Triton WR-1339, a lysosomotropic compound, is excreted into bile and alters the biliary excretion of lysosomal enzymes and lipids

In these experiments, we tested two hypothesis: first, that Triton WR-1339, a nonionic detergent which is sequestered in hepatocyte lysosomes, undergoes biliary excretion; and second, that Triton WR-1339, which also alters serum lipid levels and modifies hepatic catabolism of lipoproteins, affects the biliary output of proteins and lipids. When 3H-Triton WR-1339 was administered to rats, biochemical and morphologic studies showed that hepatocyte lysosomes sequestered Triton WR-1339: (i) the subcellular distribution of 3H was identical to that of lysosomal enzymes after liver fractionation by differential or isopycnic centrifugation, and (ii) lysosomes appeared engorged with Triton WR-1339 on electron microscopy. 3H was also excreted into bile in parallel to three lysosomal enzymes. Triton WR-1339 administration caused a coordinate increase in the biliary excretion of three lysosomal enzymes and also increased the biliary output of total protein, bile acids, and phospholipid. Triton WR-1339 administration did not affect bile flow or the biliary outputs of cholesterol, plasma membrane, and cytosolic enzymes, but did decrease biliary cholesterol saturation by 50%. These results demonstrate that an exogenous compound which is sequestered in hepatocyte lysosomes may be excreted directly into bile in parallel with endogenous lysosomal constituents. The data also show that such a lysosomotropic agent may also selectively modify the biliary excretion of proteins and lipids. The findings are consistent with the existence of a lysosome-to-bile hepatic excretory pathway and suggest that hepatocyte lysosomes may be important in modulating biliary protein and lipid secretion.     

Effects of acute pancreatitis on hepatic secretion of lysosomal enzymes into bile and hepatic lysosomal fragility: protective effects of a new synthetic protease inhibitor, ONO 3307.

To evaluate the effects of acute pancreatitis on hepatic function and hepatic cellular and subcellular organellar fragility, we studied 1) the hepatic secretion of lysosomal enzymes (beta-glucuronidase, beta-galactosidase, and N-acetyl-beta-glucosaminidase) into bile in the isolated perfused rat liver model; 2) the aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), and lysosomal enzyme levels in the effluent in an isolated liver model; 3) hepatic lysosomal fragility in an in vitro incubation study; and 4) protective effects of a new low molecular weight synthetic protease inhibitor, ONO 3307, against hepatic injury in doses of 2 and 5 mg/kg.h in acute pancreatitis induced by a supramaximal dose of cerulein in rats. Decreased hepatic secretion of lysosomal enzymes into bile and accelerated hepatic lysosomal fragility were observed in acute pancreatitis induced by cerulein. ONO 3307 showed a significant protective effect against this hepatic injury in acute pancreatitis, the dose of 5 mg/kg.h showing a more potent effect than the dose of 2 mg/kg.h. These results suggest that the impaired hepatic function, including depressed hepatic secretion of lysosomal enzymes, seems to be closely related to accelerated hepatic fragility and that some unknown protease, which is present in pancreatitis and is susceptible to inhibition by ONO 3307, plays a crucial pathologic role in the development of this liver injury during acute pancreatitis.     

Rat liver lysosomal enzymes: Effects of animal age and phenobarbital

The activities of two rat liver lysosomal enzymes, acid phosphatase and β-glucuronidase, undergo changes as a function of animal age. Acid phosphatase activity increases during maturation, but subsequently declines during senescence. β-glucuronidase remains unchanged during maturation, but increases during senescence such that old rats exhibit significantly higher activity than young animals. Acid phosphatase is unaffected by chronic phenobarbital administration, whereas β-glucuronidase is markedly stimulated in young and mature rats and to a lesser extent in senescent animals. The induced activities return to near control levels within two days after withdrawal of the drug, regardless of animal age. These data suggest (1) that these two hepatic lysosomal enzymes undergo different changes, and (2) that the inducibility of β-glucuronidase is diminished as a function of animal age.     

Lymphokine-induced production and release of lysosomal enzymes by macrophages.

MACROPHAGES ARE ASSOCIATED WITH MOST CHRONIC INFLAMMATORY LESIONS, AND THESE CELLS CONTAIN ENZYMES THAT ARE ABLE TO DESTROY CONNECTIVE TISSUE CONSTITUENTS. Normal lymphoid cells responding to a mitogen, phytohemagglutinin-P, release factor(s) that cause a marked increase in the size and enzyme content for mononuclear phagocytes maintained in culture. The stimulated macrophages, which by several criteria remain otherwise viable and healthy, selectively release large quantities of hydrolytic enzymes to the culture medium.     

Role of oxygen free radicals and pH on the release of cardiac lysosomal enzymes

We investigated the effect of exogenous oxygen free radicals and various pH on the release of lysosomal hydrolases from dog myocardial lysosomes. A lysosomal enriched fraction from the homogenate of dog heart was prepared, using differential centrifugation technique. Exogenous oxygen free radicals were generated using xanthine-xanthine oxidase system. The release of lysosomal hydrolases was measured from the lysosomal enriched fraction. There was about 3-fold increase in the release of cathepsin D and beta-N-acetylglucosaminidase activities in the preparations treated with xanthine-xanthine oxidase as compared to those without such treatment. The presence of superoxide dismutase, an oxygen free radical scavenger, prevented the release of cathepsin D and beta-N-acetylglucosaminidase from the lysosomes. Sonication and lubrol treatments, which are known to cause membrane disruption, also induced the release of these enzymes from lysosomal enriched fraction. However, this release was not prevented by superoxide dismutase. The changes in pH (4.5, 5.5, 6.0, 6.5, 7.4, 8.0) alone did not cause any increase in the enzyme release. The presence of oxygen free radicals at each pH resulted in a similar increase in the release of cathepsin D and beta-N-acetylglucosaminidase. These studies suggest that oxygen free radicals and not the alterations in pH are primarily responsible for the release of lysosomal hydrolases. Oxygen free radicals, in addition to their direct myocardial damaging effect, may also be responsible for the cardiac damage through the release of lysosomal enzymes.     

Mechanisms of secretion of proteins into bile: studies in the perfused rat liver

Employing the in situ perfused rat liver, we examined the origins and mechanisms of transport of proteins into bile. First, utilizing polyacrylamide gels, we noted that many biliary proteins co-migrated with dominant serum proteins. Upon liver perfusion with serum-free medium, most proteins disappeared from the biliary profile; one major biliary protein that was not present in serum, identified as secretory component, remained. Kinetic analysis of the disappearance half-lives of the biliary proteins suggested that some serum proteins enter bile by a slow (20 to 30 min; transcellular) route, while others utilize both slow and rapid (5 min; paracellular) routes. In biosynthetic labeling experiments, secretion of newly synthesized proteins into bile was delayed about 20 min when compared with secretion of proteins into the perfusion medium and comprised less than 1% of the total secreted proteins. When a new liver was inserted into the perfusion medium containing newly synthesized secreted proteins, only two proteins, hemopexin and an unidentified protein, were transported into the bile from the perfusion medium; other biliary proteins were presumed to come directly from the hepatocyte. This latter group included some proteins that were secreted into the perfusion medium as well as into bile, and others, e.g., secretory component, that were secreted only into bile. Based on our results we have defined six pathways for entry of proteins into bile.     

Effect of anti-rheumatic drugs on the release of lysosomal enzymes from human leukocytes

The release of lysosomal enzymes from human polymorphonuclear neutrophils (PMN) into the extracellular medium was selectively induced by the phagocytosis of zymosan particles. The "in vitro" effect on the release of lysosomal enzymes was determined for eight different substances used in the therapy of rheumatoid arthritis. Prednisolone and the three non-steroidal anti-rheumatic agents indomethacin, diclofenac and benoxaprofen only inhibited the release of various lysosomal enzymes at the elevated concentrations of 20-200 micrograms/ml. Of the four drugs examined which are used in basis therapy, Chloroquine and Levamisole exhibited the most clear-cut inhibition of the enzymes (at 20-200 micrograms/ml). The effect of gold salts was uncertain and D-penicillamine only inhibited the release of myeloperoxidase. The release of lysosomal enzymes from neutrophils is then largely resistant to anti-rheumatic agents--at least in the range of therapeutic concentrations. On the basis of these experimental results it seems questionable whether the relatively weak inhibition of lysosomal enzymes observed "in vitro" can contribute to the anti-rheumatic efficacy in patients of the drugs examined.     

Hepatic metabolism of colloidal gold-low-density lipoprotein complexes in the rat: evidence for bulk excretion of lysosomal contents into bile

Rats were treated with 17 alpha-ethinyl estradiol to induce high levels of low-density lipoprotein receptors in hepatocytes. When these rats were given intravenous injections of low-density lipoprotein-colloidal gold complexes, most of the gold (labeled with 195Au) appeared to be taken up by Kupffer cells, as were complexes of colloidal gold with albumin or polyvinylpyrrolidone. However, when these rats were also administered gadolinium chloride, which blocks Kupffer cell activity, most of the low-density lipoprotein-gold (but not gold complexed with albumin or polyvinylpyrrolidone) was taken up into hepatocytes by receptor-mediated endocytosis and concentrated in peribiliary lysosomes, as determined by electron microscopy. Colloidal gold taken up as a complex with low-density lipoprotein was excreted into the feces via the common bile duct at a maximal rate of about 5% daily, 4 to 12 days after injection. Thereafter, the rate of gold excretion fell off until reaching a plateau after 3 weeks. At this late time, most of the colloidal gold was shown by electron microscopy to be in Kupffer cells, whereas earlier (6 days after injection) it was contained mainly in older hepatocytic lysosomes, identified by lipofuscin granules. It is concluded that, in rats, hepatocytic lysosomes empty most of their contents into bile every week or two, apparently by exocytosis.     

Effect of cysteamine on the lysosomal enzymes of the hyperprolactinaemic rat pituitary

The effect of cysteamine on the activity of lysosomal enzymes and the prolactin content of isolated hyperprolactinaemic cells has been investigated. In broken cell preparations, cysteamine markedly stimulated acid prolactin protease activity. In intact cells, however, cysteamine inhibited acid prolactin protease activity and beta-galactosidase. Moreover, the activities of alpha-mannosidase, acid phosphatase, beta-glucuronidase, total arylsulphatase and hexosaminidase were not changed by the addition of cysteamine. Cysteamine significantly depleted the cells of prolactin, and this action was not compromized by the inclusion of either leupeptin, chloroquine or NH4Cl in the incubation media. Taken together, these results indicate that cysteamine does not promote degradation of prolactin and hence depletion of prolactin from the pituitary through a mechanism involving lysosomal enzyme degradation.     

Serum activities of lysosomal enzymes in patients with liver cell carcinoma

Serum activities of two lysosomal enzymes, -glucuronidase and acid phosphatase, were estimated in 66 patients with liver cell carcinoma, 10 with secondary liver cancer, 14 with cirrhosis of the liver, and 9 normal controls. A substantial increase in the enzyme activities was found in patients with liver cell carcinoma but not in those with secondary liver cancer. The degree of the enzyme elevations paralleled the stage of hepatoma. Although the serum activities of both enzymes were also elevated in patients with liver cirrhosis, the elevations were significantly higher in hepatoma than in liver cirrhosis. Possible mechanisms for the elevation of serum lysosomal enzyme activities in hepatoma are discussed, but further studies are necessary to elucidate the biological and clinicopathological significance of estimating serum lysosomal acid hydrolases in patients with primary liver cell carcinoma.     

Intracellular distribution and effect of the antimalarial drug mefloquine on lysosomes of rat liver

Abstract: Mefloquine was administered in a single dose (1–30 mg/100 g) to rats in order to study its subcellular distribution and effects on rat liver lysosomal structure and function. Subcellular fractionation showed a significant enrichment of mefloquine in lysosomes. Even repeated administration of mefloquine did not affect the levels of cytochrome-P-450 or its reductase, indicating, although not proving, that it is not metabolized by this mono-oxygenase system. Mefloquine caused an expansion of the lysosomal apparatus, earliest seen by 24 h and lasting for some 7 days. Initially, cytoplasmic constituents were seen inside the lysosomes. Later, the lysosomes harboured myelin-like figures (multilamellar bodies) disappearing after 7–10 days. The proteolytic and lipolytic capacity was assessed in isolated lysosomes. Mefloquine caused increased protein degradation but decreased breakdown of lipids. Concomitantly, all five major phospholipids (phosphatidyl-choline, -ethanolamine, -inositol, -serine and sphingomyelin) increased in the lysosomes. It is concluded that: (1) mefloquine is a lysosomotropic drug that accumulates in lysosomes; (2) mefloquine impairs lipid degradation with ensuing accumulation of lipids in lysosomes; and (3) lysosomal trapping explains the high volume distribution of mefloquine.     

The effect of chloroquine on lysosomal prolactin receptors in rat liver

PRL receptors have been previously identified in purified rat liver plasma membrane and Golgi vesicle preparations. In this study, we report on PRL receptors located in highly purified lysosome preparations. These lysosomal PRL receptors were characterized using Scatchard analysis and compared to other intracellular and cell surface receptors. We have identified two classes of lysosomes. Lighter lysosome-like vesicles, which are greatly enriched in acid phosphatase activity (the marker enzyme of lysosomes), contain a great deal of binding activity. This PRL binding was only slightly increased by pretreatment of animals with the lysosomotropic agent chloroquine. In contrast, mature lysosomes showed very little binding activity in control animals, but chloroquine treatment increased binding 7- to 8-fold in these mature lysosomes. We suggest that the lysosome-like structures are immature lysosomes (namely prelysosomes) toward which the hormone-receptor complex is internalized: they appear to bear little proteolytic activity. These structures could play a role in PRL receptor recycling. Lysosomal PRL receptors showed curvilinear Scatchard plots, in contrast to plasma membrane and Golgi counterparts, which were linear over the same range of hormone concentrations. The high affinity site in lysosomes had a Kd comparable to the cell surface and Golgi receptors. The number of binding sites per mg protein in prelysosomes and lysosomes was 3 times greater than that in the homogenate, but Golgi preparations were 3 times as rich as lysosomes. The great number of PRL receptors in prelysosomes could be attributed, in large part, to the low affinity sites. The internalization of PRL into rat liver was examined after in vivo injection of [125I]iodoovine PRL. The labeled hormone was found initially in the plasma membrane fraction, after which it localized preferentially in the Golgi fraction, with maximum incorporation 15 min postinjection. Substantial radioactivity was observed in both classes of lysosomes (L-1 and L-2). In contrast to the Golgi fraction, maximum incorporation of [125I]iodoovine PRL in lysosomes occurred at 30 min. This suggests either that during internalization, PRL first reaches Golgi elements and is then transferred to the lysosomal compartment, or that there are two independent pathways of internalization, one rapid toward the Golgi complex (may be a path of receptor recycling) and the other toward lysosomes (probably leading to receptor degradation).     

Enhanced biliary excretion of canalicular membrane enzymes in estrogen-induced and obstructive cholestasis, and effects of different bile acids in the isolated perfused rat liver

Backgrounds/Aims: Canalicular membrane enzymes are normally released into bile by partially known processes. This study was undertaken to investigate whether hepatocellular cholestatis induced in rats by ethynylestradiol or obstructive cholestasis produced by complete biliary obstruction for 24 h is associated with an increased release of alkaline phosphatase and gamma-glutamyl transpeptidase into bile, and to clarify how this process is affected by different bile acids.Methods: The studies were performed in the isolated perfused liver during infusion of sodium taurocholate, taurochenodeoxycholate and tauroursodeoxycholate at increasing rates.Results: Maximum sodium taurocholate, taurochenodeoxycholate and tauroursodeoxycholate secretory rates were decreased in both cholestatic groups (complete biliary obstruction>ethynylestradiol) compared with controls. Maximum biliary outputs of alkaline phosphatase and gamma-glutamyl transpeptidase were significantly increased in the ethynylestradiol group during infusion of sodium taurocholate and taurochenodeoxycholate, but not of tauroursodeoxycholate, and were increased in the complete biliary obstruction group during the infusion of sodium taurocholate and tauroursodeoxycholate but not of taurochenodeoxycholate. The biliary outputs of alkaline phosphatase and gamma-glutamyl transpeptidase showed a significant and direct linear relationship with sodium taurocholate and taurochenodeoxycholate secretory rates in both cholestatic groups. However, only in the complete biliary obstruction group did alkaline phosphatase and gamma-glutamyl transpeptidase excretion show a significant correlation with tauroursodeoxycholate secretory rates. The slope of the line, which indicated the mU of enzyme activity secreted per nmol of sodium taurocholate or taurochenodeoxycholate, was greater for gamma-glutamyl transpeptidase and alkaline phosphatase in both cholestatic groups (ethynylestradiol>complete biliary obstruction) than in the control group. Alkaline phosphatase activity in purified isolated canalicular and sinusoidal membranes was significantly increased in both cholestatic groups (complete biliary obstruction>ethynylestradiol), while gamma-glutamyl transpeptidase activity was unchanged compared with controls.Conclusion: The marked increase in sodium taurocholate and taurochenodeoxycholate-mediated release of alkaline phosphatase and gamma-glutamyl transpeptidase into bile in cholestatic rats suggests an increased lability of these intrinsic membrane proteins to the detergent effects of secreted bile acids. It remains to be elucidated whether this phenomenon, which was particularly intense in ethynylestradiol induced cholestasis, is important in the pathogenesis and perpetuation of bile secretory failure. In contrast, tauroursodeoxycholate administration did not result in enhanced biliary excretion of these membrane enzymes, in either the control group or the ethynylestradiol group, supporting the concept that this bile salt lacks the membrane toxicity of common bile acids.     

On the modulating effects of ovaries on neonatal androgen programming of rat liver enzymes.

The metabolism of 4-[4-14C]androstene-3,17-dione in rat liver microsomal and cytosol fractions was investigated in adult female rats treated with 1.45 mumole of testosterone propionate at birth. The effects of ovariectomy at 14 and 43 days of age on neonatal testosterone imprinting of enzyme levels were studied. Animals spayed 14 days after birth showed a typical masculinized hepatic enzyme activity pattern with a decreased level of the 5alpha-reductase activity and increased level of 5beta-reductase, 16alpha-hydroxylase and 17alpha- and 3beta-hydroxysteroid reductase levels. The pattern was essentially the same in testosterone propionate-treated rats spayed 43 days after birth - with the exception of a feminized 5alpha-reductase activity - whereas a completely feminized ("de-imprinted") pattern of enzyme activities was found in the rats with intact ovaries at the time of death. It is concluded that de-imprinting action of ovaries is mainly of a reversible nature.     

Mucolipidosis type IV: Abnormal transport of lipids to lysosomes

Mucolipidosis type IV (MLIV) is a lysosomal storage disorder in which various phospholipids and gangliosides accumulate. The cause of this storage has not yet been identified. Loading experiments in cultured fibroblasts with radioactive phosphatidylcholine ([14C]PC) labelled either in the acyl groups or in the choline residue, indicated increased retention of this lipid in the lysosomes of these patients. Chase experiments in intact fibroblasts, on the other hand, indicated normal degradation and discharge of the radioactive PC in MLIV lysosomes. This was further supported by measurements of the degradation of [14C]PC by isolated lysosomes which indicated normal breakdown of PC in MLIV. Cultured fibroblasts from Hunter (MPSII) patients, which contain enlarged and numerous lysosomes, did not store [14C]PC when compared to normal controls, indicating that the storage of this lipid in MLIV is not a secondary phenomenon caused by the presence of enlarged and numerous lysosomes in these cells. Incubation of [14C]PC at 18°C limits the endocytosis process only up to early endosomes. This temperature did not yield increased retention of [14C]PC in MLIV, indicating that accumulation occurs only after the PC reached late endosomes or the lysosomes.The data indicate that PC as well as other phospholipids and gangliosides accumulate in MLIV apparently owing to a defect in the endocytosis process of membranous components. This defect apparently leads to excessive transportation of these macromolecules into lysosomes rather than their recycling to the plasma membrane. The endocytosis of membrane components is different from receptor-mediated endocytosis which is not affected in MLIV. Once the membrane macromolecules reach the lysosomes in MLIV they are normally catabolized and normally discharged. This may explain the heterogeneity of the stored materials in MLIV. The normal catabolism of macromolecules in the lysosomes is reflected in the minor deterioration in the clinical manifestations of these patients.     

LTB4 production and lysosomal enzyme release by rat alveolar macrophages: effects of phagocytosis, receptor binding, and ionophore stimulation

We have previously shown that the predominant lipoxygenase product of arachidonic acid metabolism in rabbit alveolar macrophages is leukotriene (LT) B4. LTB4 was not detectable in normal unstimulated rabbit macrophages, but its production was increased following calcium ionophore A23187 stimulation, especially after in vivo activation of the immune system. In the present study, we describe that (a) rat alveolar macrophages produced LTB4 in response to natural, biological stimuli such as binding of Fc receptors and complement receptors, as well as zymosan phagocytosis and ionophore stimulation. In contrast, binding of lectin receptors such as concanavalin A and phytohemagglutinin failed to elicit significant increase of LTB4. (b) The predominant LT that was produced was LTB4 regardless of the type of stimulus. This pattern is similar to that of rabbit lung macrophages, but rat alveolar macrophages released higher quantities of LTB4, which can be easily quantitated by high-performance liquid chromatography (HPLC). (c) Phorbol myristate acetate by itself was a weak agonist for LTB4 release. Yet, in combination with a low dose of calcium ionophore A23187 it resulted in LTB4 production. (d) There was a general correlation between release of LTB4 and lysosomal enzymes. In other words, the stimulus that is effective for eliciting enzyme release was usually also effective in causing LTB4 production. (e) A considerable proportion of the LTB4 produced was retained intracellularly. This phenomenon was especially pronounced when zymosan was used as stimulus. (f) Despite the parallelism between LTB4 production and lysosomal enzyme release, the former probably does not regulate the latter. The time courses of their release are dissimilar, and nordihydroguaiaretic acid fails to inhibit lysosomal enzyme release by a dose markedly inhibiting LTB release. (g) Contrary to rabbit lung macrophages rat lung macrophages showed a predominance of lipoxygenase pathway over cyclooxygenase pathway following zymosan ingestion. However, macrophages from both species produced mainly cyclooxygenase products in response to exogenous arachidonic acid.     

Effect of bile salt on amylase release from rat pancreatic acini

The effect of different bile salts on amylase release from isolated rat pancreatic acini has been studied. The bile salt-stimulated discharge of amylase could be divided into three situations, depending on the concentration of bile salt. At low concentrations, between 1 and 100 X 10(-6)M, there was a slight increase in amylase secretion, 5-7% of total, which varied with the type of bile salt but was independent of the concentration of bile salt. The release of amylase stimulated by cholecystokinin, secretin, and carbachol was not affected by bile salts at this low concentration. At slightly higher concentrations, between 250 and 1000 X 10(-6)M, there was a large release of amylase, 10-40% of total, which was dependent on both type and concentration of bile salt. This release occurred specifically for amylase and was not followed by release of either membrane-bound dipeptidylpeptidase IV or intracellularly located lactic dehydrogenase. At higher concentrations, 2000-5000 X 10(-6)M, both amylase and dipeptidylpeptidase IV and lactic dehydrogenase were released, accompanying viability changes of the cells with uptake of trypan blue.