shah ◽  
Maik Hüttemann ◽  
Thomas H. Sanderson ◽  
Katherine Gurdziel ◽  
Douglas M. Ruden
Keyword(s):  
Gene Expression ◽  
Membrane Potential ◽  
Brain Injury ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Near Infrared ◽  
Light Exposure ◽  
Mitochondrial Dynamics ◽  
Ros Generation ◽  
Biological Sex

Traumatic brain injuries (TBIs) caused by a sudden impact to the head alter behavior and impair physical and cognitive function. Besides the severity, type and area of the brain affected, the outcome of TBI is also influenced by the patient’s biological sex. Previous studies reporting mitochondrial dysfunction mainly focused on exponential reactive oxygen species (ROS) generation, increased mitochondrial membrane potential, and altered mitochondrial dynamics as a key player in the outcome to brain injury. In this study, we evaluated the effect of a near-infrared (NIR) light exposure on gene expression in a Drosophila TBI model. NIR interacts with cytochrome c oxidase (COX) of the electron transport chain to reduce mitochondrial membrane potential hyperpolarization, attenuate ROS generation, and apoptosis. We subjected w1118 male and female flies to TBI using a high-impact trauma (HIT) device and subsequently exposed the isolated fly brains to a COX-inhibitory wavelength of 750 nm for 2 hours (hr). Genome-wide 3′-mRNA-sequencing of fly brains revealed that injured w1118 females exhibit greater changes in transcription compared to males at 1, 2, and 4 hours (hr) after TBI. Inhibiting COX by exposure to NIR downregulates gene expression in injured females but has minimal effect in injured males. Our results suggest that mitochondrial COX modulation with NIR alters gene expression in Drosophila following TBI and the response to injury and NIR exposure varies by biological sex.

Platelets apoptosis in rats after global brain ischemia

2018 ◽  
pp. 27-35
Author(s):  
А.А. Соколовская ◽  
Э.Д. Вирюс ◽  
В.В. Александрин ◽  
А.С. Роткина ◽  
К.А. Никифорова ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Ischemic Stroke ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Brain Ischemia ◽  
Mitochondrial Membrane ◽  
Male Rats ◽  
Global Brain Ischemia ◽  
Platelet Apoptosis ◽  
Global Brain

Цель исследования. Ишемические повреждения головного мозга, являются одной из наиболее частой причин инвалидности и смертности во всем мире. Недавно была установлена роль апоптоза тромбоцитов в патофизиологии инсульта, однако его механизмы до сих пор остаются невыясненными. Несмотря на различные экспериментальные модели, направленные на мониторинг апоптоза тромбоцитов, результаты, относительно изучения и выявления апоптоза тромбоцитов при ишемии головного мозга у крыс, весьма немногочисленны. Цель исследования - анализ апоптоза тромбоцитов с помощью метода проточной цитофлуориметрии на модели глобальной ишемии мозга у крыс. Методика. В экспериментах использовано 6 крыс-самцов Вистар в возрасте от 5 до 6 мес., разделенных на 2 группы: интактный контроль (К) и глобальная ишемия головного мозга. Модель глобальной ишемии головного мозга у крыс воспроизводилась путём билатеральной окклюзии общих сонных артерий на фоне гипотензии. Уровень системного артериального давления снижали посредством кровопотери до 40-45 мм рт. ст. Суспензию тромбоцитов крыс получали методом гельфильтрации с использованием сефарозы 2B. Для анализа экстернализации фосфатидилсерина (ФС) тромбоциты крыс инкубировали с Аннексином V-PE в связывающем буфере. Для оценки митохондриального мембранного потенциала (ММП) тромбоциты инкубировали с катионным красителем JC-1. После инкубации образцы немедленно анализировали на проточном цитофлуориметре FACSCalibur (Becton Dickinson, США). Результаты. Согласно полученным данным, экстернализация ФС на тромбоцитах крыс, перенесших инсульт, была значительно выше (53,45 ± 4,21%), чем в контрольной группе крыс (5,27 ± 2,40%). Данный эффект подтверждается выраженной деполяризацией митохондриальных мембран (DYm). После экспериментальной ишемии мозга почти 40% тромбоцитов было деполяризовано. Заключение. Использованный в работе подбор методов и маркеров обеспечивает понимание механизмов апоптоза тромбоцитов как в экспериментальных, так и в клинических условиях. Полученные данные позволяют сделать заключение, что апоптоз тромбоцитов является одним из факторов развития глобальной ишемии головного мозга у крыс. Результаты могут быть использованы для понимания механизмов, участвующих в развитии ишемического повреждения, что, в свою очередь, может быть использовано при разработке новых терапевтических стратегий. Aim. Stroke is one of the most common causes of disability and mortality worldwide. Multiple experimental models of stroke have focused on monitoring of platelet apoptosis. However, studies on and detection of platelet apoptosis in rats with ischemic stroke are very scarce. We investigated platelet apoptosis in rats with global brain ischemia using flow cytometry. Methods. Experiments were carried out on healthy, adult Wistar male rats weighing 300-350 g. The rats were divided into the following 2 groups: intact rats and rats with global brain ischemia. Global brain ischemia was induced by two-vessel (2-VO) carotid occlusion in combination with hypotension. Systemic blood pressure was reduced by 40-45 mm Hg by inducing haemorrhage. Platelets were isolated by gel filtration on Sepharose 2B. For evaluation of phosphatidylserine (PS) externalization, platelets were incubated with Annexin V-PE and analyzed on FACSCalibur (BD Biosciences). Mitochondrial membrane potential (DY) was measured during platelets apoptosis using JC-1, a mitochondrial membrane potential indicator. Platelets were analyzed by flow cytometry immediately after the incubation. Results. PS externalization on platelets was significantly greater after global brain ischemia (53.45 ± 4.21%) than in the control group (5.27 ± 2.40%). Pronounced depolarization of mitochondrial membrane potential (DYm) confirmed this finding. In the rat group with experimental brain ischemia, almost 40% (35.24 ± 5.21%) of platelets were depolarized. Conclusion. Our results provide insight into mechanisms involved in platelet apoptosis during ischemic stroke and can be used in further development of new therapeutic strategies.

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