The use of biomarkers in biomonitoring: a 2-tier approach assessing the level of pollutant-induced stress syndrome in sentinel organisms

The paper outlines a 2-tier approach for wide-scale biomonitoring programmes. To obtain a high level of standardization, we suggest the use of caged organisms (mussels or fish). An "early warning", highly sensitive, low-cost biomarker is employed in tier 1 (i.e. lysosomal membrane stability (LMS) and survival rate, a marker for highly polluted sites). Tier 2 is used only for animals sampled at sites in which LMS changes are evident and there is no mortality, with a complete battery of biomarkers assessing the levels of pollutant-induced stress syndrome. Possible approaches for integrating biomarker data in a synthetic index are discussed, along with our proposal to use a recently developed Expert System. The latter system allows a correct selection of biomarkers at different levels of biological organisation (molecular/cellular/tissue/organism) taking into account trends in pollutant-induced biomarker changes (increasing, decreasing, bell-shape). A selection of biomarkers of stress, genotoxicity and exposure usually employed in biomonitoring programmes is presented, together with a brief overview of new biomolecular approaches.     

Selected contribution: effect of the aldehyde acrolein on acetylcholine-induced membrane current in airway smooth muscle cells.

Acrolein administered to isolated airways has been shown to alter airway responsiveness as a consequence of its effect on Ca(2+) signaling. To examine the mechanisms involved, we studied the effect of acrolein on ACh- and caffeine-induced membrane currents (patch-clamp) in myocytes freshly isolated from rat trachea. In cells clamped at -60 mV, ACh (0.1-10 microM) induced a concentration-dependent inward current, which, in approximately 50% of the cells, was followed by current oscillations in response to high concentration of ACh (10 microM). Exposure to acrolein (0.2 microM) for 10 min significantly enhanced the amplitude of the low-ACh (0.1 microM) concentration-induced initial peak of current (318.8 +/- 28.3 vs. 251.2 +/- 40.3 pA; n = 25, P < 0.05). At a high-ACh concentration (10 microM), the frequency at which subsequent peaks occurred was significantly increased (13.2 +/- 1.1 vs. 8.7 +/- 2 min(-1); n = 20, P < 0.05). ACh-induced current was identified as a Ca(2+)-activated Cl(-) current. In contrast, similar exposure to acrolein, which does not alter caffeine-induced Ca(2+) release, did not alter caffeine-induced transient membrane currents (595 +/- 45 and 640 +/- 45 pA in control cells and in cells exposed to acrolein, respectively; n = 15). It is concluded that acrolein alters ACh-induced current as a consequence of its effect on the cytosolic Ca(2+) concentration response and that the protective role of inhibitors of Cl(-) channels in air pollutant-induced airway hyperresponsiveness should be examined.     

Cellular responses to environmental contaminants in amoebic cells of the slime mould Dictyostelium discoideum

Amoebic Dictyostelium discoideum cells were employed in a bioassay to evaluate stress responses after exposures to the polyaromatic hydrocarbon benzo[a]pyrene (B[a]P) and two heavy metals (copper and mercury). Furthermore, we developed a recombinant cell line expressing a labile Green Fluorescent Protein (GFP) variant expressed under the control of an actin promoter to monitor stress-related protein degradation. Finally, cell viability was monitored to discriminate lethal exposure concentrations. The results demonstrated that exposure to sub-micromolar concentrations of mercury rendered significant changes in all studied physiological parameters, whereas B[a]P became toxic at low micromolar, and copper at high micromolar concentrations. Exposure to 0.5 microM mercury significantly reduced lysosomal membrane stability (LMS), endocytosis rate, GFP expression, and further resulted in the elevation of cytosolic free Ca(2+) ([Ca(2+)](i)). LMS in mercury-treated cells that had been pre-incubated with a specific Ca(2+)-dependent phospholipase A2 blocking agent was however not affected by the exposure, indicating that the toxic action of mercury is linked to the activation of phospholipase A2 via a Ca(2+)-signaling pathway. Exposure to 20 microM B[a]P significantly reduced LMS, endocytosis rate, and GFP expression, however without affecting [Ca(2+)](i), suggesting a calcium-independent route of toxicity for this compound. None of the physiological parameters were significantly affected by copper exposure at concentrations <400 microM, demonstrating a high resistance to this metal. Our results further showed that neither cell growth nor viability was affected by concentrations altering the studied physiological parameters. LMS, endocytosis rate, and [Ca(2+)](I), therefore, appear sensitive biomarkers of pollutant-related stress in amoebic cells.     

Heavy metal effects on lipid peroxidation in the tissues of mytilus gallopro vincialis lam.

1. The effects of heavy metals on lipid peroxidation in the gills and digestive gland of mussels exposed for six days to Cu²⁺, Cd²⁺ or Zn²⁺ (40 μg/l/animal) were investigated.2. In the tissues of Cu-exposed mussels a significant increase of the level of malondialdehyde (MDA), which is indicative of the peroxidative process, and a decrease of the concentration of glutathione were observed.3. Moreover, in the digestive gland of mussels, copper exposure results in an increase of other carbonyl compounds and in the lysosomal accumulation of lipofuscin granules.4. The exposure of mussels to Zn or to Cd did not elicit any of the above effects.5. The results are discussed in relation to the possible role that Cu-induced lysosomal lipofuscin accumulation may play in heavy metal detoxification.     

Effect of alpha-tocopherol and some metal chelators on lipofuscin accumulation in cultured neonatal rat cardiac myocytes

The objective of this study was to test further the hypothesis that oxidative stress is a major causal factor in lipofuscin formation. We have previously shown that cultured cardiac myocytes constitute a suitable model system for the study of factors influencing lipofuscinogenesis. The specific aim of the present study was to elucidate the effects of the chain-breaking free radical scavenger alpha-tocopherol, and the chelators desferrioxamine, EDTA and DTPA on the accumulation of lipofuscin. The effects were examined at different degrees of oxidative stress, obtained by varying the ambient oxygen concentration from 5 to 40%. Lipofuscin was quantified by microspectrofluorometry. Lipofuscin-specific, yellow autofluorescence increased with time in culture, and with enhanced oxidative stress. Increasing concentration of alpha-tocopherol, up to 40 microM, had an inhibitory effect on lipofuscin accumulation that was most pronounced at high oxidative stress. Desferrioxamine and DTPA, both caused a pronounced reduction in lipofuscin formation, while EDTA had no significant effect. The findings are interpreted to support the concept that oxidative stress is a causal factor in lipofuscinogenesis, and that lipofuscin is a product of autophagocytosed, membrane-rich material subjected to free radical-induced, metal-catalyzed peroxidation, fragmentation, and polymerization within the lysosomal vacuome.     

Effect of W7 on Ca2+ uptake and Ca2+-ATPase activities of the endoplasmic reticulum of rat liver

In an initial attempt to use calmodulin antagonists as probes to study the role of calmodulin in the modulation of Ca2+ uptake activity in the endoplasmic reticulum of rat liver, we noticed that W7 had a differential effect on the Ca2+ uptake and Ca2+-ATPase activities. To test the specificity of this effect and explore the underlying mechanism, we examined the effects of W7 on Ca2+ accumulation and release by endoplasmic reticulum in both permeabilized hepatocytes and a subcellular membrane fraction (microsomes) enriched in endoplasmic reticulum. W7 reduced the steady-state Ca2+ accumulation in both preparations in a dose-dependent fashion but the half-maximal inhibitory concentrations were different for Ca2+ accumulation (90 microM) and Ca2+-ATPase activity (500 microM). Kinetic analysis indicated that the inhibition of both Ca2+ uptake and Ca2+-ATPase activity by W7 was noncompetitive with respect to Ca2+ and ATP. Addition of W7 did not enhance the rate of Ca2+ efflux from microsomes after Ca2+ influx had been terminated. The effect of W7 was apparently not related to its calmodulin antagonist properties as the phenomenon could not be demonstrated with the other more specific calmodulin antagonists, calmidazolium or compound 48/80. A similar observation with W7 has also been reported with the endoplasmic reticulum of pancreatic islets (B. A. Wolf, J. R. Colca, and M. L. McDaniel (1986) Biochem. Biophys. Res. Commun. 141, 418-425). We concluded that the effects of W7 on microsomal Ca2+ handling were not the result of increased membrane permeability to Ca2+ but rather were due to dissociation of Ca2+ uptake from Ca2+-ATPase activity.     

Lipofuscin: mechanisms of age-related accumulation and influence on cell function

The accumulation of lipofuscin within postmitotic cells is a recognized hallmark of aging occurring with a rate inversely related to longevity. Lipofuscin is an intralysosomal, polymeric substance, primarily composed of cross-linked protein residues, formed due to iron-catalyzed oxidative processes. Because it is undegradable and cannot be removed via exocytosis, lipofuscin accumulation in postmitotic cells is inevitable, whereas proliferative cells efficiently dilute it during division. The rate of lipofuscin formation can be experimentally manipulated. In cell culture models, oxidative stress (e.g., exposure to 40% ambient oxygen or low molecular weight iron) promotes lipofuscin accumulation, whereas growth at 8% oxygen and treatment with antioxidants or iron-chelators diminish it. Lipofuscin is a fluorochrome and may sensitize lysosomes to visible light, a process potentially important for the pathogenesis of age-related macular degeneration. Lipofuscin-associated iron sensitizes lysosomes to oxidative stress, jeopardizing lysosomal stability and causing apoptosis due to release of lysosomal contents. Lipofuscin accumulation may also diminish autophagocytotic capacity by acting as a sink for newly produced lysosomal enzymes and, therefore, interfere with recycling of cellular components. Lipofuscin, thus, may be much more directly related to cellular degeneration at old age than was hitherto believed.     

Orientation of synaptic plasma membrane vesicles containing calcium pump and sodium-calcium exchange activities

Sidedness of synaptic plasma membrane vesicles isolated from brain synaptosomes has been assessed by two distinct experimental approaches: first, analysis of (Na+ + K+)-ATPase, Mg2+-ATPase, and (Ca2+ + Mg2+)-ATPase activities before and after permeabilization of vesicles; second, analysis of Ca2+ fluxes via the Na+/Ca2+ exchanger, before and after modification of an imposed Na+ gradient by penetrating or nonpenetrating Na+ channel-modifying drugs. 0.05% saponin, which completely permeabilizes the vesicles, increases digitoxigenin-sensitive (Na+ + K+)-ATPase, basal Mg2+-ATPase, and (Ca2+ + Mg2+)-ATPase activities by 51.0, 47.4, and 83.6%, respectively. Saponin increases only the Vmax of the latter activity, the Km for Ca2+ (0.13 microM; the same as that for Ca2+-pumping) being unaltered by saponin. An increment of 20.5% in the Vmax of (Ca2+ + Mg2+)-ATPase activity with 10 microM A23187, reveals that the enzyme activity in nonpermeabilized vesicles is limited by the formation of a Ca2+ gradient. Thus, the saponin-induced increment in (Ca2+ + Mg2+)-ATPase due only to exposure of occluded sites (as opposed to Ca2+ gradient dissipation) is actually 52%, which is similar to values for both other ATPases, and suggests that 32-35% of plasma membranes exist in an inverted orientation. Vesicle orientation was independently assessed by the differential actions of tetrodotoxin (a membrane impermeant blocker) and veratridine (a membrane permeant agonist) on Na+-channel opening measured indirectly by dissipation of an imposed Na+ gradient utilized to drive a large 45Ca2+ accumulation via the Na+/Ca2+ exchanger. Tetrodotoxin reverses 35-44% of veratridine-mediated Na+ gradient-dissipation, the relative membrane-permeability of the two channel modifiers, suggesting that 56-65% of sealed vesicles are inverted. The concurrence of these two independent measurements of vesicle orientation reinforces their validity.     

Purification, characterization, and reconstitution of the Ca2+-transport system (high-affinity Ca2+, Mg2+-ATPase) of the human erythrocyte membrane

The Ca2+-transport system of human erythrocyte membranes was solubilized by deoxycholate in the presence of the nonionic detergent Tween 20 and was purified by calmodulin affinity chromatography. The method yields a functional enzyme, which as compared with the erythrocyte membrane was purified 207-fold based on specific activity, and about 330-fold based on protein content. The activity of the isolated enzyme can be increased about 9-fold by the addition of calmodulin, resulting in a specific activity of 10.1 mumoles/mg . min at 37 degrees C. Triton X-100 and deoxycholate stimulate the calmodulin-deficient Ca2+-ATPase in a concentration dependent manner, which results in a loss of the calmodulin-sensitivity. The Ca2+-transport ATPase could be reconstituted after solubilization of the ATPase by deoxycholate and controlled dialysis near room temperature. The system was reconstituted to form membraneous vesicles capable of energized Ca2+ accumulation. The membrane vesicles showed a protein to lipid ratio (approx. 60% protein and 40% lipid) similar to that of the original erythrocyte membrane. The stimulation by calmodulin of the calmodulin-depleted membrane-bound and partially purified Ca2+-ATPase is strongly time dependent. At a Ca2+-concentration of 40 microM and low calmodulin concentrations, approx. 120 min are required to regain full activity. This time period is decreased to about 15 min in the presence of a high excess of calmodulin. Vice versa, at fixed concentrations of calmodulin, the time necessary for regain of full activity is decreased as the Ca2+ concentrations is increased. The dependence of the Ca2+-ATPase activity on the calmodulin concentration shows strong deviation from Michaelis-Menten kinetics at Ca2+ concentrations below (4--10 microM) and above (200 microM) the optimum concentration of 40 microM. Mathematical analysis of the results at 200 microM Ca2+ leads to the assumption that 4 calmodulin molecules interact with one oligomer of Ca2+-ATPase consisting of 4 identical subunits.     

Calixarene C-91 stimulates Ca2+ accumulation in the myometrium mitochondria

Calixarenes, owing to the ability to form supramolecular complexes with biologically important molecules and ions, can influence a course of biochemical processes and, accordingly, be considered as perspective molecular platforms for creation of physiologically active compounds. The work purpose is to study calixarene C-91 influence on systems of active Ca ions transport which are localized in subcellular membrane structures (mitochondria, sarcoplasmic reticulum, plasma membrane) of myometrial cells. It has been shown, that calixarene C-91 addition to incubation medium led to an increase in Ca2+ accumulation level in mitochondria. The maximal stimulating effect was 173% and it was observed at 100 microM concentration. It is suggested, that calixarene C-91 can enter mitochondria with the subsequent precipitation of Ca ions in a matrix therefore calcium capacity increases, and as a consequence, higher Ca2+ accumulation in these structures is observed. In a wide range of concentration (1-100 microM) calixarene C-91 did not influence a level of Ca2+ accumulation in sarcoplasmic reticulum of myometrial cells. Titration of solubilized Ca2+, Mg2+-ATPase by calixarene C-91 (0,1-100 microM) did not cause changes in its activity. Thus, calixarene C-91 increases Ca2+ accumulation level in mitochondria, but practically does not influence calcium pumps activity of a plasma membrane and sarcoplasmic reticulum of myometrial cells.     

Characterization of a putative Ca2(+)-transporting Ca2(+)-ATPase in the pellicles of Paramecium tetraurelia

In Paramecium, no Ca2(+)-ATPases with the properties of Ca2+ pumps have been identified. Here we report a pellicle associated Ca2(+)-ATPase activity and a corresponding phosphoprotein intermediate characteristic of a pump. The Ca2(+)-ATPase activity requires 3 mM Mg for optimal Ca2+ stimulation (KCa = 90 nM) and is specific for ATP as substrate (Km = 75 microM). Vanadate and calmidazolium inhibit Ca2(+)-stimulated activity with an EC50 of about 2 microM and 0.5 microM, respectively. Likewise, 10 microM trifluoperazine inhibits 80% of Ca2(+)-ATPase activity, but bovine calmodulin fails to stimulate. The Ca2(+)-ATPase is not inhibited by sodium azide (10 mM), oligomycin (10 micrograms/ml) or ouabain (0.2 mM). Incubation of pellicles with [gamma-32P]ATP specifically labels a 133 kDa protein in a Ca2(+)-dependent, hydroxylamine-sensitive manner, and the level of phosphorylation is increased by 100 microM La3+. Phosphorylation of an endoplasmic reticulum-enriched fraction labels a Ca2(+)-dependent protein different from the pellicle protein, being lower in molecular mass and unaffected by La3+. Ca2+ uptake by the alveolar sacs, integral components of the pellicle membrane complex, is poorly coupled to Ca2(+)-stimulated ATP hydrolysis (Ca2+ transported/ATP hydrolysed less than 0.2) and is much less sensitive to vanadate inhibition (EC50 approx. 20 microM) compared to the total Ca2(+)-ATPase activity. Therefore, the majority of the Ca2(+)-ATPase activity is likely to be plasma membrane associated.     

Reconstitution and characterization of a nicotinic acid adenine dinucleotide phosphate (NAADP)-sensitive Ca2+ release channel from liver lysosomes of rats

Nicotinic acid adenine dinucleotide phosphate (NAADP) is capable of inducing global Ca2+ increases via a lysosome-associated mechanism, but the mechanism mediating NAADP-induced intracellular Ca2+ release remains unclear. The present study reconstituted and characterized a lysosomal NAADP-sensitive Ca2+ release channel using purified lysosomes from rat liver. Furthermore, the identity of lysosomal NAADP-sensitive Ca2+ release channels was also investigated. It was found that NAADP activates lysosomal Ca2+ release channels at concentrations of 1 nM to 1 microM, but this activating effect of NAADP was significantly reduced when the concentrations used increased to 10 or 100 microM. Either activators or blockers of Ca2+ release channels on the sarcoplasmic reticulum (SR) had no effect on the activity of these NAADP-activated Ca2+ release channels. Interestingly, the activity of this lysosomal NAADP-sensitive Ca2+ release channel increased when the pH in cis solution decreased, but it could not be inhibited by a lysosomal H+-ATPase antagonist, bafilomycin A1. However, the activity of this channel was significantly inhibited by plasma membrane L-type Ca2+ channel blockers such as verapamil, diltiazem, and nifedipine, or the nonselective Ca2+,Na+ channel blocker, amiloride. In addition, blockade of TRP-ML1 (transient receptor potential-mucolipin 1) protein by anti-TRP-ML1 antibody markedly attenuated NAADP-induced activation of these lysosomal Ca2+ channels. These results for the first time provide direct evidence that a NAADP-sensitive Ca2+ release channel is present in the lysosome of native liver cells and that this channel is associated with TRP-ML1, which is different from ER/SR Ca2+ release channels.     

High sensitivity of plasma membrane ion transport ATPases from human neutrophils towards 4-hydroxy-2,3-trans-nonenal

Lipid peroxidation results in release of 4-hydroxy-2,3-trans-nonenal (HNE), which is known to conjugate to specific amino acids of proteins and may alter their function. The effect of HNE on the activities of Na(+)/K(+)-ATPase, Mg(2+)-ATPase, Ca(2+)-ATPase, and calmodulin-stimulated Ca(2+)-ATPase has been studied both in erythrocyte ghosts and in neutrophil membrane preparations. Neutrophil Ca(2+)-ATPase was strongly inhibited by micromolar concentrations of HNE (IC(50) = 12 microM), that means in the range of pathophysiologically relevant HNE levels. The IC(50) value for neutrophil Na(+)/K(+)-ATPase was about 40 microM. HNE was considerably less effective against neutrophil Mg(2+)-ATPase and the erythrocyte ghost enzymes (IC(50) values range from 91 to 240 microM). The data suggest that HNE may play a specific role in the regulation of neutrophil calcium homeostasis in response to oxidative stress.     

动物生物标志物在土壤污染生态学研究中的应用

应用陆栖无脊椎动物的生物标志物对土壤生态系统中污染物的暴露和效应进行评价日益受到重视,并取得了显著的研究进展,文中介绍了溶酶体、胁迫蛋白和金属硫蛋白(MTs)3种主要生物标志物,体腔细胞内溶酶体膜稳定性用中性红保持时间(NRR)进行检测;胁迫蛋白类多采其中的Hsp70和Hsp60;金属硫蛋白不同同分异构体的定量分析可用于反映不同的金属污染胁迫,对3种生物标志物机理、特性、检测实例以及在污染土壤生态毒理诊断中的应用前景进行了评述。     

The photoreactivity of the retinal age pigment lipofuscin.

The presence of the age pigment lipofuscin is associated with numerous age-related diseases. In the retina lipofuscin is located within the pigment epithelium where it is exposed to high oxygen and visible light, a prime environment for the generation of reactive oxygen species. Although we, and others, have demonstrated that retinal lipofuscin is a photoinducible generator of reactive oxygen species it is unclear how this may translate into cell damage. The position of lipofuscin within the lysosome infers that irradiated lipofuscin is liable to cause oxidative damage to either the lysosomal membrane or the lysosomal enzymes. We have found that illumination of lipofuscin with visible light is capable of extragranular lipid peroxidation, enzyme inactivation, and protein oxidation. These effects, which were pH-dependent, were significantly reduced by the addition of the antioxidants, superoxide dismutase and 1,4-diazabicyclo(2,2,2)-octane, confirming a role for both the superoxide anion and singlet oxygen. We postulate that lipofuscin may compromise retinal cell function by causing loss of lysosomal integrity and that this may be a major contributory factor to the pathology associated with retinal light damage and diseases such as age-related macular degeneration.     

Autophagy, ageing and apoptosis: the role of oxidative stress and lysosomal iron

As an outcome of normal autophagic degradation of ferruginous materials, such as ferritin and mitochondrial metalloproteins, the lysosomal compartment is rich in labile iron and, therefore, sensitive to the mild oxidative stress that cells naturally experience because of their constant production of hydrogen peroxide. Diffusion of hydrogen peroxide into the lysosomes results in Fenton-type reactions with the formation of hydroxyl radicals and ensuing peroxidation of lysosomal contents with formation of lipofuscin that amasses in long-lived postmitotic cells. Lipofuscin is a non-degradable polymeric substance that forms at a rate that is inversely related to the average lifespan across species and is built up of aldehyde-linked protein residues. The normal accumulation of lipofuscin in lysosomes seems to reduce autophagic capacity of senescent postmitotic cells--probably because lipofuscin-loaded lysosomes continue to receive newly formed lysosomal enzymes, which results in lack of such enzymes for autophagy. The result is an insufficient and declining rate of autophagic turnover of worn-out and damaged cellular components that consequently accumulate in a way that upsets normal metabolism. In the event of a more substantial oxidative stress, enhanced formation of hydroxyl radicals within lysosomes jeopardizes the membrane stability of particularly iron-rich lysosomes, specifically of autophagolysosomes that have recently participated in the degradation of iron-rich materials. For some time, the rupture of a limited number of lysosomes has been recognized as an early upstream event in many cases of apoptosis, particularly oxidative stress-induced apoptosis, while necrosis results from a major lysosomal break. Consequently, the regulation of the lysosomal content of redox-active iron seems to be essential for the survival of cells both in the short- and the long-term.     

The effect of pH and/or calcium-enriched freshwater on gill Ca2+-ATPase activity and osmotic water inflow in rainbow trout (Salmo gairdneri)

Rainbow trout (Salmo gairdneri) were exposed to pH 5.0-5.1, 6.6 and/or calcium-enriched freshwater for 14 days. Hematocrit, gill Ca2+-ATPase enzyme activities, gill osmotic water inflow, plasma calcium and osmolarity were measured. No significant changes in plasma calcium ion levels were found. The typical increase in hematocrit usually associated with exposure of fish to acidified water was not found in the present study and is discussed. Plasma osmolarity decreased in fish exposed to calcium-enriched freshwater (60 mg Ca2+ X 1(-1) ) in comparison to fish exposed to control freshwater conditions (2 mg Ca2+ X 1(1) ), irrespective of the pH level. Gill Ca2+-ATPase enzyme activities were measured for both low affinity (3 mM Ca2+) and high affinity (100 microM) activity. Exposure of rainbow trout to low pH (pH 5.0-5.1) did not affect the specific activity of Ca2+-ATPase enzyme. However, low affinity Ca2+-ATPase activity in fish exposed to calcium-enriched freshwater did show a significant reduction. The increase in gill osmotic water permeability in fish exposed to calcium-enriched freshwater is interpreted as a result of the increase in osmolarity of the ambient media.     

Studies on the bivalent-cation-activated ATPase activities of highly purified human platelet surface and intracellular membranes.

Membrane-bound Ca2+-ATPases are responsible for the energy-dependent transport of Ca2+ across membrane barriers against concentration gradients. Such enzymes have been identified in sarcoplasmic reticulum of muscle tissues and in non-muscle cells in both surface membranes and endoplasmic-reticulum-like intracellular membrane complexes. In a previous study using membrane fractionation by density-gradient and free-flow electrophoresis, we reported that the intracellular membranes of human blood platelets were a major storage site for Ca2+ and involved in maintaining low cytosol [Ca2+] in the unactivated cell. In the present report we demonstrated that the intracellular membranes also exhibit a high-affinity Ca2+-ATPase which appears to be kinetically associated with the Ca2+-sequestering process. We found that both the surface membrane and the intracellular membrane exhibited a basal Mg2+-ATPase activity, but Ca2+ activation of this enzyme was confined only to the intracellular membrane. Use of Ca2+-EGTA buffers to control the extravesicle [Ca2+] allowed a direct comparison of the Ca2+-ATPase and the Ca2+-uptake process over a Ca2+ range of 0.01 microM to 1.0 mM, and it was found that both properties were maximally expressed in the range of external [Ca2+] 1-50 microM, with concentrations greater than 100 microM showing substantial inhibition. Double-reciprocal plots for the Ca2+-ATPase activity and Ca2+ uptake gave apparent Km values for Ca2+ of 0.15 and 0.13 microM respectively. However, similar plots for ATP with the enzyme revealed a discontinuity (two affinity sites, with Km 20 and 145 microM), whereas plots for the Ca2+ uptake gave a single Km value for Ca2+, 1.1 microM. Phosphorylation studies during Ca2+ uptake using [gamma-32P]ATP revealed two components of 90 and 95 kDa phosphorylated at extravesicle [Ca2+] of 3 microM. The Ca2+-ATPase activity, Ca2+ uptake and phosphorylation were all almost completely inhibited in the presence of 500 microM-Ca2+. Similar studies using mixed membranes revealed four other phosphoproteins (50, 40, 20 and 18 kDa) formed in addition to the 90 and 95 kDa components. The findings are discussed in the context of platelet Ca2+ mobilization for function and the mechanisms whereby Ca2+ homoeostasis is controlled in the unactivated cell.     

Autophagy as a second level protective process in conferring resistance to environmentally-induced oxidative stress

This conceptual paper addresses the role of lysosomal autophagy in cellular defense against environmentally-induced oxidative stress using a marine mollusc (the blue mussel) as an experimental model. It is proposed that augmented autophagic removal of oxidatively damaged organelles and proteins provides a second level or tier of defense against oxidative stress. Age pigment or lipofuscin is a product of oxidative attack on proteins and lipids and can accumulate in lysosomes, where it may generate further reactive oxygen species (ROS) and inhibit lysosomal function, resulting in autophagic failure. The previously observed protective role of augmented autophagy, induced by nutritional deprivation, against oxidative stress can be explained by this model, where autophagy boosts "cellular housekeeping" through enhanced removal of ROS-damaged proteins and organelles minimizing formation of potentially harmful stress/age pigment, and has been proposed as an anti-aging mechanism. Finally, the probable low level triggering of autophagy in mussels by fluctuating environmental regimes is considered as a potential protective mechanism that will contribute to resistance to environmentally induced oxidative stress. It is further conjectured that organisms making up functional ecological assemblages (communities) in fluctuating environments, where upregulation of autophagy should provide a selective advantage, may be pre-selected to be tolerant of pollutant-induced oxidative stress.