Department of Pharmacology, University of South Alabama School of Medicine, Mobile, Alabama 36688
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ABSTRACT |
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Cellular sodium excess is cytotoxic
because it increases both the intracellular osmotic load and
intracellular calcium concentration ([Ca2+]i). Because sodium levels rise during
hypoxia, it is thought to contribute to hypoxic injury. Thus the
present study tested the hypothesis that taurine-linked reductions in
[Na+]i reduce hypoxia-induced cell injury.
Taurine depletion was achieved by exposing isolated neonatal
cardiomyocytes to medium containing the taurine analog -Alanine. As
predicted, the
-Alanine-treated cell exhibited less hypoxia-induced
necrosis and apoptosis than the control, as evidenced by less
swelling, shrinkage, TdT-mediated dUTP nick end labeling staining, and
accumulation of trypan blue. After 1 h of chemical hypoxia,
[Na+]i was 3.5-fold greater in the control
than the taurine-deficient cell. Although more taurine was lost from
the control cell than from the
-Alanine-treated cell during hypoxia,
the combined taurine and sodium osmotic load was lower in the
-Alanine-treated cell. Taurine deficiency also reduced the degree of
hypoxia-induced calcium overload. Thus the observed resistance against
hypoxia-induced necrosis and apoptosis is probably related to
an improvement in sodium and calcium handling.
cardiomyocytes; metabolic inhibition; osmolality; osmotic stress; necrosis
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INTRODUCTION |
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ACCORDING TO THE
OSMOTIC STRESS HYPOTHESIS advanced by several investigators
(18, 26), a major cause of ischemia-induced sarcolemmal damage is cell stretching linked to osmotic swelling. Three
factors are thought to contribute to osmotic-induced cell swelling
during ischemia (4). First, the concentration of
several metabolic end products increases, causing a rise in
intracellular osmolality. Although these metabolic end products
contribute to the cell's osmotic load during the initial phase of
ischemia, with time these intermediates leak out of the cell
and their contribution to the osmotic load diminishes. Second, cellular
edema arises in part from ischemia-induced interstitial edema.
Third, disruption of cellular transporters leads to a net gain in
intracellular ions, of which sodium and Cl are the most
important. A good correlation has been found between the
ischemia-induced increase in cellular water and the rise in intracellular sodium ([Na+]i) and
Cl
concentration ([Cl
]i)
(18).
Opposing the unfavorable influence of the three hyperosmotic events is
the release of organic osmolytes from the hypoxic myocyte (9, 11,
26). One of the primary organic osmolytes in the heart is the
amino acid taurine (6). Extremely high intracellular levels of taurine are maintained by a specific -amino acid
transporter. In accordance with taurine's role as an osmolyte, the
activity of the taurine transporter in the myocyte is increased
following exposure of the cell to hyperosmotic medium (2).
On the other hand, large amounts of taurine are rapidly lost from the
myocyte during hyposmotic shock (17). Interestingly, there
is also evidence that the movement of taurine in and out of the cell is
closely tied to changes in the intracellular and extracellular
concentration of sodium (29). Therefore, changes in the
intracellular content of taurine can influence the osmotic balance of
the cell through alterations in the intracellular levels of both
taurine and sodium.
On the basis of putative importance of osmotic imbalances in the pathology of ischemia-reperfusion injury, one would predict that the movement of taurine and sodium should influence the outcome of an ischemic event. Indeed, we recently found that the drug-induced taurine-deficient heart is extremely resistant to ischemic injury (1). In that study, we proposed that taurine depletion might act by reducing the osmotic load of the ischemic heart. We also suggested that the beneficial effects of taurine depletion could be related in part to the regulation of [Na+]i, an effect thought to be linked to changes in tissue osmolality, intracellular pH (pHi), and/or the cotransport of taurine and sodium. In this regard, it is significant that [Na+]i contributes both to the osmotic load of the cell and to calcium influx via the sodium-calcium exchanger (10, 24). In addition, taurine exerts multiple effects on the heart, including alterations in both tissue osmolality and calcium movement (20). The direct and indirect effects of taurine on calcium movement could be of particular relevance because calcium overload is a major contributor to myocardial cell necrosis during ischemia (16, 24, 25). Thus taurine depletion could affect the outcome of a hypoxic or ischemic insult through a mechanism involving improved cellular calcium levels, a reduction in the osmotic load, or a combination of the two factors. To provide more information on these possibilities, we examined the effect of taurine deficiency on hypoxia-induced necrosis and apoptosis. We also examined the influence of taurine deficiency on hypoxia-induced changes in pHi, [Na+]i, [Ca2+]i, and taurine content.
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METHODS |
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Cell preparation and incubation conditions.
Rat neonatal cardiomyocytes were isolated according to the method of
McDermott and Morgan (12). The cells were suspended in
minimal essential medium containing 10% newborn calf-serum and 0.1 mM
5'-bromo-2'-deoxyuridine and plated on either glass coverslips or glass
petri dishes precoated with 0.1% gelatin. They were then placed in
standard serum-free medium containing 56 U/l insulin and 10 µg/ml
transferrin. To induce taurine deficiency, some of the cells were
incubated in standard serum-free medium supplemented with 5 mM
-Alanine. All experiments were initiated after a 2-day incubation at
37°C under a 5% CO2-20% O2 environment. To
induce chemical hypoxia, the cells were placed in Krebs-Henseleit buffer containing the metabolic inhibitors 10 mM deoxyglucose and 3 mM
Amytal. The duration of the metabolic insult was 60 min, unless
otherwise stated. The control cells (normoxic and hypoxic) were defined
as those cells that were incubated for 2 days with serum-free medium
lacking
-Alanine.
Cellular taurine content.
Taurine content of the -Alanine-treated and control cells was
determined both before and 60 min after onset of chemical hypoxia. After a 60-min incubation with medium lacking or containing metabolic inhibitors, the cells were scraped from the surface of the petri dishes
and an aliquot was removed to determine protein content. The remaining
cells were treated with 2% perchloric acid, and after neutralization,
taurine content of the extracts was determined according to the
procedure described by Mozaffari et al. (15).
Cell volume analysis.
Before chemical hypoxia, some -Alanine-treated and control cells
were incubated with medium containing 5 µM calcein-acetoxymethyl ester (AM) for 30 min. The calcein-loaded cells were then washed three
times with calcein-free medium and placed in standard serum-free medium. The volume of a series of cells was determined before and after
60 min of chemical hypoxia. To measure cell volume, a confocal
microscope was used to scan calcein fluorescence for each 1-µm
confocal slice, proceeding from the top of the cell to the bottom
(~10 slices). From the surface area of the scan and pixel thickness
of each confocal slice, the cell volume was calculated
(21). Because hypoxia does not influence the distribution of calcein in the cell, the only condition interfering with the measurement of cell volume is the complete loss of calcein. This occurred in only a few cells and was associated with the rupture of the
cell membrane. No data were obtained from these cells. The average cell
volume of the
-Alanine-treated and control cells before the chemical
hypoxic insult was 5.2 ± 0.2 × 103 and 6.5 ± 0.2 × 103 µm3, respectively. The
confocal microscope procedure was accurate within 2%. Therefore, a
deviation of >5% from the prehypoxic value was required to classify a
cell as swollen (necrotic) or shrunken (apoptotic). Each cell
served as its own control.
Cellular H+, sodium, and calcium
content.
To determine [Na+]i of the normal and
-Alanine-treated cells, the normoxic cells were first loaded with
sodium-binding benzofuran isophthalate (SBFI) by incubating the
myocytes for 1.5 h at room temperature with serum-free medium
containing 10 µM SBFI-AM. They were then washed three times with
dye-free medium and kept in the dye-free medium for at least 45 min to
facilitate the hydrolysis of the ester. The cells were then placed in
either normoxic or chemical hypoxic Krebs-Henseleit buffer. Sodium
content of the normoxic and chemically hypoxic cells was determined
fluorometrically by use of an Olympus (IMT-2) microscope, with emission
fluorescence (>420 nm) examined at two excitation wavelengths, 340 and
380 nm. The background emission was corrected at the start of the experiment. During the course of the chemical hypoxic insult, the ratio
of the fluorescence signals generated at the two excitation wavelengths
was determined. [Na+]i was calculated from
the fluorescence ratio (340 nm/380 nm) after calibration curves were
generated using the procedure of Harootunian et al. (5).
Trypan blue staining and TdT-mediated DUTP nick end labeling procedures. To measure cell viability, hypoxic and normoxic cells were incubated for 5 min at room temperature with medium containing 0.4% trypan blue. After 5 min of incubation, the cells were fixed in Krebs-Henseleit buffer containing 2% glutaraldehyde. Four separate fields in the light microscope were used to assess trypan blue staining.
End-labeled DNA fragments of apoptotic nuclei were monitored by the Klenow Frag EL DNA fragmentation detection kit (catalog no. Q1A21; Calbiochem). After either the 3-day control incubation or 1 h of chemical hypoxia, glass slides were fixed with 4% formaldehyde for 15 min and then resuspended in 80% ethanol for 20 min. After rehydration, the samples were permeabilized with proteinase K (20 µg/ml). Endogenous peroxidases were inactivated by exposing the samples to 3% H2O2 for 5 min. After the samples were rinsed, the slides were placed in Klenow buffer for 20 min. The Klenow-labeling reaction mixture was allowed to proceed for 1.5 h at 37°C. Termination of the reaction was accomplished by a 5-min incubation with buffer containing 0.5 mM EDTA (pH 8.0) followed by a 10-min exposure to blocking buffer. The samples were then exposed for 30 min to a peroxidase-streptavidin conjugate. After a 15-min incubation with 3,3'-deaminobenzidine (0.7 mg) and H2O2-urea (0.6 mg), the samples were rinsed and then counterstained with hematoxylin. The slides were briefly immersed in 100% ethanol and then in xylene. Four fields in the light microscope were counted for dark brown (apoptosis) and purple (normal) nuclei. The data were expressed as percentage of apoptotic cells.DNA ladder analysis. Cells were first scraped from the incubation dishes. After cell lysis, DNA was isolated according to the method described in the DNA apoptosis ladder kit (Boehringer-Mannheim). Samples of DNA (10 µg, amount determined by absorbance at 260 nm) were subjected to agarose gel (2%) electrophoresis. The gels were stained with ethidium bromide (2 µg/µl) and then destained for 20 min. The bands were visualized under ultraviolet light.
Statistical analysis. The statistical significance of the data was determined by using the Student's t-test for comparison within groups and analysis of variance (ANOVA) combined with Tukey's post hoc test for comparison between groups. Values of P < 0.05 were considered statistically significant.
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RESULTS |
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Previously, it was shown that rats fed a diet containing high
concentrations of the taurine analog -Alanine slowly lose a significant fraction of their intracellular taurine store
(15). As shown in Fig. 1,
incubation of isolated rat neonatal cardiomyocytes with
-Alanine
containing medium for a period of 2 days also induced a dramatic drop
(40%) in the size of the intracellular taurine pool.
-Alanine
treatment was also associated with a 30% decrease in
[Na+]i from 11.0 ± 1.4 to 7.12 ± 0.9 mM (Fig. 2). Resting
[Ca2+]i was unaffected by taurine depletion,
but the calcium transient was prolonged, as evidenced by a 70%
increase in the time from peak calcium to 90% relaxation
(TR90 was 0.7 ± 0.03 s in the control vs.
1.2 ± 0..11 s in the
-Alanine-treated group). As a result of
the osmotic imbalance created by the loss of both taurine and sodium,
the
-Alanine-treated cell also underwent a reduction in cell volume
from 6.5 ± 0.2 ×103 to 5.2 ± 0.2 ×103 µm3.
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The diminished sodium and osmotic load of the taurine-deficient cell
was maintained even after 60 min of chemical hypoxia. As shown in Fig.
2, [Na+]i increased in a nearly linear
fashion during the first 30 min of chemical hypoxia. Thereafter, the
rate of sodium accumulation accelerated in the control cell, but it
remained at a depressed level in the taurine-deficient cell. This led
to a pattern in which the [Na+]i of the two
groups of cells showed a greater divergence with time. After 60 min of
chemical hypoxia, the [Na+]i of the control
myocyte was 3.5 times greater than its concentration in the
-Alanine-treated cell.
Organic osmolytes, of which taurine is the most important (1, 6,
9, 11), serve a very crucial physiological function. By exiting
the osmotically stressed cell, organic osmolytes act as safety valves
to minimize the degree of osmotic stress. Interestingly, control cells
treated with -Alanine contain a reduced intracellular osmotic load.
Although this favorable osmotic condition would be expected to benefit
the metabolically inhibited cell, it was felt that competing influences
could diminish the beneficial effect. For example, the advantage
accrued through the reduction in hypoxia-induced sodium loading could
be lost by limitations in the amount of taurine available to exit the
-Alanine-treated cell during chemical hypoxia. To determine the
importance of these competing events, taurine content was measured both
before and after 60 min of chemical hypoxia. As expected, the taurine
content of the control myocyte fell 37% after 60 min of chemical
hypoxia (Fig. 1). By comparison, the
-Alanine-treated cell
experienced no significant decline in taurine content during chemical
hypoxia. Despite the greater loss of taurine in the control myocyte
during chemical hypoxia, the dual contribution of sodium and taurine
toward the tissue's osmolality was higher in the control cell. This
confirmed that the dominant osmotic effect in both the
taurine-deficient and control myocyte was the change in
[Na+]i. As shown in Table
1, hypoxia led to only a 6 mosmol/l
elevation in the combined sodium-plus-taurine osmotic load of the
-Alanine-treated cell, whereas the combined load in the control cell
was increased 33 mosmol/l after 60 min of chemical hypoxia, with the
difference caused by the change in sodium loading.
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Generally, a reduction in the osmotic load of the hypoxic cardiomyocyte
would be expected to reduce the degree of hypoxia-induced cell
swelling. However, only a fraction of both the control and the
-Alanine-treated cells swelled during chemical hypoxia, with the
amount of swelling averaging 12% in both groups of cells (Fig. 3). Although the degree of cell swelling
was identical in those cells that showed an increase in volume, there
was a dramatic reduction in the number of
-Alanine-treated cells
that swelled during chemical hypoxia (Fig. 3).
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Cell swelling is a characteristic feature of both hyposmotic stress and
the latter stages of cell necrosis. However, the two modes of swelling
differ. Whereas swelling caused by hyposmotic stress promotes volume
expansion in both the injured and uninjured cells, only the damaged
cells will undergo necrosis-linked swelling. Thus the observation that
taurine deficiency only affects hypoxia-induced cell swelling in a
fraction of the metabolically inhibited cells appears to be more
consistent with an effect of taurine deficiency on an early reversible
step in the necrotic pathway. To test this idea, cell viability of the
taurine-deficient and control cells was examined after 60 min of
chemical hypoxia. As shown in Fig. 4,
taurine deficiency rendered the -Alanine-treated cell resistant to
hypoxia-induced necrosis. Whereas 30% of the control cells were unable
to exclude trypan blue following 60 min of chemical hypoxia,
-Alanine treatment reduced the number of nonviable cells to about
17%. Similar values were obtained by using the propidium iodide assay
of cell death.
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Besides contributing to the osmotic load of the cell, sodium excess can
also lead to calcium overload, a major cause of hypoxia-induced cell
necrosis (16, 24, 25). Because of the importance of [Ca2+]i in cardiac pathology, the effects of
chemical hypoxia and taurine depletion on
[Ca2+]i were examined. As shown in Fig.
5, [Ca2+]i of
the control cell increased 250% from 0.15 ± 0.02 to 0.53 ± 0.06 µM after 60 min of chemical hypoxia. However, the
-Alanine-treated cell was extremely resistant to a change in calcium
content during the hypoxic insult, increasing only 20% after 60 min of
chemical hypoxia (Fig. 5).
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In the ischemic heart, sodium and calcium accumulation has been
attributed to enhanced proton (H+) production. Some of the
protons that are generated leave the cell via the sodium-H+
exchanger, leading to an accumulation of sodium. When coupled with the
sodium-calcium exchanger, sodium excess also leads to calcium overload,
which can then damage the cell (7, 10). To determine
whether taurine depletion disrupts this detrimental sequence of events,
pHi was determined both before and after 60 min of chemical
hypoxia. Under normoxic conditions, the pHi of the control
and -Alanine-treated cell was 7.16 and 7.32, respectively (Fig.
6). Chemical hypoxia reduced the
pHi to 7.03 and 7.15 in the control and
-Alanine-treated
cells, respectively.
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Hypoxia also led to an increase in the number of apoptotic cells, a
process also diminished by -Alanine treatment (Fig.
7). Whereas 29% of the control cells
were TdT-mediated dUTP nick end labeling (TUNEL) positive after 1 h of hypoxia, 19% of the
-Alanine-treated cells were TUNEL
positive. One of the characteristic features of apoptotic cells is
a reduction in cell size. Among the cardiomyocytes that underwent
apoptosis, the average reduction in cell size was 16% for the
control cells and 19% for the
-Alanine-treated cells. Therefore,
-Alanine treatment did not protect the cell by reducing the degree
of cell shrinkage. Rather,
-Alanine treatment reduced from 24% to
8% the number of cells that shrank during the hypoxic insult (Fig.
8). Figure
9 reveals that
-Alanine treatment also reduced the intensity of the DNA ladder, which indicates the extent of
DNA damage that occurred during the hypoxic insult.
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DISCUSSION |
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Previous reports have shown that large amounts of taurine are lost from the ischemic myocardium (9, 11). Similarly, taurine is lost in large quantities from cardiomyocytes during chemical hypoxia (Fig. 1). It has been proposed that this loss of taurine might benefit the cell by protecting against the development of a severe osmotic imbalance (1). In the present study, we found that the contribution of sodium to the hypoxia-induced increase in cellular osmolality was 43 mosmol/l in the control cell. Taking into account the loss of taurine, the imbalance was reduced to 33 mosmol/l. A further reduction in tissue osmolality was observed in the taurine-deficient cell.
Although taurine loss clearly limits the osmotic imbalance that
develops across the cell membrane of the hypoxic cell, three lines of
evidence suggest that the acute efflux of taurine from the compromised
cell is not a major cause of cardioprotection during chemical hypoxia.
First, the -Alanine-treated and control cells differ in their
combined [Na+]i and taurine osmotic
contributions by 42 mosmol/l. About 90% of this difference can be
attributed to the lower [Na+]i in the
-Alanine-treated cell (Table 1). Second, the small amount of taurine
that effluxes the taurine-deficient cell during chemical hypoxia (<3
mosmol/l) is incapable of significantly impacting the cell's total
osmolality. Third, taurine depletion reduces the number of cells that
shrink during the hypoxic insult but not the overall degree of
shrinkage. Fourth, Fig. 3 reveals that taurine depletion reduces the
number of cells that swell during chemical hypoxia but has no effect on
the overall degree of swelling. If the taurine effect were caused
solely by its contribution to an acute change in osmolality, the
osmotic load of all of the taurine-deficient cells should have improved
and the degree of cell swelling should have been affected. Because this
did not occur, it suggests that taurine influences the necrotic and
apoptotic pathways, presumably at a reaction that precedes the
osmotic swelling step. A likely candidate for this action is the
regulation of sodium transport. By reducing the hypoxia-induced sodium
load, the taurine-deficient cell minimizes the degree of cellular
calcium loading and thus the number of cells that succumb to necrosis and apoptosis (16, 24, 25) (Fig. 5).
Although the mechanism underlying the modulation of [Na+]i by taurine has not been firmly established, three mechanisms deserve consideration. First, taurine is transported via a sodium-taurine symporter (22, 29). According to Suleiman et al. (29), as [Na+]i increases to levels exceeding 20 mM, taurine efflux is enhanced. Figure 2 shows that chemical hypoxia raises [Na+]i sufficiently to promote taurine efflux via the symporter. However, another pathway capable of promoting taurine efflux during sodium loading utilizes phospholemman (8, 14).
Cellular taurine content is also linked to [Na+]i through changes in the pHi. Figure 6 shows that the pHi of the taurine-deficient cell is elevated in relation to the control cell. This relative alkalinization of the taurine-deficient cell is also apparent during chemical hypoxia. It remains to be determined whether taurine affects pHi by modulating the activity of the sodium-H+ exchanger or by altering H+ generation.
Another factor contributing to altered sodium transport in the taurine-deficient cell is the reduction in cellular osmolality. The cell adapts to hypertonic or hypotonic stresses by activating a series of events designed to restore volume and normalize the osmotic balance across the cell. In response to hypertonic stress, a regulatory volume increase is initiated, in which several transporters are activated that promote the cellular accumulation of both sodium and taurine (2, 13, 23). By contrast, a regulatory volume decrease occurs in response to hyposmotic stress and is associated with the loss of cellular osmolytes (23). Rasmusson et al. (17) found that the combined loss of taurine and sodium from the hyposmotically stressed chick myocyte accounts for nearly half of the rapid intracellular osmotic change that accompanies a 50% decrease in the osmolality of cell medium.
Ischemia and hypoxia lead to the accumulation of several
metabolic end products that raise the cell's osmolality (18,
26). Consequently, a regulatory volume decrease is triggered,
leading to the efflux of taurine from the cell. Although the regulatory volume decrease would also be expected to trigger the efflux of sodium
from the cell, this effect is countered by the inhibition of the sodium
pump and the promotion of flux through the sodium-H+
exchanger, leading to a net increase in
[Na+]i. When taurine levels are reduced
before the chemical hypoxic insult, the degree of sodium accumulation
by the metabolically inhibited cell is attenuated (Fig. 2). This effect
is unlikely to be caused by a change in sodium-K+ATPase
activity, because the effect of taurine on ATP generation is minimal
(15). Rather, taurine deficiency presumably reduces the
influx of sodium via the sodium-H+ exchanger and promotes
the efflux of sodium from the cell during the regulatory volume
decrease. In the control myocyte, chemical hypoxia leads to a sodium
gain equivalent to 43 mosmol/l (Table 1). By contrast, the sodium gain
in the -Alanine-treated cell adds only 8.5 momol/l to tissue
osmolality. Although a reduction in sodium influx via the
sodium-H+ exchanger may account for the decrease in sodium
accumulation in the
-Alanine-treated cell, the possibility that more
sodium exits the
-Alanine-treated cell is also a viable option.
During the chemical hypoxic insult, 10.1 mosmol/l equivalents of
taurine leave the control cell, whereas only 2.5 mosmol/l equivalents of taurine leave the
-Alanine-treated cell. It is known that the taurine-deficient cell has a propensity to retain cellular taurine
(27). Therefore, the taurine-deficient cell might limit the amount of taurine that is lost at the expense of other osmolytes. Consequently, the metabolically inhibited cell might preferentially extrude sodium and retain taurine. This interpretation would be consistent with the finding that the rise in
[Na+]i in the
-Alanine-treated cell during
chemical hypoxia is less than one would have predicted. It would be
attractive to suggest that the improvement in sodium handling is a
major cause for the cardioprotection seen in the taurine-deficient cardiomyocyte.
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ACKNOWLEDGEMENTS |
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This work was supported by American Heart Association Grant 9650002N.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. W. Schaffer, Department of Pharmacology, Univ. of South Alabama School of Medicine, Mobile, AL 36688 (E-mail: swschaffer{at}aol.com).
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.
10.1152/ajpcell.00485.2001
Received 10 October 2001; accepted in final form 19 December 2001.
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