AJP - Heart Journal of Neurophysiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 288: H2154-H2162, 2005. First published January 14, 2005; doi:10.1152/ajpheart.00987.2004
0363-6135/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/5/H2154    most recent
00987.2004v1
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 Web of Science
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 Web of Science (12)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, C.
Right arrow Articles by Tune, J. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, C.
Right arrow Articles by Tune, J. D.

Coronary arteriolar vasoconstriction to angiotensin II is augmented in prediabetic metabolic syndrome via activation of AT1 receptors

Cuihua Zhang,3,4,5 Jarrod D. Knudson,3 Srinath Setty,2 Alberto Araiza,3 Ü. Deniz Dincer,3 Lih Kuo,1 and Johnathan D. Tune3

1Department of Medical Physiology, Cardiovascular Research Institute, College of Medicine, Texas A&M University System Health Science Center, Temple; and 2Department of Integrative Physiology, University of North Texas Health Science Center, Fort Worth, Texas; and 3Department of Physiology, 4Department of Anesthesiology, and 5Department of Surgery, Louisiana State University Health Sciences Center, New Orleans, Louisiana

Submitted 24 September 2004 ; accepted in final form 10 January 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The metabolic syndrome is associated with activation of the renin-angiotensin system. However, whether the coronary vascular response to ANG II is altered under this condition is unknown. Experiments were conducted in control and chronically high-fat-fed dogs with the prediabetic metabolic syndrome both in vitro (isolated coronary arterioles, 60–110 µm) and in vivo (anesthetized and conscious). We found that plasma renin activity and ANG II levels are elevated in high-fat-fed dogs and that this increase in ANG II is associated with a significant increase in ANG II-mediated coronary vasoconstriction in isolated coronary arterioles and in anesthetized open-chest dogs. The vasoconstriction to ANG II is abolished by ANG II type 1 (AT1) receptor blockade. In conscious chronically instrumented dogs, AT1 receptor blockade with telmisartan improved the balance between coronary blood flow and myocardial oxygen consumption in the high-fat-fed dogs but not in normal control dogs, i.e., the relationship between coronary venous PO2 and myocardial oxygen consumption was shifted upward, toward normal control values. Quantitative assessment of coronary arteriolar AT1 and ANG II type 2 (AT2) receptor mRNA levels by real-time PCR revealed no significant difference between normal control and high-fat-fed dogs; however, Western blot analysis showed a significant increase in AT1 receptor protein level with no change in AT2 receptor protein density. These findings indicate that AT1 receptor-mediated coronary constriction is augmented in the prediabetic metabolic syndrome and contributes to impaired control of coronary blood flow via increases in circulating ANG II and/or coronary arteriolar AT1 receptor density.

obesity; microcirculation; blood flow; exercise

THE PREVALENCE OF OBESITY and its related disorders has reached epidemic proportion, and recent estimates predict that ~47 million Americans have the metabolic syndrome, the combined disorder of obesity, insulin resistance, dyslipidemia, and hypertension (3). Importantly, each of these disorders contributes independently to the risk of cardiovascular disease (7). Therefore, it is not surprising that cardiovascular morbidity and mortality are significantly elevated in individuals with the prediabetic syndrome (11, 12, 16, 32). Despite these alarming statistics, studies addressing the mechanisms by which the metabolic syndrome increases cardiovascular morbidity and mortality are lacking.

Recently, Setty et al. (30) demonstrated that the prediabetic metabolic syndrome significantly impairs the balance between coronary blood flow and myocardial metabolism by tonically vasoconstricting the coronary circulation. The reason for this increased vasoconstriction is presently unknown, but it could be related to chronic activation of the renin-angiotensin system (RAS) (5, 6, 9, 10). Although ANG II does not have a pronounced effect on coronary vascular resistance under normal physiological conditions (2, 17, 22), it does have significant vasoconstrictor activity when chronically elevated (2, 19–21). Other studies indicate that both hyperinsulinemia and hypercholesterolemia induce overexpression of ANG II type 1 (AT1) receptors (23, 24), the receptor subtype responsible for ANG II-mediated coronary arteriolar vasoconstriction (35). However, whether increased ANG II-mediated vasoconstriction contributes to impaired coronary flow regulation in the prediabetic metabolic syndrome has not been previously investigated.

Accordingly, the present study was designed to test whether the metabolic syndrome increases coronary constriction to ANG II via activation of AT1 receptors. Experiments were conducted in vivo in anesthetized, open-chest dogs and in vitro in isolated coronary arterioles (<100 µm) from normal control and chronically high-fat-fed dogs. We (30) recently found that this high-fat diet induces insulin resistance, increases body weight and plasma insulin concentration, and significantly impairs blood pressure regulation. In addition, studies were also conducted in conscious, chronically instrumented dogs to test the hypothesis that blockade of AT1 receptors significantly improves the balance between coronary blood flow and myocardial metabolism during exercise-induced increases in myocardial oxygen demand in dogs with metabolic syndrome. Classic and real-time PCR as well as Western blot analyses were also performed to assess coronary arteriolar AT1 and ANG II type 2 (AT2) receptor gene expression and protein level in control and high-fat-fed dogs.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
High-fat diet. This investigation was approved by the Louisiana State University (LSU) Health Sciences Center Institutional Animal Care and Use Committee and was conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1996). Animals of either sex (n = 39) were fed either normal dog chow (Teklad, ~13% calories from fat; n = 22) or a high-fat diet that provided ~60% of calories from fat (n = 17). The high-fat diet was administered in the morning and afternoon for 5–6 wk (14, 30). The morning feeding consisted of a homogenous mixture of canned dog food (748 g, Alpo), dry dog food (227 g, Teklad), and lard (57 g, Morrell). The afternoon feeding consisted of a homogenous mixture of canned dog food (748 g, Alpo), lard (454 g, Morrell), and chicken or beef baby food (71 g). The dogs were fed the same diet throughout the experimental protocol.

In vitro functional assessment of isolated coronary arterioles. Normal control (4–7 arterioles from 3 animals) and high-fat-fed (4 or 5 arterioles from 3 animals) dogs were anesthetized with pentobarbital sodium (30 mg/kg iv). The heart was excised and immediately placed in cold (5°C) saline solution. Individual coronary arterioles (60- to 110-µm in situ internal diameter and 0.6–1.0 mm in length without branches) were then dissected out for in vitro study as previously described (13, 15). Vessels were cannulated with glass micropipettes and pressurized to 60 cmH2O intraluminal pressure. The cannulated vessel was bathed in physiological salt solution containing bovine serum albumin (1%, pH 7.4; Amersham) at 37°C.

After vessels developed a stable basal tone, the concentration-diameter relationships of coronary arterioles from normal control and high-fat-fed dogs were assessed with ANG II (0.1 nM–1 µM). To determine the role of AT1 receptors in ANG II-induced vasomotor actions, vessels were treated extraluminally with the AT1 receptor inhibitor losartan (1 µM) for 30 min, and the ANG II concentration-diameter relationship (0.1 nM–1 µM) was then determined.

In vivo coronary dose-response experiments. Normal control (n = 6) and high-fat-fed (n = 6) dogs were sedated with morphine (3 mg/kg sc) and anesthetized with {alpha}-chloralose (100 mg/kg iv). The animals were then intubated and ventilated with room air and supplemental oxygen. The left anterior descending coronary artery was cannulated and connected to an extracorporeal perfusion system as previously described (34). After 15–30 min of recovery, ANG II was infused into the coronary perfusion line (0.1–30.0 ng/kg, bolus injection). The lowest coronary blood flow after each infusion was recorded, and the coronary blood flow was allowed to stabilize for ~3 min after each dose of ANG II (n = 6). In selected animals, once the ANG II infusion protocol was complete, telmisartan (0.3 mg/kg iv) was infused to selectively inhibit AT1 receptors and the ANG II dose-response protocol was repeated (n = 5).

Blood samples were obtained from the animals before and after high-fat feeding. The samples were collected in K3EDTA Vacutainer tubes and centrifuged at 4,300 rpm for 20 min at 4°C. Plasma supernatant was collected and stored at –90°C until analyses were performed. Plasma insulin and ANG II were measured with a commercially available RIA kit (Alpco Diagnostics). Plasma cholesterol levels were measured with standard procedures by the Pathology Core of the LSU Health Sciences Center.

Surgical instrumentation and exercise protocol. Experiments were performed on adult mongrel dogs of either sex taught to run on a motorized treadmill. The surgical procedures performed in this study were previously described by Setty et al. (30). Briefly, a catheter was placed in the aorta to measure blood pressure and to obtain arterial blood samples. A catheter was also placed in the coronary sinus via the right atrial appendage for coronary venous blood sampling. Flow transducers (Transonic Systems) were placed around the circumflex coronary artery and the root of the aorta. The animals were allowed at least 7 days of postsurgical recovery.

Coronary blood flow, aortic flow (cardiac output minus coronary flow), aortic pressure, and heart rate were continuously measured while the dogs were resting in a sling and then during three levels of treadmill exercise: 1) 2 mph, 0% grade; 2) 3 mph, 5% grade; and 3) 4 mph, 10% grade. The animals were exercised at similar levels (i.e., speed and % grade) with and without selective AT1 receptor blockade with telmisartan (0.3 mg/kg iv; control n = 8, control + telmisartan n = 4, high-fat diet control n = 5, high-fat diet + telmisartan n = 5). Each exercise period was ~2 min in duration, and the animals were allowed to rest sufficiently between each level for hemodynamic variables to return to baseline. Arterial and coronary venous blood samples were collected when hemodynamic variables were stable at each exercise level and were immediately sealed and placed on ice. The samples were analyzed in duplicate for pH, PCO2, PO2, hematocrit, and oxygen content with an Instrumentation Laboratories automatic blood gas analyzer (GEM Premier 3000) and CO-oximeter (682) system.

Isolation and quantitation of total RNA. Coronary arterioles from control and high-fat-fed dogs were isolated (internal diameter ~100 µm) from the epicardial surface of the left ventricle, placed in liquid N2, and stored at –80°C. Total RNA was extracted by the procedure provided with the SV Total RNA Isolation System (Promega). At the end of the isolation, RNA samples were dissolved in nuclease-free water (pH 7.5) and the optical density (OD) values of each sample were determined spectrophotometrically with a UV-visible spectrophotometer (Bio-Rad) at wavelengths of 260 nm ({lambda}260) and 280 nm ({lambda}280). The amount of RNA in each sample was then determined with the formula [RNA] = OD{lambda}260 x dilution factor x 40 µg/ml, where [RNA] is RNA concentration. The OD{lambda}260-to-OD{lambda}280 ratio was used as cursory estimations of RNA quality (1).

Preparation of first-strand cDNA via reverse transcriptase reactions. RNA samples were used as templates for synthesis of first-strand cDNAs as described previously (8). Briefly, 1 µl of oligo(dT)15 primer (Promega) was added to equivalent amounts of total RNA obtained from coronary arterioles isolated from control (n = 4) and chronically high-fat-fed (n = 4) dogs. The mixtures were then placed into a thermocycler (My Cycler, Bio-Rad) and held at 70°C for 5 min. The samples were then transferred into an ice bath for 5 min to permit selective binding of the oligo(dT)15 to the poly(A) tail of the mRNA. First-strand cDNA was then synthesized with an ImProm-II Reverse Transcriptase kit (Promega).

Amplification of cDNA. Real-time and classic PCR were used to simultaneously amplify cDNAs encoding AT1 and AT2 receptors as well as {beta}-actin. Primers were designed based on published sequences in the National Center for Biotechnology Information GenBank database (http://www3.ncbi.nlm.nih.gov/entrez/) [AT1 receptor, sense 5-ACTTTGCCACTATGGGCTGT-24 and antisense 215-TGCAGGTGACTTTTGCCATA-196 (accession no. U67200); AT2 receptor, sense 8-ggctttcccacctgagaaat-27 and antisense 187-atcttcaggacttggtcacg-168 (accession no. U67201); 2 different sets of {beta}-actin: sense 187-GGCATCCTGACCCTGAAGTA-206 and antisense 540-CAGGTCCAGACGCAAGATG-522 (accession no. AF484115) and sense 21-GACATCCGCAAGGACCTC-TA-40 and antisense 176-CACAGAGTACTTGCGCTCAG-157 (accession no. U67202)].

PCR was performed (2.4 µl of 25 mM MgCl for 50 µl of total reaction) with a Taq DNA polymerase kit (Promega). Amplification was then carried out as follows: 45-s denaturation (95°C) followed by 45-s annealing (55.6°C) and 2-min extension (72°C), repeated for a total of 35 cycles. {beta}-Actin was amplified in each set of PCR reactions and served as an internal reference during quantitation to correct for operator and/or experimental variations. PCR products were electrophoresed on a 1% agarose gel containing ethidium bromide [product size: AT1 receptor 225 bp (U67200 [GenBank] ), AT2 receptor 180 bp (U67201 [GenBank] ), {beta}-actin 354 bp (AF484115 [GenBank] ) and 156 bp (U67202 [GenBank] )]. Band intensities were measured and used as mRNA concentrations with Quantity One Software, Version 4.4.1 (Bio-Rad). All data were normalized to concomitant {beta}-actin intensity, and then the results were compared with the control samples.

Real-time PCR. Real-time PCR reactions were also performed for AT1 and AT2 receptors and {beta}-actin expression in duplicate with SYBRgreen (iQ SYBR Green Supermix, Bio-Rad) and analyzed with the iCycler IQ real-time PCR detection system (Bio-Rad). The 2 method was used to analyze the relative changes in AT1 and AT2 receptor gene expression from real-time quantitative PCR experiments (18). The data were analyzed with the 2–{Delta}{Delta}CT equation.

The mean threshold cycle (CT) values for both the target (AT1 and AT2 receptors; CT-ANG II receptor) and internal control ({beta}-actin; CT-BA) genes were determined (n = 4 each for control and high-fat diet). The fold change of each gene was normalized to {beta}-actin and, relative to the expression in control, was calculated for each duplicated sample using 2.

Western blot analysis of AT1 and AT2 receptor proteins. Coronary arterioles from control (n = 4) and high-fat-fed (n = 4) dogs were isolated (internal diameter ~100 µm) from the epicardial surface of the left ventricle, placed in liquid N2, and stored at –80°C. Arterioles were homogenized in 50 µl of buffer [50 mM Tris·HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% SDS, 1 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 5:1,000 phenylmethylsulfonyl fluoride (200 nM), 5:1,000 Na3VO4 (200 nM), and 5:1,000 NaF (200 nM)]. The homogenates were centrifuged at 30,000 g for 30 min at 4°C. The supernatants were used for the analysis. Protein concentrations were determined by micro-bicinchoninic acid protein assay (Pierce).

Equivalent amounts of protein were separated by gel electrophoresis (10% Tris·HCl Criterion Precast Gel, Bio-Rad). Proteins were transferred onto nitrocellulose membrane (0.2 µm, Schleicher & Schuell) by semidry electroblotting (Trans Blot SD, Bio-Rad) at 15 V for 1 h. The nitrocellulose membrane was soaked in Tris-buffered saline (TBS; 10 mM Tris·HCl and 150 mM NaCl) containing 5% nonfat dry milk (Bio-Rad) and 0.1% polyoxyethylene-sorbitan monolaurate (Tween 20) overnight at 4°C to block nonspecific sites. The membranes were then incubated with the AT1 or AT2 receptor antiserum (1: 500 dilution in TBS with 5% nonfat dry milk and 0.1% Tween 20; Santa Cruz Biotechnology) for 2 h at room temperature. {beta}-Actin receptor antiserum (Sigma) was also used for internal control [1:125 dilution in 0.01 M PBS (pH 7.4) containing 1% bovine serum and 15 mM sodium azide as preservative]. Blots were washed and incubated with peroxidase-conjugated donkey anti-rabbit secondary antibody (1:3,000 dilution; Santa Cruz Biotechnology). Immunoreactivity was visualized with an ECL Western blotting detection kit (Amersham). Quantitative assessment of band densities was performed by scanning densitometry.

Statistical analysis. All data are presented as means ± SE. For the in vivo studies, a paired t-test was used to compare body weight and plasma ANG II concentration between the normal control and high-fat-fed dogs with and without AT1 receptor blockade (within-group comparison). A t-test was also used to compare Western blot band densities. A two-way repeated-measures ANOVA was used to compare the effects of AT1 receptor blockade and exercise on coronary and systemic hemodynamic variables. When significance was found with ANOVA, a Student-Newman-Keuls multiple-comparison test was performed. Linear regression analysis was used to compare the slopes of coronary venous PO2 vs. myocardial oxygen consumption. If the slopes of the regression lines were not significantly different, an analysis of covariance (ANCOVA) was used to adjust the response variable for linear dependence on myocardial oxygen consumption.

At the end of each in vitro experiment, the vessel was relaxed with 100 µM sodium nitroprusside to obtain its maximal diameter. All diameter changes in response to agonists were normalized to the vasodilation in response to 100 µM nitroprusside and expressed as a percentage of maximal dilation. Statistical comparisons of vasomotor responses were performed with ANOVA and, when appropriate, tested with a Bonferroni multiple-range test. Significance was accepted at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of chronic high-fat feeding. The effects of chronic high-fat diet on key baseline hemodynamic and plasma variables before and after AT1 receptor blockade with telmisartan (0.3 mg/kg iv) are given in Table 1. Baseline coronary blood flow, mean aortic pressure, and heart rate data in Table 1 are from the same anesthetized, open-chest control or high-fat-fed dog before and after telmisartan administration (n = 5). Chronic high-fat feeding increased body weight from 26.4 ± 0.9 to 31.0 ± 1.3 kg (P < 0.001) and increased plasma ANG II concentration from 7.7 ± 1.8 to 17.2 ± 4.4 pg/ml (P < 0.05). In addition, plasma renin activity (P = 0.06) and total cholesterol (P < 0.05) were also elevated in high-fat-fed dogs. These findings are consistent with earlier studies from our laboratory (30) as well as others (9, 10, 14, 28), which have demonstrated that chronic high-fat feeding in dogs induces the prediabetic metabolic syndrome, i.e., animals are overweight or obese, hyperinsulinemic, normoglycemic, dyslipidemic, and insulin resistant and have impaired blood pressure regulation.


View this table:
[in this window]
[in a new window]
  		Table 1. Effect of chronic high-fat diet on key baseline variables

 
Effect of prediabetic metabolic syndrome on coronary reactivity in vitro. No significant differences in the baseline characteristics of the isolated coronary arterioles from control and high-fat-fed dogs were detected (Table 2). The effect of the prediabetic metabolic syndrome on the vasomotor response of arterioles to ANG II (0.1 nM–1 µM) is presented in Fig. 1. In normal controls, ANG II tended to induce vasoconstriction at lower concentrations from 0.1 to 10 nM but dilation at higher concentrations (>10 nM). In contrast, vasoconstrictions to ANG II were significantly augmented in the high-fat-fed dogs (1.0–10 nM), whereas the degree of vasodilation to higher doses of ANG II was similar. In the presence of losartan, ANG II exerted only vasodilation in arterioles from control and high-fat-fed dogs (Fig. 1B), demonstrating that augmented ANG II constriction in the prediabetic metabolic syndrome is mediated by activation of AT1 receptors.


View this table:
[in this window]
[in a new window]
  		Table 2. Baseline characteristics of isolated coronary arterioles

 


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 1. Coronary arteriolar vasoconstrictions to ANG II were significantly augmented in the high-fat-fed dogs (control n = 7, high-fat diet n = 5; A) in vitro. *P < 0.05 vs. control, same dose. This augmented vasoconstriction was completely inhibited by ANG II type 1 (AT1) receptor blockade with losartan (control n = 4, high-fat diet n = 4; B). *P < 0.05, high-fat diet vs. high-fat diet + losartan.

 
Effect of prediabetic metabolic syndrome on coronary reactivity in vivo. Figure 2 shows the in vivo dose-response relationship between coronary blood flow (%baseline) and intracoronary ANG II dose (Fig. 2A) and calculated plasma ANG II concentration (Fig. 2B). ANG II produced dose-dependent decreases in coronary blood flow in both the normal control (n = 6) and high-fat-fed (n = 6) dogs. However, the decrease in coronary blood flow to ANG II was significantly augmented in high-fat-fed dogs relative to ANG II dose (Fig. 2A) as well as to calculated plasma ANG II concentration (Fig. 2B). Coronary blood flow was reduced to ~40% of the baseline value in both normal control and high-fat-fed dogs at the highest ANG II dose. Importantly, vasoconstriction to ANG II was completely abolished by telmisartan (0.3 mg/kg iv) in both control (Fig. 2C) and high-fat-fed (Fig. 2D) dogs. In addition, resting coronary blood flow was significantly elevated in the anesthetized high-fat-fed dogs, but not in normal control dogs, after AT1 receptor blockade with telmisartan (Table 1).



View larger version (27K):
[in this window]
[in a new window]
  		Fig. 2. Dose-response relationship between coronary blood flow (%baseline) and intracoronary ANG II dose (A, C, and D) and calculated plasma ANG II concentration (B). ANG II produced dose-dependent decreases in coronary blood flow in both control (n = 6) and high-fat-fed (n = 6) dogs. However, the decrease in coronary blood flow was significantly augmented in high-fat-fed dogs at lower concentrations of ANG II. AT1 receptor blockade with telmisartan completely attenuated ANG II-mediated coronary vasoconstriction in vivo in control (n = 5; C) and high-fat-fed (n = 5; D) dogs. Untreated control (n = 6) and high-fat diet (n = 6) ANG II dose-response relationships were same as shown in Fig. 2. *P < 0.05 vs. control or high-fat diet, same dose.

 
Hemodynamic and blood gas variables at rest and during treadmill exercise in chronically instrumented dogs with and without telmisartan are given in Table 3. In normal control dogs, exercise increased coronary blood flow ~2.0-fold. This increase in coronary flow was not significantly affected by AT1 receptor blockade; however, the increase in coronary blood flow to exercise was diminished in the high-fat-fed dogs (~1.7-fold), and AT1 receptor blockade augmented this response (~1.9-fold). Mean aortic blood pressure and cardiac index tended to be lower under baseline conditions after telmisartan treatment in the high-fat-fed dogs, although AT1 receptor blockade had little overall effect on cardiovascular hemodynamics or arterial blood gases in conscious dogs.


View this table:
[in this window]
[in a new window]
  		Table 3. Hemodynamic and blood gas variables at rest and during graded treadmill exercise in control and high-fat-fed dogs with and without telmisartan

 
To determine whether AT1 receptor blockade improves the balance between myocardial oxygen delivery and metabolism, a plot of coronary venous PO2 vs. myocardial oxygen consumption was used (Fig. 3; Ref. 33). Telmisartan did not significantly alter the relationship between coronary venous PO2 and myocardial oxygen consumption in normal control animals (Fig. 3A; P value for slope = 0.39; ANCOVA intercept: control = 17.0 mmHg, control + telmisartan = 16.9 mmHg; P = 0.41); however, there was a parallel shift upward in this relationship in dogs chronically fed the high-fat diet (Fig. 3B; P value for slope = 0.21; ANCOVA intercept: high-fat diet = 15.8 mmHg, high-fat diet + telmisartan = 17.5 mmHg; P = 0.003). These findings indicate that ANG II has little effect on the balance between flow and metabolism under normal conditions, whereas ANG II mediates a significant tonic coronary vasoconstrictor influence in the prediabetic metabolic syndrome.



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 3. Coronary venous PO2 vs. myocardial oxygen consumption in control (A) and high-fat-fed (B) dogs with and without AT1 receptor blockade with telmisartan (0.3 mg/kg iv; control n = 8, control + telmisartan n = 4, high-fat diet control n = 5, high fat diet + telmisartan n = 5). AT1 receptor blockade did not significantly alter the relationship between coronary venous PO2 and myocardial oxygen consumption in normal control animals; however, there was a parallel shift upward in this relationship (P intercept = 0.003) in dogs chronically fed the high-fat diet (n = 5). These findings indicate that ANG II has little effect on the balance between flow and metabolism under normal conditions, whereas ANG II mediates a significant tonic coronary vasoconstrictor influence in the prediabetic metabolic syndrome.

 
Quantitation of AT1 and AT2 receptors. Classic and real-time PCR were used to determine the amount of coronary arteriolar (internal diameter ~100 µm) AT1 receptor transcripts in cDNA obtained from control and chronically high-fat-fed dogs. A representative gel is shown in Fig. 4A. After normalization to concomitant {beta}-actin expression, levels of mRNA encoding AT1 (102 ± 4%) and AT2 (96 ± 30%) receptors were not significantly different from control. Quantitative assessment of coronary arteriolar AT1 and AT2 receptor mRNA levels by real-time PCR revealed no significant difference between normal control and high-fat-fed dogs (mean fold change in gene expression 2= 1.06 ± 0.22 for AT1 receptors and 2= 0.89 ± 0.41 for AT2 receptors; Fig. 4B). However, Western blot analysis revealed a significant increase in AT1 receptor protein level (36 ± 12% increase compared with control, P < 0.05; Fig. 5A) with no change in AT2 receptor (104 ± 17% compared with control; Fig. 5B) from coronary arterioles isolated from chronically high-fat-fed dogs. In addition, there was no difference in {beta}-actin protein density (95 ± 17% compared with control; Fig. 5C), confirming that equal amounts of protein were loaded with each sample. These data indicate that an increase in AT1 receptor protein density is responsible for the augmented coronary arteriolar vasoconstriction in dogs with the prediabetic metabolic syndrome.



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 4. A: representative gel of classic PCR for AT1 receptors, AT2 receptors, and {beta}-actin (BA) from left ventricular coronary arterioles (~100 µm) from control (C, n = 4) and high-fat-fed (HF, n = 4) dogs. B: there was no significant alteration in the fold change of mRNA expression (expressed as % of control) for AT1 or AT2 receptors from high-fat-fed dogs (n = 4). Dashed line represents control value of 100%.

 


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 5. Representative gels and quantitation of Western blot signals for AT1 and AT2 receptors and {beta}-actin protein level in left ventricular coronary arterioles (~100 µm) from control (n = 4) and high-fat-fed (n = 4) dogs. There was a significant increase in AT1 receptor protein level in coronary arterioles from high-fat-fed dogs (A), whereas there was no significant alteration in AT2 receptor (B) or {beta}-actin (C) protein density. *P < 0.05 vs. control.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present findings indicate that coronary arteriolar constriction to ANG II is augmented in the prediabetic metabolic syndrome via activation of AT1 receptors. Consistent with earlier studies (5, 9, 10), we found that chronic high-fat feeding significantly increases circulating ANG II levels (Table 1). Importantly, this increase in ANG II is associated with a significant increase in AT1 receptor-mediated coronary vasoconstriction both in vitro (Fig. 1) and in vivo (Fig. 2). In addition, increased AT1 receptor activation in the metabolic syndrome impairs the balance between coronary blood flow and myocardial oxygen consumption (Fig. 3), i.e., telmisartan shifted the coronary venous PO2 vs. myocardial oxygen consumption relationship upward, toward normal control values. This augmented vasoconstriction is likely related to changes in circulating ANG II (Table 1) and/or to increases in coronary arteriolar AT1 receptor density (posttranscriptional modification) (Fig. 5) in dogs with the prediabetic metabolic syndrome. Together, these findings suggest that chronic activation of the RAS contributes to the coronary vascular dysfunction associated with the prediabetic metabolic syndrome (30).

ANG II-mediated coronary constriction in prediabetic metabolic syndrome. ANG II induces many vascular effects, including vasoconstriction, inflammation, vascular remodeling, thrombosis, and plaque rupture (29). Under normal physiological conditions, ANG II induces only modest constriction of large conduit vessels (2, 17, 22, 27). However, when ANG II levels are chronically elevated, as in animals (9, 10) and humans (4) with the metabolic syndrome, its coronary vasoconstrictor activity is evident. These data are supported by the present findings in that ANG II-mediated constriction was significantly increased in the high-fat-fed dogs (Figs. 1 and 2). Other studies have shown that, when the RAS is activated, inhibition of angiotensin-converting enzyme or AT1 receptors substantially augments coronary blood flow (2, 21, 31). Results from anesthetized dogs in the present investigation support this finding (Table 1). However, whether ANG II-mediated coronary vasoconstriction is augmented in the prediabetic metabolic syndrome has not been previously investigated. Our previous study (35) in isolated vessels from normal control pigs demonstrated that ANG II evokes AT1 receptor-mediated vasoconstriction at lower concentrations but produces vasodilation at higher concentrations via AT2 receptor activation in coronary arterioles. Results from the present investigation support this finding in that ANG II-mediated constriction in the high-fat-fed dogs was inhibited by the AT1 receptor blockade both in vitro (Fig. 1B) and in vivo (Fig. 2D).

AT1 and AT2 receptor gene and protein expression in prediabetic metabolic syndrome. The mechanism of the augmented ANG II-mediated vasoconstriction in the prediabetic metabolic syndrome is related to increases in circulating ANG II concentrations (Table 1) and to increases in coronary arteriolar AT1 receptor density (Fig. 5A). This increase in AT1 receptor density is the result of posttranscriptional modification, because AT1 receptor gene expression was unaltered in coronary arterioles from chronically high-fat-fed dogs (Fig. 4). It is unlikely that augmented ANG II vasoconstriction is due to alterations in AT2 receptor function, because AT2 receptor mRNA expression (Fig. 4) and protein levels (Fig. 5B) were unaltered in coronary arterioles from dogs with the prediabetic metabolic syndrome. AT1 receptor density is regulated by a variety of influences, including hyperinsulinemia and hypercholesterolemia, both of which occur in our dog model of the metabolic syndrome (Table 1; Ref. 30). Nickenig and Bohm (24) reported that hyperinsulinemia induces an overexpression of vascular AT1 receptors in rat aortic vascular smooth muscle cells, leading to an enhanced biological efficacy of ANG II. In addition, hypercholesterolemia also leads to an upregulation of AT1 receptors and an enhanced biological response to ANG II (25, 26). Further studies are needed to delineate the cellular mechanisms responsible for the posttranscriptional modification of AT1 receptor expression.

ANG II and control of coronary blood flow in prediabetic metabolic syndrome. An important finding of the present investigation is that augmented AT1 receptor activation and vasoconstriction in the prediabetic metabolic syndrome leads to an impairment of the balance between coronary blood flow and myocardial oxygen consumption (Fig. 3). This was evidenced by the fact that AT1 receptor blockade did not significantly affect the relationship between coronary venous PO2 and myocardial oxygen consumption in normal control dogs (Fig. 3A), but there was a parallel shift upward in this relationship in the high-fat-fed dogs (Fig. 3B), indicating that ANG II significantly limits myocardial oxygen supply-demand balance both at rest and during exercise in dogs with the prediabetic metabolic syndrome. It is important to point out that simple examination of the coronary flow data at rest and during exercise (Table 3) does not adequately address the findings of this investigation. Differences in variables that influence coronary blood flow, i.e., factors that affect myocardial oxygen consumption, must be taken into account. These factors were accounted for in the present study by plotting coronary venous PO2 vs. myocardial oxygen consumption, a sensitive index of the balance between coronary blood flow and myocardial metabolism (33).

In conclusion, we have demonstrated that AT1 receptor-mediated coronary constriction is augmented in our model of the prediabetic metabolic syndrome and that this change is related to increases in circulating ANG II and coronary arteriolar AT1 receptor density, with no changes in AT2 receptor density. This increased constriction likely contributes to the coronary dysfunction associated with this syndrome because AT1 receptor blockade improves the balance between coronary blood flow and myocardial oxygen consumption at rest and during exercise. Together, our findings demonstrate that there is an important link between the components of the metabolic syndrome and the regulation of the RAS that should be further explored. Therapeutic strategies to reduce AT1 receptor activation may improve cardiovascular outcomes in patients with this multifactorial disease.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by grants from the American Diabetes Association (J. D. Tune) and by National Heart, Lung, and Blood Institute Grants HL-67804 (J. D. Tune), HL-48179-09 (L. Kuo), and HL-71761 (L. Kuo).


   ACKNOWLEDGMENTS
 
We thank Dr. Wei Sun and Darice Yoshishige for expert technical assistance. Telmisartan was graciously provided by Boehringer Ingelheim.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. D. Tune, Dept. of Physiology, LSU Health Sciences Center, 1901 Perdido St., New Orleans, LA 70112-1393 (E-mail: jtune{at}lsuhsc.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Dincer UD, Guner S, Tay A, Arioglu E, Tasdelen A, Aslamaci S, and Bidasee KR. Decreased expression of {beta}1- and {beta}2-adrenoceptors in human diabetic atrial appendage. Cardiovasc Diabetol 2: 6, 2003.[CrossRef][Medline]
  2. Ertl G, Hu K, Bauer WR, and Bauer B. The renin-angiotensin system and coronary vasomotion. Heart 76: 45–52, 1996.[Web of Science][Medline]
  3. Ford ES, Giles WH, and Dietz WH. Prevalence of the metabolic syndrome among US adults: findings from the third National Health and Nutrition Examination Survey. JAMA 287: 356–359, 2002.[Abstract/Free Full Text]
  4. Giacchetti G, Faloia E, Sardu C, Camilloni MA, Mariniello B, Gatti C, Garrapa GG, Guerrieri M, and Mantero F. Gene expression of angiotensinogen in adipose tissue of obese patients. Int J Obes Relat Metab Disord 24, Suppl 2: S142–S143, 2000.[CrossRef]
  5. Giacchetti G, Sechi LA, Griffin CA, Don BR, Mantero F, and Schambelan M. The tissue renin-angiotensin system in rats with fructose-induced hypertension: overexpression of type 1 angiotensin II receptor in adipose tissue. J Hypertens 18: 695–702, 2000.[CrossRef][Web of Science][Medline]
  6. Goossens GH, Blaak EE, and van Baak MA. Possible involvement of the adipose tissue renin-angiotensin system in the pathophysiology of obesity and obesity-related disorders. Obes Rev 4: 43–55, 2003.[CrossRef][Medline]
  7. Grundy SM, Benjamin IJ, Burke GL, Chait A, Eckel RH, Howard BV, Mitch W, Smith SC Jr, and Sowers JR. Diabetes and cardiovascular disease: a statement for healthcare professionals from the American Heart Association. Circulation 100: 1134–1146, 1999.[Free Full Text]
  8. Guner S, Arioglu E, Tay A, Tasdelen A, Aslamaci S, Bidasee KR, and Dincer UD. Diabetes decreases mRNA levels of calcium-release channels in human atrial appendage. Mol Cell Biochem 263: 143–150, 2004.[CrossRef][Web of Science][Medline]
  9. Hall JE, Brands MW, Hildebrandt DA, and Mizelle HL. Obesity-associated hypertension. Hyperinsulinemia and renal mechanisms. Hypertension 19: I45–I55, 1992.[Web of Science][Medline]
  10. Hall JE, Brands MW, Zappe DH, Dixon WN, Mizelle HL, Reinhart GA, and Hildebrandt DA. Hemodynamic and renal responses to chronic hyperinsulinemia in obese, insulin-resistant dogs. Hypertension 25: 994–1002, 1995.[Abstract/Free Full Text]
  11. Henry P, Thomas F, Benetos A, and Guize L. Impaired fasting glucose, blood pressure and cardiovascular disease mortality. Hypertension 40: 458–463, 2002.[Abstract/Free Full Text]
  12. Isomaa B, Almgren P, Tuomi T, Forsen B, Lahti K, Nissen M, Taskinen MR, and Groop L. Cardiovascular morbidity and mortality associated with the metabolic syndrome. Diabetes Care 24: 683–689, 2001.[Abstract/Free Full Text]
  13. Jones CJ, Kuo L, Davis MJ, DeFily DV, and Chilian WM. Role of nitric oxide in the coronary microvascular responses to adenosine and increased metabolic demand. Circulation 91: 1807–1813, 1995.[Abstract/Free Full Text]
  14. Kaiyala KJ, Prigeon RL, Kahn SE, Woods SC, Porte D Jr, and Schwartz MW. Reduced {beta}-cell function contributes to impaired glucose tolerance in dogs made obese by high-fat feeding. Am J Physiol Endocrinol Metab 277: E659–E667, 1999.[Abstract/Free Full Text]
  15. Kuo L, Chilian WM, and Davis MJ. Interaction of pressure- and flow-induced responses in porcine coronary resistance vessels. Am J Physiol Heart Circ Physiol 261: H1706–H1715, 1991.[Abstract/Free Full Text]
  16. Lakka HM, Laaksonen DE, Lakka TA, Niskanen LK, Kumpusalo E, Tuomilehto J, and Salonen JT. The metabolic syndrome and total and cardiovascular disease mortality in middle-aged men. JAMA 288: 2709–2716, 2002.[Abstract/Free Full Text]
  17. Liang CS, Gavras H, and Hood WB Jr. Renin-angiotensin system inhibition in conscious sodium-depleted dogs. Effects on systemic and coronary hemodynamics. J Clin Invest 61: 874–883, 1978.[Web of Science][Medline]
  18. Livak KJ and Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2 method. Methods 25: 402–408, 2001.[CrossRef][Web of Science][Medline]
  19. Magrini F, Reggiani P, Fratianni G, Morganti A, and Zanchetti A. Coronary blood flow in renovascular hypertension. Am J Med 94: 45S–48S, 1993.[CrossRef][Medline]
  20. Magrini F, Reggiani P, Paliotti R, Bonagura F, Ciulla M, and Vandoni P. Coronary hemodynamics and the renin angiotensin system. Clin Exp Hypertens 15, Suppl 1: 139–155, 1993.
  21. Magrini F, Shimizu M, Roberts N, Fouad FM, Tarazi RC, and Zanchetti A. Converting-enzyme inhibition and coronary blood flow. Circulation 75: I168–I174, 1987.
  22. Myers PR, Katwa LC, Tanner M, Morrow C, Guarda E, and Parker JL. Effects of angiotensin II on canine and porcine coronary epicardial and resistance arteries. J Vasc Res 31: 338–346, 1994.[Web of Science][Medline]
  23. Nickenig G, Baumer AT, Temur Y, Kebben D, Jockenhovel F, and Bohm M. Statin-sensitive dysregulated AT1 receptor function and density in hypercholesterolemic men. Circulation 100: 2131–2134, 1999.[Abstract/Free Full Text]
  24. Nickenig G and Bohm M. Interaction between insulin and AT1 receptor. Relevance for hypertension and arteriosclerosis. Basic Res Cardiol 93, Suppl 2: 135–139, 1998.[CrossRef]
  25. Nickenig G, Jung O, Strehlow K, Zolk O, Linz W, Scholkens BA, and Bohm M. Hypercholesterolemia is associated with enhanced angiotensin AT1 receptor expression. Am J Physiol Heart Circ Physiol 272: H2701–H2707, 1997.[Abstract/Free Full Text]
  26. Nickenig G, Sachinidis A, Michaelsen F, Bohm M, Seewald S, and Vetter H. Upregulation of vascular angiotensin II receptor gene expression by low-density lipoprotein in vascular smooth muscle cells. Circulation 95: 473–478, 1997.[Abstract/Free Full Text]
  27. Porsti I, Hecker M, Bassenge E, and Busse R. Dual action of angiotensin II on coronary resistance in the isolated perfused rabbit heart. Naunyn Schmiedebergs Arch Pharmacol 348: 650–658, 1993.[CrossRef][Web of Science][Medline]
  28. Rocchini AP, Marker P, and Cervenka T. Time course of insulin resistance associated with feeding dogs a high-fat diet. Am J Physiol Endocrinol Metab 272: E147–E154, 1997.[Abstract/Free Full Text]
  29. Schiffrin EL. Vascular and cardiac benefits of angiotensin receptor blockers. Am J Med 113: 409–418, 2002.[CrossRef][Web of Science][Medline]
  30. Setty S, Sun W, and Tune JD. Coronary blood flow regulation in the prediabetic metabolic syndrome. Basic Res Cardiol 98: 416–423, 2003.[CrossRef][Web of Science][Medline]
  31. Sudhir K, MacGregor JS, Gupta M, Barbant SD, Redberg R, Yock PG, and Chatterjee K. Effect of selective angiotensin II receptor antagonism and angiotensin converting enzyme inhibition on the coronary vasculature in vivo. Intravascular two-dimensional and Doppler ultrasound studies. Circulation 87: 931–938, 1993.[Abstract/Free Full Text]
  32. Trevisan M, Liu J, Bahsas FB, and Menotti A. Syndrome X and mortality: a population-based study. Risk Factor and Life Expectancy Research Group. Am J Epidemiol 148: 958–966, 1998.[Abstract/Free Full Text]
  33. Tune JD, Gorman MW, and Feigl EO. Matching coronary blood flow to myocardial oxygen consumption. J Appl Physiol 97: 404–415, 2004.[Abstract/Free Full Text]
  34. Tune JD, Mallet RT, and Downey HF. Insulin improves contractile function during moderate ischemia in canine left ventricle. Am J Physiol Heart Circ Physiol 274: H1574–H1581, 1998.[Abstract/Free Full Text]
  35. Zhang C, Hein TW, Wang W, and Kuo L. Divergent roles of angiotensin II AT1 and AT2 receptors in modulating coronary microvascular function. Circ Res 92: 322–329, 2003.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Physiol. Rev.Home page
D. J. Duncker and R. J. Bache
Regulation of Coronary Blood Flow During Exercise
Physiol Rev, July 1, 2008; 88(3): 1009 - 1086.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
T. Kawada, T. Yamazaki, T. Akiyama, M. Li, C. Zheng, T. Shishido, H. Mori, and M. Sugimachi
Angiotensin II attenuates myocardial interstitial acetylcholine release in response to vagal stimulation
Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2516 - H2522.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
A. L. Mundy, E. Haas, I. Bhattacharya, C. C. Widmer, M. Kretz, R. Hofmann-Lehmann, R. Minotti, and M. Barton
Fat intake modifies vascular responsiveness and receptor expression of vasoconstrictors: Implications for diet-induced obesity
Cardiovasc Res, January 15, 2007; 73(2): 368 - 375.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
D. Merkus, D. B. Haitsma, O. Sorop, F. Boomsma, V. J. de Beer, J. M. J. Lamers, P. D. Verdouw, and D. J. Duncker
Coronary vasoconstrictor influence of angiotensin II is reduced in remodeled myocardium after myocardial infarction
Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2082 - H2089.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
C.-P. Cheng, H.-J. Cheng, C. Cunningham, Z. K. Shihabi, D. C. Sane, T. Wannenburg, and W. C. Little
Angiotensin II Type 1 Receptor Blockade Prevents Alcoholic Cardiomyopathy
Circulation, July 18, 2006; 114(3): 226 - 236.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. D. Knudson, U. D. Dincer, G. M. Dick, H. Shibata, R. Akahane, M. Saito, and J. D. Tune
Leptin resistance extends to the coronary vasculature in prediabetic dogs and provides a protective adaptation against endothelial dysfunction
Am J Physiol Heart Circ Physiol, September 1, 2005; 289(3): H1038 - H1046.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/5/H2154    most recent
00987.2004v1
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 Web of Science
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 Web of Science (12)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhang, C.
Right arrow Articles by Tune, J. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhang, C.
Right arrow Articles by Tune, J. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.