The role of lipid peroxidation and glutathione on the glycochenodeoxycholic acid-induced cell death in primary cultured rat hepatocytes

Intracellular accumulation of bile acids in the hepatocytes during cholestasis is thought to be pathogenic in cholestatic liver diseases. The objective of this study was to determine the role of lipid peroxidation and glutathione on the bile acid-induced hepatic cell death mechanism in primary cultured rat hepatocytes. To induce hepatic cell death, we incubated primary cultured rat hepatocytes with glycochenodeoxycholic acid (GCDC; 0~400 micrometer) for 3 hours. In electron microscopic examination and agarose gel electrophoresis, low concentration of GCDC treatment mainly induced apoptotic feature. Whereas 400 micrometer GCDC treated cells demonstrated both apoptosis and necrosis. Lipid peroxidation was increased dose-dependently in GCDC treated hepatocyte. And this was also accompanied by decreased glutathione. Therefore, oxygen free radical damage may play a partial role in GCDC-induced hepatic cell death.

Glycochenodeoxycholic acid induces cell death in primary cultured rat hepatocyte: Apoptosis and necrosis

Intracellular accumulation of bile acids in the hepatocytes during cholestasis is thought to be pathogenic in cholestatic liver injury. Due to the detergent-like effect of the hydrophobic bile acids, hepatocellular injury has been attributed to direct membrane damage. However histological findings of cholestatic liver diseases suggest apoptosis can be a mechanism of cell death during cholestatic liver diseases instead of necrosis. To determine the pattern of hepatocellular toxicity induced by bile acid, we incubated primary cultured rat hepatocytes with a hydrophobic bile acid, Glycochenodeoxycholate (GCDC), up to 5 hours. After 5 hours incubation with 400 muM GCDC, lactate dehydrogenase released significantly. Cell viability, quantitated in propidium iodide stained cells concomitant with fluoresceindiacetate was decreased time-and dose-dependently. Most nuclei with condensed chromatin and shrunk cytoplasm were heavily labelled time- and dose-dependently by a positive TUNEL reaction. These findings suggest that both apoptosis and necrosis are involved in hepatocytes injury caused by GCDC.

Differential effect of homocysteic acid and cysteic acid on changes of inositol phosphates and (Ca2+)i in rat cerebellar granule cells

The present study was undertaken to characterize homocysteic acid (HCA)-and cysteic acid (CA)mediated formation of inositol phosphates (InsP) in primary culture of rat cerebellar granule cells. HCA and CA stimulated InsP formation in a dose-dependent manner, which was prevented by the N-methyl-D-aspartate (NMDA) receptor antagonist D,L-2-amino-5-phosphopentanoic acid (APV). CA-, but not HCA-, mediated InsP formation was in part prevented by the metabotropic glutamate receptor antagonist alpha-methyl-4-carboxyphenylglycine ((+/-)-MCPG). Both HCA- and CA-mediated increases in intracellular calcium concentration were completely blocked by APV, but were not altered by (+/-)-MCPG. CA-mediated InsP formation was in part prevented by removal of endogenous glutamate. In contrast, the glutamate transport blocker L-aspartic acid-beta-hydroxamate synergistically increased CA responses. These data indicate that in cerebellar granule cells HCA mediates InsP formation wholly by activating NMDA receptor. In contrast, CA stimulates InsP formation by activating both NMDA receptor and metabotropic glutamate receptor, and in part by releasing endogenous glutamate into extracellular milieu.

Effect of adenosine on the release of (3H)-5-hydroxytryptamine during glucose/oxygen deprivation from rat hippocampal slices

The effects of adenosine, adenosine A1 receptor antagonist (DPCPX), or NMDA receptor antagonist (APV) on the spontaneous release of (3H)-5-hydroxytryptamine ((3H)-5-HT) during normoxic/normoglycemic or hypoxic/hypoglycemic period were studied in the rat hippocampal slices. The hippocampus was obtained from the rat brain and sliced 400 mum thickness with the tissue slicer. After 30 min's preincubation in the normal buffer, the slices were incubated for 30 min in a buffer containing (3H)-5-HT (0.1 muM, 74 muCi/8 ml) for uptake, and washed. To measure the release of (3H)-5-HT into the buffer, the incubation medium was drained off and refilled every ten minutes through sequence of 14 tubes. Induction of glucose/oxygen deprivation (GOD; medium depleting glucose and gassed with 95% N2/5% CO2) was done in 6th and 7th tube. The radioactivities in each buffer and the tissue were counted using liquid scintillation counter and the results were expressed as a percentage of the total radioactivities. When slices were exposed to GOD for 20 mins, the spontaneous release of (3H)-5-HT was markedly increased and this increase of (3H)-5-HT release was blocked by adenosine (10 muM) or DL-2-amino-5-phosphonovaleric acid (APV; 30 muM). Adenosine A1 receptor specific antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) exacerbate GOD-induced increase of spontaneous release of (3H)-5-HT. These results suggest that Adenosine may play a role in the GOD-induced spontaneous release of (3H)-5-HT through adenosine A1 receptor activity.

The Effect of Unilateral Orbital Enucleation on Acetylcholine and Muscarinic Receptors in Visual Pathway, and Physical Activity of Rats

Acetylcholine is one of the major neurotransmitters in visual system of rats. Cholinergic, serotonergic and gamma-aminobutyric acid(GABA) ergic projections are known as modulators of visual processing. The purpose of this study was to examine the effects of unilateral orbital enucleation on the amount of acetylcholine and its receptor bindings in dorsolateral geniculate body, superior colliculus and visual cortex of rats, and on motor activites. The amount of acetylcholine was measured by gas chromatography; whereas [3H]quinuclidinyl benzilate(QNB) bindings to muscarinic receptors were examined by autoradiography. Motor activity was measured by activity meter for 24 hours. The results of this study were as follows: 1. The amount of acetylcholine in superior colliculus significantly increased at 1 week after enucleation, but it decreased at 2 and 4 weeks after enucleation. In visual cortex, the amount of acetylcholine did not change at 1 week after enucleation, but it significantly decreased at 2 and 4 weeks after enucleation. 2. After enucleation, [3H]-QNB binding to muscarinic receptors gradually decreased over the 4 weeks in dorsolateral geniculate body, superior colliculus and visual cortex. 3. Locomotor activity of rats did not show any change at 1 week after enucleat ion, but it decreased at 2 and 4 weeks after enucleation. In conclusion, unilateral orbital enucleation resulted in a reduction in the amount of acetylcholine and muscarinic receptors in rat visual system, which was speculated to be a major cause of a decrease in physical activity.