1

ESSENTIAL ROLE OF Ca2+-DEPENDENT PHOSPHOLIPASE A2 IN ESTRADIOL-

INDUCED LYSOSOME ACTIVATION

Bruno Burlando, Barbara Marchi2, Isabella Panfoli3, Aldo Viarengo1, 1Dipartimento di Scienze e Tecnologie Avanzate, Università del Piemonte Orientale “Amedeo Avogadro”, Corso Borsalino 54, 15100 Alessandria, Italy

2Dipartimento di Biologia Sperimentale Ambientale ed Applicata, Università di Genova, Corso Europa 26, 16132 Genova, Italy

3Dipartimento di Oncologia, Biologia e Genetica, Università di Genova, Largo R. Benzi, 16132 Genova, Italy Running title: Role of PLA2 in estradiol-induced lysosome activation

Corresponding author: Bruno Burlando Dipartimento di Scienze e Tecnologie Avanzate Università del Piemonte Orientale “Amedeo Avogadro” Corso Borsalino 54, 15100 Alessandria ITALY Phone: +39.0131.283845 Fax: +39.0131.254410 E-mail: burlando@unipmn.it

Copyright 2002 by the American Physiological Society.

AJP-Cell Articles in PresS. Published on July 24, 2002 as DOI 10.1152/ajpcell.00429.2001

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ABSTRACT

The mechanism of lysosome activation by 17β-estradiol has been studied in mussel blood

cells. Cell treatment with estradiol induced a sustained increase of cytosolic free Ca2+ that was

completely prevented by pre-incubating the cells with the Ca2+ chelator, BAPTA-AM.

Estradiol treatment was also followed by destabilization of the lysosomal membranes, as

detected in terms of the lysosomes’ increased permeability to neutral red. The effect of

estradiol on lysosomes was almost completely prevented by pre-incubation with the inhibitor

of cytosolic Ca2+-dependent PLA2 (cPLA2), AACOCF3, and was significantly reduced by

pre-incubation with BAPTA-AM. In contrast, it was virtually unaffected by pre-incubation

with the inhibitor of Ca2+-independent PLA2, BEL. The Ca2+ ionophore A23187 yielded

similar effects on [Ca2+]i and lysosomes. Exposure to estradiol also resulted in cPLA2

translocation from cytosol to membranes, lysosome enlargement, and increased protein

degradation. These results suggest that the destabilization of lysosomal membranes following

cell exposure to estradiol occurs mainly through a Ca2+-dependent mechanism involving

activation of Ca2+-dependent PLA2. This mechanism promotes lysosome fusion and catabolic

activities, and may mediate short-term estradiol effects.

KEY WORDS: Lysosome membrane stability; 17 beta-estradiol; cytosolic phospholipase A2;

calcium signaling; AACOCF3; BEL.

ABBREVIATIONS

AACOCF3, arachidonyl trifluoromethyl ketone; BAPTA/AM 1,2-bis-(o-aminophenoxy)

ethane-N,N,N',N'-tetraacetic acid/tetra(acetoxymethyl) ester; BEL, bromoenol-lactone; NR,

neutral red; PLA2, phospholipase A2.

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INTRODUCTION

The gonadal steroid 17β-estradiol has been described mostly in terms of its long-term

effects related to genomic activation. However, short-term, non-genomic effects of the

hormone have also been observed in a variety of cell types. These effects are frequently

associated to increases in [Ca2+]i (1, 3, 12, 26, 36, 44) and may rely on the presence of

estradiol receptors in the plasma membrane (18, 41, 44). As they may play a significant role

in the regulatory functions of the hormone, these short-term effects warrant further study.

One short-term effect of estradiol which has not previously been studied in association

with other effects of the hormone is destabilization of lysosomal membranes (29, 45, 51).

Lysosomes are membrane-bound organelles rich in acidic hydrolases that represent the

cellular site for bulk macromolecule degradation. Lysosomal activities mediate several

processes in cell feeding, homeostasis and anti-microbial defense, which involve lysosome

fusion with endosomes and (auto)phagosomes (22, 32, 43). Lysosomal membranes are

essential for the correct functioning of the lysosome, permitting hydrolase

compartmentalisation, maintenance of an acidic internal environment and vacuole trafficking.

Because of lysosomal sensitivity to a variety of chemical agents and stressors, lysosomal

alterations are often used as a general biomarker of stress in environmental biomonitoring

(e.g. 10, 48). Lysosomal membrane destabilization involves increased fusion of lysosomes

and phagosomes and is frequently associated to cellular stress deriving from the action of

xenobiotics and pro-oxidant agents (16, 21, 27, 49, 52), or from pathological conditions (11,

14, 46). Under extreme circumstances, normally latent acidic hydrolases may leak into the

cytoplasm, causing damage to cell components and eventually leading to cell death (23, 50,

53). However, lysosomal membrane destabilisation can also occur independently from

cellular stress, such as during apoptosis (19, 34, 40) or in the short-term response to 17β-

estradiol, as mentioned above.

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This study is part of an attempt to address a broader question: is there a common

explanation for the lysosomal (hyper)activations induced by stress conditions and by

endogenous processes? In order to answer this question the mechanisms responsible for

lysosomal membrane destabilization and lysosome activation need to be further clarified. To

reach this aim, we tried to understand the effect of estradiol on lysosomes by exploring

possible links between the process of destabilization of the lysosomal membrane and other

short-term effects of the hormone. We thus sought for an estradiol-induced, Ca2+-dependent

mechanism which leads to modification of the lysosomal membranes. As both cytosolic and

exogenous PLA2 have been found to cause lysosomal membrane destabilisation (33) and

lysosomal enzyme leakage (24), we focussed on identifying a potential association between

Ca2+-dependent cytosolic PLA2 and the effect of estradiol on lysosomal membranes.

Marine mussel blood cells served as our experimental model system. These cells are

lysosome-enriched elements involved in anti-pathogen defence, and have been widely used

for investigations of lysosomes in general (7, 37, 49, 52) and for the study of membrane

destabilizing effects produced by 17β-estradiol in particular (19). The presence of 17β-

estradiol has been ascertained in mussels (39), and in addition, steroid hormones seem to play

typical roles in the lifecycle of these molluscs, for example, in gonad maturation during the

reproductive period (38).

Estradiol treatments were used in combination with two selective inhibitors: the inhibitor

of Ca2+-dependent PLA2, AACOCF3, and the inhibitor of Ca2+-independent PLA2, BEL (47).

Digital imaging of fura-2-loaded cells was used to evaluate cytosolic free Ca2+ variations,

while digital imaging of neutral red stained cells served to evaluate lysosomal membrane

destabilization. Translocation of Ca2+-dependent PLA2 from the cytosol to the membranes, an

essential step in the enzyme activation pathway (8), was detected using cell fractionation and

Western blotting. As lysosomal membrane destabilization is correlated to an increase in

protein degradation, we analyzed the catabolism of short-lived proteins, using cell labelling

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with [14C]-valine, and structural protein catabolism, using actin staining with FITC-labelled

phalloidin and confocal image analysis.

MATERIALS AND METHODS

Chemicals

Bromoenol-lactone: (E)-6-(bromomethylene)tetrahydro-3-(1-naphtalenyl)-2H-pyran-2-one

(BEL), bovine serum albumin (BSA), digitonin, 17β-estradiol, neutral red (NR), phalloidin-

FITC labelled, poly-L-lysine and Sigmacote were from Sigma Chemical Co. (St. Louis, MO);

fura-2 was from Molecular Probes (Eugene, OR); DEVD-CHO was from Biomol Research

Laboratories Inc. (Plymouth Meeting, PA); arachidonyl trifluoromethyl ketone (AACOCF3),

MG132 and 1,2-bis-(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid/tetra(acetoxymethyl)

ester (BAPTA/AM) were from Calbiochem (La Jolla, CA); [14C]-valine was from NEN Life

Sci. Prod. (Boston, MA). All other reagents were of analytical grade.

Solutions Artificial sea water (mM): 473 NaCl, 18 Na2SO4 , 16 MgCl2, 6.2 KCl, 5 CaCl2, 1.5

NaHCO3, 0.045 NaF, 0.56 KBr, 0.32 H3BO3, 0.048 SrCl2·6H2O, pH 8.0.

Physiological saline (mM): 20 Hepes, 436 NaCl, 53 MgSO4, 10 KCl, 10 CaCl2, pH 7.3.

Loading buffer (mM) : 30 Hepes, 0.5 sucrose, 2.5 MgCl2, 2.5 CaCl2, 125 NaCl, 2.5 KCl,

pH 7.3.

Animals

Mussels (Mytilus galloprovincialis Lam.) with a shell length of 4-5 cm were obtained from

a local farm (La Spezia, Italy) and acclimated for three days in an aquarium with aerated,

recirculating, artificial seawater at 15 °C.

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Blood cell collection

Mussel blood (1 ml) was withdrawn from the posterior adductor muscle using a

hypodermic syringe containing an equal volume of artificial seawater. The needle was then

removed and the syringe content was put into a siliconized (Sigmacote) Eppendorf tube.

Evaluation of lysosomal membrane stability and of lysosomal volume

Aliquots of 30 µl of blood cell suspension were placed on microscope slides coated with 2

µl poly-L-lysine (1:10 in distilled water) in order to improve cell-to-substratum adhesion.

Slides were incubated for 30 min in a light-proof humidity chamber at 15 °C, to allow the

cells to settle. Excess solution was removed and 30 µl of a 5 % neutral red (NR) stock

solution (20 mg/ml in DMSO) in artificial seawater were added. After 15 min excess dye was

washed out, 30 µl of artificial seawater were added, with or without 50 nM estradiol, and a

coverslip was applied. Some slides were preincubated with 10 µM AACOCF3 or with 20 µM

BEL for 10 min prior to estradiol treatment. At different times, slides were observed at room

temperature under an Olympus IMT-2 inverted microscope equipped with a CCD CUE video

camera, and images were recorded by a Dage MTI camera and digitised by the CUE2 imaging

system (Galai Production Ltd., Israel). After image recording, the slides were returned to the

humidity chamber. Different fields of each slide were recorded at different times.

Digitised images allowed the evaluation of NR retention time within lysosomes. The

optical density (OD) of 30 cells, randomly selected from each of 4 coverslips from different

cell preparations, was measured by first tracing the cell contour and then recording the

average OD within the selected area. An estimate of the ratio between the total cell volume

and the overall volume of lysosomes was obtained by measuring whole cell areas and overall

lysosomal areas in the 30 different cells selected as described above, using an OD threshold

function in the CUE2 imaging system.

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[Ca2+]i measurements

Aliquots of 40 µl of blood suspension were settled on coverslips as described above,

incubated with loading buffer containing 4 µM fura-2/AM for 30 min at 15 °C, rinsed with

buffer to remove extracellular dye, and bathed with 40 µl of physiological saline with or

without estradiol. Immediately after exposure to estradiol or to control physiological saline,

cells were observed at room temperature under an Olympus IMT-2 inverted microscope

equipped with an IMT2-RFL fluorescence attachment (Olympus Optical Co., Germany) and

with an MTI SIT 68 intensified camera (Oatencourt Ltd., England). Images were acquired

every minute using the CUE2 RMS 4.0 imaging system (Galai Production Ltd., Israel).

Background fluorescence was subtracted before analysis.

Fura-2 calibration was achieved by the equation from Grynkiewicz et al. (13):

[Ca2+]i= Kd (F-Fmin)/(Fmax - F) Sf2/Sb2

where Kd = 135 nM, Fmax and Fmin are maximum and minimum fluorescence intensities,

measured after cell treatment with 50 µM digitonin and 5 mM EGTA, respectively, and

Sf2/Sb2 is the ratio between the excitation efficiencies of free probe and Ca2+-bound probe at

380 nm.

Cell fractionation and Western blotting

Blood suspensions (see above) were settled in petri dishes at 15 °C, and cells were then

exposed to 50 nM estradiol for 30 min, with or without pre-incubation with 10 µM

AACOCF3 for 10 min. Cells were then washed with artificial seawater, scraped in

homogenisation buffer (50 mM NaF, 0.2 mM Na orthovanadate, 130 mM NaCl, 10 mM

phosphate buffer, pH 7.2, 5 µg/ml aprotinin, 5 µg/ml antipain, 5 µg/ml pepstatin A, 1 µg/ml

chimostatin, and 1 µg/ml leupeptin, Sigma Chemical Co.) and lysed by means of a tight

glass/glass potter. Lysates were centrifuged at 100 g for 15 min using a Sorvall RC-5B

(DuPont Instruments, Newtown, CT), the supernatant was centrifuged at 100,000 g for 1.5 h

in a Beckman L5-50B Ultracentrifuge (Beckman Instruments, Inc., Fullerton, CA), and the

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ensuing supernatant and pellet were stored at –40 °C until used. Protein determination was

performed using the bicinchoninic/BCA protein assay (Pierce Chemical Company, Rockford,

IL). Samples were electrophoresed under reducing conditions on 8.5 % SDS-PAGE and

proteins blotted to Hybond ECL filters (Amersham Pharmacia Biotech, Uppsala, Sweden)

using a Hoefer TE blotting device (Hoefer Scientific Instruments, San Francisco, CA). Filters

were incubated for 1 h at 4° C with goat polyclonal anti-cPLA2 (C-20, Santa Cruz

Biotechnology Inc, Santa Cruz, CA), diluted 1:100 in TBST-BSA 3.5%, and then incubated

for 1 h with HRP-conjugated anti-goat IgG (Santa Cruz) diluted 1:500 in TBST-BSA 1%.

Binding of antibodies was visualised by the ECL chemiluminescence detection system

(Roche, Basel, Switzerland), digitised by the the Fluor-S Max gel analyzer (Bio Rad

Laboratories), and analysed by the Quantitiy One 4.2.1 software (Bio Rad Laboratories).

Protein degradation

Cell radiolabelling. Blood cells were settled onto slides and incubated in physiological

saline with 1 µM DEVD-CHO, a caspase-3 inhibitor, 100 µM MG132, a proteasome

inhibitor, plus 1 µCi/ml [14C]-valine, (specific activity 250 mCi/mmol), in a humidity

chamber at 15°C for 2 h. The reaction was stopped by adding 2 mM of cold valine.

Thereafter, cells were treated with 50 nM estradiol at 15°C for 2.5 h and then 60 µl aliquots

were spotted on 3 MM filter paper disks (Whatman, Whatman House, UK) moistened with 5

% cold trichloroacetic acid solution (TCA) (diameter = 25 mm). Filters were then transferred

to 5 % cold TCA for 10 min, washed twice more with the same TCA before being dried, and

counted for acid-soluble radioactivity with PicoFluor-40 (Packard, Camberra, AU) in a 1600

CA Tri-Carb scintillation analyser (Packard). Radioactivity was expressed in CPM per mg of

total protein.

Actin labelling. Cells were incubated with proteasome and caspase-3 inhibitors as

described above, and then treated with 50 nM estradiol at 15 °C for 4 h. Thereafter, cells were

washed with phosphate buffered saline (PBS), fixed with 3 % paraformaldehyde in PBS at

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room temperature for 10 min followed by 0.5 % Triton X100 in PBS for 1 min, washed twice

with PBS, incubated with 0.1 mg/ml phalloidin-FITC labelled at room temperature for 30

min, and mounted in glycerol. Slides were observed under a Zeiss LSM 510 confocal

microscope (Carl Zeiss Inc., Thornwood, NY).

Statistics

Data were analysed by the Systat 8.0 software (SPSS Inc., Evanston, IL).

RESULTS

[Ca2+]i measurements and lysosomal membrane stability analysis

Loading of mussel blood cells with fura2-AM and subsequent imaging with digital

fluorescence microscopy revealed that exposure to 50 nM 17β estradiol induced a significant

increase in [Ca2+]i (Fig. 1A), which was completely prevented by cell loading with the Ca2+

chelator BAPTA-AM (15 µM for 15 min) prior to estradiol exposure (Fig. 1A).

Lysosomal membrane destabilization was evaluated by staining cells with the

lysosomotropic dye neutral red (NR). Transmission light microscopy and digital image

analysis showed no lysosome destaining in control cells within 1 h. By contrast, destaining of

cells exposed to 50 nM estradiol started at about 15 min and was highly significant after 1 h,

indicating dye leakage due to lysosomal membrane destabilization (Fig. 1B). The effect of

estradiol on lysosomes was almost entirely prevented by preincubation with AACOCF3,

significantly lowered by pre-incubation with BAPTA-AM, and almost unaffected by

preincubation with BEL (Fig. 1B). In addition, the use of fura-2 showed that AACOCF3 did

not produce any effect on cell Ca2+ and did not prevent the Ca2+ rise induced by estradiol

(Table 1).

In another set of experiments, blood cells were exposed to the Ca2+ ionophore A23187 (20

µM), which yielded effects quite similar to those induced by estradiol. By using fura-2 or

neutral red in different tests, A23187 was shown to induce an increase in [Ca2+]i and

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lysosomal membrane destabilization (Fig. 2). Also, preincubation with AACOCF3

significantly reduced the lysosome destaining caused by A23187 (Fig. 2B), without

preventing the Ca2+ rise induced by the ionophore (Table 1). Hence, data deriving from the

use of estradiol and of the Ca2+ ionophore, indicate that the destabilization of lysosomal

membranes occurs mainly through a Ca2+-dependent mechanism, involving the activation of

Ca2+-dependent PLA2 However, the failure of BAPTA to completely prevent the estradiol

effect on lysosomes suggests that some Ca2+-independent mechanism also plays a minor role.

Assessment of Ca2+-dependent PLA2 activation

The use of a goat polyclonal antibody on mussel blood cell homogenates permitted

detection of a putative homologue of mammalian cPLA2, the Ca2+-dependent cytosolic PLA2.

Western blotting after fractionation of mussel blood cell homogenates showed an increase of

the cPLA homologue in the particulate fraction of cells exposed to estradiol, indicating that

estradiol induces a translocation of Ca2+-dependent PLA2 from cytosol to membranes.

Moreover, such an effect was significantly reduced by preincubation with AACOCF3 (Fig.

3).

Assessment of lysosomal volume and protein degradation

As lysosomal membrane destabilization generally leads to an increase in lysosomal volume

and cell catabolic activities, we sought similar effects in estradiol treated cells. In one

experiment, transmission light digital imaging of NR stained cells showed an increase of the

ratio between the overall volume of lysosomes and the total cell volume after exposure to

estradiol. In this case too, preincubation with AACOCF3 successfully prevented the estradiol

effect, whereas in the presence of BEL the effect of estradiol remained almost the same (Fig.

4A). Quite similar results were found after cell exposure to A23187 (Fig. 4B).

In another experiment, the degradation of long-lived protein was evaluated by staining

actin with fluorescein-labelled phalloidin. Quantitative analysis of confocal laser micrographs

showed a sharp decrease of fluorescence in cells exposed to estradiol, indicating actin

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degradation (Fig. 5A). However, the fluorescence decrease was significantly lowered by cell

pre-incubation with AACOCF3 (Fig. 5A).

Finally, the ability of estradiol to induce an increase in protein turnover was investigated

by cell labelling with the radioactive amino acid [14C]-valine. The release of radioactive

amino acid from labelled proteins was higher after exposure to estradiol, indicating an

increase in protein breakdown (Fig. 5B), while AACOCF3 pre-incubation abolished such an

estradiol effect (Fig. 5B). In these experiments, possible bias due to extra-lysosomal proteases

was limited using specific inhibitors. The use of caspase-3 protease inhibitor was essential for

preventing the activation of caspase-3 due to lysosome labilization (15, 20), while the use of

proteasome inhibitor decreased background noise.

Results from experiments using digital imaging and protein labelling consistently indicate

that the lysosomal membrane destabilization induced by estradiol is strictly correlated to an

increase of lysosomal volume and protein degradation.

DISCUSSION

Our data have demonstrated that exposure of living mussel blood cells to 17β-estradiol

causes a destabilization of lysosomal membranes, detected in terms of increased permeability

to NR. Similar effects of estradiol on lysosomes were pointed out in previous studies (29, 51),

but the present results indicate for the first time a strict association between lysosomal

membrane destabilization and [Ca2+]i increase.

The estradiol-induced increase in [Ca2+]i can be correlated to different cell processes (36).

Moreover, different studies showed that the increase in [Ca2+]i depends on either Ca2+ entry

(1, 36) or Ca2+ release from intracellular stores (12), or else on both processes (4, 26). While

we did not establish the mechanism of [Ca2+]i elevation in mussel blood cells, we did show

that it can be abolished by loading cells with the Ca2+ chelator BAPTA prior to estradiol

stimulation, as previously shown in vascular endothelium by Goetz et al. (12). This evidence

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was essential for assessing the involvement of Ca2+ in the estradiol effect on lysosomes, as

BAPTA also prevents most lysosomal membrane destabilization in estradiol-treated cells.

Further confirmation arose from the use of the Ca2+ ionophore A23187, showing that an

increase in [Ca2+]i, similar to that caused by estradiol, is sufficient to produce a significant

destabilising effect on lysosomal membranes.

Having established the role of Ca2+ as a mediator of estradiol effects, several arguments

pointed to Ca2+-dependent PLA2 as the potential connection between Ca2+ and lysosomes.

Many cell types and organs express a cytosolic Ca2+-dependent PLA2, which selectively

releases arachidonic acid from membrane phospholipids (17). PLA2 activity is responsible for

eicosanoid synthesis, which is crucial to the initiation of the inflammatory response (2) and is

activated by Ca2+-dependent translocation from the soluble to the membrane fraction of cells,

allowing access to the arachidonoyl-containing phospholipid substrate (5, 42). The lipolytic

activity of PLA2 can affect different membrane structures, possibly also including lysosomal

membrane, as has been seen in snake venom PLA2 (33). Moreover, PLA2 plays an important

role in blood phagocytic cells through the activation of NADPH oxidase (9).

In our experiments, BAPTA considerably reduced the effects of estradiol on lysosomes,

while the inhibitor of Ca2+-dependent PLA2 (AACOCF3) almost entirely blocked these

effects without preventing an increase in [Ca2+]i. By contrast, the inhibitor of Ca2+-

independent PLA2 (BEL) had no effect. These results indicate that Ca2+-dependent cytosolic

PLA2 is the agent responsible for estradiol-induced lysosomal membrane destabilization.

However, experiments with BAPTA showed that when Ca2+ is removed from the cytosol,

estradiol is still capable of inducing a slight destaining of lysosomes, while the lysosome

destaining induced by A23187 was only partially abolished by AACOCF3. This suggests that

either estradiol or Ca2+ can induce limited membrane destabilization on lysosomes

independently from the Ca2+/PLA2 pathway. Overall, however, the data indicate that the

Ca2+-dependent PLA2 pathway is the main mechanism by which estradiol produces its effect

13

on lysosomes. A further confirmation of this mechanism comes from Western blotting

experiments, which clearly showed cPLA2 translocation to the membranes induced by

estradiol.

Membrane destabilization leads to fusion of membrane-bound organelles, in which a role

is played by arachidonic acid (6). Accordingly, experimental evidence points to the

involvement of PLA2 in lysosome/endosome fusion (25). Moreover, lysosomal membrane

destabilization is known to be correlated both to enlargement of lysosomes (16), suggesting

lysosome/endosome fusion, and to intracellular free Ca2+ elevation in fibroblasts (3). In

addition, it has been shown that lysosomal fusion involves activation of different catabolic

activities, particularly protein degradation (30). This scenario is consistent with the results of

the present study. In our experimental system, the PLA2-dependent increase in lysosome

volume following estradiol treatment is no doubt linked to increased fusion activities. Also,

the increase in lysosome volume that was induced induced by estradiol or A23187 was

completely abolished by AACOCF3. This clearly indicates that lysosome fusion is totally

dependent on PLA2 activation, in contrast to what happens in lysosome destaining during the

NR assay. In addition, our data on degradation rates of both short- and long-lived proteins

showed that estradiol can induce metabolic activation of the lysosomal machinery, while the

use of AACOCF3 confirmed the involvement of Ca2+-dependent PLA2 in this process.

In conclusion, we have identified a short-term pathway of estradiol, which consists in an

activation of Ca2+-dependent PLA2 and leads to typical morphofunctional modifications of

the lysosomal vacuolar system. Besides yielding a molecular explanation for endogenous

lysosome activation, this study also offers a new perspective for the understanding of

lysosomal alterations due to pathological conditions or the effects of xenobiotic compounds.

Lysosomal membrane destabilization can be used as a biomarker of stress, and a variety of

stressors causing lysosomal alterations, such as heavy metals and oxidants (28,31,49), are also

known to affect cell Ca2+ homeostasis (35). Therefore, an involvement of Ca2+-dependent

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PLA2 could also be postulated in these cases. Hence, the mechanism described in the present

study may be relevant to different fields of investigation, ranging from the understanding of

cellular mechanisms of lysosome activation to the application of lysosome-based biomarkers

in ecological risk assessment.

ACKNOWLEDGEMENT

This work was supported by grants from the Italian Ministry for University and Scientific

Research (MURST). B. Marchi is recipient of a MURST postdoctoral fellowship.

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20

TABLES

Table 1. Variations of [Ca2+]i in mussel blood cells exposed to different compounds and in

the presence or absence of AACOCF3.

t = 0 min t = 15 min

c ontrol 75 ± 7 78 ± 6 AACOCF3 76 ± 5 77 ± 2

p > 0.05

e stradiol 76 ± 11 221 ± 28 e stradiol+AACOCF3 78 ± 10 209 ± 26

p > 0.05

A 23187 79 ± 2 222 ± 30 A23187+AACOCF3

79 ± 2 210 ± 28 p > 0.05

Data are expressed as [Ca2+]i (nM), n = 8. P values refer to pairwise t test comparisons

between [Ca2+]i levels recorded at 15 min in the presence or absence of AACOCF3.

21

FIGURE LEGENDS

Figure 1. Effects of estradiol on the [Ca2+]i and lysosomes of mussel blood cells. (Upper panel)

Free Ca2+ measurements in fura 2-loaded cells treated with 50 nM estradiol show a sustained

Ca2+ rise starting from 10 min after exposure. Preincubation with the Ca2+ chelator BAPTA-

AM (15 µM) for 15 min completely abolishes the effect of estradiol on cytosolic Ca2+. Data

are means ± SD out of 8 Ca2+ measurements in different cells. (Lower panel) Digital imaging

measurements of the optical density of cells 60 min after staining with the lysosomotropic dye

neutral red (see Experimental Procedures). Data show significant destaining upon treatment

with estradiol (50 nM, exposure starts at t = 0). Different preincubations prior to estradiol

exposure show the following results: (a) almost complete destaining using 20 µM BEL for 10

min; (b) limited destaining using 15 µM BAPTA-AM for 15 min; (c) non-significant

destaining using 10 µM AACOCF3 for 10 min. Data are means ± SD (n = 30) expressed as

percent of control optical density. Bars with different letters indicate significant differences

according to the Bonferroni test (p < 0.01).

Figure 2. Effects of the Ca2+ ionophore A23187 on [Ca2+]i and lysosome membrane stability.

(A) Fura 2-loaded cells exposed to 20 µM A23187 show sustained [Ca2+]i rise. (B) Cells

stained with neutral red show significant destaining after treatment with A23187 (exposure

starts at t = 0), while the effect is partially prevented by pre-incubation with 10 µM

AACOCF3 for 10 min. Data as in fig. 1.

Figure 3. Western blot analysis of cPLA2 in cytosolic and particulate fractions of mussel blood

cells. Fifty micrograms of protein were solved on SDS-polyacrylamide gels, transferred to

Hybond ECL membranes and probed with goat polyclonal anti-cPLA2 (see Experimental

Procedures). (A) Exposure to estradiol induces cPLA2 translocation to membranes, as shown

by the increase of the cPLA2 band in the particulate fraction and parallel decrease in the

cytosolic one respect to control. Incubation with AACOCF3 prior to estradiol produces a

22

strong reduction of cPLA2 band increase in the particulate fraction. (B) Digital image

quantification of band peak densities.

Figure 4. Effects of estradiol (A) and of A23187 (B) on the size of lysosomes. Digital imaging

evaluation of the lysosomal/cell volume ratio shows an increase in the overall lysosomal

volume after 60 min exposure to either 50 nM estradiol or 20 µM A23187. The fraction of the

cell volume pertaining to lysosomes varies from about 23-24 % (t = 0 min) to about 36-38 %

(t = 60 min). The effects of both estradiol and A23187 on lysosome volume are abolished by

pre-incubation with AACOCF3, but not by pre-incubation with BEL. Data are means ± SD (n

= 30), * = p < 0.01 in a t-test comparison between t = 0 and t = 60 min.

Figure 5. Estradiol effect on protein degradation, as evaluated after cell labelling with phalloidin-

FITC, or with [14C]-valine. (A) Cell stained for actin using phalloidin-FITC labelled, and

incubated with estradiol, with or without AACOCF3 preincubation, show different

fluorescence intensities when observed under a confocal microscope. Treatment with 50 nM

estradiol induces significant fluorescence decrease after 4.5 h respect to control, while

AACOCF3 pre-incubation partially prevents such a fluorescence decrease. Data are means ±

SD (n = 46), all groups are significantly different from each other (p < 0.01) according to

bonferroni t-test. Insets show confocal images of phalloidin-labelled cells after the different

incubations (bar = 25 µm). (B) Cells labelled with [14C]-valine show increased release of the

radioactive aminoacid after 2.5 h of treatment with estradiol. Preincubation with AACOCF3

is able to restore [14C]-valine release to control values. Data are means ± SD (n = 6),

expressed as percent of specific activity in control cells. * = p < 0.05, bonferroni test.

23

FIGURES

50

100

150

200

250

300

0 5 10 15 20 25time (min)

[Ca2

+] (n

M)

estradiol

estradiol/ BAPTA

control A

0

40

80

120

control estradiol estradiol/ estradiol/ estradiol/

optic

al d

ensi

ty (%

)

aa

bb

c

BAPTA AACOCF3 BEL

B

Fig. 1.

24

50

100

150

200

250

0 5 10 15time (min)

[Ca2

+] (n

M)

A23187 control

A

20

40

60

80

100

0 10 20 30 40 50 60time (min)

optic

al d

ens.

(%)

controlA23187 + AACOCF3A23187 B

Fig. 2.

25

A estradiol /

AACOCF3 estradiol control

particulate cytosol

0

20

40

60

80

100

120

optic

al d

ens.

particulatecytosol

estradiolestradiol/AACOCF3control

B Fig. 3.

26

0

10

20

30

40

estradiol estr/ BEL estr /AACOCF3

lyso

som

e/ce

ll vo

lum

e (%

) * *A

0

10

20

30

40

A23187 A23187/ A23187/

lyso

som

e/ce

ll vo

lum

e (%

) t=0 min

t=60 min* *B

BEL AACOCF3

Fig. 4.

27

0

20

40

60

80

control estradiol estr/AACOCF3

fluor

esc.

int.

(A. U

.) A

0

40

80

120

control estradiol estr/AACOCF3

CPM

/ mg

prot

*B

Fig. 5.