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

Abstract

The unicellular marine phytoplankton Chattonella marina is known to exhibit potent fish-killing activity. Previous studies have demonstrated that C. marina produces reactive oxygen species (ROS), and ROS-mediated ichthyotoxic mechanism has been postulated. However, the exact toxic mechanism is still controversial. In this study, we obtained evidence that C. marina produces nitric oxide (NO) under normal growth conditions. We utilized chemiluminescence (CL) reaction between NO and luminol–H2O2 to detect NO in C. marina cell suspensions. In this assay, significant CL was 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 by C. marina was also confirmed by a spectrophotometric assay based on the measurement of the diazo-reaction-positive substances (NOx) and by fluorometric assay using highly specific fluorescent indicator of NO. The NO level in C. marina was significantly decreased by NG-nitro-l-arginine methyl ester (l-NAME), a specific NO synthase (NOS) inhibitor. The addition of l-arginine resulted in the increased NO level, whereas NaNO2 had no effect. These results suggest that a NOS-like enzyme is mainly responsible for NO generation in C. marina.