Division of Medical Genetics, Department of Pediatrics, Emory University, Atlanta, Georgia 30322
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ABSTRACT |
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Insulin stimulates K+ transport by the
Na+-K+-ATPase in human fibroblasts. In other
cell systems, this action represents an automatic response to increased
intracellular [Na+] or results from translocation of
transporters from an intracellular site to the plasma membrane. Here we
evaluate whether these mechanisms are operative in human fibroblasts.
Human fibroblasts expressed the 1 but not the
2 and
3 isoforms of
Na+-K+-ATPase. Insulin increased the influx of
Rb+, used to trace K+ entry, but did not modify
the total intracellular content of K+, Rb+, and
Na+ over a 3-h incubation period. Ouabain increased
intracellular Na+ more rapidly in cells incubated with
insulin, but this increase followed insulin stimulation of
Rb+ transport. Bumetanide did not prevent the increased
Na+ influx or stimulation of
Na+-K+-ATPase. Stimulation of the
Na+-K+- ATPase by insulin did not produce any
measurable change in membrane potential. Insulin did not affect the
affinity of the pump toward internal Na+ or the number of
membrane-bound Na+-K+-ATPases, as assessed by
ouabain binding. By contrast, insulin slightly increased the affinity
of Na+-K+-ATPase toward ouabain. Phorbol esters
did not mimic insulin action on Na+-K+-ATPase
and inhibited, rather than stimulated, Rb+ transport. These
results indicate that insulin increases the turnover rate of
Na+-K+-ATPases of human fibroblasts without
affecting their number on the plasma membrane or modifying their
dependence on intracellular [Na+].
membrane transport; phorbol esters; membrane potential; rubidium transport
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INTRODUCTION |
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INSULIN INITIATES ITS
ACTION by interacting with specific membrane receptors located on
the plasma membrane of target cells. The insulin-receptor complex
activates a number of cellular functions, among which is transmembrane
transport of ions and nutrients. In human fibroblasts, insulin
stimulates the transport of potassium, glucose, and amino acids
(11, 13, 14, 17). The stimulation of potassium
transport is due to activation of both the
Na+-K+-Cl cotransporter and the
Na+-K+-ATPase; it occurs very rapidly and
reaches a maximum after only 10 min (13). By contrast,
insulin stimulation of glucose and amino acid transport requires
significantly longer times and reaches a maximum after 30 min and
several hours, respectively (14, 17). The mechanism by
which insulin stimulates the Na+-K+-ATPase in
human fibroblasts is not known.
The Na+-K+-ATPase is composed of an - (112 kDa) and a
-subunit (35 kDa). The
-subunit contains the binding
sites for Na+, K+, ATP, and ouabain and is
commonly referred to as the catalytic subunit. There are four isoforms
of Na+-K+-ATPase
-subunits
(
1,
2,
3, and
4) (29). The
-subunit is a glycoprotein
whose function, although essential to pump activity, remains to be
determined (29). There are at least three isoforms of
Na+-K+-ATPase
-subunit (
1,
2, and
3). All these different isoforms have different tissue-specific expression (29), with
4 being expressed only in the testis (32,
33).
The mechanism of insulin stimulation of
Na+-K+-ATPase activity varies among different
cells and tissues. In rat hepatocytes (9) and BC3H-1
myocytes (26), insulin stimulates pump activity by
increasing the availability of Na+ in the cytoplasm.
Increased Na+ availability is due to increased
Na+ influx through the amiloride-sensitive
Na+/H+ exchanger (9, 26). In
3T3-L1 fibroblasts, increased Na+ entry through the
bumetanide-sensitive Na+-K+-Cl
cotransporter has been proposed to play a major role in insulin stimulation of Na+-K+-ATPase, since bumetanide
prevents the stimulation (30). In 3T3-F442A fibroblasts
and adipocytes, insulin stimulates pump activity again by increasing
Na+ entry, but this time through a Na+ channel,
which is not inhibited by amiloride (4). This channel may
correspond to the µ-conotoxin-sensitive Na+ channel
reported in skeletal muscle (20). It is not known whether increased Na+ entry raises intracellular
[Na+], since total intracellular [Na+] has
not been measured. In addition, it is unclear whether the eventual
increase in intracellular [Na+] precedes the insulin
effect on Na+-K+-ATPase.
In primary adipocytes, insulin does not increase Na+ entry
(21) and directly stimulates the 2 isoform
of the pump by increasing its affinity toward intracellular
Na+ (18). In these cells, insulin does not
increase the number of functional
Na+-K+-ATPases on the plasma membrane
(25). By contrast, in the skeletal muscle, another tissue
that expresses the
2 isoform of
Na+-K+-ATPase, insulin increases the number of
Na+-K+-ATPases in the plasma membrane
(10, 19). Immunological studies have indicated that
insulin specifically recruits preformed
2 and
1 isoforms, but not
1 isoforms, from an
intracellular pool (10), in analogy with the mechanism of
insulin action on the insulin-responsive glucose transporter (GLUT4)
(10). It is not clear why the same isoform of
Na+-K+-ATPases behaves differently in different tissues.
In this paper, we report that insulin increases the activity of the Na+-K+-ATPase of human fibroblasts without increasing intracellular [Na+] or the affinity of the pump toward intracellular [Na+]. Insulin effect occurs without an increase in the number of membrane-associated ouabain-binding sites, indicating that insulin increases the turnover rate of existing transporters.
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MATERIALS AND METHODS |
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Materials. 3-O-[U-14C]methyl-D-glucose (55 mCi/mmol) was from NEN. L-[2,3,4,5-3H]arginine monohydrochloride (57 Ci/mmol) and [21,22-3H]ouabain (32 Ci/mmol) were from Amersham. Insulin (bovine sodium, 25 U/mg) was from Calbiochem. Chemical reagents were American Chemical Society grade and were obtained from Sigma or Fisher.
Experimental techniques. Human fibroblasts were cultured in Dulbecco-Vogt medium containing 15% fetal bovine serum. For experiments, cells were seeded in 24-well plates and grown to confluence, and the medium was renewed every 3 days as well as 48 h before each experiment. On the day of the experiment, cells were washed twice and incubated for 2 h at 37°C in Tris (26 mM, pH 7.4)-buffered Earle's balanced salt solution (EBSS) containing 116 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 1 mM NaH2PO4, 0.8 mM MgSO4, and 5.5 mM D-glucose, supplemented with 1% (wt/vol) bovine serum albumin (RIA grade; Sigma). Insulin was then added to the cells, and nonradioactive Rb+ uptake was then measured for the time indicated in the presence or absence of ouabain or bumetanide. In the uptake solution, 5.4 mM KCl was replaced by 5.4 mM RbCl (Rb-EBSS) (16). Cell monolayers were rapidly washed four times with ice-cold 0.1 M MgCl2 (total washing time <10 s) (16). Ethanol (0.1 ml) was then added to each well and allowed to dry. Two milliliters of 5 mM CsCl in water were added to each well, and intracellular Na+, Rb+, and K+ contents were determined in each well by emission flame photometry (Perkin Elmer 460 Atomic Absorption Spectrophotometer). Cell monolayers were then dried, solubilized with 200 µl of 0.1% sodium deoxycholate in 1 M NaOH, and assayed for protein by using a modified Lowry procedure (31).
The membrane potential of human fibroblasts was estimated from the distribution ratio of L-arginine (5). Fibroblasts were incubated for 1 h in EBSS containing L-[3H]arginine (20 µM, 2 µCi/ml) in the absence or presence of insulin, ouabain, or valinomycin. Cells were then washed three times with ice-cold 0.1 M MgCl2, and intracellular arginine was extracted in 0.5 ml of ethanol. The ethanol extract was added to 3 ml of scintillation fluid and counted in a scintillation counter. Intracellular arginine concentration was determined by dividing for intracellular water. Membrane potential was calculated by applying the Nernst equation to the distribution ratio of L-arginine (5). Ouabain binding was measured for 30 min at 37°C. Cells were incubated for 10 min in EBSS without or with insulin, and then they were washed twice with EBSS in which KCl was replaced by choline chloride and incubated with or without insulin in the same K+-free solution for 30 min in the presence of increasing concentrations of ouabain (4-500 nM). Nonspecific binding was measured in the presence of 1 mM cold ouabain and accounted for <2% of total binding at 4 nM ouabain. After binding, cells were washed three times with ice-cold 0.1 M MgCl2, and membrane-bound radioactivity was determined by extraction in ethanol, as for arginine accumulation. Bound ouabain was then normalized to protein content. Preliminary experiments in human fibroblasts indicated that extracellular K+ inhibited ouabain binding with half-maximal inhibition observed at 1.8 ± 0.4 mM. In the absence of extracellular K+, equilibrium ouabain binding was reached at 30 min and remained constant up to 120 min (not shown).Calculations. Intracellular water was evaluated in parallel experiments from the equilibrium distribution of 3-O- methyl-D-glucose, which is reached within 10 min in cultured human fibroblasts (15). The mean cell water content was 7.0 ± 0.7 µl/mg of cell proteins in human fibroblasts and was not affected by insulin treatment.
Intracellular ion content was calculated from the total amount of nonradioactive Na+, K+, and Rb+ (as determined by flame photometry) and was then expressed as micromoles per milliliter of cell water by dividing for the corresponding intracellular water space. Ouabain-sensitive Rb+ uptake was determined by subtracting the uptake in the presence of the inhibitor from total Rb+ uptake (16). The standard error of the difference between two samples was determined as the square root of the sum of the two sample variances (16). Statistical comparisons were performed by using analysis of variance. The analysis of kinetic curves was performed by nonlinear regression analysis with the use of SigmaPlot. The equation used for the uptake of Rb+ at different intracellular [Na+] was
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(1) |
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(2) |
RNA analysis.
RNA was extracted from confluent cells or from tissues by using
guanidinium thiocyanate (6). Total RNA (5-20 µg)
was separated by formaldehyde-agarose gel electrophoresis, transferred
to nylon (ZetaProbe; Bio-Rad), and hybridized in high-stringency
conditions to cDNA encoding the rat 1 (full length),
2 (1.8-kb fragment), and
3 (278-bp
PstI-SmaI fragment corresponding to the 5' of the cDNA) isoforms of Na+-K+-ATPase labeled with
[32P]dCTP by primer extension. Blots were then
washed at 65°C in 0.5× standard sodium citrate and autoradiographed
for 16-24 h at
70°C.
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RESULTS |
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Northern blot analysis.
In rats, there are four isoforms of the -subunit of
Na+-K+-ATPase, with
4 being
expressed only in testis and essential for sperm motility (32,
33). To define which of the other three isoforms of
Na+-K+-ATPase were present in human
fibroblasts, we hybridized RNA to cDNAs coding for the
1,
2, and
3 isoforms of
Na+-K+-ATPase. Human fibroblasts expressed the
1 isoform and had a band of 3.8 kb (Fig.
1). Human fibroblasts did not have mRNA
for the
2 isoform of
Na+-K+-ATPase, while RNA from bovine brain
(used as positive control) gave hybridizing bands of 5.4 and 3.5 kb.
Bovine brain, but not human fibroblasts, expressed a 3.7-kb mRNA
corresponding to the
3 isoform of
Na+-K+-ATPase.
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Effect of insulin on intracellular ion concentrations.
Figure 2 shows the changes in
intracellular [Na+], [K+], and
[Rb+] in human fibroblasts incubated in a buffered
solution containing Rb+ instead of K+. As
expected (16), Rb+ replaced K+ in
the intracellular space (Fig. 2A). The sum of
[Rb+] and [K+] remained constant over time,
as did intracellular [Na+]. In the presence of insulin
(500 nM; Fig. 2B), the exchange of Rb+ for
K+ was faster. However, the sum of [Rb+] and
[K+] remained constant and was not significantly
different from that measured in the absence of insulin.
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Effect of insulin on the membrane potential of human fibroblasts.
Insulin modifies the membrane potential of a number of cell types
(23). It is not known whether this applies to fibroblasts. Figure 4 shows the effect of insulin (500 nM) on the membrane potential of human fibroblasts. Valinomycin (20 µM), which increases K+ permeability and the membrane
potential (5), and ouabain (0.4 mM), which decreases
intracellular K+ concentration and the membrane potential
(16), were used as internal controls. A 1-h incubation in
the presence of insulin did not affect the membrane potential. Shorter
and longer incubation times (30-120 min) with insulin also failed
to modify the membrane potential. These data are consistent with the
lack of changes in intracellular ion content shown in Fig. 2.
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Mechanism for insulin stimulation of
Na+-K+- ATPase
in human fibroblasts.
In primary adipocytes, insulin stimulates pump activity by increasing
the affinity of Na+-K+-ATPase toward internal
Na+ (18). Internal [Na+]
was varied by incubating human fibroblasts during the 5-min uptake
assay with increasing concentrations of Na+ (1-120 mM)
in the presence of monensin, a Na+ ionophore (5 µg/ml).
Na+ was replaced by choline in the incubation medium such
that the sum of [Na+] and [choline+]
remained at 120 mM. Intracellular Na+ was determined by
flame photometry and corrected for the intracellular water
content, determined in parallel trays from the equilibrium distribution of 3-O-methyl-D-glucose
(15). Rb+ uptake was determined in both the
absence and presence of 1 mM ouabain to calculate the portion of
Rb+ uptake due to Na+-K+-ATPase.
Incubation of human fibroblasts with monensin and different extracellular [Na+] varied intracellular
[Na+] from 10 to 50 mM (Fig.
5). Pump activity in cells incubated in
the absence of insulin was highly dependent on intracellular [Na+] with a K0.5 of 23.1 ± 2.8 mM, assuming a cooperative model. Addition of insulin did not
affect the K0.5, which remained at 19.9 ± 2.0 mM. The difference between control and insulin-stimulated cells was not significant (P > 0.05) when 95%
confidence intervals were used. Rb+ uptake was always
higher in insulin-stimulated cells at all intracellular [Na+] created by the experimental procedure inside the
cell. The estimated maximal transport activity by
Na+-K+-ATPase at 5.4 mM Rb+ was
3.4 ± 0.4 and 4.9 ± 0.4 µmol · ml cell
water1 · min
1 (P < 0.01) in the absence and presence of insulin, respectively. This
finding indicates that insulin-stimulated cells have an increased number or activity of Na+-K+-ATPases.
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Effect of kinase activators and inhibitors on insulin stimulation
of Rb+ influx.
Insulin interacts with specific receptors on the plasma membrane of
target cells and stimulates their phosphorylation on tyrosine residues
and kinase activity toward endogenous substrates. Previous studies
(13) indicated that genistein, a tyrosine-kinase
inhibitor, and staurosporine, a serine/threonine kinase inhibitor,
significantly reduced insulin stimulation of
Na+-K+-ATPase. These results indicate that the
kinase activity of the insulin receptor is required to stimulate ion
transport and that a serine/threonine kinase is also involved. Phorbol
esters mimic several actions of insulin, including stimulation of
glucose transport in human fibroblasts (17). Incubation of
human fibroblasts with phorbol 12,13-dibutyrate (PDBU) at a
concentration capable of fully stimulating glucose transport
(17) inhibited, rather than stimulated, ouabain-sensitive
Rb+ influx (Fig. 7). Insulin
reversed the inhibition caused by phorbol esters.
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DISCUSSION |
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Insulin interacts with specific receptors on the plasma membrane
of target cells and stimulates a number of cellular functions, including potassium (Rb+) transport. In human fibroblasts,
insulin stimulates Rb+ transport by both the
Na+-K+-Cl cotransporter and the
Na+-K+-ATPase (13). These effects
of insulin are very rapid and precede stimulation of glucose and amino
acid transport (14, 17). The specific isoform of
Na+-K+-ATPase expressed in human fibroblasts
and how it is stimulated by insulin is not known. Northern blot
analysis indicated that human fibroblasts express only the
1 isoform of Na+-K+-ATPase,
while mRNA for the
2 and
3 isoforms was
not present (Fig. 1). The
2 isoform of
Na+-K+-ATPase was found to be translocated to
the plasma membrane in response to insulin in the muscle
(10) or to increase its affinity toward intracellular
Na+ in adipocytes (18). By contrast, the
1 isoform of Na+-K+-ATPase is
usually expressed in other fibroblast-like cells, and in cells other
than human fibroblasts, its activity is stimulated by increased
intracellular [Na+] caused by hormonal exposure (9,
26).
Previous studies (13) indicate that activation of Na+-K+-ATPase by insulin is dose dependent and increases the Vmax of the pump without significantly affecting the Michaelis-Menten constant (Km) toward K+. This could be caused by increased availability of intracellular [Na+] to the pump, increased affinity of the pump toward intracellular Na+, an increased number of membrane-associated Na+-K+-ATPases, or an increased turnover rate of existing pumps. Activation of pump activity is not inhibited by blockers of the Na+/H+ exchanger (13) and, in this study, occurred in the absence of any observable increase in intracellular [Na+] (Fig. 2). However, when the activity of the Na+-K+-ATPase was inhibited by ouabain, a significant increase in Na+ accumulation was observed (Fig. 3). This increase, however, followed rather than preceded activation of Rb+ uptake by the Na+-K+-ATPase. The delay in the rise of intracellular [Na+] following insulin stimulation was not an artifact of the method used, since an immediate increase in intracellular [Na+] is seen in human fibroblasts stimulated by serum when the same method is used (13). For these reasons, the increase of intracellular [Na+] observed in the presence of ouabain is likely to represent an independent effect of insulin on a separate channel or ion transporter, rather than causing increased Na+-K+-ATPase activity. Increased Na+ influx occurred in the presence of bumetanide and ouabain (Fig. 3), indicating that it is not mediated by the Na+-K+-Cl cotransporter and does not represent a homeostatic mechanism to compensate increased Na+ extrusion by the Na+-K+-ATPase.
Insulin modifies the membrane potential of a number of target cells (23). This is accomplished by modification of ion permeability or stimulation of the Na+-K+-ATPase (12, 24, 35). In human fibroblasts, insulin failed to modify significantly the membrane potential (Fig. 4). While newer methods for the measurement of membrane potential such as the use of microelectrodes or fluorescent dyes could have been more sensitive to transient changes (8), the distribution ratio of arginine used in this study would have detected stable changes in membrane potential induced by insulin. In fact, this system could easily detect changes caused by exposure to ouabain and valinomycin (Fig. 4). These results indicate that the increased sodium extrusion caused by stimulation of the Na+-K+-ATPase was not sufficient to hyperpolarize the cell membrane or that it was compensated by the secondary increase in Na+ accumulation shown in Fig. 3. Changes in membrane potential have been proposed in the past as part of the mechanism of insulin action (23, 34). Our data indicate that stimulation of glucose and amino acid transport in human fibroblasts (14, 17) can well occur in the absence of any change in membrane potential.
Although stimulation of the Na+-K+-ATPase in human fibroblasts occurred in the absence of a significant increase in intracellular Na+, insulin could have increased the affinity of existing pumps to intracellular Na+ (18). Direct measurement of intracellular Na+ indicated that the Na+-K+-ATPase of human fibroblasts had the same affinity toward Na+ in cells stimulated or not stimulated by insulin (Fig. 5). Importantly, at the same concentration of intracellular Na+, the Na+-K+ pump was more active in insulin-treated than in control cells, indicating that insulin increased either the number of active Na+-K+-ATPases or their turnover rate. Measurement of ouabain binding indicated that insulin did not increase the number of membrane-associated Na+-K+-ATPases but slightly increased their affinity toward ouabain (Fig. 6). Because the number of Na+-K+-ATPases was not modified, by exclusion insulin is predicted to stimulate the Na+-K+-ATPase of human fibroblasts by increasing its turnover rate. Previously published data (13) and those in Fig. 6 indicate that insulin increased the turnover rate of the Na+-K+-ATPase of human fibroblasts from 10 to 18.5 cycles/s, assuming that two K+ are taken up by the cell at each cycle (16). The slight, but significant, modification of the Kd toward ouabain induced by insulin is suggestive of possible conformational changes of the Na+-K+-ATPase induced by insulin. Alternatively, since ouabain binds only during part of the Na+-K+-ATPase cycle, insulin may simply increase the rate of a limiting step of pump cycling, increasing both the rate of the overall process and the affinity for ouabain interaction.
After ligand binding, the insulin receptor becomes an active tyrosine
kinase and activates a phosphorylation cascade within target cells.
This phosphorylation cascade is essential for insulin action on glucose
transport. Previous studies in human fibroblasts have shown that
genistein, a tyrosine kinase inhibitor (1), and
staurosporine, an inhibitor of serine/threonine kinases activated by
the insulin receptor (28), block insulin effect on the
Na+-K+ pump (13). Additional data
presented in this report indicate that insulin stimulation of the
Na+-K+-ATPase was not mimicked by the protein
kinase C activator PDBU, which inhibited, rather than stimulated,
Rb+ transport (Fig. 7). This was relatively unexpected
because phorbol esters stimulate glucose transport in human fibroblasts
(17). Phorbol esters also stimulate the
Na+-K+-ATPase in some cultured skeletal muscle
cells (27) but inhibit it in others (2). In
the case of the 1-subunit of
Na+-K+-ATPase, mutagenesis of the site
responsible for protein kinase C phosphorylation results in enzyme
activation and prevents further inhibition by phorbol esters
(2), indicating that activation of protein kinase C may
have an inhibitory action on Na+-K+-ATPase. Our
data in fibroblasts, which express the
1-subunit of
Na+-K+-ATPase, are consistent with this model
and indicate that insulin can reverse the inhibition caused by phorbol
esters (Fig. 7). Wortmannin, an inhibitor of phosphatidylinositol
3-kinase, inhibits insulin stimulation of Rb+ transport in
3T3-L1 fibroblasts (30). However, it was without effect in
human fibroblasts (Fig. 7), indicating that the mechanism used by
insulin to activate Na+-K+-ATPase differs even
among similar cell types. This contrasts with insulin activation of
glucose transport, in which wortmannin acts as an inhibitor in
different cell systems (3, 7), including fibroblasts and
Chinese hamster ovary cells (22). The results with kinase
activators and inhibitors indicate that insulin stimulates ion and
glucose transport with independent mechanisms in human fibroblasts.
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ACKNOWLEDGEMENTS |
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We thank Dr. Jerry B. Lingrel, University of Cincinnati, Ohio, for
kindly providing cDNA probes for the 1,
2, and
3 isoforms of
Na+-K+-ATPase.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-48742.
Present address of F. Scaglia: Feigin Center, Ste. C235, 6621 Fannin, Department of Molecular and Human Genetics, Baylor College of Medicine, Mail code 3-3370, Houston, TX 77030.
Address for reprint requests and other correspondence: N. Longo, Division of Medical Genetics, Dept. of Pediatrics, Emory Univ., 2040 Ridgewood Drive, Atlanta, GA 30322 (E-mail: nl{at}rw.ped.emory.edu).
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. Section 1734 solely to indicate this fact.
Received 18 July 2000; accepted in final form 1 November 2000.
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