Labilization Of Membranes Research Articles

Hydrolytic enzymes of airway neutrophilic granulocytes in patients with asthma under cold stimuli exposure

Introduction . The labilization of lysosomal membranes with the release of a wide range of lysosomal enzymes of pro-inflammatory and proapoptotic action, which have a high hydrolytic potential, is one of the essential components of adaptive changes in cells under stress. The participation of hydrolytic enzymes of phagocytes in cellular reactions of inflammation and adaptation to cold stress in patients with asthma has been little studied. Aim . To study the activity of neutrophil hydrolases of bronchi in patients with asthma under cold-induced stress. Materials and methods. In patients with persistent asthma (n=13), lung function (FEV 1 ,%), airway response (∆FEV 1 IHCA ,%) to standard 3-minute isocapnic hyperventilation with cold air (IHCA) were determined by spirometry. The collection of induced and spontaneously produced sputum after the IHCA was carried out. The activity of acidic (AP) and alkaline (ALP) phosphatases, adenosine triphosphatase (ATPase) was detected in sputum neutrophils by cytochemical methods. The assessment of the reaction to the activity of hydrolases was carried out by the semi-quantitative Kaplow method with the distribution of cells into groups depending on the color intensity of the product formed during the interaction of the enzyme with the substrate, and the subsequent calculation of the average cytochemical index (ACI) of the enzymatic activity. Results . The baseline FEV 1 , averaged 97.9±5.3%, ∆FEV 1 IHCA -2.0 (-8.0; 2.0)%. Under the influence of a cold stimulus against the background of a mixed pattern of bronchial inflammation in patients, the following was recorded: an increase in the ACI AP of neutrophils (from 1.11±0.06 to 1.42±0.06; p˂0.01), an increase in the number of cells with an average degree of AP activity (from 23.4±1.94 to 40.1±4.30%; p˂0.01) and a decrease in the number of cells with a negative reaction to AP (from 15.1±3,58 to 5.2±2.78%; p˂0.05), a tendency to an increase in the number of neutrophils with a high degree of ATPase activity (from 3.50±2.70 to 7.22±2.64%) and ALP (from 13.7±4.22 to 15.7±3.46%). Conclusion . The activation of AP of neutrophils under cold-induced stress indicates the possibility of an increase in the acidity of the interstitium, stimulation of damage and vulnerability to cold effects of the desmo-epithelial barrier of the bronchi and is regarded as a criterion for a high risk of cold airway hyperresponsiveness in asthma patients with a mixed inflammation phenotype.

Features of cellular mechanisms affecting the pathogenesis and course of chronic catarrhal gingivitis

Aim. To investigate the cellular mechanisms of chronic catarrhal gingivitis to develop the appropriate treatment schemes based on the analysis of obtained data. Methods. 57 patients aged 16 to 22 years were observed and divided into the control group (18 subjects with the intact periodontium with no orthopedic constructions) and the study group of patients with chronic catarrhal gingivitis (39 subjects, group 1). The degree of gingival inflammation was evaluated by papillo-marginal alveolar index as well as by gingival bleeding index. In addition, laboratory parameters were measured: spontaneous nitroblue tetrazolium test and zymosan-stimulated nitroblue tetrazolium test. Leukocyte-modulating activity of oral fluid was determined by measurement of luminol-dependent chemiluminescence. The activity of lysosomal enzymes - acid deoxyribonuclease and acid phosphatase - was determined in whole oral fluid, supernatant fraction of oral fluid and in sediment, obtained by centrifugation. Antioxidant activity was determined in whole oral fluid by biochemiluminescent method with hydrogen peroxide. Results. The performed investigations demonstrated that the development of chronic catarrhal gingivitis is accompanied by a sharp increase of the number of neutrophils and macrophages with a significant increase of their functional activity. At the same time, more than two-fold increase of the activity of lysosomal enzymes, lysosomal membrane labilization and release of enzymes outside the organelles and cells were observed, that indicated the increase of destructive potential in chronic catarrhal gingivitis. Use of indometacin in the complex treatment of chronic catarrhal gingivitis contributed to normalization of clinical parameters, increased neutrophil functional activity and enhanced their functional reserve, reduced the activity of phlogogenic factors and normalized the antioxidant activity of oral fluid. Pro- and anti-inflammatory factors secreted into the oral cavity during inflammatory periodontal diseases collectively have leukocyte-modulating activity. Conclusion. The obtained data indicate the important role of both high oxygen-dependent and oxygen-independent biocidal activity of neutrophils and macrophages in the pathogenesis and clinical course of chronic catarrhal gingivitis.

The influence of trans-cranial electrical stimulation on the cells of the macrophagal system of the women presenting with the climacteric syndrome

The search for the harmless and effective methods for the drug-free correction of various vegetovascular manifestations associated with the climacteric syndrome (CS) is currently a serious challenge facing modern medicine. Dysfunction of the ovaries during the perimenopause is characterized by the impaired production of sex steroids. The estrogens and progesterone, in their turn, are able to interact with macrophages via the specific receptors of steroid hormones localized on the surface of these cells. In this context, the trans-cranial electrical stimulation (TES) technique is of great interest due to its strong influence on the functional state of the central nervous system, subcortical brain structures, the vegetative and hormonal balance. The immunotropic effects of TES therapy described in the literature provide a basis for anticipating its beneficial action on the functional activity of monocytes/macrophages in the women presenting with the climacteric syndrome. The objective of the present study was to evaluate the influence of trans-cranial electrical stimulation on the state and stability of lysosomal membranes of blood monocytes and peritoneal macrophages, the secretory and synthetic activity of these cells in the women exhibiting the vegetovascular manifestations of the climacteric syndrome. The influence of the transcranial electrical stimulation (TES) technique on the stability of lysosomal membranes of blood monocytes and peritoneal macrophages, the secretory and synthetic activity of these cells was studied in 23 women at the age from 45 to 52 years presenting with vegetovascular manifestations of the climacteric syndrome. The control group was comprised of 16 healthy women of the same age. The state of blood monocytes and peritoneal macrophages was evaluated before and after the course of TES therapy. To determine stability of lysosomal membranes of blood monocytes and peritoneal macrophages and calculate the stability index (PSI), the isolated cells were cultured in a medium 199 supplemented by sterile 0.5% L-glutamine and 2.5% mixed human serum previously heated at 560 °C for 30 min and at 37 °C for 12-15 hours. The micro-method was employed to determine secreted lysozyme (Lsec) as well as total lysozyme (L total=L secreted plus L intracellular) following 4-6-fold freeze-thawing of the cultured cells. The data obtained on the levels of secreted and total lysozymes were used to calculate PSI by the formula: PSI=Lsec/Ltotal´100%. The increase in PSI above the optimal value (53- 58%) was regarded as giving evidence of labilization of the lysosomal membranes and the decrease of this parameter as the indicator of membrane stabilization. Based on the difference between the total lysozyme levels before and after cultivation, the amount of newly synthesized lysozyme (L int) was determined. The results of the present study give evidence that the women with climacteric syndrome experience labilization of the blood monocytes and peritoneal macrophages; their lysozyme secretion increases while its synthesis decreases. In other words, the therapeutic application of the transcranial electrical stimulation technique contributed to the disappearance or reduction of 'hot flashes' and the change of these features to the level characteristic of the healthy women. The data obtained extend our knowledge about the mechanisms underlying the action of TES therapy on the macrophagic cells; moreover, they allow to draw the conclusion that the course of medication with the application this technique not only decreases the intensity of manifestations of the climacteric syndrome but also corrects the function of the immune system during the perimenopause.

IN VITRO EFFECT OF L-CARNITINE ON THE ACTIVITY OF LYSOSOMAL CYSTEINE PROTEASES AND THE STATE OF LYSOSOMAL MEMBRANE

The influence of L-carnitine in vitro on the lysosomal cysteine proteinase activity and stability of the lysosomal membrane of the liver homogenates of intact sexually Mature female rats of Wistar line weighing 280-330 g were studied. In the experimental groups isolated lysosomes were incubated in vitro in a solution of L-carnitine during 1, 2 and 4 hours, in the control groups in vitro incubation was carried out in a medium of isolating solution. The activity of ca-thepsins B, L and H was investigated by spectrofluorimetric method of Barrett & Kirschke in two fractions - lysosomal and outside of lysosomes. The activity of acid phosphatase was used as the main marker of a membrane labilization. In vitro incubation of lysosomes showed that carnitine at a concentration of 5 mM increases the total activity of cathepsin B in a one-hour incubation at 73,2% (p=0,008), cathepsin L in a two- and four-hour incubation - at 77,7% (p=0,005) and 42,3% (p=0,013) respectively, and reduces the overall activity of the cathepsin H in a one-hour incubation at 200,0% (p=0,008), in a two-hour - by 67,9% (p=0,05), in a four-hour -27,1% (p=0,02). In addition, incubation in 5 mM L-carnitine solution leads to an increase of unsedimentable activity and fall sedimentaries activity for cathepsin L in a two-hour, and for acid phosphatase - in a two - and four-hour exposure. 5 mM L-carnitine in one - and two-hour incubation stabilizes lysosomal membrane (whereas increase in incubation time up to 4 hours leads to its damage) and increases the selective permeability of the lysosomal membrane for the studied cathepsins, to the greatest extent - for cathepsin H.

Hypolipidemic Effect of Mannan in Mice with Acute Lipemia Induced by Poloxamer 407.

We studied biological effects of mannan, a polysaccharide immunomodulator from C. albicans, that interacts with mannose receptor in vivo. It is shown that preliminary administration of mannan (5 times in a dose of 50 mg/kg or 2 times in a dose of 100 mg/kg) to mice with acute lipemia induced by lipase inhibitor poloxamer 407 (300 mg/kg) reduces the serum concentrations of atherogenic LDL, cholesterol, and triglycerides. Administration of mannan to intact mice and animals with acute lipemia reduces triglyceride concentration and causes labilization of lysosomal membranes in the liver. Serum activity of chitotriosidase, a marker of macrophage activation, was elevated in mice with acute lipemia treated with mannan. Thus, mannan from C. albicans is a promising hypolipidemic polysaccharide compound, similar by its activity to β-glycan, a component of LPS.

Effect of L-arginine and L-name on lysosomal cysteine proteases activity and lysosomal membranes permeability in rat aorta

Aim. To study the effect of L-arginine and its analogue N-nitro-L-arginine methyl ester (L-NAME) alone and in combination on lysosomal cysteine proteolysis and lysosomal membranes state in rat aorta.Methods. The study was performed on male Wistar rats kept under standard vivarium conditions and divided into three control and three experimental groups of 8 animals each. The experimental samples included groups with L-arginine and/or L-NAME administration. The indicators were studied in the rat aorta homogenate precipitating and non precipitating fractions. Acid phosphatase activity was determined by a standardized method of «end point», the cathepsins B, L and H activity was studied by spectrofluorimetric method.Results. When simulating the changes of nitric oxide synthesis level using L-arginine, the increase of the total cathepsins activity was detected, acid phosphatase lability coefficient was reduced, what is characterized by general lysosomal membranes stabilization. L-NAME group, in contrast, is characterized by a decrease in the cathepsin B and H activity indicators, differences from arginine group were observed in the cathepsin H in lysosomal and general fractions, lysosomal membrane is labile. Combined drugs administration reduces the total cathepsins activity, while there is an increase of the acid phosphatase total activity, all indicators suggest lysosomal membranes labilization.Conclusion. L-arginine at a dose of 500 mg/kg causes increase in the total cathepsins B, L and H activity in rat aorta due to lysosomal fraction; L-arginine action leads to lysosomal membranes stabilization; L-NAME group in cathepsin H shows a decrease in the cathepsins secretion level with decreased total activity due to both factions; combined administration of arginine + L-NAME group in cathepsin B is characterized by an increase in secretion due to lysosomes membrane labilization.

Assessment of Erythrocytes as a Model for In Vitro Drug Toxicity

Mitochondria and lysosome have been used to study drug-cell interactions both in vitro and in vivo. However, their preparations to free them from other sub-cellular organelles are tedious and cumbersome. Erythrocytes (being a simple type of cells without sub-cellular organelles and very easy to obtain in pure form) was assessed for in-vitro toxicity testing of selected chemical compounds by monitoring the release of its hemoglobin content as an index of damage to cell membrane. Washed erythrocytes from male wistar rats were prepared in sodium phosphate buffer (pH 7.5) incubated with and without acetylsalicylic acid (a membrane stabilizer) and chloroquine (a membrane labilizer). The light scattering properties of the suspensions and the hemoglobin released were determined using standard methods. Acetylsalicylic acid did not significantly alter the degree of hemolysis (P > 0.05) whereas the chloroquine alone as well as the mixture of acetylsalicylic acid and chloroquine significantly increased (P < 0.05) it. Acetylsalicylic acid stabilized the erythrocytes membrane while chloroquine destroys it causing lysis of the cell membrane. Results obtained in this study suggest that the drugs have interacted with the erythrocytes in a manner that corresponds to their mode of action. Erythrocytes can therefore be used to study interactions between drug molecules and cell membrane.

Protein kinase C-δ isoform mediates lysosome labilization in DNA damage-induced apoptosis

A lysosomal pathway, characterized by the partial rupture or labilization of lysosomal membranes (LLM) and cathepsin release into the cytosol, is evoked during the early events of 20-S-camptothecin lactone (CPT)-induced apoptosis in human cancer cells, including human histiocytic lymphoma U-937 cells. These lysosomal events begin rapidly and simultaneously with mitochondrial permeabilization and caspase activation within 3 h after drug treatment. Recently, in a comparative proteomics analysis performed on highly-enriched lysosomal extracts, we identified proteins whose translocation to lysosomes correlated with LLM induction after CPT treatment, including protein kinase C-δ (PKC-δ). In this study, we show that the PKC-δ translocation to lysosomes is required for LLM, as silencing its expression with RNA interference or suppressing its activity with the inhibitor, rottlerin, prevents CPT-induced LLM. PKC-δ translocation to lysosomes is associated with lysosomal acidic sphingomyelinase (ASM) phosphorylation and activation, which in turn leads to an increase in ceramide (CER) content in lysosomes. The accumulation of endogenous CER in lysosomes is a critical event for CPT-induced LLM as suppressing PKC-δ or ASM activity reduces both the CPT-mediated CER generation in lysosomes and CPT-induced LLM. These findings reveal a novel mechanism by which PKC-δ mediates ASM phosphorylation/activation and CER accumulation in lysosomes in CPT-induced LLM, rapidly activating the lysosomal pathway of apoptosis after CPT treatment.

Proteomic analysis of enriched lysosomes at early phase of camptothecin-induced apoptosis in human U-937 cells

A lysosomal pathway, characterized by partial rupture or labilization of lysosomal membranes and cathepsin activation, is evoked during camptothecin-induced apoptosis in human cancer cells, including human histiocytic lymphoma U-937 cells. These lysosomal events begin rapidly and simultaneously with mitochondrial permeabilization and caspase activation within 3 h after drug treatment. In this study, comparative and quantitative proteome analyses were performed to identify early changes in lysosomal protein expression/localization from U-937 cells undergoing apoptosis. In 2 independent experiments, among a total of more than 538 proteins putatively identified and quantitated by iTRAQ isobaric labeling and LC-ESI-MS/MS, 18 proteins were found to be upregulated and 9 downregulated in lysosomes purified from early apoptotic compared to control cells. Protein expression was validated by Western blotting on enriched lysosome fractions, and protein localization confirmed by fluorescence confocal microscopy of representative protein candidates, whose functions are associated with lysosomal membrane fluidity and dynamics. These include sterol-4-alpha-carboxylate 3-dehydrogenase (NSDHL), prosaposin (PSAP) and protein kinase C delta (PKC-delta). This comparative proteome analysis provides the basis for novel hypothesis and rationale functional experimentation, where the 3 validated candidate proteins are associated with lysosomal membrane fluidity and dynamics, particularly cholesterol, sphingolipid and glycosphingolipid metabolism.

Biomarkers in mussels from a copper site gradient (Visnes, Norway): An integrated biochemical, histochemical and histological study

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

DNA adducts, benzo( a)pyrene monooxygenase activity, and lysosomal membrane stability in Mytilus galloprovincialis from different areas in Taranto coastal waters (Italy)

The aim of this study was to investigate the impact of environmental pollution at different stations along the Taranto coastline (Ionian Sea, Puglia, Italy) using several biomarkers of exposure and the effect on mussels, Mytilus galloprovincialis, collected in October 2001 and October 2002. Five sampling sites were compared with a “cleaner” reference site in the Aeronautics Area. In this study we also investigated the differences between adduct levels in gills and digestive gland. This Taranto area is the most significant industrial settlement on the Ionian Sea known to be contaminated by polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls, heavy metals, etc. Exposure to PAHs was evaluated by measuring DNA adduct levels and benzo( a)pyrene monooxygenase activity (B( a)PMO); DNA adducts were analyzed by 32P-postlabeling with nuclease P1 enhancement in both gills and digestive glands to evaluate differences between DNA adduct levels in the two tissues. B( a)PMO was assayed in the microsomal fraction of the digestive glands as a result of the high expression of P450-metabolizing enzymes in this tissue. Lysosomal membrane stability, a potential biomarker of anthropogenic stress, was also evaluated in the digestive glands of mussels, by measuring the latent activity of β- N-acetylhexosaminidase. Induction of DNA adducts was evident in both tissues, although the results revealed large tissue differences in DNA adduct formation. In fact, gills showed higher DNA adduct levels than did digestive gland. No significant differences were found in DNA adduct levels over time, with both tissues providing similar results in both years. DNA adduct levels were correlated with B( a)PMO activity in digestive gland in both years ( r=0.60 in 2001; r=0.73 in 2002). Increases were observed in B( a)PMO activity and DNA adduct levels at different stations; no statistical difference was observed in B( a)PMO activity over the two monitoring campaigns. The membrane labilization period in mussels from some stations was decreased in both years. No statistical differences were established in the membrane labilization times from 2001 to 2002. Our results suggest the existence of different sources and amounts of environmental contaminants at the stations investigated. The formation of DNA adducts confirms the existence of activation pathways in mussels and shows the importance of DNA adduct analysis in the gill tissue in addition to the more commonly used digestive gland; these results confirm the utility of lysosomal membrane stability as a biomarker of general stress. Overall, the integrated use of biomarkers of exposure and the effects of environmental contaminants on living marine organisms may help to better interpret the impact of pollutants in a marine coastal environment.

Beneficial effect of iloprost on the course of acute taurocholate pancreatitis in rats and its limitation by antecedent acute ethanol intake

The effects of stable prostacyclin analogue iloprost on the trypsinogen activation, labilization of lysosomal membranes, lipolytic enzymes activities, histopathological and ultrastructural changes in the pancreas of rats with severe, taurocholate acute pancreatitis (AP), preceded for 6 h by acute ethanol intake have been investigated. Iloprost (1 microg/kg b.w., i.p.) was applied every 6 hours after inducing of taurocholate AP. The antecedent intragastric 40% ethanol intake (5 g/kg b.w.) increased an index of trypsinogen activation in AP lasting 18 h. Treatment with iloprost prevented this increase in the rats with AP given earlier alcohol, and limited the labilization of lysosomal membranes in nonalcoholized rats with AP. Phospholipase A2 and lipase activities were reduced by iloprost only in the rats not given ethanol. The additional damaging effect of acute ethanol abuse prior to AP could be dependent on augmented activation of trypsinogen. The protective effect of iloprost in AP seems to be dependent on the attenuation of trypsinogen activation, decrease of total potential trypsin and the decrease of lysosomal membranes labilization. Its protective effect could be limited in taurocholate acute pancreatitis preceded by acute ethanol intake as evidenced by the differences in the cathepsin B, phospholipase A2 and lipase activities and by histopathological and ultrastructural examination.

Lysosomal Membrane Stability and Metallothioneins in Digestive Gland of Mussels ( Mytilus galloprovincialis Lam.) as Biomarkers in a Field Study

The lysosomal membrane destabilization and the metallothionein content in the digestive gland cells of mussels ( Mytilus galloprovincialis Lam.), collected along the east coast of the North Adriatic (Istrian and Kvarnerine coast, Croatia), were examined over a period of four years (1996–1999). The lysosomal membrane stability, as a biomarker of general stress, showed that the membrane labilization period in mussels from polluted, urban- and industrial-related areas was significantly decreased ( p<0.05) when compared to mussels from control, clean sea water sites. In the harbour of Rijeka, the most contaminated site, the lysosomal membrane stability was reduced by more than 70% compared to the control. This method also proved to be a useful biomarker for detection of additional stress caused by short-term hypoxia that occurred once during this study inside the polluted and periodically quite eutrophic Pula Harbour. The concentration of metallothioneins in the mussel digestive gland, as a specific biomarker of exposure to heavy metals, did not reveal significant differences ( p<0.05) between sites covered by this study.

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.

Critical evaluation of an intercalibration exercise undertaken in the framework of the MED POL biomonitoring program

The results of an intercalibration exercise among the laboratories participating in the MED POL program for monitoring biological effects of pollutants along the Mediterranean coasts are presented. Three established biomarkers, i.e. lysosomal membrane stability, metallothionein concentration and ethoxyresorufin- O-deethylase (EROD) activity, were intercalibrated. The stability of lysosomal membranes in mussels ( Mytilus galloprovincialis Lam.) was assessed with a cytochemical method. The four participating laboratories were able to discriminate between control animals (membrane labilization times ranging from 21 to 35 min) and Cu-exposed animals (40 μg/l Cu for 3 days) (labilization times ranging from 4.5 to 7.4 min). The metallothionein concentration was evaluated in digestive gland homogenates of control mussels and of animals exposed to 200 μg/l Cd for 7 days. The eight participating laboratories were able to discriminate between controls and treated samples using a spectrophotometric method. The EROD activity was evaluated by 11 laboratories. All laboratories were able to discriminate between liver microsomal preparations obtained from control and from benzo- a-pyrene exposed fish ( Dicentrarchus labrax), with values ranging from 0.5 to 15.88 pmol/min/mg protein in controls and from 5.41 to 165.13 pmol/min/mg protein in treated animals. Using S9 fractions, it was possible to correctly identify control and treated fish, with a variation similar to that found using microsomal fractions, albeit with an inevitable difference in specific activity. As a corollary, all laboratories involved produced comparable data and were able to identify pollutant-induced stress syndromes in sentinel organisms. Thus, intercalibration enables the use of biomarkers in large biomonitoring programs.

Na+channel and Na+-K+ATPase involvement in norepinephrine- and veratridine-stimulated metabolism in perfused rat hind limb

In the constant flow perfused rat hind limb, norepinephrine (NE) evoked increases in oxygen uptake (VO2) and lactate efflux (LE) were inhibited by the cardiac glycoside ouabain (1 mM), without interrupting the NE-mediated vasoconstriction. The membrane labilizer veratridine, previously shown to increase VO2 and LE, without increasing perfusion pressure, was also shown to be inhibited by the cardiac glycoside ouabain, as well as by the ouabain analogues digitoxin and digoxin. The stimulatory actions of veratridine on VO2 were inhibitable by low doses of the specific sodium channel blocker tetrodotoxin (TTX), while NE effects were unaffected, suggesting that NE may be acting via a TTX-insensitive sodium channel. It is concluded that agents such as NE (a vasoconstrictor) or veratridine (a membrane labilizer), which stimulate VO2 in the perfused rat hind limb, do so by increasing Na+ influx. The observed increases in oxygen consumption and LE are due to Na+-K+ ATPase activity to pump Na+ out of the cell at the expense of ATP turnover. Energy dissipation due to Na+ cycling may be a form of facultative thermogenesis attributable to NE that can be stimulated by membrane labilizers such as veratridine in the constant flow perfused rat hind limb.

Uptake of oxidized LDL by macrophages results in partial lysosomal enzyme inactivation and relocation.

The cytotoxicity of oxidized LDL (oxLDL) to several types of artery wall cells might contribute to atherosclerosis by causing cell death, presumably by both apoptosis and necrosis. After its uptake into macrophage lysosomes by receptor-mediated endocytosis, oxLDL is poorly degraded, resulting in ceroid-containing foam cells. We studied the influence ofoxLDL on lysosomal enzyme activity and, in particular, on lysosomal membrane stability and the modulation of these cellular characteristics by HDL and vitamin E (vit-E). Unexposed cells and cells exposed to acetylated LDL (AcLDL) were used as controls. The lysosomal marker enzymes cathepsin L and N-acetyl-beta-glucosaminidase (NAbetaGase) were biochemically assayed in J-774 cells after fractionation. Lysosomal integrity in living cells was assayed by the acridine orange (AO) relocation test. Cathepsin D was immunocytochemically demonstrated in J-774 cells and human monocyte-derived macrophages. We found that the total activities of NAbetaGase and cathepsin L were significantly decreased, whereas their relative cytosolic activities were enhanced, after oxLDL exposure. Labilization of the lysosomal membranes was further proven by decreased lysosomal AO uptake and relocation to the cytosol of cathepsin D, as estimated by light and electron microscopic immunocytochemistry. HDL and vit-E diminished the cytotoxicity of oxLDL by decreasing the lysosomal damage. The results indicate that endocytosed oxLDL not only partially inactivates lysosomal enzymes but also destabilizes the acidic vacuolar compartment, causing relocation of lysosomal enzymes to the cytosol. Exposure to AcLDL resulted in its uptake with enlargement of the lysosomal apparatus, but the stability of the lysosomal membranes was not changed.

Similarities between vasoconstrictor- and veratridine-stimulated metabolism in perfused rat hind limb

The vasoconstrictors norepinephrine (NE) and angiotensin II (AII) mediate increases in oxygen uptake (VO2) by the constant flow perfused rat hind limb that are inhibited by quinidine-like membrane-stabilizing effects (involving the interruption of action potential) of (+/-)-propranolol with little effect on vasoconstriction. The membrane labilizer veratridine, 10 microM, which has the capability of maintaining voltage-gated Na+ channels of the plasma membrane in their open state, also increases VO2 but without an increase in pressure. Thus in the present study veratridine was characterized in detail and compared with NE in the same system. Veratridine (3-100 microM) produced a dose-dependent stimulation of VO2 (from 11.8 +/- 0.3 to 20.4 +/- 0.6 mumol.h-1.g-1 (n = 5), p < 0.0001) and lactate efflux (LE) (from 7.4 +/- 0.6 to 23.0 +/- 4.7 mumol.h-1.g-1 (n = 5), p < 0.01). These increases were independent of vasoconstriction at low doses (< or = 10 microM). At higher doses of veratridine the accompanying minor vasoconstriction (from 17 +/- 1 to 30 +/- 2 mmHg (1 mmHg = 133.3 Pa) (n = 5), p < 0.005) was blocked by sodium nitroprusside (NP) while neither VO2 nor LE was greatly affected. Low Na+ perfusions (25 mM) did not affect the vasoconstrictor action of NE but markedly inhibited increases in VO2 and LE due to either veratridine or NE. Veratridine (10 microM) mediated increases in VO2 and LE were blocked by either (+/-)-propranolol (100 microM) or 150 microM quinidine. It is concluded that vasoconstrictors such as NE, which stimulate VO2 in the perfused rat hind limb, do so by a two-stage process involving an essential nitroprusside-sensitive redirection of flow followed by a mechanism involving increased ion movement across skeletal muscle cell membranes, which is blocked by membrane stabilizers. Veratridine achieves a similar increase in VO2 but may do so by directly destabilizing the skeletal muscle cell membrane without the requirement of a redirection of flow.

Cytochemical Responses in the Digestive Tissue of Mytilus edulis Complex Exposed to Microencapsulated PAHs or PCBs

Cytochemical responses in the digestive tissue of Mytilus edulis complex fed a microencapsulated mixture of PAHs (composed of phenanthrene, fluoranthene and benzo[a]pyrene) or PCBs (Aroclor 1254) were evaluated. Lysosomal membrane labilization period and lipofuscin content of digestive tissue were significantly decreased and increased, respectively, after 30 days of exposure to PAHs but not PCBs. NADPH-ferrihemoprotein reductase activity in the digestive tissue was significantly increased after 30 days exposure to both PAHs and PCBs, whereas catalase activity was significantly increased only after exposure to PCBs. Neutral lipid content and gamma-glutamyl transpeptidase activity of digestive tissue of mussels were significantly increased and decreased, respectively, after 30 days exposure to both PAHs and PCBs when compared to untreated mussels, but were not significantly different when compared to mussels treated with com oil representing the vehicle control. Exposure to PAHs induced peroxisome proliferation in the digestive tissue of M. edulis complex. The observed cytochemical responses in the digestive tissue of M. edulis complex exposed to PAHs and PCBs in the laboratory are comparable to the cytochemical responses observed in the digestive tissue of mussels collected in the field from sites contaminated with predominantly PAHs and PCBs.

Vasoconstrictor-mediated increase in muscle resting thermogenesis is inhibited by membrane-stabilizing agents

Norepinephrine and angiotensin II are potent vasoconstrictors and stimulate thermogenesis (oxygen uptake) as well as lactate and glycerol efflux in the constant-flow perfused rat hind limb at rest. However, the mechanism by which oxygen uptake (VO2) is increased is unknown, and it is not clear whether vasoconstriction is required for the increase in VO2 by the hind limb. In the present study the association between vasoconstriction and VO2 was further investigated, and a chance observation that high-dose propranolol selectively blocked vasoconstrictor-induced increase in VO2 was further explored. The norepinephrine-mediated increase in VO2 was totally blocked by either 50 microM (+)-propranolol or 50 microM (-)-propranolol (active beta-blocking enantiomer), but only (+)-propranolol reduced the vasoconstriction. Similarly, 100 microM (+/-)-propranolol (a dose likely to cause plasma membrane stabilizing effects involving interruption and (or) prevention of action potentials) blocked increases in VO2, lactate, and glycerol efflux by 5 nM angiotensin II (a nonadrenergic vasoconstrictor) with only marginal effects on pressure development. (+/-)-Propranolol (100 microM) had no effect on postequilibration red blood cell washout mediated by angiotensin II, a putative indicator of vasoconstrictor-induced redistribution of flow. Quinidine (260 microM) (an antiarrhythmic agent with membrane-stabilizing activity) inhibited only the increase in VO2, but neither nadolol (300 microM) nor atenolol (300 microM) (beta-blockers without membrane-stabilizing activity) inhibited VO2 or perfusion pressure increases produced by 5 nM angiotensin II. Veratridine (a membrane labilizer that is capable of evoking plasma membrane depolarization by maintaining voltage-gated Na+ channels in their open state) increased VO2 without vasoconstriction, and the increase in VO2 was blocked by 100 microM (+/-)-propranolol. It is concluded that the increase in hind-limb VO2 results from a destabilization of skeletal muscle plasma membranes. This can be achieved directly by veratridine or indirectly by angiotensin II, involving vasoconstriction and redistribution of flow. The findings suggest a novel mechanism for resting muscle thermogenesis.