Properties of membrane-bound bilirubin UDP-glucuronyltransferase in rough and smooth endoplasmic reticulum and in the nuclear envelope from rat liver.

We examined regulatory properties of bilirubin UDP-glucuronyltransferase in sealed RER (rough endoplasmic reticulum)- and SER (smooth endoplasmic reticulum)-enriched microsomes (microsomal fractions), as well as in nuclear envelope from rat liver. Purity of membrane fractions was verified by electron microscopy and marker studies. Intactness of RER and SER vesicles was ascertained by a high degree of latency of the lumenal marker mannose-6-phosphatase. No major differences in the stimulation of UDP-glucuronyltransferase by detergent or by the presumed physiological activator, UDPGlcNAc, were observed between total microsomes and RER- or SER-enriched microsomes. Isolated nuclear envelopes were present as a partially disrupted membrane system, with approx. 50% loss of mannose-6-phosphatase latency. The nuclear transferase had lost its latency to a similar extent, and the enzyme failed to respond to UDPGlcNAc. Our results underscore the necessity to include data on the integrity of the membrane permeability barrier when reporting regulatory properties of UDP-glucuronyltransferase in different membrane preparations.

Bilirubin kinetics in intact rats and isolated perfused liver. Evidence for hepatic deconjugation of bilirubin glucuronides.

Most previous compartmental models describing bilirubin transport and metabolism in the liver have been validated solely by analysis of the plasma disappearance of radiolabeled bilirubin in human subjects. We now have determined the transport kinetics of a bilirubin tracer pulse by analysis of plasma, liver, and bile radioactivity data from 30 intact rats. Plasma [3H]bilirubin disappearance was best described by the sum of three exponentials, and a six-compartment model, derived by simulation analysis, was necessary and adequate to describe all experimental data. Examination of the injected radiolabeled bilirubin by extraction with hexadecyltrimethylammonium bromide and thin-layer chromatography revealed that 6.6% (mean) of the original pigment had been degraded to labeled nonbilirubin derivatives during preparation of the tracer dose. This material exhibited a significantly longer half-life (mean 50.6 min) of the plasma terminal exponential than that of authentic radiobilirubin (20.6 min). In isolated perfused rat liver, the kinetics of [3H]bilirubin in perfusate and bile readily fitted the proposed model. Compatibility of the model with the data obtained, both in the isolated liver and in vivo, required that a fraction of bilirubin conjugated in the liver be deconjugated and returned to the plasma. Deconjugation of bilirubin glucuronides was evaluated directly by infusion of bilirubin monoglucuronides, containing 14C in the glucuronosyl group, into rats with an external bile fistula. Since metabolic degradation of hydrolyzed 14C-labeled glucuronic acid yields 14CO2, this was measured in expired air. Whereas 86% of the administered labeled pigment was recovered in bile, 7% of the label appeared in 14CO2. These findings directly validate a portion of the proposed kinetic model and suggest that hepatic deconjugation of a small fraction of bilirubin glucuronides is a physiological event. Deconjugation may also account, at least in part, for the presence of increased concentrations of unconjugated bilirubin in the plasma of patients with cholestasis.

Effect of endotoxin on tryptophan pyrrolase and delta-aminolaevulinate synthase: evidence for an endogenous regulatory haem fraction in rat liver.

Endotoxin was administered to rats at a dose shown previously to stimulate hepatic haem oxygenase activity and to block induction of delta-aminolaevulinate synthase, apparently by causing redistribution of haem from cytochrome P-450 to a regulatory haem pool in the liver. Within 5h of the administration of endotoxin (at a time when the effect of the compound on cytochrome P-450 is maximal) the relative saturation of tryptophan pyrrolase with intrinsic haem rose, from a basal value of 50% to 90%, indicating that 'free' haem had become available. Concurrently, the activity of delta-aminolaevulinate synthase was decreased to 25% of its basal value. Haem oxygenase reached peak activity 13h after endotoxin administration. These findings provide new evidence for the existence of an 'unassigned' hepatic haem fraction, which exchanges with cytochrome P-450 haem and regulates these three enzyme functions.

Parenchymal cells from adult rat liver in nonproliferating monolayer culture. I. Functional studies.

Parenchymal cells from adult rat liver have been established in primary monolayer culture. Donor animals are subjected to a partial hepatectomy and, 4 days later, cells are prepared by collagenase perfusion of the regenerated liver. The hepatic parenchymal cells, separated from nonparenchymal material and suspended in serum-free medium, are placed in plastic tissue culture dishes, where they form a monolayer within 24 h. The monolayer cells exhibit minimal mitotic activity and demonstrate several major metabolic functions characteristic of liver in vivo; these include albumin synthesis and secretion, gluconeogenesis from 3-carbon precursors, responsiveness to insulin and glucagon, glycogen synthesis, and activity of two microsomal enzymes. These functions are present in the monolayer cells for several days at activities similar to those observed in the liver in vivo. The findings indicate that hepatic parenchymal cells in this monolayer system are viable and behave in many respects like normal adult rat liver.

Liver sinusoidal cells. Identification of a subpopulation for erythrocyte catabolism.

Sequestration and degradation of red blood cells (RBC) are believed to occur in part in the liver, but the magnitude and cellular localization of this process remain uncertain. This problem was studied in rats by investigating isolated parenchymal and sinusoidal cell populations of the liver. After digesting the perfused liver with pronase, hepatic sinusoidal cells were isolated free of RBC and debris. Of the isolated cells, 90% were phagocytic, as judged by their uptake of colloidal (198)Au or of aggregated albumin-(131)I administered in vivo After administration of spherocytic (heat-treated) RBC, however, only about one quarter of the isolated cells were found to contain phagocytized RBC. This apparently distinct population of RBC-phagocytizing cells is designated as "erythrophagocytic (EP)" cells. The EP cell population was further characterized functionally by its specific phagocytosis of colloidal carbon and of (99m)technetium-sulfur colloid and histochemically by its peroxidase activity. The role of the EP population in the catabolism of RBC-hemoglobin was studied in isolated hepatic sinusoidal cells by assay of microsomal heme oxygenase (MHO), which is the inducible enzyme system that converts heme to bilirubin. The MHO activity of individual sinusoidal isolates was related directly to their content of EP cells Assay of the MHO activity of the whole spleen and of the total EP cell population of the liver suggested that these two tissues may be of comparable importance in their ability to degrade RBC-hemoglobin.

Soluble hepatic delta-aminolevulinic acid synthetase: end-product inhibition of the partially purified enzyme.

The present study confirms the existence of hepatic delta-aminolevulinic acid synthetase in the cytosol of the liver, suggests that this enzyme may be in transit to the mitochondria, and defines some of the characteristics of the partially purified enzyme. The substrate and cofactor requirements are similar to those of mitochondrial delta-aminolevulinic acid synthetase. Heme strongly inhibits the partially purified enzyme. A number of proteins that bind heme block this inhibition, which explains previous failures to demonstrate heme inhibition in crude systems. End-product inhibition of delta-aminolevulinic acid synthetase in the mitochondria may play an important role in the regulation of heme biosynthesis in eukaryotic cells.