Protein nitration discloses oxidative stress-mediated nitric oxide signaling pathways during metamorphosis in Ciona intestinalis

Metamorphic processes in the ascidian Ciona intestlnalls depend on a complex interplay of events, including a caspasc-dcpcndent apoptosis, which has recently been shown to be regulated by the nitric oxide (NO)-cGMP s.ignalin& pathway. Basic features or the regulatory role or NO included a correlation between endogenous NO levels, tail regression and caspase activity, as well as a very dynamic spatial expression pattern of NO and NO synthase (NOS) during larval development and metamorphosis. We provide now evidence that NO regulates Ciona metamorphosis via a complex balance of signaling pathways that are critically dependent on local redox control and temporal changes of reactive oxygen species production. The data demonstrate that NOS inhibitors along with scavengers or peroxynitrite and No2 two key NO-derived nitrative agents involved in protein nitration, markedly decrease the rate or Ciona metamorphosis. On the contrary, a NO donor as well peroxynitrite result in acceleration of the process. Increased protein nitration was observed at the late larvae stage, when larvae are competent, and proteomic methodologies identified nitrated tyrosine residues in ERK and snail. Exposure or larvae to NO resulted in a marked decrease in ERK phosphorylation. Overall, these and the previous results point to a dual mechanism of NO regulation during metamorphosis in Ciona operating on different time frames and with different out-comes. Oxidative stress and elevated ROS production emerge as a means of diverting NO signaling toward long-term effects in Ciona development that are mediated by protein tyrosine nitration and the associated modulation of phosphorylation-activated processes.