scholarly journals Biochemical Properties and Cytochemical Localization of Ouabain-insensitive, Potassium-dependent p-Nitrophenylphosphatase Activity in Rat Atrial Myocytes

1997 ◽  
Vol 45 (2) ◽  
pp. 177-187 ◽  
Author(s):  
Vadim S. Zinchuk ◽  
Toshihiro Kobayashi ◽  
Eva Garcia del Saz ◽  
Harumichi Seguchi
Keyword(s):  
Ventricular Myocytes ◽  
Reaction Medium ◽  
Biochemical Properties ◽  
Atrial Myocytes ◽  
Electron Microscopic ◽  
Cytochemical Localization ◽  
X Ray ◽  
T Tubules ◽  
P Type ◽  
Triton X

Enzyme activity that represents ouabain-insensitive, potassium-dependent p-nitrophenylphosphatase (p-NPPase) was assessed in rat atrial myocytes by biochemical and cytochemical procedures. No activity was detected in parallel experiments with ventricular myocytes. Fixed tissues were incubated in a reaction medium containing Tricine buffer, p-nitrophenylphosphate (p-NPP), KCl, MgCl2, CaCl2, CeCl3, Triton X-100, levamisole, and ouabain. Final pH was adjusted to 7.5. Biochemical studies showed that accumulation of p-nitrophenol in the medium was increased proportionally in accordance with the amount of incubated tissue. This activity was optimal with incubation at pH 7.5 and in the presence of KCl. Approximately 70% of the enzyme was inhibited by 2 mM CeCl3. Electron microscopic observations revealed reaction product (RP) at sites of ouabain-insensitive, potassium-dependent p-NPPase activity as electron-dense precipitate localized at the inner surface of the plasma membrane and at the T-tubules of atrial myocytes. Control experiments indicated that the activity was strongly inhibited by sodium orthovanadate and was repressed by omeprazole and 1,3-dicyclohexylcarbodiimide. X-ray microanalysis confirmed the presence of cerium within the cytochemical RP. The ouabain-insensitive, K-dependent p-NPPase activity detected in the present study is considered to be an isoform of a P-type, H-transporting, K-dependent adenosine triphosphatase (H,K-ATPase).

scholarly journals Myocardin is differentially required for the development of smooth muscle cells and cardiomyocytes

2011 ◽  
Vol 300 (5) ◽  
pp. H1707-H1721 ◽  
Author(s):  
Mark H. Hoofnagle ◽  
Ronald L. Neppl ◽  
Erica L. Berzin ◽  
G. C. Teg Pipes ◽  
Eric N. Olson ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Ventricular Myocytes ◽  
Embryoid Body ◽  
Embryonic Stem ◽  
Muscle Cells ◽  
Atrial Myocytes ◽  
Electron Microscopic ◽  
Virtual Absence ◽  
Vascular Smcs

Myocardin is a serum response factor (SRF) coactivator exclusively expressed in cardiomyocytes and smooth muscle cells (SMCs). However, there is highly controversial evidence as to whether myocardin is essential for normal differentiation of these cell types, and there are no data showing whether cardiac or SMC subtypes exhibit differential myocardin requirements during development. Results of the present studies showed the virtual absence of myocardin−/− visceral SMCs or ventricular myocytes in chimeric myocardin knockout (KO) mice generated by injection of myocardin−/− embryonic stem cells (ESCs) into wild-type (WT; i.e., myocardin+/+ ESC) blastocysts. In contrast, myocardin−/− ESCs readily formed vascular SMC, albeit at a reduced frequency compared with WT ESCs. In addition, myocardin−/− ESCs competed equally with WT ESCs in forming atrial myocytes. The ultrastructural features of myocardin−/− vascular SMCs and cardiomyocytes were unchanged from their WT counterparts as determined using a unique X-ray microprobe transmission electron microscopic method developed by our laboratory. Myocardin−/− ESC-derived SMCs also showed normal contractile properties in an in vitro embryoid body SMC differentiation model, other than impaired thromboxane A2 responsiveness. Together, these results provide novel evidence that myocardin is essential for development of visceral SMCs and ventricular myocytes but is dispensable for development of atrial myocytes and vascular SMCs in the setting of chimeric KO mice. In addition, results suggest that as yet undefined defects in development and/or maturation of ventricular cardiomyocytes may have contributed to early embryonic lethality observed in conventional myocardin KO mice and that observed deficiencies in development of vascular SMC may have been secondary to these defects.

X-Ray Analysis of Cytochemical Reaction Products in Synaptic Areas

1976 ◽  
Vol 34 ◽  
pp. 6-7 ◽  
Author(s):  
J. Wood
Keyword(s):  
Electron Microscopic Level ◽  
Junctional Complex ◽  
Reaction Products ◽  
Electron Microscopic ◽  
Cytochemical Localization ◽  
X Ray ◽  
The Past ◽  
Electron Dense Deposit ◽  
Dense Deposit ◽  
Cytochemical Reaction

Specific cytochemical reactions have been instrumental in the illucidation of compounds within tissues, whether these compounds are hormones, enzymes, or molecules, such as certain nerve transmitter agents. Many cytochemical reaction products depend upon some complex, which is an electron dense deposit. Several types of cytochemical procedures can be used to visualize agents related to synaptic transmission at the junctional complex. One method which has been used with considerable success has been the cytochemical localization of biogenic amines (BAs), i.e., norepinephrine (NE) and dopamine (DA). For the past few years, a chrome complex formed with certain BAs and glutaraldehyde has been utilized to localize BAs at the electron microscopic level and the specificity of the reaction has been verified biochemically.

scholarly journals Differences in the organization of actin in the growth cones compared with the neurites of cultured neurons from chick embryos.

1983 ◽  
Vol 97 (4) ◽  
pp. 963-973 ◽  
Author(s):  
P C Letourneau
Keyword(s):  
Growth Cone ◽  
Actin Filaments ◽  
Electron Microscopic Observation ◽  
Neurite Growth ◽  
Growth Cones ◽  
Chick Embryos ◽  
Heavy Meromyosin ◽  
Electron Microscopic ◽  
Cytochemical Localization ◽  
Triton X

Sensory neurons from chick embryos were cultured on substrata that support neurite growth, and were fixed and prepared for both cytochemical localization of actin and electron microscopic observation of actin filaments in whole-mounted specimens. Samples of cells were treated with the detergent Triton X-100 before, during, or after fixation with glutaraldehyde to determine the organization of actin in simpler preparations of extracted cytoskeletons. Antibodies to actin and a fluorescent derivative of phallacidin bound strongly to the leading margins of growth cones, but in neurites the binding of these markers for actin was very weak. This was true in all cases of Triton X-100 treatment, even when cells were extracted for 4 min before fixation. In whole-mounted cytoskeletons there were bundles and networks of 6-7-nm filaments in leading edges of growth cones but very few 6-7-n filaments were present among the microtubules and neurofilaments in the cytoskeletons of neurites. These filaments, which are prominent in growth cones, were identified as actin because they were stabilized against detergent extraction by the presence of phallacidin or the heavy meromyosin and S1 fragments of myosin. In addition, heavy meromyosin and S1 decorated these filaments as expected for binding to F-actin. Microtubules extended into growth cone margins and terminated within the network of actin filaments and bundles. Interactions between microtubule ends and these actin filaments may account for the frequently observed alignment of microtubules with filopodia at the growth cone margins.

scholarly journals Electron microscopic cytochemical localization of Ca-ATPase in toad urinary bladder.

1987 ◽  
Vol 35 (1) ◽  
pp. 39-48 ◽  
Author(s):  
W L Davis ◽  
R G Jones ◽  
D B Goodman
Keyword(s):  
Urinary Bladder ◽  
Atpase Activity ◽  
Reaction Medium ◽  
Ion Concentration ◽  
Toad Urinary Bladder ◽  
Electron Microscopic ◽  
Cytochemical Localization ◽  
Atpase Reaction ◽  
Amphibian Urinary Bladder ◽  
Free Media

The calcium-regulating enzyme calcium adenosine triphosphatase (Ca-ATPase) was localized in the epithelium of amphibian urinary bladder by the one-step electron microscopic cytochemical procedure. The enzyme was identified along the basolateral border of the epithelial cells that comprise the bladder mucosa. The electron-dense precipitate indicating Ca-ATPase activity was seen in association with the outer leaflet of the basolateral plasmalemmae. Intracellularly, Ca-ATPase activity was seen in association with the mitochondrial matrix of the mitochondria-rich cells. Ca-ATPase was not seen along the apical microvillated border. Enzyme activity was also not seen after incubation in substrate-free media, calcium-free media, or incubation in the presence of vanadate. However, Ca-ATPase activity was evident when the calcium in the standard reaction medium was deleted in favor of magnesium. Addition of antidiuretic hormone (ADH; vasopressin) increased both the basolateral Ca-ATPase reaction and the mitochondrial reaction. Such data appear to indicate further that changes in cytosolic calcium ion concentration take place during the response of amphibian urinary bladder to the polypeptide hormone vasopressin.

scholarly journals Hypokalemia Promotes Arrhythmia by Distinct Mechanisms in Atrial and Ventricular Myocytes

2020 ◽  
Vol 126 (7) ◽  
pp. 889-906 ◽  
Author(s):  
Kiarash Tazmini ◽  
Michael Frisk ◽  
Alexandre Lewalle ◽  
Martin Laasmaa ◽  
Stefano Morotti ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Atrial Myocytes ◽  
Early Afterdepolarizations ◽  
Membrane Hyperpolarization ◽  
Atrial Cells ◽  
Ventricular Cells ◽  
Rat Myocytes ◽  
Delayed Afterdepolarizations ◽  
T Tubules ◽  
A Current

Rationale: Hypokalemia occurs in up to 20% of hospitalized patients and is associated with increased incidence of ventricular and atrial fibrillation. It is unclear whether these differing types of arrhythmia result from direct and perhaps distinct effects of hypokalemia on cardiomyocytes. Objective: To investigate proarrhythmic mechanisms of hypokalemia in ventricular and atrial myocytes. Methods and Results: Experiments were performed in isolated rat myocytes exposed to simulated hypokalemia conditions (reduction of extracellular [K + ] from 5.0 to 2.7 mmol/L) and supported by mathematical modeling studies. Ventricular cells subjected to hypokalemia exhibited Ca 2+ overload and increased generation of both spontaneous Ca 2+ waves and delayed afterdepolarizations. However, similar Ca 2+ -dependent spontaneous activity during hypokalemia was only observed in a minority of atrial cells that were observed to contain t-tubules. This effect was attributed to close functional pairing of the Na + -K + ATPase and Na + -Ca 2+ exchanger proteins within these structures, as reduction in Na + pump activity locally inhibited Ca 2+ extrusion. Ventricular myocytes and tubulated atrial myocytes additionally exhibited early afterdepolarizations during hypokalemia, associated with Ca 2+ overload. However, early afterdepolarizations also occurred in untubulated atrial cells, despite Ca 2+ quiescence. These phase-3 early afterdepolarizations were rather linked to reactivation of nonequilibrium Na + current, as they were rapidly blocked by tetrodotoxin. Na + current-driven early afterdepolarizations in untubulated atrial cells were enabled by membrane hyperpolarization during hypokalemia and short action potential configurations. Brief action potentials were in turn maintained by ultra-rapid K + current (I Kur ); a current which was found to be absent in tubulated atrial myocytes and ventricular myocytes. Conclusions: Distinct mechanisms underlie hypokalemia-induced arrhythmia in the ventricle and atrium but also vary between atrial myocytes depending on subcellular structure and electrophysiology.

Co-localization of a kallikrein-like serine protease (arginine esterase A) and atrial natriuretic peptide in rat atrium.

1989 ◽  
Vol 37 (12) ◽  
pp. 1913-1917 ◽  
Author(s):  
J A Simson ◽  
M G Currie ◽  
L Chao ◽  
J Chao
Keyword(s):  
Molecular Weight ◽  
Atrial Natriuretic Peptide ◽  
Serine Protease ◽  
Natriuretic Peptide ◽  
Ventricular Myocytes ◽  
Electron Microscopic Level ◽  
Atrial Myocytes ◽  
Electron Microscopic ◽  
Double Label ◽  
Atrial Natriuretic

Atrial natriuretic peptide (ANP) is stored in atrial granules primarily as a larger molecular weight precursor (pro-ANP), which is believed to be rapidly converted to an active peptide of 28 amino acids during or shortly after secretion. A tissue kallikrein-like serine protease has been suggested as a potential processing enzyme. In the present immunocytochemical study, using specific monoclonal antibodies, we found that esterase A, a kallikrein-like serine protease, was demonstrable in rat atrial myocytes and in ventricular myocytes, and was capable of cleaving pro-ANP to yield a low molecular weight product. Using colloidal gold immunocytochemistry at the electron microscopic level, we have found esterase A in atrial myocytes, both in granules and in another subcellular site that corresponds to sarcoplasmic reticulum. Double-label electron microscopic immunocytochemical results indicated that esterase A can co-localize with ANP in granules of atrial myocytes.

Direct visualization of phase-separated domains in lipid membranes

1978 ◽  
Vol 36 (2) ◽  
pp. 290-291
Author(s):  
S. W. Hui ◽  
T. P. Stewart
Keyword(s):  
Lipid Membranes ◽  
Structural Information ◽  
Freeze Fracture ◽  
Biological Molecules ◽  
Vacuum Drying ◽  
Direct Visualization ◽  
X Ray Diffraction ◽  
Electron Microscopic ◽  
X Ray ◽  
Hydrocarbon Chains

Direct electron microscopic study of biological molecules has been hampered by such factors as radiation damage, lack of contrast and vacuum drying. In certain cases, however, the difficulties may be overcome by using redundent structural information from repeating units and by various specimen preservation methods. With bilayers of phospholipids in which both the solid and fluid phases co-exist, the ordering of the hydrocarbon chains may be utilized to form diffraction contrast images. Domains of different molecular packings may be recgnizable by placing properly chosen filters in the diffraction plane. These domains would correspond to those observed by freeze fracture, if certain distinctive undulating patterns are associated with certain molecular packing, as suggested by X-ray diffraction studies. By using an environmental stage, we were able to directly observe these domains in bilayers of mixed phospholipids at various temperatures at which their phases change from misible to inmissible states.

Polarity Determination in Sphalerite and Wurtzite Structures

1990 ◽  
Vol 48 (2) ◽  
pp. 500-501
Author(s):  
Stuart McKernan ◽  
C. Barry Carter
Keyword(s):  
Opposite Polarity ◽  
Single Domain ◽  
Polar Material ◽  
Electron Microscopic ◽  
Dynamical Interaction ◽  
X Ray ◽  
Polarity Determination ◽  
The Absolute ◽  
Microscopic Techniques

The determination of the absolute polarity of a polar material is often crucial to the understanding of the defects which occur in such materials. Several methods exist by which this determination may be performed. In bulk, single-domain specimens, macroscopic techniques may be used, such as the different etching behavior, using the appropriate etchant, of surfaces with opposite polarity. X-ray measurements under conditions where Friedel’s law (which means that the intensity of reflections from planes of opposite polarity are indistinguishable) breaks down can also be used to determine the absolute polarity of bulk, single-domain specimens. On the microscopic scale, and particularly where antiphase boundaries (APBs), which separate regions of opposite polarity exist, electron microscopic techniques must be employed. Two techniques are commonly practised; the first [1], involves the dynamical interaction of hoLz lines which interfere constructively or destructively with the zero order reflection, depending on the crystal polarity. The crystal polarity can therefore be directly deduced from the relative intensity of these interactions.

X-Ray Microanalysis of Nickel and Cobalt Intensified Peroxidase- Antiperoxidase For Verification of Reaction Sites

1985 ◽  
Vol 43 ◽  
pp. 468-469
Author(s):  
A. Angel ◽  
K. Miller ◽  
V. Seybold ◽  
R. Kriebel
Keyword(s):  
Reaction Mechanisms ◽  
Visual Discrimination ◽  
Osmium Tetroxide ◽  
Ultrastructural Level ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
X Ray ◽  
Insoluble Polymer ◽  
Cytochemical Reaction

Localization of specific substances at the ultrastructural level is dependent on the introduction of chemicals which will complex and impart an electron density at specific reaction sites. Peroxidase-antiperoxidase(PAP) methods have been successfully applied at the electron microscopic level. The PAP complex is localized by addition of its substrate, hydrogen peroxide and an electron donor, usually diaminobenzidine(DAB). On oxidation, DAB forms an insoluble polymer which is able to chelate with osmium tetroxide becoming electron dense. Since verification of reactivity is visual, discrimination of reaction product from osmiophillic structures may be difficult. Recently, x-ray microanalysis has been applied to examine cytochemical reaction precipitates, their distribution in tissues, and to study cytochemical reaction mechanisms. For example, immunoreactive sites labelled with gold have been ascertained by means of x-ray microanalysis.

Factors in Photographic Emulsions for Low Dose Electron Crystallography

1980 ◽  
Vol 38 ◽  
pp. 230-231
Author(s):  
T.W. Jeng ◽  
W. Chiu
Keyword(s):  
Radiation Damage ◽  
Low Dose ◽  
Damage Effect ◽  
Photographic Emulsion ◽  
Electron Crystallography ◽  
Electron Microscopic ◽  
X Ray ◽  
Signal Contrast ◽  
Diffraction Patterns ◽  
Photographic Emulsions

With the advances in preparing biological materials in a thin and highly ordered form, and in maintaining them hydrated under vacuum, electron crystallography has become an important tool for biological structure investigation at high resolution (1,2). However, the electron radiation damage would limit the capability of recording reflections with low intensities in an electron diffraction pattern. It has been demonstrated that the use of a low temperature stage can reduce the radiation damage effect and that one can expose the specimen with a higher dose in order to increase the signal contrast (3). A further improvement can be made by selecting a proper photographic emulsion. The primary factors in evaluating the suitability of photographic emulsion for recording low dose diffraction patterns are speed, fog level, electron response at low electron exposure, linearity, and usable range of exposure. We have compared these factors with three photographic emulsions including Kodak electron microscopic plate (EMP), Industrex AA x-ray film (AA x-ray) and Kodak nuclear track film (NTB3).

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