This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yang, H. Articles by Raizada, M. K. Search for Related Content PubMed PubMed Citation Articles by Yang, H. Articles by Raizada, M. K.

(Hypertension. 1996;27:1277-1283.)
© 1996 American Heart Association, Inc.

Articles

Lack of Cross Talk Between ₁-Adrenergic and Angiotensin Type 1 Receptors in Neurons of Spontaneously Hypertensive Rat Brain

Hong Yang; Di Lu; Mohan K. Raizada

From the Department of Physiology, University of Florida, College of Medicine, Gainesville.

Correspondence to Mohan K. Raizada, PhD, College of Medicine, Department of Physiology, PO Box 100274 JHMHC, 1600 SW Archer Rd, Gainesville, FL 32610. E-mail mraizada@phys.med.ufl.edu.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract Norepinephrine causes downregulation of angiotensin II (Ang II) receptors in Wistar-Kyoto rat (WKY) brain neuronal cultures. The aim of this study was to compare the cross talk between Ang II and ₁-adrenergic receptors in these neuronal cultures. Norepinephrine causes a 66% decrease in B_max of Ang II type 1 (AT₁) receptors in neuronal cultures of WKY brain. This decrease is mediated by the interaction of norepinephrine with the _1A-adrenergic receptor subtype. Norepinephrine also causes a decrease in mRNA levels for AT₁ receptors. A maximal decrease of 83% in AT₁ receptor mRNA is observed in 8 hours with 100 µmol/L norepinephrine, is blocked by 5-methyluradipil, and involves inhibition of AT₁ receptor transcription. Furthermore, decreases in the AT₁ receptor and its mRNA are associated with a significant attenuation of AT₁ receptor–mediated stimulation of norepinephrine transporter mRNA in WKY brain neurons. In contrast, norepinephrine does not decrease AT₁ receptors or mRNA and has no effect on Ang II stimulation of norepinephrine transporter mRNA in neuronal cultures of spontaneously hypertensive rat brain. Thus, these data show that norepinephrine-mediated downregulation of AT₁ receptors is associated with a parallel decrease in AT₁ mRNA and Ang II stimulation of norepinephrine transporter mRNA and involves the _1A-adrenergic receptor in neurons of WKY brain. This cross talk between the two receptors is lacking in neurons of spontaneously hypertensive rat brain.

Key Words: receptors, angiotensin II • RNA, messenger • brain • receptors, adrenergic, alpha • angiotensin II • norepinephrine • neurons

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
It is well known that the brain contains a renin-angiotensin system including both AT₁ and AT₂ receptor subtypes.^{1 2 3 4 5 6} Interaction of Ang II with its neuronal AT₁ receptor subtype initiates a cascade of cellular events that ultimately leads to such physiological effects of Ang II as BP increase, vasopressin release, sympathetic activation, dipsogenic response, and altered baroreflex function.^{1 2 3 4 5 6} The pressor effect of Ang II partly involves activation of sympathetic nerve activity and is associated with an interaction of AT₁ receptors with brain catecholamines. Evidence for this Ang II–catecholamine interaction includes the following: (1) Injection of pressor doses of Ang II into the brain stimulates the synthesis, turnover, and release of norepinephrine from hypothalamic and brain stem nuclei.^{7 8 9} This action is abolished by antagonists of norepinephrine, dopamine, and -adrenergic receptors.^{10 11 12} In addition, catecholamines regulate brain Ang II receptors in a negative feedback fashion.¹³ (2) Hypothalamic and brain stem nuclei that are involved in the Ang II–induced pressor response also contain catecholamine input/fiber tracts and Ang II and AT₁ receptors.^{14 15 16 17 18} (3) Ang II receptors are present on norepinephrine neurons.¹⁹ The relevance of this Ang II–catecholamine interaction in the central control of BP is further supported by studies with SHR, a genetic animal model of hypertension that parallels human essential hypertension.²⁰ These studies demonstrate that Ang II–mediated sympathetic activity is significantly heightened in SHR as a consequence of a hyperactive brain Ang II system.^{3 4 21 22} Thus, one could conclude from these in vivo studies that the Ang II–catecholamine interaction is pivotal in Ang II–mediated central control of BP and that elucidation of the mechanism of this interaction would be of great significance in furthering our understanding of the pathophysiology of hypertension.

Our group has been studying the cellular and molecular mechanisms of Ang II–catecholamine interactions with the use of an in vitro hypothalamus–brain stem neuronal coculture system from 1-day-old WKY and SHR. These studies have established that a negative feedback interaction exists between Ang II receptors and the catecholamine system that controls the cellular actions of Ang II in neurons. For example, interaction of Ang II with its neuronal AT₁ receptor initiates a cascade of cellular events that includes stimulation of the phospholipase C–phosphoinositide–protein kinase C signal transduction pathway and results in stimulation of the NET system and norepinephrine synthesis and release.^{4 13} The released norepinephrine interacts with the ₁-adrenergic receptors, and their chronic activation results in the downregulation of Ang II receptors, thus turning off the neuromodulatory action of Ang II.¹³ Our objectives in this study were to determine whether (1) downregulation of Ang II receptors by norepinephrine involves the AT₁ receptor subtype in neuronal cultures of WKY brain and (2) norepinephrine has a similar or distinct effect on Ang II receptors in neuronal cultures of SHR brain. Evidence shows that the interaction of norepinephrine with _1A-adrenergic receptors downregulates AT₁ receptor numbers and mRNA in WKY brain neurons, an action lacking in SHR brain neurons.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
One-day-old WKY and SHR were obtained from our breeding colony, which originated from Harlan Sprague Dawley, Indianapolis, Ind. Dulbecco's modified Eagle's medium (DMEM), plasma-derived horse serum (PDHS), and crystallized trypsin were from Central Biomedia. Norepinephrine and yohimbine were from Sigma Chemical Co. [-³²P]dCTP (3000 Ci/mmol) was from DuPont-NEN. PCR kits containing Taq DNA polymerase were purchased from Perkin-Elmer Cetus. Reverse transcriptase and a deoxynucleotide mixture containing dATP, dTTP, dGTP, and dCTP were from Stratagene. Oligo(dT)₁₅ was from Promega. RNase inhibitor was from 5'-3' Prime, Inc. Losartan potassium (formally DuP 753) was a gift from Dr A.T. Chiu of DuPont/Merck. 5-Methylurapidil and chloroethylelonidine were from RBI. Propranolol was purchased from Calbiochem. ¹²⁵I–[Sar¹,Ile⁸]Ang II (specific activity, 2200 Ci/mmol) was obtained from Dr Robert Speth, Washington State University (Pullman). All other biochemicals were from Fisher Scientific and were of molecular biology grade.

PCR primers for AT₁ and AT₂ receptors, NET, and ß-actin were synthesized by the DNA Synthesis Core Facility of the Interdisciplinary Center for Biotechnology Research, University of Florida. Sequences for these primers were as follows: AT₁ receptor: sense, 5'-TTTCTTCTCAATCTCGCCTTGGCTG-3'; antisense, 5'-GGGGATCCAGAAAGCCGTAGAACAG-3'; NET: sense, 5'-CCGCATCCATGCTTCTGGCGCGGATGAA-3'; antisense, 5'-GGGCAGGCTCAGATGGCCAGCCAGTGTT-3'; ß-actin: sense, 5'-GAGAAGATGACCCAGATCATGT-3'; antisense, 5'-ACTCCATGCCCAGGAAGGAAG-3'; and AT₂ receptor: sense, 5'-GGGAGTCTCTGACAGTTCAAT-3'; antisense, 5'-CCATTGCTAGGCTGATTACAT-3'.

Preparation of Neuronal Cultures From WKY and SHR Brains
Hypothalamus–brain stem areas of brains from 1-day-old WKY and SHR were dissected and brain cells dissociated by trypsin. The hypothalamic block contained the paraventricular nucleus and the supraoptic, anterior, lateral, posterior, dorsomedial, and ventromedial nuclei. The brain stem block contained medulla oblongata and pons. Dissociated brain cells were plated in poly-L-lysine–precoated tissue culture dishes (3x10⁶ cells per 35-mm-diameter dish) in DMEM containing 10% PDHS, and neuronal culture was established essentially as described previously.^{4 23 24} The cultures were allowed to grow for 10 to 15 days before their use in experiments. These neuronal cultures from both rat strains are comparable in terms of the number of neurons, total cellular protein per dish, and many other biochemical criteria and have been extensively used as an in vitro model in our laboratory for investigation of the cellular and molecular bases of a hyperactive brain Ang II system in SHR.⁴ Immunohistochemical analysis repeatedly has indicated that the cultures from both rat strains contain 85% to 90% neuronal cells and 10% to 15% astroglial cells.

¹²⁵I–[Sar¹,Ile⁸]Ang II Binding Assay
Neuronal cultures established in 35-mm culture dishes were incubated with indicated 100 µmol/L norepinephrine for 16 hours in the presence of 10 µmol/L sodium ascorbate. Control cultures contained 10 µmol/L sodium ascorbate essentially as described previously.^{24 25 26} AT₁ receptor binding to untreated and norepinephrine-treated intact neuronal cells attached to culture dishes was determined with the use of ¹²⁵I–[Sar¹,Ile⁸]Ang II as follows: Growth media were aspirated from cultures, and cells were washed twice with ice-cold PBS (pH 7.4) and incubated with a 0.5-mL reaction mixture containing 1.0 µmol/L ¹²⁵I–[Sar¹,Ile⁸]Ang II, 1.0% bovine serum albumin, and 1 µmol/L PD 123319 in the absence or presence of increasing concentrations of losartan (0.1 nmol/L to 10 µmol/L).²⁴ After incubation for 1 hour at room temperature, the reaction mixture was removed. Cells were washed twice with ice-cold PBS (pH 7.4) and then dissolved in 0.1N NaOH (0.5 mL per dish). Dissolved cells were transferred into 12/75-mm tubes, and radioactivity was measured in a gamma counter (DP5500, Beckman Instruments). Specific binding, expressed as femtomoles per milligram protein, was calculated by subtracting ¹²⁵I–[Sar¹,Ile⁸]Ang II bound to cells in the presence of losartan from that bound in its absence. Triplicate culture dishes for total and nonspecific binding were used, and the experiment was repeated three times. Scatchard analysis was carried out from competition-inhibition experiments for the calculation of K_d and B_max values with the EBDA-ligand program (Elsevier-Biosoft).

Measurements of AT₁ Receptor, AT₂ Receptor, NET, and ß-Actin mRNA Levels
Isolation of Poly(A⁺) RNA From Neuronal Cultures
Neuronal cultures established in 35-mm dishes were rinsed once with ice-cold PBS (pH 7.4) and lysed by 100 µL lysis buffer (10 mmol/L Tris-HCl, pH 7.5, 0.14 mol/L NaCl, 5 mmol/L KCl, and 1% NP-40). After centrifugation, 100 µL of supernatant was moved to sterile RNase-free tubes containing 3 µL of Dynabeads Oligo(dT)₂₅ (Dynal Inc) and 100 µL of 2x binding buffer (20 mmol/L Tris-HCl, pH 7.5, 1.0 mol/L LiCl, 2 mmol/L EDTA). mRNAs were allowed to bind with Dynabeads for 5 minutes at room temperature, and the poly(A⁺)-Dynabeads complex was washed and isolated as described in the protocol provided by the company and described by us previously.²⁷

RT-PCR
The poly(A⁺) RNA–Dynabeads complex was suspended in 20 µL RT solution containing 50 mmol/L Tris-HCl, pH 8.3, 75 mmol/L KCl, 3 mmol/L MgCl₂, 10 mmol/L dithiothreitol, 200 ng oligo(dT)₁₅, 200 µmol/L of each dNTP, and 40 IU Moloney murine leukemia virus reverse transcriptase, and the reaction was run for 60 minutes at 37°C. This was followed by heating the reaction for 5 minutes at 65°C. Five microliters of this RT solution was subjected to PCR for the AT₁ or AT₂ receptor or NET. Two microliters of RT solution was used for ß-actin PCR. AT₁ and AT₂ receptor PCRs in a total volume of 50 µL contained 10 mmol/L Tris-HCl, pH 9.0, 50 mmol/L KCl, 2.5 mmol/L MgCl₂, 50 µmol/L of each dNTP, 20 pmol/L of sense and antisense primers, 2 IU Taq DNA polymerase, and 0.1 µCi [³²P]dCTP. PCR was performed at 94°C for 1 minute, 58°C for 1 minute, and 72°C for 1 minute for 28 cycles. NET PCR and ß-actin PCR were performed essentially as described by us previously.²⁷ All these PCR conditions were determined quantitatively for their mRNAs by cyclic lineage analysis described previously.²⁷ ß-Actin was used as a standard marker for normalizing the measurements of AT₁, AT₂, and NET PCR products. PCR products were analyzed on a nondenaturing polyacrylamide gel as described below.

Analysis of [³²P]dCTP-Incorporated RT-PCR Products by Nondenaturing Polyacrylamide Gel Electrophoresis
After PCR, 5 µL of the sample containing ³²P-labeled PCR products was mixed with 5 µL of 2x gel loading buffer (4% Ficoll 400, 20 mmol/L EDTA, pH 8.0, 0.05% bromophenol blue, and 0.05% xylene cyanole FF) and applied to a 5% acrylamide gel (acrylamide/bis-acrylamide, 29:1) prepared in 1x TBE buffer (89 mmol/L Tris base, 89 mmol/L boric acid, and 2 mmol/L EDTA, pH 8.0). The gel was run for 1 hour at 100 V in a Bio-Rad Mini-Gel System in 1x TBE buffer. The gel was decasted, wrapped in a plastic bag, and exposed to x-ray film overnight at -70°C before development. Each experimental data point was quantified for AT₁ receptor, NET, and ß-actin mRNAs. Autoradiograms from three independent experiments were used for analysis after normalization with ß-actin.

Measurement of PCR Bands
The bands representing PCR products from the x-ray film were scanned with the use of a UVP Imagestore 5000 system (Ultra Violet Products Ltd). The images of PCR bands on X-ray film were captured, and the same level of background and size of scanning area were applied to each individual band. The density of each PCR band was then analyzed and measured with the personal computer software SW 5000 Gel Analysis, and the data were presented as OD units essentially as described previously.^{25 27 28} The OD units were presented as a ratio of the OD of AT₁ receptor, NET, or AT₂ receptor mRNA products to the OD for ß-actin mRNA product.

Nuclear Run-on Assay
The transcription rate of AT₁ receptor mRNA in untreated and norepinephrine-treated neuronal cultures was determined essentially as we described previously.²⁹ In brief, neuronal cultures were established in 100-mm-diameter tissue culture dishes and treated with 100 µmol/L norepinephrine for 16 hours at 37°C. Control cultures incubated with vehicle were used in parallel incubations. Cells (2x10⁷ cells per dish) were scraped and pelleted by centrifugation at 500g for 5 minutes. Nuclei were isolated, the RNA was labeled with [-³²P]UTP, and labeled RNA (6x10⁶ cpm/mL) was hybridized with 5 µg AT₁ receptor cDNA probe applied on a nitrocellulose membrane. Radiolabeled bands were measured as described above.

Measurement of AT₁ Receptor mRNA In Vivo
Adult male WKY (mean BP, approximately 100 mm Hg) and SHR (mean BP, approximately 190 mm Hg) were housed singly in stainless steel cages in a standard vivarium with Purina Chow pellets and tap water. They were fitted with an indwelling cannula (10 mm long, 23-gauge stainless steel) stereotaxically aimed to end in or just above the lumen of the right lateral ventricle and firmly fixed to the skull with jeweler's screws and dental acrylic. Surgery was performed with ketamine and xylazine (50 and 5 mg/kg IP) anesthesia, and rats were allowed to recover for 1 week before use in the experiments. Injections were made through an 11-mm, 30-gauge injector needle attached to a 35-mm syringe. Five microliters of either PBS or PBS containing 10 µg norepinephrine was injected into each rat. Twenty-four hours later, rats were killed, and hypothalami were dissected and homogenized in 4 mol/L guanidinium isothiocyanate, 0.01% ß-mercaptoethanol, 25 mmol/L sodium acetate, pH 6.0, and 0.5% sacrosyl. Total RNA from each sample was subjected to poly(A⁺) RNA isolation and AT₁ receptor RT-PCR as described for neuronal cultures.

Experimental Groups and Data Analysis
For ¹²⁵I–[Sar¹,Ile⁸]Ang II binding, each data point was obtained from three 35-mm dishes and each experiment was repeated three times with cells from different rat litters. Similar experimental protocols were used for AT₁ receptor, AT₂ receptor, and NET mRNA determinations. Data presented are mean±SE and normalized for ß-actin mRNA, which was quantified from the same samples for equal loading. Comparisons between experimental and control data were made with one-way ANOVA and Dunnett's test with the use of Statistica software.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Our first objective was to determine whether norepinephrine-induced downregulation of Ang II receptors observed previously^{26 30} is a result of its action on the AT₁ receptor subtype. Incubation of WKY brain neuronal cultures with norepinephrine caused a dose-dependent decrease in the binding of ¹²⁵I–[Sar¹,Ile⁸]Ang II to AT₁ receptors, with a maximal decrease of approximately 70% that was observed with 100 µmol/L norepinephrine in 16 hours. This decrease in the binding was associated with a 66% decrease in the B_max for AT₁ receptors without significantly affecting the K_d for the binding (Table). In contrast, norepinephrine failed to exert any effect on ¹²⁵I–[Sar¹,Ile⁸]Ang II binding to AT₁ receptors in neuronal cultures of SHR brain.

View this table: [in this window] [in a new window]   Table 1. Kd and BmaxValues of125I–[Sar1,Ile8]Ang II Binding to AT1 Receptors in Untreated and Norepinephrine-Treated WKY Rat Brain Neuronal Cultures

RT-PCR was used for determination of whether the norepinephrine-induced downregulation of AT₁ receptors in WKY brain neurons and the lack of its effect in SHR brain neurons are reflected at the level of AT₁ receptor mRNA. The validity of the RT-PCR technique was first established for the measurement of AT₁ receptor mRNA in neuronal cultures. Fig 1 shows a linear relationship between the PCR products for AT₁ receptor and ß-actin in both untreated and norepinephrine-treated WKY brain neurons as a function of PCR cycles. The products were measured by a UVP Imagestore 5000 system, and data are presented as the density of each band. In addition, the data also show that norepinephrine caused a decrease in the AT₁ receptor PCR product that was also linear with the PCR cycles. In contrast, norepinephrine had no effect on the ß-actin PCR product. An alinear relationship between the PCR products for AT₁ receptor and ß-actin and the volume of the RT reaction used for the PCR was also observed (Fig 2). This indicated that excellent reproducibility without a significant variability caused by multiple sampling as well as other experimental conditions in either RT or PCR were inherent in this RT-PCR technique. Finally, a linear relationship between the PCR cycles and PCR products was also observed when both AT₁ receptor and ß-actin reactions were carried out in a single tube (Fig 3). Thus, we concluded from these data that the RT-PCR conditions established for measurement of the AT₁ receptor are reflective of its mRNA levels in control and norepinephrine-treated neuronal cultures. Therefore, subsequent experiments were carried out under linear conditions for 28 PCR cycles, and the AT₁ receptor mRNA data were normalized by the levels of ß-actin PCR products.

View larger version (26K): [in this window] [in a new window]   Figure 1. Relationship between PCR cycles and PCR products for AT1 receptor (AT1-R) and ß-actin in untreated and norepinephrine (NE)–treated WKY brain neuronal cultures. Triplicate neuronal cultures were incubated without (lanes 4 through 6) or with (lanes 1 through 3) 100 µmol/L norepinephrine for 16 hours at 37°C. mRNAs were extracted and subjected to RT-PCRs as described in "Methods." PCR for the AT1 receptor (A) and ß-actin (B) mRNAs was carried out from the same samples but in separate tubes for 22 (lanes 1 and 4), 26 (lanes 2 and 5), and 30 (lanes 3 and 6) cycles. Data from two separate experiments were quantified. Insets show representative autoradiograms.

View larger version (23K): [in this window] [in a new window]   Figure 2. Relationship between RT volumes and PCR products for AT1 receptor (AT1-R) and ß-actin in untreated and norepinephrine (NE)–treated WKY brain neuronal cultures. Triplicate neuronal cultures were incubated without (lanes 1 through 3) or with (lanes 4 through 6) 100 µmol/L norepinephrine for 16 hours at 37°C. mRNAs were extracted and subjected to RT reactions as described in "Methods." Indicated volumes of RT reactions (5 µL, lanes 1 and 4; 10 µL, lanes 2 and 5; and 15 µL, lanes 3 and 6) were then subjected to PCR for both AT1 receptor and ß-actin mRNAs for 28 cycles. Data from two experiments were quantified as described in "Methods." Insets show representative autoradiograms.

View larger version (27K): [in this window] [in a new window]   Figure 3. Relationship between PCR cycles and PCR products for AT1 receptor (AT1-R) and ß-actin in untreated and norepinephrine-treated WKY brain neuronal cultures. Experimental conditions are essentially the same as described in Fig 1 legend except the PCRs for both AT1 receptor and ß-actin were carried out in the same tube.

Incubation of neuronal cultures of WKY brain with 100 µmol/L norepinephrine caused a time-dependent decrease in AT₁ receptor mRNA levels (Fig 4). A maximal decrease of 80% was observed within 8 hours. Basal AT₁ receptor mRNA levels were fivefold higher in SHR brain neurons when compared with WKY brain neurons (Fig 4). The decrease in the AT₁ receptor mRNA in WKY brain neurons also depended on norepinephrine dose, and a maximal decrease of 83% was observed with 100 µmol/L norepinephrine in 16 hours (Fig 5). The decrease was blocked by 5-methylurapidil and not by chloroethylelonidine, yohimbine, or propranolol (Fig 6), indicating the involvement of the _1A-adrenergic receptor subtype. The norepinephrine-induced decrease in the AT₁ receptor mRNA was specific for the AT₁ receptor as norepinephrine failed to influence mRNA levels for the AT₂ receptor (Fig 7). We carried out nuclear run-on assays to determine the effect of norepinephrine on the transcription rate of AT₁ receptor mRNA in WKY brain neurons. Fig 8 shows that the transcription rate of the AT₁ receptor was significantly higher in SHR brain neurons compared with WKY brain neurons. This confirms our previous observation that the increased AT₁ receptors in this strain are a result of an increase in AT₁ receptor gene transcription.⁴ Fig 8 also shows that norepinephrine caused a 70% decrease in the transcription rate in WKY brain neurons without any influence in SHR brain neurons.

View larger version (39K): [in this window] [in a new window]   Figure 4. Effect of norepinephrine on AT1 receptor mRNA levels in neuronal cultures of WKY and SHR brains as a function of time. Neuronal cultures of WKY (1 through 5) and SHR (6 through 10) brains were incubated with 100 µmol/L norepinephrine for 0 (1 and 6), 2 (2 and 7), 4 (3 and 8), 8 (4 and 9), and 24 (5 and 10) hours at 37°C. AT1 receptor mRNA was quantified as described in "Methods." Data are mean±SE (n=3). Inset shows representative autoradiogram. *Significantly different from untreated control (lane 1) (P<.05).

View larger version (25K): [in this window] [in a new window]   Figure 5. Effect of norepinephrine on AT1 receptor mRNA levels in neuronal cultures of WKY brain as a function of norepinephrine concentrations. Cultures were incubated without (1) or with (2) 1 nmol/L, 10 nmol/L (3), 100 nmol/L (4), 1 µmol/L (5), 10 µmol/L (6), 100 µmol/L (7), or 1000 µmol/L (8) norepinephrine for 16 hours at 37°C. Data are mean±SE (n=3). Inset shows representative autoradiogram. *Significantly different from untreated control (lane 1) (P<.05).

View larger version (39K): [in this window] [in a new window]   Figure 6. Effect of 1-adrenergic receptor antagonists on AT1 receptor mRNA levels in neuronal cultures of WKY brain. Cultures were incubated without (1, 3, 5, 7, and 9) or with (2, 4, 6, 8, and 10) 100 µmol/L norepinephrine in the presence of 10 µmol/L chloroethylelonidine (4), 1 µmol/L yohimbine (6), 10 µmol/L 5-methylurapidil (8), or 10 µmol/L propranolol (10). Cultures were also incubated with similar concentrations of chloroethylelonidine (3), yohimbine (5), 5-methylurapidil (7), and propranolol (9) alone for 16 hours at 37°C. Data are mean±SE (n=3). Inset shows representative autoradiogram. *Significantly different from untreated control (lane 1) (P<.05).

View larger version (35K): [in this window] [in a new window]   Figure 7. Effect of norepinephrine on AT2 receptor (AT2-R) mRNA levels in neuronal cultures of WKY brain. Cultures were incubated without (1) or with (lanes 2 through 6) 100 µmol/L norepinephrine for 0.5 (lane 2), 1 (lane 3), 2 (lane 4), 4 (lane 5), or 8 (lane 6) hours. RT-PCR was carried out essentially as described for the AT1 receptor except that specific primers for the AT2 receptors were used. This protocol is valid for measurement of AT2 receptor mRNA because the RT-PCR was linear with PCR cycles and RT reaction volume. Data are means of two experiments. Inset shows representative autoradiogram.

View larger version (29K): [in this window] [in a new window]   Figure 8. Transcription rate of AT1 receptor mRNA in WKY and SHR brain neuronal cultures treated with norepinephrine (NE). Experimental conditions are similar to those described in "Methods." Data are means of two experiments. Inset shows representative autoradiogram. C, control.

We determined the ability of Ang II to stimulate NET mRNA in untreated and norepinephrine-treated WKY brain neuronal cultures to evaluate whether downregulation of AT₁ receptor was associated with a parallel decrease in Ang II responsiveness. Fig 9 shows that Ang II caused a fourfold and sixfold stimulation of NET mRNA levels in neuronal cultures of WKY and SHR brains, respectively. Preincubation of WKY brain neurons with 100 µmol/L norepinephrine for 16 hours resulted in attenuation of Ang II stimulation of NET mRNA levels as there was no significant difference between the mRNA levels in the control and norepinephrine-treated WKY. In addition, norepinephrine failed to have any effect on the ability of Ang II to stimulate NET mRNA in SHR brain neurons.

View larger version (42K): [in this window] [in a new window]   Figure 9. Effect of norepinephrine on Ang II stimulation of NET mRNA in neuronal cultures of WKY and SHR brains. Neuronal cultures of WKY (open bars) and SHR (hatched bars) brains were incubated without (1 and 5) or with (2 and 6) 100 nmol/L Ang II for 4 hours. Cultures were also incubated with 100 µmol/L norepinephrine for 16 hours at 37°C (3, 4, 7, and 8), followed by incubation without (3 and 7) or with (4 and 8) 100 nmol/L Ang II for an additional 4 hours. NET mRNAs were measured as described in "Methods." Data are mean±SE (n=3). Inset shows representative autoradiogram. *Significantly different from control (P<.05); **significantly different from Ang II–treated cultures (lane 4) (P<.05).

Finally, we carried out in vivo experiments to validate our in vitro observation of a decrease in AT₁ receptor mRNA levels in WKY brain neurons with norepinephrine by measuring the levels of AT₁ receptor mRNA in the hypothalami of untreated and norepinephrine-treated WKY and SHR brains. AT₁ receptor mRNA levels in the hypothalamus of SHR were threefold higher compared with levels in WKY brain hypothalamus (Fig 10). In addition, 24 hours after norepinephrine injection, AT₁ receptor mRNA levels decreased by 56% in WKY brain hypothalamus. In contrast, norepinephrine failed to influence AT₁ receptor mRNA levels in the SHR hypothalamus (Fig 10).

View larger version (30K): [in this window] [in a new window]   Figure 10. Effect of norepinephrine on AT1 receptor mRNA levels in hypothalami of WKY and SHR. WKY (1 and 2) and SHR (3 and 4) brains were injected with PBS (1 and 3) or 10 µg norepinephrine (2 and 4). AT1 receptor mRNA levels were measured as described in "Methods." Top, Representative autoradiogram; bottom, measurement of PCR products. Data are mean OD±SE of three rats. *Significantly lower compared with control (lane 1) (P<.05); **significantly higher compared with control (P<.05).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Observations from this study demonstrate that previously reported negative feedback regulation of Ang II receptors by norepinephrine^{13 26 30} is a result of a decrease in AT₁ receptor gene expression in WKY brain neurons. In addition, neurons of SHR brain lack this downregulatory mechanism. The action of norepinephrine on AT₁ receptors involves the _1A-adrenergic receptor and thus provides an example of the cross talk between these two receptors in the neurons.

The decrease in AT₁ receptor mRNA in neurons of WKY brain appears to be due to the effect of norepinephrine on AT₁ receptor gene transcription, as evidenced by nuclear run-on assay, with no significant effect on the rate of AT₁ receptor mRNA degradation. Although our data are not conclusive as to whether this decrease in the mRNA precedes the action of norepinephrine on AT₁ receptor numbers, the relatively slow turnover rate for neuronal receptors¹³ and the influence of norepinephrine on AT₁ receptor transcription argue in its favor. However, other possibilities, such as independent effects of norepinephrine on the receptor and its mRNA, cannot be ruled out at the present time. An interesting aspect of these observations is that they provide a cellular basis for the regulation of Ang II actions in neurons of WKY brain. We propose the following sequence of events based on these and other data: Ang II interacts with the AT₁ receptor and stimulates norepinephrine turnover, synthesis, uptake, and release in neurons.^{4 13 31} There are two consequences of the released norepinephrine. It would be taken up by the neurons via the specific NET system,⁷ and it would interact with the ₁-adrenergic receptors. The latter would downregulate AT₁ receptor and turn off the cellular actions of Ang II. This appears to be the only known mechanism by which neuromodulatory actions of Ang II could be regulated in the brain. Finally, our observation of a lack of an effect of norepinephrine on neurons of SHR brain is of great interest given this paradigm. It would suggest that Ang II would persistently stimulate the norepinephrine system in SHR brain neurons because AT₁ receptors are not regulated by norepinephrine. Validation of these in vitro data and hypothesis is provided by our in vivo observation presented in Fig 10. This indicates that the downregulatory action of norepinephrine on AT₁ receptor mRNA also occurs in the hypothalamus of the adult WKY and that such a mechanism is lacking in SHR. In addition, these data also confirm our previous observations indicating an increase in the basal levels of AT₁ receptor mRNA in SHR brain.²⁸

Three important questions arise from these observations. (1) Would an increase in the basal levels of AT₁ receptor mRNA and its expression and a lack of their downregulation by norepinephrine observed in neuronal cultures of SHR brain be observed in the neurons of other genetically hypertensive rats? Recent data demonstrating similar observations with the neurons of stroke-prone SHR brain confirm the validity of our observations in SHR.³² In addition, comparisons of observations in SHR with both WKY and Sprague-Dawley rats as normotensive controls further support this conclusion.⁴ (2) Is the lack of the response of norepinephrine in SHR brain neurons a result of decreases in _1A-adrenergic receptor subtype or an impairment of the signaling mechanism involved in the cross talk between _1A-adrenergic and AT₁ receptors? Our data favor the latter possibility because ₁-adrenergic receptor levels are in fact increased and not decreased in SHR neurons.^{33 34} This increase is associated with a parallel increase in both _1A- and _1B-adrenergic receptors and their mRNAs.³⁵ (3) Is the lack of cross talk the basis for increased AT₁ and ₁-adrenergic receptors in SHR brain neurons? No direct evidence is yet available to answer this question. ₁-Adrenergic receptor–mediated signaling pathways should be compared in the neurons from the two rat strains to derive this information. Also, the possibility that differences in norepinephrine response element(s) in the AT₁ receptor gene may exist in neurons from the two strains also should be considered. Finally, the regulation of AT₁ receptors by norepinephrine seems to be physiologically relevant in the brain on the basis of in vivo experiments that show that both AT₁ and ₁-adrenergic receptors are increased in the hypothalamic area of SHR²⁷ and that central injections of norepinephrine cause downregulation of AT₁ receptor mRNA in WKY brain hypothalamus.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II AT1, AT2 = angiotensin type 1, type 2 BP = blood pressure NET = norepinephrine transporter OD = observed density PBS = phosphate-buffered saline PCR = polymerase chain reaction RT = reverse transcription SHR = spontaneously hypertensive rat(s) WKY = Wistar-Kyoto rat(s)

	Acknowledgments

 
This research was supported by grant HL33610 from the National Institutes of Health, Bethesda, Md. Dr Di Lu is a postdoctoral fellow of the American Heart Association, Florida Affiliate. We wish to thank Elizabeth Brown for cell culture preparation, Jennifer Brock for the preparation of the manuscript, and Kevin Fortin for graphics.

Received November 17, 1995; first decision January 8, 1996; accepted February 21, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Phillips MI. Brain angiotensin. In: Gross P, ed. Circumventricular Organs and Body Fluids. Boca Raton, Fla: CRC Press; 1987;3:163-182.

2. Saavedra JM. Brain and pituitary angiotensin. Endocr Rev. 1992;13:329-380. [Abstract/Free Full Text]

3. Timmermans PBMWM, Wang PC, Chiu AT, Herblin MF, Benfield P, Carini DJ, Lee RJ, Wexler RR, Saye JAM, Smith RD. Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol Rev. 1993;45:205-251. [Medline] [Order article via Infotrieve]

4. Raizada MK, Lu D, Sumners C. AT₁ receptors and angiotensin actions in the brain and neuronal cultures of normotensive and hypertensive rats. In: Mukhopadhyay A, Raizada MK, eds. Current Concepts: Tissue Renin-Angiotensin System as Local Regulators in Reproductive and Endocrine Organs. New York, NY: Plenum Publishing Corp; 1994:331-348.

5. Wright JN, Harding JN. Brain angiotensin receptor subtypes in the control of physiological and behavioral responses. Neurosci Biobehav Rev. 1994;18:21-53. [Medline] [Order article via Infotrieve]

6. Steckelings V, Lebrun C, Quadri F, Veltman A, Unger T. Role of brain angiotensin in cardiovascular regulation. J Cardiovasc Pharmacol. 1992;19(suppl 6):S73-S79.

7. Chevillard C, Duchene N, Rasquire R, Alexandre JM. Relation of the centrally evoked pressor effect of angiotensin II to central noradrenaline in the rabbit. Eur J Pharmacol. 1979;58:203-206. [Medline] [Order article via Infotrieve]

8. Quadri F, Badoer E, Stadler T, Unger T. Angiotensin II-induced noradrenaline release from anterior hypothalamus in conscious rats: a brain microdialysis study. Brain Res. 1991;563:137-141. [Medline] [Order article via Infotrieve]

9. Stadler T, Veltmar A, Quadri F, Unger T. Angiotensin II evokes noradrenaline release from the paraventricular nucleus in conscious rats. Brain Res. 1992;569:117-122. [Medline] [Order article via Infotrieve]

10. Brody MI, Fint GD, Biggy J, Haywood JR, Gordon FJ, Johnson AK. Role of anteroventral third ventricle (AV3V) region in experimental hypertension. Circ Res. 1978;43:102-113. [Abstract/Free Full Text]

11. Camacho A, Phillips MI. Separation of drinking and pressor responses to control angiotensin by monoamines. Am J Physiol. 1981;240:R106-R110. [Abstract/Free Full Text]

12. Jones DE. Injection of phentolamine into the anterior hypothalamus-preoptic area of rats blocks both pressor and drinking responses produced by central administration of angiotensin II. Brain Res Bull. 1984;13:127-133. [Medline] [Order article via Infotrieve]

13. Sumners C, Raizada MK. Angiotensin II receptor subtypes in neuronal cells. In: Raizada MK, Phillips MI, Sumners C, eds. Cellular and Molecular Biology of the Renin-Angiotensin System. Boca Raton, Fla: CRC Press; 1993:379-411.

14. Moore RY, Bloom FZ. Central catecholamine neuron systems: anatomy and physiology of the norepinephrine systems. Annu Rev Neurosci. 1979;2:113-168. [Medline] [Order article via Infotrieve]

15. Gehlert DR, Gackenheimer S, Reel JK, Lin HS, Steinberg MI. Nonpeptide angiotensin II receptor antagonists discriminate subtypes of [¹²⁵I] angiotensin II binding sites in the rat brain. Eur J Pharmacol. 1990;187:125-126.

16. Rowe BP, Kalivar PW, Speth RC. Autoradiographic localization of angiotensin II receptor binding sites on noradrenergic neurons of the locus coeruleus of the rat. J Neurochem. 1990;55:533-540. [Medline] [Order article via Infotrieve]

17. Obermueller N, Unger TH, Culman J, Gahike P, de Gaspero M, Bottari SP. Distribution of angiotensin II receptor subtypes in rat brain nuclei. Neurosci Lett. 1991;132:11-15. [Medline] [Order article via Infotrieve]

18. Song K, Allen A, Paxinos G, Mendelsohn FAO. Mapping of angiotensin II receptor subtype heterogeneity in rat brain. J Comp Neurol. 1992;316:467-490. [Medline] [Order article via Infotrieve]

19. Rowe BP, Grove KL, Saylor DL, Speth RC. Angiotensin II receptor subtype in the rat brain. Eur J Pharmacol. 1990;186:339-342. [Medline] [Order article via Infotrieve]

20. Trippodo NC, Frolich ED. Similarities of genetic (spontaneous) hypertension: man and rat. Circ Res. 1981;48:309-319. [Free Full Text]

21. Ding H, Zhou Q, Deng J, Lao HY, Yang K. Effect of the overactivated central renin-angiotensin system on the concentration of brain norepinephrine and epinephrine in stroke-prone spontaneously hypertensive rats and its significances. Sheng Li Hsueh Pao. 1990;42:379-384.

22. Yang K, Ding H, Zhou Q, Luo HY, Wu ZY. Central norepinephrine and angiotensin II contents in the brain regions of spontaneously hypertensive rats (SHR) and the interaction between them. Sheng Li Hsueh Pao. 1991;43:345-351.

23. Raizada MK, Muther TF, Sumners C. Increased angiotensin II specific receptors in neuronal culture of spontaneously hypertensive rat brain. Am J Physiol. 1984;247:C364-C372. [Abstract/Free Full Text]

24. Sumners C, Tang W, Zelezna B, Raizada MK. Angiotensin II receptor subtypes are coupled with distinct signal transduction mechanisms in neurons and astrocytes from rat brain. Proc Natl Acad Sci U S A. 1991;88:7567-7571. [Abstract/Free Full Text]

25. Raizada MK, Lu D, Tang W, Kurian P, Sumners C. Increased angiotensin II type 1 receptor gene expression in neuronal cultures from spontaneously hypertensive rats. Endocrinology. 1993;132:1715-1722. [Abstract/Free Full Text]

26. Sumners C, Walkins LL, Raizada MK. ₁-Adrenergic receptor-mediated down-regulation of angiotensin II receptors in neuronal cultures. J Neurochem. 1986;47:1117-1126. [Medline] [Order article via Infotrieve]

27. Lu D, Raizada MK. Delivery of angiotensin II type-1 receptor antisense inhibits angiotensin action in neurons from hypertensive rat brain. Proc Natl Acad Sci U S A. 1995;92:2914-2918. [Abstract/Free Full Text]

28. Raizada MK, Sumners C, Lu D. Angiotensin II type 1 receptor mRNA levels in the brains of normotensive and spontaneously hypertensive rats. J Neurochem. 1993;60:1949-1952. [Medline] [Order article via Infotrieve]

29. Lu D, Sumners C, Raizada MK. Regulation of angiotensin II type 1 receptor mRNA in neuronal cultures of normotensive and spontaneously hypertensive rat brains by phorbol esters and forskolin. J Neurochem. 1994;62:2079-2084. [Medline] [Order article via Infotrieve]

30. Sumners C, Raizada MK. Catecholamine-angiotensin II receptor interaction in primary cultures of rat brain. Am J Physiol. 1984;246:C502-C509. [Abstract/Free Full Text]

31. Sumners C, Raizada MK. Angiotensin II stimulates norepinephrine uptake in hypothalamus-brainstem neuronal cultures. Am J Physiol. 1986;250:C236-C244. [Abstract/Free Full Text]

32. Raizada MK, Lu D, Yang H, Yu K. AT₁ receptors and angiotensin II actions in neuronal cultures of stroke-prone spontaneously hypertensive rat brain. In: Raizada MK, Phillips MI, Sumners C, eds. Recent Advances in Cellular and Molecular Aspects of Angiotensin Receptors. New York, NY: Plenum Press Publishers. In press.

33. Bottiglieri DF, Morse CA, Baker SP, Crews FT, Sumners C, Raizada MK. Increased expression of ₁-adrenergic receptors in hypothalamus of spontaneously hypertensive rat brain. Brain Res. 1988;439:187-194. [Medline] [Order article via Infotrieve]

34. Feldstein JB, Pacitti AJ, Sumners C, Raizada MK. ₁-Adrenergic receptors in neuronal cultures from the brain: increased expression in hypertensive rat. J Neurochem. 1986;47:1190-1198. [Medline] [Order article via Infotrieve]

35. Magdalena WR. Differential Regulation of -1A and -1B Adrenergic Receptor Subtypes in Neuronal and Astroglial Cell Cultures. Gainesville, Fla: University of Florida; 1992. PhD dissertation.

This article has been cited by other articles:

				G. Nickenig and D. G. Harrison The AT1-Type Angiotensin Receptor in Oxidative Stress and Atherogenesis: Part II: AT1 Receptor Regulation Circulation, January 29, 2002; 105(4): 530 - 536. [Full Text] [PDF]

				H. Wang, M. J. Katovich, C. H. Gelband, P. Y. Reaves, M. I. Phillips, and M. K. Raizada Sustained Inhibition of Angiotensin I-Converting Enzyme (ACE) Expression and Long-Term Antihypertensive Action by Virally Mediated Delivery of ACE Antisense cDNA Circ. Res., October 1, 1999; 85(7): 614 - 622. [Abstract] [Full Text] [PDF]

				H. Yang and M. K. Raizada Role of Phosphatidylinositol 3-Kinase in Angiotensin II Regulation of Norepinephrine Neuromodulation in Brain Neurons of the Spontaneously Hypertensive Rat J. Neurosci., April 1, 1999; 19(7): 2413 - 2423. [Abstract] [Full Text] [PDF]

				N. L. Han and M. K. Sim Hypothalamic angiotensin receptor subtypes in normotensive and hypertensive rats Am J Physiol Heart Circ Physiol, August 1, 1998; 275(2): H703 - H709. [Abstract] [Full Text] [PDF]

				Y. Du, J. Qiu, S. H. Nelson, and D. H. Wang Regulation of type 1 ANG II receptor in vascular tissue: role of alpha 1-adrenoreceptor Am J Physiol Regulatory Integrative Comp Physiol, October 1, 1997; 273(4): R1224 - R1229. [Abstract] [Full Text] [PDF]

				H.-T. Li, C. S. Long, M. O. Gray, D. G. Rokosh, N. Y. Honbo, and J. S. Karliner Cross Talk Between Angiotensin AT1 and {alpha}1-Adrenergic Receptors : Angiotensin II Downregulates {alpha}1a-Adrenergic Receptor Subtype mRNA and Density in Neonatal Rat Cardiac Myocytes Circ. Res., September 19, 1997; 81(3): 396 - 403. [Abstract] [Full Text]

				D. H. Wang, J. Qiu, Z. Hu, and Y. Du Regulation of Type 1 Angiotensin II Receptor in Adrenal Gland : Role of {alpha}1-Adrenoreceptor Hypertension, September 1, 1997; 30(3): 345 - 350. [Abstract] [Full Text]

				H. Yang, D. Lu, and M. K. Raizada Angiotensin II-Induced Phosphorylation of the AT1 Receptor From Rat Brain Neurons Hypertension, September 1, 1997; 30(3): 351 - 357. [Abstract] [Full Text]

				D. Lu, M. K. Raizada, S. Iyer, P. Reaves, H. Yang, and M. J. Katovich Losartan Versus Gene Therapy : Chronic Control of High Blood Pressure in Spontaneously Hypertensive Rats Hypertension, September 1, 1997; 30(3): 363 - 370. [Abstract] [Full Text]

				H. Yang, D. Lu, G. P. Vinson, and M. K. Raizada Involvement of MAP Kinase in Angiotensin II-Induced Phosphorylation and Intracellular Targeting of Neuronal AT1 Receptors J. Neurosci., March 1, 1997; 17(5): 1660 - 1669. [Abstract] [Full Text] [PDF]

This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yang, H. Articles by Raizada, M. K. Search for Related Content PubMed PubMed Citation Articles by Yang, H. Articles by Raizada, M. K.