Comparative measurements of membrane potentials with microelectrodes and voltage-sensitive dyes

Bräuner, Thomas; Hülser, Dieter F.; Strasser, Reto J.

doi:10.1016/0005-2736(84)90535-2

Cited by 190 publications

(132 citation statements)

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“…The delocalized charge on the negative DiBAC,(3) allows membrane permeation and membrane potential-dependent distribution of the probe (1,21). This probe, once inside the cell, is thought to form dimers and may also bind to intracellular proteins (1,5).…”

Section: Discussionmentioning

confidence: 99%

Determination of the viability of trichomonas vaginalis using flow cytometry

1994

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In clinical laboratories, viability of Trichomonas vaginalis is determined by using light microscopy (differential count of motile to nonmotileorganisms). Alternative methods are proposed that utilise flow cytometry. Under an epifluorescence microscope, live organisms fluoresce intensely green with fluorescein diacetate (FDA), whereas dead cells fluoresce orange with propidium iodide (PI). Flow cytometric histograms of green versus red fluorescence reveal distinct populations for live and dead cells. The anionic oxonal probe DiBAC4(3) is a membrane potential sensitive dye that distributes between the inside of the cell and the medium. Live organisms are less fluorescent than dead organisms when stained with the oxonol probe. Valinomy: cin, dicyclohexylcarbodiimide, and vana‐date all give significant changes in the fluorescence intensities of cultures stained with the oxonol probe compared with control cultures, indicating that this probe is detecting changes in plasma membrane potential. Both FDA/PI and oxonol staining protocols allow good discrimination between populations and permit counts that are more statistically significant than those obtained by light microscopy. These methods remove the subjectiveness of microscopic counts and would increase the accuracy of susceptibility assays. © 1994 Wiley‐Liss, Inc.

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Section: Discussionmentioning

confidence: 99%

Determination of the viability of trichomonas vaginalis using flow cytometry

1994

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“…Upon membrane hyperpolarization, the dye concentrates in the cell membrane, leading to a decrease of fluorescence intensity, whereas depolarization results in a sequestration of the dye into cytosol and is associated with an increase in the fluorescence intensity (27).…”

Section: Methodsmentioning

confidence: 99%

Acidic NAADP-sensitive Calcium Stores in the Endothelium

Brailoiu

Gurzu

Gào

et al. 2010

Journal of Biological Chemistry

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Accumulating evidence implicates nicotinic acid adenine dinucleotide phosphate (NAADP) in the control of Ca2؉ -dependent functions. Little, however, is known concerning its role in the vascular endothelium, a major regulator of blood pressure. Here, we show that NAADP acetoxymethyl ester (NAADP-AM), a cell-permeant NAADP analog, increases cytosolic Ca 2؉ concentration in aortic endothelial cells. We demonstrate that these signals and those evoked by acetylcholine are blocked by disrupting acidic organelles with bafilomycin A1. In contrast, Ca 2؉ signals in response to thrombin are only partially inhibited by bafilomycin A1 treatment, and those to ATP were insensitive, suggesting that recruitment of acidic stores is agonist-specific. We further show that NAADP-evoked Ca 2؉ signals hyperpolarize endothelial cells and generate NO. Additionally, we demonstrate that NAADP dilates aortic rings in an endothelium-and NO-dependent manner. Finally, we show that intravenous administration of NAADP-AM into anesthetized rats decreases mean arterial pressure. Our data extend the actions of NAADP to the endothelium both in vitro and in vivo, pointing to a previously unrecognized role for this messenger in controlling blood pressure.Nicotinic acid adenine dinucleotide phosphate (NAADP) 4 is a potent and widespread Ca 2ϩ -mobilizing messenger first described in sea urchin eggs (1, 2). Although its mechanism of action is subject to debate, much evidence indicates that NAADP mobilizes Ca 2ϩ from acidic, lysosome-like organelles (3) through activation of novel Ca 2ϩ -permeable channels (1, 4). These channels have recently been identified as members of the two-pore channel family (5-9). Importantly, mobilization of so-called "acidic Ca 2ϩ stores" (10) by NAADP is often linked to mobilization of the well established endoplasmic reticulum Ca 2ϩ stores through the process of Ca 2ϩ -induced Ca 2ϩ release (11). NAADP is thus thought to act as trigger during agonistevoked Ca 2ϩ signaling (2). The functional positioning of NAADP-sensitive Ca 2ϩ stores upstream of those targeted by the messengers, inositol 1,4,5-trisphosphate (IP 3 ) and cyclic ADP-ribose, raises the possibility that agonist-evoked Ca 2ϩ signals previously ascribed to endoplasmic reticulum Ca 2ϩ release might also involve NAADP and acidic organelles.Using isolated cells or tissues, NAADP-induced Ca 2ϩ signaling has been implicated in the regulation of several physiological functions such as egg fertilization (12, 13), T lymphocyte activation (14), muscle contraction (15), and neuronal differentiation (16). The role of NAADP in vivo, however is not known, although a recent study demonstrated that a newly described NAADP antagonist could prevent T cell motility in an animal model of multiple sclerosis (17).In the cardiovascular system, a role for NAADP has been demonstrated in agonist-evoked Ca 2ϩ signaling in pulmonary (15, 18) and coronary arteries (19,20), cardiac myocytes (21), and the renal microcirculation (22). Direct measurements of NAADP have confirmed its messenge...

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“…The dye partition between the cell membrane and the cytosol is a function of membrane potential. Depolarization of the membrane leads to a sequestration of the dye into cytosol and is associated with an increase in the fluorescence intensity (Brauner et al, 1984), whereas during membrane hyperpolarization the dye concentrates in the cell membrane, leading to a decrease of fluorescence intensity. …”

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confidence: 99%

Excitatory effects of human immunodeficiency virus 1 Tat on cultured rat cerebral cortical neurons

et al. 2008

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HIV-1 Tat protein is one of the neurotoxins involved in the pathogenesis of HIV-1-associated neuronal disorders. Combined electrophysiological and optical imaging experiments were undertaken to investigate whether HIV-1 Tat30-86, herein referred to as Tat30-86, acted directly or indirectly via the release of glutamate or both and to test its effect on the properties of spontaneous quantal events in cultured cortical neurons. Whole-cell patch recordings were made from cultured rat cortical neurons in either current-or voltage-clamp mode. Tat30-86 (50-1000 nM) induced in a population of cortical neurons a long-lasting depolarization, which was accompanied by a decrease of membrane resistance and persisted in a Krebs solution containing tetrodotoxin (TTX, 0.5 μM). Depolarizations were slightly reduced by pretreatment with glutamate receptor antagonists CNQX (10 μM) and AP-5 (50 μM), and were markedly reduced in a Ca 2+ -free Krebs solution; the differences were statistically significant. Tat30-86-induced inward currents had a reversal potential between -30 and 0 mV. While not causing a noticeable depolarization, lower concentrations of Tat30-86 (10 nM) increased membrane excitability, as indicated by increased numbers of neuronal discharge in response to a step depolarizing pulse. Tat30-86 (10 nM) increased the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs), while not significantly affecting their amplitude. Tat30-86 (10 nM) moderately increased the frequency as well as the amplitude of spontaneous miniature inhibitory postsynaptic currents (mIPSCs). Ratiometric Ca 2+ imaging studies showed that Tat30-86 produced three types of Ca 2+ responses: 1) a fast and transitory increase, 2) Ca 2+ oscillations, and 3) a fast increase followed by a plateau; the glutamate receptor antagonists eliminated the late component of Ca 2+ response. The result suggests that Tat30-86 is an active fragment and that it excites cortical neurons directly and indirectly via releasing glutamate from adjacent neurons. Keywords calcium imaging; electrophysiology; postsynaptic currentsInfection of the central nervous system (CNS) with the human immunodeficiency virus 1 (HIV-1) may lead to cognitive, motor and behavioral dysfunctions, collectively termed HIVCorresponding author: G. Cristina Brailoiu, M.D., Department of Pharmacology, Temple University School of Medicine, 3420 N. Broad Street, Philadelphia, PA 19140 USA, Tel: 215-707-7705, Fax: 215-707-7068, E-mail: gbrailou@temple.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. (Kaul et al., 2001; Gonzales-Scara...

show abstract

Comparative measurements of membrane potentials with microelectrodes and voltage-sensitive dyes

Cited by 190 publications

References 14 publications

Determination of the viability of trichomonas vaginalis using flow cytometry

Determination of the viability of trichomonas vaginalis using flow cytometry

Acidic NAADP-sensitive Calcium Stores in the Endothelium

Excitatory effects of human immunodeficiency virus 1 Tat on cultured rat cerebral cortical neurons

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