Department of Physiology, Morehouse School of Medicine, Atlanta, Georgia 30310
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ABSTRACT |
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Hyperpolarization in human leukemia THP-1 monocytes adherent to vascular cell adhesion molecule (VCAM)-1 is due to an induction of inwardly rectifying K+ currents (Iir) (Colden-Stanfield M and Gallin EK, Am J Physiol Cell Physiol 275: C267-C277, 1998). We determined whether the VCAM-1-induced hyperpolarization is sufficient to augment the increase in intracellular free calcium ([Ca2+]i) produced by Ca2+ store depletion with thapsigargin (TG) and readdition of external CaCl2 in fura 2-loaded THP-1 monocytes. Whereas there was a 2.1-fold increase in [Ca2+]i in monocytes bound to glass for 5 h in response to TG and CaCl2 addition, adherence to VCAM-1 produced a 5-fold increase in [Ca2+]i. Depolarization of monocytes adherent to VCAM-1 by Iir blockade or exposure to high [K+] abolished the enhancement of the peak [Ca2+]i response. In monocytes bound to glass, hyperpolarization of the membrane potential with valinomycin, a K+ ionophore, to the level of hyperpolarization seen in cells adherent to VCAM-1 produced similar changes in peak [Ca2+]i. Adherence of monocytes to E-selectin produced a similar peak [Ca2+]i to cells bound to glass. Thus monocyte adherence to the physiological substrate VCAM-1 produces a hyperpolarization that is sufficient to enhance Ca2+ entry and may impact Ca2+-dependent monocyte function.
monocytic leukemia; calcium signaling; integrins; adhesion molecules; potassium channels; very late antigen-4
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INTRODUCTION |
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THE HUMAN MONOCYTIC LEUKEMIA THP-1 cell line closely resembles primary monocytes, displaying a variety of integrins on their surfaces that mediate their interactions with other cells and extracellular matrix molecules (4, 54). One of these integrins, very late antigen (VLA)-4, not only mediates the initial tethering and rolling of monocytes via interactions with vascular cell adhesion molecule (VCAM)-1 (39) and binding to the CS1 region of the extracellular matrix protein fibronectin (37) but also acts to trigger signaling events to impact cellular function (33, 45). The expression and production of tissue factor, an important procoagulant protein, are enhanced by VLA-4 integrin cross-linking or adhesion to fibronectin in human monocytes and THP-1 monocytes (38) and is possibly modulated by K+ channel activity (11). Although signaling pathways induced by VLA-4 integrin engagement have not yet been well characterized, evidence from various laboratories has demonstrated the activation of the Na+/H+ antiporter (44), Ca2+ influx and mobilization (47), tyrosine phosphorylation of cellular proteins (38), and activation of ionic channel activity (3, 5, 42, 55, 56). Recently, we demonstrated a membrane hyperpolarization produced by increased expression of inwardly rectifying K+ currents (Iir) in THP-1 monocytes adherent to activated endothelial cells or immobilized VCAM-1 (9), presumably by engaging VLA-4 integrins.
Hyperpolarization of membrane potential enhances the magnitude of Ca2+ entry by increasing the driving force for Ca2+ influx into cells that possess non-voltage-gated Ca2+ influx pathways (53). The predominant pathway is activated by depletion of intracellular Ca2+ stores (51, 52). A store-depleted Ca2+ influx pathway has been described in human monocyte-derived macrophages (35), human U937 monocytes/macrophages (13), and human U937 cells differentiated to macrophage-like cells (16) with the use of fura 2 measurements of intracellular Ca2+ and the direct electrophysiological measurement of Ca2+ release-activated Ca2+ currents (CRAC). The VCAM-1-induced Iir and subsequent hyperpolarization would most likely modulate Ca2+-dependent monocyte functions such as respiratory burst (24), integrin expression (17), and the production of nitric oxide (48), eicosanoids (26), and cytokines (40).
In the present study, we address the question of whether there is a relationship between store-depleted Ca2+ changes and membrane potential changes in THP-1 monocytes after direct engagement of VLA-4 integrins by using fluorometric and current-clamp approaches. We use thapsigargin (TG), an endoplasmic reticulum Ca2+-ATPase inhibitor, as a tool to trigger the store-operated Ca2+ influx pathway in THP-1 monocytes and demonstrate that the presence of Iir after VLA-4 engagement on THP-1 monocytes augments store-operated Ca2+ influx. Unlike most previous work on ion channel function in monocytes and/or macrophages, we provide new information about how the Iir induced by the interaction between monocytes and VCAM-1, one of their physiological substrates, alters Ca2+ influx pathway(s) that may impact Ca2+-mediated function. Preliminary results of this work have been previously reported (10).
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MATERIALS AND METHODS |
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Chemicals.
TG, valinomycin (Val), ionomycin (Sigma Chemical, St. Louis, MO), and
fura 2-AM (Molecular Probes, Eugene, OR) were stored in DMSO stock
solutions at 20°C. Aliquots of the stock solutions were diluted in
the appropriate experimental solutions immediately before use. A stock
solution of CsCl was prepared in distilled water and stored in aliquots
at
20°C until just before use.
Cell culture. The undifferentiated THP-1 human monocyte cell line (American Type Culture Collection, Manassas, VA) was cultured in suspension with RPMI culture medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT), 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml fungizone, and 2 mM L-glutamine (all from Life Technologies, Grand Island, NY). Cells were passaged every 3-4 days by centrifugation, removal of spent medium, and resuspension in fresh medium at 1-2 × 106 cells/ml. THP-1 cell cultures were passaged and used only for 1 mo after being thawed from liquid nitrogen.
Immobilization of soluble adhesion molecules. A 25-µl aliquot of recombinant soluble E-selectin (formerly known as endothelial-leukocyte adhesion molecule-1) or VCAM-1 (50 µg/ml; both generous gifts from Dr. Roy Lobb of Biogen, Cambridge, MA; see Ref. 32) in binding buffer (15 mM NaHCO3-35 mM Na2CO3, pH 9.2) was placed in the middle of glass coverslips attached to plastic dishes for coating overnight at 4°C. To prevent nonspecific binding of cells to substrata, dishes were blocked with PBS-1% BSA for 0.5 h at 4°C and washed once with complete RPMI. THP-1 monocytes (1 × 106 cells/ml) in complete RPMI were then added to the dishes for incubation at 37°C for 5 h. Nonadherent THP-1 monocytes were removed by washing with HEPES-buffered Ca2+-free saline solution twice before Ca2+ imaging experiments were performed on the remaining adherent THP-1 monocytes.
Measurement of intracellular free Ca2+. Intracellular free Ca2+ concentration ([Ca2+]i) was measured in THP-1 monocytes using the Ca2+-sensitive fluorescent dye fura 2 and digital imaging techniques (19). THP-1 monocytes were incubated on uncoated glass or immobilized adhesion molecules for 5 h before the cells were loaded with fura 2 by incubation with 5 µM fura 2-AM for 30 min at 37°C. After loading, the cells were rinsed and maintained in HEPES-buffered Ca2+-free saline supplemented with 1 mg/ml glucose and 1 mg/ml BSA, and cellular images were collected, digitized, and analyzed as previously described (29). Briefly, cells were maintained at 37°C on the stage of a Zeiss IM-35 microscope. Fluorescent images of fields of 20-30 cells were collected at 32 frames/s at excitation wavelengths of 340 and 380 nm (emission wavelength 510 nm) using a Nikon ×40 objective (PlanNeoFluar, 0.9 NA) and a GenIIsys-intensified charge-coupled device camera (Dage-MTI, Michigan City, IN). A ratio of the image of fluorescence was generated by dividing the background-subtracted image collected at an excitation wavelength of 340 nm by that collected at 380 nm using IC300 image analysis software (Inovision, Research Triangle, NC) on an O2 workstation (Silicon Graphics, Mountain View, CA). Fluorescence ratios were acquired at 15-s intervals and converted to [Ca2+]i as described in Data analysis. Ratio pairs were collected for 5 min before the addition of any agent. At the end of several experiments, cells permeabilized with 10 µM ionomycin revealed a 10- to 20-fold increase in the mean ratio of fluorescence in cells in buffer containing 1.8 mM CaCl2 compared with that in cells in Ca2+-free buffer, suggesting that intracellular fura 2 was fully Ca2+ sensitive in THP-1 monocytes.
The rise in [Ca2+]i in THP-1 monocytes adherent to uncoated glass (integrin-independent substrate), immobilized VCAM-1, or E-selectin was recorded in response to depletion of internal stores of Ca2+ by incubating the cells in HEPES-buffered Ca2+-free saline and TG (100 nM), an endoplasmic reticulum Ca2+-ATPase inhibitor (51, 52), for 5 min before 1 mM CaCl2 was added to the solution bathing the cells. As a positive control, THP-1 monocytes bound to glass were exposed to Val (20 µM), a K+ ionophore (12), immediately before the aforementioned protocol was performed to determine whether a similar level of hyperpolarization (i.e., an increased electrical driving force for Ca2+ entry) would enhance the increase in [Ca2+]i in response to TG and CaCl2. In other experiments, cells adherent to VCAM-1 were exposed to either 10 mM CsCl, which blocks Iir in THP-1 cells (9, 14), or 50 mM KCl HEPES-buffered saline (equimolar substitution of NaCl with KCl) to abolish the VCAM-1-induced hyperpolarization before the response to TG and CaCl2 addition was recorded.Measurement of resting membrane potential.
Resting membrane potential (RMP) was recorded in THP-1 monocytes bound
to glass (integrin-independent substrate) or immobilized adhesion
molecules using patch electrodes (BF100-50-10, Sutter Instruments) with
resistances of 4-7 M (20). The pipette contained (in mM) 150 KCl, 0.1 EGTA, 1 CaCl2 (free
[Ca2+] = 26.5 nM), and 10 HEPES, with pH adjusted to 7.2 with KOH, and the Ringer bath solution contained (in mM) 150 NaCl, 4.5 KCl, 2 CaCl2, 1 MgCl2, 5.5 glucose, and 10 HEPES as well as 0.01% BSA, with pH adjusted to 7.3 with NaOH. All
experiments were performed on cells at 22-24°C with an Axopatch
200A amplifier (Axon Instruments, Foster City, CA) after correction of
junction potentials. RMP was measured in current-clamp mode
(I = 0, where I is current) immediately
after attainment of the whole cell configuration. Whereas rapid
equilibration of the pipette solution with cytoplasm during the whole
cell configuration (43) may reduce the precision of the
RMP measurement, we and others have demonstrated this method to be an
accurate estimation of RMP (6, 8,
41). Total membrane capacitance, which was used to assess
cell size, was measured in the whole cell mode by integrating the
capacity transient and was then compensated electronically. In some
experiments, RMP was measured in THP-1 monocytes adherent to VCAM-1 in
the presence of CsCl (10 mM) or while being bathed in 50 mM KCl
HEPES-buffered saline. To determine the magnitude of membrane potential
change after Val exposure, RMP was measured in 2-min intervals for 30 min in THP-1 monocytes bound to uncoated glass during exposure to Val
(20 µM) following readdition of TG and CaCl2 to the
Ringer bath solution.
Data analysis. [Ca2+]i was quantitated by comparing cellular ratios of fluorescence with those in a standard curve generated from several aliquots of 1 µM fura 2-free acid in buffers of various [Ca2+]. The basal value for [Ca2+]i was calculated from the ratio pair that immediately preceded the addition of TG or, in the case of cells pretreated with CsCl, high [K+], or Val, from the ratio pair that preceded each of those treatments. The peak value was the maximum [Ca2+]i observed following the addition of CaCl2 to TG-treated cells.
Basal and peak [Ca2+]i in response to addition of TG (100 nM) and CaCl2 (1 mM) were compared in THP-1 monocytes bound to glass or adherent to immobilized adhesion molecules by using analysis of variance (ANOVA) followed by pairwise comparisons (Tukey's protected t-test). Differences between mean values were considered statistically significant when P ![]() |
RESULTS |
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Interaction of THP-1 monocytes with VCAM-1-enhanced
Ca2+ entry.
To determine whether the VCAM-1-induced hyperpolarization in THP-1
monocytes altered store-depleted Ca2+ influx, the time
course of [Ca2+]i changes in response to TG
and CaCl2 addition was followed in THP-1 monocytes adherent
to glass, E-selectin, or VCAM-1 for 5 h. Basal
[Ca2+]i in THP-1 monocytes was similar
regardless of substrate (Table 1).
Addition of TG (100 nM) to the Ca2+-free bath produced
small but significant increases in peak
[Ca2+]i in THP-1 monocytes adherent to each
substrate, with cells adherent to VCAM-1 producing the greatest
[Ca2+]i increase (Table 1). CaCl2
(1 mM) was added back to the bath to trigger store depletion-activated
Ca2+ influx in THP-1 monocytes. A 2.1-fold increase in
[Ca2+]i in THP-1 monocytes bound to glass was
achieved after a 2-min delay and was maintained over the next 10 min
(Fig. 1 and Table 1). The peak
[Ca2+]i level attained in monocytes adherent
to E-selectin was not significantly different from that observed in
monocytes bound to glass (Fig. 1 and Table 1). In contrast, a fivefold
increase in [Ca2+]i was produced in THP-1
monocytes adherent to VCAM-1 for 5 h when TG and CaCl2
were sequentially added to the bath (Fig. 1 and Table 1). The increase
in [Ca2+]i was maintained near peak levels
throughout the recording period. Control experiments, in which cells
were used regardless of substrate, confirmed the requirement of
CaCl2 readdition to produce the substantial increases in
[Ca2+]i after store depletion by TG and
indicate the activation of a Ca2+ influx pathway(s).
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Blockade of VCAM-1-induced hyperpolarization in THP-1 monocytes
prevents enhanced Ca2+ entry.
We have previously shown that the hyperpolarization produced by the
increased magnitude of Iir in THP-1 monocytes
adherent to VCAM-1 is blocked by exposure to exogenously applied CsCl
or BaCl2 (9). Because of the interference of
Ba2+ with fura 2 measurements, we examined
[Ca2+]i changes in response to TG and
CaCl2 in THP-1 monocytes adherent to VCAM-1 after
Iir blockade with CsCl. Blockade of
Iir depolarized THP-1 monocytes adherent to
VCAM-1 within 5 min to a mean RMP of 25 mV (Fig.
2A), which is identical to RMP
in THP-1 monocytes bound to glass (Fig.
3A). The enhanced peak
[Ca2+]i in response to TG and
CaCl2 addition in monocytes adherent to VCAM-1 was
completely abolished by exposure to Cs+ (Fig. 2B
and Table 2). Peak
[Ca2+]i in response to TG and
CaCl2 addition was similar in THP-1 monocytes adherent to
glass or VCAM-1 in the presence of Cs+ and was no different
from the response in cells bound to glass in the absence of
Cs+ (Table 2).
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Hyperpolarization of THP-1 monocytes increases the driving force
for Ca2+ entry.
Exposure of cells to the K+ ionophore Val forms
K+-selective pores through which K+ can flux
across the cell membrane down its chemical gradient to hyperpolarize
cells (12). Positive control experiments were done with
Val to confirm that the hyperpolarization produced by monocyte
adherence to VCAM-1 would be sufficient to increase Ca2+
entry and enhance [Ca2+]i changes in response
to TG and CaCl2 addition. RMP was measured in THP-1
monocytes bound to uncoated glass after exposure to exogenously applied
20 µM Val. RMP was hyperpolarized to 44 mV in the presence of Val
(Fig. 3A). Our previous observations in THP-1 monocytes adherent to VCAM-1 revealed a hyperpolarization to
38 mV compared with
25 mV in THP-1 monocytes bound to glass (9)
(compare Figs. 2A and 3A).
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DISCUSSION |
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Integrin-ligand interactions regulate ionic conductances to impact
cellular function. Engagement of
V
3-integrins, vitronectin receptors
expressed on vascular smooth muscle cells, causes arteriolar vasodilation, in part, by stimulating K+ channel activity
(42), whereas Ca2+ influx via L-type
Ca2+ channels is inhibited by arteriolar smooth muscle cell
contact with vitronectin or fibronectin (56). Rat PC-12
and cerebellar neuronal cell interaction with cell adhesion molecules,
L1, neural cell adhesion molecule, or N-cadherin stimulates
Ca2+ influx through voltage-dependent Ca2+
channels and is an important step in the commitment to neurite outgrowth (55). Activation of an
Iir in neuroblastoma cells adherent to
fibronectin has also been found to produce a membrane hyperpolarization
that plays a crucial role in the commitment to neuritogenesis
(3, 5). Relatively little is known about the
impact that VLA-4 integrin interaction with VCAM-1 has on ion channel
activation as it relates to cellular function in monocytes and/or
macrophages. Recently, we studied ionic currents in THP-1 monocytes
adherent to the physiological substrates, activated endothelial cells,
and, specifically, VCAM-1 and discovered that a hyperpolarization is
induced by increased Iir expression
(9). The Iir was characterized by
its activation at potentials negative to the equilibrium potential for
K+, its voltage-inactivation properties, blockade by
Cs+ and Ba2+, and its ability to set RMP to
negative levels.
Modulation of monocyte/macrophage function by Ca2+. An alteration in the driving force for Ca2+ entry would likely be affected by the VCAM-1-induced hyperpolarization to modulate Ca2+-mediated monocyte/macrophage function. Removal of extracellular Ca2+ or blockade of Ca2+ entry abolishes the initiation of phagocytosis (22), expression of inducible nitric oxide synthase (25), stimulated respiratory burst (24, 49), superoxide anion production (23), stimulated eicosanoid release (1), and cytokine production (34, 36) in monocytes and macrophages. In this present study, we provide several lines of evidence to support the hypothesis that the VCAM-1-induced hyperpolarization in THP-1 monocytes enhances the Ca2+ influx pathway activated by Ca2+ store depletion. First, adherence of THP-1 monocytes to purified VCAM-1, which hyperpolarizes the cells ~15 mV, increased [Ca2+]i in response to store depletion by TG fivefold compared with a twofold increase in cells bound to glass. Second, adherence of THP-1 monocytes to purified E-selectin, which does not alter RMP (9), did not enhance the TG-induced Ca2+ response. Third, depolarizing monocytes adherent to VCAM-1 by blocking Iir channel activity or exposing the cells to high extracellular [K+] completely abolished the augmentation of the TG-stimulated Ca2+ response. Fourth, monocytes bound to glass that were hyperpolarized by exposure to Val, a K+ ionophore, mimicked the VCAM-1-induced enhancement of the TG-stimulated increase in [Ca2+]i. Taken together, these results strongly suggest that the augmentation of the [Ca2+]i changes in response to store depletion is a result of an increased driving force for Ca2+ entry produced by the VCAM-1-induced hyperpolarization.
VCAM-1-induced Iir activity underlies the enhanced store-operated Ca2+ influx pathway. Hyperpolarization of membrane potential can be produced by K+ efflux through K+ channels to enhance the magnitude of Ca2+ entry into cells that possess non-voltage-gated Ca2+ influx pathways (53). Activation of T lymphocyte antigen is associated with a prolonged Ca2+ influx through CRAC channels that is regulated by hyperpolarization via K+ channels (31). In T lymphocytes and other cells, it is clear that the TG-sensitive Ca2+ influx pathway is modulated by transmembrane potential changes (15, 21). The magnitude of peak [Ca2+]i in response to TG is reduced in cells depolarized by exposure to a high-[K+] bath solution.
The Ca2+ entry into monocytes and macrophages is accomplished predominately by the presence of CRAC channels and nonselective cation channels in the cell membrane (30, 35). Differentiation of human monocytic U937 or promyelocytic HL-60 cells to macrophage-like cells upregulates the expression of the store-operated Ca2+ influx pathway (16). The presence of an inwardly rectifying K+ conductance in human monocytic leukemia THP-1 cells sets RMP to more negative levels as it does in most cells, including normal monocytes and macrophages (18, 27). In our study, the VCAM-1-induced Iir produced a membrane hyperpolarization that increased the driving force for store-dependent Ca2+ entry into THP-1 monocytes. Depolarizing cells adherent to VCAM-1 by blocking Iir channel activity or exposing the cells to high extracellular [K+] completely abolished the enhancement of Ca2+ entry and peak [Ca2+]i in response to TG and Ca2+ addition. Increasing the driving force for Ca2+ entry by hyperpolarizing THP-1 monocytes bound to glass with exposure to Val mimicked the enhancement of peak [Ca2+]i observed in THP-1 monocytes adherent to VCAM-1. It is not clear whether other mechanisms such as increased synthesis and insertion of CRAC channel protein into the cell membrane are involved in the enhancement of the TG-stimulated [Ca2+]i response by VCAM-1. However, it is unlikely that other K+ currents known to be present in THP-1 cells (14) played a role in producing the hyperpolarization-induced enhancement of the [Ca2+]i response to store-depletion. Basal [Ca2+]i was identical and below the threshold Ca2+ activation concentration for either of the Ca2+-sensitive K+ currents known to be present in THP-1 cells whether exposed to normal (4.5 mM KCl) or depolarizing (50 mM KCl) conditions. Additionally, the inhibition of the TG-stimulated [Ca2+]i response with Cs+ or depolarizing conditions, despite the likely activation of delayed rectifier K+ currents, argues against a contribution of other voltage-gated K+ currents.Pathophysiological relevance of enhanced Iir activity and hyperpolarization in macrophages. The interaction of monocytes with VCAM-1 on the surface of activated endothelium and at sites of inflammation such as early foam cell lesions leading to hyperpolarization may significantly impact the progression and complications of atherosclerosis. Preliminary work in our laboratory reveals that engagement of VLA-4 integrins on THP-1 monocytes by VCAM-1 induces Iir activity and enhances the Ca2+-dependent production of the chemokine interleukin (IL)-8 (7). Recently, the atherogenic role of IL-8 has gained particular attention. Cholesterol-loaded THP-1 cells possess higher IL-8 mRNA levels that correlate with increased IL-8 mRNA in a macrophage-rich area of human atheromas (46). Studies in which macrophages were isolated from human atherosclerotic plaques have provided direct evidence confirming that these cells are responsible for the enhanced IL-8 production found in plaques (2). IL-8 is chemotactic for neutrophils, endothelial cells, lymphocytes, and vascular smooth muscle cells, all contributors to atherosclerotic plaque formation (28, 50, 57).
In summary, we have demonstrated that the hyperpolarization produced by adherence of monocytes to one of its physiological substrates, VCAM-1, is sufficient to increase the electrical driving force for Ca2+ entry and enhance the store-operated Ca2+ influx pathway. The enhanced Ca2+ influx resulting from the Iir-induced hyperpolarization may, therefore, contribute to the alterations in macrophage function to impact the progression of atherosclerosis and other pathological conditions such as adult respiratory distress syndrome. ![]() |
ACKNOWLEDGEMENTS |
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We thank Drs. Pamela Gunter-Smith, Catherine Chew, and Gordon Leitch for critical evaluation of this manuscript. We also acknowledge the excellent technical assistance provided by Esther Carlisle Doele, Roberta Hawkins, and Andrew Shaw.
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FOOTNOTES |
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This work was supported by American Heart Association-Georgia Affiliate and, in part, by National Institutes of Health Grant G13-RR-03034.
Address for reprint requests and other correspondence: M. Colden-Stanfield, Morehouse School of Medicine, Dept. of Physiology, Rm. 1311, 720 Westview Dr. S.W., Atlanta, GA 30310-1495 (E-mail: stanfiel{at}msm.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. §1734 solely to indicate this fact.
Received 4 October 1999; accepted in final form 29 February 2000.
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