Biliary excretion of polystyrene microspheres depends on the type of receptor-mediated uptake in rat liver

Hepatic uptake and biliary excretion of fluorescein isothiocyanate-labeled polystyrene microspheres with a particle size of 50 nm (MS-50) were studied in rats. Liver perfusion studies revealed that not only apo-E-mediated but also asialoglycoprotein receptor-mediated uptake is involved in the mechanism of the serum protein-dependent uptake of MS-50 in the liver. The uptake of MS-50 mediated by apo-E contributes more to the total uptake of MS-50 by the hepatocytes than that via asialoglycoprotein receptor in the presence of serum in the perfusate. Furthermore, it was found that MS-50 is substantially excreted into the bile by transcytosis. The extent of exocytosis of MS-50 taken up by the hepatocytes was much higher after MS-50 was endocytosed via asialoglycoprotein receptor than after taken up via the process mediated by apo-E. On the basis of these results, a possible regulation of the intracellular sorting of ligands, depending on the receptor-mediated uptake mechanism, was inferred.     

Hormone sensitive calcium uptake by liver microsomes

The effects of glucagon and insulin on hepatic microsomal calcium uptake were investigated. Microsomes isolated from perfused rat liver accumulated calcium in the presence of ATP and oxalate. Addition of glucagon to the perfusate significantly increased calcium uptake by microsomes subsequently isolated. In contrast, addition of insulin to the perfusate resulted in a decreased microsomal calcium uptake and inhibition of the glucagon effect. Because the effects of glucagon and insulin on hepatic microsomal calcium uptake are opposite, as are the metabolic effects of these hormones, it is likely that the observed differences are of physiological importance.     

Relation between glutathione redox changes and biliary excretion of taurocholate in perfused rat liver

The influence of the intracellular glutathione status on bile acid excretion was studied in the perfused rat liver. Perturbation of the thiol redox state by short term additions of diamide (100 microM) or hydrogen peroxide (250 microM) or t-butyl hydroperoxide (250 microM) led to a reversible inhibition of biliary taurocholate release without affecting hepatic uptake; inhibition amounted to 45% for diamide and 90% for the hydroperoxides. Concomitantly, the bile acid accumulated intracellularly. Bile flow increased from 1.3 to 2.0 microliters X min-1 X g liver-1 upon infusion of taurocholate (10 microM); the latter value was suppressed to 1.2 microliters X min-1 X g liver-1 by the addition of t-butyl hydroperoxide (250 microM). Similarly, the hepatic disposition of another bile constituent, bilirubin, was suppressed by 70% upon addition of hydrogen peroxide. While the addition of hydrogen peroxide inhibited also the endogenous release of bile acids almost completely, endogenous bile flow was much less affected, decreasing from 1.3 to 1.0 microliters X min-1 X g liver-1. Measurement of [14C]erythritol clearance showed bile/perfusate ratios of about unity both in the absence and presence of hydrogen peroxide, suggesting canalicular origin of the bile under both conditions. In livers from Se-deficient rats low in Se-GSH peroxidase (less than 5% of controls), hydrogen peroxide inhibited taurocholate transport substantially less, providing evidence for the involvement of glutathione in mediating the inhibition observed in normal livers. The percentage inhibition of taurocholate release and intracellular glutathione disulfide (GSSG) content were closely correlated. The addition of t-butyl hydroperoxide caused a several-fold increase of biliary GSSG release, whereas biliary GSH release was even decreased. The results establish a role of glutathione in canalicular taurocholate disposition.     

Pharmacokinetics and disposition characteristics of recombinant decorin after intravenous injection into mice

The pharmacokinetics and disposition characteristics of recombinant decorin after intravenous administration were investigated in mice. Following bolus injection of 111In-labeled decorin at doses of 0.02 and 0.1 mg/kg, radioactivity rapidly disappeared from the circulation and approximately 70% of the dose accumulated in liver within 10 min. 111In-labeled decorin was preferentially localized in hepatic nonparenchymal cells. At a higher dose of 1 mg/kg, clearance from the circulation and hepatic uptake of [111In]decorin were slower than at lower doses. Both the accumulation in other tissues and urinary excretion of [111In]decorin were 5% or less. Pharmacokinetic analysis demonstrated that hepatic uptake clearance was large and accounted almost completely for total body clearance; in addition the clearance values decreased as the dose increased, suggesting that the hepatic uptake of decorin is mediated by a specific mechanism which becomes saturated at higher doses. In competitive inhibition experiments, hepatic uptake of 111In-labeled decorin was partially inhibited (about 20-30%) by several sulfated glycans such as glycosaminoglycans and dextran sulfate and by mannosylated bovine serum albumin (BSA), mannan and mannose to a lesser extent (about 10%). On the other hand, polyinosinic acid, polycytidylic acid and succinylated BSA were ineffective, suggesting that the scavenger receptor for polyanions in the liver is not involved in the hepatic uptake of decorin. A basic protein, protamine, and a ligand of the apoE receptor, lactoferrin, also had no effect. Taken together, the present results have demonstrated that recombinant decorin is rapidly eliminated from the blood circulation through extensive uptake by the liver, primarily by the nonparenchymal cells, following systemic administration. The sugar structure and mannose residue in decorin have also been suggested to play an important role in the hepatic uptake of decorin. These findings provide useful information for the development of decorin as a therapeutic agent.     

Uptake, metabolism and release of carnitine and acylcarnitines in the perfused rat liver

The uptake and release of carnitine and isovalerylcarnitine have been studied in the perfused rat liver. Labelled carnitine accumulates in rat livers perfused with 50 or 500 microM [3H]carnitine. When alpha-ketoisocaproate (5 mM) is added to the perfusate after 30 min of perfusion, the net uptake of carnitine in the liver stops, and there is even a decrease in liver radioactivity. The decrease in liver carnitine can be attributed to an enhanced formation and efflux to the perfusate of short-chain acylcarnitines. Thin-layer chromatography of liver and perfusate extracts showed that efflux rates for branched-chain acylcarnitines (isovalerylcarnitine) formed are at least 2.5-fold the efflux rate for carnitine. Acetylcarnitine is released about twice as fast as carnitine from the liver. Perfusion with 50 microM [3H]isovalerylcarnitine showed that the influx rate of isovalerylcarnitine exceeds that of carnitine 1.5-fold. Since the efflux rate is still higher, a net loss of carnitine from the liver to the perfusate will result when branched-chain acylcarnitines are formed in the perfused liver. The addition of 500 microM unlabelled carnitine to the perfusate does not influence the release of labelled carnitine or acylcarnitines from the liver, showing that uptake and release are independent processes. Isovalerylcarnitine accumulates faster than carnitine does, also in the perfused rat heart. A mechanism for the development of secondary carnitine deficiencies associated with organic acidemia is proposed.     

Effects of ethynyloestradiol on the metabolism of [1-14C]-oleate by perfused livers and hepatocytes from female rats

Normal female rats were given 15mug of ethynyloestradiol/kg body wt. for 14 days and were killed on day 15 after starvation for 12-14h. The livers were isolated and were perfused with a medium containing washed bovine erythrocytes, bovine serum albumin, glucose and [1-(14)C]oleic acid; 414mumol of oleate were infused/h during a 3h experimental period. The output of bile and the flow of perfusate/g of liver were decreased in livers from animals pretreated with ethynyloestradiol, whereas the liver weight was increased slightly. The rates of uptake and of utilization of [1-(14)C]oleate were measured when the concentration of unesterified fatty acid in the perfusate plasma was constant. The uptake of unesterified fatty acid was unaffected by pretreatment of the animal with oestrogen; however, the rate of incorporation of [1-(14)C]oleate into hepatic and perfusate triacylglycerol was stimulated, whereas the rate of conversion into ketone bodies was impaired by treatment of the rat with ethynyloestradiol. Pretreatment of the rat with ethynyloestradiol increased the output of very-low-density lipoprotein triacylglycerol, cholesterol, phospholipid and protein. The production of (14)CO(2) and the incorporation of radioactivity into phospholipid, cholesteryl ester and diacylglycerol was unaffected by treatment with the steroid. The net output of glucose by livers from oestrogen-treated rats was impaired despite the apparent increased quantities of glycogen in the liver. The overall effect of pretreatment with oestrogen on hepatic metabolism of fatty acids is the channeling of [1-(14)C]oleate into synthesis and increased output of triacylglycerol as a moiety of the very-low-density lipoprotein, whereas ketogenesis is decreased. The effect of ethynyloestradiol on the liver is apparently independent of the nutritional state of the animal from which the liver was obtained. It is pertinent that hepatocytes prepared from livers of fed rats that had been treated with ethynyloestradiol produced fewer ketone bodies and secreted more triacylglycerol than did hepatocytes prepared from control animals. In these respects, the effects of the steroid were similar in livers from fed or starved (12-14h) rats. Oestrogens may possibly inhibit hepatic oxidation of fatty acid, making more fatty acid available for the synthesis of triacylglycerol, or may stimulate the biosynthesis of triacylglycerol, or may be active on both metabolic pathways.     

Effect of binding to hemoglobin and albumin on pyridoxal transport and metabolism

Scatchard plot analysis indicated that pyridoxal binds to hemoglobin more than twice as tightly as it does to serum albumin. Comparison of the formation constants for hemoglobin and albumin, using standard competitive binding equations, indicated that the distribution ratio for pyridoxal between erythrocytes and plasma should be 6.5:1. This distribution was approximately the same as that observed when pyridoxal was incubated with whole human blood, suggesting that these two proteins are the primary determinants of the pyridoxal distribution in whole blood. With in situ perfused rat liver the uptake of [3H] pyridoxal from the perfusate was reduced by the inclusion of erythrocytes in the perfusate. This was reflected in the decreased production of 4-pyridoxic acid by the perfused liver from 3.8% to 1.2% of the dose by the addition of erythrocytes to the perfusate. The major labeled metabolites found in the liver were pyridoxal phosphate, pyridoxamine phosphate, and 4-pyridoxic acid for both types of perfusion. In intact animals, reduction of the erythrocytes concentrations to hematocrits of 30-40% increased the recovery in the urine of 3H from administered [3H] pyridoxal from control values of 27-35% to 40-50% of the dose within 48 h. Half of the label in urinary metabolites was in 4-pyridoxic acid.     

Metabolism of high density lipoprotein lipids by the rat liver: evidence for participation of hepatic lipase in the uptake of cholesteryl ester.

In order to determine the role of hepatic lipase in the hepatic uptake and metabolism of high density lipoprotein (HDL) triglycerides, cholesteryl esters, and phospholipids, isolated rat livers were perfused with a reconstituted HDL (rHDL) radiolabeled with [3H]triolein and [14C]cholesteryl oleate or palmitoyl-[14C]linoleoyl phosphatidylcholine. A bolus of radiolabeled rHDL was injected into the portal vein and livers were perfused for 5 min using a nonrecirculating perfusion system. Recovery of rHDL triolein in the liver as intact triolein was used to determine the amount of unmetabolized rHDL remaining in the liver. After correcting for the amount of unmetabolized rHDL remaining in the liver, about 30% of the rHDL triolein was hydrolyzed of which 19% was recovered in the liver and 11% in the perfusate. Moreover, about 7% of the rHDL phosphatidylcholine was hydrolyzed to lysophosphatidylcholine, all of which was recovered in the perfusate. Although there was no hydrolysis of rHDL cholesteryl oleate, about 30% of the cholesteryl oleate was taken up by the liver. Preperfusion of the liver with heparin to deplete the liver of hepatic lipase resulted in about a 70% reduction in rHDL triolein hydrolysis and about a 75% reduction in rHDL cholesteryl oleate uptake. Although hepatic lipase hydrolyzes both triglycerides and phosphatidylcholines, elimination of the triolein from rHDL had no effect on the uptake of rHDL cholesteryl oleate, but replacement of the rHDL phosphatidylcholine with a nonhydrolyzable phosphatidylcholine diether resulted in an 87% reduction in cholesteryl oleate uptake. These results indicate that hepatic lipase is necessary for the hepatic uptake of both HDL triglycerides and cholesteryl esters and that the uptake of cholesteryl esters is not dependent on the hydrolysis of HDL triglycerides but is dependent on the hydrolysis of HDL phospholipids.     

Utilization of exogenous free fatty acids for the production of very low density lipoprotein triglyceride by livers of carbohydrate-fed rats

High carbohydrate diets enhance the hepatic output of very low density lipoprotein triglycerides. The fatty acids of these triglycerides could come from exogenous sources (i.e., diet or adipose tissue) or from de novo fatty acid synthesis in the liver. The role of exogenous free fatty acids was evaluated in rats fed Purina Chow or diets containing 10% fructose for up to 14 wk. In carbohydrate-fed rats, serum triglycerides were twice normal, and VLDL accounted for about 60% of the increases. Pre-beta-lipoprotein was increased and alpha- and beta-lipoprotein were decreased. Phospholipid and cholesterol levels were unchanged. Livers were perfused with glucose and free fatty acids. Perfusate free fatty acids rose from 180 to 1800 micro eq/liter as the infused acids increased from 0 to 992 micro eq/3 hr; simultaneously, net free fatty acid uptake rose from < 1 to 18 micro eq/g/hr and triglyceride output by the liver doubled. However, rates of secretion of triglyceride became constant, and triglyceride accumulated in liver at uptakes of free fatty acids > 13 micro eq/g/hr. More lauric and myristic acid appeared in the perfusate than was infused, suggesting the hepatic discharge of free fatty acids. Livers of fructose-fed rats secreted twice as much oleate-(14)C-labeled triglyceride as controls at all levels of free fatty acid uptake. The ratios of the specific activities of perfusate triglyceride to free oleate-(14)C were unaffected by diet and were about 0.6 and 1.0 at low and high triglyceride secretion rates, respectively. Thus, carbohydrate feeding did not result in altered uptakes of free fatty acids or preferential secretion of triglycerides containing endogenously synthesized fatty acid. Instead, the increased secretion of triglyceride was accomplished by enhanced formation of VLDL triglyceride from exogenous free fatty acids.     

Accumulation of Ca2+ induced by cytotoxic levels of menadione in the isolated, perfused rat liver

Previous studies have indicated that the presence of cytotoxic levels of menadione (2-methyl-1,4-naphthoquinone) causes rapid changes in intracellular thiol and Ca2+ homeostasis in isolated rat hepatocytes. The present investigation was undertaken to examine these effects in the intact liver. Rat livers were therefore perfused with Krebs-Henseleit buffer containing 1.3 mM Ca2+ using a single-pass mode, and the perfusate Ca2+ level was monitored with an on-line Ca2+-selective electrode. Infusion of menadione elicited an increased O2 uptake by the liver, followed by a dose-dependent decrease in the perfusate level of Ca2+. Hepatic accumulation of Ca2+ was accompanied by stimulation of cytosolic phosphorylase a activity. Cessation of menadione infusion resulted in gradual recovery of perfusate Ca2+ to base levels. Ca2+ uptake was not accompanied by decreases in reduced pyridine nucleotide or ATP levels in the liver as evidenced by measurements either during maximal Ca2+ uptake or after recovery. However, Ca2+ uptake was correlated with decreased glutathione and increased glutathione disulfide levels in the liver, both of which reversed during recovery from Ca2+ uptake. Moreover, depletion of hepatic glutathione by pretreatment with diethylmaleate resulted in increased Ca2+ uptake during menadione infusion. The amount of protein-bound mixed disulfides showed a particularly striking relationship to Ca2+ uptake, reaching a maximal level during Ca2+ uptake and reversing toward normal value during recovery from Ca2+ accumulation. The present findings suggest that menadione-induced Ca2+ uptake is due to plasma membrane dysfunction as a result of loss of protein thiol groups critical for maintaining the plasma membrane Ca2+ extrusion mechanism. Our model offers a particularly useful opportunity to study mechanisms underlying toxic disturbances in Ca2+ homeostasis in the intact liver, since Ca2+ fluxes can be monitored under conditions in which cellular control mechanisms are not obliterated by excessive toxicity.     

Clearance and killing of Candida albicans in the perfused mouse liver

Hepatic interactions of C. albicans with perfused mouse livers were characterized and compared in normal and glucan-treated mice. Normal livers, in the absence of serum, trapped greater than 90% and killed greater than 20% of the infused yeast. Phenylbutazone had no effect. Silica treatment abolished killing and decreased trapping suggesting that candidicidal activity of the liver is mediated by Kupffer cells. Immune serum, but not normal serum, enhanced trapping and killing in normal livers. Liver hypertrophy was evident in mice treated with glucan, but no enhanced candidicidal activity was observed in the absence of humoral factors. Specific immune serum and normal serum increased killing of C. albicans in glucan stimulated livers, suggesting a requirement for serum opsonin in facilitating glucan enhanced killing. Specific immune serum potentiated the greatest increase in killing. Glucan treatment in conjunction with immune serum increased killing to approximately 40%. D-mannose, but not D-glucose or D-mannitol impaired trapping of the yeast in livers of normal mice. Together, the data suggest that hepatic trapping of C. albicans involves phagocytic events as well as interactions of the yeast with surface receptors on sinusoidal cells and support the role for the liver in restricting hematogenous dissemination of C. albicans in the infected host.     

Characterization of lipoprotein produced by the perfused rhesus monkey liver

Isolated livers from rhesus monkeys (Macaca mulatta) were perfused in order to asses the nature of newly synthesized hepatic lipoprotein. Perfusate containing [3H]leucine was recirculated for 1.5 hr, followed by an additional 2.5-hr perfusion with fresh perfusate. Equilibrium density gradient ultracentrifugation clearly separated VLDL from LDL. The apoprotein composition of VLDL secreted by the liver was similar to that of serum VLDL. The perfusate LDL contained some poorly radiolabeled, apoB-rich material, which appeared to be contaminating serum LDL. There was also some material of an LDL-like density, which was rich in radiolabeled apoE. Rate zonal density gradient ultracentrifugation fractionated HDL. All perfusate HDL fractions had a decreased cholesteryl ester/unesterified cholesterol ratio, compared to serum HDL. Serum HDL distributed in one symmetric peak near the middle of the gradient, with coincident peaks of apoA-I and apoA-II. The least dense fractions of the perfusate gradient were rich in radiolabeled apoE. The middle of the perfusate gradient contained particles rich in radiolabeled apoA-I and apoA-II. The peak of apoA-I was offset from the apoA-II peak towards the denser end of the gradient. The dense end of the HDL gradient contained lipoprotein-free apoA-I, apoE, and small amounts of apoA-II, probably resulting from the relative instability of nascent lipoprotein compared to serum lipoprotein. Perfusate HDL apoA-I isoforms were more basic than serum apoA-I isoforms. Preliminary experiments, using noncentrifugal methods, suggest that some hepatic apoA-I is secreted in a lipoprotein-free form. In conclusion, the isolated rhesus monkey liver produces VLDL similar to serum VLDL, but produces LDL and HDL which differ in several important aspects from serum LDL and HDL.     

Metabolism of cysteinyl leukotrienes in non-recirculating rat liver perfusion. Hepatocyte heterogeneity in uptake and biliary excretion

1. The uptake, metabolism and biliary excretion of the cysteinyl leukotrienes LTC4, LTD4 and LTE4, were studied in a non-recirculating rat liver perfusion system at constant flow in both antegrade (from the portal to the caval vein) and retrograde (from the caval to the portal vein) perfusion directions. During a 5-min infusion of [3H]LTC4, [3H]LTD4 and [3H]LTE4 (10 nmol/l each) in antegrade perfusions single-pass extractions of radioactivity from the perfusate were 66%, 81% and 83%, respectively. Corresponding values for LTC4 and LTD4 in retrograde perfusions were 83% and 93%, respectively, indicating a more efficient uptake of cysteinyl leukotrienes in retrograde than in antegrade perfusions. The concentrations of unmetabolized leukotrienes in the effluent perfusate were 8-12% in antegrade and 2-4% in retrograde perfusions. [14C]Taurocholate extraction from the perfusate was inhibited by LTC4 by only 3%, suggesting that an opening of portal-venous/hepatic-venous shunts does not explain the effects of perfusion direction on hepatic LTC4 uptake. 2. Following infusion of [3H]LTC4 and [3H]LTD4, in the antegrade perfusion direction, about 80% and 87%, respectively, of the radiolabel taken up by the liver was excreted into bile. In retrograde perfusions, however, only 40% and 57%, respectively, was excreted into bile and the remainder was slowly redistributed into the perfusate, indicating that leukotrienes were taken up into a hepatic compartment with less effective biliary elimination or converted to metabolites escaping biliary excretion. The metabolite pattern found in bile was not affected by the direction of perfusion. Biliary products of LTC4 were polar metabolites (31-38%), LTD4 (27-30%), LTE4 (about 1%) and N-acetyl-LTE4 (3-4%) in addition to unmodified LTC4 (17-18%). 3. LTC4 was identified as a major metabolite of [3H]LTD4 in bile, amounting to about 20% of the total radioactivity excreted into bile. This is probably due to a gamma-glutamyltransferase-catalyzed glutamyl transfer from glutathione in the biliary compartment, as demonstrated in in vitro experiments. The presence of sinusoidal gamma-glutamyltransferase activity in perfused rat liver was shown in experiments on the hydrolysis of infused gamma-glutamyl-p-nitroanilide. 90% inhibition of this enzyme activity by AT-125 did not affect the metabolism of LTC4. 4. When [3H]LTE4 was infused in the antegrade perfusion direction, biliary metabolites comprised N-acetyl-LTE4 (24%) and polar components (60%).(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of conjugation reactions in enhancing biliary secretion of bile acids.

Shortening the five-carbon carboxylic acid side chain of cholic acid by one methylene group gave rise to a bile acid (norcholate) that was not a substrate for the bile acid-conjugating enzymes. The metabolism and biliary secretion of norcholate in intact liver was examined in the isolated perfused rat liver system. When rat livers were perfused with 14-20 microM solutions of norcholate for 10 min, norcholate was found in the unconjugated form in liver, venous effluent and bile. Neither tauronorcholate nor glyconorcholate was detectable by high-pressure liquid chromatography or fast-atom-bombardment mass spectrometry. The kinetics of hepatic uptake and biliary secretion of norcholate was compared with that for cholate, taurocholate and chemically synthesized tauronorcholate. The latter three bile acids were completely cleared from the perfusate and efficiently secreted into the bile. However, norcholate was incompletely extracted from the perfusate, and this was shown to be at least partially due to its relatively lower rate of hepatic uptake. Furthermore, the rate of norcholate secretion into bile was greatly reduced relative to the secretion of cholate or chemically synthesized tauronorcholate, even though the concentration of norcholate in the liver was comparatively high. These data demonstrate that the conjugation of bile acids greatly facilitates their secretion into bile.     

Lipoproteins in a nonrecirculating perfusate of rat liver

Rat livers were perfused in a nonrecirculating system for 30-40 min with Krebs-Ringer bicarbonate-0.1% glucose solution gassed with 95% O(2)-5% CO(2) at 37 degrees C at a flow rate of 3 ml/g/min. The livers appeared normal as judged by O(2) uptake, bile flow, transaminase release, and net protein output (2.5 mg/g/hr). The perfusate was concentrated by ultrafiltration using Amicon PM-10 or PM-30 membranes. The concentrated perfusate was subjected to sequential ultracentrifugation at solution densities of 1.006, 1.04, 1.06, and 1.21, and the top fractions were analyzed for protein and lipid. The net release of protein in the four density classes, suitably corrected, averaged 39, 10, 5, and 20 micro g/g/hr. The lipid composition of the perfusate lipoprotein fractions differed from that of serum mainly in the high percentage of free cholesterol, reflecting the lack of exposure to lecithin:cholesterol acyltransferase. When rat serum was fractionated in the same way, most of the lipoprotein in the d 1.006-1.06 range had a density greater than 1.04. It was concluded from these experiments that the liver secretes very low density lipoprotein (VLDL), high density lipoprotein (HDL), and a modified form of VLDL containing less lipid. Comparison of secretion rates and serum lipoprotein levels leads to the conclusion that the latter are largely determined by catabolic rates. When labeled amino acids were present, the perfusate HDL had a higher specific activity than VLDL. Addition of carrier whole serum did not alter recovery of labeled lipoproteins, but when these were isolated from Golgi membranes after a 40-min perfusion, more than twice as much label was recovered in HDL, suggesting the presence of precursors within the Golgi. The main advantages of the nonrecirculation perfusion technique are the avoidance of catabolic reactions, simplicity, and complete control over the composition of the perfusing medium.     

Hepatic lipoate uptake

Uptake of [35S]lipoate was studied in perfused rat liver and in isolated rat hepatocytes. During single-pass perfusion of [35S]lipoate about 30% of the radioactivity is retained in the liver. A substantial amount of 5,5'-dithiobis(2-nitrobenzoic acid)-reactive material appears in the effluent perfusate, while hepatic efflux of GSH is unchanged. The hepatic uptake of lipoate, the release of thiols, and also the biliary excretion of 35S-labeled compounds are suppressed by octanoate. In isolated hepatocytes the uptake of lipoate follows saturation kinetics showing a Km value of 38 microM and a Vmax of 180 pmol/mg X 10 s. The uptake is temperature-dependent; from the Arrhenius plot an activation energy of 14.8 kcal/mol at 20 microM lipoate is calculated. At high concentrations of lipoate (above 75 microM) a nonsaturable uptake component becomes predominant. Lipoate uptake is selectively inhibited by medium-chain fatty acids. Only slight inhibition is seen in the presence of long-chain fatty acids, and there is no inhibition with acetate or lactate. Substantial inhibition is also observed with acetylsalicylic acid, but not with taurocholate, bromosulfophthalein or biotin. Lipoate uptake can be inhibited by high concentrations of phloretin (200 microM) and is rather insensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (200 microM). The results indicate that hepatic uptake of lipoate at physiological concentrations is largely carrier-mediated.     

Effects of glucagon, insulin and cyclic-AMP on mitochondrial calcium uptake in the liver.

The effects of glucagon and insulin administration $\text{in vivo}$ on hepatic mitochondrial Ca²⁺ uptake were compared with the effects of these hormones when they were added directly to the perfused liver. Glucagon administration increased mitochondrial calcium uptake both $\text{in vivo}$ and in the perfused liver. In contrast, while injection of insulin into rats stimulated, addition of insulin to the perfusate, inhibited Ca²⁺ uptake. Cyclic AMP, when added to the perfusate, also increased the uptake of Ca²⁺ by mitochondria, subsequently isolated. The possible implications of the results are discussed.     

The effect of chlordiazepoxide hydrochloride on the isolated perfused rat liver.

The effects of chlordiazepoxide-hydrochloride (CDZ) on the isolated perfused rat liver were examined. CDZ administration decreased bile flow, biliary excretion of sulfobromophthalein (BSP) and hepatic uptake of BSP. The addition of CDZ to the perfusate of livers obtained from phenobarbital (Pb) pretreated rats led to 50% greater reductions in bile flow, concentration of BSP in bile and hepatic uptake of BSP. The adverse effects of CDZ on BSP excretion per g liver, however, did not appear to be enhanced by Pb pretreatment. The complex nature of the interrelationship of the effects of Pb and of CDZ on the control liver prevented differentiation of the role of CDZ from that of a metabolite on the adverse effect on liver function.     

Uptake of asialo-glycophorin by the perfused rat liver and isolated hepatocytes.

Perfused rat livers took up asialo-glycophorin, a glycoprotein derived from human erythrocyte membranes, with a t1/2 for clearance of 7 min. As a comparison, asialo-orosomucoid was taken up by this system with a t1/2 of 3.5 min. Both proiteins were digested and their 125I labels were released to the perfusate as free 125I-. EGTA completely inhibited uptake of these glycoproteins, but not uptake of denatured bovine serum albumin. Addition of Ca2+ reversed the inhibition nearly completely. Isolated hepatocytes had an uptake rate of approximately 3 ng/min per 10(6) cells for the asialo forms of glycophorin, orosomucoid and fetuin. Cellular uptake of each of these asialoglycoproteins could be inhibited by one of the other proteins. Asialo-fetuin caused a 95% inhibition of the uptake rate of asialo-orosomucoid by the perfused liver. This fetal calf glycoprotein had a similar inhibitory effect on asialo-glycophorin, but only after an initial 40% of the asialo-glycophorin had been taken up by the liver at an almost normal rate during the first 30 min of perfsuion. The possibility of an alternative hepatic removal system for asialo-glycophorin is suggested.     

The uptake of the apoprotein and cholesteryl ester of high-density lipoproteins by the perfused rat liver

The uptake of the 125I-labeled apolipoprotein and 3H-labeled cholesteryl ester components of rat apolipoprotein E-deficient HDL by the perfused liver was studied. The uptake of the cholesteryl ester moiety was 4-fold higher than that of apolipoprotein. The concentration-dependent uptake of labeled protein was saturable and competed for by an excess of unlabeled HDL. The uptake of cholesteryl ester was not saturable over the concentration range studied. In the presence of a 50-fold excess of unlabeled HDL, the uptake of both radiolabeled components was decreased by over 75%, indicating that three-quarters of the hepatic uptake of HDL is by a receptor-mediated process. After 15 min of perfusion, 37% of the apolipoprotein radioactivity that was initially bound at 5 min was released into the perfusate as a more dense particle. After 5, 15, 30 and 60 min of perfusion the subcellular distribution of the apolipoprotein and cholesteryl ester components was analyzed by Percoll density gradient centrifugation. Over the 60 min period, there appeared to be transfer of radioactivity from the plasma membrane fraction to the lysosomal fraction. However, the internalization and degradation of cholesteryl ester was more rapid than that of the apolipoprotein. Our findings indicate that there is preferential uptake of HDL cholesteryl ester relative to protein by the liver and that the internalization of these components may occur independently.