Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5120
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ABSTRACT |
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Many membrane transport systems are altered by changes in the state of the actin cytoskeleton. Although an intact microtubule network is required for hypertonic activation of the betaine transporter (BGT1), the possible role of the actin cytoskeleton is unknown. BGT1 function in Madin-Darby canine kidney cell monolayers was assessed as Na+-dependent uptake of GABA, following disassembly of F-actin by cytochalasin D (1.0 µM) or latrunculin A (0.6 µM). Both drugs significantly increased (P < 0.001) the activation of BGT1 transport by 24-h hypertonicity (500 mosmol/kgH2O). In contrast, the hypertonic upregulation of Na+-dependent alanine uptake remained unaltered by cytochalasin D. Disruption of F-actin did not interfere with downregulation of BGT1 transport when cells were transferred from hypertonic to isotonic medium. Immunofluorescence staining revealed colocalization of BGT1 and F-actin at the plasma membrane of hypertonic cells. Surface biotinylation revealed no major change in BGT1 protein abundance after cytochalasin D action, suggesting that stimulation of hypertonic activation of BGT1 transport is due to increased activity of existing BGT1 transporters.
cytochalasin D; latrunculin A; phalloidin; osmotic stress; alanine; GABA
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INTRODUCTION |
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WHEN EXPOSED TO HYPERTONIC stress, cells regulate their volume initially by ion accumulation. During prolonged hypertonicity, this step is followed by amino acid accumulation and eventually by accumulation of organic solutes or osmolytes. The intracellular accumulation of osmolytes does not disrupt metabolic pathways or interrupt protein synthesis, in contrast to the accumulation of ions and amino acids. Betaine is an osmolyte that enters cells via the betaine transporter (BGT1) that also transports GABA (21). BGT1 is found in many mammalian tissues, including the brain (5), and is abundant in cells of the inner renal medulla where it is located in the basolateral membrane (27, 36). Renal medullary cells are routinely exposed to the high hypertonicity that is an important component of the urinary concentrating mechanism. The activity of BGT1 is upregulated in response to prolonged (24 h) hypertonic stress (26, 36), and this is an important part of the overall adaptation that allows the medullary cells to balance the osmotic stress across the plasma membrane (6).
The activities of many membrane transporters have been shown to be affected by changes in the state of the actin cytoskeleton (16), suggesting a possible regulatory role for actin (24). For example, the open probability of the voltage-dependent Na+ channel in rabbit cardiac myocytes is decreased when the actin cytoskeleton was disrupted by cytochalasin D (33), and in rat hippocampal neurons the Ca2+ influx by the Ca2+ transporter was decreased by cytochalasin D (13). In contrast, the conductance of the cystic fibrosis transmembrane regulator in 3T3 fibroblasts (11), insertion of aquaporins into epithelial cell apical membranes (12), and the open probability of the epithelial Na+ channel (4) were increased by cytochalasin D. Previous studies in this laboratory showed that microtubule disruption by nocodazole or colchicine prevented the typical hypertonic upregulation of BGT1 transport in Madin-Darby canine kidney (MDCK) cells (3). Microtubules are an important component of the trafficking mechanism that delivers newly synthesized BGT1 transporters from intracellular compartments to the plasma membrane. The present study extends these observations by focusing on the role of the actin cytoskeleton in hypertonic upregulation of BGT1 in MDCK cells.
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METHODS |
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Cell culture and transport measurement. MDCK cells (CCL-34, American Type Culture Collection, Rockville, MD) were used between passages 10 and 30 and were grown as monolayers in a 1:1 mixture of DMEM:Ham's F-12K containing 10% bovine calf serum, 10 mM HEPES, 25 mM NaHCO3 (pH 7.4), and penicillin G (100 U/ml), as in previous studies (3, 18). Cultures were maintained at 37°C in an atmosphere of 5% CO2 in air. Cells were grown on glass coverslips for immunofluorescence studies and in six-well plates for transport measurements.
Hypertonic stress was induced by replacing normal growth medium with growth medium made hypertonic by addition of sucrose to achieve a final osmolality of 500 mosmol/kgH2O, as in previous studies (3). The osmolality of all solutions used for transport was matched to the osmolality of the growth medium by addition of sucrose where necessary. The transport function of endogenous BGT1 in cell monolayers was determined by measuring cell uptake of [3H]GABA, as described previously in detail (3, 18). Briefly, [3H]GABA uptake was determined both in medium containing Na+ and also in medium in which Na+ was replaced by methyl-D-glucamine-HCl. The difference, which is referred to as the Na+-dependent component, represents transport specifically via BGT1. Drugs were used from stock solutions that were diluted at least 1:1,000 after addition to cell culture medium. Cytochalasin D (Sigma Aldrich, St. Louis, MO) and latrunculin A (Calbiochem, San Diego, CA) were dissolved in dimethylsulfoxide and were added to the growth medium when the cells were switched from isotonic to hypertonic conditions. Phalloidin (Sigma Aldrich) was dissolved in methanol, and treatment of cells was always begun in isotonic medium for 24 h before a switch to hypertonic medium containing phalloidin. This allows adequate time for phalloidin to accumulate within the cells (22, 23) before onset of hypertonic stress.Visualization of actin in cultured cells. The actin cytoskeleton was visualized by staining cells with Texas red-phalloidin (Molecular Probes, Eugene, OR), as described previously (28). Cell monolayers on glass coverslips were rinsed in PBS of appropriate osmolality and were fixed for 15 min in 4% paraformaldehyde in PBS. The cells were rinsed three times, permeabilized by immersion for 2 min in 0.2% Triton X-100 in PBS, and washed three times by 5-min immersion in PBS. After incubation for 30 min at 37°C in Texas red-phalloidin, diluted 1:400 in PBS, the cells were rinsed three times by immersion in PBS and mounted in fluoromount-G (Southern Biotechnology, Birmingham, AL). Images were recorded on CD-RW using an RT Color Spotdigital camera (Diagnostic Instruments, Sterling Heights, MI) mounted on an Optiphot-2 Nikon epifluorescent microscope with an oil-immersion ×60 lens. The images were imported into Adobe Photoshop 5.0 (Adobe Systems, San Jose, CA).
Colocalization of actin and BGT1 proteins. Cell monolayers were rinsed in PBS as above, and some were extracted with a cytoskeleton-stabilizing buffer containing 1% Triton X-100, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl2, 1 mM EGTA, and protease inhibitors (Halt kit, Pierce, Rockford, IL) for 15 min on ice (34). All samples were fixed by immersion for 10 min in cold methanol, as described previously (3). A 30-min preincubation in 2% gelatin in PBS was followed by a 2-h incubation in affinity-purified anti-dog BGT1 rabbit antibody (3) (1:100 dilution) and an anti-actin mouse monoclonal antibody (clone AC-40, Sigma) (1:200) diluted in 1% gelatin/PBS. Primary antibodies were detected by a 1-h incubation in affinity-purified, FITC-conjugated goat anti-rabbit IgG (1:100) and TRITC-conjugated goat anti-mouse IgG (1:50) (Jackson ImmunoResearch, West Grove, PA) in 1% gelatin/PBS. All incubations were at 37°C. Cells were mounted in fluoromount-G, and images were acquired with a Zeiss LSM 510 confocal microscope using a ×40 water-immersion lens with 1.2 numerical aperture and 22-µm working distance. Excitation wavelengths were 488 (FITC) and 543 nm (TRITC), and emission was collected at 500-530 nm (FITC) and 565-615 nm (TRITC). The instrument gain and offset were the same for all samples.
Surface biotinylation.
The procedure was similar to that described previously for MDCK
(27) and opossum kidney (37) cells. Cell
monolayers in 100-mm dishes were used 3 days after plating, just before
confluence, to allow access of reagents to the basolateral membrane
where BGT1 is primarily located (27). The cells were
washed four times in ice-cold PBS containing 0.1 mM CaCl2
and 1.0 mM MgCl2 (PBSCM), pH 7.4, and incubated with PBSCM
(2 ml/dish) containing biotin-X-NHS (Calbiochem) at a final
concentration of 1.0 mg/ml for 30 min at 4°C with constant rocking.
After aspiration of the biotin solution, the cells were washed five
times in ice-cold PBSCM. When appropriate, all the preceding steps were
performed with hypertonic solutions (500 mosmol/kgH2O). The
cells were lysed with 1 ml/dish of 1% Triton X-100 solution containing
150 mM NaCl, 5 mM EDTA, 10% glycerol, 50 mM Tris, pH 7.4, and protease
inhibitors (Halt cocktail, Pierce). Cells were collected by scraping,
sonicated briefly (5 s) to disrupt DNA, incubated for 30 min at 4°C
with rotation, and centrifuged at 13,000 g for 15 min. The
supernatant was adjusted to a protein concentration of 1 mg/ml, and
biotinylated proteins were precipitated by addition of 100 µl of a
slurry of streptavidin-coupled agarose (Pierce) followed by incubation
with rotation at room temperature for 1 h. To prevent nonspecific
binding, the agarose beads were previously incubated with PBSCM
containing 2% bovine serum albumin, followed by four washes
with PBSCM. The Triton supernatant was centrifuged to pellet the
beads, and the supernatant was stored at 80°C. The beads were
washed four times in the Triton lysis buffer, mixed with an equal
volume of 2× electrophoresis sample buffer (3), heated at
65°C for 15 min, and subjected to gel electrophoresis and Western
blotting, as described previously (3). Rabbit antibodies
to dog BGT1 and mouse E-cadherin, generously provided by Drs. Moo Kwon
(Johns Hopkins University School of Medicine) and James Marrs (Indiana
University School of Medicine), were used at dilutions of 1:1,500 and
1:10,000, respectively. Mouse monoclonal antibody to actin (Sigma) was
used at 1:1,000. Secondary antibodies conjugated to horseradish
peroxidase and ECL reagents (Amersham) were used for detection. Short
exposures of the Western blots that were not saturating were used for
quantitation. Films were scanned with a Bio-Rad GS-670 imaging
densitometer (3).
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RESULTS |
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In MDCK cells maintained in isotonic medium, there was no
significant Na+-dependent uptake of GABA, indicating the
absence of BGT1 transport activity. However, as expected, after 24-h
treatment of MDCK cells with hypertonic medium (500 mosmol/kgH2O), there was activation of BGT1 transport, as
indicated by pronounced Na+-dependent uptake of GABA (Fig.
1). When the 24-h hypertonic stress was
performed in the presence of 1.0 µM cytochalasin D, there was
significant stimulation (109 ± 45%) of the hypertonic activation of BGT1. In contrast, cytochalasin D did not change GABA uptake in
cells in isotonic medium. The final concentration of cytochalasin D
(1.0 µM) was in the range shown previously to be effective for disrupting the actin cytoskeleton in epithelial and other cell types
(7, 23, 31, 35).
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MDCK cells grown on glass filters were stained with Texas
red-phalloidin to visualize the actin cytoskeleton after exposure to
hypertonic medium in the presence and absence of cytochalasin D
for 24 h. Filamentous actin in controls (Fig.
2A) was completely disrupted in cells treated with cytochalasin D (Fig.
2B). Most of the actin was clumped together around the cell
periphery, as shown previously (28). Additional studies
(not shown) revealed that cytoskeletal disruption was established
within 1 h after addition of the drug, as reported previously in
muscle cells (31).
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Latrunculin A also disrupts the actin cytoskeleton, but the mechanism
is different from that for cytochalasin D. Latrunculin A sequesters
G-actin to inhibit actin polymerization, whereas cytochalasin D changes
the polymerization and depolymerization rates of F-actin (22,
25). A final concentration of 0.6 µM latrunculin A was used
because an imaging study of the dose dependence showed that 0.6 µM
disrupted the actin cytoskeleton without causing rounding up and loss
of cells from the culture dish. This is also within the concentration
range used previously (17). Exposure of MDCK cells to 0.6 µM latrunculin A for 24 h produced a similar response in BGT1
transport compared with cytochalasin D. Activation of BGT1 transport by
hypertonic stress was more pronounced in the presence of latrunculin A,
an increase of 52 ± 8% compared with hypertonicity alone (Fig.
3). Latrunculin A had no effect on GABA
uptake in cells maintained in isotonic medium.
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In contrast to latrunculin A and cytochalasin D, phalloidin stabilizes
the actin cytoskeleton (22, 23). A dose-dependent study
confirmed that 4 µM phalloidin was an effective concentration in
hypertonic cells (23). This concentration was used to
study the effect of actin stabilization on BGT1 transport in MDCK
cells. Activation of BGT1 transport in cells exposed to hypertonic
medium containing 4 µM phalloidin for 24 h was not different
compared with activation by hypertonic medium alone (Fig.
4).
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Because cytochalasin B was shown previously to inhibit glucose
transporters (29), it was important to verify that the
stimulation of BGT1 transport by cytochalasin D and latrunculin A was
due primarily to actin destabilization. The possible direct action of
these drugs on BGT1 transport was tested by including them in the GABA
uptake medium instead of the cell growth medium. The hypertonic
activation of BGT1 transport remained unchanged during this relatively
brief 10-min exposure to either drug (Fig.
5), indicating that neither cytochalasin
D nor latrunculin A had any direct effect on the BGT1 transporter.
Phalloidin also had no effect on BGT1 transport when tested in this way
(Fig. 5).
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To determine whether an intact cytoskeleton is a specific requirement
for hypertonic regulation of the BGT1 transporter, alanine uptake in
MDCK cells was studied. We showed previously that amino acid system A,
a major route for alanine uptake in MDCK cells, is upregulated within
5 h after exposure to hypertonic stress (9). Cells
were treated with hypertonic cell growth medium containing 1.0 µM
cytochalasin D for 5 h before determining alanine uptake. Compared
with hypertonic medium alone, the presence of cytochalasin D had no
significant effect on the upregulation of Na+-dependent
alanine uptake (Fig. 6). Similarly a 24-h
treatment with cytochalasin D, corresponding to the time course of BGT1 upregulation, also did not change Na+-dependent alanine
transport (not shown). This indicates that the stimulatory effect of
actin disassembly is selective for the hypertonic upregulation of the
BGT1 transporter.
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The action of cytochalasin D to augment hypertonic activation of BGT1
transport could be due to increased insertion of new proteins or to
decreased retrieval of existing proteins from the plasma membrane. The
latter possibility was tested indirectly by monitoring downregulation
of BGT1 transport after recovery in isotonic medium. MDCK cells were
hypertonically stressed for 24 h, returned to isotonic medium, and
the downregulation of Na+-dependent GABA uptake was
determined after recovery for 24 and 48 h in the presence or
absence of 1.0 µM cytochalasin D. Compared with the GABA uptake in
cells switched to isotonic medium in the absence of cytochalasin D, the
presence of cytochalasin D in the isotonic medium for either 24 or
48 h had no effect on downregulation of BGT1 transport (Fig.
7). This strongly suggests that the
stimulatory action of cytochalasin D on the hypertonic activation of
BGT1 transport is not due to blocking retrieval of BGT1 transporters from the membrane.
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The next experiments focused on the upregulation of BGT1
transport and used surface biotinylation to determine whether
cytochalasin D increased the abundance of BGT1 protein in the plasma
membrane. As expected, no BGT1 protein was detected at the cell surface under isotonic conditions, but there was a marked increase in surface
BGT1 after hypertonic stress (Fig. 8).
Cytochalasin D produced no major change in surface BGT1 abundance under
either isotonic or hypertonic conditions. Blots were stripped and
reprobed with antibody to E-cadherin, a loading control. E-cadherin was biotinylated under all conditions and its abundance did not change (Fig. 8). In each sample lane, the densities of the BGT1 and E-cadherin signals were quantitated by densitometric scans. On the basis of six
separate experiments, the BGT1/cadherin ratio was 3.9 ± 0.6 (means ± SE) for hypertonic cells and 4.1 ± 1.1 for
hypertonic cells treated with cytochalasin D (P > 0.05, paired t-test). These findings suggest that there was
no significant increase in surface abundance of BGT1 protein following
cytochalasin D treatment.
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The possibility that biotin may have access to an intracellular pool of
BGT1 in hypertonic or drug-treated cells was tested by probing for
actin, an intracellular protein (37). Although actin was
very abundant in the initial Triton lysate (before streptavidin treatment), almost no actin was detectable in the protein fractions recovered on streptavidin beads (Fig. 9).
This indicates that biotinylation of intracellular proteins was
negligible and was not affected by cytochalasin D. Thus data in Fig. 8
are not complicated by labeling of intracellular BGT1, which might mask
a change in surface labeling.
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The possibility of a physical interaction between F-actin and BGT1 in
MDCK cells was tested by colocalization using dual antibody staining.
Under isotonic conditions, there was little evidence of stress fibers
in confluent cells, most of the F-actin stain was cytosolic (Fig.
10A). The same was true for
BGT1 staining (Fig. 10B), and this was confirmed by the
merged image (Fig. 10C). Triton extraction removed most of
the cytosolic staining, only cortical F-actin remained (Fig. 10,
D and E), and there was little or no overlap
(Fig. 10F). After 24 h of hypertonic stress, most of
the F-actin was distributed around the cell periphery in the cortical pool (Fig. 10G). As expected, most of the BGT1 stain was in
the plasma membrane (Fig. 10H) where there was pronounced
colocalization with cortical actin (Fig. 10I). Triton
extraction of hypertonic cells removed all the BGT1 without changing
the distribution of F-actin, and no overlapping signals were detected
(Fig. 10, J-L). This suggests that any direct
physical interactions between F-actin and BGT1 are weak because they
are disrupted by 1% Triton. A similar conclusion is indicated by
experiments with cytochalasin D. Inclusion of cytochalasin D during
hypertonic stress disrupted the cortical F-actin and the cell
boundaries were much less distinct (Fig. 10M). In contrast,
there was no noticeable change in BGT1 distribution (Fig.
10N) resulting in a marked decrease in colocalization (Fig. 10O). As expected, the combination of cytochalasin D and
Triton treatment removed more F-actin than either treatment alone (Fig. 10P) and all of the BGT1 (Fig. 10Q).
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Antibody staining of BGT1 in plasma membranes was heterogeneous between different cells in the same field of view (Fig. 10H) making quantitation difficult. Overall, there appeared to be no clear-cut differences in the intensity of the BGT1 fluorescence in the plasma membranes shown in Fig. 10H (hypertonic) and Fig. 10N (hypertonic + cytochalasin D). This is at least consistent with the biotinylation data that provide a much better quantitative comparison of surface BGT1 (Fig. 8).
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DISCUSSION |
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Cytochalasin D and latrunculin A disrupt the actin cytoskeleton through different mechanisms (22, 25). However, treatment of MDCK cells with either drug during hypertonic stress for 24 h produced the same response, the stimulation of BGT1 transport activity. The low level of BGT1 transport under isotonic conditions remained unchanged following drug treatment. The action of cytochalasin D on the cytoskeleton was established within 1 h and was maintained for up to 24 h. When included in the uptake medium, none of the tested drugs had an acute effect on BGT1 transport under hypertonic conditions, which rules out a possible direct action on the transporter. The response to cytochalasin D appeared to be selective for BGT1 because cytochalasin D did not stimulate the upregulation of Na+-dependent alanine uptake after 5 h. Alanine enters MDCK cells principally via system A (ATA2), which is also upregulated by 5-6 h of hypertonic stress in both epithelial (9) and endothelial cells (1, 2). The unchanged alanine transport following cytochalasin D treatment also provides indirect evidence that the drug does not interfere with maintenance of the transmembrane Na+ gradient. In summary, the stimulation of BGT1 transport by cytochalasin D and latrunculin A is most likely due to disruption of the cytoskeleton.
Several studies suggest that the organization of the actin cytoskeleton is important for cell volume regulation and is altered by changes in cell size (15). For example, the cytoskeleton of Chinese hamster ovary cells showed marked reorganization within 10 min after increasing medium osmolality to 500 mosmol/kgH2O (30). Because immunochemical staining revealed the presence of intracellular BGT1 proteins under isotonic conditions (3), the question arose that some of these preexisting proteins might gain access to the plasma membrane during the initial exposure to hypertonicity and while the cytoskeleton was being reorganized. This initial response to hypertonicity may be independent of the presence of depolymerizing drugs. This possibility was investigated by using the F-actin-stabilizing drug phalloidin. The presence of phalloidin did not inhibit the normal increase in BGT1 transport activity in response to 24-h hypertonic stress (Fig. 4), suggesting that the increase in BGT1 transport due to 24-h hypertonicity is unlikely to be caused simply by the initial disruption of the actin cytoskeleton. Induction of BGT1 transport during hypertonicity occurs principally through activation of BGT1 gene transcription by the transcription factor tonicity-responsive enhancer binding protein, as reported previously (14, 32).
The mechanism by which the cytoskeleton is involved in upregulation of BGT1 transport has not been established. It does not appear to be required for downregulation of BGT1 transport when cells were returned to isotonic medium after 24 h of hypertonicity (Fig. 7). There are at least two possible mechanisms to consider. First, the actin cytoskeleton may act as a barrier or fence that restricts the access of BGT1 proteins to the plasma membrane, as suggested for aquaporin-2 (12). Disruption of the actin cytoskeleton causes breaks in the fence, which may allow more BGT1 proteins to be inserted into the plasma membrane during upregulation by hypertonic stress. This mechanism seems very unlikely because it would lead to an increased abundance of BGT1 in the plasma membrane. The biotinylation studies revealed no major change in surface expression of BGT1 protein following exposure to cytochalasin D. Furthermore, the upregulation of Na+-alanine cotransport was not stimulated by cytochalasin D, and it is difficult to understand why removal of a potential barrier would only affect upregulation of BGT1.
The second, and more likely, explanation is that the actin cytoskeleton may serve to inhibit BGT1 transport activity through direct interactions with the transporter protein. In MDCK cells, for example, the Na+-K+-ATPase in the basolateral membrane is linked to actin through ankyrin and spectrin (10, 19). The apical Na+/H+ exchanger isoform NHE3 also interacts with the actin cytoskeleton (20). Disruption of the actin cytoskeleton by cytochalasin D may allow BGT1 proteins already in the plasma membrane to function at an increased rate. Although BGT1 protein colocalized with cortical F-actin in MDCK cells after hypertonic stress, the apparent association was completely disrupted by treatment with either cytochalasin D or Triton X-100 (Fig. 10). This suggests that any direct interactions are weak (8, 38) or that only a small fraction of BGT1 interacts with F-actin that cannot be detected by immunofluorescence. Actin and actin-binding proteins may also regulate BGT1 by changing membrane fluidity or by influencing signaling pathways (19). Additional work will be required to identify the specific mechanism of cytochalasin D action on BGT1 upregulation and to determine if actin-BGT1 interactions have a significant role in normal adaptation to hypertonic stress.
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ACKNOWLEDGEMENTS |
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We are indebted to Dr. F. Pavalko and Dr. S. Norvell for continued advice and assistance.
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FOOTNOTES |
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This work was presented in abstract form at the American Society of Nephrology, 35th Annual Meeting, Philadelphia, PA, 2002. This work was also supported in part by a grant from the National Kidney Foundation of Indiana and a Grant-in-Aid for Research from Indiana University-Purdue University at Indianapolis (S. A. Kempson) and by the American Heart Association and the Showalter Foundation (S. Chu).
Address for reprint requests and other correspondence: S. A. Kempson, Dept. of Cellular and Integrative Physiology, Indiana Univ. School of Medicine, Indianapolis, Indiana 46202-5120 (E-mail: skempson{at}iupui.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.
First published January 14, 2003;10.1152/ajprenal.00289.2002
Received 12 August 2002; accepted in final form 6 January 2003.
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