Lysosomal Fragility Research Articles

Changes in lysosomal properties of alveolar macrophages of rats treated with chlorphentermine

Rats were treated with chlorphentermine (30 mg/kg) 5 days per week for 4 weeks. Alveolar macrophages washed from the lungs were greatly enlarged and filled with lamellated inclusions. Lysosomal acid hydrolase activity was increased in these cells when compared to macrophages from untreated rats. Chlorphentermine treatment appeared to have marked effects on lysosomal stability. The latency of acid phosphatase was increased. The effects on β- N-acetyl-glucosaminidase were somewhat different, however, with the latency of this enzyme characterized by an increased lysosomal fragility. These findings indicated that in addition to producing morphological changes within alveolar macrophages, chlorphentermine administration leads to lysosomal changes as well.

Analytical subcellular fractionation of jejunal biopsy specimens: enzyme activities, organelle pathology and response to gluten withdrawal in patients with coeliac disease.

1. Portions of jejunal biopsy specimens from patients with coeliac disease were homogenized in isotonic sucrose solution and subjected to analytical subcellular fractionation by sucrose-density-gradient centrifugation. 2. The activities and distribution of marker enzymes for the principal subcellular organelles were determined by highly sensitive enzymic micro-assays employing fluorigenic and radiolabelled substrates. 3. In untreated coeliac disease the brush border showed decreased activities of five marker enzymes, which were reduced to up to one-eighth of control values. On sucrose density gradients the distinct brush-border peak at density 1.21 was replaced by a broad peak at density 1.16. 4. Two out of three lysosomal enzymes showed increased activities and there was evidence of enhanced fragility of both lysosomes and peroxisomes. 5. There were reduced activities of both mitochondrial and cytosol malate dehydrogenase, with increased activities of cytosol lactate dehydrogenase. 6. The endoplasmic reticulum and basal-lateral membranes were relatively unaffected except that the equilibrium densities were slightly increased and decreased respectively. 7. The patients were treated by gluten withdrawal and subsequent biopsies examined as above. Biopsies from patients treated for 2–4 weeks and who had not yet achieved a morphological improvement showed a partial return to normal of the pathological organelles. 8. Treated patients with partial villus atrophy showed progressively improved enzyme activities and a partial reappearance of the brush-border peak on the sucrose density gradients. 9. Patients whose intestinal mucosa had returned to morphological normality after prolonged gluten withdrawal showed a persistent defect in the brush-border enzyme activities. This was particularly striking for β-glucosidase, which was persistently deficient from the brush-border although approximately one-third of the total activity of this enzyme is normally found in this organelle. 10. The properties and enzyme activities of lysosomes, peroxisomes, endoplasmic reticulum, mitochondria and cytosol were essentially normal in the biopsies from fully treated patients.

Lysosomal stability, superoxide dismutase and zinc deficiency in regenerating rat liver

1. Lysosomal stability was slightly increased in regenerating livers from rats receiving a zinc-deficient (less than 0.5 mg/kg) diet for 10 d before surgery. 2. The activity of superoxide dismutase (EC 1.15.1.1) was significantly reduced in the same tissues. 3. The results do not support the view that increased lysosomal fragility represents a major biochemical consequence of nutritional Zn deficiency in animals.

Interferon Enhances the Fragility of Lysosomes in L-929 Mouse Fibroblasts

Infection of interferon-treated L-929 mouse fibroblasts with vaccinia WR virus is followed by severe cytolysis within 3 to 4 h. It is shown that this cytolysis cannot be caused by enzymes released from lysosomes into the cytosol. However, there is evidence that homologous interferon has a noxious effect on lysosomes. This phenomenon appears to be another aspect of the anticellular functions of interferon.

Anti-endotoxin actions of methylprednisolone in the isolated perfused cat liver.

The efficacy of the synthetic glucocorticoid, methylprednisolone, was examined in vitro using an isolated cat liver perfused with a blood-free medium. Addition of endotoxin (75 microgram/g tissue) to the perfusate did not change perfusion pressure or total oxygen consumption. However, cellular integrity was severely compromised as reflected by increases in perfusate lactate dehydrogenase and cathepsin D activities, increases in tissue lysosomal fragility, and enlargement and vacuolization of lysosomes. Addition of methylprednisolone (1 x 10(-3) M) to the perfusion medium prevented the endotoxin-induced changes in hepatocyte integrity. It is suggested that a major action of endotoxin in the liver is to increase lysosomal fragility, and the protective action of methylprednisolone appears to be related to its lysosomal stabilizing action. The potent anti-endotoxin action of glucocorticoids in vivo may be due in part to the stabilization of lysosomal membranes in tissues such as the liver.

Preservation of hepatic cell integrity by aprotinin in hypoxia

Perfused cat livers subjected to 2.5 hr of hypoxia exhibited dramatic increases in perfusate cathepsin D and lactic acid dehydrogenase (LDH) activities, amino nitrogen concentrations, and a 60% depression in the clearance rate of carbon particles by the reticuloendothelial system (RES). Addition of aprotinin (250 KIU/ml) to the perfusate prior to hypoxia prevented the increases in circulating cathepsin D, LDH, and amino-nitrogen observed at 150 minutes. In addition, aprotinin prevented the reduction in carbon clearance during severe hypoxia. However, aprotinin had no effect on the percent free cathepsin D activity indicating that this agent did not directly prevent increases in lysosomal fragility occurring in response to hypoxia. Thus, addition of pharmacologic doses of aprotinin to the perfusate protected RES cells, and markedly reduced the release of cytoplasmic and lysosomal enzymes. The prevention of cell membrane dissolution appears to be a critical factor in hepatic preservation, and may be related to the inhibition of proteolysis by aprotinin. These effects may help explain the therapeutic effectiveness of this agent in shock and in myocardial ischemia.

Protective action of timolol in acute myocardial ischemia

Timolol, a β-adrenoceptor blocking agent with little or no cardiodepressant activity, was studied in acute myocardial ischemia in cats. Timolol, at a dose of 25 μg/kg, blocked 75 to 80% of the cardiac response to isoproterenol. This dose also significantly heart rate in cats subjected to acute myocardial ischemia by ligation of the left coronary artery. Timolol significantly prevented the spread of ischemic damage in the myocardium as assessed by (a) curtailing the increase in plasma creatine phosphokinase (CPK) activity, (b) preventing the loss of CPK from the ischemic portion of the myocardium, and (c) restoring the elevated S-T segment of the electrocardiogram toward normal. Timolol did not significantly retared the increase in fragility of lysosomes in ischemic myocardial tissue. The mechanism of the protective effect to timolol on the ischemic myocardium appears to be via reducing myocardial oxygen demand by decreasing heart rate.

Enhanced lysosome fragility in the anoxic perfused guinea pig heart: Effects of glucose and mannitol

Guinea pig hearts were perfused under aerobic and anoxic conditions in the presence and absence of glucose or mannitol. Samples of the coronary perfusate were collected and assayed for creatine phosphokinase (CPK), hydroxybutyrate dehydrogenase (HBDH), leucyl-β-naphthylamidase (LBN) and lysosomal enzyme activities. After various periods of perfusion samples of left ventricle were homogenized and the specific activities (mu/mg protein) of N-acetyl-β-glucosaminidase, N-acetyl-β-galactosaminidase, β-glucuronidase and cathepsin C were determined. Estimates of lysosomal integrity were made from measurements of the latency and sedimentability of the marker enzymes. In control aerobic studies, with or without glucose or mannitol, there was an initial wash-out of CPK, HBDH and LBN with no further enzyme release for up to 6 h. The specific activities and the latency and sedimentability values of the lysosomal enzymes in cardiac homogenates were generally unaffected by aerobic perfusions. In contrast, during anoxic perfusions in the absence of glucose large amounts of CPK, HBDH and LBN were released into the coronary perfusate. The latency and sedimentability of each of the lysosomal enzymes was considerably decreased as a result of anoxic perfusion indicating enhanced lysosome fragility. These changes were significant after 30 min and progressed with the duration of anoxia. The specific activities of the lysosomal enzymes were significantly increased after 6 h anoxic perfusion. Inclusion of glucose in the anoxic perfusion medium did not prevent the release of CPK, HBDH and LBN in the early stages of anoxia but a reduction in myocardial enzyme release was observed in the later stages. The presence of glucose considerably delayed the onset of lysosomal membrane labilization but caused further increases in the specific activities of three of the acid hydrolases. Equimolar concentrations of mannitol had a limited effect on lysosome stability indicating that the protective effect of glucose was mainly due to its metabolic action.

Lysosomal disruption in the pathogenesis of hepatic damage in primary and secondary haemochromatosis.

The disruption of lysosomes with release of their content of lytic enzymes was an early concept for the possible role of these organelles in the pathogenesis of tissue damage. Many examples are known of primary lysosomal storage diseases due to a congenital deficiency of certain acid hydrolases. It is suggested that iron overload due to either primary haemochromatosis or transfusional siderosis is a form of acquired secondary lysosomal storage disease. Subcellular fractionation experiments and electron microscopic studies have shown that liver tissue from patients with iron overload has iron-laden lysosomes. Similar results have been found in iron-overloaded rats. In patients, but not in experimental animals, enzymic analyses have shown increased activities of acid hydrolases and strikingly enhanced lysosomal fragility in liver homogenates. When it has been possible to deplete the patients of the excessive iron, these parameters have returned to normal. The possible mechanisms by which the iron compounds disrupt lysosomes, including distension with ferritin or haemosiderin or free-radical-mediated membrane damage, will be discussed.

Cardiac lysosomal derangements in mouse heart after long-term exposure to nonmetabolizable sugars.

Hearts of late fetal mice were maintained in organ culture in the presence of 30-100 mM sucrose or mannitol. Activities of several lysosomal enzymes (cathepsin D, beta-acetylglucosaminidase, acid phosphatase) were increased by up to 30% after 18-24 hours and by up to 50% after 48-72 hours, as compared to enzyme activities in litter-matched hearts maintained in control medium or medium supplemented with equimolar urea. Simultaneously, the ratio of nonsedimentable to sedimentable enzyme activity was significantly increased, suggesting increased lysosomal fragility. Light and electron microsopic examination of the hearts revealed marked vacuolization in myocytic, interstitial, and endothelial cells. The vacuoles were limited by single membranes, often contained particulate or amorphous cellular debris resulting from autophagocytosis, and in cytochemical preparations frequently exhibited an electron-dense reaction product indicative of acid phosphatase activity. Hydrocortisone failed to prevent the marked lysosomal activation induced by the sugars. In conclusion, prolonged exposure to nonmetabolizable sugars induces severe lysosomal derangements with prominent autophagy, in fetal mouse heart maintained in organ culture.

Functional and biochemical evidence of damage to enterocytes induced by triparanol: role of lysosomes and the effect of gluten-free diet.

1. Functional and biochemical studies were performed on the small intestine of control rats, and the results were compared with similar studies on animals given triparanol at a dosage of 0.114 mmol/kg daily for 10 days. The animals given triparanol were fed with either standard rat food or a gluten-free diet. 2. By using a recirculating-perfusion technique in vivo, it was shown that absorption of galactose from an 8 mmol/l solution was impaired in the ileum but not in the jejunum of the triparanol-treated rats. 3. Assays of marker enzymes for the principal subcellular organelles were performed on isolated jejunal and ileal enterocytes. In the ileum there was a striking decrease in lysosomal enzyme activities and a smaller but significant decrease of lactate dehydrogenase, catalase and malate dehydrogenase activities. In the jejunum there was no significant change in the activities of these enzymes. 4. Measurements of lysosomal integrity indicated that ileal lysosomal fragility was markedly increased and that jejunal lysosomes were affected to a much smaller extent. 5. These effects of triparanol could not be ameliorated by feeding with a gluten-free diet.

Effects of Prednisolone on the Small Intestinal Mucosa of the Rat

1. The effects in rats of an oral pharmacological dose of prednisolone on mucosal function, enzymology, lysosomal membrane fragility, morphology and cell kinetics have been examined in proximal jejunum and distal ileum. 2. The maximum absorptive capacity for galactose was significantly greater in both the jejunum and the ileum of the steroid-treated animals. This was due to an increase in carrier-mediated transport in the individual enterocytes and not to a change in the cell population. The Michaelis constant for galactose was not significantly altered by prednisolone. 3. Activities of brush-border and mitochondrial enzymes and of RNA were increased in isolated enterocyte preparations from the jejunum and ileum of the steroid-treated group. 4. Lysosomal membrane fragility was unaltered in the prednisolone-treated group. 5. Morphometrical observations confirmed that the size of the enterocyte population was unaltered by prednisolone. Studies on cell kinetics indicate that the effects of prednisolone are due to a direct action on the enterocyte and not secondary to changes in migration rate.

Studies on the hepatotoxicity of Amanita phalloides in the rat : II. Biochemical analysis of the lysosomal changes

Marked changes in the integrity and fragility of liver lysosomes have been detected in rats poisoned with a lethal dose of a crude aqueous extract of Amanita phalloides. In the course of the poisoning, four lysosomal hydrolases (acid phosphatase, β- N-acetylglucosaminidase, β-galactosidase and β-glucuronidase) were found to undergo a progressive reduction in both their structure-linked latency and sedimentability. Concurrently, there occurred in the blood plasma a conspicuous increase of acid hydrolase activities, which distinctly preceded the subsequent increase of both aspartate aminotransferase and isocitrate dehydrogenase. Observations on the fractional free activity of the acid hydrolases at different substrate concentrations seem to rule out the possibility that a graded change in the permeability of the lysosomal membrane underlies the activation of lysosomal enzymes. Rather, activation seems to be an all-or-none kind of phenomenon. When submitted to various labilizing procedures in vitro, the liver lysosomal population from poisoned rats exhibited a modified fragility in comparison with control lysosomes. Factors such as the increased size of hydrolase-bearing particles, changes in the properties of the limiting membrane, or modifications in the intra as well as extralysosomal environment are likely to account for these results. The reliability of the observed lysosomal changes as indexes of the modifications that take place in vivo and the relevance of the latter to the production of the liver injury by Amanita phalloides are discussed.

Differential effects of lipids on the osmotic fragility of lysosomes.

The effect of a wide range of concentrations of oleic acid, oleyl alcohol and oleic acid methyl ester on lysosomal stability has been studied under both hypotonic and isoosmotic medium conditions. Both oleic acid and oleyl alcohol exhibited a biphasic interaction pattern with lysosomes; stabilizing at low concentrations and labilizing at high concentrations. Lysosome labilization by the ester required an initial lag period.

Evidence for the selective release of lysosomal proteinases in fasted rabbits

The enzyme responsible for the conversion of "neutral" to "alkaline" fructose 1,6-bisphosphatase (EC 3.1.3.11) by removal of a 7000 dalton peptide (converting enzyme, Proteinase I) has been shown to be localized in rat liverlysosomes. Lysosomes also contain a specific proteinase (Proteinase II) that catalyzes the release of a small peptide from the NH2-terminus of the native subunits. In fasted rabbits Proteinase II is released into the cytoplasm, together with Cathepsin A, but Proteinase I remains associated with the lysosomal fraction. Increased osmotic fragility of liver lysosomes in fasted rabbits has also been observed, but this increased fragility does not result in the release of Proteinase I. The appearance of Proteinase II in the cytoplasm may be due either to its selective release from the lysosomes, without release of Proteinase I, or its localization in a different lysosomal fraction. Changes in lysosomal structure induced by fasting may play a dual role in : 1) the mobilization of amino acids for gluconeogenesis and 2) the modulation of activity of gluconeogenic enzymes.

On lysosomal fragility and induction of liver hexose-monophosphate dehydrogenases in the fasted-refed rat

Young adult male rats were fasted for 3 days, then fed a glucose-rich diet, ad libitum. At the end of the fasting period, the specific activity of liver glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was decreased to 60% of control (nonfasted) levels. After 24 to 72 h of refeeding, the specific activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase increased seven- and twofold, respectively. During the fasting period, the liver lysosome fragility increased, as judged by increased release of bound acid phosphatase and β- N-acetylglucosammidase activity during standard homogenization. Three hours after feeding a carbohydrate-rich diet, a further increase in liver lysosomal fragility was observed that returned to control values prior to the induction of the dehydrogenases. Similarly, the susceptibility of liver lysosomes from fasted rats to increased fragility by the intraperitoneal injection of glucose or galactose was also observed. Prior starvation was not a requisite for labilization of lysosomal membranes by injected glucose, but induction of the pentose phosphate shunt dehydrogenase was not observed. In a group of 6-week old male rats fed a commercial pellet diet throughout, the injection of insulin caused no change in liver lysosomal fragility, though hypoglycemia resulted. Similar animals made diabetic by treatment with Streptozotocin and diabetic rats given insulin, showed no change in liver lysosmal fragility based on the percentage of free to total activities of β- N-acetylglucosaminidase, β-glucuronidase, β-galactosidase, and Cathespin D. However, when adult female rats were fasted for 24 h, then injected with sufficient insulin to produce hypoglycemia, liver lysosomal fragility, based on the release of β- N-acetylglucosaminidase during homogenization, increased nearly threefold. These studies demonstrate that stimulated lysosomal fragility can be initiated by refeeding fasted animals a carbohydrate-rich diet, by intraperitoneal injections of fasted rats with glucose or galactose, or by administering insulin alone to fasted rats. However, hyperglycemia induced by diabetogenic doses of Streptozotocin, or hypoglycemia induced in well-fed animals by insulin injection failed to elicit an enhanced liver lysosomal fragility. Whether induction of the enzymes of lipogenesis by rat liver is dependent upon a prior lysosomal membrane labilization remains to be determined.

The use of cultured cells for in situ measurment of lysosomal fragility

The use of cultured cells for in situ measurment of lysosomal fragility

Influence of starvation on the activities and localization of cathepsin D and other lysosomal enzymes in hearts of rabbits and mice

An investigation was made in mice and rabbits of the effects of starvation on the activities and cellular localization of lysosomal enzymes in cardiac muscle of the left ventricle. The specific activity of cathepsin D was increased in the atrophying hearts of mice, by 14% after one day and by 26% after 3 days. Simultaneously, the proportion of the enzyme activity that was nonsedimentable during centrifugation at 40 000 g rose significantly, suggesting increased fragility of lysosomes or release of enzyme from lysosomes into the cytosol. In the same hearts protein concentration and the activities of acid phosphatase and beta-acetylglucosaminidase remained unchanged. Changes were similar in rabbit hearts except that specific cathepsin D activity was increased more (46%) and changes in the proportion of the activity that was nonsedimentable were less marked. Immunofluorescent staining for rabbit cathepsin D revealed that this acid proteinase was localized in lysosome-like particles in both interstitial cells and muscle fibres in hearts of normal animals. After 3 days starvation there was no detectable change in interstitial cells, but in the muscle fibres particulate staining was greater and, in addition, intense diffuse cytoplasmic staining was observed. The results indicate that cardiac muscle, like liver but unlike skeletal muscle, develops a significant increase in the activity of the lysosomal protease cathepsin D during starvation. The change occurs specifically in myocytes rather than in interstitial cells. The results suggest a possible role for cathepsin D in contributing to the development of cardiac atrophy and net protein catabolism during prolonged starvation.

Biochemical and structural changes of rat liver lysosomes by ethionine

1.(1) The administration of ethionine to rats (1 g/kg body wt) was shown to induce marked yet reversible changes in liver lysosomes, detectable both biochemically and histochemically.2.(2) In liver homogenates the free activity of the lysosomal marker acid phosphatase, still unchanged at three hours, was consistently increased at six hours, and by 13 hours returned to normal levels. The free activity of β-N-acetylglucosaminidase, another lysosomal hydrolase, was similarly increased at six hours.3.(3) Six hours after ethionine, the proportion of acid phosphatase activity recovered in the soluble phase of the homogenate was in most cases significantly increased. The levels of soluble activity, however, were distincly lower than those for free activity.4.(4) The ratio of free to total activity for acid phosphatase and β-N-acetyl-glucosaminidase is not modified by varying the substrate concentration in the medium; thereby an acquired permeability of the lysosomal membrane to the substrates is likely ruled out as a mechanism for the increased value of free activity.5.(5) The fragility of liver lysosomes from rats six hours after ethionine was investigated by exposing the whole homogenate to mechanical or hypoosmotic shock as well as a lysosome-rich fraction (M + L) to hypoosmotic shock or preincubation at pH 5, 37° C. In all cases both hydrolases were shown to be activated to a higher degree than control preparations. Moreover, an enhanced osmotic fragility of lysosomes in liver homogenates was also observed at three and 13 hours after ethionine. These findings suggest the presence in the liver of ethionine-treated animals of an increased proportion of particles particularly fragile.6.(6) Possible mechanism(s) by which ethionine administration causes the enhanced lysosomal fragility are briefly considered. The relationship between the augmented fragility of lysosomes and the increased value of free and soluble activities of their hydrolases is discussed.

Enzyme Activities and Properties of Lysosomes and Brush Borders in Jejunal Biopsies from Control Subjects and Patients with Coeliac Disease

1. Reduced activities of four enzymes from brush borders were found in intestianl biopsies from patients with untreated coeliac disease. The activities returned towards control values after treatment by gluten withdrawal. Parallel changes were noted for the cytosol enzyme lactate dehydrogenase. 2. Measurement of brush-border integrity by differential centrifugation of biopsy extracts indicated increased fragility of the brush borders in biopsies from untreated patients. Normal values were obtained for biopsies from treated patients. 3. Increased activites of six acid hydrolases (lysosomal enzymes) were found in biopsies from untreated coeliac patients. Normal values were obtained for biopsies from treated patients. 4. Assement of lysosomal integrity by assay of latent N-acetyl-beta-glucosaminidase and of sedimentable activity of four acid hydrolases demonstrated increased lysosomal fragility in untreated coeliac mucosa. These lysosomal changes return to within the normal range after treatment by gluten withdrawal. 5. The lysosomal changes are consistent with their having a pathogenic role in the enterocyte damage of coeliac disease. Possible mechanisms for the lysosomal changes are discussed.