Biomedical subjects

M R Vieytes

Publications and source records attributed to M R Vieytes.

At least 19 recordsLinked to original sources

PubMed2006

Changes in membrane potential: an early signal triggered by neurologically active phycotoxins.

Most common phycotoxin poisoning syndromes have important neurological symptoms. However, little is known of the cellular and molecular targets of many of the phycotoxins that produce those human intoxications. We explore the effect of representative toxins on the membrane potential in human neuroblastoma cells by using a fluorimetric assay. Results presented in this study demonstrate that maitotoxin, palytoxins, brevetoxins, and ciguatoxins triggered a dose-dependent membrane depolarization. Mechanisms responsible for the toxins-induced changes in membrane potential are always related to a direct action of the compounds on membrane ion fluxes. This initial screening of the phycotoxins effect is the starting point to lately develop functional methods of detection.

Cell Line, Tumor↗

PubMed2005

Mast cell exocytosis can be triggered by ammonium chloride with just a cytosolic alkalinization and no calcium increase.

A human mast cell line (HMC-1) has been used to study the effect of cytosolic alkaline pH in exocytosis. Compound 48/80, concanavalin A, and thapsigargin do not induce histamine release in HMC-1 cells. Although thapsigargin does not activate histamine release, it does show a large increase in cytosolic Ca(2+), and no change in cytosolic pH. However, when HMC-1 cells were activated with ionomycin, a significant histamine release takes place, and this effect is higher in the presence of thapsigargin. Both drugs show an additive effect on cytosolic Ca(2+) levels. Ammonium chloride (NH(4)Cl) does activate cytosolic alkalinization and histamine release, with no increase in cytosolic Ca(2+). NH(4)Cl does block the release of internal Ca(2+) by thapsigargin, not by ionomycin, and decreases Ca(2+) influx stimulated by these drugs. Under conditions in which the alkalinization induced by NH(4)Cl is blocked by acidification with sodium propionate, histamine release is inhibited. The release of histamine is also observed when NH(4)Cl is added after propionate addition, regardless of the final pH value attained. Our results show that a shift in pH alkaline values, even with final pH below 7.2 is enough to activate histamine release. A shift to less acidic values is a sufficient signal to activate the cells.

Ammonium Chloride↗

PubMed2005

Actin cytoskeleton of rabbit intestinal cells is a target for potent marine phycotoxins.

Biotoxins produced by harmful marine microalgae (phycotoxins) can be accumulated into seafood, representing a great risk for public health. Some of these phycotoxins are responsible for a variety of gastrointestinal disturbances; however, the relationship between their mechanism of action and toxicity in intestinal cells is still unknown. The actin cytoskeleton is an important and highly complicated structure in intestinal cells, and on that basis our aim has been to investigate the effect of representative phycotoxins on the enterocyte cytoskeleton. We have quantified for the first time the loss of enterocyte microfilament network induced by each toxin and recorded fluorescence images using a laser-scanning cytometer and confocal microscopy. Our data show that pectenotoxin-6, maitotoxin, palytoxin and ostreocin-D cause a significant reduction in the actin cytoskeleton. In addition, we found that the potency of maitotoxin, palytoxin and ostreocin-D to damage filamentous actin is related to Ca(2+) influx in enterocytes. Those results identify the cytoskeleton as an early target for the toxic effect of those toxins.

Acrylamides↗

PubMed2005

Effect of okadaic acid on glucose regulation.

Okadaic acid is the main toxin responsible for the natural phenomena known as diarrheic shellfish poisoning (DSP). This toxin is a tumor promoter C38 polyether fatty acid that contains acidic and hydrophobic moieties and is cyclic. Okadaic acid is a potent inhibitor of important classes of protein serine/threonine phosphatases such as protein phosphatase 1 and 2A. The toxin binds in a hydrophobic groove adjacent to the active site of the protein phosphatases and interacts with basic residues within the active site. Therefore okadaic acid causes increases in phosphorylation of proteins that affect a diverse array of cellular processes. For instance, this toxin modulates metabolic parameters in intact cells. In this sense it stimulates lipolysis, and inhibits fatty acid synthesis in adipocytes however increases glucose output and gluconeogenesis in hepatocytes. Additionally, okadaic acid reaches cytotoxic concentrations in the intestinal tissues in accordance with the diarrhea. Recent studies suggested that toxic effects of okadaic acid might be related to modification of nutrients, ionic and water absorption across the small intestine presumably by altering the transporter system. The subject of this review is limited to the effect of okadaic acid on glucose regulation and its cellular as well as clinical implications.

Animals↗

PubMed2004

Calcium-pH crosstalks in rat mast cells: modulation by transduction signals show non-essential role for calcium in alkaline-induced exocytosis.

Alkalinization of cytosolic pH with ammonium chloride (NH4Cl) was reported to be a stimulus for mast cell degranulation. This paper studied the modulatory role of drugs that target protein kinase C (PKC), adenosine 3',5'-cyclic monophosphate (cAMP), tyrosine kinase (TyrK) and phosphatidylinositol 3-kinase (PI3K) on this effect. We used Go6976 (100 nM) and low concentrations of GF109203X (Gf) (50 nM) to inhibit calcium-dependent PKC isozymes. For calcium-independent isozymes, we used 500 nM Gf, and 10 microM rottlerin to specifically inhibit PKC delta, and chelerythrine as non-specific PKC inhibitor. Genistein (10 microM) and lavendustin A (1 microM) were used as unspecific TyrK inhibitors, and 10 nM wortmannin as a PI3K inhibitor. Chelerythrine and 50 nM Gf inhibit histamine release in the presence of external calcium. The inhibition caused by wortmannin was strictly internal calcium-dependent. cAMP-active drugs did not modify the response to NH4Cl. The effect of NH4Cl on histamine release was triggered by a transient elevation on cytosolic pH, which was simultaneous to an elevation on cytosolic calcium and followed by a probable Ca2+-H+ exchange after addition of external calcium. EGTA inhibit the response to suboptimal concentrations of NH4Cl, and BAPTA increased the effect of NH4Cl. There is a clear relationship between NH4Cl-mediated calcium release and histamine release, since those drugs that inhibit this release also inhibit NH4Cl-mediated histamine release; nevertheless, NH4Cl-mediated histamine release was possible in the absence of any calcium release, as shown with BAPTA. This data, in combination with the results with PKC inhibitors, suggest that calcium is not only unnecessary to trigger cell activation, but also that it may be a negative modulator of NH4Cl-mediated exocytosis.

Alkalies↗

PubMed2004

Detection of sodium channel activators by a rapid fluorimetric microplate assay.

Marine toxins such as brevetoxins and ciguatoxins are produced by dinoflagellates and can accumulate in seafood. These toxins affect humans through seafood consumption. Intoxication is mainly characterized by gastrointestinal and neurological disorders and, in most severe cases, by cardiovascular problems. To prevent the consumption of food contaminated with these toxins, shellfish have been tested by mouse bioassay. However, this method is expensive, time-consuming, and ethically questionable. The objective of this study was to use a recently developed fluorimetric microplate assay to rapidly detect brevetoxins and ciguatoxins. The method is based on the pharmacological effect of brevetoxins and ciguatoxins known to activate sodium channels and involves (i). the incubation of excitable cells in 96 well microtiter plates with the fluorescent dye bis-oxonol, whose distribution across the membrane is potential-dependent, and (ii). dose-dependent cell depolarization by the toxins. Our findings demonstrate that measuring changes in membrane potential induced by brevetoxins and ciguatoxins allowed their quantitation. Active toxins could be reliably detected at concentrations in the nanomolar range. The simplicity, sensitivity, and possibility of being automated provide the basis for development of a practical alternative to conventional testing for brevetoxins and ciguatoxins.

Animals↗

PubMed2003

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.

Actins↗

PubMed2003

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.

Actins↗

PubMed2003

Dimethylsphingosine increases cytosolic calcium and intracellular pH in human T lymphocytes.

N,N-Dimethyl-D-erythro-sphingosine (DMS) is the N-methyl derivative of sphingosine; both are activators of sphingosine-dependent protein kinases. The aim of this work was to study the effect of DMS on cytosolic calcium and intracellular pH (pHi) in human T lymphocytes. The variations of calcium and pH were determined by fluorescence digital imaging using Fura-2-AM and BCECF-AM, respectively. DMS increased both pHi and Ca(2+)-cytoslic in human T lymphocytes. These effects were dose-dependent. This drug induced a fast increase in pHi and a release of calcium from different intracellular calcium pools than thapsigargin. DMS also induced a Ca(2+)-influx different from the store-operated calcium channels, since drug effect was not modified by 30 microM SKF 96365. The influx of calcium induced by DMS was completely blocked by preincubation in the presence of nickel, or lanthanum, while the increase in pHi was no affected. However, the presence of cadmium reduced but does not block Ca(2+)-influx. The inhibition of G-protein by 100 ng/mL pertussis toxin, and the inhibition of tyrosine kinases by genistein significantly reduced the cytosolic calcium increase induced by DMS by an inhibition of both, release of calcium from intracellular pools and influx from extracellular medium. The inhibition of pools emptiness by these drugs was related with the inhibition that they induce in the DMS cytosolic alcalinization. In summary, DMS increases pHi and as consequence releases calcium from intracellular pools, and it increases calcium-influx through a channel different from store-operated channel (SOC). Both cytosolic calcium and pHi increase are modulated by G-proteins and tyrosine kinases.

Calcium↗

PubMed2003

Glucose uptake in enterocytes: a test for molecular targets of okadaic acid.

The main diarrheic shellfish poisoning (DSP) toxin is okadaic acid (OA). Although OA is a protein phosphatase 1 and 2A inhibitor less is known about the involvement of the toxin in diarrhea. The initial statement was that OA, by altering the phosphorylation state of proteins, might modify glucose uptake and consequently ionic and water reabsorption across the small intestine. This report presents studies of glucose transport in isolated rabbit enterocytes by using a fluorescent derivative of D-glucose. The dye allowed examining the relation between the toxic effect of OA and cellular mechanisms involved in glucose transport. The central findings are: (i) OA potentiates decrease on glucose uptake due to protein kinase A (PKA) inhibitors such as H89; and (ii) the increase of sugar uptake induced by the protein kinase C (PKC) inhibitor chelerythrine is enhanced by OA. Importance of this work is justified by the need to determine molecular targets of diarrheic toxins in intestinal cells.

4-Chloro-7-nitrobenzofurazan↗

PubMed2002

Characterization of distinct apoptotic changes induced by okadaic acid and yessotoxin in the BE(2)-M17 neuroblastoma cell line.

Apoptotic changes induced by okadaic acid and yessotoxin in BE(2)-M17 neuroblastoma cells have been evaluated and quantified by combining classical methods and fast and sensitive fluorimetric microplate assays. The phosphatase inhibitor okadaic acid induced rapid time- and dose-dependent apoptotic changes in this cell line, which were evident after 1h at concentrations equal or higher than 500 nM. Decreased mitochondrial membrane potential by okadaic acid (IC(50)=350 nM at 1h) was followed by cell detachment (IC(50)=400 nM at 1h), changes in total nucleic acids content (50% of controls after 1h with 1000 nM okadaic acid), caspase-3 activation (3- to 4-fold increase at 6h) and increased Annexin-V binding (1.5-fold at 6h). Yessotoxin induced similar changes in BE(2)-M17 cells, although significant differences were found in the time-course and degree of apoptotic events induced by this phycotoxin, indicating a lower potency for yessotoxin when compared with okadaic acid. This is the first report on apoptogenic activity of yessotoxin.

Annexin A5↗

PubMed2002

Fluorescent microplate cell assay to measure uptake and metabolism of glucose in normal human lung fibroblasts.

This is the first report of a fluorimetric microplate assay to assess glucose uptake and metabolism in eukaryotic cells. The assay was carried out incubating normal human lung fibroblasts in the wells of microtiter trays with a fluorescent D-glucose derivative, 2-N-7-(nitrobenz-2-oxa-1,3-diazol-4-yl)amino-2-deoxy-D-glucose (2-NBDG). This dye could be incorporated by glucose transporting systems in living cells. Substrate uptake was determined by analysing the data obtained with a fluorescence microplate reader. Variables studied in the development of the assay included dye concentration and incubation period. We found that this cell assay is very sensitive, reproducible, provides fast results and graphical display of data. It requires small sample volumes and allows handling of a large number of samples simultaneously. Okadaic acid was used to assess this microplate assay in the field of cytotoxicity. This diarrhetic shellfish toxin is a tumour promoter and a specific inhibitor of protein phosphatases 1 and 2A. The exposition of cells to okadaic acid (0.1 nM-1 microM) at different time intervals causes a decrease in intracellular glucose (40-50% over controls). Results obtained with okadaic acid are the starting point to evaluate application of the method to routine toxicity probes.

4-Chloro-7-nitrobenzofurazan↗

PubMed2001

Modulation of cytosolic calcium levels of human lymphocytes by yessotoxin, a novel marine phycotoxin.

Yessotoxin (YTX) is a polyether toxin of marine origin that has been classified among the diarrheic shellfish poisoning (DSP) toxins group due to its lipophilic nature. However, unlike other DSP toxins, YTX does not produce diarrhea and its mechanisms of action are unknown. We studied the effect of YTX on the cytosolic calcium levels of freshly isolated human lymphocytes by means of fluorescence imaging microscopy. We showed that YTX produced a calcium influx through nifedipine and SKF 96365 (1-[beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenyl]-1H-imidazole hydrochloride)-sensitive channels. This Ca2+ entry was not affected by the DSP toxin okadaic acid, which inhibits protein phosphatases. In addition, YTX also produced an inhibition of capacitative calcium entry activated by thapsigargin or by preincubation in a Ca2+-free medium. This capacitative calcium entry was not sensitive to nifedipine. Furthermore, the inhibitory effect of YTX was dependent on the time of addition of the toxin. We suggest that YTX may interact with calcium channels in a way similar to that described for other polyether marine compounds such as brevetoxins and maitotoxin, although an involvement of other second messengers is also likely.

Biological Transport↗

PubMed2001

Modified mass action law-based model to correlate the solubility of solids and liquids in entrained supercritical carbon dioxide.

The solubility of solids and liquids in supercritical CO2 with added entrainers was modeled with a modified version of the equation of Chrastil to include the effect of entrainers. By considering the formation of the solute-entrainer-solvent complexes an equation is obtained which predicts an exponential increase of solubility with fluid density and/or entrainer concentration. The correlating model was tested by non-linear regression through a computerized iterative process for several systems where an entrainer was present. Four experimental parameters are easily regressed from experimental data, hence the corresponding properties of components such as chemical potentials or critical parameters are not needed. Instead of its simplicity, this thermodynamical model provided a good correlation of the solubility enhancement in the presence of entrainer effect.

Carbon Dioxide↗

PubMed2001

A fluorimetric method based on changes in membrane potential for screening paralytic shellfish toxins in mussels.

To prevent the consumption of bivalves contaminated with paralytic shellfish poisoning (PSP), toxin levels in seafood products are estimated by using the official mouse bioassay. Because of the limitations of this bioassay other methods of monitoring toxins are clearly needed. We have developed a test to screen for PSP toxins based on its functional activity; the toxins bind to the voltage-gated Na+ channels and block their activity. The method is a fluorimetric assay that allows quantitation of the toxins by detecting changes in the membrane potential of human excitable cells. This assay gives an estimate of toxicity, since each toxin present in the sample binds to sodium channels with an affinity which is proportional to its intrinsic toxic potency. The detection limits for paralytic shellfish toxins were found to be 1 ng saxitoxin equivalents/ml compared to the regulatory limit threshold of 400 ng/ml (equivalent to 80 microg/100 g) used in most countries. Our results indicate that this fluorescent assay is a specific, very sensitive, rapid, and reliable method of monitoring PSP toxin levels in samples from seafood products and toxic algae.

Animals↗

PubMed2001

HCO3(-) ions increase mast cell sensitivity to thapsigargin-induced Ca(2+) entry.

In rat mast cells Ca(2+) entry is modified by the presence or absence of other ions in the external medium. HCO3(-) ions, which modify mast cell degranulation, seemed to modulate the Ca(2+) entry elicited by the intracellular Ca(2+)-ATPase inhibitor thapsigargin. In this work we studied the regulation of the Ca(2+) entry by HCO3(-) and its relationship with exocytosis. The Ca(2+) entry was activated by thapsigargin and Ca(2+) in mast cells bathed by a HCO3(-)-buffered medium or a HCO3(-)-free medium. Both Ca(2+) entry and exocytosis were enhanced by the presence of HCO3(-) ions. Nondegranulated mast cells showed a low Ca(2+) entry either in the presence or absence of HCO3(-). Thus, mast cells with a high [Ca(2+)](i) increase in a HCO3(-)-buffered medium undergo degranulation. In the same cells a second Ca(2+) entry was significantly higher than the first Ca(2+) entry in a HCO3(-)-free medium, while in a HCO3(-)-buffered medium the first and second Ca(2+) entries reached similar [Ca(2+)](i) levels. Although the second Ca(2+) entry is high in a HCO3(-)-free medium, degranulation is still low. Our results demonstrate that HCO3(-) ions increase the capacitative Ca(2+) entry and the sensitivity of mast cells to intracellular Ca(2+) in order to induce degranulation.

Animals↗

PubMed2001

Confocal microscopy study of the different patterns of 2-NBDG uptake in rabbit enterocytes in the apical and basal zone.

D-Glucose uptake in isolated rabbit enterocytes was studied using confocal microscopy and 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NDBG), a D-glucose fluorescent analogue, by analysing the fluorescence of apical and basal enterocyte zones. Under normal conditions, apical fluorescence was always higher than basal, presumably due to the location of the Na+-D-glucose cotransporter in the brush-border membrane. After blocking this transporter with phlorizin, apical and basal fluorescence values were similar. This suggests that both brush-border and basolateral membranes participate in phlorizin-insensitive D-glucose transport, since transport across only one membrane cannot explain the uniform overall fluorescence observed. Similarly, after inhibiting the Na+-D-glucose cotransporter by incubating the enterocytes in a medium containing 0.5 mM Na+, neither apical nor basal fluorescence predominated. In contrast, with 130.5 mM extracellular Na+, apical fluorescence was clearly higher than basal fluorescence. These results suggest that phlorizin-insensitive, Na+-independent 2-NDBG uptake occurred through both brush-border and basolateral membranes, probably via the glucose uniporters GLUT2 and GLUT5, suggesting that the latter is a D-glucose transporter.

4-Chloro-7-nitrobenzofurazan↗

PubMed2001

Apoptotic events induced by the phosphatase inhibitor okadaic acid in normal human lung fibroblasts.

We have studied different biochemical indicators of apoptosis in okadaic acid-treated normal human lung fibroblasts (NHLF). Apoptosis was identified by fluorimetric microplate measurements of DNA content, caspase-3 activation and changes in mitochondrial and plasma membrane after 1-48-h treatments with 1-1000 nM okadaic acid. Cells exposed to okadaic acid showed activation of caspase-3, decreased DNA content (<50% of controls at >100 nM okadaic acid after 12 h of incubation) and translocation of phosphatidylserine to the outer leaflet of the plasma membrane, as indicated by the increase in Merocyanine 540 fluorescence after 4 h of incubation with more than 250 nM okadaic acid. Decreased mitochondrial membrane potential (53-98% of controls) was observed with MitoTracker Red CMXRos in all cases, which indicated an active role of mitochondria during the early phase of apoptosis. However, reactive oxygen species were significantly reduced in okadaic acid-treated fibroblasts (50-70% of controls at 1000 nM after 3 h of incubation), which indicates that ROS cannot be considered as a hallmark of apoptosis in okadaic acid-treated cells. These results provide evidence of apoptotic events induced by okadaic acid in NHLF, which can be detected by means of sensitive and reliable fluorimetric microplate assays.

Apoptosis↗