Lysosomal Fragility Research Articles

The lysosomal permability test modified for toxicity testing with cultured heart endothelioid cells

A modified lysosomal fragility test is described which is suitable for use with cultured cells. The permeability (fragility) of the lysosomal membranes of the cells to the substrate beta-glycerophosphate is measured by assessing the degree of particulate lysosomal straining seen after exposing the cells to the Gomori acid phosphatase staining reaction under carefully controlled conditions. Monolayer cultures of endothelioid cells from the hearts of neonatal rats have been used in all experiments. The time-course of lysosomal straining for cells exposed to various treatments (normal saline, isotonic sucrose, 0.25 m sucrose, distilled water, acetate buffer pH 5.0, cold acetone, neutral formalin, acetic-ethanol, Triton X-100, hydrocortisone, choloroquine and vitamin A) was compared with that of control cells stained under identical conditions. Statistical differences in staining between the test and control cells were determined by the Wilcoxin Signed Rank Test and also by regression analysis following a transformation designed to allow for the saturation character of the reaction. The success of the modified technique depends upon meticulous methodology. It is capable of demonstrating both lysosomal membrane labilization and stabilation, second- and third-stage lysosomal activation, and apparent lysosomal enzyme loss or destruction in situ. The technique also allows the degree of reversible or first-stage lysosomal activation to be subdivided on an almost continous basis and is suitable for investigating the effects of drugs and other agents on the integrity of the lysosome in situ.

Toxicity, clearance, and metabolic effects of pactamycin in combination with bacterial lipopolysaccharide

Combinations of pactamycin and Escherichia coli 026: B6 Boivin lipopolysaccharide (LPS) or Salmonella minnesota R595 lipid A, as a bovine serum albumin-lipid A complex, administered simultaneously, killed BALB c mice synergistically. The enhanced lethality of the combination was not the result of a reduced rate of elimination of LPS or pactamycin from the circulation. Pactamycin administered iv, but not ip, accumulated in the lungs immediately after injection; thereafter the drug progressively disappeared from lung tissue. Concurrent administration of LPS did not alter the accumulation of pactamycin in the lungs nor its rate of elimination from blood and lung. The amount of pactamycin recoverable after incubation with liver or lung homogenates was markedly decreased at both 4 and 37°C. No products of the antibiotic were detected chromatographically after incubation with homogenates. Pactamycin and LPS in combination increased the in vitro fragility of hepatic and renal lysosomes, as measured by the release of cathepsin and β-glucuronidase; the effect of the combination was approximately equivalent to the sum of effects of LPS and pactamycin alone. Concanavalin A and cyclic adenosine 3′,5′-monophosphate did not modify the lethality of pactamycin and LPS, singly or in combination, whereas caffeine, ethylenediamine tetraacetate, or 6α-methylprednisolone protected mice from the lethal action of LPS and the synergistic combination but not that of pactamycin. Lethal doses of LPS and the synergistic combination, but not pactamycin, provoked marked hypothermia and elevated concentrations of blood urea nitrogen. These results indicate that pactamycin rendered the mice more susceptible to LPS and that LPS was the causal lethal agent.

Relation of lysosomal fragility in CLL lymphocytes to PHA reactivity.

AFTER addition of various mitogens such as phytohaemagglutinin (PHA) in vitro lymphocytes shift from a “resting” state to one of rapid enlargement, culminating in DNA synthesis and mitosis. The mechanism initiating transformation and proliferation is not yet fully understood. Some observations, however, suggest that alterations of cellular membranes might be involved in these processes. In their cytochemical studies, Allison and Mallucci1 observed increased permeability of lysosomes in normal human peripheral blood lymphocytes after exposure to PHA for 5 h. Hirschhorn et al.2 demonstrated that during the early phase of stimulation 30–120 min after addition of PHA in normal human blood lymphocytes a redistribution of lysosomal acid hydrolases from a sedimentable into a non-sedimentable form as well as an increased release by agents which disrupt membranes, such as streptolysin S and filipin, of lysosomal enzymes from a subcellular fraction rich in lysosomes are taking place. It has been suggested that these alterations, both reflecting enhanced fragility of lysosomal membranes, might be due to increased endocytosis induced by the mitogen and the hypothesis was raised that lysosomal hydrolases might be involved in intracellular processes leading to lymphocyte activation2. We have therefore studied the fragility of lysosomes in unstimulated and PHA-treated lymphocytes from patients with chronic lymphocytic leukaemia (CLL), because lymphocytes in most patients with this disease show a diminished and/or delayed or even no response to PHA3 and other mitogens4,5.

Influence of some hydrosoluble substances with vitamin P activity on the fragility of lysosomes in vitro

A method is described for measuring in vitro the stabilizing capacity of hydrosoluble pharmaceutical products on the lysosomes and this has been applied to substances with vitamin P action. The flavonoids [rutin, tri(hydroxyethyl)rutosid, magnesium flavonic chelates] exert a stabilizing influence on the lysosomes. The anthocyanosides on the other hand show no such effect because they exhibit weak chelating power in our experimental conditions. To this end the chelating capacity for copper, the influence on the —SH groups of cystein and the stabilizing power in the presence of Na 2 EDTA of vitamin P substances have been studied. No parallelism seems to exist between the test of stabilization applied in vitro and the methods usually employed in vivo to find a vitamin P action.

Enhanced fragility of neural lysosomes from chicks suffering from galactose toxicity.

Abstract—The stability of neural lysosomes to osmotic and temperature shock and the free (non‐sedimentable) activities of selected lysosomal hydrolases from chicks suffering from galactose neurotoxicity were investigated. The neural lysosomes from chicks fed galactose demonstrated enhanced fragility to both elevated temperature and hypo‐osmotic media in comparison to the behavior of neural lysosomes isolated from control animals. The increased lability to osmotic shock could be duplicated by preincubation of normal lysosomes in solutions of galactose or galactitol. Further, the increased fragility induced in vivo by galactose feeding could be reversed by removing the chicks from the diet for 8 h, and such removal was accompanied in the brain by large reductions in levels of galactose and galactitol. The free activities of both β‐galactosidase (EC 3.2.1.23) and β‐N‐acetyl hexosaminidase (EC 3.2.1.30) were elevated above those of controls, and the percentage increases were proportional to the combined brain levels of galactose and galactitol. Our data suggest that increased fragility of lysosomes is a function of the accumulation of galactose and galactitol in the brains of chicks fed toxic amounts of galactose. Alteration of lysosomal integrity represents an attractive role for galactitol, as well as galactose, in the causation of galactose neurotoxicity in chicks.

Effects of selected chelating agents and metals on the stability of liver lysosomes

Diethyldithiocarbamate (DDC), penicillamine and sodium ethyldiaminetetraacetate (Na-EDTA) in 1 mM concentrations significantly stabilized the membrane of isolated liver lysosomes, as shown by a decreased release of β-glucuronidase upon incubation at 37° and pH 5·0. Thiol compounds which are readily auto-oxidized (cysteine, glutathione and dithiothreitol) did not affect lysosomal fragility when subjected to 1 mM concentration; at a higher concentration (5 mM), these substances increased the release of β-glucuronidase. Stabilization by DDC and penicillamine, both of which contain sulphydryl-like groups, may be related to their nonauto-oxidizability, although their chelation effect cannot be excluded. Various chelating agents relatively specific for Fe 2+ or Fe 3+, such as l,10-phenanthroline, 5,6-dimethyl-l,10-phenanthroline, 2-2′-dipyridyl, Desferal, and pyrocatechol-3,5-disulfonic acid (Tiron), had no effect on lysosomal stability. Zinc ions (0·05–2·5 mM) significantly stabilized lysosomal membranes; this stabilization was concentration dependent, was not affected by thiol compounds, and was potentiated by equimolar complex with 8-hydroxyquinoline (8-HQ) ; 8-HQ alone (1 mM) labilized lysosomes. Calcium ions had no effect, whereas Cu 2+ and Hg 2+ labilized the lysosomes. Labilization by Cu 2+ was reversed by chelating agents and decreased by thiol compounds. Incubation of lysosomes in nitrogen and dark prevented labilization by copper, and did not affect stabilization by zinc. The effects of certain chelating agents (8-HQ, DDC, penicillamine, EDTA) or metals (Zn 2+, Cu 2+, Hg 2+) on the stability of the lysosomal membranes are discussed in terms of the reactivity of these agents with components of the membrane, or interference with metal-catalyzed lipid peroxidation.

Lysosomal fragility induced by prostaglandin F 2 .

PROSTAGLANDINS have been suggested as mediators of inflammatory reactions because they increase vascular permeability1,2, are found in inflammatory exudates3, are released during antigen-antibody reactions4 and have leucotactic properties5. For this reason and because lysosomes have been assigned an important role in the pathogenesis of diverse tissue injury reactions6, we have studied the effect of prostaglandins on lysosomal fragility.

Changes in liver lysosome fragility, erythrocyte membrane stability, and local and systemic lysosomal enzyme levels in adjuvant-induced polyarthritis

Adjuvant-induced polyarthritis in rats is characterized by the development of liver lysosome fragility, and elevated plasma and local hind paw activities of lysosomal acid hydrolases. During the first few days of the disease, there is a marked increase in liver lysosome fragility and a sharp elevation in plasma β-glucuronidase and acid phosphatase. Commencing on day 4, both liver lysosome fragility and plasma levels of acid hydrolases decrease. Liver lysosome fragility and plasma acid hydrolases again increase on day 9 and continue to rise. Between days 4 and 9, when liver lysosomes appear to be stabilized and plasma levels of acid hydrolases are near control values, the onset of erythrocyte membrane stabilization occurs which endures throughout the disease. Acid phosphatase activity in the injected hind paw increases immediately after induction of polyarthritis as does the swelling, whereas enzyme activity in and swelling of the contralateral hind paw increase 9 or 10 days later. Paramethasone, phenylbutazone and indomethacin, administered orally for various periods of time, attenuated the swelling of primary and secondary lesions and reduced the severity of the biochemical lesions examined.

Functional characteristics and lysosomal integrity of the isolated perfused cat pancreas

The isolated cat pancreas perfused with a physiological salt solution plus low molecular-weight dextran functions satisfactorally for at least 2 1 2 hours. Evidence for stability and functional integrity of the preparation included measurements of vascular resistance, arterial and venous pH, PO 2, and PCO 2, pancreatic juice volumes and trypsin concentrations, and studies of acinar cell ultrastructure. Total specific β-glucuronidase activity of the unstimulated perfused pancreas was comparable to that seen in the intact cat pancreas. However, pancreozymin and secretin decreased the total β-glucuronidase activity of the perfused pancreas. Pancreozymin also increased the release of β-glucuronidase from lysosomal suspensions of liver and pancreas. β-Glucuronidase activity rose in the pancreatic juice, duodenal secretion, perfusate, and the bathing medium of the hormonally stimulated perfused pancreas concomitant with a decreased number of zymogen granules and lysosomes in the acinar cells. The data are consistent with the hypothesis that pancreozymin increases the fragility of pancreatic lysosomes liberating β-glucuronidase into the pancreatic duct, extracellular space, and intravascular compartment. Lysosomal enzymes released into the duct may have an auxiliary digestive function in the duodenum, and the increased extracellular lysosomal enzyme activity may play a role in the pathophysiology of pancreatitis or other pancreatic disease states in which the pancreatic lysosomes become compromised.

Amino acid incorporation by isolated rat liver mitochondria during liver regeneration.

Intact mitochondria, isolated from regenerating rat liver 2-3 days after partial hepatectomy, are 2.5-3 times more active in amino acid incorporation than mitochondria from control livers. Liver mitochondria from sham-operated animals showed normal amounts of incorporation. Sterile procedures insured low levels of bacterial contamination; cycloheximide was used to eliminate any contribution by contaminating microsomes. Mitochondria from control and 3-day-old regenerating livers were nearly identical in their concentration of several respiratory chain components, P/O ratios, specific O(2) consumption, and cytochrome c oxidase activities. Small differences were observed in respiratory control ratios but these were shown to be unrelated to the differences observed in amino acid incorporating ability. Differential contamination by lysosomes and differences in lysosome fragility were also shown not to be factors in the increased incorporation by regenerating liver mitochondria. Thus, mitochondria from rapidly growing and dividing mammalian tissues are more active in protein synthesis than mitochondria from tissues that grow and divide more slowly.

Properties of muscle and liver lysosomes in adrenalectomized rats.

AbstractThe stability of lysosomes from liver and muscle of adrenalectomized, young rats has been tested by measuring the release of acid hydrolases (RNase, cathepsin D, β‐glucuronidase and aryl sulphatase) from lysosomes under various conditions. Adrenalectomy increased the proportion of enzyme released from the lysosomal particles, as measured in tissue homogenates or fractions. The release, which may have occurred in vivo or during homogenization, probably reflects an increased lysosomal fragility. In muscle (but not in liver) an increase in the total activtiy of acid hydrolases could also be observed after adrenalectomy. The effects of adrenalectomy could not be corrected by saline treatment, and were not due to reduced food intake. All parameters were restored to normal by administration of 300 μg of corticosterone daily for 5 days, and higher doses (3 mg per day) led to further reduction in enzyme release. The stability of muscle and liver lysosomes in vitro was tested by exposing particles to osmotic stress, vitamin A alcohol and acid medium (pH 5). Liver and muscle lysosomes from adrenalectomized rats were less stable when exposed to osmotic stress or vitamin A, but hepatic lysosome were more stable towards acid incubation than particles prepared from normal rats. Differential centrifugation of liver homogenates indicated that more acid hydrolases were associated with the heavier fractions in homogenates from operated animals. An increased particle size in tissues from the adrenalectomized animals may possibly account for the observed changes in lysosomal stability.

Nutrition and lysosomal activity. The effect of dietary cod-liver oil on the distribution of polyunsaturated fatty acids in the kidney lysosomes of rats receiving deficient or adequate intakes of vitamin E.

1. In an attempt to explain the antagonism between polyunsaturated fatty acids (PUFA) and vitamin E in the promotion of post-mortem autolysis and increased lysosomal fragility in the kidney of rats, studies were made by gas chromatography of the incorporation of PUFA into the lysosomes.2. Kidneys were taken from rats that had received various diets, which differed in their fat components and which were with or without vitamin E. Since the inclusion of cod-liver oil in the diet reduces the period of dietary preparation necessary for rapid kidney autolysis, the effect of this oil on the PUFA distribution in the lysosomes was specially studied.3. In purified preparations of kidney lysosomes from rats that had received substantial amounts of cod-liver oil for several weeks, C 20:5 acid was incorporated mainly at the expense of C 18:2 (linoleic) and C 20:4 (arachidonic) acids. In less purified lysosomal fractions the incorporation of C 20:5 and C 22:6 acids and the corresponding reductions in linoleic and arachidonic were well advanced after 10 days, but were not maximal until about 30 days. The same changes took place in the reverse direction, with about the same rapidity, when rats that had previously been given cod-liver oil were changed to a diet containing lard.4. The percentage of PUFA in the kidney lysosomes of rats not dosed with vitamin E was not significantly different from that of rats given adequate doses.5. Thus the increased tendency to kidney autolysis, and the reduced stability of the lysosomes, caused by the feeding of cod-liver oil were associated with the partial replacement of linoleic acid and of the endogenous arachidonic acid by an acid, usually foreign to the rat, which is even more unsaturated. Since vitamin E did not prevent the entry of this acid into the lysosomes its potency in retarding autolysis and stabilizing the lysosomes must be exerted at some point subsequent to the incorporation of PUFA.6. Since change in the percentage of lysosomal PUFA in response to dietary changes is slow, this suggests that the lysosornal lipidsescape usage in general metabolism as an immediate source of calories and that they have a half-life of at least 15 days. Individual fatty acids, however, may differ in their half-lives.

Lysosomal function in the corpus luteum of the sheep.

SUMMARY A subcellular fraction containing particles showing the characteristics of lysosomes has been isolated from the corpus luteum of the sheep. Histochemical and biochemical observations have demonstrated that the lysosomes increase in size and fragility late in the oestrous cycle. Similar changes were not found in the corpora lutea of pregnant animals. The observed increase in lysosomal fragility is one of the earliest changes associated with luteal regression, and is thought to be of functional significance in the involution of the lutein cells. The possible modification of lysosomal function by the production of a cell-specific lytic factor from the uterus of non-pregnant sheep is discussed.

Decreased lysosome fragility in response to wounding in the skin and oral mucosa of the mouse.

DURING a comparative histochemical investigation of repair in the skin and oral mucosa of the mouse, a marked change in the distribution of acid phosphatase activity in response to wounding was noted in the epithelial cells.

Effect of X-Irradiation on the Fragility of Rat Spleen Lysosomes

The effect of various treatments on the release of acid phosphatase from spleen homogenates of irradiated rats was studied. Enzyme release increased 3 and 24 hr after a single dose of 1000 r of whole-body x irradiation. Spleen homogenate irradiated in vitro did not show increased acid phosphatase release. Rats injected with thyrotropic hormone or spleen homogenate treated with L- thyroxine in vitro showed an effect on enzyme release similar to that of x irradiation. Thus, the x-ray effect on this enzyme release cannot be attributed to a direct action on membranes of acid phosphatase-containing particles of the cells but is apparently mediated through a hormonal mechanism. (auth)

ACID RIBONUCLEASE IN LIVER CELL SAP.

THE acid ribonuclease of rat liver cytoplasm is located mainly in particles (‘lysosomes’), and shows ‘total’ activity only if a treatment such as freezing and thawing is applied1,2. However, as much as one-quarter of the cytoplasmic activity is found in the supernatant fraction from liver homogenates, even with a centrifugal force as high as 105,000g. This supernatant-fraction activity, expressed per gm of tissue, is markedly increased in the case of hepatomas3,4, precancerous liver from rats fed an azo dye3, or liver from adrenalectomized rats5. De Duve1 showed that autolysis likewise lead to increased supernatant-fraction activity of lysosomal enzymes, but remarked that the results “have been obtained on homogenates and it remains to be seen to what extent they apply to the cells themselves”; obviously an increase in supernatant-fraction activity could be attributed to increased fragility of lysosomes.