Department of Physiology, College of Medicine, The University of Tennessee Health Science Center, Memphis, Tennessee 38163
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ABSTRACT |
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The polyamines spermidine and spermine
and their precursor, putrescine, are required for the growth and
proliferation of eukaryotic cells. This study compares and contrasts
growth arrest caused by polyamine depletion in the untransformed IEC-6
cell line with that in the p53-mutated colon cancer Caco-2 cell line. Cells were grown in the presence or absence of
-difluoromethylornithine (DFMO), a specific inhibitor of ornithine
decarboxylase, the first rate-limiting enzyme in the synthesis of
polyamines. Depletion of polyamines inhibited the growth of both cell
lines equally and over the same time frame. However, whereas IEC-6
cells were arrested in the G1 phase of the cell cycle,
there was no accumulation of Caco-2 cells in any particular phase. In
IEC-6 cells, growth arrest was accompanied by elevated levels of p53
and p21Waf1/Cip1 (p21). There were no changes in p53 levels
in Caco-2 cells. Levels of p21 increased in Caco-2 cells on day
2 without any effect on cell cycle progression. The amount of
cyclin-dependent kinase (Cdk)2 protein was unchanged by polyamine
depletion in both cell lines. However, the activity of Cdk2 was
significantly inhibited by DFMO in IEC-6 cells. These data suggest that
in the untransformed IEC-6 cells the regulation of Cdk2 activity and
progression through the cell cycle are p53- and p21 dependent. Growth
arrest in the p53-mutated Caco-2 line after polyamine depletion occurs
by a different, yet unknown, mechanism.
ornithine decarboxylase; putrescine; spermine; p53; p21Waf1/Cip1; Cdk2; cell cycle
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INTRODUCTION |
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THE POLYAMINES SPERMIDINE and spermine and their precursor diamine putrescine are required for the growth and proliferation of eukaryotic cells (24, 32). Intracellular levels of polyamines are regulated closely by both synthesis and uptake. Under most conditions polyamine levels are dependent primarily on the activity of ornithine decarboxylase (ODC), the first rate-limiting enzyme in the polyamine synthetic pathway (30). In nonproliferating cells, ODC activity is low and rapidly increases in response to growth factors, hormones, nutrients, and other inducers of growth (24, 32). Our laboratory has examined the role of polyamines in the growth and repair of gastrointestinal epithelia by studying these processes both in rats (36, 39) and in cultured IEC-6 cells, a nontransformed line of intestinal epithelial cells derived from adult rat crypt cells (27). These studies have shown that polyamines are essential for the repair of mucosal stress ulcers of both the stomach and duodenum. Polyamines are involved in both the early phase of mucosal restitution that depends on cell migration (21) as well as the later phase dependent on cell division (30, 36).
Recently we have found that polyamine depletion arrests IEC-6
cells in the G1 phase of the cell cycle (28).
Cell cycle arrest was accompanied by increased levels of p53, p21
(p21Waf1/Cip1), and p27 (p27Kip1). A
phylogenetically conserved family of protein kinases called cyclin-dependent kinases (Cdks), which include a catalytic subunit and
a requisite positive regulatory subunit termed a cyclin, mediates progression through the cell cycle (25). Cdk activities
are regulated and coordinated to govern progression through the cell cycle. In most cases, positive regulation is mediated at the level of
cyclin accumulation (14, 22). Negative regulation of Cdk activity occurs by phosphorylation of the catalytic subunit or by
binding to Cdk inhibitory proteins (23). Increased levels of Cdk inhibitory proteins bind to cyclin-Cdk complexes and inactivate them. The prototypical Cdk inhibitory protein p21 was discovered because of its stimulation by wild-type p53 and lack of stimulation by
mutant p53 (8). Induction of p21 has been shown to be both p53 dependent and p53 independent depending on the type of stimulus (18). Polyamine depletion of IEC-6 cells by a 4-day
exposure to -difluoromethylornithine (DFMO) increased the levels of
both p21 and p27. The increase in p21 was significantly greater than the increase in p27 (28). Tian and Quaroni
(33) recently have found that p21 is also the main Cdk
inhibitory protein involved in growth arrest during the early stages of
differentiation of intestinal epithelial cells. Activation of p53 turns
on the transcription of p21, which binds to and inhibits Cdks,
resulting in the accumulation of the hypophosphorylated form of
retinoblastoma protein. This is the form of retinoblastoma protein that
binds the transcription factor E2F, which is essential for cell cycle
progression from G1 to S. Thus with the inactivation of
Cdks, E2F release is prevented and cells accumulate in G1
(8).
Polyamines are required for the growth of cancer cells as well as normal cells. A number of human colon cancer cell lines such Caco-2, DLD-1, and HT-29 are p53 mutated, yet they respond to polyamine depletion with growth arrest similar to normal intestinal cells. To better understand the process involved in cell cycle arrest in the normal IEC-6 cell line following polyamine depletion, we have examined the levels of cell cycle regulatory proteins in the Caco-2 line following treatment with DFMO. We chose this cell line because it readily differentiates (26), and a number of investigators use it as a model for intestinal epithelial cells. The primary question we asked was whether growth arrest in Caco-2 cells proceeded via the same mechanisms as in IEC-6 cells and thus whether the cell cycle arrest observed in IEC-6 cells was p53 dependent.
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MATERIALS AND METHODS |
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Materials.
Medium and other cell culture reagents were obtained from GIBCO-BRL
(Grand Island, NY). Fetal bovine serum (FBS), dialyzed fetal bovine
serum (dFBS, 1,000 molecular weight cut-off), myelin basic protein, and
propidium iodide were from Sigma (St. Louis, MO).
[-32P]ATP and an enhanced chemiluminescence Western
blot detection system were purchased from DuPont-New England Nuclear
(Boston, MA). DFMO was a gift from Merrell Dow Research Institute of
Marion Merrell Dow (Cincinnati, OH). Cdk2, Cdk4, p21, p27, and p53
antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA). The IEC-6 cell line (ATCC CRL 1592) was obtained from the American Type Culture Collection (Manassas, VA) at passage 13. The IEC-6 cell
line was derived from normal rat intestine and was developed and
characterized by Quaroni et al. (27). The cells are
nontumorigenic and retain the undifferentiated character of epithelial
stem cells. The Caco-2 cell line (ATCC HTB37) was obtained from ATCC at
passage 18. All other chemicals were of the highest purity commercially available.
Cell culture. The IEC-6 cell stock was maintained in T150 flasks in a humidified 37°C incubator in an atmosphere of 90:10 air-CO2. The medium consisted of DMEM with 5% heat-inactivated FBS and 10 µg insulin and 50 µg gentamicin sulfate/ml. The stock was passaged weekly at 1:10 and fed three times per week, and passages 15-20 were used. Caco-2 cells were maintained as stock in T150 flasks in minimum essential medium containing 10% heat-inactivated FBS and 50 µg gentamicin sulfate/ml. The stock was incubated at 37°C in 95:5 air-CO2. Stock was passaged weekly at 1:5 and fed three times per week, and passages 23-29 were used for the experiments. For the experiments, cells were taken up with 0.05% trypsin plus 0.53 mM EDTA in Hanks' balanced salt solution without calcium and magnesium and counted in a hemocytometer.
Growth studies. The cells were plated at a density of 6.25 × 104 cells/cm2 in T25 flasks in DMEM containing 5% dFBS plus 10 µg/ml insulin and 50 µg/ml gentamicin sulfate (DMEM-dFBS) with or without 5 mM DFMO or 5 mM DFMO plus 5 µM spermidine. Each experiment contained three each of untreated, DFMO-treated, and DFMO-spermidine-treated flasks. Every other day the cells in three flasks from each group were taken up with trypsin-EDTA, and the number of cells in an aliquot was determined using a Coulter counter. Each experiment was carried out in triplicate, the controls were combined, and the data were expressed as percentages of control.
Cell cycle analysis.
Cells were plated in DMEM-dFBS with or without DFMO in T25 flasks at a
density of 6 × 104 cells/cm2. They were
taken up with trypsin-EDTA on days 2 and 4 postplating. The cells were collected by centrifugation for 5 min at
100 g, washed with ice-cold PBS-1% BSA (washing buffer),
resuspended in 0.5 ml, and fixed with 1 ml 70% ethanol at 20°C,
added dropwise. The cells were washed three times in washing buffer at
4°C and resuspended in 1 ml containing 100 µg RNase (Worthington
RASE) and 5 µg propidium iodide. They were then incubated at 37°C
for 15 min in the dark, washed three times with 3 ml washing buffer, resuspended in 1 ml, and analyzed by flow cytometry.
Preparation of cell extract.
The IEC-6 and Caco-2 cells were plated (day 0) in 60-mm
dishes at a density of 6.25 × 104
cells/cm2 in DMEM containing 5% dFBS plus 10 µg/ml
insulin and 50 µg/ml gentamicin sulfate (DMEM-dFBS) with or without 5 mM DFMO and/or 10 µM putrescine. On day 4, medium was
removed, cells were washed twice with Dulbecco's phosphate-buffered
saline (DPBS), and 500 µl of cold immunoprecipitation buffer (IPB; 10 mM Tris · HCl at pH 7.4, 1% Triton X-100, 150 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl
fluoride, 10 mM NaF, 200 µM Na3VO4, 80 µg/ml leupeptin, 40 µg/ml aprotenin) was added. The cell
suspension was rotated at 4°C for 30 min, and the extract was cleared
by centrifugation at 10,000 g for 5 min. The extracts were
stored at 80°C until use. Protein concentration was determined by
the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL)
with BSA as a standard.
Immunoprecipitation. Cell lysates were matched for protein and precleared with 20 µl of protein A/G agarose for 1 h at 4°C. The precleared supernatants were further incubated overnight with 2 µg of antibody recognizing Cdk2, p53, agarose-conjugated p21, and p27. Cdk2 and p53 immunocomplexes were further incubated with protein A/G agarose. Immunocomplexes were captured by centrifugation, washed twice with IPB, and then used to measure either Cdk2 or Cdk4 activities by in vitro kinase assay or Cdk2, p21, p27, or p53 protein levels by Western blotting.
Polyamine analysis.
The cellular polyamine content was determined by the method of Tsai and
Lin (34, 38) with HPLC. The IEC-6 and Caco-2 cells were
plated (day 0) in 60-mm dishes at a density of 6.25 × 104 cells/cm2 in DMEM containing 5% dFBS plus
10 µg/ml insulin and 50 µg/ml gentamicin sulfate (DMEM-dFBS) with
or without DFMO (5 mM). On day 4, medium was removed,
monolayers were washed three times with ice-cold DPBS, 0.5 M perchloric
acid was added, and monolayers were frozen at 80°C until
extraction, dansylation, and HPLC analysis. Polyamine content was
analyzed by comparing ratios of polyamines/1,10-diaminodecane peak area
with standard curve. The level of polyamines was expressed as nanomoles
per milligram protein. Values less than 0.31 µM were considered undetectable.
Cdk activity. IEC-6 and Caco-2 cell extracts (100 µg) were incubated with 20 µg of anti-Cdk2 antibodies. Immune complexes were recovered with protein A/G-agarose beads and washed twice with IPB buffer and once with kinase assay buffer [25 mM HEPES (pH 7.4), 10 mM MgCl2, and 1 mM EGTA]. Pellets were resuspended in 40 µl of kinase buffer containing 5 µg histone H1 (Cdk2 substrate) and incubated at 30°C for 30 min. The kinase reaction was terminated by addition of SDS sample loading buffer [50 mM Tris (pH 6.8), 100 mM dithiothreitol, 2% SDS, 0.1% bromphenol blue, and 10% glycerol]. The samples were then heated to 95°C for 5 min and resolved by 10% SDS-PAGE. The gels were dried, and the phosphorylated histone H1 protein substrate was visualized by autoradiography and quantitated by densitometry.
Deoxycholate activation assay.
Cdk2 immune complexes captured as described earlier were incubated with
either 20 mM HEPES (pH 7.4) alone [ deoxycholate (DOC)] or 20 mM
HEPES containing 0.8% (+ DOC) on ice for 20 min. DOC was removed by
washing three times with buffer (IPB) and once with kinase assay
buffer. Cdk2 activity was then assayed by phosphorylation of histone H1
substrate as previously described.
Western blot analysis. Immunoprecipitated proteins were separated on 15% SDS-PAGE and transferred to Immobilon-P (Millipore) transfer membranes for Western blotting. Equal loading of protein was confirmed by staining the membrane by Ponceau S. The membranes were then washed with PBS and probed with an antibody directed against one of the proteins (Cdk2, p21, p27, or p53). The immunocomplexes were visualized by the enhanced chemiluminescence detection system and quantitated by densitometric scanning.
Statistics. Numerical data are means ± SE of three experiments. Blots shown are representative of three experiments. The significance of the differences between means was determined by ANOVA and Dunnett's post hoc testing. Values of P < 0.05 were considered significant.
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RESULTS |
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Polyamine depletion and growth arrest.
DFMO, an enzyme-activated inhibitor of ODC, is known to cause polyamine
depletion of eukaryotic cells including IEC-6 and Caco-2 cells
(21). Results in Table 1
show that 5 mM DFMO treatment depleted IEC-6 and Caco-2 cells of
putrescine and spermidine within 2 days, whereas the spermine level was
reduced to almost 40% compared with control by day 4 (Table
1). In IEC-6 cells, DFMO and subsequent polyamine depletion
arrest growth without causing apoptosis (28, 37).
Figure 1 shows that Caco-2 cells grown in
the presence of DFMO grow slowly, plateauing around day 6 at
approximately one-third of the number of cells present in control
cultures or in cultures treated with DFMO to which 5 µM spermidine
has been added. These data are essentially the same for IEC-6
cells in identical experiments (Fig. 1). Percentages of cells in
various phases of the cell cycle are relatively equal regardless of the treatment group of the Caco-2 cells. These results are depicted in
Table 2 for days 2 and
4. The relatively equal percentages for each phase of the
cycle among the groups of Caco-2 cells is readily apparent and can be
contrasted with similar data for the IEC-6 cell line. In IEC-6 cells,
unlike Caco-2 cells, growth arrest was accompanied by cell cycle arrest
at the G1 phase. This cell cycle arrest following polyamine
depletion was prevented if exogenous spermidine was added to the medium
containing DFMO. Thus virtually identical growth arrest in the two cell
lines, following polyamine depletion, is accompanied by cell cycle
arrest in G1 in the normal IEC-6 line, whereas there is no
cell G1 phase cycle arrest in the Caco-2 line.
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p53 levels.
In IEC-6 cells, polyamine depletion significantly increased the level
of p53, which was prevented by addition of 10 µM putrescine (Fig.
2). Figure 2 also shows, as expected,
that the p53 protein in Caco-2 cells was unaffected by DFMO. Although
p53 is mutated and inactive, it is still present in Caco-2 cells and is
therefore recognized by the antibody.
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Cdk2 and Cdk4 activities.
As can be seen in Fig. 3, Cdk2 activity
(histone H1 phosphorylation) changed with progression through the cell
cycle in both IEC-6 and Caco-2 cells. In both cell lines the activity
of Cdk2 decreased on day 3 as cell numbers increased. The
decreases in enzyme activities were independent of protein levels,
which remained unchanged. Cdk4 activities and protein levels were also
measured, and both were considerably less than the corresponding
measurements for Cdk2. In both cell lines there were no changes in Cdk4
activities (data not shown). Polyamine depletion decreased Cdk2
activity by 50% in IEC-6 cells (Fig. 4).
This decrease was prevented by addition of 10 µM putrescine. There
was little effect of polyamine depletion on Cdk2 (histone H1
phosphorylation) activity in Caco-2 cells.
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Levels of Cdk inhibitory proteins.
Western blots of p21 from IEC-6 cells showed significant increases in
protein levels at both 2 and 4 days after plating cells in the
presence of DFMO compared with control (Fig.
6). In Caco-2 cells p21 increased after 2 days and incubation in the presence of DFMO but remained at control
levels after 4 days. There was little effect of polyamine depletion on
p27 levels in either cell line, although there was a tendency for an
increase in IEC-6 cells (Fig. 7).
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DISCUSSION |
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Polyamines have an important role in cell proliferation, cell migration, and apoptosis and consequently the integrity of the intestinal epithelium. To delineate the mechanism of action of polyamines, we depleted cells of polyamines to study the interaction of polyamines with cellular regulatory proteins involved in growth-related processes in IEC-6 and Caco-2 cells. Previous work from our laboratory has shown that growth arrest in IEC-6 cells in response to polyamine depletion was caused by cell cycle arrest in the G1 phase and was accompanied on day 4 by increases in p53 and p21 (28). In the current study, we tested the hypothesis that cell cycle arrest in IEC-6 cells was mediated by p53 by comparing growth arrest in IEC-6 cells with that in the p53-mutated Caco-2 cell line. We also examined the role of Cdk2 in the growth arrest caused by polyamine depletion in both cell lines.
The current study showed that the result of growth arrest in response to polyamine depletion in Caco-2 cells was virtually the same as that in IEC-6 cells. However, we found no evidence of a block at any point in the cell cycle for Caco-2 cells. Significant increases in p21 levels occurred on days 2 and 4 of DFMO treatment in IEC-6 cells, but an increase in p21 in Caco-2 cells was observed on day 2 only. Cdk2 activity was strongly inhibited by 50% on day 2 of polyamine depletion in IEC-6 cells, and the use of DOC to remove inhibitory proteins resulted in the recovery of activity. Cdk2 activity in Caco-2 cells was increased on day 2 in the presence of DOC.
The p53 protein has been referred to as "the cellular gatekeeper for
growth and division" (16). Because of its relatively short half-life, around 20 min, p53 is normally present in cells at
relatively low concentrations. The pathways leading to increased levels
of p53 are not known, but these signals respond to several varieties of
stress. p53 is activated rapidly in response to DNA damage of several
different types. Damage induced by -radiation rapidly increases the
level of p53 and activates it as a transcription factor
(16). Linke et al. (17) have shown that the
depletion of ribonucleotides, compounds necessary for DNA synthesis and repair, also activates p53. Polyamines also are involved in the structure of DNA and are associated with specific base sequences of
nucleic acids (6, 18). Polyamines cause the condensation of DNA (10) and induce a conformational shift from the
right-handed B form to the left-handed Z form (2). The
binding of spermine to the major groove of B-DNA induces a bend in the
helical axis (1). Other studies have shown that the B-Z
transition and the induction of a bend in the helical axis may be
important for chromatin condensation (31) and gene
expression (29). Thus polyamine depletion may induce p53
through pathways similar to those elicited by the absence of other
compounds essential to DNA structure. Hypoxia is a third type of
stimulus that increases p53 levels and activates the protein
(11). Thus, regardless of the pathways that result in p53
activation, it is likely that the cell interprets polyamine depletion
as a stress signal.
In response to stress, cells arrest either at the transition from
G1 to S phase or at the transition from G2 to M
phase of the cell cycle (4). Whether arrest occurs at
G1 or at G2 depends on the growth conditions,
the type of cell, and the type of damage or stress. Arrest in
G1-S has been shown to be due to p53-regulated synthesis of
p21 (3, 35) and the inhibition of the cyclin-Cdk complexes
required for progression through the cell cycle. Bunz et al.
(4) compared the cell cycle response to -radiation in
colon cancer cells with intact p53 to those with a mutated and inactive
p53. They found that the cells with a normal p53 arrested in the
G2 phase of the cell cycle and that this arrest was
sustained only when p53 was present and able to transcriptionally activate p21. Our own findings are similar in that polyamine depletion resulted in cell cycle arrest at the G1 phase in IEC-6
cells with a normal p53, but not in p53-mutated Caco-2 cells.
Activation of p53 in IEC-6 cells was accompanied by activation of p21
and inhibition of Cdk2, which is the Cdk involved in progression
through the cell cycle at the G1-S boundary (7,
13). These results indicate that cell cycle arrest in response
to polyamine depletion is p53 dependent.
Other investigators have examined the effects of polyamine depletion on the cell cycle. Koza and Herbst (15) found that HeLa cells depleted of polyamines were arrested throughout the G1 phase. In Chinese hamster ovary (CHO) cells depleted of polyamines, the cell cycle was lengthened in both the G1 and S phases, with the greater block occurring in G1 (12). Recently, Fredlund and Oredsson (9) found that DFMO increased the length of the S phase within one cell cycle after seeding CHO cells in the presence of the inhibitor. After 2 days, DFMO produced lengthening of the G1 phase. Thus our finding that polyamine depletion in IEC-6 cells results in arrest at the G1-S checkpoint agrees in general with these other reports.
Ding et al. (5) examined the cellular mechanisms regulating differentiation in Caco-2 cells. Interestingly, differentiation was preceded by inhibition of Cdk2 and Cdk4 activity, which led to cell cycle arrest in G1. They also treated Cdk immune complexes with DOC and found that it restored Cdk2 activity, which was in agreement with an increased binding of p21 to Cdk2 that they observed on the third day postconfluence. In IEC-6 cells we found that DOC treatment of Cdk immune complexes from cells incubated for 2 days in DFMO restored Cdk2 activity to normal levels, indicating that inhibition was caused by the binding of an inhibitory protein. Our data also implicated p21. Thus the p21 inhibition of Cdk2, which mediates G1 arrest in polyamine-depleted IEC-6 cells, is also active in Caco-2 cells, where it plays a role in the G1 arrest occurring during differentiation. Obviously, during differentiation of Caco-2 cells p21 is activated by mechanisms independent of p53.
Our previous paper was the first, to our knowledge, to detect and describe signaling events following polyamine depletion in any cell type (28). In IEC-6 cells we showed that DFMO increased p53, p21, and, to a lesser extent, p27 after 4 days. In the current study we have shown that the inhibition of Cdk2 activity is involved in the G1 arrest observed in IEC-6 cells. Furthermore, studies with DOC indicate that the inhibition is due to the binding of protein, which we believe is due, at least in part, to p21. The experiments described here in the p53-mutated Caco-2 cell line strongly suggest that in normal cells p53 mediates the G1 arrest in response to polyamine depletion. In Caco-2 cells there was no p53 activation. The increase in p21 levels on day 4 appears to be independent of p53 and may be associated with Caco-2 differentiation. The activation of Cdk2 by DOC on day 2 in control and DFMO-treated Caco-2 cells (Fig. 5) and the low levels of p21 in the same cultures (Fig. 6) suggest that p21 does not regulate Cdk2 and that other Cdk2 inhibitory proteins might be involved in Caco-2 cell proliferation.
Perhaps the most significant aspect of these results is that the absence of polyamines in normal cells actually sets in motion a well-defined signal transduction pathway that results in the cell cycle arrest and the inhibition of growth that may be a type of stress response. The point, however, is that cells actively respond to the depletion of polyamines, and the arrest of growth is not due simply to the failure of events to occur in the absence of a required compound.
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ACKNOWLEDGEMENTS |
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This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-16505 to L. R. Johnson.
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FOOTNOTES |
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Address for reprint requests and other correspondence: L. R. Johnson, Dept. of Physiology, College of Medicine, The Univ. of Tennessee Health Science Center, Memphis, Tennessee 38163 (E-mail: ljohn{at}physio1.utmem.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 28 September 2000; accepted in final form 16 February 2001.
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