Extracellular organic compounds from the ichthyotoxic red tide alga Heterosigma akashiwo elevate cytosolic calcium and induce apoptosis in Sf9 cells

Toxin(s) from the ichthyotoxic red tide alga Heterosigma akashiwo have been responsible for the destruction of millions of dollars of finfish aquaculture around the globe. Mechanisms of toxicity may include the production of reactive oxygen species (ROS) or organic toxins. The purpose of this study was to investigate the bioactivity of extracellular organic compounds from cultures of H. akashiwo. Cytosolic free calcium levels ([Ca2+]i) in Spodoptera frugiperda (Sf9) insect cells infected with baculoviruses encoding the M1 muscarinic receptor were monitored. Exposure of cells to Heterosigma organics increased [Ca2+]i up to 120 nM above basal levels (two-fold increase). Within minutes following exposure of the cells to the organics, the increase in [Ca2+]i peaked and was followed by a slightly reduced, yet sustained plateau. This plateau was maintained for the duration of an experiment (>15 min) and was inhibitable by lanthanum. Furthermore, stimulation of Ca2+ release from intracellular stores by carbachol (muscarinic agonist) or thapsigargin (sarco-endoplasmic reticulum Ca2+-ATPases, SERCA inhibitor) potentiated the [Ca2+]i response induced by the organics resulting in a maximal increase of >250 nM above basal levels (three-fold increase). However, the [Ca2+]i response to Heterosigma organics was strictly dependent on the presence of extracellular calcium. Flow cytometric analyses revealed that these organics induced apoptosis of these same cells. Collectively, our data indicate that extracellular organics from cultures of H. akashiwo acutely increase [Ca2+]i in cells by inhibiting the plasma membrane Ca2+-ATPase transporter and ultimately induce apoptotic cell death. These organics may play a significant role in the ichthyotoxic and allelopathic behaviour of this alga.