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CELLULAR AND MOLECULAR
Department of Pharmacology (B.T.A., G.G.R., E.K.J.) and Center for Clinical Pharmacology (B.T.A., E.K.J.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
Received October 19, 2003; accepted December 5, 2003.
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Abstract |
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). AT1 receptors are coupled to several types of G
subunits (Sasamura et al., 2000), whereas AT2 receptors are predominantly coupled to Gi/o (Zhang and Pratt, 1996; Hansen et al., 2000). Recent studies indicate that AT2 receptors signal via three main pathways: 1) generation of the vasodilator nitric oxide (Carey et al., 2000); 2) activation of phospholipase A2 and subsequent formation of arachidonic acid metabolites (Harwalkar et al., 1998); and 3) activation of phosphoprotein phosphatases (Tsuzuki et al., 1996; Fischer et al., 1998).
Studies in Ang II receptor knockout mice have revealed different physiological roles for AT1 and AT2 receptors in the regulation of the cardiovascular system. AT2 receptor knockout mice have increased blood pressure compared with controls and have enhanced responses to treatment with Ang II (Akishita et al., 1999; Siragy et al., 1999). Conversely, AT1A/AT1B receptor double knockout mice have lower blood pressure compared with controls and lack the typical increase in blood pressure to exogenous Ang II (Oliverio et al., 1998). Additionally, the AT2 receptor acts as a vasodepressor receptor in the hypertensive (mRen-2)27 transgenic rat (Nishioka et al., 1998), and recent studies indicate that AT2 receptors mediate sustained vasorelaxation in Wistar-Kyoto rats (WKY) (Widdop et al., 2002). Furthermore, experiments conducted in rats that are not genetically modified indicate that AT2 receptors indeed counteract the effects of AT1 receptors on vascular contraction and growth (Endo et al., 1998; Akishita et al., 2000; Carey et al., 2001; Moore et al., 2001). Therefore, the AT2 receptor buffers AT1 receptor-mediated effects on vascular smooth muscle cells, and, as demonstrated by studies in knockout mice, changes in the relative activity of the two signaling pathways significantly alter vascular responses to Ang II.
Vascular growth, contraction, and generation of superoxide in response to Ang II may be determined in part by activation of phospholipase D (PLD). In vascular smooth muscle, Ang II activates the ERK1/2 pathway (Kubo et al., 1999), which in turn contributes to Ang II-induced growth (Chen et al., 1992) and contraction (Touyz et al., 1999; Muthalif et al., 2000). Recent work from our laboratory has shown that PLD, via the formation of phosphatidic acid, plays a critical role in this signal transduction process due to a specific role of phosphatidic acid in the translocation of Raf-1 to the membrane (for review, see Andresen et al., 2002). Likewise, phosphatidic acid is involved in generation of superoxide (Palicz et al., 2001), and inhibition of PLD in vascular smooth muscle attenuates Ang II-mediated generation of superoxide (Touyz and Schiffrin, 1999).
Despite the key role of PLD in Ang II-mediated signal transduction processes in vascular smooth muscle cells and the evidence that AT2 receptors buffer the vascular responses mediated by AT1 receptors, there is currently no information on the effects of AT2 receptor signaling on AT1 receptor-induced PLD activity. The fact that the AT2 receptor is expressed primarily during development and wound healing and only sparsely in a few organs of adult animals (Nouet and Nahmias, 2000) explains, at least in part, the lack of attention received by AT2 receptor signaling in the regulation of PLD and other regulatory pathways. Previous studies indicate that Ang II-mediated PLD activity is increased in spontaneously hypertensive rats (SHR) compared with WKY smooth muscle cells, and the AT1 receptor is responsible for activating PLD (Freeman and Tallant, 1994; Andresen et al., 2001). We hypothesized that this difference in responses to Ang II may be due to differences in the levels of receptors on WKY and SHR cells.
In the present study, we show that AT2 receptors are expressed by cultured preglomerular vascular smooth muscle cells (PGSMCs) obtained from WKY and SHR. The AT1 to AT2 receptor ratio was 1:1 in WKY and 3:1 in SHR PGSMCs. These findings provided an ideal model system for investigating the role of endogenous AT2 receptors in modulating AT1 receptor-induced activation of PLD.
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Materials and Methods |
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Cell Culture. All cell culture reagents were obtained from Invitrogen (Carlsbad, CA). Six 13- to 15-week-old SHR and WKY rats from Taconic Farms (Germantown, NY) were used to acquire the PGSMCs as reported in detail previously (Jackson et al., 1997). Briefly, 1% Fe2O3 Dulbecco's modified Eagle's medium was forcefully injected into isolated kidneys through the renal artery. The iron-loaded kidney was removed from the rat, the cortex was minced, the microvessels (accurate, interlobular, and afferent arteriolar) were obtained using a magnet, and the microvessels were incubated in collagenase type IV (0.6 mg/ml). PGSMVs were obtained by tissue culture and purified by differential plating. Experiments were conducted between passage 1 and 10, and the PGSMCs were grown in Dulbecco's modified Eagle's medium/F-12 supplemented with 10% fetal bovine serum and 1x penicillin/streptomycin/amphotericin B. Phospholipase D activity was measured as described previously (Shome et al., 2000), and phospholipase D activity is reported as phosphatidylethanol formed as a percentage of total lipid.
Quantification of mRNA. Total RNA was isolated from confluent 100-mm plates using the RNeasy mini kit protocol (QIAGEN, Valencia, CA), and 10 µl of total RNA was used in the Advantage RT-for-PCR kit (BD Biosciences Clontech, Palo Alto, CA). The resulting cDNA was used to amplify AT1 receptor, AT2 receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. GAPDH was used as an internal control in each reaction. The primers for the AT1 receptors were designed to amplify both AT1A and AT1B generating the same size band (Table 1), and the amplification cycles were designed so that all the reactions would occur simultaneously. The products were run on 2% agarose gels subsequently stained with ethidium bromide. Digital images of the gels were analyzed using Molecular Analyst for Windows NT (Bio-Rad, Hercules, CA).
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Western Blots. Confluent PGSMCs were frozen in liquid nitrogen and ground into a powder on liquid nitrogen. Samples were placed in a tube with 0.5 ml of lysis buffer (50 mM Tris, pH 7.0, 2% SDS, 10% glycerol, 2 µg/ml antipain, 1 µg/ml aprotinin, 2 µg/ml leupeptin, and 1 mg/ml phenylmethylsulfonyl fluoride) and homogenized. The homogenate was centrifuged at 12,000 rpm at 4°C for 10 min, and the supernatant was recovered. Protein concentration was determined by the copper bicinchoninic acid method, and samples were stored at -20°C.
125I-Sar1-Ile8-Ang II Binding. PGSMCs were grown to confluence in 6-well plates, washed, and serum-starved overnight. The next day, the cells were washed twice for 5 min in 1.5 ml binding buffer (50 mM Na2HPO4, 150 mM NaCl, 10 mM MgCl2, and 0.05% bovine serum albumin, pH 7.1) and treated in 1 ml of binding buffer with 30 pmol (final concentration, 30 nM) 125I-Sar1-Ile8-Ang II. Various amounts of Ang II, L-158,809 (an AT1 receptor antagonist) or PD-123,319 (an AT2 receptor antagonist) were added at room temperature for 60 min. The PGSMCs were quickly washed with 1 ml of binding buffer and lysed in 10% SDS. Protein concentration was determined by the bicinchoninic acid method, and the amount of 125I-Sar1-Ile8-Ang II bound was determined by gamma counting. To convert the binding data to number of cells, the number of cells in each sample was determined by counting multiple aliquots of WKY and SHR PGSMCs and determining the total protein in aliquots from the same samples. Thus, the following simple formula was experimentally determined to convert the binding data to cell number: micrograms of protein in sample x (number of cells/microgram of protein). For WKY and SHR PGSMCs, the number of cells per microgram of protein was 6064 ± 234 and 9212 ± 370, respectively. This formula, the specific activity of 125I-Sar1-Ile8-Ang II and the results from the gamma counter were used to determine the amount of 125I-Sar1-Ile8-Ang II bound per cell.
Data and Statistical Analysis. For all mathematical operations containing two independent data sets with a measurable error, the following error propagation formulas were applied. If f and g are two means and fe and ge are their respective error, then the error for f/g is [fe x g - f x ge]/g2, and the error for f ± g is fe + ge. For multiple comparisons, the data were analyzed by analysis of variance with Fisher's least significant difference post hoc test; for individual comparisons, a t test was used to determine significance. Data points are indicated to be significant only if P < 0.05. Statistical analysis was conducted using the NCSS 2000 software package (Kaysville, UT), and dose-response curves were analyzed using the curve fit routines of GraphPad Prism 4 (GraphPad Software Inc., San Diego, CA).
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Results |
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Because we lacked the Western blot conformation of the RT-PCR studies for the AT2 receptor and because receptor mRNA does not necessarily correlate with receptor expression, binding experiments with the Ang II antagonist 125ISar1-Ile8-Ang II were conducted to determine the number of Ang II receptors on the cell surface (Fig. 3). Displacement of 125I-Sar1-Ile8-Ang II with unlabeled Ang II (Fig. 3A) indicated that the expression of Ang II receptors in SHR PGSMCs was marginally greater than that in WKY PGSMCs, 1356 ± 86 versus 897 ± 50 receptors per cell, respectively. To examine the expression of AT1 versus AT2 receptors, we used similar 125I-Sar1-Ile8-Ang II displacement protocols with AT1 and AT2 receptor-specific antagonists. The data obtained with the AT1 receptor antagonist L-158,809 (Fig. 3B) indicated that the level of expression of AT1 receptors was significantly greater in SHR than in WKY PGSMCs, 692 versus 356 receptors per cell, respectively. In contrast, displacement with the AT2 receptor-specific antagonist PD-123,319 (Fig. 3C) showed that SHR PGSMCs expressed significantly fewer AT2 receptors than WKY PGSMCs, 248 versus 482 receptors per cell, respectively. Importantly, the combination of the displacement with L-158,809 and PD-123,319 resulted in a total Ang II receptor number per cell of 838 ± 147 and 940 ± 149 for WKY and SHR PGSMCs, respectively. In WKY PGSMCs, displacement with Ang II and the antagonists yielded internally consistent results for the total number of receptors. However, in SHR PGSMCs, the L-158,809 plus PD-123,319 did not displace all of the bound 125I-Sar1-Ile8-Ang II, suggesting an additional binding site in SHR PGSMCs. Regardless, as indicated in Table 2, the ratio of the levels of expression of AT1 and AT2 receptors was similar to the AT1 receptor mRNA to AT2 receptor mRNA ratio calculated from the RT-PCR studies. Therefore, we conclude that SHR PGSMCs express significantly more AT1 receptors than AT2 receptors, resulting in an increased AT1 to AT2 receptor ratio compared with WKY PGSMCs.
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Effect of AT2 Receptors on Ang II-Mediated PLD Activity. We previously reported that Ang II is a more potent activator of PLD in SHR than in WKY PGSMCs (Andresen et al., 2001). In this regard, the concentration-response curves to Ang II were characterized by an EC50 value of 3.9 and 47 nM for SHR and WKY PGSMCs, respectively (see Fig. 4). The increased potency of Ang II-mediated PLD activity in SHR PGSMCs may be due to the greater AT1 to AT2 receptor ratio observed in the SHR cells. Accordingly, in the present study, we examined the role of AT1 versus AT2 receptors in mediating Ang II-induced PLD activity.
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As shown in Fig. 5A, in both WKY and SHR PGSMCs, basal PLD activity was not significantly affected by L-158,809 (an AT1 receptor antagonist), PD-123,319 (an AT2 receptor antagonist) or CGP-42112A (an AT2 receptor agonist). In both WKY and SHR PGSMCs, a high, saturating concentration of Ang II (1 µM) increased PLD activity (Fig. 5B), and this response was blocked by L-158,809, but not by PD-123,319. These data confirm that AT1 receptors, not AT2 receptors, mediate Ang II-induced PLD activity.
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To examine the hypothesis that the AT1 to AT2 receptor ratio determines the effects of Ang II on PLD activation, we examined Ang II-induced PLD activity concentration-response curves in the absence and presence of the specific AT2 receptor antagonist PD-123,319. The rationale for these experiments was that in the presence of the AT2 receptor antagonist, the differences between SHR and WKY PGSMCs should be significantly reduced by PD-123,319 if the AT2 receptor importantly modulates the effects of the AT1 receptor on PLD activity. As shown in Fig. 6A, addition of the AT2 receptor antagonist shifted the Ang II EC50 of WKY PGSMCs from 63 to 4.2 nM, a value that is consistent with the EC50 of 8.8 nM calculated for the SHR cells (Fig. 6B). The effect of PD-123,319 on SHR cells was not significant (Fig. 6B), a finding consistent with the reduced AT2 receptor expression in SHR cells.
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The hypothesis that AT2 receptor signaling attenuates Ang II-mediated PLD activation was further examined using the AT2 receptor agonist CGP-42112A. As with PD-123,319, CGP-42112A alone has no significant effect on PLD activity (Fig. 5A). However, CGP-42112A significantly altered the Ang II-mediated PLD activity concentration-response curves of WKY and SHR PGSMCs (Fig. 6, A and B). The WKY curve was shifted significantly downwards resulting in a 60% decrease in efficacy (Fig. 6A), whereas the SHR concentration-response curve was shifted significantly rightward resulting in an EC50 of 31 nM, compared with an EC50 of 8.8 nM in the absence of the agonist (Fig. 6B).
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Discussion |
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Because an AT2 receptor antagonist enhances and an AT2 receptor agonist inhibits Ang II-mediated PLD activation, these studies indicate that the mechanism by which AT2 receptors inhibit AT1 receptor-mediated PLD activation involves AT2 receptor-mediated signaling rather than via heterodimerization and subsequent formation of inactive AT1-AT2 receptor complexes as has been previously suggested (AbdAlla et al., 2001). An important issue is the mechanism by which AT1 and AT2 receptors signal to PLD and how those signals interact. In this regard, our previously published studies demonstrate that AT1 receptors signal to PLD in PGSMCs from both SHR and WKY via a RhoA-dependent mechanism (Andresen et al., 2001); however, the signal transduction cascade activated by the AT2 receptor responsible for inhibiting AT1 receptor-mediated PLD activity remains undefined.
Vascular smooth muscle cells in vivo express high levels of AT2 receptors during fetal development (Hutchinson et al., 1999) and after vascular injury (Suzuki et al., 2002). Moreover, AT2 receptors mediate vasodilation in selected vascular beds (Widdop et al., 2002). These findings have motivated researchers to examine signaling mechanisms of AT2 receptors in vascular smooth muscle cells in vitro. However, efforts in this regard have been hampered by the fact that most vascular smooth muscle cells in vitro express low to undetectable levels of native AT2 receptors (Stoll et al., 1995). Consequently, investigators have relied upon in vitro model systems involving overexpression of AT2 receptors in vascular smooth muscle cells using various transfection protocols. Unfortunately, this approach has limitations in that overexpression of receptors may lead to artifacts, such as receptor oligomerization (Ramsay et al., 2002) and engagement of illicit signal transduction pathways (Wellner-Kienitz et al., 2001). Our model system is unique in that PGSMCs express both AT1 and AT2 receptors without transfection; moreover, the ratio of AT1 to AT2 receptors is dependent on the strain from which the PGSMCs are obtained. Therefore, PGSMCs from SHR and WKY afford an ideal model system for investigating the role and signal transduction pathways of AT2 receptors in vascular biology.
It should be noted that previous reports did not identify AT2 receptors in the renal vasculature (Song et al., 1994; Song et al., 1995; Haddad and Garcia, 1996; Gao et al., 2003). Indeed, we have been unable to detect AT2 receptors in freshly isolated preglomerular microvessels from either WKY or SHR using either Western blots or RT-PCR (Gao et al., 2003). There are at least two explanations for the presence of AT2 receptors in cultured PGSMCs, but not freshly isolated microvessels. It is possible that the phenotype of PGSMCs changes in vitro such that Ang II receptor expression patterns are altered in culture. However, it is equally possible that PGSMCs that migrate from isolated preglomerular microvessels into a culture dish and survive differential plating are a subset of cells that do not represent the average phenotype of the larger population of PGSMCs in the preglomerular vasculature. Thus, it is important to note that the inability to detect AT2 receptors in intact preglomerular microvessels does not rule out the expression of AT2 receptors in a subset of PGSMCs in vivo.
Regardless of whether AT2 receptor-expressing PGSMCs are induced or selected, the experiments reported here demonstrate conclusively that AT2 receptors attenuate the ability of Ang II to activate PLD. This conclusion has important implications. Previous studies show that the expression of AT2 receptors is increased in injured arteries (Viswanathan and Saavedra, 1992). Because PLD activity is critical to activation of the ERK cascade, which leads to vascular proliferation, and AT2 receptors inhibit PLD activation, AT2 receptors may play a significant role in the attenuation of the proliferation of vascular smooth muscle cells and the development of vaso-occlusion after vascular injury. Additionally, because PLD is involved in generation of reactive oxygen species, AT2 receptor-mediated inhibition of PLD activity may be partially responsible for the observed AT2 receptor-mediated reduction of the generation of superoxide induced by Ang II (Rueckschloss et al., 2002). Because our studies demonstrate that the genetic background strongly influences the ratio of AT1 to AT2 receptors, this may explain why some patients experience restenosis after angioplasty, whereas others do not. This may also explain why some patients are genetically susceptible to vaso-occlusive disorders, whereas others are resistant. Finally, our data suggest that WKY and SHR PGSMCs may provide a useful model system for elucidating the genetic mechanisms that regulate the relative expression of AT1 and AT2 receptors.
In conclusion, cultured PGSMCs express both AT1 and AT2 receptors. AT1 receptors mediate the activation of PLD by Ang II, whereas AT2 receptors inhibit AT1 receptor signaling to PLD. The differences between SHR and WKY PGSMCs regarding their Ang II-mediated PLD activity responses are likely a result of an altered ratio of AT1 to AT2 receptors in these two strains. Our work suggests that genetic mechanisms regulate the AT1 to AT2 receptor ratio in vascular smooth muscle cells, and such genetic mechanisms may determine in part the susceptibility of human beings to vasoocclusive disorders. The WKY and SHR PGSMCs thus provide an excellent model system for analysis of the genetic basis for Ang II receptor levels and for examining the contribution of endogenous AT2 receptor signaling to the effects of Ang II stimulation.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: Ang II, angiotensin II; AT1, angiotensin II receptor, type I; AT2, angiotensin II receptor, type II; WKY, Wistar-Kyoto rat; PLD, phospholipase D; SHR, spontaneously hypertensive rat; PGSMC, preglomerular vascular smooth muscle cell; RT-PCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
1 Current address: Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892-4340. 
Address correspondence to: Dr. Edwin K. Jackson, The Center for Clinical Pharmacology, 623 Scaife Hall, University of Pittsburgh School of Medicine, 3550 Terrace St., Pittsburgh, PA 15261. E-mail: edj{at}pitt.edu
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) had slightly more total Ang II binding sites than WKY PGSMCs (
). B, displacement of 125I-Sar1-Ile8-Ang II with L-158,809 indicates that SHR PGSMCs had 2-fold more AT1 receptors than WKY; inset is the WKY curve shown with a reduced scale and 95% confidence intervals (dashed lines). C, displacement with PD-123,319 indicates that WKY PGSMCs had 2-fold more AT2 receptors than SHR. Inset, SHR curve shown with a reduced scale and 95% confidence intervals (dashed lines). n = 3 for all experiments, and the experiments were conducted in parallel.
3. Analysis of variance indicates that the curves are significantly different (P < 0.05).
