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NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION
Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, Mississippi 39216
Submitted 1 March 2004 ; accepted in final form 17 May 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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renin-angiotensin system; ribonucleic acid half-life; mean arterial pressure; plasma renin activity
Important regulatory factors for the regulation of angiotensinogen include glucocorticoids, estrogen, mediators of inflammatory reactions, glucagon, and prostaglandins (2, 7, 9, 25, 27). More recently, a polysomal protein was identified, which stabilizes the mRNA coding for angiotensinogen by binding to its untranslated 3'-end and which is 5- to 11-fold overexpressed in the spontaneously hypertensive rat (SHR) strain (11). However, it remains unclear whether this increase in RNA stabilization is related to the development of hypertension in the SHR. To evaluate the role of the mRNA-stabilizing protein on tissue angiotensinogen expression and blood pressure regulation in the SHR, we examined the effects of three antisense oligodeoxynucleotides (AONs) directed against the 5'-end of the cDNA coding for the polysomal protein. The effects of AONs on mean arterial pressure, plasma angiotensinogen, plasma renin activity, and tissue mRNA levels coding for angiotensinogen and the mRNA-stabilizing protein were examined in SHRs at the age of 7 wk. At this age blood pressure linearly increases in the SHR, and the mRNA-stabilizing protein is significantly (5- to 12-fold) overexpressed in various tissues with importance for the pathogenesis of hypertension. In addition, we compared the effects of AONs with the effects of converting enzyme inhibition and ANG II type 1 receptor antagonism in SHRs at the same age.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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All animal experiments in this study were approved by the Institutional Animal Care and Use Committee, and animal care complied with the Guide for the Care and Use of Laboratory Animals. Studies were conducted in male SHR or normotensive Wistar-Kyoto (WKY) rats at the age of 7 wk. Animals were kept under 24-h dark-light cycles, fed a standard rat chow, and had free access to water. Animals were purchased from Harlan Sprague Dawley (Indianapolis, IN).
Animal Instrumentation and Blood Pressure Measurement
Blood pressure was measured in conscious chronically instrumented rats after catheterization of the femoral vein and carotid artery. Two days before experimentation, animals were briefly anesthetized with isoflurane (4%) and surgically instrumented with a femoral venous (drug application) and carotid arterial (measurement of mean arterial blood pressure and blood withdrawal) catheter (PE-50) under aseptic conditions. Catheters were exteriorized at the nape of the neck and filled with heparin. The arterial line was connected to a pressure transducer and a blood pressure recorder.
Experimental Protocol I: Effect of AONs
Experiments were carried out in a blinded fashion using a color-code system for the administration of drugs and the recording of data. The experimental protocol was started after stabilization of blood pressure for 20–30 min. Subsequently, three different AONs were administered intravenously in rats of the respective experimental groups (n = 8). Rats of the control group received scrambled oligodeoxynucleotides. Animals were infused with one bolus per day with increasing dosages for 3 days (day 1: 450 µg/kg; day 2: 900 µg/kg; day 3: 1,800 µg/kg). Subsequent to each infusion, blood pressure was monitored for 5 h. Infusion volumes were 0.075–0.300 ml; sterile saline was used as vehicle. At the end of the experiment (day 4), blood samples were taken for the analysis of plasma angiotensinogen concentrations and plasma renin activity, and tissue samples were collected for the analysis of mRNA coding for angiotensinogen or the polysomal protein.
The following oligodeoxynucleotides were used in the experiment.
AONs. The AONs were 1) RNASTAAS1, 5'-G*G*C*GTAATCATGGTCATA*G*C*-3' (position 31–50); 2) RNASTAAS2, 5'-T*G*G*TCATAGCTGTTCCTG*T*G*-3' (position 21–40); and 3) RNASTAAS3, 5'-T*T*G*TCATCCGCAGGCAGG*T*C*-3'(position 143–162)
Corresponding scrambled oligodeoxynucleotides. The scrambled oligodeoxynucleotides corresponding to AONs 1–3, respectively, were 4) SCR1, 5'-T*A*C*TAGTTGGGAGCAGTC*C*A*-3'; 5) SCR2:5'-G*T*C*GGTTATTGCGTCTAC*T*G*-3'; and 6) SCR3:5'-C*A*T*CCGCTGGACCTAGTG*G*T*-3'.
Oligodeoxynucleotides were 20 mers. The first and the last three nucleotides (marked by an asterisk) were phosphothioated to increase stability and lipophilicity (tissue penetration).
Experimental Protocol II: Angiotensin-Converting Enzyme Inhibition and ANG II Type 1 Receptor Antagonism
Additional groups of rats were examined to compare the blood pressure response between AONs, converting enzyme inhibition, and ANG II type 1 receptor antagonism. At the age of 7 wk SHRs were treated for 4 days with the converting enzyme inhibitor captopril (100 mg·kg–1·day–1; n = 4) or the ANG II type I receptor antagonist L-158809 (1.5 mg·kg–1·day–1; n = 4). Drugs were administered with the drinking water. After 3 days, rats were catheterized with an arterial catheter for measurement of mean arterial pressure.
Isolation of Total RNA
Total RNA was prepared from several tissues with critical importance for the development of hypertension such as renal cortex, renal medulla, heart, brain, and liver 4 days after the administration of AONs. Total RNA was extracted from tissues employing the LiCl-urea method (15), which typically results in practically DNA-free RNA preparations. Briefly, tissues were homogenized in 3 M LiCl and 6 M urea, and total RNA was allowed to precipitate overnight at 4°C. Pellets obtained by centrifugation were washed with LiCl-urea and dissolved in 50 mM sodium acetate, pH 5.0, with 0.1% SDS. After phenolization, the RNA was ethanol precipitated overnight, washed with 80% ethanol, vacuum dried, and resuspended in diethylpyrocarbonate-treated water. The integrity of the mRNA was routinely checked on ethidium bromide-stained agarose gels. RNA content was estimated from the absorbance at 260 nm. RNA preparations were stored at –80°C until subjected to real-time RT PCR quantification.
Real-time RT-PCR
For quantitative analysis of mRNAs coding for angiotensinogen or the polysomal protein, we employed real-time RT PCR with SYBR Green as intercalating dye (24). Corresponding amounts of total RNA (4 µg) were used for first-strand synthesis. The first-strand cDNA synthesis protocol consists of denaturation at 65°C for 10 min, annealing at 25°C for 2–3 min, and extension of the cDNA sequence at 42°C, employing a poly-dt-15mer oligodeoxynucleotide and Avian Megalovirus reverse transcriptase (Promega). First-strand cDNA was further purified after alkaline hydrolysis of RNA by ispropanol precipitation. Aliquots of 1 µl of the cDNA preparations were subsequently used for further real-time PCR amplification during 40 cycles each consisting of denaturation for 30 s at 95°C and extension of the specific primers at 65°C for 1 min. Real-time PCR was carried out using an iCycler (Bio-Rad, Hercules, CA) and a SYBR Green reaction mix (Supermix, Bio-Rad). Quantification was achieved by comparing the number of cycles needed for the fluorescence of the PCR products to reach a predefined threshold value. A difference of one cycle between control and experimental sample represents a difference in the starting mRNA concentrations of a factor of two. Data were normalized for the expression of 18S rRNA using primers for amplification as published recently (24).
Primer pairs for real-time PCR. For angiotensinogen, the forward primer was 5'-GCAAAAATCAGTGCCTTCACCC-3', and the reverse primer was 5'-AAACAAACCCTCACCCCAGGAG-3'. The primer pair covers position 1547–1674 of the angiotensinogen gene, resulting in a DNA fragment of 127 bp.
For mRNA-stabilizing protein, the forward primer was 5'-TGTGATGTACGATACATTCACA-3', and the reverse primer was 5'-ACCAGTATCGACAAGGACAC-3'. The primer pair covers position 21–170 of the cDNA coding for the mRNA-stabilizing protein, resulting in a DNA fragment of 150 bp.
For 18S rRNA, the forward primer was 5'-CTTAGAGGGACAAGTGGCG-3', and the reverse primer was 5'-GGACATCTAAGGGCATCACA-3'.
Polysomal Protein Isolation
Polysome extraction and protein purification were carried out as described previously (11). Briefly, hepatic tissue was homogenized in a Dounce homogenizer and subjected to three differential centrifugation steps before the collection of pellets by ultracentrifugation (210,000 g for 3 h). Polysomal pellets were washed once, and the RNA-protein complexes were subsequently dissociated in the presence of 0.5 M KCl. The 0–40% ammonium sulfate fraction of this protein fraction was used for cross-link analysis.
Cross-Link Assay
Cross-link assays were conducted to specifically measure protein expression of the polysomal protein in polysomal protein extracts. Cross-link analysis was performed as described previously (6) using a [32P]UTP-labeled RNA fragment of 269 nucleotides coding for the 3'-untranslated region of angiotensinogen mRNA, including two putative UCCUU binding motifs and 5–10 µg of the individual protein preparations corresponding to 10 mg liver for each sample. All samples contained heparin 5 mg/ml to block unspecific binding and cross-linking. After an incubation period of 30 min at 30°C, RNA/protein complexes were irradiated for 20 min in a UV cross-linker (Spectronics). After a complete RNA digest [32P]UTP-labeled proteins were separated on a standard 12% SDS polyacrylamide gel and subjected to autoradiography.
Determination of Plasma Angiotensinogen Concentrations
Angiotensinogen plasma concentrations were measured indirectly by quantitative conversion of angiotensinogen to ANG I with an excess of hog renin and subsequent radioimmunoassay of ANG I (20).
Determination of Plasma Renin Activity
Plasma renin activity was measured after self-incubation of plasma at 37°C by an ANG I radioimmunoassay (3) utilizing an antibody obtained from Chemicon (Los Angeles, CA).
Statistics
Data are shown as means ± SE of four or eight rats per experimental group. Mean values were compared by ANOVA and, if applicable, by Bonferroni's method (P 
0.05, P 0.01, or P 0.005). Statistical significances (P values) are provided for comparisons vs. control.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Intravenous administration of AONs caused a significant decrease in mean arterial pressure, which became apparent with a time lag of 4–5 h (Fig. 1, A-C). RNASTAAS2 was the most effective, resulting in a decrease of blood pressure from 147 ± 6 mmHg in controls to 106 ± 4 mmHg after administration of the highest dose of RNASTAAS2 (P 0.005) (Fig. 1B). Oligodeoxynucleotide RNASTAAS1 also had a significant blood pressure-lowering effect (119 ± 4 mmHg, P 0.005) at the end of the experimental period (Fig. 1A), although less effective than RNASTAAS2. The least effect (132 ± 5 mmHg, P 0.05) was measured for RNASTAAS3, which targeted sequences further downstream of the untranslated 5'-end of the protein. Corresponding scrambled oligodeoxynucleotide sequences (SCR1, SCR2, SCR3) had no significant effect on mean arterial pressure. Mean arterial pressure in untreated SHR was 149 ± 7 mmHg and 152 ± 6, 146 ± 5, and 149 ± 6 mmHg after the administration of the highest dose of SCR1, SCR2, and SCR3, respectively.
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0.01) after the infusion of RNASTAAS2. The net decrease in mean arterial pressure was –14 ± 6 mmHg (P 0.01) after administration of the highest dose of RNASTAAS2 in WKY compared with –39 ± 6 mmHg (P 0.005) in SHR (Fig. 2B).
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To analyze whether antisense inhibition of the polysomal protein indeed lowers circulating angiotensinogen concentrations, we measured plasma angiotensinogen in blood samples taken at the end of the experiment. In accordance with the decreases in mean arterial pressure, all AONs administered in the study lowered angiotensinogen plasma concentrations (Fig. 3). In control SHR, plasma angiotensinogen concentrations were 780 ± 55 pmol/ml. RNASTAAS1 decreased plasma concentrations of angiotensinogen to 608 ± 42 pmol/ml (P 0.005), RNASTAAS2 to 424 ± 48 pmol/ml (P 0.005), and RNASTAAS3 to 709 ± 41 pmol/ml (P 0.05) at the end of the experiment.
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To evaluate whether the decreases in plasma angiotensinogen are related to changes in the activity of the circulating renin-angiotensin system, we determined, in addition, plasma renin activity in the same plasma samples (Fig. 4). Control SHR had an average plasma renin activity of 13.5 ± 2.1 ng ANG I·ml–1·h–1. RNASTAAS1 significantly reduced plasma renin activity to 9.1 ± 1.4 ng ANG I·ml–1·h–1 (P 0.005), RNASTAAS2 significantly reduced plasma renin activity to 6.9 ± 1.8 ng ANG I·ml–1·h–1 (P 0.005), but RNASTAAS3 had no significant effect (12.1 ± 3.1 ng ANG I·ml–1·h–1).
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Tissue mRNA concentrations were measured by real-time RT PCR. The expression of the polysomal protein was significantly reduced in all tissues examined in the current study (Table 1). The most predominant effects were measured for RNASTAAS2 in heart (18.1 ± 4.9% of control), renal cortex (29.7 ± 6.2%), and liver (37.3 ± 5.3%); weaker effects were obtained in brain (41.6 ± 2.8%) and renal medulla (52.5 ± 8.3%). All the changes obtained with RNASTAAS2 were highly significant (P 0.005). RNASTAAS 1 and RNASTAAS3 exhibited weaker effects (for details see Table 1), which were in accordance with the lower efficacy of these oligodeoxynucleotides to lower blood pressure.
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To determine whether the AON-induced inhibition of RNA expression indeed results in a reduced protein expression of the protein, we measured polysomal protein levels for the mRNA-stabilizing protein in an RNA-protein cross-link assay in the presence of an excess of [32P]UTP labeled 3'-untranslated region of angiotensinogen mRNA (Fig. 5). Semiquantitative densitometric analysis of the autoradiographs demonstrated a reduction of protein expression to <50% of the control group in livers of SHRs that received RNASTAAS2. As observed at the RNA level the effects of RNASTAAS1 and RNASTAAS3 were less expressed. A reasonably good correlation existed between protein and mRNA data.
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To determine the functional relevance of the polysomal protein for its interference with angiotensinogen expression, we measured hepatic angiotensinogen mRNA concentrations at the end of the experiment employing real-time RT PCR. Hepatic angiotensinogen mRNA was significantly (P 0.005) decreased to 68.0 ± 5.9, 49.1 ± 6.1, and 79.2 ± 5.8% of the control for RNASTAAS1, RNASTAAS2, and RNASTAAS 3, respectively (Table 1). These results related well to the decreases in plasma angiotensinogen mentioned above and confirm the constitutive type of secretion for angiotensinogen. Angiotensinogen expression in extrahepatic tissues was inhibited as well with the strongest effects observed for RNASTAAS2 (for details, see Table 1).
Antihypertensive Effects of Angiotensin Converting Enzyme Inhibition or AT1 Blockade on Mean Arterial Pressure
To determine whether antihypertensive effects as observed during antisense deoxynucleotide inhibition can be similarly produced by an inhibition of the renin-angiotensin system, we compared them to the effect of captopril (100 mg·kg–1·day–1) or L-158809 (1.5 mg·kg–1·day–1) after 4 days of treatment (Fig. 6). Administration of captopril resulted in a decrease in mean arterial pressure from 149 ± 7 mmHg in untreated SHR to 117 ± 8 mmHg (–32 mmHg, P 0.005). L-158809 decreased mean arterial pressure to 113 ± 9 mmHg (–36 mmHg, P 0.005). The effect of the pharmaceutical compounds was comparable to the effect of the most potent AON RNASTAAS2 (–41 mmHg, P 0.005), while the effects of RNASTAAS1 and RNASTAAS3 were considerably weaker (–28 mmHg, P 0.005 and –15 mmHg, P 0.01) (Fig. 6).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The experiments were carried out in SHR at the age of 7 wk because blood pressure linearly increases at this age and the mRNA-stabilizing protein is significantly (5- to 12-fold) overexpressed in various tissues with importance for the pathogenesis of hypertension such as liver, heart, brain, blood vessels, and adrenal glands. Blood pressure effects became evident with a time lag of 4–5 h, which is in accordance with the relatively short half-life of 80 min for angiotensinogen mRNA under normal physiological conditions (10), suggesting a half-life for the mRNA coding for the mRNA-stabilizing protein of 
3 h.
The measured decreases in blood pressure were in good correlation with an inhibition of plasma renin activity and plasma angiotensinogen concentrations at the end of the experiment. They also related well to a reduction of the expression of the polysomal protein at the mRNA and protein level. The effects seemed to be dose dependent and lasted for >24 h. A stimulation of renin secretion as an anticipated feedback response to the decrease in plasma concentrations of ANG II could not completely counterbalance the effects of the AONs. Similar observations have been made in other laboratories with pharmaceutical drugs successfully applied to inhibit the activity of the renin-angiotensin system, such as converting enzyme inhibitors or ANG II type 1 receptor antagonists (13, 17, 26). In our current study captopril treatment (100 mg·kg–1·day–1) and L-158809 treatment (1.5 mg·kg–1·day–1) for three consecutive days lowered mean arterial pressure by 32 and 36 mmHg, respectively, demonstrating a similar effectiveness as observed for the AONs designed to inhibit the expression of the mRNA-stabilizing protein.
The good correlation between mean arterial pressure, renin-angiotensin system, and polysomal protein expression supported our hypothesis that suppression of the polysomal protein results in an inhibition of the renin-angiotensin system via reduced angiotensinogen synthesis. In the rat as in the mouse, angiotensinogen has an influence on the formation rate of ANG I and ANG II. In genetically altered mice, Kim and colleagues (5) reported an increase in blood pressure of 8 mmHg per angiotensinogen gene copy, resulting in a difference of 16 mmHg between the two-copy and four-copy mouse with angiotensinogen plasma concentrations being elevated 1.45-fold. Although plasma renin activity was not measured in this study, blood pressure data suggest that feedback stimulation (in the 0-copy and 1-copy mouse) or inhibition (in the 3-copy or 4-copy mouse) of renin release could not counterbalance changes in the activity of the renin-angiotensin system. In the rat, Tomita and colleagues (30) demonstrated that AONs directed against angiotensinogen significantly lowered blood pressure for several days. Also in this study performed in the rat, a feedback stimulation of renin release was not able to shift blood pressure back to values observed in normotensive rats. From these studies it is evident that angiotensinogen plasma concentrations should be considered as a rate-limiting factor for the generation of ANG I and ANG II as our laboratory has demonstrated in one of our previous studies (8). Therefore, regulatory mechanisms for angiotensinogen production may have to be interpreted as important candidates participating in the complex pathogenesis of hypertension.
The expression of mRNA was measured by real-time RT PCR utilizing highly purified mRNA preparations and cDNA synthesis products with virtually no DNA or RNA contamination as starting material for PCR amplification. It is, therefore, unlikely that remnants of genomic DNA participated in amplification process. In addition, the inhibition of the polysomal protein at the mRNA level related well to an inhibition at the protein level. Protein expression was measured in a cross-link assay under inhibition (heparin) of unspecific binding. This assay and related band shift techniques were used in the past (11) to isolate the protein from polysome extracts and to identify a binding region (UCCUU) that is expressed twice in the 3'-untranslated tail of angiotensinogen mRNA. In this assay, proteins that specifically interact with the 3'-untranslated region of angiotensinogen are cross-linked and after enzymatic digestion of the mRNA fragment become radioactively labeled by covalently linked [32P]UMP. This analytical approach, which is relatively specific for the interaction between the polysomal protein and the 3'-untranslated region of angiotensinogen mRNA, demonstrated that AON administration resulted in an inhibition of mRNA-stabilizing protein expression at both the mRNA and the protein level.
The polysomal protein that controls the half-life of angiotensinogen mRNA may be considered as one of the critical factors that participate in blood pressure control because it has been shown to be 5- to 12-fold upregulated in the SHR in various tissues during pathogenesis of hypertension. Until recently it was not clear whether the elevated concentrations have some functional impact on the development of hypertension or whether they represent a nonfunctionally associated phenomenon. Our current study clearly addresses this point by demonstrating that AON inhibition of the polysomal protein causes a decrease in the mRNA coding for the mRNA-stabilizing protein and for angiotensinogen and leads to a sustained decrease in mean arterial pressure. The effect seems to be relatively specific for an inhibition of the renin-angiotensin system given the good correlation between polysomal protein and angiotensinogen mRNA and the decrease in blood pressure. Screening of the database genomatix for potential interactions of the designed AONs with major promoter sequences did not reveal any significant homologies, supporting the view of relative specificity of the observed effects. In addition, our current study does not support the view of any importance of immunostimulatory activity related to any of the oligodeoxynucleotides used in our experiments, since potential DNA motifs (12, 28) present in RNASTAAS1 and SCR2 exhibit no correlation with mean arterial pressure. It is, therefore, unlikely that the hypotensive effects of the RNASTAAS2 are related to some potential immunostimulatory actions of the experimental compounds.
In our current study, a decrease in plasma renin activity to 50% of the control reduced mean arterial pressure by 40 mmHg. Given the state of expended volume in the SHR and the low renin levels, this rat strain may be prone to react more sensitively to an inhibition of the activity of the renin-angiotensin system via angiotensinogen synthesis than a normotensive strain. This way of interpretation is in accordance with the higher efficiency of ANG II receptor blockers or converting enzyme inhibitors in hypertensive than in normotensive individuals and with the lower efficacy of RNASTAAS2 in the WKY rat. In a previous study we demonstrated (8) that 10–15 min after angiotensinogen antibody administration a decrease to 50% in plasma renin activity was related to a decrease in blood pressure of approximately the same magnitude in the adult Sprague-Dawley rat.
Because of the lack of more functional studies aiming at renal function at this time, we cannot completely rule out additional mechanisms that may play a role in the documented effects of the AONs. However, the good correlation between angiotensinogen, polysomal protein, and mean arterial pressure suggests the renin-angiotensin system as the main target of the AONs, whereas others may have to be considered as secondary. These issues will be addressed in the near future in more detailed functional studies focusing on renal physiology and related topics.
Data from the current study demonstrate that a polysomal protein may have to be considered as an important regulator for the synthesis and secretion of angiotensinogen and, thereby, blood pressure. The results of our current study are based on acute effects of AONs. However, blood pressure regulation is a complex multifactorial process, and at this time it is difficult to extrapolate long-term effects of AON inhibition of the polysomal protein. Future studies, including continuous administration of AONS, need to be carried out to address these important issues.
Perspectives
ANG II has been implicated in the etiology of genetic hypertension in humans and animals. Recent studies indicated that, in addition to renin, substrate concentrations have to be considered as rate limiting at least in humans, rats, and mice. This point of view is supported by the fact that a strong association exists between mutations of the angiotensinogen gene and the pathogenesis of hypertension and other cardiovascular diseases in humans and rats. Our current study characterizes the importance of a polysomal protein for the regulation of angiotensinogen mRNA half-life and, therefore, angiotensinogen expression in hepatic and extrahepatic tissues. Antisense inhibition of the polysomal protein significantly lowered angiotensinogen and mean arterial pressure. Future studies will target mechanisms of interference of the polysomal protein expression with renal function and chronic aspects of the manipulation of angiotensinogen expression. These critical experiments will further characterize important mechanisms of actions for the polysomal protein and may lead to the development of a new class of drugs for the treatment of hypertension and related cardiovascular diseases.
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GRANTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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