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European Journal of Heart Failure 2002 4(6):699-705; doi:10.1016/S1388-9842(02)00166-6
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© 2002 European Society of Cardiology

Comparison of the antagonistic effects of different angiotensin II receptor blockers in human coronary arteries

Emil Pantev, Emelie Stenman, Angelica Wackenfors, Lars Edvinsson and Malin Malmsjö*

Division of Experimental Vascular Research, Department of Internal Medicine, Lund University Hospital Lund, Sweden

* Corresponding author. Experimental Vascular Research, BMC A13, SE-221 84 Lund, Sweden. Tel.: +46-733-565650; fax: +46-46-222-0616. E-mail address: malin.malmsjo{at}med.lu.se


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background: Angiotensin II (Ang II) is a potent vasoconstrictor and a deleterious factor in cardiovascular pathophysiology. Ang II receptor blockers (ARBs) have recently been introduced into clinical practice for treatment of hypertension and congestive heart failure.

Aims: This study was undertaken to evaluate the inhibitory effects of ARBs on vasoconstriction in humans.

Methods: Vasomotor tone was analyzed in endothelium denuded, human coronary artery (HCA) segments. Ang II type 1 (AT1) and type 2 (AT2) receptor mRNA expression was examined by reverse transcriptase-polymerase chain reaction (RT-PCR).

Results: Ang II was a potent vasoconstrictor (pEC50=7.7). At 1 nM of the AT1 receptor antagonists, candesartan and valsartan, the maximum contraction was depressed to 57 and 50% of Ang II, respectively, indicating insurmountability. Although generally considered surmountable, the presence of 100 nM losartan elicited a depression of the Ang II response to 32%. Its active metabolite, EXP 3174 (1 nM), abolished the Ang II contraction. The AT1 receptor antagonists had the following order of blocking effect; EXP 3174>candesartan=valsartan>losartan. The AT2 receptor antagonist, PD 123319 (100 nM) significantly attenuated the Ang II contraction (Emax=62% of Ang II). RT-PCR of HCA smooth muscle cells demonstrated expression of both AT1 and AT2 receptor mRNA.

Conclusions: Ang II contraction in HCA is mediated mainly by AT1 but also involves AT2 receptors. The active metabolite of losartan, EXP 3174, is the most efficacious AT1 receptor antagonist in HCA.

Key Words: Coronary circulation • Vasoconstriction • Angiotensin II • AT1 receptor • AT2 receptor

Received December 3, 2001; Revised February 22, 2002; Accepted May 1, 2002


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Angiotensin II (Ang II) is a major effector molecule in the renin–angiotensin system and exerts a wide range of cardiovascular, renal and endocrine actions. Ang II formation has been demonstrated, not only in the systemic circulation, but also in a number of local tissues, including the human vasculature [1,2]. Two angiotensin receptors have been identified in man, the Ang II type 1 (AT1) and type 2 (AT2) receptors [3,4], which are members of the G protein coupled, seven transmembrane domain receptor family. Ang II-induced vasoconstriction in human arteries has so far mainly been attributed to AT1 receptors on smooth muscle cell [5–8]. The role for AT2 receptors in the vasculature is less well known, although the discovery of the highly selective, non-peptidergic, AT2 receptor antagonist, PD 123319, has made it possible to distinguish a dilatory response. This response is due to an endothelial release of nitric oxide and prostaglandins [9]. Adaptation to pathological conditions involves increased expression of AT2 receptors in vascular smooth muscle cells, and its contribution to the Ang II-induced vasoconstriction is therefore greater in disease [10].

During the last decade, much effort has been put into the development of non-peptide AT1 receptor antagonists [11]. In preclinical studies, these antagonists are routinely tested in vascular tissues for their ability to affect the contractile Ang II-induced concentration–response curves [5,6,12–14]. Based on their capability to depress the maximal contractile response to Ang II, the antagonists are commonly divided into two categories; surmountable and insurmountable. Losartan is an imidazole derivative and the first non-peptidergic Ang II receptor antagonist to be introduced into clinical practice [15–18]. This antagonist possesses surmountable properties since it only produces a parallel rightward shift of the concentration–response curve, without depressing the maximal response. Candesartan, valsartan and the active metabolite of losartan, EXP 3174, depress the maximal response to Ang II and are therefore referred to as insurmountable [19].

Although Ang II receptor blockers now are available for the treatment of hypertension and congestive heart failure, data on the pharmacological characteristics in the human vasculature is scarce. The present study was designed to evaluate the inhibitory effect of the clinically used Ang II receptor antagonists, losartan, candesartan and valsartan, on vasoconstriction of endothelium denuded human coronary arteries (HCAs). The relative contribution of AT1 and AT2 receptors to the Ang II-induced vasoconstriction was assessed using the specific AT2 receptor inhibitor PD 123319.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Patients
HCA were harvested in the operating room from organs explanted in the process of a heart transplant procedure. The patients were between 19 and 64 years of age and suffered from end-stage heart failure due to cardiomyopathy.

2.2. In vitro pharmacology
Epicardial segments from medium sized (2–3 mm outer diameter) coronary arteries were immersed in cold oxygenated buffer solution (for composition, see below) and transported to the laboratory for dissection from adhering tissues under a microscope. Immediately prior to the experiment, the endothelium was mechanically removed by gentle rubbing in the artery lumen with a thin wooden stick. The vessels were then cut into cylindrical segments (2-mm long) and mounted on two L-shaped metal prongs, one of which connected to a force displacement transducer (FT03C) for continuous recording of the isometric tension, and the other to a displacement device [20]. The mounted artery segments were immersed in temperature controlled (37 °C) tissue baths that contained a bicarbonate based buffer solution of the following composition (mM): NaCl 119, NaHCO3 15, KCl 4.6, MgCl2 1.2, NaH2PO4 1.2, CaCl2 1.5 and glucose 5.5. The solution was continuously gassed with 5% CO2 in O2 resulting in a pH of 7.4. Eight ring segments were studied simultaneously in separate tissue baths. The segments were allowed to stabilize at a resting tension of approximately 3 mN for 1 h before the experiments were started. The contractile capacity of each vessel segment was examined by exposure to a K+ rich (60 mM) buffer solution in which NaCl was exchanged for an equimolar concentration of KCl (for composition, see above). When two reproducible contractions had been achieved, the vessels were exposed to further studies. PD 123319 (100 nM), losartan (100 nM), candesartan (1 and 10 nM), valsartan (0.1 and 1 nM) or EXP 3174 (0.01, 0.1 and 1 nM) was added 1 h prior to Ang II. Concentration–response curves were constructed by addition of increasing concentrations of Ang II (0.01 nM to 10 µM).

2.3. Reverse transcriptase-polymerase chain reaction (RT-PCR)
2.3.1. RNA extraction
The HCAs were carefully dissected and the endothelium was removed (see above). The arteries were snap-frozen in liquid nitrogen immediately after acquisition. Total cellular RNA was extracted using TRIzol reagent (Gibco BRL) following the supplier's instructions. The resulting RNA pellet was washed with 70% ice-cold ethanol, air-dried and re-dissolved in 10-µl diethyl-pyrocarbonate (DEPC) treated water. The RNA concentration was determined spectrophotometrically considering a ratio of OD260:280>=1.6 as pure.

2.3.2. RT-PCR
Reverse transcription and subsequent PCR were carried out in a Perkin-Elmer DNA Thermal Cycler (Perkin-Elmer, Foster City, CA). Specific primers for the human AT1 and AT2 receptors were constructed based on published nucleotide sequences [21,22].
Product length (bp)
AT1 forward 5'-GATGATTGTCCCAAAGCTGG-3'
AT1 reverse 5'-TAGGTAATTGCCAAAGGGCC-3' 255
AT2 forward 5'-AAGAAGAAATCCCTGGCAAGC-3'
AT2 reverse 5'-CTTGGTCACGGGTTATCCTGT-3' 302

First-strand cDNA synthesis was carried out with the TaqMan® Reverse Transcription Reagents kit (Perkin-Elmer) in a 20-µl volume using random hexamers as primers. The reaction mixture was incubated at 42 °C for 30 min, heated to 99 °C for 5 min, and thereafter chilled to 4 °C. The DNA amplification was performed using the GeneAmp RNA PCR kit (Perkin-Elmer) and AmpliTaq Gold® DNA polymerase (Applied Biosystems, Foster City, CA). Amplification was performed using a modified profile (9 min at 95 °C followed by 40 cycles of 1 min 95 °C, 30 s 60 °C, 30 s 72 °C, and a final extension step of 7 min at 72 °C). The products were separated on a 2% agarose gel containing 1.0 µg/ml ethidium-bromide and photographed. A 100-bp DNA ladder (Promega Co.) was used as molecular weight marker. A no-template control experiment, where nuclease free water was added instead of cDNA prior to the PCR reaction, was run to exclude contamination of foreign nucleic acids.

2.4. Ethics
The project was approved by the Ethics Committee of Lund University in Sweden (LU 379-93) and conforms with the principles outlined in the Declaration of Helsinki.

2.5. Drugs
Ang II was purchased from Sigma Co., USA, and was dissolved in 0.1% bovine serum albumin. PD 123319 ((S)-1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid trifluoroacetate), losartan, candesartan, valsartan and EXP 3174 were generous gifts from Assoc. Prof. Peter Morsing, AstraZeneca, Sweden. These substances were dissolved in 0.9% saline. If not stated otherwise, all reagents for the RT-PCR assay were purchased from Gibco, BRL (USA).

2.6. Calculations and statistics
The negative logarithm of the Ang II concentration that elicited 50% contraction (pEC50) was determined by fitting the data to the Hill equation. Emax refers to the maximal Ang II contraction presented as percent of the contraction evoked by 60 mM K+ (% of K+). All different types of experiments could not be performed in arteries from only 1 patient, since the amount of arteries that were obtained from the transplanted hearts was limited. The data could therefore not be paired. In addition, the maximum response to Ang II varied from 11 to 144% of K+. The results from the controls and the different antagonist concentrations could therefore not be added up. The problem was solved by calculating the Ang II contraction, in the presence of each antagonist, as percent of the maximal Ang II response in the absence of antagonist, i.e. the control, and the results were presented as Emax (% of Ang II). Each experiment was repeated in 5–10 vessels segments from different patients. Statistical significance was accepted when P<0.05, using Student's t-test. All differences referred to in the text are statistically verified and values are presented as mean values±S.E.M. RT-PCR was carried out in blood vessels from 3 patients with similar results.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Contractile responses
Viability and contractility of the HCA was examined by addition of 60 mM K+ (11.3±2.9 mN). Ang II was a potent and efficacious vasoconstrictor (pEC50=7.7±0.2 and Emax=75±11% of K+).

3.1.1. Candesartan
Increasing concentrations of candesartan resulted in a progressive reduction in the maximal response to Ang II (Fig. 1a). At a concentration of 1 nM the Emax value was reduced to 65±14% of Ang II while the pEC50 value was not altered (pEC50=7.8±0.1). 10 nM of candesartan almost abolished this response (Emax=2±2% of Ang II), indicating insurmountability.


Figure 1
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  		Fig. 1 Concentration–response curves to increasing concentrations of Ang II in the absence (control, Emax fixed to 100%) and presence of (a) 1 and 10 nM candesartan (b) 0.1 and 1 nM valsartan (c) 100 nM losartan and (d) 0.01, 0.1 and 1 nM EXP 3174, in endothelium denuded HCAs. Contractions in the presence of inhibitors are expressed as percentage of the Ang II control. Data are shown as mean values±S.E.M.

 
3.1.2. Valsartan
Preincubation with 0.1 nM of valsartan only slightly attenuated the Ang II contraction (Emax=85±12% of Ang II, pEC50=8.0±0.5), while 1 nM reduced the maximum response to 50±15% (pEC50=7.6±0.4, Fig. 1b). Valsartan thus possessed similar insurmountable properties to those of candesartan.

3.1.3. Losartan
In the presence of 0.1 µM losartan the Ang II concentration response curve did not reach a maximum, although at an Ang II concentration of 10 µM the contraction amounted to 32±11% of Ang II (Fig. 1c). In approximation, the Ang II concentration–response curve was shifted 2.5 logarithms to the right.

3.1.4. EXP 3174
The active metabolite of losartan, EXP 3174, proved to be a very efficacious insurmountable antagonist since already at a concentration of 0.01 nM, the maximum Ang II response was reduced to 69±20% of Ang II without affecting the pEC50 value (7.6±0.4). 0.1 nM of EXP 3174 inhibited the major part of the Ang II contraction (Emax=34±13% of Ang II, pEC50=7.5±0.6), while it was almost completely abolished at a concentration of 1 nM (Emax=2±2% of Ang II, Fig. 1d).

3.1.5. AT2 receptor antagonism
Preincubation with 100 nM PD 123319 attenuated the contractile effect of Ang II (Emax=62±11% of Ang II, pEC50=7.8±0.2, Fig. 2), indicating presence of contractile AT2 receptors in HCA smooth muscle cells.


Figure 2
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  		Fig. 2 Concentration–response curves to increasing concentrations of Ang II in the absence (control, Emax fixed to 100%) and presence of 100 nM PD 123319 in endothelium denuded HCAs. Contractions in the presence of PD 123319 are expressed as percentage of the Ang II control. Data are shown as mean values±S.E.M.

 
3.2. RT-PCR
Agarose gel electrophoresis of PCR products from endothelium denuded HCA demonstrated products of the expected size for the corresponding mRNA encoding human AT1 (255 bp) and AT2 (302 bp) receptors (Fig. 3). Bands for the AT1 receptor mRNA was especially prominent. No bands were detected in the no-template control experiments, excluding contamination of foreign nucleic acids.


Figure 3
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  		Fig. 3 Electrophoresis of RT-PCR products corresponding to mRNA encoding human AT1 and AT2 in endothelium denuded HCAs. The amplified products were of the predicted size for AT1 (255 bp) and AT2 (302 bp) when compared to a 100-bp DNA ladder (to the left). Samples were analyzed on a 2% agarose/ethidium-bromide gel at 5 V/cm, and photographed.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In the present study, the clinically used AT1 receptor antagonists were evaluated for their inhibitory effects at the Ang II-induced vasoconstriction in HCA. Interestingly, the active metabolite of losartan, EXP 3174, was the most efficacious blocker. This was true even when the EXP 3174 effects, at the clinically obtained plasma-concentration of after administration of a recommended dose of losartan (0.6 nM), was compared to that of candesartan (0.9 nM) [12,16,23]. At these concentrations candesartan only inhibited approximately 35% of the Ang II-induced vasoconstriction while EXP 3174 totally abolished it. The contractile effect of Ang II showed to be mediated both by AT1 and AT2 receptors in HCA. AT2 receptors are generally believed to mediate endothelium-dependent dilatation [9]. In this study, the HCA were obtained from patients that were transplanted due to heart failure. Contractile properties of AT2 receptors, located on smooth muscle cells, might in this case have been a consequence of a pathophysiological adaptation to heart failure and are not necessarily expressed in healthy HCA.

HCA were obtained from explanted hearts during transplantation. The contractile response to Ang II showed to be inhomogeneous in these arteries. Ang II-induced contraction varied between 11 and 144% of K+. Contributing causes to this variation may be differences in age, health status and medication. Unfortunately, the number of transplanted patients is too small to allow correlations of these different factors to the Ang II response. Therefore, to eliminate such a source of error, the effect of each receptor antagonist was related to the Ang II contraction in each patient, where the Ang II effect was normalized to 100%.

Before the experiments were started, the endothelium was removed mechanically to minimize the influence that a varying endothelium function would imply in these arteries from patients with heart failure. Previous results from our group have shown that the endothelium is poor in HCAs that are obtained in the procedure of heart transplantation. The arteries did not dilate when exposed to 1-µM acetylcholine, which was probably due to coronary artery disease [24].

At the recommended doses of candesartan in humans (8 and 16 mg [23]) the free plasma concentration at half drug peak concentration is estimated to be approximately 0.3 nM [12]. At a similar concentration (1 nM) the maximum response to Ang II was reduced to 65% indicating insurmountability. The clinically used dose of losartan (50 mg [16]) corresponds to a free plasma concentration, at half drug peak, of approximately 5 nM for losartan and 0.6 nM of its metabolite EXP 3174 [12]. The antagonist concentrations used in this study have been chosen in accordance to the evaluated plasma-concentrations observed in previous work. In the presence of 10 nM losartan, the concentration–response curve for Ang II was shifted 1.6 logarithms to the right in HCA [24]. In the present experiments, addition of 100 nM losartan resulted in an {approx}2.5 logarithm shift. Although losartan is generally believed to be a surmountable AT1 receptor antagonist, this seems not to be the case in HCA where the maximum response of Ang II is decreased by 26% by 10 nM losartan [24]. One possible explanation is that losartan is hydrolyzed to EXP 3174, occurring either while present in the tissue bath (1 h prior to the addition of Ang II), at the time of preparation from dry substance or during storage in solution. EXP 3174 by itself displayed prominent inhibitory effects, with 1 nM almost completely abolishing the Ang II contraction. This supports the suggestion that EXP 3174 exerts the major part of the losartan-induced AT1 receptor blocking effect in humans after oral administration of a dose of 50 mg [12]. The effect of EXP 3174 is more prominent than that for candesartan at clinically relevant plasma-concentrations. 0.9 nM candesartan (obtained after administration of 8–16 mg candesartan [12]) only inhibited approximately 35% of the Ang II constriction while 0.6 nM EXP 3174 (obtained after administration of 50 mg losartan [12]) totally abolished it. Inhibitory effects of AT1 receptor antagonists on the Ang II contraction and the simultaneous presence of AT1 receptor mRNA in HCA smooth muscle cells, enabled us to conclude that Ang II-induced contraction was mediated by AT1 receptors.

The order of blocking effect of the AT1 receptor antagonists tested in this study was; EXP 3174>candesartan=valsartan>losartan. This pharmacological profile differs slightly to what have previously been reported in other vascular preparations and in isolated cells; candesartan>>EXP 3174>valsartan>losartan [7,12,25,26]. It should be noted that few of these observations have been made in human vasculature and none using coronary tissue. Indeed, Ytterberg et al. confirmed that this order of potency also applies to human subcutaneous arteries, where EXP 3174 was more efficacious as compared to candesartan [7]. Another explanation to the difference in AT1 receptor blocker effect is the presence of contractile AT2 receptors in addition to the AT1 receptors in our vascular preparation. More unlikely, a not previously described receptor, with a different antagonist profile might be responsible for the observed effects.

Contractile AT2 receptors were present in the HCA. RT-PCR of HCA smooth muscle cells revealed AT2 receptor mRNA expression and the Ang II-induced contraction was in part antagonized by 100 nM PD 123319. This concentration has previously been used in human arteries to selectively inhibit AT2 receptors [5]. Furthermore, 1 µM PD 123319 has been shown to have no effect at the AT1 receptor mediated constriction [27]. AT2 receptors are generally believed to mediate endothelium-dependent dilatation [9]. Conversely, AT2 receptors have been visualised by immunohistochemistry in the media of arteries [28]. Human adult hearts have been shown to express AT2 receptors [29]. A contractile effect of AT2 receptors in the HCA as compared to human subcutaneous [7] and omental arteries [8] may be due to different embryological origin where the coronary arteries arise from the epicardial lining and the peripheral arteries develop from the mesectoderm of the neural crest [30]. Expression of AT2 receptors can be modulated by pathological states associated with tissue remodeling and inflammation. AT2 receptors have been implicated in pathological conditions associated with cardiovascular remodeling. In neointima formation after vascular injury, AT1 receptor expression is changed to that of the AT2 subtype [31]. In diabetes [32], postmyocardial infarction [33], ischemia [34] and hypertension [35], AT2 receptor expression may be enhanced. The patients in this study suffered from heart failure, a condition shown to induce increased AT2 receptor expression in human hearts [29,36–38]. Due to natural causes, human studies are hampered by the lack of control material and therefore it cannot be concluded whether pathology is the cause of this AT2 receptor presence although, involvement of AT2 receptors in Ang II contraction has been shown to be associated with a pathophysiological adaptation to hypertension in rats [39]. During neointima formation after vascular injury and atherosclerosis, AT2 receptors are re-expressed in cells proliferating in interstitial regions or the neointima [10]. Presence of AT2 receptors on smooth muscle cells in HCA may have pathophysiological negative effects due to contractile properties. Of greater importance may be that AT2 receptors inhibit Ang II-induced mitogenesis and synthesis of extracellular matrix proteins, resulting in attenuation of tissue remodeling and vascular hypertrophy [10].

In conclusion, the studied AT1 receptor antagonists inhibit the Ang II contraction in endothelium denuded HCA in the following order; EXP 3174>candesartan=valsartan>losartan. The Ang II-induced contraction was totally inhibited by EXP 3174 at a concentration roughly equivalent to that measured in plasma after oral administration of 50 mg of losartan. Ang II was observed to elicit vasoconstriction not only by affecting AT1, but also through AT2 receptors. The HCA were derived from patients with heart failure and it remains to be elucidated whether the contractile AT2 receptors were expressed as a consequence of a pathological adaptation of the arteries.


   Acknowledgements
 
This study has been supported by the Swedish Hypertension Society, the Royal Physiographic Society (Lund), the Swedish Research Council (grant no. 5958). We thank Assoc. Prof. Peter Morsing, AstraZeneca, Sweden, for supplying us with candesartan, valsartan, losartan, EXP 3174 and PD 123319.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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