Azaspiracid-1, a potent, nonapoptotic new phycotoxin

with several cell targets

Yolanda Romana, Amparo Alfonsoa, M. Carmen Louzaoa, Laura A. de la Rosaa,Francisco Leirab, Juan M. Vieitesb, Mercedes R. Vieytesc, Katsuya Ofujid,

Masayuki Sataked, Takeshi Yasumotoe, Luis M. Botanaa,*aDepartamento de Farmacologıa, Facultad de Veterinaria, USC, 27002 Lugo, Spain

bANFACO-CECOPESCA, Campus Universitario, Lagoas (Marcosende), 36310 Vigo, SpaincDepartamento de Fisiologıa, Facultad de Veterinaria, USC, 27002 Lugo, Spain

dGraduate School of Agricultural Science, Tohoku University, Sendai 981-8555, JapaneJapan Food Research Laboratories, Tama, Tokyo 206-0025, Japan

Received 16 August 2001; accepted 20 December 2001

Abstract

This paper reports on potential cellular targets of azaspiracid-1 (AZ-1), a new phycotoxin that causes diarrhoeic and neurotoxic symptoms

and whose mechanism of action is unknown. In excitable neuroblastoma cells, the systems studied were membrane potential, F-actin levels

and mitochondrial membrane potential. AZ-1 does not modify mitochondrial activity but decreases F-actin concentration. These results

indicate that the toxin does not have an apoptotic effect but uses actin for some of its effects. Therefore, cytoskeleton seems to be an

important cellular target for AZ-1 effect. AZ-1 does not induce any modification in membrane potential, which does not support for

neurotoxic effects. In human lymphocytes, cAMP, cytosolic calcium and cytosolic pH (pHi) levels were also studied. AZ-1 increases

cytosolic calcium and cAMP levels and does not affect pHi (alkalinization). Cytosolic calcium increase seems to be dependent on both the

release of calcium from intracellular Ca2 + pools and the influx from extracellular media through Ni2 + -blockable channels. AZ-1-induced

Ca2 + increase is negatively modulated by protein kinase C (PKC) activation, protein phosphatases 1 and 2A (PP1 and PP2A) inhibition and

cAMP increasing agents. The effect of AZ-1 in cAMP is not extracellularly Ca2 + dependent and unsensitive to okadaic acid (OA).

D 2002 Elsevier Science Inc. All rights reserved.

Keywords: Azaspiracid; Okadaic acid; Saxitoxin; Lymphocyte; cAMP; Actin; Cytosolic calcium; Cytosolic pH; Potential

1. Introduction

Phycotoxins constitute a large source of active phar-

macological tools, with a very high variety of mechanisms

of action. They can be potent phosphatase inhibitors [such

as okadaic acid (OA)] [1], block sodium channels in several

different receptors within the channel (such as brevetoxins

[2] or saxitoxin (STX) [3]), inhibit sodium–potassium

ATPase (such as the case of palytoxin) [4] or increase

calcium uptake (such as the case of maitotoxin) [5]. In

some cases, the particular mechanism of action of some

phycotoxins renders lead structures to develop new drugs,

such as conotoxins [6]. However, despite all the information

already gathered about many phycotoxins, they pose a

constant challenge to scientists, because new compounds

are being reported with new and unknown mechanisms of

actions. Examples of these are yessotoxins [7,8], which

were recently discovered and still have unknown mecha-

nisms of action [9].

A new and very recent group of marine toxins that were

implicated in a mussel poisoning in the Netherlands in

November 1995 [10] are azaspiracids (AZ) [11]. They have

spiral ring assemblies, a cyclic amine and a carboxylic acid,

and these characteristics make them unique within the

nitrogen-containing marine toxins. So far, five different

compounds have been described: AZ-1, AZ-2 (8-methyla-

zaspiracid) and AZ-3 (22-dimethylazaspiracid) [12] and

more recently AZ-4 and AZ-5, which are 3-hydroxy-

0898-6568/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved.

PII: S0898 -6568 (02 )00015 -3

* Corresponding author. Tel./fax: +34-982-252-242.

E-mail address: luis.botana@lugo.usc.es (L.M. Botana).

www.elsevier.com/locate/cellsig

Cellular Signalling 14 (2002) 703–716

dimethylazaspiracid and 24-hydroxy-demethylazaspiracid,

respectively [13] (Scheme 1). The toxic episodes caused

by AZs show gastrointestinal illnesses, resembling there-

fore the potential symptoms of OA and diarrhoeic shell-

fish toxins. However, despite this fact, the mouse

bioassay shows clear neurotoxic symptoms (respiratory

difficulties, spasms, paralysis of the limbs and death),

which is quite different from the typical diarrhoetic

shellfish poisoning (DSP) bioassay. AZ has been reported

to cause multiple organ damage in mice, with fluid

accumulation in the small intestine, serious affectation

of the lamina propia and liver and spleen damage as

well as damage on lymphoid tissue in both T and B

lymphocytes [14].

So far, there is no information whatsoever about either

the cellular target of these toxins or their mechanism of

action, and no study has been performed. Because these

toxins are a serious threat to human health and show a

new strategy to modify the function of biological systems,

we decided to explore the possible targets of their action.

In order to seek for AZ cellular targets, we decided to

check for modulatory activities in several systems: mem-

brane potential in excitable cells (in order to check for

possible neurotoxic mechanisms), changes in calcium lev-

els (because calcium is a signal in any mammalian cell,

and for that we used human lymphocytes, which have the

advantage of being very stable cells and provide direct

information from healthy human donors), cAMP levels

(which, in the same fashion as calcium, is a universal

signal transducer), cytosolic pH (pHi) (which is a broad

source of information about changes in cytosolic ions and

are in the end will be related to possible losses of ions and

fluid as is the case of diarrhoea), mitochodrial membrane

potential (which is a marker of apoptosis effects related to

long-term effects of the toxin) [15] and finally F-actin pool

formation (which is also related to the potential changes

that may take place in the mechanisms of phycotoxin-

induced diarrhoea) [16,17].

2. Materials and methods

2.1. Chemicals and solutions

STX was obtained as previously reported [18,19]. AZ-1

was obtained and isolated by Satake et al. [11]. Veratridine

(VTD), gramicidin, pertussis toxin (PT), wortmannin and

N 6,20-O-dibutyryladenosine 30:50-cyclic monophosphate

(db-cAMP) were from Sigma (Spain). Bis-(1,3-diethylthio-

barbituric acid) trimethine oxonol (bis-oxonol), 20,70-bis(car-

boxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester

(BCECF-AM), Oregon Green-514 phalloidin, Mitotracker

Red CMXRos, recombinant fluorescein- and rhodamine-

labelled protein kinase A (FlCRhR), Influx Pinocytic Cell-

Loading Reagent and Fura2 acetoxymethyl ester were from

Molecular Probes (The Netherlands). Percoll was from

Pharmacia (Sweden). [1-[b-[3-(4-methoxyphenyl)propoxy]-

4-methoxyphenethyl]-1H-imidazole HCl] (SKF96365),

12-O-tetradecanoylphorbol 13-acetate (PMA), OA, [40,5,

7-trihydroxyisoflavone] (genistein), forskolin (FSK), [9-(tet-

rahydro-2-furanyl)-9H-purine-6-amine] (SQ22,536) and

[N-[2( p-bromocinnamylamino)ethyl]-5-isoquinolinesulfo-

namide 2HCl] (H89) were from Alexis (Switzerland). Go

6976 was from Calbiochem (Spain). All other chemicals

were reagent grade and purchased from Sigma and Merck.

The composition of saline solution used to lymphocyte

purification (PBS) was 145.2-mMNa+, 4.7-mMK+, 8.2-mM

HPO42�, 1.5-mM H2PO4�, 141.2-mM Cl � and 2-mM

ethylenediaminetetraacetic acid (EDTA). The composition

of physiological saline solution used for microscopy experi-

ments was 142.3-mM Na+, 5.94-mM K+, 1-mM Ca2+,

1.2-mM Mg2+, 126.1-mM Cl�, 22.85-mM CO3�, 1.2-mM

PO4H2�, 1.2-mM SO4

2� and 1-mg/ml glucose, giving a final

osmotic pressure of 300 ± 5-mOsm/kg H2O. Ca2+-free solu-

tion was made by omitting Ca2+ from the medium. In all the

experiments, the incubation medium was equilibrated with

CO2 and the final pH was adjusted to 7.2.

2.2. Human lymphocytes isolation

Peripheral human lymphocytes were isolated from fresh

human blood from healthy donors. The blood was diluted

1:1 with PBS plus 2-mM EDTA and 4 ml of diluted blood

was carefully placed over 3 ml of 57.5% isotonic Percoll.

After centrifugation (25 min, 1000� g), Percoll was elim-

inated by washing three times with PBS plus 2-mM EDTA

at 400� g for 5 min. Lymphocytes purity was always higher

than 85%.Scheme 1. Structures of AZs.

Y. Roman et al. / Cellular Signalling 14 (2002) 703–716704

2.3. Measurement of intracellular cAMP levels

Image processing. Purified lymphocytes were loaded

with FlCRhR by the following protocol: 10-ml HypertonicLoading Medium (Influx Pinocytic Cell-Loading Reagent)

containing 0.4-ml FlCRhR were added to 300,000 purified

lymphocytes. After gentle resuspension, the cells were

incubated for 10 min at 37 �C. Then, 3-ml Hypotonic

Lysis Medium [water-diluted saline solution (6:4)] was

added and incubated for 1.5 min at 37 �C. Loaded cells

were quickly washed three times (400� g, 2 min) with

saline solution. Aliquots of 20-ml loaded cells were

allowed to attach to poly-L-lysine-coated 22-mm glass

coverslips for 10 min. The glass coverslips were inserted

into a 37 �C thermostated chamber (Intracell, UK) and

cells were viewed with a Nikon Diaphot 200 microscope

equipped with epifluorescence optics (Nikon 40� immer-

sion UV-Fluor objective). The chamber was used in the

open bath configuration and additions made by aspiration

and addition of fresh bathing solution. Intracellular cAMP

variations were obtained from the images collected by

duplicate emission fluorescence with Life Science Resour-

ces equipment. The light source was a 175-W xenon

lamp, and the light reached the objective through optic

fiber. The excitation wavelength for FlCRhR was 490 nm

and the emission wavelengths were 530 and 580 nm.

FlCRhR is a single-excitation dual-emission dye, similar

to native protein kinase A, whose emission spectrum

changes with cAMP binding. In this way, the FlCRhR

530/580 emission ratio increases upon intracellular free

cAMP concentration elevation [20]. db-cAMP (200 mM)

was added at the end of each experiment to test cAMP

fluorosensor functionality. Graphics represent the average

of three or four experiments done in duplicate (40 cells

per single experiment).

2.4. Cell labelling and determination of pHi and [Ca2+]i

Purified lymphocytes were incubated with the pH and

Ca2+-sensitive fluorescent dyes 0.2-mM BCECF-AM and

2-mM Fura2-AM for 10 min at 37 �C in 1 ml of Ca2+ and

BSA-containing saline solution. After incubation, cells were

washed and attached to a glass coverslip coated with

0.001% polylysine. The coverslips were mounted on a

thermostated chamber at 37 �C (Intracell) to which 500 mlof Umbreit solution was added. Drugs were added by

exchanging the bathing solution.

pHi and Ca2+ ([Ca2+]i) were simultaneously monitored

in individual cells by measuring the BCECF and Fura2

fluorescence, respectively, with the ratio imaging micro-

scopy equipment (see cAMP measurements). BCECF

fluorescence was recorded by alternating the excitation

at 490 and 440 nm and measuring the emission at 535 nm.

Fura2 fluorescence was recorded by alternating the excita-

tion at 340 and 380 nm and measuring the emission at

520 nm.

The calibration of fluorescence vs. pHi was made using

nigericin in K+ solution as per Thomas et al. [21]. Briefly, a

calibration curve was obtained with four known values of

pH, measuring the fluorescence ratio obtained in the pres-

ence of nigericin for each pH value. The [Ca2+]i was

obtained from the ratio 340/380 of the images collected

according to the method of Grinkiewicz et al. [22].

2.5. Neuroblastoma cell culture

Neuroblastoma cell line BE(2)-M17 (ATCC Number

CRL-2267) was purchased from the European Collection

of Cell Cultures (UK) and seeded in 25-cm2 flasks at a

seeding density of 4� 104 cells/cm2. Cells were cultured

on EMEM (EBSS):Ham’s F12 (1:1) with 2-mM gluta-

mine, 1% nonessential amino acids, 15% foetal bovine

serum, 50-mg/l gentamicine and 50-mg/l amphotericine B

(Biochrom KG, Germany). The culture medium was

renewed on a 2–3-day basis and the cells incubated at

37 �C/5% CO2 until 70–90% confluence was reached.

Later on, cells were subcultured by transferring cells

released by the application of 0.1% trypsin. For micro-

plate assays, the attached cells were trypsinized after

reaching optimum confluence, and the concentration of

the culture was adjusted at 12,500 cells/ml, dispensing the

cell suspension in 96-well microtiter plates at 2500 cells/

well (200 ml). Following an additional incubation of 48 h

(37 �C/5% CO2), cells grown in microtiter plates were

used for fluorimetric microplate assays.

2.6. Cell membrane potential

Changes in membrane potential of neuroblastoma cells

were monitored with the fluorescent dye bis-oxonol. Bis-

oxonol is a permeant lipophilic anion whose distribution

across the cell membrane is dependent on membrane poten-

tial. Fluorescence emission of this dye increases with

membrane depolarization. Detection of changes in the

membrane potential induced by toxins in excitable cells is

as follows. Suspension of 0.5� 106 human neuroblastoma

cells were incubated with 2 nM of the fluorescent dye bis-

oxonol. During the experiment, bis-oxonol fluorescence was

registered at 37 �C [wavelengths 540 (excitation) and 560

(emission) nm] in a stirred quartz microcuvette with a

Shimadzu RF-500 spectrofluorometer. Stabilization of the

baseline (equilibrium distribution of the probe) lasted for

5–10 min before the experiment began. Toxins were added

from stock solutions with a pipette directly into the cuvette

through a small hole on top of the cuvette lid. At the end of

each experiment, 10-mg/ml gramicidin was added to induce

complete cell depolarization.

2.7. Mitochondrial membrane potential

Changes in mitochondrial membrane potential in BE(2)-

M17 cells were evaluated in triplicate experiments using

Y. Roman et al. / Cellular Signalling 14 (2002) 703–716 705

the fluorescent probe Mitotracker Red CMXRos. Follow-

ing 1 and 24 h of drug exposure of the cells, the plates

were centrifuged, 50 ml of the culture medium were

removed and 50 ml of Mitotracker Red CMXRos diluted

in the same medium were added to each well to give a

final concentration of 1 mM.After an additional 37 �C/45-min

incubation, the plates were centrifuged, the culture medium

was removed and the cells were washed once with Hank’s

solution and centrifuged. Fluorescence was measured as

previously described, and results of triplicate experiments

were expressed as average percentages of fluorescence

values obtained in controls.

2.8. Determination of F-actin

The effect on F-actin levels in BE(2)-M17 cells was

also evaluated on triplicate experiments after 1 and 24 h

of exposure of the cells to phytoplanktonic toxin. Fol-

lowing incubation, F-actin was measured as follows: 50 mlof 18.5% formaldehyde were added to each well (except

blanks) and cells were fixed for 30 min at room temper-

ature. Following a washing step with Hank’s balanced salt

solution (200 ml), cells were permeabilized (except

blanks) with 200 ml of 0.1% Triton-X 100 for 15 min

Fig. 1. (A) AZ- and STX-induced changes in membrane potential.

Neuroblastoma cells were equilibrated with a final concentration of 2-nM

bis-oxonol. Once a steady baseline was obtained, cell suspensions were

treated with 10- or 100-nM AZ-1 or 160-ng/ml STX. In order to show

complete depolarization, cells were treated with 10-mg/ml gramicidin at the

end of the experiment. (B) AZ-induced changes in membrane potential

following treatment with VTD. Neuroblastoma cells were equilibrated with

a final concentration of 2-nM bis-oxonol. Once a steady baseline was

obtained, cell suspensions were treated with 40-mM VTD. Once the VTD-

induced depolarization reached a steady level, cells were treated with

10- and 100-nM AZ-1, 160-ng/ml STX or DMSO in controls. In order to

show complete depolarization, cells were treated with 10-mg/ml gramicidin

at the end of the experiment.

Fig. 2. (A) AZ-induced changes in membrane potential following treatment

with STX. Neuroblastoma cells were equilibrated with a final concentration

of 2-nM bis-oxonol. Once a steady baseline was obtained, cell suspensions

were treated first with 16-ng/ml STX and then cells were treated with

100-nM AZ-1. In order to show complete depolarization, cells were treated

with 10-mg/ml gramicidin at the end of the experiment (representative

trace). (B) AZ-induced changes in membrane potential following treatment

with VTD. Neuroblastoma cells were equilibrated with a final concen-

tration of 2-nM bis-oxonol. Once a steady baseline was obtained, cell

suspensions were treated with 40-mM VTD. Once the VTD-induced

depolarization reached a steady level, cells were treated with 16-ng/ml

STX. Once STX-induced hyperpolarization reached a steady level, cells

were treated with 100-nM AZ-1. In order to show complete depolarization,

cells were treated with 10-mg/ml gramicidin at the end of the experiment

(representative trace).

Y. Roman et al. / Cellular Signalling 14 (2002) 703–716706

at room temperature. Triton-X was removed by washing

with Hank’s balanced salt solution (two times, 200 ml

each), and 500-nM Oregon Green-514 phalloidin solution

in Hank’s was added to each well (50 ml). Cells were

stained with the fluorochrome for 30 min at room

temperature and finally washed with Hank’s balanced salt

solution (two times, 200 ml each). Fluorescence of stained

Fig. 4. Effect of different channels blockers on the [Ca2+]i modifications

elicited by 200-nM AZ-1 in human lymphocytes bathed by a Ca2+-

containing medium. Fura-loaded cells were attached to a glass coverslip,

bathed by Ca2+-containing saline solution and cytosolic Ca2+ was

monitored (A) Effect of La3+. Cells were preincubated for 5 min in the

presence or absence of 100-mM La3+. (B) Effect of SKF96365. Cells were

preincubated for 5 min in the presence or absence of 30-mM SKF96365.

(C) Effect of Ni2+. Cells were preincubated for 5 min in the presence or

absence of 1 mM Ni2+. Mean ± S.E.M. of three experiments.

Fig. 3. Effect of AZ-1 on the [Ca2 + ]i of human lymphocytes. Fura-loaded

cells were attached to a glass coverslip, bathed by Ca2 + -containing saline

solution and cytosolic Ca2 + was monitored. (A) Effect of different

concentrations of AZ-1 in cells bathed by Ca2 + -free saline solution. Next,

the bathing solution was exchanged for a Ca2 + -containing one. (B) Effect

of 200-nM AZ-1 in cells bathed by a Ca2 + -containing medium. Mean of

three experiments.

Y. Roman et al. / Cellular Signalling 14 (2002) 703–716 707

F-actin was measured in a LS-50B fluorescence micro-

plate reader (Perkin-Elmer, UK) set at 511 (excitation)

and 528 (emission) nm. Results of triplicate experiments

were expressed as a percentage of fluorimetric values

observed in controls after blank subtraction.

2.9. Data analysis

cAMP, [Ca2 + ]i and pHi values of all cells observed in

each experiment were averaged. All the experiments were

carried out at least three times in duplicate. Results were

analysed using the Student’s t test for paired data. A

probability level of .05 or smaller was used for statistical

significance. Data were normalized and results were

expressed as the mean ± S.E.M.

3. Results

3.1. Effect on membrane potential

Our objective was to test a possible neurological effect

of AZ in human cells. The membrane potential-sensitive

dye bis-oxonol was used to examine if AZ altered mem-

brane potential in neuroblastoma cells. Negative charge of

bis-oxonol prevents accumulation in the mitochondria.

Therefore, the probe only indicates changes in membrane

potential [23]. The introduction of bis-oxonol in a cell

suspension shows an increase in fluorescence, which

becomes stable in 5–10 min. Information obtained with

bis-oxonol is qualitative. In order to plot the results, we

averaged the fluorescence values obtained each 30 s from

Fig. 5. Effect of PMA, Go 6976, OA and PT on the [Ca2+]i modifications elicited by 200-nM AZ-1 in human lymphocytes bathed by a Ca2+-containing

medium. Fura-loaded cells were attached to a glass coverslip, bathed by Ca2+-containing saline solution and cytosolic Ca2+ was monitored. (A) Effect of PMA.

Cells were preincubated for 5 min in the presence or absence of 100-ng/ml PMA. (B) Effect of Go 6976. Cells were preincubated for 5 min in the presence or

absence of 100-nM Go 6976. (C) Effect of OA. Cells were preincubated for 5 min in the presence or absence of 1-mM OA. (D) Effect of PT. Cells were

preincubated for 3 h at 37 �C in the presence or absence of 50-ng/ml PT in a Ca2+-containing medium. Mean ± S.E.M. of three experiments.

Y. Roman et al. / Cellular Signalling 14 (2002) 703–716708

the stability of fluorescence after adding bis-oxonol. There-

fore, all experiments show the total average using as arbitrary

values those obtained in the fluorometer and calculating each

value’s fraction using the baseline as a control. In order to

show complete depolarization, the figures below show depol-

arization obtained with 10-mg/ml gramicidin at the end of

the experiment.

Fig. 1A illustrates the effect of STX and 10- and 100-nM

AZ-1 on membrane potential in human neuroblastoma cells.

STX is a known sodium channel modulator that does not

induce any direct change in the fluorescence of membrane

potential dye [24]. AZ-1 does not induce any significant

change in this fluorescence. We next examined the AZ

effect in VTD-depolarized neuroblastoma cells. VTD is

known to depolarize excitable cells by opening the sodium

channel and by blocking its inactivation [25]. Applying to

the cells VTD in the first place and then AZ, we obtained

results presented in Fig. 1B. Addition of 40-mM VTD to

neuroblastoma cells increased the fluorescence. The later

addition of the sodium channel blocker STX selectively

reduced VTD-induced depolarization, showing a decrease

of fluorescence. However, AZ-1 did not modify the VTD-

induced depolarization.

As an initial approach to test if the effect of AZ-1 is

directly related to the site 1 of sodium channel (which is

the STX receptor [26]), we examined the action of STX on

the AZ effect in neuroblastoma cells. The association of

STX and AZ-1 does not induce any change in the fluor-

escence of membrane potential dye (Fig. 2A). As an

alternative, we examined whether STX modifies the effect

of AZ on VTD-induced depolarization. We found that in

the presence of STX, AZ-1 does not modify the effect that

STX induces in the depolarization observed in the presence

of VTD (Fig. 2B).

3.2. Effect in cytosolic calcium and pHi

The effects of AZ-1 on the basal state of [Ca2+]i and pHi

of freshly isolated human lymphocytes were also studied. In

cells maintained 10 min in a calcium-free solution and later

with the calcium concentration restored, different concen-

trations of AZ-1 induced a significant concentration-

dependent rise in [Ca2+]i (Fig. 3A). In these conditions,

no effect on pHi was observed (data not shown). As Fig. 3A

shows, concentrations of 200- and 1000-nM AZ-1 produce a

modest increase in [Ca2+]i in a calcium-free medium,

whereas an important [Ca2+]i increase is observed when

calcium concentration is restored. This seems to suggest that

AZ-1 releases Ca2+ from internal stores and also origins

Ca2+ influx.

For the rest of the assays, we selected the 200-nM

concentration because this is enough to produce a sig-

nificant effect. When this AZ-1 concentration is added to

cells in a calcium-containing medium (Fig. 3B), an

increase in [Ca2+]i of the same magnitude can be observed

as when calcium concentration is restored in a calcium-

free medium. Again, no effect in on pHi is detected (data

not shown).

With the aim of characterizing this AZ-1-induced [Ca2+]i

increase, we used different calcium channels blockers.

La3+ is an efficient and specific blocker of store-operated

Ca2+ channels [27,28]. Neither 10 nor 100 mM (Fig. 4A) of

La3+ were able to block the toxin-induced influx. A similar

result was observed with 30-mM SKF96365 (Fig. 4B), an

imidazole reagent that is a potent blocker of Ca2+ and Mn2+

uptake activated by of stores depletion [29]. Finally, we tried

Ni2+, a divalent cation known to block calcium channels

and that does not enter into the cells [30]. When the cells are

Fig. 6. Effect of wortmannin and genistein on the [Ca2+]i modifications

elicited by 200-nM AZ-1 in human lymphocytes bathed by a Ca2+-

containing medium. Fura-loaded cells were attached to a glass coverslip,

bathed by Ca2+-containing saline solution and cytosolic Ca2+ was

monitored. (A) Effect of wortmannin. Cells were preincubated for 5 min

in the presence or absence of 10-nM wortmannin. (B) Effect of genistein.

Cells were preincubated for 5 min in the presence or absence of 10-mMgenistein. Mean ± S.E.M. of three experiments.

Y. Roman et al. / Cellular Signalling 14 (2002) 703–716 709

preincubated with 1-mM Ni2+, the effect of AZ-1 is com-

pletely inhibited (Fig. 4C).

Next, we studied the signaling pathway of AZ-1 effect in

[Ca2+]i. To this purpose, we employ different modulators of

intracellular signals. Lymphocytes preincubation with the

direct protein kinase C (PKC) activator PMA [30] induces a

significant inhibition of AZ-1-induced [Ca2+]i increase

(Fig. 5A). However, as Fig. 5B shows, the PKC inhibitor

Go 6976 [31] does not modify the cellular response. In this

way, we analysed the consequences of protein phosphatases

1 and 2A (PP1 and 2A) inhibition by using OA. Fig. 5C

shows that 1-mM OA inhibits the AZ-1 effect.

To further investigate the signal transduction pathways

mediating AZ-1 action, we used PT. Preincubation of

lymphocytes with 50-ng/ml PT for 3 h at 37 �C produces

a nonsignificant decrease in the [Ca2+]i rise induced by

AZ-1 (Fig. 5D). Therefore, PT-sensitive and -insensitive G

proteins might mediate AZ-1-induced Ca2+ influx.

We used 10-nM wortmannin to block phosphoinosi-

tide-3 kinase [32] and 10-mM genistein to block tyrosine

kinases [33], as shown in Fig. 6A and B, respectively.

The results shown in this figure show that these proteins

seem not to be involved in the pathway or modulation of

AZ-1-induced [Ca2+]i increase, although results show that

the inhibition of tyrosine kinases produces a nonsignifi-

cant decrease in AZ-1 effect.

On the other hand, we studied the role of changes in

cAMP levels in the toxin effect. cAMP has been described

Fig. 7. Effect of up- and down-regulation of cAMP pathway on the [Ca2+]i modifications elicited by 200-nM AZ-1 in human lymphocytes bathed by a

Ca2+-containing medium. Fura-loaded cells were attached to a glass coverslip, bathed by Ca2+-containing saline solution and cytosolic Ca2+ was monitored.

(A) Effect of forskolin. Cells were preincubated for 5 min in the presence or absence of 30-mM forskolin. (B) Effect of db-cAMP. Cells were preincubated

for 5 min in the presence or absence of 250-mM db-cAMP. (C) Effect of SQ22,536. Cells were preincubated for 5 min in the presence or absence of 10-mMSQ22,536. (D) Effect of H89. Cells were preincubated for 5 min in the presence or absence of 1-mM H89 in a Ca2+-containing medium. Mean ± S.E.M. of

three experiments.

Y. Roman et al. / Cellular Signalling 14 (2002) 703–716710

as an important modulator in Ca2+ signals, with different

actions in transformed and normal lymphocytes. The addi-

tion of forskolin, an adenylyl cyclase (AC) activator, prior to

the stimulation of lymphocytes with AZ-1, results in a total

inhibition of AZ-1-induced [Ca2+]i elevation (Fig. 7A). A

similar inhibition is observed when cAMP is directly

increased by the preincubation of cells with 250-mM db-

cAMP (Fig. 7B), a cAMP analogue. However, in this case,

the inhibition is lower and only takes place after 800 s.

Furthermore, neither the inhibition of AC with 10-mMSQ22,536 (Fig. 7C) nor the inhibition of cAMP-dependent

kinase PKA with 1-mM H89 (Fig. 7D) has had any effect in

the toxin-induced response.

3.3. F-actin cytoskeleton

Results obtained in fluorimetric microplate assays indic-

ate that F-actin cytoskeleton is one important cellular target

Fig. 8. (A) F-actin levels inBE(2)-M17cells exposed for 1 h to 1–10.000-nM

AZ-1 (average ± S.D., n = 3; results are expressed as percentage of

fluorimetric values for bound Oregon Green-514 phalloidin with respect to

controls). *P < .05 with respect to controls. (B) F-actin levels in BE(2)-M17

cells exposed for 24 h to 1–10.000-nM AZ-1 (average ± S.D., n= 3; results

are expressed as percentage of fluorimetric values for bound Oregon Green-

514 phalloidin with respect to controls). *P< .05 with respect to controls.

Fig. 9. (A) Changes in mitochondrial membrane potential of BE(2)-M17

cells incubated for 1 h with 1–10.000-nM AZ-1 (average ± S.D., n= 3;

results are expressed as percentage of Mitotracker Red CMXRos-retained

fluorescence in cells with respect to controls). (B) Changes in mitochon-

drial membrane potential of BE(2)-M17 cells incubated for 24 h with

1–10.000-nM AZ-1/ST4 (average ± S.D., n= 3; results are expressed as

percentage of Mitotracker Red CMXRos-retained fluorescence in cells with

respect to controls).

Y. Roman et al. / Cellular Signalling 14 (2002) 703–716 711

for AZ. Neuroblastoma cells exposed to AZ-1 show a

reproducible time- and dose-dependent decrease of F-actin

pools, as shown in Fig. 8A and B. A significant decrease

(P < .05) of F-actin levels was first observed in BE(2)-M17

cells incubated for 1 h with the highest concentration of

AZ-1 used in the assay (10 mM, 37 ± 16% of values in

controls). The IC50 values for AZ-1-induced depolymeriza-

tion of F-actin changed to 7.5 mM at 24 h, showing

significant differences with controls at concentrations higher

than 5 mM (44 ± 17% of F-actin levels in controls at 7.5-mMAZ-1), which demonstrates a time-dependent effect of the

toxin. F-actin cytoskeleton was completely disrupted after

24-h exposure of the cells at 10-mM AZ-1, showing baseline

levels for F-actin in all cases.

3.4. Mitochondrial membrane potential

Fluorimetric microplate measurements of mitochondrial

membrane potential in AZ-1-cultured cells did not show

significant differences with controls at both 1 and 24 h from

1 nM to 10 mM of AZ-1 (Fig. 9A and B), suggesting that

AZ-1 does not trigger the apoptotic cascade at these levels.

3.5. Effect on cAMP levels

The effect of AZs in cAMP levels was studied using

as a cellular model human lymphocytes. The experiments

were carried out using AZ-1. Fig. 10 shows the changes

in cAMP levels in the presence of different amounts of

AZ-1 and a clear increase in cytosolic cAMP levels can

be observed (a control of 200-mM db-cAMP is used at the

end of each experiment to check FlCRhR functionally).

This effect is dose dependent because 500-nM AZ-1

induces half the increase obtained with 1000 nM. The

effect of external calcium on the increase of cAMP levels

caused by AZ-1 was studied by eliminating calcium from

the extracellular media, as shown in Fig. 11. The increase

induced in cAMP levels by AZ-1 is slightly higher in a

Ca2 + -free medium although not significant. The implica-

tion of AC in AZ-1 effect was checked by preincubation

in the presence of 1-mM SQ22,536. This drug is a known

AC inhibitor [34]. Fig. 12 shows AZ-1 effect on cAMP

levels after SQ22,536 preincubation. In these conditions,

AZ-1 does not induce any cAMP increase and does not

modify the decrease of cAMP caused by SQ22,536 (the

initial sharp decrease after addition of AZ-1 is an artifact

caused by the addition of the liquid to the cell suspen-

Fig. 10. Dose-dependent effect of AZ-1 on cAMP levels in human

lymphocytes. The arrow indicates the addition of 0.5- or 1-mM AZ-1. The

addition of 200-mM db-cAMP indicates FlCRhR functionally.

Fig. 11. Effect of AZ-1 on cAMP levels in human lymphocytes in a

calcium-free and in a calcium-containing medium. The arrow indicates the

addition of 1-mM AZ-1. The addition of 200-mM db-cAMP indicates

FlCRhR functionally.

Fig. 12. Effect of AZ-1 on cAMP levels in human lymphocytes when AC

was inhibited. The cells were incubated for 10 min in the presence of 1-mMSQ22,536 and then 1-mM AZ-1. The addition of 200-mM db-cAMP

indicates FlCRhR functionally.

Y. Roman et al. / Cellular Signalling 14 (2002) 703–716712

sion). Although the effect seems to be a slight decrease, it

is nonsignificant.

Because the diarrhoeic toxin OA may coexist with AZs,

we studied the combined effect of both drugs together, as

shown in Fig. 13. In the presence of 1-mM OA, cAMP levels

do increase (manuscript submitted), and the profile shows a

biphasic response in cAMP production, first an initial fast

peak and second a plateau. Nevertheless, the addition of

AZ-1 after the OA plateau induces an increase in cAMP

levels or the same magnitude as that induced by the AZ-1

control. Therefore, the toxins OA and AZ-1 do not interact

in their effects on cAMP.

4. Discussion

The objective of this work was to identify key signal

transduction pathways and cellular targets of AZ that has

been recently isolated and partially characterized as a toxic

natural shellfish contaminant and linked to toxic episodes in

humans following the consumption of mussels [11]. The

mechanism of action of AZ is still unknown and toxico-

logical data are very scarce, although Ito et al. [14] have

reported multiple organ damage in mice caused by AZ, but

this is the first report on the toxicity of AZ in cultured

eukaryotic cells. AZ was found to be a potent toxin initially

associated to DSP. Although human symptoms such as

nausea, vomiting, severe diarrhoea and stomach cramps

were reminiscent of DSP, extracts of mussels containing

AZ toxins have shown also neurotoxic symptoms such as

respiratory difficulties, spasm, paralysis and death [14]. It

was clear that this poisoning was a new type, and it was

named AZ poisoning (AZP) [12]. The mechanism of action

of this toxin is unknown though. In order to seek for the

mechanistic characteristics of the AZ action, we designed a

neurological approach based on nonintrusive measurement

of changes in membrane potential. For this matter, neuronal

cultures can represent an excellent model to distinguish the

mechanisms of action of different neurotoxins [35]. We

have also previously demonstrated the effectiveness of the

optical probe bis-oxonol for detection and even quantitation

of other natural toxins, namely STX and analogues, that

interact with the sodium channel and the genesis of the

membrane potential [24].

The studies with neuroblastoma provided the first evid-

ence that AZ-1 does not produce a membrane hyperpolari-

zation. Because AZ intoxication has been reported, among

other symptoms, as neurotoxic, it could be possible that

other AZ analogues have this effect, but our results rule out

AZ-1 as a candidate. We are currently checking for possible

neurotoxic effects of other AZ analogues. On the other

hand, it might be possible that other neurotoxins did coexist

with AZ when first checked by mouse bioassay. Application

of VTD evoked depolarization in neuroblastoma cells and,

as expected, this activity was suppressed by STX. The

mixture of AZ analogues strongly decreases the depolariza-

tion induced by VTD, but this depolarization is not affected

by AZ-1 alone. The possible interaction between AZ and

STX on sodium channel was also studied. STX did not

modify the hyperpolarization induced by AZ-1. This sug-

gests that the site 1 of voltage-dependent sodium channel,

which is the receptor to STX [36], is not the receptor target

for AZ.

We have demonstrated that AZ-1 disrupts cytoskeletal

structure in neuroblastoma cells inducing a time- and dose-

dependent decrease of F-actin pools, which suggests that

the F-actin cytoskeleton is one important cellular target for

AZ. In recent years, an increasing number of actin-targeted

marine natural products have been identified [37], including

OA and pectenotoxins. OA-induced changes in F-actin

have been extensively reported [38–41], and we have

observed that OA disrupts cytoskeletal structure in the

same neuroblastoma cell line used in this work (unpub-

lished results). However, the IC50 values for AZ-induced

changes on F-actin pools are significantly higher (500-fold

at 24 h) than those corresponding to OA, which suggests a

lower toxicity for AZ when compared to the phosphatase

inhibitor OA [42].

The relevance of cytoskeletal changes on AZ toxicity

should be further confirmed as F-actin represents a complex

polymer system that plays essential roles in many cellular

processes [43]. The actin cytoskeleton can be considered as

a highly dynamic structure, and it has a key role in signal

transduction and other regulatory pathways within eukary-

otic cells [44]. Previous reports [17] have suggested that the

actin-disrupting effect of OA in cultured cells could be

associated with its diarrhogenic effect, reflecting the loosen-

ing of tight junctions in vivo. This link between F-actin

changes and diarrhogenic activity could also be relevant to

explain AZ toxicity as symptoms of AZ poisoning resemble

those of DSP, including vomiting, diarrhoea and stomach

Fig. 13. Effect of OA and AZ-1 on cAMP levels in human lymphocytes.

The cells were incubated in the presence of 1-mM OA for 10 min and then

1-mM AZ-1 was added. The addition of 200-mM db-cAMP indicates

FlCRhR functionally.

Y. Roman et al. / Cellular Signalling 14 (2002) 703–716 713

cramps [14]. However, the cytoskeleton also participates in a

wide range of cellular processes such as apoptotic cell death,

F-actin being one of the substrates of the executioners of the

apoptotic cascade, a group of cysteine proteases called

caspases [45–47]. The apoptotic machinery is triggered by

different stimuli, resulting, in most cases, in the loss of

mitochondrial membrane potential. This induces the release

of proapoptotic factors to the cytoplasm and the activation of

caspases, with final disassembly of cellular structures [48].

As previously reported [15,49], the mitochondria play a

central role in the apoptotic cascade, and changes in mito-

chondrial membrane potential are a good estimate of the

apoptotic activity of different xenobiotics. In this paper, we

have demonstrated that AZ at 0.001–10 mM does not induce

apoptosis in BE(2)-M17 cells, as indicated by the mainten-

ance of mitochondrial membrane potential and opposite to

previously described changes in mitochondrial membrane

potential of BE(2)-M17 cells exposed to OA (unpublished

results). Thus, cytoskeletal changes induced by AZ can be

considered as specific events, which are not associated with

the triggering of the apoptotic process, although some

previous works [50] have reported apoptotic cell death in

the absence of mitochondrial changes, which can be con-

sidered as an exception to the general mechanism of

apoptosis. The cytoskeleton should therefore be considered

as a key target of AZ-induced toxicity in further works

related to the mechanism of toxicity of this phycotoxin.

Control of intracellular free calcium concentration is a

critical component of cellular homeostasis for all cell types.

Therefore, the [Ca2+]i increase originated by receptor stimu-

lation is necessary for some T-cell functions, and a sustained

increase in [Ca2+]i concentration induces cell death gen-

erally by apoptosis [51]. However, the mechanism of the

Ca2+ entry into electrically nonexcitable cells remains in

general unclear. It is firmly established that the store-

operated pathway is the predominant Ca2+ entry. However,

other currents have been described [52].

Our results show that AZ-1 induces a significant con-

centration-dependent increase in [Ca2+]i levels, both in a

calcium-free and in a calcium-containing medium. The

results obtained in a calcium-free medium might indicate a

Ca2+ release from internal stores. Following this Ca2+

depletion, we can observe a Ca2+ influx from the extrac-

ellular medium. We have not found that AZ-1 may to have a

direct effect on pHi.

La3+ is an important and efficient blocker of store-

operated Ca2+ entry (SOCE) [28,53]. SKF96365 is also an

effective inhibitor of this pathway but does not appear to be

not entirely specific, showing an inhibitory effect on voltage-

operable Ca2+ channels and ability to release Ca2+ from

internal stores [54]. Ni2+ is a low selective blocker involved

in SOCE [52] and also in voltage-regulated Ca2+ channels

[55] or in Ca2+ influx induced by a testosterone receptor in

plasmatic membrane [56]. Our results pointing to AZ-1-

stimulated [Ca2+]i increase seem to be mediated by Ni2+-

blockable channels, not inhibited by the effective blockers of

SOCE: SKF96365 or La3+. Therefore, AZ-1-induced Ca2+

influx seems not to be dependent on store depletion.

In the present study, PKC activation with PMA and PP1

and PP2A inhibition with OA abolish AZ-1-evoqued

[Ca2+]i increase. Therefore, a protein sensitive to PKC

phosphorylation and to PP1 and PP2A dephosphorylation

may have an inhibitory effect on AZ-1-induced response.

Several studies have demonstrated the modulatory effect of

PKC activation in Ca2+ influx. In this sense, in human

lymphocytes, an inhibitory effect of PKC in Ca2+ influx

induced by thapsigargin has been described [30]. Besides, in

Jurkat cells, this inhibitory effect is described in Ca2+ influx

and in Ca2+ release from internal stores, when cells are

stimulated with thapsigargin or through the T-cell receptor

[51]. However, this action mechanism is not yet fully

understood and the multiple targets proposed for PKC

(phospholipase C and receptors) fail to explain all the

processes observed. In addition to this controversy, it is

not clear whether PKC acts in Ca2+ efflux [57]. Consistent

with our observations, a target protein sensitive to PP1 and

PP2A dephosphorylation has been described as an inhibitor

of capacitative Ca2+ entry in rat thymic lymphocytes [58].

AZ-1 actions are not modified by genistein or wortman-

nin and only partially modified by PT. PT is a complex

structure where two subunits are separated. So, PT holo-

toxin consists of the S1 polypeptide, which contains ADP-

ribosyl transferase activity over Gi/o proteins, and the

b-oligomer, which interacts with cell surface structures

and facilitates entry of the S1 subunit. Our experiments

pointing to both PT-sensitive and -insensitive G proteins

might mediate AZ-1-stimulated Ca2+ influx. Interaction

between G proteins and Ca2+ influx has been described

intensively. Indeed, in some cell types, a G protein-activated

Ca2+ channel has been demonstrated [52]. Besides, in T-

cells, a selective loss of PT-sensitive G proteins from the

plasma membrane has been described after antibody

induced internalization, although, apparently, in this paper

the modulation process is independent of the presence of G-

protein [59]. However, recently, in some nonexcitable cell

types, PT-sensitive G proteins have been related to

agonist-induced [Ca2+]i increase [60–63]. Finally, in

our results, we must consider that PTX might directly

act through a plasmatic membrane interaction with its b

oligomer [64–66]. In any case, further studies would be

needed to clarify this point.

Finally, this report suggests a prominent role of cAMP

increasing agents in the regulation of AZ-1-induced [Ca2+]i

increase. Hence, AC, cAMP and PKA might be negative

modulatory signals of the toxin-induced response. cAMP is

an important and ubiquitous second messenger, and it is

interesting having in mind that some PKA-independent

actions have been reported [67,68]. For this reason, Ca2+

and cAMP relationship has been investigated intensively.

Despite the different results obtained, probably due to the

variety of the stimuli and cells types studied, a cross-talk

between this pathways seems to be inferred in lymphocytes.

Y. Roman et al. / Cellular Signalling 14 (2002) 703–716714

Therefore, it has been described that increases in [cAMP]

inhibit Ca2+ influx [67,69–71] and that different agonist

triggered an increase in Ca2+ influx associated with an

inhibition of AC [72,73]. All these reports are in good

agreement with our results. PKA is also presumed to be a

fine modulator of Ca2+ entry. Therefore, PKA activation and

also PKA inhibition inhibit thapsigargin-induced Ca2+ influx

[67]. In our findings, only PKA activation inhibits AZ-1-

induced Ca2+ uptake, without any effect of PKA inhibition.

In summary, we demonstrate that AZ-1 induces a [Ca2+]i

increase in human lymphocytes and has no effect in pHi.

AZ-1-induced [Ca2+]i increase consists of two parts: a

small stores depletion and Ca2+ influx from extracellular

medium through Ni2+-blockable channels. This AZ-1 effect

is negatively regulated by PKC activation, PP1 and PP2A

inhibition as well as by cAMP increasing agents. Phos-

phoinositide-3 kinases and tyrosine kinases seem not to be

involved in toxin effect, but a PT-sensitive G protein might

be part of its pathway.

AZ induces a dose-dependent increase in cAMP levels

in human lymphocytes. Human lymphocytes were used

because they were obtained from healthy humans, and

these cells withstand well the procedure to load the cAMP

indicator. This is not the case for neuroblastoma cells.

Also, the cAMP production machinery is also very active

in lymphocytes.

Cells regulate cAMP levels through a balance between

ACs (synthesis) and phosphodiesterases (hydrolysis), both

enzymatic groups including several isozyme families selec-

tively expressed. When AC was inhibited by SQ22,536

[34], AZ-1 did not induce any cAMP increase. This could

indicate that the AZ effect is due to an increase in AC

activity. Therefore, in the presence of SQ22,536, it has no

effect. It is also feasible that AZ inhibits phosphodiesterases.

No increase in cAMP is observed because in the presence of

SQ22,536 AC is inhibited, there is no cAMP synthesis, and

its intracellular levels are not increased even phosphodies-

terases could be inhibited.

On the other hand, an increase in cytosolic calcium is

often related to the inhibition of cAMP accumulation [74]. In

this case, because AZ induces an increase in cytosolic

calcium concentration, we studied the variations of cAMP

in a calcium-free medium in order to prevent any external

calcium influx. In calcium-free conditions, the effect of AZ in

cAMP accumulation was not significantly modified, which

suggests an independence of cAMP and calcium effects.

The cAMP signal is increased by OA. Our data show that

in human lymphocytes this toxin increases cAMP, an effect

that was also observed after b-stimulation in lymphoma cells

[75]. Nevertheless, in other cellular models, OA was

described as a potent phosphodiesterase stimulator [76].

Because OA and AZ may coexist in nature, we have

checked the combined effect of both toxins, and results

show that the addition of AZ after OA stimulation induces

the same increase as the control, indicating that both toxins

might interact at the same transduction level.

Acknowledgments

This work could not have been done without the

invaluable collaboration of the staff at the General Hospital

of Lugo from the Centro de Transfusion de Galicia (Drs.

Marcelo Alvarenga and Jose A. Gonzalez, Ms. Sara Gomez,

Ana Perez, Dolores Sanchez, Mr. J.M. Rodrıguez, Ms.

Dolores Atanes and Noelia Lauda). This work was funded

with grants FEDER-CICYT-1FD97-0153, Xunta Galicia

(PGIDT99INN26101 and PGIDT00MAR26101PR) and

MCYT(DGI) BMC2000-0441. L.A. de la Rosa is supported

by a scholarship from CONACYT, Mexico.

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