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(Circulation. 1997;95:503-510.)
© 1997 American Heart Association, Inc.

Articles

Regulation of Spontaneous Transient Outward Potassium Currents in Human Coronary Arteries

Rostislav Bychkov, PhD; Maik Gollasch, MD, PhD; Christian Ried, MS; Friedrich C. Luft, MD; Hermann Haller, MD

the Franz Volhard Clinic and the Max-Delbruck Center for Molecular Medicine, Virchow Clinic, Humboldt University of Berlin, Germany.

Correspondence to Friedrich C. Luft, MD, Franz Volhard Clinic, Wiltberg Str 50, Berlin, Germany. E-mail fcluft{at}orion.rz.mdc-berlin.de

	Abstract

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Background Spontaneous transient outward potassium currents (STOCs) induce myogenic relaxation in small cerebral vessels. We found STOCs in human coronary artery vascular smooth muscle cells (VSMCs) and studied their regulation.

Methods and Results K⁺ currents were recorded in human coronary VSMCs by current- and voltage-clamp techniques. STOCs were recorded in the presence of 200 µmol/L Cd²⁺ and 10 µmol/L verapamil, which block voltage-dependent Ca²⁺ channels. STOCs were inhibited by iberiotoxin (100 nmol/L), a selective blocker of Ca²⁺-activated potassium channels (BK_Ca), and disappeared in a Ca²⁺-free bath. Iberiotoxin depolarized the VSMCs within 20 minutes from -44±7 to -18±5 mV (n=17). The Ca²⁺ ionophore A23187 increased intracellular Ca²⁺ and stimulated whole-cell BK_Ca current. Depletion of Ca²⁺ from the sarcoplasmic reticulum with caffeine (4 mmol/L) abolished STOCs for several minutes. Ryanodine (50 µmol/L) transiently stimulated STOCs but then completely inhibited STOCs within 10 minutes. The firing frequency of STOCs was directly correlated with intracellular Na⁺ concentrations from 0 to 24 mmol/L. Lowering intracellular Na⁺ to zero abolished STOCs. We next gave monensin (30 µmol/L) to increase intracellular Na⁺. This maneuver resulted in an increase in whole-cell current fluctuations and STOCs. Monensin-induced STOCs were abolished by either lowering extracellular Ca²⁺ to zero or chelating Ca²⁺ intracellularly with BAPTA-AM (30 µmol/L).

Conclusions STOCs resulted from BK_Ca activity and were dependent on extracellular Ca²⁺ but not significantly on voltage-dependent Ca²⁺ channels. STOCs were dependent on intracellular Na⁺ and intracellular calcium store refilling state. We suggest that Ca²⁺ entry into the cell through reverse-mode Na⁺/Ca²⁺ exchange determines calcium store refilling, which in turn regulates STOC generation in human coronary VSMCs.

Key Words: potassium • calcium • cells • muscle, smooth

	Introduction

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Potassium channels are a diverse group that fulfill important cellular functions, including regulation of membrane excitability, resting membrane potential, action potential waveforms and frequency, excitation threshold, and vascular tone. We recently characterized five different kinds of vascular K⁺ currents in human coronary artery VSMCs.¹ During our studies, we observed STOCs, which had not been described previously in human coronary arteries. STOCs influence myogenic arterial constriction and thus affect resistance and flow.² Recently, Nelson et al³ were able to show in small cerebral arteries that STOCs are most likely responsible for a specific function, namely vasodilation. Their seminal work indicated that fast, fluctuant increases in local subsarcolemmal Ca²⁺ concentrations called "Ca²⁺ sparks" are important in STOC generation. Functional coupling of ryanodine-sensitive release units of the sarcoplasmic reticulum and the plasmalemmal BK_Ca channels is involved in the production of STOCs. We studied the regulation of STOCs in human coronary VSMCs and found that mainly Ca²⁺ entry into the cell through reverse-mode Na⁺/Ca²⁺ exchanger determines calcium store refilling, which in turn regulates STOC generation in human coronary VSMCs.

	Methods

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Human coronary arteries were obtained from hearts of patients with heart failure (dilated cardiomyopathy without significant arteriosclerosis) after orthotopic heart transplantation and from one nonfailing donor heart that could not be transplanted for technical reasons. The tissue was immediately placed in cold (8°C) Hanks' solution (in mmol/L: NaCl 119, KCl 4.7, KH₂PO₄ 1.2, NaHCO₃ 25, MgSO₄ 1.2, glucose 11.1, EDTA 0.026, and CaCl₂ 2.5, plus 5% CO₂ and 95% O₂) during transportation to the laboratory for further dissection. Left coronary arteries, right coronary arteries, or branches from left, right, or circumflex coronary arteries (diameter, 1.5 mm) were dissected and cleaned of adhering tissue and fat in Hanks' solution. All coronary arteries were stored in Hanks' solution at 4°C before use and were studied within 48 hours. Smooth muscle cells were isolated as previously described for porcine and human coronary artery, with some modifications.^{1 4} The vessels were cut into small segments (4 to 8 mm long) and placed in a Ca²⁺-free Hanks' solution (in mmol/L: NaCl 137, KCl 5.4, KH₂PO₄ 0.44, NaH₂PO₄ 0.42, MgCl₂ 2, Ca²⁺ 0.05, glucose 11.1, and HEPES 10; pH 7.4 with NaOH) for 2 to 4 minutes at room temperature (20°C to 24°C). After a longitudinal section was cut, the segments were washed twice in this solution. The segments were then placed in the Ca²⁺-free solution containing 3 mg/mL collagenase (Sigma, type IA), 10 mg/mL BSA (Sigma), and 1 mg/mL elastase (Sigma, type IIA) and were incubated for 30 to 50 minutes with gentle agitation in the Ca²⁺-free Hanks' solution. The cells were stored in Hanks' solution (Ca²⁺, 0.12 mmol/L) containing 1 mg/mL BSA or in a solution containing (in mmol/L) NaCl 90, KH₂PO₄ 1.2, MgCl₂ 5, glucose 5, taurine 20, and HEPES 5; pH 7.1 with NaOH at 4°C. Whole-cell K⁺ currents were measured according to the conventional patch-clamp method or the perforated-patch method with nystatin. The pipette solution contained (in mmol/L) potassium aspartate 80, KCl 50, NaCl 12, MgCl₂ 1, magnesium ATP 3, EGTA 10, potassium HEPES 5; pH 7.4. The extracellular solution contained (in mmol/L) NaCl 140, CaCl₂ 1.8, MgCl₂ 1, KCl 5.4, CdCl₂ 0.1 (or 0.2), glucose 10, and sodium HEPES 10; pH 7.4. Nystatin (Sigma) was dissolved in DMSO and diluted into the pipette solution to give a final concentration ranging from 50 to 100 µg/mL. A detailed description of the patch-clamp technique is given elsewhere.^{5 6}

Materials
Iberiotoxin was obtained from RBI. EGTA, HEPES, A23187, ryanodine, and caffeine were purchased from Sigma-Aldrich. All salts were obtained from Merck. BAPTA-AM 30 mmol/L, a Ca²⁺ chelator that diffuses through membranes (Calbiochem), was used to chelate intracellular Ca²⁺.

Statistical Analysis
All values are given as mean±SEM. We performed parametric (Student's t test) and nonparametric (Mann-Whitney test) and linear regression analysis as appropriate. A value of P<.05 was considered significant. The terms increase and decrease are used only when the results were statistically significant.

	Results

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Electrophysiological Properties of STOCs
To investigate the ionic mechanism involved in the generation of STOCs, we performed perforated patch-clamp experiments with nystatin (n=124); 70% of all cells investigated possessed STOCs. The VSMCs exhibiting STOCs had resting membrane potential of -52±15 mV (n=21). Spontaneous transient negative hyperpolarization potentials (negative voltage spikes) were observed in our cells under current-clamp conditions. The decay phase of the negative voltage spikes was fitted by means of a multiexponential function. The duration of negative spikes varied from 50 to >100 ms, and their amplitude varied from 2 to 15 mV (Fig 1A). Iberiotoxin (100 nmol/L) completely blocked generation of negative spikes and depolarized the VSMCs within 20 minutes from -44±7 to -18±5 mV (n=17).

View larger version (32K): [in this window] [in a new window]   Figure 1. Spontaneous transient voltage hyperpolarizations and STOCs in human coronary VSMCs. A, Spontaneous transient voltage changes from -45 to -60 mV recorded from single VSMC with nystatin whole-cell patch-clamp method. Hyperpolarization duration ranges from 50 to 300 ms. B, STOCs recorded at steady-state membrane potentials of -30 and -10 mV, respectively. Data were filtered at 10 kHz and sampled at 1 kHz. STOC amplitude and frequency increased with membrane depolarization. Inset, Four amplitude levels of STOCs are visible. Duration of STOCs was in the range of 80 to 200 ms. C, AUC was calculated for characterization of STOC amplitudes and firing frequencies at different membrane potentials. Every point in graph is an average value for five different cells. Lengths of recordings used for plots were 5 to 7 minutes (A). Experimental points were fitted with linear regression (r=.9): AUC=21.7 pA·s+0.36(pA·s/mV)·Vm, where Vm is membrane potential in mV.

Measurement of the amplitudes from individual STOCs revealed that each cell generated STOCs with several amplitudes and uniform duration of 121±35 ms (n=7) at all ranges of physiological potentials from -60 to +20 mV (Fig 1B). STOC amplitude and frequency increased with increasing membrane potential (Fig 1B). Fig 1B, bottom, shows a composite picture of the discharges. A superimposition of STOCs, which occurred when the next discharge appeared during the decay phase of the previous discharge (latency period, <100 ms), increased the STOC duration up to 200 to 400 ms. The individual STOC duration was stable and was independent of the membrane potential. To show the integral characteristics, including discharge frequency and amplitude changes at different membrane potentials, we calculated the AUC of current traces recorded in steady-state potentials. AUC values were obtained from traces recorded at different holding potentials from five different cells. The values are plotted as a function of membrane potential (Fig 1C). The reversal potential of STOCs obtained by linear fit was -65 mV.

Pharmacological Characterization of STOCs
Iberiotoxin (100 nmol/L) (Fig 2A, top), a specific blocker of BK_Ca, abolished STOCs completely (n=9). STOCs were also blocked completely with the addition of BAPTA-AM 30 µmol/L (n=10) (Fig 2A, middle). Removal of external Ca²⁺ depressed STOCs completely (n=12) within 1 minute (Fig 2A, bottom), suggesting that Ca²⁺ influx from external sources is important for the generation of STOCs. STOCs were observed not only in steady-state holding potentials but also superimposed onto K⁺ currents elicited by ramps (from -100 to +100 mV) or by step voltage pulses in the perforated patch configuration (Fig 2B, top left). The STOCs had a duration of 100 ms, and their amplitude increased when the membrane potential became increasingly positive. Threshold activation of macroscopic current fluctuations (STOCs superimposed onto currents elicited by voltage step pulses or ramps) was about -59±8 mV (n=17). Spontaneous fluctuations of whole-cell currents were abolished when the cells were placed in a Ca²⁺-free solution (n=14) (Fig 2B, bottom left). The AUC, calculated for every trace of current induced by ramp pulses with a rate of 0.2 Hz, was plotted as a function of time (Fig 2B, top right). AUC fluctuations disappeared completely in Ca²⁺-free solution (Fig 2B, bottom right). BAPTA-AM and iberiotoxin also decreased the amplitude of whole-cell currents and blocked STOCs superimposed on potassium currents elicited by ramps or voltage pulses (data not shown). The calcium ionophore A23187 (30 µmol/L) appeared to induce superposition of STOCs several minutes after application (data not shown). Ca²⁺ influx with the calcium ionophore also increased the whole-cell current amplitude from 220±26 to 367±31 pA, with a shift in the threshold activation from -30±11 to -60±9 mV (n=12). STOCs were never observed after full activation of calcium-dependent potassium currents induced by Ca²⁺ influx through the ionophore, either during steady-state potentials or during whole-cell current configurations elicited by voltage pulses.

View larger version (27K): [in this window] [in a new window]   Figure 2. Pharmacological characterization and dependency of STOCs on external Ca2+. After internal Ca2+ storage depletion of isolated smooth muscle cells in Ca2+-free external solution for 1 hour, cells were exposed in experimental chamber to Ca2+ (1.8 mmol/L)–containing solution containing 200 µmol/L CdCl2 to block Ca2+ influx through voltage-dependent Ca2+ channels. A, Top, STOCs were blocked by iberiotoxin (100 nmol/L), which blocks BKCa. A, Middle, Membrane-diffusible calcium chelator BAPTA-AM (30 µmol/L) blocked STOCs, further suggesting generation of STOCs through BKCa by transient increase of internal Ca2+. A, Bottom, Removal of external Ca2+ depressed STOCs completely within 1 minute, indicating that Ca2+ influx from external solution was responsible for STOCs. B, Left, STOCs elicited by voltage ramp pulses disappeared after Ca2+ was removed from external solution. B, Right, Corresponding plot of AUC of STOCs as a function of time shows that STOCs depend on Ca2+ influx even when voltage-dependent Ca2+ channels are blocked. After removal of external Ca2+ (arrow), AUC was markedly decreased. Holding potential was -80 mV. Voltage ramp pulses depolarizing cell membrane from -100 to +100 mV within 400 ms were applied. Rate of pulses, 0.2 Hz. Similar results were observed for STOCs superimposed on whole-cell outward currents evoked by step pulses from -80 to -30 mV. Rate of pulses, 0.3 Hz.

Influence of Ryanodine and Caffeine on STOCs
We next examined a potential role for internal Ca²⁺ stores in STOC generation in more detail. To prevent Ca²⁺ influx through voltage-dependent Ca²⁺ channels⁷ and to discriminate a pure effect of Ca²⁺ release from internal stores, all experiments were performed with 200 µmol/L Cd²⁺ and 10 µmol/L verapamil in the bath solution. Cd²⁺ ions reduced the frequency of STOCs in human VSMCs by 18% (n=6) but had no effect on duration and amplitude of STOCs (Fig 3A). The durations were 99±35 ms and 104±30 ms before and after Cd²⁺, respectively (n=6). Subsequent application of verapamil (10 µmol/L), an organic voltage-dependent L-type Ca²⁺ channel blocker, to the bath solution had no effect or transiently decreased STOC amplitudes (compare Fig 3A versus 3B). The frequency and duration of STOCs, however, were not further reduced by verapamil (duration of STOCs in the presence of Cd²⁺ plus verapamil, 116±40 ms; n=6). Ryanodine (50 µmol/L) produced a transient stimulation (n=6) of STOC firing frequency (Fig 3A) and transiently increased the whole-cell potassium current. STOCs were completely blocked by 50 µmol/L ryanodine within 10 minutes. A ryanodine washout for >20 minutes never produced recovery of STOC firing frequency. To further test the notion of Ca²⁺ release from internal stores, we added caffeine (4 mmol/L) in the presence of Cd²⁺ and verapamil. The first application of caffeine (n=9) transiently increased steady-state current (hump), followed by a temporary inhibition of STOCs (Fig 3B). STOCs recovered to control values within 7 to 12 minutes after the removal of caffeine from the bath. After the internal Ca²⁺ stores were replenished and STOCs had recovered, caffeine again had a transient stimulatory effect on steady-state current (hump) (Fig 3B). Caffeine 4 mmol/L was not able to induce a transient stimulatory effect on steady-state current (hump) during caffeine-induced temporary inhibition of STOCs (not shown).

View larger version (79K): [in this window] [in a new window]   Figure 3. Effect of ryanodine and caffeine on STOCs. External Ca2+ was 1.8 mmol/L. A, Effects of ryanodine 50 µmol/L on STOCs in presence of Cd2+ 200 µmol/L and verapamil 10 µmol/L. B, Effects of caffeine 4 mmol/L on STOCs in presence of Cd2+ 200 µmol/L and verapamil 10 µmol/L. [Na+]i was 24 mmol. Presence of drugs is indicated by horizontal lines.

Intracellular Na⁺ and Intracellular and Extracellular Ca²⁺ and STOCs
To test for a possible role of the Na⁺/Ca²⁺ exchanger in the generation of STOCs, we used the sodium ionophore monensin to increase intracellular Na⁺ concentration.⁸ Monensin 30 mmol/L (n=9) increased STOC frequency and the duration of serial STOC discharges (Fig 4A, top and middle). The increased intracellular Na⁺ concentration did not affect the duration of STOC decay. Monensin also increased the amplitude of whole-cell outward currents evoked by voltage pulses from 245±26 to 394±47 pA (n=10) at +40 mV. When extracellular Ca²⁺ was reduced to zero (Fig 4A, bottom), monensin no longer had a stimulatory effect on STOCs. The same effect on the monensin-induced increase in STOCs was produced by BAPTA-AM (data not shown). The relationship between STOC AUC and the membrane potential with monensin was compared with the same relationship under control conditions (Fig 4B). A significantly steeper relationship with monensin compared with controls was identified.

View larger version (30K): [in this window] [in a new window]   Figure 4. Effect of internal Na+ concentration on STOCs. A, Top, Extracellular application of Na+ ionophore monensin (30 µmol/L) stimulates STOC frequency. Extracellular solution contained 0.2 mmol/L CdCl2 to block Ca2+ influx through voltage-dependent Ca2+ channels. External Ca2+ was 1.8 mmol/L. A, Bottom, After removal of external Ca2+, stimulatory effect of monensin disappeared. Steady-state membrane potential was -10 mV. All three current traces were recorded on same cell. B, Plot of AUC for STOC as function of steady-state membrane potential before () and after monensin (30 µmol/L, , n=5 to 7) showed stimulatory effect of monensin on STOC activity, indicating that increase in internal Na+ stimulates STOCs dependent on extracellular Ca2+ influx (see A, bottom). Data were fitted by linear regression (r=.9 in both cases) with AUC=14.1(pA·s)+0.22(pA·s/mV)·Vm and AUC=57.6(pA·s)+0.88(pA·s/mV)·Vm for data obtained before and after application of monensin, respectively, where Vm is membrane potential in mV. Lengths of recordings used for plots were 4 to 7 minutes. Slopes of the two relationships are significantly different. Reversal potentials calculated by these fits were -64.1 and -65.4 mV, respectively, indicating a specific effect of monensin on STOCs. C, Top, Dependency of STOCs on Na+ concentration in internal pipette solution is shown in three representative cells. No STOCs were observed when internal Na+ was 0 mmol/L. C, Bottom, STOCs were observed more frequently with 24 mmol/L internal Na+ than with 12 mmol/L Na+. Steady-state membrane potential, -10 mV. D, Even when internal Na+ was 0 mmol/L, application of monensin 30 µmol/L induced fluctuations of outward current with a reversal potential of -70 mV. These currents disappeared after removal of Ca2+ from external solution. Holding potential, -80 mV; voltage ramp pulses depolarizing cell membrane within 400 ms from -100 to +100 mV. Rate of pulses, 0.5 Hz.

We next tested the dependency of STOC generation on the intracellular Na⁺ concentration. STOCs were not observed when intracellular Na⁺ was adjusted to zero (n=14) (Fig 4C). STOC firing frequency increased significantly when the pipette solution contained 12 mmol/L Na⁺ (n=10). A further increase was observed when the pipette solution contained 24 mmol/L Na⁺ (n=14). An increase in the intracellular Na⁺ concentration with monensin induced whole-cell outward current increases from 218±42 to 367±24 pA, even when the initial intracellular Na⁺ concentration was zero (n=9) (Fig 4D). During the first several minutes after application of monensin to the bath solution, the current/voltage relationship elicited by ramp voltage pulses exhibited spontaneous changes in amplitude and a shift of current activation threshold to negative potentials from -32±7 to -61±9 mV. Some cells with 0 mmol/L intracellular Na⁺ concentration (4 of 18 cells investigated) exhibited STOCs because of increases in the intracellular Na⁺ concentration elicited by monensin. The stimulatory effect of monensin on spontaneous current fluctuations in the cells containing 0 mmol/L Na⁺ was blocked by BAPTA-AM (n=6) and by perfusion of the cells with a Ca²⁺-free bath solution (n=7).

	Discussion

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The important observations of this study are that STOCs are present in human coronary VSMCs and that mainly Ca²⁺ entry into the cell through reverse-mode Na⁺/Ca²⁺ exchanger determines calcium store refilling, which in turn regulates STOC generation. We identified a novel pathway for regulation of STOCs, and therefore arterial tone, that differs from those reported in several other, nonhuman, smooth muscle preparations.^{3 9 10 11 12 13} We showed that STOCs depend on intracellular Ca²⁺ and obtained strong evidence that the BK_Ca channel is responsible for STOC generation. However, blockade of voltage-dependent Ca²⁺ channels by Cd²⁺ and verapamil had only a minor inhibitory effect on STOCs. Thus, STOCs were related to BK_Ca channels but were not dependent on Ca²⁺ entry through voltage-dependent Ca²⁺ channels. We next used ryanodine and caffeine to further elucidate the role of internal Ca²⁺ stores.^{3 9 10 11 12} We observed that ryanodine initially increased STOCs but eventually resulted in a decrease in STOC firing frequency and block. Caffeine led to a temporary blockade of STOCs. After recovery of STOCs, caffeine again transiently increased the steady-state current (hump), indicating that the internal Ca²⁺ stores had been replenished and STOCs were merely dependent on the refilling state of the internal Ca²⁺ stores.

Since STOCs were not dependent on voltage-dependent Ca²⁺ channels but rather were solely dependent on internal Ca²⁺ stores, we reasoned that an additional transporter for Ca²⁺ ions must be responsible for modulating membrane conductance and for the generation of STOCs.¹⁴ The Na⁺/Ca²⁺ exchanger, which is present in cardiac, neuronal, retinal, and smooth muscle cells,^{8 15 16 17} would explain Ca²⁺ entry into our cells. This exchanger is known to be electrogenic and is a major route for transmembrane Ca²⁺ fluxes. To test for a possible role of the Na⁺/Ca²⁺ exchanger in the generation of STOCs, we first lowered intracellular Na⁺ to zero, which abolished STOCs. We next identified a direct correlation between STOC firing and intracellular Na⁺ concentration. We then used the sodium ionophore monensin to increase intracellular Na⁺ concentration.⁸ A fundamental feature of the Na⁺/Ca²⁺ exchanger is that an elevated [Na⁺]_i concentration promotes the reverse mode of the Na⁺/Ca²⁺ exchanger. Thus, when intracellular Na⁺ is exchanged for extracellular Ca²⁺, a change in STOC firing frequency would be expected. The modulation of the STOC firing frequency by monensin provides strong evidence that intracellular Ca²⁺ values are influenced by an increase in the intracellular Na⁺ concentration.

An increase in intracellular Na⁺ with monensin induced whole-cell outward current increases and oscillations, even when the initial intracellular Na⁺ concentration was zero. The stimulatory effect of monensin was blocked by perfusion of the cells with a Ca²⁺-free bath solution and by BAPTA-AM. Our results demonstrate the existence of a functional Na⁺/Ca²⁺ exchanger operating under physiological conditions in VSMCs from human coronary arteries. Na⁺/Ca²⁺ exchange is able to replenish Ca²⁺-depleted sarcoplasmic reticulum and could regulate STOC generation via this pathway. Our data can be explained by a model in which BK_Ca channels and sarcoplasmic reticulum are linked to a Na⁺/Ca²⁺ exchange–dependent calcium compartment.^{18 19} We suggest that mainly Ca²⁺ entry into the cell through reverse-mode Na⁺/Ca²⁺ exchange determines calcium store refilling, which in turn regulates spontaneous STOC generation in human coronary VSMCs. We believe that STOCs are likely to be important in protecting human coronary arteries from vasoconstrictor influences. Elucidation of their regulation may provide new insight into the control of coronary tone and may also have future therapeutic implications.

	Selected Abbreviations and Acronyms

 

AUC = area under the curve BKCa = Ca2+-activated potassium channel STOC = spontaneous transient outward K+ current VSMC = vascular smooth muscle cell

	Acknowledgments

 
This work was supported by the Deutsche Forschungsgemeinschaft (Ha 1388/2-3) and by a grant-in-aid from the Bundesministerium fur Bildung und Forschung. Prof R. Hetzer of the Deutsches Herzzentrum, Berlin, kindly supplied us with tissue from human hearts during orthotopic heart transplants.

Received June 24, 1996; revision received September 3, 1996; accepted September 6, 1996.

	References

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