Departments of 1 Medicine and 2 Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, Ohio 45267-0585; 3 Department of Molecular and Cell Biology, University of California, Berkley, California 94720; 4 University of Tubingen, 72076 Tubingen, Germany; and 5 Veterans Affairs Medical Center at Cincinnati, Cincinnati, Ohio 45220
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ABSTRACT |
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The
apical Cl/HCO
/HCO
/HCO
Cl/HCO
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INTRODUCTION |
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THE GASTRIC EPITHELIUM
COMPRISES several structurally and physiologically distinct cell
populations that are essential for the digestive process and for
mucosal protection. Secretion of acid into the lumen of the stomach is
mediated by the apical H-K-ATPase of the gastric parietal cell
(1, 27, 28). Acid is generated within the parietal
cell by the conversion of H2O and CO2 to
H+ and HCO, which
is required for sustained secretion of HCl across the apical membrane
(7, 20). The available evidence suggests that basolateral
Cl
/HCO
Recent studies have identified a family of anion exchangers, referred
to as the SLC26A family, that includes at least nine distinct genes
(2, 5, 9, 10, 14, 16, 17, 19, 22-25, 29, 31, 33, 36,
38). Three well-known members of this family are SLC26A3
[downregulated in adenoma (DRA) or congenital Cl
diarrhea], SLC26A4 (pendrin), and SLC26A6 [putative anion transporter (PAT1) or Cl
/formate exchanger] (5, 10,
16, 24). Each of these transporters is located apically in a
limited number of epithelial tissues and can function as a
Cl
/HCO
) exchanger
(19, 33, 36). In addition, PAT1 has been shown to mediate
Cl
/formate exchange (14), and pendrin is
thought to mediate Cl
/I
exchange
(25). DRA is expressed predominately in the colon, with
lower levels in the small intestine (19), PAT1 is
expressed at high levels in the small intestine (36) and
renal proximal tubule (14), and pendrin is expressed in
thyroid follicular cells (22) and in kidney collecting
ducts (23, 31).
In addition to the kidney and small intestine, PAT1 is also expressed
in the stomach (36). In the current studies, we
investigated the cell distribution and membrane localization of PAT1 in
the stomach. Given its apical location in other tissues, we anticipated that it might contribute to the HCO/HCO
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EXPERIMENTAL PROCEDURES |
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Animals
Black Swiss mice (30-35 g) were used for PAT1 localization studies in stomach, duodenum, and colon. Wild-type and gastric H-K-ATPase-null mice (30) were used for colocalization studies of PAT1 and gastric H-K-ATPase in the stomach. Animals had free access to water and food during their stay. For acid stimulation, animals were fasted overnight, injected in the morning with a single dose of intraperitoneal histamine (2 µg/g body wt), and killed 30 min later. All animal protocols were approved by the institutional review committee.Immunocytochemical Staining
Antibodies.
A PAT1-specific antibody, raised against the amino-terminal sequence
RRDYHMERPLLNQE (36), was used for these studies. For gastric H-K-ATPase, a monoclonal antibody to the -subunit, 2G11, was
used for double-labeling studies (3). The
-subunit
Na-K-ATPase antibody was a generous gift from Dr. J. Lingrel
(University of Cincinnati).
Immunofluorescence labeling studies.
Animals were euthanized with an overdose of sodium pentobarbital 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, duodena, and colons were removed, cut in tissue blocks, and
left in the same 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.
Preparation of Purified Tubulovesicles From Rabbit Gastric Parietal Cells
Tubulovesicles were isolated from the gastric homogenates of New Zealand White rabbits and purified by differential centrifugation and density gradient flotation as described by Tyagarajan et al. (34).Immunoblot Analysis of Gastric H-K-ATPase
Tubulovesicle membranes from rabbit parietal cells were prepared as above, resolved by SDS-PAGE (30 µg/lane), and transferred to nitrocellulose membranes. The membrane was blocked with 5% milk proteins and then incubated for 6 h with the H-K-ATPaseFunctional
Cl/HCO
RT-PCR of PAT1 in Rabbit Gastric Parietal Cells
Rabbit gastric parietal cells were isolated as described (21) and processed for total RNA isolation. The resulting RNA was poly(A+) selected using Oligotex latex beads (Qiagen) and then reverse transcribed at 47°C by using SuperScript II reverse transcriptase (Life Technologies) and oligo(dT) primers. The oligonucleotide primers (AAG GCC AGC CTG ACT GCA ATA C sense and GGG AGA TTG AAG TGG AAG TGT ACA TC antisense), which encode nucleotides 2162-2550, were designed based on mouse PAT1 cDNAs (GenBank accession no. AY032863). This area is conserved between mouse and human PAT1 (GenBank accession no. AF279265). The expected size of PCR product is 388 bp. These primers are specific for PAT1 and do not recognize other members of SLC26A family. Amplification of the PAT1 cDNA by PCR was performed according to established methods. Each PCR contained 10 µl cDNA, 5 µl 10× PCR buffer (with 20 mM MgCl2), 1 µl 10 mM dNTPs, 10 pmol each primer, and 2.5 units Taq DNA polymerase in a final volume of 50 µl. Cycling parameters were 95°C, 45 s; 47°C, 45 s; and 72°C, 2 min.Materials
Nitrocellulose filters and other chemicals were purchased from Sigma. RadPrime DNA labeling kit was purchased from GIBCO-BRL. 36Cl was purchased from New England Nuclear (Boston, MA). Valinomycin was dissolved in 95% ethanol and added to the membranes in a 1/100 dilution.Statistical Analyses
Values are expressed as means ± SE. The substantial difference between mean values was examined by using ANOVA. P < 0.05 was considered statistically significant. ![]() |
RESULTS |
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Immunocytochemical Staining of PAT1 in the Gastrointestinal Tract
In this series of experiments, the localization of PAT1 in mouse colon, duodenum, and stomach was examined by immunocytochemistry. PAT1 expression was virtually undetectable in the colon (Fig. 1A), consistent with Northern blot hybridization experiments (36). PAT1 is expressed on the apical membrane of the villi of the duodenum, with lower expression levels on the apical membrane of crypt cells (Fig. 1, B and C). These results are consistent with the localization of PAT1 on brush-border membranes of small intestine and confirm previous studies from our laboratory (36). Immunocytochemical staining in the stomach indicated that PAT1 is localized in the glandular portion of the stomach with no labeling in surface cells (Fig. 1, D and E). This reaction was specific, because the labeling was completely prevented by preadsorption of the immune serum (Fig. 1F).
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Colocalization of PAT1 With D. Biflorus Agglutinin or With the Gastric H-K-ATPase
Mouse stomach sections were stained with PAT1 antibody and with D. biflorus agglutinin, a parietal cell marker (18). As indicated in Fig. 2, when the dual images of cells that were stained with PAT1 (Fig. 2A) and D. biflorus agglutinin (Fig. 2C) were acquired, it became evident that cells that were stained with D. biflorus agglutinin were the same cells that expressed PAT1 (Fig. 2B). These results indicate the exclusive expression of PAT1 in parietal cells.
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To determine the localization of PAT1 with respect to gastric
H-K-ATPase in parietal cells, stomach sections were double labeled with
antibodies directed against PAT1 and the -subunit of the gastric
H-K-ATPase. Figure 3 [lower
(A) and higher (B) magnifications] shows that
the patterns of distribution of gastric H-K-ATPase and PAT1 are
identical when a dual image was acquired. These results indicate that
gastric H-K-ATPase and PAT1 are present in the same intracellular pool
in parietal cells, namely tubulovesicular and/or secretory canalicular
membranes.
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Expression of PAT1 is Reduced in Gastric H-K-ATPase Knockout Mice
The colocalization of PAT1 and the gastric H-K-ATPase suggests that PAT1 expression should be reduced in parietal cells in which the tubulovesicle and canalicular membranes are reduced. Parietal cells from gastric H-K-ATPase
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Functional Presence of
Cl/HCO
Tubulovesicles were isolated from rabbit parietal cells, subjected to
several purification steps as described before (34), and
examined for the expression of H-K-ATPase. Figure
5A shows an immunoblot
analysis of tubulovesicle membranes using the gastric H-K-ATPase
-subunit antibody. As indicated, the fraction containing pure
tubulovesicle membranes (lane 3) showed the highest gastric H-K-ATPase abundance when compared with the initial homogenate (lane 1) and semipurified membranes (lane 2). To examine the
presence of basolateral membrane contamination in tubulovesicle
proteins, immunoblot analysis of
-subunit Na-K-ATPase was performed
on the initial homogenate (Fig. 5B, corresponding to
lane 1 in A) and purified tubulovesicles (Fig.
5B, corresponding to lane 3 in A). As
shown in Fig. 5B, Na-K-ATPase labeling was very faint in
tubulovesicle membranes vs. the initial homogenate, indicating the
absence of a significant amount of basolateral contamination in
tubulovesicle membranes. Because our PAT1 polyclonal antibody that was
raised in rabbit does not recognize the rabbit PAT1 in stomach,
duodenum, or kidney (data not shown), we examined the mRNA expression
of PAT1 in rabbit stomach parietal cells. Parietal cells were isolated
as before (21) and subjected to RT-PCR as described in
EXPERIMENTAL PROCEDURES. The ethidium bromide
staining of the agarose gel demonstrated the amplification of a
~390-bp band (Fig. 5C). The product was gel purified and
subjected to sequence analysis, which verified the sequence as rabbit
PAT1.
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Figure 5D shows a functional assay examining the presence of
Cl/HCO
1 · min
1 in
the presence of DIDS. Whether there is more than one anion exchanger or
both Cl
/OH
and
Cl
/HCO
In the last series of experiments, we examined the distribution of
PAT1, vis-à-vis H-K-ATPase, in mouse parietal cells in response
to stimulation of acid secretion. Mice were treated with intraperitoneal histamine (2 µg/g body wt) after an overnight fast
and euthanized 30 min later. Stomach sections were double labeled with
PAT1 and H-K-ATPase antibodies and analyzed by high-resolution confocal
microscopy. The results demonstrate remarkably similar labeling pattern
for PAT1 and H-K-ATPase during the resting and the stimulated states
(Fig. 6), with both transporters showing a diffuse pattern in a majority of cells during the resting state (Fig.
6A) but a reticular pattern during the stimulated state (Fig. 6B).
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DISCUSSION |
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The results of the above experiments indicate that PAT1 is
expressed on apical membranes in the small intestine, as shown previously (34), and in the glandular portion of the
stomach (Fig. 1). The expression of PAT1 in the stomach is limited to the parietal cells (Fig. 2) and mirrors the expression pattern of the
H-K-ATPase, indicating that PAT1 is present in secretory canalicular
and/or tubulovesicular membranes (Fig. 3). PAT1 expression was
significantly decreased in parietal cells of gastric H-K-ATPase knockout mice (Fig. 4), in which H-K-ATPase-containing membranes are
sharply reduced, and functional studies using purified tubulovesicles demonstrated the presence of a DIDS-sensitive
Cl/HCO
It is difficult to conceive of a physiological function for
Cl/HCO
/HCO
/HCO
/HCO
In the studies described here, we showed that purified tubulovesicles
contain robust Cl/HCO
/HCO
/HCO
/HCO
/HCO
/HCO
/HCO
Although further studies will be needed to determine the physiological
function of Cl/HCO
/HCO
for cytoplasmic HCO
In conclusion, PAT1 or SLC26A6 expression in the stomach occurs
exclusively in the parietal cells. PAT1 colocalizes with the gastric
H-K-ATPase and mediates Cl/HCO
from secretory membranes during the passage
of parietal cells from the stimulated state to the resting state and
for maintaining the normal resting state of tubulovesicles.
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ACKNOWLEDGEMENTS |
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These studies were supported by a Merit Review Grant from the Department of Veterans Affairs, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-54430, a Cystic Fibrosis Foundation Grant, grants from Dialysis Clinic Incorporated (to M. Soleimani), and by NIDDK Grants DK-50594 (to G. E. Shull) and DK-38972 (to J. G. Forte).
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. Soleimani, Division 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).
1 The volume density of total secretory membranes and tubulovesicle membranes were 3.5-fold and 22-fold greater, respectively, in wild-type parietal cells than in H-K-ATPase-deficient parietal cells; M. Miller, L. M. Judd, I. R. van Driel, A. Andringa, M. Flagella, S. M. Bell, P. J. Schultheis, and G. E. Shull (unpublished observations).
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.
July 17, 2002;10.1152/ajpgi.00137.2002
Received 9 April 2002; accepted in final form 12 July 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Allen, A,
Flemstrom G,
Garner A,
and
Kivilaakso E.
Gastroduodenal mucosal protection.
Physiol Rev
73:
823-857,
1993
2.
Bissig, M,
Hagenbuch B,
Stieger B,
Koller T,
and
Meier PJ.
Functional expression cloning of the canalicular sulfate transport system of rat hepatocytes.
J Biol Chem
269:
3017-3021,
1994
3.
Chow, DC,
and
Forte JG.
Characterization of the -subunit of the gastric H,K-ATPase by an inhibitory monoclonal antibody.
Am J Physiol Cell Physiol
265:
C1562-C1570,
1993
4.
Cox, KH,
Adair-Kirk TL,
and
Cox JV.
Variant AE2 anion exchanger transcripts accumulate in multiple cell types in the chicken gastric epithelium.
J Biol Chem
271:
8895-8902,
1996
5.
Everett, LA,
Glaser B,
Beck JC,
Idol JR,
Buchs A,
Heyman M,
Adawi F,
Hazani E,
Nassir E,
Baxevanis AD,
Sheffield VC,
and
Green ED.
Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS).
Nat Genet
17:
411-422,
1997[ISI][Medline].
6.
Feldman, M.
Gastric secretion: normal and abnormal.
In: Gastrointestinal and Liver Disease: Pathophysiology/Diagnosis/Management (2nd ed.), edited by Feldman MD,
Scharschmidt BF,
Sleisenger MH,
and Klein SWB. Philadelphia, PA: Saunders, 1998, p. 587-603.
7.
Flemstrom, G.
Gastric and duodenal mucosal bicarbonate secretion.
In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by Johnson LR,
Jacobson ED,
Christensen J,
Alpers D,
and Walsh JH.. New York: Raven, 1994, p. 1285-1309.
8.
Garner, A,
Flemstrom G,
Allen A,
Heylings JR,
and
McQueen S.
Gastric mucosal protective mechanisms: roles of epithelial bicarbonate and mucus secretions.
Scand J Gastroenterol Suppl
101:
79-86,
1984[Medline].
9.
Hastabacka, J,
de la Chapelle A,
Mahtani MM,
Clines G,
Reeve-Daly MP,
Daly M,
Hamilton BA,
Kusumi K,
Trivedi B,
Weaver A,
Coloma A,
Lovett M,
Buckler A,
Kaitila I,
and
Lander ES.
The diastrophic dysplasia gene encodes a novel sulfate transporter: positional cloning by fine-structure linkage disequilibrium mapping.
Cell
78:
1073-1087,
1994[ISI][Medline].
10.
Hoglund, P,
Haila S,
Socha J,
Tomaszewski L,
Saarialho-Kere U,
Karjalainen-Lindsberg ML,
Airola K,
Holmberg C,
Chapelle A,
and
Kere J.
Mutations of the down-regulated in adenoma (DRA) gene cause congenital chloride diarrhea.
Nat Genet
14:
316-319,
1996[ISI][Medline].
11.
Ito, S,
Munro DR,
and
Schofield GC.
Morphology of the isolated mouse oxyntic cell and some physiological parameters.
Gastroenterology
73:
887-898,
1977[ISI][Medline].
12.
Jons, T,
and
Drenckhahn D.
Anion exchanger 2 (AE2) binds to erythrocyte ankyrin and is colocalized with ankyrin along the basolateral plasma membrane of human gastric parietal cells.
Eur J Cell Biol
75:
232-236,
1998[ISI][Medline].
13.
Kauffman, GL, Jr.
Gastric mucus and bicarbonate secretion in relation to mucosal protection.
J Clin Gastroenterol
3, Suppl2:
45-50,
1981[ISI][Medline].
14.
Knauf, F,
Yang CL,
Thomson RB,
Mentone SA,
Giebisch G,
and
Aronson PS.
Identification of a chloride-formate exchanger expressed on the brush border membrane of renal proximal tubule cells.
Proc Natl Acad Sci USA
98:
9425-9430,
2001
15.
Lamprecht, G,
Seidler U,
and
Classen M.
Intracellular pH-regulating ion transport mechanisms in parietal cell basolateral membrane vesicles.
Am J Physiol Gastrointest Liver Physiol
265:
G903-G910,
1993
16.
Lohi, H,
Kujala M,
Kerkela E,
Saarialho-Kere U,
Kestila M,
and
Kere J.
Mapping of five new putative anion transporter genes in human and characterization of SLC26A6, a candidate gene for pancreatic anion exchanger.
Genomics
70:
102-112,
2000[ISI][Medline].
17.
Lohi, H,
Kujala M,
Makela S,
Lehtonen E,
Kestila M,
Saarialho-Kere U,
Markovich D,
and
Kere J.
Functional characterization of three novel tissue-specific anion exchangers: SLC26A7, A8 and A9.
J Biol Chem
277:
14246-14254,
2002
18.
Lorenz, RG,
and
Gordon JI.
Use of transgenic mice to study regulation of gene expression in the parietal cell lineage of gastric units.
J Biol Chem
268:
26559-26570,
1993
19.
Melvin, JE,
Park K,
Richardson L,
Schultheis PJ,
and
Shull GE.
Mouse down-regulated in adenoma (DRA) is an intestinal Cl/HCO
20.
Paradiso, AM,
Townsley MC,
Wenzl E,
and
Machen TE.
Regulation of intracellular pH in resting and in stimulated parietal cells.
Am J Physiol Cell Physiol
257:
C554-C561,
1989
21.
Rossmann, H,
Bachmann O,
Wang Z,
Shull GE,
Obermaier B,
Stuart-Tilley A,
Alper SL,
and
Seidler U.
Differential expression and regulation of AE2 anion exchanger subtypes in rabbit parietal and mucous cells.
J Physiol
534:
837-848,
2001
22.
Royaux, IE,
Suzuki K,
Mori A,
Katoh R,
Everett LA,
Kohn LD,
and
Green ED.
Pendrin, the protein encoded by the Pendred syndrome gene (PDS), is an apical porter of iodide in the thyroid and is regulated by thyroglobulin in FRTL-5 cells.
Endocrinology
141:
839-845,
2000
23.
Royaux, IE,
Wall SM,
Karniski LP,
Everett LA,
Suzuki K,
Knepper MA,
and
Green ED.
Pendrin, encoded by the Pendred syndrome gene, resides in the apical region of renal intercalated cells and mediates bicarbonate secretion.
Proc Natl Acad Sci USA
98:
4221-4226,
2001
24.
Schweinfest, CW,
Henderson KW,
Suster S,
Kondoh N,
and
Papas TS.
Identification of a colon mucosa gene that is down-regulated in colon adenomas and adenocarcinomas.
Proc Natl Acad Sci USA
90:
4166-4170,
1993[Abstract].
25.
Scott, DA,
Wang R,
Kreman TM,
Sheffield VC,
and
Karniski LP.
The Pendred syndrome gene encodes a chloride-iodide transport protein.
Nat Genet
21:
440-443,
1999[ISI][Medline].
26.
Shorrock, CJ,
and
Rees WD.
Overview of gastroduodenal mucosal protection.
Am J Med
84:
25-34,
1988[Medline].
27.
Shull, GE.
cDNA cloning of the beta-subunit of the rat gastric H,K-ATPase.
J Biol Chem
265:
12123-12126,
1990
28.
Shull, GE,
and
Lingrel JB.
Molecular cloning of the rat stomach (H+-K+)-ATPase.
J Biol Chem
261:
16788-16791,
1986
29.
Silberg, DG,
Wang W,
Moseley RH,
and
Traber PG.
The down regulated in adenoma (dra) gene encodes an intestine-specific membrane sulfate transport protein.
J Biol Chem
270:
11897-11902,
1995
30.
Spicer, Z,
Miller ML,
Andringa A,
Riddle TM,
Duffy JJ,
Doetschman T,
and
Shull GE.
Stomachs of mice lacking the gastric H,K-ATPase alpha-subunit have achlorhydria, abnormal parietal cells, and ciliated metaplasia.
J Biol Chem
275:
21555-21565,
2000
31.
Soleimani, M.
Molecular physiology of the renal chloride-formate exchanger.
Curr Opin Nephrol Hypertens
10:
677-683,
2001[ISI][Medline].
32.
Soleimani, M,
and
Bizal GL.
Functional identity of a purified proximal tubule anion exchanger protein: mediation of chloride/formate and chloride/bicarbonate exchange.
Kidney Int
50:
1914-1921,
1996[ISI][Medline].
33.
Soleimani, M,
Greeley T,
Petrovic S,
Wang Z,
Amlal H,
Kopp P,
and
Burnham CE.
Pendrin: an apical Cl/OH
/HCO
34.
Tyagarajan, K,
Chow DC,
Smolka A,
and
Forte JG.
Structural interactions between alpha- and beta-subunits of the gastric H,K-ATPase.
Biochim Biophys Acta
1236:
105-113,
1995[ISI][Medline].
35.
Waisbren, SJ,
Geibel JP,
Modlin IM,
and
Boron WF.
Unusual permeability properties of gastric gland cells.
Nature
368:
332-335,
1994[ISI][Medline].
36.
Wang, Z,
Petrovic S,
Mann E,
and
Soleimani M.
Identification of an apical Cl/HCO
37.
Wang, Z,
Schultheis PJ,
and
Shull GE.
Three N-terminal variants of the AE2 Cl/HCO
38.
Zheng, J,
Shen W,
He DZ,
Long KB,
Madison LD,
and
Dallos P.
Prestin is the motor protein of cochlear outer hair cells.
Nature
405:
149-55,
2000[ISI][Medline].