1 Department of Pharmacology, University of South Alabama College of Medicine, Mobile, Alabama 36688; and 2 Department of Internal Medicine, University of Iowa Health Sciences Center, Iowa City, Iowa 52242
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ABSTRACT |
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The present study evaluated the necessity of store-operated Ca2+ entry in mediating thrombin-induced 20-kDa myosin light chain (MLC20) phosphorylation and increased permeability in bovine pulmonary artery endothelial cells (BPAECs). Thrombin (7 U/ml) and thapsigargin (1 µM) activated Ca2+ entry through a common pathway in confluent BPAECs. Similar increases in MLC20 phosphorylation were observed 5 min after thrombin and thapsigargin challenge, although thrombin produced a sustained increase in MLC20 phosphorylation that was not observed in response to thapsigargin. Neither agonist increased MLC20 phosphorylation when Ca2+ influx was inhibited. Thrombin and thapsigargin induced inter-endothelial cell gap formation and increased FITC-dextran (molecular radii 23 Å) transfer across confluent BPAEC monolayers. Activation of store-operated Ca2+ entry was required for thapsigargin and thrombin receptor-activating peptide to increase permeability, demonstrating that activation of store-operated Ca2+ entry is coupled with MLC20 phosphorylation and is associated with intercellular gap formation and increased barrier transport of macromolecules. Unlike thrombin receptor-activating peptide, thrombin increased permeability without activation of store-operated Ca2+ entry, suggesting that it partly disrupts the endothelial barrier through a proteolytic mechanism independent of Ca2+ signaling.
thapsigargin; thrombin; myosin light chain kinase; receptor-operated calcium channels; lung
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INTRODUCTION |
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ELEVATED CYTOSOLIC Ca2+ concentration ([Ca2+]i) promotes inter-endothelial cell gap formation and increased vascular permeability (7, 9, 11, 12, 15-17, 19, 21-23, 25, 26, 35, 38, 41). Both Ca2+ release from intracellular stores and Ca2+ influx across the cell membrane contribute to an increase in [Ca2+]i (1, 2, 25, 30-32, 34, 42). Ca2+ release is accomplished by generation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which stimulates Ins(1,4,5)P3 receptors located predominantly in the smooth endoplasmic reticulum membrane (1, 2, 32). Ca2+ influx may be accomplished by a number of different mechanisms (1, 14, 15, 17, 24, 26, 28, 30) including 1) membrane potential-regulated Ca2+ leak, 2) Na+/Ca2+ exchange, 3) activation of mechanosensitive cation channels, 4) ligand stimulation of receptor-coupled cation channels (ROCs), and 5) activation of capacitative or store-operated Ca2+ entry channels (SOCs). In regard to endothelial barrier properties, recent data indicate that Ca2+ influx in response to agents activating SOCs promotes intercellular gap formation and increases transport of macromolecules across pulmonary arterial endothelial monolayers (4, 5, 19, 24, 25, 41). The specific molecular target affected by Ca2+ entry via SOCs to regulate permeability is unclear.
Endothelial myosin light chain (MLC) kinase (MLCK) can be activated by Ca2+/calmodulin to initiate phosphorylation of 20-kDa MLC (MLC20), leading to actomyosin interaction and endothelial tension development (9, 10, 13, 20, 27, 36, 37, 44, 45). It is unknown whether Ca2+-stimulated MLCK activation and MLC20 phosphorylation are regulated in a preferential manner by Ins(1,4,5)P3-mediated Ca2+ release from intracellular stores or Ca2+ influx across the plasmalemma. However, a recent report (43) indicated that Ca2+ entry associated with SOC activation may be linked to MLC20 phosphorylation (43). This observation coupled with the known role of MLCK in permeability regulation suggests that a complex relationship exists among MLC20 phosphorylation status, Ca2+ influx through SOCs, and endothelial barrier function.
Thrombin is a receptor-coupled inflammatory agonist that elicits Ins(1,4,5)P3-dependent Ca2+ release coincident with Ca2+ influx in endothelial cells (14, 28). In addition, thrombin promotes endothelial cell shape change and in vitro barrier disruption in part by influencing MLCK activity and MLC20 phosphorylation (21, 22, 27, 33, 36). Our present studies tested whether thrombin-induced MLC20 phosphorylation and endothelial barrier disruption are regulated by Ca2+ influx through SOCs. Our data indicate that Ca2+ influx is coupled to increased MLC20 phosphorylation in response to thrombin, although the influx pathway activated by thrombin is receptor gated as well as store operated. Although direct activation of the thrombin receptor increased permeability dependent on store-operated Ca2+ entry, thrombin also increased permeability through a proteolytic mechanism.
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METHODS |
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Isolation and culture of bovine pulmonary artery endothelial cells. Bovine pulmonary artery endothelial cells (BPAECs) were isolated and cultured according to methods previously described (39). In addition, cells were purchased from Clonetics (San Diego, CA). All cells were routinely identified as endothelial by morphological assessment ("cobblestone" appearance at confluence), factor VIII antigen staining, and uptake of acetylated 1,1'-dioctadecyl-1,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled low-density lipoprotein. Cells were studied between passages 6 and 10.
[Ca2+]i estimation with fura 2 epifluorescence. BPAECs were seeded onto chambered glass coverslips (Nalge Nunc, Naperville, IL) and grown to confluence. [Ca2+]i was estimated with the Ca2+-sensitive fluorophore fura 2-AM (Molecular Probes, Eugene, OR) according to methods previously described (19, 25, 40, 41). Briefly, BPAECs were washed with 2 ml of a HEPES (Fisher Scientific, Atlanta, GA)-buffered physiological salt solution (PSS) containing (in g/l) 6.9 NaCl, 0.35 KCl, 0.16 KH2PO4, 0.141 MgSO4, and 2.0 D-glucose and 25 mM HEPES. The loading solution (2 ml) consisted of PSS plus 3 µM fura 2-AM, 0.003% pluronic acid, and 2 mM or 100 nM CaCl2. BPAECs were loaded for 20 min in a CO2 incubator at 37°C. The cells were washed again with PSS (2 ml) and treated with deesterification medium (PSS plus 2 mM or 100 nM CaCl2) for an additional 20 min. After deesterification, [Ca2+]i was estimated with an Olympus IX70 inverted microscope at ×400 and a xenon arc lamp photomultiplier system (Photon Technologies, Monmouth Junction, NJ). Data were acquired with PTI Felix software. Epifluorescence (signal averaged) was measured from three to four endothelial cells in a confluent monolayer, and the changes in [Ca2+]i are reported as the fluorescence ratio of the Ca2+-bound (340-nm) to Ca2+-unbound (380-nm) excitation wavelengths emitted at 510 nm.
MLC20 phosphorylation assay. BPAECs were seeded in T-75 flasks and grown to confluence. MLC20 phosphorylation was assessed as previously described (8, 36) with minor modifications. Briefly, confluent BPAECs were transferred to micropore filters and labeled with 1.5 ml of [35S]methionine (555 µCi/ml) in D/Val MEM with 20% calf bovine serum for 48 h at 37°C and 5% CO2. After a 5- or 15-min incubation with vehicle control, thapsigargin (1 µM), or thrombin (7 U/ml) in Ca2+-containing (2 mM) or -depleted (100 nM) PSS, the reactions were stopped by snap-freezing in a dry ice-methanol bath, and the cells were lysed. The lysate was sedimented at 1,000 g for 5 min at 4°C, and the supernatant was incubated with 20 µl of rabbit anti-human platelet myosin antibody (2 mg/ml) at 4°C for 1 h. The mixture was centrifuged at 100,000 g for 1 min, and the pellet was collected, washed with lysing buffer, and recentrifuged. The pellet was then washed with 0.5 ml of a 50:50 mixture of lysing buffer and PBS. The pellet was resuspended in 35 µl of urea lysing buffer for two-dimensional gel electrophoresis to isolate unphosphorylated and phosphorylated MLC20 isoforms. Isoelectric focusing and second dimension SDS-PAGE were performed as described (36). The resulting bands were quantitated by densitometry. MLC20 exhibited un-, mono-, and diphosphorylated states. Stoichiometry of moles phosphate per mole of MLC20 was calculated as described (36).
Assessment of endothelial cell shape change and intercellular gap formation. BPAECs were seeded onto 35-mm plastic culture dishes and grown to confluence. The cells were washed twice with PBS and once with PSS plus 2 mM or 100 nM CaCl2 and then equilibrated with 2 ml of PSS for 2 min before the experiment was started. At the end of each experiment, the monolayers were fixed in 3% glutaraldehyde for 10 min and prepared for microscopy as previously described (19, 25). Gap formation was assessed by a pathologist blinded to the experimental protocols, and micrographs were taken of representative areas in the monolayer.
Estimation of diffusive capacity.
BPAECs were seeded onto Transwell inserts (6.5-mm diameter, 0.4-µm
pore size; Costar) at a density of 2.27 × 103
cells/mm2 in a final volume of 100 µl of DMEM plus 10%
fetal bovine serum. The inserts were placed into 24-well plates
containing 600 µl of growth medium, and the cells were allowed to
grow for 5 days. After the cells achieved confluence, the growth medium
in the upper chamber was replaced with 100 µl of a 1 mg/ml
FITC-dextran solution in Krebs-Henseleit PSS. The insert was then moved
to a fresh lower well containing 600 µl of PSS. The cells were
equilibrated with these solutions at 37°C in a CO2
incubator for 15 min. After equilibration, the Transwell insert was
placed into another lower chamber containing 600 µl of PSS, and the
FITC-dextran was allowed to diffuse across the monolayer for 30 min.
This procedure was repeated three times so that a total time of 2 h for assessing monolayer integrity was employed. Samples of the lower
chamber (50 µl) were taken in triplicate and placed in 96-well
cluster plates for measuring fluorescence intensity (Perkin-Elmer
luminescence spectrometer LS 50B) excitation at 480 nm and emission at
530 nm. Fluorescence values were then converted to milligrams of
FITC-dextran per milliliter with a standard curve that was generated
concurrent with the measurements of monolayer integrity. With these
values, diffusive capacity (PS; in nanoliters per minute) was
calculated by determining the net rate of FITC-dextran flux
(Js) generated for each concentration difference
(C) across the monolayer with the equation PS = Js/
C.
Statistical methods. Data are reported as means ± SE. Comparisons were made with either paired or unpaired Student's t-test or one-way or two-way analysis of variance with repeated measures as appropriate. A Student-Newman-Keuls post hoc test was applied. Differences were considered significant at P < 0.05.
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RESULTS |
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The Ca2+ influx pathway activated by
thrombin is under dual regulation by receptor coupling and store
depletion.
Changes in [Ca2+]i elicited by thrombin
compared with those elicited by thapsigargin, an activator of SOCs,
were assessed in confluent fura 2-loaded BPAECs (Fig.
1). Figure 1A shows that thrombin produced a change in [Ca2+]i
characterized by an abrupt and transient "spike" in the
fluorescence ratio, representing a rapid Ca2+ release from
intracellular stores. After the spike, a sustained elevation in
[Ca2+]i above baseline was observed. In
addition, the average sustained [Ca2+]i level
demonstrated some variability over time as indicated by the wide SE
range, which was attributed to oscillatory changes in
[Ca2+]i at different rates of cycling for
individual cells in the measurement field (40).
Thapsigargin elicited a more gradual increase in [Ca2+]i that likewise remained elevated above
basal levels on activation of SOCs. To assess the Ca2+
influx component of the thrombin response, experiments were repeated with fura 2-loaded BPAECs incubated in a low extracellular
Ca2+ concentration ([Ca2+]o; Fig.
1B). Reducing [Ca2+]o to 100 nM
slightly attenuated the thapsigargin-induced Ca2+ release
and did not affect the peak [Ca2+]i response
to thrombin. However, the sustained elevation in
[Ca2+]i seen previously for both agonists was
absent, thereby indicating that Ca2+ influx was a vital
component to the total change in [Ca2+]i
generated in response to thapsigargin and thrombin.
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Ca2+ influx is coupled to
MLC20 phosphorylation.
We observed two major isoforms of MLC in BPAECs; the smaller isoform
was 16 kDa, and the larger more basic isoform was 20 kDa. Similar to
human umbilical vein endothelial cells and platelets, the 16-kDa
isoform existed at two distinct isoelectric points (data not shown).
The 20-kDa isoform exhibited three distinct isoelectric points
corresponding to un-, mono-, and diphosphorylated states. Figure
2 illustrates the effects of thrombin and
thapsigargin on MLC20 phosphorylation state with
respect to time and [Ca2+]o. Basal
stoichiometry of 0.39 ± 0.06 mol phosphate/mol
MLC20 increased 135 and 120% 5 min after application of
thrombin and thapsigargin, respectively. Whereas the response to SOC
activation with thapsigargin was transient, the response to thrombin
persisted at 15 min. When studies were repeated in 100 nM extracellular Ca2+, the basal stoichiometry decreased 15% to 0.33 ± 0.05 mol phosphate/mol MLC20, suggesting that basal leak
of Ca2+ across the endothelial plasmalemma contributed to
MLC20 phosphorylation status. Prevention of
Ca2+ entry effectively eliminated the thrombin- and
thapsigargin-induced increase in MLC20 phosphorylation,
thereby clamping endothelial MLC20 phosphorylation levels
below values normally existing in the presence of physiological
[Ca2+]o. These data indicate that, at least
over the time period studied, Ca2+ influx was tightly
coupled to the level of MLC20 phosphorylation induced by
SOC activation alone (e.g., thapsigargin) and by thrombin-stimulated Ca2+ entry.
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Activation of store-operated Ca2+
entry increased endothelial cell permeability.
Figure 3 shows phase-contrast micrographs
for BPAEC monolayers challenged with thrombin or thapsigargin. Although
untreated (control) cells, in either the presence or absence of
extracellular Ca2+, displayed close cell-cell apposition
without apparent intercellular gaps (Fig. 3, A and
B), thrombin challenge of BPAEC monolayers for 5 min
resulted in obvious gap formation regardless of
[Ca2+]o studied (Fig. 3, C and
D). Thapsigargin likewise elicited gap formation (Fig.
3E) but only when [Ca2+]o was
conducive to Ca2+ influx in response to the opening of SOCs
(Fig. 3F). Figure 4 illustrates that the gap formation produced by thrombin and
store-operated Ca2+ entry was sufficient to allow for an
increase in diffusive capacity to macromolecular FITC-dextran. In the
presence of 2 mM extracellular Ca2+, permeability was
increased 60% with thrombin and 35% with thapsigargin. Although the
increase in permeability to thapsigargin was eliminated in 100 nM
extracellular Ca2+, thrombin increased permeability
40% under these conditions, suggesting that its effect was
independent of store-operated Ca2+ entry.
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DISCUSSION |
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Store-operated Ca2+ influx in pulmonary artery endothelium is alone sufficient to stimulate endothelial cell shape change and increase macromolecular permeability (4, 5, 19, 24, 25, 41). However, humoral inflammatory agonists do not selectively activate store-operated Ca2+ entry without first binding receptors coupled to G proteins, thereby activating multiple intracellular signaling pathways. Thus the role of store-operated Ca2+ influx in permeability regulation should be investigated further in conjunction with receptor-coupled inflammatory mediators. Thrombin is one such mediator coupled to Gq proteins that stimulates cultured pulmonary artery endothelial cell shape change to increase macromolecular permeability by activating multiple, parallel intracellular signaling pathways concurrent with increased [Ca2+]i (21, 22, 27, 33, 36). As described here and consistent with previous observations (1, 14, 21, 22), the thrombin-induced increase in [Ca2+]i occurs in response to Ins(1,4,5)P3-mediated Ca2+ release and sustained Ca2+ influx, which are distinguished with the Ca2+ fluorophore fura 2 (14, 21, 22, 40). With respect to Ca2+ release, our data indicate that extracellular Ca2+ levels do not influence peak responses to thrombin. However, the sustained increase in [Ca2+]i elicited by thrombin is dependent on Ca2+ influx because a nominally Ca2+-free extracellular environment prevents this increase from occurring.
Endothelial cells possess several discernible Ca2+ entry pathways (1, 2, 7, 26, 28), and electrophysiological characterization of Ca2+ and other cationic currents suggests that multiple Ca2+ entry channels may be activated by thrombin (28). Presently, the molecular identities of Ca2+ entry pathway(s) in endothelial cells are poorly understood. The transient receptor potential (trp) gene family is likely responsible for forming membrane Ca2+ channels (3, 24, 25, 28), although there are many uncertainties concerning Trp proteins. It is unclear whether Trp proteins are responsible for the Ca2+ release-activated current measured in nonexcitable cells (6, 18). Likewise, it is not known how Trp channels expressed in endothelial cells, principally Trp1 and Trp4, are specifically gated in terms of G protein coupling and Ca2+ store regulation (3, 6, 26, 28).
A recent work (3) indicated that some Trp monomeric isoforms exhibit preferential regulation by Gq protein activation, whereas others demonstrate regulation by store depletion. That report also suggested that Ca2+ influx pathways in nonexcitable cells are composed of Trp proteins forming multimeric channel complexes, consistent with the notion that Trp channel complexes composed of both Gq protein-regulated monomers and store-operated monomers could be dually regulated. Our present data support this concept as evidenced by the results from the heparin microinjection experiments and from the experiments where BPAECs were challenged with thrombin after thapsigargin pretreatment. When Ins(1,4,5)P3-mediated Ca2+ store depletion was prevented by heparin blockade of the Ins(1,4,5)P3 receptor and the store-operated Ca2+ influx pathway was inhibited, an increase in [Ca2+]i equivalent to that in the absence of heparin blockade was observed. Thus thrombin activated a receptor-coupled Ca2+ influx channel that mediated the sustained increase in [Ca2+]i in the absence of Ins(1,4,5)P3-dependent store depletion.
This receptor-coupled pathway may not be distinct from the store-operated pathway; e.g., store depletion and G protein stimulation may activate the same pool of membrane Ca2+ influx channels. When the BPAEC store-operated pathway was stimulated with thapsigargin, the level of sustained [Ca2+]i generated was not altered by thrombin. Had a different and distinctly regulated population of membrane Ca2+ channels sensitive to thrombin been activated, the overall level of [Ca2+]i would have increased, especially in combination with thapsigargin-induced sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibition. One possible explanation for these data is that the receptor-coupled Ca2+ entry pathway [elucidated during heparin blockade of the Ins(1,4,5)P3 receptor] is the same pathway stimulated by store depletion. Ca2+ entry in BPAECs, therefore, would occur via a channel complex under redundant regulation by membrane G proteins and internal store filling coupled to the Ins(1,4,5)P3 signaling cascade. We do not rule out an alternative possibility that activation of a distinct population of membrane SOCs with thapsigargin causes simultaneous or time-dependent inhibition of receptor-coupled Ca2+ influx channels, which would also account for our experimental observations. A clear determination of a redundancy in channel regulation can only be achieved with subsequent identification and characterization of membrane proteins forming native endothelial Ca2+ channels and development of specific inhibitors to both receptor-coupled and store-operated Ca2+ entry events.
Even though Ca2+ entry in response to thrombin occurs through receptor- and store-operated pathways, a clear link between the store-operated pathway and endothelial cell shape change has previously been elucidated (4, 5, 19, 24, 25, 41). What has not been made clear, however, is the relationship between store-operated Ca2+ entry and the intracellular events downstream from Ca2+ entry leading to shape change and permeability alterations. We hypothesized that store-operated Ca2+ entry-induced increases in endothelial permeability involved activation of the Ca2+/calmodulin-regulated MLCK and subsequent MLC20 phosphorylation, leading to increased actomyosin interactions and tension development (9, 13, 43, 44). Consistent with this hypothesis, we observed that activation of store-operated Ca2+ entry by thapsigargin was tightly coupled to a significant increase in MLC20 phosphorylation, suggestive of an increase in MLCK activity; Ca2+ influx was associated with a similar degree of MLC20 phosphorylation stimulated by thrombin.
The thrombin-induced increase in MLC20 was sustained for at
least 15 min. This particular finding differed from the
thapsigargin-induced MLC20 phosphorylation profile, showing
a dramatic increase in phosphorylation that dissipated to near basal
levels after 15 min. These data are consistent with the recent findings
of Shasby et al. (36), who demonstrated that
thrombin-mediated signaling inhibits protein phosphatase 1 activity,
resulting in a prolonged increase in MLC20 phosphorylation.
This signaling response is unique to thrombin compared with other
receptor-dependent (e.g., histamine) and -independent (e.g.,
ionomyosin, thapsigargin, Staphylococcus aureus -toxin
clamped) Ca2+ agonists. Thus Gq protein-coupled
inflammatory agonists increase and maintain MLCK activity and
MLC20 phosphorylation via activation of several
Ca2+-dependent and Ca2+-independent processes,
consistent with a previous work (10) indicating that
endothelial MLCK activity is a function of multiple signal inputs.
Finally, the present studies indicated that activation of store-operated Ca2+ entry in response to thapsigargin and TRAP is sufficient to increase endothelial cell permeability. However, the role of MLC20 in linking store-operated Ca2+ entry to barrier disruption remains unclear. Both thrombin and thapsigargin increased permeability over a 1-h time course, even though MLC20 phosphorylation had returned to near baseline values in response to thapsigargin. Although these data would support a role for MLC20 phosphorylation in the initial response to activation of store-operated Ca2+ entry, MLC20 phosphorylation was not associated with the prolonged permeability response. Future studies will be required to comprehensively discriminate between the effect of MLC20 phosphorylation with increased centripetally directed tension and the loss of cell-cell and cell-matrix adhesion in the regulation of the sustained permeability response to Ca2+ agonists (12, 27).
In summary, our present studies addressed key issues relating to Ca2+ regulation and control of endothelial cell shape and barrier function. Receptor-coupled, G protein-linked inflammatory mediators may activate Ca2+ influx pathways under dual regulation by plasmalemmal effectors and the filling state of internal Ca2+ pools. Our data also extend the previous observation that store-operated Ca2+ entry can initiate intercellular gap formation, in part, by an associated transient increase in MLC20 phosphorylation. Furthermore, in addition to store-operated Ca2+ entry, thrombin activates proteolytic signaling pathways that can act independently to produce changes in pulmonary endothelial cell shape and increased permeability. Subsequent work is necessary to fully elucidate the exact nature of the relationship between endothelial store-operated Ca2+ entry, MLCK activity, and MLC20 phosphorylation status as related to endothelial Ca2+ homeostasis and barrier function (29).
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ACKNOWLEDGEMENTS |
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This work supported by National Heart, Lung, and Blood Institute Grants HL-56050 and HL-60024 (to T. Stevens); a Parker B. Francis Foundation Fellowship (to T. Stevens); and an American Heart Association Southern Research Consortium Fellowship (to T. M. Moore).
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. Stevens, Dept. of Pharmacology, Univ. of South Alabama College of Medicine, Mobile, AL 36688 (E-mail: tstevens{at}jaguar1.usouthal.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 9 July 1999; accepted in final form 27 April 2000.
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