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MS# 01-887R2
Increased Dietary Salt Activates Rat Aortic Endothelium
Wei-Zhong Ying
and
Paul W. Sanders
Nephrology Research and Training Center, Comprehensive Cancer Center,
and
Cell Adhesion and Matrix Research Center;
Division of Nephrology,
Department of Medicine, and Department of Physiology & Biophysics,
University of Alabama at Birmingham,
Birmingham, AL 35294-0007
and
Department of Veterans Affairs Medical Center,
Birmingham, AL 35233
Address correspondence to: Paul W. Sanders, M. D.
Division of Nephrology/Department of Medicine
642 Lyons-Harrison Research Building
701 South 19th Street
University of Alabama at Birmingham
Birmingham, AL 35294-0007
Telephone: (205) 934-3589
FAX: (205) 975-6288
E-mail:[email protected]
Ying and Sanders
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MS# 01-887R2
ONLINE METHODS
Animal preparation
Studies were conducted using 40 male Sprague-Dawley rats, 28 day of age, obtained
from Charles River Laboratories (Wilmington, MA). Animals were chosen at this age because
of our previous experience that showed normal renal function and blood pressure responses to
dietary salt for up to two weeks of observation. 1 The rats were given 0.3% NaCl diet (AIN76A, Dyets, Inc., Bethlehem, PA) and water ad libitum for 4 d before initiating the experiment.
The rats were then continued on a diet (AIN-76A, Dyets, Inc.) that contained 0.3%, 1.0%, 3.0%,
or 8.0% NaCl. These formulated diets were identical in protein and electrolyte composition,
except for NaCl content. On the fourth day of study, rats were anesthetized with pentobarbital
sodium, 50 mg/kg, intraperitoneally. The aortas were perfused in situ with a cold isotonic
heparinized perfusion solution that contained 90 mmol/L NaCl, 50 mmol/L sodium fluoride, 1
mmol/L Na3VO4, and 10 mmol/L sodium pyrophosphate. Fifty ml of solution was perfused over
2 min. In some experiments, five min prior to harvesting the aorta, a 1-ml bolus of either
tetraethylammonium chloride (TEA) (Sigma Chemical Co., St. Louis, MO), 15 mmol/L in
isotonic saline, or saline alone was injected intravenously in the tail vein over 5 minutes. This
dose is less than the amount (2-6 mg/kg) administered intravenously to human volunteers with
congestive heart failure; no toxic effects were reported in that study. 2 The aorta was perfused in
situ with the perfusion solution that also contained 3 mmol/L TEA in those animals that received
TEA intravenously. The aorta was harvested under sterile conditions.
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MS# 01-887R2
Western blot analysis (p38 MAPK, p42/44 MAPK, p46/54 JNK/SAPK, ATF-2, and Elk-1)
Harvesting of aortic tissue, generation of protein lysates, and Western blotting proceeded
as described previously, 3-5 with slight modifications.
The lysis buffer contained sodium
pyrophosphate, 2.5 mmol/L, and Na3VO4, 1 mmol/L. Protein concentration was determined
using a kit (Micro BCA Protein Assay Reagent Kit, Pierce, Rockford, IL). Samples containing
60 µg of total protein were used in the immunoblot assays. The primary antibodies were used at
1:1000 dilution and recognized specifically total and phosphorylated forms of p38 MAPK,
p42/44 MAPK and p46/54 JNK/SAPK (Cell Signaling Tech, Inc., Beverly, MA). Peroxidaseconjugated anti-IgG (Bio-Rad, Hercules, CA), 1:10,000 dilution, served as the secondary
antibody. Bands were detected by ECL enhanced chemiluminescence (Amersham Pharmacia
Biotech, Piscataway, NJ). Total and phosphorylated forms of the activating transcription factor,
ATF-2, were detected using a kit (PhosphoPlus® ATF-2 (Thr71) Antibody Kit, Cell Signaling
Tech.). The phospho-specific antibody recognized ATF-2 phosphorylated only at the threonine
at position 71. Phosphorylated Elk-1 was detected using a monoclonal antibody directed against
Elk-1 phosphorylated at the serine at position 383 (Phospho-Elk-1 (Ser383) 2B1 monoclonal
antibody, Cell Signaling Tech.).
Activity Assays for p38 MAPK and p42/44 MAPK
Two hundred µl of aortic tissue lysate, 1 µg/µl, were incubated with 15 µl of suspended
beads that bound anti-phospho-p42/44 MAPK (T202/Y204) or anti-phospho-p38 MAPK
(T180/Y182) monoclonal antibodies, with gentle rocking overnight at 4ºC. Kinase assays were
performed using 200 µmol/L ATP and 2 µg of substrate protein (ATF-2 or Elk-1 fusion protein).
Substrate phosphorylation was detected and quantified using antibodies that specifically
recognized phosphorylated ATF-2 or Elk-1.
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MS# 01-887R2
Immunohistochemical Staining for phospho-p38 MAPK and phospho-p42/44 MAPK
Aortic tissue was isolated, fixed in phosphate-buffered 10% (vol/vol) formalin (Sigma
Diagnostics, St. Louis, MO). Immunohistochemistry analysis proceeded in standard fashion,
using affinity-purified rabbit polyclonal anti-phospho-p38 MAPK (1:25 dilution in
TBS/Triton/BSA buffer) or anti-phospho-p42/44 MAPK (1:100 dilution in TBS/Triton/BSA
buffer) (both from Cell Signaling Technology, Beverly, MA). Antibody binding was detected
using a biotinylated anti-rabbit secondary antibody and avidin-biotin immunoperoxidase
complex methodology (ABC-Vectastain Elite Kit, Vector Laboratories, Inc., Burlingame, CA).
The slides were counterstained with hematoxylin.
In vitro incubation studies
Aortic tissue, which was harvested simultaneously from rats on the various NaCl diets,
was cut into 2-3 mm ring segments. The samples were initially incubated with serum-free
medium (RPMI 1640; Life Technologies, grand Island, NY) alone or medium that contained 50
µmol/L PD-098059 (2’-amino-3’-methoxyflavone), a potent and specific cell-permeable
inhibitor of activation of MAPK kinase-1 (MEK1), 6 10 µmol/L SB-203580 (4-(4-fluorophenyl)2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole), a highly specific and cell-permeable
inhibitor of p38 MAPK- 7, 8 or both inhibitors. The inhibitors (both from Calbiochem, San
Diego, CA), were dissolved in dimethyl sulfoxide (DMSO) and were diluted in medium to a
final concentration of 50 µmol/L immediately prior to use; the final concentration of DMSO was
0.1% (vol/vol).
Control incubation medium contained 0.1% (vol/vol) DMSO without the
inhibitors. After a 30-min incubation period, the medium was removed and refreshed with
serum-free medium alone or medium that contained the same concentrations of PD-098059, SB203580 or both inhibitors. Incubation continued for 24 h at 37ºC. The medium was harvested
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MS# 01-887R2
and assayed for TGF-ß1 and NOx. Total and active levels of TGF-ß1 were determined by
enzyme-linked immunoassay (TGF-ß1 Emax TM ImmunoAssay System, Promega Inc.,
Madison, WI), as performed previously. 3-5 Production of nitrite and nitrate was quantified in
standard fashion using nitrate reductase and Griess reagent. 5, 9, 10 Experiments were also
performed using aortic segments that had endothelium mechanically removed by gently rubbing
the lumenal surface of each ring segment with a sterile wooden probe. 5
Statistical analysis
Data were presented as mean ± standard error. Significant differences among data sets
were determined using either unpaired t test or one-way analysis of variance using multiple
comparisons by Fisher's protected least significant difference method, where appropriate. A P
value less than 0.05 assigned statistical significance.
REFERENCES
1.
Chen PY, Sanders PW: L-arginine abrogates salt-sensitive hypertension in Dahl/Rapp
rats. J Clin Invest 1991;88:1559-1567
2.
Relman AS, Epstein FH: Effect of tetraethylammonium on venous and arterial pressure
in congestive heart failure. Proc Soc Exp Biol Med 1949;70:11-14
3.
Ying W-Z, Sanders PW: Dietary salt modulates renal production of transforming growth
factor-ß in rats. Am J Physiol 1998;274(Renal Physiol. 43):F635-F641
4.
Ying W-Z, Sanders PW: Dietary salt enhances glomerular endothelial nitric oxide
synthase through TGF-ß1. Am J Physiol 1998;275(Renal Physiol. 44):F18-F24
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5.
Ying W-Z, Sanders PW: Dietary salt increases endothelial nitric oxide synthase and
TGF-ß1 in rat aortic endothelium. Am J Physiol 1999;277(Heart Circ. Physiol.
46):H1293-H1298
6.
Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR: PD 098059 is a specific
inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo.
J Biol Chem 1995;270:27489-27494
7.
Jiang Y, Gram H, Zhao M, New L, Gu J, Feng L, Di Padova F, Ulevitch RJ, Han J:
Characterization of the structure and function of the fourth member of the p38 group
mitogen-activated protein kinase, p38. J Biol Chem 1997;272:30122-30128
8.
Gum RJ, McLaughlin MM, Kumar S, Wang Z, Bower MJ, Lee JC, Adams JL, Livi GP,
Goldsmith EJ, Young PR: Acquisition of sensitivity of stress-activated protein kinases to
the p38 inhibitor, SB 203580, by alteration of one or more amino acids within the ATP
binding pocket. J Biol Chem 1998;273:15605-15610
9.
Chen PY, Sanders PW: Role of nitric oxide synthesis in salt-sensitive hypertension in
Dahl/Rapp rats. Hypertension 1993;22:812-818
10.
Chen PY, Gladish RG, Sanders PW: Vascular smooth muscle nitric oxide synthase
anomalies in Dahl/Rapp salt-sensitive rats. Hypertension 1998;31:918-924
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