A comparative study for PSP toxins quantification by using MBA and HPLC official methods in shellfish.

Commission Regulation (EC) N° 2074/2005 recognises the biological method as the reference method for Paralytic Shellfish Poisoning (PSP) toxins detection in molluscs. It was amended by Commission Regulation (EC) N° 1664/2006 that accepted the so-called Lawrence method as an alternative to the reference method. The goal of this study was to compare AOAC Official Methods of Analysis 959.08 (Biological method) and 2005.06 (Prechromatographic Oxidation and Liquid Chromatography with fluorescence detection) in samples with different toxin profiles. The influence of extraction solvent in the total samples toxicity was also evaluated. A total of 40 samples including mussels, clams, scallops, razor-clams, cockles, oysters and barnacles were analysed by both official methods. Samples were selected with Alexandrium and Gymnodinium toxic profiles, from different origin and including several presentations: fresh, frozen, canned and boiled. Acetic and hydrochloric acid extractions were performed in all samples and the extracts were simultaneously analysed by both methods. Most samples were naturally contaminated and two samples were spiked. Comparison of both official methods, mouse bioassay (MBA) with HCl extraction and Liquid Chromatography with fluorescence detection (HPLC-FLD) with acetic acid extraction, led to an 85% of consistent results regarding compliance with legal limit, including samples below and above it. The linear correlation coefficient was r² = 0.69 and the paired t test (two tails, α = 0.05) indicated that there were not significant differences among both sets of data. Nevertheless, toxicity differences were found in several samples. In 15 out of 18 shellfish with a Gymnodinium toxic profile, higher toxicity levels were obtained by MBA. This fact was more evident in 7 samples, partially related to the lack of standards and the impossibility of analysing dc-NEO, C1, 2 and GTX6 at the beginning of the study. However, other factors concerning the extraction and SPE clean-ups steps may also contribute. By contrast, 9 samples presented a much higher total toxicity by HPLC-FLD than by MBA. These higher results obtained by HPLC-FLD could not only be due to the use of the highest toxicity equivalency factor (TEF) for isomers oxidated into products that coelute when total toxicity of these samples were calculated. Further analyses of results obtained by HPLC-FLD and by MBA with both extracts were done separately.

Update of risk assessments of main marine biotoxins in the European Union.

This review is an up-to-date compilation of the available literature, including scientific papers, reviews, and EFSA's opinions, on toxicity and risk assessment data on the main marine biotoxins of importance in the European Union, including the legislated ones and the ones of recent appearance which are not legislated. Information about the hazard identification and hazard characterisation of okadaic acid, dynophysistoxins, pectenotoxins, yessotoxins, azaspiracids, domoic acid, saxitoxins, tetrodotoxins, brevetoxins, ciguatoxins, cyclic imines and palytoxins is reviewed and presented in the form of a collection of risk assessments. It is concluded that the importance of having an appropriate exposure assessment reiterates the urgency of establishing a database with representative and comparable data on exposure to food items possiby containing marine biotoxins. It is also concluded that a revision of the present regulation of marine biotoxins in the EU legislation could be considered, as some regulated toxins have been shown not to pose a risk for EU's population (as yessotoxin) and some non regulated toxins have been shown to be harmful and/or to occur in the EU (as tetrodotoxin, palytoxin, and some cyclic imines) while they are not regulated.

Rapid isolation of single-chain antibodies by phage display technology directed against one of the most potent marine toxins: Palytoxin.

Several recombinant antibodies against one of the most potent marine toxins, Palytoxin (PlTX), were obtained using two naive human semi-synthetic phage display libraries (Tomlinson I and J) as an effective method for generating specific anti-toxin single-chain variable fragment (scFv) antibodies. After four rounds of panning and selection on free palytoxin adsorbed immunotubes, individual clones were isolated, sequenced and characterized by Enzyme-Linked Immunosorbent Assay (ELISA). Four phage-antibody clones specifically recognized the toxin. A competitive ELISA assay was optimized with one of these phage antibodies giving a very reproducible standard curve with a linear regression (R(2)=0.9945), showing a working range of 0.0005-500ngmL(-1). Several spiked shellfish samples were analysed by competitive ELISA to determine the accuracy of the assay, with a mean recovery rate of 90%. This study demonstrates that phage display libraries provide a valuable system for the easy and rapid generation of specific antibody fragments directed against difficult antigenic targets, such as free small molecules. Large-scale, low-cost production of anti-palytoxin scFv antibodies in Escherichia coli (E. coli) is an exciting prospect for the development of rapid and simple detection methods. Our results suggest that anti-palytoxin phage antibodies could be a valuable tool with competitive ELISA to detect palytoxin in natural shellfish samples.

Lipophilic toxins analyzed by liquid chromatography-mass spectrometry and comparison with mouse bioassay in fresh, frozen, and processed molluscs.

The search for alternative methods to the mouse bioassay (MBA) has intensified over recent years. The present work analyzes seven different species of shellfish (clams, small scallops, small clams, mussels, oysters, cockles, and edible whelks) in fresh, frozen boiled, and canned presentations using liquid chromatography-mass spectrometry (LC-MS/MS), and the results are compared with the same samples analyzed through MBA. The toxins studied were OA, DTX1, DTX2, YTX, PTX2, and AZA1, which are legislated in the EU, and SPX1, which is not regulated yet. Consistent results between LC-MS/MS and MBA were found in 69% of the samples, whereas 26% of MBA showed "false-positive" results with respect to the toxins analyzed. No "false negatives" were observed. The possibility of LC-MS/MS as an alternative or complementary technique to MBA is discussed.

Characteristics of palytoxin-induced cytotoxicity in neuroblastoma cells.

Cation fluxes appear to play a key role in palytoxin-induced signal. There are other cellular targets that have not been described as well as the biochemical signaling cascades that transmit palytoxin-stimulated signals remain to be clarified. Since modifications of cations, mainly calcium, are generally associated to cell death or apoptosis, we wanted to further evaluate the effect of palytoxin on cell death. Then, in vitro cytotoxic effects of palytoxin were characterized on human neuroblastoma cells. By using several techniques, we studied markers of cell death and apoptosis, such as cell detachment, mitochondrial membrane potential, caspases, DNA damage, LDH leakage, propidium iodide uptake, F-actin depolymerization and inhibition of cellular proliferation. Results show that palytoxin triggers a series of toxic responses; it inhibits cell proliferation, induces cell rounding, detachment from the substratum and F-actin disruption. Among the apoptotic markers studied we only detected fall in mitochondrial membrane potential. Neither caspases activation nor chromatin condensation or DNA fragmentation were observed in palytoxin-treated cells.

In vitro approaches to evaluate palytoxin-induced toxicity and cell death in intestinal cells.

Palytoxin isolated from the genus Palythoa is the most potent marine toxin known. The aim of the present study was to quantify palytoxin-induced cellular injury in the human intestinal cell line Caco-2. Cellular damage was measured by evaluating cell proliferation, cell membrane permeability, cell morphology and apoptotic markers. Furthermore, changes in F-actin were studied after exposure of cells to increasing amounts of palytoxin. The results show that cell proliferation decreased in a concentration-dependent manner with a mean IC(50) value of about 0.1 nM. A noticeable increase of cell detachment correlated with cell rounding and F-actin depolymerization was observed in palytoxin-treated cells. Moreover LDH was released from the cells in a dose and time dependent manner, although under these conditions there was no propidium iodide uptake. On the other hand, palytoxin impaired mitochondrial activity but other apoptotic markers, such as DNA fragmentation or caspases activation, were not observed. The results obtained in this paper suggest that the effects of palytoxin in Caco-2 cells were very potent and unspecific, since a primary necrosis and a secondary apoptosis seem to occur under these conditions.

Cytoskeletal disruption is the key factor that triggers apoptosis in okadaic acid-treated neuroblastoma cells.

Okadaic acid (OA) is a tumour promoter that induces apoptosis in several cell models. Following previous findings, the objective of this work was to elucidate the pathways involved in OA-triggered apoptosis in BE(2)-M17 cells by using a combination of pharmacological agents and apoptosis-related assays. OA-induced apoptosis involves disruption of F-actin cytoskeleton, activation of caspase-3, collapse of mitochondrial membrane potential, DNA fragmentation and decreased levels of monomeric Bcl-2 and Bax proteins. All the agents tested were unable to obliterate changes in F-actin levels, caspase-3 activation or DNA fragmentation, but all of them prevented OA-induced decrease of mitochondrial potential and changes in Bax/Bcl-2 levels. Taken together, these results demonstrate that collapse of mitochondrial membrane potential is accessory in the execution of apoptosis, which is directly dependent on cytoskeletal changes. Mitochondrial changes are mediated by complex associations among the Bcl-2 proteins. Cytochrome c release from mitochondria is a late event, occurring 24 h after OA exposure. Moreover, okadaic acid triggers activation of upstream caspases resembling the extrinsic pathway of apoptosis.

Development of a F actin-based live-cell fluorimetric microplate assay for diarrhetic shellfish toxins.

A new cytotoxicity assay for detection and quantitation of diarrhetic shellfish toxins (DSP) is presented. This assay is based upon fluorimetric determination of F-actin depolymerization induced by okadaic acid (OA)-class compounds in the BE(2)-M17 neuroblastoma cell line. No interferences were observed with other marine toxins such as saxitoxin, domoic acid, or yessotoxin, thus indicating a good specificity of the assay as expected by the direct relationship between protein phosphatase inhibition and cytoskeletal changes. The proposed method is rapid (<2h) and shows a linear response in the range of 50-300 nM OA. The detection limit of the assay for crude methanolic extracts of bivalves lies between 0.2 and 1.0 microg OA per gram of digestive glands, depending on the type of samples (fresh or canned), thus being similar to that of the mouse bioassay. The performance of this assay has been evaluated by comparative analysis of 32 toxic mussel samples by the F-actin assay, mouse bioassay, HPLC and PP2A inhibition assay. Results obtained by the F-actin method showed no differences with HPLC and significant correlation with PP2A inhibition assay (r(2)=0.71). No false negative results were obtained with this new cell assay, which also showed optimum reproducibility.

Hypertonicity-induced intracellular pH changes in rat mast cells.

In a non-isotonic environment, cells can shrink or swell and return to their normal shape by activating ion transport pathways. Changes in intracellular pH (pHi) after osmotic stress have been identified in several cells. In order to study the mechanisms that regulate cytosolic pH of rat mast cells in a hypertonic medium, we used the pH sensitive dye, BCECF. Under these hypertonic conditions, pHi undergoes an alkalinization following an initial acidification. The alkalinization is mediated by a Na+/H+ exchanger, since it is inhibited by amiloride and lack of extracellular sodium. Under these conditions, the alkalinization is increased with the PKC activators, TPA and OAG, and partially blocked with trifluoperazine, an unspecific protein kinase C (PKC) and Ca2+ calmodulin-dependent protein kinases (Ca2+/CaM K) inhibitor. There is also an anion exchanger, blocked with DIDS but not activated by PKC, that participates in the observed alkalinization. However, Na+/H+ exchanger is the main mechanism involved in the alkalinization of pHi of mast cells in a hyperosmotic environment.

Crosstalk between cytosolic pH and intracellular calcium in human lymphocytes: effect of 4-aminopyridin, ammoniun chloride and ionomycin.

Stimulation of lymphocytes by specific antigens is followed by the activation of different signal transduction mechanisms, such as alterations in the cytoplasmic levels of Ca(2+), H(+) and variations in membrane potential. To study interrelationships among these parameters, changes in pHi and Ca(2+) were measured with the fluorescent probes BCECF and Fura-2 in freshly isolated blood human lymphocytes. Moreover, membrane potential qualitative alterations were recorded with the fluorescent dye bis-oxonol. In a bicarbonate-free medium, cell alkalinization with NH(4)Cl slightly decreased intracellular Ca(2+) concentration ([Ca(2+)](i)) due to efflux of Ca(2+) from the cell. In contrast, an elevation of pHi induced with 4-AP increased [Ca(2+)](i), either in the presence or absence of external Ca(2+). The increase in Ca(2+)-free medium is likely to be due to Ca(2+) release from thapsigargin and caffeine-independent intracellular stores. Both 4-AP or NH(4)Cl induced a plasma membrane depolarisation, although with different kinetics. The ionosphere ionomycin increased pHi, Ca(2+) levels and also induced membrane depolarisation. Together, these observations demonstrate a lack of correlation between the magnitude of changes in pHi and Ca(2+).

Calcium-pH crosstalks in rat mast cells: cytosolic alkalinization, but not intracellular calcium release, is a sufficient signal for degranulation.

The aim of this work was to study the relationship between intracellular alkalinization, calcium fluxes and histamine release in rat mast cells. Intracellular alkalinization was induced by nigericin, a monovalent cation ionophore, and by NH(4)Cl (ammonium chloride). Calcium cytosolic and intracellular pH were measured by fluorescence digital imaging using Fura-2-AM and BCECF-AM. In rat mast cells, nigericin and NH(4)Cl induce a dose-dependent intracellular alkalinization, a dose-dependent increase in intracellular calcium levels by releasing calcium from intracellular pools, and an activation of capacitative calcium influx. The increase in both intracellular calcium and pH activates exocytosis (histamine release) in the absence of external calcium. Under the same conditions, thapsigargin does not activate exocytosis, the main difference being that thapsigargin does not alkalinize the cytosol. After alkalinization, histamine release is intracellular-calcium dependent. With 2.5 mM EGTA and thapsigargin the cell response decreases by 62%. The cytosolic alkalinization, in addition to the calcium increase it is enough signal to elicit the exocytotic process in rat mast cells.

Functional compartments in rat mast cells for cAMP and calcium on histamine release.

The crosstalk between 3', 5'-cyclic adenosine monophosphate (cAMP), intracellular calcium, and histamine release in rat mast cells using the stimulatory effect of three different drugs, thapsigargin, sodium fluoride (NaF), and compound 48/80 were studied. Each of these drugs induces histamine release by different mechanisms. The transducting pathways modulating cAMP and intracellular calcium levels were modified by using, cholera toxin (CTX) which ADP-rybosylates Gs-protein, pertussis toxin (PTX) which ADP-rybosylates Gi-protein, and okadaic acid (OA) which inhibits phosphatases 1 and 2a. Our results show that CTX increased cAMP levels and inhibited histamine release elicited by thapsigargin and compound 48/80. The inhibitory effect of CTX on histamine release was potentiated by OA in the presence of compound 48/80 but was decreased in the presence of thapsigargin. Calcium uptake was stimulated by NaF and compound 48/80. The previous treatment with OA increased calcium uptake when combined with compound 48/80 but not with NaF. Treatment with NaF highly stimulated calcium uptake and cAMP levels only when combined with OA and CTX. These results suggest that the modulatory effect of intracellular calcium and cAMP on histamine release depend more on the crosstalk of the activated signal transducting pathway than on the final level of calcium or cAMP, further supporting the theory that rat mast cells are divided into functionally distinct compartments.

Evidence for an electrogenic, negatively protein-kinase-A-modulated, Na+-dependent HCO3- transporter in human lymphocytes.

We studied the effects of external HCO3- on pHi regulation in human lymphocytes after an acid load. Cells were acidified by preincubation with NH4Cl and pHi recovery was measured with the fluorescent dye BCECF. Cells recovering in HCO3--containing medium reached a higher final pHi, the H+ efflux rate was increased and shifted to alkaline pHi compared to that of cells recovering in HCO3--free solution. The resting pHi was higher in a HCO3--containing solution. Experiments carried out in the presence of amiloride, DIDS and in the absence of external Na+ suggest the existence of two major mechanisms acting in the pHi recovery of lymphocytes after an acid load: an amiloride-sensitive Na+/H+ exchanger and a DIDS-sensitive Na+-dependent HCO3- transporter. The last mechanism could be a Na+/HCO3- cotransporter based on membrane potential changes determined with the potential-sensitive fluorescent probe bis-oxonol. Preincubation of cells with forskolin and H-89 showed protein-kinase-A-dependent downregulation of the amiloride-insensitive recovery of pHi in human lymphocytes. In summary, this paper provides functional evidence for the existence of a Na+/HCO3--dependent mechanism involved in pHi recovery in human lymphocytes following an acid load, that is electrogenic and downregulated by PKA.

Membrane potential changes associated with calcium signals in human lymphocytes and rat mast cells.

Human lymphocytes and rat mast cells, two non-excitable cellular models, were used to investigate membrane potential changes accompanying Ca2+ signals. Cells were stimulated with agents known to induce both Ca2+ release from internal stores and influx of extracellular Ca2+, namely thapsigargin, ionomycin and compound 48/80. Thapsigargin and ionomycin were used to activate lymphocytes, while compound 48/80 was used to stimulate mast cells. Membrane potential changes and Ca2+ concentration were monitored with the fluorescent dyes bis-oxonol and fura-2, respectively. In lymphocytes, thapsigargin induced a hyperpolarization temporally correlated with the increase in intracellular Ca2+ concentration. This hyperpolarization is due to activation of a K+ conductance which consists of two phases, a first phase independent on external Ca2+ and a second one blocked in a Ca2+-free medium. Ionomycin induced a Ca2+-dependent depolarization attributed to a massive influx of external Ca2+. On the other hand, stimulation of mast cells with compound 48/80 produced a fast hyperpolarization and an increase in intracellular Ca2+ levels. Besides different time-courses, this hyperpolarization differs from that induced by thapsigargin in lymphocytes in two aspects, it is mainly due to a Cl(-)-entry current and exit of K+ and it is completely inhibited in the absence of extracellular Ca2+. Compound 48/80-induced histamine release is not related to membrane potential changes.

Inhibition of Na+/K+ ATPase under hypertonic conditions in rat mast cells.

Ionic fluxes that contribute to changes in membrane potential and variations of pHi (intracellular pH) are not well known in mast cells, although they can be important in the stimulus-secretion coupling. Cellular volume regulation implies changes in the concentration of intracellular ions, such as sodium and potassium and volume changes can be imposed varying the tonicity of the medium. We studied the physiology of sodium and examined the effect of ouabain on [22Na] entry in mast cells in isotonic and hypertonic media. We also recorded changes in membrane potential and pHi using the fluorescent dyes bis-oxonol (Bis-(1,3-diethylthiobarbituric acid) trimethineoxonol) a n d BCECF (2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester) in hypertonic conditions. The results show that [22Na] influx increases four fold in hypertonic solutions and it is mediated mainly by an amiloride-sensitive Na+/H+ exchanger. This transporter is involved in the shrinkage-activated cellular alkalinization and the pHi recovery is accelerated by inhibition of the Na+/K+ ATPase with ouabain in the absence of extracellular calcium. Under hypertonic conditions 22Na influx is apparently not increased by ouabain, while the Na+/K+ ATPase inhibitor clearly increases [22Na] uptake and also induces membrane depolarization in isotonic conditions. All together, these findings suggest that Na+/K+ ATPase is partially inhibited in hypertonic conditions.

The epithelial sodium-hydrogen antiporter Na+/H+ exchanger 3 accumulates and is functional in recycling endosomes.

Na+/H+ exchangers (NHEs) mediate electroneutral exchange of Na+ for H+ and thereby play a central role in pH regulation and Na+ homeostasis. NHE3, the predominant epithelial isoform, is found in apical membranes of renal and intestinal epithelial cells, where it contributes to NaCl (re)absorption. NHE activity has been detected in endomembrane vesicles of epithelial cells, but the precise compartment involved and its functional role have not been defined. Many aspects of the targeting machinery that defines the compartmentation and polarity of epithelia are also functional in nonepithelial cells. We therefore compared the targeting of NHE1, the basolateral isoform, with that of NHE3 in Chinese hamster ovary cells. To circumvent the confounding effects of endogenous exchangers, epitope-tagged constructs of NHE1 and NHE3 were stably expressed in antiport-deficient (AP-1) cells. While NHE1 was found almost exclusively in the surface membrane, NHE3 was also found intracellularly, accumulating in a juxtanuclear compartment. Confocal microscopy showed this compartment to be distinct from the Golgi, trans-Golgi network, and lysosomes. Instead, NHE3 colocalized with transferrin receptors and with cellubrevin, markers of recycling endosomes. The activity of NHE3 in endomembranes was assessed by targeting pH-sensitive probes to the recycling endosomes using a chimeric cellubrevin construct with an accessible extracellular epitope. Fluorescence ratio imaging indicated that cellubrevin resides intracellularly in an acidic compartment. In AP-1 cells, endosomal acidification was unaffected by omission of Na+ but was dissipated entirely by concanamycin, a blocker of H(+)-ATPases. In contrast, the cellubrevin compartment was more acidic in NHE3 transfectants, and the acidification was only partially reduced by concanamycin. Moreover, removal of extracellular Na+ resulted in a significant alkalization of the endocytic compartment. These results indicate that NHE3 is present and active in recycling endosomes. By recruiting NHE3 to the plasma membrane, modulation of vesicular traffic could contribute to the regulation of Na+ reabsorption across epithelia.

Identification of sites required for down-regulation of Na+/H+ exchanger NHE3 activity by cAMP-dependent protein kinase. phosphorylation-dependent and -independent mechanisms.

We recently identified a region within the cytoplasmic C-terminal tail of the Na+/H+ exchanger NHE3 isoform (residues 579 to 684) which is essential for inhibition of transport activity by cAMP-dependent protein kinase (PKA) (Cabado, A. G., Yu, F. H., Kapus, A., Gergely, L., Grinstein, S., and Orlowski, J. (1996) J. Biol. Chem. 271, 3590-3599). To further define determinants of PKA regulation, six serine residues located in potential recognition sequences for PKA within, or adjacent to, this region (positions 552, 605, 634, 661, 690, and 691) were altered either independently or in various combinations using site-directed mutagenesis. Wild type and mutant NHE3s tagged with the influenza virus hemagglutinin epitope were stably expressed in exchanger-deficient Chinese hamster ovary cells (AP-1) for functional studies. Of the individual mutations examined, only substitutions at Ser605 or Ser634 affected sensitivity to forskolin, an activator of adenylate cyclase, although partial inhibition of NHE3 activity by forskolin remained. By contrast, simultaneous mutation of both these serines completely abolished cAMP-mediated inhibition of NHE3 without greatly affecting basal transport activity. Two-dimensional analysis of tryptic digests of immunoprecipitated NHE3 labeled in vivo with [32P]orthophosphate revealed several phosphopeptides under basal conditions. Phosphorylation was increased approximately 3-fold in one of these peptides following forskolin treatment, and this change was eliminated by mutation of residue Ser605. Thus, phosphorylation of Ser605 is essential for cAMP-mediated inhibition of NHE3. In addition, Ser634 is also required for the effect of cAMP, even though this residue does not become phosphorylated upon activation of PKA.

Distinct structural domains confer cAMP sensitivity and ATP dependence to the Na+/H+ exchanger NHE3 isoform.

Agents known to increase cAMP levels in renal and intestinal epithelia decrease sodium absorption by inhibiting NHE3, an isoform of the Na+/H+ exchanger expressed at high levels in apical membranes of these cells. In contrast, the ubiquitous, housekeeping isoform of the exchanger (NHE1) is stimulated by cAMP in some cell types. Optimal activity of NHE3 as well as NHE1 requires the presence of ATP. To gain insight into the molecular mechanisms of ATP dependence and cAMP regulation of NHE3, a series of mutations were constructed by progressively truncating segments of the C-terminal cytoplasmic domain of the transporter at amino acid positions 684, 638, and 579 (named NHE3delta684, NHE3delta638, and NHE3delta579). In addition, chimeric antiporters were constructed with the N-terminal transmembrane domain of NHE3 linked to the entire cytoplasmic region of NHE1 (chimera NHE3/1) or vice versa (chimera NHE1/3). These constructs were heterologously expressed in antiport-deficient Chinese hamster ovary cells, and their activities were assessed by fluorimetric measurements of intracellular pH and by radioisotope determinations of Na+ influx. Forskolin, which directly stimulates adenylate cyclase, inhibited NHE3 as well as NHE1/3, but not NHE3/1, suggesting that the cytoplasmic domain of NHE3 was sufficient to confer sensitivity to inhibition by cAMP. Forskolin also inhibited the truncated mutant NHE3delta684 to an extent similar to that for wild type NHE3. However, the inhibitory effect was greatly reduced in NHE3delta638 and more profound truncations (NHE3delta579 obliterated the effect of forskolin. These findings suggest that a region found between amino acids 579 and 684 is essential for the cAMP response of NHE3. In contrast, comparable ATP dependence was observed in all exchanger constructs examined. These observations indicate that ATP dependence is conferred by a region of the molecule in or adjacent to the transmembrane domain, which is most conserved between isoforms. It is concluded that different sites, and therefore different mechanisms, underlie inhibition of NHE3 by cAMP and by depletion of ATP.

Effect of lyophilization on the stability of gonyautoxins obtained from contaminated mussels.

This study describes the stability of gonyautoxins (GTX) and C toxins obtained from contaminated mussels and stored at different temperatures in lyophilized samples. Analyses of extracts from mussels contaminated with paralytic shellfish poison (PSP) indicated the presence of gonyautoxins as the major component in red tides of the North-West coast of Spain. These GTX and C toxins were extracted from contaminated mussels (Mytilus galloprovincialis Lmk) and partially purified by chromatography on Bio-Gel P-2 and Bio-Rex 70. The stability of these toxins was analysed by high performance liquid chromatography. GTX 4 and GTX 6 are the most stable toxins among GTX. We conclude that the lyophilization procedure is not the safest way to process most of the gonyautoxins. However, the lyophilization procedure made the C toxins unstable, so clearly this procedure must be rejected.

Effect of ion composition on the changes in membrane potential induced with several stimuli in rat mast cells.

We studied, in different ionic conditions, the effect of various agents on the membrane potential of rat peritoneal mast cells using the fluorescent probe bisoxonol. Ouabain and ionophore A23187 lead to a fast depolarization of the plasma membrane of mast cells, while compound 48/80 and thapsigargin induced membrane hyperpolarization, which was more pronounced in the case of compound 48/80. When using compound 48/80, the amount of gramicidin necessary to depolarize the cells was twice the amount required in resting cells, which indicates that compound 48/80 increases considerably the activity of the Na+/K+ pump. On the other hand, the ionophore A23187 elicited a clear depolarization which was oblated in the absence of intracellular calcium. The increase in the osmolarity of the medium causes a depolarization in the plasma membrane of mast cells. Hypertonicity-stimulated depolarization is inhibited by removing sodium and potassium.