Nitric oxide synthase-like enzyme mediated nitric oxide generation by harmful red tide phytoplankton, Chattonella marina

The unicellular marine phytoplankton Chattonella marina is knownto exhibit potent fish-killing activity. Previous studies havedemonstrated that C. marina produces reactive oxygen species(ROS), and ROS-mediated ichthyotoxic mechanism has been postulated.However, the exact toxic mechanism is still controversial. Inthis study, we obtained evidence that C. marina produces nitricoxide (NO) under normal growth conditions. We utilized chemiluminescence(CL) reaction between NO and luminol–H₂O₂ to detect NOin C. marina cell suspensions. In this assay, significant CLwas observed in C. marina in a cell-number-dependent manner,and this was diminished by the addition of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide(carboxy-PTIO), a specific NO scavenger. The NO generation byC. marina was also confirmed by a spectrophotometric assay basedon the measurement of the diazo-reaction-positive substances(NO_x) and by fluorometric assay using highly specific fluorescentindicator of NO. The NO level in C. marina was significantlydecreased by N^G-nitro-L-arginine methyl ester (L-NAME), a specificNO synthase (NOS) inhibitor. The addition of L-arginine resultedin the increased NO level, whereas NaNO₂ had no effect. Theseresults suggest that a NOS-like enzyme is mainly responsiblefor NO generation in C. marina.