Unité Mixte de Recherche 6548, Centre National de la Recherche Scientifique, Université de Nice-Sophia Antipolis, 06108 Nice Cedex 2, France
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ABSTRACT |
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We characterized
Cl conductance activated by extracellular ATP in an
immortalized cell line derived from rabbit distal bright convoluted
tubule (DC1). 125I
efflux experiments showed
that ATP increased 125I
loss with an
EC50 = 3 µM. Diphenylamine-2-carboxylate
(10
3 M) and NPPB (10
4 M) abolished the
125I
efflux. Preincubation with 10 µM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester or 10
7 M thapsigargin inhibited
the effect of ATP. Ionomycin (2 µM) increased
125I
efflux with a time course similar to
that of extracellular ATP, suggesting that the response is dependent on
the intracellular Ca2+ concentration
([Ca2+]i). The ATP agonist potency order was
ATP
UTP > ATP
S. Suramin (500 µM) inhibited the
ATP-induced 125I
efflux, consistent with P2
purinoceptors. 125I
effluxes from cells grown
on permeable filters suggest that ATP induced an apical efflux that was
mediated via apical P2 receptors. Whole cell experiments showed that
ATP (100 µM) activated outwardly rectifying Cl
currents
in the presence of 8-cyclopentyl-1,3-dipropylxanthine, excluding the
involvement of P1 receptors. Ionomycin activated Cl
currents similar to those developed with ATP. These results demonstrate the presence of a purinergic regulatory mechanism involving ATP, apical
P2Y2 receptors, and Ca2+ mobilization for apical
Cl
conductance in a distal tubule cell line.
kidney; intracellular calcium
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INTRODUCTION |
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ION CHANNELS IN
EPITHELIAL cells play an essential role among various mechanisms
of transcellular Cl transport. As in secretory epithelia,
where they have been extensively studied, Cl
channels
with diverse and distinct properties have been described for the
kidney. In previous papers we have shown that primary cultures of the
rabbit distal tubule express three different Cl
conductances regulated by cAMP, cytosolic Ca2+, or by cell
swelling (3, 30, 36). Moreover,
proof of the existence of both A1 and A2 receptors in the basolateral
membrane of distal tubule cells has been made possible by using the DC1 cell line immortalized from primary cultures of these cells
(31).
Adenosine activates an apical cystic fibrosis transmembrane conductance
regulator (CFTR) Cl conductance by a pathway involving
A2A receptors, G proteins, adenylate cyclase, and protein kinase A
(31). It has also been postulated that adenosine induces
an increase in calcium influx via A1 receptors, which, in turn,
stimulates swelling-activated Cl
channels
(29). In attempting to elucidate properties of purinergic receptors in the distal tubule, we were able to identify, in a preceding paper (1), a P2Y2 receptor linked to a
Ca2+-dependent signaling transduction mechanism.
Consequently, in the present study, we have examined the effect of ATP
on the Cl
conductance in the DC1 cell line.
Previous studies in secretory epithelia have shown that extracellular
ATP stimulates Cl secretion across the apical membrane
(15, 33, 35). The majority of
authors concur with the existence of P2Y2 receptors in secretory
epithelia, the stimulation of which increases cytosolic Ca2+, which, in turn, activates Ca2+-sensitive
Cl
channels. Various studies performed in cultured
epithelial cells of the kidney, such as proximal tubule
(5) and collecting duct cells (18), as well
as Madin-Darby canine kidney (MDCK) (40), LLC-
PK1 (38), and A6 (24,
26) cells, have demonstrated the presence of ATP receptors
responsible for the triggering of ion transport mechanisms. This is
also the case for isolated Necturus maculosus and
rabbit proximal tubules (4, 6). Although it seems clear that the receptor involved is a P2Y-type receptor, conflicting conclusions have arisen concerning the location of this
receptor, the location and nature of the channels involved in mediating
its effects, and the mechanism of the signaling pathway involved. For
example, in N. maculosus proximal tubule
(4), a basolateral P2Y1 receptor increases a
Ca2+-insensitive basolateral Cl
conductance
and mobilizes Ca2+ independently from internal stores. In
MDCK cells, two apical subtypes of purinoceptors could control
transepithelial ion transport by means of an increase in cytosolic
Ca2+ (P2Y1) or prostaglandin synthesis (P2Y2)
(39). Moreover, in primary cultures of rabbit cortical
collecting and connecting tubules (18), ATP binds to an
apical P2Y2 receptor, thereby inhibiting Na+ and
Ca2+ reabsorption independently of a Ca2+
signaling mechanism.
In the present study, we demonstrate that DC1 cells, immortalized from
cultured DCTb cells, express the P2Y2 receptor in their apical
membranes. Stimulation of this receptor by ATP activates a
Ca2+-sensitive Cl conductance in the apical
membrane via a pathway involving the liberation of Ca2+
from internal stores.
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MATERIALS AND METHODS |
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Cultures
The DC1 renal cell line was obtained from primary cultures of rabbit distal convoluted tubule after transfection of cells with the pSV3 neo plasmid and G418 selection. The technique of transformation of primary cultures is described in a previous paper (31). Cultures were seeded on collagen-coated 35-mm petri dishes or on collagen-coated permeable Millipore filters filled with a culture medium composed of equal quantities of DMEM and Ham's F-12 (GIBCO, Grand Island, NY). The medium was supplemented with 15 mM NaHCO3, 20 mM HEPES at pH 7.4, 2 mM glutamine, 5 mg/l insulin, 50 nM dexamethasone, 10 µg/l epidermal growth factor, 5 mg/l transferrin, 30 nM sodium selenite, and 10 nM triiodothyronine. Cultures were maintained at 37°C in a 5% CO2-95% air, water-saturated atmosphere. DC1 cells were used between passages 12 and 25.125I Efflux From DC1 Monolayers
Calculations.
From backaddition of the radioactivity in the efflux samples to the
radioactivity remaining in the cells, the apical and basolateral efflux
rate constants were calculated according to the following equations.
The equations thus give the fraction of total radioactivity lost per
unit time
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Whole Cell Experiments
Whole cell currents were recorded from DC1 cells (3-4 days of age) grown on collagen-coated supports maintained at 33°C for the duration of the experiments. The ruptured-patch whole-cell configuration of the patch-clamp technique was used. Patch pipettes (resistance 2-3 MData acquisition and analysis.
Voltage-clamp commands, data acquisition, and data analysis were
controlled via a computer equipped with a Digi data 1200 interface
(Axon Instruments). pCLAMP software (versions 5.51 and 6.0, Axon
Instruments) was used to generate whole cell current-voltage relationships, with the membrane currents resulting from voltage stimuli filtered at 1 kHz, sampled at 2.5 kHz, and stored directly onto
the computer's hard disk. Cells were held at a holding potential of
50 mV, and 400-ms pulses from
100 to +120 mV were applied in
increments of 20 mV every 2 s.
Chemical Compounds
Diphenylamine-2-carboxylate (DPC) from Aldrich was prepared as a 1 M stock solution in DMSO and dissolved at 1 mM in the incubation medium. DIDS from Sigma Chemical was dissolved directly to a final concentration of 1 mM. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX; Sigma Chemical) was prepared as a 10 mM stock solution in DMSO. Suramin was kindly provided by Bayer-Pharma, France (Puteaux). Ionomycin (Sigma Chemical) was dissolved at 2 mM in ethanol and used at a final concentration of 2 µM in solutions. The following compounds were purchased from Sigma-Aldrich: ATP, adenosine 5'-O-[thiotriphosphate] (ATP ![]() |
RESULTS |
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125I Efflux Stimulated by Extracellular
ATP
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Figure 2 shows that the application of
the chloride channel blockers DPC (1 mM) or DIDS (1 mM) completely
abolished the ATP-evoked 125I efflux.
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Involvement of intracellular Ca2+ in the ATP
response.
We have previously shown that purinoceptor stimulation in DC1 cells
increases the intracellular Ca2+ concentration
([Ca2+]i) (1). Consequently, the
involvement of cytosolic Ca2+ in the Cl
permeability of DC1 cells was investigated here. Preincubations for 1 h
with the membrane-permeable, Ca2+-chelating agent
1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM; 10 µM) resulted in the
complete inhibition of the extracellular ATP-induced rise in
125I
efflux (Fig.
3). TG, a well-known inhibitor of the
endoplasmic reticulum Ca2+-ATPase that causes depletion of
intracellular Ca2+ stores, was used to further investigate
the way in which depleting cytosolic Ca2+ could affect the
level of 125I
efflux. Treatment with TG
(10
7 M for 30 min) significantly inhibited the ATP-evoked
125I
efflux (Fig. 3), which strongly suggests
that the ATP-induced chloride efflux is regulated by
[Ca2+]i.
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Characterization of purinergic receptors.
To analyze the nature of the purinoceptor involved in the increase in
125I efflux described here, the effects of
various other nucleotides were tested. The histogram in Fig.
5A shows the magnitude of the 125I
efflux response obtained 1 min after
exposure of cells to ATP or ATP analogs. ATP
S (10 µM), a
nonhydrolyzable ATP analog, enhanced 125I
efflux, although the amplitude of the response was less than that
obtained with 10 µM ATP. UTP (50 µM) or ATP at the same
concentration was equipotent in stimulating
125I
efflux. The order of agonist potency was
thus measured to be ATP
UTP > ATP
S.
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Localization of purinergic receptors and ATP-activated
Cl conductance.
In a second set of experiments, DC1 cells were grown on permeable
Millipore supports, which permitted separate measurements to be made of
125I
effluxes across the apical and
basolateral membranes, as well as determination of the location of P2
receptors. Figure 6 shows the degree of
125I
efflux measured across both membranes.
Under control conditions (i.e., during the first 4 min before the
application of ATP), apical and basolateral
125I
effluxes were independent of time. The
basolateral efflux rate constant [(6.09 ± 0.27) × 10
2 min
1, n = 18] exceeded
by a factor of 1.25 the apical rate constant [(4.13 ± 0.28) × 10
2 min
1, n = 18].
Figure 6 shows the 125I
effluxes across both
membranes when ATP was applied either to the apical (Fig.
6A) or to the basolateral (Fig. 6B) side of the monolayer. ATP induced an increase in apical
125I
efflux only when added to the apical
bathing medium, which is to say that basolateral efflux was not
modified by ATP.
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ATP-Activated Whole Cell Currents
To further characterize the regulation of ClWhole cell currents were recorded with a low-[Ca2+]
pipette solution containing 140 mM NMDGCl (see MATERIALS AND
METHODS). Extracellular solutions were made hyperosmotic (140 mM
NMDGCl + 50 mM mannitol) to prevent the development of
swelling-activated Cl currents. After successful gigaseal
formation, the whole cell configuration was obtained in 10% of cells
tested. Voltage-clamp experiments were performed by holding the
membrane potential at
50 mV and applying voltage steps of 400-ms
duration every 2 s from
100 to 120 mV in 20-mV increments. In a
large majority of cells, the voltage-step protocol elicited small
currents (Fig. 7A) that
changed linearly with the membrane voltage and reversed near 0 mV (Fig.
7D). The amplitude of the currents was 59 ± 11 pA
(n = 8) at +100 mV. Because of its small amplitude, the
nature of the current was not analyzed further, although its reversal potential indicates that Cl
may well have been the charge
carrier.
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In previous reports (3), it was shown that intracellular
Ca2+ was implicated in the regulation of Cl
channels in cultured DCTb cells. For this reason, therefore, the role
of ATP and ionomycin in the whole cell currents of DC1 cells was evaluated.
The application of ATP (100 µM) to the bath medium induced an
increase in membrane currents within 1 min, even in the presence of
DPCPX, which thus excludes the involvement of P1 receptors in this
process (Fig. 7B). The kinetics of the macroscopic current were clearly time dependent for depolarizing potentials and exhibited a
slowly developing component. The corresponding current-voltage relationships for steady-state activated currents measured at 380 ms
are given in Fig. 7D. The ATP-activated currents measured in
symmetrical Cl solutions reversed at
2.59 ± 1.82 mV (n = 4), which is close to the equilibrium potential
for Cl
. The steady-state current exhibited marked outward
rectification, with an inward current at
100 mV of
53.2 ± 10.8 pA and outward current at +100 mV of 144.1 ± 26.1 pA
(n = 4). The currents were transient in nature and
returned to control levels within 3 min after the exposure of cells to
ATP (Fig. 7C).
To determine the possible role of cytosolic Ca2+ in the
development of ATP-induced Cl currents, experiments were
performed by using pipette solutions containing the Ca2+-
chelating agent EGTA (5 mM) to suppress intracellular free
Ca2+. In 100% of the cells tested, the ATP-induced
activation of Cl
currents was blocked (data not shown).
Ionomycin-activated whole-cell currents were also studied. Elevation of
[Ca2+]i by the application of 2 µM
ionomycin to the bath solution activated outwardly rectifying
Cl currents that exhibited delayed activation kinetics at
depolarizing voltages, in a manner very similar to those developed with
extracellularly applied ATP (Fig.
8B). The reversal potential of
these currents was
1.8 ± 0.9 mV. One minute after the exposure
of cells to ionomycin, the amplitude of the steady-state current
recorded at +100 mV was 2.7 times the current at
100 mV (i.e.,
35.2 ± 12.2 vs. 95.3 ± 8.6 pA at
100 mV and +100 mV,
respectively; n = 4). The current amplitude increased 3 min after the commencement of exposure to ionomycin (Fig.
8C).
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DISCUSSION |
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In the present study, the effects of extracellular ATP on
125I efflux and on whole cell
Cl
currents in DC1 cells were investigated. Results
clearly showed that extracellular ATP stimulated Cl
secretion via the activation of apical P2Y2 receptors and intracellular Ca2+-dependent signaling pathways, which in turn activated
Ca2+-dependent Cl
channels in the apical
membrane of the DC1 cells.
The activation of Cl conductances via P2 purinoceptors
has been reported for various epithelial tissues such as airway
epithelium (34, 41), colonic T84
(10) or HT-29 cells (19), epididymal cells
(8), endometrial epithelium (7), and the
distal colon (21). Although many studies have been devoted
to the characterization and the localization of P2 purinoceptors in
renal tissue (4, 6, 18,
39), fewer works have investigated the role of these receptors in the control of transmembrane ion movements. Of the studies
that have investigated these effects, data were mainly obtained by
using MDCK cells (40), A6 cell lines (24,
26), N. maculosus proximal tubule
(4), and cultured rabbit connecting tubules
(18). We have previously shown that the DC1 cell line immortalized from rabbit DCTb expresses characteristics of the native
epithelium (23). Notably, it was found that DC1 cells exhibited CFTR Cl
channels in the apical membrane and
that this conductance could be enhanced by extracellularly applied
adenosine acting via A2A receptors located in the basolateral membrane
that were coupled to the adenylate cyclase second-messenger system
(31).
In a companion paper to this report (1), we have also
shown that extracellular ATP raises [Ca2+]i
via P2Y2 receptor activation. The present data demonstrate that
exposure of 125I-loaded DC1 cells led to a
concentration-dependent increase in 125I
efflux with a half-maximal effect at an ATP concentration of ~3 µM.
Comparison of the effects of ATP with the effects of other nucleotides
yielded the potency sequence UTP
ATP > ATP
S, which corresponds to the conventional pharmacology of the P2Y2-type receptor
(28).
In accordance with literature data (7, 8,
15, 35, 41, 42),
our observations suggest that
[Ca2+]i-activated signaling pathways were
important for stimulation of Cl conductances by ATP.
First, the stimulation of 125I
efflux by ATP
was completely inhibited by the pretreatment of cells with BAPTA-AM or
TG, suggesting that ATP receptors in DC1 cells utilized
Ca2+ as a second messenger. Second, the elevation of
[Ca2+]i by ionomycin mimicked the response to
ATP. Finally, the order of nucleotide potencies for the elevation of
[Ca2+]i and stimulation of
125I
efflux was equivalent.
It could be postulated that the stimulation of Cl
secretion by ATP is mediated by P2Y2-linked Ca2+
mobilization, which in turn activates Ca2+-dependent
Cl
channels. This notion is supported by the results
obtained from whole cell patch-clamp experiments, which demonstrated
that the currents activated by ATP and ionomycin shared similar
characteristics. This finding suggests that the two agents act on the
same Cl
conductance by a common mechanism, i.e, by
raising intracellular Ca2+. The ATP-activated whole cell
currents exhibited delayed activation at depolarized voltages and
outward rectification. Similar properties have been described for the
Ca2+-dependent Cl
channel described in
cultured DCTb (3) and in various epithelial cells
(12). However, compared with ionomycin-induced currents, ATP-sensitive Cl
currents were of smaller amplitude and
decreased rapidly after the nucleotide application. This could be
explained by the fact that the rise in cytosolic Ca2+
induced by ATP was transient and probably occurred in a more physiological range than that obtained with ionomycin (1,
30). In contrast to other reports (10,
16), the mechanism underlying Cl
secretion
by ATP is probably not related to the activation of Ca2+-sensitive K+ channels because barium
and charybdotoxin were not effective in blocking the ATP-induced
Cl
secretion.
As stated above, we have previously demonstrated that DC1 cells possess
P1 purinoceptors (31). Thus ATP might activate a Cl conductance either directly by binding to a P2Y2
receptor or indirectly by its conversion by ectoenzymes to adenosine,
which binds to P1 receptors. Because the response to ATP is preserved in the presence of DPCPX, an adenosine receptor antagonist, it is
unlikely that hydrolysis of ATP to adenosine is responsible for the
response initiated by raised [Ca2+]i.
Most of the literature data agree that the increase in Cl
conductance via P2Y2 receptors is mediated by an increase in
intracellular Ca2+ (7, 13,
16, 41). However, a study has indicated that such an activation of Cl
conductance is not dependent on
an increase in cytosolic Ca2+ (4). Taken
together, these results indicate that extracellular ATP might modulate
different types of Cl
channels, probably via different
mechanisms. For instance, in MDCK cells, Zegarra-Moran et al.
(40) suggest that ATP stimulates Cl
secretion via a P2Y1 receptor linked to increased cytoplasmic Ca2+ and via P2Y2 receptors coupled to prostaglandin
secretion in the absence of any change in cytosolic Ca2+.
In primary cultures of tracheal epithelial cells, Hwang et al. (15) proposed an interesting model in which ATP stimulates
Cl
secretion through Ca2+-activated
Cl
channels via P2Y2 receptors coupled to pathways,
resulting in increased Ca2+. This group also activated CFTR
Cl
channels via P2Y3 receptors coupled to a
cAMP-dependent pathway. Chan et al. (8) described another
model, using epididymal cells, which suggested that both
Ca2+- and cAMP-dependent Cl
conductances were
activated by extracellular ATP via a unique P2 receptor and that
activation of a cAMP cascade by ATP is Ca2+ and calmodulin
dependent. These three examples underlie the complexity of the
regulation of Cl
transport by extracellular ATP. Although
DC1 cells express at least three types of Cl
conductances
[i.e., CFTR Cl
channels, volume-sensitive
Cl
channels, and Ca2+-dependent
Cl
channels (3, 30,
36)], the present data indicate that ATP is involved only
in the control of the calcium-sensitive Cl
conductance,
probably via the activation of P2Y2 receptors only. In contrast to the
studies cited above, an action of ATP on CFTR Cl
channels
was never observed, although it can be clearly demonstrated that
adenosine activates an apical CFTR Cl
conductance via a
pathway involving A2A receptors (31).
In addition to Ca2+-dependent Cl channels, it
has also been postulated that ATP could activate a volume-sensitive
Cl
conductance in human bronchial cells
(41). This action will be Ca2+ dependent and
mediated by the P2Y2 receptor. In DC1 cells, ATP did not stimulate a
Cl
conductance with characteristics of volume-sensitive
currents, although adenosine acting via A1 receptors is capable of this (29). Thus considering that ectonucleotidase activity
exists in many tissues, including kidney (20,
32), one could pose the question of whether some of the
effects of ATP on epithelial Cl
conductances reported in
the literature could be attributable to adenosine.
One of the findings of the present work was that the action of ATP in
increasing Cl conductance was observed only when ATP was
applied to the apical membrane of the cell monolayer. Moreover, ATP
triggered a Cl
efflux only through the apical membrane.
This result strongly suggests that P2Y2 receptors are located on the
apical membrane, together with the Ca2+- sensitive
Cl
conductance. The location of P2 receptors in
epithelial cells, as judged from literature reports, is not clear-cut.
It can be generally concluded that P2Y2 receptors in respiratory cells
are located in the apical membrane (9, 15,
22), whereas other subtypes of purinoceptors are probably
present in the basolateral membrane (15, 22).
In other epithelial cells P2Y2 receptors are found either exclusively
in the basolateral membrane (42) or in both the
basolateral and apical membranes (7, 13,
16, 18).
In DC1 cells, the apical location of P2Y2 receptors raises the question of the in vivo sources of luminal nucleotides that could bind to apical purinoceptors. An autocrine mechanism can be postulated for the release of ATP from epithelial cells (6, 25, 37). In the kidney, ATP is also produced by paracrine mechanisms, particularly involving macula densa cells. In this case, ATP is released into the interstitial fluid bathing the juxtaglomerular structures (17), and can therefore reach the distal tubule.
The physiological responses induced by ATP on renal function remain
largely unexplored. Up until the present time, ATP has been shown to
affect renal microvascular function and, more specifically, influences
the tubuloglomerular feedback mechanism while indirectly affecting
epithelial transport mechanisms. Besides this role, the action of ATP
on Cl secretion in the distal tubule has not yet been
placed in a physiological context. However, the bright part of the
rabbit distal tubule, which expresses apical CFTR Cl
channels (27, 36) and Na+
channels (23), could behave as a Cl
secretory epithelium (27, 36). Therefore,
activation of the apical Ca2+-dependent Cl
conductance would contribute to the regulation of transepithelial Cl
transport. Because these cells possess an apical
Cl
/HCO3
exchanger (2), one
could postulate that the secretion of Cl
through the
apical membrane might indirectly activate the
Cl
/HCO3
exchanger by increasing the
apical inward Cl
gradient, thereby causing the secretion
of HCO3
ions.
In conclusion, the results for these experiments are summarized in the
schematic diagram presented in Fig. 9:
ATP stimulates apical Ca2+-sensitive Cl
conductances in DC1 cells via an apical P2Y2 receptor, which triggers
an increase in cytosolic Ca2+. In addition, basolateral
adenosine could also stimulate apical CFTR Cl
channels
via an A2 receptor coupled to cAMP production (31).
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FOOTNOTES |
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Address for reprint requests and other correspondence: P. Poujeol, UMR, CNRS 6548, Bâtiment Sciences Naturelles Université de Nice-Sophia Antipolis, Parc Valrose, 06108 Nice Cedex 2, France (E-mail:poujeol{at}unice.fr).
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.
Received 20 September 1999; accepted in final form 29 February 2000.
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