Gian Carlo Lunazzi

Cellular localization of sulfobromophthalein transport activity in rat liver

The movement of sulfobromophthalein is measured in rat liver plasma-membrane vesicles by direct dual-wavelength spectrophotometry. The technique is based on the principle that the dye, when entering a more acidic compartment, changes its absorption in the visible region. From this study it may be concluded that, among the different cellular subfractions, only liver plasma-membrane vesicles can catalyze electrogenic transport of sulfobromophthalein. Plasma membranes from erythrocytes are unable to perform such a function. The movement follows the distribution pattern of (Na+ + K+)-ATPase and it is therefore concluded that this process occurs exclusively at the sinusoidal membrane level. Inhibition studies confirm that the process is catalyzed by bilitranslocase.

Isolation of bilitranslocase, the anion transporter from liver plasma membrane for bilirubin and other organic anions

Publisher Summary This chapter discusses the isolation of bilitranslocase, the anion transporter from liver plasma membrane for bilirubin and other organic anions. Bilitranslocase––a carrier protein involved in sulfobromophthalein (BSP) and other organic anion transport––has been isolated from rat liver plasma membranes. A more direct demonstration of the origin and involvement of bilitranslocase in the transport function has been obtained from immunochemical studies. The only way to trace bilitranslocase during a purification procedure is to follow the BSP binding capacity at the different purification steps. This can be done either by gel filtration or by membrane ultrafiltration. In both cases, the principle is to separate physically the protein-dye complex and to measure the amount of the dye bound in equilibrium with the free form. This chapter also discusses the estimate of BSP binding to protein by gel filtration on BioGel P-2.

Sex steroid modulation of the hepatic uptake of organic anions in rat

To investigate the role of sex steroids in the sex-related difference in the hepatic uptake of organic anions, sulphobromophthalein (bromsulphalein, BSP) transport was measured in hepatocytes isolated from rats either deprived of hormonal influence by castration at prepubertal age or after hormonal substitution. In control animals, the kinetics of BSP uptake showed the presence of two components: one saturable (0-3 microM), with high affinity and low capacity, and the other linear (9-30 microM), probably related to the non-specific component of BSP uptake. Sex difference was detected only in the saturable portion of the uptake process as the apparent Km was significantly lower in females than in males (3.8 +/- 0.7 vs. 6.1 +/- 1.8 microM, mean +/- S.D. of six animals, P less than 0.01). In contrast, no difference was observed in Vmax (2.3 +/- 0.3 vs. 2.2 +/- 0.7 nmol BSP.(mg protein)-1.min-1). Castration was associated with the disappearance of the saturable uptake site and abolished the sex difference. Progesterone treatment of castrated males failed to restore the saturable kinetics of BSP uptake. In contrast, administration of oestradiol to castrated males or testosterone to castrated females did restore the saturable kinetics of the high-affinity BSP uptake. Km and Vmax were comparable to those of adult females and males, respectively, with the exception of testosterone which induced a Vmax value higher than that observed in the other groups of animals. These data suggest that the influence of oestrogen and testosterone is necessary for the expression of the high-affinity, low-capacity carrier-mediated process of hepatic BSP uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

RECONSTITUTION OF SULFOBROMOPHTHALEIN TRANSPORT IN ERYTHROCYTE MEMBRANES INDUCED BY BILITRANSLOCASE

Bilitranslocase, the protein responsible for the anion translocation at the sinusoidal plasma membrane level in liver, was shown to be able to reconstitute the transport of sulfobromophthalein in liposomes in the past. The protein preparation used in those experiments consisted of two subunits of 35.5 and 37 kDa. The isolated 37 kDa protein, when inserted in erythrocyte membrane vesicles, confers to the particles the ability to carry out an electrogenic transport of sulfobromophthalein. The effect is specific and can be inhibited by monospecific polyclonal antibodies raised against the protein. In may be concluded that the 37 kDa protein band, present in previous preparations of bilitranslocase, is not only a necessary but also a sufficient component of the transport system for bilirubin and functional analogues.

Difference in hepatic uptake of tetra- and Di-bromosulfophthalein in rat: Role of hydrophobicity, binding to plasma proteins and affinity for plasma membrane carrier protein

The relative role of hydrophobicity, binding to plasma proteins and affinity for one of the plasma membrane transport proteins in the hepatic uptake of 3,4,5,6-tetra- (BSP) and 3,6-di- (DBSP) bromosulfophthalein was investigated in the rat. In terms of physicochemical characteristics, the two molecules show different pKa values and degrees of hydrophobicity, as determined from the n-octanol:water partition coefficient. In the intact animal, the plasma clearance and the plasma removal rate after a dose of 1.5 mumol/kg i.v. were significantly (P < 0.001) faster for BSP than DBSP, while no difference was found in the plasma distribution volume. The dissociation constant (Kd) of the high affinity binding sites of plasma proteins also differed for the two anions, being significantly lower for BSP than DBSP (0.95 +/- 0.02 vs 1.44 +/- 0.14 microM, P < 0.001). [35S]BSP uptake by liver plasma membrane vesicles was saturable with an apparent Km of 5.20 +/- 0.80 microM, and was competitively inhibited by DBSP (Ki 18.2 +/- 1.2 microM) indicating a common uptake system. The Kd value for binding of the organic anions to purified bilitranslocase, a plasma membrane protein involved in the electrogenic transport of pthaleins, was also significantly lower for BSP than DBSP (1.10 +/- 0.12 vs 3.02 +/- 0.27 microM, N = 3, P < 0.001), indicating a higher affinity of the former ligand for the carrier protein. No difference was observed in the capacity of the high affinity binding sites (32 +/- 3 vs 33 +/- 3 nmol/mg protein, BSP and DBSP, respectively). These data indicate that BSP and DBSP are two different cholephilic organic anions which share a common uptake mechanism, at least partly mediated by bilitranslocase. The greater affinity of BSP than DBSP for the carrier protein may account for the faster plasma disappearance rate of BSP observed in vivo, in spite of the higher plasma protein binding.

Serum free fatty acids and bilirubin concentration during fasting in patients with Gilbert's syndrome and normal controls

SummaryThe increments in serum concentrations of unconjugated bilirubin and free fatty acids (FFA) were measured 24 and 48h after reduction of the caloric intake (400 cal/day) in 17 patients with Gilbert’s syndrome (GS) and in 12 healthy control subjects. In males, both normal and with GS, the rise in serum bilirubin was statistically higher (P<0.01) as compared to females. On the contrary, no sex difference was found in FFA concentrations. A linear correlation (p<0.01) between bilirubin and FFA serum levels was present in normal males and in patients with Gilbert’s syndrome of both sexes. Because bilirubin and FFA partly share a common, bilitranslocase-mediated, hepatic uptake mechanism, data reported support the hypothesis that a bilitranslocase function may be one of the metabolic defects in Gilbert’s syndrome.

The Hepatic Uptake of Organic Anions: An Overview

The complex process by which organic anions are efficiently taken up, stored, metabolically modified, and eventually excreted into the bile has been the subject of intensive investigation.