H874 -H882, 2008). The ROS responsible for this effect is not known. The present study focuses on superoxide (O 2 ·Ϫ ), hydrogen peroxide (H2O2), and hydroxyl radical (HO˙), each of which has been proposed as the signaling ROS. Feedback activation of mitoK ATP provides an ideal setting for studying endogenous ROS signaling. Respiring rat heart mitochondria were preincubated with ATP and diazoxide, together with an agent being tested for interference with this process, either by scavenging ROS or by blocking ROS transformations. The mitochondria were then assayed to determine whether or not the persistent phosphorylated open state was achieved. Dimethylsulfoxide (DMSO), dimethylformamide (DMF), deferoxamine, Trolox, and bromoenol lactone each interfered with formation of the ROS-dependent open state. Catalase did not interfere with this step. We also found that DMF blocked cardioprotection by both ischemic preconditioning and diazoxide. The lack of a catalase effect and the inhibitory effects of agents acting downstream of HO˙excludes H2O2 as the endogenous signaling ROS. Taken together, the results support the conclusion that the ROS message is carried by a downstream product of HO˙and that it is probably a product of phospholipid oxidation. mitochondria; cardioprotection; reactive oxygen species; KATP channels; cardiac ischemia; ROS signaling REACTIVE OXYGEN SPECIES (ROS) are second messengers of preconditioning (21) and have long been known to be required for cardioprotective signaling (3,10,18,34,43,49). The mechanism of increased ROS is reasonably well understood: signaling from the plasma membrane leads to opening of the mitochondrial ATP-sensitive K ϩ channel (mitoK ATP ; Ref. 19), and the increased K ϩ influx into the matrix causes an increase in ROS, which derive during normoxia from complex I of the respiratory chain (1).The ROS transformations that take place in mitochondria are summarized in Figs. 1 (27). The consequences of ROS signaling for mitochondrial physiology are that two mitochondrial protein kinase-Cε (PKCε) are activated by oxidation of their thiol groups (28). Activation of PKCε1, which is associated with mitoK ATP at the mitochondrial inner membrane, leads to opening of mitoK ATP (13,25). Activation of PKCε2 leads to inhibition of the mitochondrial permeability transition (MPT; Ref. 12). Progress is being made in these areas and in the molecular identification of mitoK ATP (17). However, the ROS responsible for activating these PKCεs in vitro or in vivo is still not known. Because the signaling ROS not only originates in mitochondria but also acts on mitochondria, it should be possible to narrow the search for the signaling ROS by studies on isolated rat heart mitochondria. The objective of these studies was to identify the point in the reaction sequence of ROS transformations at which the ROS signal is formed and, ultimately, to determine the identity of the ROS signal itself.We previously described feedback activation of mitoK ATP , in which mitoK ATP opening by a K ATP channel opener ...

No abstract

No abstract