Andrea Romani

Loss of membrane protein thiols and lipid peroxidation in allyl alcohol hepatotoxicity

The data reported suggest that--following initiation of lipid peroxidation--membrane protein thiols can be attacked by lipid-derived radicals and/or reactive, lipid-soluble aldehydes like 4-hydroxynonenal and other hydroxyalkenals originated within the lipid core of cell membranes, resulting in a membrane protein thiol loss which is in turn associated with the development of hepatocellular injury.

Glucose 6-phosphate stimulation of MgATP-dependent Ca2+ uptake by rat kidney microsomes

(1) The features of MgATP-dependent Ca2+ accumulation under stimulation with glucose 6-phosphate were studied in rat kidney microsomes. (2) Ca2+ accumulated in the presence of MgATP alone does not exceed approx. 2 nmol/mg protein. (3) Glucose 6-phosphate markedly stimulates Ca2+ accumulation, up to steady-state levels approx. 15-fold higher than in its absence. (4) The hydrolysis of glucose 6-phosphate by glucose-6-phosphatase is essential for the stimulation, as shown by inhibiting the glucose 6-phosphate hydrolysis with adequate concentrations of vanadate. Inorganic phosphate is accumulated in microsomal vesicles during glucose 6-phosphate-stimulated Ca2+ uptake in equimolar amounts with respects to Ca2+. (5) Increasing concentrations of glucose 6-phosphate result in increasing stimulations of Ca2+ uptake, until a maximal Ca2(+)-loading capacity of approx. 27 nmol/mg microsomal protein is reached. It is suggested that the enlargement of the kidney microsomal Ca2+ pool induced by glucose 6-phosphate (an important metabolite in kidney) might play a role in the regulation of Ca2+ homeostasis in kidney tubular cells.

Stimulatory effect of glucose 6-phosphate on the non-mitochondrial Ca2+ uptake in permeabilized hepatocytes and Ca2+ release by inositol trisphosphate.

The relationships between Ca2+ transport and glucose-6-phosphatase activity, previously studied in isolated liver microsomes, were investigated in permeabilized hepatocytes in the presence of mitochondrial inhibitors. It was found that the addition of glucose 6-phosphate to the cells markedly stimulates the MgATP-dependent Ca2+ uptake. A progressive increase in the stimulation of Ca2+ uptake was seen with increasing amounts of glucose 6-phosphate up to 5 mM concentrations. Vanadate, when added in adequate concentrations (20-40 microM) to the hepatocytes inhibits both the glucose-6-phosphatase activity and the stimulation of Ca2+ uptake by glucose 6-phosphate, while not affecting the MgATP-dependent Ca2+ uptake. The addition of inositol 1,4,5-trisphosphate to permeabilized hepatocytes in which Ca2+ had been accumulated in the presence of MgATP and glucose 6-phosphate, results in a rapid release of Ca2+.

Ca2+ mobilization by vasopressin and glucagon in perfused livers. Effect of prior intoxication with bromotrichloromethane

Perfused livers isolated from rats treated with BrCCl3 for up to 15 min were used as an experimental tool to investigate the role of the hepatic endoplasmic reticulum in Ca2+ mobilization elicited by vasopressin and glucagon. BrCCl3-treatment caused extensive impairment (37 to 92%) of Ca2+ pumps of isolated liver microsomes, while Ca2+ pumps of mitochondria and plasma membrane vesicles remained undamaged. In perfused livers of BrCCl3-treated rats, the efflux of Ca2+ and the concomitant stimulation of O2 consumption and glucose release induced by vasopressin were decreased. The extent of the decrease paralleled the duration of BrCCl3-treatment. The decrease of Ca2+ efflux following vasopressin addition was closely correlated with the decrease of active Ca2+ accumulation by isolated microsomes (r = 0.99, P less than 0.001). The Ca2+ efflux elicited by glucagon was also decreased after BrCCl3-treatment, whereas stimulation of O2 consumption and glucose release were retained. The possibility that BrCCl3-treatment might impair the production of the intracellular Ca2+-mobilizing messenger IP3 is unlikely, since vasopressin still induced the formation of inositol phosphates, including IP3, in isolated hepatocytes obtained from BrCCl3-treated rats. Thus, this work supports the hypothesis that the Ca2+ stored in the liver ER is the major pool of intracellular Ca2+ available for mobilization by vasopressin, glucagon and other effectors.

4-Hydroxynonenal and Other Aldehydes Produced in the Liver In Vivo After Bromobenzene Intoxication

4-Hydroxynonenal (4-HNE) has been identified as one of the most reactive products in a series of toxic aldehydes originating from lipid peroxidation of cellular membranes. The possibility that this aldehyde plays a role as one of the mediators of the cellular injury induced by pro-oxidants is currently investigated. Mice intoxicated with bromobenzene showed levels of lipid peroxidation in the liver that exceed those induced by hepatotoxic haloalkanes CCl4 and BrCCl3. Hence, we have searched for the presence of 4-HNE and other lipid peroxidation products in the liver of bromobenzene-poisoned mice. We looked for 4-HNE in liver extracts as either free aldehyde or its 2,4-dinitrophenylhydrazone derivative. Using thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC) we obtained well resolved peaks, corresponding to the standard aldehyde or its 2,4-dinitrophenylhydrazone derivative, respectively. 2,4-dinitrophenylhydrazone derivatization was also used to determine the total carbonyl content in the liver of the intoxicated animals. Three fractions of hydrazones, according to their different polarity (“polar”; “non-polar carbonyls, fraction I”; and “non-polar carbonyls, fraction II”), were obtained using TLC. The UV-visible spectra were recorded for quantitative evaluation. Further fractionation of “non-polar carbonyls, fraction II” provided a fraction containing several alkanals and alk-2-enals, which were analyzed and identified by HPLC. Furthermore, protein bound carbonyls were determined in the liver of the intoxicated animals.

MgATP-dependent, glucose 6-phosphate-stimulated liver microsomal Ca2+ accumulation: Difference between rough and smooth microsomes

Some features of the MgATP-dependent Ca2+-accumulating capacity of rough as compared to smooth liver microsomal fraction were studied. Smooth microsomes accumulate somewhat higher amounts of Ca2+ than rough ones in the presence of MgATP. In the presence of glucose 6-phosphate, which markedly stimulates MgATP-dependent Ca2+ accumulation in both fractions, smooth microsomes exhibit a much higher Ca2+-accumulating capacity than rough ones. Possible reasons of the differences observed between the two fractions were investigated. Smooth microsomes exhibit a higher Ca2+-dependent ATPase activity, suggesting a higher Ca2+ inward transport into smooth vesicles. Also, following the inhibition of active Ca2+ transport by means of vanadate, smooth microsomes appear to release the Ca2+ previously accumulated--both in the absence (i.e., with MgATP only) and in the presence of glucose 6-phosphate--at a lower rate than rough ones. This indicates a lower passive backflux of Ca2+ accumulated in smooth vesicles. On the basis of these data, differences can be envisaged with respect to cellular Ca2+ handling by different domains of endoplasmic reticulum in the liver.

Detection of 4-Hydroxynonenal and Other Lipid Peroxidation Products in the Liver of Allyl Alcohol-Intoxicated Mice

Allyl alcohol has long been known to produce periportal necrosis of the liver in rats and mice (Miessner 1891; Piazza 1915). Also it is known that allyl alcohol is metabolized by the cytosolic enzyme alcohol dehydrogenase to acrolein (Rees and Tarlow 1967; Serafini-Cessi 1972). The latter is considered as one of the most important toxic metabolites responsible for the damage induced by allyl alcohol in liver and other tissues. Acrolein is in fact the most toxic member of the class of 2-alkenals (Beauchamp et al. 1985; Schauenstein et al. 1977), α-β unsaturated aldehydes which also include crotonaldehyde, pentenal, hexenal and so on. Acrolein is a powerful electrophile which reacts even spontaneously with nucleophiles such as sulphydryl groups (Esterbauer et al. 1975). The reaction is markedly accelerated by the activity of GSH-transferases. Cellular GSH is primarily involved in the reaction and the result is a dramatic loss of GSH stores (Hanson and Anders 1978; Zitting and Heinonen 1980; Dawson et al. 1984; Ohno et al. 1985; Jaeschke et al. 1987). The covalent binding of allyl alcohol metabolites to liver cells has been demonstrated with various techniques (Reid 1972).

Liver cytosolic non-dialysable factor(s) can counteract GTP-dependent Ca2+ release in rat liver microsomal fractions

Readdition to rat liver microsomes of dialysed liver post-microsomal supernatant resulted in an almost complete inhibition of the Ca2+-releasing effect of GTP. Such inhibition was heat-labile, and was associated with non-ultrafiltrable supernatant components with a molecular weight higher than 30,000 D. A preliminary fractionation of liver supernatant showed that the inhibitory effect is recovered in the 40-50% ammonium sulfate-precipitated proteins, with an approx. 10-fold enrichment. The active ammonium sulfate fraction did not modify the GTP-induced Ca2+ increase of passive Ca2+ efflux from microsomes, nor did it affect microsomal GTP hydrolysis, which is likely required for its Ca2+ releasing effect. The active ammonium sulfate fraction appears to markedly favour the translocation of GTP-released Ca2+ into a microsomal GTP-insensitive pool. Separation of liver microsomes in smooth and rough fractions revealed that such GTP-insensitive Ca2+ pool is almost completely associated with smooth microsomes.