Hepatocyte horseradish peroxidase uptake is saturable and inhibited by mannose-terminal glycoproteins.

In the liver, horseradish peroxidase (HRP) is thought to be taken up via mannose receptor-mediated endocytosis by non-parenchymal cells (NPC) and via fluid-phase endocytosis by hepatocytes. When we attempted to inhibit NPC uptake of HRP with mannan in the whole perfused rat liver, > 80% of HRP uptake was eliminated. Liver cell fractionation revealed that mannan not only inhibited HRP uptake by NPC (91%) but also by hepatocytes (81%). In isolated hepatocytes, HRP uptake was linear over 60 min and saturable in the range of 0 to 200 mg/l (Vmax = 4.3 ng.mg protein-1.min-1; Km = 8.3 mg/l). Mannan inhibited uptake competitively (Ki = 2.0-2.5 mg/l). At high concentrations of HRP, a nonsaturable component of HRP uptake became evident (k = 2.8 pg.mg protein-1.min-1.mg HRP-1.l-1). Hepatocyte uptake of HRP was inhibited by other glycoproteins and glycopeptides with mannose-terminal groups, as well as by mannan, but not by asialofetuin (ASF) or bovine serum albumin. Hepatocyte uptake of 125I-labeled ASF, which is taken up via the asialoglycoprotein receptor, was saturable and not inhibited by mannan. HRP binding to hepatocytes, determined at 4 degrees C, was also inhibited by mannan. Quantification of contamination of the parenchymal cell fraction by NPC by cell counting and by pronase digestibility suggested our results could not be explained by contamination of hepatocytes by NPC. At concentrations used for most morphological studies (1,000-10,000 mg/l), fluid-phase endocytosis accounts for much of HRP uptake. However, at low concentrations, a saturable low-capacity mechanism is responsible for most HRP uptake by the hepatocyte.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasopressin and A23187 stimulate phosphorylation of myosin light chain-1 in isolated rat hepatocytes.

Vasopressin (VP) and other hormones that elevate intracellular Ca2+ concentration also increase tight junctional permeability in the liver cell. Data derived from study of other tissues suggest that microfilaments are instrumental in regulating tight junctional permeability. By analogy to microfilament contraction in smooth muscles, it is likely that the transduction pathway for these hormones involves Ca(2+)-stimulated complex formation between calmodulin and myosin light chain (MLC) kinase with activation of this latter enzyme. MLC kinase then phosphorylates MLC, which, in the presence of actin, exerts adenosinetriphosphatase activity and produces microfilament contraction. This transduction pathway in the hepatocyte remains speculative. To demonstrate the likelihood of this pathway, we stimulated isolated hepatocytes with 10(-8) M VP and assayed MLC phosphorylation. We did this by immunoprecipitation of myosin from homogenates of liver cells prelabeled with [32P]-orthophosphate. We used a polyclonal antibody raised in rabbits against rat liver cell myosin. Our data demonstrate that VP is a potent stimulator of MLC phosphorylation. Maximal rises in intracellular Ca2+ and maximal MLC phosphorylation occur within 40 s of VP administration. The dose-response curve for MLC phosphorylation by VP is similar to that for tight junctional permeabilization in perfused liver with maximal effect at about 10(-8) M VP. The calcium ionophore A23187 also stimulated MLC phosphorylation. MLC phosphorylation, therefore, is at least coincident with, and probably responsible for, tight junctional permeabilization caused by elevation of intracellular Ca2+ in the liver cell.

Function of rat hepatocyte tight junctions: studies with bile acid infusions.

To determine the effects of alteration of biliary paracellular permeability on bile flow and composition, we measured the biliary outputs of compounds highly concentrated in bile, all infused at a constant rate in the isolated rat liver perfused with Krebs-Henseleit buffer in a one-pass fashion. Paracellular permeability was increased by infusing 10(-8) M vasopressin (VP). The cholephilic compounds were three cations of various molecular weights, tributylmethylammonium (TBuMA), N-acetylprocainamide ethobromide (APAEB), and propidium iodide, and two anions, taurocholate (TC), a micelle-forming bile acid, and taurodehydrocholate (TDHC), an nonmicelle former. When TC was infused and paracellular permeability increased with VP, neither bile flow nor TC output changed, whereas outputs of cations fell. When TDHC was infused, TDHC output fell, as did outputs of all cations. The decrements in cation outputs exceeded that of TDHC and were inversely related to the molecular weight of the cation. To document that these changes were not related to reduced uptake of these compounds, we tested the uptakes of TBuMA, APAEB, and TDHC into isolated hepatocytes. In no case did 10(-8) M VP significantly reduce uptake. The data demonstrate that micelle-forming bile acids, with their high effective molecular weights, do not efflux from the biliary tree when permeability is increased with VP, whereas nonmicelle-forming bile acids do. Cations efflux more readily than anions, and within this group efflux rate is inversely related to molecular weight. The data confirm the size and charge selectivity of biliary tree permeability.(ABSTRACT TRUNCATED AT 250 WORDS)