Departments of Physiology and Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109-0622
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ABSTRACT |
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Insulin attenuates vascular contraction via inhibition of
voltage-operated Ca2+ channels and
by enhancement of endothelium-dependent vasodilation. Thus it has been
suggested that hypertension-associated insulin resistance results from
an insensitivity to the hormone's effects on vascular reactivity. This
hypothesis has been strengthened by reports that thiazolidinediones, a
class of insulin-sensitizing agents, lower blood pressure and improve
insulin responsiveness in hypertensive, insulin-resistant animal
models. We tested the hypothesis that troglitazone enhances the
vasodilating effect of insulin via inhibition of voltage-operated
Ca2+ channels in vascular smooth
muscle cells. Rat thoracic aortic rings (no endothelium) were suspended
in tissue baths for isometric force measurement. Rings were incubated
with 0.1 DMSO vehicle (control), troglitazone
(105 M), insulin
(10
7 U/l), or both
troglitazone and insulin (1 h) and then contracted with phenylephrine
(PE), KCl, or BAY K 8644. Troglitazone increased the
EC50 values for PE and KCl.
Contractions to BAY K 8644 in troglitazone-treated rings were virtually
abolished. Insulin alone had no effect on contraction. However, when
insulin was combined with troglitazone, the
EC50 values for PE and KCl were
further increased. Additionally, the maximum contractions
to both PE (14 ± 4% of control) and KCl (12 ± 2% of control)
were reduced. Measurement of Ca2+
concentration ([Ca2+])
with fura 2-AM in dispersed vascular smooth muscle cells indicated that
neither insulin nor troglitazone alone altered PE-induced increases in
intracellular [Ca2+].
However, troglitazone and insulin together caused a significant reduction in PE-induced increases in intracellular
[Ca2+] (expressed as
percentage of preincubation stimulation to PE: 47 ± 10%, treated;
102 ± 13%, vehicle). These results demonstrate that troglitazone
inhibits Ca2+ influx and that it
acts synergistically with insulin to attenuate further vascular
contraction via inhibition of voltage-operated Ca2+ channels.
vascular smooth muscle; calcium channels
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INTRODUCTION |
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CLINICAL AND EPIDEMIOLOGICAL studies have provided evidence that a correlation exists between insulin resistance and essential hypertension (2, 3, 14). This association has prompted investigations into the role of insulin in the modulation of blood pressure. Investigators have demonstrated that insulin can directly alter activity of isolated cells by acting on transmembrane cation transporters, such as the Ca2+-ATPase, Na+-K+-ATPase, Na+/H+ antiport, and Ca2+/Na+ exchange systems (9, 10, 13, 22, 29). Several studies have also shown that insulin can directly attenuate vasoconstrictor responses of vascular smooth muscle via inhibition of voltage-operated Ca2+ channels (22, 25), as well as by enhancing endothelium-dependent vasodilation through the nitric oxide (NO)-guanosine 3',5'-cyclic monophosphate pathway (20, 23, 27). Recently, studies have suggested that insulin resistance is involved in the development and maintenance of hypertension in such animal models as the obese Zucker rat (3, 30), fructose-fed rat (17), and spontaneously hypertensive rat (SHR; 7, 12). Thus many investigators believe that a resistance to the vasodilating effects of insulin may lead to the development of hypertension (18, 21). This hypothesis has been strengthened by reports that thiazolidinediones, a class of insulin-sensitizing agents, lower blood pressure in addition to improving insulin responsiveness in hypertension (1, 5). More recently, pioglitazone and troglitazone, two related insulin- sensitizing agents, were demonstrated to inhibit vascular smooth muscle contraction, as well as reduce L-type voltage-gated Ca2+ current in A7r5 vascular smooth muscle cells (11, 21, 28). The current study was performed to test the hypothesis that the insulin-sensitizing agent troglitazone can enhance the vasodilating effects of insulin via inhibition of voltage-gated Ca2+ channels in vascular smooth muscle cells. To eliminate the potential involvement of endothelium-derived factors (18, 20, 23), we conducted these studies in endothelium-denuded segments of rat aorta.
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METHODS |
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The methods and procedures described in the present report were reviewed by the animal protocol review committee of the University of Michigan Medical School and are in accordance with institutional guidelines.
Isolated tissue bath protocol. Adult
male Sprague-Dawley rats (weight, 275 g; Harlan Industries and Charles
Rivers) were anesthetized with pentobarbital sodium (50 mg/kg ip) and
exsanguinated. Thoracic aortas were removed and placed into cold
physiological salt solution (PSS; in mmol/l: 130 NaCl, 4.7 KCl, 1.6 CaCl2 · H2O,
1.18 KH2PO4, 1.17 MgSO4, 5.5 dextrose, 14.9 NaHCO3, and 0.03 CaNa2EDTA). The vessels were
cleaned of connective tissue and cut into 4-mm cylindrical segments
under a dissecting microscope. The endothelium was removed from the
arterial ring preparations by cannulating the lumen with microforceps
and gently rolling the vessel between the forceps and palm. Finta et
al. (4) have previously reported that this rubbing procedure removes at
least 95% of the endothelium. The absence of endothelium was confirmed
by lack of a response to the endothelium-dependent vasodilator
acetylcholine (106 mol/l)
in rings contracted with phenylephrine (PE; 5 × 10
8 mol/l).
Muscle bath experiments. Vessel
segments were mounted in 50-ml jacketed organ baths containing PSS
after the lumen was cannulated with two wire hooks; one hook was
fastened to a stationary stainless steel rod, the other to an isometric
force transducer. PSS was maintained at 37°C and aerated with 95%
O2-5%
CO2 throughout the experiment.
Rings were placed under optimal resting tension (passive tension placed
on tissue that results in maximal isometric performance, determined as
4 g for aortic rings) and equilibrated for ~60 min with washing.
Tissues were incubated with indomethacin (5 × 106 mol/l) during the
incubation period in order to block cyclooxygenase activity. After the
equilibration period, drugs were added directly to the muscle bath.
Tissues were incubated with insulin and/or troglitazone for 1 h
before evaluation of contractile activity. These agents remained in the
bath while the aortic rings were contracted in a cumulative fashion
with PE, the depolarizing agent KCl, or the voltage-gated Ca channel
agonist BAY K 8644. Isometric tension was measured as grams of force
and then normalized to maximal contraction to either PE or KCl.
Isolation of vascular smooth muscle cells and fura 2-AM loading. Single vascular smooth muscle cells were isolated via enzymatic dispersion from arteries removed from rats according to the methods of Wilde et al. (26) as modified by Tostes et al. (24). Briefly, thoracic aortas were quickly excised from pentobarbital-anesthetized rats (50 mg/kg, ip) and placed into 0.1 mmol/l Ca2+ Hanks' balanced salt solution containing (in mmol/l) 140 NaCl, 5.4 KCl, 0.44 KH2PO4, 0.42 NaH2PO4, 4.17 NaHCO3, 0.026 CaNa2EDTA, 0.10 CaCl2 · H2O, 5.0 HEPES, and 5.5 dextrose (pH 7.35). Vessels were placed into 5 ml of 0.1 mmol/l Ca2+ Hanks' PSS containing 0.05 g bovine serum albumin (type I), 5 mg soybean trypsin inhibitor, 5 mmol/l taurine, 2 mg dithiothreitol, 3 mg type I collagenase, and 3 mg papain (all from Sigma, St. Louis, MO). Vessels were incubated in a shaking water bath at 37°C for 45 min. After incubation, vessel fragments were removed by pipette and placed in 15 ml plastic capped tubes and resuspended in 0.1 mmol/l Ca2+ Hanks' solution containing albumin, trypsin inhibitor, and taurine as described above. The aortic fragment was then loaded with the intracellular, fluorescent Ca2+ indicator fura 2 by incubation with the membrane-permeant, acetoxymethyl ester form, fura 2-AM, at a concentration of 5 µmol/l for 60 min. Excess fura 2 was removed from the cell suspension by washing the tissue with 0.1 mmol/l Ca2+ Hanks' balanced salt solution.
Vascular smooth muscle cells were released by gentle pipette agitation. Loaded cells were allowed 30 min for complete cytosolic deesterification of the fura 2-AM. Small aliquots of fura 2-loaded cells were placed on coverslips in a controlled-atmosphere (95% O2-5% CO2), controlled-temperature (37°C) superfusion chamber. This bath was positioned on the stage of a Leitz Diavert inverted microscope. The superfusion system delivered the PSS containing Ca2+ (1.6 mmol/l) at a rate of 3 ml/min.
Measurement of intracellular Ca.
Intracellular Ca2+ concentration
([Ca2+]) was monitored
by alternately exciting fura 2-AM at 340 and 380 nm. The fluorescence
emission, taken as the ratio of emissions at 340 and 380 nm excitation
wavelength (340/380 ratio), was recorded. Previous studies have
demonstrated the increase in fura 2-AM 340/380 ratio has a linear
relationship with the increase in the intracellular free
[Ca2+] (16, 24, 26).
Isolated vascular smooth muscle cells did not exhibit autofluorescence.
Maximum and minimum levels for intracellular [Ca2+] were determined
at the end of each experimental protocol by treating the cells with
buffer containing 1.6 mmol/l Ca2+
with 1 µmol/l ionomycin and 15 mmol/l EGTA (0 mmol/l
Ca2+). The 340/380 ratios were
used to calculate intracellular free [Ca2+] as described
previously by Tostes et al. (24). Vascular smooth muscle cells were
initially stimulated with PE (3 × 106 mol/l) and after the
fura 2-AM 340/380 ratio reached plateau, PE was washed out with fresh
PSS. Cells were then incubated with vehicle (0.1% DMSO), troglitazone,
insulin, or a combination of troglitazone and insulin. After the
incubation period, cells were again stimulated with PE (3 × 10
6 mol/l). The effects of
the various incubations on the fura 2-AM 340/380 ratio in response to
PE are expressed as percentage of response to PE before the specific incubation.
Drugs. Troglitazone was obtained as a gift from Warner-Lambert Pharmaceuticals (Ann Arbor, MI). BAY K 8644 was purchased from Calbiochem. Bovine pancreatic insulin, indomethacin, PE, acetylcholine, sodium nitroprusside, and all other chemicals were purchased from Sigma. Troglitazone was prepared in DMSO, and BAY K 8644 and indomethacin were prepared in 95% ethanol. All other drugs were prepared in distilled water. Each drug was prepared on the day of the experiment from its powder form or was diluted from a frozen stock solution.
Statistics. Data are means ± SE.
Statistical analyses were performed by Student's
t-test. The criterion for statistical
significance was P 0.05. The
Bonferroni correction was applied in multiple-testing procedures.
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RESULTS |
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A number of studies have suggested that insulin can lower
[Ca2+] and inhibit the
contraction of isolated vascular smooth muscle cells (9, 12, 29, 30).
Incubation of endothelium-denuded rings of rat thoracic aorta with
insulin (107 U/l) for 1 h
had no effect on the cumulative concentration-response curve to PE
(10
9 to 3 × 10
6 mol/l; Fig.
1). However, incubation with troglitazone
(10
5 mol/l) significantly
shifted the concentration response to PE downward and to the right at
concentrations ranging from 3 × 10
9 to 3 × 10
8 mol/l compared with
control (incubated with 0.1% DMSO vehicle) and insulin-incubated
tissues. These results suggest that troglitazone attenuates contractile activity mediated by receptor activation with
the
-adrenergic agonist (Fig. 1). The addition of insulin combined
with troglitazone further inhibited PE-induced contractions at a much
broader range of concentrations (3 × 10
9 to 3 × 10
6 mol/l). In a separate
set of experiments, a concentration-dependent relationship between
troglitazone and inhibition of PE-mediated contraction was established
by varying troglitazone
(10
6 to
10
5 mol/l) while
maintaining insulin at 10
7
U/l. The EC50 values for PE
(concentration required to reach half-maximal response;
log mol/l) were increased for tissues incubated with
troglitazone compared with the control and insulin-incubated aortic
rings. At 10
6 mol/l, 3 × 10
6 mol/l, and
10
5 mol/l troglitazone, the
EC50 (
log mol/l)
values were 7.45 ± 0.12, 7.07 ± 0.06, and 6.29 ± 0.10, respectively. Control and insulin-incubated tissues had similar
EC50 values of 7.56 ± 0.11 and
7.26 ± 0.19 mol/l, respectively (Fig.
2).
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To investigate the changes in
[Ca2+] paralleling the
alterations in vascular reactivity induced by troglitazone and insulin, we used fura 2 epifluorescence microscopy to measure changes in the
340/380 ratio of isolated rat aortic smooth muscle cells. Vascular
segments stimulated with PE (3 × 106 mol/l) were shown to
result in maximum contractile force production in the isolated tissue
bath experiments (Fig. 1), and this concentration of the agonist was
used to evaluate changes in the 340/380 ratio. Basal
[Ca2+] levels were
38.6 ± 2.2 nmol/l (n = 22 experiments) and the initial exposure to PE (3 × 10
6 mol/l) increased
[Ca2+] levels to 86.5 ± 6.2 nmol/l (n = 22;
P < 0.05). After this
initial exposure to PE, the isolated smooth muscle cells were washed
and then incubated with either vehicle (0.1% DMSO), insulin
(10
7 U/l), troglitazone
(10
5 mol/l), or a
combination of insulin (10
7
U/l) and troglitazone (10
5
mol/l). After these incubations PE (3 × 10
6 mol/l) was added again,
and the magnitude of the change in the 340/380 ratio was measured and
expressed as a percentage of the initial exposure to PE (Fig.
3). Exposure to vehicle (0.1% DMSO) did
not alter the magnitude of the PE-stimulated increase in intracellular [Ca2+] (101.67 ± 12.47%; Fig. 3). Although there was a tendency for insulin
and troglitazone alone to reduce the increased level of the 340/380
ratio produced by PE stimulation, these differences were not
statistically significant (expressed as a percentage of response to PE
before incubation: insulin = 85.14 ± 13.77%; troglitazone = 83.67 ± 17.74%; Fig. 3). However, the increases in the 340/380 ratio for
cells stimulated with PE and incubated with both troglitazone and
insulin (46.75 ± 10.31%) were significantly reduced compared with
control (101.67 ± 12.4%; P < 0.05; Fig. 3).
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To provide a more precise understanding of how troglitazone and insulin may act to modulate vascular reactivity, cumulative concentration-response curves were then performed with KCl. Elevating K+ provides a means for assessing vasoconstriction mediated by voltage-gated Ca2+ channels. As with PE, insulin had no effect on contractile responses to KCl (Fig. 4). However, troglitazone appeared to be much more effective in shifting the cumulative concentration-response curve for KCl (15 to 100 mmol/l) to the right compared with control than was observed for PE-induced contraction (Fig. 4). Troglitazone in combination with insulin significantly shifted the concentration-response curve to the right compared with control at all concentrations of KCl tested (Fig. 4). Thus troglitazone is more efficacious in reducing contraction induced by voltage-gated Ca2+ channels than those regulated by ligand-receptor complexes. As observed for PE, insulin and troglitazone synergistically inhibited KCl-induced contractile responses.
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In order to address the possibility that troglitazone acts specifically
on voltage-gated Ca2+ channels,
cumulative concentration-response curves were performed to BAY K 8644, a specific agonist for voltage-gated
Ca2+ channels. Again, insulin had
no effect on contractile activity. Troglitazone almost completely
abolished the response to BAY K 8644 (3 × 108 to 3 × 10
7 mol/l) compared with
control, providing further evidence that troglitazone depresses
contractile activity by acting as an antagonist of voltage-gated
Ca2+ channels (Fig.
5).
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DISCUSSION |
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Whereas a correlation between insulin resistance and hypertension has been observed, the exact nature of this relationship is unclear (2, 14). Insulin has been shown in previous studies to modulate the activity of transmembrane cation transporters such as Ca2+-ATPase, Na+-K+-ATPase, Na+/H+ antiport, and Ca2+/Na+ exchange systems (9, 10, 13, 22, 29). Recent studies have suggested that insulin resistance is involved in the development and maintenance of hypertension in such animal models as the obese Zucker rat (3, 30), fructose-fed rat (17), and SHR (7, 12). This has prompted investigators to hypothesize that hypertension in insulin-resistant individuals is the result of an inability of insulin to directly attenuate vasoconstriction (18, 20, 30). This hypothesis has been strengthened by reports that thiazolidinediones, a class of insulin-sensitizing agents, inhibit contraction of vascular tissue and lower blood pressure in the SHR (1). The current study supports previous reports that the insulin-sensitizing agent troglitazone inhibits contraction of rat vascular smooth muscle via reduction of intracellular Ca2+, independent of NO release from the endothelium (21). We also show for the first time that insulin and troglitazone synergistically inhibit contractility in an endothelium-denuded aortic ring preparation.
Previous studies have shown that insulin attenuates the contractility of isolated smooth muscle cells and intact vessels (9, 22, 27). The vasodilating effects of insulin have been attributed to stimulation of NO production by the endothelium (20, 23, 27) and inhibition of Ca2+ entry into vascular smooth muscle cells (9). Insulin alone did not have an effect on PE- or KCl-induced contractions in our experiments. The lack of an insulin effect on vascular reactivity may be attributed to denudation of the vascular rings. By removing the endothelium, we can assume that production of the endothelium-derived relaxing factor NO or a hyperpolarizing factor is negligible. Thus any attenuation of vascular reactivity mediated by the effects of insulin on synthesis and release of these substances was eliminated. In addition, the lack of an insulin effect on vascular reactivity suggests that the inhibition of Ca2+ entry into smooth muscle cells by insulin in our aortic ring preparation is not sufficient to reduce contraction. However, our results do support the view that insulin can modulate contractile responses of rat aortic smooth muscle when used in combination with troglitazone.
Previous studies by Zhang et al. (31) demonstrated that pioglitazone,
also an insulin-sensitizing agent, may lower blood pressure by its
inhibitory effects on current mediated by what is considered a distinct
voltage-gated Ca2+ channel. In our
study, troglitazone, which is of the same chemical class as
pioglitazone, decreased both the sensitivity to KCl as well as the
maximum response. Whereas troglitazone also reduced sensitivity to PE,
it did not cause a reduction in maximal force production. Studies by
Godfraind et al. (6) suggest that
Ca2+ antagonists are less potent
against Ca2+ entry stimulated by
receptor than by high K+-induced
depolarization. Because PE-induced activation of -adrenoreceptors causes both release of Ca2+ from
intracellular stores as well as influx from the extracellular medium
via the phosphoinositide pathway, we would expect this type of
contraction to be less sensitive to troglitazone. However, agonist-induced contraction is believed to rely partially on
depolarization and activation of potential-operated
Ca2+ channels in the plasma
membrane, thus providing a possible explanation for the decrease in
sensitivity to PE in the presence of troglitazone (Fig. 1). Although
fura 2 measurements did not demonstrate a significant reduction by
troglitazone in the increased level of intracellular [Ca2+] stimulated by a
high concentration of PE (3 × 10
6 mol/l, a concentration
producing maximal force in isolated aortic rings, see Fig. 1), it is
possible that the rightward shift in the concentration-response effect
for contraction reflects a decrease in intracellular
[Ca2+] at lower
concentrations of PE (Fig. 1). Intracellular
[Ca2+] is
significantly reduced in the presence of both troglitazone and insulin.
To elucidate further the mechanism by which troglitazone and insulin modulate vascular reactivity, we contracted aortic rings in the muscle bath by the cumulative addition of increasing concentrations of KCl. Elevating K+ provides a means for assessing contraction mediated by voltage-induced influx of extracellular Ca2+ through channels in the plasma membrane. To gain a more accurate assessment of the effects of troglitazone on vascular reactivity, we characterized responses to BAY K 8644, a specific agonist of L-type Ca2+ channels. Responses to BAY K 8644 were almost completely abolished by troglitazone, suggesting that troglitazone acts specifically on voltage-gated Ca2+ channels. These results are consistent with findings by Song et al. (21) demonstrating that troglitazone inhibits L-type Ca2+ currents in freshly dissociated rat-tail artery and in aortic and cultured vascular smooth muscle cells.
The fact that insulin alone had no effect on vascular reactivity but in combination with troglitazone produced a profound inhibition of vascular smooth muscle contraction led us to speculate about the possibility of one Ca2+ channel possessing two different gating mechanisms. We propose that insulin has a small inhibitory effect on gating of the Ca2+ channel by both agonists and membrane potential, whereas troglitazone potently inhibits activation of the Ca2+ channel by voltage. When combined, there is an enhancement of the inhibitory effects of insulin on the Ca2+ channel, in addition to troglitazone's own antagonizing effects, resulting in attenuated contractile responses to both PE and KCl. Indeed, investigators have been able to modulate the degree of inhibition of norepinephrine-induced contraction by nisoldipine, a voltage-gated Ca2+ channel antagonist that can distinguish between receptor-mediated and voltage-dependent responses (6).
Another tenable explanation for greater attenuation of aortic contraction in the presence of insulin and troglitazone may be attributed to stimulation of Ca2+-ATPase by insulin. Previous studies by Zemel et al. (29) provide evidence that insulin increases Ca2+ efflux as the rate of spontaneous relaxation after PE washout. Whereas the increase in Ca2+-ATPase activity caused by insulin alone may not be sufficient to shift the response curves to KCl or PE, troglitazone may facilitate the effects of insulin, thus providing an explanation for the greater inhibition of contractile activity observed in the presence of troglitazone and insulin.
Stimulation of the action of vasodilating factors by troglitazone may
provide an alternative mechanism for the synergism with insulin. Itoh
et al. (8) demonstrated the presence of -gene transcripts for
peroxisome proliferator-activated receptors (the purported target
receptors for thiazolidinediones) in rat cultured vascular smooth
muscle cells. Troglitazone was found to cause a significant increase of
basal secretion of an endothelium-derived relaxing factor (19). Because
the current study was conducted in endothelium-denuded aortic rings, it
is unlikely that troglitazone increased an endothelium-derived relaxing
factor. However, it is conceivable that troglitazone resulted in the
production of a smooth muscle relaxing factor. Consistent with this
observation, Muniyappa et al. (15) have provided evidence that
insulin-like growth factor 1 can increase arterial smooth muscle NO
production. The notion that insulin and troglitazone activated
transcription factors to induce NO production at the level of the
smooth muscle cell is doubtful considering the relatively short
exposure time (1 h).
In summary, we have demonstrated that the insulin-sensitizing agent troglitazone decreases contractile responses of endothelium-denuded rat aortic rings to both agonist-mediated and depolarization-induced contraction via reduction of intracellular [Ca2+]. Furthermore, insulin and troglitazone have synergistic effects on attenuating vascular reactivity. These data support the notion that resistance to the direct effects of insulin on the vasculature may be an important contributor to the development of hypertension in insulin resistant states.
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ACKNOWLEDGEMENTS |
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This study was supported by National Heart, Lung, and Blood Institute Grant HL-18575.
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FOOTNOTES |
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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: J. M. Richey, 7813 Medical Science Bldg. II, Dept. of Physiology, Univ. of Michigan, Ann Arbor, MI 48105-0622.
Received 20 April 1998; accepted in final form 15 July 1998.
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