Ouabain protects rat hearts against ischemia-reperfusion injury via pathway involving src kinase, mitoK_ATP, and ROS

Abstract

We showed recently that mitochondrial ATP-dependent K(+) channel (mitoK(ATP)) opening is required for the inotropic response to ouabain. Because mitoK(ATP) opening is also required for most forms of cardioprotection, we investigated whether exposure to ouabain was cardioprotective. We also began to map the signaling pathways linking ouabain binding to Na(+)-K(+)-ATPase with the opening of mitoK(ATP). In Langendorff-perfused rat hearts, 10-80 microM ouabain given before the onset of ischemia resulted in cardioprotection against ischemia-reperfusion injury, as documented by an improved recovery of contractile function and a reduction of infarct size. In skinned cardiac fibers, a ouabain-induced protection of mitochondrial outer membrane integrity, adenine nucleotide compartmentation, and energy transfer efficiency was evidenced by a decreased release of cytochrome c and preserved half-saturation constant of respiration for ADP and adenine nucleotide translocase-mitochondrial creatine kinase coupling, respectively. Ouabain-induced positive inotropy was dose dependent over the range studied, whereas ouabain-induced cardioprotection was maximal at the lowest dose tested. Compared with bradykinin (BK)-induced preconditioning, ouabain was equally efficient. However, the two ligands clearly diverge in the intracellular steps leading to mitoK(ATP) opening from their respective receptors. Thus BK-induced cardioprotection was blocked by inhibitors of cGMP-dependent protein kinase (PKG) or guanylyl cyclase (GC), whereas ouabain-induced protection was not blocked by either agent. Interestingly, however, ouabain-induced inotropy appears to require PKG and GC. Thus 5-hydroxydecanoate (a selective mitoK(ATP) inhibitor), N-(2-mercaptopropionyl)glycine (MPG; a reactive oxygen species scavenger), ODQ (a GC inhibitor), PP2 (a src kinase inhibitor), and KT-5823 (a PKG inhibitor) abolished preconditioning by BK and blocked the inotropic response to ouabain. However, only PP2, 5-HD, and MPG blocked ouabain-induced cardioprotection.

Interactions of K+ ATP channel blockers with Na+/K+-ATPase

“…From this point of view, the important findings of the present study are: (1) 5-HD concentrations that may have significant inhibitory effects on Na + /K + -ATPase (Figs. 1 and 4) far exceed those (30-300 lM) that effectively block the mitochondrial K ATP channels [5][6][7]26]. (2) There is no influence of 5-HD on the ouabain sensitivity of the purified Na + /K + -ATPase (Fig.…”

“…Although initially thought to be a blocker of the plasma membrane K ATP channel [24], 5-HD has subsequently been shown to act as a highly selective inhibitor of the mitochondrial K ATP channel [25][26][27][28]; and as such, 5-HD has been used to explore the relation of the ouabain-sensitive Na + /K + -ATPase to mitochondrial K ATP channels [5][6][7]. From this point of view, the important findings of the present study are: (1) 5-HD concentrations that may have significant inhibitory effects on Na + /K + -ATPase (Figs.…”

“…In studies addressing the relation of Na + /K + -ATPase to mitochondrial K ATP channels, ouabain and 5-HD are often used together [5][6][7]. To explore the possibility of the interactive effects of the two inhibitors on Na + /K + -ATPase, the effect of a partially inhibitory concentration of 5-HD , and decanoic acid on Na + /K + -ATPase activity of purified kidney enzyme at optimal and suboptimal ATP concentrations.…”

“…In recent years, it has been recognized that Na + /K + -ATPase of intact cells may also function as a signal transducer; i.e., the ouabain-inhibited fraction of the enzyme interacts with neighboring membrane proteins such as Src and EGFR, causing the activations of multiple signal transduction pathways that link the initial cell membrane events to intracellular organelles and the nucleus [3,4]. It is now well established that some of these ouabain-activated pathways lead to functional communication between Na + /K + -ATPase and mitochondria by mechanisms that involve the mitochondrial ATP-sensitive K + channel (K ATP channel), but are not fully characterized [5][6][7]. In studies designed to explore the pathway that relays the ouabain message from Na + /K + -ATPase to mitochondria, 5-hydroxy-decanoate (5-HD) has been used as a well established blocker of mitochondrial K ATP channel [5][6][7].…”

“…It is now well established that some of these ouabain-activated pathways lead to functional communication between Na + /K + -ATPase and mitochondria by mechanisms that involve the mitochondrial ATP-sensitive K + channel (K ATP channel), but are not fully characterized [5][6][7]. In studies designed to explore the pathway that relays the ouabain message from Na + /K + -ATPase to mitochondria, 5-hydroxy-decanoate (5-HD) has been used as a well established blocker of mitochondrial K ATP channel [5][6][7]. The tacit assumption in such studies is that 5-HD has no significant direct interaction with Na + /K + -ATPase.…”