Electron Probe Analysis of Vertebrate Smooth Muscle: Distribution of Ca and Ci

1976 ◽  
Vol 34 ◽  
pp. 334-335 ◽  
Author(s):  
Avril V. Somlyo ◽  
H. Shuman ◽  
A.P. Somlyo
Keyword(s):  
Smooth Muscle ◽  
Electron Probe ◽  
Preliminary Report ◽  
Cell Damage ◽  
Electron Probe Analysis ◽  
Perinuclear Space ◽  
Probe Analysis ◽  
High K ◽  
Low K ◽  
Initial Results

This is a preliminary report of electron probe analysis of rabbit portal-anterior mesenteric vein (PAMV) smooth muscle cryosectioned without fixation or cryoprotection. The instrumentation and method of electron probe quantitation used (1) and our initial results with cardiac (2) and skeletal (3) muscle have been presented elsewhere.In preparations depolarized with high K (K2SO4) solution, significant calcium peaks were detected over the sarcoplasmic reticulum (Fig 1 and 2) and the continuous perinuclear space. In some of the fibers there were also significant (up to 200 mM/kg dry wt) calcium peaks over the mitochondria. However, in smooth muscle that was not depolarized, high mitochondrial Ca was found in fibers that also contained elevated Na and low K (Fig 3). Therefore, the possibility that these Ca-loaded mitochondria are indicative of cell damage remains to be ruled out.

Electron Probe Analysis of Muscle

1982 ◽  
Vol 40 ◽  
pp. 398-401
Author(s):  
A. V. Somlyo ◽  
H. Shuman ◽  
A. P. Somlyo
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Resting State ◽  
Binding Sites ◽  
Electron Probe ◽  
Frog Skeletal Muscle ◽  
Electron Probe Analysis ◽  
Probe Analysis

Electron probe analysis of frozen dried cryosections of frog skeletal muscle, rabbit vascular smooth muscle and of isolated, hyperpermeab1 e rabbit cardiac myocytes has been used to determine the composition of the cytoplasm and organelles in the resting state as well as during contraction. The concentration of elements within the organelles reflects the permeabilities of the organelle membranes to the cytoplasmic ions as well as binding sites. The measurements of [Ca] in the sarcoplasmic reticulum (SR) and mitochondria at rest and during contraction, have direct bearing on their role as release and/or storage sites for Ca in situ.

scholarly journals ELECTRON MICROSCOPY AND ELECTRON PROBE ANALYSIS OF MITOCHONDRIAL CATION ACCUMULATION IN SMOOTH MUSCLE

1974 ◽  
Vol 61 (3) ◽  
pp. 723-742 ◽  
Author(s):  
A. P. Somlyo ◽  
A. V. Somlyo ◽  
C. E. Devine ◽  
P. D. Peters ◽  
T. A. Hall
Keyword(s):  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Vascular Smooth Muscle ◽  
Electron Probe ◽  
Average Distance ◽  
Main Pulmonary Artery ◽  
Mitochondrial Swelling ◽  
Electron Probe Analysis ◽  
X Ray ◽  
Probe Analysis

The contractile responses to barium and the ultrastructure and ionic composition of mitochondria were studied in vascular smooth muscle. In normal rabbit portal anterior mesenteric vein (PAMV) and main pulmonary artery (MPA) smooth muscle mitochondria were frequently associated with the surface vesicles. The average distance between the outer mitochondrial and inner surface vesicle membrane was 4–5 nm. Ba contractures of MPA were tonic and of PAMV were phasic. Incubation of MPA and PAMV with Ba resulted in the accumulation of mitochondrial granules, followed in the MPA by massive mitochondrial swelling. Oligomycin and anoxia inhibited the appearance of mitochondrial electron-opaque granules and prevented the Ba-induced mitochondrial swelling in the MPA. Electron probe analysis of mitochondria in PAMV incubated with Ba and containing granules showed characteristic Ba signals over the mitochondria. Electron probe X-ray microanalysis also showed a highly significant (P < 0.001) correlation of P with mitochondrial Ba, in an estimated elemental ratio of approximately 3 Ba/4 P. Mitochondrial granules were still prominent after block staining of the osmium-fixed, Ba-loaded PAMV, but electron probe microanalysis showed no Ba, but only U, emissions. Tissues incubated with strontium had electron-opaque mitochondrial granules and deposits in the sarcoplasmic reticulum. X-ray microanalysis of mitochondria containing granules showed the presence of characteristic Sr and Ca emissions. The presence of Sr was similarly verified in the sarcoplasmic reticulum. These findings indicate the energy dependent uptake of divalent cations, in association with phosphate, by mitochondria in vascular smooth muscle in situ and the possibility that mitochondria may contribute to the regulation of intracellular divalent cation levels in smooth muscle.

Electron probe analysis of cultured vascular smooth muscle

1980 ◽  
Vol 103 (2) ◽  
pp. 313-322 ◽  
Author(s):  
M. R. James-Kracke ◽  
B. F. Sloane ◽  
H. Shuman ◽  
R. Karp ◽  
A. P. Somlyo
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Electron Probe ◽  
Electron Probe Analysis ◽  
Probe Analysis

Electron Probe Analysis and Ultrastructure of Cultured, Freeze Dried Vascular Smooth Muscle

1975 ◽  
Vol 33 ◽  
pp. 558-559 ◽  
Author(s):  
R. E. Garfield ◽  
A. P. Somlyo
Keyword(s):  
Smooth Muscle ◽  
Electron Probe ◽  
Cultured Cells ◽  
Divalent Cations ◽  
Electron Probe Analysis ◽  
X Ray ◽  
Whole Cells ◽  
Probe Analysis ◽  
Freeze Dried ◽  
Ultrathin Sections

Electron probe x-ray microanalysis at a spatial resolution of 1000 Å or better is now feasible and has been used to demonstrate mitochondrial deposits of divalent cations in ultrathin sections of fixed tissues. Monovalent and unbound ions, however, are lost and/or translocated during chemical fixation, necessitating the use of unfixed ultrathin frozen dried sections for electron probe analysis. The use of whole, frozen-dried cultured cells for electron probe analysis is easier than cryo-ultramicrotomy and presents some advantages such as rapid freezing rates attainable and imaging at conventional (75-80 kV) voltages. Smooth muscle cells from guinea pig aortas were cultured on Formvar-coated nylon grids (Fig 1). Grids with attached cells were blotted on filter paper and plunged into liquid nitrogen and dried frozen in a vacuum evaporator.In dried whole cells examined at 75 or 80 kV, the cell outline and processes were distinct and the nuclei and mitochondria containing electron opaque granules identifiable (Fig 2,3).

scholarly journals Electron probe analysis of vascular smooth muscle. Composition of mitochondria, nuclei, and cytoplasm.

1979 ◽  
Vol 81 (2) ◽  
pp. 316-335 ◽  
Author(s):  
A P Somlyo ◽  
A V Somlyo ◽  
H Shuman
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Electron Probe ◽  
Smooth Muscles ◽  
Electron Probe Analysis ◽  
Muscle Composition ◽  
Mesenteric Vein ◽  
Osmotic Equilibrium ◽  
Probe Analysis

Electron probe analysis of dry cryosections was used to determine the composition of the cytoplasm and organelles of rabbit portal-anterior mesenteric vein (PAMV) smooth muscle. All analytical values given are in mmol/kg wt +/- SEM. Cytoplasmic concentrations in normal, resting muscles were: K, 611 +/- 1.7; Na, 167 +/- 2.7; Cl, 278 +/- 1.0; Mg, 36 +/- 1.1; Ca, 1.9 +/- 0.5; and P, 247 +/- 1.1. Hence, the sum of intracellular Na + K exceeded cytoplasmic Cl by 500 mmol/kg dry wt, while the calculated total, nondiffusible solute was approximately 50 mmol/kg. Cytoplasmic K and Cl were increased in smooth muscles incubated in solutions containing an excess (80 mM) of KCl. Nuclear and cytoplasmic Na and Ca concentrations were not significantly different. The mitochondrial Ca content in normal fibers was low, 0.8 +/- 0.5, and there was no evidence of mitochondrial Ca sequestration in muscles frozen after a K contracture lasint 30 min. Transmitochondrial gradients of K, Na, and Cl were small (0.9--1.2). In damaged fibers, massive mitochondrial Ca accumulation of up to 2 mol/kg dry wt in granule form and associated with P could be demonstrated. Our findings suggest (a) that the nonDonnan distribution of Cl in smooth muscle is not caused by sequestration in organelles, and that considerations of osmotic equilibrium and electroneutrality suggest the existence of unidentified nondiffusible anions in smooth muscle, (b) that nuclei do not contain concentrations of Na or Ca in excess of cytoplasmic levels, (c) that mitochondria in PAMV smooth muscle do not play a major role in regulating cytoplasmic Ca during physiological levels of contraction but can be massively Ca loaded in damaged cells, and (d) that the in situ transmitochondrial gradients of K, Na, and Cl do not show these ions to be distributed according to a large electromotive Donnan force.

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