Membrane Potential Changes Visualized in Complete Growth Media through Confocal Laser Scanning Microscopy of bis-Oxonol-Loaded Cells

DallˈAsta, Valeria; Gatti, Rita; Orlandini, Guido; Rossi, Patrizia A.; Rotoli, Bianca Maria; Sala, Roberto; Bussolati, Ovidio; Gazzola, Gian C.

doi:10.1006/excr.1996.3469

Cited by 56 publications

(64 citation statements)

References 26 publications

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“…Standard curves were generated in endosomes treated with either nigericin (pH) or gramicidin [Na] Intracellular voltage measurements were made using the slow voltage-sensitive dye, DiSBAC 2 3, a bis-oxonol. The negatively charged intracellular dye associates with the positively charged side of organelle membranes, concentrates in membranes with enhanced electrical activity, and provides a prolonged measurement of intracellular structures that show charge movement during the stimulation period (23). As with Lysosensor Blue, 10 M TTX treatment, but not 0.3 M TTX, decreased densitometric and particle measurements (Fig.…”

Section: High-dose Ttx Blocks Lps-stimulated Acidification Of Endosommentioning

confidence: 94%

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Expression of the Voltage-Gated Sodium Channel NaV1.5 in the Macrophage Late Endosome Regulates Endosomal Acidification

Carrithers¹,

Dib‐Hajj

Carrithers³

et al. 2007

The Journal of Immunology

119

122

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Voltage-gated sodium channels expressed on the plasma membrane activate rapidly in response to changes in membrane potential in cells with excitable membranes such as muscle and neurons. Macrophages also require rapid signaling mechanisms as the first line of defense against invasion by microorganisms. In this study, our goal was to examine the role of intracellular voltage-gated sodium channels in macrophage function. We demonstrate that the cardiac voltage-gated sodium channel, NaV1.5, is expressed on the late endosome, but not the plasma membrane, in a human monocytic cell line, THP-1, and primary human monocyte-derived macrophages. Although the neuronal channel, NaV1.6, is also expressed intracellularly, it has a distinct subcellular localization. In primed cells, NaV1.5 regulates phagocytosis and endosomal pH during LPS-mediated endosomal acidification. Activation of the endosomal channel causes sodium efflux and decreased intraendosomal pH. These results demonstrate a functionally relevant intracellular voltage-gated sodium channel and reveal a novel mechanism to regulate macrophage endosomal acidification.

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Section: High-dose Ttx Blocks Lps-stimulated Acidification Of Endosommentioning

confidence: 94%

“…Intracellular voltage was assessed by a modification of the method of Dall'Asta et al (23). Cells were primed with IFN-␥ as described and then loaded for 60 min (25°C) with bis-(1,3-diethylthiobarbituric acid) trimethine oxonol (DiSBAC 2 3) (1 M) in HBSS supplemented with 2% FBS.…”

Section: Live Cell Voltage Dye and Ph Measurementsmentioning

confidence: 99%

Expression of the Voltage-Gated Sodium Channel NaV1.5 in the Macrophage Late Endosome Regulates Endosomal Acidification

Carrithers¹,

Dib‐Hajj

Carrithers³

et al. 2007

The Journal of Immunology

119

122

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“…DiBAC 4 (3) is a bisbarbituric acid oxolol compound that partitions into the membrane as a function of membrane potential. Fluorescence intensity was monitored at 10-s intervals with a confocal microscope (LSM510-Zeiss; Zeiss) using the excitation and emission wavelengths of 470 and 525 nm, respectively (40).…”

Section: Animalsmentioning

confidence: 99%

Morphine peripheral analgesia depends on activation of the PI3Kγ/AKT/nNOS/NO/K _ATP signaling pathway

Cunha

Roman‐Campos

Lotufo

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

189

188

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Morphine is one of the most prescribed and effective drugs used for the treatment of acute and chronic pain conditions. In addition to its central effects, morphine can also produce peripheral analgesia. However, the mechanisms underlying this peripheral action of morphine have not yet been fully elucidated. Here, we show that the peripheral antinociceptive effect of morphine is lost in neuronal nitric-oxide synthase null mice and that morphine induces the production of nitric oxide in primary nociceptive neurons. The activation of the nitric-oxide pathway by morphine was dependent on an initial stimulation of PI3Kγ/AKT protein kinase B (AKT) and culminated in increased activation of K ATP channels. In the latter, this intracellular signaling pathway might cause a hyperpolarization of nociceptive neurons, and it is fundamental for the direct blockade of inflammatory pain by morphine. This understanding offers new targets for analgesic drug development.M orphine is one of the most prescribed and effective drugs used for treatment of postoperatory and acute severe pain. Nevertheless, its use is frequently limited by undesirable side effects including respiratory depression, tolerance, and addiction. The discovery that morphine can also produce peripheral analgesia in the setting of inflammatory pain opened the possibility of developing peripheral restricted opioids devoid of central side effects (1).Morphine peripheral analgesia was discovered by its direct effect on already established inflammatory hypernociception induced by prostaglandin E 2 (PGE 2 ) injected in rat hind paws (1). Therefore, in contrast to aspirin-like drugs whose analgesic mechanism depends on prevention of nociceptor sensitization by inhibiting synthesis of prostaglandins, opioids are able to directly block ongoing nociceptor sensitization. However, the molecular mechanisms triggered by morphine to promote this action have not been fully elucidated. The present study reports on a series of experiments using behavioral, biochemical, and electrophysiological approaches to address this issue. The following major findings are reported herein: (i) the activation of peripheral opioid receptors in primary nociceptive neurons by morphine triggers a cascade of intracellular signaling events initiated by PI3Kγ/Protein kinase B (AKT); (ii) this is accompanied by activation of neuronal nitric oxide synthase (nNOS) and nitric oxide (NO) production, which (iii) induces an increase in K ATP channel currents; and (iv) it causes a hyperpolarization of nociceptive neurons. Results and DiscussionBased on the evidence that cAMP was the key intracellular second messenger involved in PGE 2 -induced nociceptor sensitization (2) and that opioid-receptor activation in vitro was coupled to adenylyl-cyclase inhibition (3), it was initially suggested that these drugs counteracted inflammatory hypernociception directly through inhibition of PGE 2 -induced adenylyl-cyclase activation (1, 4). Subsequent in vitro studies, which confirmed the ability of opioids to inhibit ad...

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“…Two different cell lines, PC12 and NIH3T3, were studied in a self-built flow chamber that allows long-term observation of living adherent cells under controlled microenvironmental conditions and repeated culture medium changes without modification of a preset observation field (Dall'Asta et al 1997). Particular attention was devoted to ascertaining the relationship between annexin V-FITC positivity and maintenance of cell adhesion.…”

mentioning

confidence: 99%

Comparison of Annexin V and Calcein-AM as Early Vital Markers of Apoptosis in Adherent Cells by Confocal Laser Microscopy

Gatti

Belletti

Orlandini

et al. 1998

J Histochem Cytochem.

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SUMMARYAlthough morphological criteria for apoptosis are in general reliable, no systematic comparison of the techniques employed thus far has yet been performed. In this study, using confocal laser microscopy, we compared the performance of annexin V-FITC and calcein-AM for early detection of apoptosis in living adherent cells. Experiments were carried out on two distinct cell lines, PC 12 and NIH3T3, endowed with different shape and adhesion properties. The apoptotic process was followed for a prolonged period in the same cells of a predetermined field by means of a special flow chamber. Our results show that both probes allowed the detection of apoptotic cells in either cell line. However, some cells that clearly exhibited apoptotic changes on calcein visualization were annexin-negative. In NIH3T3 cells, annexin negativity of apoptotic cells was correlated with the preservation of cell shape and adhesion properties. These findings show that, at least in PC12 and NIH3T3 cells, annexin might be less sensitive than calcein-AM for early apoptosis detection and, for NIH3T3 cells, suggest that phosphatidilserine exposure is in some way linked to changes in cell shape and/or adhesion to culture substrate.

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Membrane Potential Changes Visualized in Complete Growth Media through Confocal Laser Scanning Microscopy of bis-Oxonol-Loaded Cells

Cited by 56 publications

References 26 publications

Expression of the Voltage-Gated Sodium Channel NaV1.5 in the Macrophage Late Endosome Regulates Endosomal Acidification

Expression of the Voltage-Gated Sodium Channel NaV1.5 in the Macrophage Late Endosome Regulates Endosomal Acidification

Morphine peripheral analgesia depends on activation of the PI3Kγ/AKT/nNOS/NO/K _ATP signaling pathway

Comparison of Annexin V and Calcein-AM as Early Vital Markers of Apoptosis in Adherent Cells by Confocal Laser Microscopy

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Membrane Potential Changes Visualized in Complete Growth Media through Confocal Laser Scanning Microscopy of bis-Oxonol-Loaded Cells

Cited by 56 publications

References 26 publications

Expression of the Voltage-Gated Sodium Channel NaV1.5 in the Macrophage Late Endosome Regulates Endosomal Acidification

Expression of the Voltage-Gated Sodium Channel NaV1.5 in the Macrophage Late Endosome Regulates Endosomal Acidification

Morphine peripheral analgesia depends on activation of the PI3Kγ/AKT/nNOS/NO/K ATP signaling pathway

Comparison of Annexin V and Calcein-AM as Early Vital Markers of Apoptosis in Adherent Cells by Confocal Laser Microscopy

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Morphine peripheral analgesia depends on activation of the PI3Kγ/AKT/nNOS/NO/K _ATP signaling pathway