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Cellular localization of mitochondria contributes to K v channel-mediated regulation of cellular excitability in pulmonary but not mesenteric circulation

Cellular localization of mitochondria contributes to K v channel-mediated regulation of cellular excitability in pulmonary but not mesenteric circulation. Am J Physiol Lung Cell Mol Physiol 296: L347–L360, 2009. are proposed to be a major oxygen sensor in hypoxic pulmonary vasoconstriction (HPV), a unique response of the pulmonary circulation to low oxygen tension. Mitochondrial factors including reactive oxygen species, cytochrome c , ATP, and magnesium are potent modulators of voltage-gated K (cid:1) (K v ) channels in the plasmalemmal membrane of pulmonary arterial (PA) smooth muscle cells (PASMCs). Mitochondria have also been found close to the plasmalemmal membrane in rabbit main PA smooth muscle sections. Therefore, we hypothesized that differences in mitochondria localization in rat PASMCs and systemic mesenteric arterial smooth muscle cells (MASMCs) may contribute to the divergent oxygen sensitivity in the two different circulations. Cellular localization of mitochondria was compared with immunoﬂuorescent labeling, and differences in functional coupling between mitochondria and K v channels was evaluated with the patch-clamp technique and speciﬁc mitochondrial inhibitors antimycin A (acting at complex III of the mitochondrial electron transport chain) and oligomycin A (which inhibits the ATP synthase). It was found that mitochondria were located signiﬁcantly closer to the plasmalemmal membrane in PASMCs compared with MASMCs. Consistent with these ﬁndings, the effects of the mitochondrial inhibitors on K v current ( I Kv ) were signiﬁcantly more potent in PASMCs than in MASMCs. The cytoskeletal disruptor cytochalasin B (10 (cid:2) M) also altered mitochondrial distribution in PASMCs and signiﬁcantly attenuated the effect of antimycin A on the voltage-dependent parameters of I Kv . These ﬁndings suggest a greater structural and functional coupling between mitochondria and K v channels speciﬁcally in PASMCs, which could contribute to the regulation of PA excitability in HPV.

THE PULMONARY CIRCULATION represents a vascular bed with unique properties. Under physiological conditions this vasculature is a highly oxygenated, low-pressure system. When oxygen tension drops during, for example, chronic obstructive pulmonary disease, a response known as hypoxic pulmonary vasoconstriction (HPV) occurs, diverting the blood flow to the higher oxygenated areas to match ventilation to perfusion. The mechanisms involved in this response are complex and cur-rently incompletely understood (14,48,52). Such responses do not occur in the systemic circulation, which dilates when oxygen tension is reduced to maximize blood supply to all parts of the body.
Smooth muscle cell contraction is critical to vasoconstriction; however, to date no definitive mechanism can account for how these cells sense changes in oxygen tension or how increased contractility occurs. The response is considered to be a complex, multifactorial phenomenon ultimately contributing to elevated intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ), essential for formation of the calcium-calmodulin complex and activation of the myosin light chain kinase. Hypoxic inhibition of voltage-gated K ϩ (K v ) channels, which results in membrane depolarization and voltage-dependent Ca 2ϩ influx in pulmonary arterial (PA) smooth muscle cells (PASMCs), was originally suggested as one of the important factors leading to increased [Ca 2ϩ ] i during HPV (3,36,47,51). Currently, mitochondria, the main consumers of cellular oxygen during respiration, are considered to be one of the putative oxygen sensors in pulmonary smooth muscle cells (18,22,50,53), and it has been postulated that K v channels may be regulated by an effector of mitochondrial origin (3,22). This idea is supported by the observations that 1) inhibitors of the mitochondrial electron transport chain (mETC) such as rotenone and antimycin A, acting at complexes I and III, respectively, and the mitochondrial uncoupler FCCP mimic the effect of hypoxia on K v current (I Kv ) in PASMCs (3,57) and ductus arteriosus (23) and 2) mice lacking the active gp91 phox subunit of the membrane-bound NADPH oxidase preserve the effect of hypoxia on I Kv in PASMCs (5). Numerous products of mitochondrial metabolism have also been shown to activate K v channels, including cytochrome c (34) and hydrogen peroxide in pulmonary and coronary arterial smooth muscle cells (39,40), while reducing agents inhibit K v channels (27,28,38). In addition, we recently reported (9) that that the voltage-dependent characteristics of I Kv in PASMCs are potently modulated via a mitochondrion-mediated Mg 2ϩ -dependent mechanism.
Although products of mitochondrial metabolism are currently established to have effects on K v channels, the exact reasons why these mediators and channels existing in all circulatory beds should behave differently in the pulmonary circulation in response to hypoxia remain unknown. One possibility could be functional differences of the mitochondria present in different vascular beds (22,37). Another possibility could be that the intracellular locality of mitochondria in relation to other organelles and the cellular membrane may vary in the different vascular beds. Using electron microscopy imaging, Vallières et al. (46) demonstrated the presence of mitochondria close to the plasmalemmal membrane in rabbit main PA smooth muscle sections. Such close proximity between mitochondria and the plasmalemmal membrane, if it specifically exists in PASMCs, could be responsible for the opportunistic detection of a mitochondrion-dependent mediator by K v channels.
No systematic comparison of mitochondrial distribution in smooth muscle cells (SMCs) isolated from resistance PAs and systemic arteries has yet been performed. Therefore, the main aim of this study was to investigate the differences in cellular localization of mitochondria in PASMCs and mesenteric arterial SMCs (MASMCs, as an example of systemic circulation) with fluorescence confocal imaging. The functional differences between the inhibition of mitochondria and I Kv characteristics in both cell types were investigated with the patch-clamp technique using the inhibitor of complex III of the mETC antimycin A and the inhibitor of the mitochondrial F 0 /F I ATP synthase oligomycin. These inhibitors were chosen because they have a most pronounced effect on I Kv in rat PASMCs (9).

MATERIALS AND METHODS
Materials. All standard chemicals were purchased from BDH Merck or Fisher. Collagenase type XI and type P were obtained from Sigma and Roche, respectively. Papain, dithiothreitol, mETC inhibitor antimycin A (a mixture of antimycins from Streptomyces species), and ATP synthase inhibitor oligomycin were all obtained from Sigma; cytochalasin B was purchased from Calbiochem. Brefeldin A BODIPY 558/568, MitoTracker Green FM, di-8-ANEPPS, and BAPTA-AM were purchased from Invitrogen.
Cell isolation. Male Wistar rats (225-300 g) were killed by cervical dislocation as approved by the local United Kingdom Home Office inspector, in accordance with Schedule 1 as prescribed and approved by the Home Office and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Pub. No. 85-23, revised 1996). Small intrapulmonary arteries (3rd-5th order) were microdissected. Isolation of PASMCs (using 1 mg/ml type XI collagenase, 0.5 mg/ml papain, and 1 mM dithiothreitol and 20 min incubation at 37°C) were performed as previously described (43). SMCs from small mesenteric arteries (MA) (3rd-4th order) (MASMCs) were isolated in a similar manner except that 2 mg/ml collagenase and 1 mg/ml papain were used and the tissue incubation in the enzyme solution was increased to 30 min.
Confocal microscopy. Small aliquots of the suspension of freshly isolated PASMCs or MASMCs were transferred to the experimental chambers and diluted with a saline buffer composed of (mM) 120 NaCl, 6 KCl, 0.5 CaCl 2, 1.2 MgCl2, 12 glucose, and 10 HEPES, pH adjusted to 7.4 with NaOH. The sarcoplasmic reticulum (SR) in the SMCs was stained by 30-min incubation of the myocytes with 2 M brefeldin A BODIPY 558/568 [absorbance (Abs)/emission (Em) ϭ 559 nm/568 nm], which was previously demonstrated to stain the SR in rat gastric myocytes (54). The validity of this staining was further confirmed in myocytes in which intracellular calcium stores were visualized with the low-affinity [k d(Ca) ϭ 42 M] fluorescent Ca 2ϩ indicator fluo-3FF (12). This approach was demonstrated previously to reveal a well-developed subplasmalemmal SR network in both vascular (11) and visceral (12)  For all desired laser lines the illumination intensity was set with an acoustooptical tunable filter. Averaging of two frames was used to reduce noise. The gains were such that the red and green fluorescent signals were approximately of the same intensity but below the saturation of the photomultiplier. To avoid any bleed-through, imaging was performed with the multitrack mode of an LSM 510 (sequential capturing of images with 2 separate photomultipliers). A threshold was set to remove subsignal noise from the images. The adequacy of the imaging protocol applied to the doublelabeled myocytes was confirmed by control experiments on the single-labeled cells. It is worth noting that, although the z-sectioning was performed through the entire cell volume, the distribution of the SR and mitochondria was analyzed only in x-y images taken from the half of each SMC located closer to the objective lens. This shortens the length of the optical path and thus minimizes erroneous measurements caused by waveguide dispersion of light, which occurs when the wave propagates through any inhomogeneous structure such as a cell.
Electrophysiology. Cells were placed in a chamber with a volume of 100 -200 l and continually superfused (ϳ1 ml/min) with a physiological saline solution (PSS) or a test solution via a five-barrel pipette at room temperature. PSS contained (mM) 140 NaCl, 5 KCl, 1.5 CaCl 2, 1.2 MgCl2, 10 HEPES, and 10 glucose, pH ϭ 7.2. Control pipette solution contained (mM) 140 KCl, 0.5 MgCl 2, 10 HEPES, 10 EGTA, and 0.5 CaCl 2, pH ϭ 7.2, and was used for recording unless otherwise stated. Cells were dialyzed with pipette solution for 5 min before currents were recorded. The effects of inhibitors were studied a minimum of 5 min after addition. Paxilline (1 M) and glibenclamide (10 M) to block Ca 2ϩ -activated and ATP-sensitive K ϩ currents, respectively, were present in all extracellular recording solutions, isolating the voltage-dependent K ϩ channels from other K ϩ conductances as previously described (43). To evaluate cell size, whole cell capacitance transient currents were measured with a 10-mV hyperpolarizing step after stable whole cell access was achieved in each cell. Cell membrane capacitance (C m) was then calculated as an area under a capacitance transient and expressed in picofarads.
I Kv amplitude was measured with a 200-ms voltage step applied from the holding potential of Ϫ80 mV to membrane potentials between Ϫ100 and ϩ50 mV in 10-mV increments with a frequency of 0.1 Hz. After membrane depolarization cells were repolarized to Ϫ20 (PASMCs) or 0 (MASMCs) mV for 160 ms to measure tail currents, which were used to assess changes in the steady-state activation of IKv. A more positive step potential in MASMCs was required to increase the size of tail currents because of the significantly smaller current amplitude in these cells. Current-voltage (I-V) curves were constructed from the tail current measures 2-3 ms after repolarization and fitted with the following equation: where Va and ka are the half-activation potential and the e-fold steepness of the activation dependence (the slope factor of activation), respectively, and Vm is membrane potential. IKv block was calculated as a percentage of the current at ϩ50 mV in control conditions from the same cell.
I Kv inactivation was measured with a two-pulse protocol consisting of a 10-s conditioning pulse stepping in 10-mV increments from Ϫ100 to ϩ40 mV, followed by a test potential to ϩ60 mV for 200 ms. Current at the end of the test pulse was measured, normalized to the maximal current recorded, and plotted versus conditioning potential. The resulting I-V curve was fitted to the following equation: where Vh and kh are the half inactivation potential and the e-fold steepness of the inactivation dependence (the slope factor of inactivation), respectively, and A denotes the noninactivating component of the current. Data analysis and statistics. Data were analyzed and presented with pCLAMP 8 (Axon Instruments), Microsoft Excel, and Microcal Origin 6.0 software. Data are expressed as means Ϯ SE. Statistical comparisons were performed with a paired or unpaired two-tailed t-test, with P Ͻ 0.05 deemed significant unless stated otherwise. When normality test failed (Shapiro-Wilk test) the Wilcoxon signed rank test and the Mann-Whitney test were used for comparison of paired and unpaired data, respectively. Paired statistical tests were used to compare the difference in parameters obtained in the control condition and in the presence of mitochondrial inhibitor in the same cell, whereas unpaired tests were used to compare differences between two groups of cells (e.g., PASMCs vs. MASMCs).

Differences in spatial distribution of mitochondria in
PASMCs and MASMCs. The distribution of mitochondria [stained with MitoTracker Green FM (24)] in relation to the SR [stained with brefeldin A BODIPY 558/568 (54)] was compared in SMCs isolated from rat small PAs and MAs. The association between mitochondria and SR elements was assessed with the confocal z-sectioning protocol described in MATERIALS AND METHODS. This approach revealed that in PASMCs, on movement from the cell bottom toward the middle of the myocyte depth (Fig. 1A), the fluorescent signal from MitoTracker Green appeared in the x-y confocal optical slices (Fig. 1C) before that of brefeldin A BODIPY (Fig. 1B). In PASMCs, in individual x-z and y-z optical cross sections of the myocyte MitoTracker Green fluorescence (mitochondria) was detected distinctly closer to the cell periphery than that of brefeldin A BODIPY (SR) (Fig. 1, D and E). In MASMCs, however, brefeldin A BODIPY fluorescence staining the SR appeared before or simultaneously with MitoTracker Green fluorescence (Fig. 2B). Such opposing juxtaposition between the SR and mitochondria in MASMCs compared with PASMCs becomes more evident from the comparison of individual x-z and y-z optical cross sections of the myocyte Mito- To quantify these differences, the total area occupied by pixels showing MitoTracker Green and brefeldin A BODIPY fluorescence was analyzed in four PASMC and MASMCs and plotted against the optical slice number in Fig. 3. This analysis clearly shows the existence of an area close to the surface of PASMCs that is enriched with mitochondria (proportional to the area with MitoTracker Green fluorescence), while in MASMCs a similar area is predominantly occupied with the SR (proportional to the area with brefeldin A BODIPY fluorescence). Figure 3 also shows diagrammatic representations of these areas, comparing PASMCs with MASMCs.
The existence of the subplasmalemmal pool of mitochondria in PASMCs, but not in MASMCs, was further studied by double staining of both types of cells with MitoTracker Green and di-8-ANEPPS, selective fluorescent probes for mitochondria and the plasmalemmal membrane, respectively (Fig. 4). Figure 4A shows x-y confocal images taken though the middle of a representative PASMC. As can be seen clearly in Fig. 4A, MitoTracker Green fluorescence often appeared to be "fused" (within ϳ200 nm, the limit of lateral resolution of our confocal microscope) with di-8-ANEPPS fluorescence shown in red (plasmalemma). Conversely, in MASMCs, a clear gap between the two fluorescent signals was observed (Fig. 4B). To quantitatively compare relative distances between mitochondria and the plasma membrane in the two cell types, the spatial profiles of fluorescent signals recorded for di-8-ANEPPS (red) and MitoTracker Green (green) were constructed across the shortest distance between the center of each mitochondrion and the plasma membrane (shown by yellow lines in Fig. 4, C and D, for PASMC and MASMC, respectively). Histograms of distribution of mitochondria according to their distance from the plasma membrane measured in six PASMCs and six MASMCs are compared in Fig. 4E. The histogram bin size was set to 0.5 m, which is 2.5-fold greater than the lateral optical resolution of our imaging system determined with 0.2-m fluorescent beads. This analysis clearly shows that in PASMCs the majority of peripheral mitochondria are localized between 0.5 and 1.5 m from the plasma membrane, compared with ϳ1.5-3.5 m in MASMCs. On average, the mean distance between peripheral mitochondria and the plasma membrane calculated as described above was twofold greater in MASMCs (2.4 Ϯ 0.05 m, n ϭ 405) than in PASMCs (1.2 Ϯ 0.05 m, n ϭ 235) (P Ͻ 0.0001; Fig. 4F).
Comparison of I Kv characteristics in PASMCs and MASMCs. In view of the locality of the mitochondria to the plasma membrane demonstrated above, it could be postulated that the mitochondrion-mediated effects on K v channels that occur in PASMCs may exist in MASMCs but would be less effective. Consequently, I Kv was isolated pharmacologically (as described in MATERIALS AND METHODS), and its electrophysiological properties were compared in PASMCs and MASMCs under identical experimental conditions.
With the I Kv activation protocol featured in Fig. 5A, whole cell I Kv was recorded in both PASMCs and MASMCs. The amplitude of the current recorded from a representative MASMC (Fig. 5C) was considerably smaller than in a PASMC (Fig. 5B). On average, the differences in I Kv amplitude were significantly different between Ϫ40 and ϩ50 mV. For example, at 0 and ϩ50 mV, the mean amplitude of I Kv was equal to 69 Ϯ 5 and 270 Ϯ 17 pA (n ϭ 60), respectively, in MASMCs and 446 Ϯ 20 and 1,369 Ϯ 59 pA (n ϭ 184), respectively, in PASMCs (P Ͻ 0.0001). On the other hand, the size of MASMCs, calculated from C m , was significantly larger than that of PASMCs [16.3 Ϯ 0.6 pF (n ϭ 60) vs. 11.1 Ϯ 0.3 pF (n ϭ 184); P Ͻ 0.0001]. The smaller I Kv and the larger cell size in MASMCs result in significant differences in the whole cell current density in the two cell types (Fig. 5D), which was 10- to 18-fold greater between Ϫ30 and 0 mV and 8-to 9-fold greater between 0 and ϩ50 mV in PASMCs compared with MASMCs.
Comparison of the steady-state activation of I Kv measured from the normalized I-V curves (which were derived from the I Kv tail current measurements) from the representative PASMC and MASMC shown in Fig. 5, B and C, indicates that I Kv activation in MASMC was shifted by ϳ6 mV to more positive membrane voltages and was steeper than in PASMC (Fig. 5E). These differences in the activation parameters were significant ( Table 1).
Comparison of I Kv inactivation measured with the voltage protocol shown in Fig. 6A from the representative PASMC (Fig. 6B) and MASMC (Fig. 6C) demonstrates that I Kv inactivation in PASMCs is relatively slow and incomplete compared with that in MASMCs. The normalized I Kv recorded during the test pulse was plotted against the prepulse potential, and resulting dependencies were fitted to the equation described in MATERIALS AND METHODS, yielding the parameters of I Kv inactivation, the half-inactivation potential V h , the slope factor k h , and the noninactivating component A (Fig. 6D). Statistical comparison of these parameters showed that I Kv in MASMCs is inactivated at more negative membrane potentials, its dependence on the conditional prepulse potential is steeper, and it has less noninactivated current than I Kv in PASMCs; these differences in inactivation characteristics were significant (Table 1).
Therefore, direct comparison of the steady-state activation and inactivation of I Kv measured under identical experimental conditions in both cell types clearly demonstrates that PASMCs have significantly greater current amplitude and more available and less inactivated K v channels in the negative range of membrane potentials compared with MASMCs.
Mitochondrial inhibition regulates I Kv more potently in PASMCs. Inhibition of the mETC was previously shown to inhibit I Kv in PASMCs (3,23,57). We recently showed (9) that steady-state activation and I Kv block were significantly affected by mitochondrial inhibition in PASMCs, with the most pronounced effects observed for antimycin A (inhibiting the mETC at complex III) and oligomycin (blocking the ATP synthase). These two mETC inhibitors therefore were chosen to verify whether functional coupling between mitochondria and K v channels in PASMCs is greater than in MASMCs, as would be predicted based on the difference in the distribution of mitochondria in the two cell types as demonstrated in Figs. 1-4.
The different sites of action of antimycin A and oligomycin in mitochondria are schematically depicted in Fig. 7A. The differences in the mean parameters for I Kv activation and inactivation due to mitochondrial inhibition with antimycin A and oligomycin in PASMCs and MASMCs are comprehensively analyzed in Table 2, while Fig. 7,B-D, compares the relative changes in I Kv activation, inactivation, and block in PASMCs to those in MASMCs. Although both inhibitors caused significant negative shifts in I Kv inactivation (Fig. 7C) and significant decreases in I Kv amplitude at ϩ50 mV (Fig. 7D) in both cell types, all these effects were significantly smaller in MASMCs than in PASMCs (Fig. 7, B-D). Similarly, antimycin A had a significantly greater effect in PASMCs than in MASMCs (Fig. 7B). Oligomycin, on the other hand, caused significant differences in I Kv activation only in PASMCs and not in MASMCs (Fig. 7B and Table 2). Furthermore, inhibitorinduced changes in other parameters, such as k a , k h , and A, were less pronounced and overall not significantly different in MASMCs from those in PASMCs (Table 2). Thus differences in potency of the effects of the two different mitochondrial inhibitors in two cell types correlate with the difference in mitochondrial distribution described above.
Disruption of cytoskeleton with cytochalasin B alters both peripheral mitochondria distribution and antimycin A-dependent modulation of I Kv . A link between mitochondria and the cytoskeleton has been demonstrated previously in cardiomyocytes (2), neurons (25), human coronary arterioles (19), and pulmonary endothelial cells (1) but not in PASMCs. If the cytoskeleton is involved in juxtaposition of mitochondria and the plasma membrane in PASMCs, then disruption of the cytoskeleton should alter the association between mitochondria and the plasma membrane and at the same time attenuate the effect of the mitochondrial inhibitors on I Kv . The effect of treatment of PASMCs with 10 M cytochalasin B, a disrupter of F-actin microfilaments (1,17,19), on distribution of mitochondria was visualized with MitoTracker Green and confocal imaging. Figure 8, A and B, compare distribution of mitochondria (shown in green) in relation to the plasma membrane (stained with di-8-ANEPPS and shown in red) in a single PASMC before and 10 min after incubation with cytochalasin B, respectively. To reduce possible errors resulting from changes in the cell shape due to a disruption of the cytoskeleton, partially contracted PASMCs were used in these experiments. As can be seen in Fig. 8, cytochalasin B did not significantly change the cell shape; however, the pattern of green fluorescence across the whole cell was altered, suggesting that the mitochondria are associated with F-actin filaments in PASMCs. Importantly, the distribution of mitochondria at the submembrane regions was altered, as can be seen at an increased magnification in Fig. 8, A and B, bottom. Similar effects were observed in the other eight PASMCs studied.
The above results suggest that if K v channels in the plasma membrane and peripheral mitochondria are functionally coupled in PASMCs as the electrophysiological evidence indicates ( Fig. 7; Ref. 9), then the cell treatment with cytochalasin B should also modulate the effect of the mitochondrial inhibitors on I Kv characteristics. To test this prediction, the effect of 10 M cytochalasin B on antimycin A-induced changes in I Kv activation, inactivation, and block was assessed. Cytochalasin B caused a decrease in I Kv amplitude by 35.7 Ϯ 6.1% (n ϭ 5, P Ͻ 0.01) but itself did not have any significant effect on the steady-state activation or inactivation parameters of I Kv (Table 3). Importantly, the effect of antimycin A on I Kv activation was completely abolished (Fig. 8C) and antimycin A-induced changes in I Kv inactivation were significantly attenuated (Fig.  8D) in the presence of cytochalasin B. Thus the ability of cytochalasin B to inhibit the antimycin A-induced changes in the voltage-dependent I Kv characteristics correlates well with cytochalasin B-mediated changes in mitochondrial distribution in the vicinity of the plasma membrane, providing direct evidence for close functional coupling between K v channels and peripheral mitochondria in PASMCs. Pretreatment of cells with the cytoskeletal disruptor did not, however, significantly change the antimycin A-mediated inhibition of I Kv (Fig. 8E), suggesting that other factors are likely to contribute to the I Kv block caused by the mitochondrial inhibitors.

Intracellular Mg 2ϩ is involved in antimycin A-mediated changes in I Kv inactivation.
We recently demonstrated (9) that dialysis of PASMCs with Na 2 ATP and EDTA, which are more potent chelators of Mg 2ϩ than Ca 2ϩ , compared with EGTA   Considering that the effect of the mitochondrial inhibitors on the steady-state inactivation of I Kv has not been studied previously, it is important to demonstrate whether changes in I Kv inactivation induced by the mitochondrial inhibitors are also mediated by intracellular Mg 2ϩ . To test this possibility, the effect of antimycin A and oligomycin was studied in cells dialyzed with pipette solutions containing 5 mM EDTA or 5 mM Na 2 ATP, respectively. Figure 9, A and B, clearly demonstrate that the relative changes in V h caused by both inhibitors were significantly reduced under these conditions. We previously found (9) that cell dialysis with increased Mg 2ϩ concentration also mimicked the effects of the mitochondrial inhibitors on I Kv activation and block. When the steady-state inactivation of I Kv was measured in cells dialyzed with 5 mM instead of 0.5 mM MgCl 2 in the pipette solution but in the absence of mitochondrial inhibitors, a significant leftward shift by ϳ7 mV in the inactivation dependence was observed. The mean V h was changed from Ϫ23.9 Ϯ 1.1 mV for 0.5 mM MgCl 2 (n ϭ 73) to Ϫ30.7 Ϯ 2.8 mV for 5 mM MgCl 2 (n ϭ 12, P Ͻ 0.026). The noninactivating component A of I Kv was also decreased under this condition, from 0.28 Ϯ 0.01 (0.5 mM) to 0.21 Ϯ 0.02 (5 mM) (P Ͻ 0.03). No significant changes in k h were observed [11.2 Ϯ 0.4 (n ϭ 72) in 0.5 mM MgCl 2 vs. 10.4 Ϯ 0.5 mV (n ϭ 12) in 5 mM MgCl 2 ]. Furthermore, antimycin A-induced changes in V h were significantly attenuated (Fig. 9C), whereas changes in k h and A remained similar (compare Tables 2 and 4) under these conditions.

Subplasmalemmal localization of mitochondria provides structural basis for functional interaction with K v channels in
PASMCs. Although the amount of intracellular diffusible mediators in close vicinity to the K v channel cannot be precisely quantified, the overall local cellular changes could be substantial, providing that diffusion processes in the vicinity of the K v channels are limited. The existence of a submembrane compartment formed by the SR and called the "superficial barrier" has been previously proposed in systemic vascular SMCs [recently reviewed by Poburko et al. (35)]. Indeed, a tight juxtaposition between the plasmalemmal membrane and the SR has been directly visualized in vascular (11) and visceral (12) SMCs. In myocytes isolated from intralobar rat PAs, we previously demonstrated (42) the presence of a restricted diffusion space by measuring the changes in the K ϩ gradient during the development of I Kv . This phenomenon has not been reported in other vascular SMCs. Comparison of the relative distribution of mitochondria and the SR in PASMCs and MASMCs clearly demonstrates the presence of a distinctive population of mitochondria close to the plasmalemmal membrane and relatively separate from the SR in PASMCs but not in MASMCs, where mitochondria are more closely associated with the SR (Figs. 1-4). This novel and important observation demonstrates the presence of a subplasmalemmal pool of mitochondria in PASMCs that are potentially functionally associated with the K v channels, as our electrophysiological evidence shows. Such compartmentalization, similar to that formed by the SR in other types of vascular SMCs (35), could be primarily responsible for the lack of sensitivity of I Kv to hypoxia in mesenteric arteries previously reported by others (31,56,58). Notably, close association between the plasma membrane and mitochondrion has previously been shown by electron microscopy (46). Although the full physiological significance of the existence of the distinctive subplasmalemmal population of mitochondria in PASMCs remains to be established, it is possible that a close juxtaposition of the plasma membrane and mitochondria in PASMCs might be important in polarization of mitochondrion-mediated signals [e.g., reactive oxygen species (ROS) release] in a similar manner to the "mitochondrial belt" involvement in cytosolic Ca 2ϩ signal polarization in pancreatic acinar cells (45).
The ability of the F-actin filament disrupter cytochalasin B to alter mitochondrial distribution in PASMCs suggests that the cytoskeleton contributes to cellular organization of mitochondria in PASMCs. Whether mitochondria are similarly associated with the cytoskeleton in MASMCs remains to be established. Interestingly, inhibition of F-actin filament polymerization with cytochalasin suppressed mitochondrial ROS production induced by stretch in bovine pulmonary endothelial cells (1) and by flow in human coronary arterioles (19). Whether the cytoskeleton is involved in the generation of ROS in PASMCs has not been yet investigated. Our data clearly suggest that in PASMCs mitochondria are spatially arranged in the distinctive layer separate from the SR, forming a compartment with re-  A: diagrammatic representation of sites of action of antimycin A and oligomycin in mitochondrial membrane. B and C: comparison of antimycin A-and oligomycin-induced changes in half-activation and half-inactivation potentials. Relative changes in half-activation and half-inactivation potential in 8 and 12 (B) and 8 and 11 (C) PASMCs and in 11 and 9 (B) and 9 and 9 (C) MASMCs for antimycin A and oligomycin, respectively, are shown. D: comparison of decrease in IKv amplitude in the presence of antimycin A or oligomycin for PASMCs (n ϭ 9 and 12, respectively) and MASMCs (n ϭ 11 and 9, respectively). *P Ͻ 0.05, **P Ͻ 0.01, ***P Ͻ 0.001, difference in parameters between control condition and presence of an inhibitor by paired statistical test. #P Ͻ 0.05, ##P Ͻ 0.01, ###P Ͻ 0.001, difference between 2 cell types by unpaired statistical test. stricted diffusion in the subplasmalemmal space, thus providing a structural basis for a better functional coupling between mitochondria and K v channels in PASMCs than in systemic arterial myocytes. The correlation between the disruption of the distribution of peripheral mitochondria by cytochalasin B and significant attenuation of the effects of antimycin A on I Kv activation and inactivation directly supports this conclusion.

Mechanisms involved in K v channel regulation by mitochondria in PASMCs.
The electrophysiological evidence presented in Fig. 9, demonstrating that addition of Mg 2ϩ -chelating agents into the pipette solution inhibits changes in V h caused by antimycin A and oligomycin, clearly suggests that an increase in intracellular concentration of Mg 2ϩ plays a key role in the modulation of I Kv inactivation by mitochondrial inhibitors. The ability of the increased pipette Mg 2ϩ concentration first to mimic and then to attenuate the effect of antimycin A (Fig. 9C) also supports this notion. We previously demonstrated (9) that similar pipette interventions significantly attenuated changes in the I Kv activation and I Kv block caused by antimycin A and oligomycin in PASMCs, thus suggesting that changes in mitochondrion-mediated changes in intracellular Mg 2ϩ concentration represent one of the major mechanisms controlling the functional activity of K v channels. On the other hand, the failure of cytochalasin B to attenuate I Kv block, while inhibiting changes in I Kv activation and inactivation (Fig. 8, C-E), suggests the presence of other factors (e.g., ROS) that may influence the ability of Mg 2ϩ to interact with K v channels. The identification of these factors and the establishment of their importance in mitochondrion-mediated and Mg 2ϩ -dependent modulation of I Kv in PASMCs in normoxic and hypoxic conditions will entail further experimental work.
Stronger functional coupling exists between mitochondria and K v currents in PASMCs than in MASMCs. To analyze the differences in functional coupling between mitochondria and K v channels in PASMCs and MASMCs, the inhibitors of complex III (antimycin A) and of the ATP synthase (oligomycin) were used because, as we previously reported (9), their effects on I Kv activation and I Kv block in PASMCs are substantially stronger compared with other mETC inhibitors (rotenone, myxothiazol, and cyanide) and the mitochondrial uncoupler CCCP. Comparison of the effects of antimycin A and oligomycin on I Kv activation and I Kv block in the two cell types clearly demonstrated significantly lesser effects in MASMCs than in PASMCs (Fig. 7, B and D). Notably, the effect of oligomycin on I Kv activation was not significant in MASMCs. In addition, we also compared the effect of the two inhibitors on I Kv inactivation, which represents another important voltage-dependent characteristic of K v channels that, together with the steady-state activation, determines the availability of K v channels at membrane potentials negative to 0 mV (26). Similar to I Kv activation and I Kv block, changes in I Kv activation induced by both inhibitors were also significantly smaller in MASMCs than in PASMCs (Fig. 7C), as would be predicted from the different distribution of mitochondria in PASMCs and MASMCs.
The tighter spatial and functional coupling between K v channels and mitochondria, one of the proposed oxygen sensors (18,49), in PASMCs can contribute to the difference in sensitivity of I Kv to hypoxia in the pulmonary and systemic  arterial SMC reported previously (22,31,56,58). This could be further enhanced by the difference in the functional activity of mitochondria. Thus Michelakis et al. (22) demonstrated a significantly greater rate in basal production of mitochondrial ROS in PASMCs than in renal arterial SMCs, while Rathore et al. (37) found that mitochondrial ROS were selectively increased by hypoxia in PASMCs but not in MASMCs. It is currently difficult to directly measure changes in the concentration of mitochondrial ROS or other mitochondrial metabolites such as ATP, cytochrome c, or magnesium ions [all of which modulate the activity of K v channels in PASMCs (3,8,9,34,38,59)] in submembrane compartments in intact cells. In this instance, ion channel characteristics, which can be measured, quantified, and even calibrated with different intracellular solutions, can serve as useful markers. Indeed, ATPsensitive K ϩ (K ATP ) channels and Ca 2ϩ -sensitive K ϩ (BK Ca ) channels have been studied to assess changes in ATP concentration and [Ca 2ϩ ] i , respectively, directly beneath the plasma membrane (13,44). Similarly, as our results demonstrate, voltage-dependent parameters of I Kv , a dominant K ϩ current in PASMCs, can serve as useful markers of changes in the confined subplasmalemmal environment in close vicinity to the K v channel in PASMCs.
Distinct differences in K v channel activity between PASMCs and MASMCs. To investigate mechanisms of HPV, which are specific to the pulmonary circulation, MAs are often used as a negative control. Although the voltage-dependent characteristics of I Kv , such as steady-state activation and inactivation, have been reported previously in rat PASMCs (27,43) and MASMCs (15,21,30), no direct comparison between the two cell types has been performed under identical experimental conditions to determine whether there is a significant difference in I Kv properties in the two cell types. Also, in this study, I Kv was isolated in both PASMCs and MASMCs by elimination of BK Ca and K ATP currents with paxilline and glibenclamide, respectively. In other studies a low extracellular Ca 2ϩ concentration (15,21) and a high concentration of ATP in the pipette (15,21,30), respectively, were used in MASMCs for this purpose. It is noteworthy that the amplitude and the parameters for activation and inactivation of I Kv measured under our experimental conditions in MASMCs were not dissimilar to those reported by others. For example, the mean midpoints for I Kv activation and inactivation of Ϫ10 and Ϫ39.4 mV (30), Ϫ11 and Ϫ40 mV (21), and Ϫ3.5 and Ϫ33.9 mV (15) are comparable with the Ϫ9.4 and Ϫ37 mV, respectively, we report in the present study (Table 1), suggesting that that our experimental conditions and pharmacological agents used for current isolation do not have a substantial effect on the voltage-dependent properties of I Kv in MASMCs.
Comparison of the I Kv amplitude, cell size, and steady-state activation and inactivation characteristics of the current measured in identical experimental conditions revealed significant differences in all three groups of characteristics in PASMCs and MASMCs. The most significant differences were found in the whole cell current amplitude (larger in PASMCs) and the cell size (larger for MASMCs), giving between 8-and 18-fold differences in the range of membrane potential between Ϫ30 and ϩ50 mV of cells with PASMCs. Also, the midpoint for I Kv inactivation was significantly shifted to more negative potentials by ϳ13 mV in MASMCs compared with PASMCs. Although a relatively small shift to more positive membrane potentials (ϳ4 mV) was seen in the midpoint for I Kv activation, the effect was nevertheless significant. Finally, the differences in the slope factors for both activation and inactivation dependencies were also significantly different between PASMCs and MASMCs.
Since native K v channels are believed to be heteromultimers that are formed by more than one K v 1.x ␣-subunit in both PASMCs (6,7,33,60) and MASMCs (10,20,30,55), the differences in the voltage-dependent characteristics may be due to a different heteromultimeric assembly of the K v channels in the two cell types. In addition, differences in levels of expression or isoform of accessory K v ␤-subunits, found in both cell types (7,10,30,33,60), could also contribute to the differences in I Kv parameters we observed between two cell types. Also, the presence of functional K v channels formed by the K v 2.1 ␣-subunit, a member of another K v channel family, cannot be ruled out (6,10,20,29,60). Indeed, between two and four small-amplitude single-channel K v currents were found in rat (60), human (32), and mouse (16) PASMCs. Although heterogeneity in distribution of different K v currents was also observed in different PASMCs (4,43), the presence of multiple voltage-dependent K v components in a single cell was not obvious because of the homogeneity of the activation and inactivation dependencies (Figs. 5 and 6). It is possible that K v channel heterogeneity observed at the single-channel level may contribute to some variability in the voltage-dependent parameters for I Kv between cells.
It is worth mentioning that the 2-to 3-fold difference in single-channel conductance for the delayed currents encoded by K v 1.x and K v 2.x ␣-subunits is unlikely to explain the at least 10-fold increase in the whole cell I Kv density between Ϫ40 and 0 mV in PASMCs compared with MASMCs. Similarly, the differences in the I Kv activation and inactivation parameters alone between the two cell types could only predict a 2.6-to 3-fold increase in the steady-state open probability of K v channels available in the same voltage range (calculated with the mean values for I Kv activation and inactivation parameters listed in Table 1 and the theoretical model described in Ref. 26). It is therefore likely that the number of functional K v channels expressed per unit of the cell membrane area should also be greater in PASMCs than in MASMCs.
In conclusion, our findings demonstrate the presence of a distinctive layer of mitochondria at the cell periphery in PASMCs, forming a restricted diffusion cytosolic space between the mitochondria and plasmalemmal membrane and thus providing a structural basis for a close functional interaction between the K v channels and mitochondria as supported by the electrophysiological evidence. The smaller density of K v channels in MASMCs alongside the greater diffusion space between the mitochondria and the cell membrane may provide one plausible explanation for the differences in cellular responses during hypoxia.