Labilization Of Membranes Research Articles

The effect of beta-thia-iminoprostacyclin in taurocholate acute pancreatitis in rats: the role of antecedent acute ethanol abuse.

The promoting effect of acute ethanol (E) abuse and protective effect of prostaglandin derivatives in acute pancreatitis (AP) remain obscure. The aim of this study was to assess the effect of previous intake of high-dose E on trypsinogen (Tn) activation and labilization of pancreatic lysosomal membranes (PLM), in taurocholate AP in rats, considering treatment with stable beta-thia-iminoprostacyclin (T). In 60 male Wistar rats taurocholate AP was induced or a sham operation was performed. Half of them received 40% E (5 g/kg body weight), 6 h earlier. T (0.3 mg/kg body weight i.g.) was applied before E or before the induction of AP. Free active (FAT) and total potential (TPT) trypsin, free (F) and total (T) cathepsin B, phospholipase A2 (PLA2), and lipase (L) activities were assayed. Percentage FAT/TPT was an index of Tn activation and fractional free (% F/T) activity of cathepsin B was an index of PLM fragility. FAT increased after 12 h of AP, and in E rats this increase was even more evident. Pretreatment and treatment with T partly prevented this increase, however, this effect was abolished or limited in rats previously given E-the changes were not effected by T. PLA2 and L activities in AP were not diminished after T. The promoting effect of acute E abuse prior to AP could be dependent on augmented activation of Tn and labilization of PLM. The protective effect of T seems to be dependent on the decrease in Tn activation in pancreatitic tissue. The potential therapeutic effect of this drug in AP could be limited by previous acute E intake, as evidenced by differences in histopathological changes.

Glycerol and Polylysine Synergize in Their Ability to Rupture Vesicular Membranes: A Mechanism for Increased Transferrin–Polylysine-Mediated Gene Transfer1

The presence of about 1.2Mglycerol during transfection with DNA/transferrin–polylysine and DNA/polylysine complexes dramatically increases transgene expression in a variety of cell types, provided that the complexes have an excess of polylysine. We have characterized this phenomenon using a human melanoma cell line (H225). The addition of 1.2Mglycerol to the transfection medium has no influence on the internalization of DNA complexes or on the promoter activity used to direct reporter gene expression. Neither pre- nor postincubation of the cells with glycerol results in a notable increase in transgene expression. Bafilomycin A1and chloroquine, two drugs affecting the endosomal pathway, both influenced transgene expression, indicating that glycerol acts on internal vesicles. Glycerol and polylysine synergized in their ability to lyse erythrocytes as well as internal vesicles (microsomes) isolated from H225 cells, indicating that the glycerol effect is due to a labilization of vesicular membranes, which facilitates membrane disruption by polylysine. Our current model suggests that the excess of polylysine in the DNA complexes disrupts vesicular membranes in the presence of glycerol, thus allowing the release of DNA complexes into the cytoplasm.

Hepatic mitochondrial and lysosomal alterations in acute experimental pancreatitis with ethanolic coetiology in rats

In order to assess the cumulative effects of antecedent acute ethanol intake and acute pancreatitis on the liver, the mitochondrial respiratory functions and lysosomal membrane integrity of the liver were evaluated in taurocholate pancreatitis (AP) in rats, induced 6 hr after intragastric ethanol 40% (5 g/kg body wt). The oxygen consumption rate, RCR (respiratory control ratio), and ADP/O ratio were measured according to Estabrook. Fractional free activity of lysosomal hydrolases was assayed. RCR with glutamate + malate was most decreased at 12 hr of AP with partial improvement after 18 hr. The ADP/O ratio dropped maximally after 18 hr of AP. The fragility of lysosomal membranes increased significantly at 18 hr of AP. The antecedent ethanol intake abolished the partial restoration of RCR after 18 hr; however, it did not affect the ADP/O ratio or the integrity of lysosomal membranes impaired in AP at this time. In conclusion, the antecedent acute ethanol abuse could aggravate the liver mitochondrial deterioration, but not the lysosomal membrane labilization seen in AP.

Approach to Estimation of Pesticide Occupational Hazard

In the present work, mussels (Mytilus edulis) were transplanted into a copper (Cu) gradient in Visnes (Norway) for a period of 3 weeks during November 2003. Sites 1 and 2 showed similar low levels of Cu, site 3 had intermediate Cu levels and site 4 was the most polluted with Cu as confirmed by AAS of digestive gland tissue. Values of lysosomal membrane labilization period were significantly lower at sites 3 and 4 compared to sites 1 and 2. The volume density and size of lysosomes was significantly decreased at site 4. The volume density of neutral lipids was also significantly lower at site 4 compared with the rest of sites. The volume density of lipofuscins showed significantly higher values at sites 2 and 3 compared to the reference site 1. Similar results were obtained regarding bioavailable metal levels measured by autometallography. All together, results are indicative of exocytosis of metal-containing lysosomes and lipofuscins to the digestive tubule lumen in mussels from site 4. In fact, autometallographic metal deposits were detected in digestive tubule lumen, brown cells and stomach in site 4 mussels. In agreement, there was a loss of digestive cells in mussels from site 4 (atrophy of the digestive epithelium) and cell type replacement (diminished volume density of digestive cells and increased volume density of basophilic cells). In conclusion, selected biomarkers indicated that mussels transplanted to sites closest to the Cu mine showed significant differences in metal accumulation pattern and in organization of the digestive gland tissue. Finally, female mussels closest to the Cu mine showed advanced gametogenesis with higher gonad index and vitellogenin-like protein levels than mussels at sites 1 and 2.

An in situ study of beta‐glucosidase activity in normal and gaucher fibroblasts with fluorogenic probes

Beta-glucosidase activity was evaluated in situ by means of fluorogenic probes in normal human fibroblasts and fibroblasts from homozygous carriers of the Gaucher trait. Probe internalization, targeting to lysosomes and post-cleavage probe retention were the primary concerns. Internalization and targeting were attempted by in situ photosensitized labilization of lysosomal membranes, lysosomotropic detergents and the use of low density lipid (LDL) or the receptor ligand apolipoprotein E (ApoE). Post-cleavage increase of fluorescence with fluoresceinyl (bis) beta-glucopyranoside was appreciably above the rather large pre-cleavage emission. In cells incubated overnight with nonylumbelliferyl-beta-glucoside (UG9) in the presence of bovine serum albumin and in the absence of ApoE, the probe was dealt with as a cytotoxic agent, accumulating in a paranuclear cap, most likely comprising elements of the endoplasmic reticulum (ER) and Golgi apparatus. Targeting of UG9 to lysosomes occurred within 1 to 3 h of preincubation in the presence of ApoE. There was some evidence of specificity, as Gaucher fibroblasts exhibited weaker cleavage of UG9 (by 50 per cent or more) compared to normal fibroblasts, but in the Gaucher cells there was some residual beta-glucosidase activity. Cleavage of UG9 was nearly totally suppressed in Gaucher cells treated with the beta-glucosidase inhibitor, conduritol B epoxide, for 24 h to 7 days. Suppression in the control fibroblasts was evident but to a lesser degree. The in situ method of fluorogenic assay established for beta-glucosidase deficiency, is in principle applicable to enzyme deficiencies in other lysosomal storage diseases, or to evaluate enhanced enzyme activity following gene therapy.

Postmortem changes in the rat kidney. II. Histopathological, electron microscopical, and enzyme histochemical studies of postmortem changes at 0.DEG.C..

In the first part of this study, we reported that the postmortem changes in rat kidneys at room temperature proceed most rapidly in the epithelial cells of the distal tubule and thick ascending limb of the loop of Henle, and that one of the factors which determine the rapidity of the postmortem changes at room temperature is the intensity of K-NPPase (Na, K-ATPase) activity. In the present study, the postmortem changes in rat kidneys maintained at 0°C for 20, 48, and 72hr after death were studied histopathologically, electron microscopically, and histo- and cytochemically to obtain further information on the mechanisms of postmortem changes. At 0°C, the postmortem changes in the distal tubules and thick ascending limb of the loop of Henle were delayed considerably, and only mild changes were observed electron microscopically even 72hr after death. Contrary to the results at room temperature, the postmortem changes in the proximal tubules were quicker than those in the distal tubules at 0°C, and the proximal tubules show obvious cytolysis 48hr or more after death. Electron microscopy revealed swelling of the lysosomes, and in enzyme cytochemistry, reaction products indicating ACPase activity increased in the cytoplasm around the lysosomes 48hr after death. These findings suggest that the lysosomal enzymes leak out from the lysosomes at least 48hr after death at 0°C. The lysosomal enzymes were considered to have activity even at 0°C, because ACPase activity could be detected cytochemically at 0°C in the lysosomes. The cytolysis observed in the proximal convoluted tubules at 0°C was not seen in the kidneys flushed with chlorpromazine, a membrane stabilizer, and was severe in the kidneys flushed with Triton X-100, a membrane labilizer. From these results, the postmortem cytolysis observed especially in the proximal convoluted tubules 48hr or more after death even at 0°C is considered to be autolysis caused by the lysosomal enzymes which leak out from the lysosomes. Together with the result of the previous part of this study, it is concluded that there are at least two biological factors which determine the rapidity of the postmortem changes in the cells of the rat kidney, the intensity of cellular Na, K-ATPase activity and the lability of the lysosomal membrane.

Postmortem changes in the rat kidney. I. Histopathological, electron microscopical, and enzyme histochemical studies of postmortem changes at room temperature.

In the first part of this study, we reported that the postmortem changes in rat kidneys at room temperature proceed most rapidly in the epithelial cells of the distal tubule and thick ascending limb of the loop of Henle, and that one of the factors which determine the rapidity of the postmortem changes at room temperature is the intensity of K-NPPase (Na, K-ATPase) activity. In the present study, the postmortem changes in rat kidneys maintained at 0°C for 20, 48, and 72hr after death were studied histopathologically, electron microscopically, and histo- and cytochemically to obtain further information on the mechanisms of postmortem changes. At 0°C, the postmortem changes in the distal tubules and thick ascending limb of the loop of Henle were delayed considerably, and only mild changes were observed electron microscopically even 72hr after death. Contrary to the results at room temperature, the postmortem changes in the proximal tubules were quicker than those in the distal tubules at 0°C, and the proximal tubules show obvious cytolysis 48hr or more after death. Electron microscopy revealed swelling of the lysosomes, and in enzyme cytochemistry, reaction products indicating ACPase activity increased in the cytoplasm around the lysosomes 48hr after death. These findings suggest that the lysosomal enzymes leak out from the lysosomes at least 48hr after death at 0°C. The lysosomal enzymes were considered to have activity even at 0°C, because ACPase activity could be detected cytochemically at 0°C in the lysosomes. The cytolysis observed in the proximal convoluted tubules at 0°C was not seen in the kidneys flushed with chlorpromazine, a membrane stabilizer, and was severe in the kidneys flushed with Triton X-100, a membrane labilizer. From these results, the postmortem cytolysis observed especially in the proximal convoluted tubules 48hr or more after death even at 0°C is considered to be autolysis caused by the lysosomal enzymes which leak out from the lysosomes. Together with the result of the previous part of this study, it is concluded that there are at least two biological factors which determine the rapidity of the postmortem changes in the cells of the rat kidney, the intensity of cellular Na, K-ATPase activity and the lability of the lysosomal membrane.

Comparative study of toxic lead effect on gill and haemoglobin of tilapia fish

This study was designed to determine the 72-h LC50 of lead for tilapia fish (Oreochromis hornorum), as well as the effect of exposure to sublethal lead concentrations (15, 23, 31, 39 and 47% of the LC50) on gill tissue lysosomal membranes of the fish and thaemoglobin concentration in blood. The LC50 value was found to be 202 mg Pb2+ l-1. Exposure to sublethal lead concentrations for 72 h showed significant increases in the lability of gill lysosomal membranes, measured by the release of acid phosphatase. Changes in membrane lability and in haemoglobin concentration were dependent on the amount of lead used during the exposure. We considered that the membrane lability is an adequate parameter to assay for monitoring lead contamination in water, because it is more sensitive than the haemoglobin concentration in blood.

Lysosomal enzyme release in the bluegill sunfish (Lepomis macrochirus Rafinesque) exposed to cadmium.

Lysosomal membrane lability and enzyme activities were measured in gill and liver tissues of the bluegill sunfish (Lepomis macrochirus). Exposure to sublethal cadmium concentrations for 10 or 21 days resulted in significant increases in the lability of liver lysosomal membranes, as measured by release of N-acetyl-β-D-glucosaminidase (NAG) and acid phosphatase (ACP). Acid phosphatase and NAG activities in liver were decreased by 10 day exposure to cadmium. However, after 21 days of exposure, liver NAG activity was elevated. Cadmium exposure did not affect the lysosomal lability index or the total enzyme activity of lysosomes isolated from gill tissue.In vitro exposure of isolated liver lysosomes to cadmium demonstrated that the increase in membrane lability was not a direct effect of cadmium on the lysosome. Stress induced by crowding the fish had no effect on liver lysosomal membrane lability. Starved bluegill sunfish had significantly destabilized lysosomes, as measured by both NAG and ACP. However, starvation did not affect the total activity of either enzyme. Neither size nor sex had any effect on lysosomal membrane lability, which indicates that field experiments will not be confounded by these factors. The problem of metal bioconcentration in fish has created the need for diagnostic tests which can be utilized to understand the biological effects of these contaminants on fish. The lysosomal membrane lability assay currently holds promise for use as a health monitoring tool.

Structural and enzymic disorganization of biological membranes in liver cells of rats with thermal burns

The state after extensive and deep thermal burns is characterized by the development of dystrophic and destructive processes, whose intensity varies in different organs outside the burned zone. These changes are based on disturbances of the normal properties of cell membranes [2, 3, 5, 6]. This is mainly the case with cell and lysosomal membranes. Meanwhile we know that in response to the harmful action of several toxic external environmental factors, universal labilization of membranes develops in the body [4]. Such a response may perhaps be characteristic also of the post-burned state, but no information on this matter can be found in the literature.

Studies on gentamicin-induced labilization of rat kidney lysosomes in vitro: Possible protection by selenium

The labilizing effect of gentamicin, an aminoglycoside antibiotic, on isolated rat kidney lysosomes was investigated. The light-scattering behavior of lysosomal suspensions and the release of lysosomal acid hydrolase enzymes (acid phosphatase, β-glucuronidase and muramidase) from incubated lysosomal suspensions, in the presence of gentamicin, were used as indices of lysosomal membrane labilization. Gentamicin was found to cause a decrease in light absorbance and a release of lysosomal acid hydrolases, which indicate lysosomal membrane swelling. In the presence of selenium, in the form of potassium selenate, the decrease in light absorbance of lysosomal suspensions and the release of lysosomal acid hydrolases from isolated lysosome particles were reduced markedly. This suggests that selenium protects against gentamicin-induced lysosomal membrane labilization. The possible mechanisms of protection by selenium are discussed.

The Effect of Retinol on the Hyperthermal Response of Normal Tissue in Vivo

The effect of prior administration of retinol, a membrane labilizer, on the in vivo hyperthermal response of lysosomes was investigated in the mouse spleen using a quantitative histochemical assay for the lysosomal enzyme acid phosphatase. A dose of retinol which had no effect when given alone enhanced the thermal response of the lysosome, causing an increase in lysosomal membrane permeability. In contrast, the same dose of retinol had no effect on the gross hyperthermal response of mouse intestine; a tissue which is relatively susceptible to hyperthermia. Thermal damage to intestine was assayed directly by crypt loss 1 day after treatment or assessed as thermal enhancement of X-ray damage by counting crypt microcolonies 4 days after a combined heat and X-ray treatment. Thus, although the hyperthermal response of the lysosome could be enhanced by the administration of retinol, thermal damage at a gross tissue level appeared to be unaffected, suggesting that lysosomal membrane injury is unlikely to be a primary event in hyperthermal cell killing.

Antiatelectatic function of lung surfactant

Activity and intracellular distribution of lysosomal glucosidases (beta-glucosidase and beta-galactosidase) of the cornea as well as membrane permeability were studied in experimental herpetic keratitis. It was demonstrated that the action of herpes simplex entailed labilization of the lysosomal membranes of the cornea together with the decreased strength of lysosomal association with the enzymes thereby favouring the increased enzyme-substrate contact and excess destruction of cellular and exocellular glycosaminoglycans. The data obtained suggest that lysosomes participate in the pathogenesis of herpetic keratitis.

Effect of lysosome modification on the heat potentiation of radiation damage and direct heat death of BP-8 sarcoma cells

It has been suggested that lysosomes represent the intracellular target responsible for heat potentiation of radiation damage and direct heat death in mammalian cells. To investigate the hypothesis that heat damages cells by lysosmal activation, BP- 8 murine sarcoma celle were treated with trypan blue. This agent accumulates within lysosomes and causes pronounced inhibition of lysosmal enzymes. To evaluate the possibility that heat damage is mediated by lysosmal membrane instability and leakage of hydrolases into the cytoplasm, BP- 8 cells were treated with a membrane labilizer (retinol) or stabilizer (hydrocortisone). The pre-treated cells were then subjected to hyperthermia ( 1 hr incubation at 37°–43°C), or to X-irradiation at normal ( 37°C) vs elevated ( 41.5°C) temperatures. The results indicate that pretreatment with trypan blue, retinol or hydrocortisone does not exert any effect on the heat response of BP- 8 sarcoma cells. Therefore, it would seem unlikely that lysosomes are responsible for either thermal enhancement of radiation damage or direct heat-induced cell death.

In vitro toxicity tests of zinc phosphate cement

BHK-21 (C-13) cells were exposed to freshly mixed zinc phosphate cement for 0.5, 1 and 2 h in a simulated cavity apparatus. The effect on the cells was evaluated by quantitative measurement of acid phosphatase and succinic dehydrogenase reaction product in order to determine organelle membrane permeability. After 0.5 h cell-material contact there was no significant effect. After 1 h cell-material contact, there was slight labilization of the lysosomal membranes, and a significant decrease in succinic dehydrogenase activity. The mean overall stain density in the experimental cells was 109% and 57% of the control values for acid phosphotase and succinic dehydrogenase respectively. After 2 h cell-material contact, there was significant labilization of the lysosomal membranes, and also labilization of the mitochondrial membranes. The mean overall stain densities were 114% and 75% of the control values for acid phosphatase and succinic dehydrogenase respectively. The relevance of these findings to the in vivo situation is discussed.

Acute mitochondrial toxicity of caffeine in cultured heart cells.

The cytotoxic effect of caffeine on heart cells was evaluated in primary cultures of rat heart muscle (M) and endothelioid (E) cells. The cultures were exposed to 5, 10, or 20 mM caffeine for 6, 12, or 24 hours. As an index of cytotoxicity produced by the treatments, in situ changes in mitochondrial membrane fragility were measured by a sensitive cytochemical technique. The 24 hr treatment with 20 mM caffeine was lethal to both E and M cell cultures; only the 12 hr treatment of M cell cultures with 20 mM caffeine produced significant labilization of mitochondrial membranes. The results of tests with other concentrations and durations of treatment were not statistically significant.

Effect of subextremal and extremal factors on liver lysosomes

The state of the lysosomal apparatus of the liver in rats was studied under the influence of subextremal and extremal factors (intensive physical exertion, starvation, and a combination of the two). When extremal stimuli act on the body, acid hydrolase activity in the liver rises and labilization of the lysosomal membranes takes place. Activation of the lysosomal apparatus corresponds to the degree of stress on the body and is an advantageous process aimed at the adaptive reorganization of the structures and metabolism of the cells.

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.

Studies on vinblastine-induced autophagocytosis in the mouse liver.I. The relation of lysosomal changes to general injurious effects.

Intraperitoneal injection of two doses of vinblastine (10 mg/kg and 50 mg/kg) induced a prominent formation of autophagic vacuoles in mouse liver parenchymal cells in 4 h. Clearly recognizable organelles were seen inside these vacuoles. The amount of autophagy was dependent on doses in that autophagosomes almost disappeared within 12 h with the low dose, whereas with the high dose the -ells were filled with residual bodies. Defecation of lysosomal material was ovserved into the sinusoidal lumen 12 h after injection with high dose. The total activities of lysosomal enzymes acid phosphatase, beta-galactosidase, and beta-acetylglucosaminidase did not change in the liver after vinblastine administration. The soluble activities of beta-galactosidase and beta-glucosaminidase were elevated with the high dose 12 h after injection, indicating labilization of the lysosomal membranes. Simultaneously the activities of the three lysosomal enzymes were elevated in the serum. The injurious effect of VBL appeared in light-and electron microscopic levels indicating diffuse necrosis of the liver lobules with the high gose within 12 h, fat accumulation in the cells, accumulation of secretory vesicles containing very low density lipoprotein particles, partial dilatation, vesiculation of endoplasmic reticulum and dilatation of Golgi cisternae. The serum GOT and CPK levels were also elevated

Effect of prostaglandin F2? on lysosomal membranes of the eye tissues

Both in experimentsin vitro and after intravenous injection of prostaglandin (PG) F2α labilization of the lysosomal membranes takes place in the sclera and ciliary body but not in the cornea of the rabbit eye. Under the influence of PG, glycosidase activity appears in the vitreous body, where it cannot be found normally.