Isolated Hamster Liver Research Articles

Biogenic aldehyde(s) derived from the action of monoamine oxidase may mediate the antidipsotropic effect of daidzin

Daidzin, a major active principle of an ancient herbal treatment for ‘alcohol addiction’, was first shown to suppress ethanol intake in Syrian golden hamsters. Since then this activity has been confirmed in Wistar rats, Fawn hooded rats, genetically bred alcohol preferring P rats and African green moneys under various experimental conditions, including two-level operant, two-bottle free-choice, limited access, and alcohol-deprivation paradigms. In vitro, daidzin is a potent and selective inhibitor of mitochondrial aldehyde dehydrogenase (ALDH-2). However, in vivo, it does not affect overall acetaldehyde metabolism in golden hamsters. Using isolated hamster liver mitochondria and 5-hydroxytryptamine (5-HT) and dopamine (DA) as the substrates, we demonstrated that daidzin inhibits the second but not the first step of the MAO/ALDH-2 pathway, the major pathway that catalyzes monoamine metabolism in mitochondria. Correlation studies using structural analogs of daidzin led to the hypothesis that the mitochondrial MAO/ALDH-2 pathway may be the site of action of daidzin and that one or more biogenic aldehydes such as 5-hydroxyindole-3-acetaldehyde (5-HIAL) and/or DOPAL derived from the action of monoamine oxidase (MAO) may be mediators of its antidipsotropic action.

The mitochondrial monoamine oxidase-aldehyde dehydrogenase pathway: a potential site of action of daidzin.

Recent studies showed that daidzin suppresses ethanol intake in ethanol-preferring laboratory animals. In vitro, it potently and selectively inhibits the mitochondrial aldehyde dehydrogenase (ALDH-2). Further, it inhibits the conversion of monoamines such as serotonin (5-HT) and dopamine (DA) into their respective acid metabolites, 5-hydroxyindole-3-acetic acid (5-HIAA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in isolated hamster or rat liver mitochondria. Studies on the suppression of ethanol intake and inhibition of 5-HIAA (or DOPAC) formation by six structural analogues of daidzin suggested a potential link between these two activities. This, together with the finding that daidzin does not affect the rates of mitochondria-catalyzed oxidative deamination of these monoamines, raised the possibility that the ethanol intake-suppressive (antidipsotropic) action of daidzin is not mediated by the monoamines but rather by their reactive biogenic aldehyde intermediates such as 5-hydroxyindole-3-acetaldehyde (5-HIAL) and/or 3,4-dihydroxyphenylacetaldehyde (DOPAL) which accumulate in the presence of daidzin. To further evaluate this possibility, we synthesized more structural analogues of daidzin and tested and compared their antidipsotropic activities in Syrian golden hamsters with their effects on monoamine metabolism in isolated hamster liver mitochondria using 5-HT as the substrate. Effects of daidzin and its structural analogues on the activities of monoamine oxidase (MAO) and ALDH-2, the key enzymes involved in 5-HT metabolism in the mitochondria, were also examined. Results from these studies reveal a positive correlation between the antidipsotropic activities of these analogues and their abilities to increase 5-HIAL accumulation during 5-HT metabolism in isolated hamster liver mitochondria. Daidzin analogues that potently inhibit ALDH-2 but have no or little effect on MAO are most antidipsotropic, whereas those that also potently inhibit MAO exhibit little, if any, antidipsotropic activity. These results, although inconclusive, are consistent with the hypothesis that daidzin may act via the mitochondrial MAO/ALDH pathway and that a biogenic aldehyde such as 5-HIAL may be important in mediating its antidipsotropic action.

Mitochondrial fixation for the detection of cytochrome oxidase activity using microwave irradiation.

We examined cell fixation with microwave irradiation (MWI) used in cytochemistry. MWI was applied to blocks of about 1 mm3 of mouse parotid glands at 500 W for about 5 sec in a fixative at 37 degrees C. The activities of endogenous peroxidase and mitochondrial cytochrome oxidase were demonstrated by using the DAB method with 3,3'-diaminobenzidine (DAB) and 0.01% H2O2. Under electron microscopy, peroxidase activity was localized in the nuclear envelope, endoplasmic reticulum and secretory granules. However, mitochondria cytochrome oxidase activity seemed to be rather weak against the MWI at 37 degrees C. Moreover, suspension of isolated hamster liver mitochondria was fixed by MWI and also demonstrated cytochrome oxidase activity by using the cytochemical methods with DAB, cytochrome c, catalase and sucrose. Such mitochondrial fractions were subjected to 6-second MWI given 10 or 18 times with an interval of 10 seconds with and without a chilled water bath. The final temperature of each fixative was kept at about 10 degrees C or rose to about 37 and 55 degrees C. When we took care to keep the temperature below 10 degrees C, the DAB reaction products accumulated in the mitochondrial intermembrane-intracristal space. No mitochondrial deposits were observed when the temperatures of the fixatives rose to 37 and 55 degrees C. These results indicated that peroxidase was very resistant to the heat with MWI fixation. Cytochrome oxidase is sensitive to the heat with MWI, so, a chilled water bath had to be used.

Effects of dietary cholesterol on hepatic production of lipids and lipoproteins in isolated hamster liver

The effect of 2-week 2% cholesterol vs. chow feeding on regulation of hepatic lipoprotein, lipids and apoprotein (Apo), and biliary lipids production was evaluated by the isolated perfused hamster liver model. Cholesterol feeding did not change very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) particle size but significantly increased the hepatic production of VLDL-cholesterol fourfold, VLDL-triglyceride two and one-half-fold but not phospholipid in VLDL. It also increased LDL-cholesterol fourfold but not triglyceride or phospholipid in LDL, whereas lipids in HDL remained unchanged. Gradient sodium dodecyl sulfate-polyacrylamide gel electrophesis (SDS-PAGE) and Western blot analysis (density of apoprotein/density of albumin/g liver) indicated that cholesterol feeding enhanced Apo B tenfold, Apo A-I fivefold but not Apo E in VLDL. Apo E and Apo B did not change in LDL. Apo E but not Apo A-I increased (threefold) in HDL by cholesterol feeding. Cholesterol feeding decreased bile salt secretion 28% but increased cholesterol secretion 118% in bile, whereas phospholipid and bile volume remained unchanged. Increased Apo A-I in VLDL suggested that Apo A-I is involved in enhanced hepatic export of cholesterol and triglyceride. Different patterns of lipid and Apos in VLDL and LDL after cholesterol feeding also suggested separate VLDL and LDL export mechanisms. Elevated Apo E but not lipids in HDL after cholesterol feeding suggests that hepatic HDL may function as a carrier of newly synthesized hepatic Apo E into the circulation for transfer to other lipoproteins (chylomicron [CM], CMr) to facilitate hepatic cholesterol uptake and clearance. (Hepatology 1996 Aug;24(2):424-34)

Effects of Dietary Cholesterol on Hepatic Production of Lipids and Lipoproteins in Isolated Hamster Liver

The effect of 2-week 2% cholesterol vs. chow feeding on regulation of hepatic lipoprotein, lipids and apoprotein (Apo), and biliary lipids production was evaluated by the isolated perfused hamster liver model. Cholesterol feeding did not change very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) particle size but significantly increased the hepatic production of VLDL-cholesterol fourfold, VLDL-triglyceride two and one-half-fold but not phospholipid in VLDL. It also increased LDL-cholesterol fourfold but not triglyceride or phospholipid in LDL, whereas lipids in HDL remained unchanged. Gradient sodium dodecyl sulfate-polyacrylamide gel electrophesis (SDS-PAGE) and Western blot analysis (density of apoprotein/density of albumin/g liver) indicated that cholesterol feeding enhanced Apo B tenfold, Apo A-I fivefold but not Apo E in VLDL. Apo E and Apo B did not change in LDL. Apo E but not Apo A-I increased (threefold) in HDL by cholesterol feeding. Cholesterol feeding decreased bile salt secretion 28% but increased cholesterol secretion 118% in bile, whereas phospholipid and bile volume remained unchanged. Increased Apo A-I in VLDL suggested that Apo A-I is involved in enhanced hepatic export of cholesterol and triglyceride. Different patterns of lipid and Apos in VLDL and LDL after cholesterol feeding also suggested separate VLDL and LDL export mechanisms. Elevated Apo E but not lipids in HDL after cholesterol feeding suggests that hepatic HDL may function as a carrier of newly synthesized hepatic Apo E into the circulation for transfer to other lipoproteins (chylomicron [CM], CMr) to facilitate hepatic cholesterol uptake and clearance.

Inhibition of in vitro RNA synthesis by hycanthone, oxamniquine and praziquantel

The schistosomicides, hycanthone, oxamniquine and praziquantel, were found to inhibit the in vitro RNA synthesis using isolated hamster liver nuclei. Preincubation of the nuclei with these drugs revealed that the inhibitory effect of oxamniquine was irreversible and progressed with time, whereas that of hycanthone and praziquantel was reversible. On the other hand, hycanthone and praziquantel have a high affinity for DNA but oxamniquine does not. The data indicate that the mechanism of inhibition by oxamniquine is different from that of hycanthone and praziquantel.

The metabolism of l[3- 3H]lactate by isolated hamster liver cells

The rate of tritium removal from l[3- 3H]lactate by hamster liver cells is faster than the analytical rate of lactate utilization, or the rate of 14C disappearance from l[U- 14C]lactate, with the result that the 3H/ 14C ratio in residual lactate from l-[U- 14C,3- 3H]lactate decreases. However, addition of low concentrations (0.1 to 1.0 mM) of l-cycloserine, a glutamate pyruvate transaminase inhibitor, nearly equalizes the rates of isotope utilization from l-[3- 3H]lactate and l-[U- 14C]lactate. The results suggest a very limited rate of recycling of phosphoenolpyruvate back to pyruvate during gluconeogenesis from lactate in fasted hamster liver cells.

Cholestasis induced by sodium taurolithocholate in isolated hamster liver.

The mechanism of cholestasis (decreased bile flow) induced by taurolithocholate in the isolated perfused hamster liver was investigated. Taurocholate was infused to maintain bile acid output, and sulfobromophthalein (BSP) was administered to establish a BSP transport maximum in bile. The effects of taurolithocholate on bile flow and on the biliary secretion of BSP and bile acid anions were determined.A significant dose-response correlation was found between taurolithocholate and the degree of cholestasis. No significant hepatic morphologic alterations were observed. At low doses, cholestasis was reversible. A multiple regression equation was developed to validate the steroid dehydrogenase determination of total bile acids in bile that contained BSP. During cholestasis, output of bile acid was maintained by a significantly increased concentration of bile acid. Hepatic removal rate and transport maximum of BSP were significantly decreased, whereas BSP concentration, conjugation, and hepatic content were unaffected. The concentrating capacity for BSP in bile appeared to be the rate-limiting factor in BSP transport. Individual bile acids were determined by gas-liquid chromatography. Of the injected taurolithocholate, 40-50% was recovered in bile as lithocholic acid, 30% was converted to chenodeoxycholic acid, and only traces of lithocholic acid were detected in the perfusate after 4 hr. Cholic and chenodeoxycholic acids comprised 75-89%, and lithocholic acid comprised 11-25% of bile acids in bile after taurolithocholate injection; only traces of deoxycholic acid were seen. Small amounts of taurolithocholate sulfate were detected in bile by thinlayer chromatography. The outputs of sodium and potassium in bile were significantly diminished during cholestasis.A substantial fraction (75%) of basal bile flow in the isolated hamster liver was estimated to be independent of bile acid secretion. Cholestasis occurred after taurolithocholate, whereas bile acid secretion was maintained. The results indicate that the most likely mechanism for acute cholestasis induced by taurolithocholate in isolated hamster liver was interference with the bile acid-independent fraction of canalicular or ductular bile flow or both.

Bile secretion in isolated hamster liver.

ARTICLESBile secretion in isolated hamster liverJ. E. King, S. Oshiba, and L. J. SchoenfieldJ. E. King, S. Oshiba, and L. J. SchoenfieldPublished Online:01 Apr 1970https://doi.org/10.1152/jappl.1970.28.4.495MoreSectionsPDF (1 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation More from this issue > Volume 28Issue 4April 1970Pages 495-500 Copyright & PermissionsCopyright © 1970 the American Physiological Societyhttps://doi.org/10.1152/jappl.1970.28.4.495PubMed5437440History Published online 1 April 1970 Published in print 1 April 1970 Metrics

Plasminogen activator in bile stimulated by sodium taurocholate in isolated hamster livers.

SummaryPlasminogen activator activity (bilokinase) was demonstrated for the first time in fistula bile of the hamster as well as in the bile of isolated hamster liver. Sodium taurocholate was shown to stimulate the release of bilokinase into bile. Bilokinase in bile is analogous to urokinase in urine, but its role in coagulation homeostasis or in gallstone formation remains to be determined.Acknowledgment is made of the expert assistance of Mr. Emery Van Hook in preparing the isolated liver system and of Miss Linda L. Livingston in laboratory determinations.