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Bkaily, Ghassan; Pothier, Pierre; D’Orléans-Juste, Pedro; Simaan, May; Jacques, Danielle; Jaalouk, Doris; Belzile, François; Hassan, Ghada S.; Boutin, Chantal; Haddad, Georges; Neugebauer, Witold

doi:10.1023/a:1006840228104

Cited by 87 publications

(54 citation statements)

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“…Laser scanning confocal microscopy imaging using sensitive Ca 2+ fluorescent indicators accurately allowed visualization of rapid Ca 2+ fluctuations in endothelial cells [20, 22, 23, 24, 25, 27, 28, 29, 34]. The use of ratiometric dyes or a combination of dyes allowing emitted fluorescence ratioing permitted quantification of intracellular [Ca 2+ ] i by excluding artifact measurements, which are not related to [Ca 2+ ] i changes, such as dye bleaching and release, or nonhomogeneous cellular thickness and dye loading [20, 21, 22, 24].…”

Section: Discussionmentioning

confidence: 99%

“…The development of laser scanning confocal microscopy coupled to high-performance software may give physiologists the capability to overcome many limitations of standard microscopic imaging measurement [20, 21]. Laser scanning confocal microscopy, in combination with sensitive fluorescent dyes, has been shown to be both a powerful means to monitor [Ca 2+ ] i changes within endothelial cells of the vascular wall [20, 22, 23, 24, 25, 26, 27, 28, 29]. …”

Section: Introductionmentioning

confidence: 99%

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

Marie

Bény²

2002

J Vasc Res

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The aim of the study was to assess, in intact murine thoracic aorta in vitro, the distribution of endothelial cells responsive to endothelium-dependent vasodilators ACh, ATP, bradykinin and substance P, using laser line confocal microscopy in combination with two Ca²⁺ fluorescent dyes, Fluo-4 and Fura-red. We observed that 82 ± 3% of endothelial cells responded to ATP, 33 ± 5% to Ach, whereas less than 3% of them responded to bradykinin or substance P. In order to determine whether the findings of pharmacological tests agree with confocal microscopy data, endothelium-dependent vasodilators induced relaxation was evaluated using isometric tension measurement. We show a marked correlation between a higher number of activated endothelial cells, using confocal microscopy, and a greater degree of endothelium-dependent relaxation using isometric tension measurement (p = 0.00286). Our results suggest that endothelial cells responding to endothelium-dependent vasodilators are not homogeneously distributed in intact murine thoracic aorta. This could be due to nonhomogeneous distribution of surface receptors or to differences in post-receptor coupling mechanisms.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Marie

Bény²

2002

J Vasc Res

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“…The intranuclear calcium concentration was measured with a spectrofluorometer (model LS50, PerkinElmer Life Sciences) and the fluorescent signal appropriately calibrated (16). In another set of experiments, confocal microscopy was used to monitor spatiotemporal movements of nuclear Ca 2ϩ induced by SNP within a single nucleus using the calcium indicator dye fluo-4-AM as described (16,25). Intensity of fluorescence of the calcium-fluo-4 complex was converted into absolute calcium concentration as reported (26).…”

Section: R/ Htc4 Cells (Not Shown)mentioning

confidence: 99%

“…Moreover, it is now becoming obvious that nuclear and cytosolic calcium are regulated independently (25,27), and a number of Ca 2ϩ channels and pumps have been identified on the nuclear envelope (18). We therefore examined whether changes in nuclear Ca 2ϩ could be elicited by SNP stimulation by means of live imaging confocal microscopy and spectrofluorometry using the fluorescent Ca 2ϩ -sensitive probes fluo-4-AM and FIGURE 4.…”

Section: No Evokes Nuclear Signaling Cascade Leading To Inos Expressimentioning

confidence: 99%

Nitric Oxide Signaling via Nuclearized Endothelial Nitric-oxide Synthase Modulates Expression of the Immediate Early Genes iNOS and mPGES-1

Gobeil

Zhu

Brault

et al. 2006

Journal of Biological Chemistry

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Stimulation of freshly isolated rat hepatocytes with lysophosphatidic acid (LPA) resulted in LPA 1 receptor-mediated and nitricoxide-dependent up-regulation of the immediate early genes iNOS (inducible nitric-oxide synthase (NOS)) and mPGES-1 (microsomal prostaglandin E synthase-1). Because LPA is a ligand for both cell surface and intracellular receptor sites and a potent endothelial NOS (eNOS) activator, we hypothesized that NO derived from activated nuclearized eNOS might participate in gene regulation. Herein we show, by confocal microscopy performed on porcine cerebral endothelial cells expressing native LPA 1 -receptor and eNOS and on HTC4 rat hepatoma cells co-transfected with recombinant human LPA 1 -receptor and fused eNOS-GFP cDNA, a dynamic eNOS translocation from peripheral to nuclear regions upon stimulation with LPA. Nuclear localization of eNOS and its downstream effector, soluble guanylate cyclase, were demonstrated in situ in rat liver specimens by immunogold labeling using specific antibodies. Stimulation of this nuclear fraction with LPA and the NO donor sodium nitroprusside resulted, respectively, in increased production of nitrite (and eNOS phosphorylation) and cGMP; these separate responses were also correspondingly blocked by NOS inhibitor L-NAME and soluble guanylate cyclase inhibitor ODQ. In addition, sodium nitroprusside evoked a sequential increase in nuclear Ca 2؉ transients, activation of p42 MAPK, NF-B binding to DNA consensus sequence, and dependent iNOS RNA. This study describes a hitherto unrecognized molecular mechanism by which nuclear eNOS through ensuing NO modulates nuclear calcium homeostasis involved in gene transcription-associated events. Moreover, our findings strongly support the concept of the nucleus as an autonomous signaling compartment.Nitric oxide (NO) is a short-lived uncharged free radical involved in numerous complex biological processes such as blood pressure regulation, vascular inflammation, cell-mediated cytotoxicity, and survival (1, 2). One of the most biologically relevant actions of NO is its binding to the heme moiety in the heterodimeric enzyme soluble guanylate cyclase (sGC), 3 which leads to the production of the intracellular second messenger molecule cGMP and activation of cGMP-dependent protein kinases G (PKG). However, NO can also interact with and modify the bioactivity of a number of protein macromolecules through a series of reversible and nonreversible chemical reactions (e.g. S-, N-, and hemenitrosylation, tyrosine and tryptophan nitration) providing multifaceted regulatory mechanisms for cellular functions (3,4). NO is synthesized from L-arginine by a tripartite family of nitric-oxide synthase (NOS) isozymes composed of NOS-1 (nNOS), NOS-2 (iNOS), and NOS-3 (eNOS) originally found in brain, macrophages, and endothelium, respectively, and later discovered, for each NOS, in many other cell types (5).NOS isozymes, especially eNOS, are highly regulated by a number of mechanisms. Emerging findings show that eNOS bioactivity and mobilization to...

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“…Transfection was performed with FuGENE-HD (Roche Applied Science) according to the manufacturer's instructions. Primary culture of mouse cardiomyocytes was carried out following the isolation procedure described (19). Hearts from neonatal mice were washed with suspension minimum essential medium (Invitrogen), sliced into small pieces with scissors, and transferred into a cell dispenser containing 0.1% trypsin at 37°C.…”

Section: Methodsmentioning

confidence: 99%

α-Kinase Anchoring Protein αKAP Interacts with SERCA2A to Spatially Position Ca2+/Calmodulin-dependent Protein Kinase II and Modulate Phospholamban Phosphorylation

Singh

Salih

Tuana

2009

Journal of Biological Chemistry

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The sarco-endoplasmic reticulum calcium ATPase 2a (SERCA2a) is critical for sequestering cytosolic calcium into the sarco-endoplasmic reticulum (SR) and regulating cardiac muscle relaxation. Protein-protein interactions indicated that it exists in complex with Ca 2؉ /calmodulin-dependent protein kinase II (CaMKII) and its anchoring protein ␣KAP. Confocal imaging of isolated cardiomyocytes revealed the colocalization of CAMKII and ␣KAP with SERCA2a at the SR. Deletion analysis indicated that SERCA2a and CaMKII bind to different regions in the association domain of ␣KAP but not with each other. Although deletion of the putative N-terminal hydrophobic amino acid stretch in ␣KAP prevented its membrane targeting, it did not influence binding to SERCA2a or CaMKII. Both CaMKII␦ C and the novel CaMKII␤ 4 isoforms were found to exist in complex with ␣KAP and SERCA2a at the SR and were able to phosphorylate Thr-17 on phospholamban (PLN), an accessory subunit and known regulator of SERCA2a activity. Interestingly, the presence of ␣KAP was also found to significantly modulate the Ca 2؉ /calmodulin-dependent phosphorylation of Thr-17 on PLN. These data demonstrate that ␣KAP exhibits a novel interaction with SERCA2a and may serve to spatially position CaMKII isoforms at the SR and to uniquely modulate the phosphorylation of PLN.The phosphorylation/dephosphorylation cycle is critical for controlling a diverse series of signaling processes in cell biology (1, 2). Specificity of the phosphorylation/dephosphorylation event is in part achieved by selective employment of a protein kinase/phosphatase cascade and subcellular targeting (1, 2). Both spatial and temporal specificity of signaling events is achieved by the compartmentalization of the signaling complexes through adaptor or anchoring proteins (1, 2). Recent studies have highlighted novel aspects of integrating spatially and temporally the cAMP signaling cascades via a diverse family of protein kinase A anchoring proteins (AKAPs) 2 (3). TheAKAPs are responsible for positioning the signaling complex via protein-protein interactions for effective and time-sensitive compartmentalization of the cAMP signal (4). Although the intracellular targeting of protein kinase A to the effectors is being unraveled, little is known about the targeting of CaMKII activity, which is ubiquitously expressed and serves important roles in calcium signaling to guide synaptic transmission (2, 5, 6), gene transcription (7), cell growth (8), and excitation-contraction coupling (9 -11). Although four different isoforms of CaMKII (␣, ␤, ␦, and ␥) are expressed in a tissuespecific manner, cardiac tissue is shown to have predominance of CaMKII␦ C (cytosolic) and CaMKII␦ B (nuclear) isoforms, which serve roles in excitation-contraction coupling and cell growth, respectively (7, 12). Studies have also revealed a significant level of a muscle-specific CaMKII ␤ isoform (CaMKII␤ 4 ) in skeletal and cardiac muscle (11,(13)(14)(15). In addition, the CAMK2A gene that encodes CaMKII␣ kinase in brain expresses an al...

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Cited by 87 publications

References 0 publications

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

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

Nitric Oxide Signaling via Nuclearized Endothelial Nitric-oxide Synthase Modulates Expression of the Immediate Early Genes iNOS and mPGES-1

α-Kinase Anchoring Protein αKAP Interacts with SERCA2A to Spatially Position Ca2+/Calmodulin-dependent Protein Kinase II and Modulate Phospholamban Phosphorylation

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