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      Cholesterol depletion alters coronary artery myocyte Ca 2+ signalling in a stimulus-specific manner

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          Abstract

          Although there is evidence that caveolae and cholesterol play an important role in myocyte signalling processes, details of the mechanisms involved remain sparse. In this paper we have studied for the first time the clinically relevant intact coronary artery and measured in situ Ca 2+ signals in individual myocytes using confocal microscopy. We have examined the effect of the cholesterol-depleting agents, methyl-cyclodextrin (MCD) and cholesterol oxidase, on high K +, caffeine and agonist-induced Ca 2+ signals. We find that cholesterol depletion produces a stimulus-specific alteration in Ca 2+ responses; with 5-HT (10 μM) and endothelin-1 (10 nM) responses being selectively decreased, the phenylephrine response (100 μM) increased and the responses to high K + (60 mM) and caffeine (10 mM) unaffected. Agonist-induced Ca 2+ signals were restored when cholesterol was replenished using cholesterol-saturated MCD. In additional experiments, enzymatically isolated myocytes were patch clamped. We found that cholesterol depletion caused a selective modification of ion channel function, with whole cell inward Ca 2+ current being unaltered, whereas outward K + current was increased, due to BK Ca channel activation. There was also a significant decrease in cell capacitance. These data are discussed in terms of the involvement of caveolae in receptor localisation, Ca 2+ entry pathways and SR Ca 2+ release, and the role of these in agonist signalling.

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          Most cited references49

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          Phenotypic heterogeneity of the endothelium: II. Representative vascular beds.

          Endothelial cells, which form the inner cellular lining of blood vessels and lymphatics, display remarkable heterogeneity in structure and function. This is the second of a 2-part review on the phenotypic heterogeneity of blood vessel endothelial cells. The first part discusses the scope, the underlying mechanisms, and the diagnostic and therapeutic implications of phenotypic heterogeneity. Here, these principles are applied to an understanding of organ-specific phenotypes in representative vascular beds including arteries and veins, heart, lung, liver, and kidney. The goal is to underscore the importance of site-specific properties of the endothelium in mediating homeostasis and focal vascular pathology, while at the same time emphasizing the value of approaching the endothelium as an integrated system.
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            PTRF-Cavin, a conserved cytoplasmic protein required for caveola formation and function.

            Caveolae are abundant cell-surface organelles involved in lipid regulation and endocytosis. We used comparative proteomics to identify PTRF (also called Cav-p60, Cavin) as a putative caveolar coat protein. PTRF-Cavin selectively associates with mature caveolae at the plasma membrane but not Golgi-localized caveolin. In prostate cancer PC3 cells, and during development of zebrafish notochord, lack of PTRF-Cavin expression correlates with lack of caveolae, and caveolin resides on flat plasma membrane. Expression of PTRF-Cavin in PC3 cells is sufficient to cause formation of caveolae. Knockdown of PTRF-Cavin reduces caveolae density, both in mammalian cells and in the zebrafish. Caveolin remains on the plasma membrane in PTRF-Cavin knockdown cells but exhibits increased lateral mobility and accelerated lysosomal degradation. We conclude that PTRF-Cavin is required for caveola formation and sequestration of mobile caveolin into immobile caveolae.
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              Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities.

              Caveolin-1 is the principal structural protein of caveolae membranes in fibroblasts and endothelia. Recently, we have shown that the human CAV-1 gene is localized to a suspected tumor suppressor locus, and mutations in Cav-1 have been implicated in human cancer. Here, we created a caveolin-1 null (CAV-1 -/-) mouse model, using standard homologous recombination techniques, to assess the role of caveolin-1 in caveolae biogenesis, endocytosis, cell proliferation, and endothelial nitric-oxide synthase (eNOS) signaling. Surprisingly, Cav-1 null mice are viable. We show that these mice lack caveolin-1 protein expression and plasmalemmal caveolae. In addition, analysis of cultured fibroblasts from Cav-1 null embryos reveals the following: (i) a loss of caveolin-2 protein expression; (ii) defects in the endocytosis of a known caveolar ligand, i.e. fluorescein isothiocyanate-albumin; and (iii) a hyperproliferative phenotype. Importantly, these phenotypic changes are reversed by recombinant expression of the caveolin-1 cDNA. Furthermore, examination of the lung parenchyma (an endothelial-rich tissue) shows hypercellularity with thickened alveolar septa and an increase in the number of vascular endothelial growth factor receptor (Flk-1)-positive endothelial cells. As predicted, endothelial cells from Cav-1 null mice lack caveolae membranes. Finally, we examined eNOS signaling by measuring the physiological response of aortic rings to various stimuli. Our results indicate that eNOS activity is up-regulated in Cav-1 null animals, and this activity can be blunted by using a specific NOS inhibitor, nitro-l-arginine methyl ester. These findings are in accordance with previous in vitro studies showing that caveolin-1 is an endogenous inhibitor of eNOS. Thus, caveolin-1 expression is required to stabilize the caveolin-2 protein product, to mediate the caveolar endocytosis of specific ligands, to negatively regulate the proliferation of certain cell types, and to provide tonic inhibition of eNOS activity in endothelial cells.
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                Author and article information

                Journal
                Cell Calcium
                Cell Calcium
                Cell Calcium
                Elsevier
                0143-4160
                1532-1991
                January 2010
                January 2010
                : 47
                : 1
                : 84-91
                Affiliations
                [a ]The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, Merseyside, UK
                [b ]Department of Human Anatomy & Cell Biology, University of Liverpool, Crown Street, Liverpool L69 3BX, UK
                Author notes
                [* ]Corresponding author. Tel.: +44 151 7945329; fax: +44 151 7945321. c.prendergast@ 123456liv.ac.uk
                [1]

                Tel.: +44 151 7945329; fax: +44 151 7945321.

                Article
                YCECA1125
                10.1016/j.ceca.2009.11.009
                2824115
                20022108
                a3ebdec3-0487-4948-8844-e61d4737a1de
                © 2010 Elsevier Ltd.

                This document may be redistributed and reused, subject to certain conditions.

                History
                : 17 June 2009
                : 23 November 2009
                : 26 November 2009
                Categories
                Article

                Molecular biology
                coronary artery,lipid rafts,cholesterol,vascular smooth muscle
                Molecular biology
                coronary artery, lipid rafts, cholesterol, vascular smooth muscle

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