Effects of digitonin on the intracellular content of rat hepatocytes: implications for its use in the study of intralobular heterogeneity.

Abstract

Anterograde or retrograde perfusion of rat liver with digitonin selectively permeabilizes the periportal or the perivenous zone of the hepatic lobule. Digitonin perfusion is used to analyze the effluents released by permeabilized hepatocytes or, combined with collagenase perfusion, to obtain cell suspensions enriched in either periportal or perivenous hepatocytes. Despite the wide use of digitonin to study lobular heterogeneity, its affects on rat hepatocytes are not well documented. We therefore analyzed the effects of digitonin perfusion on the intracellular content of rat hepatocytes by combining electron microscopy, histoenzymology, immunohistochemistry, and in situ hybridization. At the concentration currently used for the study of lobular heterogeneity, digitonin perfusion induced a marked cytosolic clarification of permeabilized hepatocytes, while most organelles except mitochondria were well preserved. In the digitonin-altered zones, there was no histochemical detection of non-membrane-bound enzymes (lactate dehydrogenase, glutamate dehydrogenase), wheteas membrane-bound

Gluconeogenesis, glucose handling, and structural changes in livers of the adult offspring of rats partially deprived of protein during pregnancy and lactation.

“…Digitonin-affected cells are permeabilized and lose their cytosolic contents, unlike mitochondria, which are grossly swollen. Cells which have not been reached by the digitonin front are histologically (12, 13), electron-microscopically (13), and functionally (8) normal in terms of gluconeogenesis from lactate. Demarcation of digitonin-destroyed from digitonin-unaffected cells is sharp (8,12,13).…”

“…Cells which have not been reached by the digitonin front are histologically (12, 13), electron-microscopically (13), and functionally (8) normal in terms of gluconeogenesis from lactate. Demarcation of digitonin-destroyed from digitonin-unaffected cells is sharp (8,12,13). Flow was resumed in the normal antegrade direction, and portal and hepatic vein sample pairs were taken at 60, 70, and 80 min for estimation of net glucose production.…”

Zonation of gluconeogenesis, ketogenesis and intracellular pH in livers from normal and diabetic ketoacidotic rats: evidence for intralobular redistribution of metabolic events in ketoacidosis

“…Digitonin binds plasma membrane cholesterol avidly on a firstpass basis, permeabilizing cells along the digitonin front, which moves retrogradely along the sinusoid more slowly than the medium. The affected cells lose most of their cytosolic components, including enzymes not bound to organelles [12]. Mitochondria become grossly swollen ; metabolic functions such as gluconeogenesis cease in affected cells.…”

“…Mitochondria become grossly swollen ; metabolic functions such as gluconeogenesis cease in affected cells. Cells that have not been reached by the digitonin front are normal histologically, electron microscopically [12,13] and functionally in terms of gluconeogenesis from lactate [8]. The demarcation of digitonin-permeabilized from digitonin-unaffected cells is sharp [12,13].…”

Hepatic intralobular mapping of fructose metabolism in the rat liver

“…The functional and microscopic effects of digitonin have been previously summarized [10,[13][14][15]. Briefly, a sharply demarcated zone of destruction of hepatocytes, sinusoidal and Kupffer cells, centred on the central venules, is produced.…”