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(Circulation. 2002;105:2666.)
© 2002 American Heart Association, Inc.

Basic Science Reports

Diazoxide Opens the Mitochondrial Permeability Transition Pore and Alters Ca²⁺ Transients in Rat Ventricular Myocytes

Hideki Katoh, MD, PhD; Nobuhiro Nishigaki, PhD; Hideharu Hayashi, MD, PhD

From the Department of Internal Medicine III, Hamamatsu University School of Medicine, Hamamatsu, Japan.

Correspondence to Hideki Katoh, MD, PhD, Department of Internal Medicine III, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Hamamatsu, Japan 433-3192. E-mail hkatoh{at}hama-med.ac.jp

	Abstract

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Background— The mitochondrial K_ATP channel (mitoK_ATP) has been implicated as an end effector or trigger of ischemic preconditioning (IP). Although a mitoK_ATP opener, diazoxide, mimics IP, mechanisms for the cardioprotective action remain unclear.

Methods and Results— We measured Ca²⁺ transients (CaTs) and mitochondrial inner membrane potential (_m) with confocal microscopy and the fluorescent probes fluo-4 and tetramethylrhodamine ethyl ester perchlorate in rat ventricular myocytes. Diazoxide increased the amplitudes and diastolic levels of CaTs dose dependently. The effects of diazoxide on CaTs were inhibited by the mitoK_ATP antagonist sodium 5-hydroxydecanoic acid (100 µmol/L), whereas application of diazoxide caused little change in _m. After sarcoplasmic reticulum function was disabled with ryanodine and thapsigargin, the effects of diazoxide on CaTs were still observed. The opening of the mitochondrial permeability transition pore was monitored with fluorescent calcein. Diazoxide accelerated the leakage of calcein from mitochondrial matrix (16% of control; P<0.05), and this effect was inhibited by cyclosporin A (2 µmol/L). Cyclosporin A also abolished the effects of diazoxide on CaTs. Diazoxide oxidized flavoprotein fluorescence reversibly, and this effect was partially blunted by cyclosporin A (by 24%; P<0.05).

Conclusions— We conclude that in rat ventricular myocytes, diazoxide modulates the opening of the mitochondrial permeability transition pore, resulting in an increase in CaTs independent of the changes in _m. The action of diazoxide on the mitochondrial permeability transition pore also affects the mitochondrial redox state.

Key Words: myocytes • ischemia • calcium

	Introduction

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Ischemic preconditioning (IP) is a phenomenon in which transient nonlethal periods of ischemia increase the resistance to a subsequent prolonged ischemic period.¹ Various substances and signaling pathways could be involved in IP, including sarcolemmal and mitochondrial ATP-sensitive potassium channels (sarcoK_ATP and mitoK_ATP).² Emerging evidence has suggested the contribution of mitoK_ATP as an end effector or trigger of IP.^3–6 Although it has been demonstrated that diazoxide, a selective mitoK_ATP opener,^5,7 mimicked the effects of IP and that sodium 5-hydroxydecanoic acid (5-HD), a mitoK_ATP channel inhibitor, abolished the protective action of IP and diazoxide,^2,3,5 the precise mechanisms for the cardioprotective action of diazoxide remain elusive. It has been speculated that the opening of mitoK_ATP dissipates the mitochondrial inner membrane potential (_m), leading to a reduction in mitochondrial Ca²⁺ uptake.^5,8

Mitochondria play pivotal roles in the maintenance of cellular Ca²⁺ homeostasis.⁹ Ca²⁺ uptake occurs through the Ca²⁺ uniporter driven by _m. The mechanisms for Ca²⁺ efflux are Na⁺/Ca²⁺ exchange (NCX) and Ca²⁺/H⁺ exchange.¹⁰ The opening of the proteinaceous pore in the mitochondrial membrane, ie, the mitochondrial permeability transition pore (mPTP), causes permeabilization of the mitochondrial membrane and provides another pathway for Ca²⁺ efflux.¹¹ Cyclosporin A (CsA) is known as a specific inhibitor of mPTP. Classically, irreversible opening of mPTP has been implicated as an early event in lethal cell damage (ie, apoptosis).^12,13 Recent studies, however, suggested that mPTP could contribute to Ca²⁺ homeostasis under physiological conditions.¹⁴

In this study, we aimed to investigate the changes in _m and Ca²⁺ transients (CaTs) by diazoxide in intact myocytes and showed that diazoxide modulated mPTP and increased Ca²⁺ transients via a CsA-sensitive pathway.

	Methods

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Cells and Solutions
Rat ventricular myocytes were prepared as described previously.¹⁵ Myocytes were placed in a chamber and superfused with standard Tyrode solution. All experiments were conducted at 22°C. Standard Tyrode solution contained (mmol/L) 140 NaCl, 6 KCl, 1 MgCl₂, 5 HEPES, 5.8 glucose, and 1 CaCl₂ (pH 7.4 with NaOH). Cells were field stimulated (at 1 Hz) with 2-ms voltage pulses of 1.5 times the threshold amplitude.

Measurement of CaTs and Ca²⁺ Sparks
Cells were loaded with 20 µmol/L fluo 4-AM for 20 minutes. Fluorescence imaging was performed with a laser scanning confocal microscope (LSCM; Oz, Noran) coupled to an inverted microscope (Axiovert S100, Zeiss) with a 63x water immersion objective (numerical aperture [NA]=1.3; Zeiss), excitation wavelength of 488 nm, and emission at >510 nm.

Image acquisition for quantitative analysis of CaTs was made in the line-scan mode (scanned at 250 lines/s), and CaTs were derived from averaged fluorescence intensities along the scanned line. In the measurement of Ca²⁺ sparks, [Ca²⁺]_i was calculated, and visually identified Ca²⁺ sparks were accepted if local Ca²⁺ changes exceeded 60 nmol/L with duration at half-amplitude >8 ms.¹⁵

Measurement of Membrane Potential in Mitochondria
To measure _m, cells were loaded with 100 nmol/L tetramethylrhodamine ethyl ester perchlorate (TMRE) for 20 minutes. Images of TMRE fluorescence at 514-nm excitation and >590-nm emission were obtained. TMRE is distributed between cellular compartments according to Nernst’s equation.¹⁶ For the measurement of time-dependent TMRE fluorescent changes, images were recorded every 30 seconds (illumination time was 480 ms/image). Fluorescence was integrated over regions of interest (60x60 pixels), placed over the bright portion of myocytes.

Imaging of mPTP Opening With Calcein
Cells were loaded with 1 µmol/L calcein-AM for 20 minutes, and the quenching of cytosolic calcein was achieved by addition of 5 mmol/L CoCl₂ to the solution.¹⁷ Images of calcein fluorescence (excitation at 488 nm and emission at 505 to 550 nm) were recorded, and fluorescent intensity of the region of interest was integrated.

Measurement of Flavoprotein Fluorescence
Mitochondrial redox state was assessed by measurement of fluorescence of FAD-linked enzymes.⁵ Endogenous flavoprotein fluorescence images were recorded (excitation wavelength at 488 nm and emission at >505 to 550 nm).

Statistical Analyses
Results are expressed as mean±SEM for the indicated number of myocytes from at least 3 animals. Statistical significance was determined by paired t test or ANOVA. Values of P<0.05 were considered significant.

	Results

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Diazoxide Increases CaTs and Ca²⁺ Sparks
Figure 1A demonstrates that perfusion of diazoxide (200 µmol/L) for 10 minutes increased diastolic level and the amplitudes of CaTs. Dose-dependent effects of diazoxide on CaTs are summarized in Figure 1B, which shows that concentrations of diazoxide >200 µmol/L altered CaTs. Thus, subsequent experiments were conducted with 200 µmol/L diazoxide, unless otherwise stated. Diazoxide also increased resting Ca²⁺ spark frequency from 37.9±12.8 sparks · pL^-1 · s^-1 to 56.9±13.8 sparks · pL^-1 · s^-1 (P<0.05; n=5; Figures 1C and 1D).

View larger version (45K): [in this window] [in a new window]   Figure 1. Effects of diazoxide on Ca2+ transients and resting spark frequency. A, Cells were exposed to diazoxide (200 µmol/L) and field stimulated at 1 Hz. CaTs were recorded at 10 minutes after application of diazoxide. B, Dose-dependent effects of diazoxide on CaTs. Diazoxide was applied cumulatively to solution. Data represent mean±SEM from 4 experiments. C, Confocal line scan images of Ca2+ sparks were converted to [Ca2+]i by pseudoratio method.15 D, Pooled data from 5 experiments. F/F0 indicates normalized fluorescent intensity, where F0 indicates basal fluorescence; ctl, control; Dz, diazoxide; and wo, washout. *P<0.05 vs control (paired t test).

Next, we investigated whether SR Ca²⁺ content and the activity of sarcolemmal NCX were affected by diazoxide. After field stimulation was omitted, caffeine (10 mmol/L) was applied rapidly (Figure 2A), and the amplitude of caffeine transients (CafTs) was measured as an estimate of SR Ca²⁺ content. Figure 2B demonstrated that diazoxide increased the amplitude of CafTs slightly but significantly (by 5.0±0.2% versus control; P<0.05; n=8). The declining phase of CafTs was fitted to single exponential decay to index Ca²⁺ efflux via NCX. There were no effects of diazoxide on Ca²⁺ efflux via NCX, because the time constants of the CafT decline were not changed by diazoxide (Figure 2C).

View larger version (14K): [in this window] [in a new window]   Figure 2. Diazoxide increased SR Ca2+ content but did not alter sarcolemmal NCX activity. A, CafTs (caffeine transients). After cessation of field stimulation, caffeine (10 mmol/L) was applied rapidly to evoke CafTs. B, Pooled data of amplitude of CafTs (as index of SR Ca2+ content). C, Pooled data of time constant of decay of CafTs (as index of Ca2+ efflux via sarcolemmal NCX). Data are mean±SEM from 8 experiments. ctl indicates control; Dz, diazoxide; and WO, washout. *P<0.05 vs control (paired t test).

To address whether increased CaTs could be attributed to the direct effects of diazoxide on SR, we applied diazoxide under conditions such that SR function was inhibited. Cells were pretreated with ryanodine (5 µmol/L) and thapsigargin (5 µmol/L) for 15 minutes, and then diazoxide was applied. The effect of diazoxide on CaTs was not attenuated under these conditions (Figures 3A and 3B), which indicates that the effects of diazoxide on CaTs were not mediated directly via SR.

View larger version (19K): [in this window] [in a new window]   Figure 3. SR did not contribute to effects of diazoxide. A, Application of diazoxide for 10 minutes increased [Ca2+]i (in which SR function was abolished by 5 µmol/L thapsigargin and 5 µmol/L ryanodine). B, Summarized pooled data from 4 experiments. Diastolic levels of F/F0 were compared. F/F0 indicates normalized fluorescent intensity, where F0 indicates basal fluorescence; ctl, control; Dz, diazoxide; and wo, washout. Data represent mean±SEM. *P<0.05 vs control (paired t test).

Diazoxide Does Not Alter Mitochondrial Membrane Potential
It has been speculated that the opening of mitoK_ATP would dissipate _m, leading to decreased driving force for the Ca²⁺ uniporter.⁸ The reduction of mitochondrial Ca²⁺ uptake could increase cytosolic Ca²⁺. Thus, we monitored the changes in _m before and after application of diazoxide. As shown in Figure 4A, diazoxide (500 µmol/L) did not alter _m. Application of FCCP (2 µmol/L) after washout of diazoxide decreased the TMRE signal remarkably (the same results were observed in 3 other cells). We next investigated whether 5-HD could inhibit the effect of diazoxide on CaTs. In control experiments, reapplication of diazoxide after the first exposure and washout caused the same increase in CaTs as the initial application (data not shown). When cells were treated with 5-HD (100 µmol/L) for 10 minutes before the second application, subsequent application of diazoxide in the presence of 5-HD did not increase CaTs (Figures 4B and 4C). These results indicated that although opening of mitoK_ATP could be involved, depolarization of _m was not correlated with the observed increase in CaTs.

View larger version (25K): [in this window] [in a new window]   Figure 4. Diazoxide did not alter mitochondrial membrane potential, but effects of diazoxide on CaTs were inhibited by 5-HD. A, Representative recording of TMRE signal after application of diazoxide (500 µmol/L) and FCCP (2 µmol/L). TMRE signal was expressed as % of initial intensity. Drugs were added as indicated in bars. B, After application and washout of diazoxide, cells were treated with 5-HD (100 µmol/L) for 10 minutes, and diazoxide was reapplied with 5-HD. C, Pooled data from 4 cells. F/F0 indicates normalized fluorescent intensity, where F0 indicates basal fluorescence; ctl, control; and Dz, diazoxide. Data represent mean±SEM. *P<0.05 vs control (paired t test).

CsA-Sensitive Pathway and CaTs
A previous study with isolated mitochondria showed that diazoxide released Ca²⁺ from mitochondria via a CsA-sensitive mechanism.⁸ Thus, we examined whether CsA inhibits the effects of diazoxide on CaTs. After exposure and washout of diazoxide, cells were treated with 2 µmol/L CsA for 15 minutes. Reapplication of diazoxide with CsA did not increase CaTs, which indicates that a CsA-sensitive mechanism was responsible for the increase in CaTs (Figures 5A and 5B). Because CsA is a potent inhibitor of mPTP, we investigated the effects of diazoxide on mPTP by measuring calcein signal as an index for mPTP opening.¹⁷ Perfusion of Tyrode solution for 10 minutes decreased the calcein signal by 6.3%, and perfusion of Tyrode plus CsA (2 µmol/L) caused only a slight decrease in signal intensity (by 1.9%; data not shown). Diazoxide decreased the calcein signal by 16% (P<0.05), and there was little change when diazoxide was applied with CsA (2.0%; Figure 5C). These results suggested that there was a CsA-sensitive leakage of calcein from mitochondria and that diazoxide accelerated this leakage significantly.

View larger version (23K): [in this window] [in a new window]   Figure 5. CsA inhibits effects of diazoxide on Ca2+ transients. A, After application and washout of diazoxide, cells were incubated with CsA (2 µmol/L) for 10 minutes. Subsequent application of diazoxide with CsA did not increase CaTs. B, Pooled data from 4 experiments. Data represent mean±SEM. P<0.05 vs control (paired t test). C, Time courses of changes in calcein signal during perfusion of control Tyrode solution () and during perfusion of diazoxide in the presence () and absence () of CsA (2 µmol/L). n=4. *P<0.05 vs diazoxide plus CsA () and P<0.05 vs Tyrode’s () (ANOVA). F/F0 indicates normalized fluorescent intensity, where F0 indicates basal fluorescence; ctl, control; and Dz, diazoxide.

CsA-Sensitive Pathway and Flavoprotein Oxidation
To investigate the possible link between diazoxide and the mitochondrial metabolic state, we measured flavoprotein fluorescence to index the mitochondrial redox state. The redox signal was calibrated by exposure of the cells to 2,4-DNP (100 µmol/L) followed by sodium cyanide (2 mmol/L).⁵ As shown in Figure 6A, diazoxide reversibly induced oxidation of flavoprotein to 37% of the DNP value, and CsA (2 µmol/L) partially attenuated the oxidative effect of diazoxide by 24% (P<0.05, n=4; Figure 6B).

View larger version (20K): [in this window] [in a new window]   Figure 6. Oxidative effect of diazoxide (Dz) is partially inhibited by CsA (2 µmol/L). A, Representative recording of changes in flavoprotein signal. Signal was normalized as 100% for 2,4-DNP (100 µmol/L)-induced maximum oxidation and 0% for sodium cyanide (CN; 2 mmol/L)-induced complete reduction. B, Pooled data from 4 experiments (mean±SEM). *P<0.05 vs CsA(-) (paired t test).

	Discussion

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In this study, we demonstrated that diazoxide increased CaTs, CafTs, and Ca²⁺ spark frequency without causing significant changes in _m, and CsA attenuated the effects of diazoxide on CaTs. Diazoxide caused flavoprotein oxidation, which was partially inhibited by CsA. These results indicate the possible involvement of a CsA-sensitive mechanism for the effects of diazoxide on cytosolic Ca²⁺ homeostasis and the mitochondrial redox state.

Diazoxide and CaTs
Here, we showed that diazoxide increased CaTs dose dependently and that >200 µmol/L was required, which was close to the reported concentration range of diazoxide for cardioprotection (10 to 100 µmol/L). However, a higher concentration of diazoxide could have unexpected toxic effects on mitochondrial function as a metabolic inhibitor and a protonophore.¹⁸ We have observed that even at 10 µmol/L, diazoxide has effects in skinned myocytes (unpublished data). Therefore, the reduced accessibility of diazoxide to mitochondria in intact myocytes might account for the relatively higher concentration. An increase in [Ca²⁺]_i by diazoxide has been reported previously.^6,19

Several mechanisms could be involved in the increase in CaTs, such as (1) increased Ca²⁺ entry via sarcolemmal Ca²⁺ channels, (2) decreased Ca²⁺ efflux via NCX and Ca²⁺ pump, and (3) increased Ca²⁺ release from SR. Here, we demonstrated that diazoxide did not alter Ca²⁺ efflux via NCX (Figure 2C). In rat myocytes, SR dominates for the regulation of [Ca²⁺]_i during twitch, and only small portions of Ca²⁺ were extruded via the Ca²⁺ pump.²⁰ It has been shown that diazoxide does not affect I_Ca.¹⁹ Therefore, neither the changes in Ca²⁺ influx or Ca²⁺ efflux via sarcolemma could contribute to the increased CaTs. In addition, there was no direct action of diazoxide on SR (Figure 3). Thus, diazoxide does not likely modulate these Ca²⁺ handling systems. Wang et al⁶ reported that blockade of CaTs by verapamil in diazoxide-pretreated hearts abolished cardiac protection and suggested the possible involvement of sarcolemmal Ca²⁺ channels for activation of mitoK_ATP by diazoxide. Because we showed that Ca²⁺ release from SR was unlikely to be involved as a source of elevated Ca²⁺, the changes in I_Ca (as a trigger of Ca²⁺-induced Ca²⁺ release) by diazoxide might not alter CaTs significantly. The different experimental conditions (myocytes or Langendorff-perfused hearts and the existence of ischemia during protocols) may explain these differences.

Diazoxide and Mitochondrial Membrane Potential
Previous reports suggested that opening mitoK_ATP depolarized _m and reduced mitochondrial Ca²⁺ uptake.^5,8 Our results, however, did not support this hypothesis, because there were no detectable changes in _m by diazoxide. There are possible explanations for undetected changes in _m. First, the K⁺ influx through mitoK_ATP could be compensated rapidly enough that we could not follow the changes in _m.²¹ Second, depolarization of _m was too small to be detected by TMRE. Lawrence et al²² reported that similar concentrations of diazoxide as used in this study (100 to 200 µmol/L) did not change _m but protected myocytes from metabolic inhibition. Another study, however, demonstrated that cardioprotective effects of diazoxide during ischemia/reperfusion have been associated with mild depolarization of _m.²³ In the experiments with isolated mitochondria and neonatal cardiac myocytes, diazoxide depolarized _m by 15 mV,⁹ whereas Kowaltowski et al¹⁸ reported that estimated changes of _m from calculated K⁺ influx through mitoK_ATP were only 1 to 2 mV. Different experimental conditions and different methods could account for these discrepancies.

Diazoxide and mPTP Opening
The loading of cells with calcein-AM plus cobalt to assess mPTP opening was initially reported in hepatocytes¹⁷ but not in cardiac myocytes. To confirm that the calcein signal originated from mitochondria, we compared the fluorescent intensities before and after saponin permeabilization of the sarcolemma and found that reduction of the calcein signal was <10%. We have also observed that calcein fluorescence decreased when cells were coloaded with TMRE, in agreement with an original report¹⁷ (data not shown). Taken together, these results indicate it is likely that the calcein signal mainly originated from mitochondria. We showed that diazoxide accelerated the calcein leakage from mitochondria, and these effects were attenuated by CsA, which suggests the possible action of diazoxide on mPTP.

In our experimental conditions, CsA alone did not alter CaTs (data not shown), and CsA per se had few effects on SR and NCX in rat myocytes.²⁴ Thus, our results that CsA inhibited the effect of diazoxide on CaTs support the idea that mPTP might contribute to the increase in CaTs.

The opening of mPTP has been associated with dissipation of _m, matrix swelling, and uncoupling of oxidative phosphorylation and plays a key role in apoptosis by releasing cytochrome c.^13,25 It is recognized that in ischemia/reperfusion injury, some cells undergo apoptotic cell death as opposed to necrosis and that mPTP may act as the trigger for apoptosis. In this regard, closing but not opening the mPTP is ideal to prevent cell death. This might be the case in irreversible opening of the mPTP. Diazoxide was reported to inhibit the release of cytochrome c and prevented apoptosis in myocytes exposed to hydrogen peroxide.²⁶ However, isolated mitochondria have been reported to release cytochrome c in response to K_ATP openers.²⁷

Evidence has revealed the existence of the flickering of mPTP (low-conductance state).^12,28–30 A brief and reversible mPTP opening does not cause mitochondrial swelling, is not related to the release of cytochrome c, and might serve as a physiological means of ridding mitochondria of excess metabolites or ions, in particular Ca²⁺. Our results indicate that diazoxide could induce a low-conductance state of mPTP and release Ca²⁺ from mitochondria, which is a different phenomenon from irreversible mPTP opening. Previous studies demonstrated that a mPTP-related Ca²⁺ release occurred in conditions in which mitochondria were relatively Ca²⁺ overloaded.⁸ However, it is not likely that mitochondrial Ca²⁺ concentrations ([Ca²⁺]_m) were heavily loaded in the present study. Although Ca²⁺ efflux pathways with low-conductance mPTP remain unresolved, both mPTP per se and the reversed mode of the uniporter might be involved.^10,28

Investigation of [Ca²⁺]_m is essential to elucidate the action of diazoxide on mitochondria. However, [Ca²⁺]_m measurement with confocal microscopy and a Ca²⁺ indicator (eg, Rhod-2) in intact myocytes has not been established.³¹

The mechanism for the action of diazoxide on mPTP is poorly understood. It has been suggested^14,28 that low-conductance mPTP is principally operated by matrix pH (elevated pH opens mPTP) rather than Ca²⁺. In relation to this, it has been postulated that opening of mitoK_ATP causes K⁺ influx, and this charge could be compensated for by proton extrusion, leading to raised matrix pH, which triggers low-conductance mPTP. Additional studies are necessary to clarify the precise mechanisms involved.

Regardless of the duration of opening, the mPTP opening must be accompanied by the depolarization of _m. However, Petronilli et al³² reported that a short mPTP opening was detected only by the calcein-plus-cobalt method, whereas TMRM distribution required longer mPTP opening. Another problem is the production of reactive oxygen species, which are potent mPTP-inducing agents,^12,13,30,33 by exposure to the laser. Thus, we shortened laser excitation duration, and the intervals for each recording were prolonged to 30 seconds.

The released Ca²⁺ from mitochondria could fill the SR and provide sufficient changes of [Ca²⁺]_i in a tiny localized space to activate ryanodine receptors. This could explain the increased SR Ca²⁺ contents and Ca²⁺ spark frequency after the addition of diazoxide. Ichas et al¹⁴ suggested the interaction between mitochondria and SR within restricted space as the mechanism termed mitochondrial Ca²⁺-induced Ca²⁺ release.

MPTP and Mitochondrial Redox State
We demonstrated that diazoxide increased flavoprotein oxidation and that CsA partially inhibited this effect of diazoxide (Figures 6A and 6B). Thus, although the exact process is unknown, the effects of diazoxide on mPTP could be related to the mitochondrial redox state. Because [Ca²⁺]_m stimulates key enzymes for NADH regulation,³⁴ the reduced [Ca²⁺]_m may inhibit NADH oxidation. Thus, the diazoxide-induced low-conductance mPTP could serve as an endogenous uncoupler of oxidative phosphorylation.³⁵

In summary, we provide evidence that diazoxide modifies the low-conductance state of mPTP and that there are possible links between mPTP, [Ca²⁺]_i, and the mitochondrial redox state in intact myocytes. Additional studies are required to elucidate the underlying mechanisms between the diazoxide-induced mPTP opening and cardioprotective actions.

Received January 21, 2002; revision received March 13, 2002; accepted March 14, 2002.

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