The use of confocal microscopy in the investigation of cell structure and function in the heart, vascular endothelium and smooth muscle cells

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

doi:10.1007/978-1-4615-6353-2_18

Cited by 50 publications

(157 citation statements)

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“…Increasing IP3 production, in turn, increases [Ca 2+ ] n through an IP3R-mediated release of Ca 2+ from perinuclear stores in both atrial and ventricular myocytes [49,86,87]. ET-1 has also been shown to increase [Ca 2+ ] n , presumably released from stores within the nuclear cisternae, in nuclei isolated from chick heart, cultured human aortic smooth muscle cells [15,88] and rat heart [12]. Similarly, the ETB-selective agonist IRL-1620 increases [Ca 2+ ] n in nuclei isolated from rat heart [12] and liver [89].…”

Section: Discussionmentioning

confidence: 99%

Intracrine endothelin signaling evokes IP3-dependent increases in nucleoplasmic Ca2+ in adult cardiac myocytes

Merlen¹,

Farhat²,

Luo³

et al. 2013

Journal of Molecular and Cellular Cardiology

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Endothelin receptors are present on the nuclear membranes in adult cardiac ventricular myocytes. The objectives of the present study were to determine 1) which endothelin receptor subtype is in cardiac nuclear membranes, 2) if the receptor and ligand traffic from the cell surface to the nucleus, and 3) the effect of increased intracellular ET-1 on nuclear Ca 2+ signalling. Confocal microscopy using fluorescently-labeled endothelin analogs confirmed the presence of ETB at the nuclear membrane of rat cardiomyocytes in skinned-cells and isolated nuclei. Furthermore, in both cardiac myocytes and aortic endothelial cells, endocytosed ET:ETB complexes translocated to lysosomes and not the nuclear envelope. Although ETA and ETB can form heterodimers, the presence or absence of ETA did not alter ETB trafficking. Treatment of isolated nuclei with * Abbreviations: The abbreviations used are: 2-APB, 2-aminoethoxydiphenyl borate; A, Angiotensin II; αAR, α-adrenergic receptor;[Ca 2+ ] n , nucleoplasmic free calcium concentration; CaMKII, Ca 2+ /calmodulin-dependent protein kinase II; DMNB, 4,5-dimethoxynitrobenzyl group; DNA, deoxyribonucleic acid; DCC, 1,3-dicyclohexylcarbodiimide; DCM, dichloromethane; DTT, dithiothreitol; ECE1-a, endothelin converting enzyme 1a; cET-1, caged ET-1; caged ET-1, [Trp-ODMNB 21 ]ET-1; ET-1, endothelin 1; ET-2, endothelin 2; ET-3, endothelin 3; ETA, endothelin type A receptor; ETB, endothelin type B receptor; ETR, endothelin receptor; GPCR, G protein-coupled receptor; HDAC5; histone deacetylase 5; IP3, inositol 1,4,5-trisphosphate; IP3R, inositol 1,4,5-trisphosphate receptor; ISO, isoproterenol; MEF2, myocyte enhancing factor-2; PBS, phosphate buffered saline; PMSF, phenylmethanesulphonylfluoride; PNGase F, peptide N-glycosidase F; qPCR, quantitative real-time polymerase chain reaction; RNA, ribonucleic acid; RyR, ryanodine receptor; TCA, trichloroacetic acid; TFA, trifluoroacetic acid; TX-100, Triton X-100. Address correspondence to: Bruce G. Allen, Montreal Heart Institute, 5000 Belanger St,. Montréal, Québec, Canada, H1T 1C8., Telephone: (514) ] n whereas extracellular application of ETA and ETB receptor antagonists did not. These data suggest that 1) the endothelin receptor in the cardiac nuclear membranes is ETB, 2) ETB traffic directly to the nuclear membrane after biosynthesis, 3) exogenous endothelins are not ligands for ETB on nuclear membranes, and 4) ETB associated with the nuclear membranes regulate nuclear Ca 2+ signalling.

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Section: Discussionmentioning

confidence: 99%

Intracrine endothelin signaling evokes IP3-dependent increases in nucleoplasmic Ca2+ in adult cardiac myocytes

Merlen¹,

Farhat²,

Luo³

et al. 2013

Journal of Molecular and Cellular Cardiology

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“…The idea that nuclear Ca 2+ levels are regulated independently from those in the cytosol emerged as soon as imaging technology advanced to allow monitoring of Ca 2+ changes simultaneously in different cellular compartments (Bkaily et al, 1997). Using both wide-field and confocal fluorescence imaging of living cells, several groups began to report that nuclear Ca 2+ concentration differed from that in the cytosol.…”

Section: Measuring Nuclear Ca 2+ Signals: Key Considerationsmentioning

confidence: 99%

An update on nuclear calcium signalling

Bootman

Fearnley

Smyrnias

et al. 2009

Journal of Cell Science

186

194

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Over the past 15 years or so, numerous studies have sought to characterise how nuclear calcium (Ca 2+ ) signals are generated and reversed, and to understand how events that occur in the nucleoplasm influence cellular Ca 2+ activity, and vice versa. In this Commentary, we describe mechanisms of nuclear Ca 2+ signalling and discuss what is known about the origin and physiological significance of nuclear Ca 2+ transients. In particular, we focus on the idea that the nucleus has an autonomous Ca 2+ signalling system that can generate its own Ca 2+ transients that modulate processes such as gene transcription. We also discuss the role of nuclear pores and the nuclear envelope in controlling ion flux into the nucleoplasm. Journal of Cell Science 2338(which would cause its nuclear export) and through a physical interaction that occludes a nuclear-export sequence (NES) in the NFAT protein.An example of a nuclear-localised transcription factor that is regulated by both nuclear and cytosolic Ca 2+ signals is cyclic AMP response element-binding protein (CREB). The signalling mechanisms that underlie CREB activation have been worked out particularly well in neurons, in which it has been established that depolarisation leads to the rapid phosphorylation of CREB on Ser133 by Ca 2+ -calmodulin-dependent kinase IV, which provides a necessary, but not sufficient, signal for CREB-mediated gene transcription (Dolmetsch et al., 2001). The triggering event is an increase in cytosolic Ca 2+ levels in the immediate vicinity of L-type Ca 2+ channels or N-methyl-D-aspartate receptors that are found in the synapse, at a site that is distal to the nucleus (Shaywitz and Greenberg, 1999). As CREB is only found in the nucleus, the phosphorylation of Ser133 is mediated by one of several Ca 2+ -sensitive signal transduction cascades that convey information from the remote synapses into the nucleus. In the nucleus, phosphorylated CREB binds additional proteins to form a transcriptionally active complex (Shaywitz and Greenberg, 1999). Although the synaptic Ca 2+ signal does not need to propagate to the nucleus to induce phosphorylation of Ser133 on CREB, an increase in the level of nuclear Ca 2+ is required for CREB-mediated gene transcription to occur. This is because additional Ca 2+ -dependent phosphorylation of CREB and its co-activators is required (Chawla et al., 1998;Kornhauser et al., 2002). Indeed, nuclear injection of an artificial Ca 2+ buffer prevents CREB-mediated gene transcription, but not transcription induced by the serum-response element, which is sensitive to cytosolic Ca 2+ buffering (Hardingham et al., 1997).Another well-known target of nuclear Ca 2+ signalling is the transcriptional regulator downstream regulatory element antagonist modulator (DREAM). It is well established that DREAM represses the expression of prodynorphin, an opiate-receptor precursor, and that mice that lack DREAM have a constitutive analgesic condition (Cheng et al., 2002). DREAM contains four Ca 2+ -binding EF hands (a widely expressed structural mo...

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“…The cells were cultured on 35-mm glass-bottom dishes (Plastek Cultureware, MatTek, Ashland, MA) and then loaded with the Ca 2ϩ -sensitive fluorescent dye fluo 3-AM (Molecular Probes) at a final concentration of 13.5 M in Tyrode-BSA solution for 45 min, as described previously (6).…”

Section: Ca 2ϩ Influx Experiments Using Confocal Laser Scanning Micromentioning

confidence: 99%

Caveolin-1-deficient aortic smooth muscle cells show cell autonomous abnormalities in proliferation, migration, and endothelin-based signal transduction

Hassan¹,

Williams²,

Frank³

et al. 2006

American Journal of Physiology-Heart and Circulatory Physiology

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. Caveolin-1-deficient aortic smooth muscle cells show cell autonomous abnormalities in proliferation, migration, and endothelin-based signal transduction. Am J Physiol Heart Circ Physiol 290: H2393-H2401, 2006. First published January 13, 2006 doi:10.1152/ajpheart.01161.2005.-We previously showed that ablation of caveolin-1 (Cav-1) gene expression in mice promotes neointimal hyperplasia in vivo, a phenomenon normally characterized by smooth muscle cell (SMC) migration and proliferation. Whether these defects are cell autonomous, i.e., due to loss of Cav-1 within SMCs or loss of Cav-1 expression in other adjacent cell types in vivo, remains unknown. Cav-1 has been shown to associate with receptors for many vasoactive factors on the SMC surface. Therefore, Cav-1 might be an important regulator of SMC proliferation, migration, and signal transduction. To mechanistically dissect the role of Cav-1 in SMC signaling, we isolated SMCs from the aortas (AoSMCs) of Cav-1-deficient (Cav-1 Ϫ/Ϫ ) mice and characterized these cells with respect to their proliferation, migration, and Ca 2ϩ response to an important vasoactive factor, endothelin-1 (ET-1). 5-Bromo-2Ј-deoxyuridine incorporation and a wound-healing assay showed an increase in proliferation and migration rates in Cav-1 Ϫ/Ϫ compared with wild-type (Cav-1 ϩ/ϩ ) AoSMCs. Cav-1 Ϫ/Ϫ AoSMCs demonstrated upregulation of phosphorylated ERK1/2, cyclin D1, and proliferating cell nuclear antigen and reduced expression of the cyclin-dependent kinase inhibitor p27Kip1 . The Ca 2ϩ response was examined in the presence of ET-1 and assessed by confocal microscopy with the Ca 2ϩ -sensitive fluorescent probe fluo 3. When treated with ET-1, Cav-1 Ϫ/Ϫ AoSMCs exhibited a faster and larger increase in free intracellular Ca 2ϩ than Cav-1 ϩ/ϩ cells. The ET-1-induced response in Cav-1 Ϫ/Ϫ cells was mediated by the ETB receptor, as shown using the ETB receptor antagonist BQ-788 and the ETA receptor antagonist BQ-123. In Cav-1 Ϫ/Ϫ cells, ETA receptor expression was reduced and ETB receptor expression was upregulated. Therefore, Cav-1 ablation increased the ET-1-induced Ca 2ϩ response in SMCs by altering the type and expression level of the ET receptor (i.e., receptor isoform switching). These data suggest a novel regulatory role for Cav-1 in SMCs with respect to their proliferation, migration, and Ca 2ϩ -mediated signaling. vascular disease; calcium response; endothelin receptors; neointimal hyperplasia; caveolae; caveolin SMOOTH MUSCLE CELLS (SMCs) are used to construct the walls of hollow organs, such as the stomach, intestine, bladder, uterus, and blood vessels (44). Their proliferation, migration, and contraction processes are highly controlled by many factors, ensuring proper functioning of the corresponding organ (31). In the vasculature, abnormalities in these processes are associated with many different classes of vascular disease, such as hypertension, atherosclerosis, and restenosis. However, many of the factors that contribute to the development of vascular disease remain unknown.C...

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The use of confocal microscopy in the investigation of cell structure and function in the heart, vascular endothelium and smooth muscle cells

Cited by 50 publications

References 42 publications

Intracrine endothelin signaling evokes IP3-dependent increases in nucleoplasmic Ca2+ in adult cardiac myocytes

Intracrine endothelin signaling evokes IP3-dependent increases in nucleoplasmic Ca2+ in adult cardiac myocytes

An update on nuclear calcium signalling

Caveolin-1-deficient aortic smooth muscle cells show cell autonomous abnormalities in proliferation, migration, and endothelin-based signal transduction

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