Mitochondria-Derived Reactive Oxygen Species Dilate Cerebral Arteries by Activating Ca2+ Sparks
Mitochondria regulate intracellular calcium (Ca2+) signals in smooth muscle cells, but mechanisms mediating these effects, and the functional relevance, are poorly understood. Similarly, antihypertensive ATP-sensitive potassium (KATP) channel openers (KCOs) activate plasma membrane KATP 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 (−15%, 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 Ca2+ sparks and large-conductance Ca2+-activated potassium (KCa) channels, leading to transient KCa current activation. KCO-induced mitochondrial depolarization and transient KCa current activation were attenuated by 5-HD and glibenclamide, KATP channel blockers. MnTMPyP, an antioxidant, and Ca2+ spark and KCa channel blockers reduced diazoxide-induced vasodilations by >60%, 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 KCa currents. In contrast, micromolar CCCP, or rotenone, an electron transport chain blocker, induced a large mitochondrial depolarization (−84%, TMRM), reduced ROS, and inhibited transient KCa currents. In summary, data demonstrate that mitochondria-derived ROS dilate cerebral arteries by activating Ca2+ sparks, that some antihypertensive KCOs dilate by stimulating this pathway, and that small and large mitochondrial depolarizations lead to differential regulation of ROS and Ca2+ sparks.
Systemic blood pressure and tissue blood flow are modulated by changes in the diameter of small arteries and arterioles. A fundamental determinant of arterial diameter is the intracellular calcium concentration ([Ca2+]i) of smooth muscle cells.1 This spatially averaged [Ca2+]i, termed global [Ca2+]i, arises due to extracellular Ca2+ influx and intracellular Ca2+ release.1 A nanomolar elevation in smooth muscle cell global [Ca2+]i stimulates Ca2+/calmodulin-dependent myosin light chain kinase, leading to vasoconstriction, whereas a reduction in global [Ca2+]i results in dilation.1 Another intracellular Ca2+ signal, termed a Ca2+ spark, is a localized [Ca2+]i transient that is generated in response to the opening of multiple ryanodine-sensitive Ca2+ release (RyR) channels on the sarcoplasmic reticulum (SR).1 Although Ca2+ sparks elevate [Ca2+]i in the local vicinity of the release site to micromolar concentrations, Ca2+ sparks do not contribute directly to global [Ca2+]i because of their transient and localized nature.1,2 Rather, sparks activate several nearby sarcolemma large-conductance Ca2+-activated potassium (KCa) channels.1 Ca2+ spark-induced transient KCa currents induce membrane hyperpolarization, leading to a reduction in voltage-dependent Ca2+ channel activity, a decrease in global [Ca2+]i, and relaxation.1 Several signaling elements, including protein kinases and carbon monoxide,3 modulate arterial diameter by regulating Ca2+ sparks and the effective coupling of Ca2+ sparks to KCa channels.1
Mitochondria also regulate local and global Ca2+ signaling in smooth muscle cells,4–7 although the underlying mechanisms and the physiological function of such modulation are poorly understood. Mitochondria potential is ≈150 to 200 mV more negative than the cytosol, and changes spontaneously and in response to stimuli. Spontaneous changes in mitochondrial potential, termed flickers, occur in mitochondria of several cell types, including smooth muscle cells.8 Mitochondria potential is also altered by stimuli, including hypoxia.9 Thus, in arterial smooth muscle cells, changes in mitochondria potential may modulate local and global Ca2+ signals, leading to functional consequences.
It has been known for several years that synthetic openers of ATP-sensitive K+ (KATP) channels depolarize mitochondria, including those in cardiac myocytes.10,11 Recently, diazoxide, a KATP channel opener, was shown to depolarize mitochondria and reduce cytosolic Ca2+ removal in femoral artery smooth muscle cells.6 Investigating the regulation of mitochondrial potential and local and global Ca2+ signaling in arterial smooth muscle cells by KATP channel openers may provide insights into the mechanisms by which mitochondria and changes in mitochondria potential modulate contractility, particularly because these compounds are vasodilators.12
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 Ca2+ spark frequency and increase the effective coupling of Ca2+ sparks to KCa 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 Ca2+ sparks. This study identifies a novel mechanism by which mitochondria regulate local and global Ca2+ signaling and arterial diameter.
Materials and Methods
Animal procedures used were approved by the Animal Care and Use Committee at the University of Tennessee. Sprague-Dawley rats (200 to 250g) of either sex were euthanized and the brain removed. Cerebral arteries (50 to 200 μm in diameter) were harvested, cleaned, and maintained in ice-cold (4°C) physiological saline solution (PSS) containing (in mmol/L): 112 NaCl, 4.8 KCl, 26 NaHCO3, 1.8 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, and 10 glucose, gassed with 74% N2−21% O2−5% CO2 (pH 7.4). Smooth muscle cells were isolated as described previously.13
Tetramethylrhodamine, Methyl Ester Imaging
Experiments were performed using HEPES-buffered PSS containing (in mM): 134 NaCl, 6 KCl, 2 CaCl2, 1 MgCl2, 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 >560 nm was captured using a Zeiss LSM5 confocal microscope. To measure temporal changes in TMRM intensity, cells were excited with 535 nm light, and background corrected fluorescence collected every 1 second at 610 nm using a Dage MTI iCCD camera and Ionwizard software (Ionoptix).
Potassium currents were measured using the conventional whole-cell or perforated patch-clamp configuration (Axopatch 200B, Clampex 8.2). Bath solution was HEPES-buffered PSS. For perforated patch-clamp configuration, the pipette solution contained (in mmol/L): 110 KAsp, 30 KCl, 10 NaCl, 1 MgCl2, 10 HEPES, and 0.05 EGTA (pH 7.2, KOH). For conventional whole-cell configuration, the pipette solution contained (in mmol/L): 140 KCl, 1.9 MgCl2, 0.037 CaCl2, 0.1 EGTA, 10 HEPES, and 2 Na2ATP (pH 7.2, KOH). Transient KCa currents and single KCa channel activity (NPo) were measured at −40 and 0 mV, respectively. At least 5 minutes of continuous data were analyzed offline to calculate transient KCa current properties, or KCa channel NPo.
Reactive Oxygen Species Measurements
Endothelium-denuded artery segments were incubated for 60 minutes at room temperature in HEPES-buffered PSS containing H2DCFDA (10 μmol/L), followed by wash. Dichlorofluoroscein (DCF) was excited with 488 nm light, and emitted light >530 nm was collected using a Zeiss LSM5 confocal microscope.
Endothelium-denuded artery segments were incubated for 20 minutes in HEPES-buffered PSS with or without diazoxide (100 μmol/L). Arteries were embedded in OTC, snap-frozen, sectioned, and then incubated with dihydroethidium (DHE; 10 μmol/L) for 30 minutes at 37°C. Sections were illuminated with 488 nm light, and emitted light >590 nm was collected using a Noran Oz confocal microscope. Tissue processing did not elevate ROS (see online data supplement, available at http://circres.ahajournals.org).
Confocal Ca2+ Imaging
Cells were incubated in HEPES-buffered PSS containing fluo-4AM (10 μmol/L) for 25 minutes at room temperature followed by a 30 minute wash. Cells were imaged using a Noran Oz laser scanning confocal microscope by illuminating with 488 nm light and collecting emitted light >500 nm. Images were recorded every 8.3 ms. Ca2+ sparks and global Ca2+ were analyzed offline as described elsewhere and in the online data supplement.4,14
Pressurized Artery Diameter Measurements
Endothelium-denuded artery segments were cannulated at each end in a perfusion chamber containing PSS (Living Systems Instrumentation). The chamber was continuously perfused with PSS and maintained at 37°C. Intravascular pressure was monitored using a pressure transducer. Wall diameter was measured at 1 Hz using a CCD camera attached to a Nikon TS100-F microscope and the edge-detection function of IonWizard.
Values are expressed as mean±SEM. Student t test was used for comparing paired and unpaired data from two populations, and ANOVA and Student–Newman–Keuls tests were used for multiple group comparisons. Evaluation of whether distributions were Gaussian was by the method of Kolmogorov and Smirnov. Simultaneous spark and transient KCa current amplitude data were fit with a first-order polynomial linear function and the slope±SEM of each fit was compared using a Student t test. No differences were observed between genders and data were pooled. P<0.05 was considered significant.
An expanded Materials and Methods section is provided in the online data supplement.
Regulation of Arterial Smooth Muscle Mitochondrial Potential by KATP Channel Modulators
Punctuate TMRM fluorescence in cerebral artery smooth muscle cells was reduced by rotenone, an electron transport chain (ETC) complex I blocker (10 μmol/L), indicating mitochondria specific loading (Figure 1). Rotenone (10 μmol/L), or CCCP, (10 μmol/L) rapidly reduced mean TMRM fluorescence by 69±5 (n=24) and 84±3 (n=40) %, respectively (P<0.05 for each), similarly to previous reports.4 Diazoxide (10 to 500 μmol/L) induced a concentration-dependent steady-state reduction in TMRM fluorescence of between 8±1 and 15±1% (P<0.05 for each). Levcromakalim (100 μmol/L), another KATP channel opener, reduced TMRM fluorescence by 8±1% (P<0.05). In contrast, the KATP channel opener pinacidil (100 μmol/L) or 15 mmol/L K+, which induces a similar cell membrane hyperpolarization as sarcolemmal KATP (sarcKATP) channel activation (≈15 mV12), did not alter TMRM fluorescence (P>0.05). Glibenclamide (10 μmol/L), a sarcKATP channel and mitochondrial KATP (mitoKATP) channel blocker, and 5-HD (500 μmol/L), a mitoKATP channel blocker, did not alter TMRM fluorescence when applied alone. However, glibenclamide (1 μmol/L) attenuated diazoxide- and levcromakalim-induced mitochondrial depolarization, and 5-HD (500 μmol/L) blocked diazoxide-induced mitochondrial depolarization. These data indicate that diazoxide and levcromakalim depolarize mitochondria in cerebral artery smooth muscle cells by a mechanism that does not involve sarcolemmal KATP channel activation or membrane hyperpolarization, but by a mechanism that is inhibited by KATP channel blockers.
KATP Channel Openers That Depolarize Mitochondria Activate Transient KCa Currents
Diazoxide induced concentration-dependent (10 to 100 μmol/L) transient KCa current activation in voltage-clamped (−40 mV) smooth muscle cells. For example, diazoxide (100 μmol/L) increased mean transient KCa current frequency from 0.58±0.1 to 0.96±0.14 Hz, or to ≈203% of control (Figure 2). Diazoxide (100 μmol/L) also increased mean transient KCa current amplitude from 27±3 to 35±4 pA, or to ≈135% of control (Figure 2). Levcromakalim (100 μmol/L) was a less effective transient KCa current activator than diazoxide but increased frequency to ≈187% of control. Diazoxide similarly activated transient KCa currents when applied in the presence of diltiazem (50 μmol/L), indicating stimulation was independent of voltage-dependent Ca2+ channel activity. When applied alone, glibenclamide (10 μmol/L) and 5-HD (500 μmol/L) did not alter transient KCa currents, but both KATP channel blockers reduced diazoxide-induced transient KCa current activation. In the continued presence of diazoxide, rotenone (10 μmol/L) rapidly reduced mean transient KCa current frequency and amplitude to ≈22% and 48% of the control values before diazoxide application, respectively. In contrast to the stimulatory effects of diazoxide and levcromakalim, pinacidil (100 μmol/L) did not alter transient KCa currents. These data indicate KATP channel openers that depolarize mitochondria also stimulate transient KCa currents, and this effect is reversed by KATP channel inhibitors and an ETC blocker.
Diazoxide Elevates Ca2+ Spark Frequency and Effective Coupling to KCa Channels
To investigate mechanisms mediating transient KCa current activation by KATP channel openers, simultaneous measurements of sparks and transient KCa currents were obtained in voltage-clamped (−40 mV) cells. Diazoxide (100 μmol/L) increased mean Ca2+ spark frequency to ≈179% of control (Figure 3). Diazoxide also increased the effective coupling of Ca2+ sparks to KCa channels (P<0.05), although mean Ca2+ spark amplitude and the percentage of Ca2+ sparks that activated a transient KCa current (control, 93±4%; diazoxide, 94±2%) did not change (P>0.05). In the same cells, diazoxide did not change global [Ca2+]i (F/F0, 107±5% of control, P>0.05). Data indicate diazoxide elevates Ca2+ spark, and thus, transient KCa current frequency, by a mechanism that does not involve an elevation in global [Ca2+]i, and suggest diazoxide elevates transient KCa current amplitude by increasing KCa channel sensitivity to Ca2+ sparks.
To determine whether diazoxide enhances KCa channel coupling to Ca2+ sparks due to a direct or indirect mechanism, KCa channel activity (NPo) was measured using the perforated patch-clamp configuration. Ca2+ sparks, and thus, transient KCa currents, were abolished with thapsigargin, a SR Ca2+-ATPase inhibitor.1 Diazoxide (100 μmol/L) increased mean KCa channel NPo to ≈187% of control (online Figure I). In contrast, when applied in the continued presence of CCCP (1 μmol/L), to depolarize mitochondria, diazoxide did not alter KCa channel activity. Data suggest diazoxide does not activate KCa channels directly, but elevates the effective coupling of Ca2+ sparks to KCa channels by a mitochondria-dependent mechanism.
Diazoxide Elevates ROS, Whereas Rotenone and CCCP Reduce ROS
Because mitochondria are a major source of ROS,15 we tested the hypothesis that mitochondrial depolarization alters ROS generation in cerebral artery smooth muscle cells. Diazoxide (100 μmol/L) elevated the fluorescence of DCF, an ROS indicator, in smooth muscle cells of endothelium-denuded cerebral arteries to 143±11% of control (Figure 4). DCF fluorescence did not change after vehicle (DMSO, time control) or pinacidil (100 μmol/L). Catalase (2000 U/mL), or MnTMPyP (10 μmol/L), a superoxide dismutase and catalase mimetic, blocked diazoxide-induced DCF fluorescence elevations. In contrast to the effect of diazoxide, rotenone (10 μmol/L), or CCCP (1 μmol/L), caused a profound reduction in DCF fluorescence to ≈53% and ≈37% of control, respectively. Rotenone also prevented diazoxide-induced DCF fluorescence elevations, indicating diazoxide generates mitochondria-derived ROS. In agreement, apocynin (25 μmol/L), an NAD(P)H oxidase blocker, oxypurinol (10 μmol/L), a xanthine oxidase inhibitor, and 17-octadecanoic acid (10 μmol/L), a cytochrome P450 blocker, did not alter diazoxide (100 μmol/L)-induced DCF fluorescence elevations (see online data supplement). Diazoxide (100 μmol/L) also elevated fluorescence intensity of DHE, another ROS indicator, in smooth muscle cells to 137±9% of control (Figure 4). Data indicate diazoxide elevates mitochondria-derived ROS, pinacidil does not alter ROS, and rotenone and CCCP reduce ROS in cerebral artery smooth muscle cells.
Diazoxide Stimulates Transient KCa Currents by Elevating ROS
To determine whether diazoxide activates transient KCa currents by elevating ROS, the conventional whole-cell configuration of patch-clamp was used. Inclusion of the antioxidants superoxide dismutase (SOD), and catalase (300 U/mL of each) in the pipette solution abolished diazoxide-induced elevations in transient KCa current frequency and amplitude (Figure 5). In contrast, when boiled SOD and catalase (92°C, 30 minutes) were included in the pipette solution, diazoxide increased transient KCa current frequency to ≈213% of control. Collectively, data indicate that diazoxide elevates transient KCa current frequency and amplitude by inducing an elevation in mitochondria-derived ROS and suggest diazoxide does not activate RyR channels directly.
Diazoxide Dilates Cerebral Arteries Because of RyR and KCa Channel Activation
To investigate whether mitochondria-derived ROS induce vasodilation by activating RyR and KCa channels, diameter regulation of endothelium-denuded pressurized (60 mm Hg) cerebral arteries was measured. Arterial diameter regulation by a KATP channel opener was measured in control and then again in the same artery after MnTMPyP, thapsigargin, ryanodine, a RyR channel inhibitor, or iberiotoxin, a selective KCa channel blocker.
Diazoxide (100 μmol/L) and pinacidil (100 μmol/L) reversibly increased mean diameter by 25±3 μm (n=29) and 77±6 μm (n=21), respectively, from a baseline diameter of 142±3 μm, (Figure 6 and online data supplement). MnTMPyP (10 μmol/L), thapsigargin (100 nM), ryanodine (10 μmol/L), and iberiotoxin (100 nM) reduced mean diazoxide-induced dilations to between ≈23% and 36% of those obtained in control in the same arteries. In control, diazoxide induced reproducible dilations (second application was 98±4% of first, n=5, P>0.05, online Figure II), indicating attenuated dilations with blockers were not attributable to receptor desensitization. In contrast, thapsigargin, ryanodine, iberiotoxin, or MnTMPyP did not alter dilations induced by pinacidil (100 μmol/L), which did not depolarize mitochondria, elevate ROS, or activate transient KCa currents. Data suggest diazoxide dilates pressurized cerebral arteries by inducing an elevation in ROS and by activating RyR and KCa channels. In contrast, pinacidil-induced dilations do not occur because of ROS elevations, or RyR or KCa channel activation.
Differential Regulation of Transient KCa Currents by Small and Large Mitochondrial Depolarizations
We sought to determine mechanisms by which KATP channel openers elevate ROS, and to investigate mechanisms that lead to differential regulation of ROS and transient KCa currents by KATP channel openers and CCCP or rotenone. We tested the hypothesis that a small mitochondrial depolarization, such as that induced by diazoxide, elevates ROS and activates transient KCa currents, whereas a large mitochondrial depolarization reduces ROS and inhibits transient KCa currents. Thus, concentration-dependent regulation of mitochondrial potential, ROS, and transient KCa currents by CCCP, which elevates H+ permeability, were measured.
Figure 7A illustrates concentration-dependent regulation of TMRM fluorescence intensity by CCCP in a smooth muscle cell. A low concentration of CCCP (1 nM) decreased mean TMRM fluorescence intensity by 13±1%, similar to the effect of 100 μmol/L diazoxide (Figure 7B). In the same cell, a higher CCCP concentration (10 μmol/L) reduced TMRM fluorescence intensity by 84±3% (Figures 1 and 7⇓A, n=40, P<0.05). In agreement with our hypothesis, 1 nM CCCP elevated DCF fluorescence intensity to 140±5% of control, whereas micromolar CCCP profoundly reduced DCF intensity (Figures 4 and 7⇓C). Consistent with these observations, 1 nM CCCP increased mean transient KCa current frequency to 218±53% of control, whereas in the same cells, 10 μmol/L CCCP abolished transient KCa currents (Figure 7D and 7E). CCCP (1 nM) did not activate transient KCa currents by elevating global [Ca2+]i, because global [Ca2+]i did not change (see online data supplement). These data suggest a small mitochondrial depolarization activates transient KCa currents by elevating ROS, whereas a large mitochondrial depolarization inhibits these events by reducing ROS.
The principal novel findings of this study are: (1) Mitochondria-derived ROS activate Ca2+ sparks and transient KCa currents in arterial smooth muscle cells. (2) This pathway is activated by antihypertensive KATP channel openers and leads to vasodilation. (3) Small and large mitochondrial depolarizations lead to differential regulation of ROS, Ca2+ sparks, and transient KCa currents.
KATP channel openers depolarize mitochondria in several cell types, including cardiac myocytes.10,11 We show that structurally distinct KATP channel openers depolarize mitochondria in arterial smooth muscle cells. Similarities and differences are apparent when comparing the effects of different KATP channel openers on mitochondrial potential in smooth muscle cells to findings in cardiac myocytes, where responses are well characterized. In heart cells, diazoxide, levcromakalim, and pinacidil depolarize cardiac myocyte mitochondria, whereas in smooth muscle cells, pinacidil had no effect.11 However, KATP channel opener-induced mitochondrial depolarization is similarly reversed by glibenclamide and 5-HD in both cell types. One explanation for dissimilar mitochondrial pharmacology between cardiac and smooth muscle cells is that the identity or amino acid sequence of targets for KATP channel openers may differ slightly.
Inner mitochondrial membrane KATP channels have been isolated from several tissues, including heart and brain, and incorporated into proteoliposomes and lipid bilayers for characterization.10,11,16 MitoKATP channel-independent targets for KATP channel openers have also been described in mitochondria. In studies using submitochondrial particles isolated from pig heart, pinacidil and diazoxide did not alter mitochondrial potential but inhibited NADH dehydrogenase in ETC complex I and succinate dehydrogenase in ETC complex II, respectively.17 5-HD may also be a substrate for acyl-coA synthetase.17 Although mitoKATP has been characterized in isolated mitochondrial membranes from several cell types, there are no direct measurements of a smooth muscle mitoKATP channel. CCCP at 1 nM produced a similar mitochondrial depolarization, ROS elevation, and transient KCa current activation as 100 μmol/L diazoxide, indicating mitochondrial depolarization is the trigger for these changes. Rotenone reduced ROS, prevented the diazoxide-induced ROS elevation, and blocked transient KCa currents, either when applied alone4 or together with diazoxide. Thus, the ETC appears to be the source of ROS generated in response to a small mitochondrial depolarization, and ETC inhibition reduces ROS and transient KCa currents. If diazoxide elevated ROS generation through complex II block, then pinacidil should have some effect by blocking the ETC upstream at complex I. In addition, KATP channel opener-induced mitochondrial depolarization and transient KCa current activation were inhibited by KATP channel blockers. Although effects mediated via ETC inhibition cannot be ruled out, diazoxide may act via an ETC-independent pathway in smooth muscle cells. Conceivably, diazoxide may activate mitoKATP channels to elicit mitochondrial depolarization, ROS generation, and transient KCa current activation.
Transient and sustained mitochondrial depolarizations occur under resting conditions and in response to stimuli.8,9 In the vasculature and in airway smooth muscle, mitochondrial generation of ROS can occur in response to several stimuli, including flow, temperature, and carbon monoxide, a heme-oxygenase–derived vasodilator.18–20 Data suggest regulation of Ca2+ sparks by mitochondria-derived ROS is an ongoing feedback process that can be up- or downregulated by changes in mitochondrial potential. A small mitochondrial depolarization leads to an increase in ROS generation and Ca2+ spark activation. In contrast, a large mitochondrial depolarization reduces ROS and inhibits Ca2+ sparks, consistent with the concept that under resting conditions, 1% to 2% of O2 used in the ETC is incompletely reduced, leading to O2−· generation.15 ETC blockers reduce ROS in pulmonary artery smooth muscle cells but increase ROS in renal artery smooth muscle cells, effects that may be attributable to different mitochondria in these cell types.9 Data presented here suggest the degree of mitochondrial depolarization also regulates both the magnitude and direction of ROS generation and thus, Ca2+ spark frequency in arterial smooth muscle cells.
Several proteins that regulate Ca2+ sparks and transient KCa currents, including RyR channels, KCa channels, and the SR Ca2+-ATPase, are redox sensitive.21,22 Consistent with our findings, oxidizing agents activate RyR channels.21 In contrast, ROS inhibit bovine and pig vascular smooth muscle SR Ca2+-ATPases.23,24 Oxidizing and reducing agents have been shown to both activate and inhibit smooth muscle KCa channels.25–29 ROS also activate a number of other signal transduction pathways that may modulate RyR and KCa channels, including those mediated by protein kinases.22 At a physiological voltage of −40 mV, mitochondria-derived ROS activated Ca2+ sparks and increased KCa channel sensitivity to Ca2+ sparks, resulting in transient KCa current frequency and amplitude elevations. Ca2+ spark activation occurred in the absence of a change in global Ca2+, suggesting ROS may mediate local communication between mitochondria and RyR channels. Supporting this local signaling concept, mitochondria and the SR are found within 20 nm of each other in smooth muscle cells.30 A large mitochondrial depolarization blocks Ca2+ sparks and transient KCa currents because of permeability transition pore (PTP) opening.4 Data here suggest that a large mitochondrial depolarization also inhibits transient KCa currents by reducing mitochondrial-derived ROS, which explains why PTP blockers only partially attenuate rotenone-induced transient KCa current inhibition.4
Previous studies that investigated the regulation of diazoxide-induced dilations by KCa channel blockers most commonly used vascular rings in which tone was established with a vasoconstrictor (eg, norepinephrine31). In these investigations, KCa channel blockers did not significantly attenuate diazoxide-induced relaxations.31,32 However, vasoconstrictors strongly inhibit Ca2+ sparks, and intravascular pressure activates Ca2+ sparks.13,33,34 In a nonpressurized vascular ring exposed to a vasoconstrictor, Ca2+ spark frequency would be low, and mechanisms that dilate via Ca2+ spark activation would be blunted. In our study, cerebral arteries were pressurized to physiological pressure (60 mm Hg). Under these conditions, Ca2+ sparks occur frequently in smooth muscle cells (≈1 Hz), and Ca2+ spark and transient KCa current activation leads to vasodilation.1,13 Indeed, topical application of ROS dilates cerebral arteries in vivo via KCa channel activation.35 The remaining dilation induced by diazoxide in the presence of MnTMPyP, or Ca2+ spark or KCa channel blockers, is most likely attributable to sarcKATP channel activation, particularly because each blocker had similar effects.
KATP channel openers have been used in a wide variety of therapeutic applications.36 Because of relatively high specificity for pancreatic β-cell plasma membrane KATP channels, diazoxide is used to attenuate excessive insulin secretion but is also used to reduce blood pressure in severe hypertension. Nicorandil, a KATP channel opener and NO donor, is used in angina treatment. Our study reveals a novel vasodilatory pathway activated by KATP channel openers that could be exploited. Mitochondrial potential and transient KCa currents were most sensitive to diazoxide and insensitive to pinacidil, indicating distinct pharmacology when compared with arterial smooth muscle cell sarcKATP channels.12
In summary, this study describes a novel dilatory signaling pathway activated by mitochondria-derived ROS. We show ROS activate Ca2+ sparks and transient KCa currents in arterial smooth muscle cells, leading to vasodilation, and we illustrate that this pathway can be stimulated by KATP channel openers. Data also suggest small and large mitochondrial depolarizations lead to differential regulation of ROS and Ca2+ sparks, indicating the degree to which mitochondria depolarize determines not only signal magnitude, but also direction.
This study was supported by grants from the National Institutes of Health and American Heart Association National Center to J.H.J. Q.X. and S.Y.C. are recipients of American Heart Association Postdoctoral Fellowships. We thank Dr C.W. Leffler for critical reading of the manuscript.
Original received March 24, 2005; resubmission received June 14, 2005; accepted July 6, 2005.
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