AJP - Heart Watch the video to see how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 274: H719-H727, 1998;
0363-6135/98 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lu, D.
Right arrow Articles by Raizada, M. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lu, D.
Right arrow Articles by Raizada, M. K.
Vol. 274, Issue 2, H719-H727, February 1998

SPECIAL COMMUNICATION
Attenuation of ANG II actions by adenovirus delivery of AT1 receptor antisense in neurons and SMC

Di Lu, Hong Yang, and Mohan K. Raizada

Department of Physiology, University of Florida, Gainesville, Florida 32610

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Both central and peripheral renin-angiotensin systems (RAS) are important in the development and establishment of hypertension. Thus, introducing genes relevant to RAS into neuronal and vascular smooth muscle (VSM) cells, two major targets for angiotensin (ANG) II action, is a prerequisite in considering a gene therapy approach for the control of ANG-dependent hypertension. In this study, we explored the use of adenoviral (Ad) vector to transfer AT1 receptor antisense cDNA (AT1R-AS) into neuronal and VSM cells with the anticipation of attenuation of ANG II-mediated cellular actions. Incubation of neurons and VSM cells with viral particles containing AT1R-AS (Ad-AT1R-AS) resulted in a robust expression of AT1R-AS in a majority (~80%) of the cells. The expression was persistent for at least 28 days and was associated with decreases in the immunoreactive AT1 receptor protein and the maximal binding for AT1 receptor in a time- and dose-dependent manner in both cell types. ANG II stimulation of [3H]thymidine incorporation in VSM cells and norepinephrine transporter gene expression in neuronal cells were attenuated by Ad-AT1R-AS infection. Uninfected cells or cells infected with adenovirus particles containing a mutant AT1 receptor sense cDNA showed no effects on either AT1 receptor or on attenuation of ANG II's cellular affects. These observations show, for the first time, that adenovirus can be used to deliver AT1 receptor mutant sense and antisense cDNAs into two major ANG II target tissues. This consequently influences AT1 receptor-mediated cellular actions of ANG II.

gene transfer; adenoviral vector; AT1 receptor; antisense cDNA; neurons; vascular smooth muscle cell; angiotensin; thymidine; norepinephrine

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

IT IS WELL ESTABLISHED THAT both the brain and the peripheral angiotensin (ANG) systems play important roles in the development and establishment of hypertension (9, 12, 17, 50). Evidence for this has been derived, for the most part, from studies with animal models of hypertension. For example, experiments with the spontaneously hypertensive rat (SHR), a genetic model for human essential hypertension, have indicated that inhibitors of angiotensin-converting enzyme, such as captopril, are excellent antihypertensive agents (9, 12, 17, 50). They affect both peripheral and brain ANG systems (2, 9, 12, 17, 48, 50). In addition, losartan, an AT1 receptor-specific antagonist, has been proven to be an effective antihypertensive agent (27, 42-44). Its peripheral or central administration has been shown to be highly effective in lowering the blood pressure in the SHR (25, 30, 44, 45). Studies with the renovascular hypertensive rat, a model for human renovascular hypertension, have indicated that both the central and the peripheral ANG are important and that the establishment of high blood pressure could be attenuated by angiotensin-converting enzyme or AT1 receptor antagonists (2, 9, 12, 17, 25, 30, 44, 45, 48, 50).

Recent clinical trials have indicated that the AT1 receptor antagonists could be the drug of choice for the treatment of ANG-dependent hypertension, since they are highly effective in both young and old patients, equally potent for all three forms (mild, moderate, and severe) of hypertension, and do not produce adverse effects that are characteristic of other drugs targeted for the ANG system. In spite of a promising future, they suffer from a major drawback of "compliance," typical of any pharmacological agent, since they must be administered daily to control blood pressure. In addition, pharmacological intervention is temporary and does not provide a cure for this pathophysiological state. One way to resolve these problems would be to devise strategies that interfere with the AT1 receptor function at a genetic level by taking advantage of gene transfer and gene delivery technologies. The view that genetic intervention could be used successfully in the long-term control of ANG II actions is supported by the following: 1) an association of the AT1 receptor encoding gene polymorphism with hypertension in both humans and rats has been indicated (3, 33); 2) disruption of the AT1 receptor expression in mice causes blunting of pressor responses to ANG II (14); and 3) antisense oligonucleotides to the AT1 receptor reduce high blood pressure in the SHR (29, 49).

On the basis of the above discussion, it is evident that gene transfer strategies targeted to deliver the AT1 receptor antisense into cells could be used in the control of cellular and physiological actions of ANG II. This view is further strengthened by recent observations that adenoviral (Ad)-based vectors can be successfully used to deliver various physiologically relevant genes into cells in vitro and in vivo (4, 7, 10, 6). We decided to first study the usefulness of Ad vector in the control of the AT1 receptor and ANG II-mediated cellular actions in two major ANG II target tissues, i.e., neurons and vascular smooth muscle (VSM) cells, as a prelude to our long-term objective to control hypertension in adult animals. The observations presented here demonstrate that viral particles containing AT1 receptor antisense cDNA (Ad-AT1R-AS) can successfully deliver AT1R-AS into both neurons and VSM cells. As a consequence, the AT1 receptor-mediated cellular actions of ANG II are attenuated.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

One-day-old Wistar-Kyoto normotensive rats (WKY) were obtained from our breeding colony, which originated from Harlan Sprague Dawley (Indianapolis, IN). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, and 1× crystallized trypsin were from GIBCO-BRL (Grand Island, NY). Plasma-derived horse serum and ANG II were from Sigma Chemical (St. Louis, MO). Losartan potassium was a gift from Du Pont-Merck (Wilmington, DE), and PD-123319 was from RBI (Natick, MA). [32P]dCTP (3,000 Ci/mmol), [3H]norepinephrine (NE; 10.4 Ci/mmol), and [3H]thymidine (TdR; 10 Ci/mmol) were purchased from NEN (Boston, MA). The reverse transcriptase (RT)-polymerase chain reaction (PCR) kit was from Perkin-Elmer Cetus (Norwalk, CT). Ribonuclease (RNase) inhibitor, dNTP, rhodamine-dUTP, and other reagents for RT in situ PCR were purchased from Boehringer-Mannheim Biochemicals (Indianapolis, IN). Poly(A)+ RNA purification kit containing Dynal-bead was from Dynal (Lake Success, NY). DNA T4 ligase and other cloning reagents were purchased from Promega (Madison, WI), and restriction enzymes were from New England Biolab (Beverly, MA). Rabbit anti-AT1 receptor polyclonal antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibody to the AT1 receptor was supplied by Dr. G. Vinson, University of London. Characterization of this antibody has previously been established (1). All other reagents were purchased from Fisher Scientific (Pittsburgh, PA) and were the highest quality available.

Primers for AT1B receptor, AT2 receptor, and NE transporter (NET) were based on published sequences (16, 19-22, 26, 28) and were synthesized in the DNA Synthesis Facility of the Interdisciplinary Center for Biotechnology Research, University of Florida. The sequences of these primers have been described previously (19-22).

Construction of Ad-AT1R Mutant Sense and Antisense Recombinants

AT1 receptor cDNA was generated by RT-PCR with the use of AT1B receptor-specific primers essentially as described previously (20, 22) except that one base "c" was deleted from the sense primer to produce a nonsense mRNA that could not be translated into an active AT1 receptor protein. This served as a sense control and was called Ad-AT1R-mS. The sequence for this sense primer was 5'-ATGGCCodot TTAACTCTTCTGCTGAAGATGGTATCAAAA-3'; odot  designates the position of the missing c. The sequence for the antisense primer was AGTGTGCTTTGAACCTGTCACTCCACCTCAAAACAA-3. The cDNA was ~1.2 kb in length, included the entire coding region of the receptor (nuclear transcript -132 to ±1,128), and was further characterized by restriction enzyme analysis and sequencing (20, 22).

The DNA vector of adenovirus was kindly provided by Dr. Bruce Trapnell of Genetic Therapy (Gaithersburg, MD). The rationale for the choice of this vector was based on the reports of its ability to deliver genes under various in vivo conditions (4, 6, 7, 10, 24, 46). The Ad vector was digested with a restriction enzyme, EcoR V, and ligated with the AT1B receptor mutant sense cDNA (Ad-AT1R-mS) or antisense cDNA (Ad-AT1R-AS) using standard protocol (46). After transformation into bacteria, the colonies were selected, and orientations, forward (mutant sense) or reversal (antisense), of AT1B receptor cDNA insert were analyzed by Rca I restriction enzyme analysis. The map of Ad vector is shown in Fig. 1A. Rca I digestion of the construct provided the anticipated 1-, 1.9-, and 4.4-kb bands corresponding to AT1BR-AS and the 1-, 2.8-, and 4.4-kb bands corresponding to AT1BR-mS. The orientation and configuration of the representative AT1BR cDNAs was further confirmed by sequencing of the joint regions between the vector and AT1B receptor cDNA. Viral particles containing Ad-AT1BR-mS and Ad-AT1BR-AS were generated by Genetic Therapy, essentially as outlined elsewhere (24, 46). Quantitative preparation of the viral particles for all of our experiments was carried out by the Vector Core Laboratory of the Gene Therapy Center at the University of Florida, College of Medicine.


View larger version (23K):
[in this window]
[in a new window]
  		Fig. 1.   A: map of viral particles containing AT1 receptor antisense cDNA (Ad-AT1R-AS). ITR, inverted terminal repeats; MLP, major late promoter; TPL, tripartite leader. Restriction sites of Rca I, Fok I, and cloning site EcoR V are indicated. B: restriction enzyme, Rca I, analysis of Ad-AT1R-AS. Lane 1, molecular weight markers; lane 2, Ad-AT1R-AS; lane 3, Ad-AT1R-S; lane 4, adenovirus (Ad) without AT1R-AS. C: restriction enzyme analysis of AT1R-AS. AT1R-AS was digested out of Ad-AT1R-AS using EcoR V restriction enzyme and was analyzed by Fok I restriction enzyme. Lane 1, EcoR V digestion of Ad-AT1R-AS; lane 2, Fok I digestion of AT1R-AS.

Preparation of Neuronal Cells in Primary Culture

The hypothalamus-brain stem areas of 1-day-old WKY rat brains were dissected, and brain cells were dissociated by trypsin (30, 39). The hypothalamic block contained the paraventricular nucleus and the supraoptic, anterior, lateral, posterior, dorsomedial, and ventromedial nuclei, whereas the brain stem block contained the medulla oblongata and pons. Trypsin-dissociated brain cells were plated in poly-L-lysine-precoated 35-mm tissue culture dishes (3 × 106 cells/dish) or 8-well Nunc slides (4 × 103/well) in DMEM containing 10% plasma-derived horse serum, and neuronal cultures were established essentially as described previously (30, 39). The cultures were allowed to grow for 5 days before the addition of Ad-AT1R-mS or Ad-AT1BR-AS virus at a final concentration of 1 × 109 viral particles/ml of the culture media. Cultures were allowed to grow for an additional 7 days before their use in the experiments, unless stated otherwise.

Preparation of VSM Cells in Culture

VSM cells of rat origin were kindly provided by Dr. T. Inagami of Vanderbilt University (Nashville, TN). They were dissociated by trypsin and plated onto 35-mm-diameter tissue culture dishes (1 × 105 cells) or onto 8-well Nunc slides (4 × 103 cells/well). Cultures were grown for 3 days in DMEM with 10% fetal bovine serum before infection with 5 × 109 viral particles/ml containing Ad-AT1R-mS or Ad-AT1R-AS. Cultures were allowed to grow for seven additional days before experiment, unless stated otherwise.

Measurement of mRNAs for AT1R-AS, AT2 Receptor, and NET by RT-PCR

Isolation of poly(A)+ RNA from neuronal and VSM cell cultures. Neuronal cultures were used for the measurement of mRNA for AT1R-AS, AT1 and AT2 receptors, and NET, whereas VSM cells were used for AT1R-AS and AT1 receptor mRNA. Cultures were established in 35-mm-diameter culture dishes, rinsed three times with phosphate-buffered saline, pH 7.4, and lysed by the addition of 0.5 ml of a solution made with 4 mol/l guanidium isothiocyanate, 0.01% beta -mercaptoethanol, 25 mmol/l sodium acetate, and 0.5% sarcosyl at room temperature for 10 min. The lysis solution was moved to sterile RNase-free tubes, mixed with an equal volume of H2O-saturated phenol, and incubated on ice for 15 min. This was followed by addition of 1/10 volume of chloroform. The total RNA was precipitated by isopropanol, and the resulting pellet was resuspended in Dynal-beads (dT)-25 binding buffer. Poly(A)+ RNA was isolated with the use of Dynal-beads exactly as described in the protocol provided by the company.

RT-PCR. Poly(A)+ Dynal-bead complex was suspended in 50 µl of RT solution containing 50 mmol/l tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 8.3), 75 mmol/l KCl, 3 mmol/l MgCl2, 10 mmol/l dithiothreitol (DTT), 200 ng oligo(dT) 15 (Promega, Madison, WI), and 200 nM of each dNTP. After the reaction was heated at 75°C for 10 min and cooled slowly to room temperature, 50 units Moloney murine leukemia virus RT were added and the reaction was run for 60 min at 37°C. Then the reaction was heated for 3 min at 95°C. Five microliters of this RT solution were subjected to PCR with the use of specific primers for AT1R-AS, AT2R, or NET. The PCR reaction in a total volume of 50 µl contained 20 mmol/l Tris · HCl (pH 8.75), 10 mmol/l KCl, 10 mmol/l (NH4)2SO4, 2 mmol/l MgSO4, 0.1% Triton X-100, 0.1% bovine serum albumin, 50 µmol/l of each dNTP, 20 pmol/l of sense and antisense primers, 2 units Taq DNA polymerase, 0.05 plaque-forming units DNA polymerase, and 1 µCi [32P]dCTP. PCR was performed essentially as described previously (20, 22) with each cycle at 94°C for 1 min, 58°C for 1 min, and 72°C for 2 min. PCR products were quantified on nondenaturing polyacrylamide gels as described below. This protocol has been well established to compare the relative levels of mRNA for AT1, AT2 receptors, and NET mRNA (15, 19, 21).

Nondenaturing polyacrylamide gel electrophoresis of 32P-labeled RT-PCR products. After PCR, 5 µl of samples containing 32P-labeled PCR products were mixed with 5 µl of 2× gel loading buffer [4% Ficoll 400, 20 mmol/l EDTA (pH 8.0), 0.2% sodium dodecyl sulfate (SDS), 0.05% bromphenol blue, and 0.05% xylene cyanol] and applied to a 5% acrylamide gel (29:1 acrylamide-to-bisacrylamide ratio) in a mini-cell prepared in 1× of buffer containing 89 mmol/l Tris base, 89 mmol/l boric acid, and 2 mmol/l EDTA, pH 8.0. The gel was run for 45 min at 200 V in a Bio-Rad Mini-Gel System (Bio-Rad Laboratories, Hercules, CA). Gel was decasted and wrapped in a plastic bag and exposed to an X-ray film overnight at -70°C. X-ray film was developed, and bands representing PCR products were quantitated by a Microscan 1000 Gel Analyzer essentially as described previously (19, 20, 22). beta -Actin mRNA levels were measured in parallel samples for normalization (19, 20, 22).

Measurement of 125I-[Sar1-Ile8]ANG II Binding to AT1 and AT2 Receptors

Neuronal cultures grown in 35-mm culture dishes were used for measurement of AT1 and AT2 receptor, essentially as described previously (38, 53). VSM cells were used for AT1 receptor measurement. Untreated and Ad-AT1R-mS- or Ad-AT1R-AS-treated cultures were rinsed with phosphate-buffered saline (pH 7.2) and incubated with 1 nmol/l 125I-[Sar1-Ile8]ANG II for 60 min at room temperature in the absence and presence of various concentrations of losartan (0-10,000 nmol/l). Binding assays were carried out essentially as described previously (38, 53). Scatchard analysis of the competition-inhibition data was used to calculate dissociation constant (Kd) and maximal binding (Bmax) values using the EBDA-Ligand program (Elsevier-Biosoft, Orlando, FL), essentially as described previously (38, 53). Binding of 125I[Sar1-Ile8]ANG II to AT2 receptor was measured essentially as described previously with the use of PD-123319, an AT2 receptor-specific antagonist (15, 38).

Measurement of TdR Incorporation

Untreated and Ad-AT1R-mS- or Ad-AT1R-AS-treated VSM cells in culture were preincubated with 100 nmol/l ANG II for 24 h. This was followed by labeling the cells with 0.5 µCi/ml [3H]TdR for 60 min at 37°C. Triplicate culture dishes were used for each treatment. The amount of [3H]TdR incorporated into cellular macromolecules was measured essentially as described previously (32).

Measurement of [3H]NE Uptake

Neuronal cultures were treated either with Ad-AT1R-mS or Ad-AT1R-AS for 10 days, followed by incubation with 100 nM ANG II for 4 h at 37°C. Specific uptake of [3H]NE was determined in triplicate culture dishes essentially as described previously (38).

Measurement of AT1 Receptor Protein by Immunoblotting

Immunoblotting of AT1 receptor from the neurons was carried out essentially as described previously (51, 52). Briefly, neurons grown on 100-mm dishes were treated with Ad-AT1R-AS and lysed by adding 1 ml lysis buffer (25 mmol/l Tris · HCl, pH 7.4, 150 mmol/l NaCl, 1% Triton X-100, 1% deoxycholic acid, 1 mmol/l sodium orthovanadate, 10 mmol/l sodium fluoride, 10 mmol/l sodium pyrophosphate, 2.5 mmol/l EDTA, 1 mmol/l phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 0.8 µg/ml leupeptin) and scraping cells off the culture dish. Cell lysates were centrifuged at 6,000 g for 10 min at 4°C, and the protein contents of the resulting supernatants were determined using a Bio-Rad Bradford protein assay kit (Bio-Rad, Hercules, CA). Lysates containing 400 µg protein were subjected to a immunoprecipitation protocol as follows. Lysates were incubated with 1 µg rabbit anti-AT1 receptor antibody overnight at 4°C. Immunoprecipitates were collected on protein A/G PLUS-agarose, washed three times with lysis buffer, suspended in 20 µl Laemmli's sample buffer in a boiling water bath for 3 min, electrophoresed in 10% SDS-polyacrylamide gel electrophoresis, and transferred onto nitrocellulose membrane. The membrane was blocked by 5% nonfat dry milk in 20 mmol/l Tris · HCl, pH 8.0, 150 mmol/l NaCl, and 0.05% Tween 20 for 1 h followed by incubation for 1 h at room temperature with mouse anti-AT1 receptor antibody. Protein-bound antibody was detected by incubation of the membrane with horseradish peroxidase-labeled second antibody and enhanced by chemiluminescence assay reagents. The bands recognized by the primary antibody were visualized by exposure to film (51, 52).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Neuronal and VSM cells were incubated with Ad-AT1R-AS, and the expression of AT1R-AS was determined by RT-PCR. The expression of AT1R-AS transcript was evident as early as 24 h after infection of cultures and reached maximal levels in 7-14 days (Fig. 2), at which time it was ~10-fold higher than its expression levels after 1 day. The expression of AT1R-AS, although minimal, was still evident by 28 days in both neuronal and VSM cells.


View larger version (29K):
[in this window]
[in a new window]
 

View larger version (30K):
[in this window]
[in a new window]
  		Fig. 2.   Expression of AT1R-AS in neuronal (A) and vascular smooth muscle (VSM; B) cells as a function of time. Neuronal and VSM cell cultures were infected with 1 × 109 or 5 × 109 viral particles/ml of Ad-AT1R-AS on days 5 or 3 after plating, respectively. At indicated time periods, RNA was isolated and subjected to a semiquantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) analysis essentially as described in MATERIALS AND METHODS. Top: representative autoradiogram. Bottom: quantitation of PCR bands representing AT1R-AS essentially as described previously (15). Data are means ± SE (n = 4).

Next, we studied the effects of AT1R-AS expression on AT1 receptor numbers. Figure 3, A and B, shows competition-inhibition experiments of 125I-[Sar1-Ile8]ANG II binding to neuronal and VSM cell cultures. Treatment of cultures with Ad-AT1R-AS resulted in a significant decrease in the binding in both cell types at each concentration of losartan. An average decrease of 55% was observed in Ad-AT1R-AS-treated cultures. In contrast, cultures treated with Ad-AT1R-mS showed levels of 125I-[Sar1-Ile8]ANG II binding comparable with control cultures. Scatchard analysis of the data revealed that this decrease was a result of a 54 and 60% decrease in the Bmax for 125I-[Sar1-Ile8]ANG II binding to neuronal and VSM cell AT1 receptors, respectively, rather than an effect on the Kd values. For example, Bmax values of Ad-AT1R-mS- and Ad-AT1R-AS-treated neuronal cultures were 128 ± 6.2 and 51 ± 3.4 fmol/mgprotein, respectively, whereas they were 244 ± 11 and 112 ± 9 fmol/mg protein, respectively, for VSM cells. The effect of AT1R-AS expression on 125I-[Sar1-Ile8]ANG II binding to AT1 receptor was studied as a function of time after a 24-h infection of neuronal cells. Figure 4A shows a time-dependent effect of infection with Ad-AT1R-AS and Ad-AT1R-mS on AT1 receptor binding. A 34% decrease in the binding was observed after 3 days of Ad-AT1R-AS infection, which reached a maximal decrease of 52% by day 7. This level of inhibition was maintained throughout the duration of the experiment (21 days). A similar time course of a decrease of AT1 receptor binding was observed with VSM cell cultures (Fig. 4B). No effect of Ad-AT1R-mS infection on AT1 receptor binding was observed in either cell type.


View larger version (20K):
[in this window]
[in a new window]
 

View larger version (17K):
[in this window]
[in a new window]
  		Fig. 3.   Competition of 125I-[Sar1-Ile8]ANG II binding to Ad-AT1R-AS-treated neuronal and VSM cell cultures by losartan. Neuronal (A) and VSM (B) cell cultures were treated with Ad-AT1 mutant sense (Ad-AT1R-mS) or with Ad-AT1R-AS, and binding of 125I-[Sar1-Ile8]ANG II to AT1 receptors was determined essentially as described in MATERIALS AND METHODS. Data are means ± SE (n = 4).


View larger version (16K):
[in this window]
[in a new window]
 

View larger version (14K):
[in this window]
[in a new window]
  		Fig. 4.   Effect of Ad-AT1R-AS infection on binding of 125I-[Sar1-Ile8]ANG II to AT1 receptors in neuronal and VSM cells in culture. Neuronal (A) and VSM (B) cells in cultures were treated with either Ad-AT1R-AS or Ad-AT1R-mS. At indicated time periods after infection, binding of 125I-[Sar1-Ile8]ANG II in the absence (total) or presence of 1 µmol/l losartan (nonspecific) was carried out. Data are means ± SE of specific binding (n = 4). * Significantly different (P < 0.05) from Ad-AT1R-mS-treated cultures.

Immunoblotting, with the use of AT1 receptor-specific antibody, was carried out to determine the effects of Ad-AT1R-AS treatments on AT1 receptor protein levels. Figure 5 shows that the AT1 receptor protein levels in Ad-AT1R-AS-treated neurons were significantly reduced by day 3, and a 80% decrease was observed in 14 days. A similar but less dramatic decrease in the AT1 receptor protein was observed in the Ad-AT1R-AS-treated VSM cells (Fig. 5B). The decrease in 125I-[Sar1-Ile8]ANG II binding in Ad-AT1R-AS-treated neurons was viral dose dependent. A dose of 1 × 109 viral particles/ml resulted in a 55% decrease in the binding (Fig. 6). In contrast, Ad-AT1R-mS showed no such inhibition of binding at comparable doses. A similar observation was found in Ad-AT1R-AS-treated VSM cells (data not shown).


View larger version (22K):
[in this window]
[in a new window]
 

View larger version (24K):
[in this window]
[in a new window]
  		Fig. 5.   Effect of Ad-AT1R-AS on AT1 receptor protein levels in neuronal and VSM cells in culture. Neuronal cells (A) and VSM cells (B) were treated with Ad-AT1R-AS essentially as described in the legend to Fig. 4. AT1 receptor protein levels were measured as described in MATERIALS AND METHODS. A and B, top: representative autoradiogram. A and B, bottom: quantitation of bands corresponding to AT1 receptor protein. Data are means ± SE (n = 3). * Significantly different (P < 0.05) from Ad-AT1R-S-treated cells.


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 6.   Dose-dependent decrease of 125I-[Sar1-Ile8]ANG II binding to AT1 receptors by Ad-AT1R-AS in neuronal cells. Neuronal cells were treated with indicated concentrations of Ad-AT1R-AS or with Ad-AT1R-mS for 14 days. Binding of 125I-[Sar1-Ile8]ANG II to AT1 receptors was measured essentially as described in the legend to Fig. 4. Data are means ± SE (n = 3). * Significantly different (P < 0.05) from Ad-AT1R-mS-treated cells.

Cellular effects of ANG II were studied to determine if decreases in AT1 receptors were associated with a parallel decrease in the responsiveness of these cells to ANG II. Figure 7 shows that ANG II-stimulated [3H]TdR incorporation was decreased by 68% in VSM cells that were treated with Ad-AT1R-AS for 7 days. Treatment of cultures with Ad-AT1R-mS had no such inhibitory action of [3H]TdR incorporation. In neuronal culture, ANG II stimulates NET mRNA and uptake of NE, an effect mediated by AT1 receptor (19). Incubation of neuronal cultures with 100 nM ANG II caused a 7.5-fold increase in NET mRNA (Fig. 8A) and a 4-fold increase in [3H]NE uptake (Fig. 8B). Treatment of cultures with Ad-AT1R-AS for 7 days resulted in a significant attenuation of ANG II stimulation of both NET mRNA and [3H]NE uptake (Fig. 8, A and B). This attenuation was specific since treatment with Ad-AT1R-mS was without an effect.


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 7.   Effect of Ad-AT1R-AS and Ad-AT1R-mS on ANG II stimulation of [3H]thymidine (TdR) incorporation in VSM cells. VSM cells were infected with either Ad-AT1R-mS or with Ad-AT1R-AS for 7 days essentially as described in MATERIALS AND METHODS. Untreated cultures were used as controls. Cultures were preincubated without or with 100 nM ANG II for 24 h posttreatment, followed by determination of [3H]TdR incorporation, as described in MATERIALS AND METHODS. Data are presented as means ± SE (n = 4). * Significantly different (P < 0.05) from cells that were not treated with ANG II. ** Significantly different (P < 0.05) from ANG II-treated cells. cpm, Counts/min.


View larger version (30K):
[in this window]
[in a new window]
 

View larger version (16K):
[in this window]
[in a new window]
  		Fig. 8.   A: effect of Ad-AT1R-AS and Ad-AT1R-mS on ANG II-stimulated norepinephrine transporter (NET) mRNA levels in neuronal cells. Neuronal cells were treated with either Ad-AT1R-mS or with Ad-AT1R-AS for 7 days essentially as described in MATERIALS AND METHODS. Untreated cultures (1, 2) were used as controls. After incubation for 4 h without or with 100 nM ANG II, NET mRNA levels were measured as described in MATERIALS AND METHODS. Top: representative autoradiogram. Bottom: quantitation of bands corresponding to NET mRNA. Data are means ± SE (n = 4). * Significantly different (P < 0.05) from samples without ANG II. ** Significantly different (P < 0.05) from Ad-AT1R-mS treated or untreated cells. B: effect of Ad-AT1R-AS and Ad-AT1R-mS on ANG II-stimulated [3H]norepinephrine (NE) uptake in neuronal cultures. Experimental conditions were essentially as described in legend to Fig. 7. Untreated and Ad-AT1R-mS- or Ad-AT1R-AS-treated neuronal cultures were incubated without or with 100 nM ANG II for 4 h. Specific [3H]NE uptake was measured as described in MATERIALS AND METHODS. Data are means ± SE (n = 4). * Significantly different (P < 0.05) from untreated controls. ** Significantly different (P < 0.05) from ANG II-treated control and Ad-AT1R-mS-treated cultures.

Finally, the specificity of Ad-AT1R-AS-mediated effects on the AT1 receptor system was studied. Because neuronal cultures express both AT1 and AT2 receptors, we decided to determine the effects of Ad-AT1R-AS expression on AT2 receptor. Figure 9 shows that neither the binding of 125I-[Sar1-Ile8]ANG II to AT2 receptor nor AT2 receptor mRNA was changed in Ad-AT1R-AS-treated neuronal cultures when compared with uninfected cultures or cultures infected with Ad-AT1R-mS.


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 9.   Effects of Ad-AT1R-AS on AT2 receptor binding and AT2 receptor mRNA in neuronal cultures. Neuronal cultures were incubated without or with Ad-AT1R-AS for indicated time periods. Binding of 125I-[Sar1-Ile8]ANG II to specific AT2 receptors was determined as described in MATERIALS AND METHODS. Data are presented as percent of specific binding in Ad-AT1R-AS-treated cultures. Specific binding in untreated cultures was used as 100%. RNA was isolated from parallel cultures, and levels of AT2 receptor mRNA were analyzed as described in MATERIALS AND METHODS. Data were normalized for beta -actin and presented as a percent of untreated controls. Inset, representative autoradiogram. Data are means ± SE (n = 4).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The observations presented in this report demonstrate that 1) Ad vector can be used to deliver AT1R-AS into two major ANG II target cells, i.e., neurons and VSM cells and 2) the delivery of AT1R-AS results in its expression and is associated with a significant attenuation of cellular actions of ANG II mediated by the AT1 receptor. This effect appears to be specific, since Ad particles containing a mutant form of AT1R-S show no such attenuation of ANG II actions.

Retroviral vector has been used in the past by us to deliver AT1R-AS in cultured cells with great success (20, 22). In addition, this vector has been useful in the delivery of AT1R-AS in developing normotensive rats and SHR (15). These studies have established that delivery of AT1R-AS in developing rats selectively attenuates development of high blood pressure in SHR on a long-term basis (15). This has led us to propose that a viral-mediated gene delivery system holds great potential for a long-term control of hypertension. However, there are inherent limitations with the use of retroviral vector. Retroviruses are not only known to produce mutagenic and oncogenic effects in the host cells, but they have limited ability to infect nondividing cells (8, 11). This restricts their usefulness in adult animals. Because our long-term objective has been to use a gene therapy approach in the chronic control of ANG II-dependent hypertension in adult animals and because most of the ANG II-responsive tissues contain nondividing cells and are terminally differentiated, we decided to investigate the possibility of using the Ad vector for this purpose. In fact, Ad vector has been used extensively to successfully deliver genes both in vitro and in vivo (4, 6, 7, 10), although its usefulness in the delivery of the components of the ANG II system has not been explored. The observations presented in this study demonstrate that infection of both neurons and VSM cells with Ad-AT1R-AS results in a robust expression of AT1R-AS within 24 h that lasts at least 28 days. The expression of AT1R-AS is associated with decreases in the AT1 receptor and appears to be specific for AT1R-AS. 1) Cells treated with Ad particles containing a mutant form of AT1 receptor sense cDNA do not show either any effect on AT1 receptor or levels of ANG II stimulation of TdR incorporation in VSM cells or on the NET system in the neurons. 2) Neuronal cells express both AT1 and AT2 receptor subtypes that bind ANG II with comparable affinity (39, 40). In spite of this, AT1R-AS does not influence expression of AT2 receptor. 3) Although stimulation of alpha 1-adrenergic receptors by NE increases c-fos mRNA in neuronal cultures, there is no significant change in NE-induced c-fos mRNA observed in Ad-AT1R-AS-treated neurons compared with those treated with Ad-AT1R-S (unpublished data). 4) Basal levels of mRNA for other genes, such as NET and beta -actin, were comparable between untreated or Ad-AT1R-mS-treated and Ad-AT1R-AS-treated neurons.

Expression of AT1R-AS is associated with the attenuation of the AT1 receptor-mediated cellular responses of ANG II in both cell types. AT1R-AS expression attenuates ANG II-induced [3H]TdR incorporation in VSM cells. This observation is consistent with the previous report (23) showing that losartan attenuates ANG II-stimulated TdR incorporation and VSM cell proliferation. Similarly, AT1R-AS expression attenuates ANG II stimulation of NET mRNA and [3H]NE uptake, two established neuromodulatory actions of ANG II in neurons (19). Although the relationship between the AT1R-AS expression and the AT1 receptor function is quite certain, the mechanism of their relationship remains to be established. It is tempting to suggest that attenuation of the AT1 receptor function is directly related to AT1R-AS-mediated impairment of the AT1 receptor gene expression. This would be consistent with other observations demonstrating the inhibition of targeted genes by the introduction of antisense through virally mediated or other delivery systems (5, 36). However, lacking direct evidence in favor of this, other possibilities could not be ruled out at the present time.

Rat tissues, including neurons and VSM cells, express both AT1A and AT1B receptor subtypes (18, 31, 35, 37). Experiments presented in this study were performed with the use of antisense cDNA to the AT1B receptor subtype. Because the AT1 receptor-mediated cellular actions of ANG II are attenuated by 60-70%, this would suggest that AT1BR-AS was able to influence both AT1A and AT1B receptor subtypes to some degree. This conclusion is consistent with the reported cDNA sequence homology of 95-98% between the two receptor subtypes and our previous observations (13).

Finally, the relevance of this in vitro study to the in vivo situation in the control of physiological actions of ANG II remains to be proven. Previous studies with developing rats have indicated that retrovirally mediated delivery of AT1R-AS chronically attenuates development of high blood pressure in SHR (15). This indicates that, if AT1R-AS cDNA can be delivered successfully in ANG II target tissues in vivo, it has the potential to attenuate ANG II's actions on blood pressure. Thus this study sets the stage for in vivo experiments in adult animals by demonstrating that the Ad gene delivery system can transfer AT1R-AS into tissues relevant to ANG II actions.

    ACKNOWLEDGEMENTS

We thank Elizabeth Brown for the preparation of vascular smooth muscle cells and neuronal cultures and Jennifer Brock for the preparation of the manuscript.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-56921.

Address for reprint requests: M. K. Raizada, Dept. of Physiology, College of Medicine, Univ. of Florida, PO Box 100274, Gainesville, FL 32610.

Received 8 September 1997; accepted in final form 28 October 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Barker, S., W. Marchant, M. M. Ho, J. R. Puddefoot, J. P. Hinson, A. J. L. Clark, and G. P. Vinson. A monoclonal antibody to a conserved sequence in extracellular domain recognizes the angiotensin II AT1 receptor in mammalian tissues. J. Mol. Endocrinol. 11: 241-245, 1993[Abstract].

2.   Berecek, K. H., T. Okuno, T. Nagahama, and S. Oparil. Altered vascular reactivity and baroreflex sensitivity induced by chronic central administration of captopril in the spontaneously hypertensive rat. Hypertension 5: 687-700, 1983.

3.   Bonnardeaux, A., E. Davies, X. Jeunemaitre, I. Fery, A. Charru, E. Clauser, L. Tiret, F. Cambien, P. Corvol, and F. Soubrier. Angiotensin II type 1 receptor gene polymorphisms in human essential hypertension. Hypertension 24: 63-69, 1994[Abstract/Free Full Text].

4.   Chang, M. W., E. Barr, M. M. Lu, K. Barton, and J. M. Leiden. Adenovirus-mediated over-expression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty. J. Clin. Invest. 96: 2260-2268, 1995.

5.   Chuah, M. K., T. Vandendriessche, H. K. Chang, B. Ensoli, and R. A. Morgan. Inhibition of human immunodeficiency virus type 1 by retroviral vectors expressing antisense-TAR. Hum. Gene Ther. 5: 1467-1475, 1994[Medline].

6.   Cirielli, C., T. Riccioni, C. Yang, R. Pili, T. Gloe, J. Chang, K. Inyaku, A. Passaniti, and M. C. Capogrossi. Adenovirus-mediated gene transfer of wild-type p53 results in melanoma cell apoptosis in vivo and in vitro. Int. J. Cancer 63: 673-679, 1995[Medline].

7.   Doran, S. E., B. J. Roessler, J. W. Hartman, J. T. Hoff, D. S. Shewach, and B. L. Davidson. Adenovirus-mediated in vivo gene transfer into the central nervous system of non-human primate. Clin. Neurosurg. 41: 242-257, 1994[Medline].

8.   Finkel, T., and S. E. Epstein. Gene therapy for vascular disease. FASEB J. 9: 843-851, 1995[Abstract].

9.   Giudicelli, J. F., J. L. Freslon, S. Glasson, and C. Richer. Captopril and hypertension: development in SHR. Clin. Exp. Hypertens. 2: 1083-1096, 1980.

10.   Guzman, R. J., E. A. Hirschowitz, S. L. Brody, R. G. Crystal, S. E. Epstein, and T. Finkel. In vivo suppression of injury-induced vascular smooth muscle cell accumulation using adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene. Proc. Natl. Acad. Sci. USA 91: 10732-10736, 1994[Abstract/Free Full Text].

11.   Hanaria, E. G., J. Kavanagh, G. Hortobagyi, R. E. Giles, R. Champlin, and A. B. Deisseroth. Recent advances in the application of gene therapy to human disease. Am. J. Med. 99: 537-552, 1995[Medline].

12.   Harrap, S. B., W. M. Van der Merwe, S. A. Griffin, F. Macepherson, and A. F. Lever. Brief angiotensin converting enzyme inhibitor treatment in young spontaneously hypertensive rats reduces blood pressure long-term. Hypertension 16: 603-614, 1990[Abstract/Free Full Text].

13.   Inagami, T., N. Iwai, K. Sasaki, Y. Yamano, S. Bardhan, S. Chaki, D. F. Guo, and H. Furuta. Cloning, expression, and regulation of angiotensin II receptors. In: Cellular and Molecular Biology of the Renin-Angiotensin System, edited by M. K. Raizada, M. I. Phillips, and C. Sumners. Boca Raton, FL: CRC, 1993, p. 273-292.

14.   Ito, M., M. I. Oliverio, P. J. Mannon, C. F. Bert, N. Maeda, O. Smithies, and T. M. Coffman. Regulation of blood pressure by type 1A angiotensin II receptor gene. Proc. Natl. Acad. Sci. USA 92: 3521-3535, 1995[Abstract/Free Full Text].

15.   Iyer, S. N., D. Lu, M. J. Katovich, and M. K. Raizada. Chronic control of high blood pressure in the spontaneously hypertensive rat by delivery of angiotensin type 1 receptor antisense. Proc. Natl. Acad. Sci. USA 93: 9960-9965, 1996[Abstract/Free Full Text].

16.   Kambayashi, Y., S. Bardhan, K. Takahashi, S. Tsuzuki, H. Inui, T. Hamakubo, and T. Inagami. Molecular cloning of a novel angiotensin II receptor isoform involved in phosphotyrosine phosphatase inhibition. J. Biol. Chem. 268: 24543-24546, 1993[Abstract/Free Full Text].

17.   Lee, R. M. K. W., K. H. Berecek, J. Tsoporis, R. McKenzie, and C. R. Triggle. Prevention of hypertension and vascular changes by captopril treatment. Hypertension 17: 141-150, 1991[Abstract/Free Full Text].

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

19.   Lu, D., M. R. Paddy, N. E. Rowland, and M. K. Raizada. Regulation of norepinephrine transport system by angiotensin II in neuronal cultures of normotensive and spontaneously hypertensive rat brains. Endocrinology 137: 763-772, 1996[Abstract].

20.   Lu, D., and M. K. Raizada. Delivery of angiotensin II type 1 receptor antisense inhibits angiotensin action in neurons form hypertensive rat brain. Proc. Natl. Acad. Sci. USA 92: 2914-2918, 1995[Abstract/Free Full Text].

21.   Lu, D., H. Yang, and M. K. Raizada. Angiotensin II regulation of neuromodulation: downstream signalling mechanism from activation of mitogen-activated protein kinase. J. Cell Biol. 135: 1609-1617, 1996[Abstract/Free Full Text].

22.   Lu, D., K. Yu, and M. K. Raizada. Retrovirus-mediated transfer of an angiotensin type I receptor (AT1R) antisense sequence decreases AT1Rs and angiotensin II action in astroglia and neuronal cells in primary cultures from the brain. Proc. Natl. Acad. Sci. USA 92: 1162-1166, 1995[Abstract/Free Full Text].

23.   Makita, S., S. Nakamura, H. Yoshida, and K. Hiramori. Effect of angiotensin II receptor blocker on angiotensin II stimulated DNA synthesis of cultured human aortic smooth muscle cells. Life Sci. 56: 383-388, 1995.

24.   Mittereder, N., S. Yei, C. Bachurski, J. Cuppolatti, J. A. Whitsett, P. Tolstoshev, and B. C. Trapnell. Evaluation of the efficacy and safety of in vitro adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator cDNA. Hum. Gene Ther. 5: 717-729, 1994[Medline].

25.   Mizuno, K., S. Niimura, M. Tani, H. Haga, T. Gomibuchi, H. Sanada, and S. Fukuchi. Anti-hypertensive and hormonal activity of MK954 in spontaneously hypertensive rats. Eur. J. Pharmacol. 215: 305-308, 1992[Medline].

26.   Murphy, T. J., R. W. Alexander, K. K. Griendling, M. S. Runge, and K. E. Bernstein. Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor. Nature 351: 223-236, 1991.

27.   Nelson, E., K. Arcuri, L. Ikeda, D. Snavely, and C. Sweet. Efficancy and safety of losartan in patients with essential hypertension (Abstract). Am. J. Hypertens. 5: 19A, 1992.

28.   Pacholczyk, T., R. D. Blakely, and S. G. Amara. Expression cloning of a cocaine- and antidepressant-sensitive human noradrenaline transporter. Nature 350: 350-354, 1991[Medline].

29.   Phillips, M. I., D. Wielbo, and R. Gyurko. Antisense inhibition of hypertension: a new strategy for renin-angiotensin candidate gene. Kidney Int. 46: 1554-1556, 1994[Medline].

30.   Raizada, M. K., D. Lu, and C. Sumners. AT1 receptors and angiotensin actions in the brain and neuronal cultures of normotensive and hypertensive rats. In: Current Concepts: Tissue Renin-Angiotensin System as Local Regulators in Reproductive and Endocrine Organs, edited by A. Mukhopadhyay, and M. K. Raizada. New York: Plenum, 1994, p. 331-348.

31.   Raizada, M. K., D. Lu, W. Tang, P. Kurian, and C. Sumners. Increased angiotensin type 1 receptor gene expression in neuronal cultures from spontaneously hypertensive rats. Endocrinology 132: 1715-1722, 1993[Abstract].

32.   Raizada, M. K., J. W. Yang, and R. E. Fellows. Binding of 125-I insulin to specific receptors and stimulation of nucleotide incorporation in cells cultured from rat brain. Brain Res. 200: 389-400, 1980[Medline].

33.  Rolfs, A., I. Weber-Rolfs, V. Regitz-Zagrosek, H. Kallisch, K. Riedel, and E. Fleck. Genetic polymorphisms of the angiotensin II type 1 (AT1) receptor gene. Eur. Heart J. 15, Suppl. D: 108-112, 1994.

34.   Sambrook, J., E. F. Fritsch, and T. Maniatis. Molecular Cloning, A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989.

35.   Shanmugam, S., P. Corvol, and J. M. Gase. Ontogeny of the two angiotensin II type 1 receptor subtypes in rats. Am. J. Physiol. 267 (Endocrinol. Metab. 30): E828-E836, 1994[Abstract/Free Full Text].

36.   Skotzko, M., L. Wu, W. F. Anderson, E. M. Gordon, and F. L. Hall. Retroviral vector-mediated gene transfer of antisense cyclin G1 inhibits proliferation of human osteogenic sarcome cells. Cancer Res. 55: 5493-5498, 1995[Abstract/Free Full Text].

37.   Steckelings, V., S. P. Bottari, and T. Unger. Angiotensin receptor subtypes in the brain. Trends Pharmacol. Sci. 13: 365-368, 1992[Medline].

38.   Sumners, C., T. F. Muther, and M. K. Raizada. Altered norepinephrine uptake in neuronal cultures from spontaneously hypertensive rat brain. Am. J. Physiol. 248 (Cell Physiol. 17): C488-C497, 1985[Abstract/Free Full Text].

39.   Sumners, C., and M. K. Raizada. Angiotensin II receptor subtypes in neuronal cells. In: Cellular and Molecular Biology of Renin-Angiotensin System, edited by M. K. Raizada, M. I. Phillips, and C. Sumners. Boca Raton, FL: CRC, 1993, p. 379-411.

40.   Sumners, C., M. K. Raizada, J. Kang, D. Lu, and P. Posner. Receptor mediated effects of angiotensin II on neurons. Front. Neuroendocrinol. 15: 203-230, 1995.

41.   Sumners, C., W. Tang, B. Zelenza, and M. K. Raizada. Angiotensin II receptor subtypes are coupled with distinct signal transduction mechanisms in neurons and astrocytes from rat brain. Proc. Natl. Acad. Sci. USA 88: 7567-7571, 1991[Abstract/Free Full Text].

42.  Timmermans, P. B. M. W. M., D. J. Carini, A. T. Chiu, J. V. Duncia, W. A. Price, Jr., G. J. Wells, P. C. Wong, R. R. Wexler, and A. L. Johnson. Angiotensin II receptor antagonists: from discovery to anti-hypertensive drugs. Hypertension 18, Suppl. III: 136-142, 1991.

43.   Timmermans, P. B. M. W. M., P. C. Wong, A. T. Chiu, and W. F. Herblin. Non-peptide angiotensin II receptor antagonists. Trends Pharmacol. Sci. 12: 55-62, 1991[Medline].

44.   Tofovic, S. P., A. S. Pong, and E. K. Jackson. Effects of angiotensin subtype 1 and subtype 2 receptor antagonists in normotensive and hypertensive rats. Hypertension 18: 774-784, 1991[Abstract/Free Full Text].

45.   Toney, G. M., and J. P. Porter. Effects of blockade of AT1 and AT2 receptors in brain on the central angiotensin II pressor response in conscious spontaneously hypertensive rats. Neuropharmacology 32: 581-589, 1993[Medline].

46.   Trapnell, B. C. Adenoviral vectors for gene transfer. Adv. Drug Delivery Res. 12: 185-199, 1993.

47.   Tsunoda, K., K. Abe, T. Hagino, K. Omata, S. Misawa, Y. Imai, and K. Yoshiaga. Hypotensive effect of losartan, a non-peptide angiotensin II receptor antagonist in essential hypertension. Am. J. Hypertens. 1: 28-32, 1993.

48.   Unger, T., I. Kaufman-Buhler, B. A. Scholkens, and D. Ganten. Brain-converting enzyme inhibition: a possible mechanism for the anti-hypertensive action of captopril in spontaneously hypertensive rats. Eur. J. Pharmacol. 70: 467-478, 1981[Medline].

49.   Wielbo, D., C. Sernia, R. Gyurko, and M. I. Phillips. Antisense inhibition of hypertension in the spontaneously hypertensive rat. Hypertension 25: 314-319, 1995[Abstract/Free Full Text].

50.   Wu, J.-N., and K. H. Berecek. Prevention of genetic hypertension by early treatment of spontaneously hypertensive rats with the angiotensin converting enzyme inhibitor captopril. Hypertension 22: 139-146, 1993[Abstract/Free Full Text].

51.   Yang, H., D. Lu, G. P. Vinson, and M. K. Raizada. Involvement of MAP kinase in angiotensin II-induced phosphorylation and intracellular targeting of neruonal AT1 receptors. J. Neurosci. 17: 1660-1669, 1997[Abstract/Free Full Text].

52.   Yang, H., D. Lu, K. Yu, and M. K. Raizada. Regulation of neuromodulatory actions of angiotensin II in the brain neurons by the ras-dependent mitogen-activated protein kinase pathway. J. Neurosci. 16: 4047-4058, 1996[Abstract/Free Full Text].

53.   Zelezna, B., B. Rydzewski, D. Lu, J. A. Olson, K. T. Shiverick, W. Tang, C. Sumners, and M. K. Raizada. Angiotensin II induction of plasminogen activator inhibitor-1 gene expression in astroglial cells of normotensive and spontaneously hypertensive rat brain. Mol. Endocrinol. 6: 2009-2017, 1992[Abstract].

AJP Heart Circ Physiol 274(2):H719-H727
0363-6135/98 $5.00 Copyright © 1998 the American Physiological Society


This article has been cited by other articles:


Home page
 Circ. Res.Home page
H. Wang, M. J. Katovich, C. H. Gelband, P. Y. Reaves, M. I. Phillips, and M. K. Raizada
Sustained Inhibition of Angiotensin I-Converting Enzyme (ACE) Expression and Long-Term Antihypertensive Action by Virally Mediated Delivery of ACE Antisense cDNA
Circ. Res., October 1, 1999; 85(7): 614 - 622.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
M. K. Raizada, M. J. Katovich, H. Wang, K. H. Berecek, and C. H. Gelband
Is antisense gene therapy a step in the right direction in the control of hypertension?
Am J Physiol Heart Circ Physiol, August 1, 1999; 277(2): H423 - H432.
[Full Text] [PDF]

Home page
 HypertensionHome page
C. H. Gelband, P. Y. Reaves, J. Evans, H. Wang, M. J. Katovich, and M. K. Raizada
Angiotensin II Type 1 Receptor Antisense Gene Therapy Prevents Altered Renal Vascular Calcium Homeostasis in Hypertension
Hypertension, January 1, 1999; 33(1): 360 - 365.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lu, D.
Right arrow Articles by Raizada, M. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lu, D.
Right arrow Articles by Raizada, M. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online