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(Circulation Research. 2001;89:365.)
© 2001 American Heart Association, Inc.

Integrative Physiology

Elevated Blood Pressure in Transgenic Mice With Brain-Specific Expression of Human Angiotensinogen Driven by the Glial Fibrillary Acidic Protein Promoter

Satoshi Morimoto, Martin D. Cassell, Terry G. Beltz, Alan Kim Johnson, Robin L. Davisson, Curt D. Sigmund

From the Departments of Internal Medicine and Physiology & Biophysics (S.M., C.D.S.), Psychology (T.G.B., A.K.J.), and Anatomy and Cell Biology (M.D.C., R.L.D.), University of Iowa, Iowa City, Iowa.

Correspondence to Curt D. Sigmund, PhD, Departments of Internal Medicine and Physiology and Biophysics, 2191 Medical Laboratory, University of Iowa College of Medicine, Iowa City, IA 52242. E-mail curt-sigmund{at}uiowa.edu

	Abstract

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In addition to the circulatory renin (REN)–angiotensin system (RAS), a tissue RAS having an important role in cardiovascular function also exists in the central nervous system. In the brain, angiotensinogen (AGT) is expressed in astrocytes and in some neurons important to cardiovascular control, but its functional role remains undefined. We generated a transgenic mouse encoding the human AGT (hAGT) gene under the control of the human glial fibrillary acidic protein (GFAP) promoter to experimentally dissect the role of brain versus systemically derived AGT. This promoter targets expression of transgene products to astrocytes, the most abundant cell type expressing AGT in brain. All transgenic lines exhibited hAGT mRNA expression in brain, with variable expression in other tissues. In one line examined in detail, transgene expression was high in brain and low in tissues outside the central nervous system, and the level of plasma hAGT was not elevated over baseline. In the brain, hAGT protein was mainly localized in astrocytes, but was present in neurons in the subfornical organ. Intracerebroventricular (ICV) injection of human REN (hREN) in conscious unrestrained mice elicited a pressor response, which was abolished by ICV preinjection of losartan. Double-transgenic mice expressing the hREN gene and the GFAP-hAGT transgene exhibited a 15–mm Hg increase in blood pressure and an increased preference for salt. Blood pressure in the hREN/GFAP-hAGT mice was lowered after ICV, but not intravenous losartan. These studies suggest that AGT synthesis in the brain has an important role in the regulation of blood pressure and electrolyte balance.

Key Words: blood pressure • renin-angiotensin system • brain • astrocyte • transgenic mouse

	Introduction

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Angiotensin II (Ang II) has several actions on the central nervous system that may influence blood pressure (BP) regulation. These include increased sympathetic outflow and vasopressin release, as well as altered drinking and salt appetite.¹ Ang II is formed by the enzymatic action of renin (REN) on angiotensinogen (AGT), followed by the action of angiotensin-converting enzyme (ACE) on angiotensin I (Ang I). Several lines of evidence point to a contribution of an overactive brain REN-angiotensin system (RAS) to the hypertensive state in experimental models such as spontaneously hypertensive rats (SHRs), DOCA-salt hypertensive rats, Dahl-salt sensitive rats, and renal hypertensive rats.^2–8 For example, REN activity, AGT, Ang II, and Ang II binding are increased in the brains of SHRs.^2,9–11 Acute and chronic intracerebroventricular (ICV) injection of ACE inhibitor, angiotensin antagonist, and antisense oligonucleotides to Ang II type 1 receptor (AT₁) mRNA and AGT mRNA attenuates the development of hypertension in SHR.^5,12,13

Overactivity of the brain RAS may play a role in the hypertension observed in a number of transgenic animal models.^14,15 For example, transgenic mice overexpressing both the human REN (hREN) and human AGT (hAGT) genes are chronically hypertensive.¹⁵ ICV injection of the selective AT₁ antagonist, losartan, in these mice causes a greater fall in BP than in control nontransgenic mice.¹⁶ It was hypothesized that overexpression of the brain RAS in this model caused systemic hypertension by a mechanism involving a central Ang II–dependent increase in circulating arginine vasopressin. Like the SHR model, however, this transgenic model exhibits both altered endocrine RAS (elevated circulating Ang II) and tissue RAS (elevated tissue Ang II) function, making it difficult to experimentally distinguish between them.

In the brain, AGT expression is localized mainly in astrocytes (glia).¹⁷ Glial AGT is detectable throughout the brain, with the highest expression in the hypothalamus and preoptic areas and moderate to high expression in the mesencephalon and myelencephalon. We have reported that transgenic mice expressing hAGT or a hAGT promoter–controlled reporter gene express the transgene in astrocytes in all regions of the brain and in selected neurons in important cardiovascular control centers.¹⁸ Neuronal expression of AGT has also been reported in the paraventricular nucleus and supraoptic nucleus in rats.¹⁹ Thus, although there is convincing evidence for primary expression of AGT mRNA and protein in the brain, its role in cardiovascular control remains unclear. Accordingly, because AGT synthesis is most abundant in astrocytes, we generated a transgenic mouse model expressing hAGT under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter to accomplish the following two specific goals: (1) to examine whether AGT synthesized in brain is able to raise BP and alter drinking and (2) to create a model of neurogenic hypertension resulting from brain-specific overexpression of the RAS.

	Materials and Methods

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Generation of Transgenic Mice
The transgene consists of a 2.2-kb fragment of human GFAP promoter fused to the hAGT gene. Human GFAP promoter was excised with EcoRI from the plasmid gfa2 and subcloned into pBluescriptIISK+/- to form pGFAP-SK.²⁰ The hAGT gene was cloned as a BglII-NheI fragment into the BamHI-SpeI sites in pGFAP-SK to form pGFAP-hAGT.²¹ This fusion results in the utilization of DNA present in intron I as a 5'-untranslated region. Translation initiation starts in hAGT exon II. All cloning junctions were confirmed by sequencing. The transgene was excised by BssHII, purified by agarose gel electrophoresis, recovered using the Qia-Quick purification kit (Qiagen), and microinjected into 1-cell fertilized mouse (C57BL/6J x SJL/J; B6SJL F₂) embryos.²² All transgenic mice were maintained by breeding with B6SJL F₁ mice, and received standard mouse chow (LM-485; Teklad Premier Laboratory Diets) and water ad libitum. Care of the mice met the standards set forth by the NIH in their guidelines for the care and use of experimental animals. All procedures were approved by the University of Iowa Animal Care and Use Committee.

Analysis of Nucleic Acids
Genomic DNA was purified from tail biopsies and analyzed by Southern blot.²¹ Ten micrograms of genomic DNA was digested with EcoRI and probed with a segment of human AGT intron II. There was no cross-reactivity of this probe with mouse genomic DNA. A 9.5-kb band indicates the presence of the transgene. Polymerase chain reaction was performed using primers specific for hAGT, which amplified a 539-bp segment of exon II.²³

Tissues were harvested and snap-frozen in liquid N₂, and RNA was purified using TriReagent (Molecular Research Center). RNase protection assays were performed using the RPAIII kit (Ambion Inc). Total RNA (10 µg) was hybridized to hAGT and mouse ß-actin probes labeled with [-³²P]UTP by in vitro transcription and purified through a Sephadex G-50 spin column (Boehringer Mannheim). Protected fragments for hAGT and ß-actin were 518 and 245 nucleotides, respectively.

Plasma AGT Assay
Plasma mouse AGT (mAGT) and hAGT protein were determined as described previously.^21,24 RIA was performed on plasma using the Ang I ¹²⁵I-labeled RIA kit (NEN Life Science Products Inc). Samples were diluted with reagent blank to remain on the linear portion of the standard curve. Plasma AGT was extrapolated using the 1:1 molar relationship between Ang I and AGT.

Immunohistochemistry
Mice were killed and then perfused transcardially with 20 mL PBS followed by 50 mL 4% formaldehyde. The brain was removed, postfixed overnight (4°C) in the same fixative, and placed in 30% sucrose solution. The following day, the brain was frozen and cut coronally (30 µm, Reichert-Jung Cryostat). Sections were permeabilized with 0.1% Triton X-100 in PBS (25°C) and incubated 16 to 18 hours with a rabbit polyclonal antibody against hAGT (4°C, 1:100 dilution, provided by Dr Duane Tewksbury [Marshfield Medical Research Foundation, Marshfield, Wis]). Sections were also incubated with mouse monoclonal antisera against GFAP (1:1000 dilution, Chemicon International Inc) or microtubule-associated protein-2 (MAP-2, 1:400 dilution, Chemicon International Inc).^25,26 Samples were washed in 0.1% Triton X-100, incubated with fluorescein-conjugated anti-rabbit IgG (1:100 dilution, Chemicon International Inc) for 1 hour (25°C), rinsed, and then incubated with rhodamine-conjugated anti-mouse IgG (1:100 dilution, Chemicon International Inc).

Physiology
Mice were anesthetized with ketamine (90 mg/kg, IP) and acepromazine maleate (1.8 mg/kg, IP), shaved, and surgically instrumented with catheters in the carotid artery and jugular vein.¹⁶ Catheters were tunneled subcutaneously, exteriorized, and sutured in place between the scapulae. After catheterization, some mice were instrumented with ICV cannulae for microinjection into the lateral ventricle. Anesthetized mice were placed in a stereotaxic apparatus (David Kopf Instruments), and a 9-mm guide cannula (25 gauge, Small Parts Inc) was implanted.¹⁶ Injections were made using a 33-gauge stainless steel injector (Small Parts) connected to tubing (PE-10, drawn over heat) fitted with a 10-µL microsyringe (Hamilton Company). ICV and intravenous infusions were performed over 30 seconds. Compounds were dissolved in artificial cerebrospinal fluid (ACSF, in mmol/L, NaCl 136, KCl 5.6, NaHCO₃ 16.2, NaH₂PO₃ 1.2, MgCl₂ 1.2, and CaCl₂ 2.2, pH 7.4) and saline, delivered in 0.5- and 10-µL volumes, respectively. Correct insertion of the ICV cannula was determined by the pressor and bradycardiac response to ICV Ang II (200 ng).¹⁶ Mice that did not respond (one GFAP-hAGT, three systemic hAGT, and one nontransgenic mouse) were excluded from further studies. Methylene blue dye (5 µL) was injected after finishing experiments, and the brains were fixed and sectioned. All mice that exhibited central Ang II–induced cardiovascular responses were confirmed to have correct cannula placement by histological examination.

After surgery, mice were allowed 48 hours to recover and were then tested in a conscious, unrestrained state in their home cages. Mice were allowed a 30-minute adaptation period after connection of the arterial line to a Transpac pressure transducer (Abbott Laboratory), venous line, or insertion of the microinjector into the guide cannula. Baseline BP and heart rate (HR) were measured continuously for 1 hour per day for 3 consecutive days. Each infusion was performed on different days after measuring basal BP and HR, and mice were monitored at least 1 hour after drug infusion. Mice were infused with ICV (10, 30, and 100 ng) or intravenous (500 ng) hREN (gift of Walter Fischli, Hoffman-LaRoche, Basel, Switzerland). Some mice were pretreated with intravenous or ICV losartan (10 µg, Merck Research Laboratories). Controls included ICV injection of ACSF and intravenous injection of saline. All hemodynamic data were collected and analyzed on a computer using Chart version 4.0.1 in Powerlab.

Measurement of Drinking Volume and Salt Preference
Mice were housed individually in metabolic cages and fed standard chow and tap water ad libitum. After a 3-day acclimatization period, water intake was measured daily for 3 days. To determine salt preference, mice were fed salt-deficient chow (Teklad Premier Laboratory Diets). Two burettes containing either 0.3 mol/L hypertonic saline or tap water were provided to each mouse in a metabolic cage. Access to either burette was randomized and ad libitum. After a 3-day acclimatization period, intake of the hypertonic saline and tap water was measured daily for 3 days. The preference for hypertonic saline was calculated as a percentage determined by dividing the volume of saline consumed by the total volume of fluid consumed.

Statistical Analysis
Data are expressed as mean±SEM. Group comparisons were made with unpaired t tests and confirmed with repeated-measures ANOVA followed by the Student modified t test with Bonferroni correction. A value of P<0.05 was considered statistically significant.

	Results

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Transgenic mice were generated with a construct containing the human GFAP promoter fused to a genomic clone encompassing exons II, III, IV, and V; the intervening introns; and the native 3'-end of the hAGT gene containing the poly(A) site (Figure 1A). Of 53 live-born offspring, three transgenic founders were identified (Figures 1B and 1C). All founders were successfully bred to establish lines and each transmitted the transgene to 50% of both males and females, indicating insertion in autosomes.

View larger version (47K): [in this window] [in a new window]   Figure 1. Schematic map and identification of transgenic founders. A, Schematic map of the transgene. The GFAP promoter is the open box and exons of hAGT gene are filled boxes. The start site of transcription is indicated as +1. The thick bar below the map represents the probe used for the Southern blot. BSS indicates BssHII. B, Transgenic founders and their offspring were identified by a diagnostic 539-bp band after polymerase chain reaction. C, Southern blot of DNAs digested with EcoRI and probed with segment indicated in panel A. A 9.5-kb band was diagnostic of the transgene. M indicates 100-bp ladder marker; C, distilled water instead of DNA; N, genomic DNA from nontransgenic mouse; and P, plasmid DNA containing the transgene.

When controlled by its own endogenous promoter, hAGT expression was observed in the liver, kidney, and other tissues including the brain (Figure 2A). This transgene is termed the systemic hAGT transgene. However, expression of hAGT in GFAP-hAGT transgenic mice was more restricted, with the highest level of expression in brain, and lower-level expression in liver, heart, aorta, submandibular gland, diaphragm, and adipose tissue (Figure 2B). Note the clear shift in the ratio between liver and brain expression comparing systemic hAGT mice (Figure 2A) with GFAP-hAGT mice (Figure 2B). In all three lines, hAGT mRNA expression was consistently observed in the brain, but exhibited variable expression of hAGT mRNA in tissues outside the brain (Table 1).

View larger version (80K): [in this window] [in a new window]   Figure 2. Expression of hAGT in systemic hAGT and GFAP-hAGT transgenic mice. RNase protection assays of RNA from a male systemic hAGT (A) and male GFAP-hAGT (line 10176/3, B) mouse. - indicates liver of nontransgenic mouse; Br, brain of transgenic mouse; Lv, liver; K, kidney; H, heart; Lu, lung; Ag, adrenal gland; Ao, aorta; Sp, spleen; Sg, submandibular gland; D, diaphragm; Wa, white adipose tissue; Ba, brown adipose tissue, Sm, skeletal muscle; and T, testes.

View this table: [in this window] [in a new window]   Table 1.  Summary of hAGT Expression in GFAP-hAGT Transgenic Mice

The liver is the primary site of AGT release into the circulation. Therefore, given the presence of "ectopic" hAGT mRNA in liver of all three lines, we were concerned that hAGT protein may be released into the systemic circulation. Therefore, we measured circulating levels of mAGT and hAGT to determine whether transgene expression outside the brain resulted in a significant increase of AGT protein in the systemic circulation. The specificity of the assay was confirmed by the observation of baseline levels of mAGT, but high levels of hAGT protein in the plasma of systemic hAGT transgenic mice (Table 2). Plasma hAGT levels in GFAP-hAGT transgenic mice were extremely low compared with those of systemic hAGT mice. A statistically significant increase above baseline was only reached in the 10187/1 line, which exhibited the highest extrabrain transgene expression. Line 10176/3, which exhibited the most selective transgene expression in brain and had baseline plasma hAGT, was chosen for further analysis.

View this table: [in this window] [in a new window]   Table 2.  Plasma AGT Levels

To examine the cell-specific expression of the transgene in the brain, double-labeling for hAGT and GFAP (a glial marker) or MAP-2 (a neuronal marker) was performed (Figure 3).^25,26 hAGT staining was observed in the cell bodies and processes of astrocytes in all regions of the brain as confirmed by costaining with GFAP, but not with MAP-2 (Table 3). The only exception to this was the subfornical organ (SFO), where hAGT colocalized with both GFAP and MAP-2 (Figures 3F through 3I).

View larger version (89K): [in this window] [in a new window]   Figure 3. Cell-specific expression of GFAP-hAGT. Representative photomicrographs of double-labeling for hAGT and GFAP or hAGT and MAP-2 in brain. In cerebral cortex, GFAP-positive cells (B) but not MAP-2–positive cells (D) were costained with hAGT (A and C) in GFAP-hAGT mice. In SFO, both GFAP-positive cells (G) and MAP-2–positive cells (I) were double-labeled with hAGT (F and H). No hAGT staining was observed in nontransgenic mice (E). A and B, C and D, F and G, and H and I, respectively, are pairs of the same sections.

View this table: [in this window] [in a new window]   Table 3.  Distribution of hAGT Staining in Brain of GFAP-hAGT Mice

We next measured BP and HR to investigate whether functional hAGT protein was expressed in the brain in GFAP-hAGT mice. Consistent with the species specificity of the REN-AGT enzymatic reaction, there was no difference in baseline mean arterial pressure or HR among GFAP-hAGT transgenic mice (120±5 mm Hg, 598±16 bpm), systemic hAGT transgenic mice (118±5 mm Hg, 604±18 bpm), and nontransgenic mice (117±5 mm Hg, 617±21 bpm). In GFAP-hAGT mice, ICV infusion of purified hREN increased mean arterial pressure in a dose-dependent manner (Figure 4A). The pressor response to ICV hREN exhibited delayed onset and was of long duration, appearing in 5.7±1.0 minutes, reaching a peak in 24.3±4.5 minutes, and lasting 56.5±9.2 minutes. By comparison, the BP-elevating effect of ICV Ang II, initiated after 36±4 seconds, peaked quickly (4.1±0.5 minutes) and lasted only 15.4±1.0 minutes (P<0.0001 at all time points). The HR change by ICV hREN was variable and did not achieve statistical significance. Neither ICV nor intravenous injection of losartan (10 µg) on its own caused a significant change in mean arterial pressure or HR, but the pressor response to ICV hREN (30 ng) was prevented by pretreatment with ICV losartan. Interestingly, administration of a large dose of hREN (500 ng) intravenously did not raise BP in GFAP-hAGT, whereas that same dose caused a 35-mm Hg rise in mean arterial pressure in systemic hAGT mice, further confirming the lack of hAGT protein in the systemic circulation of GFAP-hAGT mice. In nontransgenic mice, neither ICV hREN (30 ng) nor intravenous hREN (500 ng) altered mean arterial pressure or HR (data not shown).

View larger version (18K): [in this window] [in a new window]   Figure 4. Pressor responses to acute infusion of purified hREN in GFAP-hAGT (A, n=6) and systemic hAGT (B, n=6) transgenic mice. *P<0.01 compared with ACSF infusion; **P<0.01 compared with saline injection. MAP indicates mean arterial pressure.

Finally, to examine the effect of chronic production of hAGT in astrocytes, we bred mice expressing hREN systemically with GFAP-hAGT mice. This hREN transgene is expressed in renal juxtaglomerular cells, in extrarenal tissues, and at a low level in the brain.^16,23 Double-transgenic mice containing this hREN transgene and the systemically expressed hAGT exhibit hypertension that can be partially corrected by centrally administered losartan.¹⁶ The hREN/GFAP-hAGT transgenic mice exhibited moderately but significantly elevated mean arterial pressure on each of 3 days of measurement (mm Hg, 48 hours after surgery, 136.5±4.3 versus 117.2±2.8, P=0.006; 72 hours after surgery, 134.8±5.3 versus 120.4±1.8, P=0.026; and 96 hours after surgery, 134.1±4.1 versus 120.8±2.6, P=0.010) (compiled in Figure 5A). HR was not significantly different between groups (hREN/GFAP-hAGT, 611±17 bpm, n=4; nontransgenic, 605±17, n=5). ICV injection of losartan caused a 15–mm Hg fall in mean arterial pressure in the double-transgenic mice (Figure 5B), reducing mean arterial pressure to baseline, but only a 5–mm Hg decrease in control mice. The HR change induced by ICV losartan was variable and not significant. Importantly, intravenous administration of the same amount of losartan injected ICV did not significantly affect mean arterial pressure (6±1 mm Hg for losartan versus 7±1 mm Hg for saline) or HR in either group.

View larger version (14K): [in this window] [in a new window]   Figure 5. Arterial pressure in hREN/GFAP-hAGT transgenic mice. A, Resting mean arterial pressure in hREN/GFAP-hAGT mice (black bar, n=4) and controls (white bar, n=5). *P<0.01 vs controls. B, BP response to ICV losartan (10 µg) in hREN/GFAP-hAGT mice (black bar, n=4) and controls (white bar, n=5). **P<0.05 vs controls. C, Drinking volume in hREN/GFAP-hAGT mice (black bar, n=15) and controls (white bar, n=17). D, Salt preference in hREN/GFAP-hAGT mice (black bar, n=14) and controls (white bar, n=17). *P<0.04 vs controls. MAP indicates mean arterial pressure.

We also measured drinking volume and salt preference comparing hREN/GFAP-hAGT with nontransgenic littermates. There was no significant difference in baseline water intake between groups (Figure 5C). In measuring salt preference, hREN/GFAP-hAGT mice drank (in mL/day per 10 g body weight) 1.3±0.2 of water and 0.8±0.1 of saline compared with 1.6±0.2 water and 0.6±0.1 saline in controls. When examined as a percentage of total volume intake, hREN/GFAP-hAGT mice exhibited a significantly greater preference for saline, drinking nearly 40% of their total volume as saline as compared with 26% in control mice (Figure 5D).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The major site of AGT production in mammals is the liver, where it is released into the systemic circulation. AGT expression is also evident in several extrahepatic tissues including the heart, kidney, adipose tissue, and brain. There is a longstanding hypothesis that AGT synthesis in extrahepatic tissues may provide the precursor to locally produced Ang II, which may serve important functions at or near its site of synthesis. However, it remains unclear whether AGT synthesis in these tissues generates Ang II, which is important for cardiovascular function. We recently demonstrated that localized overproduction of AGT in the kidney causes systemic hypertension, providing strong evidence favoring the importance of extrahepatic AGT in cardiovascular function.²⁷ In this report, we demonstrate that hREN/GFAP-hAGT double-transgenic mice are moderately hypertensive and have increased salt preference. The increased BP is due to stimulation of central AT₁s caused by elevated production of central Ang II. These data clearly demonstrate that AGT protein synthesized in the brain is the substrate for local Ang II synthesis, which can then influence arterial pressure and electrolyte homeostasis.

In the brain, AGT mRNA and protein is most abundant in neuroglia (astrocytes) in the hypothalamus and preoptic nuclei, and at lower levels in the mesencephalon, myelencephalon, and cerebellum.^17,28 In addition, AGT can be detected in neurons in the cerebral cortex, midbrain, and medulla, including areas important for cardiovascular function.¹⁷ Imboden et al¹⁹ reported that neurons containing both AGT and Ang II are present in hypothalamic paraventricular and supraoptic nuclei in rats. We reported that AGT-expressing neurons were identifiable in SFO, mesencephalic trigeminal nucleus, and parabrachial nucleus in hAGT transgenic mice.¹⁸ Despite strong evidence for the presence of AGT in the brain, its function remains poorly defined because of a lack of direct evidence demonstrating that Ang II is generated from AGT synthesized in the brain. We therefore generated a novel transgenic mouse model expressing hAGT under the control of the GFAP promoter to begin to unravel the functional significance of brain AGT. The GFAP promoter was chosen because it was shown to effectively restrict expression to astrocytes throughout the brain.²⁰ Use of the GFAP promoter eliminated neuronal hAGT expression in the parabrachial complex and mesencephalic nucleus, where hAGT is expressed when its endogenous promoter is used.¹⁸ Three individually derived transgenic lines exhibited hAGT mRNA expression in the brain, with variable expression in other tissues. In one line, transgene expression was observed primarily in the brain, and hAGT protein was expressed almost exclusively in astrocytes. Only in the SFO was AGT found to be colocalized with both GFAP- and MAP-2–staining cells. The colocalization of MAP-2 with hAGT is suggestive of neuronal localization of hAGT in the SFO. Neuronal expression of AGT has been reported previously in SFO, and both ACE and Ang II have been localized to neurons in this structure.^18,29 However, MAP-2 is present in modified astrocytes in the neurohypophysis, and these cells are sensitive to water deprivation.³⁰ It is possible that the cells containing both MAP-2 and hAGT in the SFO are not neurons but modified astrocytes.

ICV injection of hREN increased arterial pressure in GFAP-hAGT transgenic mice, and the pressor response to hREN was prevented by ICV pretreatment with the AT₁ antagonist losartan. The pressor response may be due to processing of brain or ventricular hAGT by hREN and the stimulation of AT₁s. The absence of a pressor response after intravenous hREN infusion argues that AGT synthesized in brain is not released into the circulation and supports our biochemical data showing that the level of plasma hAGT was not above baseline. This suggests that tissues outside the brain do not release significant amounts of AGT into the circulation, consistent with our previous examination of extrahepatic AGT in liver-specific hAGT knockout mice.³¹ Nevertheless, it is surprising that a large dose of hREN administered intravenously (15 times greater than administered ICV) did not exert any pressor effect, as we would have anticipated that plasma hREN would gain access to the circumventricular organs (CVOs). It remains unclear whether a sufficient dose of hREN (after intravenous administration) was present in the CVOs to mediate a pressor response.

It has been previously reported that in addition to processing by REN and ACE, AGT can be directly converted to Ang II by cathepsin G or tonin in the brain.^32,33 At least in the context of this model, we feel this to be unlikely as single GFAP-hAGT and single systemic hAGT mice (which exhibit hAGT expression in astrocytes) exhibit normal resting arterial pressure.²¹ Thus, unless the species specificity of the AGT cleavage reaction extends to non-REN enzymes, such as cathepsin and tonin, then cleavage of AGT by endogenous non-REN enzymes in the brain may not occur in mice.

Double-transgenic mice containing both the hREN and GFAP-hAGT transgenes are modestly hypertensive. ICV losartan lowered arterial pressure in the double-transgenic mice, whereas the same dose administered intravenously did not affect BP. AT₁s are widely distributed in the brain, including regions controlling cardiovascular function, hypothalamic nuclei such as the paraventricular, and brainstem nuclei such as the ventrolateral medulla and the nucleus of the solitary tract.^34–37 In addition to these intrinsic brain sites, AT₁s are densely localized at cardiovascular control centers located at the blood-brain interface surrounding the cerebro- ventricles, namely CVOs such as SFO, organum vasculosum of the lamina terminalis, and area postrema.³⁴ ACE is observed throughout the brain, including all of those areas containing AT₁s, and has been reported in cerebrospinal fluid.^38,39 In GFAP-hAGT transgenic mice, hAGT-containing cells are widely distributed in areas containing AT₁s. Therefore, our data are consistent with the notion that Ang II, produced via processing of hAGT by hREN and ACE, stimulates AT₁s in the brain to increase arterial pressure and alter sodium appetite. Indeed, the inhibition of the pressor response to hREN, and the lowering of BP in the hREN/GFAP-hAGT mice after ICV losartan strongly suggests that the increase in BP is mediated by an AT₁-dependent mechanism caused by chronic overproduction of Ang II in brain. Nevertheless, it is important to point out that the relative contribution of AT₁s in the CVOs versus parenchyma of the brain remains undefined and will require additional experiments. Moreover, it is known that central Ang II increases arterial pressure by enhancing sympathetic nerve activity and vasopressin secretion, causing antidiuresis, and increases drinking and salt intake.^1,40 Central infusion of Ang II and losartan also changes the sensitivity of the arterial baroreflex.^41,42 Each of these mechanisms will have to be examined to determine which is responsible for the elevation in BP in the hREN/GFAP-hAGT model.

In the brain, it is unclear where the REN-AGT cleavage reaction occurs. Are both proteins synthesized in the same cells or are the proteins secreted into the extracellular space or cerebroventricles? It is interesting to note that we recently reported that an altered form of REN mRNA is expressed in the brain of transgenic mice carrying a large hREN transgene encoded on a P1 artificial chromosome.⁴³ Importantly, the same form of REN mRNA was found in human fetal brain.⁴³ If translated, this mRNA would encode an intracellular (nonsecreted) form of the protein, suggesting the intriguing possibility of an intracellular pathway of angiotensin production in brain. We are currently testing this hypothesis using transgenic mice designed to specifically express only the predicted nonsecreted form of REN in brain.

Our data are in general agreement with experiments performed in rats in which direct administration of antisense oligonucleotides or RNAs directed against AGT into cerebroventricles lower BP.⁵ Similarly, lowered BP and diabetes insipidus were reported in transgenic rats containing an antisense RNA directed at AGT under the control of a glial-specific promoter.⁴⁴ Although a 90% decrease in brain AGT was reported, it is unclear whether AGT in both glial cells and neurons was targeted. Transgenic rats containing the antisense are more sensitive to the dipsogenic actions of central Ang II, and when microinjected into the rostral ventrolateral medulla were more sensitive to the pressor actions of Ang II.^45,46 AT₁ density was increased in structures inside the blood-brain barrier, but decreased in the CVOs, suggesting a functional role for AT₁s within the blood-brain barrier in the control of water intake and BP.

In conclusion, the results of both overexpression and underexpression experiments suggest that AGT produced in the brain may play an important role in central regulation of BP and electrolyte balance.

	Acknowledgments

 
This work was funded by NIH Grants HL58048, HL61446, and HL55006. S.M. is funded by a postdoctoral fellowship from Uehara Memorial Foundation in Japan. Transgenic mice were generated and maintained at the University of Iowa Transgenic Animal Facility, which is supported in part by the College of Medicine and the Diabetes and Endocrinology Research Center. We thank Norma Sinclair, Lucy Robbins, Patricia Lovell, Debbie Davis, and Xiaoji Zhang for their excellent technical assistance.

Received May 16, 2001; accepted June 21, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Wright JW, Harding JW. Regulatory role of brain angiotensins in the control of physiological and behavioral responses. Brain Res Brain Res Rev. . 1992; 17: 227–262.
2. Yongue BG, Angulo JA, McEwen BS, Myers MM. Brain and liver angiotensinogen messenger RNA in genetic hypertensive and normotensive rats. Hypertension. . 1991; 17: 485–491.
3. Israel A, Saavedra JM. High angiotensin converting enzyme (kininase II) activity in the cerebrospinal fluid of spontaneously hypertensive adult rats. J Hypertens. . 1987; 5: 355–357.
4. Gehlert DR, Speth RC, Wamsley JK. Quantitative autoradiography of angiotensin II receptors in the SHR brain. Peptides. . 1986; 7: 1021–1027.
5. Gyurko R, Wielbo D, Phillips MI. Antisense inhibition of AT₁ receptor mRNA and angiotensinogen mRNA in the brain of spontaneously hypertensive rats reduces hypertension of neurogenic origin. Regul Pept. . 1993; 49: 167–174.
6. Pochiero M, Nicoletta P, Losi E, Bianchi A, Caputi AP. Cardiovascular responses of conscious DOCA-salt hypertensive rats to acute intracerebroventricular and intravenous administration of captopril. Pharmacol Res Commun. . 1983; 15: 173–182.
7. Itaya Y, Suzuki H, Matsukawa S, Kondo K, Saruta T. Central renin-angiotensin system and the pathogenesis of DOCA-salt hypertension in rats. Am J Physiol Heart Circ. . 1986; 251: H261–H268.
8. Lark LA, Weyhenmeyer JA. The antihypertensive effect of acute intracerebroventricular administration of captopril in Dahl salt-sensitive rats. Eur J Pharmacol. . 1992; 222: 33–37.
9. Schelling P, Meyer D, Loos HE, Speck G, Phillips MI, Johnson AK, Ganten D. A micromethod for the measurement of renin in brain nuclei: its application in spontaneously hypertensive rats. Neuropharmacology. . 1982; 21: 455–463.
10. Printz MP, Healy DP. Changes in brain angiotensinogen during development of hypertension. Clin Exp Hypertens A. . 1983; 5: 1037–1046.
11. Salaymeh B, Lee MF, Weyhenmeyer JA. A comparison of CNS angiotensin II binding in the hypothalamus-thalamus-septum-midbrain region of developing spontaneously hypertensive and normotensive rats. J Hypertens. . 1986; 4: 617–622.
12. Stamler JF, Brody MJ, Phillips MI. The central and peripheral effects of Captopril (SQ 14225) on the arterial pressure of the spontaneously hypertensive rat. Brain Res. . 1980; 186: 499–503.
13. Phillips MI, Mann JF, Haebara H, Hoffman WE, Dietz R, Schelling P, Ganten D. Lowering of hypertension by central saralasin in the absence of plasma renin. Nature. . 1977; 270: 445–447.
14. Moriguchi A, Ferrario CM, Brosnihan KB, Ganten D, Morris M. Differential regulation of central vasopressin in transgenic rats harboring the mouse Ren-2 gene. Am J Physiol Regul Integr Comp Physiol. . 1994; 267: R786–R791.
15. Merrill DC, Thompson MW, Carney C, Schlager G, Robillard JE, Sigmund CD. Chronic hypertension and altered baroreflex responses in transgenic mice containing the human renin and human angiotensinogen genes. J Clin Invest. . 1996; 97: 1047–1055.
16. Davisson RL, Yang G, Beltz TG, Cassell MD, Johnson AK, Sigmund CD. The brain renin-angiotensin system contributes to the hypertension exhibited by mice containing both human renin and human angiotensinogen transgenes. Circ Res. . 1998; 83: 1047–1058.
17. Thomas WG, Sernia C. Immunocytochemical localization of angiotensinogen in the rat brain. Neuroscience. . 1988; 25: 319–341.
18. Yang G, Gray TS, Sigmund CD, Cassell MD. The angiotensinogen gene is expressed in both astrocytes and neurons in murine central nervous system. Brain Res. . 1999; 817: 123–131.
19. Imboden H, Harding JW, Hilgenfeldt U, Celio MR, Felix D. Localization of angiotensinogen in multiple cell types of rat brain. Brain Res. . 1987; 410: 74–77.
20. Brenner M, Kisseberth WC, Su Y, Besnard F, Messing A. GFAP promoter directs astrocyte-specific expression in transgenic mice. J Neurosci. . 1994; 14: 1030–1037.
21. Yang G, Merrill DC, Thompson MW, Robillard JE, Sigmund CD. Functional expression of the human angiotensinogen gene in transgenic mice. J Biol Chem. . 1994; 269: 32497–32502.
22. Sigmund CD. Manipulating genes to understand cardiovascular diseases: major approaches for introducing genes into cells. Hypertension. . 1993; 22: 599–607.
23. Sigmund CD, Jones CA, Kane CM, Wu C, Lang JA, Gross KW. Regulated tissue- and cell-specific expression of the human renin gene in transgenic mice. Circ Res. . 1992; 70: 1070–1079.
24. Hatae T, Takimoto E, Murakami K, Fukamizu A. Comparative studies on species-specific reactivity between renin and angiotensinogen. Mol Cell Biochem. . 1994; 131: 43–47.
25. Debus E, Weber K, Osborn M. Monoclonal antibodies specific for glial fibrillary acidic (GFA) protein and for each of the neurofilament triplet polypeptides. Differentiation. . 1983; 25: 193–203.
26. Caceres A, Binder LI, Payne MR, Bender P, Rebhun L, Steward O. Differential subcellular localization of tubulin and the microtubule-associated protein MAP2 in brain tissue as revealed by immunocytochemistry with monoclonal hybridoma antibodies. J Neurosci. . 1984; 4: 394–410.
27. Davisson RL, Ding Y, Stec DE, Catterall JF, Sigmund CD. Novel mechanism of hypertension revealed by cell-specific targeting of human angiotensinogen in transgenic mice. Physiol Genomics. . 1999; 1: 3–9.
28. Stornetta RL, Hawelu-Johnson CL, Guyenet PG, Lynch KR. Astrocytes synthesize angiotensinogen in brain. Science. . 1988; 242: 1444–1446.
29. Pickel VM, Chan J, Ganten D. Dual peroxidase and colloidal gold-labeling study of angiotensin converting enzyme and angiotensin-like immunoreactivity in the rat subfornical organ. J Neurosci. . 1986; 6: 2457–2469.
30. Matsunaga W, Miyata S, Kiyohara T. Redistribution of MAP2 immunoreactivity in the neurohypophysial astrocytes of adult rats during dehydration. Brain Res. . 1999; 829: 7–17.
31. Stec DE, Davisson RL, Haskell RE, Davidson BL, Sigmund CD. Efficient liver-specific deletion of a floxed human angiotensinogen transgene by adenoviral delivery of Cre recombinase in vivo. J Biol Chem. . 1999; 274: 21285–21290.
32. Ferrario CM, Santos RA, Brosnihan KB, Block CH, Schiavone MT, Khosla MC, Greene LJ. A hypothesis regarding the function of angiotensin peptides in the brain. Clin Exp Hypertens A. . 1988; 10: 107–121.
33. Ferrario CM, Schiavone MT. The renin angiotensin system: importance in physiology and pathology. Cleve Clin J Med. . 1989; 56: 439–446.
34. Kakar SS, Riel KK, Neill JD. Differential expression of angiotensin II receptor subtype mRNAs (AT-1A and AT-1B) in the brain. Biochem Biophys Res Commun. . 1992; 185: 688–692.
35. Johren O, Inagami T, Saavedra JM. AT1A, AT1B and AT2 angiotensin II receptor subtypes in rat brain. Neuroreport. . 1995; 6: 2549–2552.
36. Rowe BP, Grove KL, Saylor DL, Speth RC. Angiotensin II receptor subtypes in the rat brain. Eur J Pharmacology. . 1990; 186: 339–342.
37. Bunnemann B, Fuxe K, Ganten D. The brain renin-angiotensin system: localization and general significance. J Cardiovasc Pharmacol. . 1992; 19: S51–S62.
38. Mendelsohn FA, Chai SY, Dunbar M. In vitro autoradiographic localization of angiotensin-converting enzyme in rat brain using 125I-labelled MK351A. J Hypertens. . 1984; 2: S41–S44.
39. Unger T, Bader E, Ganten D, Lang RE, Rettig R. Brain angiotensin: pathways and pharmacology. Circulation. . 1988; 77 (suppl I): I-40–I-54.
40. Unger T, Rascher W, Schuster C, Pavlovitch R, Schomig A, Dietz R, Ganten D. Central blood pressure effects of substance P and angiotensin II: role of the sympathetic nervous system and vasopressin. Eur J Pharmacol. . 1981; 71: 33–42.
41. DiBona GF, Jones SY, Sawin LL. Effect of endogenous angiotensin II on renal nerve activity and its cardiac baroreflex regulation. J Am Soc Nephrol. . 1998; 9: 1983–1989.
42. Gaudet E, Godwin SJ, Head GA. Effects of central infusion of ANG II and losartan on the cardiac baroreflex in rabbits. Am J Physiol Heart Circ Physiol. . 2000; 278: H558–H566.
43. Sinn PL, Sigmund CD. Identification of three human renin mRNA isoforms resulting from alternative tissue-specific transcriptional initiation. Physiol Genomics. . 2000; 3: 25–31.
44. Schinke M, Baltatu O, Bohm M, Peters J, Rascher W, Bricca G, Lippoldt A, Ganten D, Bader M. Blood pressure reduction and diabetes insipidus in transgenic rats deficient in brain angiotensinogen. Proc Natl Acad Sci USA. . 1999; 96: 3975–3980.
45. Monti J, Schinke M, Bohm M, Ganten D, Bader M, Bricca G. Glial angiotensinogen regulates brain angiotensin II receptors in transgenic rats TGR(ASrAOGEN). Am J Physiol Regul Integr Comp Physiol. . 2001; 280: R233–R240.
46. Baltatu O, Fontes MA, Campagnole-Santos MJ, Caligiorni S, Ganten D, Santos RA, Bader M. Alterations of the renin-angiotensin system at the RVLM of transgenic rats with low brain angiotensinogen. Am J Physiol Regul Integr Comp Physiol. . 2001; 280: R428–R433.

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