acid-phosphatase and leupeptin

The effects of the lysosomotropic agents chloroquine and leupeptin on the taurocholate-stimulated biliary excretion of horseradish peroxidase (HRP) was studied in bile fistula rats. HRP (0.5 mg/100 g body wt) was injected into the portal vein during taurocholate (0.4 mumol/min/100 g body wt) or saline infusion. HRP appeared in bile showing both an early (approx. 5 min) and a late (approx. 25 min) excretion peak. The late peak, which represented about 95% of the total HRP excreted, is due to transcellular vesicular transport. The early peak is mainly due to paracellular leakage although a rapid vesicular transport also contributes. Taurocholate infusion significantly increased the biliary output of HRP (both peaks) and of the endogenous lysosomal enzyme acid phosphatase. Pretreatment with chloroquine or leupeptin inhibited the taurocholate-stimulated late excretion of HRP into bile, without affecting its early excretion. The lysosomotropic agents did not affect the biliary excretion of bile salts but significantly inhibited the taurocholate-stimulated biliary excretion of acid phosphatase. The results are consistent with a role of lysosomes in the taurocholate-stimulated major transcellular vesicular transport of HRP into bile.

Topics: Acid Phosphatase; Animals; Bile Acids and Salts; Biliary Tract; Chloroquine; Horseradish Peroxidase; Leupeptins; Lysosomes; Male; Rats; Rats, Wistar; Taurocholic Acid

Administration of phalloidin in vivo to rats causes marked changes in the distribution of actin and myosin in hepatocytes, which accompanies reduced bile flow. We have found that in hepatocytes treated with phalloidin for 3 and 7 days, cellular myosin content increased about 1.5-fold and 4.7-fold, respectively. In addition, total cell protein content and several marker enzyme activities were also elevated by 30-120% depending on the duration of phalloidin treatment. These observations allow us to speculate that phalloidin somehow elicits inhibition of cellular protein degradation, which results in the increase of these protein levels. To examine this possibility further, we analyzed leupeptin-induced density shift of phagolysosomes. In normal liver, the injection of leupeptin/E64c caused an increase in the density of both heterolysosomes and autolysosomes, due to retarded digestion of sequestered proteins as a result of the inhibition of lysosomal cathepsins. Accumulation, in these denser autolysosomes, of lactic dehydrogenase, pyruvate kinase, aldolase, and myosin was demonstrated by enzyme assays and immunoblot analysis. In the phalloidin-treated liver, the increase in the density of autolysosomes and the accumulation of above cytoplasmic enzymes were markedly inhibited. However, phalloidin did not affect the shift in the density of heterolysosomes. From these data, we concluded that autolysosome formation was specifically hindered in phalloidin-treated rat hepatocytes, which results in the reduction of autophagic protein degradation and eventual increase in intracellular protein levels.

Topics: Acid Phosphatase; Actins; Animals; Cathepsins; Female; Fructose-Bisphosphate Aldolase; Immunoenzyme Techniques; L-Lactate Dehydrogenase; Leupeptins; Liver; Lysosomes; Microscopy, Electron; Myosins; Oligopeptides; Phalloidine; Pyruvate Kinase; Rats; Rats, Inbred Strains

1. The effects of potent protease inhibitors in vitro (leupeptin, pepstatin and E-64[N-[L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine]) on intracellular cathepsin B (EC 3.4.22.1), hemoglobin (Hb)-hydrolase and acid phosphatase (EC 3.1.3.2) from cultured B16 melanoma variants (B16-F1, F10 and BL6) were studied. 2. E-64 induced all the cultured B16 melanoma variants to decrease the activity of intracellular cathepsin B but did not have this effect with Hb-hydrolase or acid phosphatase. Furthermore, E-64 decreased the activity of cathepsin B in both the lysosomal and cytosol fractions. 3. Leupeptin induced all the cultured B16 melanoma variants to increase the activities of intracellular cathepsin B and Hb-hydrolase but not that of acid phosphatase. An increase in the level of cathepsin B activity was most significant in B16-BL6 followed by F10 and then F1 variants. 4. Leupeptin induced all the cultured B16 melanoma variants to increase the cathepsin B activity in the lysosomal fraction. Our data differed from the results of Tanaka et al. (1981) in that leupeptin induced rat cultured hepatocytes to inhibit the activity of intracellular cathepsin B and increase the Hb-hydrolase activity, especially in the cytosol fraction.

Topics: Acid Phosphatase; Animals; Cathepsin B; Enzyme Induction; Leucine; Leupeptins; Melanoma, Experimental; Mice; Oligopeptides; Pepstatins; Peptide Hydrolases; Protease Inhibitors; Subcellular Fractions; Tumor Cells, Cultured

To determine the physiological role of the thiol proteases in T4 and T3 release from thyroglobulin, experiments were performed with 131I-prelabelled rat thyroid lobes incubated in vitro in the presence and absence of leupeptin, an inhibitor of thiol proteases. Basal secretion of [131I]T4 and [131I]T3 from rat thyroid lobes prelabelled in vivo was quite low, but in the presence of 10 mU/ml bovine TSH a marked stimulatory effect was observed. The stimulatory effect of TSH was completely abolished by leupeptin. This was associated with marked inhibition of lysosomal proteolytic activity, suggesting that the inhibitory effect of leupeptin on T4 and T3 secretion could be attributed to its inhibitory action on proteolysis of thyroglobulin. Further evidence for an inhibitory effect of leupeptin on intralysosomal hydrolysis of thyroglobulin was obtained when thyroid lobes were incubated with 131I- in the presence and absence of leupeptin and TSH. The crude lysosomal preparation was fractionated on a Percoll density gradient, which separates 131I-containing particles into a dense peak containing purified lysosomes and a buoyant peak containing pinocytotic vesicles. A marked increase in the 131I-content of the dense peak was observed in the presence of TSH + leupeptin. Analysis of the 131I in the dense fraction by sucrose density gradient centrifugation and by SDS-polyacrylamide gel electrophoresis demonstrated that leupeptin inhibited degradation of 19S thyroglobulin, especially the formation of [131I]peptides of MW less than 14K.

Topics: Acid Phosphatase; Animals; Centrifugation, Density Gradient; Cysteine Endopeptidases; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Leupeptins; Lysosomes; Male; Rats; Rats, Inbred Strains; Thyroid Gland; Thyroxine; Triiodothyronine

Administration of leupeptin to rats induces the accumulation of numerous autophagic vacuoles in the liver. Furuno et al. (Furuno, K., Ishikawa, T., and Kato, K. (1982) J. Biochem. (Tokyo) 91, 1485-1494) have recently devised a method for Percoll density gradient equilibrium fractionation of crude lysosomal fractions to isolate a highly enriched preparation of autophagic vacuoles. This system was used to determine whether cytoplasmic enzymes are normally sequestered into autophagic vacuoles in fed animals. Within 30 min following the administration of leupeptin to fed rats, several cytoplasmic enzymes could be demonstrated in vacuolar fractions heavier than mitochondria and normal lyosomes. The activities of tyrosine aminotransferase and lactic dehydrogenase as well as antigens of fructose-bisphosphate aldolase were detectable in fractions with densities of 1.115 to 1.15 g/ml containing cathepsins and acid phosphatase. The cytoplasmic enzymes in these fractions exhibited latency and were sequestered within membranous organelles. Six hours after the administration of leupeptin, the autophagic vacuoles gradually disappeared from these fractions concurrently with the loss of both cytoplasmic and lysosomal marker enzymes. For 6 h after injection of leupeptin the activities of cathepsin D and acid phosphatase increased in autophagic vacuoles and decreased in the postvacuolar lysosomal fraction. Administration of dexamethasone, which induces the synthesis of tyrosine aminotransferase and cytosolic aspartate aminotransferase, selectively increased the sequestration of these enzymes to proportional degrees. Cycloheximide administered simultaneously with leupeptin rapidly inhibited formation of autophagic vacuoles and the sequestrations of both cytoplasmic and lysosomal enzymes. However, when cycloheximide was administered 1 h after leupeptin, the formation of autophagosomes and the sequestration of cytoplasmic enzymes were inhibited but the vacuolar uptake of acid phosphatase and cathepsin D continued to increase for several hours. When cycloheximide was injected 1 h after leupeptin, losses of lactic dehydrogenase and aldolase proteins were observed in autophagic vacuoles isolated 1 and 2 h later.

Topics: Acid Phosphatase; Aminopeptidases; Animals; Autophagy; Cathepsin D; Cathepsin H; Cathepsins; Centrifugation, Density Gradient; Cycloheximide; Cysteine Endopeptidases; Cytoplasm; Dexamethasone; Fructose-Bisphosphate Aldolase; Kinetics; L-Lactate Dehydrogenase; Leupeptins; Liver; Lysosomes; Male; Oligopeptides; Organoids; Phagocytosis; Rats; Rats, Inbred Strains; Tyrosine Transaminase; Vacuoles

We have studied the morphological alterations of the lysosomal compartment in rat hepatocytes following intraperitoneal administration of leupeptin, using electron microscopy and cytochemical techniques. At 30 min after the injection, autophagic vacuoles (autophagosomes and autolysosomes), containing cytoplasmic organelles, increased in number in the vicinity of bile canaliculi and also near the Golgi apparatus. At 1 h, most of the autophagic vacuoles were autolysosomes, single membrane-limited bodies positive for acid phosphatase activity. Development of the autolysosomes was accompanied by the reciprocal disappearance of pre-existing secondary lysosomes. From 1 to 8 h, the autolysosomes varied to a great extent in both size and shape as a result of coalescence. Segregated organelles within the autolysosomes were gradually degraded into electron-lucent unidentifiable debris. At later, residual bodies were abundant in the cytoplasm, and occasionally, their contents were discharged into the space of Disse. From 9 to 12 h, the autolysosomes decreased in the volume and number and secondary lysosomes of normal shape and size appeared. The autolysosomes seem to persist for long periods because of a retarded degradation of sequestered materials in leupeptin-treated hepatocytes.

Topics: Acid Phosphatase; Animals; Autophagy; Leupeptins; Liver; Lysosomes; Male; Microbodies; Microscopy, Electron; Oligopeptides; Organoids; Rats; Rats, Inbred Strains; Time Factors