We have studied the localization of
osmium reduction products to investigate the functional state of organelles as well as organelle interrelationships during cell injury. In normal hepatocytes
osmium deposits of variable intensity are seen in nuclear envelope, endoplasmic reticulum. Golgi cisternae and vesicles and lysosomes. Buffering of
osmium with s-
collidine (pH 7.4) prevents the deposition of
osmium. Reversible (30 min) and irreversible (60 min)
ischemia without reflow causes no change in the pattern of
osmium deposition. Irreversible
ischemia followed by reflow causes decreased staining of endoplasmic reticulum (ER) and redistribution of the
osmium deposits through the cytoplasm. Reversibly injured pancreatic acinar cells in cultured explants manifest a similar loss of
osmium staining in the endoplasmic reticulum cisternae. The administration of antimicrotubule drugs induces an accentuation of
osmium staining in localized cisternal elements of hepatocytes. These heavily stained cisternae appear to give rise to the bounding membranes of
drug-induced autophagic vacuoles. Cytoplasmic organelles sequestered inside the autophagic vacuoles acquire intense staining when they begin to undergo degradation. In homogenized liver tissue all the subcellular organelles show
osmium deposits. The deposits are preferentially localized along the organelle membranes. In particular the dense deposits in the ER lumen are not seen in the subcellular fractions.
Phospholipase A2 (3 units/mg
protein) enhances the deposition of
osmium in the lumen of microsomal vesicles, whereas the presence of
detergent has no such effect. Addition of
EDTA to the homogenizing medium enhances the ultrastructural preservation of the subcellular fractions but has little effect on the deposition of
osmium. OsO4 deposition occurs at
acid pH and the intensity and pattern of the
stain can be modified in vivo and in vitro.
Osmium tetroxide deposition is induced at sites of membrane transformation (autophagic vacuoles) and degradation (lysosomes).
Calcium influx and
phospholipase activation (
ischemia, tissue homogenization,
phospholipase addition) enhance
osmium deposition and/or influence the localization of the staining pattern.