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Yessotoxin (YTX) is a marine polyether toxin previously described as a phosphodiesterase (PDE) activator in fresh human lymphocytes. This toxin induces a decrease of adenosine 3',5'-cyclic monophosphate (cAMP) levels in fresh human lymphocytes in a medium with calcium (Ca(2+) ), whereas the contrary effect has been observed in a Ca(2+) -free medium. In the present article, the effect of YTX in K-562 lymphocytes cell line has been analysed. Surprisingly, results obtained in K-562 cell line are completely opposite than in fresh human lymphocytes, since in K-562 cells YTX induces an increase of cAMP levels. YTX cytotoxicity was also studied in both K-562 cell line and fresh human lymphocytes. Results demonstrate that YTX does not modify fresh human lymphocytes viability, whereas in K-562 cells, YTX has a highly cytotoxic effect. It has been described in a previous study that YTX induces a small cytosolic Ca(2+) increase in fresh human lymphocytes but no effect was observed on Ca(2+) pools depletion in these cells. However, our results show that, in K-562 cells, YTX has no effect on cytosolic Ca(2+) levels in a medium with Ca(2+) and induces an increase on Ca(2+) pools depletion followed by a Ca(2+) influx. As far as Ca(2+) modulation is concerned these results demonstrate that YTX has a clear opposite effect in tumoural and fresh human lymphocytes. In addition, intracellular Ca(2+) reservoirs affected by YTX are different than thapsigargin-sensible pools. Furthermore, YTX-dependent Ca(2+) pools depletion was abolished by cAMP analogue (dibutyryl cAMP), phosphodiesterase-4 (PDE4) inhibitor (rolipram), protein kinase A inhibitor (H89) and oxidative phosphorylation uncoupler carbonyl cyanide p-(trifluoromethoxy) (FCCP) treatments. This evidences the crosstalks between Ca(2+) , YTX and cAMP pathways. Also, results obtain demonstrate that YTX-dependent Ca(2+) influx was only abolished by FCCP pre-treatment, which indicates a link between YTX and mitochondria in K-562 cell line. Cytosolic expression of A-kinase anchor proteins (AKAPs), the proteins which integrates phosphodiesterases (PDEs) and PKA to the mitochondria, was determined in both cell models. On the one hand, in human fresh lymphocytes, YTX increases AKAP149 cytosolic expression. This fact is accompanied with a decrease in cAMP levels, and therefore PDEs activation, which finally leads to cell survival. On the other hand, in tumoural lymphocytes, YTX has an opposite effect since decreases AKAP149 cytosolic expression and increase cAMP levels which leads to cell death. This is the first time that YTX and mitochondrial AKAPs proteins relationship is characterised.

Understanding the mechanism of action of the yessotoxin (YTX) is crucial since this drug has potential pharmacological effects in allergic processes, tumor proliferation and neurodegenerative diseases. It has been described that YTX activates apoptosis after 24h of treatment, while after 48 h of incubation with the toxin a decrease in cell viability corresponding to cellular differentiation or non-apoptotic cell death was observed. In this paper, these processes were extensively studied by using the erythroleukemia K-562 cell line. On one hand, events of K-562 cell differentiation into erythrocytes after YTX treatment were studied using hemin as positive control of cell differentiation. Cell differentiation was studied through the cyclic nucleotide response element binding (phospho-CREB) and the transferrin receptor (TfR) expression. On the other hand, using rapamycin as positive control, autophagic hallmarks, as non-apoptotic cell death, were studied after toxin exposure. In this case, the mechanistic target of rapamycin (mTOR) and light chain 3B (LC3B) levels were measured to check autophagy activation. The results showed that cell differentiation was not occurring after 48 h of toxin incubation while at this time the autophagy was triggered. Furthermore after 24h of toxin treatment none of these processes were activated. In addition, the role of the type 4A phosphodiesterase (PDE4A), the intracellular target of YTX, was checked. PDE4A-silencing experiments showed different regulation steps of PDE4A in the autophagic processes triggered either by traditional compounds or YTX. In summary, after 48 h YTX treatment PDE4A-dependent autophagy, as non-apoptotic programmed cell death, is activated.

Palytoxin (PLTX), produced by dinoflagellates from the genus Ostreopsis was first discovered, isolated, and purified from zoanthids belonging to the genus Palythoa. The detection of this toxin in contaminated shellfish is essential for human health preservation. A broad range of studies indicate that mammalian Na+,K+-ATPase is a high affinity cellular receptor for PLTX. The toxin converts the pump into an open channel that stimulates sodium influx and potassium efflux. In this work we develop a detection method for PLTX based on its binding to the Na+,K+-ATPase. The method was developed by using the phenomenon of surface plasmon resonance (SPR) to monitor biomolecular reactions. This technique does not require any labeling of components. The interaction of PLTX over immobilized Na+,K+-ATPase is quantified by injecting different concentrations of toxin in the biosensor and checking the binding rate constant (kobs). From the representation of kobs versus PLTX concentration, the kinetic equilibrium dissociation constant (KD) for the PLTX-Na+,K+-ATPase association can be calculated. The value of this constant is KD = 6.38 × 10−7 ± 6.67 × 10−8 M PLTX. In this way the PLTX-Na+,K+-ATPase association was used as a suitable method for determination of the toxin concentration in a sample. This method represents a new and useful approach to easily detect the presence of PLTX-like compounds in marine products using the mechanism of action of these toxins and in this way reduce the use of other more expensive and animal based methods.