1 Department of Medicine, University of Cincinnati, Cincinnati 45267; 2 Veterans Affairs Medical Center at Cincinnati, Cincinati, Ohio 45220; 3 Department of Medicine, University of Tubingen, 72076 Tubingen, Germany; 4 Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215; 5 Department of Medical Genetics, University of Helsinki Finland, 00014 Helsinki, Finland; and 6 Department of Biosciences at Novum, Karolinska Institute, 14157 Huddinge, Sweden
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ABSTRACT |
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The basolateral
Cl/HCO
/HCO
/HCO
/HCO
SLC26A7; AE2
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INTRODUCTION |
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GASTRIC EPITHELIUM IS
COMPRISED of several structurally and physiologically distinct
cell populations that are essential to the digestive process. Parietal
cells secrete an acidic juice, whereas mucous cells (or surface
epithelial cells) secret a bicarbonate-rich fluid that is essential in
protecting the gastric mucosa from the acid injury and ulcer (7,
9, 10, 12, 13, 37, 38). Acid secretion in parietal cells occurs
via the apical gastric H+-K+-ATPase, a P-type
ATPase that is present in tubulovesicular and canalicular membranes of
the gastric parietal cells (1, 11, 18, 38). Extrusion of
acid across the apical membrane results in the generation of
intracellular hydroxyl ions (OH), which are converted to
HCO
/HCO
, the basolateral
Cl
/HCO
, thereby
maintaining HCl secretion across the apical membrane during acid
stimulation (10, 24, 26).
The anion exchanger AE2 is located on the basolateral membrane of
gastric parietal cells (6, 20, 30, 47). On the basis of
functional studies demonstrating mediation of
Cl/HCO
/HCO
/HCO
Recent studies (3, 8, 14, 15, 21-23, 25, 34, 39, 41,
46, 49) have identified a family of anion exchangers referred to
as the SLC26A family, which include at least 10 distinct genes. Three
well-known members of this family are SLC26A3 (or DRA), SLC26A4 (or
pendrin), and SLC26A6 (PAT1 or CFEX) (8, 16, 21, 22, 46).
Each of the above transporters is located apically in a limited and
distinct number of epithelia and functions as a
Cl/OH
/HCO
A recently cloned member of the SLC26A family is SLC26A7, which has
been shown to be expressed in kidneys and testes (23). In
these studies, we investigated the gastrointestinal distribution and
functional identity of SLC26A7, because little is known about this
transporter. Our results indicate that SLC26A7 expression in the
gastrointestinal tract is limited to the stomach, with predominant
expression in parietal cells. SLC26A7 functions as a
Cl/HCO
across
the basolateral membrane of gastric parietal cells.
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MATERIALS AND METHODS |
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RT-PCR of SLC26A7 in Mouse and Rabbit Tissues
A blast search of mouse expressed sequence tag (EST) database against the human SLC26A7 sequence (GenBank accession no. AF331521) identified a mouse EST (Genbank accession no. BB666404) with a high degree of sequence homology. On the basis of the cDNA sequence of the mouse EST, the following oligonucleotide primers: 5'-CTC ACC ACC GAA CCT ATT AC-3' (sense) and 5'-AAC TCG GAT AAG CCC AAC AC-3' (antisense), were designed and used for RT-PCR on RNA isolated from the mouse kidney and other tissues. A <50-bp PCR fragment was purified and sequenced that corresponded to the human nucleotides 8-550, 199.For studies in rabbit stomach, the following mouse primers were designed and used for RT-PCR on RNA isolated from gastric parietal cells or mucous cells: 5'-TTG TTC TCG TTA AAG AGC TG-3' (sense) and 5'-ATA TTA GAC AAG CCA CCT GC-3' (antisense). These primers were designed based on the full-length mouse slc26a7 cDNA (GenBank accession no. BC026928), which was deposited in the GenBank after the original studies on mouse EST had been performed in our laboratory. The above primers encode nucleotides 831-1265. To amplify rabbit SLC26A7 by RT-PCR, a gradient-based PCR machine, which allows varying stringency conditions during the amplification process, was used with mouse specific primers. The primers were used for RT-PCR in various tissues and the purified PCR fragments were used as a probe for Northern blot hybridization in mouse tissues.
For PAT1 (SLC26A6), the following primers were designed based on a full-length mouse PAT-1 cDNA (GenBank accession no. AY 032863) and used for RT-PCR on mouse tissues: 5'-CGTCTGCACTGCTCCCTCCTCCATTG-3' (sense) and 5'-GAGTCCCAGG GCATCCATCCATG-3' (antisense). These primers encode nucleotides 45-2498 of the mouse PAT1 cDNA.
RNA Isolation and Northern Blot Hybridization
Total cellular RNA was extracted from various mouse tissues, including gastrointestinal tract segments, kidney, liver, heart, brain, and lung, using Tri Reagent (4). Hybridization was performed according to Church and Gilbert (5). The membranes were washed, blotted dry, and exposed to a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA). 32P-labeled mouse PCR fragment for SLC26A7 (corresponding to nucleotides 8-550 of human SLC26A7 cDNA) was used as a probe for Northern hybridizations. For gastric H+-K+-ATPase, a ~380 PCR fragment corresponding to nucleotides 1681-2060 of the mouseImmunocytochemistry of SLC26A7 in Stomach
Antibodies.
For SLC26A7, antibodies against human or mouse sequence were used. For
mouse, a synthetic peptide corresponding to the amino acids residues
CGAKRKKRSVLWGKMHTP of mouse slc26a7 (using the mouse EST with GenBank
accession no. BB666404) was used for polyclonal antibody generation in
two rabbits. For human, antibodies were raised against a synthetic
peptide based on human SLC26A7 sequence (23).
Gastric H+-K+-ATPase -subunit antibody was a
generous gift from Dr. Forte (Univ. of California, Berkley). For AE2,
an isoform-specific antibody was used (29, 30).
Immunoblotting of SLC26A7 in stomach. Mouse microsomal membranes from the stomach were isolated according to established methods (44). Immunoblotting experiments were carried out as previously described (46). Briefly, the solubilized membrane proteins were size fractionated on 8% SDS polyacrylamide minigels (Novex, San Diego, CA) under denaturing conditions, electrophoretically transferred to nitrocellulose membranes, blocked with 5% milk proteins, and then probed with the affinity-purified anti-SLC26A7 immune serum at an IgG concentration of 0.6 µg/ml. The secondary antibody was donkey anti-rabbit IgG conjugated to horseradish peroxidase (Pierce, Rockford, IL). The sites of antigen-antibody complex formation on the nitrocellulose membranes were visualized using a chemiluminescence method (SuperSignal Substrate; Pierce) and captured on light-sensitive imaging film (Kodak).
Immunofluorescense labeling studies.
Mice were euthanized with an overdose of pentobarbital sodium and
perfused through the left ventricle with 0.9% saline followed by cold
4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4).
Stomachs were removed, cut in tissue blocks, and left in the fixative
solution overnight at 4°C. For cryosections, tissue blocks were
removed from the fixative solution and soaked in 30% sucrose
overnight. The tissue was frozen on dry ice, and 5-µm sections were
cut with a cryostat and stored at 80°C until used.
Cloning of Human and Mouse SLC26A7
Full-length human SLC26A7 cDNA was cloned from a human kidney cDNA library by PCR using the following primers: 5'-AAA TGA CAG GAG CAA AGA G-3' and 5'-TTA TTG TAG CAG AGG TCA TC-3' (GenBank accession no. AF331521). An ~2-kb PCR fragment was obtained that contained the full-length coding region of the exchanger (corresponding to nucleotides 208-2297). Full-length mouse SLC26A7 cDNA was cloned from a mouse stomach by RT-PCR using the following primers: 5'-AGA AGT TGA CTA CTA CAG GAG G-3' (sense) and 5'-AGT TGC CAA GTC ATA TCA TTC-3' (antisense). These primers encode nucleotides 68-2208 of a mouse SLC26A7 cDNA (GenBank accession no. BC026928). Amplification of the human or mouse SLC26A7 cDNA by PCR was performed according to Clontech Advantage 2 PCR kit (Clontech, Palo Alto, CA) protocol. Each PCR reaction contained 5 µl cDNA, 5 µl 10× PCR buffer, 1 µl 10 mM dNTPs, 10 pmol of each primer, and 1 µl Advantage 2 Polymerase mix in a final volume of 50 µl. Cycling parameters were 95°C, 1 min; 95°C, 30 s; and 68°C, 4 min. After PCR, the product was gel purified (revealing a single band of ~2 kb). Sequence analysis of the PCR products verified the sequences as SLC26A7. The PCR products were ligated into pGEM-T easy vector for expression studies.Synthesis of SLC26A7 cRNA
The capped SLC26A7 was generated using mMESSAGE mMACHINE Kit (from Ambion, Austin, TX) according to the manufacturer's instructions. Briefly, the plasmids containing the full-length human or mouse cDNA were linearized, and the products were then in vitro transcribed to cRNAs, as described previously (45).Expression of n SLC26A7 in Xenopus Oocytes
Xenopus oocytes were injected with the human or mouse SLC26A7 cRNA. Briefly, 50 nl cRNA (0.5-1.0 µg/µl) were injected with a Drummond 510 microdispenser via a sterile glass pipette.pHi studies. pHi in oocytes was measured with the pH-sensitive fluorescent probe 2',7'-bis-(3-carboxypropyl)-5-(6)-carboxyfluorescein acetoxymethyl ester (BCPCF-AM; Molecular Probes), a close analog of BCECF-AM (Molecular Probes) as previously described (33, 45, 46). Oocytes were loaded with 10 µM BCPCF-AM for 5 min at room temperature, transferred on a nylon mesh in a 1-ml perfusion chamber, and perfused at a rate of 3 ml/min with the following solution (in mM): 63 NaCl, 33 NaHCO3, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES. Solutions were constantly gassed with 95% O2-5% CO2 yielding the pH of 7.5 at room temperature. Fluid was delivered to the chamber by a peristaltic pump via CO2-impermeable tubing (Cole Palmer, IL). The chamber was closed by a lid and constantly superfused with the gas mixture of 5% CO2-95% O2 to prevent CO2 loss and keep the constant pH. Ratiometric fluorescence measurements were performed on a Zeiss Axiovert S-100 inverted microscope equipped with Attofluor RatioVision digital imaging system (Attofluor, Rockville, MD). Excitation wavelengths were alternated between 440 and 488 nm, and fluorescence emission intensity was recorded at 520 nm. Data analyses were performed using Attograph and Attoview software packages provided with the imaging system. The ratios were obtained from the submembrane region of the oocytes that were visualized with an achroplan ×40/0.8 water objective with 3.6 mm working distance. Measured excitation ratios were converted to pHi by using a calibration curve that was constructed with high K+/nigericin method (33, 43, 45, 46).
To examine the ClElectrophysiology.
Membrane potentials in oocytes injected with SLC26A7 cRNA were measured
in response to sequential removal and addition of Cl
using conventional microelectrode technique and as described previously
(45). Glass microelectrodes (resistance 3-5 m
)
were filled with 3 M KCl and connected to an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA). The digitized signals were stored
and analyzed on a personal computer using Axotape (Axon Instruments).
Rabbit Gastric Cell Purification
Cells were purified exactly as described before (30). Briefly, rabbit gastric cells were enzymatically dispersed and loaded into an elutriator (JM 6-C with JE-5.0 rotor; Beckman, Munich, Germany) using a 40-ml chamber and a constant rotor speed of 1,000 rpm. The cells were eluted in six fractions with increasing flow rates (of 15, 25, 50, 65, and 120 ml/min). The cells from fractions 3 and 5 were pelleted, reelutriated in a 5-ml chamber with a constant rotor velocity of 1,780 rpm and increasing flow rates (of 7, 14, 28, 35, 65, and 100 ml/min), loaded onto a Percoll density gradient, and centrifuged at 800 g for 20 min, as described. The upper band of the gradients from fraction 3 (mucous cells) and the upper band of the fraction 5 gradients (parietal cells) were collected, washed, homogenized in cold lysis buffer, and stored at 80°C. The mucous cell fraction consisted of 90% periodic acid-Schiff granule-positive cells, whereas the parietal cell fraction showed a purity of 80-90%.Materials
[32P]dCTP was purchased from New England Nuclear (Boston, MA). Nitrocellulose filters and other chemicals were purchased from Sigma (St. Louis, MO). RadPrime DNA labeling kit was purchased from GIBCO-BRL. BCECF was from Molecular Probes. mMESSAGE mMACHINE Kit was purchased from Ambion. The human multiple tissue blots were purchased from Clontech.Statistical Analyses
Values are expressed as means ± SE. The significance of differences between mean values was examined using ANOVA. P < 0.05 was considered statistically significant. ![]() |
RESULTS |
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SLC26A7 mRNA Expression
To examine the distribution of SLC26A7 mRNA in mouse tissues, RT-PCR was performed on RNA isolated from various mouse tissues using mouse specific primers (MATERIALS AND METHODS). Figure 1A is an ethidium bromide staining of an agarose gel and shows that an expected PCR fragment (~550 bp) was identified in RNA isolated from mouse stomach and kidney. Sequencing of the purified band verified its identity as SLC26A7. No PCR fragment was identified in heart, brain, or liver. Figure 1B shows the expression of PAT1 (or SLC26A6) in the same tissues as examined by RT-PCR. As indicated, PAT1 shows a wider distribution pattern and is expressed in brain, kidney, heart, liver, and stomach. To better examine SLC26A7 mRNA expression levels in mouse tissues, RNA isolated from various tissues and segments of gastrointestinal tract was hybridized against an SLC26A7 specific cDNA probe. As indicated, SLC26A7 mRNA was abundantly expressed in the stomach, with lower levels in the kidney (Fig. 1C). These results further demonstrate that SLC26A7 mRNA expression in the gastrointestinal tract is exclusively limited to the stomach and is absent in the small and large intestines (Fig. 1C). This pattern of expression is distinct from SLC26A6 (PAT1), which is abundant in small intestine (duodenum, jejunum, and ileum) and stomach but is very low in the colon (cecum, proximal, and distal colon; see Ref. 46 and Fig. 1B).
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Differential Expression of SLC26A7 mRNA in Parietal and Mucous Cells of Stomach
To determine the cellular distribution of SLC26A7 mRNA in the stomach, RT-PCR and Northern hybridizations were performed on RNA isolated from rabbit gastric parietal and mucous cells. Figure 2A is an ethidium bromide staining of an agarose gel and shows that a PCR fragment (~430 bp) is amplified from both gastric parietal and mucous cells. Sequencing verified the identity of this band as rabbit SLC26A7. The rabbit cDNA fragment shows 88% homology to the human SLC26A7. The sequence of the fragment has been deposited in the GenBank with the accession no. AY 166770.
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Figure 2B is a representative Northern hybridization and
examines the expression of SLC26A7 in gastric epithelial cells. Figure 2B, bottom shows the 28S rRNA abundance in each
lane and indicates equity in RNA loading. SLC26A7 mRNA shows more
abundance in parietal cells than mucous cells (Fig. 2B). To
determine the degree of cross contamination of mucous cells by parietal
cells, the membrane was stripped and reprobed for gastric
H+-K+-ATPase -subunit expression. Comparison
of the mRNA levels of gastric H+-K+-ATPase
(which should only be expressed in parietal cells) and SLC26A7 shows an
identical expression pattern in both cells, consistent with mild
cross-contamination of mucous cells by parietal cells. Together, these
results indicate that SLC26A7 mRNA is predominantly expressed in
parietal cells.
Immunoblotting and Immunofluorescent Labeling of SLC26A7 in Mouse Stomach
We first examined the specificity of the antibody in microsomal membranes isolated from mouse stomach. Figure 3A demonstrates the labeling of a ~94-kDa band that was blocked with the preadsorbed immune serum.
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To determine the cellular distribution and subcellular localization of
SLC26A7, immunofluorescent staining with the purified immune serum was
performed in mouse stomach. As shown in Fig. 3B (low and
high magnifications), SLC26A7 is localized on the basolateral membrane
of cells that are mostly located in the glandular portion of the
stomach. There was also intracellular staining in a number of these
cells. Preadsorbed serum did not detect any labeling (Fig.
3C). These results are consistent with the basolateral membrane and intracellular localization of SLC26A7 in certain gastric
epithelial cells. To determine the identity of SLC26A7-expressing cells, double immunocytochemical staining with gastric
H+-K+-ATPase antibody was performed. As shown
in Fig. 4, SLC26A7 and gastric
H+-K+-ATPase localized to the same cells. These
results demonstrate that SLC26A7 is located on the basolateral membrane
of gastric parietal cells.
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Immunofluorescent Labeling of AE2 in Mouse Stomach
In this series of experiments, the cell distribution and membrane localization of AE2 in mouse stomach was examined. Figure 5 is a double immunocytochemical staining with AE2 and gastric H+-K+-ATPase antibodies. As shown in both lower magnification and higher magnification in Fig. 5, AE2 and gastric H+-K+-ATPase localized to the same cells in the middle portion of glandular cells. These results demonstrate that AE2 is located on the basolateral membrane of gastric parietal cells.
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Functional Identity of SLC26A7
In these series of experiments, the functional identity of human SLC26A7 was examined using the oocyte expression system. On the basis of structural similarity with DRA and pendrin, we speculated that SLC26A7 could function in Cl
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Next, we examined the sensitivity of SLC26A7 to inhibition by DIDS.
Oocytes that were injected with SLC26A7 were examined for
Cl/HCO
removal was
examined in the presence of increasing concentrations of DIDS (50, 150, 450 µM). The summary of the results is depicted in Fig.
6C. The results indicate that DIDS inhibits the
Cl
/HCO
Activation of SLC26A7 at Acidic pHi
The results of the above experiments indicate that SLC26A7 is a Cl
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Examination of Electrogenecity of SLC26A7
To determine whether SLC26A7 is electrogenic, membrane potentials were recorded in oocytes injected with SLC26A7 cRNA using a conventional microelectrode technique (45) under conditions that favor Cl ![]() |
DISCUSSION |
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SLC26A7 displays a unique expression pattern in the
gastrointestinal tract, with abundant mRNA levels in the stomach and no detectable levels in small and large intestines (Fig. 1). Lower levels
of SLC26A7 mRNA were detected in the kidney (Fig. 1). This pattern of
expression is distinct from the expression of PAT1 (SLC26A6), which
shows a wider tissue distribution and is expressed in the small
intestine, stomach, kidney, and heart (Fig. 1). SLC26A7 expression in
the stomach is predominantly limited to the parietal cells (Fig. 2).
Immunofluorescence labeling exclusively localized SLC26A7 to the
basolateral membrane of gastric parietal cells (Figs. 3 and 4).
Expression studies in oocytes demonstrated that SLC26A7 functions in
Cl/HCO
Acid secretion by gastric parietal cells is dependent on the activity
of the basolateral Cl/HCO
across the basolateral membrane via this exchanger are
essential for maintaining the pHi and Cl
gradient within a physiological range. This, in turn, allows the
secretion of HCl to proceed across the apical membrane during acid
stimulation. Reductions in the rate of HCO
entry will perturb intracellular ionic composition
in gastric parietal cells and will result in decreased acid secretion.
In support of this mechanism, it was found that inhibition of the basolateral Cl
/HCO
Northern hybridizations and immunolabeling studies have localized the
anion exchanger AE2 on the basolateral membrane of gastric parietal
cells (20, 29, 30, 42). On the bases of functional studies
in in vitro expression systems demonstrating mediation of
Cl/HCO
/HCO
/HCO
/HCO
/HCO
/HCO
in gastric parietal cells. Interestingly, SLC26A7
shows a pHi sensitivity profile that is distinct from AE2;
whereas AE2 is only active at neutral and alkaline pHi and
remains silent at acidic pHi (17), SLC26A7 is
active at both alkaline and acidic pHi (Figs. 6 and 7).
This latter property of SLC26A7 is in agreement with functional data on
Cl
/HCO
The most salient feature of these studies is demonstration of the
unique expression pattern of SLC26A7. SLC26A7 expression in the
gastrointestinal tract is exclusively limited to the stomach and is
absent in the small and large intestines (Figs. 1-4). Furthermore, the expression of SLC26A7 in the stomach is predominantly limited to
the basolateral membrane of parietal cells (Figs. 3-4). Coupled to
functional studies (Figs. 6 and 7), our studies demonstrate that
SLC26A7 is an electroneutral basolateral
Cl/HCO
/HCO
/HCO
cDNA analysis indicates that SLC26A7 is closely related to a family of
anion transport proteins (SLC26A) that include the sulfate-anion
transporter (Sat-1 or SLC26A1), the human diastrophic dysplasia sulfate
transporter (DTDST or SLC26A2), the downregulated in adenoma (DRA or
SLC26A3), pendrin (or SLC26A4), prestin, and PAT1 (SLC26A6) (2,
3, 8, 14, 16, 21, 22, 49). Sequence comparison revealed that
SLC26A7 has ~31, 29, and 31% homology with DRA, Pendrin, and PAT1,
respectively, at the amino acid level (3, 8, 16, 22). DRA,
pendrin, and PAT1 mediate Cl/HCO
/HCO
/OH
(28),
and sulfate/OH
exchanger (2). PAT1 can
function in Cl
/HCO
/OH
exchange modes (46) as
well as in Cl
/oxalate and Cl
/formate
exchange modes (19, 21). Whether SLC26A7 can function in
other anion exchange modes is currently under investigation. A
recent study indicates that SLC26A7 can function as a
Cl
/oxalate and a Cl
/sulfate exchanger when
expressed in oocytes (23).
In conclusion, SLC26A7 shows unique expression in the stomach (and
kidney tubules), with no expression in the intestine or other tissues.
Northern hybridizations and immunofluorescent staining studies
localized SLC26A7 to the basolateral membrane of gastric parietal
cells. Functional studies demonstrated that SLC26A7 functions as a
Cl/HCO
/base exchanger specific to
gastric parietal cells. On the basis of its unique expression and
function, we propose that SLC26A7 plays a major role in acid secretion
in gastric parietal cells.
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ACKNOWLEDGEMENTS |
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These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-52820 and DK-54430, a Merit review grant, a Cystic Fibrosis Foundation grant, and grants from the Dialysis Clinic (to M. Soleimani).
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. Soleimani, Div. of Nephrology and Hypertension, Dept. of Medicine, Univ. of Cincinnati, 231 Albert Sabin Way, MSB G259, Cincinnati, OH 45267-0585 (E-mail: Manoocher.Soleimani{at}uc.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.
10.1152/ajpgi.00454.2002
Received 23 October 2002; accepted in final form 7 January 2003.
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