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Content of fructose-2,6-bisphosphate was drastically decreased (about 5-fold) in hepatocyte suspension obtained from rats with streptozotocin-induced diabetes, as compared with that of control animals. At the same time, activity of fructose-1,6-bisphosphatase was increased 2-fold in these hepatocytes. Incubation mixture of the hepatocytes from the diabetes impaired animals contained only low amounts of lactate.

The study was carried out on primary coculture Kupffer cells and liver fibroblasts of newborn and mature rats. At first Kupffer cells and liver fibroblasts were taken on the equal quantity. There were observed significant decrease of Kupffer cells quantity and fibroblasts death after 4-5 days in coculture. Mitotic and functional activity liver fibroblasts, adhesion of Kupffer cells were increased by the using of conditioned medium of newborn rat hepatocytes. The rise of mitotic activity of liver fibroblasts and quantity of nucleolus in their nuclei forestalls of the increase Kupffer cells size, appearing of Kupffer cells with some nuclei.

The monolayer culture of 3-day-old rat hepatocytes was treated by antimicrotubule agent colchicine after activation with isoproterenol. Alterations in the amount of lysosomes with the size of 1.0-2.5 and 3-6 mkm per cell were studied with light microscope after staining with the vital dye neutral red and after identification of acid phosphatase by plumbum precipitation in the frozen preparations. It was shown that isoproterenol application (20 microM) within 1.5 h did not change significantly the number of lysosomes sized 1.0-2.5 microns but stimulated the increase in the amount of lysosomes 3-6 mkm in size. The addition of 2.5 mM colchicine, after the isoproterenol treatment, decreases greatly the number of lysosomes with the size of 1.0-2.5 and 3-6 mkm. A considerable part of the large lysosomes can be resistant to the colchicine action. It is concluded that the microtubules play an important role in lysosome functioning.

Effects of insulin was studied in the monolayer hepatocyte cultures of new born rats under anoxic conditions. Insulin 10(-7) M stabilized lysosomal membranes obtained after 20 min anoxia. I hr incubation of hepatocytes with insulin in normal conditions, which was followed by anoxia, caused a significant increase of acid phosphatase activity in the fraction enriched with lysosomes. Insulin caused more distinct stabilization effect on lysosomal membranes when its action was prolonged. On the other hand, insulin caused more than 2-fold increase of cAMP content in hepatocytes within 2 min of exposition as compared with control cultures. When exposition of the cells with insulin exceeded 2 min, lowering of cAMP content was observed. These data appear to indicate that stabilizing effect of insulin was secondary towards cAMP level increase. Indirect connection between cAMP level, insulin stabilizing action and the state of lysosomal membranes were noted in the rat hepatocyte culture.

The monolayer cultures of newborn rats hepatocytes were treated with substrate starvation (20 min.) anoxia accompanied with substrate starvation (1 h.), following rehabilitation (1 h.) and cooling (4 degrees C, 30 min). After the neutral red staining we have examined the alterations in lysosomes quantity and the development of phagocytosis; we also tested the hepatocytes distribution due to the amount of the second lysosomes per cell. We have shown that a short time pathological treatment caused a decrease in large lysosomes (1-3 microns) amount in significant portion of hepatocytes. There were neither increase in phagosomes amount per cell nor alterations in the quantity of cells, containing phagosomes. However a long time pathological treatment caused the increase in phagosomes and cells. We propose that initial stage of lysosomal apparatus alterations, connected with reduction of CAMP level, is accompanied by a stamping of large lysosomes.

A direct observation of primary cultures of 3-day-old rat hepatocytes was performed with the help of time-consuming cinematography. The process of monolayer formation has been examined. In addition, the continuous preservation of bile canaliculi was observed. A significant increase in hepatocyte mobility was marked in 6-7-day cultures. The structural analysis of cultured cells during the modelling of anoxia with substrate deficiency revealed a succession in hepatocyte and fibroblast alterations. The hepatocyte destruction was accompanied with changes in the cell form, that become rounded, and in the appearance of small formations protruding on the cell surface. The fibroblast destruction appeared after cell contracture.

Acid phosphatase activity and cAMP level were studied in primary hepatocyte culture of new born rats under conditions of anoxia and substrate deprivation (incubation of the cells in Hanks salt solution). Incubation of hepatocytes in Hanks salt solution within one hour under conditions of anoxia caused a significant increase in free (cytosolic) enzyme activity. Substitution of Hanks salt solution by normal tissue culture medium and reoxygenation after 1 hr anoxia resulted in a decrease of free acid phosphatase activity, whereas activity of the enzyme in lysosomal fraction was increased. Content of cAMP was decreased distinctly after 15 min incubation of hepatocyte culture under conditions of anoxia and substrate deprivation and was increased above control values within 5 min of reoxygenation and substitution of Hanks salt solution by normal tissue culture media. Addition of cAMP-containing liposomes to hepatocyte culture under these experimental conditions led to a decrease in free acid phosphatase activity and to an increase of the enzyme activity in lysosomal fraction. cAMP appears to modulate the lability of hepatocyte lysosomal membrane. The mechanisms involved in these processes are discussed.

A primary culture of hepatocytes from new born rats was used as an experimental model suitable for studies of the cell lysosomal apparatus and of molecular mechanisms involving cyclic nucleotides for initiation of lysosomal response to extremal conditions. Substrate deficiency and anoxia of the cultivated hepatocytes were found to cause distinct alterations in the state of lysosomal apparatus accompanied by an increase in free activity of acid phosphatase.

Activity of some lysosomal enzymes was studied in the primary heart cell culture of newborn rats under conditions of hypoxia and in isolated ischemic perfused heart, in liver and heart tissues of dogs after traumatic shock and after experimental myocardium infarction as well as in liver tissue of rats after 10 min experimental heart arrest and during the postresuscitation period. Decrease in cAMP and elevation in cGMP levels, impairment of the adenylate cyclase hormonal regulation, decrease in activity of cAMP-dependent protein kinases were shown to correlate distinctly with an increase of acid hydrolases free activity in liver and heart cell cytoplasm of dogs with traumatic shock and with experimental myocardium infarction. Free activity of acid phosphatase was increased in primary heart cell culture under conditions of hypoxia and in isolated ischemic perfused rat heart. Intraperitoneal administration of cAMP-containing liposomes into the rats with 10 min heart arrest led to stabilization of liver lysosomal membranes within 1 hr of 4 hrs of the post-resuscitation period. The mechanisms involved in stabilization of lysosomal membranes are discussed.