1 Department of Anesthesiology, The University of Maryland Baltimore, Baltimore, Maryland 21201; 2 Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, SP, Brazil; 3 Section on Neuronal Secretory Systems, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and 4 Departamento de Farmacologia, Universidade Federal de São Paulo, UNIFESP, São Paulo, Brazil
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ABSTRACT |
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This study tested
the hypothesis that the activity of the mitochondrial membrane
permeability transition pore (PTP) affects the resting mitochondrial
membrane potential () of normal, healthy cells and that the
anti-apoptotic gene product Bcl-2 inhibits the basal activity of the
PTP.
was measured by both fluorometric and nonfluorometric
methods with SY5Y human neuroblastoma cells and with GT1-7
hypothalamic cells and PC12 pheochromocytoma cells in the absence and
presence of Bcl-2 gene overexpression. The resting
of Bcl-2
nonexpressing PC12 and wild-type SY5Y cells was increased significantly
by the presence of the PTP inhibitor cyclosporin A (CsA) or by
intracellular Ca2+ chelation through exposure to the
acetoxymethyl ester of
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA-AM). The
of Bcl-2-overexpressing PC12 cells was larger
than that of Bcl-2-negative cells and not significantly increased by
CsA or by Ca2+ chelation. CsA did not present a significant
effect on the
monitored in unstressed GT1-7 cells but did
inhibit the decrease in
elicited by the addition of
t-butyl hydroperoxide, an oxidative inducer of the
mitochondrial permeability transition. These results support the
hypothesis that an endogenous PTP activity can contribute to lowering
the basal
of some cells and that Bcl-2 can regulate the
endogenous activity of the mitochondrial PTP.
calcium; mitochondrial permeability transition; energy metabolism
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INTRODUCTION |
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EXPOSURE
OF ISOLATED MITOCHONDRIA to Ca2+ ions can cause a
nonselective permeabilization of the inner mitochondrial membrane due
to the opening of the mitochondrial permeability transition pore (PTP)
(30, 53). The PTP promotes a drop in
mitochondrial membrane potential () and a loss of accumulated
Ca2+ and even induces large amplitude swelling of
mitochondria (30, 53). These phenomena are
stimulated by the presence of inorganic phosphate, oxidative stress, or
dithiol reagents and are typically inhibited by cyclosporin A (CsA)
(25, 27, 30, 53).
Although the PTP has been studied extensively using isolated mitochondria or permeabilized cells, these experiments have rarely been conducted under physiologically relevant conditions (2). In some cells and tissues, the PTP has been implicated as an early event in both apoptotic and necrotic cell death (17, 29, 30). In addition, the anti-apoptotic protein Bcl-2 inhibits the PTP and prevents mitochondrial release of cytochrome c, a trigger for apoptosis (26, 28, 52). However, few studies have detected the activity of the PTP in intact cells in the absence of potentially lethal stressful conditions (11, 16, 19, 46), e.g., in the presence of greatly elevated intracellular Ca2+ or toxic hydroperoxides.
Classically, PTP opening has been associated with generalized mitochondrial dysfunction, which is consistent with a role of the PTP in cell death but would be incompatible with a physiological role for this pore. Some studies suggest that, under certain conditions, the PTP mediates a limited transport of small ions, which could allow for the maintenance of viable mitochondrial energy-transducing activities (13, 49). This activity state of the PTP has been referred to as the "low-conductance state" (19, 20, 37) but can also be interpreted as a transient opening of the PTP that, unlike a relatively high-conductance state, is insufficient to cause high-amplitude swelling and irreversible mitochondrial destruction (38).
Recent elucidation of the multiple roles that mitochondria play in normal cellular Ca2+ homeostasis has provided additional evidence for a physiological PTP activity. Upon mobilization of Ca2+ from the endoplasmic reticulum by the second messenger inositol 1,4,5-trisphosphate (IP3), mitochondria adjacent to the Ca2+ release sites play an important role in clearance of cytosolic Ca2+ (16, 39, 43). Under these circumstances, activation of mitochondrial Ca2+ influx can modulate IP3 receptors and cytosolic Ca2+ signaling (43). Moreover, it has been shown that mitochondrial Ca2+ uptake triggers mitochondrial Ca2+ release, which, in turn, leads to an amplification of the cytosolic Ca2+ signals (19). A low-conductance PTP would present a tendency to flicker between the opened and closed states as Ca2+ is taken up and released (Ca2+-induced Ca2+ release), generating and conveying Ca2+ signals (20, 44). Thus the low-conductance PTP may be responsible for the mitochondrial participation in modulating and shaping Ca2+ transients during Ca2+ signaling.
In this report, we investigated the contribution of PTP activity to
mitochondrial in healthy, unstressed neural cells. We found that
the PTP contributes significantly toward the reduction in mitochondrial
in two of three different cell lines. The anti-apoptotic gene
product Bcl-2, which has been shown to inhibit the PTP in stressed
cells, was also found to minimize the contribution of the PTP to the
resting
of unstressed cells.
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MATERIALS AND METHODS |
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Cell cultures.
Immortalized PC12 adrenal pheochromocytoma cells, GT1-7
hypothalamic tumor cells, and SY5Y human neuroblastoma cells were maintained as described previously (34, 3).
PC12 and GT1-7 cells were transfected with the human
bcl-2 gene (Bcl-2+) or with a control retroviral
construct (Bcl-2) (24). Experiments
were performed either with cells plated on coverslips or with cells
that were grown normally, trypsinized, and suspended in the incubation
medium. All cells presented >98% viability at the time they were
used, as assayed by trypan blue staining.
Standard incubation conditions. All assays were conducted at 37°C, in medium containing 130 mM NaCl, 5.6 mM KCl, 0.8 mM MgSO4, 1 mM Na2PO4, 25 mM glucose, 20 mM HEPES (pH 7.3), 1.5 mM CaCl2, 2.5 mM NaHCO3, 1.5 mg/ml BSA, and 1 mM ascorbic acid. Cells in suspension were continuously stirred while cells on coverslips were continuously superfused with medium. All additions during experiments were made to the suspension or superfusion medium and did not involve a change in media.
Determination of mitochondrial using TMRE.
Cells were maintained during the experimental assays in media
containing tetramethylrhodamine ethyl ester (TMRE, 50 nM), a cationic
dye that is rapidly and reversibly accumulated by mitochondria, due to
their
(12, 31). As TMRE-based
measurements of
may underestimate the absolute value of the
membrane potential, these determinations were used to compare relative
levels rather than assigning specific values (41).
Moreover, because TMRE can, under some conditions, produce superoxide
radicals and even induce the permeability transition when
photodynamically excited (18), all incubations were
conducted in the dark, and light exposure was kept to the minimum
necessary for accurate measurements.
Determination of using TPP+.
Cells were incubated in standard media supplemented with 0.5 µM
tetraphenylphosphonium (TPP+), and the concentration of
TPP+ was continuously monitored in the extracellular medium
using a TPP+-selective electrode constructed according to
Kamo et al. (23). TPP+ uptake by cells treated
with antimycin A plus oligomycin was <1% of that in respiring cells,
again indicating that TPP+ measurements reflect the
mitochondrial
rather than the plasma membrane potential.
Materials. Tert-butyl hydroperoxide (t-bOOH), FCCP, antimycin A, oligomycin, and TPP+ were purchased from Sigma Chemical. The acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) was purchased from Calbiochem, CsA was from Alexis, and TMRE was obtained from Molecular Probes. FK-506 was a gift from Fujisawa, Japan. CsA and BAPTA were diluted in ethanol or DMSO. The final concentration of these vehicles was 0.001%, which was determined to have no effect on TMRE fluorescence intensity or the response of the TPP+ electrode.
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RESULTS |
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The mitochondrial in normal, intact cells was initially
evaluated by fluorescence microscopy using TMRE, a fluorescent probe of
. Although the baseline TMRE fluorescence of Bcl-2
PC12 cells remained constant over the first 5 min of measurements (Fig.
1) and for at least 15 min thereafter
(not shown), cells treated with the PTP inhibitor CsA exhibited a
substantial increase in TMRE fluorescence that appeared to reach a
plateau 10-15 min after the addition of CsA (Fig. 1).
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To ascertain that the increase in TMRE response observed could be
attributed to the PTP, we treated the cells with BAPTA-AM (Fig.
2), which chelates intracellular
Ca2+, a necessary trigger for PTP opening
(53). We observed that PC12 cells treated with BAPTA-AM
also presented an increase in TMRE response over time, similar to that
observed with CsA. Thus both intracellular Ca2+ chelation,
which prevents PTP opening, and CsA, which inhibits the PTP, increased
the resting mitochondrial in PC12 cells.
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The anti-apoptotic protein Bcl-2, which inhibits PTP opening induced by
Ca2+ and pro-oxidants, has been reported to elevate the
resting of isolated mitochondria (42,
28). As a test of the hypothesis that Bcl-2 can elevate
the resting mitochondrial
within intact, unstressed cells, TMRE
fluorescence measurements were also performed with transfected PC12
cells that overexpress the human bcl-2 gene. Quantification
of changes in fluorescence over a 20-min period during which data were
obtained is provided in Fig. 3. In the absence of CsA, TMRE fluorescence for both Bcl-2
and
Bcl-2+ cells increased slightly, with a trend toward a
greater increase in the Bcl-2+ cells (Fig. 3). The
fluorescence increase that occurred in the presence of 1 µM CsA was
significantly greater for Bcl-2
than for
Bcl-2+ cells (P < 0.05). By analyzing the
changes in fluorescence of individual cells with time, we also found
that CsA evoked an increase in
for 82.7 ± 6.0% of the
Bcl-2
cells vs. 62.4 ± 4.2% of the
Bcl-2+ cells (n = 3; P < 0.05).
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Results obtained with fluorescence microscopy and cells grown on
coverslips were expanded upon using spectrofluorometric measurements of
PC12 cells suspended in medium containing TMRE (Fig.
4A). These measurements of
are expressed as the difference in TMRE fluorescence measured
before and after the addition of the uncoupler FCCP to quantify the
response to PTP inhibitors and to exclude potential artifacts. We
observed that the difference in TMRE fluorescence in the
Bcl-2
PC12 cells was significantly increased
(P < 0.05) by a 20-min exposure to either CsA or
BAPTA-AM. The concomitant presence of both inhibitors did not present
an additive effect. Also, FK-506, an immunosuppressant similar to CsA,
which also inhibits protein phosphatase activity but does not inhibit
the PTP (15), did not significantly alter the
FCCP-sensitive fluorescence observed under these conditions.
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TMRE, as well as other cell-permeant fluorescent probes of , can
under some conditions significantly alter mitochondrial function
(41). Indeed, it has been demonstrated that TMRE-loaded mitochondria undergo PTP opening in a manner dependent on the concentration of the probe and light exposure, due to photodynamically induced free radical generation by TMRE (18). To minimize
these possible effects of TMRE, a relatively very low concentration of
dye was used (50 nM), and light exposure was kept to a minimum (see
MATERIALS AND METHODS). Further validation of our
measurements employed the use of an alternative, nonfluorometric method
for monitoring
. In Fig. 4B, mitochondrial
in
intact PC12 Bcl-2
cells was monitored by continuously
measuring the extracellular concentration of TPP+ (see
MATERIALS AND METHODS). TPP+ is a lipophilic
cation that is actively accumulated into mitochondria due to the high
inside-negative potential (>180 mV) that exists across the
mitochondrial inner membrane. Consistent with the results observed
using TMRE (Fig. 4A), we observed that the cellular
TPP+ uptake was greater in the presence of CsA or BAPTA-AM
(Fig. 4B). Furthermore, preincubation of the cells for 40 min in media containing 5 mM EGTA to reduce intracellular
Ca2+ content resulted in a TPP+ uptake similar
to that observed in the presence of CsA or BAPTA-AM (not shown). As
with the TMRE measurements, FK-506 did not present a significant effect
on the
measured by TPP+ uptake. These results are
fully consistent with the results obtained with the fluorescent
probe TMRE and support the conclusion that PTP opening lowers the
resting
in this cell line.
Using TMRE fluorescence measurements with cells in suspension, it was
also possible to verify that the resting of PC12 Bcl-2+ cells was significantly higher (P < 0.05) than that of the control cells (compare Figs.
5A and 4A). We have
previously established that bcl-2 overexpression in
transformed neural cells does not affect the content of mitochondria
present within the cells, as indicated by measurements of mitochondrial
O2 consumption and DNA levels (33,
34). Therefore, the present results expand upon previous
reports that Bcl-2 increases the
of isolated mitochondria
(42) and mitochondria present within permeabilized cells
(28) to indicate that the same effect occurs within
unstressed, intact cells. Neither CsA or BAPTA-AM added alone had any
significant effect on the TMRE fluorescence of Bcl-2-overexpressing
PC12 cells. Although the combined presence of these agents did result
in a significant increase in
F, this increase (<30%) was
substantially lower than that observed with Bcl-2
cells
(>60%). This finding suggests that the resting PTP activity in
Bcl-2+ cells is lower than in control cells. Indeed,
because the final levels of
after the addition of CsA or BAPTA
to Bcl-2
vs. Bcl-2+ cells were not
statistically different, our results suggest that the difference in
resting
of Bcl-2
and Bcl-2+ cells is
due to inhibition of the resting PTP activity by Bcl-2. Alternative
measurements of
by monitoring the cellular accumulation of
TPP+ in the suspending medium were qualitatively similar to
the results obtained from fluorescent TMRE measurements (Fig.
5B). Although either CsA or BAPTA-AM slightly increased
TPP+ uptake, their effects on Bcl-2+ cells
appeared less than those observed with Bcl-2
cells
(compare with Fig. 4B). FK-506 exhibited a slight depression of TPP+ uptake, which is also consistent with the effects
it had on TMRE-based measurements of
.
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In addition to the difference in the extent of the contribution of PTP
opening to the resting of Bcl-2
and
Bcl-2+ cells, we also detected variability in the apparent
endogenous PTP activity among different cell lines. Figure
6A describes TMRE fluorescence
responses obtained with suspensions of GT1-7 cells, a hypothalamic
transformed neural cell line. Unlike that of PC12 cells, the TMRE
fluorescence of GT1-7 cells was not significantly affected by the
intracellular Ca2+ chelator BAPTA-AM or the PTP inhibitor
CsA, even when the concentration of CsA was increased to 5 µM. The
of unstressed GT1-7 cells was also not affected by exposure
to FK-506. However, if these cells were treated with t-bOOH,
a compound capable of enhancing Ca2+-induced PTP in both
isolated mitochondria and cells by oxidizing mitochondrial pyridine
nucleotides (4, 6, 14,
21, 28, 36), then an average
value for
F was obtained (32 ± 4; Fig. 6B) that is
significantly lower than that obtained in the absence of
t-bOOH (47 ± 1; Fig. 6A). The fluorescence
values obtained in the presence of t-bOOH were significantly
increased by exposure of cells to CsA (5 µM) or BAPTA but not FK-506.
Thus GT1-7 cells do not present a detectable resting PTP activity
but do exhibit a drop in
consistent with PTP activity when
exposed to the pro-oxidant t-bOOH.
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In contrast to the insensitivity of GT1-7 cells to alterations in
resting caused by CsA or BAPTA-AM, but in agreement with the
sensitivity of PC12 cells, the resting
of human SY5Y
neuroblastoma cells was significantly elevated by the addition of the
MPT inhibitor CsA or by exposure to the intracellular Ca2+
chelator BAPTA-AM (Fig. 7). In addition,
we found that exposure of these cells to the extracellular
Ca2+ chelator EGTA for 40 min resulted in an increase in
TMRE fluorescence. Thus, with SY5Y cells, we observed an increase in
the normal
after treatment with three different conditions that
can inhibit PTP activity by two different mechanisms. As with the other
cell lines, the
of SY5Y cells was not elevated by exposure to
FK-506.
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DISCUSSION |
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Taken together, our results strongly suggest that the PTP is
active and contributes toward a decrease in mitochondrial in
resting PC12 and SY5Y cells but not in unstressed GT1-7 cells. We
have determined that the PTP influences the resting
in PC12 and
SY5Y cells by demonstrating that
can be elevated by the presence
of CsA or Ca2+ chelators but not by the immune suppressor
FK-506, which does not inhibit PTP activity in isolated mitochondria
(15). In addition, fluorescence microscopy measurements on
individual cells indicate that the increase in
induced by CsA or
BAPTA-AM in PC12 cells is a common phenomenon and occurs in the
majority of cells that are analyzed. This finding argues against the
possibility that the observed responses are due to a fraction of cells
that are undergoing PTP as part of a cell death process. The GT1-7
cell line that did not exhibit sensitivity of resting
to PTP
inhibitors nevertheless did demonstrate a CsA- and BAPTA-AM-sensitive
fraction of
in the presence of the PTP inducer
t-bOOH. The finding that t-bOOH does not
completely eliminate the
of GT1-7 cells, as it does in
hepatocytes (4, 21, 36), may
relate to the reason why GT1-7 cells do not express a detectable
endogenous PTP activity. For example, variability of endogenous and
induced PTP activity could be due to variability in cellular redox
state or sensitivity of mitochondrial pyridine nucleotides to oxidation
(14).
The PTP has been studied most extensively with isolated mitochondria
with the use of conditions in vitro that bear little resemblance to
those that exist within intact cells. Within the last few years,
however, evidence obtained with models of cell and tissue injury has
supported the involvement of the PTP in necrotic and apoptotic cell
death (8, 17, 29,
30). Moreover, it has been proposed that a
Ca2+- and proton-selective, low-conductivity state of the
PTP may be active in normal Ehrlich tumor cells, generating and
conveying electrical and Ca2+ signals (19). In
the study reported by Ichas et al. (19), the PTP is
activated upon IP3-induced Ca2+ mobilization
from the endoplasmic reticulum, whereupon PTP-mediated mitochondrial
Ca2+ efflux contributes to the amplification of cytosolic
Ca2+ signals. These findings are supported by studies
showing that binding of IP3 to its receptors results in
discrete areas of elevated intracellular Ca2+ that are
sensed by neighboring mitochondria (39). The ensuing increase in intramitochondrial Ca2+ can lead to an
activation of mitochondrial dehydrogenases and, therefore, ATP
production (40). However, the rapid stimulation of
mitochondrial Ca2+ uptake by focal spikes in
extramitochondrial Ca2+ concentrations could result in
transient reductions in mitochondrial (9,
47), which would promote activation of the PTP. Opening of
the PTP would be expected to prolong the period of
mitochondrial depolarization and induce the release of at least some
fraction of the accumulated Ca2+. Additional support for
this scenario comes from observations that CsA increases mitochondrial
Ca2+ accumulation in normal cardiomyocytes (1)
and decreases Ca2+-induced mitochondrial Ca2+
release in Ehrlich ascites tumor cells and endothelial cells (10, 50). In addition, blockade of MPT
inhibits agonist-evoked Ca2+ oscillations in glial cells,
reinforcing the hypothesis that, during physiological stimulation,
transient PTP openings support Ca2+ signaling
(46). Further evidence for the baseline activity of a
low-conductance state of the PTP has come from TPP+ uptake
measurements (5), fluorescent flow cytometry and
microscopic imaging of
in SY5Y neuroblastoma cells
(11), and rat oligodendrocyte progenitors
(46).
The activity of the MPT under physiological conditions would qualify it
as an endogenous uncoupler of oxidative phosphorylation. However, the
degree of uncoupling and energy expenditure by the movement of ions
through the PTP would, by necessity, need to be very limited so that
metabolic homeostasis could be preserved. Like the activity of
well-characterized tissue-specific uncoupling proteins
(22), the resting state PTP activity may act similarly to
increase energy consumption without obstructing ATP synthesis. Studies
are in progress to determine the extent to which the endogenous PTP
contributes to the basal rate of respiration by the PC12 and SY5Y cells
used in our experiments. Increased energy utilization associated with
the cycling of protons, Ca2+, and other ions mediated by
PTP activity should be manifested as heat generation. Indeed, CsA has
been demonstrated to decrease the heat output of normal lymphocytes
(25). In addition to the influence PTP activity can exert
on intracellular Ca2+ signaling, oxygen utilization, and
heat production, the associated reduction in would reduce the
formation of superoxide due to "leakage" of electrons from the
ubiquinone region of the electron transport (45). It is
therefore possible that a controlled endogenous PTP activity could
actually protect mitochondria against self-inflicted oxidative stress
(45).
Bcl-2 overexpression has previously been shown to increase in
isolated mitochondria (42) and permeabilized cells
(28). Our findings further indicate that Bcl-2 can elevate
the resting
of intact, unstressed cells by inhibiting the
endogenous activity of the PTP, as previously suggested by JC-1
fluorescence probe measurements of
in another strain of PC12
cells (7). It is possible that the differences in
uncoupler-sensitive TMRE fluorescence and TPP+ uptake
between Bcl-2+ and Bcl-2
cells are due to
differences in mitochondrial volume, even though maximal rates of
respiration and mitochondrial DNA contents are equivalent. However, the
observation that overexpression of Bcl-2 minimizes the effects of PTP
inhibitors on resting
constitutes evidence that Bcl-2 actually
increases
possibly via inhibition of endogenous PTP activity.
Bcl-2 has been proposed to act as a H+ channel that
contributes to rather than detracts from the mitochondrial electrochemical gradient of protons (42). However,
considering the known ability of Bcl-2 to inhibit the stress-evoked PTP
opening in isolated mitochondria and permeabilized cells
(28, 32, 42, 48)
and the ability of Bcl-2 to inhibit endogenous PTP activity in our
experiments, its endowment for MPT inhibition may be its primary
mechanism of action. Although the physiological role for Bcl-2 is
generally thought to be one of protection against cytotoxicity
(24, 26, 32, 34,
35, 48, 52), the present results
suggest that Bcl-2 may also serve as an enhancer of the efficiency of
mitochondrial energy coupling by decreasing the endogenous PTP activity.
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ACKNOWLEDGEMENTS |
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We thank S. J. Russell for the excellent technical assistance.
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FOOTNOTES |
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* A. J. Kowaltowski and S. S. Smaili contributed equally to this work.
Transfected cells were kindly provided by Dr. Dale Bredesen (Burnham Research Institute, La Jolla, CA), and FK-506 was provided by Fujisawa, Japan.
This work was supported by the FAPESP, by National Institutes of Health Grant NS-34152, and by the Bayer Corporation.
Address for reprint requests and other correspondence: G. Fiskum, Univ. of Maryland, Baltimore, Dept. of Anesthesiology, 685 W. Baltimore St., Baltimore, MD, 21201 (E-mail: gfiskum{at}anesthlab.ummc.umaryland.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 20 May 1999; accepted in final form 30 March 2000.
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