This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Harrison-Bernard, L. M. Articles by Navar, L. G. Search for Related Content PubMed PubMed Citation Articles by Harrison-Bernard, L. M. Articles by Navar, L. G. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Medline Plus Health Information High Blood Pressure

(Hypertension. 1999;33:340-346.)
© 1999 American Heart Association, Inc.

Scientific Contributions

Regulation of Angiotensin II Type 1 Receptor mRNA and Protein in Angiotensin II–Induced Hypertension

Lisa M. Harrison-Bernard; Samir S. El-Dahr; Denise F. O'Leary; L. Gabriel Navar

From the Departments of Physiology (L.M.H.-B., D.F.O., L.G.N.) and Pediatrics (S.S.E.-D.), Tulane University School of Medicine, New Orleans, La.

Correspondence to Lisa M. Harrison-Bernard, PhD, Department of Physiology SL39, Tulane University School of Medicine, 1430 Tulane Ave, New Orleans, LA 70112-2699. E-mail lharris{at}mailhost.tcs.tulane.edu

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract—Chronic elevations of circulating angiotensin II (Ang II) cause sustained hypertension and enhanced accumulation of intrarenal Ang II by an AT₁ receptor–dependent process. The present study tested the hypothesis that chronic elevations in circulating Ang II regulate AT₁ mRNA and protein expression in a tissue-specific manner. Sprague-Dawley rats were infused with Ang II (80 ng/min) or vehicle subcutaneously for 13 days via osmotic minipump. On day 12, systolic blood pressure averaged 186±12 mm Hg in Ang II–infused rats compared with rats given vehicle (121±2 mm Hg). Plasma renin activity was markedly suppressed in the Ang II–infused rats compared with vehicle-infused rats (0.1±0.01 versus 4.9±0.9 ng of Ang I · mL^-1 · h^-1; P<0.05). Semiquantitative reverse transcription polymerase chain reaction using rat AT_1A- and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH)-specific primers was followed by Southern blot hybridization using specific radiolabeled cDNA or oligonucleotide probes. The results showed that the ratios of AT_1A/GAPDH mRNA in the kidney (0.19±0.05 versus 0.26±0.03) and liver (2.8±0.9 versus 3.0±0.5) were comparable in Ang II– and vehicle-infused rats. In contrast, AT_1A/GAPDH mRNA levels were increased in the adrenal glands of Ang II–infused rats (0.49±0.04 versus 0.36±0.02; P<0.05). Western blot analysis showed that AT₁ protein levels in the kidney and liver were also similar in the two groups. Therefore, these results indicate that renal and liver AT₁ receptor gene expression is maintained in Ang II–induced hypertension. The failure to downregulate AT₁ receptor mRNA and protein levels thus allows the sustained effects of chronic elevations in Ang II to elicit progressive increases in arterial pressure.

Key Words: kidney • liver • adrenal gland • gene expression • antibodies

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Chronic administration of angiotensin II (Ang II) is a useful experimental model of Ang II–dependent hypertension that mimics 2-kidney, 1 clip (2K1C) Goldblatt hypertension. The mechanisms responsible for the progressive nature of Ang II–induced hypertension are multifarious and incompletely understood. We previously demonstrated that chronic infusion of initially a subpressor dose of Ang II into uninephrectomized rats produces a slowly developing hypertension that is associated with diminished renal renin content and mRNA, enhanced renal ACE activity, maintained renal and hepatic angiotensinogen mRNA levels, and an AT₁-mediated augmentation of intrarenal Ang II content.^{1 2 3 4} However, the contribution of the expression of the AT₁ receptor to the hypertensinogenic effect of Ang II has not been established. Because most of the hypertensinogenic actions of the renin-angiotensin system are mediated by the binding of Ang II to the AT₁ receptor, the predominant receptor subtype in adult animals, and the availability of AT₁ receptors essentially determines the efficacy of the renin-angiotensin system, we studied the tissue-specific expression of the AT₁ receptor after chronic low-dose infusions of Ang II in the rat. In particular, the regulation of AT₁ receptor mRNA and protein expression was determined in the kidney, liver, and adrenal gland.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Animals and Tissue Preparation
Male Sprague-Dawley rats (Charles River Labs, Wilmington, MA) were housed in wire cages and maintained in a temperature-controlled room regulated on a 12-hour light/dark cycle. Rats had free access to water and standard rat chow (Ralston-Purina). The experimental protocol was approved by the Tulane University Animal Care and Use Committee. Rats (230±4 g body wt; n=17) were anesthetized with sodium pentobarbital (50 mg/kg IP), and an osmotic minipump (model 2002; Alza Corp) was implanted subcutaneously at the dorsum of the neck. Rats were selected at random to receive Ang II infusion (n=8; Novabiochem) at a rate of 80 ng/min (350 ng · kg^-1 · min^-1) or vehicle (n=9; 5% acetic acid) for a period of 13 days. Systolic blood pressures were measured in conscious rats 1 day before surgery and on days 6 and 12 of the infusion using tail-cuff plethysmography (model 52-0338; Harvard Apparatus). Blood and tissue samples (kidneys, liver, adrenal glands) were harvested on day 13 after rapid decapitation. Trunk blood was collected into chilled tubes containing EDTA. Plasma was separated and stored at -20°C until assayed for plasma renin activity using a commercially available Ang I RIA kit (Incstar) as described previously.¹ Liver and kidneys were removed for protein and RNA extraction. Adrenal glands were removed for RNA extraction. Samples were snap-frozen in liquid nitrogen and stored at -80°C until processed.

AT_1A Receptor mRNA Analyses
Total RNA was extracted using the RNeasy Midi Kit (Qiagen) according to the manufacturer's specifications. The extracted RNA was quantified spectrophotometrically by absorbance at 260 nm, dissolved in sterile water, and stored at -80°C until use. Integrity of RNA was documented by agarose gel electrophoresis and observation of 28S and 18S ribosomal RNA.

RT-PCR
cDNA was synthesized by use of SuperScript II RT (GIBCO BRL). The reaction mixture contained DNase I–digested RNA (kidney, 5 µg; liver, 3 µg; adrenal, 2.5 µg), 10x PCR buffer (200 mmol/L Tris · HCl, pH 8.4, 500 mmol/L KCl, 10 mmol/L MgCl₂), 100 ng random hexamers, 10 mmol/L concentration each of deoxynucleoside triphosphate, 0.1 mmol/L DTT, and 200 U RT for a total volume of 23 µL. RT was omitted in control samples. Positive control for the RT step consisted of control RNA provided by the manufacturer. The reaction mixture was incubated at 25°C for 10 minutes and 42°C for 50 minutes, heated to 70°C for 15 minutes, and chilled on ice. RNase H (2 U) was added to each tube and incubated at 37°C for 20 minutes. RT mixture was then diluted with 10x PCR buffer, followed by the addition of upstream and downstream primers and 2.5 U Taq DNA polymerase (GIBCO BRL) for a final volume of 50 µL. The reaction was initiated by heat denaturation of RNA-cDNA hybrid at 95°C for 1 minute, annealing of the primers (50 pmol for AT_1A, 25 pmol glyceraldehyde-3-phosphate-dehydrogenase [GAPDH]), and extension at 72°C. The cycle was repeated 25 times using a programmable PCR thermocycler (Perkin-Elmer Cetus Instruments). Details of each PCR are described. PCRs for GAPDH and AT_1A were performed from the same RT products to allow the data to be expressed as a ratio to GAPDH. The appropriate PCR reaction cycle number was determined for the two targeted mRNAs by including 0.5 µCi [-³²P]dCTP in the PCR and performing 15 to 35 cycles of amplification. PCR products were run on 2% agarose gel, and the bands were excised, dried at 37°C overnight, and counted using scintillation fluid. Twenty-five–cycle amplification was on the linear portion of the plot of the log of the cpm as a function of cycle number for each PCR product. Semiquantification of AT_1A and GAPDH mRNA in the liver and kidney was carried out in the presence of 0.5 µCi [-³²P]dCTP. Products were analyzed as stated above.

Primers
AT_1A primers⁵ are from the 3'-untranslated regions of the rat AT_1A receptor cDNA. The upstream primer is complementary to nucleotides 1370 to 1389 (5'-GCACACTGGCAATGTAATGC-3'), and the downstream primer corresponds to nucleotides 1737 to 1756 (5'-GTTGAACAGAACAAGTGACC-3'). The predominant cDNA amplification product is predicted to be 385 bp. Ten µL of RT product was amplified as described above with annealing at 58°C for 45 seconds and extension at 72°C for 90 seconds. The upstream primer of GAPDH is complementary to nucleotides 524 to 547 of the rat GAPDH cDNA⁶ (5'-AATGCATCCTGCACCACCAACTGC-3'), and the downstream primer corresponds to nucleotides 1055 to 1078 (5'-GGAGGCCATGTAGGCCATGAGGTC-3'). The predicted size of the PCR product is 555 bp. Two µL of RT product was amplified as described above with annealing at 55°C and extension at 72°C for 60 seconds each.

Southern Blot Hybridization
Electrophoresis of 10 to 20 µL PCR products was carried out in 1% agarose gel. DNA was transferred to a positively charged nylon membrane (GeneScreen Plus; New England Nuclear) using capillary method. Blots were prehybridized for 1 hour at 46°C with 5x SSC, 1x Denhardt's solution, 0.05 mol/L PO₄ buffer (pH6.7), 2 mg denatured herring sperm DNA, 0.10 mmol/L dextran sulfate, and 50% formamide. Blots were hybridized overnight with 10⁷ cpm/mL oligo probe for AT_1A and GAPDH using 5x SSC, 0.5x Denhardt's solution, 0.02 M PO₄ buffer, 1 mg herring sperm DNA, 0.2 mmol/L dextran sulfate, and 50% formamide. Blots were prehybridized at 65°C for 1 hour with 1% SDS in 0.1x SSPE and hybridized overnight with 10⁷ cpm/mL GAPDH cDNA probe in 7% SDS, 0.25 mol/L sodium phosphate buffer, 2 mg denatured herring sperm DNA, 0.2 g BSA fraction V, and 0.1 mmol/L EDTA. Blots were washed successively in 1x, 0.2x, and 0.1x SSC containing 0.1% SDS at room temperature with gentle agitation (30 minutes each).

Slot Blot Analysis
Slot blots were prepared by serial 2-fold dilutions (4 to 1 µg) of total RNA from individual animals as described previously.⁴ Blots were hybridized to the AT_1A oligo probe and rehybridized with a GAPDH oligo probe to account for loading variations (2 to 3 months allowed for ³²P decay). Signals were detected by autoradiography and quantified by scanning laser densitometry (Ultroscan; Pharmacia LKB).

DNA Probe Radiolabeling
Probes were labeled with 50 µCi [-³²P]dCTP using random primers DNA labeling system (GIBCO BRL) and purified on Sephadex G-50 Nick columns (Pharmacia). The cDNA probe used was a human GAPDH cDNA (Clontech). Five pmol of the oligonucleotide (lower AT_1A or GAPDH primer) was 5'-end labeled with 50 to 70 µCi [-³²P]dATP using T4 polynucleotide kinase (20 U; GIBCO BRL) and purified on a Sephadex G25 column (Pharmacia).

AT₁ Receptor Protein Analysis
Proteins were extracted from kidney and liver samples after homogenization as described previously⁷ and assayed by the method of Lowry et al.⁸

Western Blot Analysis of AT₁ Receptor
Kidney (50 µg) or liver (25 µg) samples were electrophoretically separated by 3% to 10% stacking Tris-glycine gel at 100 V for 2 hours (10% SDS, 24 mmol/L Tris base, 192 mmol/L glycine) and transferred (20% methanol, 12 mmol/L Tris base, 96 mmol/L glycine) to nitrocellulose membrane (0.45; BioRad) for 90 minutes at 25 V according to the manufacturer's specifications (XCell II Mini-Cell; Novex). Molecular weight markers (10- to 250-kDa rainbow; Amersham; and 10-kDa protein ladder, GIBCO BRL) were used to determine approximate molecular mass. Western blot analysis was performed as described previously⁷ with minor modifications. Blots were incubated with the primary antibody (1) anti-peptide (8 to 17 amino acids, AT₁ monoclonal antibody 6313/G2, 1:50; GP Vinson) or (2) anti-peptide AT₁ polyclonal antibody 1:200 (15 to 24 amino acids, SC-1173; Santa Cruz) for 3 hours, washed, incubated with the secondary antibody conjugated to horseradish peroxidase (1:1500) for 1 hour, and washed. Detection was accomplished using enhanced chemiluminescence Western blotting (ECL; Amersham), and the blots were exposed to x-ray film (Hyperfilm-ECL; Amersham). For preadsorption studies, the polyclonal antibody and the synthetic peptide antigen (20 µg; SC-1173P) were incubated overnight at 4°C. Kidney (50 µg) and liver (25 µg) samples were separated in duplicate on each of 2 gels. One gel was stained with 0.1% Coomassie blue R250 to visualize the protein bands for total protein quantification. The proteins from the second gel were electrophoretically transferred to nitrocellulose and incubated with the primary polyclonal antibody or preadsorbed antibody as described above.

Statistical Analysis
Results are expressed as mean±SEM. For slot blots, regression lines were calculated for each tissue sample from the ratio of the densitometric values of the 3 serial dilutions, and only those samples exhibiting linearity of the hybridization signals with values of r0.9 were accepted for further consideration. For Southern and slot blot analyses, AT_1A signals were factored for GAPDH. The data were analyzed using unpaired t test or Mann-Whitney rank sum test. Statistical significance is defined at a value of P0.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Body Weight, Blood Pressure, and Plasma Renin Activity
Body weights in the two groups at the initiation of the study were similar (Ang II, 226±4 g, n=8; vehicle, 225±4 g, n=9). On day 13 of the infusion, average body weight in the Ang II–infused animals was significantly reduced compared with vehicle (292±7 versus 343±9 g). Systolic blood pressures (Figure 1) were identical in the 2 groups before implantation of the osmotic minipumps (121±2 mm Hg). On days 6 and 12 of infusion, pressures were significantly elevated to 168±3 and 186±12 mm Hg in the Ang II–infused rats compared with vehicle-infused rats (118±3 and 119±3 mm Hg). Plasma renin activity was markedly suppressed in the Ang II–infused rats compared with vehicle-infused rats (0.1±0.01 versus 4.9±0.9 ng of Ang I · mL ^-1 · h^-1, n=7 and 9).

View larger version (19K): [in this window] [in a new window]   Figure 1. Systolic blood pressure in Ang II–infused (; n=8) and vehicle-infused (; n=9) rats before, during, and 12 days after implantation of osmotic minipumps. Values are mean±SEM. *P<0.05 vs control and vehicle.

AT₁ Receptor Antibody Specificity
We documented the specificity of the anti-peptide polyclonal AT₁ receptor antibody by performing Western blot analysis of liver and kidney protein using the antibody preadsorbed with the synthetic peptide antigen. Figure 2 illustrates a Western blot autoradiograph comparing liver and kidney AT₁ receptor protein detected using the primary antibody shown on the left and preadsorbed antibody on the right. Densitometric analysis of the bands indicates that the intensity of the signal was >90% decreased using the preadsorbed antibody. Equal loading was determined by Coomassie blue staining of the duplicate gel. The results demonstrate that the polyclonal AT₁ receptor antibody is specific for the 46-kDa AT₁ receptor protein in these tissues. We previously reported that adsorption of the monoclonal AT₁ antibody by the antigenic peptide eliminates renal vascular and tubular AT₁ receptor antibody immunostaining, demonstrating that the monoclonal antibody is specific for the AT₁ receptor protein.⁷ The antigenic peptide sequences for the two antibodies correspond to portions of the AT₁ receptor protein that are identical for the AT_1A and AT_1B receptor subtypes.

View larger version (39K): [in this window] [in a new window]   Figure 2. Western blot analysis of AT1 receptor. AT1 receptor protein was detected in liver (25 µg; lanes 1 and 3) and kidney (50 µg; lanes 2 and 4) using the polyclonal AT1 antibody (lanes 1 and 2) or antibody preadsorbed with the immunogenic peptide (lanes 3 and 4). Major band is detected at 46 kDa with the primary antibody and eliminated by preadsorption.

Kidney AT₁ Receptor mRNA and Protein Expression
Western blot analysis of AT₁ receptor protein from whole kidney homogenates using the monoclonal antibody in Ang II–infused animals showed that the receptor protein levels remain unchanged during chronic Ang II–induced hypertension (42±6 versus 36±3 du; n=5) (Figure 3A). Similar results were obtained using the polyclonal antibody for Ang II–infused (9±3 du; n=4) and vehicle-infused (6±1 du; n=5) rats. Renal AT_1A mRNA expression was assessed using semiquantitative RT-PCR and Southern blot analysis (Figure 3B). Renal AT_1A mRNA levels were not significantly different between the vehicle- and Ang II–infused animals. The ratio of renal AT_1A to GAPDH densitometric signals was 0.66±0.07 in vehicle-infused (n=9) and 0.47±0.10 in Ang II–infused (n=8) animals. In addition, semiquantification of kidney AT_1A mRNA by incorporation of [-³²P]dCTP confirmed the results obtained by Southern blot analysis. The ratio of AT_1A and GAPDH PCR products (cpm) was 1.1±0.4 (n=4) in vehicle-infused and 1.1±0.3 (n=4) in Ang II–infused rats. AT_1A mRNA slot blot analysis of 4, 2, and 1 µg of total kidney RNA is shown in Figure 3C and demonstrates that AT_1A mRNA levels were not different between the 2 groups, in agreement with the RT-PCR analysis. The ratios of kidney AT_1A and GAPDH densitometric signals were 4.2±0.4 versus 4.5±0.3, 3.2±0.2 versus 3.3±0.3, and 3.6±0.5 versus 3.6±0.3 for vehicle-infused (n=9) versus Ang II–infused (n=7) animals at 4, 2, and 1 µg RNA, respectively.

View larger version (22K): [in this window] [in a new window]   Figure 3. Kidney AT1 receptor mRNA and protein expression. A, Top, Western blot of renal AT1 receptor protein levels in Ang II– and vehicle-infused rats detected using a monoclonal antibody (lanes 1 through 5, vehicle infused; lanes 6 through 10, Ang II infused). Bottom, Similar renal AT1 receptor protein density in vehicle-infused (hatched bar; n=5) and Ang II–infused (solid bar; n=5) rats. B, Top, Representative Southern blot showing AT1A and corresponding GAPDH mRNA levels (lanes 1, 3, and 5, vehicle infused; lanes 2, 4, and 6, Ang II infused). Bottom, Ratio of the densitometric analysis of the autoradiographic signals for AT1A and GAPDH in vehicle-infused (hatched bar; n=9) and Ang II–infused (solid bar; n=8) rats. C, Top, representative autoradiograph of AT1A mRNA slot blot hybridized with radiolabeled AT1A-specific oligonucleotide. Membranes were reprobed with radiolabeled GAPDH oligonucleotide probe (lanes 1, 3, and 5, vehicle infused; lanes 2, 4, and 6, Ang II infused). Bottom, Average data for AT1A mRNA detected by slot blot for vehicle-infused (; n= 9) and Ang II–infused (; n=7) rats.

Liver AT₁ Receptor mRNA and Protein Expression
Western blot analysis of liver AT₁ receptor protein from Ang II– and vehicle-infused animals showed that the receptor protein levels were not altered during Ang II–induced hypertension (52±3 versus 52±3 du, polyclonal primary antibody; 11±2 versus 10±1 du, monoclonal: Figure 4A). Semiquantification of liver AT_1A mRNA by RT-PCR demonstrated that the receptor gene expression is not altered by chronic Ang II infusion (Figure 4B). The ratio of liver AT_1A to GAPDH Southern blot densitometric signals was 3.0±0.5 in vehicle-infused (n=9) and 2.8±0.9 in Ang II–infused (n=8) animals. Semiquantification of liver AT_1A mRNA by incorporation of [-³²P]dCTP confirmed the results obtained by Southern blot analysis. The ratio of AT_1A and GAPDH PCR products (cpm) was 0.42±0.09 (n=5) in vehicle-infused and 0.30±0.03 (n=4) in Ang II-infused animals (P=0.14). Slot blot analysis of liver AT_1A mRNA in vehicle- and Ang II–infused animals is shown in Figure 4C. Liver AT_1A gene expression was not altered during Ang II–induced hypertension. The ratios of liver AT_1A and GAPDH densitometric signals were 2.9±0.3 versus 3.1±0.1, 3.0±0.2 versus 3.2±0.3, and 3.8±0.7 versus 4.1±0.5 for vehicle-infused (n=9) versus Ang II–infused (n=8) animals at 4, 2, and 1 µg RNA, respectively.

View larger version (22K): [in this window] [in a new window]   Figure 4. Liver AT1 receptor mRNA and protein expression. A, Top, Western blot of liver AT1 receptor protein in Ang II– and vehicle-infused rats detected using polyclonal (top) and monoclonal (bottom) antibodies. Blots are from separate gels run using protein from the same animals in each group (lanes 1, 3, 5, 7, and 9, vehicle infused; lanes 2, 4, 6, 8, and 10, Ang II infused). Bottom, Comparable liver AT1 receptor protein expression in vehicle-infused (hatched bar) and Ang II–infused (solid bar) rats (n=5) for the polyclonal (left) and monoclonal (right; du x5) primary antibodies. B, Top, Representative blots showing AT1A and corresponding GAPDH mRNA levels (lanes 1, 3, and 5, vehicle infused; lanes 2, 4, and 6, Ang II infused). Bottom, Ratio of the densitometric analysis of the autoradiographic signals for AT1A and GAPDH in vehicle-infused (hatched bar; n=9) and Ang II–infused (solid bar; n=8) rats. C, Top, Representative autoradiograph of AT1A mRNA slot blot hybridized with radiolabeled AT1A-specific oligonucleotide in vehicle- and Ang II–infused rats (lanes 1, 3, and 5, vehicle infused; lanes 2, 4, and 6, Ang II infused). Bottom, Graph blotting the average data for AT1A mRNA detected by slot blot for vehicle-infused (; n= 9) and Ang II–infused (; n=8) rats.

Adrenal AT_1A Receptor mRNA Expression
Adrenal AT_1A mRNA was analyzed as described above. RT-PCR and Southern blot analysis demonstrated a significant elevation in AT_1A mRNA in the adrenal gland of Ang II–infused animals (Figure 5A). The ratios of adrenal AT_1A to GAPDH Southern blot densitometric signals were 0.36±0.02 in vehicle-infused (n=8) and 0.49±0.04 in Ang II–infused (n=8) animals (P<0.01). By slot blot analysis, AT_1A/GAPDH mRNA tended to be higher but did not reach statistical significance (Figure 5B). The ratios of adrenal AT_1A and GAPDH densitometric signals were 0.58±0.05 versus 0.68±0.08, 0.75±0.05 versus 0.87±0.08, and 0.98±0.12 versus 0.87±0.08 for vehicle-infused (n=8) versus Ang II–infused (n=6) animals at 4, 2, and 1 µg RNA, respectively.

View larger version (25K): [in this window] [in a new window]   Figure 5. Adrenal AT1A receptor mRNA expression. A, Top, Representative blots showing AT1A and corresponding GAPDH mRNA levels (lanes 1, 3, and 5, vehicle infused; lanes 2, 4, and 6, Ang II infused). Bottom, Ratio of the densitometric analysis of the autoradiographic signals for AT1A and GAPDH (n=8) in vehicle-infused (hatched bar) and Ang II–infused (solid bar) rats demonstrated a significant increase in adrenal AT1A mRNA in hypertensive rats. Bottom, Representative autoradiograph of AT1A mRNA slot blot hybridized with radiolabeled AT1A-specific oligonucleotide (lanes 1, 3, and 5, vehicle infused; lanes 2, 4, and 6, Ang II infused). Bottom, Graph plotting the average data for AT1A mRNA detected by slot blot for vehicle-infused (; n= 8) and Ang II–infused (; n=6) rats.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In agreement with previous studies, chronic low-dose infusion of Ang II produced significant elevations in systolic blood pressure and reductions in plasma renin activity. After 13 days of infusion, we found that kidney and liver AT_1A mRNA and protein levels were maintained under conditions of chronic elevations in Ang II. However, in the adrenal gland, AT_1A mRNA expression was significantly elevated. These results indicate that renal AT₁ receptor gene expression is not suppressed in Ang II–induced hypertension. The failure to downregulate AT₁ receptor mRNA and protein levels thus allows the sustained effects of chronic elevations in Ang II to elicit progressive increases in arterial pressure.

The AT₁ receptor is 1 of 2 major Ang II receptor subtypes, which has been characterized pharmacologically⁹ and cloned.^{10 11} In rodents, 2 subtypes of AT₁ receptors exist (AT_1A, AT_1B).¹² The AT_1A and AT_1B receptor subtypes are >95% homologous at the amino acid level, but their tissue distributions and chromosomal locations are different. In the rat kidney, AT_1A mRNA expression has been reported to be 3- to 8-fold higher than AT_1B mRNA levels.^{5 13 14 15} Using RT-PCR techniques, Bouby et al¹⁶ reported that AT_1A mRNA was the predominant subtype in all microdissected nephron segments, with the exception of the glomerulus, where AT_1B mRNA levels were higher than AT_1A mRNA levels. In agreement with the AT₁ mRNA localization, we have previously shown by immunohistochemical analysis that the AT₁ receptor protein is present in all nephron segments.⁷ The liver is almost exclusively of the AT_1A subtype.^{13 14} Therefore, the AT_1A receptor subtype is involved in the majority of the hepatic and renal effects of Ang II. However, the adrenal gland expresses equal or 2-fold higher AT_1B mRNA levels compared with AT_1A mRNA.^{13 14} In the present study, the monoclonal and polyclonal antibodies used for Western blot analysis detect both the AT_1A and AT_1B receptors. The peptide sequences used to generate the antibodies were from the amino-terminal extracellular tail of the protein in which both the AT_1A and AT_1B amino acids sequences are identical. The results of the Western blot analysis using these 2 different AT₁ antibodies indicated that kidney and liver AT₁ protein levels were not significantly altered by chronic Ang II infusion. To determine the regulation of the predominant AT₁ receptor–subtype mRNA expression, RT-PCR and slot blot analyses were performed using an AT_1A-specific oligonucleotide. The nucleotide sequences chosen were from the 3' untranslated region of the AT_1A cDNA.⁵ Interestingly, the 5' and 3' flanking sequences of the AT_1B cDNA are only 35% identical with the AT_1A cDNA.¹⁷ Therefore, this study permitted us to assess AT₁ receptor expression at both the protein and mRNA levels to determine the contribution of regulation of the AT₁ receptor at the level of gene transcription as well as at the level of protein expression.

In vitro studies have demonstrated that AT₁ mRNA levels are altered in response to Ang II. Decreases in AT₁ mRNA have been described in aortic vascular smooth muscle cells¹⁸ and cultured rat glomerular mesangial cells,¹⁹ whereas increases were reported in cultured rabbit proximal tubule cells.²⁰ These data suggest that the regulation of AT₁ mRNA by vascular and tubular structures within the kidney may be differentially regulated and demonstrate that the regulation of the receptor by its ligand may be cell or tissue specific, or both.

Angiotensin-dependent hypertensive animal models have also been used to address the regulation of the AT₁ receptor by alterations in the components of the renin-angiotensin system. In 2K1C Goldblatt hypertensive animals, AT_1A receptor mRNA levels were not changed in the contralateral intact kidneys but were decreased in the clipped kidneys 2 days²¹ and 1 week after clipping.²² AT_1A, but not AT_1B, mRNA levels were decreased in both kidneys 4 weeks after clipping in 2K1C rats.¹⁵ Decreases in vascular and glomerular AT₁ receptor density have been reported in both the clipped and nonclipped kidney of 2K1C rats.²³ In contrast, AT₁ receptor mRNA was elevated in both the clipped and nonclipped kidneys after 10 weeks of 2K1C.²² The renal expression of the AT₁ receptor during the development of 2K1C hypertension may exhibit a temporal profile. Elevated expression of the renal AT₁ receptor during the latter stages of 2K1C hypertension may explain the maintenance of an elevated blood pressure at a time when plasma renin activity is no longer elevated. In the present study, after 2 weeks of elevations in Ang II, overall kidney AT₁ receptor mRNA and protein levels were maintained at levels similar to vehicle-infused animals, suggesting that the continued synthesis of the AT₁ receptor enabled the sustained effects of circulating Ang II on arterial blood pressure.

Previous studies have evaluated the effects of chronic infusions of Ang II on renal AT₁ receptor expression. Wang et al²⁴ reported that 2 weeks of Ang II infusion at doses that do not elevate blood pressure decreases both AT_1A and AT_1B mRNA levels and AT₁ receptor density in the rat kidney. However, Sechi et al²⁵ showed that although renal AT₁ mRNA levels were similar in rats infused with Ang II for 1 week compared with vehicle-infused rats, Ang II binding was slightly but significantly decreased by 15%. Ang II infusion has been shown to increase²⁶ and decrease²⁷ the affinity and number of glomerular Ang II–binding sites. Amiri and Garcia²⁸ demonstrated that 7-day infusions of nonpressor or pressor doses of Ang II did not alter preglomerular or glomerular Ang II receptor density. Therefore, there is continued uncertainty regarding the effects of chronic infusions of Ang II on renal AT₁ mRNA expression. This may be due to the duration and dose of Ang II infusion, level of blood pressure, the methodologies used to measure AT₁ mRNA and density, or a combination. It is possible that the direct effects of Ang II on the expression of its receptor may be offset by the ambient blood pressure. It has been shown that infusion of Ang II that did not produce a change in blood pressure did not alter AT₁ mRNA in aorta and mesenteric arteries.²⁹ However, pressor infusion of Ang II increased AT₁ mRNA in these vessels.²⁹ Less is known about the organ-specific regulation of the AT₁ receptor during Ang II infusions that produce hypertension.

Several studies, including our own, have failed to detect changes in AT₁ mRNA levels in the kidney in response to alterations in circulating Ang II levels. This failure to detect changes in the total kidney tissue may reflect opposing changes in glomerular/vascular and tubular AT₁ mRNA, as has been shown in response to low sodium diet and chronic AT₁ receptor blockade in rabbits.²⁰ However, the present study did not address the regional and segment-specific regulation of intrarenal AT₁ receptor expression during the development of Ang II–induced hypertension.

In response to Ang II, the adrenal gland has been shown to increase Ang II receptors.³⁰ In the rat adrenal cortex, AT_1A mRNA increased in response to salt depletion, whereas AT_1B mRNA decreased.^{5 31 32} However, opposite findings have also been reported in the adrenal gland in response to low salt diet.¹⁴ After 2 weeks of Ang II infusion in the rat, Iwai and Inagami³³ found that adrenal AT₁ mRNA levels were elevated but were not altered in the kidney. Our findings are in agreement with most of the studies that reported that the expression of the AT₁ receptor in the adrenal gland is upregulated by its ligand. We found a modest (36%) but significant increase in adrenal AT_1A gene expression in Ang II–infused animals with use of the sensitive technique of RT-PCR; although slot blot analysis did not detect these differences.

Significant reductions in body weight in Ang II–infused rats (350 ng · kg^-1 · min^-1, 2 weeks) have been reported to be due to increased peripheral metabolism that is independent of elevations in blood pressure.³⁴

In summary, chronic Ang II infusion in the rat that produces a significant elevation in arterial pressure is accompanied by elevations in adrenal AT_1A mRNA levels and the maintenance of kidney and liver AT₁ mRNA and protein levels. The combined effects of elevations in circulating Ang II levels and preservation of the AT₁ receptor in these organs contribute to the hypertensinogenic effects of Ang II.

	Acknowledgments

 
This work was supported by the Shaul G. Massry, MD, National Kidney Foundation Young Investigator Grant; the American Heart Association, Louisiana Affiliate (L.M.H.-B.); and National Heart, Lung, and Blood Institute Grant HL-26371 (L.G.N.). S.S.E.-D. is supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-53595 and Grant-in-Aid 96-008140 from the American Heart Association, National Center. S.S.E.-D. is a recipient of a Clinical Scientist Award from the National Kidney Foundation. The monoclonal antibody was generously provided by Gavin P. Vinson (University of London). The authors would like to acknowledge Susana Dipp, MS, for excellent technical assistance.

Received September 15, 1998; first decision October 15, 1998; accepted October 28, 1998.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. von Thun AM, Vari RC, El-Dahr SS, Navar LG. Augmentation of intrarenal angiotensin II levels by chronic angiotensin II infusion. Am J Physiol. 1994;266:F120–F128.[Abstract/Free Full Text]

2. Zou LX, Imig JD, von Thun AM, Hymel A, Ono H, Navar LG. Receptor-mediated intrarenal angiotensin II augmentation in angiotensin II-infused rats. Hypertension. 1996;28:669–677.[Abstract/Free Full Text]

3. Zou LX, Hymel A, Imig JD, Navar LG. Renal accumulation of circulating angiotensin II in angiotensin II-infused rats. Hypertension. 1996;27:658–662.[Abstract/Free Full Text]

4. von Thun AM, El-Dahr SS, Vari RC, Navar LG. Modulation of renin-angiotensin and kallikrein gene expression in experimental hypertension. Hypertension. 1994;23(suppl I):I-131–I-136.

5. Kitami Y, Okura T, Marumoto K, Wakamiya R, Hiwada K. Differential gene expression and regulation of type-1 angiotensin II receptor subtypes in the rat. Biochem Biophys Res Commun. 1992;188:446–452.[Medline] [Order article via Infotrieve]

6. Tso JY, Sun XH, Kao TH, Reece KS, Wu R. Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res. 1985;13:2485–502.[Abstract/Free Full Text]

7. Harrison-Bernard LM, Navar LG, Ho MM, Vinson GP, El-Dahr SS. Immunohistochemical localization of angiotensin II AT₁ receptor in the adult rat kidney using a monoclonal antibody. Am J Physiol. (Renal Physiol). 1997;273:F170–F177.

8. Lowry OH, Rosebrough NJ, Farr AL, Randal RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265–275.[Free Full Text]

9. Bumpus FM, Catt KJ, Chiu AT, DeGasparo M, Goodfriend T, Husain A, Peach MJ, Taylor DG Jr, Timmermans PB. Nomenclature for angiotensin receptors: a report of the Nomenclature Committee of the Council for High Blood Pressure Research. Hypertension. 1991;17:720–721.[Free Full Text]

10. Murphy TJ, Alexander RW, Griendling KK, Runge MS, Bernstein KE. Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor. Nature. 1991;351:233–236.[Medline] [Order article via Infotrieve]

11. Sasaki K, Yamano Y, Bardhan S, Iwai N, Murray JJ, Hasegawa M, Matsuda Y, Inagami T. Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type-1 receptor. Nature. 1991;351:230–233.[Medline] [Order article via Infotrieve]

12. Sasamura H, Hein L, Krieger JE, Pratt RE, Kobilka BK, Dzau VJ. Cloning, characterization, and expression of two angiotensin receptor (AT-1) isoforms from the mouse genome. Biochem Biophys Res Commun. 1992;185:253–259.[Medline] [Order article via Infotrieve]

13. Llorens-Cortes C, Greenberg B, Huang H, Corvol P. Tissular expression and regulation of type 1 angiotensin II receptor subtypes by quantitative reverse transcriptase-polymerase chain reaction analysis. Hypertension. 1994;24:538–548.[Abstract/Free Full Text]

14. Schmid C, Castrop H, Reitbauer J, Della Bruna R, Kurtz A. Dietary salt intake modulates angiotensin II type 1 receptor gene expression. Hypertension. 1997;29:923–929.[Abstract/Free Full Text]

15. Haefliger J-A, Bergonzelli G, Waeber G, Aubert J-F, Nussberger J, Gavras H, Nicod P, Waeber B. Renin and angiotensin II receptor gene expression in kidneys of renal hypertensive rats. Hypertension. 1995;26:733–737.[Abstract/Free Full Text]

16. Bouby N, Hus-Citharel A, Marchetti J, Bankir L, Corvol P, Llorens-Cortes C. Expression of type 1 angiotensin II receptor subtypes and angiotensin II-induced calcium mobilization along the rat nephron. J Am Soc Nephrol. 1997;8:1658–1667.[Abstract]

17. Elton TS, Stephan CC, Taylor GR, Kimball MG, Martin MM, Durand JN, Oparil S. Isolation of two distinct type I angiotensin II receptor genes. Biochem Biophys Res Commun. 1992;184:1067–1073.[Medline] [Order article via Infotrieve]

18. Lassegue B, Alexander RW, Nickenig G, Clark M, Murphy TJ, Griendling KK. Angiotensin II down-regulates the vascular smooth muscle AT₁ receptor by transcriptional and post-transcriptional mechanisms: evidence for homologous and heterologous regulation. Mol Pharmacol. 1995;48:601–609.[Abstract]

19. Makita N, Iwai N, Inagami T, Badr KF. Two distinct pathways in the down-regulation of type-1 angiotensin II receptor gene in rat glomerular mesangial cells. Biochem Biophys Res Commun. 1992;185:142–146.[Medline] [Order article via Infotrieve]

20. Cheng HF, Becker BN, Burns KD, Harris RC. Angiotensin II upregulates type-1 angiotensin II receptors in renal proximal tubule. J Clin Invest. 1995;95:2012–2019.

21. Della Bruna R, Bernhard I, Gess B, Schricker K, Kurtz A. Renin gene and angiotensin II AT1 receptor gene expression in the kidneys of normal and of two-kidney/one-clip rats. Pflugers Arch. 1995;430:265–272.[Medline] [Order article via Infotrieve]

22. Modrall JG, Quinones MJ, Frankhouse JH, Hsueh WA, Weaver FA, Kedes L. Upregulation of angiotensin II type 1 receptor gene expression in chronic renovascular hypertension. J Surg Res. 1995;59:135–140.[Medline] [Order article via Infotrieve]

23. Amiri F, Garcia R. Renal angiotensin II receptor regulation in two-kidney, one clip hypertensive rats: effect of ACE inhibition. Hypertension. 1997;30:337–344.[Abstract/Free Full Text]

24. Wang DH, Du Y, Yao A, Hu Z. Regulation of type 1 angiotensin II receptor and its subtype gene expression in kidney by sodium loading and angiotensin II infusion. J Hypertens. 1996;14:1409–1415.[Medline] [Order article via Infotrieve]

25. Sechi LA, Griffin CA, Giacchetti G, Valentin JP, Llorens-Cortes C, Corvol P, Schambelan M. Tissue-specific regulation of type 1 angiotensin II receptor mRNA levels in the rat. Hypertension. 1996;28:403–408.[Abstract/Free Full Text]

26. Douglas JG. Subpressor infusions of angiotensin II alter glomerular binding, prostaglandin E₂, and cyclic AMP production. Hypertension. 1987;9(suppl III):III-49–III-56.

27. Kitamura E, Kikkawa R, Fujiwara Y, Imai T, Shigeta Y. Effect of angiotensin II infusion on glomerular angiotensin II receptor in rats. Biochim Biophys Acta. 1986;85:309–16.

28. Amiri F, Garcia R. Differential regulation of renal glomerular and preglomerular vascular angiotensin II receptors. Am J Physiol. (Endocr Metab). 1996;270:E810–E815.

29. Wang DH, Du Y. Regulation of vascular type 1 angiotensin II receptor in hypertension and sodium loading: role of angiotensin II. J Hypertens. 1998;16:467–475.[Medline] [Order article via Infotrieve]

30. Hanger RL, Aguilera G, Catt KJ. Angiotensin II regulates its receptor sites in the adrenal glomerulosa. Nature (Lond). 1978;27:176–178.

31. Iwai N, Yamano Y, Chaki S, Konishi F, Bardhan S, Tibbetts C, Sasaki K, Hasegawa M, Matsuda Y, Inagami T. Rat angiotensin II receptor: cDNA sequence and regulation of the gene expression. Biochem Biophys Res Commun. 1991;177:299–304.[Medline] [Order article via Infotrieve]

32. Iwai N, Inagami T, Ohmichi N, Nakamura Y, Saeki Y, Kinoshita M. Differential regulation of rat AT1a and AT1b receptor mRNA. Biochem Biophys Res Commun. 1992;188:298–303.[Medline] [Order article via Infotrieve]

33. Iwai N, Inagami T. Regulation of the expression of the rat angiotensin II receptor mRNA. Biochem Biophys Res Commun. 1992;182:1094–1099.[Medline] [Order article via Infotrieve]

34. Cassis LA, Marshall DE, Fettinger MJ, Rosenbluth B, Lodder RA. Mechanisms contributing to angiotensin II regulation of body weight. Am J Physiol. (Endocr Metab). 1998;37:E867–E876.

This article has been cited by other articles:

				D. Zhao, D. M. Seth, and L. G. Navar Enhanced Distal Nephron Sodium Reabsorption in Chronic Angiotensin II-Infused Mice Hypertension, July 1, 2009; 54(1): 120 - 126. [Abstract] [Full Text] [PDF]

				W. Shao, D. M. Seth, and L. G. Navar Augmentation of endogenous intrarenal angiotensin II levels in Val5-ANG II-infused rats Am J Physiol Renal Physiol, May 1, 2009; 296(5): F1067 - F1071. [Abstract] [Full Text] [PDF]

				L. M. Bivol, M. Hultstrom, O. A. Gudbrandsen, R. K. Berge, and B. M. Iversen Tetradecylthioacetic acid downregulates cyclooxygenase 2 in the renal cortex of two-kidney, one-clip hypertensive rats Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2008; 295(6): R1866 - R1873. [Abstract] [Full Text] [PDF]

				A. K. Sampson, K. M. Moritz, E. S. Jones, R. L. Flower, R. E. Widdop, and K. M. Denton Enhanced Angiotensin II Type 2 Receptor Mechanisms Mediate Decreases in Arterial Pressure Attributable to Chronic Low-Dose Angiotensin II in Female Rats Hypertension, October 1, 2008; 52(4): 666 - 671. [Abstract] [Full Text] [PDF]

				C. Hu, A. Dandapat, L. Sun, M. R. Marwali, N. Inoue, F. Sugawara, K. Inoue, Y. Kawase, K.-i. Jishage, H. Suzuki, et al. Modulation of Angiotensin II-Mediated Hypertension and Cardiac Remodeling by Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 Deletion Hypertension, September 1, 2008; 52(3): 556 - 562. [Abstract] [Full Text] [PDF]

				H. Kobori, M. Nangaku, L. G. Navar, and A. Nishiyama The Intrarenal Renin-Angiotensin System: From Physiology to the Pathobiology of Hypertension and Kidney Disease Pharmacol. Rev., September 1, 2007; 59(3): 251 - 287. [Abstract] [Full Text] [PDF]

				H. Muller, N. Schweitzer, O. Johren, P. Dominiak, and W. Raasch Angiotensin II stimulates the reactivity of the pituitary-adrenal axis in leptin-resistant Zucker rats, thereby influencing the glucose utilization Am J Physiol Endocrinol Metab, September 1, 2007; 293(3): E802 - E810. [Abstract] [Full Text] [PDF]

				Y. Ozawa and H. Kobori Crucial role of Rho-nuclear factor-{kappa}B axis in angiotensin II-induced renal injury Am J Physiol Renal Physiol, July 1, 2007; 293(1): F100 - F109. [Abstract] [Full Text] [PDF]

				H. Lu, C. M. Boustany-Kari, A. Daugherty, and L. A. Cassis Angiotensin II increases adipose angiotensinogen expression Am J Physiol Endocrinol Metab, May 1, 2007; 292(5): E1280 - E1287. [Abstract] [Full Text] [PDF]

				S. D. Crowley, S. B. Gurley, M. J. Herrera, P. Ruiz, R. Griffiths, A. P. Kumar, H.-S. Kim, O. Smithies, T. H. Le, and T. M. Coffman Angiotensin II causes hypertension and cardiac hypertrophy through its receptors in the kidney PNAS, November 21, 2006; 103(47): 17985 - 17990. [Abstract] [Full Text] [PDF]

				W. J. Cheung, M.-A. H. Kent, E. El-Shahat, H. Wang, J. Tan, R. White, and F. H. H. Leenen Central and peripheral renin-angiotensin systems in ouabain-induced hypertension Am J Physiol Heart Circ Physiol, August 1, 2006; 291(2): H624 - H630. [Abstract] [Full Text] [PDF]

				C. S. Carter, G. Onder, S. B. Kritchevsky, and M. Pahor Angiotensin-Converting Enzyme Inhibition Intervention in Elderly Persons: Effects on Body Composition and Physical Performance J. Gerontol. A Biol. Sci. Med. Sci., November 1, 2005; 60(11): 1437 - 1446. [Abstract] [Full Text] [PDF]

				L. M. Bivol, O. B. Vagnes, and B. M. Iversen The renal vascular response to ANG II injection is reduced in the nonclipped kidney of two-kidney, one-clip hypertension Am J Physiol Renal Physiol, August 1, 2005; 289(2): F393 - F400. [Abstract] [Full Text] [PDF]

				J. S. Gilbert, A. L. Lang, A. R. Grant, and M. J. Nijland Maternal nutrient restriction in sheep: hypertension and decreased nephron number in offspring at 9 months of age J. Physiol., May 15, 2005; 565(1): 137 - 147. [Abstract] [Full Text] [PDF]

				C. S. Carter, M. Cesari, W. T. Ambrosius, N. Hu, D. Diz, S. Oden, W. E. Sonntag, and M. Pahor Angiotensin-Converting Enzyme Inhibition, Body Composition, and Physical Performance in Aged Rats J. Gerontol. A Biol. Sci. Med. Sci., May 1, 2004; 59(5): B416 - B423. [Abstract] [Full Text] [PDF]

				C. Suarez, G. Diaz-Torga, A. Gonzalez-Iglesias, C. Cristina, and D. Becu-Villalobos Upregulation of angiotensin II type 2 receptor expression in estrogen-induced pituitary hyperplasia Am J Physiol Endocrinol Metab, May 1, 2004; 286(5): E786 - E794. [Abstract] [Full Text] [PDF]

				M Otsuka, H Takahashi, M Shiratori, H Chiba, and S Abe Reduction of bleomycin induced lung fibrosis by candesartan cilexetil, an angiotensin II type 1 receptor antagonist Thorax, January 1, 2004; 59(1): 31 - 38. [Abstract] [Full Text] [PDF]

				F. Aguilar, M. Lo, B. Claustrat, J. M. Saez, J. Sassard, and J. Y. Li Hypersensitivity of the Adrenal Cortex to Trophic and Secretory Effects of Angiotensin II in Lyon Genetically-Hypertensive Rats Hypertension, January 1, 2004; 43(1): 87 - 93. [Abstract] [Full Text] [PDF]

				L. M. Harrison-Bernard, I. H. Schulman, and L. Raij Postovariectomy Hypertension Is Linked to Increased Renal AT1 Receptor and Salt Sensitivity Hypertension, December 1, 2003; 42(6): 1157 - 1163. [Abstract] [Full Text] [PDF]

				J.-Y. Qian, A. Haruno, Y. Asada, T. Nishida, Y. Saito, T. Matsuda, and H. Ueno Local Expression of C-Type Natriuretic Peptide Suppresses Inflammation, Eliminates Shear Stress-Induced Thrombosis, and Prevents Neointima Formation Through Enhanced Nitric Oxide Production in Rabbit Injured Carotid Arteries Circ. Res., November 29, 2002; 91(11): 1063 - 1069. [Abstract] [Full Text] [PDF]

				Y. Xu, D. Kumar, J. R. B. Dyck, W. R. Ford, A. S. Clanachan, G. D. Lopaschuk, and B. I. Jugdutt AT1 and AT2 receptor expression and blockade after acute ischemia-reperfusion in isolated working rat hearts Am J Physiol Heart Circ Physiol, April 1, 2002; 282(4): H1206 - H1215. [Abstract] [Full Text] [PDF]

				J. D. Imig, X. Zhao, J. H. Capdevila, C. Morisseau, and B. D. Hammock Soluble Epoxide Hydrolase Inhibition Lowers Arterial Blood Pressure in Angiotensin II Hypertension Hypertension, February 1, 2002; 39(2): 690 - 694. [Abstract] [Full Text] [PDF]

				L. Cervenka, H. J. Kramer, J. Maly, and J. Heller Role of nNOS in Regulation of Renal Function in Angiotensin II-Induced Hypertension Hypertension, August 1, 2001; 38(2): 280 - 285. [Abstract] [Full Text] [PDF]

				Y. Quiroz, H. Pons, K. L. Gordon, J. Rincon, M. Chavez, G. Parra, J. Herrera-Acosta, D. Gomez-Garre, R. Largo, J. Egido, et al. Mycophenolate mofetil prevents salt-sensitive hypertension resulting from nitric oxide synthesis inhibition Am J Physiol Renal Physiol, July 1, 2001; 281(1): F38 - F47. [Abstract] [Full Text] [PDF]

				H. Kobori, L. M. Harrison-Bernard, and L. G. Navar Enhancement of Angiotensinogen Expression in Angiotensin II-Dependent Hypertension Hypertension, May 1, 2001; 37(5): 1329 - 1335. [Abstract] [Full Text] [PDF]

				H. KOBORI, L. M. HARRISON-BERNARD, and L. G. NAVAR Expression of Angiotensinogen mRNA and Protein in Angiotensin II-Dependent Hypertension J. Am. Soc. Nephrol., March 1, 2001; 12(3): 431 - 439. [Abstract] [Full Text]

				J. Maly, L. Karasova, M. Simova, S. Vitko, and S. S. El-Dahr Angiotensin II-Induced Hypertension in Bradykinin B2 Receptor Knockout Mice Hypertension, March 1, 2001; 37(3): 967 - 973. [Abstract] [Full Text] [PDF]

				A. Lacroix, N. N'Diaye, J. Tremblay, and P. Hamet Ectopic and Abnormal Hormone Receptors in Adrenal Cushing's Syndrome Endocr. Rev., February 1, 2001; 22(1): 75 - 110. [Abstract] [Full Text]

				A. Nishiyama, T. Fukui, Y. Fujisawa, M. Rahman, R.-X. Tian, S. Kimura, and Y. Abe Systemic and Regional Hemodynamic Responses to Tempol in Angiotensin II-Infused Hypertensive Rats Hypertension, January 1, 2001; 37(1): 77 - 83. [Abstract] [Full Text] [PDF]

				S. Engeli, R. Negrel, and A. M. Sharma Physiology and Pathophysiology of the Adipose Tissue Renin-Angiotensin System Hypertension, June 1, 2000; 35(6): 1270 - 1277. [Abstract] [Full Text] [PDF]

				L. M. Harrison-Bernard, J. Zhuo, H. Kobori, M. Ohishi, and L. G. Navar Intrarenal AT1 receptor and ACE binding in ANG II-induced hypertensive rats Am J Physiol Renal Physiol, January 1, 2002; 282(1): F19 - F25. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Harrison-Bernard, L. M. Articles by Navar, L. G. Search for Related Content PubMed PubMed Citation Articles by Harrison-Bernard, L. M. Articles by Navar, L. G. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Medline Plus Health Information High Blood Pressure