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First published online 11 December 2007
doi: 10.1242/jcs.014522

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The mitochondrial membrane potential and Ca²⁺ oscillations in smooth muscle

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, John Arbuthnott Building, 27 Taylor Street, Glasgow, G4 0NR, UK

^* Author for correspondence (e-mail: J.McCarron{at}strath.ac.uk)

Accepted 16 October 2007

	Summary

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Ca²⁺ uptake by mitochondria might both modulate the cytosolic Ca²⁺ concentration ([Ca²⁺]_c) and depolarize the mitochondrial membrane potential (_m) to limit ATP production. To investigate how physiological Ca²⁺ signaling might affect energy production, _m was examined during Ca²⁺ oscillations in smooth muscle cells. In single, voltage-clamped smooth muscle cells, inhibition of mitochondrial Ca²⁺ accumulation inhibited inositol (1,4,5)-trisphosphate [Ins(1,4,5)P₃]-evoked Ca²⁺ release and prolonged the time required for restoration of [Ca²⁺]_c following activation of plasmalemmal Ca²⁺ currents (I_Ca). Ca²⁺ could be released from mitochondria immediately (within 15 seconds) after a [Ca²⁺]_c rise evoked by Ins(1,4,5)P₃ or I_Ca. Despite this evidence of mitochondrial Ca²⁺ accumulation, no change in _m was observed during single or repetitive [Ca²⁺]_c oscillations evoked by these conditions. Occasionally, spontaneous, repetitive, persistent Ca²⁺ oscillations were observed. In these cases, mitochondria displayed stochastic _m depolarizations, which were independent both of events in neighboring mitochondria and of the timing of the [Ca²⁺]_c oscillations themselves. Such _m depolarizations could be mimicked by increased exposure to either fluorescence excitation light or the _m-sensitive dye tetramethylrhodamine ethyl ester (TMRE) and were inhibited by antioxidants (ascorbic acid, catalase, Trolox and TEMPO) or the mitochondrial permeability transition pore (mPTP)-inhibitor cyclosporin A (CsA). Individual mitochondria within smooth muscle cells might depolarize during repetitive Ca²⁺ oscillations or during oxidative stress but not during the course of single [Ca²⁺]_c transients evoked by Ca²⁺ influx or store release.

Key words: Smooth muscle, Calcium, Mitochondria, Membrane potential

	Introduction

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Changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_c) provide signals to control crucial events such as muscle contraction, neurotransmitter release, alterations in gene transcription and cell death (Berridge et al., 2003; Bootman et al., 2001; Orrenius et al., 2003). Ca²⁺ signals, physiological and pathological, are modulated by the activity of mitochondria (Berridge, 2002; Hajnoczky et al., 2000; Jacobson and Duchen, 2004). These organelles accumulate Ca²⁺ via an electrogenic uniporter when [Ca²⁺]_c exceeds 100 nM, using the potential, _m, generated across the inner mitochondrial membrane by the activity of the electron-transport chain (Becker et al., 1980; Nicholls and Crompton, 1980). Mitochondria release Ca²⁺ more slowly back to the cytosol via a Na⁺- or H⁺-antiporter (Crompton et al., 1978; Rottenberg and Marbach, 1990). In this way, mitochondria act as localized [Ca²⁺]_c buffering units and might modulate Ca²⁺ feedback-inhibition or activation mechanisms (Collins et al., 2000; Hajnoczky et al., 1999; Mackenzie et al., 2004), For example, mitochondrial Ca²⁺ accumulation might determine whether or not a localized Ca²⁺ signaling event, such as a `puff' (Parker and Ivorra, 1990) or `spark' (Cheng et al., 1993), proceeds to a global [Ca²⁺]_c oscillation or wave.

Mitochondria, being the site of aerobic ATP production, also affect cellular signaling by modulating the cytosolic ATP:ADP ratio (Nicholls and Ferguson, 2002). ATP production is itself stimulated by the uptake of Ca²⁺. The activity of the mitochondrially located citric acid (Krebs)-cycle enzymes pyruvate dehydrogenase, oxoglutarate dehydrogenase and NAD⁺-isocitrate dehydrogenase (McCormack and Denton, 1980) as well as that of the F₁F₀-ATP synthase are stimulated by Ca²⁺ (Territo et al., 2000). Conversely, a decrease in _m, which provides the driving force for the ATP synthase, inhibits ATP production (Leyssens et al., 1996). Uptake of positively charged Ca²⁺ ions should depolarize _m to some extent and so might restrict ATP production. Therefore, Ca²⁺ signals might limit cellular energy production.

In some cells, mitochondria are located close to sites of initiation of [Ca²⁺]_c signals, such as at voltage-gated Ca²⁺ channels on the plasmalemma (Barstow et al., 2004) or at inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] receptors [Ins(1,4,5)P₃Rs] on the endoplasmic/sarcoplasmic reticulum (ER/SR) (Csordas and Hajnoczky, 2003; Hajnoczky et al., 1999; Rizzuto et al., 1998). Indeed, mitochondria might be tethered to be within 10 nm of Ins(1,4,5)P₃Rs (Csordas et al., 2006). At these locations, mitochondria are exposed to a local [Ca²⁺]_c that exceeds bulk average values (Rizzuto et al., 1998), and hence might be expected to both have a greater influence over the [Ca²⁺]_c signal itself (because of their low affinity for Ca²⁺) and, in turn, be more likely to exhibit a decrease in _m due to the larger mitochondrial Ca²⁺ current generated during these local [Ca²⁺]_c transients.

Although transient decreases in _m have been observed in isolated mitochondria that have been exposed to sudden increases in [Ca²⁺] (Brustovetsky and Dubinsky, 2000; Huser et al., 1998), it is less clear whether or not this occurs during [Ca²⁺]_c signaling in intact cells. Transient, or `flickering', _m changes have been observed in some (Buckman and Reynolds, 2001; Drummond et al., 2000; Duchen et al., 1998; Haak et al., 2002; Jacobson and Duchen, 2002; Kaftan et al., 2000; O'Reilly et al., 2004; Petronilli et al., 2001) but not all (Collins et al., 2001; Csordas and Hajnoczky, 2003) intact cell types, although their relationship to [Ca²⁺]_c changes is uncertain. Whether or not transient _m depolarizations are seen during cellular Ca²⁺ signals might depend on the cell type, the origin and the characteristics of the Ca²⁺ signal. For example, inhibition of Ca²⁺ release from the SR inhibits transient _m depolarizations in some circumstances (Jacobson and Duchen, 2002) but not in others (O'Reilly et al., 2004).

Transient _m depolarizations have also been attributed to the increased levels of oxidative species generated from a combination of intense excitation light and high concentrations of certain mitochondrial dyes (Szado et al., 2003), rather than to changes in [Ca²⁺]_c. In yet other studies, neither the intensity of the excitation light nor the concentration of mitochondrial dyes contributed to the generation of transient _m depolarizations (Buckman and Reynolds, 2001; O'Reilly et al., 2004; Vergun et al., 2003). Evidently, the mechanism(s) underlying the generation of _m depolarizations and, in particular, whether or not Ca²⁺ signals arising from either influx or release modulate _m is unclear.

Cellular Ca²⁺ signaling imposes an energy burden on the cell (in order to rebalance resting ion concentrations) and paradoxically also impedes the production of ATP by mitochondria if Ca²⁺ signals result in _m depolarization. The question of whether or not mitochondrial Ca²⁺ uptake depolarizes mitochondria was addressed in the present study. To examine how mitochondria affect Ca²⁺ signals and whether or not changes in _m occur, _m was measured during [Ca²⁺]_c transients evoked by Ca²⁺ influx across the plasmalemma and following Ca²⁺ release from the SR by localized photolysis of caged Ins(1,4,5)P₃, in voltage-clamped single smooth muscle cells. Voltage clamp was used because changes in plasmalemmal potential might compromise the ability to resolve mitochondrial _m changes (Ward et al., 2000). The use of caged Ins(1,4,5)P₃ should minimize the number of second-messenger systems that are operative, which otherwise might also alter _m independently of Ca²⁺ changes. With attenuated light intensity and low concentrations of the _m indicator tetramethylrhodamine ethyl ester (TMRE), only minimal _m changes were observed under resting conditions. _m depolarizations did not occur when [Ca²⁺]_c was increased by Ca²⁺ influx via voltage-gated Ca²⁺ channels or by release from Ins(1,4,5)P₃Rs, even though mitochondrial Ca²⁺ uptake took place. However, _m depolarizations were observed on increasing TMRE concentration or light intensity even though no change in [Ca²⁺]_c took place. These depolarizations were blocked by antioxidants or the mitochondrial permeability transition pore (mPTP)-inhibitor cyclosporin A (CsA). Very occasionally, transient _m depolarization was seen in cells undergoing sustained, spontaneous [Ca²⁺]_c oscillations. In this case, _m depolarizations were neither synchronized throughout the mitochondrial complement nor with changes in [Ca²⁺]_c. Therefore, during physiological Ca²⁺ signaling, mitochondrial Ca²⁺ uptake modifies the amplitude and duration of Ca²⁺ signals without significantly altering _m, unless [Ca²⁺]_c oscillations are repetitive and sustained.

	Results

Top Summary Introduction Results Discussion Materials and Methods References

 
Mitochondria influence on [Ca²⁺]_c increases in single smooth muscle cells
Plasmalemmal depolarization (–70 to +10 mV for 500 ms) of single colonic myocytes transiently increased [Ca²⁺]_c (Fig. 1A). This increase arises solely from Ca²⁺ influx via the voltage-dependent Ca²⁺ current (I_Ca); activation of Ca²⁺-induced Ca²⁺ release does not occur in this cell type (Bradley et al., 2002). The time required for [Ca²⁺]_c to return to half its peak value (50% recovery time) was measured following two depolarizations more than 2 minutes apart in the same cell. In control cells, the 50% recovery time of the second depolarization pulse (in the presence of the vehicle) was 89±2% of the first (in the absence of the vehicle, n=6). In a second group of cells, mitochondrial Ca²⁺ uptake was inhibited by pharmacological dissipation of _m between the two depolarization events, in order to examine the influence of mitochondrial Ca²⁺ accumulation upon I_Ca-evoked [Ca²⁺]_c increases. The proton uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 1 µM) and the ATP synthase blocker oligomycin (6 µM) slowed the 50% recovery time (134±13% of the first pulse; n=9, P<0.05 compared with vehicle-treated controls, Fig. 1A), as did mitochondrial depolarization with rotenone (5 µM) plus oligomycin (123±12%; n=8, P<0.05 compared with controls). Oligomycin alone does not alter I_Ca-evoked [Ca²⁺]_c transients in these cells (McCarron and Muir, 1999). Together, these results suggest that mitochondria regulate the time-course of recovery following [Ca²⁺]_c increases evoked by plasmalemmal depolarization.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Mitochondrial depolarization slowed the rate of [Ca2+]c decline following ICa activation and decreased the amplitude of the Ins(1,4,5)P3-induced [Ca2+]c rise. Plasmalemmal depolarization (–70 mV to +10 mV, 500 ms, A), local photolysis of caged Ins(1,4,5)P3 (UV-flash release at , B) and carbachol (CCh, 100 µM by pressure ejection, C) each transiently increased [Ca2+]c, as indicated by an increase in Fluo-4 fluorescence (controls, thin line). Inhibition of mitochondrial Ca2+ uptake by m depolarization with CCCP plus oligomycin (1 µM and 6 µM, respectively, A), increased the time for [Ca2+]c to decline to resting values (thick line), compared with vehicle-treated control (0.2% DMSO, thin line). The peak [Ca2+]c rise evoked by Ins(1,4,5)P3 release was decreased by m depolarization (B, thick line), as was the peak [Ca2+]c evoked by CCh (C, thick line), each compared with vehicle-treated control (0.2% DMSO, thin lines). By contrast, m depolarization did not alter the amplitude or rate of decline of [Ca2+]c increases evoked by caffeine (CAF; 10 mM by pressure ejection, D).

Mitochondria also regulate Ins(1,4,5)P₃-mediated Ca²⁺ release from the SR. Local photolytic release of Ins(1,4,5)P₃ from the caged compound evoked reproducible increases in [Ca²⁺]_c, the second transient was 93±5% of the first Ins(1,4,5)P₃-evoked [Ca²⁺]_c peak (amplitude of first 2.86±0.63 F/F₀, amplitude of second 2.78±0.91 F/F₀, n=9, P>0.05; where F indicates fluorescence counts and F₀ indicates baseline values before stimulation). To examine the influence of mitochondrial Ca²⁺ accumulation upon Ins(1,4,5)P₃-evoked [Ca²⁺]_c increases, mitochondrial Ca²⁺ uptake was again inhibited by pharmacological depolarization of _m using either CCCP or rotenone 60 seconds prior to the second Ins(1,4,5)P₃ release. _m depolarization substantially reduced the second Ins(1,4,5)P₃-evoked [Ca²⁺]_c rise. CCCP (1 µM) plus oligomycin (6 µM) caused a decline in the peak of the second Ins(1,4,5)P₃-evoked [Ca²⁺]_c amplitude to 47±9% of that obtained prior to application of these drugs (n=13, P<0.01, Fig. 1B). _m depolarization with rotenone (5 µM) plus oligomycin (6 µM) also reduced the peak of the second Ins(1,4,5)P₃-evoked [Ca²⁺]_c transient to 49±10% of that occurring prior to _m depolarization (n=11, P<0.01). Inhibition of mitochondrial Ca²⁺ uptake with Ru360 – a specific inhibitor of the mitochondrial uniporter (Matlib et al., 1998) – also inhibited Ins(1,4,5)P₃-evoked Ca²⁺ release to 86±8% of control (from 2.01±0.26 F/F₀ in control to 1.72±0.16 F/F₀ with Ru360; n=3, P<0.05). Again, oligomycin alone did not alter Ins(1,4,5)P₃-induced [Ca²⁺]_c transients (McCarron and Muir, 1999).

The influence of mitochondrial Ca²⁺ uptake on [Ca²⁺]_c transients evoked by the Ins(1,4,5)P₃-generating agonist carbachol (CCh, 100 µM by pressure ejection) was also examined. In vehicle-treated controls, CCh evoked a large, reproducible transient rise in [Ca²⁺]_c (Fig. 1C). There was no difference in peak [Ca²⁺]_c evoked by two successive applications of CCh, 2 minutes apart (the second peak [Ca²⁺]_c transient was 108±5% of the first, n=3, P>0.05). _m depolarization with CCCP (1 µM) plus oligomycin (6 µM) 60 seconds prior to the second CCh application decreased the evoked [Ca²⁺]_c transient amplitude to 8±7% of the first (n=3, P<0.01, Fig. 1C). _m depolarization with rotenone (5 µM) plus oligomycin (6 µM) caused the CCh-evoked [Ca²⁺]_c transient amplitude to decline to 62±9% of that prior to application of the drugs (n=3, P<0.01). On the other hand, Ca²⁺ release evoked by the RyR agonist caffeine (10 mM) was not significantly altered by _m depolarization (Fig. 1D, peak Ca²⁺ was 1.76±0.23 F/F₀ prior to, and 1.78±0.24 F/F₀ after CCCP plus oligomycin treatment, n=6, P>0.05). This latter result suggests that _m depolarization had not significantly depleted the SR of Ca²⁺. Together, these results suggest that, when polarized, mitochondria modulate [Ca²⁺]_c increases arising both from influx of extracellular Ca²⁺ and from Ca²⁺ release from intracellular stores via Ins(1,4,5)P₃R.

Relationship between [Ca²⁺]_c and _m
Ca²⁺ uptake by the mitochondrial uniporter is driven by _m (Nicholls and Crompton, 1980) and might itself depolarize _m (Duchen et al., 1998; Kaftan et al., 2000). Accordingly, _m was measured to determine whether or not depolarization occurred during transient [Ca²⁺]_c rises evoked by either I_Ca activation or Ins(1,4,5)P₃. Changes in [Ca²⁺]_c and _m were measured simultaneously with Fluo-4 AM (10 µM) and TMRE (10 nM), respectively, in single colonic myocytes. The long wavelength Ca²⁺ indicator (Fluo-4) was necessary because the requirement for ultra violet (UV)-sensitive caged Ins(1,4,5)P₃ precluded the use of ratiometric Ca²⁺ indicators such as Fura-2, which are excited by UV light. Despite the overlap of the excitation and emission spectra of Fluo-4 and TMRE, independent signals were resolved using selective dichroic mirrors, filters and excitation wavelengths (Fig. 2A) as described in the Materials and Methods. Separation of Fluo-4 and TMRE signals was established by first examining the fluorescence properties of cells loaded separately with each dye (Fig. 2B). Cells loaded with Fluo-4 alone showed barely detectable fluorescence when excited at 560 nm (the wavelength used for excitation of TMRE). Conversely, when cells were loaded with TMRE alone and excited at 475 nm, only a small fluorescence signal was detected, which was less than 5% of that obtained when either TMRE was excited at 560 nm or Fluo-4 at 475 nm (Fig. 2Bii). The separation of the signal from each indicator was confirmed in cells co-loaded with Fluo-4 and TMRE. I_Ca activation (depolarization to +10 mV from –70 mV for 500 ms) caused a large transient increase in Fluo-4 fluorescence with no change in TMRE fluorescence (Fig. 2D). Subsequent _m depolarization with CCCP (1 µM) did not alter [Ca²⁺]_c (McCarron and Muir, 1999), causing a large decrease in TMRE fluorescence with no decrease in Fluo-4 fluorescence (Fig. 2D). The extensive co-localization of TMRE with the mitochondrial stain MitoTracker Green confirmed that TMRE partitioned largely to mitochondria (Fig. 2C).

View larger version (63K): [in this window] [in a new window]   Fig. 2. Dual loading of colonic myocytes with Fluo-4 and TMRE allows simultaneous observation of [Ca2+]c and m, leaving shorter wavelengths to be used for UV-photolysis of caged compounds. (A) In spite of overlapping excitation and emission profiles (i), cross-talk between Fluo-4 and TMRE fluorescence signals was minimized using a custom-made dichroic mirror (black line, ii), dual-bandpass excitation (dark-blue line, ii) and emission (light-blue line, ii) filters and exciting at 475 and 560 nm (ii). (B) Virtual absence of overlap between Fluo-4 and TMRE channels in cells loaded with either Fluo-4 alone (10 µM, i) or, in another cell, with TMRE alone (10 nM, ii). Each cell was excited at 475 nm (left-hand panel) and 560 nm (right-hand panel). Pseudocolored images represent emission intensity from a 12-bit range (calibration in top-left corners). (C) Mitochondrial localization of TMRE (10 nM, i) was confirmed by its co-localization with the mitochondria-specific dye MitoTracker Green (25 nM, ii). (iii) Overlay of red TMRE on green MitoTracker images produces yellow regions in which the two indicators are co-localized. (iv) A bright-field image of the cell is also shown. (D) Separation of [Ca2+]c and m measurements in a voltage-clamped myocyte co-loaded with Fluo-4 (green) and TMRE (red). Plasmalemma depolarization (500 ms, –70 to +10 mV, ii) induced a large increase in Fluo-4 fluorescence with no change in TMRE fluorescence (i); subsequent CCCP (1 µM) treatment caused a large decrease in TMRE fluorescence with no decrease in Fluo-4 fluorescence. Scale bars: 10 µm.

View larger version (35K): [in this window] [in a new window]   Fig. 3. m depolarization with CCCP and m hyperpolarization with oligomycin were each detected by changes in TMRE fluorescence of individual mitochondria. (A) CCCP (1 µM, white squares, n=4) caused a large, rapid decrease in the average TMRE fluorescence compared with controls (black triangles, n=9). Oligomycin (6 µM, white circles, n=3) caused a small but significant increase in TMRE fluorescence (**P<0.01 compared with controls). (B) Images of TMRE fluorescence from approximately 50% of a single smooth muscle cell before and after CCCP (1 µM) treatment. Left-most panel: the punctuate fluorescence comes from the TMRE signals from individual mitochondria. The loss of punctuate TMRE fluorescence in successive panels occurs as a result of the loss of TMRE from mitochondria as m is depolarized with CCCP. Images were obtained at the time-points indicated by connecting lines to panel A. Scale bars: 10 µm.

View larger version (38K): [in this window] [in a new window]   Fig. 4. Ca2+accumulated by mitochondria during Ins(1,4,5)P3- or ICa-evoked [Ca2+]c transients can be released by mitochondrial depolarization in the 15 seconds after [Ca2+]c elevation. Local photolysis of caged Ins(1,4,5)P3 (UV-flash release at , A) or plasmalemmal depolarization (–70 to +10 mV, 500 ms, C) each transiently increased [Ca2+]c, as indicated by an increase in Fluo-4 fluorescence (controls, black line). In a subsequent activation by Ins(1,4,5)P3 (A) or plasmalemmal depolarization (C) in the same cells, rapid m depolarization (A,C, as shown by a decrease in TMRE fluorescence, red line) by CCCP (20 µM, by pressure-ejection puffer pipette for 2 seconds as shown), caused a transient increase in [Ca2+]c (blue line). The magnitude of mitochondrial Ca2+ release was measured from the time-integrated Fluo-4 signal for a 10-second period immediately after CCCP application minus the time-integrated signal for the control (hashed area, A,C, shown in panels B,D, n=3 individual cells for each data point).

_m during [Ca²⁺]_c transients evoked either by Ins(1,4,5)P₃ or I_Ca activation
To evaluate whether or not [Ca²⁺]_c increases altered _m of individual mitochondria in single colonic myocytes, TMRE fluorescence of individual mitochondria was monitored during increases in [Ca²⁺]_c evoked either by UV flash-photolysis of caged-Ins(1,4,5)P₃ or by I_Ca activation (depolarization to +10 mV for 500 ms). I_Ca-evoked [Ca²⁺]_c elevation caused little alteration in the TMRE fluorescence of individual mitochondria compared with separate, time-matched control cells in which I_Ca was not activated (Fig. 5A). However occasionally (5 out of 92 mitochondria examined, n=10), _m depolarization of individual mitochondria occurred (an example is shown in Fig. 5Ciii). These _m depolarizations were not correlated with either the timing of I_Ca activation or the subsequent Ca²⁺ rise and, consequently, were not considered significant.

View larger version (26K): [in this window] [in a new window]   Fig. 5. Neither Ins(1,4,5)P3- nor ICa-evoked [Ca2+]c increases significantly altered m of individual mitochondria. ICa activation (A, by depolarizing from –70 to +10 mV, 500 ms, n=9, white squares) or local photolysis of caged Ins(1,4,5)P3 (B, , n=10, white circles) each increased [Ca2+]c (Ci and Di). Neither caused significant alteration in m (as shown by TMRE fluorescence, normalized to starting values, F/F0) compared with control (n=9, black triangles, P>0.05 for all data points). Occasionally, the m of individual mitochondria decreased during the period of observation of either ICa-evoked [Ca2+]c elevation (Ci, [Ca2+]c changes; Cii, holding potential, Vm; Ciii, m of six individual mitochondria) or Ins(1,4,5)P3-evoked [Ca2+]c elevation (Di, [Ca2+]c changes; Dii, activated by photolytic release of Ins(1,4,5)P3 at ; Diii, m of eight individual mitochondria); however, this was equally as likely to occur before, during or after [Ca2+]c elevation.

_m measurement using quenching concentrations of TMRE
Hitherto in this investigation, TMRE was used at a concentration of 10 nM to prevent dye auto-quenching within the mitochondrial matrix and to enable visualization of the _m of individual mitochondria. The low concentration might have limited our ability to resolve minor _m changes across the cell. To examine this possibility, the _m of the entire mitochondrial complement was studied by loading cells with a concentration of TMRE that exceeds its `quench limit' (150 nM) and monitoring fluorescence throughout the entire cell. At this higher concentration, _m depolarization increases whole-cell TMRE fluorescence because of a reduction of fluorescence quenching as the dye re-equilibrates out of mitochondria (Duchen et al., 1998; Ward et al., 2000) (see Materials and Methods). In these conditions, no change in TMRE fluorescence was observed when [Ca²⁺]_c was elevated either by Ins(1,4,5)P₃ release (0.997±0.0014 F/F₀, n=8, compared to untreated controls 0.997±0.002 F/F₀, n=4, P>0.05, Fig. 6A), a single 500 ms plasmalemma depolarization to +10 mV (1.003±0.004 F/F₀, n=6, P>0.05 compared to untreated controls, Fig. 6B), or a 5-second train of depolarizing pulses (–70 to +10 mV, 2 Hz, 1.001±0.002 F/F₀, n=4, P>0.05 compared to untreated controls, Fig. 6C). Mitochondrial depolarization with CCCP (1 µM) plus oligomycin (6 µM), by contrast, caused a large increase in TMRE fluorescence (1.204±0.056 F/F₀, n=3, P<0.01, Fig. 6D,E). These results again suggest that a transient increase in [Ca²⁺]_c does not significantly depolarize _m in colonic smooth muscle cells.

View larger version (27K): [in this window] [in a new window]   Fig. 6. Neither Ins(1,4,5)P3 (IP3)- nor ICa-induced [Ca2+]c increases altered the m of the entire-cell mitochondrial complement when measured with TMRE in `quench' mode. m depolarization was not seen during increases of [Ca2+]c induced by either Ins(1,4,5)P3 (released by localized photolysis of caged Ins(1,4,5)P3, at , A), following a single plasmalemmal depolarization (+10 mV for 500 ms, B) or following a train of plasmalemmal depolarizations (+10 mV for 250 ms, 2 Hz, 5 seconds, C) in myocytes co-loaded with Fluo-4 (10 µM) and TMRE (150 nM, see text). By contrast, the mitochondrial inhibitors CCCP plus oligomycin (1 and 6 µM, respectively, D) significantly increased TMRE fluorescence when compared with untreated controls (E); **P<0.01; n.s. (not significant) P>0.05.

_m fluctuations in cells during repetitive [Ca²⁺]_c oscillations
Very occasionally (<1%), cells exhibited spontaneous, repetitive [Ca²⁺]_c oscillations of variable frequency and amplitude (Fig. 7). _m depolarization, and often subsequent repolarization of individual mitochondria, was observed in these cells. The _m depolarization apparently occurred regardless of the _m of neighboring mitochondria. Fig. 7Aii and iv show enlarged regions of cells in which single mitochondria depolarized and repolarized independently of neighboring mitochondria, although the distance between the organelles could be as little as 5 µm. Regions in which fluorescence decreased from one frame to another are marked with a blue arrow; those in which fluorescence increased with a white arrow. This observation suggests that, rather than acting as an interconnected electrical network, these _m depolarizations were independent of similar events in neighboring mitochondria. The _m depolarizations also do not appear to be linked to the occurrence of individual [Ca²⁺]_c oscillations (shown in Fig. 7Ai,iii). Fig. 7B shows examples of TMRE fluorescence from individual mitochondria alongside the corresponding [Ca²⁺]_c changes. _m depolarizations appeared to be equally as likely when [Ca²⁺]_c was at resting or peak values, suggesting that these depolarizations did not occur as a direct consequence of the flux of positively charged Ca²⁺ ions into mitochondria.

View larger version (44K): [in this window] [in a new window]   Fig. 7. m changes in cells undergoing spontaneous, repetitive [Ca2+]c oscillations. (A) During spontaneous, repetitive [Ca2+]c oscillations (Fluo-4 fluorescence normalized to baseline values, F/F0, i and iii), individual mitochondria (visualized as punctate TMRE fluorescence, shown in close-up still frames at the time points indicated during [Ca2+]c oscillations, ii and iv) depolarized. Localized fluorescence decreases (depolarizations) are indicated by blue arrows and localized fluorescence increases (repolarizations) are shown by white arrows (images pseudocolored to indicate fluorescence intensity, v). (B) TMRE-fluorescence changes in three individual mitochondria (lower panels) from three separate cells were of varying magnitude and duration, and did not correspond directly to changes in [Ca2+]c (upper panels, Fluo-4 fluorescence, F/F0). Scale bars, 5 µm.

Cells displaying spontaneous Ca²⁺ oscillations were rare (<1%), precluding the study of the mechanisms underlying _m depolarization. Interestingly, mimicking the spontaneous Ca²⁺ oscillations by repetitively applying either Ins(1,4,5)P₃ (by photolysis) or CCh did not produce _m depolarization (not shown, repeated application of Ins(1,4,5)P₃ at a holding plasma membrane potential, V_m, of –70 mV: n=7; repeated application of Ins(1,4,5)P₃ at a V_m of –20 mV: n=4; repeated application of CCh at a V_m of –70 mV: n=4; sequential application of Ins(1,4,5)P₃, CCh and I_Ca activation by depolarization of V_m to +10 mV for 500 ms: n=3). However, flickering _m depolarizations could be evoked by increasing either the intensity of excitation light or the concentration of TMRE used, or both. When used at a concentration of 150 nM, transient decreases in the TMRE fluorescence (_m depolarizations) of individual mitochondria were observed, without any changes in [Ca²⁺]_c. (Although _m depolarization increases whole-cell fluorescence when the concentration of TMRE is above the quench limit, individual mitochondria will still display a decrease in fluorescence intensity upon _m depolarization because of dye re-equilibration, see Materials and Methods.) As the TMRE concentration was decreased from 150 nM to 10 nM, the number of _m depolarizations decreased from 1.009±0.092 (150 nM TMRE) to 0.074±0.004 (10 nM TMRE) per mitochondrion per 10-minute imaging period (1 frame s^–1, n=5, P<0.01, Fig. 8A). Thus, 0.74% of mitochondria displayed _m depolarizations per minute when loaded with 10 nM TMRE, whereas some 10% depolarized per minute when loaded with 150 nM TMRE. The occurrence of _m depolarizations also increased with increasing illumination light intensity when examined at lower TMRE concentrations (10 or 50 nM, Fig. 8A). These transient _m depolarizations were attenuated by a mixture of antioxidants containing ascorbic acid (1 mM), catalase (250 units/ml), (R)-Trolox methyl ether (Trolox, 1 mM) and 2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPO, 500 µM). In these experiments _m depolarizations decreased from 1.061±0.091 (control) to 0.280±0.035 in the presence of antioxidants (depolarizations per mitochondrion per 10-minute imaging period, n=5, P<0.01, Fig. 8B). The mPTP inhibitor CsA (2 µM) also decreased _m depolarization frequency from 1.061±0.091 to 0.427±0.028 (n=4, P<0.01, Fig. 8B). These _m depolarizations might arise from an oxidant-mediated transient opening of the mPTP (Huser et al., 1998; Jacobson and Duchen, 2002).

View larger version (15K): [in this window] [in a new window]   Fig. 8. m depolarization frequency increased with [TMRE] or intensity of excitation light and was inhibited by antioxidants or cyclosporin A. (A) The frequency of m depolarizations plotted against [TMRE] (10-150 nM) with (shaded bars) and without (white bars) the attenuation of excitation light by a neutral density filter (*P<0.05, **P<0.01). (B) The mPTP inhibitor cyclosporin A (CsA, 2 µM) or a combination of antioxidants [ascorbic acid (1 mM), catalase (250 units/ml), Trolox (1 mM) and TEMPO (500 µM)] each decreased the number of m depolarizations observed at 150 nM TMRE (**P<0.01). Cells were co-loaded with TMRE and Fluo-4 (10 µM) and fluorescent images acquired at 1 Hz.

	Discussion

Top Summary Introduction Results Discussion Materials and Methods References

Mitochondrial Ca²⁺ uptake should depolarize _m to restrict the ability of mitochondria to produce ATP. Ca²⁺ signaling could, in theory at least, limit the primary mechanism for ATP production. It is therefore important to resolve whether or not _m depolarization occurs following increases in [Ca²⁺]_c. The present study found no detectable alterations in _m during large, transient [Ca²⁺]_c rises evoked by Ca²⁺ influx across the plasmalemma in single colonic myocytes (Figs 5 and 6). The limit of resolution in the present study was 5 mV. Therefore, _m depolarization during Ca²⁺ uptake was smaller than this value.

Isolated mitochondria exposed to bolus additions of Ca²⁺ undergo transient _m depolarizations (Brustovetsky and Dubinsky, 2000; Huser et al., 1998). However, whether or not the local [Ca²⁺]_c experienced by mitochondria in situ, in intact cells, is sufficient to cause _m depolarizations comparable to those that occur in isolated mitochondria is unresolved. _m depolarization has been observed in response to the [Ca²⁺]_c rise produced by trains of plasmalemmal depolarization in single toad gastric myocytes (Drummond et al., 2000). No alterations in _m were observed in the present study to trains of depolarization (Fig. 6C). Mitochondria might be arranged differently in the two cell types and positioned further from voltage-gated Ca²⁺ channels in colonic myocytes than in gastric myocytes, and hence exposed to lower local [Ca²⁺]_c. Alternatively, the inclusion of mitochondrial substrates (pyruvate and malate) in the pipette filling solution in the present study, which are required to maintain the driving force for _m production, might have contributed to the differences in the two studies. Indeed, partial metabolic limitation of the electron-transport chain resulted in a greater _m depolarization during Ca²⁺ entry evoked by tetanic stimuli in motor neuron terminals (Talbot et al., 2007).

The present study also found no significant alteration in _m during [Ca²⁺]_c transients evoked by Ca²⁺ release from internal stores via Ins(1,4,5)P₃Rs (Figs 5 and 6). Other studies have, like ours, failed to measure any significant _m depolarization in response to transient [Ca²⁺]_c increases produced by Ins(1,4,5)P₃-generating agonists, e.g. in HeLa or RBL-2H3 cells (Collins et al., 2001; Csordas and Hajnoczky, 2003). Although Ca²⁺ uptake by mitochondria must lead to _m depolarization, its magnitude is small and unresolved because either the input resistance of the inner mitochondrial membrane was too low for a significant _m to occur with the current carried by Ca²⁺ or because a compensatory efflux of positively charged species (i.e. protons) offsets the depolarization evoked by Ca²⁺ influx.

Mitochondrial ion fluxes and energy metabolism have recently been modeled in electrically paced cardiac myocytes (Nguyen et al., 2007). This model shows that a rapid rise in mitochondrial [Ca²⁺], during cytosolic [Ca²⁺] transients, is accompanied by increases in mitochondrial [ATP], [NAD⁺], [P_i] and [Na⁺] and decreases in mitochondrial [ADP], [NADH] and _m. The magnitude of the drop in _m is 8 mV with a time constant of recovery of <0.1 seconds (Nguyen et al., 2007). A transient _m depolarization of 8 mV is around the limit of resolution in the present study and is unlikely to be detected. Mitochondrial energy metabolism was also modeled in hypothetical microdomains of high [Ca²⁺]_c (up to 20 µM) in cardiac dyadic space. Here, _m was predicted to transiently depolarize to 0 mV due to mitochondrial Ca²⁺ uptake. Recovery was complete within 0.3 seconds (Nguyen et al., 2007). It is unlikely, therefore, in the current study, that [Ca²⁺]_c in microdomains near mitochondria reached these concentrations (20 µM), or _m depolarization would have been observed.

Very occasionally, spontaneous [Ca²⁺]_c oscillations were observed in colonic myocytes. Because these spontaneous Ca²⁺ oscillations were rare (occurring in only 3 out of 314 cells), they would appear to be an unusual form of Ca²⁺ signaling in this cell type. In cells showing spontaneous Ca²⁺ oscillations, depolarization of the _m of individual mitochondria occurred (Fig. 7). The _m depolarizations were randomly distributed throughout the cell, did not affect the _m of neighboring mitochondria and appeared independent of the timing of individual [Ca²⁺]_c oscillations.

Random depolarization of _m was also observed with increasing intensity of excitation light or increased TMRE concentration (see also Collins et al., 2002; Duchen et al., 1998; Huser and Blatter, 1999; Huser et al., 1998; Jacobson and Duchen, 2002) (Fig. 8). The _m depolarizations were decreased in frequency by antioxidants (Jacobson and Duchen, 2002). Hence, these _m depolarizations seem likely to have arisen from light- or dye-induced oxidant production. Oxidants activate the mPTP, which might depolarize _m (Duchen, 2000). Indeed, _m depolarization was blocked by the mPTP inhibitor CsA in the present study (Fig. 8B).

No significant transient _m depolarizations were observed during or after either I_Ca- or Ins(1,4,5)P₃-evoked increases in [Ca²⁺]_c because, in these experiments, the TMRE concentration used was low (10 nM), excitation light was attenuated with a neutral density filter and the duration of the imaging period was limited to 30 seconds. Each of these measures should limit oxidant production. The _m depolarizations that did occur during spontaneous [Ca²⁺]_c oscillations were not linked temporally to the [Ca²⁺]_c changes. It might be that these _m depolarizations arise from a gradual accumulation of [Ca²⁺]_m, perhaps resulting in activation of the mPTP directly by Ca²⁺ or indirectly via a Ca²⁺-dependant increase in oxidant production (Brookes et al., 2004). The rare appearance of spontaneous Ca²⁺ oscillations precluded further examination of these possibilities.

The localized, individual nature of _m depolarizations that occurred in cells with Ca²⁺ oscillations, or during light- and dye-induced _m depolarizations, suggests that mitochondria do not form an interconnected electrical network in smooth muscle. Had mitochondria done so, depolarization of _m at one site should have depolarized _m at distant sites. Mitochondria might exist as an interconnected network in some (Diaz et al., 2000; Rizzuto et al., 1998; Szado et al., 2003) but not in other (Buckman and Reynolds, 2001; Collins et al., 2001; Jacobson and Duchen, 2002; Montero et al., 2002; O'Reilly et al., 2004) cell types. In the present study, mitochondria appear to be individual units of approximately 1-5 µm in length. Mitochondrial modulation of Ca²⁺ signaling might primarily be a localized, subcellular effect.

The present findings demonstrate that mitochondria contribute to the decline in [Ca²⁺]_c after I_Ca activation and promote Ca²⁺ release via Ins(1,4,5)P₃Rs on the SR. During these [Ca²⁺]_c increases, _m – the driving force for ATP production – was not significantly altered; hence, one mitochondrial function – that of ATP production – is not negatively affected by another, i.e. Ca²⁺ uptake and modulation of [Ca²⁺]_c. Some alteration in _m could occur with prolonged repetitive Ca²⁺ signaling, presumably as a consequence of a gradual, cumulative mitochondrial Ca²⁺ overloading or oxidant production. Mitochondrial Ca²⁺ uptake provides a fine-tuning of Ca²⁺ signaling. For example, a slight alteration of Ins(1,4,5)P₃R activity by mitochondrial Ca²⁺ accumulation could modulate the threshold for global [Ca²⁺]_c wave initiation. Mitochondria accomplish this activity without compromising their ability to produce ATP.

	Materials and Methods

Top Summary Introduction Results Discussion Materials and Methods References

 
Preparation of freshly dissociated cells
Single smooth muscle cells were enzymatically dissociated from the colon of male guinea pigs (Cavia porcellus, 500 g) as described previously (McCarron and Muir, 1999). Animals were humanely killed in accordance with the guidelines of the Animal (Scientific Procedures) Act UK 1986 by stunning and immediate cervical dislocation and exsanguination. All experiments and loading of cells with fluorescent dyes were carried out at room temperature (22±1°C).

Electrophysiology
Membrane currents were measured using conventional tight-seal whole-cell recording. The extracellular solution contained (mM): Na glutamate 80, NaCl 40, tetraethylammonium chloride (TEA) 20, MgCl₂ 1.1, CaCl₂ 3, HEPES 10, glucose 30 (pH 7.4 with NaOH) plus TMRE (10 nM) unless otherwise indicated. The pipette solution contained (mM): Cs₂SO₄ 85, CsCl 20, MgCl₂ 1, HEPES 30, MgATP 3, sodium pyruvate 2.5, malic acid 2.5, NaH₂PO₄ 1, creatine phosphate 5 and NaGTP 0.5, with caged inositol trisphosphate [Ins(1,4,5)P₃] trisodium salt 0.025 added for experiments involving photolytic release of Ins(1,4,5)P₃. The high concentration of HEPES was to ensure adequate control of pH during _m depolarization; sodium pyruvate and malic acid to maintain mitochondrial activity; and phosphocreatine and ATP to maintain [ATP] during the experiments. Whole-cell currents were measured using an Axopatch 200B (Molecular Devices, Sunnyvale, CA, USA), low-pass filtered at 500 Hz (8-pole bessel filter; Frequency Devices, Haverhill, MA, USA), digitally sampled at 1.5 kHz using a digidata interface and pClamp (version 8; Molecular Devices), and stored for analysis.

Imaging
Cells were co-loaded with the Ca²⁺-sensitive dye Fluo-4 AM (10 µM) and the _m-sensitive dye TMRE (10 nM unless stated otherwise) for 30 minutes in the presence of wortmannin (10 µM; to prevent contraction). Qualitatively identical results were obtained in the absence of wortmannin. TMRE is a lipophilic, cationic fluorescent dye that accumulates within mitochondria according to their _m in a Nernstian fashion (Duchen et al., 1998; Scaduto and Grotyohann, 1999). _m depolarization causes a decrease in mitochondrial [TMRE] regardless of the concentration of the indicator used; however, TMRE fluorescence of the whole cell may increase or decrease on _m depolarization, depending on the concentration of TMRE used (Ward et al., 2000). This is because, above a certain concentration (e.g. 100 nM in the cells used in this study), TMRE self-quenches within mitochondria to reduce the fluorescence emanating from the indicator. In these circumstances, _m depolarization will decrease the mitochondrial [TMRE] to decrease or cause no change in the fluorescence of individual mitochondria, but whole-cell TMRE fluorescence will transiently increase. Dye that was previously quenched (non-fluorescent) within mitochondria moves to the cytosol to increase fluorescence there before re-equilibrating across the plasmalemma (Ward et al., 2000). In this `quenched' mode, TMRE cannot be used quantitatively to monitor _m of individual mitochondria. At a concentration below 100 nM, TMRE fluorescence does not self-quench within mitochondria and decreases with _m depolarization. The _m of individual mitochondria can be resolved with TMRE concentrations below the quench limit (e.g. 10 nM). In this study, a sub-quenching concentration of TMRE (10 nM) was used to monitor the _m of individual mitochondria and a quenching concentration of TMRE (150 nM) to monitor the _m of the entire mitochondrial complement of the cell.

Two-dimensional images of [Ca²⁺]_c and _m were obtained using a wide-field digital imaging system. Single cells were excited at 475 nm and 560 nm (Polychrome IV monochromator, TILL Photonics, Martinsried, Germany), and light was passed via a fiber-optic guide through a dual 483/553 nm bandpass filter (bandpass 15 and 20 nm, respectively), a field stop diaphragm, through an ND4 neutral density filter (unless otherwise stated) and reflected off a custom-made dual long-pass dichroic mirror (transmissive in the ranges 505-540 nm and 577-640 nm, and reflective from 490 to below 300 nm; Chroma, Rockingham, VT, USA) and then through an oil immersion objective (x40 UV 1.3 NA; Nikon UK, Surrey, UK). Emitted light was guided through a dual bandpass 518/594 nm barrier filter (bandpass 25 and 18 nm, respectively) to an intensified, cooled, frame transfer CCD camera (Pentamax Gen IV; Roper Scientific, Trenton, NJ, USA) controlled by MetaFluor software (Universal Imaging, Downingtown, PA, USA). Full-frame images (150x150 pixels), with a pixel size of 563 nm at the cell, were acquired sequentially with an exposure period of 100 ms for Fluo-4 and then 50 ms for TMRE (resultant acquisition rate for the pair was 5 Hz). In some experiments, TMRE fluorescence alone was recorded at a frequency of 100 Hz (10 ms exposure) with the camera under the control of WinView32 operating in high-speed mode. No difference in the frequency of _m depolarizations was noted between 5 and 100 Hz sampling frequency.

Localized flash photolysis
The output of a xenon flashlamp (Rapp Optoelecktronic, Hamburg, Germany), used to uncage Ins(1,4,5)P₃, was passed through a UG-5 filter to select UV light, focused and merged into the excitation light path via a fiber-optic bundle and long pass dichroic mirror at the lens part of the epi-illumination attachment of the microscope. The diameter of the fiber optic together with the lens magnification determined the area (spot size 10 µm diameter) of Ins(1,4,5)P₃ photolysis.

Data analysis
Images were analyzed using MetaMorph 6.2 (Universal Imaging). [Ca²⁺]_c measurements were made from the Fluo-4 fluorescence changes in the entire cell. Fluorescence signals were expressed as ratios (F/F₀) or change of ratios (F/F₀) of fluorescence counts (F) relative to baseline values before stimulation (F₀). Peak Fluo-4 F/F₀ and the time required for Fluo-4 F/F₀ to decline to 50% of the peak value were calculated before and after treatment with either CCCP (1 µM) plus oligomycin (6 µM) or rotenone (5 µM) plus oligomycin (6 µM). CCCP dissipates transmembrane proton gradients, oligomycin prevents ATP consumption by inhibiting the mitochondrial ATP synthase and rotenone is a complex I inhibitor that will also depolarize _m. The use of CCCP and rotenone with oligomycin will depolarize mitochondria without ATP depletion (Budd and Nicholls, 1996); 0.2% dimethyl sulfoxide (DMSO) served as the vehicle control. TMRE fluorescence was measured over regions of interest drawn around individual mitochondria or the whole cell as described in the text. Each of these fluorescence signals from mitochondria and whole cells were normalized to starting baseline values (taken as 1) and the gradual decline in the signals due to dye photobleaching was corrected for by subtracting a linear extrapolation of bleaching from each region (thus making the baseline fluorescence values 0). To create summarized results and allow statistical analysis, a rolling `box-car' average (5 seconds) of corrected TMRE F/F₀ was created and sampled every 2.5 seconds for most experiments (30-second data-collection period). With longer data-collection periods during the examination of oxidant-induced _m depolarization (60-600 seconds), the rolling average was increased to a 20-second window, sampled every 10 seconds. Results are express