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CARDIOVASCULAR
Department of Pediatrics, Georgetown University Medical Center, Washington, DC (C.S.E., L.B.K.); and Department of Internal Medicine, Division of Endocrinology, University of Missouri-Columbia School of Medicine, Columbia, Missouri (A.A.G., B.T.A.)
Received for publication
May 25, 2007
Accepted
October 1, 2007.
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Abstract |
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), attenuation of vascular relaxation (Touyz et al., 2002a), and blood pressure control (Muthalif et al., 2000a,b; Hu et al., 2007); however, this is not a universal mechanism (Watts et al., 1998; Touyz et al., 2002a). In addition, the molecular mechanism underlying the role of ERK in control of vascular contraction is not completely understood; currently there are two proposed mechanisms. One pathway is an ERK-mediated phosphorylation of the 20-kDa myosin light chain regulatory subunit (MLC20) at the same position as myosin light chain kinase (D'Angelo and Adam, 2002; Roberts, 2004). A second pathway involves ERK-mediated phosphorylation of caldesmon (Adam and Hathaway, 1993; D'Angelo et al., 1999). Phosphorylation of MLC20 has been proposed to be more relevant than caldesmon (D'Angelo and Adam, 2002), but activation of ERK does not always lead to phosphorylation of MLC20 (Tsai and Jiang, 2005). Importantly, both of these targets would increase contraction through modulating the actin-myosin interaction via classically described interactions (for review, see Webb, 2003).
There is no unified theory to describe the molecular mechanisms underlying G protein-coupled receptor (GPCR) activation of ERK. Multiple mechanisms have been proposed, including transactivation (Daub et al., 1996), β-arrestin (Luttrell et al., 2001), or phospholipase D (PLD)-mediated scaffolding (Rizzo et al., 1999), and nonreceptor kinase-mediated activation of ERK (Ettehadieh et al., 1992; Carroll and May, 1994). These proposed mechanisms are not necessarily mutually exclusive (Luttrell et al., 1999; Shah and Catt, 2002); however, the transactivation theory has become predominant, and it has been shown to occur in many (Wetzker and Bohmer, 2003) but not all (Shah et al., 2004) tissues. in brief, transactivation involves GPCR-activating receptor tyrosine kinases that then signal through their respective signaling cascade, leading to ERK activation. In thoracic aortic smooth muscle cells (TASMCs) angiotensin II (Ang II), a GPCR agonist, activates ERK through transactivation of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors (Eguchi et al., 1998).
Ang II is a vasoactive hormone that can cause multiple cardiovascular and renal disorders in experimental models. One such disorder, hypertension, can be caused by Ang II, and interference with either Ang II production (via angiotensin-converting enzyme inhibitors) or signaling (via Ang II receptor blockers) is common antihypertensive therapy. Renal transplantation studies demonstrate that hypertension can be transferred to a normotensive host if receiving a kidney from a hypertensive donor (Grisk and Rettig, 2001). Recently, similar experiments using genetically modified mice demonstrate that kidney angiotensin type 1 receptors are responsible for Ang II-mediated hypertension and associated cardiovascular disorders (Crowley et al., 2006). This is further supported by the finding that the renal vascular bed is the most responsive to Ang II in vivo (Jackson and Herzer, 2001). Therefore, if Ang II-mediated ERK activation is involved in blood pressure regulation, as has been shown previously (Muthalif et al., 2000b), then inhibition of ERK activation, either by inhibiting MEK or through blocking transactivation of EGF or PDGF receptor kinase activity, has to reduce Ang II-mediated increase in blood pressure, if, and only if, transactivation is a universal mechanism.
In this study, we tested the above-mentioned statement and the hypothesis that Ang II-mediated ERK activation is different in TASMCs compared with renal microvascular smooth muscle cells (RµVSMCs). Previous pharmacological data separated Ang II and EGF signaling to ERK in RµVSMCs, but such studies did not directly examine transactivation (Andresen et al., 2003). Here, we confirm previous studies (Muthalif et al., 2000a,b; Hu et al., 2007) that indicate a role for MEK/ERK in Ang II-mediated blood pressure regulation in vivo, and we demonstrate that this is independent of EGF and PDGF receptor activity. Furthermore, the physiological data are supported via cell culture studies demonstrating that Ang II signals to ERK through a Src family member-dependent, yet nontransactivation-mediated pathway in RµVSMCs, compared with a Src-independent transactivation pathway in TASMCs.
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The rat was allowed to equilibrate for 1 h postsurgery. Once equilibrated, basal blood pressure was recorded for 20 min before a bolus injection of 2.5 µg/kg Ang II via the jugular catheter. Following the injection, blood pressure was recorded for the next hour, after which either the drug or vehicle was injected i.p. The injections were one of the following: DMSO as a vehicle (Sigma-Aldrich, St. Louis, MO), 15 mg/kg PD98059, an MEK-specific inhibitor; 20 mg/kg AG1478, an EGF receptor-specific inhibitor; and 1.5 mg/kg AG1296, a PDGF receptor-specific inhibitor (all obtained from Calbiochem, San Diego, CA). Thirty minutes after the i.p. injection, a second 2.5-µg/kg dose of Ang II was administered via the jugular catheter. The rat was euthanized 30 min after the second Ang II injection by overdose with pentobarbital (100 mg/kg i.v.). The raw data were calculated by determining the change (
) of Ang II-mediated systolic blood pressure by subtraction of the baseline from the maximal systolic blood pressure. The percentage of change was also calculated as -fold change times 100, which incorporates the baseline into the value. All analysis was conducted with the systolic blood pressure values.
Cell Culture. All cells were obtained from Dr. Edwin K. Jackson (University of Pittsburgh, Pittsburgh, PA) and grown in Dulbecco's modified Eagle's medium/Ham's F-12 with 10% fetal bovine serum plus antibiotics (100 U/ml penicillin G, 100 U/ml streptomycin sulfate, and 250 ng/ml amphotericin B). All cell culture materials were obtained from Invitrogen (Carlsbad, CA).
Activation of ERK. TASMCs and RµVSMCs were grown in 60-mm tissue culture plates, serum-starved overnight (at least 12 h), and then treated with the vehicle or drug. The drugs were all dissolved in DMSO, and the cells were pretreated as follows: 20 µM PD98059 was applied for 30 min, 100 nM PP2 was applied for 30 min, 1 µM AG1478 was applied for 2 h, 10 µM AG1296 was applied for 2 h, or DMSO was applied for 2 h. These concentrations and durations of exposure to drug were chosen because each has been demonstrated to specifically inhibit their respective target. The cells were then stimulated with 1 µM Ang II, 50 ng/ml EGF, or 50 ng/ml PDGF for 5 min, washed with ice-cold PBS, and then lysed in 250 µl of a radioimmunoprecipitation assay lysis buffer (1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, 100 mM NaCl, 20 mM Tris, pH 7.5, 2 mM EDTA, 10 mM MgCl2, 10 mM NaF, and 40 mM β-glycerol phosphate, supplemented with 1 mM phenylmethylsulfonyl fluoride and 2 mM Na3VO4). The samples were clarified by centrifuging the lysate at 14,000 rpm and removing the supernatant, and then Laemmli sample buffer was added, and the samples were boiled and stored at –20°C before use. The samples were separated on a 10% SDS-polyacrylamide gel electrophoresis gel and transferred to nitrocellulose obtained from Bio-Rad (Hercules, CA). ERK activation was measured via Western blot with phosphospecific ERK1/2 antibodies (Cell Signaling Technology Inc., Danvers, MA), followed by stripping the membrane with 62.5 mM Tris, pH 6.7, 100 mM 2-mercaptoethanol, and 2% SDS and reprobing for total ERK1/2 (Cell Signaling Technology Inc.). Secondary antibodies and enhanced chemiluminescence were obtained from Pierce Chemical (Rockford, IL), and the images were captured on Fujifilm Super RX X-ray film obtained from Crystalgen (Plainview, NY) and developed with a Kodak RPX-OMAT processor (model M6B; Eastman Kodak, Rochester, NY).
Confocal Imaging. TASMCs and RµVSMCs were grown on 22-mm number 1 coverslips (Fisher Scientific, Pittsburgh, PA) coated with poly-D-lysine (Sigma-Aldrich) in six-well plates. The cells were serum-starved overnight (at least 12 h) and stimulated as described above. After stimulation, the cells were washed with ice-cold PBS, fixed with 4% paraformaldehyde in PBS for 30 min at 4°C, and permeabilized with 0.1% Triton X-100 for 2 min. The cells were then washed with PBS, and blocking was performed with 3% BSA (Sigma-Aldrich) in PBS. After blocking, the E10 antiphosphorylated ERK (mouse monoclonal) primary antibody (Cell Signaling Technology Inc.) was diluted at 1:500 in a 3% BSA in PBS solution, and it was applied to the cells for 1 h. The cells were then washed with PBS and stained with a 3% BSA in PBS solution containing 5 µM Hoechst (Invitrogen), 10 units Alexa 488-labeled phalloidin (Invitrogen), and 2.6 µg/ml Cy5-labeled F(ab')2 fragment donkey anti-mouse (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) for 1 h. The samples were washed with PBS, and then they were mounted on glass slides (Fisher Scientific) with Prolong antifade mounting medium (Invitrogen). Imaging was conducted with an Olympus BX61 Fluoview FV500 laser scanning confocal microscope (Olympus, Melville, NY) with a Plan Apo 60 x 1.40 numerical aperture oil immersion objective. Hoechst was visualized using a 405-nm diode laser, Alexa 488-labeled phalloidin was visualized using a 488-nm argon laser, and the Cy5-labeled E10 antibody was visualized using a 633-nm He-Ne laser. All images were acquired individually to eliminate the possibility of bleedthrough and set to a resolution of 1024 x 1024. Each image is an average of four repetitive scans.
Statistics and Data Analysis. All graphs were generated with GraphPad Prism 4 for Windows (GraphPad Software Inc., San Diego, CA). Paired t tests were used to determine whether the change in the increase in blood pressure was different between the two injections of Ang II for each treatment group with reverse experiment tester (Boss Statistics) (Lew, 2006) and NCSS 2007 (NCSS, Kaysville, UT). Reverse tester has two functions, the t test and a noninferiority test that tests for similarity between groups instead of a difference. This was used to determine whether the two Ang II-generated peaks in blood pressure were significantly similar in each group, and if the drug treatments were significantly similar to vehicle injection. A repeated measures ANOVA with Tukey-Kramer multiple comparison test was used to compare all the raw data, and a second ANOVA with Tukey-Kramer multiple comparison test was used to compare the differences in systolic blood pressure between the four groups via NCSS 2007. All measurements are represented as mean ± S.E.M., followed by the n. Western blots of p44 ERK1 and p42 ERK2 (referred to as ERK) were grouped together and quantified using ImageJ (http://rsb.info.nih.gov/ij/) 1.36b for Windows. Phosphorylated ERK was normalized to total ERK and that ratio was analyzed via an ANOVA with Tukey-Kramer Multiple-comparison post-hoc test to compare the levels of Ang II-mediated ERK activation. The red channel in the confocal images was enhanced using the curves option in Photoshop 7.0 (Adobe Systems, Mountain View, CA), so that they are more visible on the black background. All TASMCs were altered with the same curve, and a similar curve was used on all the RµVSMCs. MetaMorph software (Molecular Devices, Sunnyvale, CA) was used to filter the Hoechst staining in Fig. 5 via use of the median filter to reduce background signal while maintaining visual localization of the nuclei.
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). In addition, PD98059 was used instead of U0126 because U0126 seems to be nonspecific in vivo in regard to muscle contraction (Watts, 2000). The EGF receptor inhibitor AG1478 was used at a dose sufficient to clear tumors from mice when administered i.p. (He et al., 2001), and the PDGF receptor inhibitor AG1296 was used at a dose calculated from previous in vivo studies (Dell et al., 2006). On occasion, injection of the drug or vehicle transiently lowered blood pressure due to a stress response, but it had no appreciable effect on baseline blood pressure over time. Repeated measures ANOVA demonstrated a significant difference between the groups (drug treatments) (p = 0.016531) with a power of 0.804994, with no difference between the first and second stimulation with Ang II (period); however, there is a significant interaction between the period and drug treatments (p = 0.014309) with a power of 0.823593. Tukey-Kramer multiple comparison test indicates that treatment with DMSO, AG1478, or AG1296 had no significant effect (p > 0.05) on the response to the second dose of Ang II, but that PD98059 did have a significant effect (p < 0.05) (Table 1). There are differences among period 1, the first stimulation with Ang II, between the groups; however, this seems to be due to random distribution of the individual rats; overall, the 16 period 1 data points do confer to a normal distribution. In addition, paired t test also indicates that there is only a significant difference between the PD98059-treated samples (Table 1). Furthermore, the DMSO and AG1296 treatments were significantly similar to their controls using a paired noninferiority test (Table 1). To graphically display this, the maximum change in systolic blood pressure caused by the first dose of Ang II was subtracted from the maximal change in systolic blood pressure of the second dose of Ang II for comparison across groups (Fig. 1). ANOVA analysis of this normalized data demonstrated a significant difference between the treatment groups (p = 0.014457) with a power of 0.822299, and Tukey-Kramer multiple comparison test indicates that only inhibition of MEK reduced Ang II-mediated increases in systolic blood pressure (p < 0.05). Furthermore, the AG1478 and AG1296 groups are similar to DMSO (for p < 0.05, the power is 0.705 for AG1478 and 0.9707 for AG1296; n = 4). The data indicate that MEK, and presumably ERK, play a role in Ang II-mediated increases in blood pressure. Moreover, transactivation of EGF and PDGF receptors does not contribute to the acute increase in blood pressure in response to Ang II administration.
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Since in RµVSMCs Ang II and EGF signaling to ERK can be pharmacologically separated (Andresen et al., 2003) and the kidney plays a large role in Ang II-mediated changes in blood pressure (Jackson and Herzer, 2001; Crowley et al., 2006), we examined the role of transactivation of EGF and PDGF receptors in Ang II-mediated ERK activation in RµVSMCs (Fig. 2, A and B). As shown in Fig. 2, A and B, in TASMCs Ang II-mediated activation of ERK is sensitive to inhibition of MEK and EGF and PDGF receptors, which confirms previous reports (Eguchi et al., 1998). However, in RµVSMCs Ang II-mediated ERK activation is sensitive to inhibition of MEK, but it is insensitive to inhibition of EGF and PDGF receptors. Quantification and ANOVA analysis of the Ang II-treated samples (n = 3) indicates that basal ERK levels are slightly, yet not statistically, increased in RµVSMCs compared with TASMCs (Fig. 2B). Ang II significantly increases ERK phosphorylation in TASMCs and RµVSMCs to a similar extent, and PD98059 reduces Ang II-mediated ERK phosphorylation to basal levels in both TASMCs and RµVSMCs. Inhibition of EGF and PDGF receptors only inhibits Ang II-mediated activation of ERK in TASMCs, and this inhibition reduces phosphorylated ERK levels to baseline (Fig. 2B). Treatments with EGF and PDGF demonstrate that PD98059 works similarly in both TASMCs and RµVSMCs (Fig. 2A). In addition, treatments with AG1478 and AG1296 inhibit EGF- and PDGF-mediated activation of ERK, respectively, in both cell types (Fig. 2A). These data demonstrate that EGF and PDGF receptors are present and signal to ERK in RµVSMCs. Therefore, Ang II is not signaling through transactivation of EGF and PDGF receptors in the RµVSMCs.
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Multiple studies indicate that Src, or a Src family kinase, plays a role in Ang II-mediated ERK activation (Eguchi et al., 1998; Matrougui et al., 2000; Touyz et al., 2001; Shah and Catt, 2002; Shah et al., 2004), and preliminary data indicated that a Src family kinase is involved in Ang II signaling to ERK in RµVSMCs. Therefore, we used the Src family kinase inhibitor PP2 to examine the role of Src family members in Ang II, EGF, and PDGF-mediated activation of ERK in TASMCs and RµVSMCs (Fig. 2, C and D). As shown in the Western blot (Fig. 2C) 100 nM PP2 only inhibited Ang II-mediated activation of ERK in RµVSMCs. Quantification and ANOVA analysis of the western blots (n = 3) demonstrates that a Src family member is required for Ang II-mediated activation of ERK in RµVSMCs, but not TASMCs (Fig. 2D). Moreover, EGF and PDGF are not sensitive to 100 nM PP2 in either cell type.
Preliminary data suggested that in RµVSMCs, Ang II caused phosphorylated ERK to localize on actin filaments. Therefore, we examined the cellular location of phosphorylated ERK in TASMCs and RµVSMCs after stimulation with Ang II, EGF, or PDGF for 5 min (Figs. 3 and 4). Hoechst stain was used to identify the nucleus, and phalloidin was used to identify the actin filaments. As shown in Fig. 3, Ang II, EGF, and PDGF all traffic phosphorylated ERK to the nucleus and perinuclear region of TASMCs. Ang II directed phosphorylated ERK to actin filaments in approximately 25% of the TASMCs observed. In contrast, all agonists directed phosphorylated ERK to actin filaments and the nucleus and perinuclear region in RµVSMCs (Fig. 4). Ang II directed phosphorylated ERK to the actin filaments in all of the RµVSMCs cells observed (Figs. 4, E–H, and 5), whereas EGF and PDGF directed phosphorylated ERK to the actin filaments in approximately 50% of the RµVSMCs observed. In addition, as shown in Fig. 5, the alignment of the actin filaments is not in an orientation from the cell surface toward the nucleus, suggesting that active ERK is not using the actin filaments merely as a route to traffic to the nucleus. Therefore, the localization of active ERK is clearly different in RµVSMCs compared with TASMCs, and in RµVSMCs the localization of active ERK suggests that it may mediate phosphorylation of the actin-myosin contractile apparatus.
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).
The role of ERK in vascular contraction has been described in multiple models (Touyz et al., 1999, 2002a; Muthalif et al., 2000a; Roberts, 2004), including a model of Ang II-dependent hypertension (Muthalif et al., 2000b). Using male Sprague-Dawley rats with induced hypertension via chronic Ang II infusion, Muthalif et al. (2000b) reported a decrease in mean arterial blood pressure of 20 mm Hg 30 min after a single injection of a comparable dose of PD98059. As shown in Table 1 and Fig. 1, injection of 15 mg/kg PD98059 significantly reduced transient Ang II-mediated increase in systolic blood pressure by 23 ± 8 mm Hg, which is drastically different from the DMSO-treated controls increase of 1 ± 5 mm Hg. Our method was designed specifically to isolate changes in systolic blood pressure derived from vasoconstriction, which is exemplified by the rapid increase in blood pressure followed by an equally rapid decline and return to baseline. This allowed us to conclude that the major cause of the increase in blood pressure is due to vasoconstriction and that MEK, and presumably ERK, provide for roughly 20% of the contractile response to Ang II in this model. Because of the similarity between our studies and the studies by Muthalif et al. (2000b), we suggest that MEK is involved in hypertension. However, both studies were conducted in anesthetized rats and remain unconfirmed in conscious animals.
The molecular mechanisms underlying Ang II-mediated phosphorylation of ERK do not fit into a unified mechanism. Most studies of smooth muscle cellular biology are conducted in TASMCs; however, these vessels are conduit vessels that have little physiological role in controlling blood pressure, especially Ang II-mediated changes in blood pressure (Jackson and Herzer, 2001). Through using TASMCs, and other cell lines, it has been established that Ang II signals to ERK through transactivation of receptor tyrosine kinases (Eguchi et al., 1998). This pathway relies on the ability of EGF and PDGF receptor inhibitors to block Ang II-mediated activation of ERK; yet, as shown in Fig. 2, this is not the case in RµVSMCs. Because the renal vasculature plays the largest role in modulating transient vascular changes in response to Ang II (Jackson and Herzer, 2001), the lack of transactivation in the RµVSMCs explains the lack of significant effect of AG1478 and AG1296 on transient Ang II-mediated changes in systolic blood pressure. It is important to note that these experiments examine transient increases in blood pressure; thus, these data do not preclude a role for EGF and PDGF receptors in the maintenance or progression of hypertension. Further studies in other models are required to definitively comment on the role of EGF and PDGF receptors in the maintenance or progression of hypertension.
Beyond transactivation, there exist multiple mechanisms that can mediate signaling from a GPCR, such as the angiotensin type 1 receptor, to ERK. Likely candidates are β-arrestin-mediated internalization of the receptor and scaffolding of signaling components (Luttrell et al., 2001), PLD generation of phosphatidic acid (Rizzo et al., 1999), and Src (Ettehadieh et al., 1992) and protein kinase C (Carroll and May, 1994). Previous studies in RµVSMCs indicate that PLD is involved in Ang II-mediated activation of ERK (Andresen et al., 2003), and as shown in Fig. 2, a Src family member plays a role in Ang II-mediated ERK activation. These data agree with reports that Src is involved in Ang II-mediated preglomerular vascular contraction (Che and Carmines, 2005). However, these data are contrary to multiple papers, indicating that transactivation is Src-dependent (Eguchi et al., 1998; Shah and Catt, 2002; Touyz et al., 2002b). This may be because the Wistar-Kyoto seems to be less sensitive to Src inhibition (Touyz et al., 2002b). In addition, multiple reports use concentrations of PP2 in the micromolar range. In addition to transactivation, Src can directly activate ERK (Ettehadieh et al., 1992) and may be involved in β-arrestin-mediated activation of ERK (Luttrell et al., 1999). Therefore, Fig. 2 demonstrates that the transactivation pathway is Src-independent, but it does little to further elucidate the pathway used by Ang II in RµVSMCs. Overall, since the PLD/phosphatidic acid mechanism only provides for the recruitment and scaffolding of the ERK signaling cascade in RµVSMCs (for review, see Andresen et al., 2002), a Src family member most probably provides the mechanism to activate ERK.
The β-arrestin and PLD mechanisms are linked to internalization, where actin is known to play a role and internalization can occur on actin tracks (Engqvist-Goldstein and Drubin, 2003). Actin tracks are generally oriented in a pattern that point from the plasma membrane toward the center of the cell. These tracks allow for cargo to traffic to the nucleus or a recycling center. Although our data indicate that ERK is localized on actin filaments, they do not seem to be used to internalize cargo. This is concluded from the following observations. First, the colocalization of ERK with actin filaments in the RµVSMCs is not aligned in a pattern that resembles internalization (Fig. 5) in that the actin is not ordered from the membrane to a central point in the cell. Second, although a time course of ERK activation was not addressed in this study, Fig. 2B indicates that at 5 min the level of activated ERK is similar in TASMCs and RµVSMCs. Generally, ERK is internalized and within the nuclear/perinuclear region within 5 min. This is observed in TASMCs (Fig. 3), but not RµVSMCs (Fig. 4), indicating that either the RµVSMCs are "slow" traffickers of ERK, or that ERK is purposefully targeted to actin filaments. Therefore, the data suggest that phosphorylated ERK is targeted to actin filaments in RµVSMCs. Further time course and live cell experiments are needed to confirm this conclusion. Targeting phosphorylated ERK to actin filaments provides a potential mechanism for ERK-mediated phosphorylation of MLC20 (Roberts, 2004), and thus vascular contraction and increases in blood pressure.
Unexpectedly, stimulation with EGF or PDGF also placed phosphorylated ERK on actin filaments. This indicates that there is a general mechanism for trafficking ERK that is independent of the mechanism of ERK activation in RµVSMCs and that this mechanism is most probably different or at least slower than that in TASMCs. In addition, the data suggest that agonists that activate ERK, such as EGF, could also contribute to modulating blood pressure, as has been demonstrated previously (Carmines et al., 2001). Interestingly, infusion of EGF in rats slowly and marginally increases blood pressure, but decreases blood pressure in monkeys (Keiser and Ryan, 1996). However, a more physiological dose of EGF did not alter blood pressure in rats, but it did alter arteriolar constriction and cause renal alterations (Harris et al., 1988). In this study, we found that inhibition of EGF receptors is neither significantly different nor similar to the controls, suggesting that EGF receptors may have a role in blood pressure regulation, but they are not significantly involved in Ang II-mediated transient elevations of blood pressure. Further investigation is warranted to determine the role of EGF and EGF receptors in both transient and long-term blood pressure regulation. Inhibition of PDGF receptors has been shown to be a potent therapy for treatment of pulmonary hypertension, whereas no effect was observed on mean arterial pressure (Schermuly et al., 2005). Thus, PDGF has a negligible role, if any, in regulating systemic blood pressure. Importantly, the studies conducted here use only Ang II as an agonist in the physiological experiments, so the role of EGF or PDGF cannot be assessed. Only the role of the EGF and PDGF receptors in Ang II-mediated rapid changes in blood pressure can be discerned.
In conclusion, this is the first report to document that signaling to ERK in smooth muscle cells within resistance vessels from the kidney is different from that of the smooth muscle cells from the thoracic artery from the same strain of rats. Specifically, in RµVSMCs, Ang II-mediated activation of ERK is independent of receptor tyrosine kinases, but is dependent on a Src family kinase. In TASMCs, the transactivation pathway is dominant, but it has little to no role in regulating transient Ang II-mediated changes in blood pressure. A notable difference between TASMCs and RµVSMCs is that agonists that activate ERK direct phosphorylated ERK to actin filaments and the nuclear/perinuclear region in RµVSMCs, whereas in TASMCs phosphorylated ERK is directed to only the nuclear/perinuclear region. This may play a role in ERK-mediated increased blood pressure. Thus, the molecular mechanisms underlying Ang II-mediated activation of ERK and ERK trafficking to actin filaments in RµVSMCs should be investigated further.
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Acknowledgements |
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C.S.E. and L.B.K. contributed equally to this work and share equal authorship as first authors.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: ERK, extracellular signal-regulated kinase; MLC20, 20-kDa myosin light chain regulatory subunit; GPCR, G protein-coupled receptor; PLD, phospholipase D; TASMC, thoracic aorta smooth muscle cell; Ang II, angiotensin II; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; RµVSMC, renal microvascular smooth muscle cell; PE, polyethylene; DMSO, dimethyl sulfoxide; PD98059, 2'-amino-3'-methoxyflavone; AG1478, 4-(3-chloroanilino)-6,7-dimethoxyquinazoline; AG1296, 6,7-dimethoxy-3-phenylquinoxaline; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine; PBS, phosphate-buffered saline; BSA, bovine serum albumin; ANOVA, analysis of variance; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio) butadiene.
Address correspondence to: Dr. Bradley T. Andresen, Internal Medicine/Endocrinology, D109 HSC Diabetes Center, One Hospital Dr., Columbia, MO 65212. E-mail: andresenb{at}health.missouri.edu
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