Species-specific difference in distribution of voltage-gated L-type Ca2+ channels of cardiac myocytes

2000 ◽  
Vol 279 (6) ◽  
pp. C1963-C1969 ◽  
Author(s):  
Yoshiko Takagishi ◽  
Kenji Yasui ◽  
Nicholas J. Severs ◽  
Yoshiharu Murata
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Cardiac Myocytes ◽  
Immunogold Electron Microscopy ◽  
Surface Plasma ◽  
Gold Particles ◽  
Intracellular Ca ◽  
Rat Myocytes ◽  
T Tubules ◽  
Species Specific

Ca2+influx via sarcolemmal voltage-dependent Ca2+ channels (L-type Ca2+ channels) is the fundamental step in excitation-contraction (E-C) coupling in cardiac myocytes. Physiological and pharmacological studies reveal species-specific differences in E-C coupling resulting from a difference in the contribution of Ca2+ influx and intracellular Ca2+ release to activation of contraction. We investigated the distribution of L-type Ca2+ channels in isolated cardiac myocytes from rabbit and rat ventricle by correlative immunoconfocal and immunogold electron microscopy. Immunofluorescence labeling revealed discrete spots in the surface plasma membrane and transverse (T) tubules in rabbit myocytes. In rat myocytes, labeling appeared more intense in T tubules than in the surface sarcolemma. Immunogold electron microscopy extended these findings, showing that the number of gold particles in the surface plasma membrane was significantly higher in rabbit than rat myocytes. In rabbit myocyte plasma membrane, the gold particles were distributed as clusters in both regions that were associated with junctional sarcoplasmic reticulum and those that were not. The findings are consistent with the idea that influx of Ca2+ via surface sarcolemmal Ca2+ channels contributes to intracellular Ca2+ to a greater degree in rabbit than in rat myocytes.

scholarly journals Analysis of GLUT4 Distribution in Whole Skeletal Muscle Fibers: Identification of Distinct Storage Compartments That Are Recruited by Insulin and Muscle Contractions

1998 ◽  
Vol 142 (6) ◽  
pp. 1429-1446 ◽  
Author(s):  
Thorkil Ploug ◽  
Bo van Deurs ◽  
Hua Ai ◽  
Samuel W. Cushman ◽  
Evelyn Ralston
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Plasma Membrane ◽  
Surface Membrane ◽  
Muscle Fibers ◽  
Muscle Contractions ◽  
The Other ◽  
Immunogold Electron Microscopy ◽  
Double Labeling ◽  
T Tubules

The effects of insulin stimulation and muscle contractions on the subcellular distribution of GLUT4 in skeletal muscle have been studied on a preparation of single whole fibers from the rat soleus. The fibers were labeled for GLUT4 by a preembedding technique and observed as whole mounts by immunofluorescence microscopy, or after sectioning, by immunogold electron microscopy. The advantage of this preparation for cells of the size of muscle fibers is that it provides global views of the staining from one end of a fiber to the other and from one side to the other through the core of the fiber. In addition, the labeling efficiency is much higher than can be obtained with ultracryosections. In nonstimulated fibers, GLUT4 is excluded from the plasma membrane and T tubules. It is distributed throughout the muscle fibers with ∼23% associated with large structures including multivesicular endosomes located in the TGN region, and 77% with small tubulovesicular structures. The two stimuli cause translocation of GLUT4 to both plasma membrane and T tubules. Quantitation of the immunogold electron microscopy shows that the effects of insulin and contraction are additive and that each stimulus recruits GLUT4 from both large and small depots. Immunofluorescence double labeling for GLUT4 and transferrin receptor (TfR) shows that the small depots can be further subdivided into TfR-positive and TfR-negative elements. Interestingly, we observe that colocalization of TfR and GLUT4 is increased by insulin and decreased by contractions. These results, supported by subcellular fractionation experiments, suggest that TfR-positive depots are only recruited by contractions. We do not find evidence for stimulation-induced unmasking of resident surface membrane GLUT4 transporters or for dilation of the T tubule system (Wang, W., P.A. Hansen, B.A. Marshall, J.O. Holloszy, and M. Mueckler. 1996. J. Cell Biol. 135:415–430).

Molecular identification of a TTX-sensitive Ca2+current

2001 ◽  
Vol 280 (5) ◽  
pp. C1327-C1339 ◽  
Author(s):  
Silvia Guatimosim ◽  
Eric A. Sobie ◽  
Jader dos Santos Cruz ◽  
Laura A. Martin ◽  
W. J. Lederer
Keyword(s):  
Cardiac Myocytes ◽  
Ion Selectivity ◽  
Inactivation Kinetics ◽  
Experimental Conditions ◽  
Guinea Pig Heart ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Intracellular Ca ◽  
Rat Myocytes

The TTX-sensitive Ca2+ current [ I Ca(TTX)] observed in cardiac myocytes under Na+-free conditions was investigated using patch-clamp and Ca2+-imaging methods. Cs+ and Ca2+were found to contribute to I Ca(TTX), but TEA+ and N-methyl-d-glucamine (NMDG+) did not. HEK-293 cells transfected with cardiac Na+ channels exhibited a current that resembled I Ca(TTX) in cardiac myocytes with regard to voltage dependence, inactivation kinetics, and ion selectivity, suggesting that the cardiac Na+ channel itself gives rise to I Ca(TTX). Furthermore, repeated activation of I Ca(TTX) led to a 60% increase in intracellular Ca2+ concentration, confirming Ca2+ entry through this current. Ba2+ permeation of I Ca(TTX), reported by others, did not occur in rat myocytes or in HEK-293 cells expressing cardiac Na+channels under our experimental conditions. The report of block of I Ca(TTX) in guinea pig heart by mibefradil (10 μM) was supported in transfected HEK-293 cells, but Na+current was also blocked (half-block at 0.45 μM). We conclude that I Ca(TTX) reflects current through cardiac Na+ channels in Na+-free (or “null”) conditions. We suggest that the current be renamed I Na(null) to more accurately reflect the molecular identity of the channel and the conditions needed for its activation. The relationship between I Na(null)and Ca2+ flux through slip-mode conductance of cardiac Na+ channels is discussed in the context of ion channel biophysics and “permeation plasticity.”

scholarly journals The R-SNARE Endobrevin/VAMP-8 Mediates Homotypic Fusion of Early Endosomes and Late Endosomes

2000 ◽  
Vol 11 (10) ◽  
pp. 3289-3298 ◽  
Author(s):  
Wolfram Antonin ◽  
Claudia Holroyd ◽  
Ritva Tikkanen ◽  
Stefan Höning ◽  
Reinhard Jahn
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Subcellular Fractionation ◽  
Immunogold Electron Microscopy ◽  
Fusion Reactions ◽  
Coated Pits ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Golgi Network

Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown in which intracellular fusion step it operates. Using subcellular fractionation and quantitative immunogold electron microscopy, we found that endobrevin/VAMP-8 is present on all membranes known to communicate with early endosomes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of thetrans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potently inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevin/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of early and late endosomes.

Parvovirus Infects Cardiac Myocytes in Hydrops Fetalis

2003 ◽  
Vol 6 (5) ◽  
pp. 414-420 ◽  
Author(s):  
Aiveen O'Malley ◽  
Carole Barry-Kinsella ◽  
Caroline Hughes ◽  
Peter Kelehan ◽  
Deirdre Devaney ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cardiac Myocytes ◽  
Cardiac Tissue ◽  
Parvovirus B19 ◽  
Negative Control ◽  
Hydrops Fetalis ◽  
Immunogold Electron Microscopy ◽  
Erythroid Precursor ◽  
Transmission Electron ◽  
Parvovirus B19 Infection

Parvovirus infection during pregnancy is an important cause of hydrops fetalis. It is attributed to anemia caused by viral-induced destruction of red blood cells. Infection of other organs has been reported including the heart, liver, and lungs. Few of these reports, however, convincingly demonstrate virions within the functional parenchyma of the tissue. This is of particular concern regarding myocardium in the context of hydrops fetalis which is, in part, due to cardiac failure. The problem in routine pathology practice is that most fetuses with the infection are macerated. This, in part, probably explains the paucity of published information on cardiac involvement. This study examined five cases of fatal hydrops fetalis with variable maceration with serologically proven parvovirus B19 infection. Transmission electron microscopy of cardiac tissue demonstrated intranuclear virions in both erythroid precursor cells and in cardiac myocytes in three of these cases. In each of these, immunogold electron microscopy provided confirmatory evidence of parvovirus infection. Virions were not identifiable where maceration had caused disintegration of nuclei in the myocytes. In addition, virions were absent in the three negative control cases where retroplacental hemorrhage was confirmed as the cause of death. This study suggests that parvovirus infection of cardiac myocytes may play a more important role in causing hydrops fetalis than previously realized. It also demonstrates that maceration should not discourage the use of electron microscopy.

scholarly journals Quantitative immunocytochemical analysis of the induction of cytochrome P450IIB in rat hepatocytes.

1992 ◽  
Vol 40 (1) ◽  
pp. 73-82 ◽  
Author(s):  
Y Fukui ◽  
A Yamamoto ◽  
R Masaki ◽  
K Miyauchi ◽  
Y Tashiro
Keyword(s):  
Electron Microscopy ◽  
Particle Density ◽  
Protein A ◽  
Liver Microsomes ◽  
Rat Hepatocytes ◽  
Immunoblot Analysis ◽  
Immunogold Electron Microscopy ◽  
Thin Sections ◽  
Gold Particles ◽  
Rough Er

We examined whether induction of the phenobarbital (PB)-inducible form of cytochrome P450 (P450IIB) in rat hepatocytes could be analyzed quantitatively by immunogold electron microscopy. Rats received intraperitoneal injections of PB every 24 hr and livers at the various stages of PB induction were fixed by perfusion with a mixture of paraformaldehyde (4%) and glutaraldehyde (0.1%) and embedded in LR White. Ultra-thin sections were cut and labeled by the protein A-gold procedure using affinity-purified anti-P450IIB antibody which was previously immunoabsorbed with liver microsomes from a control rat (not treated with PB). We counted the number of gold particles per micron of the rough ER membranes (particle density). Before PB treatment, the particle density of the rough ER in rat hepatocytes was practically zero and increased markedly at 48 and 72 hr after PB treatment. The rough microsomes were prepared from these PB-treated rat livers. The amount of P450IIB was estimated by immunoblot analysis and the number of gold particles bound to the rough microsomal membrane was determined by the same post-embedding immunogold procedure. The particle density of the rough microsomes increased in parallel with the increase in the amount of P450IIB, indicating good correlation of the two variables. Thus, the induction of cytochrome P450IIB can be quantitatively and reliably investigated by immunogold electron microscopy.

scholarly journals Immunogold Electron Microscopic Demonstration of Distinct Submembranous Localization of the Activated γPKC Depending on the Stimulation

2007 ◽  
Vol 56 (3) ◽  
pp. 253-265 ◽  
Author(s):  
Miho Oyasu ◽  
Mineko Fujimiya ◽  
Kaori Kashiwagi ◽  
Shiho Ohmori ◽  
Hirotsugu Imaeda ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Fluorescent Microscopy ◽  
Confocal Laser ◽  
Immunogold Electron Microscopy ◽  
Subcellular Targeting ◽  
Electron Microscopic ◽  
Proper Distance

We examined the precise intracellular translocation of γ subtype of protein kinase C (γPKC) after various extracellular stimuli using confocal laser-scanning fluorescent microscopy (CLSM) and immunogold electron microscopy. By CLSM, treatment with 12- O-tetradecanoylphorbol-13-acetate (TPA) resulted in a slow and irreversible accumulation of green fluorescent protein (GFP)-tagged γPKC (γPKC–GFP) on the plasma membrane. In contrast, treatment with Ca2+ ionophore and activation of purinergic or NMDA receptors induced a rapid and transient membrane translocation of γPKC–GFP. Although each stimulus resulted in PKC localization at the plasma membrane, electron microscopy revealed that γPKC showed a subtle but significantly different localization depending on stimulation. Whereas TPA and UTP induced a sustained localization of γPKC–GFP on the plasma membrane, Ca2+ ionophore and NMDA rapidly translocated γPKC–GFP to the plasma membrane and then restricted γPKC–GFP in submembranous area (<500 nm from the plasma membrane). These results suggest that Ca2+ influx alone induced the association of γPKC with the plasma membrane for only a moment and then located this enzyme at a proper distance in a touch-and-go manner, whereas diacylglycerol or TPA tightly anchored this enzyme on the plasma membrane. The distinct subcellular targeting of γPKC in response to various stimuli suggests a novel mechanism for PKC activation.

scholarly journals Localization of myosin IC and myosin II in Acanthamoeba castellanii by indirect immunofluorescence and immunogold electron microscopy.

1990 ◽  
Vol 111 (5) ◽  
pp. 1895-1904 ◽  
Author(s):  
I C Baines ◽  
E D Korn
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Indirect Immunofluorescence ◽  
Synthetic Peptide ◽  
Myosin Ii ◽  
Phosphorylation Site ◽  
Acanthamoeba Castellanii ◽  
Immunogold Electron Microscopy ◽  
Membrane Phospholipids ◽  
Immunogold Cytochemistry

Polyclonal antisera have been raised against purified Acanthamoeba myosin II and to a synthetic 26 amino acid peptide that corresponds in sequence to the phosphorylation site of Acanthamoeba myosin IC. These antisera are specific for their respective antigens as determined by immunoblotting after SDS-PAGE of total cell lysates. By using the antisera, localization studies were performed by indirect immunofluorescence and by immunogold electron microscopy. Myosin II occurred in the cell cytoplasm and appeared to be concentrated in the cortex. Immunogold cytochemistry revealed at high resolution that myosin II is organized into rodlike filaments approximately 200 nm long. The antibody raised against the myosin IC synthetic peptide recognized both the plasma membrane and the membrane of the contractile vacuole. The plasma membrane staining was labile to treatment with saponin suggesting an intimate association of the myosin IC with membrane phospholipids. Immunogold cytochemistry with the antimyosin IC synthetic peptide showed that the myosin IC is closely associated with the membrane bilayer.

scholarly journals Periplakin, a Novel Component of Cornified Envelopes and Desmosomes That Belongs to the Plakin Family and Forms Complexes with Envoplakin

1997 ◽  
Vol 139 (7) ◽  
pp. 1835-1849 ◽  
Author(s):  
Christiana Ruhrberg ◽  
M.A. Nasser Hajibagheri ◽  
David A.D. Parry ◽  
Fiona M. Watt
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Sequence Analysis ◽  
Cdna Sequence ◽  
Terminal Differentiation ◽  
Immunogold Electron Microscopy ◽  
Epidermal Keratinocytes ◽  
Cornified Envelope ◽  
Keratin Filaments

The cornified envelope is a layer of transglutaminase cross-linked protein that is assembled under the plasma membrane of keratinocytes in the outermost layers of the epidermis. We have determined the cDNA sequence of one of the proteins that becomes incorporated into the cornified envelope of cultured epidermal keratinocytes, a protein with an apparent molecular mass of 195 kD that is encoded by a mRNA with an estimated size of 6.3 kb. The protein is expressed in keratinizing and nonkeratinizing stratified squamous epithelia and in a number of other epithelia. Expression of the protein is upregulated during the terminal differentiation of epidermal keratinocytes in vivo and in culture. Immunogold electron microscopy was used to demonstrate an association of the 195-kD protein with the desmosomal plaque and with keratin filaments in the differentiated layers of the epidermis. Sequence analysis showed that the 195-kD protein is a member of the plakin family of proteins, to which envoplakin, desmoplakin, bullous pemphigoid antigen 1, and plectin belong. Envoplakin and the 195-kD protein coimmunoprecipitate. Analysis of their rod domain sequences suggests that the formation of both homodimers and heterodimers would be energetically favorable. Confocal immunofluorescent microscopy of cultured epidermal keratinocytes revealed that envoplakin and the 195-kD protein form a network radiating from desmosomes, and we speculate that the two proteins may provide a scaffolding onto which the cornified envelope is assembled. We propose to name the 195-kD protein periplakin.

Hepatic Subcellular Compartmentation of Cytoplasmic Phosphoenolpyruvate Carboxykinase Determined by Immunogold Electron Microscopy

1995 ◽  
Vol 1 (4) ◽  
pp. 151-161
Author(s):  
Kuixiong Gao ◽  
Emma Lou Cardell ◽  
Randal E. Morris ◽  
Bruce F. Giffin ◽  
Robert R. Cardell
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Subcellular Distribution ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Smooth Endoplasmic Reticulum ◽  
Immunogold Electron Microscopy ◽  
Immunogold Staining ◽  
Thin Sections ◽  
Gold Particles

Phosphoenolpyruvate carboxykinase (PEPCK) is the rate-limiting gluconeogenic enzyme and in liver occurs in a lobular gradient from periportal to pericentral regions. The subcellular distribution of cytoplasmic PEPCK molecules within hepatocytes and its relationship to organelles have not been determined previously. In this study, we have used immunogold electron microscopy to evaluate the subcellar distribution of the enzyme, in addition to brightfield and epipolarized light microscopy. Cryosections (10 μm) of perfusion-fixed rat liver were collected on silanated slides and immunostained using goat anti-rat PEPCK followed by 5-nm gold-labeled secondary and tertiary antibodies. Additionally, free-floating vibratome sections (25, 50, and 100 μm) of perfusion-immersion-fixed rat liver were immunogold stained using goat anti-rat PEPCK and 5-nm gold-labeled secondary antibody, with and without silver enhancement. The immunogold labeled sections from both procedures were embedded in epoxy resin for the preparation of thin sections for electron microscopy. The results showed that the gold-labeled antibodies penetrated the entire thickness of cryosections, resulting in a high signal for PEPCK, but membranes in general, the smooth endoplasmic reticulum in particular, were not identifiable as electron dense unit membranes. On the other hand, the vibratome sections of well-fixed tissue allowed good visualization of the ultrastructure of cellular organelles, with the smooth endoplasmic reticulum appearing as vesicles and tubules with electron dense unit membranes; however, the penetration of the gold-labeled antibody was limited to cells at the surface of the vibratome sections. In both procedures, PEPCK, as indicated by gold particles, is predominantly in the glycogen areas of the cytosome and not in mitochondria, nuclei, Golgi apparatus, or other cell organelles. Hepatocytes in periportal regions have a compact subcellular distribution of PEPCK shown by gold particles; hepatocytes in pericentral regions have a diffuse subcellular distribution of PEPCK and thus more scattered gold particles. When normal serum replaced the first antibody in the immunogold staining procedures, the background was very low.

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