The accumulation of hydrophobic bile acids plays a role in the induction of apoptosis and necrosis of hepatocytes during cholestasis. The aim of this study was to determine in freshly isolated rat hepatocytes the roles of oxidant stress and the mitochondrial permeability transition (MPT) in bile acid-induced apoptosis. Hepatocytes isolated from adult male Sprague-Dawley rats were incubated for 4 hours in buffer containing the hydrophobic bile acid, glycochenodeoxycholic acid (GCDC, 0-500 mol/L) or the hydrophilic bile acid, glycocholic acid (GCA), and either the antioxidants, alpha tocopherol, ebselen, or idebenone (a coenzyme Q analogue); or the MPT blockers, cyclosporin A, or bongkrekic acid, or a caspase-8 inhibitor.

The two main advantages of cryofixation over chemical fixation methods are the simultaneous stabilization of all cellular components and the much faster rate of fixation. The main drawback pertains to the limited depth (less than 20 microns surface layer) to which samples can be well frozen when freezing is carried out under atmospheric conditions. High-pressure freezing increases the depth close to 0.6 mm to which samples can be frozen without the formation of structurally distorting ice crystals. This review discusses the theory of high-pressure freezing, the design of the first commercial high-pressure freezing apparatus (the Balzers HPM 010), the operation of this instrument, the quality of freezing, and novel structural observations made on high-pressure-frozen cells and tissues.

Hydrophobic bile acids may cause hepatocellular necrosis and apoptosis during cholestatic liver diseases. The mechanism for this injury may involve mitochondrial dysfunction and the generation of oxidant stress. The purpose of this study was to determine the relationship of oxidant stress and the mitochondrial membrane permeability transition (MMPT) in hepatocyte necrosis induced by bile acids. The MMPT was measured spectrophotometrically and morphologically in rat liver mitochondria exposed to glycochenodeoxycholic acid (GCDC). Freshly isolated rat hepatocytes were exposed to GCDC and hepatocellular necrosis was assessed by lactate dehydrogenase release, hydroperoxide generation by dichlorofluorescein fluorescence, and the MMPT in cells by JC1 and tetramethylrhodamine methylester fluorescence on flow cytometry. GCDC induced the MMPT in a dose-and Ca 2ϩ -dependent manner. Antioxidants significantly inhibited the GCDC-induced MMPT and the generation of hydroperoxides in isolated mitochondria. Other detergents failed to induce the MMPT and a calpain-like protease inhibitor had no effect on the GCDC-induced MMPT. In isolated rat hepatocytes, GCDC induced the MMPT, which was inhibited by antioxidants. Blocking the MMPT in hepatocytes reduced hepatocyte necrosis and oxidant stress caused by GCDC. Oxidant stress, and not detergent effects or the stimulation of calpain-like proteases, mediates the GCDC-induced MMPT in hepatocytes. We propose that reducing mitochondrial generation of reactive oxygen species or preventing increases in mitochondrial Ca 2ϩ may protect the hepatocyte against bile acid-induced necrosis. 1-3). Although hydrophobic bile acids cause injury to isolated hepatocytes (4), cultured hepatocytes (5), and the intact liver (6), the mechanisms of this toxicity are not fully understood. Both hepatocellular necrosis at higher bile acid concentrations (4) and apoptosis at lower concentrations (7) have been demonstrated and are proposed as playing a role in cholestatic liver injury. Hepatocyte necrosis is characterized by cellular swelling, loss of mitochondrial respiratory function, depleted cellular ATP levels, and formation of plasma membrane blebs that rupture and release cellular contents (8, 9). In cholestatic liver ABSTRACT519

The polar distribution of Na(+)-K(+)-ATPase to the basolateral membrane of proximal tubule cells is essential for the efficient and vectorial reabsorption of Na+ and may be dependent on the formation of a metabolically stable, detergent-insoluble complex of Na(+)-K(+)-ATPase with the actin membrane cytoskeleton. The present studies utilized immunocytochemical techniques to demonstrate and quantify the apical redistribution of Na(+)-K(+)-ATPase during mild ischemia (15 min) that occurred in proximal (1.3 +/- 0.9 vs. 4.5 +/- 1.1 particles/100 microns surface membrane, P less than 0.01) but not distal tubule cells. Treatment of control apical membranes with 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A2C), a fluidizing agent, markedly increased membrane fluidity without any effect on Na(+)-K(+)-ATPase activity. In brush-border membrane vesicles isolated after ischemia, however, A2C further increased an already elevated Na(+)-K(+)-ATPase activity. During ischemia, total cellular Na(+)-K(+)-ATPase activity remained unaltered, but the Triton X-100-soluble (noncytoskeleton associated) fraction of Na(+)-K(+)-ATPase increased significantly following 15 and 30 min. There was a corresponding decrease in the Triton X-100-insoluble fraction of Na(+)-K(+)-ATPase, with the ratio of detergent-soluble to -insoluble Na(+)-K(+)-ATPase increasing from 13 +/- 2 to 32 +/- 5% (P less than 0.01) during 30 min of ischemia. Western blot analysis of the Triton X-100-soluble fraction, following 30 min of ischemic injury, revealed the presence of Na(+)-K(+)-ATPase, actin, fodrin, and uvomorulin. However, in a fraction highly enriched for Na(+)-K(+)-ATPase, neither actin, fodrin, nor uvomorulin was detected.(ABSTRACT TRUNCATED AT 250 WORDS)