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CARDIOVASCULAR
Department of Pharmacology, Faculty of Pharmaceutical Sciences and High Technology Research Center, Kobe Gakuin University, Kobe, Japan (K.Y., M.H., H.H., M.T., H.O.); and Department of Pharmacology, Faculty of Pharmaceutical Sciences, Mukogawa Women's University, Nisinomiya, Japan (S.K., M.K.)
Received August 10, 2003; accepted October 14, 2003.
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Abstract |
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). Ang II production in the proximity to its receptors on the target cells constitutes the local renin-angiotensin system, which regulates the cardiovascular functions in both autocrine and paracrine manners. The actions of Ang II are primarily mediated by two receptors, Ang II type 1 (AT1) and type 2 (AT2). The activation of the AT1 receptor mediates vasoconstriction, proliferation of vascular smooth muscle cell, and production of extracellular matrix proteins by vascular smooth muscle cells (de Gasparo et al., 2000). In contrast, the AT2 receptor has been considered to mediate vasodilation, antiproliferation, and proapoptosis in the vasculature, presumably mediated by the activation of the nitric oxide (NO) system via bradykinin production (de Gasparo et al., 2000).
Although a small number of AT2 receptors are present in the vessels, the physiological actions of Ang II via AT2 receptors have been difficult to determine, because the AT2 subtype has a low degree of expression compared with the AT1 subtype (Viswanathan et al., 1991). Nevertheless, most previous studies using normotensive or hypertensive animals (Scheuer and Perrone, 1993; Siragy and Carey, 1999), as well as knockout or transgenic mice for the AT2 receptor gene (Hein et al., 1995; Ichiki et al., 1995; Akishita et al., 1999; Tsutsumi et al., 1999), have demonstrated that the AT2 receptor mediates a depressor response to Ang II. Recently, it became evident that the AT2 receptor levels in the vasculature were increased under some pathological conditions, such as hypertension and vascular injury (Otsuka et al., 1998; Hutchinson et al., 1999; Touyz et al., 1999). This evidence suggests that the up-regulation of vascular AT2 receptors under pathological conditions is one of the compensatory mechanisms of vessels counteracting the AT1-mediated contractile response to Ang II to protect vessels from the mechanical overload. To test this hypothesis, we used a rat model of pressure-induced left ventricular hypertrophy (Doggrell and Brown, 1998) produced by suprarenal abdominal aortic coarctation (banding) to determine how the increased transmural pressure influences the vascular AT2 receptor expression and thereby changes the contractile response to Ang II.
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Materials and Methods |
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Animals and Operation. All animal experiments were performed according to the guidelines of the Kobe Gakuin University Experimental Animal Care and Use Committee. Male Wistar rats (4 weeks old; Japan SLC, Hamamatsu, Japan) were divided into three groups: 1) untreated, 2) sham-operated, and 3) pressure-overloaded rats. Pressure-overload was produced by abdominal aortic banding, which has been primarily used as a model of cardiac hypertrophy (Doggrell and Brown, 1998). Briefly, animals were anesthetized with sodium pentobarbital (50 mg/kg i.p.), and the aorta was exposed through a midline abdominal incision. For the banding model, a blunt 22-gauge needle was placed adjacent to the abdominal aorta between the renal arteries just below the renal bifurcations, and a ligature was tightened around the aorta and adjacent needle. The sham procedure for control rats included injection of the same amount of anesthetic, in incision of approximately the same size, and the placement of a loosely tied ligature at the exact same position on the abdominal aorta.
Losartan was dissolved in saline and administered i.p. at a dose of 1 mg/kg once a day for 7 days. Candesartan was suspended in 10% gum arabic and administered orally at a dose of 2 mg/kg once a day for 7 days.
Blood Pressure Measurement. The patency of the aortic banding was assessed by measuring the blood pressure of carotid artery under pentobarbital anesthesia (50 mg/kg i.p. pentobarbital sodium). To measure the blood pressure, the left carotid artery was cannulated with a PE-10 polyethylene catheter, and pulse waveforms were monitored by a polygraph system (Nihon Kohden, Tokyo, Japan).
Assays of Plasma Renin Concentration (PRC) and Plasma Renin Activity (PRA). Blood was collected from the abdominal aorta into a syringe containing 1/10 volume of 3.8% sodium citrate under ether anesthesia. After centrifugation at 800g for 15 min, plasma samples were collected and stored at –90°C until assays. PRC or PRA in plasma samples were determined by radioimnmunoassay of Ang I liberated in the presence or absence of plasma from bilaterally nephrectomized rats, respectively, as described previously (Ohtani et al., 1989).
Analysis of AT1 and AT2 Receptor mRNAs by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Animals were sacrificed under ether anesthesia 4, 7, 14, and 28 days after aortic banding or sham operation. Age-matched untreated rats were also sacrificed as a control. The excised thoracic aorta (approximately 10 mg) was stripped of adventitia and then homogenized in acid guanidinium-phenol-chloroform to extract total RNA, as described previously (Yayama et al., 2003). To detect AT1 and AT2 receptor mRNAs, we used two methods: one was RT-PCR followed by Southern blotting with respective specific probes, and second was quantitative real-time RT-PCR. Reverse transcription was performed in a reaction volume of 4 µl containing 0.3 µg of RNA, 1.55 µl of dNTP (10 mM), 0.8 µl of MgCl2 (20 mM), 0.4 µl of 10x buffer (500 mM KCl in 0.1 mol/l Tris-HCl, pH 8.3), 0.05 µl of 10 unit enzyme (Moloney murine leukemia virus reverse transcriptase), and 0.2 µl of reverse primer (50 pmol/µl). The mixture was incubated at 42°C for 15 min, 95°C for 5 min, and then 4°C for 5 min to allow synthesis of the first-strand cDNA. The cDNA was amplified in a 20-µl reaction mixture containing 10 pmol of the forward primer, 1.6 µl of 10x buffer, 4 µl of MgCl2 (25 mM), and 0.5 units of Taq DNA polymerase at 95°C for 9 min, at 94°C for 1 min, and annealing at 54°C (AT1 receptor), 56°C (AT2 receptor) or 60°C (glyceraldehyde-3-phosphate dehydrogenase; GAPDH) for 2 min, and then extension at 72°C for 3 min followed by at 72°C for 10 min. The amplification cycles were 35 for the AT1 receptor, 40 for the AT2 receptor and 25 for GAPDH. After amplification, the PCR products were electrophoresed on a 1.5% agarose gel, and then denatured, neutralized, and transferred onto a nylon membrane by capillary blotting and cross-linked by UV irradiation. The transferred membranes were detected by Southern blot hybridization with 32P-labeled cDNA probes specific for each DNA. The blot signals were detected using a Fujix Bio Imaging analyzer BAS 2000 (Fujifilm, Tokyo, Japan). The forward and reverse primers for the AT1 receptor were 5'-CACCTATGTAAGATCGCTTCT-3' and 5'-GCACAATCGCCATAATTATCC-3', for the AT2 receptor 5'-CTGACCCTGAACATGTTTGCA-3' and 5'-GGTGTCCATTTCTCTAAGAGA-3', and for GAPDH 5'-GTGCCAAAAGGGTCATCATCT-3' and 5'-CAGCATCAAAGGTGGAGGAAT-3', respectively.
For quantitative measurement of the AT2 receptor mRNA, 0.3-µg RNA samples from the thoracic aorta were reverse-transcribed using a reverse primer (5'-ATACCCATCCAGGTCAGAGCAT-3') as described above. The cDNA products in 2 µl were mixed with 18 µl of TaqMan master mixture (2 µlof10x buffer, 2.8 µl of MgCl2 (20 mM), 2 µl of reverse primer (3 µM), 2 µl of forward primer (3 µM), 2 µl of TaqMan probe (2 µM), 0.4 µl of 10 mM dUTP, 0.1 µl of uracil N-glycosylase (1 unit/µl), and 0.1 µl of Taq DNA polymerase (5 unit/µl)). PCR was carried out in an ABI Prism 7700 system (Applied Biosystems, Tokyo, Japan) under the following conditions: 50°C for 2 min, 95°C for 10 min, 40 cycles at 95°C for 15 s, and 60°C for 1 min. The PCR results were analyzed with the Sequence Detector 1.6 program (PerkinElmer Instruments, Norwalk, CT). For the AT2 receptor, the following primers and probes were used: forward primer, 5'-CCCGTGACCAAGTCTTGAAGAT-3'; reverse primer, 5'-ATACCCATCCAGGTCAGAGCAT-3'; TaqMan probe, 5'-FAM-TGGCATTCATCATTTGCTGGCTTCC-TAMRA-3'. For GAPDH, the following primers and probes were used: forward primer, 5'-CGTGTTCCTACCCCCAATGT-3'; reverse primer, 5'-TGATGTCATCATACTTGGCAGGTT-3'; TaqMan probe, 5'-FAM-CGTTGTGGATCTGACATGCCGCC-TAMRA-3'.
Organ Chamber Experiments. Rats were anesthetized with pentobarbital sodium (50 mg/kg i.p.), and then the thoracic aorta was dissected free, excised, and placed in Krebs-Henseleit solution of the following composition: 118.4 mmol/l NaCl, 4.7 mmol/l KCl, 2.5 mmol/l CaCl2, 1.2 mmol/l KH2PO4, 1.2 mmol/l MgSO4, 25 mmol/l NaHCO3, and 11.1 mmol/l glucose. The aortas were cleaned of adherent connective tissue and cut into rings (3 mm in length). Each ring was fixed vertically under a resting tension of 1.0 g in a 10-ml organ bath filled with the solution (37°C, pH 7.4) described above. In some rings, the endothelium was mechanically removed by gentle rubbing with moistened cotton. The bath solution was continuously aerated with a gas mixture of 95% O2, 5% CO2 and then the rings were allowed to equilibrate for 90 min before the start of the experiments. Isometric tension change was measured with a force displacement transducer (model t-7; NEC San-Ei, Tokyo, Japan) coupled to a dual-channel chart recorder (model 8K21; NEC San-Ei). After reaching equilibrium, cumulative concentration-response curves were constructed for Ang II (10–10–10–7 M) in each ring. In some experiments, various agents, such as AT2 receptor antagonist PD123319 (1 x 10–6 M), bradykinin type 2 (B2) receptor antagonist icatibant (1 x 10–6 M), and L-NAME (1 x 10–4 M), were added 30 min before the cumulative addition of Ang II. The contractile responses obtained were expressed as a percentage of the maximal constriction evoked by 40 mM KCl. No significant changes in the contractile response to 40 mM KCl were observed in aortic rings from rats 1 week after banding compared with those from age-matched untreated rats. Denudation of the endothelium was confirmed pharmacologically by the disappearance of the 1 x 10–7 M acetylcholine-induced relaxation response during constriction evoked by 3 x 10–7 M phenylephrine.
Assay of cGMP in Aortic Rings. Aortic rings were fixed in a 10-ml organ bath filled with Krebs-Henseleit solution as described above. After allowing equilibration for 1 h, IBMX (5 x 10–5 M) was added to the organ bath. Twenty-five minutes after the addition of IBMX, the rings were stimulated with Ang II (1 x 10–7 M) for 5 min. In some experiments, L-NAME (1 x 10–4 M) was added at the same time as the IBMX addition. Thereafter, the rings were frozen in dry ice-acetone and homogenized in ice-cold 6% trichloroacetic acid. cGMP was extracted, acetylated, and quantified by radioimmunoassay using the cyclic GMP 125I Biotrak assay system (Amersham Biosciences Inc., Piscataway, NJ) and is expressed as picomoles per milligram of protein. Protein contents of the rings were measured by the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA) using 
-globulin as a standard.
Statistical Analysis. All data are expressed as mean ± S.E. Statistical comparisons of PRC, PRA, blood pressure, AT2 receptor mRNA levels, and cGMP contents under various treatments were performed with one-way analysis of variance with pairwise comparison by the Bonferroni-Dunn method. Comparison of concentration-response curves of Ang II was carried out by repeated measures analysis of variance followed by the Bonferroni-Dunn method. Differences were considered significant for p < 0.05.
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PRC and PRA were significantly increased at 2 and 4 days after aortic banding and returned to the levels of sham-operated animals at 7 days; both levels remained low 14 and 28 days after banding (Fig. 1).
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AT1 and AT2 Receptor mRNA Levels in the Thoracic Aorta after Aortic Banding. To determine whether the pressure-overload alters the vascular expression of AT1 and AT2 receptor mRNAs, we examined the mRNA levels of these receptors in the thoracic aorta by RT-PCR. As shown in Fig. 2, signals corresponding to AT1 receptor mRNA could easily be detected in the thoracic aorta of sham-operated animals, whereas only faint signals were detectable for AT2 receptor mRNA. However, the levels of AT2 receptor mRNA, but not AT1 subtype, were increased 4, 7, 14, and 28 days after the banding of the abdominal aorta (Fig. 2). These observations were confirmed by quantitative real-time PCR: the levels of AT2 receptor mRNA increased more than 300% after 4, 7, and 14 days compared with those of age-matched untreated rats or sham-operated rats (Fig. 2). High levels of AT2 receptor mRNA were also observed after 28 days.
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Involvement of the AT1 Receptor in the Up-Regulation of AT2 Receptor mRNA in the Thoracic Aorta after Aortic Banding. To examine whether the pressure-overload-induced increase in AT2 receptor mRNA is mediated by Ang II itself, we studied the effects of the AT1 receptor antagonists losartan and candesartan on the up-regulation of AT2 receptor mRNA in the thoracic aorta. As shown in Fig. 3, the up-regulation of AT2 receptor mRNA 7 days after aortic banding was completely inhibited by successive administration of losartan (1 mg/kg/day i.p.) or candesartan (2 mg/kg p.o.) for 7 days. In contrast, the administration of losartan or candesartan did not affect the blood pressure elevation in the thoracic aorta after aortic banding: 128.3 ± 5.9 mm Hg in untreated rats with aortic banding (n = 5) versus 126.7 ± 5.6 mm Hg in losartan-treated rats with aortic banding (n = 5; p > 0.1) and 127.4 ± 5.2 mm Hg in candesartan-treated rats with banding (n = 5; p > 0.1).
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AT2 Receptor-Dependent Decrease in the Contractile Response to Ang II in Ring Preparations of Pressure-Overloaded Thoracic Aorta in Vitro. The contractile response to Ang II was compared between the ring preparations of the thoracic aorta from sham-operated (control rings) and aortic banding rats (pressure-loaded rings). We examined thoracic aortas from rats 7 days after banding or sham operation, because high levels of AT2 receptor mRNA were detected in thoracic aortas of banding rats during this period. The contractions evoked by Ang II were significantly decreased in the pressure-loaded rings at higher concentrations of Ang II, such as 3 x 10–8 and 1 x 10–7 M, compared with control rings (Fig. 4A). Blockade of the AT2 receptor by PD123319 (1 x 10–6 M) increased the contractile response to Ang II in the pressure-loaded rings (Fig. 4C), but not in control rings (Fig. 4B).
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Then we compared the contractile response to Ang II between the ring preparations from rats treated or untreated with losartan for 7 days after aortic banding, because of the observation that the administration of losartan abolished the up-regulation of AT2 receptor mRNA in the thoracic aorta after pressure-overload. Aortic rings were prepared from rats 24 h after the final administration of losartan at day 6. As shown in Fig. 5, losartan administration to sham-operated animals did not alter responses to Ang II in aortic rings compared with those from vehicle-treated sham animals. In contrast, the decrease in the response to Ang II by banding was significantly prevented in aortic rings from losartan-treated rats after banding compared with vehicle-treated banding animals (Fig. 5).
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Endothelium-Dependent Decrease in the Contractile Response to Ang II in Ring Preparations of Pressure-Overloaded Thoracic Aorta in Vitro. The contractile response to Ang II was markedly augmented by removal of the endothelium in either control (Fig. 6A) or pressure-loaded rings (Fig. 6B). When the concentration-response curves were compared between control and pressure-loaded rings after removal of endothelium, decreased response to Ang II in pressure-loaded rings was not observed, and rather the response was augmented significantly in the pressure-loaded rings at lower concentrations of Ang II between 1 x 10–10 and 1 x 10–9 M (Fig. 6C). PD123319 did not influence the response to Ang II in the endothelium-denuded rings of either control or pressure-loaded rings (data not shown).
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To determine the involvement of endothelium-derived NO in the decreased response to Ang II in pressure-loaded rings, the ring preparations with intact endothelium were treated with a NO synthase inhibitor L-NAME (1 x 10–4 M) for 30 min before the cumulative addition of Ang II. As shown in Fig. 7, L-NAME pretreatment augmented the contractile response of Ang II in both control and pressure-loaded rings (Fig. 7, A and B), and no significant differences were observed in the response to Ang II between these L-NAME-pretreated rings (Fig. 7C).
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Previous studies have suggested a potential role of the bradykinin/NO system in AT2 receptor-mediated aortic functions in mice (Tsutsumi et al., 1999). Therefore, we examined the effect of the bradykinin B2 receptor antagonist icatibant on the decreased response to Ang II in the pressure-loaded rings with intact endothelium and found that the pretreatment with icatibant (1 x 10–6 M) for 15 min did not influence the contractile response to Ang II in either control or pressure-loaded rings (data not shown). In both control and pressure-loaded rings, acetylcholine (1 x 10–8 M) exhibited a relaxation response during constriction evoked by 3 x 10–7 M phenylephrine, whereas no relaxation was observed by bradykinin up to the concentration of 3 x 10–6 M (data not shown).
cGMP Levels in Aortic Rings. Basal cGMP levels were approximately 2 times greater in pressure-loaded rings than in control rings (8.46 ± 1.39 versus 4.83 ± 1.14 pmol/mg protein; n = 4, p < 0.05) (Fig. 8). Stimulation with Ang II (1 x 10–7 M) for 5 min did not significantly affect the cGMP levels in either control or pressure-loaded rings. Treatment with L-NAME (1 x 10–4 M) for 30 min markedly reduced the cGMP levels not only in control rings (p < 0.001), but also in pressure-loaded rings (p < 0.001). Ang II (1 x 10–7 M) did not influence the decreased levels of cGMP in L-NAME-pretreated rings.
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Discussion |
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).
Several lines of evidence have suggested the existence of a link between the effects of transmural pressure and Ang II in blood vessels. Noda et al. (1994) indicated that mechanical stretch and Ang II synergistically stimulated cultured rat aortic smooth muscle cells to induce a marked increase in the expression of parathyroid hormone-related peptide mRNA. Bardy et al. (1996) reported that the increased transmural pressure in the aorta might have caused the local generation of Ang II, which acted synergistically with the transmural pressure to enhance vascular fibronectin expression via the AT1 receptor. Recently, Bonnet et al. (2001) demonstrated that the AT2 receptor mRNA was up-regulated in rat mesenteric arteries after a pressure dose of Ang II infusion for 2 weeks; they suggested the involvement of AT1-receptor mediation in this Ang II effect, because AT1 receptor antagonist inhibited the Ang II-induced up-regulation of the AT2 receptor. In the aortic banding model, the decreased blood pressure distal to the banding stimulates the kidney to release renin, resulting in increased circulating levels of Ang II. However, as shown in this study and by other investigators (Baker et al., 1990; Doggrell and Brown, 1998), the fact that the elevation of plasma renin is observed only within a few days after aortic banding does not account for the increased levels of the AT2 receptor mRNA over 3 weeks. Therefore, a rapid increase in AT2 receptor mRNA levels within 4 days seems to depend on a transient elevation of plasma renin, but the sustained increase in AT2 receptor mRNA may probably be due to locally generated Ang II in the pressure-overloaded aorta.
In ring preparations of rat thoracic aortas that were dissected 7 days after aortic banding, the contractile response to Ang II was significantly decreased compared with that in control rings. The response to Ang II in the pressure-loaded rings was increased in the presence of the AT2 receptor antagonist PD123319, but not in the control rings, suggesting a potential role of the AT2 receptor in the decreased aortic response to Ang II. Indeed, the reduction of Ang II responsiveness in pressure-loaded rings was prevented by the administration of losartan, which was found to inhibit the up-regulation of AT2 receptor mRNA after aortic banding. Because Ang II binds to the AT1 and AT2 receptor subtypes with similar affinity (Nouet and Nahmias, 2000), the contractile response of the aorta to Ang II seems to be dependent on the relative expression level and/or responsiveness of both receptors. Thus, it seems likely that the decreased response to Ang II in the pressure-overloaded aortas depends on, at least in part, the up-regulation of the AT2 receptor.
There is evidence that Ang II binding to AT2 receptors decreases the Gq-coupled phospholipase C activation by the AT1 receptor (Gyurko et al., 1992), suggesting that the activation of AT2 receptors on aortic smooth muscle cells directly interacts with the signaling pathway of AT1 receptors. However, a decreased response to Ang II was not observed in the endothelium-denuded rings of pressure-overloaded aortas, and PD123319 did not alter the response to Ang II in the pressure-loaded rings, as well as in the control rings, after removal of the endothelium. Thus, it seems that the contraction of aortic smooth muscle cells by Ang II via the AT1 receptor is attenuated by a vasorelaxation factor(s) derived from the endothelium via AT2 receptor stimulation. A potential factor is NO, because of the observation that, after the inhibition of NO synthase by L-NAME, the response to Ang II became comparable in both control and pressure-loaded rings.
The basal levels of cGMP in pressure-loaded aortic rings were significantly higher than those in control rings. Because the cGMP levels in pressure-loaded rings were significantly reduced after L-NAME treatment, it is likely that the increased levels of basal cGMP in pressure-loaded rings result from the enhanced production of NO by the endothelium of pressure-loaded aortas. In fact, the protein and mRNA levels of endothelial NO synthase are up-regulated in pressure-overloaded thoracic aortas after banding of the abdominal aorta (Bouloumie et al., 1997; Barton et al., 2001). Thus, the increased levels of basal cGMP in pressure-loaded rings may participate with, at least in part, the attenuation of the AT1 receptor-mediated contractile response.
Given that the increased expression of AT2 receptors in pressure-loaded aortas is functionally coupled to the NO-cGMP system, it is logical to expect that the stimulation by Ang II results in the elevation of cGMP levels in pressure-loaded rings, because of observations that the contractile response to Ang II in pressure-loaded rings was significantly increased by PD123319. However, stimulation by Ang II did not affect the cGMP levels in pressure-loaded rings, as in control rings. These results do not support the idea that the decreased response to Ang II observed in pressure-loaded rings simply depends on the NO-cGMP system via the activation of the AT2 receptor. Thus, it is reasonable to consider mechanisms other than the NO-cGMP system to explain the AT2 receptor-mediated reduction of Ang II-responsiveness in pressure-loaded aortas. Together, the decreased contractile response to Ang II in thoracic aorta by pressure-overload seems to result from at least two different mechanisms: one is the pressure-overload-induced activation of the NO-cGMP system, and another the AT2 receptor-dependent vasodilatory mechanisms, such as the activation of phospholipase A2 and release of arachidonic acid (Zhu et al., 1998).
It has recently been demonstrated that the AT2 receptor-mediated vasodilator response to Ang II is mediated by kinin in various vessels, such as the rat aorta (Gohlke et al., 1998), canine coronary microvessels (Seyedi et al., 1995), and rat mesenteric artery (Katada and Majima, 2002). These studies suggest that the AT2 receptor is coupled to the local generation of kinin in the vascular wall, which stimulates NO production in endothelial cells via the bradykinin B2 receptor. However, as shown in the present study, the decreased response to Ang II in pressure-loaded rings was not affected by the B2 receptor antagonist icatibant. Furthermore, both control and pressure-loaded rings did not exhibit a relaxation response to exogenous bradykinin, even at high concentrations, in contrast to the sensitive relaxation by acetylcholine, indicating that the rat thoracic aorta is essentially insensitive to bradykinin. This observation is supported by a previous study (Wirth et al., 1996) that rat aorta shows vasorelaxation in response to acetylcholine, but not to bradykinin. Thus, it is unlikely that the kinin-NO-cGMP system plays a role in the signaling cascade of the AT2-receptor in the rat thoracic aorta.
Recent studies on the vascular AT2 receptor have focused on the pathophysiological roles under hypertensive conditions in which vascular AT2 receptors are up-regulated, such as in the mesenteric arteries of young spontaneously hypertensive rats (SHR) (Touyz et al., 1999) and in the thoracic aorta of SHR (Otsuka et al., 1998). Barber et al. (1999) demonstrated that an AT2 receptor agonist induced a depressor response during simultaneous AT1 receptor blockade in SHR, suggesting that the AT2 receptor opposes the action of AT1 receptor in blood pressure regulation, at least in SHR. Carey et al. (2000) demonstrated a depressor effect of Ang II in the presence of AT1 receptor blocker via AT2 receptor stimulation. These studies strongly suggest that the AT2 receptor acts as a vasodilatory pathway counterregulatory to the vasoconstrictor actions of Ang II through the AT1 receptor. The present study supports this concept by the finding that the up-regulation of the aortic AT2 receptor under pressure-overload contributes to the attenuation of the AT1 receptor-mediated aortic constriction. Thus the up-regulation of the vascular AT2 receptor under pressure-overload seems to be one of the compensatory responses of vessels counter-acting the AT1 receptor-dependent vasoconstriction to relieve the mechanical overload. However, the beneficial effects of AT1 receptor antagonists through the activation of the AT2 receptor (Liu et al., 1997; Gigante et al., 1998; Carey et al., 2001) may be limited under pathological conditions in which the expression of AT2 receptors is controlled by the activation of the AT1 receptor, as shown in the present study.
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Footnotes |
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ABBREVIATIONS. Ang II, angiotensin II; AT1, angiotensin II type 1; AT2, angiotensin II type 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NO, nitric oxide; L-NAME, NG-nitro-L-arginine methyl ester; IBMX, isobutylmethylxanthine; PRC, plasma renin concentration; PRA, plasma renin activity; RT-PCR, reverse transcription-polymerase chain reaction; SHR, spontaneously hypertensive rat; PD123319, (1-[[4-(dimethulamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid ditrifluoroacetate).
Address correspondence to: Dr. Hiroshi Okamoto, Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Ikawadanicho, Nishi-ku, Kobe 651-2180, Japan. E-mail: p-okamoto{at}kobegakuin.ac.jp
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