1 Physiologie Cellulaire, Unité Mixte de Recherche Centre National de Recherche Scientifique 6558 and 2 Institut de Biologie Moléculaire et d'Ingéniérie Génétique, Université de Poitiers, 86022 Poitiers, France
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ABSTRACT |
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Gap junctional channels are essential for normal cardiac impulse propagation. In ventricular myocytes of newborn rats, channel opening requires the presence of ATP to allow protein kinase activities; otherwise, channels are rapidly deactivated by the action of endogenous protein phosphatases (PPs). The lack of influence of Mg2+ and of selective PP2B inhibition is not in favor of the involvements of Mg2+-dependent PP2C and PP2B, respectively, in the loss of channel activity. Okadaic acid (1 µM) and calyculin A (100 nM), both inhibitors of PP1 and PP2A activities, significantly retarded the loss of channel activity. However, a better preservation was obtained in the presence of selective PP1 inhibitors heparin (100 µg/ml) or protein phosphatase inhibitor 2 (I2; 100 nM). Conversely, the stimulation of endogenous PP1 activity by p-nitrophenyl phosphate, in the presence of ATP, led to a progressive fading of junctional currents unless I2 was simultaneously added. Together, these results suggest that a basal phosphorylation-dephosphorylation turnover regulates gap junctional communication which is rapidly deactivated by PP1 activity when the phosphorylation pathway is hindered.
connexin; patch clamp; protein phosphorylation
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INTRODUCTION |
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CELLS OF A VAST MAJORITY of tissues directly exchange ions, second messengers, and small metabolites through intercellular channels clustered in specialized membrane areas called gap junctions. Intercellular channels are formed from two oligomeric assemblies of integral membrane proteins, termed connexons, which span two adjacent cell plasma membranes and join in a narrow extracellular gap. Each connexon is formed by the oligomerization of six protein subunits (connexins) that delineate an aqueous pore. Connexins are homologous proteins encoded by a multigene family and are named according to their predicted molecular weight (3). Connexin 43 (Cx43), widely distributed in different cell types, is the main gap junction protein expressed in ventricular myocytes, although Cx40 and Cx45 have also been reported to be expressed (21). Cx43 is a phosphoprotein, and a dephosphorylated state (41 kDa) and two phosphorylated forms of the protein (43 and 47 kDa, respectively) have been identified (for review, see Ref. 21). In many tissues, e.g., neonatal heart cells in primary culture, Cx43 is predominantly phosphorylated, and it was suggested that changes in the connexin phosphorylation state could modulate the gap junctional intercellular communication (GJIC) capacity (reviewed in Refs. 22 and 25). However, the fact that GJIC reduction may also occur without (9, 19) or before (23) Cx43 dephosphorylation leads to the possibility that the target protein for protein phosphatases (PPs) might be an accessory protein, capable of interacting with Cx43 and possibly involved in its function and regulation. Protein phosphorylation, then, directly or through protein-protein interactions, appears to be implicated in the regulation of a broad variety of junctional communication processes.
A nucleophilic agent, 2,3-butanedione monoxime (BDM), considered to have a "chemical phosphatase" activity (8) or to enhance the activity of endogenous phosphatases (38), interrupted GJIC, and a part of its action seemed to result from a dephosphorylation process (32). To sustain a sufficient state of phosphorylation, high energy phosphates have to be present. In intact cardiac myocytes, for instance, under physiological conditions, the intracellular ATP concentration is kept at a level ranging from 3.0 to 7.5 mM (1).
Under voltage-clamp conditions, ATP has to be present at comparable levels in the solution in contact with the intracellular medium to preserve cell-to-cell communication in both ventricular myocytes (28, 31) and astrocytes (30). ATP is known for its ability to maintain the activity of some membrane channels by a direct binding; its presence also allows the maintenance of a low Ca2+ concentration at the inner surface of the plasma membrane and makes possible protein kinase (PK) activities to counteract a tonic PP activity. The last-mentioned action appeared responsible for the interruption of cell-to-cell communication in ventricular myocytes of newborn rat. Indeed, in these cells, the rapid decline of channel activity in ATP-deprived conditions can be both mimicked by PK inhibition and virtually abolished by nonspecific inhibition of phosphoserine/threonine PPs (31), suggesting that the channel rundown results from dephosphorylation of either the channel-forming protein itself or an associated regulatory protein, induced by endogenous, plausibly membrane-bound PP(s).
Phosphoserine/threonine PPs are classically divided into two classes:
type 1 phosphatases (PP1) are inhibited by two heat-stable proteins,
termed protein phosphatase inhibitor 1 and inhibitor 2 (I1 and I2), and
preferentially dephosphorylate the -subunit of phosphorylase kinase,
whereas PP2 are insensitive to I1 and I2 and preferentially
dephosphorylate the
-subunit of phosphorylase kinase
(7). PP2 can be further subdivided into PP2A
(okadaic sensitive), PP2B (Ca2+ dependent), and PP2C
(Mg2+ dependent).
The aim of the present study was to identify the PP responsible, in rat ventricular myocytes in culture, for the interruption of cell-to-cell coupling when not counterbalanced by PK activities.
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METHODS |
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Cell preparation. Cardiomyocytes were obtained from neonatal (1- to 2-day-old) Wistar rats that were killed by cervical dislocation and then decapitated. Heart ventricles were minced into small pieces (~1 mm3), washed in a Ca2+- and Mg2+-free medium (Spinner's solution) that contained (in mM) 116 NaCl, 5.3 KCl, 8 NaH2PO4, 0.3 NaHCO3, 10 HEPES, and 5.6 D-glucose (pH 7.4), and incubated in the same solution, supplemented with 0.02% crude trypsin (Boehringer-Mannheim, Meylan, France). Five successive incubations at 37°C, for 8-10 min each, upon continuous stirring, were carried out. The successive enzymatic releases, except the first, were cooled at 4°C and centrifuged at 500 g for 10 min. The cell pellets were resuspended in Ham's F-10 culture medium (GIBCO, Cergy-Pontoise, France) supplemented with 10% fetal calf serum (Boehringer), 10% heat-inactivated horse serum (GIBCO), penicillin G (100 IU/ml; Sigma, Saint Quentin Fallavier, France), and streptomycin (50 IU/ml; Sigma). They were preplated in large polystyrene dishes (Nunclon, Roskilde, Denmark) to allow the attachment of nonmuscle cells. The cardiac myocytes in the supernatants were counted, diluted with culture medium to reach a final concentration of 300,000 cells/ml, and seeded (~55,000 cells/cm2) in 35-mm plastic petri dishes (Nunclon). Finally, the cells were incubated at 37°C in a CO2 incubator (5% CO2-95% ambient air, giving a pH of 7.4). After 24 h, the culture medium was replaced by a culture medium devoid of fetal calf serum. The experiments were performed after 2 or 3 days of culture; the dishes were transferred onto the stage of an inverted microscope, and the cells were observed by phase-contrast microscopy. The spontaneous synchronized mechanical activity was used as evidence to avoid confusion with nonmuscle cells.
Evaluation of the junctional conductance.
The macroscopic gap junctional conductance
(Gj) was determined using a dual-voltage
clamp technique applied to myocyte cell pairs. Both cells of a pair
were at first clamped at a common holding potential
(Vh; close to 70 mV), then a pulse (e.g.,
10 mV) was applied to one cell while the other was maintained at Vh to generate a transjunctional voltage
difference (Vj). Therefore, when cells in
contact were connected by open junctional channels, this voltage
gradient induced a junctional current (Ij)
flowing through them from one cell to its neighbor and was compensated by an opposite current supplied by the feedback amplifier connected to
the cell maintained at Vh.
Gj was then calculated by dividing Ij by the amplitude of the
Vj pulse. Current and potential records were
digitized, stored, and analyzed with a personal computer by means of a
software package (pCLAMP; Axon Instruments, Burlingame, CA).
Gj was expressed as means ± SE.
Ca2+ imaging experiments. A possible involvement of a rise in the intracellular free Ca2+ concentration ([Ca2+]i) during antimycin A action was examined by photometry of the light emission of the fluorescent Ca2+ indicator indo 1. The use of this compound allows us to rule out the possibility of a partial loss of the Ca2+ indicator (through a permeabilized membrane or through incidental bleaching) that could mask an eventual increase of free Ca2+ concentration. Indeed, the ratio of the fluorescent signals from the free and from the Ca2+-bound molecules does not depend on dye loading or leakage, nor on cell thickness, and possible changes in [Ca2+]i during the experiment can be studied by determining the fluorescent signal ratio F405/F485.
Briefly, cells were loaded for 30 min at 37°C in the dark with 3 µM of the ester form of indo 1 (indo 1-AM, dissolved in DMSO) with the addition of 0.03% Pluronic acid F-127 (diluted in Tyrode solution) to facilitate the loading of the Ca2+ indicator. Cells were then washed off carefully several times with Tyrode to prevent further loading and were placed again in the cell incubator for 15 min to complete ester hydrolysis. Fluorescence was recorded using an epifluorescence inverted microscope (Olympus IX 70). Excitation was set in a range of 351-364 nm, and the fluorescent emission of both the free (485 nm) and Ca2+-bound (405 nm) forms of the dye were directed by a dichroic filter to two photomultiplier tubes.Solutions.
Gj measurements were made at room temperature
(22-24°C) after replacing the culture medium with a Tyrode
solution that contained (in mM) 144 NaCl, 5.4 KCl, 1 MgCl2,
2.5 CaCl2, 0.3 NaH2PO4, 5 HEPES,
and 5.6 glucose (buffered to pH 7.4 with NaOH). The test solutions were
either added to the culture dishes or applied by directing a streamline
flow onto the investigated cells from the opening of a stainless steel
capillary (internal diameter 30 µm), positioned in the bath near the
cell pair. Washing of the test fluids was similarly performed by
switching to a Tyrode solution flowing out of a second capillary joined
to the first at the opening. Low resistance (1.5-2.5 M) patch
pipettes were backfilled with a filtered solution that contained (in
mM) 140 KCl, 0-5 MgATP, 5 EGTA, 10-15 glucose, 0.1 GTP, and
10 HEPES (buffered to pH 7.2 with KOH) and connected the cells to their
respective feedback amplifiers (Biologic RK 300, Grenoble, France). H-7
and calyculin A were dissolved in DMSO before addition to bath
solution. Antimycin A and cyclosporin A were dissolved in ethanol
before addition to solutions, whereas okadaic acid (ammonium salt),
heparin, I2, and p-nitrophenyl phosphate (Boehringer) were
directly added to the solutions. All chemicals, unless otherwise
stated, were obtained from Sigma.
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RESULTS |
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The degree of cell-to-cell communication between cardiac cells is
closely determined by the metabolic state of the cells.
Both myocytes of the pair were clamped at Vh,
then a 10-mV Vj was applied; when cells were
interconnected, this difference resulted in a current flowing through
junctional channels, compensated by an opposite current supplied by the
feedback amplifier connected to the cell maintained at the holding
potential. In whole cell conditions, Gj measured
between neonatal rat cardiomyocytes was well maintained when
experiments were carried out with 5 mM ATP in the patch pipette
solution, whereas it rapidly decreased to complete closure of the
channels within 12-20 min when ATP was absent (31).
At a relatively low ATP concentration (1 mM), Gj was progressively reduced, as illustrated in Fig.
1A, to ~12.5% of its
initial value after 15 min. The degree of GJIC after 5, 10, and 15 min of recording was compared when pipette filling solutions
contained, respectively, 0, 1, 2, and 5 mM ATP and were expressed in
percent of the original conductances (Fig. 1B). ATP at 2 mM
allowed almost the whole level of cell-to-cell communication to be
retained.
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Cyclosporin A failed to prevent the decay of GJIC induced by
nonspecific inhibition of PK, whereas okadaic acid or calyculin A
allowed the preservation of the main part of cell-to-cell
communication.
Cruciani et al. (9) assessed the PP activities in hamster
V79 fibroblasts expressing Cx43 and studied the effects of different PP
inhibitors. The Ca2+-dependent PP2B (calcineurin) was found
in a considerable amount in the cells, and PP2B inhibitors delayed the
normalization of GJIC in 12-O-tetradecanoylphorbol
13-acetate (TPA)-treated cells. The possible involvement of PP2B in the
rundown of Gj of rat cardiac myocytes in
ATP-deprived conditions was examined with a PP2B inhibitor, cyclosporin
A (1 µM). As illustrated in Fig. 3, the
complete interruption of GJIC was not prevented by the presence of
cyclosporin A.
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PP1 inhibitors prevent the rundown of junctional conductance in
ATP-deprived conditions.
Heparin, a sulfated polysaccharide, inhibits different PKs, including
casein kinase 2 (14) as well as the dephosphorylation of
different proteins by PP1 (11). In the absence of ATP,
phosphorylation by PKs are interrupted, and heparin (100 µg/ml) was
used, in these conditions, as a PP1 inhibitor. In its presence, the
degree of GJIC slightly decreased with time, but an important
intercommunication persisted (77 ± 15% of the initial
Gj after 5 min) in ATP-derived conditions (Fig.
5A). Thus the inhibition of
PP1 activities allowed an important proportion of junctional channels
to stay open in spite of the interruption of phosphorylating
reactions.
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Inhibitions of both the phosphorylation and PP1 pathways preserved
cell-to-cell communication.
In the presence of ATP, Gj was progressively
reduced to a stable level corresponding to ~13% of its initial value
within 10 min by H-7, a broad spectrum inhibitor of serine/threonine
PKs, used at a relatively high concentration (1 mM) with the intent to
inhibit a range of PKs (31). H-7, although structurally
unrelated to ATP, is considered to compete with ATP for free enzymes
(18). When both heparin (100 µg/ml) and H-7 (1 mM) were
present in the external bath, the degree of intercellular communication
remained almost unmodified, as shown Fig.
6. The ability of a PP inhibitor to
prevent H-7 effects allows the possibility of other mechanisms of
action of H-7 (e.g., a deleterious effect on membranes or cytoskeleton) to be excluded and shows that this compound alters the junctional conductance by impeding the phosphorylation pathway. It also shows that
the simultaneous hindrance of both phosphorylation and
dephosphorylation allows preservation of cell-to-cell communication, at
least for the duration of the experiment.
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Stimulation of PP1 activity triggers the interruption of
cell-to-cell communication despite the presence of ATP.
With the aim of confirming that PP1 activity was responsible for the
closure of junctional channels when protein phosphorylation was
depressed, the effects of the enhancement of PP1 activity have been
examined by stimulation of the endogenous PP1 activity. The compound
p-nitrophenyl phosphate, able to specifically stimulate PP1
activity (12) although it acts as a diversion substrate for other PPs (27), appears to be a suitable tool for the
identification of the active PP form. The addition of
p-nitrophenyl phosphate (1 mM, Fig.
7A) to the ATP-containing
solution that filled the patch pipettes induced a progressive fading of
the junctional currents.
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DISCUSSION |
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The Gj measured among neonatal rat cardiomyocytes in conventional double whole cell mode was well maintained when ATP was present in the patch pipette solution, whereas it rapidly vanished to complete interruption of the GJIC if ATP was absent. An ATP intracellular concentration of 2 mM allowed the preservation of a stable level of cell-to-cell communication. When Gj between paired cardiomyocytes was determined after excision of one of the cells, ATP had to be present in the bath solution in a comparable concentration level to preserve cell-to-cell communication, but Sugiura et al. (28) suggested that Gj was regulated through a specific ligand-receptor interaction between ATP and gap junctional proteins rather than through the promotion of protein phosphorylation.
When in vivo ATP synthesis was interrupted by exposure to antimycin A, an inhibitor of mitochondrial respiration, the GJIC was completely abolished. Prolonged exposure (16 h) to antimycin A also interrupted the cell-to-cell diffusion of Lucifer yellow between rat astrocytes, and this effect was interpreted as a consequence of an increase in cytosolic Ca2+ concentration (30). Antimycin A is, indeed, considered to deplete mitochondrial Ca2+ (35). In the present study, short-time treatments elicited a relatively important increase in [Ca2+]i that, however, remained less important than the rise observed when the cells were exposed to a high-K+ solution, a treatment known to have no effect on the cell-to-cell diffusion of a fluorescent dye in these cells (32).
In ventricular myocytes of neonatal rats, several observations (the effects of ATP replacement by some other nucleotides, of broad-spectrum inhibition of endogenous serine/threonine PKs, and of introduction into the cells of a nonspecific exogenous PP) led to the possibility that the presence of high energy nucleotides is essential to allow PK activities to counteract the tonic activities of endogenous PP(s) (31). Although several PKs have an established role in this process, less is known about the involvement of PPs. The four main phosphoserine/threonine protein phosphatases, PP1, PP2A, PP2B, and PP2C, have been previously detected in heart (for review, see Ref. 16). The main aim of the present study was to identify the dephosphorylating enzyme whose activity is responsible for the interruption of cell-to-cell communication among rat ventricular myocytes.
The lack of influence of Mg2+ on the loss of channel activity is not in favor of the involvement of PP2C in the rundown of Ij (31). Cyclosporin A, a calcineurin (PP2B) inhibitor, was able to delay the gap junction plaque recovery of hamster V79 fibroblasts expressing Cx43 after TPA exposure (9); however, in the present study, it did not prevent the rundown of Ij occurring in ATP-deprived conditions.
Several works have suggested that PP1 and/or PP2A are the PPs involved
in the regulation of gap junctional communication. Exposure of rat
liver epithelial cells to 18-glycyrrhetinic acid rapidly and
reversibly elicited an interruption of cell-to-cell communication
together with a Cx43 dephosphorylation, unless okadaic acid or
calyculin A, inhibitors of PP1 and PP2A, were present (13). A part of the junctional uncoupling action of BDM
seemed to result from a dephosphorylation process (32),
and it was suggested (38) that this compound could enhance
the activity of endogenous phosphatases PP2A, and, to a lesser extent,
PP1. In Madin-Darby canine kidney cells, okadaic acid pretreatment (60 nM) potentiated the TPA-induced increase in phosphorylated Cx43,
suggesting that okadaic acid-sensitive PP(s) participate in the
dephosphorylation of Cx43 (2). In a
communication-deficient cell line (SKHep1) transfected with a cDNA
encoding human Cx43, okadaic acid (300 nM) shifted the frequency
histograms of unitary conductances in the same direction as
phosphorylating agents such as TPA, 8-bromoadenosine 3',5'-cyclic
monophosphate, or forskolin (24), suggesting that the
gating properties of gap junctional channels would depend on PP1 and/or
PP2A activities.
In the present study, okadaic acid slowed down the decay of Gj in ATP-deprived conditions but was less efficient than calyculin A to preserve the cell-to-cell coupling, suggesting that PP1 might be the main protein phosphatase involved in the modulation of the channel. The effectiveness of two selective PP1 inhibitors, I2 and heparin, to preserve cell-to-cell coupling in ATP-deprived conditions has been examined. I2 is a heat- and acid-stable 22.8-kDa protein that specifically inhibits PP1 in vitro (6, 27) as well as in intact cells (10). Heparin is known to almost completely inhibit PP1, particularly the membrane and nuclear fractions (5). Both of these compounds allowed, in whole cell conditions, the preservation of cell-to-cell communication in the absence of ATP. Similarly, when the phosphorylation pathway was in the presence of ATP, hindered by H-7, the degree of cell-to-cell coupling remained almost unaltered, provided that heparin was present.
To verify that PP1 activity is responsible for the closure of gap junctional channels, we examined the consequences of its selective activation by p-nitrophenyl phosphate in the presence of ATP and verified that this treatment led to a complete interruption of the cell-to-cell communication except when I2 was concomitantly added to selectively prevent the effects of PP1 activation. The activity of junctional channels of rat ventricular myocytes appears regulated by ongoing phosphorylation/dephosphorylation under basal conditions, and PP1 activity leads to the rapid closure of all intercellular channels when it is not counterbalanced by PK activities. PP1 is a broad specificity phosphatase, able to regulate many biological processes, including, for example, synaptic plasticity, cell cycle, gene transcription, glycogen metabolism, and muscle contraction (for review see Ref. 27).
It has also been reported that this enzyme can modulate the activity of
a number of membrane channels, including, for example, that of
voltage-gated Na+ (26), Ca2+ (N-
and P-types, see Ref. 29), or K+ (outward
K+, see Ref. 10) channels as well as that of a
Ca2+-sensitive nonspecific cation channel (36)
or glutamate receptor channels, including
N-methyl-D-aspartate (4, 34) or
DL--amino-3-hydroxy-5-methylisoxazole-4-propionic acid (37) receptors.
Cx43, as with a large majority of cell proteins, is predominantly phosphorylated on serine residues, and important changes in its state of phosphorylation can be resolved by differences in electrophoretic mobilities. Preliminary results showed that several treatments aimed to shift the protein phosphorylation/dephosphorylation balance toward dephosphorylation reduced the degree of junctional coupling of cardiac myocytes without modifying the extent of Cx43 phosphorylation examined by Western blot analysis (9a). Similar observations have been reported in other Cx43-expressing cells, such as T51B rat liver epithelial cells (19), V79 hamster fibroblasts (9), or rat astrocytes (23). Limited changes in the degree of phosphorylation of Cx43, particularly on tyrosine residues, may, however, occur without shift of the electrophoretic mobility of the protein. Sustained disruptions of GJIC have, indeed, been found to be correlated with an enhanced Cx43 phosphorylation on tyrosine (for review, see Ref. 22). In the present study, an enhanced phosphorylation is not expected in ATP-depleted conditions. On the other hand, the tyrosine kinase inhibitor genistein had no effect on the junctional coupling of these cells (33).
In conclusion, the activity of junctional channels of rat ventricular myocytes appears to be regulated by ongoing phosphorylation/dephosphorylation under basal conditions, and PP1 activity leads to a rapid interruption of the intercellular communication if PK activities do not balance its action. Constitutively active PP1, plausibly anchored in the vicinity of junctional channels, might then be a target in signaling pathways for many extracellular molecules (e.g., growth factors, cytokines, and neurotransmitters), allowing modulation of the degree of cell-to-cell communication.
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ACKNOWLEDGEMENTS |
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The authors are most grateful to Profs. J. Délèze and M. Mesnil (Laboratoire de Physiologie Cellulaire, Poitiers) for valuable criticism of this manuscript and to Prof. N. J. Severs (Imperial College School of Medicine, Royal Brompton Hospital, London, United Kingdom) for careful reading of the manuscript.
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FOOTNOTES |
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This study was supported in part by grants from the European Community Research, Development and Technologies Action QLG1-CT-1999-00516 and from the Fondation Langlois.
F. Duthe and I. Plaisance are recipients of studentships from the Association de Recherche contre le Cancer and the Région Poitou-Charentes, respectively.
Address for reprint requests and other correspondence: J. C. Hervé, Laboratoire de Physiologie Cellulaire, Unité Mixte de Recherche Centre National de la Recherche Scientifique 6558, 40, Ave. du R. Pineau, 86022 Poitiers, France (E-mail: Jean.Claude.Herve{at}univ-poitiers.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. Section 1734 solely to indicate this fact.
Received 17 January 2001; accepted in final form 30 May 2001.
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