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...
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Frog cutaneous-sternal muscle preparations were used to examine mechanisms by which extracellularly recorded abnormal miniature postsynaptic signals are generated. The frequency of these signals was found to increase after denervation as well as after the addition of 4-aminoquinolinic acid (0.25 mmol/liter) and emetine (10 mmol/liter) to the solution perfusing the preparation. Graphic delineation, using a 3-electrode technique, of the sites where signals were arising in the nerve terminal showed that neurotransmitter (acetylcholine) quanta responsible for the generation of postsynaptic signals emerged in limited areas of the myoneural junction between terminal portions of the active zones instead of being released from the latter. It is concluded that the source of such quanta is most likely to be the Schwann cell and that the mechanism via which the neurotransmitter is released from this cell is distinct from the mechanism of its release from the nerve terminal.
Experiments on neuromuscular synapses from frog skin/chest muscle preparations in conditions of extracellular recording addressed changes in the spontaneous and evoked transmitter secretion after long-term (1.5-6 h) maintenance of preparations in calcium-free solution containing EGTA. Use of three microelectrodes for recording of single-quantum postsynaptic signals showed that calcium-free solution altered the characteristic topography of transmitter secretion in nerve terminals, with widening and fusion of groups of transmitter release. These changes persisted after preparations were returned to the initial solution. These data suggest that calcium-free solutions lead to disorganization of the active zones of nerve endings, At initially low extracellular Ca ion concentrations (0.15-0.4 mM), disorganization of active zones induced by prolonged maintenance of preparations in calcium-free solutions led to decreases in the mean amplitude of endplate currents (EPC) because of decreases in their quantum composition, increases in the time course of transmitter secretion, and decreases in the frequency of miniature endplate currents. The relationship between quantum composition of EPC and the extracellular Ca ion concentration showed a sharp displacement towards higher concentrations, without significant changes in the slope of the relationship. At high initial Ca concentrations (1.8 mM), long-term exposure to calcium-free solutions led to a less marked decrease in EPC amplitude. It is suggested that the extra- and intracellular Ca ion concentrations support the maintenance of the characteristic morphofunctional organization of the apparatus responsible for transmitter secretion in frog nerve endings. Disorganization of the active zones leads to disruption of elements involved in transmitter secretion and decreases in the efficiency of secretion.
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