Electron microscopic and biochemical research on the vacuoles formed in the erythrocytes of frogs as affected by neutral red and novocaine

No lysosomes were found in the frog intact erythrocytes with electron microscope. Under the influence of neutral red (NR-8.7.10(-5) M) and novocaine (N-4.6.10(-3) M) segregation zones (vacuoles) including these substances are formed. Using electron microscopy and morphometry the action of NR and N for 5 minutes up to 48 hours was found to provoke the formation of four types of vacuoles differing in their morphology: with electron-transparent content, with amorphous inclusions and membrane whorls. The dynamics of vacuole formation, of their changes and amount were followed depending on the time of exposition of these substances. Biochemical investigation of both NR and N isolated vacuoles showed in these some activities of lysosomal marker enzymes--acid phosphatase and N-acetyl-beta,D-glucosaminidase. Ultrastructural investigation of acid phosphatase localization in the isolated vacuoles revealed the histochemical reaction product mainly in electron-translucent vacuoles (primary lysosomes) and partly in electron dense ones (secondary lysosomes). On the ground of the above studies a conclusion is made that in frog erythrocytes treated with NR and N lysosome formation is induced to be followed by the induced autophagocytosis and heterophagocytosis. Some possible ways of the vacuolar system formation in frog erythrocytes and the origin of lysosomal hydrolases are discussed.