Mitochondria-Derived Reactive Oxygen Species Dilate Cerebral Arteries by Activating Ca ²⁺ Sparks

Abstract

Abstract-Mitochondria regulate intracellular calcium (Ca 2ϩ ) signals in smooth muscle cells, but mechanisms mediating these effects, and the functional relevance, are poorly understood. Similarly, antihypertensive ATP-sensitive potassium (K ATP ) channel openers (KCOs) activate plasma membrane K ATP channels and depolarize mitochondria in several cell types, but the contribution of each of these mechanisms to vasodilation is unclear. Here, we show that cerebral artery smooth muscle cell mitochondria are most effectively depolarized by diazoxide (Ϫ15%, tetramethylrhodamine [TMRM]), less so by levcromakalim, and not depolarized by pinacidil. KCO-induced mitochondrial depolarization increased the generation of mitochondria-derived reactive oxygen species (ROS) that stimulated Ca 2ϩ sparks and large-conductance Ca 2ϩ -activated potassium (K Ca ) channels, leading to transient K Ca current activation. KCO-induced mitochondrial depolarization and transient K Ca current activation were attenuated by 5-HD and glibenclamide, K ATP channel blockers. MnTMPyP, an antioxidant, and Ca 2ϩ spark and K Ca channel blockers reduced diazoxide-induced vasodilations by Ͼ60%, but did not alter dilations induced by pinacidil, which did not elevate ROS. Data suggest diazoxide drives ROS generation by inducing a small mitochondrial depolarization, because nanomolar CCCP, a protonophore, similarly depolarized mitochondria, elevated ROS, and activated transient K Ca currents. In contrast, micromolar CCCP, or rotenone, an electron transport chain blocker, induced a large mitochondrial depolarization (Ϫ84%, TMRM), reduced ROS, and inhibited transient K Ca currents. In summary, data demonstrate that mitochondriaderived ROS dilate cerebral arteries by activating Ca 2ϩ sparks, that some antihypertensive KCOs dilate by stimulating this pathway, and that small and large mitochondrial depolarizations lead to differential regulation of ROS Here, we demonstrate that a small mitochondrial depolarization, such as that induced by diazoxide, leads to the generation of reactive oxygen species (ROS) that elevate Ca 2ϩ spark frequency and increase the effective coupling of Ca 2ϩ sparks to K Ca channels in arterial smooth muscle cells, resulting in vasodilation. Data also indicate that small and large mitochondrial depolarizations lead to differential regulation of ROS and Ca 2ϩ sparks. This study identifies a novel mechanism by which mitochondria regulate local and global Ca 2ϩ signaling and arterial diameter. Materials and Methods Tissue PreparationAnimal procedures used were approved by the Animal Tetramethylrhodamine, Methyl Ester ImagingExperiments were performed using HEPES-buffered PSS containing (in mM): 134 NaCl, 6 KCl, 2 CaCl 2 , 1 MgCl 2 , 10 HEPES, and 10 glucose (pH 7.4, NaOH). Cells were incubated in HEPES-buffered PSS containing tetramethylrhodamine (TMRM) (100 nM) for 20 minutes, followed by a 15-minute wash. TMRM localization was identified by excitation with 543 nm light, and emission light Ͼ560 nm was captured using a Zeiss LSM5 con...

The mitochondrial function of the cerebral vasculature in insulin-resistant Zucker obese rats

“…Additionally, mitochondria in these fields are interspersed with SR, cellular structures associated with calcium release and storage (11,13,14). We and others have shown that mitochondrial activation in VSM of large cerebral arteries leads to the generation of calcium sparks from the SR, which promotes vasorelaxation (11,30,47). A new finding is that the mitochondrial profile seen in large surface arteries is also found in parenchymal arterioles in our microvessel preparation.…”

“…We also examined the effects of the nonselective nitric oxide (NO) synthase (NOS) inhibitor N -nitro-L-arginine methyl ester (L-NAME) and diazoxide (DZ), a mitochondrial ATP-sensitive potassium (mitoK ATP ) channel opener, on mitochondrial respiration. L-NAME was used to test the role of NO, and DZ was used because of its known depolarizing effect on mitochondria (47). We used freshly isolated cerebral vessels to determine levels of mitochondrial and related nonmitochondrial proteins.…”

Genetic ablation of caveolin-1 modifies Ca2+spark coupling in murine arterial smooth muscle cells

“…Cav-1 is found in the cytosol (22) and, conceivably, may interact with and inhibit RyR channels. Cav-1 is also found in mitochondria, which regulate Ca 2ϩ sparks in cerebral artery smooth muscle cells (7,41). Cav-1 abolishment may also activate RyR channels indirectly by modulating the activity of signaling pathways that regulate Ca 2ϩ sparks (e.g., see Refs.…”

Cross-talk between calcium and reactive oxygen species signaling

“…For example, O 2 · -generated from X/XO elicits a slowly developing decrease of Ca 2+ spark frequency down to 56% of control in permeabilized rat ventricular myocytes [62] . In contrast, mitochondria-derived ROS, generated from diazoxide (an ATP-sensitive K + channel opener)-induced mitochondrial depolarization, elevates Ca 2+ spark frequency and enhances the coupling of sparks to Ca 2+ -sensitve K + channels in smooth muscle cells [63] . In permeabilized rat skeletal muscle fibers, 50 µmol/L H 2 O 2 was also found to increase Ca 2+ spark frequency [64] .…”