Lucia Battiston

Specific sequence-directed anti-bilitranslocase antibodies as a tool to detect potentially bilirubin-binding proteins in different tissues of the rat.

The hypothesis that the uneven distribution of bilirubin in the organism, which occurs in hyperbilirubinemia, could reflect an uneven distribution of bilirubin‐binding proteins was tested by searching for peptides containing the bilirubin‐binding motif identified in bilitranslocase (Battiston et al., 1998). In the rat, positive proteins bands were found to be present only in the liver, gastric mucosa and central nervous system. The electrophoretic mobilities of the positive compounds in the liver and stomach were identical to that of purified bilitranslocase (38 kDa). In the brain, on the contrary, two peptides were found with molecular masses of 79 and 34 kDa, respectively. Their distribution pattern in the central nervous system was different for each of them.

Hepatic glutathione determination after ethanol administration in rat: Evidence of the first-pass metabolism of ethanol

As a fraction of ingested ethanol is metabolized by gastric mucosa, different amounts of alcohol should reach the liver when the same dose is administered by oral or intravenous route. Therefore, we investigated the time-course of hepatic reduced glutathione (GSH) concentrations after intra-peritoneal or intra-gastric load of the same amount of ethanol in the rat. The test was also performed in fasted and Cimetidine-treated rats. The oral ethanol administration was followed by a less pronounced decrease and by a quicker recovery of hepatic content of GSH than after intraperitoneal route. In the fasted rat, basal hepatic GSH significantly decreased; after alcohol administration the decrease of hepatic GSH was more severe and prolonged than in the fed rat. Cimetidine was shown to be a potent inhibitor of gastric ADH. Pre-treatment with Cimetidine did not change the basal levels of hepatic GSH, but after oral ethanol load, the decrease of the hepatic GSH content was significantly (p < 0.005) more pronounced than in controls. This study demonstrates the beneficial effects of gastric ethanol metabolism on the liver. The reduced gastric ethanol metabolism, induced by fasting or by Cimetidine resulted in a decreased content and delayed recovery of liver GSH content.

Gastric uptake of nicotinic acid by bilitranslocase

Sabina Passamonti*, Lucia Battiston, Gian Luigi SottocasaBilitranslocase is a 38 kDa membrane protein [1] localised atthe vascular surface of the liver cells. The isolated protein isendowed with transport function of sulphobromophthalein(SBP), as directly demonstrated in reconstituted systems.Thus, its proposed physiological role is to mediate the tra⁄cof SBP and related organic anions across the sinusoidal do-main of the hepatocyte plasma membrane. On account of theknown competition between SBP and bilirubin for the ¢rststep of liver uptake observed in vivo, bilirubin had beenpointed out as the most obvious physiological substrate forbilitranslocase. A strong indication in this sense has come withthe ¢nding, within bilitranslocase primary structure, of thesequence EDSQGQHLSSF [2], which has the peculiarity ofbeing the central portion of a motif highly conserved in K-phycocyanins, biliproteins found in cyanobacteria. Whereasin K-phycocyanins this motif is involved in binding the pros-thetic group phycocyanobilin [3], a compound structurally re-lated to bilirubin, in bilitranslocase it has been shown to act asthe bilirubin-binding site [2]. Indeed, the a⁄nity of the carrierfor the pigment is high enough to ensure bilirubin extractionfrom the blood stream (K

Arylsulfonylation of bilitranslocase in plasma membranes from rat liver enables to discriminate between natural and artificial substrates

The serine protease inhibitor phenylmethylsulfonyl fluoride is shown to cause partial inhibition of bilitranslocase transport activity in rat liver plasma membrane vesicles. This condition can be fully reversed by means of pyridine-2-al-doxime methiodide, indicating that the carrier has undergone sulfonylation. Protection against inactivation is afforded by both bilirubin, the natural substrate, and nicotinic acid, but, unexpectedly, by neither sulfobromophthalein, the chromophoric substrate employed in bilitranslocase transport activity assay, nor rifamycin SV, a competitive inhibitor of sulfobromophthalein transport. From these protection experiments, the Ka for the complex of bilitranslocase with either bilirubin or nicotinic acid has been estimated to be 2.1 and 10.8 nM. respectively. Tentatively, the target for phenylmethylsulfonyl fluoride on bilitranslocase is identified as a recognition site for the physiological substrates.

Arginine residues are involved in the transport function of bilitranslocase

Specific guanido group reagents inhibit bilitranslocase transport activity in rat liver plasma membrane vesicles. Their reaction is shown to be affected by sulfobromophthalein, Thymol blue and bilirubin, which are translocated by bilitranslocase across the plasma membrane. It is concluded that the transport function of bilitranslocase depends on arginine residues, which are involved in the interaction with the molecules to be translocated.

On the mechanism of bilitranslocase transport inactivation by phenylmethylsulphonyl fluoride

Bilitranslocase is a plasma membrane carrier involved in the uptake of bilirubin and other organic anions from the blood into the liver cell. In the membrane, the carrier occurs as two interchangeable metastable forms, with high and low affinity for the substrates, respectively. The latter form can be specifically produced by either cysteine- or arginine modification. In liver plasma membrane vesicles, the serine-specific reagent phenylmethylsulphonyl fluoride is a partial inhibitor of bilitranslocase-mediated BSP transport rate. In this work, phenylmethyl-sulphonyl fluoride is shown to reduce the carrier maximal transport rate, without affecting its affinity for that substrate. In addition, it is found that the chemical modification caused by this reagent neither influences the equilibrium between the high- and the low-affinity forms nor prevents their free interconversion. From the effects of combined derivatizations of cysteine(s), arginine(s) and serine(s), it is concluded that the functionally relevant aminoacid residues lie in a close spatial arrangement. Also, in this study, the PMSF-modified serine(s) is shown to be involved in bilirubin binding by bilitranslocase.

Alcohol hepatic toxicity in rat: evidence of the utility of gastric ethanol metabolism

Abstract Since a fraction of ingested ethanol (EtOH) is metabolized by gastric mucosa, different amounts of alcohol should reach the liver, when the same dose is administered by oral or intravenous route. Accordingly, we demonstrated that the hepatic depletion of glutathione induced by EtOH is lower when it is administered orally rather than intraperitoneally (i.p.). In the present study we investigated, after EtOH load, the time-course of common serum liver damage tests and of α-glutathione-S-transferase (αGST) levels as a new indicator of hepatocellular injury in rats. The tests were also performed in Cimetidine-treated rats. Oral EtOH administration was followed by a less pronounced decrease and by a quicker recovery of hepatic glutathione than after i.p. route. After oral EtOH load, Cimetidine, a potent inhibitor of gastric alcohol-dehydrogenase, produced a decrease of hepatic glutathione significantly (P

Hepatic glutathione after ethanol administration in rat: effects of cimetidine and omeprazole.

As a fraction of ingested ethanol (EtOH) is metabolized by gastric mucosa, different amounts of alcohol reach the liver, when the same dose is administered by oral or intravenous route. In previous experiments, we demonstrated that the decrease of hepatic reduced glutathione (GSH) is less pronounced and is followed by a quicker recovery after oral than after intraperitoneal administration of the same amount of EtOH. Therefore, the time-course of hepatic GSH concentration seems to be an indirect assay of EtOH metabolism by the liver. On the basis of these findings, any condition causing a reduced function of gastric alcohol dehydrogenase (ADH) should show up as a more severe depletion of hepatic GSH. In the same rat experimental model we determined the effects of cimetidine and omeprazole administration on gastric ADH activity and on the time-course of hepatic GSH after EtOH load. Cimetidine was shown to inhibit gastric ADH with a Ki of 0.167 +/- 0.009 mmol l-1; accordingly, the pretreatment with this drug (20 mg kg-1 b.w. per day for 1 week) determined, after oral EtOH load, a marked reduction of hepatic GSH, likewise after intraperitoneal administration. Omeprazole exerted only a marginal inhibition on gastric ADH and this drug (0.3 mg kg-1 b.w. per day for 1 week) did not modify the time-course of hepatic GSH concentrations after EtOH load. This study indicates that the inhibition of gastric ADH, when associated with EtOH intake, induces depletion of the hepatic GSH concentration and, therefore, possible liver damage.