Abstract The neuronal angiotensin II (Ang II) type 1 (AT1) receptor is coupled to the Ras-Raf-1–mitogen-activated protein (MAP) kinase signal-transduction pathway (Yang H, Lu D, Yu K, Raizada MK. Regulation of neuromodulatory actions of angiotensin II in the brain neurons by the Ras-dependent mitogen-activated protein kinase pathway. J Neurosci. 1996;16:4047-4058). In this study we compared the effects of angiotensin II (Ang II) on AT1 receptor phosphorylation and the ability of the phosphorylated receptor to bind Ang II in neuronal cultures of Wistar-Kyoto rat (WKY) and spontaneously hypertensive rat (SHR) brains to further our understanding of the Ang II signaling mechanism. Ang II caused a time-dependent phosphorylation of AT1 receptors in both WKY and SHR brain neurons. The level of phosphorylation was higher in the SHR brain neurons; this finding was consistent with increased AT1 receptors in these cells. MAP kinase was involved in this phosphorylation, a conclusion supported by the following evidence: (1) exogenous MAP kinase phosphorylated the AT1 receptor; (2) PD98059, a MAP kinase kinase inhibitor, attenuated Ang II–stimulated AT1 receptor phosphorylation; and (3) MAP kinase and AT1 receptors were coimmunoprecipitated in Ang II–stimulated neurons. Finally, MAP kinase phosphorylation was associated with the loss of 125I-[Sar1-Ile8]-Ang II binding ability of the AT1 receptor in both strains of neurons. These observations show that Ang II stimulates phosphorylation of the neuronal AT1 receptor by a mechanism involving MAP kinase and that the phosphorylated neuronal AT1 receptor does not exhibit Ang II binding activity in the brains of either WKY or SHR.
Angiotensin II stimulates tyrosine hydroxylase, dopamine β-hydroxylase, and NET by activating the AT1 receptor subtype in neuronal cultures.1 2 The neuromodulatory actions of the AT1 receptor, a member of the GPCR superfamily, are associated with the regulation of turnover, synthesis, and release of catecholamines in brain neurons.3 4 The physiological relevance of these actions of Ang II in brain neurons is further evidenced by observations that neurons from SHR brain express higher levels of AT1 receptors and a parallel increase in the Ang II–induced NET system.2 4 5 6 7 In recent studies, we have established that Ang II stimulation of neuromodulation involves activation of the Ras-Raf-1–MAP kinase signal-transduction pathway.8 Activation of MAP kinase initiates a cascade of downstream signaling events involving SRE and AP1 binding activities that ultimately leads to increased gene expression for tyrosine hydroxylase, dopamine β-hydroxylase, and NET.9
Despite our understanding of the signal-transduction pathways involved in the cellular and physiological actions of the AT1 receptor in neurons, little is known about the mechanism by which these signals are terminated. Studies with other GPCRs have indicated that an agonist-induced phosphorylation of the GPCR plays a key role in the desensitization of the receptor and termination of physiological actions.10 11 Many specific GRKs have been characterized in recent years that specifically phosphorylate agonist-bound GPCRs.10 11 In view of the potential role of phosphorylation in the regulation of the functions of GPCRs, the present study was conducted with the following two objectives in mind: (1) to determine whether Ang II stimulates phosphorylation of the AT1 receptor and if so, whether MAP kinase is involved in this phosphorylation; and (2) to compare the rates and mechanisms of Ang II–induced phosphorylation of AT1 receptors in neurons of WKY and SHR brains. In view of our previous observation that both AT1 receptors and AT1 receptor-mediated neuromodulation are increased in SHR brain neurons,4 5 we hypothesized that the rate of phosphorylation of the AT1 receptor would either not change or be decreased in these neurons compared with their normotensive WKY counterparts. Our observations show that Ang II stimulates AT1 receptor phosphorylation in the neurons of both strains of rats, an effect mediated by the activation of MAP kinase. The phosphorylation rate of the AT1 receptor in both WKY and SHR brain neurons was comparable.
One-day-old WKY and SHR were obtained from our breeding colony, which originated from Harlan Sprague Dawley (Indianapolis, Ind). DMEM, PDHS, and trypsin (150 IU/mg) were from Central Biomedia. [γ-32P]ATP (3000 Ci/mmol), [32P]orthophosphate (1 mCi=37 MBq), and chemiluminescence assay reagents were from Du Pont/NEN. Nitrocellulose membranes were from Micron Separations, Inc. Ang II and MBP were purchased from Sigma Chemical Co. Losartan potassium (Dup 753) was a gift from Du Pont/Merck (Wilmington, Del). PD98059, H89, bis-indolylmaleimide, and lavendustin A were purchased from Calbiochem. PD123319 was from RBI. Polyclonal anti-rabbit AT1 receptor antibody (306,Sc-579) was purchased from Santa Cruz Biotechnology (catalog No. 579). This antipeptide antibody was raised against an AT1 receptor peptide corresponding to amino acids306-359. The antibody was specific for the AT1 receptor, did not cross-react with the AT2 receptor, and was mouse, rat, and human reactive. Anti-MAP kinase (C-14) polyclonal antibody that specifically recognizes ERK-2 (and to a much lesser extent, ERK-1) and protein A/G PLUS–agarose were purchased from Santa Cruz Biotechnology. An anti-rat MAP kinase polyclonal antibody (ERK-1 through ERK-3), which recognized both the p42 and p44 isoforms of MAP kinase, and activated mouse GST-p42 MAP kinase were from Upstate Biotechnology, Inc. All other reagents were purchased from Fisher Scientific and were of the highest quality available.
Hypothalamus-Brainstem Neuronal Cells in Primary Culture
Hypothalamus-brainstem areas of 1-day-old WKY and SHR brains were dissected and brain cells were dissociated by trypsin. The hypothalamic block contained the paraventricular nucleus and the supraoptic, anterior, lateral, posterior, dorsomedial, and ventromedial nuclei. The brainstem block contained the medulla oblongata and pons. Dissociated brain cells were plated in poly-l-lysine–precoated tissue-culture dishes (2×107 cells per 100-mm-diameter dish) in DMEM containing 10% PDHS. Neuronal cultures were established essentially as previously described.5 12 The cultures were allowed to grow for 15 days before use in experiments. Immunohistochemical and biochemical analyses have repeatedly indicated that these cultures contain 90% neuronal cells and 15% astroglial cells and that cultures are comparable from the two strains of rats.5 12
Neuronal cultures, established in 100-mm culture dishes, were treated with Ang II. The cell lysates were prepared by adding 1 mL lysis buffer (25 mmol/L Tris-HCl, pH 7.4; 25 mmol/L NaCl; 1% Triton X-100; 1% deoxycholic acid; 1 mmol/L sodium orthovanadate; 10 mmol/L sodium fluoride; 10 mmol/L sodium pyrophosphate; 0.5 mmol/L EGTA; 1 mmol/L PMSF; 10 μg/mL aprotinin; and 0.8 μg/mL leupeptin) and scraping the cells off the culture dish. The lysates were centrifuged at 6000g for 10 minutes, and the protein content of the supernatant was assessed by the Bradford protein assay (Bio-Rad Laboratories). Supernatants containing 400 μg protein were subjected to an immunoprecipitation protocol as follows. Samples were incubated with 1 μg rabbit anti-AT1 receptor antibody overnight at 4°C, and AT1 receptor immunoreactivities were collected on protein A/G PLUS–agarose.8 13 The resulting immunoprecipitates were washed three times with lysis buffer and used in further experiments.
Each immunoprecipitate was suspended in 20 μL Laemmli’s sample buffer in a boiling water bath for 3 minutes, then centrifuged. The resulting supernatant (10 μL) was electrophoresed in 10% SDS–polyacrylamide gel and proteins were transferred onto a nitrocellulose membrane. The membrane was blocked by 5% nonfat dry milk in TBST (20 mmol/L Tris-HCl, pH 8.0; 150 mmol/L NaCl; and 0.05% Tween 20) for 1 hour followed by incubation for 1 hour at room temperature with either rabbit anti-MAP kinase antibody or anti-AT1 receptor antibody. Protein-bound antibody was detected by incubation of the membrane with horseradish peroxidase–labeled second antibody and enhanced by chemiluminescence assay reagents. The bands recognized by the primary antibody were visualized by exposing the membrane to x-ray film.8
Phosphorylation of AT1 Receptor by Exogenous MAP Kinase
Phosphorylation of the AT1 receptor, after its immunoprecipitation from the neuronal cell lysate by anti-rabbit AT1 receptor antibody, was carried out with the use of exogenous MAP kinase in a protocol based on that described by Paxton et al.14 Briefly, neuronal cell lysates were prepared as described above. Lysate containing equal amounts of protein (400 μg) was incubated with 1 μg rabbit anti-AT1 receptor antibody overnight at 4°C, and AT1 receptor immunoprecipitate was collected on protein A/G PLUS–agarose. This preparation was rinsed three times with lysis buffer and once with kinase assay buffer (50 mmol/L HEPES, pH 7.5; 0.1 mmol/L EDTA; and 0.015% Triton X-100). Immunoprecipitate was suspended in 10 μL kinase assay buffer. For measurement of phosphorylation, 10 μL of AT1 receptor immunoprecipitate (≈30 fmol/mg 125I-[Sar1-Ile8]-Ang II binding activity) was incubated with 0.3 IU MAP kinase, 0.1 mg/mL BSA, and 0.2% β-mercaptoethanol in a final volume of 20 μL. The reaction was started by the addition of 10 μL ATP mixture (0.3 mmol/L ATP, 30 mmol/L MgCl, and 200 μCi [γ-32P]ATP per milliliter in kinase assay buffer) and run for 0 to 15 minutes at 30°C. After the reaction was stopped by adding phosphoric acid, samples were blotted onto Whatman GF/B filter paper followed by washing the paper four times with ice-cold 0.5% phosphoric acid and finally once with acetone essentially as described elsewhere.14 The paper was allowed to dry and radioactivity quantitated in a Beckman liquid scintillation counter. Reaction mixtures that contained 3 μg MBP instead of AT1 receptor immunoprecipitate were used as the standard for the MAP kinase phosphorylation assay. In some experiments, the kinase reaction was stopped by the addition of 5× Laemmli’s sample buffer instead of phosphoric acid; samples were heated and centrifuged, and the supernatant was electrophoresed in 10% SDS–polyacrylamide gel. Proteins were then transferred to polyvinylidene difluoride membrane and subjected to autoradiography. After autoradiography, the same membrane was used for immunoblotting with AT1 receptor antibody. Autoradiograms were scanned using a UVP Imagestore 5000 system, and receptor phosphorylation data were presented as the observed density of the 32P-labeled band that was normalized for AT1 receptor protein by densitometry using the SW 5000 gel analysis program.
Labeling of Neuronal Cells With [32P]Orthophosphate and Analysis of Phosphorylated AT1 Receptor
Neuronal cultures were established for 15 days in 100-mm culture dishes. Growth medium was removed and cultures were incubated with phosphate-free DMEM containing dialyzed PDHS for 4 hours at 37°C, followed by prelabeling the cells with 1 mCi/mL [32P]orthophosphate for 4 hours at 37°C. Ang II was added and incubation continued for various time periods. Cultures were immediately rinsed with ice-cold PBS three to four times, and lysates were prepared in lysis buffer as described above. Cell lysates were centrifuged at 6000g for 10 minutes and the supernatants used to immunoprecipitate AT1 receptors essentially as described above. Agarose beads containing AT1 receptor were collected at 3000g for 10 minutes, washed three times in lysis buffer, resuspended in 20 μL Laemmli’s sample buffer, and heated to 100°C for 3 minutes. The supernatant was electrophoresed in 10% SDS–polyacrylamide gel, and proteins were transferred to polyvinylidene difluoride membrane. The membrane was dried and subjected to autoradiography. Densities of the 32P-labeled bands corresponding to AT1 receptors were scanned with the use of the UVP Imagestore 5000 system and quantitated by the SW 5000 gel analysis program. Data were normalized to the densities of total AT1 receptor in each lane as determined by immunoblotting.
Binding of 125I-[Sar1-Ile8]-Ang II to AT1 Receptor Immunoprecipitates
Cell-free lysates (400 μg protein) were subjected to immunoprecipitation by rabbit anti-AT1 receptor antibody as described above. Immunoprecipitates containing AT1 receptor were collected on protein A/G PLUS–agarose and washed three times with lysis buffer and once with kinase buffer. Immunoprecipitates were subjected to phosphorylation by exogenous MAP kinase essentially as described above. Immune complexes were rinsed with the binding buffer (PBS, pH 7.2, containing 1.0% BSA) and used for quantitation of 125I-[Sar1-Ile8]-Ang II binding as described previously.15 In brief, immune complexes suspended in 0.5 mL binding buffer were incubated with 1 nmol/L 125I-[Sar1-Ile8]-Ang II in the presence of 10 μmol/L PD123319 for 1 hour at room temperature to determine total binding. In addition, increasing concentrations of losartan (0.01 nmol/L to 100 nmol/L) were used for competition-inhibition experiments. All reactions were run in triplicate. The binding reaction was terminated by filtration and collection of 125I-[Sar1-Ile8]-Ang II bound to receptors on Whatman GF/B filters presoaked with 0.3% polyethyleneimine. Filters were washed three times with ice-cold PBS, pH 7.2, to remove unbound radioligand, and bound radioactivity was counted by a Beckman DP5500 gamma counter. Binding was expressed as femtomoles 125I-[Sar1-Ile8]-Ang II bound per milligram of the cellular protein used to immunoprecipitate the receptor. Specific binding was calculated by subtracting the 125I-[Sar1-Ile8]-Ang II bound to complex in the presence of losartan from that bound in its absence. Scatchard analysis of the competition-inhibition experiments was conducted to calculate Kd and Bmax values using the EBDA-ligand program (Elsevier-Biosoft).26
Each experiment was conducted in triplicate culture dishes. Cells in these dishes were derived from multiple brains of 1-day-old rats. Each experiment was repeated three times unless indicated otherwise. Images from autoradiograms were captured in the UVP Imagestore 5000 system, and radioactive bands were quantitated essentially as described elsewhere.8 Data from at least three autoradiograms were quantitated and corrected for equal loading with the use of AT1 receptor antibody, 125I-[Sar1-Ile8]-Ang II binding activity to the AT1 receptor, or another standard protein. They are presented as mean±SE. Statistical analysis was performed using ANOVA and Dunnett’s tests.
Previous studies have established that neuronal cultures of SHR brain express two to four times higher levels of AT1 receptors than neuronal cultures of WKY brain.4 5 This increase is associated with an increased ability of Ang II to stimulate NET.2 Our initial objective in this study was to compare the effect of Ang II on AT1 receptor phosphorylation in these two strains of rat brain neurons. Prelabeling of neuronal cultures with [32P]orthophosphate followed by stimulation with Ang II and immunoprecipitation analysis of 32P-labeled AT1 receptor showed a radiolabeled band of ≈49 kD in both WKY and SHR brain neurons (Fig 1A⇓). Ang II caused a time-dependent increase in the incorporation of 32P into this band, and a sixfold increase was observed in WKY brain neurons in 15 minutes. This increase was followed by ≈50% decrease in 32P incorporation by 30 minutes. Basal levels of phosphorylation of 32P into AT1 receptors were 65% higher in SHR brain neurons than in WKY brain neurons in spite of a comparable time course of Ang II stimulation (Fig 1B⇓). As a result, the degree of stimulation (approximately sixfold) of Ang II– induced AT1 receptor phosphorylation was comparable in both strains. This conclusion was confirmed when AT1 receptor phosphorylation data were normalized with the levels of AT1 receptor immunoreactivity in both strains of neurons (Fig 1C⇓). The ≈49-kD band is AT1 receptor protein, on the basis of the ability of the AT1 receptor-specific antibody to recognize this protein16 and the observation that the immunoprecipitated protein specifically binds 125I-[Sar1-Ile8]-Ang II (see discussion to follow). Fig 2⇓ shows that Ang II–induced phosphorylation of neuronal AT1 receptor was completely blocked by 10 μmol/L losartan but not by PD123319, an AT2 receptor subtype antagonist. This observation suggested that the occupancy by Ang II is important for stimulation of the AT1 receptor phosphorylation.
Next we studied the involvement of MAP kinase in Ang II–stimulated phosphorylation of the AT1 receptor. The rationale for this experiment was based on our previous studies, which showed that Ang II stimulates MAP kinase in neurons.8 In addition, MAP kinase has been implicated in the phosphorylation of estrogen and epidermal growth factor receptors.17 18 AT1 receptors were immunoprecipitated from WKY and SHR brain neurons, and these immunoprecipitates were subjected to an in vitro phosphorylation assay with exogenous MAP kinase. Fig 3A⇓ shows that exogenous MAP kinase catalyzed the phosphorylation of immunoprecipitated AT1 receptors in a time-dependent manner and that the phosphorylation in both strains of neurons was comparable. MBP was used as an internal standard for MAP kinase substrate. SDS-PAGE analysis of in vitro phosphorylated immunoprecipitated AT1 receptors was carried out to confirm the identity of phosphorylated protein. Fig 3B⇓ shows that the ≈49-kD 32P-labeled band was recognized by AT1 receptor antibody on the immunoblot. In addition, incorporation of 32P into this band increased over time in both WKY and SHR brain. This finding was consistent with the in vitro phosphorylation data (Fig 3A⇓). These observations indicate that exogenous MAP kinase could phosphorylate the AT1 receptor. Neuronal cultures were treated with PD98059, a relatively selective inhibitor of MAP kinase kinase, in an attempt to provide in vivo support for the above conclusion. Cultures were prelabeled with [32P]orthophosphate for 4 hours followed by coincubation without or with Ang II in the presence of 10 μmol/L PD98059. This concentration of PD98059 has previously been shown to inhibit MAP kinase kinase activity.19 In addition, a 90% inhibition of neuronal MAP kinase activity using this treatment was observed. Fig 4⇓ shows that PD98059 significantly inhibited Ang II–stimulated phosphorylation of AT1 receptors in both WKY and SHR brain neurons. Other protein kinase inhibitors were used to further establish the specificity of MAP kinase in Ang II–induced phosphorylation of the AT1 receptor. Fig 5⇓ shows that basal AT1 receptor phosphorylation was not affected to any significant degree by inhibitors of protein kinase A (H89) and protein tyrosine kinase (lavendustin). However, Ang II stimulation of AT1 receptor phosphorylation was partially blocked by the PKC inhibitor bis-indolylmaleimide. This observation was consistent with the report that PKC phosphorylation sites are present in the AT1 receptor and that Ang II stimulates neuronal PKC.20 21 Inhibition of protein tyrosine kinase or protein kinase A had little effect on phosphorylation.
Neuronal extracts were subjected to the immunoprecipitation protocol with the AT1 receptor antibody followed by immunoblotting with MAP kinase antibody to determine whether the AT1 receptor coprecipitates with MAP kinase. Fig 6⇓ shows that a significant amount of MAP kinase appears to coimmunoprecipitate with AT1 receptor protein in control neurons. Treatment of WKY brain neurons with 100 nmol/L Ang II resulted in a time-dependent increase in coimmunoprecipitation of AT1 receptors with MAP kinase. The predominant band that coimmunoprecipitated with the AT1 receptor was the p42 isoform. With Ang II, maximal coimmunoprecipitation approximately fourfold over control levels was observed in 5 minutes. A similar stimulation of coimmunoprecipitation of AT1 receptor with MAP kinase was observed in Ang II–treated SHR brain neurons.
Finally, the effect of AT1 receptor phosphorylation by MAP kinase on the binding of 125I-[Sar1-Ile8]-Ang II was measured to determine whether phosphorylated receptors retain Ang II binding activity. Fig 7⇓ shows Scatchard analyses of competition-inhibition data for nonphosphorylated and phosphorylated AT1 receptors from WKY and SHR brain neurons. Bmax values for nonphosphorylated and phosphorylated receptors were 58±6 and 7±4 fmol/mg protein, respectively, for WKY brain neurons. The Bmax values for nonphosphorylated and phosphorylated AT1 receptor from SHR brain neurons were 83±7 and 9±5 fmol/mg protein, respectively. Thus, phosphorylation resulted in an approximate 89% decrease in the Bmax in both WKY and SHR brain neurons. Kd values for nonphosphorylated receptors (0.5±0.03 nmol/L for both WKY and SHR) were comparable to those for MAP kinase–phosphorylated receptors (0.4±0.02 nmol/L for both WKY and SHR). These data show that MAP kinase–phosphorylated AT1 receptors are much less able to bind 125I-[Sar1-Ile8]-Ang II in both WKY and SHR brain neurons.
The observations presented in this study demonstrate that (1) Ang II stimulates phosphorylation of neuronal AT1 receptors, (2) phosphorylation is comparable and proportional to the levels of AT1 receptors in WKY and SHR brain neurons, (3) MAP kinase is a key protein kinase in this Ang II–induced AT1 receptor phosphorylation, and (4) phosphorylated receptors lack 125I-[Sar1-Ile8]-Ang II binding activity.
Ang II–stimulated phosphorylation of neuronal AT1 receptors is consistent with a similar observation in vascular smooth muscle cells.14 The site of this phosphorylation could be localized in the intracellular domain, consistent with the presence of threonine, serine, and tyrosine residues in this region of the receptor.20 21 Although the precise mechanism of this phosphorylation remains to be fully worked out, our data suggest a key role for MAP kinase: (1) Exogenous MAP kinase phosphorylates AT1 receptors in both WKY and SHR brain neurons, (2) the MAP kinase kinase inhibitor PD98059 attenuates Ang II–stimulated phosphorylation (phosphorylation is not affected by inhibitors of protein tyrosine kinase or protein kinase A), (3) the MAP kinase coimmunoprecipitates with AT1 receptor, and (4) the MAP kinase recognition sequence (amino acids232-233) is present in the AT1b receptor subtype. Both the AT1a and AT1b subtypes of AT1 receptors are present in relatively equal proportions in WKY and SHR brain neuronal cells.5 These observations provide strong support for a direct involvement of MAP kinase in Ang II–induced phosphorylation of AT1 receptors. However, alternate possibilities should not be totally discounted at the present. For example, the involvement of another GRK (such as βARK1) in the phosphorylation of AT1 receptor cannot be ruled out. It is likely that Ang II stimulation of MAP kinase results in the phosphorylation and activation of other GRKs, which in turn phosphorylate the AT1 receptor. This view is supported by recent data demonstrating AT1 receptor phosphorylation by βARK1.22 In addition, the MAP kinase recognition sequence is present in βARK1.23
An important question that arises from these studies concerns the physiological implications of AT1 receptor phosphorylation. It is reasonable to suggest that the phosphorylation event may be responsible for Ang II– induced desensitization and internalization of AT1 receptors from the neuronal plasma membrane. This view is supported by our data on the loss of 125I-[Sar1-Ile8]-Ang II binding ability of phosphorylated AT1 receptors and consistent with the proposed role of phosphorylation in the agonist-induced desensitization of other GPCRs, such as β- and α-adrenergic receptors.10 11 Preliminary data indicating that Ang II induces internalization of cell-surface AT1 receptors further support this proposal.27
Neuronal cells from the SHR brain express twofold to fourfold higher functional AT1 receptors than WKY brain neurons.5 This increase is associated with a 65% higher basal level of phosphorylated AT1 receptor in these cells. However, the rate of phosphorylation, degree of stimulation by Ang II, and involvement of MAP kinase were comparable in the two strains of neurons. This observation indicates that the Ang II–mediated phosphorylation mechanism of the AT1 receptor is similar in WKY and SHR brain neurons. In addition, phosphorylation results in a comparable loss of 125I-[Sar1-Ile8]-Ang II binding to AT1 receptors in both strains of neurons. Thus, in spite of higher levels of AT1 receptors and enhanced Ang II–induced neuromodulatory actions in SHR brain neurons,4 5 the rate of their inactivation is comparable, supporting our previous hypothesis that chronic neuromodulatory actions of Ang II on neurons may be distinctly regulated.
Ang II stimulates MAP kinase in neuronal cells in an Ras-Raf-1–dependent signaling pathway.8 This stimulation has been associated with Ang II regulation of chronic neuromodulation.8 9 Involvement of MAP kinase in AT1 receptor-mediated regulation of neuromodulation is a unique event for a stimulatory GPCR that may involve interaction of one or more factors with the Gβγ subunit of Gq.24 25 Thus, it is intriguing to suggest that activation of MAP kinase by Ang II potentially leads to both the stimulatory action of the AT1 receptor on neuromodulation and desensitization followed by internalization of AT1 receptors in neurons. A precise time sequence of signaling events will have to be elucidated and specific signaling molecules need to be identified to explain the specificity of these two opposing effects of Ang II mediated by AT1 receptors.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|βARK1||=||β-adrenergic receptor kinase 1|
|AT1, AT2||=||Ang II type 1, Ang II type 2|
|DMEM||=||Dulbecco’s modified Eagle’s medium|
|ERK||=||extracellular signal-related kinase|
|GPCR||=||G protein–coupled receptor|
|GRK||=||G protein receptor kinase|
|MBP||=||myelin basic protein|
|PAGE||=||polyacrylamide gel electrophoresis|
|PDHS||=||plasma-derived horse serum|
|PKC||=||protein kinase C|
|SHR||=||spontaneously hypertensive rat(s)|
This research was supported by grant HL-33610 from the National Institutes of Health, Bethesda, Md. We wish to thank Elizabeth Brown for cell-culture preparation, Jennifer Brock for the preparation of the manuscript, and Kevin Fortin for editorial suggestions.
- Received September 19, 1996.
- Revision received October 21, 1996.
- Accepted February 5, 1997.
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