Enrichment of vital adult cardiac muscle cells by continuous silica sol gradient centrifugation

Maisch, Bernhard

doi:10.1007/bf01908052

Cited by 15 publications

(6 citation statements)

References 23 publications

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“…The amounts of esterified and free indicators were determined by liquid scintillation counting of the chloroform and methanollwater extracts, respectively. Intracellular concentrations were determined assumiIig a tissue density of 1.067 g ml-I (17), intracellular water fraction of 70% (18,19), and a wet weight to dry weight ratio of 3.8 (20).…”

Section: Indicator Loadingmentioning

confidence: 99%

Resting and end‐diastolic [Ca²⁺]i measurements in the langendorff‐perfused ferret heart loaded with a ¹⁹F NMR indicator

Harding

Smith

Metcalfe

et al. 1993

Magnetic Resonance in Med

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Intracellular free calcium concentration ([Ca2+]i) was measured in Langendorff-perfused ferret hearts (30 degrees C, pH 7.4) by loading paced hearts with the 19F NMR calcium indicator, the 5,5'-difluoro derivative of 1,2-bis(o-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid (5FBAPTA), to an initial cytosolic concentration of approximately 120 microM. Increasing the pacing frequency raised the end-diastolic [Ca2+]i from 299 +/- 44 nM (mean +/- SEM) at 0.2 Hz to 522 +/- 54 nM at 1.0 Hz and 691 +/- 166 nM at 2.0 Hz. Raising [Ca]o from 1.8 to 7.0 mM at a pacing frequency of 1.0 Hz increased end-diastolic [Ca2+]i to 625 +/- 39 nM. In unpaced hearts perfused with diltiazem (100 microM), [Ca2+]i fell rapidly to a steady-state value of < 100 nM after 60 min. Raising [Ca]o from 1.8 to 7.0 mM had no detectable effect on resting [Ca2+]i. The time course of the [Ca2+]i transient was measured in hearts paced at 1.1 Hz and perfused with 1.8 mM [Ca]o. The peak [Ca2+]i was approximately 2 microM at approximately 150 msec after the pacing pulse, and peak developed LVP occurred at 550 msec compared with 280 msec in control hearts not loaded with 5FBAPTA. Comparisons with data obtained by other techniques, including fluorescent [Ca2+]i indicators, imply that although the end-diastolic [Ca2+]i values obtained with 5FBAPTA in beating hearts are elevated by the concentrations of intracellular 5FBAPTA required for signal detection, the changes in [Ca2+]i observed in response to experimental interventions are qualitatively consistent with previous data.

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Section: Indicator Loadingmentioning

confidence: 99%

Resting and end‐diastolic [Ca²⁺]i measurements in the langendorff‐perfused ferret heart loaded with a ¹⁹F NMR indicator

Harding

Smith

Metcalfe

et al. 1993

Magnetic Resonance in Med

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“…Viable myocytes were further purified by Percoll density gradient centrifugation. 27 By visual count, Ͼ95% of the cells were found to be myocytes. Cav-enriched membranes were prepared by detergent dissolution or by sonication.…”

Section: Preparation Of Cav-enriched Membrane Fragments From Rat Hearmentioning

confidence: 99%

Dystrophin Associates With Caveolae of Rat Cardiac Myocytes

Doyle

Goings

Upshaw-Earley

et al. 2000

Circulation Research

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Abstract-The possibility of an interaction between the cytoskeletal protein dystrophin and cell surface caveolae in the mammalian myocardium was investigated by several techniques. Caveolin (cav)-3-enriched, detergent-insoluble membranes isolated from purified ventricular sarcolemma by density-gradient fractionation were found to contain dystrophin and dystroglycan. Further purification of cav-3-containing membranes by immunoprecipitation using anti-cav-3-coated magnetic beads yielded dystrophin but not always dystroglycan. Electron microscopic analysis of precipitated material revealed caveola-sized vesicular profiles that could be double-labeled with anti-dystrophin and anti-cav-3 antibodies. In contrast, immunoprecipitation of membranes with anti-dystrophin-coated beads yielded both cav-3 and dystroglycan. Electron microscopic analysis of this material showed heterogeneous membrane profiles, some of which could be decorated with anti-cav-3 antibodies. To confirm that dystrophin and cav-3 were closely associated in cardiac myocytes, we verified that dystrophin was also present in immunoprecipitated cav-3-containing membranes from detergent extracts, as well as in sonicated extracts of purified ventricular myocytes. Confocal immunofluorescence microscopy of ventricular and atrial cardiac myocytes showed that the cellular distributions of cav-3 and dystrophin partially overlapped. Immuno-electron micrographs of thin sections of rat atrial myocytes revealed a fraction of dystrophin molecules that are in apparently close apposition to caveolae. These results suggest that a subpopulation of dystrophin molecules interacts with cardiac myocyte caveolae in vivo and that some of the dystrophin is engaged in linking cav-3 with the dystroglycan complex. Key Words: heart Ⅲ myocyte Ⅲ caveolae Ⅲ dystrophin T he flask-shaped plasma membrane-associated vesicles called caveolae are non-clathrin-coated organelles first described by Palade in 1953. 1 Recently, caveolae have been proposed to be multifunctional organelles that, in diverse cell types, participate in one or more of the following: as sites for concentration and subsequent internalization of small molecules and as binding sites for Ca 2ϩ ions, 2 as the loci of multiple signal-transducing molecules, 3 and as sites of cytoskeletal attachment to the plasma membrane. 4 Presumptive caveolar properties or "markers" include the caveolar coat protein caveolin (cav), an 18-to 24-kDa protein (depending on the isoform), 5 and a high concentration of cholesterol and sphingolipids. 6 Four isoforms of caveolin encoded by 3 distinct genes have so far been identified. Cav-1␣, 7 also known as VIP21 or VCAV, 8 is found in a variety of tissues, as is cav-1␤, a truncated version also derived from the same gene. 9 cav-2, the isoform most abundant in fat cells, coexpresses with cav-1 in many nonmuscle tissues, 10 whereas cav-3, 11 or MCAV, 12 is found predominantly if not exclusively in muscle cells. Thus, in the heart, cav-3 is the isoform expressed in cardiac myocytes and vascular smooth...

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“…A nonmyocyte-enriched fraction was obtained by centrifuging the supernatant 570 g for 13 min. Viable myocytes were further purified by Percoll density gradient centrifugation (11).…”

Section: Preparation Of Heart Cellsmentioning

confidence: 99%

Natriuretic peptide receptor-B in adult rat ventricle is predominantly confined to the nonmyocyte population

Doyle

Upshaw-Earley

Bell

et al. 2002

American Journal of Physiology-Heart and Circulatory Physiology

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We assessed the cellular localization and relative concentration of the C-type natriuretic peptide (CNP) guanylate cyclase-B (GC-B) receptor in the adult rat heart ventricle by several techniques. In frozen sections of the ventricle, anti-receptor antibody stained the vasculature and cells interstitial to myocytes, but not the myocytes themselves. The same antibody detected GC-B in immunoblots of protein extracts of nonmyocytes, but not myocytes and recognized an equivalent protein in extracts of cultured cardiac fibroblasts, but not A7r5 rat smooth muscle cells. In functional assays, CNP-induced cGMP accumulation per milligram cell protein was an order of magnitude greater in cultured cardiac fibroblasts than in A7r5 smooth muscle cells and two orders of magnitude greater than in freshly isolated cardiac myocytes. Modulation of cGMP accumulation by phosphodiesterases (PDEs) was cell specific as determined by antagonist pharmacological profiles, PDE1 in fibroblasts, PDE2 in A7r5 cells, and PDE3 in myocytes, suggesting that significant but low-level cGMP response to CNP measured in heart myocytes is not due to nonmyocyte contamination. Fibroblasts of cardiac origin do not show an interactive relationship between receptor responsiveness to CNP, cGMP levels, and proliferation-related mitogen-activated signal transduction pathways. Whereas previous reports suggest CNP exerts significant effects in neonatal rat cardiomyocytes, our results suggest that fibroblasts are likely the most responsive cell type (cGMP production) in the adult rat heart. cardiomyocytes; fibroblasts; smooth muscle; guanylate cyclase C-TYPE NATRIURETIC PEPTIDE (CNP) is a member of the family of NP hormones that also includes atrial NP (ANP) and brain NP (BNP) (26). ANP is synthesized in cardiac myocytes, where it is secreted constitutively in both the ventricle and atrium as well as in a regulated manner from granules in the atrium. First discovered in the brain, CNP has also been found in other tissues as well, e.g., in bone, reproductive tissue, and heart tissue (6, 22). CNP is secreted by endothelial cells in the heart (25), but its role in myocardial function is much less clear than that of ANP. There are presently three known NP receptors (NPR): NPR-A, -B, and -C. In an anomaly of nomenclature, NPR-A binds ANP and BNP in the nanomolar range and CNP in the micromolar range, whereas NPR-B binds CNP in the nanomolar range but binds ANP and BNP only in the micromolar range (17). Both of these transmembrane receptors express guanylyl cyclase (GC) activity in their cytoplasmic domains and are also known as GC-A and GC-B (27). NPR-C, found most abundantly in the kidney, has a truncated cytoplasmic tail, has no cyclase activity, and is believed to be involved predominantly in NP clearance (21), although a role in G i -dependent signaling has recently been proposed (15).mRNA for all three receptor types was detected by RT-PCR in myocytes isolated from the adult rat ventricle, but expression at the protein level was convincingly shown for GC-A, but n...

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Enrichment of vital adult cardiac muscle cells by continuous silica sol gradient centrifugation

Cited by 15 publications

References 23 publications

Resting and end‐diastolic [Ca²⁺]i measurements in the langendorff‐perfused ferret heart loaded with a ¹⁹F NMR indicator

Resting and end‐diastolic [Ca²⁺]i measurements in the langendorff‐perfused ferret heart loaded with a ¹⁹F NMR indicator

Dystrophin Associates With Caveolae of Rat Cardiac Myocytes

Natriuretic peptide receptor-B in adult rat ventricle is predominantly confined to the nonmyocyte population

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Enrichment of vital adult cardiac muscle cells by continuous silica sol gradient centrifugation

Cited by 15 publications

References 23 publications

Resting and end‐diastolic [Ca2+]i measurements in the langendorff‐perfused ferret heart loaded with a 19F NMR indicator

Resting and end‐diastolic [Ca2+]i measurements in the langendorff‐perfused ferret heart loaded with a 19F NMR indicator

Dystrophin Associates With Caveolae of Rat Cardiac Myocytes

Natriuretic peptide receptor-B in adult rat ventricle is predominantly confined to the nonmyocyte population

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Resting and end‐diastolic [Ca²⁺]i measurements in the langendorff‐perfused ferret heart loaded with a ¹⁹F NMR indicator

Resting and end‐diastolic [Ca²⁺]i measurements in the langendorff‐perfused ferret heart loaded with a ¹⁹F NMR indicator