Protective effect of betaine on changes in the levels of lysosomal enzyme activities in heart tissue in isoprenaline-induced myocardial infarction in Wistar rats

Myocardial infarction is one of the most common manifestations of cardiovascular disease. In the present study, we investigated the protective effect of betaine, a potent lipotropic molecule, on changes in the levels of lysosomal enzymes and lipid peroxidation in isoprenaline-induced myocardial infarction in Wistar rats, an animal model of myocardial infarction in man. Male albino Wistar rats were pretreated with betaine (250 mg/kg body weight) daily for a period of 30 days. After the treatment period, isoprenaline (11 mg/100 g body weight) was intraperitoneally administered to rats at intervals of 24 h for 2 days. The activities of lysosomal enzymes (β-glucuronidase, β-galactosidase, β-glucosidase, and acid phosphatase) were significantly (p < 0.05) increased in plasma with a concomitant decline in the activities of these enzymes in heart tissue of isoprenaline-administered rats. Also, the level of lipid peroxidation was higher in heart lysosomes of isoprenaline-injected rats. Pretreatment with betaine daily for a period of 30 days to isoprenaline-induced rats prevented the changes in the activities of these lysosomal enzymes. Oral treatment with betaine (250 mg/kg body weight) to normal control rats did not show any significant effect in all the biochemical parameters studied. Thus, the results of our study show that betaine protects the lysosomal membrane against isoprenaline-induced myocardial infarction. The observed effects might be due to the free radical-scavenging and membrane-stabilizing properties of betaine.     

S-allylcysteine ameliorates isoproterenol-induced cardiac toxicity in rats by stabilizing cardiac mitochondrial and lysosomal enzymes

This study was aimed to evaluate the preventive role of S-allylcysteine (SAC) on mitochondrial and lysosomal enzymes in isoproterenol (ISO)-induced rats. Male albino Wistar rats were pretreated with SAC (50, 100 and 150 mg/kg) daily for a period of 45 days. After the treatment period, ISO (150 mg/kg) was subcutaneously injected to rats at an interval of 24 h for two days. The activities of heart mitochondrial enzymes (isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase and alpha-ketoglutarate dehydrogenase) and respiratory chain enzymes (NADH dehydrogenase and cytochrome C oxidase) were decreased significantly (p<0.05) in ISO-induced rats. The activities of lysosomal enzymes (beta-glucuronidase, beta-N-acetyl glucosaminidase, beta-galactosidase, cathepsin-D and acid phosphatase) were increased significantly (p<0.05) in serum and heart of ISO-induced rats. Pretreatment with SAC (100 mg/kg and 150 mg/kg) for a period of 45 days increased significantly (p<0.05) the activities of mitochondrial and respiratory chain enzymes and decreased the activities of lysosomal enzymes significantly (p<0.05) in ISO-induced rats. Oral administration of SAC (50, 100 and 150 mg/kg) for a period of 45 days to normal rats did not show any significant (p<0.05) effect in all the parameters studied. The altered electrocardiogram (ECG) of ISO-treated rats was also restored to near normal by treatment with SAC (100 and 150 mg/kg). These results confirm the efficacy of SAC in alleviating ISO-induced cardiac damage.     

Grape seed proanthocyanidins ameliorates isoproterenol-induced myocardial injury in rats by stabilizing mitochondrial and lysosomal enzymes: an in vivo study

This study was designed to examine the effects of grape seed proanthocyanidins (GSP) against myocardial injury (MI) induced by isoproterenol (ISO), in a rat model. Induction of rats with ISO (85 mg/kg body weight, subcutaneously) for 2 days resulted in a significant decrease in the activities of heart mitochondrial enzymes (isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase and alpha-ketoglutarate dehydrogenase) and respiratory chain enzymes (NADH dehydrogenase and cytochrome c oxidase). The activities of lysosomal enzymes (alpha-d-glucuronidase, alpha-d-N-acetylglucosaminidase, cathepsin-D, acid phosphatases and alpha-d-galactosidase) were increased significantly in the heart and serum of ISO-induced rats. The prior administration of GSP for 6 days a week for 5 weeks significantly increased the activities of mitochondrial and respiratory chain enzymes and significantly decreased the activities of lysosomal enzymes in the heart tissues of ISO-induced rats, which proves the stress stabilizing action of GSP. Oral administration of grape seed proanthocyanidins alone (50, 100 and 150 mg/kg) to normal rats did not show any significant effect in all the parameters studied. These biochemical functional alterations were supported by the macroscopic enzyme mapping assay of ischemic myocardium. Thus, this study shows that 100 and 150 mg/kg of GSP gives protection against ISO-induced MI and demonstrates that GSP has a significant effect in the protection of heart.     

Preventive effect of (-)epigallocatechin-gallate (EGCG) on lysosomal enzymes in heart and subcellular fractions in isoproterenol-induced myocardial infarcted Wistar rats

This study was aimed to evaluate the preventive role of (-)epigallocatechin-gallate (EGCG) on lysosomal enzymes in isoproterenol (ISO)-induced myocardial infarcted rats. Male albino Wistar rats were pretreated with EGCG (30 mg/kg) daily for a period of 21 days. After the treatment period, ISO (100 mg/kg) was subcutaneously injected to rats at intervals of 24h for 2 days. The activities of lysosomal enzymes (beta-glucuronidase, beta-N-acetylglucosaminidase, beta-galactosidase, cathepsin-B and cathepsin-D) were increased significantly (P<0.05) in serum and the heart of ISO-induced rats. ISO-induction also resulted in decreased stability of membranes, which was reflected by decreased activities of beta-glucuronidase and cathepsin-D in mitochondrial, nuclear, lysosomal and microsomal fractions. Pretreatment with EGCG daily for a period of 21 days to ISO-induced rats prevented the changes in the activities of these enzymes. Oral treatment with EGCG (30 mg/kg) to normal control rats did not show any significant effect in all the biochemical parameters studied. Thus, the results of our study shows that EGCG protects the lysosomal membrane against ISO-induced cardiac damage. The observed effects might be due to the free radical scavenging and membrane stabilizing properties of EGCG.     

Curcumin treatment modulates collagen metabolism in isoprot3erenol induced myoxardial necrosis in rats

This study was carried out to evaluate whether curcumin, a potent antioxidant, had any specific role in the synthesis and degradation of collagen in rat heart with mocardial necrosis, induced by isoproterenol.HCI (ISO). Myocardial necrosis was induced by administration of ISO (30 mg/100 g body weight subcutaneously twice at an interval of 24 h) and studies on collagen metabolism were carried out with curcumin (200 mg/kg) pre-and co-treatment with ISO. The incorporation of ₁₄C-proline into collagen was studied as an index of collagen synthesis. The heart weight /body weight ratio,heart RNA/DNA ratio and protein were found to increase significantly in ISO administered animals. Curcumin pre- and co-treatment with ISO reversed these changes and attenuated the development of cardiac hypertrophy two weeks after the second dose of ISO. Increased fractional synthesis rate and enhanced degradation of newly synthesized collagen were observed in ISO administered animals. Curcumin pre- and co-treatment with ISO was noticed to decrease the degree of degradation of the existing collagen matrix and collagen synthesis, two weeks after the second dose of ISO. The observed effects could be due to free radical scavenging capacity and inhibition of lysosomal enzyme release by curcumin.     

Effect of sodium fluoride and magnesium chloride on different hydroxyproline fractions in rat liver

Sodium fluoride (NaF) is used for prevention of caries in the form of fluoridated drinking water, fluoride tablets etc. In the present study, the effect of NaF-induced alterations in hydroxyproline (Hyp) and collagen was investigated in rat liver. The effect of pretreatment with MgCl2 on NaF-induced changes in liver Hyp and collagen was also studied. The NaF treatment at 5, 10 and 20 mg/kg body wt (reported LD50 of NaF being 24 mg/kg body wt through intraperitoneal route) caused a significant decrease in free Hyp (P < 0.05), when compared to control rats. The rats treated with 20 mg/kg body wt of NaF showed a significant increase in protein-bound Hyp (P < 0.001), as compared to control group, while of NaF treatment at 5 and 10 mg/kg body wt caused no significant change in protein-bound Hyp. All the doses of NaF had no significant effect on peptide-bound and total Hyp and total collagen. Treatment of with MgCl2 alone (30 mg/kg body wt) or with NaF (10 mg/kg body wt) caused a significant decrease in free Hyp (P < 0.05). MgCl2 alone and with NaF caused a significant increase (P < 0.05) in total collagen content. Thus, the present study demonstrated that NaF had no significant effect on total Hyp and collagen, indicating that its use in various products may not interfere with the liver collagen.     

Therapeutic effects of Semecarpus anacardium Linn. nut milk extract on the changes associated with collagen and glycosaminoglycan metabolism in adjuvant arthritic Wistar rats

The effect of milk extract of Semecarpus anacardium Linn. nut milk extract (SA) was studied to gain some insight into this intriguing disease with reference to collagen metabolism. Arthritis was induced in rats by injecting Freund's complete adjuvant containing 10mg of heat killed mycobacterium tuberculosis in 1 ml paraffin oil (0.1 ml) into the left hind paw of the rat intradermally. After 14 days of induction, SA (150 mg/kg body weight/day) was administered orally by gastric intubations for 14 days. Decreased levels of collagen and glycosaminoglycans (GAGS) components (chondroitin sulphate, heparan sulphate, hyaluronic acid) and increase in the levels of connective tissue degrading lysosomal glycohydrolases such as acid phosphatase, beta-glucuronidase, beta-N-acetyl glucosaminidase and cathepsin-D observed in arthritic animals were reverted back to near normal levels upon treatment with SA. The drug effectively regulated the uriniray markers of collagen metabolism namely hexosamine, hexuronic acid, hydroxyproline and total GAGS. Electron microscopic studies also revealed the protective effect of SA. Hence, it can be suggested that SA very effectively regulate the collagen metabolism that derange during arthritic condition.     

Toxic effects of different concentrations of dimethoate on lysosomal enzymes of female albino rats.

The effect of three different concentrations of dimethoate on the activity of certain lysosomal enzymes, viz. beta-glucuronidase, beta-N-acetylglucosaminidase, cathepsin B and cathepsin D in serum, skin, liver, kidney and spleen and the stability of liver and kidney lysosomes was studied in female albino rats. The activity of beta-glucuronidase, beta-N-acetylglucosaminidase, cathepsin D was found to increase in serum and tissues in higher concentration (2.25 mg/100 g body weight) of dimethoate treated rats. A significant increase in the rate of release of beta-glucuronidase was found in the liver and kidney of higher concentration of dimethoate treated rats compared to controls. The results demonstrate that the activity of lysosomal enzymes increased in higher concentration of dimethoate treated rats than the lower concentration (0.56 mg/100 g body weight) of dimethoate treated rats.     

Preventive effect of caffeic acid on lysosomal dysfunction in isoproterenol‐induced myocardial infarcted rats

We evaluated the preventive effect of caffeic acid (CA) on lysosomal enzymes in isoproterenol (ISO)‐treated myocardial infarcted rats. Male albino Wistar rats were pretreated with CA (15 mg/kg) daily for a period of 10 days. After the pretreatment period, ISO (100 mg/kg) was subcutaneously injected to rats twice at an interval of 24 h. The activity of serum creatine kinase‐MB and lactate dehydrogenase was increased significantly (P < 0.05) in ISO‐induced myocardial infarcted rats. The levels of plasma thiobarbituric acid reactive substances and lipid hydroperoxides were significantly (P < 0.05) increased, and the level of plasma‐reduced glutathione was significantly (P < 0.05) decreased in ISO‐induced myocardial infarcted rats. The activities of lysosomal enzymes (β‐glucuronidase, β‐N‐acetylglucosaminidase, β‐galactosidase, cathepsin‐B and cathepsin‐D) were increased significantly (P < 0.05) in the serum and heart of ISO‐induced myocardial infarcted rats. ISO induction also resulted in decreased stability of membranes, which was reflected by lowered activities of β‐glucuronidase and cathepsin‐D in different fractions except cytosol. Pretreatment with CA (15 mg/kg) to ISO‐treated rats significantly (P < 0.05) prevented the changes in the activities of cardiac marker enzymes, the levels of lipid peroxidation products, reduced glutathione and the activities of lysosomal enzymes in the serum, heart, and subcellular fractions. Oral treatment with CA (15 mg/kg) to normal control rats did not show any significant effect. Thus, the results of our study showed that CA prevented the lysosomal membrane damage against ISO‐induced myocardial infarction. The observed effects of CA are due to membrane‐stabilizing, antilipo peroxidative, and antioxidant effects. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:115–122, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20319     

Increased degradation of dermal collagen in diabetic rats.

The effect of alloxan induced diabetes on the dermal collagen content of albino rats was studied in relation to few lysosomal enzymes. Diabetes decreased the dermal collagen content. The specific activities of the lysosomal enzymes studied in the diabetic rat skin were elevated. It has been established that lysosomal enzymes degrade the connective tissue components. Thus, it may be suggested that the increase in the lysosomal enzymes studied should have facilitated the decrease in dermal collagen content of diabetic rats by increasing the degradation of dermal collagen.     

Phosphomannose binding proteins: the phosphomannosyl receptor and insulin like growth factor II receptor

Proteins that bind phosphomannose residues in glycoproteins exhibit widely different functions. They are found as receptors of lysosomal enzymes, as ligatin that binds peripheral glycoproteins and as a lectin in parasites. The identity of the phosphomannosyl receptor for lysosomal enzymes and the insulin like growth factor II receptor raises interesting questions regarding their function.     

Centripetal cholesterol flow from the extrahepatic organs through the liver is normal in mice with mutated Niemann-Pick type C protein (NPC1)

Niemann-Pick type C (NPC) protein functions to move unesterified cholesterol from the lysosomal compartment to other intracellular sites for further metabolism and/or excretion. This cholesterol is brought into the cell through the coated-pit pathway and accumulates in the lysosomes when NPC protein is mutated. The present study quantitated the alternative uptake process that brings cholesterol into the cell through the scavenger receptor, class B, type I (SR-BI) pathway in animals with this mutation. In homozygous NPC mice, the tissues of the extrahepatic compartment accumulated an excess of 14 mg of cholesterol each day per kg body weight, and synthesis increased by a similar amount (to 111 mg/day per kg) to compensate for this functional loss of sterol through lysosomal sequestration. An amount of cholesterol (108 mg/day per kg) nearly equal to that synthesized in the extrahepatic compartment was carried through the circulation by high density lipoprotein (HDL) and taken up by the liver. The rate of hepatic cholesterol excretion from the NPC mice as fecal acidic (65 mg/day per kg) and neutral (85 mg/day per kg) sterols was elevated 61% above control values and was accounted for by the total amount of cholesterol brought to the liver in HDL and synthesized in the hepatocytes. These studies demonstrated that while cholesterol entering tissues of the NPC animals through the coated-pit pathway became sequestered in the lysosomal compartment and was metabolically inactive, cholesterol that was newly synthesized or that entered cells through the SR-BI pathway was metabolized and excreted normally.     

Swainsonine, a potent mannosidase inhibitor, elevates rat liver and brain lysosomal alpha-D-mannosidase, decreases Golgi alpha-D-mannosidase II, and increases the plasma levels of several acid hydrolases

Swainsonine, a toxic plant alkaloid reported to be the agent that induces in animals a neurological condition very similar to the hereditary lysosomal storage disease mannosidosis, and to inhibit the formation of complex glycoproteins of the asparagine-linked class, was recently shown [D.R.P. Tulsiani, T.M. Harris, and O. Touster, (1982) J. Biol. Chem. 257, 7936-7939] to be a highly potent and specific inhibitor of Golgi mannosidase II in addition to being a strong inhibitor of lysosomal mannosidase. In the present study the effect of administered swainsonine on tissue enzyme levels was investigated. The activity of Golgi mannosidase II was markedly decreased (22% of control) without changes occurring in the activities of several other Golgi enzymes. However, the effects of swainsonine on lysosomal enzymes was unexpected. In liver, acid mannosidase increased markedly, instead of decreasing as would be expected from a compound reported to induce a mannosidosis-like condition. Similarly, the principal change in brain was a substantial increase in lysosomal mannosidase levels. In plasma, most lysosomal enzymes increased. These results indicate that the pathological effects of swainsonine are not solely attributable to its being an inhibitor of lysosomal alpha-D-mannosidase and are probably a consequence of abnormal processing of glycoproteins.     

Fibroblast chemotaxis and prolidase activity modulation by insulin-like growth factor II and mannose 6-phosphate

Chemotactic locomotion of fibroblasts requires extensive degradation of extracellular matrix components. The degradation is provided by a variety of proteases, including lysosomal enzymes. The process is regulated by cytokines. The present study shows that mannose 6-phosphate and insulin-like growth factor II (IGF-II) enhance fibroblast chemotaxis toward platelet-derived growth factor (PDGF). It is suggested that lysosomal enzymes (bearing mannose 6-phosphate molecules) are involved in chemotactic activity of the cells. The suggestion is supported by the observation that a-mannosidase and cathepsin D inhibitor - pepstatin are very potent inhibitors of fibroblast chemotaxis. Simultaneously, mannose 6-phosphate stimulates extracellular collagen degradation. The final step in collagen degradation is catalyzed by the cytosolic enzyme - prolidase. It has been found that mannose 6-phosphate stimulates also fibroblast prolidase activity with a concomitant increase in lysosomal enzymes activity. The present study demonstrates that the prolidase activity in fibroblasts may reflect the chemotactic activity of the cells and suggests that the mechanism of cell locomotion may involve lysosomal enzyme targeting, probably through IGF-II/mannose 6-phosphate receptor.     

Dextran is resistant to lysosomal digestion in kidney tubules

Low molecular weight dextran (Rheomacrodex) was infused into dextran resistant rats in a dose of 5 g/kg body weight. The kidneys were studied by electron microscopy at different time intervals after infusion using a special fixative for the demonstration of dextran. The lysosomes of proximal tubule cells gradually accumulated dextran which remained in small amounts even after 10 days. In separate kidney slice experiments the ability of dextran-loaded proximal tubule lysosomes to digest absorbed proteins was determined using 125I-labelled lysozyme. There were no changes in lysosomal protein digestion. Labelled dextran was resistant to digestion in vitro by homogenates of rat or rabbit kidney cortex or isolated rat lysosomal enzymes. It is concluded that the protein absorption pathway and lysosomal protein catabolism is unchanged after tubular uptake of dextran despite pronounced ultrastructural alterations to the lysosomal system and that dextran is resistant to lysosomal digestion in renal proximal tubules.     

Subcellular localization of mannose 6-phosphate glycoproteins in rat brain.

The intracellular transport of soluble lysosomal enzymes relies on the post-translational modification of N-linked oligosaccharides to generate mannose 6-phosphate (Man 6-P) residues. In most cell types the Man 6-P signal is rapidly removed after targeting of the precursor proteins from the Golgi to lysosomes via interactions with Man 6-phosphate receptors. However, in brain, the steady state proportion of lysosomal enzymes containing Man 6-P is considerably higher than in other tissues. As a first step toward understanding the mechanism and biological significance of this observation, we analyzed the subcellular localization of the rat brain Man 6-P glycoproteins by combining biochemical and morphological approaches. The brain Man 6-P glycoproteins are predominantly localized in neuronal lysosomes with no evidence for a steady state localization in nonlysosomal or prelysosomal compartments. This contrasts with the clear endosome-like localization of the low steady state proportion of mannose-6-phosphorylated lysosomal enzymes in liver. It therefore seems likely that the observed high percentage of phosphorylated species in brain is a consequence of the accumulation of lysosomal enzymes in a neuronal lysosome that does not fully dephosphorylate the Man 6-P moieties.     

Preventive role of gallic acid on alcohol dependent and cysteine protease-mediated pancreas injury

In order to investigate an association between alcohol consumption and lysosomal cysteine protease induced pancreatic injury and preventive effect of gallic acid as dose-dependent, we determined myeloperoxidase and malondialdehyde levels, serum amylase activities and cathepsin B and L activities in the cytosolic and lysosomal fractions of pancreatic tissue in the ethanol (8?g/kg) and ethanol plus gallic acid (at different doses 50, 100 and 200?mg/kg) given rats. Absolute ethanol (8?g/kg) was given by oral gavage. Gallic acid was dissolved in the saline (2?ml/kg) and administered before 30?min the oral administration of ethanol. Pancreatic myeloperoxidase and also malondialdehyde levels and serum amylase activities were measured. Besides, histological investigations were made. Cathepsin B activities in the cytosolic fraction were decreased by gallic acid (200?mg/kg) and increased in ethanol given rats. Cytosolic/lysosomal ratio of cathepsin B and L were found to be low in the all doses of gallic acid as compared to ethanol group. Serum amylase, pancreatic myeloperoxidase activities and malondialdehyde levels in the ethanol group were higher than in the control group. These were not statistically significant for myeloperoxidase and malondialdehyde. Also, our histopathologic results indicated that ethanol administration increased pancreatic tissue injury. Gallic acid especially at 200?mg/kg improved ethanol-mediated pancreatic tissue damage.In conclusion, gallic acid treatments were decreased release of lysosomal cathepsin B and L enzymes into cytoplasmic fraction and prevented alcohol mediated pancreatic tissue injury. Preventive effect of gallic acid might be dose-dependent.     

Effect of styrene on testicular enzymes of growing rat.

Effect of styrene (100 or 200 mg/kg body wt/day) for 60 days was observed on testicular enzymes of postnatally maturing rats. A significant decrease in epididymal spermatozoa count was observed only at 200 mg/kg body weight dose. Activities of testicular sorbitol dehydrogenase and acid phosphatase decreased while activities of lactate dehydrogenase, beta-glucuronidase, glucose-6-phosphate dehydrogenase, and gamma-glutamyl transpeptidase significantly increased only in animals exposed to styrene at a dose of 200 mg/kg body weight. The results suggest that exposure to high dose of styrene during developmental period alters the activities of enzymes associated with specific cell type of testis.     

The mannose 6-phosphate receptor and the biogenesis of lysosomes

Localization of the 215 kd mannose 6-phosphate receptor (MPR) was studied in normal rat kidney cells. Low levels of receptor were detected in the trans Golgi network, Golgi stack, plasma membrane, and peripheral endosomes. The bulk of the receptor was localized to an acidic, reticular-vesicular structure adjacent to the Golgi complex. The structure also labeled with antibodies to lysosomal enzymes and a lysosomal membrane glycoprotein (lgp120). While lysosome-like, this structure is not a typical lysosome that is devoid of MPRs. The endocytic marker alpha 2 macroglobulin-gold entered the structure at 37 degrees C, but not at 20 degrees C. With prolonged chase, most of the marker was transported from the structure into lysosomes. We propose that the MPR/lgp-enriched structure is a specialized endosome (prelysosome) that serves as an intermediate compartment into which endocytic vesicles discharge their contents, and where lysosomal enzymes are released from the MPR and packaged along with newly synthesized lysosomal glycoproteins into lysosomes.