Elemental distribution in striated muscle and the effects of hypertonicity: Electron probe analysis of cryo sections

Abstract

A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusible ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A. V. Somlyo, and A. P. . Ultramicros. 1:317-339.) of freeze-dried ultrathin cryo sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of C1 in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition of striated muscle.There was no evidence of sequestered C1 in the terminal cisternae of resting muscles, although calcium (66 mmol/kg dry wt -+ 4.6 SE) was detected. The values of [C1]l determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles and over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial C1 ratio of 2.4 -+ 0.88 SD.The elemental concentrations (mmol/kg dry wt -+ SD) of muscle fibers measured with 0.5-9-/zm diameter electron probes in normal frog striated muscle were: P, 302 -4.3; S, 189 -+ 2.9; C1, 24 -1.1; K, 404 +-4.3, and Mg, 39 +-2.1. It is concluded that: (a) in normal muscle the "excess Cl" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic Cl in low [K]o solutions exceeds that predicted by a passive electrochemical distribution.Hypertonic 2.2 x NaC1, 2.5 • sucrose, or 2.2 • Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P -~(6 Ca + 1 Mg)/6 P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules. 828THe JOURNAL OF CELL BIOLOGY" VOLUME 74, 1977" pages 828-857 on May 11, 2018 jcb.rupress.org Downloaded from http://doi.org/10. 1083/jcb.74.3.828 Published Online: 1 September, 1977 KEY WORDS electron probe analysis 9 cryoultramicrotomy 9 striated muscle hypertonicity 9 sarcoplasmic reticulum The distribution of diffusible ions in, respectively, the extracellular space, cytoplasm, and cellular organelles, represents one of the most general problems of cell function. The main techniques available for determining the concentrations of ions in different compartments are the measurement of radio isotope ion fluxes in intact tissues and organeUe fractionation. These techniques are, however, subject to the respective uncertainties of the ultrast...

Activity-Dependent Calcium Sequestration in Dendrites of Hippocampal Neurons in Brain Slices

“…skeletal muscle (Somlyo et al, 1977). Again, there was no increase in mitochondrial C a and only a modest but statistically significant rise in cytoplasmic total C a.…”

“…EDX analysis of both preparations revealed that the concentrations of total C a, as well as of other elements, in the three target compartments of CA3 apical dendrites (Table 1) were comparable to basal concentrations in other excitable and nonexcitable cells (Somlyo et al, 1977(Somlyo et al, , 1985Andrews et al, 1988). The mean concentration of Ca within dendritic ER was 5.1 Ϯ 1.1 mmol/kg dry weight (mean Ϯ SEM; Table 1), although, as discussed below, this morphologically identified set of ER consists of at least two functionally distinct populations that, under basal conditions, coincidentally display similar low Ca concentrations.…”

“…Because neuronal cell bodies and processes, including axons, dendrites, and synapses, are present within 5-20 m of the exposed surface of such slices (L. D. Pozzo-Miller, T. S. Reese, and S. B. Andrews, unpublished observations), these components are structurally well preserved (Figs. 1, 2) and-most importantly for this study -also maintain on a millisecond time scale (Heuser et al, 1979) their native distributions of intracellular diff usible ions (Somlyo et al, 1977).…”

“…When viewed in terms of total Ca-binding capacity, the ER and cytoplasmic buffers appear to be approximately equal, mainly because the volume fraction of the cytoplasmic compartment is so much larger that a sixfold increase in total cytoplasmic Ca, as observed after four tetani, is roughly equivalent to the concomitant 20-fold increase in the ER. Nevertheless, the Ca load attained by some ER cisterns is extraordinary, reaching levels (Ͼ100 mmol/kg dry weight) that are unknown outside of the sarcoplasmic reticulum of skeletal muscle (Somlyo et al, 1977). Even so, this large accumulation is apparently within the range of normal physiological activity for these dendrites, as indicated by the modest, parallel elevation of cytoplasmic total Ca in comparison with unstimulated dendrites, and by the reversibility of ER and cytoplasmic Ca sequestration when slices were allowed a recovery period.…”

Calcium release and ionic changes in the sarcoplasmic reticulum of tetanized muscle: an electron-probe study.

“…Thus, until recently, the question of whether the SR of resting muscle is a truly intracellular compartment or one in ionic communication with and similar in ionic contents to the extracellular space was the subject of some debate (for review, see references 74,96) . The availability of electron-probe microanalysis at high spatial resolution combined with rapid freezing techniques to preserve the in vivo distribution of diffusible elements (for review, see references 39,44,59,60,93) made it possible to measure the inorganic content of cell organelles in situ .…”