Departments of 1 Anesthesiology and 2 Physiology and Biophysics, Mayo Clinic and Foundation, Rochester, Minnesota 55905
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ABSTRACT |
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The purpose of this study was to examine whether the nitric oxide donor S-nitrosoglutathione (GSNO) relaxes canine tracheal smooth muscle (CTSM) strips by decreasing Ca2+ sensitivity [i.e., the amount of force for a given intracellular Ca2+ concentration ([Ca2+]i)]. We further investigated whether GSNO decreases Ca2+ sensitivity by altering the relationship between regulatory myosin light chain (rMLC) phosphorylation and [Ca2+]i and the relationship between force and rMLC phosphorylation. GSNO (100 µM) relaxed intact CTSM strips contracted with 45 mM KCl by decreasing Ca2+ sensitivity in comparison to control strips without significantly decreasing [Ca2+]i. GSNO reduced the amount of rMLC phosphorylation for a given [Ca2+]i but did not affect the relationship between isometric force and rMLC phosphorylation. These results show that in CTSM strips contracted with KCl, GSNO decreases Ca2+ sensitivity by affecting the level of rMLC phosphorylation for a given [Ca2+]i, suggesting that myosin light chain kinase is inhibited or that smooth muscle protein phosphatases are activated by GSNO.
nitric oxide; airway; trachea; myosin light chain phosphorylation
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INTRODUCTION |
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NITRIC OXIDE (NO) is involved in a variety of physiological processes such as neurotransmission, platelet function, and regulation of smooth muscle tone as well as pathophysiological mechanisms such as cytotoxicity (30). Although the role of NO and NO donors in the regulation of airway smooth muscle (ASM) tone is not fully understood, NO and NO donors are increasingly recognized as physiologically important in mediating effects such as smooth muscle relaxation (47, 52).
The best-known mechanism by which NO and NO donors relax smooth muscle is their effect on cytosolic cGMP levels. Activation of soluble guanylate cyclase promotes the production of cGMP (18, 23), which subsequently activates a cGMP-dependent protein kinase (9). The activated cGMP-dependent protein kinase relaxes smooth muscle by reducing the intracellular Ca2+ concentration ([Ca2+]i) (16, 28, 29) and, in some smooth muscle types, the amount of force for a given [Ca2+]i (i.e., Ca2+ sensitivity) (5, 28, 34, 49). Regarding the role of cGMP in ASM, recent work from our laboratory (21) showed that cGMP relaxes canine tracheal smooth muscle (CTSM) not only by decreasing [Ca2+]i but also by decreasing Ca2+ sensitivity during contraction with the physiological agonist acetylcholine (ACh).
There is also increasing evidence that cGMP-independent mechanisms play an important role in NO-mediated effects (3, 8, 52). A previous work from our laboratory (37) showed that the NO donor S-nitrosoglutathione (GSNO) relaxed CTSM contracted with ACh. However, this relaxation was only partly mediated by an increase in cGMP levels. It was demonstrated that relaxation involved a cGMP-independent component mediated by reversible oxidation of thiols on unspecified proteins that regulate contraction.
Smooth muscle tone is regulated by the binding of agonists to receptors, thereby increasing [Ca2+]i. The increase in [Ca2+]i activates a Ca2+- and calmodulin-dependent myosin light chain (MLC) kinase (MLCK), which subsequently phosphorylates the 20-kDa regulatory MLC (rMLC) (15). In addition to increasing [Ca2+]i, receptor agonists also activate mechanisms that increase Ca2+ sensitivity (10, 24, 34). Changes in Ca2+ sensitivity can be related to alterations in the relationship between rMLC phosphorylation and force or the relationship between [Ca2+]i and rMLC phosphorylation. Consequently, GSNO might relax ASM by decreasing either [Ca2+]i or Ca2+ sensitivity.
The purpose of the present study was to investigate the effect of GSNO on Ca2+ sensitivity in CTSM contracted with KCl and to determine whether the decrease in Ca2+ sensitivity is mediated via alterations in rMLC phosphorylation, thereby changing the relationship between [Ca2+]i and rMLC phosphorylation or between force and rMLC phosphorylation.
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METHODS |
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Tissue Preparation
Mongrel dogs of either sex were anesthetized with an intravenous injection of pentobarbital sodium (30 mg/kg) and exsanguinated. The extrathoracic trachea (5-10 cm) was excised and immersed in chilled physiological salt solution (PSS) composed of (in mM) 110.5 NaCl, 25.7 NaHCO3, 5.6 dextrose, 3.4 KCl, 2.4 CaCl2, 1.2 KH2PO4, and 0.8 MgSO4. Fat, connective tissue, and epithelium were removed with tissue forceps and scissors.In studies examining the relationship between force and [Ca2+]i, the muscle strips (width 0.8-1.0 mm, length 4-5 mm) were mounted in a 0.1-ml quartz cuvette and continuously superfused at 2 ml/min with aerated (94% O2-6% CO2) PSS (37°C). One end of the strip was anchored via a stainless steel microforceps to a stationary metal rod; the other end was attached to a calibrated force transducer (model AE801, Aksjeselkapet Mikro Elektronik) via a stainless steel microforceps. During a 2-h equilibration period, the strips were stretched to optimal length in a stepwise fashion. The strips were incrementally stretched and activated with 1 µM ACh, and the isometric force was measured. Stretching was continued until the maximal force induced by ACh was obtained. After this protocol, each strip was maintained at this optimal length for the remainder of the experiment.
For studies measuring rMLC phosphorylation, separate muscle strips (width 3-5 mm, length 1.0-1.6 cm) were suspended in 25-ml water-jacketed tissue baths filled with PSS (37°C) and aerated with 94% O2-6% CO2. One end of each strip was anchored to a metal hook at the bottom of the tissue bath; the other end was attached to a calibrated force transducer (model FT-03, Grass Instruments, Quincy, MA). During a 2-h equilibration period, the strips were contracted isometrically for 30 s every 5 min by supramaximal electrical field stimulation (model S88D, Grass Instruments). The strips were stretched after each stimulation until optimal length was achieved (22).
Fura 2 Loading
Muscle strips were incubated for 3 h in PSS (22°C) containing 5 µM fura 2-AM and aerated with 94% O2-6% CO2 (35, 36). Fura 2-AM was dissolved in dimethyl sulfoxide (DMSO) and 0.02% cremophor. After fura 2 loading, the strips were washed with PSS (37°C) for 30-50 min to remove extracellular fura 2-AM and DMSO and to allow deesterification of any remaining cytosolic fura 2-AM. All further maneuvers were performed at 37°C.Fura 2 Fluorescence Measurements
Fura 2 fluorescence intensity was measured with a photometric system (model ph2, Scientific Instruments, Heidelberg, Germany) that measures the optical and mechanical parameters of isolated tissue simultaneously. This system has been previously described in detail (13). Light from a xenon high-pressure lamp was filtered to restrict excitation light to 340-, 360-, and 380-nm wavelengths. Excitation light at these three wavelengths was alternated every 2 ms and focused by a high-aperture objective onto the muscle strips. Fluorescence emitted from the strips passed through a filter at 500 ± 5 nm and was detected by a photomultiplier assembly. Illumination intensity of the excitation light passing through the cuvette was detected by an absorbance monitor. The photomultiplier signal was normalized by this absorbance to minimize the influence of fluctuations in the intensity of the light source. The emission fluorescence intensities due to excitation at 340 (F340)-, 360-, and 380 (F380)-nm wavelengths were measured and stored on a personal computer. The ratio of fluorescence intensity at 340-nm excitation to that at 380-nm excitation (F340/ F380) was used as an index of [Ca2+]i (35). Isometric force was measured simultaneously with a force transducer (model AE801, Aksjeselkapet Mikro Elektronik).rMLC Phosphorylation Measurements
rMLC phosphorylation was measured by two-dimensional gel electrophoresis (21, 31). Muscle strips (3-5 mm width, 1.0-1.6 cm length, 25-70 mg wet weight) were flash-frozen by rapid immersion in a dry ice-acetone slurry containing 6% (wt/vol) trichloroacetic acid (Experimental Protocols
Two experimental protocols were conducted in separate sets of CTSM strips. All strips were stretched to optimal length before the experiment. All strips were incubated with 10 µM indomethacin to prevent formation of prostanoids that could affect cyclic nucleotide levels (20, 52).Effects of GSNO on the relationship between force and
[Ca2+]i.
CTSM strips were maximally contracted with 45 mM KCl, producing steady-state increases in both F340/ F380
and force. The strips were then washed with Ca2+-free PSS
containing 2 mM EGTA until both F340/ F380
and force decreased to steady-state nadirs (these values were always
less than those measured in the resting muscle superfused with normal PSS). All subsequent F340/ F380 and force
responses were normalized such that steady-state values obtained after
washout in Ca2+-free PSS-EGTA and during contraction with
45 mM KCl in regular PSS represented 0 and 100% values, respectively
(22). The strips were then superfused with nominally
Ca2+-free PSS (without EGTA) containing 45 mM KCl (Fig.
1) for 5 min to activate Ca2+
channels. In two different strips from the same dog, cumulative concentration-response curves to CaCl2 (0.02, 0.05, 0.10, 0.20, 0.50, 1.0, 1.5, and 2.4 mM final concentrations) were generated for each strip; one concentration-response curve was generated during
exposure to 100 µM GSNO. This GSNO concentration was chosen because
our previous work (37) demonstrated a significant amount of relaxation
at this concentration. The order of exposure to GSNO was randomized to
control for any systematic effects of time on
F340/ F380 and force.
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Effects of GSNO on rMLC phosphorylation. For rMLC phosphorylation studies, pilot experiments were first performed to determine the time point at which the increase in rMLC phosphorylation after the addition of extracellular Ca2+ in the presence of 45 mM KCl was maximal. These pilot studies demonstrated that in the presence of 45 mM KCl, rMLC phosphorylation reached peak values 1 min after the addition of extracellular Ca2+. Eight strips were stimulated with 45 mM KCl until a steady-state level in force was reached and were then washed with Ca2+-free PSS-EGTA. The force responses were normalized as described in Effects of GSNO on the relationship between force and [Ca2+]i. After steady-state resting values were obtained by washing the strips in Ca2+-free PSS (without EGTA), Ca2+ channels were activated with 45 mM KCl in nominally Ca2+-free PSS for 5 min. Then four of the strips were exposed to four different CaCl2 concentrations [0 (baseline), 0.5, 1.0, or 2.4 mM CaCl2, 1 strip/concentration] for 1 min, and the strips were then flash-frozen. The other four strips were exposed to the same Ca2+ concentrations (1 strip/concentration) but in the presence of 100 µM GSNO. These four CaCl2 concentrations were chosen so that 0 mM represented the baseline and 2.4 mM CaCl2 represented the extracellular Ca2+ concentration in normal PSS (start and end points of dose-response curve, respectively; see Effects of GSNO on the relationship between force and [Ca2+]i), and 0.5 and 1.0 mM CaCl2 represented the middle of the concentration-response curve (concentrations that produced most profound increase in force).
Materials
Fura 2-AM was purchased form Molecular Probes (Eugene, OR). All other drugs and chemicals were purchased from Sigma. Stock solutions of fura 2-AM were prepared in DMSO; all other solutions and drugs were prepared in distilled water. Nominally Ca2+-free PSS was made by excluding CaCl2 from the regular PSS.Statistical Analysis
Data are expressed as means ± SE; n is the number of dogs studied. The effects of GSNO on force and [Ca2+]i and the relationship between force and [Ca2+]i were determined by one-way repeated-measures analysis of variance (ANOVA) with post hoc analysis with the Student-Newman-Keuls method.The effects of GSNO on the relationships between force and [Ca2+]i, [Ca2+]i and rMLC phosphorylation, and rMLC phosphorylation and force were determined by interpolation with nonlinear polynomial regression of the measured control values. These interpolated values were then compared by paired or unpaired t-test when appropriate. A P value < 0.05 was considered significant.
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RESULTS |
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Effects of GSNO on the Relationship Between [Ca2+]i and Force
Figure 1 shows a representative example of the effects of GSNO on the increases in force and F340/ F380 induced by CaCl2 in CTSM strips stimulated with 45 mM KCl. Cumulative addition of 0.02-2.4 mM CaCl2 to the nominally Ca2+-free PSS containing 45 mM KCl caused concentration-dependent increases in both force and F340/ F380 (control; Fig. 1, A and B, left). In the presence of 100 µM GSNO, the increase in force induced by the addition of CaCl2 was significantly attenuated (Fig. 1A, right). In contrast, there was no concomitant attenuation of the Ca2+-induced increase in F340/F380 in the presence of GSNO (Fig. 1B, right). These data are summarized in Figs. 2 and 3, respectively.
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Although GSNO had no significant effect on
F340/ F380 in comparison to control
conditions (2.4 mM CaCl2 produced increases in
F340/ F380 of 68 ± 4 and 61 ± 3% in
control conditions and the presence of GSNO, respectively; Fig. 2),
force development was significantly reduced (2.4 mM CaCl2
produced increases in force of 125 ± 6 and 52 ± 7% in control
conditions and the presence of GSNO, respectively; P < 0.05;
Fig. 3). Thus GSNO relaxed CTSM strips primarily by reducing the amount
of force developed for a given
[Ca2+]i (Fig.
4).
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Effects of GSNO on the Relationship Between rMLC Phosphorylation and Force
CaCl2 caused a concentration-dependent increase in both rMLC phosphorylation and force. Figure 5 summarizes the effects of GSNO on the relationship between rMLC phosphorylation and force. As described in Effects of GSNO on the Relationship Between [Ca2+]i and Force, GSNO decreased the amount of force produced but also decreased the amount of rMLC phosphorylation. These GSNO-induced reductions in force and rMLC phosphorylation were proportional. Thus GSNO did not affect the relationship between force and rMLC phosphorylation.
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Effects of GSNO on the Relationship Between rMLC Phosphorylation and F340/F380
Data from both experimental protocols illustrating the effect of GSNO on the amount of rMLC phosphorylation produced for a given F340/F380 are shown in Fig. 6. Although GSNO had no significant effect on the increase in F340/F380 induced by CaCl2 (Fig. 2), the increase in rMLC phosphorylation was markedly attenuated (Fig. 6). These data suggest that GSNO relaxes CTSM strips by reducing the amount of rMLC phosphorylation for a given [Ca2+]i.
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DISCUSSION |
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In a previous study, Hirasaki et al. (16) have characterized the effect of NO and NO donors on [Ca2+]i and force and suggested decreased Ca2+ sensitivity as a potential mechanism for NO action in ASM. However, the underlying cellular targets for NO and NO donors, especially GSNO, have not been identified. Furthermore, the previous study focused on the effects of NO and NO donors on [Ca2+]i and force during muscarinic-receptor stimulation. It is likely that the intracellular regulatory pathways activated by receptor stimulation and their interactions with NO and NO donors are quite complex and remain to be fully elucidated. These interactions confound direct examination of the relationship between Ca2+ and force (i.e., Ca2+ sensitivity) and the effects of NO and NO donors. Therefore, to bypass a receptor-mediated mechanism for elevating [Ca2+]i, we used KCl in the present study, thus allowing us to focus on pathways distal to elevated [Ca2+]i.
The major finding of this study is that GSNO inhibits contraction of CTSM strips stimulated with isotonic KCl primarily by decreasing Ca2+ sensitivity. This reduction in Ca2+ sensitivity is due to a decrease in the level of rMLC phosphorylation produced for any given [Ca2+]i. In contrast, the relationship between rMLC phosphorylation and force is not altered by GSNO. These data suggest that GSNO affects regulatory mechanisms that do not involve actomyosin cross-bridge cycling.
S-nitroso compounds are produced endogenously by the reaction of NO with intracellular thiols (45). For example, S-nitrosothiols, particularly GSNO, are present in human airway lining fluid at nanomolar to micromolar concentrations (11). S-nitrosothiols may be physiologically important. For example, the formation and degradation of S-nitrosothiols such as GSNO may allow for storage and transport of NO (32). S-nitrosothiols may mediate their effects by yielding free NO or by transnitrosylation of specific protein thiols (44, 46).
Synthetic S-nitrosothiols relax both vascular smooth muscle (26) and ASM (19) contracted with physiological agonists. In a recent study of CTSM, we (37) showed that GSNO caused relaxation during both ACh- and KCl-induced contractions that was largely independent of an increase in cGMP. This relaxation was completely reversed by the reducing agent dithiothreitol, suggesting that intracellular thiol oxidation was involved.
Numerous studies in vascular smooth muscle (28, 29) as well as in ASM (11, 16, 19) have shown that various NO donors mediate relaxation in part by decreasing [Ca2+]i (for a review, see Ref. 11). One study on expressed cardiac L-type Ca2+ channels in human embryonic kidney cells (17) showed that S-nitrosocysteine, S-nitroso-N-acetylpenicillamine, and GSNO all inhibited fluxes through these channels. These findings suggest that S-nitrosothiol compounds relax smooth muscle in part by reducing [Ca2+]i. It is also possible that GSNO relaxes ASM by decreasing the amount of force produced for a given [Ca2+]i (i.e., by reducing Ca2+ sensitivity), but this possible mechanism has not been studied. For example, a decrease in Ca2+ sensitivity has been described as one mechanism that contributes to relaxation of vascular smooth muscle by other NO donors (5, 28, 34, 49).
Stimulation of smooth muscle with isotonic KCl increases [Ca2+]i by promoting Ca2+ influx through voltage-operated Ca2+ channels (25). The binding of Ca2+ to calmodulin increases MLCK activity and phosphorylation of the 20-kDa rMLC. rMLC phosphorylation allows the cyclic attachment and detachment of the myosin head to actin, the hydrolysis of ATP by actin-activated myosin ATPase activity, and contraction (43). Additionally, there is evidence that actomyosin ATPase activity and cross-bridge cycling may be under the biochemical regulation of another Ca2+- and phosphorylation-dependent system (14, 41). Biochemical studies, including in vitro motility assays, have suggested that the actin-associated protein caldesmon may regulate actomyosin ATPase activity (33, 42). For example, although unphosphorylated caldesmon inhibited actomyosin ATPase activity in vitro, no inhibition was observed with phosphorylated caldesmon (33). It has also been shown that unphosphorylated caldesmon (1, 38, 40) inhibits isometric force in skinned smooth muscle preparations and is phosphorylated during smooth muscle contraction (7, 12). Thus although rMLC phosphorylation plays a central role in smooth muscle contraction, other mechanisms in addition to rMLC phosphorylation may be important in regulating ASM tone. Based on this understanding, the effects of GSNO on Ca2+ sensitivity can be categorized into mechanisms that reduce the level of rMLC phosphorylation for a given [Ca2+]i and mechanisms that reduce the amount of force for a given level of rMLC phosphorylation.
The results of our study show that the reduction in Ca2+ sensitivity was due to a decrease in the amount of rMLC phosphorylation produced for a given [Ca2+]i. This is consistent with recent evidence (50) that nitrovasodilators such as nitroglycerin and sodium nitroprusside relax the swine carotid artery by decreasing the level of rMLC phosphorylation. These effects may be mediated by decreased MLCK activity or increased smooth muscle protein phosphatase (SMPP) activity. There is currently no information on these potential mechanisms in ASM. These mechanisms were not examined in the present study but need to be a focus of future studies. In vascular smooth muscle, it appears that MLCK activity is not significantly altered by NO donors and, in fact, increases with exposure to NO donors in the absence of agonist stimulation (50). Therefore, we hypothesize that GSNO decreases Ca2+ sensitivity by activating SMPP. Although there is no evidence for SMPP activation in ASM, there is recent evidence (27, 51) in other smooth muscle types for NO-induced activation of the particular SMPP that are involved in MLC dephosphorylation. It must, however, be emphasized that the effects of NO and/or cGMP on protein phosphatases in general are complex at best. For example, NO also inhibits protein tyrosine phosphatases (4). However, tyrosine phosphorylation of calmodulin is known to activate SMPP (6). Therefore, if anything, NO inhibition of tyrosine phosphatase will only lead to maintained tyrosine phosphorylation of calmodulin and thus result in increased MLC dephosphorylation. These complex interactions remain to be examined.
It is well accepted that NO- and NO donor-mediated relaxation of different smooth muscle types involves elevated cGMP (16, 20, 23, 52). Previous studies (21, 28, 31, 39) have shown that cGMP-dependent effects on Ca2+ sensitivity contribute to this relaxation. However, there is now clear evidence in various smooth muscle types (2, 11, 37, 48) that there are also cGMP-independent mechanisms that mediate, at least in part, NO- and/or NO donor-induced relaxation. In a previous study from our laboratory, we (37) showed that GSNO relaxes CTSM by a cGMP-independent mechanism. Although we observed small increases in cGMP during superfusion with GSNO, incubation of the tissue with methylene blue, an inhibitor of soluble guanylate cyclase, inhibited the increase in cGMP while relaxation was still present. Additionally, even after removal of GSNO and a concomitant decrease in cGMP to baseline levels, relaxation persisted. Therefore, it is possible that in the present study, the GSNO-induced relaxation as well as a decrease in rMLC phosphorylation occurs through a cGMP-independent mechanism. However, this does not rule out a cGMP-dependent effect on Ca2+ sensitivity. For example, Wu et al. (51) found that 8-bromo-cGMP increases SMPP activity, also contributing to decreased rMLC phosphorylation. Jones et al. (21) demonstrated that the cGMP analog 8-bromo-cGMP decreases Ca2+ sensitivity and rMLC phosphorylation. Interestingly, this effect of 8-bromo-cGMP only occurred during agonist-induced stimulation, but 8-bromo-cGMP had no effect on the free Ca2+ concentration-response curve generated in the absence of agonist stimulation. This result makes it unlikely that during KCl-induced contraction, the effect of GSNO on Ca2+ sensitivity and rMLC phosphorylation is mediated by a cGMP-dependent mechanism.
In summary, the results of this study demonstrate that GSNO relaxes CTSM contracted with KCl by reducing Ca2+ sensitivity. The decrease in Ca2+ sensitivity is mediated only by an effect on the level of rMLC phosphorylation for a given [Ca2+]i without affecting the relationship between isometric force and rMLC phosphorylation, suggesting that MLCK is inhibited and/or SMPP is activated by GSNO. The lack of an effect of GSNO on the relationship between rMLC phosphorylation and force suggests that actomyosin ATPase activity is not affected. This is surprising given the reactive thiols on the myosin head. The underlying mechanisms are unclear. The role of cGMP in these effects needs to be further elucidated.
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ACKNOWLEDGEMENTS |
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We thank Kathleen Street for excellent technical assistance.
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FOOTNOTES |
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This work was supported by Deutsche Forschungsgemeinschaft Research Training Grant Pa 168-1, a Research Fellowship from Abbott Laboratories (to C. Pabelick), and National Heart, Lung, and Blood Institute Grants HL-45532 and HL-54757.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: K. A. Jones, Anesthesia Research, Mayo Clinic, Rochester, MN 55905 (E-mail: jones.keith{at}mayo.edu).
Received 10 May 1999; accepted in final form 11 October 1999.
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