Neuron-specific expression of human angiotensinogen in brain causes increased salt appetite
Satoshi Morimoto1,
Martin D. Cassell2 and
Curt D. Sigmund1
1 Departments of Internal Medicine and Physiology and Biophysics, the University of Iowa College of Medicine, Iowa City, Iowa 52242
2 Department of Anatomy and Cell Biology, the University of Iowa College of Medicine, Iowa City, Iowa 52242
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ABSTRACT
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The brain renin-angiotensin system (RAS) has an important role in the regulation of cardiovascular function. In the brain, angiotensinogen (AGT) is expressed mainly in astrocytes (glia) and in some neurons in regions controlling cardiovascular activities. Because of the inability to dissect the functional role of astrocyte- vs. neuron-derived AGT in vivo by pharmacological approaches, the exact role of neuron-derived AGT in the regulation of blood pressure (BP) and fluid and electrolyte balance remains unclear. Therefore, we generated a transgenic mouse model overexpressing human AGT under the control of a neuron-specific (synapsin I) promoter (SYN-hAGT). These mice exhibited high-level expression of human AGT mRNA in the brain, with lower expression in the kidney and heart. Human AGT was not detected in plasma, but in the brain it was expressed exclusively in neurons. Intracerebroventricular (30 ng) but not intravenous (500 ng) injection of purified human renin (hREN) caused a pressor response, which was prevented by intracerebroventricular preinjection of the angiotensin II type 1 receptor antagonist losartan, indicating an AT1 receptor-dependent functional role of neuron-derived AGT in the regulation of BP in response to exogenous REN. Double transgenic mice expressing both the hREN gene and SYN-hAGT transgene exhibited normal BP and water intake but had an increased preference for salt. These data suggest that neuronal AGT may play an important role in regulating salt intake and salt appetite.
blood pressure; salt preference; renin-angiotensin system; neuron; transgenic mouse
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INTRODUCTION
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THE PEPTIDE angiotensin II (ANG II) is the main circulating effector hormone of the renin-angiotensin system (RAS). Forty years ago, it was shown that ANG II is able to act not only on peripheral vascular structures but also on the central nervous system (CNS) to increase blood pressure (BP) (1). Since then, evidence has accumulated that central administration of ANG II has several actions including increasing sympathetic outflow, vasopressin release, water intake, and salt appetite (34). Existence of a local RAS in the CNS has been suggested, as all components of this system have been found in the brain (34). In addition, several lines of evidence point to the contribution of an overactive brain RAS to the hypertensive state and increased salt appetite under basal conditions and increased water intake after water deprivation in spontaneously hypertensive rats, a representative rat model of essential hypertension (3, 7, 11, 12, 16, 37).
However, it still remains unclear whether RAS components synthesized within the brain have an important role in the regulation of BP and fluid and electrolyte homeostasis. Therefore, to determine the functional role of brain-specific overexpression of angiotensinogen (AGT), we recently generated a transgenic mouse model expressing human AGT (hAGT) under the control of an astrocyte-specific (glial fibrillary acidic protein, GFAP) promoter (GFAP-hAGT) (23). These mice exhibit hAGT expression almost exclusively in astrocytes with exceptional neuronal expression in the subfornical organ in the brain. These mice are normotensive because of the strict species-specificity of the enzymatic reaction between AGT and renin (REN) (13). Double transgenic mice expressing both the GFAP-hAGT transgene and a human REN (hREN) genomic sequence spanning a region from
900 bp in the 5'-flanking region to 400 bp 3'-flanking in the region of the gene (900-hREN) exhibit chronic BP elevation and increased preference for salt (23, 27, 32). Accordingly, we concluded that AGT synthesized in the brain can be processed to ANG II which has an important role in the regulation of BP and electrolyte balance.
In the brain, AGT is expressed mainly in astrocytes (glia) and is also expressed in some neurons in important cardiovascular control regions (15, 31). However, the functional role of neuron-derived AGT remains unknown due to the inability to dissociate it from glial-derived AGT in vivo by classic pharmacological approaches. Accordingly, we generated a novel transgenic mouse model expressing hAGT under the control of a neuron-specific promoter, synapsin I (SYN) promoter, to examine the role of neuron-derived AGT in the regulation of BP and fluid and electrolyte balance.
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MATERIALS AND METHODS
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Generation of transgenic mice.
The transgene consists of a 2.2-kb fragment of rat SYN promoter fused to the hAGT gene. The rat SYN promoter was amplified by PCR from pBL4.3 Syn-CAT to contain EcoRV and BamHI ends (14). The modified SYN promoter was cloned into the EcoRV-BamHI sites of pBluescript II SK+/- to create pSYN-SK. The hAGT coding region was cloned from a previously described genomic clone as a BglII-to-NheI fragment into the unique BamHI and SpeI sites in pSYN-SK to form the plasmid pSYN-hAGT (36). The BglII site in hAGT resides within intron I, 70 bp upstream of exon II, and in this construct forms the 5'-untranslated region of the gene. Translation initiation of hAGT starts 4 bp into exon II. All cloning junctions were confirmed by sequence analysis. An EcoRV-to-NotI segment of pSYN-hAGT was excised and purified by agarose gel electrophoresis. Transgene DNA was microinjected into one-cell fertilized mouse embryos obtained from superovulated C57BL/6J x SJL/J (B6SJL F2) mice using standard procedures (26). All transgenic mice were heterozygous for the transgenes, and were maintained by breeding with B6SJL F1 mice. All mice received standard mouse chow (LM-485; Teklad Premier Laboratory Diets) and water ad libitum. Fifteen- to 20-wk-old mice were used for all experiments. Care of the mice used in the experiments met or exceeded the standards set forth by the National Institutes of Health in their Guidelines for the Care and Use of Laboratory Animals. All procedures were approved by the University Animal Care and Use Committee at the University of Iowa.
Analysis of nucleic acids.
To identify founder animals and transgenic offspring, PCR analysis was performed on tail genomic DNA using hAGT-specific primer sets as described (36). To confirm the results of PCR, Southern blot analysis was performed on genomic DNA isolated from spleen. Ten micrograms of DNA was digested with EcoRI and probed with a segment of hAGT intron II. An 11-kb band was diagnostic of the presence of the transgene.
Tissues were harvested and snap-frozen in liquid nitrogen, and RNA was purified using TriReagent (Molecular Research Center). RNase protection assays were performed using the RPA III kit (Ambion, Austin, TX). 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 mouse ß-actin were 518 and 245 nucleotides, respectively.
Plasma AGT assay.
Plasma mouse AGT (mAGT) and hAGT protein were determined on the basis of the strict species-specificity of the biochemical reaction between REN and AGT as previously described (13, 36). Approximately 0.5 ml of blood was collected from the chest cavity immediately after CO2 asphyxiation and placed in chilled tubes containing 2.5 µl of 0.5 M EDTA. The specimens were then immediately centrifuged at 12,000 rpm for 10 min at 4°C. The obtained plasma was immediately frozen and kept at -80°C until radioimmunoassay (RIA) was performed using the ANG I 125I-labeled RIA kit (NEN Life Science Products). To perform the assay, thawed samples were split into three tubes containing the reagent supplied by the kit and were incubated for 2 h at different conditions: tube A, incubated at 4°C; tube B, incubated at 37°C; tube C, incubated at 37°C with excess amount of purified hREN (gift of Drs. Walter Fischli and Klaus Lindpaintner at F. Hoffmann-La Roche, Basel, Switzerland). 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. mAGT is the value obtained from tube B subtracted by tube A, and hAGT is the value from tube C subtracted by tube B.
Immunohistochemistry.
Visualization of localization of hAGT protein in the brain was performed using immunohistochemistry and a rabbit polyclonal hAGT antibody (1:100 dilution, generous gift from Dr. Duane Tewksbury, Marshfield Medical Research Foundation) as described previously (23). Sections were also stained with mouse monoclonal antisera against either GFAP (glial marker, 1:1,000 dilution, Chemicon International) or neuron-specific nuclear protein (NeuN, neuronal marker, 1:500, Chemicon International) (6, 24).
BP measurement.
Measurement of arterial pressure (AP) and heart rate (HR) and intracerebroventricular (ICV) and intravenous (IV) infusion studies were performed in conscious, unrestrained mice as described previously (23). Briefly, baseline AP and HR were measured continuously for 1 h/day for 3 consecutive days starting 2 days after surgery. Effects of ICV (10, 30, and 100 ng) and IV (500 ng) injection of purified human REN were examined, each on separate days. After a 10-min pretreatment with 10 µg IV or ICV losartan (gift of Merck Research Laboratories, Rahway, NJ), mice were infused with ICV (30 ng) or IV (500 ng) hREN. Controls included ICV injection of artificial cerebrospinal fluid (ACSF) and IV injection of saline. All hemodynamic data were collected and analyzed on a computer using Chart v. 4.0.1 in Powerlab.
Measurement of drinking volume and salt preference.
The volume of water consumed and salt preference were measured as described previously (23); water intake was measured individually in metabolic cages with standard chow and tap water ad libitum daily for 3 days after acclimatization of the mice for 3 days. To determine salt preference, mice were fed salt-deficient chow and given 0.3 mol/l hypertonic saline and tap water in separate burettes ad libitum for 3 days after acclimatization of the mice for 3 days. Salt preference was calculated as a percentage determined by dividing the volume of saline consumed by the total volume of fluid consumed.
Urine analysis.
Urinary volume was measured by collecting urine individually in metabolic cages with standard chow ground with tap water (100 g chow/150 ml water) and tap water ad libitum daily for 3 days. The use of wet chow avoided contamination of the urine with food. Urinary osmolality was measured by using a vapor-pressure osmometer (Wescor). Urinary concentration of Na and K was determined by using a flame photometer (Instrumentation Laboratory).
Statistical analysis.
Data are means ± SE. Group comparisons were made with unpaired t-tests and confirmed with repeated-measures ANOVA followed by the Students modified t-test with Bonferroni correction. A value of P < 0.05 was considered statistically significant.
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RESULTS
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Transgenic mice were generated with a construct (SYN-hAGT) containing the rat SYN 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 (Fig. 1A). Four transgenic founders were identified by PCR analysis for SYN-hAGT (Fig. 1B). Only one founder (10479/3) was successfully bred to establish transgenic lines; and this line transmitted the transgene to
50% of both males and females, indicating insertion in autosomes (Fig. 1C).

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Fig. 1. Schematic map and identification of transgenic founders. A: a schematic map of the transgene. The synapsin I (SYN) promoter is shown as the hatched box, and exons of human angiotensinogen (hAGT) gene are shown in 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. B: PCR analysis on DNA isolated from tail biopsy. Transgenic founders and their transgenic offspring were identified by a diagnostic 539-bp band. C: Southern blot analysis on DNA isolated from spleen. Genomic and plasmid DNAs were digested with EcoRI and probed with the segment indicated in A. An 11-kb band was diagnostic of the transgene. M, 100-kb ladder marker; C, distilled water instead of DNA; N, genomic DNA of a known nontransgenic mouse; P, plasmid DNA containing the transgene.
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RNase protection assay was used to examine the tissue-specific expression of the transgene (Fig. 2). Line 10479/3 of the SYN-hAGT transgenic mice exhibited high-level expression of hAGT mRNA in the brain with lower expression in kidney and heart. No expression was observed in other tissues including peripheral nerves (mix of vagal and sciatic nerves). To examine the cell-specific expression of the transgene in the brain, double-labeling for hAGT and GFAP (a glial marker) or NeuN (a neuronal marker) was performed (Fig. 3). hAGT staining was observed in the cell bodies and processes of neurons in all regions of the brain as confirmed by costaining with NeuN, but not with GFAP (Table 1).

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Fig. 2. Expression of hAGT in SYN-hAGT transgenic mice: RNase protection assays of RNA from a male SYN-hAGT mouse. The hAGT and ß-actin transcripts are indicated on left. Top: +, liver of a transgenic mouse expressing hAGT from its endogenous promoter; -, liver of a known nontransgenic mouse; Br, brain; Li, 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; T, testes; P, peripheral nerve (mix of vagal and sciatic nerves). No expression was observed in uterus or ovaries in female SYN-hAGT transgenic mice.
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Fig. 3. Cell-specific expression of SYN-hAGT. Representative photomicrographs of double-labeling for hAGT and NeuN or hAGT and glial fibrillary acidic protein (GFAP) in cerebral cortex (top) and nucleus of the solitary tract (bottom). NeuN-positive (B, G) but GFAP-negative cells (D, I) were costained with hAGT (A, C, F, H) in SYN-hAGT mice. No hAGT staining was observed in nontransgenic mice (E). A and B, C and D, F and G, and H and I are pairs of the same sections.
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Given the presence of "ectopic" hAGT mRNA expression in extra-brain tissues, we were concerned that a significant amount of hAGT protein might be released into the systemic circulation. Therefore, we measured AGT in plasma. The fidelity and specificity of the assay was confirmed by the observation of basal levels of plasma mAGT but extremely high levels of hAGT in transgenic mice expressing hAGT under the control of its own promoter (termed systemic-hAGT transgenic mice) (Table 2). Plasma hAGT levels in SYN-hAGT transgenic mice were not elevated above baseline compared with nontransgenic littermates. Accordingly, it is unlikely that a significant amount of hAGT protein synthesized in extra-brain tissues is released into the systemic circulation in SYN-hAGT transgenic mice.
We next measured AP and HR in catheterized conscious unrestrained mice. Consistent with the species-specificity of the REN-AGT enzymatic reaction, there was no difference in baseline mean AP (MAP) or HR between SYN-hAGT (117 ± 8 mmHg, 606 ± 23 beats/min, n = 6) and nontransgenic littermates (118 ± 6 mmHg, 610 ± 19 beats/min, n = 8). In SYN-hAGT transgenic mice, ICV infusion of purified hREN increased MAP in a dose-dependent manner, which reached a plateau of 20 mmHg with a dose of 30 ng of hREN (Fig. 4A). The pressor response to 30 ng ICV hREN exhibited delayed onset and was of long duration; appearing in 6.4 ± 1.8 min, reaching a peak in 13.0 ± 3.0 min, and lasting 29.6 ± 5.5 min (n = 6). By comparison, the duration of the pressor response (in minutes) to ACSF, 10 ng hREN, and 100 ng hREN was 8.6 ± 3.4, 9.8 ± 2.0, and 29.9 ± 5.7, respectively. In contrast, the BP-elevating effect of ICV ANG II initiated after 1.1 ± 0.4 min peaked quickly (5.5 ± 1.2 min) and lasted only 14.3 ± 2.9 min (n = 6, P < 0.05 at all time points). The HR change by ICV hREN was variable and did not achieve statistical significance. Neither ICV nor IV injection of losartan (10 µg) on its own caused a significant change in MAP 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) IV did not raise BP in SYN-hAGT, further confirming the lack of hAGT protein in the systemic circulation of these mice (Fig. 4B). In nontransgenic littermates, neither ICV hREN (30 ng) nor IV hREN (500 ng) altered MAP or HR (data not shown). These data suggest that the pressor response to ICV hREN infusion in SYN-hAGT mice was due to AT1 receptor stimulation in the brain, caused by local overproduction of ANG II, most likely derived from the cleavage of hAGT released from neurons.

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Fig. 4. Pressor responses to human renin (hREN) infusion in SYN-hAGT transgenic mice. Pressor response to acute infusion of purified hREN in catheterized freely moving SYN-hAGT transgenic mice. A: responses to intracerebroventricuar infusion. B: responses to intravenous infusion. * P < 0.01 compared with artificial cerebrospinal fluid (ACSF) infusion; n = 6 for each experiment. MAP, mean arterial pressure.
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Finally, to examine the effect of chronic production of neuron-derived hAGT, we bred SYN-hAGT mice with mice expressing hREN. Two different hREN transgenic models were used. Both constructs contain the entire hREN genomic coding sequence but differ greatly in the amount of flanking DNA and therefore in their regulation. 900-hREN mice (line number 9) contain 900 bp of 5'-flanking and 400 bp of 3'-flanking DNA and exhibit juxtaglomerular (JG)-cell-specific expression of hREN in kidney, but ectopic expression in several extra-renal tissues (27). Expression of hREN is evident in the brain, but its expression in kidney is not tightly regulated (5, 32). Double transgenic mice containing this construct are markedly hypertensive (22). PAC-hREN mice (line 6407/1) contain 75 kb of 5'- and 70 kb of 3'-flanking DNA encoded on a P1 artificial chromosome. Expression is highly restricted to known REN-expressing tissues including brain, and in kidney is very tightly regulated in response to physiological cues (28, 29). Double transgenic mice containing this construct are only modestly hypertensive because of compensatory downregulation of hREN expression from the transgene (28).
Neither MAP (Fig. 5) nor HR (data not shown) were significantly different between 900-hREN/SYN-hAGT (R+/SA+), PAC-hREN/SYN-hAGT (P+/SA+), and nontransgenic littermate controls. We also measured baseline drinking volume, salt preference, and urinary volume in these mice (Table 3). There was no significant difference in baseline water intake between groups, in accordance with the data of urinary volume, osmolality, and excretion of Na and K, which were not different between groups. To measure salt preference, mice were given a choice of tap water or hypertonic saline in randomized burettes, and volumes of each were measured (Table 4). There was no difference in body weight among experimental and control mice. In addition, there was no significant difference in the intake of water in any group. In contrast, an increase in the intake of hypertonic saline was evident in 900-hREN/SYN-hAGT mice, and the preference for salt was significantly greater in both 900-hREN/SYN-hAGT and PAC-hREN/SYN-hAGT mice compared with controls. Salt-preference measurements were not made in single transgenic mice because of the proven species-specificity of ANG II production in the model.

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Fig. 5. Arterial pressure in 900-hREN/SYN-hAGT and PAC-hREN/SYN-hAGT transgenic mice. Resting MAP in 900-hREN/SYN-hAGT mice (R+ /SA+, crosshatched bar), PAC-hREN/SYN-hAGT mice (P+/SA+, black bar), and their littermates having no transgenes (open bar). The n value in each experiment is indicated at the base of each bar.
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DISCUSSION
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In the brain, AGT is most abundantly expressed in astrocytes; high expression is found in the hypothalamus and preoptic nuclei, and low expression is found in the mesencephalon, myelencephalon, and cerebellum (30, 31). In addition, AGT can be detected in neurons in the cerebral cortex, midbrain, and medulla, including areas important for cardiovascular function (31). Imboden et al. (15) 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 are observed in subfornical organ, mesencephalic trigeminal nucleus, and parabrachial nucleus in the transgenic mice expressing hAGT under the control of its endogenous promoter (35). In the present study, to determine the functional significance of neuron-derived AGT in the regulation of BP and fluid and electrolyte homeostasis, we generated transgenic mice overexpressing hAGT under the control of the SYN promoter. These mice exhibited high-level expression of hAGT mRNA in the brain and expressed hAGT protein exclusively in neurons.
To reconstitute a functional human RAS, a source of both hREN and hAGT is required. We previously reported the characterization of transgenic mice containing an hREN genomic sequence spanning a region from
900 bp in the 5'-flanking region to 400 bp 3'-flanking region of the gene (900-hREN) (27). While these mice exhibit JG-cell-specific expression of the transgene in kidney, they also exhibit high-level expression in a number of ectopic sites, and the transgene is poorly regulated in response to physiological cues (19, 32). We also generated transgenic mice expressing hREN from a P1 artificial chromosome (PAC-hREN). PAC160 contains the hREN coding region and a large segment of flanking DNA upstream and downstream of the gene (28). Expression of hREN in PAC160 mice was restricted to kidney, and only a very low-level expression was detected in ectopic sites. Moreover, transgene expression was tightly regulated in response to physiological cues. Interestingly, transcription of hREN mRNA in brain (but not kidney) of PAC160 mice occurs from an alternative transcription start site located 6.2 kb upstream of the classic promoter, thus encoding an alternative exon I that splices directly to exon II (29). This form of mRNA lacks the normal translation initiation codon in exon I, thus forcing the use of an in-frame ATG present in exon II. The predicted translation product lacks the signal peptide and the first 15 amino acids of the propeptide, suggesting the formation of an intracellular REN which was reported to have REN activity in vitro (20). Importantly, we reported that an identical form of REN mRNA has been found in RNA isolated from bona fide human fetal brain (29).
In the present study, the SYN-hAGT transgenic mice exhibited increased preference for salt when bred not only with 900-hREN mice but also with PAC-hREN mice. These data indicate that neuron-derived AGT protein can influence salt appetite. The salt preference in the R+/SA+ and P+/SA+ mice was similar in magnitude to that previously reported by us in R+/GA+ (38.5 ± 4.6%, n = 14, P < 0.05 vs. nontransgenic control) and P+/GA+ mice (45.8 ± 5.0, n = 12, P < 0.005 vs. nontransgenic control) which express hAGT under the control of the GFAP promoter (23). Microinjection of ANG II and an angiotensin-converting enzyme (ACE) inhibitor into regions near the organum vasculosum of the lamina terminalis (OVLT), respectively, increases and decreases salt appetite (8, 9). Lesioning of the most ventral part of the ventral median preoptic nucleus reduces salt appetite after treatments with chronic oral ACE inhibitor or sodium depletion (10). It is tempting to speculate that increased concentration of ANG II from local production and cleavage of neuron-derived AGT in these areas may be involved in the mechanism of increased salt preference in our models. Further experiments including microinjection and/or lesioning studies will be needed to address this directly.
AT1 receptors are widely distributed in the brain, including regions controlling cardiovascular function; e.g., hypothalamic nuclei such as the paraventricular nucleus, and brain stem nuclei such as the ventrolateral medulla and the nucleus of the solitary tract (2, 17, 18, 25). In addition to these intrinsic brain sites, AT1 receptors are highly localized to circumventricular organs such as the subfornical organ, OVLT, and area postrema (18). ACE is observed throughout the brain, including all these areas containing AT1 receptors, and has been reported in cerebrospinal fluid (21, 33). In SYN-hAGT transgenic mice, ICV injection of purified hREN caused a transient pressor response which was prevented by ICV preinjection of losartan. Therefore, it appears that neuron-derived AGT at least in circumventricular regions is capable of elevating BP through an AT1 receptor-dependent mechanism when a bolus of exogenous REN is directly injected into the lateral ventricle. The SYN-hAGT transgenic mice exhibited expression of hAGT protein throughout the brain both in circumventricular organs and within the blood-brain barrier. Consequently, we had expected that the SYN-hAGT transgenic mice would exhibit chronic BP elevation when combined with hREN transgenic mice due to continued overproduction of ANG II as occurred in double transgenic mice expressing 900-hREN and hAGT specifically targeted to glial cells (GFAP-hAGT) of the brain (23). Unexpectedly, however, both 900-hREN/SYN-hAGT and PAC-hREN/SYN-hAGT mice exhibited normal BP under baseline conditions. It remains unclear whether the absence of BP elevation is a manifestation of lower levels of hREN production in critical BP-regulating centers of the brain in the 900-hREN and PAC-hREN mice. Although this remains possible, it would not explain our previous observation that double transgenic 900-hREN/GFAP-hAGT mice exhibit increased BP (23). We also cannot rule out the possibility that higher levels of hAGT protein are released from glial cells in the GFAP-hAGT than from neurons in the SYN-hAGT mice.
Alternatively, we must acknowledge that whereas our GFAP-hAGT model accurately emulates the widespread expression of glial AGT in brain, the SYN-hAGT model described herein has a much more widespread expression pattern than normally exhibited by AGT in neurons. Indeed, this is an experimental limitation due to the absence of an appropriate molecular reagent (a promoter) that would allow us to specifically target only those neurons normally expressing AGT. However, as the neurons normally expressing AGT should be among those expressing the transgene, our data suggest the possibility that glial AGT and neuronal AGT may serve distinct functions. This may be analogous to the distinct functional roles served by the differentially expressed, but highly homologous AT1A and AT1B receptors in brain. We previously reported that the AT1A receptor signals the pressor action to central ANG II, whereas AT1B receptors signal the drinking response to central ANG II (4).
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Note Added in Proof
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Since the submission and acceptance of this paper, M. Bader and D. Ganten (Circ Res 90: 810, 2002) reported that the upstream transcription start site for renin in the brain is located 6.2 kb upstream of the classic renin promoter.
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ACKNOWLEDGMENTS
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We thank Norma Sinclair, Patricia Lovell, Brandon Campbell, Debbie Davis, and Xiaoji Zhang for excellent technical assistance. We thank M. W. Kilimann for the gift of the synapsin promoter.
The work described herein was funded by National Institutes of Health Grants HL-58048, HL-61446, and HL-55006. C. D. Sigmund was an Established Investigator of the American Heart Association. S. Morimoto is funded by Postdoctoral Fellowship from American Heart Association Heartland Affiliate. 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. DNA sequencing was performed at the University of Iowa DNA Core Facility.
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FOOTNOTES
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Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: C. D. Sigmund, Transgenic Animal Facility and Center on Functional Genomics of Hypertension, Depts. of Internal Medicine and Physiology and Biophysics, 3181 MEBRF, Univ. of Iowa College of Medicine, Iowa City, IA 52242 (E-mail: curt-sigmund{at}uiowa.edu).
10.1152/physiolgenomics.00007.2002.
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