Calcium Imaging of Murine Thoracic Aorta Endothelium by Confocal Microscopy Reveals Inhomogeneous Distribution of Endothelial Cells Responding to Vasodilator Agents

Marie, I.; Bény, Jean‐Louis

doi:10.1159/000063691

Cited by 39 publications

(56 citation statements)

References 30 publications

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“…The concentration of cytosolic free calcium was determined by measuring the calcium indicator Fluo-4 as described previously (26), with modifications. Briefly, cells were grown in 96-well plates to 70% confluence.…”

Section: Cytotoxicity Assaysmentioning

confidence: 99%

The H35A Mutated Alpha-Toxin Interferes with Cytotoxicity of Staphylococcal Alpha-Toxin

Liang

Yan

2009

Infect Immun

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Staphylococcal alpha-toxin is an important virulence factor for Staphylococcus aureus to cause severe infections. In this study, we explored whether the toxoid of alpha-toxin may be utilized to block the toxicity of wild-type alpha-toxin. We created a series of H35A mutated alpha-toxin expression strains and revealed that the H35A mutation eliminates the activity of alpha-toxin using a human lung epithelial cell line (A549). More importantly, we found that either the pretreatment or simultaneous treatment of the epithelial cells with alpha-toxin-H35A completely disrupted the cytotoxicity of alpha-toxin. Specifically, we demonstrated that alpha-toxin-H35A can effectively interfere with the pore formation and the internalization of alpha-toxin using cytotoxicity and immunofluorescence assays. In addition, we found that the removal of either the 30-amino-acid (aa) or 99-aa C-terminal region of alpha-toxin-H35A reactivated its cytotoxicity, indicating that interactions between the alanine residue at position 35 and these C-terminal regions may be associated with interrupting the toxic activity of alpha-toxin-H35A. Taken together, these results suggest that the alpha-toxin-H35A protein may be developed as a potential alternative therapeutic agent for treating early stages of S. aureus infections.

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Section: Cytotoxicity Assaysmentioning

confidence: 99%

The H35A Mutated Alpha-Toxin Interferes with Cytotoxicity of Staphylococcal Alpha-Toxin

Liang

Yan

2009

Infect Immun

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“…ATP is an autocrine/paracrine circulating hormone that relaxes arterial smooth muscle by acting on the endothelium (4,15,17,22,25,32,45). In endothelial cells, ATP triggers intracellular…”

mentioning

confidence: 99%

“…ATP is an autocrine/paracrine circulating hormone that relaxes arterial smooth muscle by acting on the endothelium (4,15,17,22,25,32,45). In endothelial cells, ATP triggers intracellular Ca 2ϩ release and sustained store-operated Ca 2ϩ entry, which contributes to NO synthesis and a subsequent smooth muscle relaxation (3,10,13,14,34,43).…”

mentioning

confidence: 99%

Ca²⁺-independent PLA₂ controls endothelial store-operated Ca²⁺ entry and vascular tone in intact aorta

Boittin¹,

Gribi²,

Serir³

et al. 2008

American Journal of Physiology-Heart and Circulatory Physiology

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Boittin FX, Gribi F, Serir K, Bény JL. Ca 2ϩ -independent PLA2 controls endothelial store-operated Ca 2ϩ entry and vascular tone in intact aorta. Am J Physiol Heart Circ Physiol 295: H2466-H2474, 2008. First published October 24, 2008 doi:10.1152/ajpheart.00639.2008.-During an agonist stimulation of endothelial cells, the sustained Ca 2ϩ entry occurring through store-operated channels has been shown to significantly contribute to smooth muscle relaxation through the release of relaxing factors such as nitric oxide (NO). However, the mechanisms linking Ca 2ϩ stores depletion to the opening of such channels are still elusive. We have used Ca 2ϩ and tension measurements in intact aortic strips to investigate the role of the Ca 2ϩ -independent isoform of phospholipase A 2 (iPLA2) in endothelial store-operated Ca 2ϩ entry and endothelium-dependent relaxation of smooth muscle. We provide evidence that iPLA2 is involved in the activation of endothelial store-operated Ca 2ϩ entry when Ca 2ϩ stores are artificially depleted. We also show that the sustained store-operated Ca 2ϩ entry occurring during physiological stimulation of endothelial cells with the circulating hormone ATP is due to iPLA2 activation and significantly contributes to the amplitude and duration of ATP-induced endothelium-dependent relaxation. Consistently, both iPLA 2 metabolites arachidonic acid and lysophosphatidylcholine were found to stimulate Ca 2ϩ entry in native endothelial cells. However, only the latter triggered endothelium-dependent relaxation through NO release, suggesting that lysophosphatidylcholine produced by iPLA 2 upon Ca 2ϩ stores depletion may act as an intracellular messenger that stimulates store-operated Ca 2ϩ entry and subsequent NO production in endothelial cells. Finally, we found that ACh-induced endothelium relaxation also depends on iPLA 2 activation, suggesting that the iPLA2-dependent control of endothelial store-operated Ca 2ϩ entry is a key physiological mechanism regulating arterial tone. endothelial cells; calcium imaging; calcium-independent isoform of phospholipase A2; store-operated channels; endothelium-dependent relaxation IN A WIDE VARIETY of cells, agonists such as neurotransmitters, hormones, or growth factors trigger cellular responses through an increase of the cytosolic Ca 2ϩ concentration. This increase can result from both Ca 2ϩ release from intracellular Ca 2ϩ stores and Ca 2ϩ influx through plasma membrane channels. Many agonists trigger Ca 2ϩ release through the stimulation of phospholipase C, which involves the generation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ] and diacylglycerol. The binding of Ins(1,4,5)P 3 to its receptor located on the endoplasmic reticulum triggers cytosolic Ca 2ϩ release, and the subsequent depletion of intracellular Ca 2ϩ stores activates Ca 2ϩ entry through store-operated channels. This phenomenon is called capacitative or store-operated Ca 2ϩ entry (28).A sustained phase of capacitive Ca 2ϩ entry through storeoperated channels following agonist-induced Ca 2ϩ release...

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“…Oscillations were defined as repetitive fluctuations in fluorescence a minimum of 20% above the basal Ca 2+ dependent fluorescent values. The presence of spontaneous oscillations in A7r5 cells was confirmed using different Ca 2+ -sensitive fluorophores, including Fluo-4, Fluo-3, Calcium Crimson, Calcium Orange, Calcium Green, Oregon Green, and ratiometric methods using Fluo-4 and Fura-Red [20,21]. The oscillations were synchronized among confluent cells.…”

Section: Spontaneous Ca 2+ Oscillations In the Nucleusmentioning

confidence: 80%

“…-sensitive indicators used in these experiments were fluo-4 and fura-red (Molecular Probes, Eugene, OR) [20,21]. While other indicators were tested (fluo-3, calcium orange, calcium green, calcium crimson, indo-1, and oregon Green; Molecular Probes), the ratiometric fluo-4/fura-red combination provided the most consistent results as fura-red fluorescence emission is quenched when Ca 2+ increases, while fluo-4 fluorescence increases, thus artifacts due to movement or dye sequestration are avoided [31].…”

Section: +mentioning

confidence: 99%

Spontaneous Ca2+ oscillations in subcellular compartments of vascular smooth muscle cells rely on different Ca2+ pools

et al. 2004

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Spontaneous Ca 2+ oscillations in vascular smooth muscle cells have been modeled using a single Ca 2+ pool. This report describes spontaneous Ca 2+ oscillations dependent on two separate Ca 2+ sources for the nuclear versus cytoplasmic compartments. Changes in free intracellular Ca 2+ were monitored with ratiometric Ca 2+-fluorophores using confocal microscopy. On average, spontaneous oscillations developed in 79% of rat aortic smooth muscle cells that were synchronous between the cytoplasm and nucleus. Reduction of extracellular Ca 2+ (< 1 µM) decreased the frequency and amplitude of the cytoplasmic oscillations with 48% of the oscillations asynchronous between the nuclear and cytoplasmic compartments. Similar results were obtained with the Ca 2+ channel blockers, nimodipine and diltiazem. Arg-vasopressin (AVP) induced a rapid release of intracellular Ca 2+ stores that was greater in the nuclear compartment (4.20 ± 0.23 ratio units, n = 56) than cytoplasm (2.54 ± 0.28) in cells that had spontaneously developed prior oscillations. Conversely, cells in the same conditions lacking oscillations had a greater AVP-induced Ca 2+ transient in the cytoplasm (4.99 ± 0.66, n = 17) than in the nucleus (2.67 ± 0.29). Pre-treatment with Ca 2+ channel blockers depressed the AVP responses in both compartments with the cytoplasmic Ca 2+ most diminished. Depletion of internal Ca 2+ stores prior to AVP exposure blunted the nuclear response, mimicking the response of cells that lacked prior oscillations. Spontaneous oscillating cells had a greater sarcoplasmic reticulum network than cells that did not oscillate. We propose that spontaneous nuclear oscillations rely on perinuclear sarcoplasmic reticulum stores, while the cytoplasmic oscillations rely on Ca 2+ influx.

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Calcium Imaging of Murine Thoracic Aorta Endothelium by Confocal Microscopy Reveals Inhomogeneous Distribution of Endothelial Cells Responding to Vasodilator Agents

Cited by 39 publications

References 30 publications

The H35A Mutated Alpha-Toxin Interferes with Cytotoxicity of Staphylococcal Alpha-Toxin

The H35A Mutated Alpha-Toxin Interferes with Cytotoxicity of Staphylococcal Alpha-Toxin

Ca²⁺-independent PLA₂ controls endothelial store-operated Ca²⁺ entry and vascular tone in intact aorta

Spontaneous Ca2+ oscillations in subcellular compartments of vascular smooth muscle cells rely on different Ca2+ pools

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Calcium Imaging of Murine Thoracic Aorta Endothelium by Confocal Microscopy Reveals Inhomogeneous Distribution of Endothelial Cells Responding to Vasodilator Agents

Cited by 39 publications

References 30 publications

The H35A Mutated Alpha-Toxin Interferes with Cytotoxicity of Staphylococcal Alpha-Toxin

The H35A Mutated Alpha-Toxin Interferes with Cytotoxicity of Staphylococcal Alpha-Toxin

Ca2+-independent PLA2 controls endothelial store-operated Ca2+ entry and vascular tone in intact aorta

Spontaneous Ca2+ oscillations in subcellular compartments of vascular smooth muscle cells rely on different Ca2+ pools

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Ca²⁺-independent PLA₂ controls endothelial store-operated Ca²⁺ entry and vascular tone in intact aorta