Release Of Lysosomal Cathepsin Research Articles

Lysosomal disruption by orthopedic wear particles induces activation of the NLRP3 inflammasome and macrophage cell death by distinct mechanisms.

Wear particles from orthopedic implants cause aseptic loosening, the leading cause of implant revisions. The particles are phagocytosed by macrophages leading to activation of the nod-like receptor protein 3 (NLRP3) inflammasome and release of interleukin-1β (IL-1β) which then contributes to osteoclast differentiation and implant loosening. The mechanism of inflammasome activation by orthopedic particles is undetermined but other particles cause the cytosolic accumulation of the lysosomal cathepsin-family proteases which can activate the NLRP3 inflammasome. Here, we demonstrate that lysosome membrane disruption causes cathepsin release into the cytoplasm that drives both inflammasome activation and cell death but that these processes occur independently. Using wild-type and genetically-manipulated immortalized murine bone marrow derived macrophages and pharmacologic inhibitors, we found that NLRP3 and gasdermin D are required for particle-induced IL-1β release but not for particle-induced cell death. In contrast, phagocytosis and lysosomal cathepsin release are critical for both IL-1β release and cell death. Collectively, our findings identify the pan-cathepsin inhibitor Ca-074Me and the NLRP3 inflammasome inhibitor MCC950 as therapeutic interventions worth exploring in aseptic loosening of orthopedic implants. We also found that particle-induced activation of the NLRP3 inflammasome in pre-primed macrophages and cell death are not dependent on pathogen-associated molecular patterns adherent to the wear particles despite such pathogen-associated molecular patterns being critical for all other previously studied wear particle responses, including priming of the NLRP3 inflammasome.

Lysosomal Cathepsin Release Is Required for NLRP3-Inflammasome Activation by Mycobacterium tuberculosis in Infected Macrophages.

Lysosomal cathepsin B (CTSB) has been proposed to play a role in the induction of acute inflammation. We hypothesised that the presence of active CTSB in the cytosol is crucial for NLRP3-inflammasome assembly and, consequently, for mature IL-1β generation after mycobacterial infection in vitro. Elevated levels of CTSB was observed in the lungs of mice and rabbits following infection with Mycobacterium tuberculosis (Mtb) H37Rv as well as in plasma from acute tuberculosis patients. H37Rv-infected murine bone marrow-derived macrophages (BMDMs) displayed both lysosomal leakage, with release of CTSB into the cytosol, as well as increased levels of mature IL-1β. These responses were diminished in BMDM infected with a mutant H37Rv deficient in ESAT-6 expression. Pharmacological inhibition of cathepsin activity with CA074-Me resulted in a substantial reduction of both mature IL-1β production and caspase-1 activation in infected macrophages. Moreover, cathepsin inhibition abolished the interaction between NLRP3 and ASC, measured by immunofluorescence imaging in H37Rv-infected macrophages, demonstrating a critical role of the enzyme in NLRP3-inflammasome activation. These observations suggest that during Mtb infection, lysosomal release of activated CTSB and possibly other cathepsins inhibitable by CA07-Me is critical for the induction of inflammasome-mediated IL-1β processing by regulating NLRP3-inflammasome assembly in the cytosol.

Macrophage digesting dysfunction by Angiotensin II through lysosomal cathepsin B‐mediated Nlrp3 inflammasome activation

Angiotensin II (Ang II) is an octapeptide hormone that plays a significant role in mediating hypertension. Hypertension is considered a chronic inflammatory disease; however, the molecular basis underlying the sterile inflammatory response involved in the process of hypertension remains unclear. The present study aims to investigate the role of macrophage Nlrp3 inflammasomes in engulfing and digesting microbes, which are associated with macrophage function, an early onset of hypertension‐associated macrophage injury. Our findings demonstrate that AngII stimulation decreased the NO release and macrophage digestion in cultured RAW264.7 cell. Accompanied with these changes, AngII markedly increased the Nlrp3 inflammasome formation and activation. As AngII‐induced up‐regulation of Nlrp3 inflammasome in macrophage was attributed to lysosome dysfunction induced cathepsin B release. In addition, Losartan, a nonpeptide Ang II receptor antagonist, decreased AngII‐induced Nlrp3 inflammasome activation, lysosomal membrane permeability, lysosomal cathepsin B release, and macrophage digestion dysfunction in macrophage. Similarly, AngII induced macrophage digestion microbes and NO production, which were blocked by Nlrp3 gene silencing and ATI gene silencing. It is concluded that Angiotensin II enhanced lysosomal membrane permeabilization and consequent release of lysosomal cathepsin B, resulting in activation of the macrophage Nlrp3 inflammasome, which may contribute to NO mediated the development of digesting microbes dysfunction.Support or Funding InformationThis study was supported by the National Natural Science Foundation of China (81603587 and 81603668) and by the Guangdong Science and Technology Department (2017A020211016).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cathepsin B-Mediated NLRP3 Inflammasome Formation and Activation in Angiotensin II -Induced Hypertensive Mice: Role of Macrophage Digestion Dysfunction

Background/Aims: Angiotensin II (Ang II) is an octapeptide hormone that plays a significant role in mediating hypertension. Although hypertension is considered a chronic inflammatory disease, the molecular basis of the sterile inflammatory response involved in hypertension remains unclear. Methods: We investigated the role of macrophage NLRP3 inflammasomes in engulfing and digesting microbes, a key macrophage function, and in early onset of hypertension-associated macrophage injury using biochemical analyses, gene silencing, molecular biotechnology, immunofluorescence, and microbiology. Results: Ang II stimulation decreased nitric oxide (NO) release and macrophage digestion in cultured THP-1 cells and markedly increased NLRP3 inflammasome formation and activation. NO release and macrophage digestion were restored by NLRP3 inflammasome inhibition with isoliquiritigenin and gene silencing. This Ang II-induced upregulation of NLRP3 inflammasomes in macrophages was attributed to lysosomal damage and release of cathepsin B. Mechanistically, losartan, a nonpeptide Ang II receptor antagonist, decreased Ang II-induced NLRP3 inflammasome activation, lysosomal membrane permeability, lysosomal cathepsin B release, and macrophage digestion dysfunction. Similarly, Ang II-induced macrophage microbe digestion and NO production, which were blocked by ATI gene silencing. In addition, in vivo experiments showed that the bacteria scavenging function was clearly decreased in macrophages from Ang II-induced hypertensive mice. Conclusion: Angiotensin II enhances lysosomal membrane permeabilization and the consequent release of lysosomal cathepsin B, resulting in activation of the macrophage NLRP3 inflammasome. This may contribute to NO mediation of dysfunction in digesting microbes.

Characterization of NLRP3-like gene from Apostichopus japonicus provides new evidence on inflammation response in invertebrates

Inflammatory/defensive response after pathogen invasion is considered a local defense reaction in vertebrates. Inflammation response in Apostichopus japonicus was hardly determined due to scarce information available for nucleotide binding domain-like receptor family, pyrin domain-containing (NLRP) family. In the present study, invertebrate NLRP homologue was identified from A. japonicus (designated as AjNLRP3-like) by rapid amplification of cDNA ends. Full-length cDNA of AjNLRP3-like measured 2970 bp with 2265 bp open reading frame encoding a 754-amino acid (aa) residue protein. Structural analysis revealed that AjNLRP3-like processed characteristic domains of pyrin (32–102aa) and NACHT (183–339aa). Multiple sequence alignment and phylogenetic analysis supported that AjNLRP3-like belongs to a new member of NLRP3 protein subfamily. Spatial expression analysis revealed that AjNLRP3-like was ubiquitously expressed in all examined tissues with larger magnitude in coelomocytes. Both Vibrio splendidus challenge in vivo and lipopolysaccharide stimulation in vitro significantly upregulated mRNA expression of AjNLRP3-like when compared with the control group. NLRP3-mediated inflammation response depended on release of lysosomal cathepsin B (CTSB) and subsequent activation of high-mobility group box (HMGB) in vertebrates. We investigated expression profiles of AjNLRP3-like and AjHMGB after AjCTSB knock-down and discovered that AjNLRP3-like was depressed by 0.66-fold and 0.47-fold, whereas AjHMGB was depressed by 0.70-fold and 0.50-fold at 24 and 48 h in AjCTSB-silenced group, respectively. Similarly, down-regulation of AjHMGB was also observed after AjNLRP3-like knock-down. This study therefore suggests that A. japonicus feature similar inflammatory events as those in vertebrates, and activation of AjNLRP3-like depends on AjCTSB expression and release of AjHMGB.

Sevoflurane postconditioning attenuates reactive astrogliosis and glial scar formation after ischemia–reperfusion brain injury

Cerebral ischemia leads to astrocyte’s activation and glial scar formation. Glial scar can inhibit axonal regeneration during the recovery phase. It has demonstrated that sevoflurane has neuroprotective effects against ischemic stroke, but its effects on ischemia-induced formation of astrogliosis and glial scar are unknown. This study was designed to investigate the effect of sevoflurane postconditioning on astrogliosis and glial scar formation in ischemic stroke model both in vivo and in vitro. The results showed that 2.5% of sevoflurane postconditioning could significantly reduce infarction volume and improve neurologic deficits. And it could also decrease the expression of the glial scar marker glial fibrillary acidic protein (GFAP), neurocan and phosphacan in the peri-infarct region and markedly reduce the thickness of glial scar after ischemia/reperfusion (I/R). Consistent with the in vivo data, in the oxygen and glucose deprivation/reoxygenation (OGD/Re) model, sevoflurane postconditioning could protect astrocyte against OGD/Re-induced injury, decrease the expression of GFAP, neurocan and phosphacan. Further studies demonstrated that sevoflurane postconditioning could down-regulate the expression of Lamp1 and active cathepsin B, and block I/R or OGD/Re-induced release of cathepsin B from the lysosomes into cytoplasm. In order to confirm whether inhibition of cathepsin B could attenuate the formation of glial scar, we used cathepsin B inhibitor CA-074Me as a positive control. The results showed that inhibition of cathepsin B could decrease the expression of GFAP, neurocan and phosphacan. Taken together, sevoflurane postconditioning can attenuate astrogliosis and glial scar formation after ischemic stroke, associating with inhibition of the activation and release of lysosomal cathepsin B.

Lysosomal dysfunction in neurodegenerative diseases.

Dane literaturowe z ostatnich lat jednoznacznie wskazują na udział lizosomów w programowanej śmierci komórki. Dysfunkcje lizosomów upośledzają fuzję autofagosomów z lizosomami, co prowadzi do wakuolizacji cytoplazmy. Obecność wakuoli autofagalnych obładowanych uszkodzonymi organellami i nieprawidłowymi białkami jest cechą charakterystyczną wielu chorób neurodegeneracyjnych. Agregacja niezdegradowanego materiału zaburza homeostazę komórki powodując śmierć neuronu w wyniku apoptozy i/lub nekrozy. Ponadto indukowany kalpainami lub spowodowany mutacjami rozpad błony lizosomu uwalnia katepsyny, które indukują szlak śmierci komórki. W artykule przedstawiono mechanizm śmierci komórki nerwowej, łączący zaburzenie szlaku autofagalno-lizosomalnego z dysfunkcjami lizosomów, zwany lizosomalnym szlakiem śmierci neuronu.

Endothelial Nlrp3 inflammasome activation associated with lysosomal destabilization during coronary arteritis

Inflammasomes play a critical role in the development of vascular diseases. However, the molecular mechanisms activating the inflammasome in endothelial cells and the relevance of this inflammasome activation is far from clear. Here, we investigated the mechanisms by which an Nlrp3 inflammasome is activated to result in endothelial dysfunction during coronary arteritis by Lactobacillus casei (L. casei) cell wall fragments (LCWE) in a mouse model for Kawasaki disease. Endothelial dysfunction associated with increased vascular cell adhesion protein 1 (VCAM-1) expression and endothelial–leukocyte adhesion was observed during coronary arteritis in mice treated with LCWE. Accompanied with these changes, the inflammasome activation was also shown in coronary arterial endothelium, which was characterized by a marked increase in caspase-1 activity and IL-1β production. In cultured endothelial cells, LCWE induced Nlrp3 inflammasome formation, caspase-1 activation and IL-1β production, which were blocked by Nlrp3 gene silencing or lysosome membrane stabilizing agents such as colchicine, dexamethasone, and ceramide. However, a potassium channel blocker glibenclamide or an oxygen free radical scavenger N-acetyl-l-cysteine had no effects on LCWE-induced inflammasome activation. LCWE also increased endothelial cell lysosomal membrane permeability and triggered lysosomal cathepsin B release into cytosol. Silencing cathepsin B blocked LCWE-induced Nlrp3 inflammasome formation and activation in endothelial cells. In vivo, treatment of mice with cathepsin B inhibitor also abolished LCWE-induced inflammasome activation in coronary arterial endothelium. It is concluded that LCWE enhanced lysosomal membrane permeabilization and consequent release of lysosomal cathepsin B, resulting in activation of the endothelial Nlrp3 inflammasome, which may contribute to the development of coronary arteritis.

TNFα-induced lysosomal membrane permeability (LMP) is downstream of MOMP and triggered by caspase-mediated p75 cleavage and ROS formation

When NF-κB activation or protein synthesis is inhibited, tumor necrosis factor alpha (TNFα) can induce apoptosis through Bax- and Bak-mediated mitochondrial outer membrane permeabilization (MOMP) leading to caspase-3 activation. Additionally, previous studies have implicated lysosomal membrane permeability (LMP) and formation of reactive oxygen species (ROS) as early steps of TNFα-induced apoptosis. However, how these two events connect to MOMP and caspase-3 activation has been largely debated. Here, we present the novel finding that LMP induced by the addition of TNFα plus cycloheximide (CHX), the release of lysosomal cathepsins and ROS formation do not occur upstream but downstream of MOMP and require the caspase-3-mediated cleavage of the p75 NDUFS1 subunit of respiratory complex I. Both a caspase non-cleavable p75 mutant and the mitochondrially localized antioxidant MitoQ prevent LMP mediated by TNFα plus CHX and partially interfere with apoptosis induction. Moreover, LMP is completely blocked in cells deficient in both Bax and Bak, Apaf-1, caspase-9 or both caspase-3 and -7. Thus, after MOMP, active caspase-3 exerts a feedback action on complex I to produce ROS. ROS then provoke LMP, cathepsin release and further caspase activation to amplify TNFα apoptosis signaling.

Squamous Cell Carcinoma Antigen 1 Promotes Caspase-8-Mediated Apoptosis in Response to Endoplasmic Reticulum Stress While Inhibiting Necrosis Induced by Lysosomal Injury

Squamous cell carcinoma antigen 1 (SCCA1) is a member of the serine protease inhibitor (serpin) family of proteins, whose target proteases include the cathepsins. Initially identified as a serological marker for advanced squamous cell carcinomas of the cervix, SCCA1 has also been found to be associated with other cancer types of epithelial or endodermal origins such as lung cancer, head and neck cancer, melanoma, and hepatocellular carcinoma. While the biological function of SCCA1 remains largely unclear, it is believed to limit cellular damage resulting from lysosomal cathepsin release. Here, we show that SCCA1 acts as a molecular switch that inhibits cell death induced by lysosomal injury resulting from DNA alkylating agents and hypotonic shock, whereas it promotes a caspase-8-mediated apoptosis in response to endoplasmic reticulum (ER) stress. In response to ER stress, SCCA1 blocks both lysosomal and proteasomal protein degradation pathways and enhances the interaction between sequestosome 1/p62 and caspase-8, which leads to the aggregation of intracellular caspase-8 and its subsequent cleavage and activation. Hence, on one hand, SCCA1 inhibits cell death induced by lysosomal injury while, on the other hand, it sensitizes cells to ER stress by activating caspase-8 independently of the death receptor apoptotic pathway.

Zinc(II) ion mediates tamoxifen-induced autophagy and cell death in MCF-7 breast cancer cell line

Treatment of MCF-7 cells with tamoxifen induced vacuole formation and cell death. Levels of the autophagy marker, microtubule-associated protein light chain 3 (LC3)-II also increased, and GFP-LC3 accumulated in and around vacuoles in MCF-7 cells exposed to tamoxifen, indicating that autophagy is involved in tamoxifen-induced changes. Live-cell confocal microscopy with FluoZin-3 staining and transmission electron microscopy with autometallographic staining revealed that labile zinc(II) ion (Zn(2+)) accumulated in most acidic LC3(+) autophagic vacuoles (AVs). Chelation of Zn(2+) with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) blocked the increase in phospho-Erk and LC3-II levels, and attenuated AV formation and cell death. Conversely, the addition of ZnCl(2) markedly potentiated tamoxifen-induced extracellular signal-regulated kinase (Erk) activation, autophagy and cell death, indicating that Zn(2+) has an important role in these events. Tamoxifen-induced death was accompanied by increased oxidative stress and lysosomal membrane permeabilization (LMP) represented as release of lysosomal cathepsins into cytosol. Treatment with the antioxidant N-acetyl-L-cysteine (NAC) blunted the increase in Zn(2+) levels and reduced LC3-II conversion, cathepsin D release and cell death induced by tamoxifen. And cathepsin inhibitors attenuated cell death, indicating that LMP contributes to tamoxifen-induced cell death. Moreover, TPEN blocked tamoxifen-induced cathepsin D release and increase in oxidative stress. The present results indicate that Zn(2+) contributes to tamoxifen-induced autophagic cell death via increase in oxidative stress and induction of LMP.

Bobel-24 and Derivatives Induce Caspase-Independent Death in Pancreatic Cancer Regardless of Apoptotic Resistance

The poor prognosis of pancreatic cancer and poor sensitivity to current therapeutics, associated with resistance to apoptosis, urge the search for new drugs. We previously described the induction of caspase-independent mithochondrial death in leukemia cells by Bobel-24 (AM-24) and derivatives. Here, we explored whether these compounds induce a similar cytotoxicity in human pancreatic carcinoma cell lines (NP18, NP9, NP31, and NP29). Bobel-24 or Bobel-16 induced cytotoxicity and DNA synthesis inhibition in all cell lines and apoptosis in all lines, except for NP9. Caspase and/or poly(ADP-ribose) polymerase-1 (PARP-1) activity inhibition experiments showed that cytotoxicity was mainly induced through apoptosis in NP18 and through a caspase-independent process in NP9. Moreover, in NP29 or NP31 cell lines, both caspase-dependent and caspase-independent cell death mechanisms coexisted. Cell death was associated with reactive oxygen species (ROS) production, mitochondrial depolarization, cytochrome c and apoptosis-inducing factor (AIF) release, AIF nuclear translocation, and lysosomal cathepsin release. Inhibition of ROS production, mitochondrial pore permeability, PARP-1, or phospholipase A2 partially prevented cell death. Moreover, cathepsin B inhibition or down-regulation by small interfering RNA partially blocked cell death. In conclusion, Bobel-24 and derivatives trigger caspase-independent lysosomal and mitochondrial death in all tested human pancreatic cancer lines, irrespective of their degree of apoptotic sensitivity, becoming the only active cytotoxic mechanism in the apoptosis-resistant NP9 line. This mechanism may overcome the resistance to apoptosis observed in pancreatic carcinoma when treated with current genotoxic drugs.

Cysteine Cathepsins Trigger Caspase-dependent Cell Death through Cleavage of Bid and Antiapoptotic Bcl-2 Homologues

As a model for defining the role of lysosomal cathepsins in apoptosis, we characterized the action of the lysosomotropic agent LeuLeuOMe using distinct cellular models. LeuLeuOMe induces lysosomal membrane permeabilization, resulting in release of lysosomal cathepsins that cleave the proapoptotic Bcl-2 family member Bid and degrade the antiapoptotic member Bcl-2, Bcl-xL, or Mcl-1. The papain-like cysteine protease inhibitor E-64d largely prevented apoptosis, Bid cleavage, and Bcl-2/Bcl-xL/Mcl-1 degradation. The pancaspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone failed to prevent Bid cleavage and degradation of anti-apoptotic Bcl-2 homologues but substantially decreased cell death, suggesting that cathepsin-mediated apoptosis in these cellular models mostly follows a caspase-dependent pathway. Moreover, in vitro experiments showed that one or more of the cysteine cathepsins B, L, S, K, and H could cleave Bcl-2, Bcl-xL, Mcl-1, Bak, and BimEL, whereas no Bax cleavage was observed. On the basis of inhibitor studies, we demonstrate that lysosomal disruption triggered by LeuLeuOMe occurs before mitochondrial damage. We propose that degradation of anti-apoptotic Bcl-2 family members by lysosomal cathepsins synergizes with cathepsin-mediated activation of Bid to trigger a mitochondrial pathway to apoptosis. Moreover, XIAP (X-chromosome-linked inhibitor of apoptosis) was also found to be a target of cysteine cathepsins, suggesting that cathepsins can mediate caspase-dependent apoptosis also downstream of mitochondria.

Deadly Redundancy

Stimuli such as tumor necrosis factor (TNF) activate cell death or apoptosis by activating the cysteine proteases known as caspases. Such receptor stimulation enhances formation of the apoptosome, a protein complex that allows activation of caspase 9. But is this the only way to skin the cat? Evidently not, according to an international collaboration of leading labs in the apoptosis field. Gyrd-Hansen et al . used mouse embryo fibroblasts (MEFs) lacking components of apoptotic pathways to show that the cells required caspase 9 to undergo TNF-induced death but did not need the protein Apaf-1, a component of the apoptosome required for activation of caspase 9 by that complex. Activation of caspase 9 in this Apaf-1-independent context appeared to require its cleavage and activation by another caspase, caspase 8. Further studies with knockout MEFs showed that permeabilization of mitochondria was also not required for this apoptosome-independent cell death in response to TNF. Instead, cell death was associated with permeabilization of lysosomal membranes, an alternative means of cell destruction that depends on release of lysosomal cathepsins. Cells with complete apoptotic machinery appeared to use both the mitochondrial and the lysosomal pathways to execute cell death in response to TNF. Protection from TNF-induced death was achieved only after gene ablation or pharmacological treatments that inhibited both pathways. The authors propose that such redundant signaling of apoptosis may be common and that death-resistant cancer cells may have perturbations in multiple pathways. Understanding the underlying pathways thus may allow more effective strategies to deliver therapeutic treatments that reactivate cell death in tumor cells. M. Gyrd-Hansen, T. Farkas, N. Fehrenbacher, L. Bastholm, M. Høyer-Hansen, F. Elling, D. Wallach, R. Flavell, G. Kroemer, J. Nylandsted, M. Jäättelä, Apoptosome-independent activation of the lysosomal cell death pathway by caspase-9. Mol. Cell. Biol. 26 , 7880-7891 (2006). [Abstract] [Full Text]

Digesting the Remodeled Heart

Of the many proteinases present in the myocardium, members of the matrix metalloproteinase (MMP) family have received most attention. However, the cysteine proteases and their inhibitors have now been implicated in the pathogenesis of cardiac remodeling in the hypertrophied and failing heart. Cathepsins, cysteine proteases of the papain family, are optimally active in the acidic pH of lysosomes and are responsible for the physiological digestive turnover of cellular molecules and organelles. We now know that the levels of cathepsins and their endogenous inhibitors, cystatins, can be regulated by a number of signaling molecules, and cathepsins can be released from lysosomes into the extracellular space. Cysteine proteinases may degrade extracellular matrix proteins, such as elastin and fibrillar collagens.1 Nonlysosomal cathepsin targets include degradation of matrix and bone via elastinolytic/collagenolytic activity, activation of pro-MMP, induction of anoikis-dependent apoptosis via matrix degradation, activation of caspases, and cleavage of focal adhesion kinases. A decade ago, measurement of cathepsin activity in heart tissue of different pathologies was used as a measure of lysosomal activity and …

Lysosomal activity of pulmonary alveolar macro phages in acute experimental pancreatitis in rats with reference to positive PAF-antagonist (BN 52021) effect

The activation of pulmonary alveolar macrophages (PAM's), might play an important role in severe complications of acute pancreatitis. The aim of our study was to assess the labilization of macrophage lysosomal membranes and release of lysosomal cathepsin B (CB) and N-acetyl-beta-D-hexosaminidase (NAH) into bronchoalveolar lavage fluid (BALF) during taurocholate acute pancreatitis (AP) in rats treated with PAF-antagonist--BN 52021. Total activity of CB increased by 374% after 6 h and by 237% after 12 h of AP in lysosomal enriched fraction of PAM's. Fractional free activity of CB increased to 40% after 6 h and to 38% after 12 h of AP. Free activity of CB was increased 5 fold in the supernatant of macrophage homogenate, and 10 fold in the supernatant of BALF after 6 h of AP. The values of NAH activity roughly paralleled that of CB. Treatment with BN 52021 (5 mg x kg(-1) every 6 h i.v.) partially normalized the measured parameters. Our results indicate that the PAF-antagonist BN 52021 reduced the increase of total and free activity of lysosomal hydrolases of PAM's and partly prevented the labilization of their lysosomal membranes. Therefore, an important mechanism of BN 52021 beneficial effect in pulmonary complications of acute pancreatitis could be dependent on the stabilization of PAM's lysosomes.

Lysosomal release of Cathepsin D precedes relocation of Cytochrome C and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress

Apoptosis was induced in human foreskin fibroblasts by the redox-cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Most of the cells displayed ultrastructure typical of apoptosis after 8 h of exposure to naphthazarin. Apoptosis was inhibited in fibroblasts pretreated with the cathepsin D inhibitor pepstatin A. Immunofluorescence analysis of the intracellular distribution of cathepsin D revealed a distinct granular pattern in control cells, whereas cells treated with naphthazarin for 30 min exhibited more diffuse staining that corresponded to release of the enzyme from lysosomes to the cytosol. After 2 h, release of cytochrome c from mitochondria to the cytosol was indicated by immunofluorescence. The membrane-potential–sensitive probe JC-1 and flow cytometry did not detect a permanent decrease in mitochondrial transmembrane potential (ΔΨ m) until after 5 h of naphthazarin treatment. Our findings show that, during naphthazarin-induced apoptosis, lysosomal destabilization (measured as release of cathepsin D) precedes release of cytochrome c, loss of ΔΨ m, and morphologic alterations. Moreover, apoptosis could be inhibited by pretreatment with pepstatin A.

The State of Lysosomes and Protein Turnover in Rat Liver Effect of Excess Vitamin A

Administration of excess vitamin A to rats induces labilization of liver lysosomal membranes, as shown by the release of lysosomal cathepsins upon tissue homogenization. The effect of lysosomal labilization on liver protein turnover was investigated. The apparent turnover rate of liver proteins in the hypervitaminotic animals, as measured by a double isotope-labeling technique (Glass and Doyle (1972) J, Biol. Chem, 247, 5234-5242), was found to be the same as that in control animals. Neutral and alkaline fructose-1, 6-biphosphatase [EC 3.3.3.11] activities in the liver were also found to be unchanged in hypervitaminosis A. These data indicate that the rate of intracellular protein degradation is not determined by the level of "free cathepsins. Protein synthesis in the livers of the hypervitaminotic animals was partially imparied, as shown by the shift of polysomal profiles toward lighter aggregates.

EFFECT OF GAMMA RADIATION ON CHICKEN LIVER CATHEPTIC ACTIVITY AND RELEASE OF LYSOSOMAL CATHEPSIN D

Journal of Food ScienceVolume 40, Issue 1 p. 47-49 EFFECT OF GAMMA RADIATION ON CHICKEN LIVER CATHEPTIC ACTIVITY AND RELEASE OF LYSOSOMAL CATHEPSIN D MUMTAZ ALI, MUMTAZ ALI Dept. of Food Science, University of British Columbia, Vancouver, Canada V6T 1W5Search for more papers by this authorJ. F. RICHARDS, J. F. RICHARDS Dept. of Food Science, University of British Columbia, Vancouver, Canada V6T 1W5Search for more papers by this author MUMTAZ ALI, MUMTAZ ALI Dept. of Food Science, University of British Columbia, Vancouver, Canada V6T 1W5Search for more papers by this authorJ. F. RICHARDS, J. F. RICHARDS Dept. of Food Science, University of British Columbia, Vancouver, Canada V6T 1W5Search for more papers by this author First published: January 1975 https://doi.org/10.1111/j.1365-2621.1975.tb03732.xCitations: 7 One of the authors (Mumtaz Ali) was supported by the Canadian International Development Agency during the course of this study. The research reported herein was made possible by a contract with Atomic Energy of -Canada Ltd. and a National Research Council Operating Grant. The authors thank Dr. R.C. Whiting for reviewing the manuscript. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume40, Issue1January 1975Pages 47-49 RelatedInformation