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. This yielded ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane were preserved and intracellular compartmentalization of diffusible ions was quantitated. Quantitative electron probe analysis of freeze-dried ultrathin cryo sections provided 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 Cl in [frog] skeletal muscle and the effects of hypertonic solutions on the subcellular composition of striated muscle. There was no evidence of sequestered Cl in the terminal cisternae of resting muscles, although Ca (66 mmol/kg dry wt .+-. 4.6 SE) was detected. The values of intracellular Cl concentration 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 Cl, with a cytoplasmic/mitochondrial Cl ratio of 2.4 .+-. 0.88 SD. The elemental concentrations (mmol/kg dry wt .+-. SD) of muscle fibers measured with 0.5-9 .mu.m diameter electron probes in normal frog striated muscle were: P, 302 .+-. 4.3; S, 189 .+-. 2.9; Cl, 24 .+-. 1.1; K, 404 .+-. 4.3, and Mg, 39 .+-. 2.1. In normal muscle the excess Cl measured with previous bulk chemical analyses and flux studies was not compartmentalized in the SR or in other cellular organelles and the cytoplasmic Cl in low extracellular K concentration solutions exceeded that predicted by a passive electrochemical distribution. Hypertonic 2.2 .times. NaCl, 2.5 .times. sucrose, or 2.2 .times. Na isethionate produced: 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 granules of Ca, Mg, and P .simeq. (6 Ca + 1 Mg)/6 P in the longitudinal SR. Hypertonicity produced compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules.