An increased cytosolic Ca2+ concentration ([Ca2+]c) is the major trigger for smooth muscle contraction; relaxation follows the return of [Ca2+]c to resting levels. Mechanisms which restore resting [Ca2+]c include a Ca2+ pump and a Na+-Ca2+ exchanger in the plasma membrane and a sarcoplasmic reticulum (SR) Ca2+ pump. The contribution of each varies with the smooth muscle, the mode of its activation and the experimental approach (see Kamishima & McCarron, 1998 for references). Despite reports of their low affinity for Ca2+, mitochondria are now also accepted as important regulators of [Ca2+]c (e.g. Fry et al. 1989; Rizzuto et al. 1993; Friel & Tsien, 1994; Werth & Thayer 1994; Budd & Nicholls, 1996; Drummond & Fay, 1996; Herrington et al. 1996). The strategic localization of mitochondria close to Ca2+ release or influx pathways may expose the mitochondria to microdomains of high Ca2+ and permit them to accumulate the ion even during small increases in the bulk average [Ca2+]c (Rizzuto et al. 1993).
Following large increases in [Ca2+]c, smooth muscle contraction can be fully sustained even after [Ca2+]c has been restored almost to resting levels. Maintained small increases in [Ca2+]c (e.g. following depolarization) may arise from a continued Ca2+ influx or, in some cells, from mitochondrial Ca2+ efflux (Friel & Tsuen, 1994; Herrington et al. 1996; Park et al. 1996).
In contrast, little is known of the mitochondrial regulation of the InsP3-sensitive store in excitable cells. Accordingly, in the present study, the role of mitochondria in regulating the increases in [Ca2+]c evoked by its influx through voltage-dependent Ca2+ channels and by release from the InsP3-sensitive store has been examined in single smooth muscle cells. Ca2+ removal from the cytosol, following depolarization-evoked Ca2+ entry, was found to involve a rapid mitochondrial Ca2+ uptake. Following its subsequent efflux from mitochondria, this Ca2+ elevated [Ca2+]c for a prolonged period. Mitochondria also regulated the InsP3-sensitive store since inhibition of mitochondrial Ca2+ uptake resulted in a signficantly reduced magnitude of the InsP3-evoked Ca2+ transient. This reduction occurred even though the Ca2+ content of the internal store remained unchanged. A preliminary account of these findings has been presented to the Physiological Society (McCarron & Muir, 1998).
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From male guinea-pigs (500-700 g) stunned by a blow to the head and killed by exsanguination, a segment of distal colon (∼5 cm) was removed and transferred to a Sylgard-coated (Dow Corning) Petri dish containing an oxygenated (95 % O2-5 % CO2) physiological saline solution (PSS) of the following composition (mM): NaCl, 118.4; NaHCO3, 25; KCl, 4.7; NaH2PO4, 1.13; MgCl2, 1.3; CaCl2, 2.7; and glucose, 11 (pH 7.4). A longitudinal incision was made in the colon from the anal end, the submucosa removed and the circular muscle dissected from the longitudinal layer (Lim & Muir, 1983). Unless otherwise stated all experimental procedures were carried out at room temperature (20-22°C).
Strips of colonic circular muscle (2 × 0.5 cm) were placed in a solution containing (mM): NaCl, 137; KCl, 5; MgCl2, 1; CaCl2, 1.8; Hepes, 10; and glucose, 11 (pH adjusted to 7.4 with NaOH). Single cells were dissociated using a two-step enzymatic process. The muscle was initially digested (30 min at 35°C) with papain (1-4 mg ml−1) and dithioerythritol (0.5 mg ml−1) in a low Ca2+ solution containing (mM): sodium glutamate, 80; NaCl, 54; KCl, 5; MgCl2, 1; CaCl2, 0.1, Hepes, 10; glucose, 10; and 0.2, EDTA (to remove heavy metals); the pH adjusted at room temperature to 7.3 using NaOH. During a second incubation, the tissue was further digested (30 min at 35°C) in the low Ca2+ saline containing collagenase (type H or F; 1-3 mg ml−1) then rinsed several times with the enzyme-free low Ca2+ solution then again rinsed in Eagle's minimum essential spinner medium (S-MEM) lacking Ca2+ (GibcoBRL). Single smooth muscle cells were dispersed by trituration with a Pasteur pipette, stored at 4°C and used the same day.
Membrane currents were measured using conventional, whole-cell, tight seal recording. The composition of the extracellular solution was (mM): sodium glutamate, 80; NaCl, 40; tetraethylammonium chloride, 20; MgCl2, 1.1; CaCl2, 3; Hepes, 10; and glucose, 10 (pH 7.4 with NaOH). Unless otherwise stated, the pipette solution contained (mM): Cs2SO4, 85; CsCl, 20; MgCl2, 1; MgATP, 3; pyruvic acid, 2.5; malic acid, 2.5; NaH2PO4, 1; creatine phosphate, 5; GTP, 0.5; Hepes, 30; and fura-2 pentapotassium salt, 0.050 (if rhod-2 or fluo-3 was used fura-2 was omitted from the pipette solution). Whole-cell currents were amplified by an Axopatch 1D (Axon Instruments, Foster City, CA, USA), filtered at 500 Hz (8-pole bessel filter; Frequency Devices, Haverhill, MA, USA), and sampled at 1.5 kHz using a digidata interface pCLAMP software (version 6.0.1, Axon Instruments). In most cases, the duration of each voltage pulse was 1.6 s. To permit longer current recordings, the sampling frequency was reduced from 1.5 to 0.5 kHz after the first 1.2 s of the depolarization. Therefore, it was possible to obtain a current recording of 20 s. In some experiments recording time was increased further using Axotape (Axon Instruments).
[Ca2+]c was measured using the membrane-impermeable fura-2 (potassium salt, 50 μM) introduced into the cell from the patch pipette. Fluorescence measurements were made using a microfluorimeter consisting of an inverted fluorescence microscope (Nikon diaphot) and a photomultiplier tube with a bi-alkali photocathode. The excitation wavelengths (340 and 380 nm, 7 nm bandpass) were provided by a PTI deltascan (Photon Technology International Inc, East Sheen, London, UK). The cell was illuminated every 10 ms for 8.5 ms with each wavelength. [Ca2+]c measurements were made therefore at a frequency of 50 Hz. The excitation light passed through a 425 nm short pass filter (76 % transmission at 340 nm and 80 % transmission at 380 nm) and a field stop diaphragm was used to reduce background fluorescence. A 400 nm long pass dichroic mirror (94 % transmission at 510 nm) reflected the excitation wavelengths onto the cell. A 570 nm short pass dichroic mirror (82 % transmission at 510 nm) passed the emission light through a 505 nm barrier filter (60 nm bandpass, 88 % transmission at 510 nm) onto the photomultiplier for photon counting. Longer wavelengths from bright field illumination with a 610 nm Shott glass filter (90 % transmission) were reflected onto a CCD camera (Sony model XC-75) mounted onto the viewing port of the delta scan allowing the cell to be monitored during the course of the experiments. All interference filters and dichroic mirrors were obtained from Glen Spectra (London, UK). Background fluorescence was measured with the pipette attached to the cell but before rupturing the membrane. This background was subtracted from the fluorescence counts obtained during the experiments. The Kd for fura-2 was determined as 280 nM from an in vitro calibration. Rmin and Rmax were also determined from in vitro calibrations and decreased by 15 % to adjust for cell viscosity (Poenie, 1990).
Mitochondial matrix Ca2+ concentration [Ca2+]m was measured using the rhod-2 introduced into the cells in the AM form (5 μM). The AM ester of rhod-2 is positively charged and so will tend to partition into mitochondria in response to the large negative membrane potential (-150 mV to -180 mV) where the dye is cleaved and trapped. After loading, in whole-cell mode, cells were dialysed for at least 15 min to remove residual dye from the cytosol. However, to ensure [Ca2+]m was measured, at the end of each experiment carbonyl cyanide m-chlorophenylhydrazone (CCCP), which dissipates the mitochondrial membrane potential and therefore prevents mitochondrial Ca2+ accumulation, was applied. Rhod-2 was excited with light at 488 nm (7 nm bandpass) reflected off a 557 nm long pass dichroic mirror (94 % transmission). Emission light was passed through a 565 nm long pass filter (90 % transmission) to the photomultiplier, again operating in photon counting mode. Bright field illumination was not used during these experiments and background fluorescence was not subtracted.
When caged InsP3 was used, the longer wavelength non-ratiometric Ca2+ indicator fluo-3 (150 μM) was used to avoid photolysis of the caged compounds by the excitation light. The indicator was excited at 488 nm (bandpass 9 nm) through the epi-illumination port of the microscope (using one arm of a bifurcated quartz fibre optic bundle). Excitation light was reflected off a 505 nm long pass dichroic mirror and emission light was guided through a 535 nm barrier filter (bandpass 35 nm) to a photomultiplier in photon counting mode. To photolyse caged compounds, the output of a xenon flashlamp (Hi-Tech) was passed though a UG-5 filter to select ultraviolet light and merged into the excitation light path of the microfluorimeter using the second arm of the quartz bifurcated fibre optic bundle. The nominal flash lamp energy was 230 J with a flash duration of about 1 ms.
Analysis of [Ca2+]c decline
The rate of [Ca2+]c decline (dCa/dt) was measured as a function of the [Ca2+]c. Due to noise inherent in the high temporal resolution [Ca2+]c measurements, the raw data were smoothed using polynomial fits to the data beginning on the first data point after repolarization. The polynomial (5th-9th order) fit to the data was selected by the highest r2 value. The derivative was subsequently obtained by averaging the slopes of two adjacent data points. The rate of decline was expressed as a function of the [Ca2+]c.
When appropriate, results were expressed as means ±s.e.m. of n cells and the statistical test applied was Student's t test with P < 0.05 considered significant.
Drugs and chemicals
Fura-2 pentapotassium salt, fluo-3 pentapotassium salt and rhod-2 AM were purchased from Molecular Probes, Inc. (Eugene, OR, USA). Caged Ins(1,4,5)P3-trisodium salt and thapsigargin were purchased from Calbiochem-Novabiochem Ltd. Apart from these, all other reagents were purchased from Sigma, UK.
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Mitochondria are clearly important in the regulation of [Ca2+]c in colonic myocytes. The present findings show that during depolarization-evoked increases in [Ca2+]c, mitochondria rapidly accumulate and subsequently export Ca2+ as [Ca2+]c approached resting levels. Mitochondrial accumulation of Ca2+ contributed significantly to the decline of [Ca2+]c above a cytosolic Ca2+ concentration of 300 nM, consistent with the notion of a ‘set-point’ at which mitochondrial uptake and efflux are balanced (Nicholls, 1978). The efflux of Ca2+ from mitochondria, at low [Ca2+]c, led to a small elevation of [Ca2+]c that persisted for some tens of seconds after the depolarization had ended, co-incident with the slow rate of muscle relaxation.
Mitochondria also regulate the release of Ca2+ from the InsP3-sensitive store in colonic myocytes. Inhibition of mitochondrial Ca2+ accumulation decreased the magnitude of the InsP3-evoked Ca2+ transient. Mitochondrial inhibitors are known to decrease Ca2+ release from the InsP3-sensitive store in non-excitable cells, an effect attributed to mitochondrial regulation of cytosolic Ca2+ or ATP levels. For example, in lymphocytes and fibroblasts mitochondrial inhibitors decreased store-operated Ca2+ entry at the plasma membrane (Hoth et al. 1997; Landolfi et al. 1998). This has led to the proposal that mitochondrial Ca2+ uptake maintains low subsarcolemma Ca2+ levels, enabling prolonged store-operated Ca2+ entry. Additionally, in Xenopus oocytes and cultured oligodendrocytes, inhibition of mitochondrial uptake has led to alterations of local Ca2+ in the vicinity of the InsP3 receptor so modulating receptor opening in response to InsP3 (Jouaville et al. 1995; Simpson & Russell, 1996). Mitochondrial inhibitors of Ca2+ uptake may decrease store-operated Ca2+ entry or Ca2+ pump activity in the internal store by localized ATP depletion, alteration of the ATP/ADP ratio or increases in ADP. These activities may also modify InsP3-evoked Ca2+ release (Gamberucci et al. 1994; Mariot & Mason, 1995; Innocenti et al. 1996; Landolfi et al. 1998).
In the present study, mitochondrial depolarization with CCCP significantly reduced the InsP3-evoked Ca2+ transient. However, neither the Ca2+ store content, as evidenced by the magnitude of ionomycin-evoked Ca2+ transients, nor the SR Ca2+ pump activity, as measured by the magnitude of the undershoot, were altered by the mitochondrial inhibitors. These results suggest that neither store refilling nor ATP levels were significantly altered by disruption of mitochondria. Rather, the results are consistent with the regulation of Ca2+ near the InsP3 receptor by the drugs. Hence following InsP3-evoked Ca2+ release, local high concentrations of [Ca2+]c around the InsP3 receptor may have decreased the release of Ca2+ from the SR because of the Ca2+ dependency of the InsP3 receptor (Bezprozvanny et al. 1991; Hirose et al. 1998). Mitochondrial Ca2+ uptake, following InsP3-evoked Ca2+ release, by maintaining a local low [Ca2+]c, facilitates the release of Ca2+ by the InsP3 receptors.
Mitochondria remain important in Ca2+ homeostasis despite their uniporter having a low affinity for Ca2+ (Gunter & Pfeiffer, 1990). At least two explanations may reconcile this apparent anomaly; (a) the uniporter's affinity in vivo may be much higher than that determined from in vitro studies (see Gunter & Gunter, 1994 for references) and (b) higher concentrations of Ca2+ may reside nearer the mitochondria than elsewhere. Indeed, the uptake of Ca2+ by mitochondria in HeLa cells and hepatocytes, despite their low affinity for Ca2+, was attributed to microdomains of high Ca2+ near InsP3 receptors and/or Ca2+ channels in the plasma membrane (Rizzuto et al. 1994). Interestingly, the distribution of mitochondria within the cell may be related to the metabolic requirements of the cell and is far from random. In the endothelial cell line (ECV304), for example, less than 4 %, and in HeLa cells 65 % of mitochondria were within 700 nm of the endoplasmic reticulum. In ECV304 cells, 14 % and in HeLa cell less than 6 % of the mitochondria lay within 700 nm of the inner surface of the plasma membrane (Lawrie et al. 1996). Examination the of spatial relationship between the mitochondria and endoplasmic reticulum (ER) in HeLa cells revealed that the former comprised a large interconnected tubular network with numerous close contacts with the ER (Rizzuto et al. 1998). One functional consequence of this anatomical arrangement was that, on the release of Ca2+ from the ER by InsP3, the mitochondria were exposed to a higher concentration of Ca2+ than the cytosol (Rizzuto et al. 1998). The close apposition of mitochondria and SR in colonic myocytes is also suggested from the present results. [Ca2+]c overshoots, after depolarizations, were increased by blocking SR Ca2+ accumulation. Thus, both the accumulation of Ca2+ by the mitochondria and its efflux from them to the SR, may be facilitated by the close apposition of mitochondria and SR. The most obvious consequence of this strategic localization, as revealed by the present study, is the control by mitochondria of InsP3-evoked Ca2+ release as a result of their regulating the local [Ca2+]c in the vicinity of the InsP3 receptor.
Evidence for the involvement of mitochondria in the present study, relied, in part, on the use rhod-2 AM, a lipophilic cationic dye which largely accumulates in the organelle (e.g. Babcock et al. 1997; Peng et al. 1998). Although a fraction of the dye would have remained in the cytosol, even after dialysis, there is little likelihood that the depolarization-evoked rhod-2 signal was cytosolic in origin. Immediately after the test depolarization, CCCP was applied and the depolarization repeated some 3-4 min later. CCCP blocked the depolarization-evoked rhod-2 signal (Fig. 5); had the latter been cytosolic in origin it would have been increased by the drug (Figs 3 and 4).
The view that mitochondria are important in the regulation of cellular Ca2+ in the present study also depends on the selectivity of CCCP as an inhibitor of mitochondrial Ca2+ accumulation. One possible complication of its use is that SR Ca2+ accumulation may require a SR proton gradient which could also be disrupted by the protonophore. Although the plasmalemma Ca2+ pump transports H+ as a counter ion (see Carafoli, 1992 for references), whether or not the SR Ca2+ uptake involves such a mechanism is unclear (but see Inesi, 1985). Active transport of Ca2+ from the SR is electrogenic and could develop large membrane voltages (Meissner, 1981). A secondary passive outflow of K+ or H+ from the SR, following Ca2+ accumulation, maintains pump activity by balancing charge movements across the SR membrane. Under these conditions an increased proton permeability (with CCCP) would increase, rather than decrease, Ca2+ accumulation by the SR. Additionally, were CCCP to inhibit Ca2+ accumulation by the SR, then the effects of CCCP and thapsigargin on the Ca2+ transient should be similar. This was not observed; thapsigargin increased depolarization-evoked [Ca2+]c overshoots and blocked [Ca2+]c undershoots, while CCCP blocked only the overshoots. Nor are the effects of CCCP likely to have arisen from a change in cytosolic pH or the ATP: ADP ratio. Throughout the present investigation, cells were dialysed with a high concentration of Hepes buffer (30 mM) together with 3 mM ATP and 5 mM creatine phosphate to minimize these possibilities.
An increase in mitochondrial-matrix Ca2+ co-ordinates an increased production of ATP by mitochondria (McCormack et al. 1990; Denton & McCormack 1990; Duchen 1992; Hajnoczky et al. 1995; Rutter et al. 1996; Peuchen et al. 1996). During periods of elevated [Ca2+]c the resulting increased contractile behaviour will be matched by an increased ATP supply. The persistent elevation of [Ca2+]c, as a result of its efflux from the mitochondria, may also form part of a ‘pulse stretching mechanism’ (Park et al. 1996) that may be of particular significance in smooth muscle. During sustained activation, the largest increase in [Ca2+]c and myosin phosphorylation occurs generally in the first 60 s, after which both decline even though tone remains. This, the ‘latch state’, is characterized by a decreased rate of cross bridge cycling and ATP utilization and it enables smooth muscle to maintain force at relatively low [Ca2+]c. The mitochondrial export of Ca2+ may prolong the contractile response observed to transient activation during the latch state.
When resting [Ca2+]c was greater than 90 nM, Ca2+ transients undershot following depolarization. At higher [Ca2+]c, when presumably the cell is activated, the undershoot may act as a negative feedback system reducing contractile behaviour. Conversely the overshoot, at low [Ca2+]c when the cell is quiescent, by increasing [Ca2+]c may act as a positive feedback system to increase contractile activity.
Together the data presented here suggest that mitochondria play a central role in regulating Ca2+ in smooth muscle. Mitochondrial Ca2+ uptake and export, while presumably altering mitochondrial ATP production, may also regulate InsP3-evoked Ca2+ release by controlling the local Ca2+ concentration around the InsP3 receptor and possibly force production through persistent elevations in [Ca2+]c.