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Characterization of the binding of human low-density lipoprotein to primary monolayer cultures of rat hepatocytes.

ABSTRACT: The binding of human low-density lipoprotein labelled with 125I to rat hepatocytes in monolayer culture was measured at 4 degrees C. Evidence for two different specific binding sites was obtained. Binding to Site 1 was characterized by: being displaced by dextran sulphate or heparin; being dependent on Ca2+; having a Kd value of about 15 micrograms of protein/ml; not being significantly displaced by a 20-fold excess unlabelled low-density lipoprotein that had been reductively methylated; being displaced by approx. 40% by a 20-fold protein excess of unlabelled human high-density lipoprotein, HDL3, and increasing with time in culture when newborn-calf serum was present in the medium. The binding to Site 2 had the following properties: it was not displaced by sulphated polysaccharides; it was only partially Ca2+-dependent, and the presence of EDTA increased the Kd value; the apparent Kd value in the presence of Ca2+ was approx. 30 micrograms of protein/ml, which was significantly higher than for Site 1; it was displaced by approx. 30% with a 20-fold excess of low-density lipoprotein that had been methylated; it was displaced by unlabelled HDL3 to a similar extent to Site 1; it did not increase significantly with time in culture. The characteristics of binding to Sites 1 and 2 are discussed in relation to the receptors for low-density lipoproteins that have previously been described in various cell types. It is proposed that the experimental system described in this paper is suitable for studying the regulation of the binding of low-density lipoproteins to hepatocytes.

SUBMITTER: Salter AM

PROVIDER: S-EPMC1147449 | biostudies-other | 1986 Dec

REPOSITORIES: biostudies-other

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Project description:In this study the effects of bile acids and other organic anions on the secretion of very-low-density lipoproteins (VLDLs) were evaluated in rat hepatocytes in primary culture. Incubation of cells with portal blood concentrations (10-200 microM) of bile acids resulted in dose-dependent suppression of secretion of VLDL-associated [3H]triglyceride (TG) formed from [3H]glycerol, and also of TG mass. The degree of the inhibition was highly correlated with intracellular bile acid concentration. Prolonged incubation with 100 microM extracellular taurocholic acid (TC) decreased the secretion of [3H]TG and TG mass to 35% and 50% of the controls respectively. Cellular content of mass and of [3H]TG during prolonged incubation with TC were about 20% and 60% higher than the controls respectively. The inhibitory effect remained for at least 24 h in the presence of TC without altering VLDL-lipid and VLDL-apolipoprotein compositions or the size distribution of the particles. Secretion of apoB-100 and of apoB-48 was inhibited to a similar extent. Cells largely lost their capacity to accumulate bile acids intracellularly after 48 h in culture. In these cells TC was unable to exert its suppressive effects. Taurine and glycine conjugates of all common bile acids were capable of suppressing [3H]TG secretion. Trihydroxylated (cholic acid) and various dihydroxylated (deoxycholic, chenodeoxycholic and ursodeoxycholic acids) bile acids had similar capacities in this respect, suggesting that their common sterol-3 alpha-OH structure is required for the suppressive effect. Neither non-bile acid organic anions, e.g. bilirubin ditaurate and dibromosulphthalein, nor dianionic bile acid metabolites, e.g. sulphated taurolithocholic acid and lithocholate-3-O-glucuronide, showed any effect on [3H]TG secretion. These results indicate that bile acids might play a physiological role in regulating VLDL production by the liver, especially in the postprandial state when their enterohepatic circulation is stimulated.

Project description:Ursodeoxycholic acid (UDCA), in contrast to both chenodeoxycholic acid (CDCA), its 7 alpha-epimer, and lithocholic acid, enhanced receptor-dependent low-density lipoprotein (LDL) uptake and degradation in isolated hamster hepatocytes. The increase in cell-associated LDL was time- and concentration-dependent, with a maximum effect observed at approx. 60 min with 1 mM-UDCA. This increase was not associated with a detergent effect of UDCA, as no significant modifications were observed either in the cellular release of lactate dehydrogenase or in Trypan Blue exclusion. The effect of UDCA was not due to a modification of the LDL particle, but rather was receptor-related. UDCA (1 mM) maximally increased the number of 125I-LDL-binding sites (Bmax.) by 35%, from 176 to 240 ng/mg of protein, without a significant modification of the binding affinity. Furthermore, following proteolytic degradation of the LDL receptor with Pronase, specific LDL binding decreased to the level of non-specific binding, and the effect of UDCA was abolished. Conversely, the trihydroxy 7 beta-hydroxy bile acid ursocholic acid and its 7 alpha-epimer, cholic acid, induced a significant decrease in LDL binding by approx. 15%. The C23 analogue of UDCA (nor-UDCA) and CDCA did not affect LDL binding. On the other hand, UDCA conjugated with either glycine (GUDCA) or taurine (TUDCA), increased LDL binding to the same extent as did the free bile acid. The half maximum time (t1/2) to reach the full effect was 1-2 min for UDCA and TUDCA, while GUDCA had a much slower t1/2 of 8.3 min. Ketoconazole (50 microM), an antifungal agent, increased LDL binding, but this effect was not additive when tested in the presence of 0.7 mM-UDCA. The results of the studies indicate that, in isolated hamster hepatocytes, the UDCA-induced increase in receptor-dependent LDL binding and uptake represents a direct effect of this bile acid. The action of the bile acid is closely related to its specific structural conformation, since UDCA and its conjugates are the only bile acids shown to express this ability thus far. However, certain agents other than bile acids, such as ketoconazole, have a similar effect. Finally, the studies suggest that the recruitment of LDL receptors from a latent pool in the hepatocellular membrane may be the mechanism by which UDCA exerts its direct effect.

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Characterization of the binding of human low-density lipoprotein to primary monolayer cultures of rat hepatocytes.

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