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.

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.

The bilirubin-binding motif of bilitranslocase and its relation to conserved motifs in ancient biliproteins.

In the primary structure of bilitranslocase, currently under study in our laboratory, an aminoacid motif was identified and found to be conserved in a number of alpha-phycocyanines, ancient biliproteins present in cyanobacteria. To test the possibility that such a motif could be at least part of the binding site for bilirubin, epitope-specific antibodies were raised. The target corresponds to the sequence 65-75 of bilitranslocase and covers the central portion of the motif identified. The antibodies were shown: 1) to inhibit the electrogenic BSP transport by plasmamembrane vesicles; 2) to react with purified bilitranslocase; and 3) to identify only one protein band with electrophoretic mobility identical to bilitranslocase in Western blots of solubilised plasmamembrane vesicles. The presence of either bilirubin or nicotinate during pre-incubation with the antibodies decreases concentration-wise the inhibition kinetics. From these experiments a dissociation constant of 2.2 +/- 0.3 and 11.3 +/- 1.3 nM for bilirubin-bilitranslocase and nicotinate-bilitranslocase complexes were calculated.

Bilitranslocase can exist in two metastable forms with different affinities for the substrates--evidence from cysteine and arginine modification.

Bilitranslocase is an organic anion carrier involved in bilirubin and phthalein uptake by the liver. In rat liver plasma membranes, its function is assayed by recording the electrogenic sulfobromophthalein movement. This has been found to be inhibited by both cysteine-specific and arginine-specific reagents. Inhibition is both partial and it occurs to the same extent, i.e. approximately 50%. The effects are not additive. Here we describe the mechanism underlying the above observations. It is concluded that bilitranslocase occurs in two possible states, featured by high and low affinity for the substrates (for sulfobromophthalein, Km = 5 microM and 37 microM, respectively). Cysteine- or arginine-reactive reagents, by reacting selectively with the low-affinity form, entrap it and shift the equilibrium between the two forms, so that, at completion, only the low-affinity form is present. The substrate concentration in the standard transport assay is 39 microM, a value at which the modified low-affinity form operates in the range of half-maximal velocity. This explains both the apparent half-inhibition measured after the chemical treatments and the lack of additivity. In addition, the substrates are shown to enhance the rate of conversion from the low-affinity to the high-affinity form of the translocator, thus favouring its high-affinity form under physiological conditions.

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.

Lansoprazole and ethanol metabolism: comparison with omeprazole and cimetidine.

Since some antisecretory drugs such as cimetidine and ranitidine, interfere with ethanol metabolism by inhibition of hepatic and/or gastric alcohol dehydrogenase, we investigated the effect of lansoprazole, a new protonic pump inhibitor, on gastric and hepatic alcohol dehydrogenase activity. We also compared the lansoprazole effect with that of omeprazole and cimetidine, respectively. Ethanol blood concentration after oral intake or intravenous administration of ethanol was estimated either in normal male human volunteers or in male rats before and after one week pretreatment with lansoprazole, omeprazole and cimetidine. Furthermore, the in vitro effect of these drugs was studied on both human and rat gastric and hepatic alcohol dehydrogenases. Finally, we measured the effect of the treatment on the reduced hepatic glutathione to test the effects of the drugs on first-pass metabolism of ethanol. The results reported in this paper indicate that lansoprazole, as well as omeprazole, does not affect ethanol metabolism, and that protonic pump inhibitors seem to be safer than imidazole-derived drugs in subjects unable to reduce ethanol intake during conditions requiring acid secretion inhibition.

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.

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.

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.