Release Of Hydrolytic Enzymes Research Articles

Immunomodulation by 17β-estradiol in bivalve hemocytes

In mammals, estrogens have dose- and cell-type-specific effects on immune cells and may act as pro- and anti-inflammatory stimuli, depending on the setting. In the bivalve mollusc Mytilus, the natural estrogen 17beta-estradiol (E(2)) has been shown to affect neuroimmune functions. We have investigated the immunomodulatory role of E(2) in Mytilus hemocytes, the cells responsible for the innate immune response. E(2) at 5-25 nM rapidly stimulated phagocytosis and oxyradical production in vitro; higher concentrations of E(2) inhibited phagocytosis. E(2)-induced oxidative burst was prevented by the nitric oxide (NO) synthase inhibitor N(G)-monomethyl-L-arginine and superoxide dismutase, indicating involvement of NO and O(2)(-); NO production was confirmed by nitrite accumulation. The effects of E(2) were prevented by the antiestrogen tamoxifen and by specific kinase inhibitors, indicating a receptor-mediated mechanism and involvement of p38 MAPK and PKC. E(2) induced rapid and transient increases in the phosphorylation state of PKC, as well as of a aCREB-like (cAMP responsive element binding protein) transcription factor, as indicated by Western blot analysis with specific anti-phospho-antibodies. Localization of estrogen receptor-alpha- and -beta-like proteins in hemocytes was investigated by immunofluorescence confocal microscopy. The effects of E(2) on immune function were also investigated in vivo at 6 and 24 h in hemocytes of E(2)-injected mussels. E(2) significantly affected hemocyte lysosomal membrane stability, phagocytosis, and extracellular release of hydrolytic enzymes: lower concentrations of E(2) resulted in immunostimulation, and higher concentrations were inhibitory. Our data indicate that the physiological role of E(2) in immunomodulation is conserved from invertebrates to mammals.

Effect of E. faecalis on the release of serine proteases elastase and cathepsin G, and collagenase‐2 (MMP‐8) by human polymorphonuclear leukocytes (PMNs)

To measure the release of hydrolytic enzymes [elastase, cathepsin G and collagenase-2 (MMP-8)] from human polymorphonuclear leukocytes (PMNs) during interaction with strains of Enterococcus faecalis isolated from endodontic infections. Six E. faecalis strains isolated from treatment resistant cases of apical periodontitis were included in the study. Overnight cultures of the microbes were used for the experiments. PMNs were isolated using the Ficoll Paque technique, and their vitality was assessed throughout the experiments by the Trypan Blue exclusion test. A known amount of microbes and PMNs were mixed in PBS at +37 degrees C in air, and the release of elastase, cathepsin G and MMP-8 was measured at 0, 20, 60 and 120 min after initiation of incubation. The activities of elastase and cathepsin G were analysed by spectrophotometer assays using specific synthetic peptide substrates, and MMP-8 by western immunoblotting quantitated by computer densitometry. PMNs incubated in buffer without any added microbes served as negative controls, cells incubated with 5 ng mL(-1) phorbol myristic acetate (PMA) served as a positive control. The 95% confidence interval was used to compare the relative amount of elastase and cathepsin G released from the samples. One E. faecalis strain induced a similar or higher elastase, cathepsin G and MMP-8 release than the positive control, whereas the other five strains induced only moderate or no release of the three enzymes examined as compared with the negative and positive controls. Western immunoblot revealed that released MMP-8 had molecular sizes of 60 and 75 kDa representing active and latent forms of MMP-8. In addition, >110 kDa high molecular size and a fragmented 20-30 kDa MMP-8 species could be observed. The majority of the E. faecalis strains induced little or no release of hydrolytic enzymes from the PMN cells. The finding may partly explain the clinical observation that root canal infections dominated by E. faecalis are usually symptom free.

Proteolytic cleavage of von Willebrand factor by ADAMTS‐13 prevents uninvited clumping of blood platelets

Proteolytic cleavage of von Willebrand factor by ADAMTS‐13 prevents uninvited clumping of blood platelets

Rapid effects of 17β-estradiol on cell signaling and function of Mytilus hemocytes

Estrogens affect the functioning of several non-reproductive tissues, the immune system in particular. In mammalian immunocytes, 17β-estradiol (E 2) has both dose- and cell-type specific effects and the responses to E 2 seem to be mediated by rapid, non-genomic mechanisms; these may be initiated at either membrane or cytosolic locations, and can result in both direct local effects, such as modification of ion fluxes, and regulation of gene transcription secondary to activation of different kinase cascades, including mitogen activated protein kinases (MAPKs). In this work, the short-term effects of E 2 and the possible mechanisms of estrogen-mediated cell signaling were investigated in the hemocytes, the immune cells of the bivalve mollusc, the mussel Mytilus galloprovincialis Lam. The results show that E 2 (25 nM) caused a rapid and significant increase in hemocyte cytosolic [Ca 2+]; lower concentrations (5 nM) showed a smaller, not significant effect. Both E 2 concentrations affected the phosphorylation state of the components of tyrosine kinase-mediated signal transduction MAPK- and STAT- (signal transducers and activators of transcription) like proteins within 5–15 min from E 2 addition. A greater effect and clearer time course were observed with 25 nM E 2: in particular, E 2 induced a transient increase in p-ERK 2 MAPK and a persistent increase in p-p38 MAPK. Moreover, both STAT3 and STAT5 were tyrosine phosphorylated in response to E 2. E 2 (5 nM) induced both morphological (as evaluated by SEM) and functional changes (such as extracellular release of hydrolytic enzymes, lysosomal membrane destabilisation, and stimulation of the bactericidal activity) within 10–30 min from addition. Lysosomal membrane destabilisation induced by both E 2 concentrations was abolished by hemocyte preincubation with the p38 MAPK inhibitor SB203580, and significantly reduced by PD98059 and Wortmannin (inhibitors of ERK MAPK and PI3-K, respectively), this suggesting that rapid activation of kinase cascades is involved in mediating the effects of E 2 in mussel hemocytes. The antiestrogen Tamoxifen prevented or strongly reduced most, but not all, the effects of E 2. Western blotting with heterologous anti-ERα–anti-ERβ-antibodies revealed the presence of immunoreactive ERα- and ERβ-like proteins in hemocyte protein extracts. Overall, our data support the hypothesis that the rapid effects and mechanisms of action of 17β-estradiol are extremely conserved and that they may play a crucial role in endocrine–immune interactions in invertebrates.

Susceptibility of lysosomes to rupture is a determinant for plasma membrane disruption in tumor necrosis factor alpha-induced cell death.

Since a release of intracellular contents can induce local inflammatory responses, mechanisms that lead to loss of plasma membrane integrity in cell death are important to know. We showed previously that deficiency of the plasma membrane Ca2+ ATPase 4 (PMCA4) in L929 cells impaired tumor necrosis factor alpha (TNF-alpha)-induced enlargement of lysosomes and reduced cell death. The lysosomal changes can be determined by measuring the total volume of intracellular acidic compartments per cell (VAC), and we show here that inhibition of the increase in VAC due to PMCA4 deficiency not only reduced cell death but also converted TNF-alpha-induced cell death from a process involving disruption of the plasma membrane to a cell demise with a nearly intact plasma membrane. The importance of the size of lysosomes in determining plasma membrane integrity during cell death was supported by the observations that chemical inhibitors that reduce VAC also reduced the plasma membrane disruption induced by TNF-alpha in wild-type L929 cells, while increases in VAC due to genetic mutation, senescence, cell culture conditions, and chemical inhibitors all changed the morphology of cell death from one with an originally nearly intact plasma membrane to one with membrane disruption in a number of different cells. Moreover, the ATP depletion-mediated change from apoptosis to necrosis is also associated with the increases of VAC. The increase in lysosomal size may due to intracellular self-digestion of dying cells. Big lysosomes are easy to rupture, and the release of hydrolytic enzymes from ruptured lysosomes can cause plasma membrane disruption.

Distinct characteristics of two intestinal protein compartments discriminated by using fenbendazole and a benzimidazole resistant isolate of Haemonchus contortus

The intestine of Haemonchus contortus is hypersensitive to the effects of the anthelmintic fenbendazole (FBZ). The effects are postulated to stem from disruption of microtubules and interference with apical secretory vesicle transport, followed by release of digestive enzymes into the intestinal cell cytoplasm. Here, FBZ caused marker proteins for both apical (pepsinogen-like protease, PEP-1) and basal (cystatin-like protein) protein compartments to became homogeneously distributed in the cytoplasm of H. contortus intestinal cells. The observations with PEP-1 support the hypothesis that release of hydrolytic enzymes into the intestinal cell cytoplasm contributes to the mechanism of benzimidazole efficacy. A benzimidazole resistant isolate of H. contortus expressed type 1 and 2 intestinal β-tubulin transcripts that would encode predominately tyr200 and phe200 variants, respectively. This isolate was resistant to the known intestinal cell alterations induced by FBZ treatment in the susceptible isolate, including inhibition of apical vesicle transport. These results implicate type 1 β-tubulin in mediating apical vesicle transport in intestinal cells and suggest that the tyr200 variant is a determinant of FBZ resistance in intestinal cells. In contrast, the basal protein compartment demonstrated sensitivity to FBZ treatment in these otherwise “resistant” worms. Hence, distinct FBZ-sensitive components appear to be involved in distributing intestinal proteins into the described apical and basal compartments of normal worms.

Programmed cell death of plant tracheary elements differentiating in vitro

We used various microscopic and labeling techniques to examine events occurring during the programmed cell death (PCD) of plant tracheary elements (TEs) developing in vitro. TEs differentiating in vitro synthesize a secondary cell wall which is complex in composition and pattern at approximately 72 h after hormone manipulation. The timing of PCD events was established relative to this developmental marker. Cytoplasmic streaming continues throughout secondary wall synthesis, which takes 6 h to complete in a typical cell. Vital dye staining and ultrastructural analysis show that the vacuole and plasma membrane are intact during secondary cell wall synthesis, but the cytoplasm becomes less dense in appearance, most likely through the action of confined hydrolysis by small vacuoles which are seen throughout the cell at this time. The final, preeminent step of TE PCD is a rapid collapse of the vacuole occurring after completion of secondary cell wall synthesis. Vacuole collapse is an irreversible commitment to death which results in the immediate cessation of cytoplasmic streaming and leads to the complete degradation of cellular contents, which is probably accomplished by release of hydrolytic enzymes sequestered in the vacuole. This event represents a novel form of PCD. The degradation of nuclear DNA is detectable by TUNEL, an in situ labeling method, and appears to occur near or after vacuole collapse. Our observations indicate that the process of cellular degradation that produces the hollow TE cell corpse is an active and cell-autonomous process which is distinguishable morphologically and kinetically from necrosis. Although TE PCD does not resemble apoptosis morphologically, we describe the production of spherical protoplast fragments by cultured cells that resemble apoptotic bodies but which are not involved in TE PCD. We also present evidence that, unlike the hypersensitive response (HR), TE PCD does not involve an oxidative burst. While this evidence does not exclude a role for reactive oxygen intermediates in TE PCD, it does suggest TE PCD is mechanistically distinct from cell death during the HR.

The biochemistry of the acrosome reaction.

The binding of the spermatozoon to the oocyte zona pellucida (ZP) occurs via specific receptors localized over the anterior head region of the spermatozoon. Zona pellucida binding stimulates the spermatozoa to undergo the acrosome reaction resulting in the release of hydrolytic enzymes and in the exposure of new membrane domains, both of which are essential for fertilization. We suggest that ZP binds to at least two different receptors in the plasma membrane. One (R) is a Gi-coupled receptor that activates phospholipase C (PLC) beta 1. The other (TK) is a tyrosine kinase receptor coupled to PLC gamma. Binding to R would regulate adenylyl cyclase (AC) leading to elevation of cAMP and protein kinase (PKA) activation. The PKA activates a voltage-dependent Ca2+ channel in the outer acrosomal membrane which releases Ca2+ from the interior of the acrosome to the cytosol. This is the first, relatively small, rise in [Ca2+]i (I) which leads to activation of the PLC gamma. The products of phosphatidyl-inositol bisphosphate (PIP2) hydrolysis by PLC diacylglycerol (DAG) and inositol-trisphosphate (IP3) will lead to PKC translocation to the plasma membrane and its activation. PKC opens a voltage-dependent Ca2+ channel (L) in the plasma membrane, leading to the second (II) higher increase in [Ca2+]i. The Gi or TK can also activate an Na+/H+ exchanger leading to alkalization of the cytosol. PKC also activates phospholipase A2 (PLA2) to generate arachidonic acid (AA) from membrane phospholipids. AA will be converted to prostaglandins (PG) and leukotriens (LT) by the enzymes cyclooxygenase (COX) and lipoxygenase (LOX) respectively. The increase in [Ca2+]i and pH leads to membrane fusion and acrosomal exocytosis.

Side Effects of Rimsulfuron on the Microbial Biomass of a Clay‐Loam Soil

AbstractThe effect of Rimsulfuron treatment on soil microbial biomass was studied in a clay‐loam soil under field and laboratory conditions. Field trials were performed at the usual field rate (0.005 ppm; FR) and 10 times the field rate (10FR). In addition to FR and 10FR, one hundred (100FR) times the field rate was also assayed in laboratory experiments. Determinations of microbial biomass‐C content, fluorescein diacetatehydrolysis activity (FDA hydrolysis) and protease activity were carried out 0, 1, 2, 4, 7, 10, 15, 25, and 45 d after herbicide application. In the field trials there were no significant side effects on the investigated parameters at either rate. Conversely, laboratory experiments showed decreases in microbial biomass‐C levels and increases in FDA‐hydrolysis and protease activity at 10FR and 100FR within the first 10 d. As increases in the activity of FDA hydrolysis and protease were concomitant with the decreases in the microbial biomass‐C content a toxic effect on soil microrganisms with a consequent release of hydrolytic enzymes from cell lysis has been hypothesized. This effect was transitory and only at the higher rates of rimsulfuron was it significant.

Dual role of Kupffer cell activation and endothelial cell damage in reperfusion injury to livers stored for transplantation surgery.

In rat models of liver preservation, the primary event leading to liver graft failure after cold storage is a reperfusion injury causing damage to sinusoidal endothelial cells and activation of Kupffer cells (KC). After storage for longer than 16 h in University of Wisconsin solution, reperfusion induces rapid endothelial cell killing. Kupffer cell activation also occurs as indicated by cell surface ruffling, degranulation, release of hydrolytic enzymes, generation of oxygen radicals, and increased phagocytosis. Down-regulation of KC activity with nisoldipine or pentoxifylline improves graft survival. Moreover, pretreatment of donors with small amounts of endotoxin to activate KC causes a drastic reduction of graft survival. Together, KC activation and endothelial damage cause marked microcirculatory disturbances after transplantation characterized by reduced and uneven blood flow and increased leucocyte and platelet adhesion. Such events culminate in inflammation, necrosis and fulminant graft failure. Modification of reperfusion conditions can reduce the extent of injury. In particular, flushing livers with Carolina rinse solution (CRS) at the end of storage reduces endothelial cell killing, suppresses KC activation, improves the microcirculation, and increases graft survival. Active ingredients in CRS include antioxidants (allopurinol, desferrioxamine and glutathione), adenosine and slightly acidic pH (6.5). Other potentially important ingredients are nicardipine, a calcium channel blocker, and fructose, glucose and insulin to promote glycolysis. The cytoprotective amino acid, glycine, further improves the performance of Carolina rinse solution. Reperfusion-induced changes to nonparenchymal cells play an essential role in damage to livers preserved for transplantation surgery. Understanding the role of sinusoidal endothelial cells and KC in this injury has led to promising new strategies to prolong organ storage and reduce graft failure.

Protein processing in lysosomes: the new therapeutic target in neurodegenerative disease

A little recognised feature of neurons is their large complement of lysosomes. Studies of the accumulation of the abnormal isoform of the prion protein (PrP SC) in the prion encephalopathies and the formation of β/A4 protein from its precursor in Alzheimer's disease suggest that generation of these key proteins takes place in lysosome-related organelles. The release of hydrolytic enzymes from lysosomes may be a primary cause of neuronal damage. Although molecular genetic approaches have identified protein mutations central to the main neurodegenerative disease, cell biological observations are now beginning to unravel the intracellular pathways involved in the molecular pathogenesis of neurodegeneration: as a result, it is now appropriate to consider therapeutic manipulation of the lysosomal system as an approach to treatment.

Lysosomes as key organelles in the pathogenesis of prion encephalopathies.

The causation, structural origin, and mechanism of formation of spongiform lesions in transmissible encephalopathies are unknown. We have used immunogold electron microscopy to locate ubiquitin conjugates, hsp 70, and beta-glucuronidase (markers of the lysosomal compartment) and prion protein (PrP) in both control and scrapie-infected mouse brain. In scrapie-infected brain, lysosomes and lysosome-related structures (multivesicular and tubulovesicular dense bodies) are present in abnormally high numbers in neuronal cell processes. These structures contain PrP, together with the lysosomal markers ubiquitin conjugates, hsp 70, and beta-glucuronidase, which could also be identified spilling from tubulovesicular dense bodies into areas of early rarefaction in neuronal processes; we suggest that these areas of rarefaction are the precursor lesions of spongiform change. We advance the hypothesis that spongiform change is brought about by cytoskeletal disruption in neuronal processes caused by liberation of hydrolytic enzymes from lysosomes overloaded with the abnormal isoform of PrP (PrPsc). We suggest that the lysosomal system is probably acting as the bioreactor for processing of normal PrP to the abnormal isoform. The continuous production of increasing quantities of abnormal PrPsc in lysosome-related bodies will eventually cause disruption of the lysosomal membrane with destruction of the neuronal cytoskeleton and the initiation of vacuolation. Later, death of the cell will be associated with release of the PrPsc isoform into the extracellular environment. Repeated rounds of phagocytosis, lysosomal biogenesis of PrPsc, lysosomal membrane rupture, hydrolytic enzyme release, and neuronal lysis will lead to an exponential increase in cell damage and cell death.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathobiochemical response of tracheobronchial lymph nodes following intratracheal instillation of Polyvinylchloride dust in rats

PVC dust, following a single intratracheal instillation (25 mg/rat), was substantially cleared through the lymphatic circulation and progressively accumulated in the tracheobronchial lymph nodes (TBLN) in a time-dependent manner for up to 1 year. The tissue response in TBLN during 60-270 days post-instillation of PVC dust was characterized by progressive increase in total organ fresh weight, dry weight, DNA, RNA and protein contents, concurrent with the proliferation of macrophages and hyperplasia of reticular cells. Active phagocytosis and enhanced hydrolytic activity in TBLN was evident around 270 days post-instillation by the appearance of PVC-laden macrophages near and within the dust foci, and increased activity of acid phosphatase, DNAse, RNAse and beta-glucuronidase. PVC dust caused degeneration of macrophages, and consequent release of hydrolytic enzymes resulted in limited cytotoxicity without inducing reticulination and fibrosis in the TBLN. The histology and clinical biochemistry of liver, kidney, spleen and serum were not altered and there were no detectable PVC particles in these tissues at up to 365 days. It is therefore concluded that lymphatic clearance of intratracheally instilled PVC dust results in its accumulation and mild foreign body reaction in TBLN which is non-fibrogenic at up to 365 days post-instillation.

IgM anti-Fc gamma R autoantibodies trigger neutrophil degranulation.

Anti-Fc gamma R IgM monoclonal antibodies (mAbs) isolated from lipopolysaccharide-stimulated spleen cells from tightskin (TSK) mice were found to be polyspecific, reacting with a wide variety of molecules, including double-stranded DNA, topoisomerase, RNA polymerase, and different collagen types. Approximately 60% of the polyspecific IgM mAbs have anti-Fc gamma R specificity. These anti-Fc gamma R mAbs induce the release of hydrolases from both azurophil and specific granules of human neutrophils. 25-45% of the total cellular content (determined in Nonidet P-40 lysates) of neutrophil elastase, 10-25% of beta-glucuronidase, and 30-50% of alkaline phosphatase was released after incubation with the mAbs. The degranulation process was accompanied by dramatic morphological changes shown by scanning and transmission electron microscopy. The release of hydrolytic enzymes stimulated by the IgM anti-Fc gamma R mAbs was inhibited by preincubation of neutrophils with Fab fragments of either anti-human Fc gamma RII (IV.3) or anti-human Fc gamma RIII (3G8) mAbs. The binding of the anti-Fc gamma R TSK mAbs to human neutrophils was inhibited by Fab fragments of mAb 3G8. However, we found that the TSK anti-Fc gamma R mAbs do not bind to human Fc gamma RII expressed in either CHO cells or the P388D1 mouse macrophage cell line. Since the enzyme release could be inhibited by Fab fragments of mAb IV.3, we suggest that the signal transduction may require Fc gamma RII activation subsequent to crosslinking of the glycan phosphatidyl inositol-anchored Fc gamma RIII-1. These data demonstrate for the first time that polyspecific autoantibodies with Fc gamma R specificity can trigger neutrophil enzyme release via human Fc gamma RIII-1 in vitro and indicate a possible role for such autoantibodies in autoimmune inflammatory processes.

Leiomyosarcoma of the scrotum

mucous membranes, t,2 In this case, the diagnosis was based on the characteristic clinical features and biopsy of the buccal mucosa. Although antimalarials have been used in the treatment of several dermatoses, they do not seem to have been used in the treatment of lichen planus? lsaacson et aP reviewed the possible mechanisms of action of antimalarials in inflammatory disorders. These include: inhibition of complement activation in vitro, interference with complement-dependent antigen-antibody reaction, reduction of formation of immune complexes, and binding to and sterilizing lysosomal membranes retarding the release of hydrolytic enzymes. They also inhibit chemotaxis of polymorphonuclear leukocytes, macrophages, and eosinophils and suppress cell-mediated immune reactions. It is through one or more of these mechanisms that antimalarial drugs may help in the management of lichen planus, in which various immunologic abnormalities have been found? In this patient, chloroquine phosphate induced remission and the nails were restored to normal. However, 10 weeks after discontinuation of treatment, her nail lesions recurred.

The release of hydrolases from Trichomonas vaginalis and Tritrichomonas foetus

Trichomonas vaginalis and Tritrichomonas foetus were found to release large amounts of β- N-acetylglucosaminidase (EC 3.2.1.30), α-mannosidase (EC 3.2.1.24), β-glucosidase (EC 3.2.1.21), acid phosphatase (EC 3.1.3.2) and proteinases during axenic growth in vitro. The enzymes were released continually throughout the growth phase, with the extracellular activity being of the same order as that within the cells. There was differential release of proteinases from Trichomonas vaginalis. The subcellular localization of the hydrolases was determined by differential and isopycnic centrifugation. The intracellular enzymes were shown to be mostly located within particle populations. Centrifugation on Percoll gradients allowed the separation of sub-populations of the particles in T. vaginalis; two distinct sub-populations were apparent with equilibrium densities in 20% (v/v) Percoll of 1.035 and 1.050 g cm −3 respectively. The higher density particles were rich in the hydrolases released most abundantly, suggesting a possible link between enzyme release and these organelles. Distinct subpopulations of hydrolase-containing particles were not detected in Tritrichomonas foetus. The results demonstrate that hydrolytic enzyme release represents a major activity during trichomonad growth.

Prostaglandins and leukotrienes in pulmonary O2 toxicity.

O2 radicals can initiate peroxidation of cellular membranes, resulting in the release of hydrolytic enzymes, decreased membrane fluidity, and compromised membrane integrity. Free radicals also may cause the release of vasoactive substances, including prostaglandins (PG) and leukotrienes (LT). These products of the arachidonic acid cascade have been shown to produce vasoconstriction, vasodilation, platelet aggregation, increased capillary permeability, plasma extravasation, and edema formation. Of interest is that a similar pathophysiology is seen after O2 exposure. Employing the isolated perfused lung (IPL) prepared from lungs of rats exposed to 100% O2, the role of PG and LT in O2-induced lung damage was studied.

29] Sponge implants as models

Publisher Summary This chapter discusses sponge implants as models. The inflammatory process is a response to tissue injury involving the infiltration of cells, the accumulation of fluids, the production of mediators, and the release of hydrolytic enzymes, all of which take place in a sequential and well-ordered series of events. To study this phenomenon in the tissues of the whole animal is a very desirable but extremely complicated procedure. Tissue fluids are not always accessible for biochemical examination, and the cellular infiltration is most commonly assessed by time consuming histologic methods. The techniques of sponge implantation described in this chapter represent a convenient system for providing ready access to the cells and fluids of an ongoing inflammatory response. Using these techniques, it has been shown that indomethacin reduces leukocyte migration into subcutaneously implanted sponges but that this effect was distinct from the effect of indomethacin in reducing the content of prostaglandin in the exudate.

The role of lipid peroxidation in liver damage

The consequences of the peroxidative breakdown of membrane lipids have been considered in relation to both the subcellular and tissue aspects of liver injury. Mitochondrial functions can be impaired by lipid peroxidation probably through the oxidation of pyridine nucleotides and the consequent alteration in the uptake of calcium. Several enzymatic functions of the endoplasmic reticulum are also affected as a consequence of peroxidative events and among these are the activities of glucose 6-phosphatase, cytochrome P-450 and the calcium sequestration capacity. Moreover, a release of hydrolytic enzymes from lysosomes and a decrease in the fluidity of plasma membranes can contribute to the liver damage consequent to the stimulation of lipid peroxidation. Extensive studies carried out in vivo and integrated with the use of isolated hepatocytes have shown that lipid peroxidation impairs lipoprotein secretion mainly at the level of the dismission from the Golgi apparatus, rather than during their assembly. However, such an alteration appears to give a late and not essential contribution to the fat accumulation. A more critical role is played by peroxidative reactions in the pathogenesis of acute liver necrosis induced by several pro-oxidant compounds as indicated by the protective effects against hepatocyte damage exerted by antioxidants. In addition, even in the cases where lipid peroxidation has been shown not to be essential in causing cell death there is evidence that it can still act synergistically with other damaging mechanisms in the amplification of liver injury.

Chemically modified low density lipoproteins as inducers of enzyme release from macrophages

Macrophages carry receptors on their surface for acetylated low density lipoprotein (ac-LDL). Receptor-mediated endocytosis of ac-LDL is followed by intracellular cholesterol accumulation. We investigated whether occupation of these binding sites evokes the release of hydrolytic enzymes from mouse peritoneal macrophages cultured for up to 48 h. ac-LDL at concentrations ranging from 25–250 μg protein ml was noted to promote in a dose-dependent fashion secretion of the neutral proteinase elastase (EC 3.4.21.37) and the lysosomal acid hydrolases N-acetyl-β-glucosaminidase (EC 3.2.1.30), β-glucuronidase (EC 3.2.1.31), β-galactosidase (EC 3.2.1.23), α-mannosidase (EC 3.2.1.24) and cathepsin D (EC 3.4.23.5). This stimulatory effect was non-cytotoxic. LDL modified by treatment with malondialdehyde was also capable of augmenting enzyme liberation into culture supernates. These findings may have implications for some aspects of the atherosclerotic process.