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Luis M Botana

University of Santiago de Compostela, Pharmacology, Faculty Member

University of Santiago de Compostela, Farmacología, Faculty Member

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Fogarty Fellow at Johns Hopkins Asthma and Allergy Center, Baltimore, MD, US. 1990-1991

Director or EU-RL for marine toxins. 2004-2009

Director of Dept Pharmacology of USC. 2003-2011

Full Professor since 1998.
Phone: +34982822233
Address: Departamento de Farmacología
Facultad de Veterinaria
Campus de Lugo-USC 27002
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Anna M. Czarnecka

Medical University of Warsaw

Centro de Investigacion y Estudios Avanzados del IPN

King Saud University

École Normale Supérieure de Lyon

Dr Naveen Kodlady

Gujarat Ayurved University,Jamnagar

Universidad Nacional Autónoma de México

Mike Williamson

The University of Sheffield

Sandhya Sree Malleboyina

Jntuh

University of Karachi

Shivaji Chaudhry

Indira Gandhi National Tribal University, Amarkantak , India

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Papers by Luis M Botana

First description of adenosine production by Gnomoniopsis smithogilvyi, causal agent of chestnut brown rot

World journal of microbiology & biotechnology incorporating the MIRCEN Journal of applied microbiology and biotechnology/World journal of microbiology & biotechnology, Mar 28, 2024

Gnomoniopsis smithogilvyi (Gnomoniaceae, Diaporthales) is the main causal agent of chestnut brown... more Gnomoniopsis smithogilvyi (Gnomoniaceae, Diaporthales) is the main causal agent of chestnut brown rot on sweet chestnut worldwide. The rotting of nuts leads to alterations in the organoleptic qualities and decreased fruit production, resulting in significant economic losses. In 2021, there was an important outbreak of chestnut rot in southern Galicia (Spanish northwest). The profile of secondary metabolites from G. smithogilvyi was studied, especially to determine its capability for producing mycotoxins, as happens with other rotting fungi, due to the possible consequences on the safety of chestnut consumption. Secondary metabolites produced by isolates of G. smithogilvyi growing in potato dextrose agar (PDA) medium were identified using liquid chromatography coupled with high-resolution mass spectrometry. Three metabolites with interesting pharmacological and phyto-toxicological properties were identified based on their exact mass and fragmentation patterns, namely adenosine, oxasetin, and phytosphingosine. The capacity of G. smithogilvyi to produce adenosine in PDA cultures was assessed, finding concentrations ranging from 176 to 834 µg/kg. Similarly, the production of mycotoxins was ruled out, indicating that the consumption of chestnuts with necrotic lesions does not pose a health risk to the consumer in terms of mycotoxins.

Activation of anion channels in human cells after long term exposure to the marine toxin azaspiracid

Proceedings of 1st International Electronic Conference on Toxins, 2021

Acute and chronic in vivo toxicity of the marine toxin palytoxin

Palytoxin (PLTX) is a marine toxin that nowadays is recognized amongst the most toxic compounds i... more Palytoxin (PLTX) is a marine toxin that nowadays is recognized amongst the most toxic compounds isolated from natural products. Originally, the toxin was only identified in a single tidal pool of the island of Maui (Hawaii). Currently, this compound is considered as an emergent toxin in Europe and its prevalence in continental European waters has increased during the last years. The high toxicity of palytoxin is related with the binding to the Na+-K+ ATPase, converting this ubiquitously distributed enzyme in a permeant cation channel [1-3]. Several reports have shown that this toxin is responsible for human fatal intoxications, either after inhalation of toxin-containing marine aerosols or after ingestion of marine products contaminated with PLTX, such as crabs, groupers, mackerel, and parrotfish. So far, different groups have explored the acute oral toxicity of PLTX in mice however, discrepancies in the PLTX source as well as in the monitoring time for the toxic effects yielded con...

Tavarua Deoxyriboside A and Jasplakinolide as Potential Neuroprotective Agents: Effects on Cellular Models of Oxidative Stress and Neuroinflammation

ACS Chemical Neuroscience, 2020

The oceans harbour a great reservoir of molecules with unknown bioactivities, which could be usef... more The oceans harbour a great reservoir of molecules with unknown bioactivities, which could be useful for the treatment of illnesses that nowadays have no cure, such as neurodegenerative diseases. In this work, we evaluated the neuroprotective potential of the marine Fijian compounds tavarua deoxyriboside A and jasplakinolide against oxidative stress and neuroinflammation, crucial mechanisms in neurodegeneration. Both metabolites protected SH-SY5Y human neuroblastoma cells from H2O2 damage, improving mitochondrial function and activating the antioxidant systems of cells. These effects were mediated by their ability of inducing Nrf2 translocation. In BV2 microglial cells activated with lipopolysaccharide, Fijian metabolites also displayed promising results, decreasing the release of pro-inflammatory mediators (ROS, NO, cytokines) through the reduction of gp91 and NFkB-p65 expression. Finally, we performed a coculture among both cell lines, in which treatment with compounds protected SH-SY5Y cells from activated microglia, corroborating their neuroprotective effects. These results 2 suggest that tavarua deoxyriboside A and jasplakinolide could be used as candidate molecules for further studies against neurodegeneration.

In Vitro Effects of Chronic Spirolide Treatment on Human Neuronal Stem Cell Differentiation and Cholinergic System Development

ACS chemical neuroscience, Jan 14, 2018

Spirolides (SPX) are marine toxins, produced by dinoflagellates that act as potent antagonists of... more Spirolides (SPX) are marine toxins, produced by dinoflagellates that act as potent antagonists of nicotinic acetylcholine receptors. These compounds are not toxic for humans, and since there are no reports of human intoxications caused by this group of toxins they are not yet currently regulated in Europe. Currently 13-desmethyl spirolide C, 13,19-didesmethyl spirolide C, and 20-methyl spirolide G are commercially available as reference materials. Previous work in our laboratory has demonstrated that after 4 days of treatment of primary mice cortical neurons with 13-desmethyl spirolide C, the compound ameliorated the glutamate induced toxicity and increased acetylcholine levels and the expression of the acetylcholine synthesizing enzyme being useful both in vitro and in vivo to decrease the brain pathology associated with Alzheimer's disease. In this work, we aimed to extend the study of the neuronal effects of spirolides in human neuronal cells. To this end, human neuronal prog...

Synergistic Effect of Transient Receptor Potential Antagonist and Amiloride against Maitotoxin Induced Calcium Increase and Cytotoxicity in Human Neuronal Stem Cells

ACS chemical neuroscience, Jan 16, 2018

Maitotoxins (MTX) are among the most potent marine toxins identified to date causing cell death t... more Maitotoxins (MTX) are among the most potent marine toxins identified to date causing cell death trough massive calcium influx. However, the exact mechanism for the MTX-induced calcium entry and cytotoxicity is still unknown. In this work, the effect of MTX-1 on the cytosolic free calcium concentration and cellular viability of human neuronal stem cells was evaluated. MTX elicited a concentration-dependent decrease in cell viability which was already evident after 1 h of treatment with 0.25 nM MTX; however, at a concentration of 0.1 nM, the toxin did not cause cell death even after 14 days of exposure. Moreover, the toxin caused a concentration dependent rise in the cytosolic calcium concentration which was maximal at toxin concentrations of 1 nM and dependent on the presence of extracellular calcium on the bathing solution. Several pharmacological approaches were employed to evaluate the role of canonical transient potential receptor channels (TRPC) on the MTX effects. The results p...

Autumnalamide targeted proteins of the immunophilin family

Immunobiology, 2017

Previous works with autumnalamide reported that Store Operated Calcium (SOC) channels were blocke... more Previous works with autumnalamide reported that Store Operated Calcium (SOC) channels were blocked through mitochondrial modulation. In the present paper we studied the effect of autumnalamide on ion-omycin Ca 2+ fluxes. Thus, autumnalamide did not modify ionomycin-sensitive intracellular pools while the ionomycin-induced Ca 2+ influx was blocked with similar potency whether the incubation was done before or after ionomycin-sensitive pools depletion. Nevertheless, autumnalamide was not able to inhibit ionomycin-induced Ca 2+ influx once the membrane channels were activated. Moreover, the compound efficiently inhibited flufenamic acid (FFA) Ca 2+ release induced in this organelle but no the next influx. Since in previous work the effect of autumnalamide was inhibited by cyclosporine A (CsA), structures that target this drug were studied. Therefore, the affinity of autumnalamide for cyclophilin D (Cyp D) was examined. The K D obtained for Cyp D-autumnalamide was 1.51 ± 1.399. Moreover, the K D for Cyp Aautumnalamide was calculated. The peptide had a similar order of Cyp A binding affinity than CsA (8.08 ± 1.23 and 6.85 ± 1.1 M respectively). After testing autumnalamide-binding capacity for Cyp A, the activity of this compound on Cyp A pathway was tested. Thus, the effect on interleukin (IL)-2 release on activated T-lymphocytes was checked. Autumnalamide was able to reduce IL-2 levels near to T cells in resting conditions. Next, the effect over calcineurin and NFATc1 was also evaluated. While CsA inhibits both calcineurin and NFATc1, autumnalamide did not produce any effect. From these results we can conclude that, autumnalamide targeted mitochondrion and prevent T-cells from IL-2 production through the modulation of SOC Ca 2+ channels.

Detection of palytoxin-like compounds by a flow cytometry-based immunoassay supported by functional and analytical methods

Analytica Chimica Acta, 2016

Palytoxin (PLTX) is a complex marine toxin produced by zoanthids (i.e. Palythoa), dinoflagellates... more Palytoxin (PLTX) is a complex marine toxin produced by zoanthids (i.e. Palythoa), dinoflagellates (Ostreopsis) and cyanobacteria (Trichodesmium). PLTX outbreaks are usually associated with Indo-Pacific waters, however their recent repeated occurrence in Mediterranean-European Atlantic coasts demonstrate their current worldwide distribution. Human sickness and fatalities have been associated with toxic algal blooms and ingestion of seafood contaminated with PLTX-like molecules. These toxins represent a serious threat to human health. There is an immediate need to develop easy-to-use, rapid detection methods due to the lack of validated protocols for their detection and quantification. We have developed an immuno-detection method for PLTX-like molecules based on the use of microspheres coupled to flow-cytometry detection (Luminex 200™). The assay consisted of the competition between free PLTX-like compounds in solution and PLTX immobilized on the surface of microspheres for binding to a specific monoclonal anti-PLTX antibody. This method displays an IC 50 of 1.83 ± 0.21 nM and a dynamic range of 0.47-6.54 nM for PLTX. An easy-to-perform extraction protocol, based on a mixture of methanol and acetate buffer, was applied to spiked mussel samples providing a recovery rate of 104 ± 8% and a range of detection from 374 ± 81 to 4430 ± 150 µg kg-1 when assayed with this method. Extracts of Ostreopsis cf. siamensis and Palythoa tuberculosa were tested and yielded positive results for PLTX-like molecules. However, the data obtained for the coral sample suggested that this antibody did not detect 42-OH-PLTX efficiently. The same samples were further analyzed using a neuroblastoma cytotoxicity assay and UPLC-IT-TOF spectrometry, which also pointed to the presence of PLTX-like compounds. Therefore, this single detection method for PLTX provides a semi-quantitative tool useful for the screening of PLTX-like molecules in different matrixes.

Amiloride-dependent transport is the main mechanism implicated in sodium iinflux regulation in rat mast cells

Journal of Cellular Physiology, 1993

Mast cell sodium regulation is a largely unknown field. In our effort to study the mechanisms by ... more Mast cell sodium regulation is a largely unknown field. In our effort to study the mechanisms by which mast cells regulate 5odium levels, we have examined the effect of amiloride and ouabain on "Na entry in rat mast cells in isotonic and hypertonic conditions. Ouabain (0.5 mM) enhances sodium uptake by 32% in isotonic conditions. Hypertonicity increases by 400% the uptake of sodium through an amiloride (1 mM) dependent mechanism. Ouabain has no appreciable effect on the entry of "Na in hypertonic conditions.

PKC and cAMP positively modulate alkaline-induced exocytosis in the human mast cell line HMC-1

Journal of Cellular Biochemistry, 2006

We study in HMC-1 the activation process, measured as histamine release. We know that ammonium ch... more We study in HMC-1 the activation process, measured as histamine release. We know that ammonium chloride (NH(4)Cl) and ionomycin release histamine, and the modulatory role of drugs targeting protein kinase C (PKC), adenosine 3&amp;amp;amp;amp;amp;amp;amp;amp;#39;,5&amp;amp;amp;amp;amp;amp;amp;amp;#39;-cyclic monophosphate (cAMP), tyrosine kinase (TyrK) and phosphatidylinositol 3-kinase (PI3K) on this effect. We used Gö6976 (100 nM) and low concentration of GF 109203X (GF) (50 nM) to inhibit Ca(2+)-dependent PKC isozymes. For Ca(2+)-independent isozymes, we used 500 nM GF and 10 microM rottlerin (specifically inhibits PKCdelta). Phorbol 12-myristate 13-acetate (PMA) (100 ng/ml) was used to stimulate PKC, and genistein (10 microM) and lavendustin A (1 microM) as unspecific TyrK inhibitors. STI571 10 microM was used to specifically inhibit the activity of Kit, the receptor for stem cell factor, and 10 nM wortmannin as a PI3K inhibitor. Activation of PKC with PMA enhances histamine release in response to NH(4)Cl and ionomycin. PMA increases NH(4)Cl-induced alkalinization and ionomycin-induced Ca(2+) entry. Inhibition of PKCdelta strongly inhibits Ca(2+) entry elicited by ionomycin, but failed to modify histamine release. The effect of cAMP-active drugs was explored with the adenylate cyclase activator forskolin (30 microM), the inhibitor SQ22,536 (1 microM), the cAMP analog dibutyryl cAMP (200 microM), and the PKA blocker H89 (1 microM). Forskolin and dibutyryl cAMP do increase NH(4)Cl-induced alkalinization, and potentiate histamine release elicited by this compound. Our data indicates that alkaline-induced exocytosis is modulated by PKC and cAMP, suggesting that pH could be a modulatory signal itself.

Rat Pleural and Peritoneal Mast Cells Stimulated at Different Cellular Levels: Difference in and Influence of Purification Media

International Archives of Allergy and Immunology, 1993

We checked the effect that purification through different media can produce on rat mast cell resp... more We checked the effect that purification through different media can produce on rat mast cell response, considering different populations of pleural and peritoneal mast cells. We compared the media Ficoll, Percoll, bovine serum albumin and sucrose with the seldom used Nycodenz. Histamine release was elicited with compound 48/80, polymyxin B, ionophore A23187 and calcium on cells preincubated with FNa. High osmolarity media induced the most important changes on the cell response as compared to low osmolarity media. Our results show that Ficoll and sucrose induced the most notable changes in the response. Also, the more sensitive stimulus to the purification procedure is sodium fluoride-calcium. The response to ionophore A23187 did not show any important alteration.

Comparative Cytotoxicity of Gambierol versus Other Marine Neurotoxins

Chemical Research in Toxicology, 2011

Many microalgae produce compounds that exhibit potent biological activities. Ingestion of marine ... more Many microalgae produce compounds that exhibit potent biological activities. Ingestion of marine organisms contaminated with those toxins results in seafood poisonings. In many cases, the lack of toxic material turns out to be an obstacle to make the toxicological investigations needed. In this study, we evaluate the cytotoxicity of several marine toxins on neuroblastoma cells, focusing on gambierol and its effect on cytosolic calcium levels. In addition, we compared the effects of this toxin with ciguatoxin, brevetoxin, and gymnocin-A, with which gambierol shares a similar ladder-like backbone, as well as with polycavernoside A analogue 5, a glycosidic macrolide toxin. For this purpose, different fluorescent dyes were used: Fura-2 to monitor variations in cytosolic calcium levels, Alamar Blue to detect cytotoxicity, and Oregon Green 514 Phalloidin to quantify and visualize modifications in the actin cytoskeleton. Data showed that, while gambierol and ciguatoxin were successful in producing a calcium influx in neuroblastoma cells, gymnocin-A was unable to modify this parameter. Nevertheless, none of the toxins induced morphological changes or alterations in the actin assembly. Although polycavernoside A analogue 5 evoked a sharp reduction of the cellular metabolism of neuroblastoma cells, gambierol scarcely reduced it, and ciguatoxin, brevetoxin, and gymnocin-A failed to produce any signs of cytotoxicity. According to this, sharing a similar polycyclic ether backbone is not enough to produce the same effects on neuroblastoma cells; therefore, more studies should be carried out with these toxins, whose effects may be being underestimated.

Changes in Membrane Potential: An Early Signal Triggered by Neurologically Active Phycotoxins

Chemical Research in Toxicology, 2006

Most common phycotoxin poisoning syndromes have important neurological symptoms. However, little ... more 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.

Human Muscarinic Acetylcholine Receptors Are a Target of the Marine Toxin 13-Desmethyl C Spirolide

Chemical Research in Toxicology, 2010

Spirolides are a group of cyclic imine marine toxins recently described. Although no human intoxi... more Spirolides are a group of cyclic imine marine toxins recently described. Although no human intoxication has been related to their presence in shellfish yet, the possible toxicological consequences to human health are actually unknown. The elucidation of the spirolide mechanism/s of action would help to estimate the threat to human consumers. Previous toxicological studies in mice suggested the involvement of acetylcholine receptors. In this work, the effects of the 13-desmethyl C spirolide on the activity and the expression of muscarinic acetylcholine receptors (mAChR) were analyzed using a human neuroblastoma cell model. The 13-desmethyl C spirolide inhibited the acetylcholine-induced calcium signal with a reduction of the maximum response to acetylcholine in the presence of the toxin. The 13-desmethyl C spirolide also reduced binding of the mAChR specific antagonist [(3)H]QNB to neuroblastoma cells. The effect of the 13-desmethyl C spirolide persisted after toxin removal and was inhibited by protection of the primary binding site with high concentrations of atropine suggesting an interaction of the spirolide with the orthologous binding site of mAChR. Moreover, the toxin induced a change in the characteristics of the membrane-associated M3 mAChRs, although it did not alter the total levels of M3 mAChR protein. The 13-desmethyl C spirolide targets mAChRs causing a reduction of function, a decrease of specific antagonist binding to mAChRs, and alteration of membrane-bound receptors that might have important toxicological implications.

Protein Synthesis Inhibition and Oxidative Stress Induced by Cylindrospermopsin Elicit Apoptosis in Primary Rat Hepatocytes

Chemical Research in Toxicology, 2013

The increasing presence of cyanotoxin producers in several regions of the world is hazardous for ... more The increasing presence of cyanotoxin producers in several regions of the world is hazardous for humans and animals. Cylindrospermopsin (CYN) is nowadays recognized as a widely distributed freshwater cyanobacterial toxin. This toxin has been shown to induce protein synthesis inhibition as well as inhibition of glutathione synthesis. Given that the liver seems to be the main target of cylindrospermopsin, in this work we used cultures of primary rat hepatocytes to study the type of cell death induced by CYN nanomolar concentrations. The involvement of reactive oxygen species in toxin induced cell death, the relationship between protein synthesis inhibition and toxicity, and the cell endogenous antioxidant response regulation were studied. We show that cylindrospermopsin induces apoptosis in primary rat hepatocytes. At the concentrations used in this work, protein synthesis inhibition and oxidative stress were involved in the cytotoxic effect elicited by the toxin. Finally, activation of the cell antioxidant response was observed at the transcriptional and translational levels.

Ouabain-induced enhancement of rat mast cells response

Cellular Signalling, 2001

The digitalic glicoside ouabain induces potentiation of rat mast cell histamine release in respon... more The digitalic glicoside ouabain induces potentiation of rat mast cell histamine release in response to several stimuli, which is mediated by Na+/Ca2+ exchanger. In this work, we studied the effect of ouabain on cytosolic calcium, intracellular pH and histamine release with Ca2+ ionophore A23187 in conditions designed to maximize ouabain-induced potentiation of rat mast cells response. The effect of protein kinase C (PKC), cAMP and phosphatase inhibition was also tested. Ouabain induced an enhancement in histamine release, cytosolic calcium and intracellular pH. The adenylate cyclase activator forskolin reduced the effect of ouabain on histamine release and intracellular pH, but enhanced the effect on cytosolic calcium. PKC activator PMA enhanced the effect of ouabain on histamine release and cytosolic calcium, without affecting intracellular pH. A PKC inhibitor, GF-109203X, reduced ouabain-induced enhancement of histamine release and intracellular pH, but increased the enhancement on cytosolic calcium. Finally, inhibition of protein phosphatases 1 and 2A with okadaic acid, increased the effect of ouabain on histamine release and intracellular pH, but reduced cytosolic calcium in presence of ouabain. This result suggest that ouabain-induced potentiation of rat mast cell histamine release with A23187 is modulated by kinases, and this modulation may be carried out by changes in intracellular alkalinization. However, the mechanism underlying cellular alkalinization remains to be elucidated.

Yessotoxin induces ER-stress followed by autophagic cell death in glioma cells mediated by mTOR and BNIP3

Cellular Signalling, 2014

Yessotoxin at nanomolar concentrations can induce programmed cell death in different model system... more Yessotoxin at nanomolar concentrations can induce programmed cell death in different model systems. Paraptosis-like cell death induced by YTX in BC3H1 cells, which are insensitive to several caspase inhibitors, has also been reported. This makes yessotoxin of interest in the search of molecules that target cancer cells vulnerabilities when resistance to apoptosis is observed. To better understand the effect of this molecule at the molecular level on tumor cells, we conducted a transcriptomic analysis using 3 human glioma cell lines with different sensitivities to yessotoxin. We show that the toxin induces a deregulation of the lipid metabolism in glioma cells as a consequence of induction of endoplasmic reticulum stress. The endoplasmic reticulum stress in turn arrests the cell cycle and inhibits the protein synthesis. In the three cell lines used we show that YTX induces autophagy, which is involved in cell death. The sensibility of the cell lines used towards autophagic cell death was related to their doubling time, being the cell line with the lowest proliferation rate the most resistant. The involvement of mTOR and BNIP3 in the autophagy induction was also determined.

Sodium, PMA and Calcium Play an Important Role on Intracellular pH Modulation in Rat Mast Cells

Cellular Physiology and Biochemistry, 1998

In a series of experiments aimed to understand the signaling pathways that regulate intracellular... more In a series of experiments aimed to understand the signaling pathways that regulate intracellular pH (pHi) in rat mast cells, the effect of different intracellular mechanisms on the activity of the Na+/H+ exchanger was studied. After promoting an artificial acidification with sodium propionate we determined the variations on pHi rate recovery. pHi was measured with the dye 2, 7-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester. We studied the effect that the inhibition of some cellular exchangers with different drugs induced on pHi. When the Na+/H+ exchanger was inhibited in the presence of amiloride, the recovery rate constant was twofold smaller than the control value. After the recovery, the final pH was lower than the initial value when the cells were treated either with amiloride or with 4, 4&#39;-diisothiocyanatostilbene-2,2&#39;-disulfonic acid (an anionic antiport inhibitor). No effect was observed when the Na+/K+-ATPase or the Na+/Ca2+ exchanger were inhibited. The suppression of intracellular and extracellular calcium did not induced any change in pHi. The addition of thapsigargin, an activator of capacitative calcium influx, or the phorbol esther 12-O-tetradecanoylphorbol-13-acetate (PMA), a protein kinase C (PKC) activator, increased the activity of the antiporter. Both effects were abrogated by inhibition of the Na+/K+-ATPase with ouabain. The increase in cAMP levels did not affect the effect of PMA on pHi recovery, but it blocked the effect of thapsigargin. Our results indicate that rat mast cells regulate pHi by the combination of some anionic exchanger and the Na+/H+ antiporter. And also that the modulation of this exchanger is the consequence of the connection between different intracellular mechanisms, Na+/K+-ATPase-PKC-calcium, among which cAMP seems not to have a direct role.

Effects of a Synthetic Analog of Polycavernoside A on Human Neuroblastoma Cells

Cellular Physiology and Biochemistry, 2007

Background: Polycavernoside A is a glycosidic marine toxin first extracted from the red alga Poly... more Background: Polycavernoside A is a glycosidic marine toxin first extracted from the red alga Polycavernosa tsudai in 1991 when 3 people died after the ingestion of this food. Polycavernoside A is an interesting molecule because of its complex macrolide structure and strong bioactivity. However, the target site of this toxin has not been characterized. Methods: We studied the effects of a synthethic analog of polycavernoside A on human neuroblastoma cells by measuring changes in membrane potential with bis-oxonol and variations in intracellular calcium levels with fura-2. Fluorescent phalloidin was utilized for assaying activity on actin cytoskeleton. Results: Data showed that this polycavernoside A analog induced a membrane depolarization and an increase in cytosolic calcium levels. Conclusion: These results provide the first insight into the mode of action of polycavernoside A, suggesting that: i) this toxin triggers an initial extracellular calcium entry neither produced across L-type voltage-gated calcium channels nor activation of muscarinic receptors ii) there is a depolarization induced by the toxin and due to the extracellular calcium entry.

Mitogen‐activated protein kinases regulate palytoxin‐induced calcium influx and cytotoxicity in cultured neurons

British Journal of Pharmacology, 2007

Background and purpose:Palytoxin (PLT) is a potent toxin that binds to the Na,K‐ATPase. Palytoxin... more Background and purpose:Palytoxin (PLT) is a potent toxin that binds to the Na,K‐ATPase. Palytoxin is highly neurotoxic and increases the cytosolic calcium concentration ([Ca2+]c) while decreasing intracellular pH (pHi) in neurons (Vale et al., 2006; Vale‐Gonzalez et al., 2007). It is also a tumour promoter that activates several protein kinases.Experimental approach:The role of different protein kinases in the effects of palytoxin on [Ca2+]c, pHi and cytoxicity was investigated in cultured neurons.Key results:Palytoxin‐induced calcium load was not affected by inhibition of calcium‐dependent protein kinase C (PKC) isoforms but it was partially ameliorated by blockade of calcium‐independent PKC isozymes. Inhibition of the extracellular signal‐regulated kinase (ERK) 2 eliminated the palytoxin‐induced rise in calcium and intracellular acidification, whereas inhibition of MEK greatly attenuated the palytoxin effect on calcium without modifying the PLT‐evoked intracellular acidification. ...

First description of adenosine production by Gnomoniopsis smithogilvyi, causal agent of chestnut brown rot

World journal of microbiology & biotechnology incorporating the MIRCEN Journal of applied microbiology and biotechnology/World journal of microbiology & biotechnology, Mar 28, 2024

Gnomoniopsis smithogilvyi (Gnomoniaceae, Diaporthales) is the main causal agent of chestnut brown... more Gnomoniopsis smithogilvyi (Gnomoniaceae, Diaporthales) is the main causal agent of chestnut brown rot on sweet chestnut worldwide. The rotting of nuts leads to alterations in the organoleptic qualities and decreased fruit production, resulting in significant economic losses. In 2021, there was an important outbreak of chestnut rot in southern Galicia (Spanish northwest). The profile of secondary metabolites from G. smithogilvyi was studied, especially to determine its capability for producing mycotoxins, as happens with other rotting fungi, due to the possible consequences on the safety of chestnut consumption. Secondary metabolites produced by isolates of G. smithogilvyi growing in potato dextrose agar (PDA) medium were identified using liquid chromatography coupled with high-resolution mass spectrometry. Three metabolites with interesting pharmacological and phyto-toxicological properties were identified based on their exact mass and fragmentation patterns, namely adenosine, oxasetin, and phytosphingosine. The capacity of G. smithogilvyi to produce adenosine in PDA cultures was assessed, finding concentrations ranging from 176 to 834 µg/kg. Similarly, the production of mycotoxins was ruled out, indicating that the consumption of chestnuts with necrotic lesions does not pose a health risk to the consumer in terms of mycotoxins.

Activation of anion channels in human cells after long term exposure to the marine toxin azaspiracid

Proceedings of 1st International Electronic Conference on Toxins, 2021

Acute and chronic in vivo toxicity of the marine toxin palytoxin

Palytoxin (PLTX) is a marine toxin that nowadays is recognized amongst the most toxic compounds i... more Palytoxin (PLTX) is a marine toxin that nowadays is recognized amongst the most toxic compounds isolated from natural products. Originally, the toxin was only identified in a single tidal pool of the island of Maui (Hawaii). Currently, this compound is considered as an emergent toxin in Europe and its prevalence in continental European waters has increased during the last years. The high toxicity of palytoxin is related with the binding to the Na+-K+ ATPase, converting this ubiquitously distributed enzyme in a permeant cation channel [1-3]. Several reports have shown that this toxin is responsible for human fatal intoxications, either after inhalation of toxin-containing marine aerosols or after ingestion of marine products contaminated with PLTX, such as crabs, groupers, mackerel, and parrotfish. So far, different groups have explored the acute oral toxicity of PLTX in mice however, discrepancies in the PLTX source as well as in the monitoring time for the toxic effects yielded con...

Tavarua Deoxyriboside A and Jasplakinolide as Potential Neuroprotective Agents: Effects on Cellular Models of Oxidative Stress and Neuroinflammation

ACS Chemical Neuroscience, 2020

The oceans harbour a great reservoir of molecules with unknown bioactivities, which could be usef... more The oceans harbour a great reservoir of molecules with unknown bioactivities, which could be useful for the treatment of illnesses that nowadays have no cure, such as neurodegenerative diseases. In this work, we evaluated the neuroprotective potential of the marine Fijian compounds tavarua deoxyriboside A and jasplakinolide against oxidative stress and neuroinflammation, crucial mechanisms in neurodegeneration. Both metabolites protected SH-SY5Y human neuroblastoma cells from H2O2 damage, improving mitochondrial function and activating the antioxidant systems of cells. These effects were mediated by their ability of inducing Nrf2 translocation. In BV2 microglial cells activated with lipopolysaccharide, Fijian metabolites also displayed promising results, decreasing the release of pro-inflammatory mediators (ROS, NO, cytokines) through the reduction of gp91 and NFkB-p65 expression. Finally, we performed a coculture among both cell lines, in which treatment with compounds protected SH-SY5Y cells from activated microglia, corroborating their neuroprotective effects. These results 2 suggest that tavarua deoxyriboside A and jasplakinolide could be used as candidate molecules for further studies against neurodegeneration.

In Vitro Effects of Chronic Spirolide Treatment on Human Neuronal Stem Cell Differentiation and Cholinergic System Development

ACS chemical neuroscience, Jan 14, 2018

Spirolides (SPX) are marine toxins, produced by dinoflagellates that act as potent antagonists of... more Spirolides (SPX) are marine toxins, produced by dinoflagellates that act as potent antagonists of nicotinic acetylcholine receptors. These compounds are not toxic for humans, and since there are no reports of human intoxications caused by this group of toxins they are not yet currently regulated in Europe. Currently 13-desmethyl spirolide C, 13,19-didesmethyl spirolide C, and 20-methyl spirolide G are commercially available as reference materials. Previous work in our laboratory has demonstrated that after 4 days of treatment of primary mice cortical neurons with 13-desmethyl spirolide C, the compound ameliorated the glutamate induced toxicity and increased acetylcholine levels and the expression of the acetylcholine synthesizing enzyme being useful both in vitro and in vivo to decrease the brain pathology associated with Alzheimer's disease. In this work, we aimed to extend the study of the neuronal effects of spirolides in human neuronal cells. To this end, human neuronal prog...

Synergistic Effect of Transient Receptor Potential Antagonist and Amiloride against Maitotoxin Induced Calcium Increase and Cytotoxicity in Human Neuronal Stem Cells

ACS chemical neuroscience, Jan 16, 2018

Maitotoxins (MTX) are among the most potent marine toxins identified to date causing cell death t... more Maitotoxins (MTX) are among the most potent marine toxins identified to date causing cell death trough massive calcium influx. However, the exact mechanism for the MTX-induced calcium entry and cytotoxicity is still unknown. In this work, the effect of MTX-1 on the cytosolic free calcium concentration and cellular viability of human neuronal stem cells was evaluated. MTX elicited a concentration-dependent decrease in cell viability which was already evident after 1 h of treatment with 0.25 nM MTX; however, at a concentration of 0.1 nM, the toxin did not cause cell death even after 14 days of exposure. Moreover, the toxin caused a concentration dependent rise in the cytosolic calcium concentration which was maximal at toxin concentrations of 1 nM and dependent on the presence of extracellular calcium on the bathing solution. Several pharmacological approaches were employed to evaluate the role of canonical transient potential receptor channels (TRPC) on the MTX effects. The results p...

Autumnalamide targeted proteins of the immunophilin family

Immunobiology, 2017

Previous works with autumnalamide reported that Store Operated Calcium (SOC) channels were blocke... more Previous works with autumnalamide reported that Store Operated Calcium (SOC) channels were blocked through mitochondrial modulation. In the present paper we studied the effect of autumnalamide on ion-omycin Ca 2+ fluxes. Thus, autumnalamide did not modify ionomycin-sensitive intracellular pools while the ionomycin-induced Ca 2+ influx was blocked with similar potency whether the incubation was done before or after ionomycin-sensitive pools depletion. Nevertheless, autumnalamide was not able to inhibit ionomycin-induced Ca 2+ influx once the membrane channels were activated. Moreover, the compound efficiently inhibited flufenamic acid (FFA) Ca 2+ release induced in this organelle but no the next influx. Since in previous work the effect of autumnalamide was inhibited by cyclosporine A (CsA), structures that target this drug were studied. Therefore, the affinity of autumnalamide for cyclophilin D (Cyp D) was examined. The K D obtained for Cyp D-autumnalamide was 1.51 ± 1.399. Moreover, the K D for Cyp Aautumnalamide was calculated. The peptide had a similar order of Cyp A binding affinity than CsA (8.08 ± 1.23 and 6.85 ± 1.1 M respectively). After testing autumnalamide-binding capacity for Cyp A, the activity of this compound on Cyp A pathway was tested. Thus, the effect on interleukin (IL)-2 release on activated T-lymphocytes was checked. Autumnalamide was able to reduce IL-2 levels near to T cells in resting conditions. Next, the effect over calcineurin and NFATc1 was also evaluated. While CsA inhibits both calcineurin and NFATc1, autumnalamide did not produce any effect. From these results we can conclude that, autumnalamide targeted mitochondrion and prevent T-cells from IL-2 production through the modulation of SOC Ca 2+ channels.

Detection of palytoxin-like compounds by a flow cytometry-based immunoassay supported by functional and analytical methods

Analytica Chimica Acta, 2016

Palytoxin (PLTX) is a complex marine toxin produced by zoanthids (i.e. Palythoa), dinoflagellates... more Palytoxin (PLTX) is a complex marine toxin produced by zoanthids (i.e. Palythoa), dinoflagellates (Ostreopsis) and cyanobacteria (Trichodesmium). PLTX outbreaks are usually associated with Indo-Pacific waters, however their recent repeated occurrence in Mediterranean-European Atlantic coasts demonstrate their current worldwide distribution. Human sickness and fatalities have been associated with toxic algal blooms and ingestion of seafood contaminated with PLTX-like molecules. These toxins represent a serious threat to human health. There is an immediate need to develop easy-to-use, rapid detection methods due to the lack of validated protocols for their detection and quantification. We have developed an immuno-detection method for PLTX-like molecules based on the use of microspheres coupled to flow-cytometry detection (Luminex 200™). The assay consisted of the competition between free PLTX-like compounds in solution and PLTX immobilized on the surface of microspheres for binding to a specific monoclonal anti-PLTX antibody. This method displays an IC 50 of 1.83 ± 0.21 nM and a dynamic range of 0.47-6.54 nM for PLTX. An easy-to-perform extraction protocol, based on a mixture of methanol and acetate buffer, was applied to spiked mussel samples providing a recovery rate of 104 ± 8% and a range of detection from 374 ± 81 to 4430 ± 150 µg kg-1 when assayed with this method. Extracts of Ostreopsis cf. siamensis and Palythoa tuberculosa were tested and yielded positive results for PLTX-like molecules. However, the data obtained for the coral sample suggested that this antibody did not detect 42-OH-PLTX efficiently. The same samples were further analyzed using a neuroblastoma cytotoxicity assay and UPLC-IT-TOF spectrometry, which also pointed to the presence of PLTX-like compounds. Therefore, this single detection method for PLTX provides a semi-quantitative tool useful for the screening of PLTX-like molecules in different matrixes.

Amiloride-dependent transport is the main mechanism implicated in sodium iinflux regulation in rat mast cells

Journal of Cellular Physiology, 1993

Mast cell sodium regulation is a largely unknown field. In our effort to study the mechanisms by ... more Mast cell sodium regulation is a largely unknown field. In our effort to study the mechanisms by which mast cells regulate 5odium levels, we have examined the effect of amiloride and ouabain on "Na entry in rat mast cells in isotonic and hypertonic conditions. Ouabain (0.5 mM) enhances sodium uptake by 32% in isotonic conditions. Hypertonicity increases by 400% the uptake of sodium through an amiloride (1 mM) dependent mechanism. Ouabain has no appreciable effect on the entry of "Na in hypertonic conditions.

PKC and cAMP positively modulate alkaline-induced exocytosis in the human mast cell line HMC-1

Journal of Cellular Biochemistry, 2006

We study in HMC-1 the activation process, measured as histamine release. We know that ammonium ch... more We study in HMC-1 the activation process, measured as histamine release. We know that ammonium chloride (NH(4)Cl) and ionomycin release histamine, and the modulatory role of drugs targeting protein kinase C (PKC), adenosine 3&amp;amp;amp;amp;amp;amp;amp;amp;#39;,5&amp;amp;amp;amp;amp;amp;amp;amp;#39;-cyclic monophosphate (cAMP), tyrosine kinase (TyrK) and phosphatidylinositol 3-kinase (PI3K) on this effect. We used Gö6976 (100 nM) and low concentration of GF 109203X (GF) (50 nM) to inhibit Ca(2+)-dependent PKC isozymes. For Ca(2+)-independent isozymes, we used 500 nM GF and 10 microM rottlerin (specifically inhibits PKCdelta). Phorbol 12-myristate 13-acetate (PMA) (100 ng/ml) was used to stimulate PKC, and genistein (10 microM) and lavendustin A (1 microM) as unspecific TyrK inhibitors. STI571 10 microM was used to specifically inhibit the activity of Kit, the receptor for stem cell factor, and 10 nM wortmannin as a PI3K inhibitor. Activation of PKC with PMA enhances histamine release in response to NH(4)Cl and ionomycin. PMA increases NH(4)Cl-induced alkalinization and ionomycin-induced Ca(2+) entry. Inhibition of PKCdelta strongly inhibits Ca(2+) entry elicited by ionomycin, but failed to modify histamine release. The effect of cAMP-active drugs was explored with the adenylate cyclase activator forskolin (30 microM), the inhibitor SQ22,536 (1 microM), the cAMP analog dibutyryl cAMP (200 microM), and the PKA blocker H89 (1 microM). Forskolin and dibutyryl cAMP do increase NH(4)Cl-induced alkalinization, and potentiate histamine release elicited by this compound. Our data indicates that alkaline-induced exocytosis is modulated by PKC and cAMP, suggesting that pH could be a modulatory signal itself.

Rat Pleural and Peritoneal Mast Cells Stimulated at Different Cellular Levels: Difference in and Influence of Purification Media

International Archives of Allergy and Immunology, 1993

We checked the effect that purification through different media can produce on rat mast cell resp... more We checked the effect that purification through different media can produce on rat mast cell response, considering different populations of pleural and peritoneal mast cells. We compared the media Ficoll, Percoll, bovine serum albumin and sucrose with the seldom used Nycodenz. Histamine release was elicited with compound 48/80, polymyxin B, ionophore A23187 and calcium on cells preincubated with FNa. High osmolarity media induced the most important changes on the cell response as compared to low osmolarity media. Our results show that Ficoll and sucrose induced the most notable changes in the response. Also, the more sensitive stimulus to the purification procedure is sodium fluoride-calcium. The response to ionophore A23187 did not show any important alteration.

Comparative Cytotoxicity of Gambierol versus Other Marine Neurotoxins

Chemical Research in Toxicology, 2011

Many microalgae produce compounds that exhibit potent biological activities. Ingestion of marine ... more Many microalgae produce compounds that exhibit potent biological activities. Ingestion of marine organisms contaminated with those toxins results in seafood poisonings. In many cases, the lack of toxic material turns out to be an obstacle to make the toxicological investigations needed. In this study, we evaluate the cytotoxicity of several marine toxins on neuroblastoma cells, focusing on gambierol and its effect on cytosolic calcium levels. In addition, we compared the effects of this toxin with ciguatoxin, brevetoxin, and gymnocin-A, with which gambierol shares a similar ladder-like backbone, as well as with polycavernoside A analogue 5, a glycosidic macrolide toxin. For this purpose, different fluorescent dyes were used: Fura-2 to monitor variations in cytosolic calcium levels, Alamar Blue to detect cytotoxicity, and Oregon Green 514 Phalloidin to quantify and visualize modifications in the actin cytoskeleton. Data showed that, while gambierol and ciguatoxin were successful in producing a calcium influx in neuroblastoma cells, gymnocin-A was unable to modify this parameter. Nevertheless, none of the toxins induced morphological changes or alterations in the actin assembly. Although polycavernoside A analogue 5 evoked a sharp reduction of the cellular metabolism of neuroblastoma cells, gambierol scarcely reduced it, and ciguatoxin, brevetoxin, and gymnocin-A failed to produce any signs of cytotoxicity. According to this, sharing a similar polycyclic ether backbone is not enough to produce the same effects on neuroblastoma cells; therefore, more studies should be carried out with these toxins, whose effects may be being underestimated.

Changes in Membrane Potential: An Early Signal Triggered by Neurologically Active Phycotoxins

Chemical Research in Toxicology, 2006

Most common phycotoxin poisoning syndromes have important neurological symptoms. However, little ... more 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.

Human Muscarinic Acetylcholine Receptors Are a Target of the Marine Toxin 13-Desmethyl C Spirolide

Chemical Research in Toxicology, 2010

Spirolides are a group of cyclic imine marine toxins recently described. Although no human intoxi... more Spirolides are a group of cyclic imine marine toxins recently described. Although no human intoxication has been related to their presence in shellfish yet, the possible toxicological consequences to human health are actually unknown. The elucidation of the spirolide mechanism/s of action would help to estimate the threat to human consumers. Previous toxicological studies in mice suggested the involvement of acetylcholine receptors. In this work, the effects of the 13-desmethyl C spirolide on the activity and the expression of muscarinic acetylcholine receptors (mAChR) were analyzed using a human neuroblastoma cell model. The 13-desmethyl C spirolide inhibited the acetylcholine-induced calcium signal with a reduction of the maximum response to acetylcholine in the presence of the toxin. The 13-desmethyl C spirolide also reduced binding of the mAChR specific antagonist [(3)H]QNB to neuroblastoma cells. The effect of the 13-desmethyl C spirolide persisted after toxin removal and was inhibited by protection of the primary binding site with high concentrations of atropine suggesting an interaction of the spirolide with the orthologous binding site of mAChR. Moreover, the toxin induced a change in the characteristics of the membrane-associated M3 mAChRs, although it did not alter the total levels of M3 mAChR protein. The 13-desmethyl C spirolide targets mAChRs causing a reduction of function, a decrease of specific antagonist binding to mAChRs, and alteration of membrane-bound receptors that might have important toxicological implications.

Protein Synthesis Inhibition and Oxidative Stress Induced by Cylindrospermopsin Elicit Apoptosis in Primary Rat Hepatocytes

Chemical Research in Toxicology, 2013

The increasing presence of cyanotoxin producers in several regions of the world is hazardous for ... more The increasing presence of cyanotoxin producers in several regions of the world is hazardous for humans and animals. Cylindrospermopsin (CYN) is nowadays recognized as a widely distributed freshwater cyanobacterial toxin. This toxin has been shown to induce protein synthesis inhibition as well as inhibition of glutathione synthesis. Given that the liver seems to be the main target of cylindrospermopsin, in this work we used cultures of primary rat hepatocytes to study the type of cell death induced by CYN nanomolar concentrations. The involvement of reactive oxygen species in toxin induced cell death, the relationship between protein synthesis inhibition and toxicity, and the cell endogenous antioxidant response regulation were studied. We show that cylindrospermopsin induces apoptosis in primary rat hepatocytes. At the concentrations used in this work, protein synthesis inhibition and oxidative stress were involved in the cytotoxic effect elicited by the toxin. Finally, activation of the cell antioxidant response was observed at the transcriptional and translational levels.

Ouabain-induced enhancement of rat mast cells response

Cellular Signalling, 2001

The digitalic glicoside ouabain induces potentiation of rat mast cell histamine release in respon... more The digitalic glicoside ouabain induces potentiation of rat mast cell histamine release in response to several stimuli, which is mediated by Na+/Ca2+ exchanger. In this work, we studied the effect of ouabain on cytosolic calcium, intracellular pH and histamine release with Ca2+ ionophore A23187 in conditions designed to maximize ouabain-induced potentiation of rat mast cells response. The effect of protein kinase C (PKC), cAMP and phosphatase inhibition was also tested. Ouabain induced an enhancement in histamine release, cytosolic calcium and intracellular pH. The adenylate cyclase activator forskolin reduced the effect of ouabain on histamine release and intracellular pH, but enhanced the effect on cytosolic calcium. PKC activator PMA enhanced the effect of ouabain on histamine release and cytosolic calcium, without affecting intracellular pH. A PKC inhibitor, GF-109203X, reduced ouabain-induced enhancement of histamine release and intracellular pH, but increased the enhancement on cytosolic calcium. Finally, inhibition of protein phosphatases 1 and 2A with okadaic acid, increased the effect of ouabain on histamine release and intracellular pH, but reduced cytosolic calcium in presence of ouabain. This result suggest that ouabain-induced potentiation of rat mast cell histamine release with A23187 is modulated by kinases, and this modulation may be carried out by changes in intracellular alkalinization. However, the mechanism underlying cellular alkalinization remains to be elucidated.

Yessotoxin induces ER-stress followed by autophagic cell death in glioma cells mediated by mTOR and BNIP3

Cellular Signalling, 2014

Yessotoxin at nanomolar concentrations can induce programmed cell death in different model system... more Yessotoxin at nanomolar concentrations can induce programmed cell death in different model systems. Paraptosis-like cell death induced by YTX in BC3H1 cells, which are insensitive to several caspase inhibitors, has also been reported. This makes yessotoxin of interest in the search of molecules that target cancer cells vulnerabilities when resistance to apoptosis is observed. To better understand the effect of this molecule at the molecular level on tumor cells, we conducted a transcriptomic analysis using 3 human glioma cell lines with different sensitivities to yessotoxin. We show that the toxin induces a deregulation of the lipid metabolism in glioma cells as a consequence of induction of endoplasmic reticulum stress. The endoplasmic reticulum stress in turn arrests the cell cycle and inhibits the protein synthesis. In the three cell lines used we show that YTX induces autophagy, which is involved in cell death. The sensibility of the cell lines used towards autophagic cell death was related to their doubling time, being the cell line with the lowest proliferation rate the most resistant. The involvement of mTOR and BNIP3 in the autophagy induction was also determined.

Sodium, PMA and Calcium Play an Important Role on Intracellular pH Modulation in Rat Mast Cells

Cellular Physiology and Biochemistry, 1998

In a series of experiments aimed to understand the signaling pathways that regulate intracellular... more In a series of experiments aimed to understand the signaling pathways that regulate intracellular pH (pHi) in rat mast cells, the effect of different intracellular mechanisms on the activity of the Na+/H+ exchanger was studied. After promoting an artificial acidification with sodium propionate we determined the variations on pHi rate recovery. pHi was measured with the dye 2, 7-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester. We studied the effect that the inhibition of some cellular exchangers with different drugs induced on pHi. When the Na+/H+ exchanger was inhibited in the presence of amiloride, the recovery rate constant was twofold smaller than the control value. After the recovery, the final pH was lower than the initial value when the cells were treated either with amiloride or with 4, 4&#39;-diisothiocyanatostilbene-2,2&#39;-disulfonic acid (an anionic antiport inhibitor). No effect was observed when the Na+/K+-ATPase or the Na+/Ca2+ exchanger were inhibited. The suppression of intracellular and extracellular calcium did not induced any change in pHi. The addition of thapsigargin, an activator of capacitative calcium influx, or the phorbol esther 12-O-tetradecanoylphorbol-13-acetate (PMA), a protein kinase C (PKC) activator, increased the activity of the antiporter. Both effects were abrogated by inhibition of the Na+/K+-ATPase with ouabain. The increase in cAMP levels did not affect the effect of PMA on pHi recovery, but it blocked the effect of thapsigargin. Our results indicate that rat mast cells regulate pHi by the combination of some anionic exchanger and the Na+/H+ antiporter. And also that the modulation of this exchanger is the consequence of the connection between different intracellular mechanisms, Na+/K+-ATPase-PKC-calcium, among which cAMP seems not to have a direct role.

Effects of a Synthetic Analog of Polycavernoside A on Human Neuroblastoma Cells

Cellular Physiology and Biochemistry, 2007

Background: Polycavernoside A is a glycosidic marine toxin first extracted from the red alga Poly... more Background: Polycavernoside A is a glycosidic marine toxin first extracted from the red alga Polycavernosa tsudai in 1991 when 3 people died after the ingestion of this food. Polycavernoside A is an interesting molecule because of its complex macrolide structure and strong bioactivity. However, the target site of this toxin has not been characterized. Methods: We studied the effects of a synthethic analog of polycavernoside A on human neuroblastoma cells by measuring changes in membrane potential with bis-oxonol and variations in intracellular calcium levels with fura-2. Fluorescent phalloidin was utilized for assaying activity on actin cytoskeleton. Results: Data showed that this polycavernoside A analog induced a membrane depolarization and an increase in cytosolic calcium levels. Conclusion: These results provide the first insight into the mode of action of polycavernoside A, suggesting that: i) this toxin triggers an initial extracellular calcium entry neither produced across L-type voltage-gated calcium channels nor activation of muscarinic receptors ii) there is a depolarization induced by the toxin and due to the extracellular calcium entry.

Mitogen‐activated protein kinases regulate palytoxin‐induced calcium influx and cytotoxicity in cultured neurons

British Journal of Pharmacology, 2007

Background and purpose:Palytoxin (PLT) is a potent toxin that binds to the Na,K‐ATPase. Palytoxin... more Background and purpose:Palytoxin (PLT) is a potent toxin that binds to the Na,K‐ATPase. Palytoxin is highly neurotoxic and increases the cytosolic calcium concentration ([Ca2+]c) while decreasing intracellular pH (pHi) in neurons (Vale et al., 2006; Vale‐Gonzalez et al., 2007). It is also a tumour promoter that activates several protein kinases.Experimental approach:The role of different protein kinases in the effects of palytoxin on [Ca2+]c, pHi and cytoxicity was investigated in cultured neurons.Key results:Palytoxin‐induced calcium load was not affected by inhibition of calcium‐dependent protein kinase C (PKC) isoforms but it was partially ameliorated by blockade of calcium‐independent PKC isozymes. Inhibition of the extracellular signal‐regulated kinase (ERK) 2 eliminated the palytoxin‐induced rise in calcium and intracellular acidification, whereas inhibition of MEK greatly attenuated the palytoxin effect on calcium without modifying the PLT‐evoked intracellular acidification. ...