Mitochondria-produced superoxide mediates angiotensin II-induced inhibition of neuronal potassium current

Abstract

Reactive oxygen species (ROS), particularly superoxide (O₂^·−), have been identified as key signaling intermediates in ANG II-induced neuronal activation and sympathoexcitation associated with cardiovascular diseases, such as hypertension and heart failure. Studies of the central nervous system have identified NADPH oxidase as a primary source of O₂^·− in ANG II-stimulated neurons; however, additional sources of O₂^·−, including mitochondria, have been mostly overlooked. Here, we tested the hypothesis that ANG II increases mitochondria-produced O₂^·− in neurons and that increased scavenging of mitochondria-produced O₂^·− attenuates ANG II-dependent intraneuronal signaling. Stimulation of catecholaminergic (CATH.a) neurons with ANG II (100 nM) increased mitochondria-localized O₂^·− levels, as measured by MitoSOX Red fluorescence. This response was significantly attenuated in neurons overexpressing the mitochondria-targeted O₂^·−-scavenging enzyme Mn-SOD. To examine the biological significance of the ANG II-mediated increase in mitochondria-produced O₂^·−, we used the whole cell configuration of the patch-clamp technique to record the well-characterized ANG II-induced inhibition of voltage-gated K⁺ current ( I_Kv) in neurons. Adenovirus-mediated Mn-SOD overexpression or pretreatment with the cell-permeable antioxidant tempol (1 mM) significantly attenuated ANG II-induced inhibition of I_Kv. In contrast, pretreatment with extracellular SOD protein (400 U/ml) had no effect. Mn-SOD overexpression also inhibited ANG II-induced activation of Ca²⁺/calmodulin kinase II, a redox-sensitive protein known to modulate I_Kv. These data indicate that ANG II increases mitochondrial O₂^·−, which mediates, at least in part, ANG II-induced activation of Ca²⁺/calmodulin kinase II and inhibition of I_Kv in neurons.