Published online before print April 26, 2004, doi: 10.1161/01.CIR.0000128696.12245.57
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(Circulation. 2004;109:2296-2301.)
© 2004 American Heart Association, Inc.
Clinical Investigation and Reports |
Angiotensin II Type 2 Receptor–Mediated Vasodilation in Human Coronary Microarteries
From the Department of Pharmacology (W.W.B., I.M.G., P.R.S., A.H.J.D.) and the Department of Thoracic Surgery and Heart Valve Bank (J.P.v.K.), Lausanne), Erasmus MC, Rotterdam, the Netherlands; and the University Institute of Pathology (C.C.B., L.J.-J.), Lausanne, Switzerland.
Correspondence to Prof Dr A.H.J. Danser, Department of Pharmacology, Room EE1418b, Erasmus MC, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. E-mail a.danser{at}erasmusmc.nl
Received August 13, 2002; revision received December 2, 2003; accepted February 18, 2004.
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Abstract |
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Background— Angiotensin (Ang) II type 2 (AT2) receptor stimulation results in coronary vasodilation in the rat heart. In contrast, AT2 receptor–mediated vasodilation could not be observed in large human coronary arteries. We studied Ang II–induced vasodilation of human coronary microarteries (HCMAs).
Methods and Results— HCMAs (diameter, 160 to 500 µm) were obtained from 49 heart valve donors (age, 3 to 65 years). Ang II constricted HCMAs, mounted in Mulvany myographs, in a concentration-dependent manner (pEC50, 8.6±0.2; maximal effect [Emax], 79±13% of the contraction to 100 mmol/L K+). The Ang II type 1 receptor antagonist irbesartan prevented this vasoconstriction, whereas the AT2 receptor antagonist PD123319 increased Emax to 97±14% (P<0.05). The increase in Emax was larger in older donors (correlation 
Emax versus age, r=0.47, P<0.05). The PD123319-induced potentiation was not observed in the presence of the NO synthase inhibitor L-NAME, the bradykinin type 2 (B2) receptor antagonist Hoe140, or after removal of the endothelium. Ang II relaxed U46619-preconstricted HCMAs in the presence of irbesartan by maximally 49±16%, and PD123319 prevented this relaxation. Finally, radioligand binding studies and reverse transcription–polymerase chain reaction confirmed the expression of AT2 receptors in HCMAs.
Conclusions— AT2 receptor–mediated vasodilation in the human heart appears to be limited to coronary microarteries and is mediated by B2 receptors and NO. Most likely, AT2 receptors are located on endothelial cells, and their contribution increases with age.
Key Words: angiotensin • bradykinin • microcirculation • nitric oxide • vasodilation
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Introduction |
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Angiotensin (Ang) II type 2 (AT2) receptors are believed to mediate vasodilation, although data to support this concept in humans are not available. Neither in vitro studies investigating Ang II–induced vasoconstriction in isolated human coronary arteries1 and saphenous veins2 nor in vivo studies investigating Ang II–induced vasoconstriction in the forearm vascular bed of healthy volunteers3,4 provided evidence for AT2 receptor–mediated vasodilation. In contrast, both in vitro and in vivo studies in rats and mice support this notion.5–10 One explanation for the discrepancy between the lack of AT2 receptor–mediated vasodilation in human coronary arteries1 and the occurrence of such dilation in the rat coronary vascular bed8 is that AT2 receptors are located in coronary microarteries only. In the present study, we therefore investigated AT2 receptor–induced vasodilation in human coronary microarteries (HCMAs) mounted in Mulvany myographs. We also investigated whether endothelial NO and/or bradykinin type 2 (B2) receptors mediate such vasodilation in HCMAs, because studies in animals support this possibility.10–13 Finally, we verified, both through radioligand binding studies and reverse transcription–polymerase chain reaction (RT-PCR), whether HCMAs express AT2 receptors.
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Methods |
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Human Tissue Collection
HCMAs were obtained from 49 heart-beating organ donors (22 men, 27 women; age, 3 to 65 years; mean, 45 years) who died of noncardiac causes (3 cerebrovascular accident, 9 head trauma, 21 subarachnoid bleeding, 4 post–anoxic encephalopathy, 12 intracranial bleeding) <24 hours before the heart was taken to the laboratory. Hearts were provided by the Rotterdam Heart Valve Bank after removal of the heart valves for transplantation purposes. The Ethics Committee of the Erasmus MC approved the study. The hearts were stored in an ice-cold sterile organ-protecting solution after circulatory arrest. After arrival at the laboratory, a tertiary branch of the left anterior descending coronary artery (diameter, 160 to 500 µm; mean, 360 µm) was removed and stored overnight in a cold (4°C), oxygenated Krebs bicarbonate solution of the following composition (mmol/L): NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, and glucose 8.3; pH 7.4. In addition, HCMAs, right epicardial coronary arteries, and/or pieces of left ventricular tissue from 29 hearts were frozen in liquid nitrogen for mRNA determinations or radioligand binding studies.
Myograph Studies
After overnight storage, HCMAs were cut into segments of 
2 mm length and mounted in a Mulvany myograph (J.P. Trading) with separated 6-mL organ baths containing oxygenated Krebs at 37°C. The Krebs was continuously aerated with 95% O2 and 5% CO2, and tissue responses were measured as changes in isometric force, with the use of a Harvard isometric transducer. After a 30-minute stabilization period, the optimal internal diameter was set to a tension equivalent to 0.9 times the estimated diameter at 100 mm Hg effective transmural pressure, as described by Mulvany and Halpern.14 In some vessels, the endothelium was removed by gently rubbing a hair through the lumen of the mounted artery. Endothelial integrity or removal was verified by observing relaxation (or lack thereof) to 10 nmol/L substance P after preconstriction with 10 nmol/L of the thromboxane A2 (TxA2) analogue U46619 (Sigma). Subsequently, to determine the maximum contractile response, the tissue was exposed to 100 mmol/L KCl. The segments were then allowed to equilibrate in fresh organ bath fluid for 30 minutes. Next, segments were preincubated for 30 minutes with the Ang II type 1 (AT1) receptor antagonist irbesartan (1 µmol/L, a gift of Bristol-Myers Squibb),1 the AT2 antagonist PD123319 (1 µmol/L, a gift of Parke-Davis),15 the B2 receptor antagonist Hoe140 (1 µmol/L, a gift of Hoechst)16 and/or L-NAME (100 µmol/L, Sigma). Thereafter, concentration-response curves (CRCs) were constructed to Ang II, either directly or after preconstriction with 10 nmol/L U46619 to 60% of the maximum contractile response. A higher concentration of U46619 (30 nmol/L) was required in segments that had been preincubated with irbesartan because irbesartan antagonizes TxA2 receptors.17 The cyclo-oxygenase inhibitor indomethacin (5 µmol/L) was present during the entire experiment to suppress spontaneously occurring contractions and relaxations.
Cyclic GMP Measurement
To study Ang II–induced cGMP production, vessel segments (5 to 10 mg) were exposed to 1 µmol/L Ang II in 10 mL oxygenated Krebs bicarbonate solution for 1 minute at 37°C in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (100 µmol/L), after a 30-minute preincubation without (control) or with 1 µmol/L PD123319 or irbesartan. Tissues were then frozen in liquid nitrogen and stored at –80°C. To determine cGMP, frozen tissues were homogenized in 0.5 mL 0.1 mol/L HCl with the use of a stainless steel ultraturrax (Polytron). Homogenates were centrifuged at 3300g, and cGMP was measured in 300 µL supernatant by ELISA after acetylation (R&D Systems). Results are expressed as picomoles per milligram of protein. The lower limit of detection was 0.1 pmol/mg protein.
Radioligand Binding Studies
Sarcolemmal membrane fractions were prepared from HCMAs and porcine adrenal glands as described before.18 The adrenals were obtained from three 2- to 3-month-old pigs that had been used in in vivo experiments investigating the effects of calcitonin gene–related peptide receptor (ant)agonists.19 125I-Ang II, prepared with the chloramine T-method (specific activity, 2200 Ci/mmol),20 was used as the radioligand. Assays were run for 60 minutes at 18°C in 30 µL Tris buffer (50 mmol/L), 40 µL membrane fraction (containing 100 µg protein, determined by the Bradford assay as described before15), and 30 µL radioligand (final volume, 100 µL). Nonspecific binding, AT1 receptor–specific binding, and AT2 receptor–specific binding were determined by repeating the experiment in the presence of Ang II (at a concentration 100 times the concentration of 125I-Ang II), irbesartan (0.3 pmol/L to 0.3 mmol/L), and PD123319 (0.3 pmol/L to 0.3 mmol/L), respectively. Incubation was stopped by adding 4 mL ice-cold PBS (pH 7.4). Samples were then filtered through a Whatman GF/B filter. Filters were washed twice with 4 mL ice-cold PBS, and filter-bound radioactivity was measured in a gamma-counter.
AT1 and AT2 Receptor mRNA
Total RNA was isolated from HCMAs, right epicardial coronary arteries, and left ventricular tissue through the use of the Trizol reagent (Gibco-BRL). RT-PCR was performed according to standard procedures and 35 cycles of amplification, using primer sequences as follows: AT1 receptor sense 5'-CTT TTC CTG GAT TCC CCA C-3', and antisense 5'-CTT CTT GGT GGA TGA GCT TAC-3', AT2 receptor sense 5'-GTG ACC AAG TCC TGA AGA TG-3' and antisense 5'-CAC AAA GGT CTC CAT TTC TC-3', resulting in amplification products of 304 and 335 bp, respectively. Positive and negative controls were mRNAs extracted from human liver, a human breast carcinoma cell line (MCF7), and a human colon carcinoma cell line (SW480).21 The absence of nonspecific amplification was verified by running RT-PCR and PCR amplifications without adding tissue extracts. As controls for RNA quality, amplification reactions were performed by using pairs of primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).22 Amplified transcripts were analyzed on 2% agarose gels.
Data Analysis
Data are given as mean±SEM. Contractile or relaxant responses are expressed as a percentage of the contraction to 100 mmol/L K+ or U46619. CRCs were analyzed as described to obtain pEC50 (–10logEC50) values.1 Statistical analysis was made by paired t test, once 1-way ANOVA, followed by Dunnett’s post hoc evaluation, had revealed that differences existed between groups. A value of P<0.05 was considered significant.
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Results |
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Myograph Studies
Ang II constricted HCMAs in a concentration-dependent manner (pEC50=8.6±0.2, n=22), with a maximal response (Emax) of 79±13% (Figure 1). Irbesartan nearly completely blocked the Ang II–mediated constriction. PD123319 increased Emax to 97±14% (P<0.05). PD123319 did not affect the potency of Ang II (pEC50=8.7±0.2, n=22), although in 11 experiments a leftward shift of the Ang II CRC (ie, an increase in the pEC50 value of
0.2) was observed in the presence of the AT2 receptor antagonist. The PD123319-dependent increase in Emax was larger in older donors (r=0.47, P<0.05; Figure 2). The increase in Emax was largest in the 11 experiments in which PD123319 induced a leftward shift of the Ang II CRC: +34±10% versus +2.2±8.4% in the experiments in which PD123319 induced either no (ie, pEC50 <0.2; n=7) or a rightward (ie, pEC50 decreased by 0.2; n=4) shift of the Ang II CRC.
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L-NAME increased baseline contraction to 20% to 30% of the maximum response to 100 mmol/L K+ and prevented the PD123319-induced potentiation of Ang II (Figure 3). Potentiation was also not observed after removal of the endothelium and in the presence of Hoe140 (Figure 3).
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After preconstriction with U46619 (to 60% of the maximum response to 100 mmol/L K+), Ang II caused a marginal further increase (P=NS) in contraction (Figure 4). This response was unaltered by PD123319 and reversed into a relaxation (by maximally 49±16%) in the presence of irbesartan. PD123319 fully prevented the latter relaxation. Without Ang II, U46619-induced preconstrictions in the presence of irbesartan remained stable for at least 60 minutes (data not shown). Thus, the Ang II–induced relaxations under these conditions cannot be attributed to TxA2 receptor antagonism by irbesartan.17
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Cyclic GMP Measurement
Ang II did not significantly increase microvascular cGMP (Figure 5; n=8, P=0.11, versus control) either alone or in the presence of PD123319 or irbesartan.
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Radioligand Binding Studies
The total amount of protein in the HCMA sarcolemmal membrane fraction (500 µg), prepared from vessel segments of 19 subjects, was too small to study a wide range of conditions. We therefore used sarcolemmal membrane fractions prepared from 6 porcine adrenal glands to obtain the most optimal conditions to demonstrate the presence of AT2 receptors in HCMAs. After a 1-hour incubation with 125I-Ang II (final concentration in the incubation mixture, 0.5 nmol/L), total and nonspecific 125I-Ang II binding to porcine adrenal membranes amounted to 4660±150 and 2100±80 cpm/100 µg protein (n=8), respectively. PD123319 and irbesartan abolished specific binding in a concentration-dependent manner (Figure 6A). The inhibitor concentration required to reduce specific binding by 50% (IC50) was 50±1 nmol/L for PD123319. This value mimics the IC50 of PD123319 obtained in previous experiments with cells expressing AT2 receptors only.23 In contrast, the IC50 of irbesartan in the present study (20±1 µmol/L) exceeded its IC50 in cells exclusively expressing AT1 receptors by 3 orders of magnitude.24 Taken together, these data suggest that our porcine adrenal membrane fraction contained predominantly AT2 receptors. A PD123319 concentration of 10 µmol/L is required to fully block 125I-Ang II binding to these receptors.
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On the basis of these findings, as well as on previous studies investigating irbesartan concentrations that selectively block AT1 receptors,24,25 we incubated HCMA membranes with 0.5 nmol/L 125I-Ang II in the absence or presence of 50 nmol/L Ang II, 10 µmol/L PD123319, or 1 µmol/L irbesartan. Ang II reduced 125I-Ang II binding from 1813 to 1175 cpm/100 µg protein. PD123319 and irbesartan both reduced specific binding by 50%, thereby indicating that HCMAs contain AT1 as well as AT2 receptors (Figure 6B).
AT1 and AT2 Receptor mRNA
RT-PCR revealed expression of AT1 and AT2 receptors in HCMAs, large epicardial coronary arteries, and/or left ventricular tissue from 5 hearts (Figure 6C). Similar data were obtained in additional HCMAs from 7 hearts (data not shown).
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Discussion |
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This study is the first to show AT2 receptor–mediated vasodilation in human blood vessels. Evidence for this effect was obtained in two ways. First, the AT2 receptor antagonist PD123319, at a concentration that has been reported to result in complete blockade of AT2 receptor–mediated effects,15 increased the maximal contractile response to Ang II, thereby indirectly demonstrating that AT2 receptor stimulation counteracts AT1 receptor–mediated vasoconstriction. Second, during AT1 receptor blockade with irbesartan (allowing selective AT2 receptor stimulation), Ang II relaxed preconstricted HCMAs, and this was prevented by PD123319. Such vasodilation was not observed in quiescent HCMAs in the presence of irbesartan, probably because vasodilator responses are more difficult to detect without preconstriction. On the basis of these data, it is clear that at least in HCMAs, the net contractile effect of Ang II is determined by the magnitude of the response mediated through AT1 (contraction) and AT2 (relaxation) receptors.
In addition to its effect on Emax, PD123319 caused a leftward shift of the Ang II CRC in 50% of the experiments. Such increased potency of Ang II in the presence of PD123319 is not due to an effect of the AT2 receptor antagonist on Ang II metabolism.25,26 It could point to more efficient AT1 receptor signal transduction during AT2 receptor blockade. Furthermore, a recent study has suggested that AT1 and AT2 receptors form heterodimers.27 An alternative explanation for the increased potency might therefore be that in some donors AT1 receptor–AT2 receptor heterodimers exist that bind Ang II with higher affinity during AT2 receptor blockade. The underlying assumption for this explanation is, however, that AT1 and AT2 receptors in these donors are located on the same cell.
The increase in Emax was larger in older donors, suggesting that the contribution of AT2 receptors increases with age. Although AT2 receptor density increases under pathological conditions,11 the donors in the present study died of noncardiac causes and did not use cardiovascular medication. Thus, it is unlikely that the increased Emax during AT2 receptor blockade in older donors simply reflects the occurrence of cardiovascular disorders in these subjects. It might reflect a general decrease of vascular function with age.
In an earlier study in large epicardial human coronary arteries, we were unable to detect AT2 receptor–mediated vasodilation,1 whereas vasodilation did occur in the rat coronary vascular bed.8 The present study solves this discrepancy by demonstrating that AT2 receptor–mediated vasodilation is limited to coronary microarteries. It is notable that AT2 receptor expression in HCMAs could be demonstrated by both RT-PCR and radioligand binding experiments. Unexpectedly, AT2 receptor mRNA was also detected by RT-PCR in large coronary arteries. This would imply that either the AT2 receptor density in large coronary arteries is too low to allow detection of vasodilation in the organ bath setup or that AT2 receptors in these arteries mediate other (nondilatory) effects, for example, effects on vascular growth and remodeling.28,29 AT2 receptor expression has been demonstrated before in the human myocardium, including the coronary vascular bed.30,31
The mechanism underlying AT2 receptor–mediated vasodilation in HCMAs is currently unknown. AT2 receptors themselves may act as AT1 receptor antagonists independent of Ang II.27 This would require their occurrence on the same cell, as discussed above. Furthermore, B2 receptors, NO, cGMP, Ca2+-activated K+ channels, and/or phosphatases have been implicated in AT2 receptor–induced effects.6,7,10–13,32,33 Our data with L-NAME and Hoe140 in HCMAs support a role for B2 receptors and NO. Because the vasodilator effects in HCMAs were observed in the presence of indomethacin, prostaglandins do not appear to be involved. The lack of effect of PD123319 in deendothelialized segments confirms the contribution of endothelial B2 receptor–induced NO release and simultaneously suggests that AT2 receptors in HCMAs are located on endothelial cells. In agreement with this concept, cultured human coronary artery endothelial cells do express AT2 receptors.34
Taken together, the most likely scenario to explain our results is that Ang II stimulates endothelial AT2 receptors in HCMAs. This results in endothelial B2 receptor activation and NO release. NO subsequently activates guanylyl cyclase in vascular smooth muscle cells, thereby counteracting the contractile responses mediated by the AT1 receptors on these cells. Guanylyl cyclase generates cGMP, and although the Ang II–induced (AT2 receptor–mediated) increase in the microvascular cGMP content in the present study was not significant, the tendency of PD123319 (but not irbesartan) to block this increase mimics similar observations in rat aorta and rat uterine arteries.10,33 The lack of significance in the present experiments probably relates to the modest (30%) increase in cGMP content induced by Ang II as compared with other agonists. For instance, in our experimental setup, 1 µmol/L bradykinin increased microvascular cGMP 7±2-fold (n=4, data not shown).
In conclusion, AT2 receptor–mediated vasodilation occurs in the coronary microcirculation of nondiseased human hearts in an endothelium-dependent manner and is mediated by B2 receptors and NO. This finding could be of clinical relevance, not only because cardiac AT2 receptors are upregulated under pathological conditions,30 but also because animal studies have shown that the beneficial effects of AT1 receptor antagonists, in contrast to those of ACE inhibitors, depend on AT2 receptor stimulation.35,36
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 M. Paul, A. Poyan Mehr, and R. Kreutz Physiology of local Renin-Angiotensin systems. Physiol Rev, July 1, 2006; 86(3): 747 - 803. [Abstract] [Full Text] [PDF]
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W. Chai, I. M. Garrelds, R. de Vries, W. W. Batenburg, J. P. van Kats, and A.H. Jan Danser Nongenomic Effects of Aldosterone in the Human Heart: Interaction With Angiotensin II Hypertension, October 1, 2005; 46(4): 701 - 706. [Abstract] [Full Text] [PDF]
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 K. M. Gauthier, D. X. Zhang, E. M. Edwards, B. Holmes, and W. B. Campbell Angiotensin II Dilates Bovine Adrenal Cortical Arterioles: Role of Endothelial Nitric Oxide Endocrinology, August 1, 2005; 146(8): 3319 - 3324. [Abstract] [Full Text] [PDF]
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 J. Zheng, Y. Wen, D.-b. Chen, I. M. Bird, and R. R. Magness Angiotensin II Elevates Nitric Oxide Synthase 3 Expression and Nitric Oxide Production Via a Mitogen-Activated Protein Kinase Cascade in Ovine Fetoplacental Artery Endothelial Cells Biol Reprod, June 1, 2005; 72(6): 1421 - 1428. [Abstract] [Full Text] [PDF]
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 J. Zheng, I. M. Bird, D.-B. Chen, and R. R. Magness Angiotensin II regulation of ovine fetoplacental artery endothelial functions: interactions with nitric oxide J. Physiol., May 15, 2005; 565(1): 59 - 69. [Abstract] [Full Text] [PDF]
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R. M. Carey Cardiovascular and Renal Regulation by the Angiotensin Type 2 Receptor: The AT2 Receptor Comes of Age Hypertension, May 1, 2005; 45(5): 840 - 844. [Full Text] [PDF]
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T. L. Pallone Microvascular Effects of Aldosterone and Angiotensin Type 2 Receptors Hypertension, May 1, 2005; 45(5): 845 - 846. [Full Text] [PDF]
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 D. J. Campbell, H. Krum, and M. D. Esler Losartan Increases Bradykinin Levels in Hypertensive Humans Circulation, January 25, 2005; 111(3): 315 - 320. [Abstract] [Full Text] [PDF]
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