Acid phosphatase activity and the state of lysosomal membranes in a primary culture of cardiomyocytes of newborn rats during hypoxia

Acid phosphatase activity, ATP and cyclic nucleotide levels were studied in primary cardiomyocyte cultures of newborn rats under hypoxic conditions. It was shown that incubation of cardiac cells in Eagle's medium supplemented with 10% calf serum during hypoxia (5% O2, 5% CO2, 90% N2) caused an hour later a decrease in lysosomal acid phosphatase activity, whereas free enzyme activity increased. The exposure of cells in Hanks salt solution to one hour hypoxia resulted in a 3-fold decrease of ATP level and further increase in free acid phosphatase activity. It was found that cAMP and cGMP levels do not depend on the presence of hypoxia. The mechanisms involved in the regulation of lysosomal membrane state are discussed.     

Induction of liver lysosomal enzymes during the autophagic phase following phenobarbital treatment of rat

Phenobarbital was given to male rats as a single injection and as repetitive injections for 7 days. The effects of treatment on the lysosomal hydrolases acid phosphatase, cathepsin D, and aryl sulfatase were analyzed at different intervals ranging from 1 to 15 days after seven injections, and from 1 to 48 h after a single injection. In both cases, microsomal protein and NADPH-cytochrome c reductase were measured to ensure proper induction. After a single injection, a slight decrease in hydrolytic activities was observed. Repetitive administration of phenobarbital gave rise to a marked decrease of lysosomal enzyme activities 1 day after cessation of treatment. This decrease was followed by a continuous increase in activity up to day 3 and 4. One or 2 weeks after treatment, enzyme activities declined to control values. The increase in activity of lysosomal hydrolytic enzymes was correlated with the onset of induced autophagy of endoplasmic reticulum membranes described as occurring in liver upon cessation of phenobarbital exposure. It is concluded that phenobarbital treatment per se decreases lysosomal enzyme activities, whereas the induced autophagy following cessation of exposure is associated with enhanced levels of lysosomal hydrolases in rat liver.     

The influence of tumor growth on natural cytotoxicity and activity of some lysosomal enzymes of human effector cells and the C3HA mouse splenocytes

In the course of malignant growth processes in patients with lung cancer, a decrease of natural cytotoxic activity of peripheral blood lymphocytes was observed. This process was accompanied by changes of activities of two lysosomal enzymes, arylsulfatase and acid phosphatase, suggesting participation of these enzymes in manifestation of effector functions of lymphocytes in cancer patients. The level of activity of granular enzyme, beta-glucuronidase, remained unchanged at all stages of disease. A study of natural killer activity of C3HA mice splenocytes after inoculation of transplantable hepatoma 22-a cells revealed a relative stability of the level of their cytotoxicity, and of the activities of lysosomal enzymes--arylsulfatase, acid phosphatase, alpha-mannosidase, acid lipase, N-acetyl-beta-D-glucosidase, and beta-galactosidase, beginning from the 3rd day after hepatoma implantation.     

Control of adaptations in protein levels in response to exercise

The nature of the contractile stimuli to which a skeletal muscle is subjected determines which proteins will increase in skeletal muscle. Rates of muscle protein synthesis decrease during an exercise bout for durations of less than 30 min. Synthesis has been reported to increase, remain unchanged, or decrease during exercise bouts lasting from 30 min to 7 h. Protein synthesis rates apparently increase when exercise exceeds 7 h. After short bouts of exercise, protein synthesis rates in muscles appear to decrease in the first hour after exercise, but in the second hour after exercise increase to levels greater than normal. We hypothesize that decreases in ATP and pH levels in muscle during contractile activity may dampen a calcium-mediated stimulation of translation of RNA. That the content of alpha-actin mRNA in muscles of immobilized limbs is unchanged when actin synthesis initially decreases suggests that a decrease in the translation of alpha-actin mRNA is the facilitating step in the decrease in actin synthesis. Rates of muscle protein degradation decrease during exercise if exercise duration is less than 12 h, but increase when exercise is continuous for a day. After intense exercise, rates of protein degradation in skeletal muscle may be increased. An increased ratio of NAD(P)H:NAD(P) in muscle during short-term exercise may decrease degradation. Increased lysosomal enzyme activity in muscle occurs during the postexercise period.     

Streptozocin model of diabetes mellitus in the mollusc Anodonta cygnea: functional state of the adenylyl cyclase mechanisms of action of peptides of the insulin superfamily and their effect on carbohydrate metabolism enzymes

In terms of development of evolutionary biomedicine using invertebrate animals as models for study of molecular grounds of various human diseases, for the first time the streptozocin (ST) model of insulin-dependent diabetes in the mollusc Anodonta cygnea has been developed. This model is based on the following authors' data: (1) redetection of insulin-related peptides (IRP) in mollusk tissues: (2) discovery of the adenylyl cyclase signal mechanism (ACSM) of action of insulin and other peptides of the insulin superfamily in tissues of mammals, human, and mollusc. A. cygnea; (3) concept of molecular defects in hormonal signal systems as causes of endocrine diseases. Studies on the ST model have revealed in mollusc smooth muscle on the background of hyperglycemia at the 2nd, 4th, and 8th day after the ST administration a decrease of the ACSM response to activating action of insulin, IGF-1, and relaxin. These functional disturbances were the most pronounced at the 2nd day of development and rather less marked at the 4th and 8th day. Analysis of data on effect of hormonal and non-hormonal (NaF, GIDP, and forskolin) ACSM activators has shown that the causes of impair of signal-transducing function of this mechanism are (1) a hyperglycemia-induced increase of the basal AC activity and as a consequence--a decrease of the enzyme catalytic potentials in response to hormone; (2) a decrease of functions of Gs-protein and of its coupling with AC. Besides, administration of ST produced in the mollusc muscles an attenuation of regulation by insulin of carbohydrate metabolism enzyme (glucose-6-phosphate dehydrogenase, glycogensynthase). The pattern of disturbances in the studied parameters in the mollusc is very similar to that revealed by the authors in rat and human muscle tissues in type 1 diabetes.     

Glutathione system of erythrocyte and plasma in peptic ulcer

A complex study of the blood glutathione system has been carried out for the first time in patients with peptic (gastric and duodenal) ulcer. In erythrocytes and blood plasma of patients with the complicated peptic ulcer and postgastroresection syndromes there was the increase of conjugated dienes (and in the second group the increase in antioxidant activity). Under these conditions the main change was the sharp and identical decrease in glutathione peroxidase activity. In patients with uncomplicated peptic ulcer there was sharp increase in erythrocite and plasma glutathione reductase activity and plasma GSH. In operated but basically healthy patients plasma glutathione peroxidase remained decreased but plasma GSH sharply increased. Evidently complicated peptic ulcer is characterized by decreased functioning of the glutathione system. Activation of this system and the decrease or disappearance of manifestations of oxidative stress are associated with a favorable course of this disease, especially at uncomplicated peptic ulcer. The revealed changes significantly differ from those observed in patients with viral hepatitis, blle excretory diseases and strokes.     

Increases in the activity of lysosomal DNAase in mouse liver during the development of Friend's viral leukemia]

A developing Friend's viral leukemia is accompanied by a 2,0--2,5-fold increase in the activity of lysosomal DNAse (DNAse II) in mouse liver as compared to normal. The increase in activity is observed on the 10--12th day after inoculation of virus-containing material and reaches its maximum on the 20th post-inoculation day. The increase in DNAse II activity is due to activation of the lysosomal system of Kupffer's and endothelial cells of the liver. The activity of mitochondrial DNAse (DNAse I) in the livers of leukemic mice showed no deviations from the normal level. A possible role of DNAse II in the protective response of the organism is discussed.     

Effects of AET and MEA on the lysosomal hydrolase activities in the mouse organs.

The adult male Swiss mice were injected intraperitoneally with AET (2-aminoethylisothiouronium Br.HBr) or MEA (cysteamine HCl), in a toxic dose of 400 mg/kg body weight. The acid phosphatase (E.C. 3.1.3.2) and arylsulphatase (E. C. 3.1.6.1) activities in crude homogenates of liver and kidneys were assessed every fourth hour throughout a 24-h period. Different patterns of temporal changes in the acid phosphatase and arylsulphatase activities in liver and kidneys expressed in nkat per 1 mg of protein, 1 g of fresh tissue and per the whole organ weight, were found. The extent and timing of the alterations in the activity of each of the lysosomal hydrolases were dependent on the particular organ chosen and aminothiol compound given.     

Activity and distribution of lysosomal enzymes during collagenous matrix-induced cartilage,bone, and bone marrow development

The appearance of the lysosomal enzymes acid phosphatase, arylsulfatase, and β-glucuronidase was studied during endochondral bone and bone marrow formation induced by implantation of demineralized bone matrix. The activities of acid phosphatase and β-glucuronidase gradually increased from the stage of mesenchymal cell proliferation on Day 3 onward to reach a peak on Day 13, during maximal bone remodeling. However, arysulfatase activity exhibited a sharp increase on Day 9, associated with the onset of cartilage hypertrophy and chondrolysis. The peak of arylsulfatase activity was also attained on Day 13. The activities of all three enzymes declined on Day 15 but acid phosphatase again exhibited an increase during hematopoietic bone marrow differentiation on Days 19–21. Histochemical and ultrastructural studies revealed intense lysosomal enzyme activity in macrophage-like cells on Day 7 and thereafter. During chondrolysis and bone remodeling, these cells were present in a perivascular location. Osteoclasts also exhibited strong reactivity for the lysosomal enzymes. Due to its characteristic temporal appearance during development of endochondral bone, arysulfatase may be used as a marker enzyme for chondrolysis and bone resorption.     

Alterations in the heart lysosomal stability in isoproterenol induced myocardial infarction in rats

The alterations in the heart lysosomal stability following isoproterenol induced myocardial infarction were studied in albino rats. The rate of release of beta-glucuronidase at various time intervals at 37 degrees C from lysosome rich fraction was taken as a measure of lysosomal stability. As compared to the control day one, three and five samples exhibited a significant increase in beta-glucuronidase activity at all the time intervals. The subcellular distribution of beta-glucuronidase was also studied and the soluble and total activities exhibited an increase at peak infarction stage and returned to normal during the recovery. The decrease in the lysosomal stability might be attributed to the increased beta-glucuronidase activity observed following myocardial infarction.     

Stobadine inhibits lysosomal enzyme release in vivo and in vitro

This study investigated the ability of stobadine, an effective cardioprotective drug with antiarrhythmic, antihypoxic and oxygen free radical scavenging properties, to protect cells against cyclophosphamide-induced toxic and cytotoxic damage in vivo and in vitro. Cyclophosphamide-induced toxic damage in female ICR mice was accompanied by marked increase in the activity of lysosomal enzymes in the spleen and kidney. Administration of stobadine prior to cyclophosphamide inhibited these biochemical changes. The in vivo protective effect of stobadine was comparable with its in vitro effect established in HeLa cells.     

Streptozotocin model of diabetes mellitus in the mollusc <Emphasis Type="Italic">Anodonta cygnea</Emphasis>: functional state of the adenylyl cyclase mechanisms of action of insulin superfamily peptides and their effect on carbohydrate metabolism enzymes

In terms of development of evolutionary biomedicine using invertebrate animals as models for study of molecular grounds of various human diseases, for the first time the streptozotocin (ST) model of insulin-dependent diabetes in the mollusc Anodonta cygnea has been developed. This model is based on the following authors’ data: (1) redetection of insulin-related peptides (IRP) in mollusc tissues: (2) discovery of the adenylyl cyclase signal mechanism (ACSM) of action of insulin and other peptides of the insulin superfamily in tissues of mammals, human, and mollusc A. cygnea; (3) concept of molecular defects in hormonal signal systems as causes of endocrine diseases. Studies on the ST model have revealed in mollusc smooth muscle on the background of hyperglycemia at the 2nd, 4th, and 8th day after the ST administration a decrease of the ACSM response to activating action of insulin, IGF-1, and relaxin. These functional disturbances were the most pronounced at the 2nd day of development and rather less marked at the 4th and 8th day. Analysis of data on effect of hormonal and non-hormonal (NaF, GIDP, and forskolin) ACSM activators has shown that the causes of impair of signal-transducing function of this mechanism are (1) a hyperglycemia-induced increase of the basal AC activity and as a consequence—a decrease of the enzyme catalytic potentials in response to hormone; (2) a decrease of functions of G_s-protein and of its coupling with AC. Besides, administration of ST produced in the mollusc muscle an attenuation of regulation by insulin of carbohydrate metabolism enzyme (glucose-6-phosphate dehydrogenase, glycogensynthase). The pattern of disturbances in the studied parameters in the mollusc is very similar to that revealed by the authors in rat and human muscle tissues in type 1 diabetes.     

Activity of lysosomal beta-glucuronidase in leukocytes of rats exposed to benzene and sodium selenate.

Chronic exposure to benzene results in rats in the decrease of the lymphocyte count in the peripheral blood, the decrease of the beta-glucuronidase (BG) activity both in lymphocytes and neutrophilic granulocytes as well as in the damage to lysosomal apparatus of lymphocytes expressed in diffusion of the enzyme within the cell cytoplasm. Administration of selenium (sodium selenate) in dosis of 1.0 microgram/Kg during consecutive 10 days prior the exposure to benzene resulted in prevention of benzene-induced decrease of the BG activity in granulocytes and of a damage to lymphocyte lysosomes. Application of selenium in dosis of 5.0 microgram/Kg during the same time prior the exposure to benzene prevented the benzene-induced lymphocytopenia, induced the reactive increase of the granulocyte number, and caused, moreover, the prevention of the BG activity decrease in granulocytes. Simultaneously the increase of the BG-positive lymphocyte percentage was noted which was related to the increase of cells exhibiting the cytoplasmatic and extralysosomal localization of the enzyme. The results suggest that only smaller doses of sodium selenate prevented the damage to lysosomal membrane of lymphocytes induced by toxic effect of benzene.     

Effects of fasting and insulin on hepatic phosphofructokinase (PFK)

Fasting of susceptible mice for 24 hr led to a sharp decrease of apparent hepatic PFK activity which was increased to near fed levels by incubation of cytosolic fractions with GSH. Incubation of the cytosols with GSSG reduced the PFK to a lower level-(GSSG insensitive activity). The action of insulin was to increase total PFK with a marked increase of the GSSG insensitive activity.     

Changes in enzyme activities of thymidine kinase and 5'-nucleotidase for dTMP during hormonal regeneration of seminal vesicles of mice.

An increase of thymidine kinase [EC 2.7.1.21] activity and decrease of 5'-nucleotidase [EC 3.1.3.5] activity for dTMP were found during hormonal regeneration of the seminal vesicles by daily or single administration of testosterone propionate into mice castrated 2 weeks previously. Actinomycin D injected on day 0 of testosterone treatment completely inhibited both the increase of thymidine kinase and the decrease of 5'-nucleotidase. When injected on day 2, actinomycin D decreased thymidine kinase activity below the control level and 5'nucleotidase activity was not restored to the normal level. The activity of 5'-nucelotidase in a mixed sample, in which seminal vesicles of castrated mice and those of testosterone-treated mice were homogenized together, was intermediate between the activities determined separately. This indicates the absence of any inhibitor of 5'nucleotidase in the regenerating vesicles. Changes in total activity of 5'nucleotidase and total protein content in extracts during various treatments showed that the decrease in specific activity of 5'-nucleotidase in the first 2 days of testosterone treatment was not due to inhibition of enzyme activity but to dilution of the enzyme with other proteins which increased in content more rapidly than 5'-nucleotidase.     

Study of lysosomal enzymes in chick embryo fibroblasts infected with microorganisms of family Halprowiaceae (Chlamydiaceae)

It is shown that infection of chick embryo fibroblasts with agents of paratrachoma and meningopneumonia Halprowiaceae (Chlamydiaceae) causes a sharp decrease of the activities of lysosomal enzymes, e.g. acidic alpha-glucosidase, beta-glucuronidase, beta-galactosidase, alpha-mannosidase, acid phosphatase, etc. The activity of cytosol enzymes (neutral alpha-glucosidase, amylo-1,6-glucosidase) does not change, however. A decrease in the activities of lysosomal enzymes in infected fibroblasts occurs some time later after inoculation and is due to a release of lysosomal enzymes from the fibroblasts into the culture medium, without loss of cell integrity. No changes in the activity of lysosomal enzymes in fibroblasts and culture medium is observed in the case of inoculation of cells with a killed agents, as well as after contact of cells with a suspension of normal chick embryo yolk sacs. The release of lysosomal enzymes from halprowiae-infected chick embryo fibroblasts probably occurs by the exocytosis.     

Phenylarsine oxide-induced increase in alveolar macrophage surface receptors: evidence for fusion of internal receptor pools with the cell surface

Rabbit alveolar macrophages which were treated at 0 degrees C with phenylarsine oxide and then incubated at 37 degrees C for 10 min exhibited a two- to threefold increase in surface receptor activity for macroglobulin.protease complexes, diferric transferrin, and mannose-terminal glycoproteins. Analysis of the concentration-dependence of ligand binding indicated that changes in ligand-binding activity were due to changes in receptor number rather than alterations in ligand-receptor affinity. Surface receptor number could also be increased by treatment of cells with three other sulfhydryl reagents, N-ethylmaleimide, p-chloromercurobenzoate, and iodoacetic acid. The increase in receptor activity was maximal after 10 min and decreased over the next hour. This decrease in cell-associated receptor activity was due to the release of large membrane vesicles which demonstrated a uniform buoyant density by isopycnic sucrose gradient centrifugation. Treatment of cells with phenylarsine oxide did not decrease the cellular content of lactate dehydrogenase or beta-galactosidase, indicating that cell integrity was maintained and lysosomal enzyme release did not occur. Our studies indicate that phenylarsine oxide treatment in the presence of extracellular Ca2+ results in the fusion of receptor-containing vesicles with the cell surface.     

The effect of TEPC-183 plasmacytoma growth on beta-glucuronidase activity in serum and tissues of tumor-bearing mice

beta-Glucuronidase activity increased in the serum of BALB/c mice during the growth of the IgM-secreting plasmacytoma, TEPC-183. The increase appeared to correlate with tumor burden. The beta-glucuronidase activity in tissue homogenates of spleen, liver, and kidney from tumor-bearing mice also increased significantly compared to the levels found in corresponding tissues from normal control mice. Assays of lysosomal and microsomal fractions from livers of TEPC-bearing mice indicated that approximately 70% of the enzyme activity was associated with the lysosomal fraction and the remainder with the microsomal fraction. A similar distribution was found in homogenates prepared from the plasmacytoma itself. In contrast to this the beta-glucuronidase activity in livers from normal BALB/c mice is nearly equally distributed between lysosomal and microsomal fractions.     

Different appearance of hepatic collagenase and lysosomal enzymes in recovery of experimental hepatic fibrosis.

1. Both activities of hepatic collagenase and lysosomal enzymes (acid phosphatase, beta-glucuronidase and N-acetyl-beta-D-glucosaminidase) have been observed in the recovery from experimental hepatic fibrosis in rats treated with carbon tetrachloride for 6 to 20 weeks, and compared with the disappearance of newly formed collagen fibers in the recovery process. 2. In the process of experimental hepatic fibrosis, collagenase activity reached maximum on sethe accumulation of collagen fibers in reversible hepatic fibrosis, but decreased to the same level as that of non-treated rat liver in cirrhotic stage. In the reocvery from reversible hepatic fibrosis, collagenase activity reached maximum on second day after the discontinuation of carbon tetrachloride, and decreased to the same extent of that of non-treated rat liver on seventh day. 3. Lysosomal enzyme activity was parallel to the activity of hepatic collagenase and to the accumulation of collagen fibers in the process of hepatic fibrosis. In the recovery stage, lysosomal enzyme activity in mesenchymal cells within the septa increased markedly on second day after the discontinuation of toxic agent but turned to the same level of that of non-treated rat liver seven days later, which was consistent with the appearance and disappearance of collagenase activity. On the other hand the appearance of lysosomal enzymes activities in Kupffer cells and hepatocytes was different from that of collagenase activity. That is lysosomal enzyme activity in Kupffer cells decreased in early days but increased five days later, and the enzyme activity in hepatocytes markedly decreased but gradually recovered to normal level seven days later. 4. The appearance of collagenase was observed at the beginning of the recovery stage. It indicates that mammalian collagenase initiates the collagen degradation and lysosomal enzymes might have a role in the subsequent degradation of collagen.     

Role of phospholipids in changes in lysosomal membrane stability in conditions of chronic alcoholic intoxication

Pronounced destabilization of liver lysosomal membranes has been revealed in rats in conditions of 30-day-long alcohol intoxication. Noticeable fractional changes in phospholipid composition of lysosomal membranes have been found. Significant increase in lysophosphatidylethanolamine and lysophosphatidylcholine levels have been observed. Type A2 phospholipase activity was found in lysosomal fractions, with the enzyme activity Ca2+-dependent, optimal at pH 8 and increasing many-fold following alcohol intoxication. The changes in lysosomal membrane phospholipids appear to be related to phospholipase A2 activation.