Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation Research
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation Research. 2002;90:617-624
Published online before print February 7, 2002, doi: 10.1161/01.RES.0000012460.85923.F0
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow All Versions of this Article:
90/5/617    most recent
01.RES.0000012460.85923.F0v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lazartigues, E.
Right arrow Articles by Davisson, R. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lazartigues, E.
Right arrow Articles by Davisson, R. L.
Related Collections
Right arrow ACE/Angiotension receptors
Right arrow Other hypertension
Right arrow Genomics
Right arrow Hypertension - basic studies
Right arrow Autonomic, reflex, and neurohumoral control of circulation
(Circulation Research. 2002;90:617.)
© 2002 American Heart Association, Inc.

Integrative Physiology

Brain-Selective Overexpression of Angiotensin (AT1) Receptors Causes Enhanced Cardiovascular Sensitivity in Transgenic Mice

Eric Lazartigues, Shannon M. Dunlay, Angela K. Loihl, Puspha Sinnayah, Julie A. Lang, Joshua J. Espelund, Curt D. Sigmund, Robin L. Davisson

From the Department of Anatomy and Cell Biology (E.L., S.M.D., A.K.L., P.S., J.A.L., J.J.E., R.L.D.) and the Departments of Internal Medicine and Physiology and Biophysics (C.D.S.), the University of Iowa College of Medicine, Iowa City, Iowa.

Correspondence to Robin L. Davisson, PhD, Department of Anatomy and Cell Biology, 1-251 Bowen Science Bldg, University of Iowa College of Medicine, Iowa City, IA 52242. E-mail robin-davisson{at}uiowa.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
To examine the physiological importance of brain angiotensin II type 1 (AT1) receptors, we developed a novel transgenic mouse model with rat AT1a receptors targeted selectively to neurons of the central nervous system (CNS). A transgene consisting of 2.8 kb of the rat neuron-specific enolase (NSE) 5' flanking region fused to a cDNA encoding the full open-reading frame of the rat AT1a receptor was constructed and transgenic mice (NSE-AT1a) were generated. Two of six transgenic founder lines exhibited brain-selective expression of the transgene at either moderate or high levels. Immunohistochemistry revealed widespread distribution of AT1 receptors in neurons throughout the CNS. This neuron-targeted overexpression of AT1a receptors resulted in enhanced cardiovascular responsiveness to intracerebroventricular (ICV) angiotensin II (Ang II) injection but not to other central pressor agents, demonstrating functional overexpression of the transgene in NSE-AT1a mice. Interestingly, baseline blood pressure (BP) was not elevated in either transgenic line. However, blockade of central AT1 receptors with ICV losartan caused significant falls in basal BP in NSE-AT1a mice but had no effect in nontransgenic controls. These results suggest that whereas there is an enhanced contribution of central AT1 receptors to the maintenance of baseline BP in NSE-AT1a mice, particularly effective baroreflex buffering prevents hypertension in this model. Used both independently, and in conjunction with mice harboring gene-targeted deletions of AT1a receptors, this new model will permit quantitative and relevant investigations of the role of central AT1a receptors in cardiovascular homeostasis in health and disease.


Key Words: renin-angiotensin system • blood pressure • hypertension • central nervous system • neurons


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
In addition to its classical role as an endocrine system, evidence suggests that all components of the renin-angiotensin system (RAS) exist in individual tissues,1–3 resulting in the local synthesis, release, and action of angiotensin II (Ang II). The brain RAS4–6 is hypothesized to contribute to normal regulation of blood pressure (BP) and volume homeostasis through its effects on sympathetic outflow7 and vasopressin synthesis and release.8,9

Ang II type 1 (AT1) receptors are densely localized in several regions of the central nervous system (CNS) involved in cardiovascular regulation, including structures of the lamina terminalis of the forebrain,10–12 brain stem sites such as the nucleus of the solitary tract, ventrolateral medulla,6,13,14 and hypothalamic paraventricular and supraoptic nuclei.12 Several AT1-rich sites lack a blood-brain-barrier, eg, subfornical organ, organum vasculosum of the lamina terminalis, area postrema, and median preoptic nucleus, thus allowing direct interfacing with circulating Ang II. Brain AT1 receptor activation by both locally and systemically derived Ang II is thought to modulate normal processing of sensory input and neurohumoral outflow.15,16 Moreover, studies showing increased density or activity of AT1 receptors in these cardiovascular control regions in various genetic and experimental hypertension models have implicated central AT1 receptors as causal in the development and maintenance of hypertension.17–19

Classical physiological and pharmacological approaches have demonstrated the importance of the brain RAS in regulating cardiovascular homeostasis. Recently developed transgenic strategies now permit further dissection of the precise mechanisms by which this occurs. Using gene targeting, we recently examined the relative contribution of AT1 receptor subtypes, AT1a and AT1b, to central Ang II responses.20 Although these studies did clarify that Ang II–elicited BP increase can be selectively ascribed to the AT1a subtype, whereas AT1b receptors are necessary for the drinking response, they did not allow us to definitively establish the relative roles of central versus peripheral AT1 receptors because the genes were deleted globally.

The specific objective of this study was to develop a novel transgenic model with CNS-targeted AT1 receptors, with the overall goal of unraveling the relative contribution of brain versus peripheral vascular and extravascular AT1 receptors to BP regulation. We chose to overexpress the AT1a receptor based on our earlier findings that this subtype, but not AT1b, is critical in mediating central Ang II BP responses.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Generation of Transgenic Mice and Animal Husbandry
A fusion transgene (NSE-AT1a) consisting of 4 kb of the rat neuron-specific enolase (NSE) 5' flanking region (kind gift of J.G. Sutcliffe, Scripps Research Institute, La Jolla, Calif)21–23 and a cDNA encoding the full open-reading frame of the rat AT1a receptor (kind gift of K.E. Bernstein, Emory University, Atlanta, Ga)24 was constructed (Figure 1A). The transgene segment was obtained by digestion with SalI, separated by agarose gel electrophoresis, and recovered using the Qia-Quick gel purification kit (Qiagen). Transgenic mice were generated by microinjection into fertilized C57BL/6JXxSJL/J (B6SJL F2) mouse embryos, and positive founders (see next paragraph) were bred for study as described previously.25 All mice were fed standard mouse chow (LM-485; Teklad Premier Laboratory Diets) and water ad libitum. Care of the mice used in the experiments exceeded 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 Animal Care and Use Committee at the University of Iowa.



View larger version (50K):
[in this window]
[in a new window]
  		Figure 1. Structure of the NSE-AT1a transgene and detection of transgenic founders. A, Fusion construct contains elements essential to CNS-specific expression of the transgene, including the neuron-specific enolase (NSE) promoter, intron sequence to aid in processing/transport of the mRNA, and a polyA addition to target appropriate 3' end processing. The AT1a cDNA contains the full open-reading frame of the receptor. The probe used for Southern blot analysis is indicated. B, Representative Southern blot showing various NSE-AT1a transgenic founders, including the two lines used in physiological studies herein (6085-2 and 6084-2). Genomic DNA (10 µg) from tail biopsies was digested with SacI, separated on 0.8% agarose gel, and hybridized to a random primer-labeled probe (indicated in panel A). The probe detects a 4.4-kb band diagnostic of the transgene. Relative transgene copy number can be estimated because the probe used detects both mouse (M) AT1 receptor subtypes (a and b) as well as the exogenous rat (R) AT1 receptor. Positive transgenic founders are indicated by asterisks (*).

Analysis of Nucleic Acids
Genomic DNA was purified from tail biopsies and subjected to either Southern blot analysis for identification of positive founders (Figure 1A) or to PCR for identification of transgenic offspring, both described in detail previously.26,27 Transgene expression was analyzed using RNase protection assay (RPA) and a cDNA probe derived from a region of rat AT1a sequence with relatively lower nucleotide homology to the mouse sequence compared with the rest of the gene (184–586 relative to transcription start site). The probe was PCR-amplified from rat genomic DNA, cloned into pCRII (Invitrogen), and single-stranded antisense RNA was generated by an in vitro transcription reaction containing T7 polymerase and {alpha}-32P[UTP]. To obtain sufficient RNA from peripheral neural tissues, ganglia and nerve bundles were widely harvested and combined from cervical, para-aortic, and extremity sites. RPAs were performed using the HybSpeed RPA kit (Ambion) following the manufacturer’s instructions and as described previously.27 Quantification was performed using a PhosphorImager SI system (Molecular Dynamics) in conjunction with ImageQuant software (version 5.0). AT1a mRNA levels were normalized by dividing the AT1a signal by the mouse ß-actin signal.

Detection of AT1 Receptor Protein
Analysis of AT1 receptor levels in brain of transgenic and nontransgenic mice was performed by Western blot analysis as described previously28 using a rabbit polyclonal antibody (Santa Cruz, sc-579, 1:100 dilution). Intensities of the bands were analyzed by densitometry using ImageQuant software (see previous paragraph).

Immunofluorescent localization of AT1 receptor protein in brain of transgenic mice (6084-2 n=5; 6085-2 n=5) and nontransgenic controls (n=5) was performed using a rabbit polyclonal antibody targeted against the carboxyl terminus of the rat AT1a receptor (kindly provided by Dr M. McKinley, Howard Florey Institute, Parkville, Victoria, Australia). As described in detail previously,29 brains were perfused, postfixed overnight, and cryosectioned (30 µm, coronal). Free-floating sections were subjected to immunofluorescence staining using the tyramide signal amplification method as described previously30 and incubated with AT1 receptor antibody for 48 hours (1:5000 dilution). In addition to specificity controls, a dilution series for AT1 antibody was carried out (1:5000, 1:10 000, 1:20 000), and comparable AT1 immunofluorescence was observed at all concentrations. Finally, primary antibody was omitted in some incubations (transgenic and control) to check for autofluorescence. Confocal microscopy (Zeiss LSM 510) was used to analyze immunohistochemical staining (488 nm for visualization of fluorescein). Anatomical analyses were performed with the aid of a mouse brain atlas.31

Physiological Studies
NSE-AT1a transgenics from two different founder lines (6085-2, n=9; 6084-2, n=7) and nontransgenic controls (n=12) were surgically instrumented with intracerebroventricular (ICV) and left common carotid arterial cannulae for central administration of drugs and measurement of cardiovascular parameters, respectively, as described previously.27 In the first set of experiments, the dose-dependent effects of Ang II (50 to 200 ng; 200 nL) were determined. Doses were given randomly, only two consecutive injections were performed in each mouse, and at least 30 minutes was allowed between injections. Baroreflex sensitivity was estimated using linear regression analysis for the central Ang II–induced cardiovascular changes in heart rate and mean arterial pressure ({Delta}HR/{Delta}MAP) as described previously.27,32 To confirm AT1 receptor involvement, mice (6084-2, n=5; 6085-2, n=5; nontransgenics, n=5) were pretreated ICV with either saline or the AT1 receptor antagonist losartan (20 µg) followed 20 minutes later by ICV injection of the highest dose of Ang II (200 ng). Finally, to determine the selectivity of the cardiovascular responses to Ang II, we also examined the effects of another central pressor agent, the cholinergic agonist carbachol (50 ng ICV), in a separate group of transgenic (6085-2, n=4; 6084-2, n=4) and nontransgenic (n=4) mice.

Statistics
Data are expressed as mean±SEM. Data were analyzed by Student’s t test or ANOVA (following Bartlett’s test of homogeneity of variance) followed by Newman-Keuls correction for multiple comparisons. Statistical analyses were performed using Prism (version 3.0) software package (GraphPad Software Inc).


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Six NSE-AT1a transgenic founders (of 59 pups born) containing varying copies of the transgene were identified by the presence of a 4.4-kb hybridization band on Southern blots (Figure 1B). All six founders were successfully bred to establish transgenic lines, with transmission of the transgene to {approx}50% of male and female offspring. By using RPA, three separate transgenic lines with differing levels of transgene mRNA in brain were identified (6084-2, 6055-1, and 6085-2) (Figures 2A and 2B). Line 6084-2 exhibited the highest level of transgene expression, while lines 6085-2 and 6055-1 exhibited more moderate levels.



View larger version (48K):
[in this window]
[in a new window]
  		Figure 2. Brain-specific expression of the transgene. A, Representative RNase protection assay of rat AT1a transgene expression in 20 µg of brain RNA from each of the positive transgenic founder lines and nontransgenic controls (non-trans.). Selectivity of the AT1a probe for the transgene is indicated by the lack of bands in nontransgenics. B, Summary of transgenic AT1a mRNA levels (normalized to ß-actin signal) in different mice (n=3) from each line. Quantification was performed as described in Materials and Methods. Values are expressed as mean±SEM. C and D, Representative RNase protection assays of 20 µg of total tissue RNA isolated from NSE-AT1a (line 6085-2) transgenic and nontransgenic mice. The rat AT1a and ß-actin protected transcripts are indicated, as are the free probes. Rat kidney was used as a positive control. All panels are 24-hour exposures.

Focusing on these 3 lines, we next determined whether the transgene was indeed expressed selectively in brain. Total RNA was purified from a wide variety of tissues of both male and female transgenic and nontransgenic mice and was subjected to RPA using the same transgene-specific probe. A representative RPA of a positive NSE-AT1a (line 6085-2) mouse and a nontransgenic littermate is shown in Figures 2C and 2D. High-level expression of the transgene was detected in brain but in no other tissues examined in line 6085-2, and the lack of expression in brain or any other tissues in nontransgenic controls further confirmed selectivity of the probe. Extremely low-level expression of rat AT1a receptors was detected in the adrenal gland of 6085-2 mice, but interestingly, the transgene was undetectable in peripheral neural tissue. This observation was confirmed using RT-PCR (data not shown). The same brain-restricted (and very low-level adrenal) expression pattern was observed in line 6084-2, and no sex-related differences in expression level or pattern were observed in either line. In contrast, line 6055-1 exhibited widespread expression of the transgene with varying levels between males and females, thus further studies were restricted to lines 6084-2 and 6085-2.

To determine whether NSE-AT1a transgene expression was also reflected at the protein level, Western blot analysis was performed on brains from 6084-2 and 6085-2 mice and nontransgenic littermates. Because all available antibodies cross-react with both mouse and rat AT1 receptors, we compared brain AT1 protein levels in transgenic mice with baseline levels of the protein in nontransgenic controls. As shown in Figure 3, both 6084-2 and 6085-2 mice exhibit enhanced AT1 receptor protein ({approx}70-kDa band) in brain compared with nontransgenic animals, and the relative levels are correlated to mRNA levels for these two lines (see Figure 2).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 3. AT1 receptor levels are elevated in brain of NSE-AT1a mice. A, Representative Western blot analysis of 50-µg brain protein from NSE-AT1a transgenic (6084-2 and 6085-2) and nontransgenic mice, using a rabbit polyclonal antibody that cross-reacts with both mouse and rat AT1 receptors. Rat kidney and brain tissues were used as positive controls for rat AT1 receptor protein. The immunoreactive AT1 band ({approx}70 kDa) is marked by an arrow. B, Summary of AT1 receptor levels in different mice (n=5) from each transgenic line and nontransgenic controls. Quantification was performed as described in Materials and Methods. Data are expressed as mean±SEM. ***P<0.001, **P<0.01, and *P<0.05.

Distribution of transgene expression in brain of 6084-2 and 6085-2 mice was determined using immunohistochemistry and confocal microscopy. The AT1 receptor antibody used for these studies was specifically developed for use in brain immunocytochemistry at light and electron microscopic levels.33 Although the antibody does not distinguish between endogenous and transgenic (rat) AT1 receptors, a comparison of distribution and fluorescence intensity between transgenic and nontransgenic mice allowed us to qualitatively estimate relative levels of transgene expression. Widespread AT1 receptor immunofluorescence was detected throughout the brain of both transgenic lines, with a similar distribution pattern in each line (see online data supplement available at http://www.circresaha.org). This is in contrast to the rather discrete localization of AT1 receptors to a few areas of the brain, most notably components of the lamina terminalis of the forebrain, brain stem nuclei such as the nucleus of the solitary tract, ventrolateral medulla, and hypothalamic areas, in nontransgenic mice and in rats using this antibody.33 In addition to a more widespread distribution, abundance of AT1 receptors was markedly elevated in both transgenic lines compared with controls and generally higher in 6084-2 versus 6085-2 mice. This is illustrated in typical confocal images (Figures 4A through 4F) of two representative regions, the lateral hypothalamic area (left panels) and the supraoptic nucleus (right panels), where AT1 receptor staining is highly abundant in 6084-2 (Figures 4A and 4B), abundant in 6085-2 (Figures 4C and 4D) and either undetectable or at low levels in nontransgenic (Figures 4E and 4F) mice. Figures 4G and 4H) demonstrates low levels of background staining in sections from nontransgenics, indicating that AT1 immunostaining observed in transgenic animals is not due to nonspecific activity of the antibody. A complete lack of immunofluorescence in sections (transgenic and controls; data not shown) incubated without the primary antibody further confirms the specificity of staining.



View larger version (106K):
[in this window]
[in a new window]
  		Figure 4. NSE-AT1a mice exhibit enhanced AT1 receptor immunostaining in widespread regions of the brain. Confocal images of AT1 receptor immunoreactivity in representative brain regions, including the lateral hypothalamic area (left panels) and the supraoptic nucleus (right panels) from 6084-2 (A and B), 6085-2 (C and D), and nontransgenic (E and F) mice. Coronal sections (30 µm) were incubated with a primary antibody targeted to the carboxyl terminus of the AT1a receptor. Specificity of the AT1 antibody was confirmed in control experiments in which sections from nontransgenic (G) and 6084-2 transgenic (H) mice were coincubated in the same antibody bath. fx indicates fornix; LHt, lateral hypothalamus; opt, optic tract; PaLM, paraventricular hypothalamic lateral magnocellular nucleus; PaMM, paraventricular hypothalamic medial magnocellular nucleus; PaMP, paraventricular hypothalamic medial parvicellular nucleus; and SON, supraoptic nucleus.

Strict neuron-specific localization of the transgene in brain of NSE-AT1a mice is difficult to demonstrate because of the cross-reactivity of the AT1 antibody with both the transgene and endogenous AT1 receptors that may be localized to glial cells.34–36 However, we performed morphological analyses to confirm localization of AT1 immunostaining in neurons. As seen in representative examples in Figures 4A, 4C, and 4E), AT1 staining was observed in cell bodies and fibers with morphological characteristics typical of neurons. This neuronal pattern of staining was observed throughout the brain in both transgenic lines.

To demonstrate functional expression of exogenous AT1a receptors in NSE-AT1a transgenic mice, we examined the dose-dependent effects of central acute administration of Ang II on MAP and HR in conscious freely moving mice. Resting MAP and HR values before Ang II are summarized (see Table). The only significant alteration in basal cardiovascular values was a significant tachycardia observed in 6084-2 mice. There were no significant differences in baseline MAP between either transgenic line and nontransgenic littermates, indicating that brain-selective overexpression of AT1a receptors did not cause hypertension under conditions of basal RAS activation. However, both transgenic lines exhibited significantly enhanced cardiovascular sensitivity to central Ang II compared with controls (Figure 5). In nontransgenics, Ang II caused typical pressor and bradycardic responses observed previously in mice21 and in many other species,6,32,37 but the magnitude of these responses was dramatically increased in both transgenic lines (Figure 6). This is in contrast to the equivalent cardiovascular effects (P>0.05 for all comparisons) produced by the central pressor agent, carbachol,38 in nontransgenic ({Delta}MAP, 14±1 mm Hg; {Delta}HR, -61±11 bpm, n=4), 6084-2 ({Delta}MAP, 17±3 mm Hg; {Delta}HR, -74±21 bpm), and 6085-2 ({Delta}MAP, 13±1 mm Hg; {Delta}HR, -73±27 bpm, n=4) mice. In addition, central Ang II–induced baroreflex responses were significantly enhanced in both 6085-2 and 6084-2 (-3.62±0.46 and -6.3±0.47 bpm/mm Hg, respectively; P<0.05) compared with nontransgenics (-1.98±0.28 bpm/mm Hg). To confirm that the enhanced cardiovascular sensitivity to central Ang II in NSE-AT1a transgenics is indeed due to AT1 receptors and not some other mechanism, we examined the effects of losartan on Ang II–induced cardiovascular responses in separate animals (Figure 6B). Interestingly, losartan itself caused a significant decrease in baseline MAP in both transgenic lines (6085-2: {Delta}MAP -18±2 mm Hg; 6084-2: {Delta}MAP -11±2 mm Hg; P<0.01 versus control) but had no effect on MAP in nontransgenic mice (1±2 mm Hg). The Ang II–induced pressor and bradycardic responses were completely abolished by pretreatment with the AT1 antagonist in both transgenic lines and in controls, confirming that AT1 receptors are essential in mediating the Ang II–induced cardiovascular effects in all three groups.


View this table:
[in this window]
[in a new window]
  		Table 1. Baseline MAP and HR in NSE-AT1a Transgenic and Nontransgenic Controls



View larger version (28K):
[in this window]
[in a new window]
  		Figure 5. NSE-AT1a transgenics exhibit cardiovascular hypersensitivity to ICV Ang II. A typical example of the effects of Ang II (200 ng, 200 nL) administered ICV on cardiovascular responses in nontransgenic and NSE-AT1a (6085-2) transgenic mice. Mice were instrumented with ICV and carotid arterial cannulae and allowed 4 days of recovery. Experiments were performed in conscious, freely moving animals. Arrows indicate time of Ang II injection. PP indicates pulsatile pressure; MAP, mean arterial pressure; and HR, heart rate.



View larger version (34K):
[in this window]
[in a new window]
  		Figure 6. Cardiovascular hypersensitivity in NSE-AT1a mice is prevented by losartan. A, Summary of the effects of Ang II (50, 100, 200 ng; 200 nL) administered ICV on MAP and HR in nontransgenic (open bars, n=12), 6084-2 (shaded bars, n=7), and 6085-2 (solid bars, n=9) transgenic mice. B, Effects of Ang II (200 ng, ICV) on MAP and HR after pretreatment with either saline or the AT1 receptor antagonist losartan were examined in separate nontransgenic (n=5), 6084-2 (n=5), and 6085-2 (n=5) transgenic mice. Data are expressed as absolute change ({Delta}mean±SEM). ***P<0.001, **P<0.01, and *P<0.05 vs nontransgenics; {dagger}P<0.01 vs saline-treated.

In addition to markedly different effects of central Ang II on the magnitude of MAP and HR responses in NSE-AT1a and control mice, the time course of these cardiovascular changes differed between the groups. In control mice, the increase in BP and decrease in HR were similar in duration (4.42±0.27 and 6.43±1.2 minutes, respectively), indicative of activation of baroreflex mechanisms. In contrast, both transgenic lines exhibited much longer-lasting bradycardic responses relative to the duration of the pressor responses. Whereas the Ang II–induced increases in BP were of significantly longer duration in both transgenic lines compared with controls (6085-2: 7.47±0.39 minutes; 6084-2: 6.73±0.88 minutes; P<0.05), the bradycardic response persisted long after the BP had returned to baseline in these mice (6085-2: 22.71±3.2 minutes; 6084-2: 27.56±4.39 minutes; P<0.001 versus control).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Intrinsic tissue RAS, defined as tissue-based systems with the potential for local Ang II production and activity, have been hypothesized to play critical roles in cardiovascular regulation that are distinct from that mediated by the classical systemic RAS. However, the importance of this mechanism remains unresolved because of the difficulty in experimentally dissecting tissue and systemic RASs. The brain RAS is particularly controversial in this regard because of the direct interfacing of central Ang II receptors with circulating Ang II at brain regions lacking a blood-brain-barrier. As an initial step toward dissecting the relative physiological significance of brain versus peripheral vascular and extravascular Ang II receptors, we report herein the development of a novel transgenic mouse model with functional expression of exogenous AT1a receptors selectively in the brain. Our long-term goal is to utilize these mice to examine the physiological consequences of overexpression of AT1a receptors selectively in brain of normal mice (with intact endogenous AT1 receptors) and in mice otherwise devoid of AT1a receptors through breeding onto the AT1a-null background.27,39

Of six transgenic founders harboring the rat AT1a receptor under the control of the NSE promoter, two individually derived lines exhibited exogenous AT1a mRNA almost exclusively in the brain. No other tissues were positive for rat AT1a mRNA except low-level expression in the adrenal gland, which was not surprising given that medullary chromaffin cells stain positive for NSE and are of neuroectodermal origin.40 We chose the NSE promoter because NSE is a distinct isoform of the glycolytic enzyme enolase found only in terminally differentiated neurons and neuroendocrine cells, whereas a different enolase isozyme (non–neuronal enolase) is expressed in neuroblasts and all other non–neuronal cells of the CNS.40 Hence, NSE-driven transgenes are reported to be neuron-selective.21,22,41 Although we could not rule out the possibility of AT1a transgene expression in astrocytes or other non–neuronal cells, our morphological analyses suggest significantly enhanced AT1 receptor staining in neurons of NSE-AT1a mice.

Consistent with the finding that NSE is one of the most abundantly expressed and widely distributed proteins in the adult mammalian brain,42 NSE-AT1a transgenics exhibited AT1a immunostaining at high levels throughout most brain regions. The losartan-sensitive enhanced cardiovascular responsiveness of both transgenic lines to acute central Ang II is presumably the functional consequence of overexpression of AT1a receptors observed in cardiovascular control regions of these mice. These data provide important evidence for functional expression of the transgene. Interestingly, the AT1a expression level is not directly correlated with cardiovascular sensitivity in the two transgenic lines. This may be interesting with regard to differential regulation and/or neural circuitry associated with transgene expression in these two different transgenic lines. The mechanism of enhanced cardiovascular sensitivity to central Ang II in NSE-AT1a mice is the subject of ongoing investigations.

Overexpression of AT1a receptors in brain stem sites known to regulate sympathetic outflow43 and in hypothalamic nuclei involved in vasopressin synthesis and release44,45 raises the possibility that one or both of these mechanisms is involved in the hyperresponsiveness of NSE-AT1a mice. The relative effect of systemically versus centrally administered Ang II in the context of AT1a overexpression observed in circumventricular organs of these mice is also of considerable ongoing interest.

Interestingly, despite increased Ang II–elicited pressor and bradycardic responses, NSE-AT1a transgenics are not hypertensive at rest. At first, this may seem to contradict earlier held notions that increased levels of AT1 receptor protein, mRNA, or binding activity found in central cardiovascular control regions of various genetic and experimental models of hypertension were responsible for the increased BP in these models.17,18 However, the present data and earlier findings can be reconciled by postulating that increased brain AT1 receptor levels alone do not cause centrally mediated hypertension, but activation of the RAS, ie, increased production of Ang II, either centrally or peripherally, is also required. We are currently investigating this possibility by examining the effects of sustained RAS activation on baseline BP in NSE-AT1a mice using radiotelemetry.

Another explanation for normal resting BP in mice with functional overexpression of brain AT1a receptors is that baroreflex activity is particularly effective in maintaining BP homeostasis in the face of increased receptor numbers. This is supported by our finding that NSE-AT1a mice exhibit significant depressor responses to ICV losartan, whereas central blockade of AT1 receptors has no effect on baseline BP in control mice. Furthermore, centrally induced baroreflex responses seem enhanced in both transgenic lines compared with controls as suggested by our baroreflex estimation. Taken together, these results suggest that although there is an enhanced contribution of central AT1 receptors to the maintenance of baseline BP in NSE-AT1a transgenic mice, effective baroreflex buffering prevents hypertension in this model. However, a complete understanding of baroreflex function in this model will require direct investigation of HR changes elicited over a range of centrally and peripherally induced changes in BP.32

Analysis of baroreflex function in NSE-AT1a transgenics is also of interest given the persistent bradycardia observed in these mice long after return of BP to baseline on ICV Ang II. Indeed, the temporal profile of this response suggests that at least a portion of the Ang II–induced bradycardia is not directly the result of baroreflex activity. One possible mechanism may involve the well-known sympathoinhibitory effects of vasopressin via actions at the area postrema.46 It is plausible that overexpression of AT1a receptors in NSE-AT1a mice leads to enhanced Ang II–induced release of vasopressin, which in turn acts on area postrema neurons that interact with other hindbrain sites known to mediate sympathoinhibition. Alternatively, the long-lasting Ang II–elicited bradycardia may involve direct activation of areas involved in HR control, eg, the nucleus ambiguus, ventrolateral medulla, and nucleus tractus solitarius. Indeed, these areas exhibit high-level overexpression of AT1 receptors in the NSE-AT1a transgenic mice.

The functional consequences of ectopic and noncardiovascular region–localized expression of AT1a receptors in brain of NSE-AT1a transgenic mice remain to be determined. Central AT1 receptors have been implicated in a wide variety of responses that are apparently independent of BP regulation, including anxiety,47 exploratory behavior,48 dopamine formation and release,49 and development, plasticity, and maturation of the brain.50 This, coupled with the widespread distribution of AT1 receptors in brain of NSE-AT1a transgenics, makes these mice potentially useful models for exploring other noncardiovascular-related functions of AT1 receptors.

In summary, we have developed a useful transgenic model that exhibits functional overexpression of AT1a receptors selectively in the brain. Used both independently, and in conjunction with mice harboring gene-targeted deletions of AT1a receptors, this new model will permit quantitative and relevant investigations of the role of central AT1a receptors in cardiovascular homeostasis in health and disease.


*    Acknowledgments
 
The work described herein was funded by grants from the NIH (HL14388-30 and HL63887-02). E. Lazartigues is funded by a postdoctoral fellowship (20572Z) from the American Heart Association. The authors would like to thank M. Cassell (University of Iowa) for assistance with neuroanatomical analyses and valuable discussions and Merck and DuPont Laboratories for providing losartan. 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.

Received August 30, 2001; revision received January 23, 2002; accepted January 25, 2002.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Phillips MI, Speakman EA, Kimura B. Levels of angiotensin and molecular biology of the tissue renin angiotensin systems. Regul Pept. 1993; 43: 1–20.[CrossRef][Medline] [Order article via Infotrieve]
  2. Lee MA, Bohm M, Paul M, Ganten D. Tissue renin-angiotensin systems: their role in cardiovascular disease. Circulation. 1993; 87: 7–13.
  3. Ganong WF. Origin of the angiotensin II secreted by cells. Proc Soc Exp Biol Med. 1994; 205: 213–219.[Abstract]
  4. Lippoldt A, Paul M, Fuxe K, Ganten D. The brain renin-angiotensin system: molecular mechanisms of cell to cell interactions. Clin Exp Hypertens. 1995; 17: 251–266.[Medline] [Order article via Infotrieve]
  5. Muratani H, Teruya H, Sesoko S, Takishita S, Fukiyama K. Brain angiotensin and circulatory control. Clin Exp Pharmacol Physiol. 1996; 23: 458–464.[Medline] [Order article via Infotrieve]
  6. Phillips MI, Sumners C. Angiotensin II in central nervous system physiology. Regul Pept. 1998; 78: 1–11.[CrossRef][Medline] [Order article via Infotrieve]
  7. Scroop GC, Lowe RD. Efferent pathways of the cardiovascular response to vertebral artery infusions of angiotensin in the dog. Clin Sci (Colch). 1969; 37: 605–619.[Medline] [Order article via Infotrieve]
  8. 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.[CrossRef][Medline] [Order article via Infotrieve]
  9. Paul M, Bader M, Steckelings UM, Voigtlander T, Ganten D. The renin-angiotensin system in the brain: localization and functional significance. Arzneimittelforschung. 1993; 43: 207–213.[Medline] [Order article via Infotrieve]
  10. Saavedra JM, Correa FM, Kurihara M, Shigematsu K. Increased number of angiotensin II receptors in the subfornical organ of spontaneously hypertensive rats. J Hypertens. 1986; 4: S27–S30.
  11. McKinley MJ, Badoer E, Vivas L, Oldfield BJ. Comparison of c-fos expression in the lamina terminalis of conscious rats after intravenous or intracerebroventricular angiotensin. Brain Res Bull. 1995; 37: 131–137.[CrossRef][Medline] [Order article via Infotrieve]
  12. Lenkei Z, Corvol P, Llorens-Cortes C. The angiotensin receptor subtype AT1A predominates in rat forebrain areas involved in blood pressure, body fluid homeostasis and neuroendocrine control. Brain Res. 1995; 30: 53–60.[Medline] [Order article via Infotrieve]
  13. Muratani H, Ferrario CM, Averill DB. Ventrolateral medulla in spontaneously hypertensive rats: role of angiotensin II. Am J Physiol. 1993; 264: R388–R395.[Medline] [Order article via Infotrieve]
  14. Lind RW, Swanson LW, Ganten D. Organization of angiotensin II immunoreactive cells and fibers in the rat central nervous system: an immunohistochemical study. Neuroendocrinology. 1985; 40: 2–24.[Medline] [Order article via Infotrieve]
  15. Keil LC, Summy-Long J, Severs WB. Release of vasopressin by angiotensin II. Endocrinology. 1975; 96: 1063–1065.[Abstract]
  16. Ito S, Sved AF. Blockade of angiotensin receptors in rat rostral ventrolateral medulla removes excitatory vasomotor tone. Am J Physiol. 1996; 270: R1317–R1323.[Medline] [Order article via Infotrieve]
  17. Gutkind JS, Kurihara M, Saavedra JM. Increased angiotensin II receptors in brain nuclei of DOCA-salt hypertensive rats. Am J Physiol. 1988; 255: H646–H650.[Medline] [Order article via Infotrieve]
  18. Gutkind JS, Kurihara M, Castren E, Saavedra JM. Increased concentration of angiotensin II binding sites in selected brain areas of spontaneously hypertensive rats. J Hypertens. 1988; 6: 79–84.[Medline] [Order article via Infotrieve]
  19. Nazarali AJ, Gutkind JS, Correa FM, Saavedra JM. Enalapril decreases angiotensin II receptors in subfornical organ of SHR. Am J Physiol. 1989; 256: H1609–H1614.[Medline] [Order article via Infotrieve]
  20. Davisson RL, Oliverio MI, Coffman TM, Sigmund CD. Divergent functions of angiotensin II receptor isoforms in the brain. J Clin Invest. 2000; 106: 103–106.[Medline] [Order article via Infotrieve]
  21. Forss-Petter S, Danielson PA, Catsicas S, Battenberg E, Price J, Nerenberg M, Sutcliffe JG. Transgenic mice expressing ß-galactosidase in mature neurons under neuron-specific enolase promoter control. Neuron. 1990; 5: 187–197.[CrossRef][Medline] [Order article via Infotrieve]
  22. Race RE, Priola SA, Bessen RA, Ernst D, Dockter J, Rall GF, Mucke L, Chesebro B, Oldstone MBA. Neuron-specific expression of a hamster prion protein minigene in transgenic mice induces susceptibility to hamster scrapie agent. Neuron. 1995; 15: 1183–1191.[CrossRef][Medline] [Order article via Infotrieve]
  23. Mucke L, Masliah E, Johnson WB, Ruppe MD, Alford M, Rockenstein EM, Forss-Petter S, Pietropaolo M, Mallory M, Abraham CR. Synaptotrophic effects of human amyloid ß protein precursors in the cortex of transgenic mice. Brain Res. 1994; 666: 151–167.[CrossRef][Medline] [Order article via Infotrieve]
  24. Murphy TJ, Alexander RW, Griendling KK, Runge MS, Bernstein KE. Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor. Nature. 1991; 351: 233–236.[CrossRef][Medline] [Order article via Infotrieve]
  25. Sigmund CD. Major approaches for generating and analyzing transgenic mice: an overview. Hypertension. 1993; 22: 599–607.[Abstract/Free Full Text]
  26. Davisson RL, Kim HS, Krege JH, Lager DJ, Smithies O, Sigmund CD. Complementation of reduced survival, hypotension, and renal abnormalities in angiotensinogen-deficient mice by the human renin and human angiotensinogen genes. J Clin Invest. 1997; 99: 1258–1264.[Medline] [Order article via Infotrieve]
  27. Davisson RL, Yang GY, Beltz TG, Cassell MD, Johnson AK, Sigmund CD. The brain renin-angiotensin system contributes to the hypertension in mice containing both the human renin and human angiotensinogen transgenes. Circ Res. 1998; 83: 1047–1058.[Abstract/Free Full Text]
  28. 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.[Abstract/Free Full Text]
  29. 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.[CrossRef][Medline] [Order article via Infotrieve]
  30. Adams JC. Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. J Histochem Cytochem. 1992; 40: 1457–1463.[Abstract]
  31. Franklin KBJ, Paxinos G. The Mouse Brain in Stereotaxic Coordinates. San Diego, Calif: Academic Press, Inc; 1997.
  32. Barron KW, Trapani AJ, Gordon FJ, Brody MJ. Baroreceptor denervation profoundly enhances cardiovascular responses to central angiotensin II. Am J Physiol. 1989; 257: H314–H323.[Medline] [Order article via Infotrieve]
  33. Giles ME, Fernley RT, Nakamura Y, Moeller I, Aldred P, Ferraro T, Penschow JD, McKinley MJ, Oldfield BJ. Characterization of a specific antibody to the rat angiotensin II AT1 receptor. J Histochem Cytochem. 1999; 47: 507–516.[Abstract/Free Full Text]
  34. Leung KH, Chang RS, Lotti VJ, Roscoe WA, Smith RD, Timmermans PB, Chiu AT. AT1 receptors mediate the release of prostaglandins in porcine smooth muscle cells and rat astrocytes. Am J Hypertens. 1992; 5: 648–656.[Medline] [Order article via Infotrieve]
  35. Rydzewski B, Zelezna B, Tang W, Sumners C, Raizada MK. Angiotensin II stimulation of plasminogen activator inhibitor-1 gene expression in astroglial cells from the brain. Endocrinology. 1992; 130: 1255–1262.[Abstract]
  36. Tallant EA, Diz DI, Ferrario CM. Identification of AT1 receptors on cultured astrocytes. Adv Exp Med Biol. 1996; 396: 121–129.[Medline] [Order article via Infotrieve]
  37. Bunnemann B, Fuxe K, Ganten D. The renin-angiotensin system in the brain: an update. Regul Pept. 1993; 46: 487–509.[CrossRef][Medline] [Order article via Infotrieve]
  38. Buccafusco JJ. The role of central cholinergic neurons in the regulation of blood pressure and in experimental hypertension. Physiol Rev. 1996; 48: 179–211.
  39. Ito M, Oliverio MI, Mannon PJ, Best CF, Maeda N, Smithies O, Coffman TM. Regulation of blood pressure by the type 1A angiotensin II receptor gene. Proc Natl Acad Sci U S A. 1995; 92: 3521–3525.[Abstract/Free Full Text]
  40. Schmechel D, Marangos PJ, Brightman M. Neuron-specific enolase is a molecular marker for peripheral and central neuroendocrine cells. Nature. 1978; 276: 834–836.[CrossRef][Medline] [Order article via Infotrieve]
  41. Fattori E, Lazzaro D, Musiani P, Modesti A, Alonzi T, Ciliberto G. IL-6 expression in neurons of transgenic mice causes reactive astrocytosis and increase in ramified microglial cells but no neuronal damage. Eur J Neurosci. 1995; 7: 2441–2449.[CrossRef][Medline] [Order article via Infotrieve]
  42. Sakimura K, Araki K, Kushiya E, Takahashi Y. Partial purification and characterization of messenger RNA coding 14-3-2 protein from rat brain. J Neurochem. 1982; 39: 366–370.[Medline] [Order article via Infotrieve]
  43. Tagawa T, Dampney AL. AT1 receptors mediate excitatory inputs to rostral ventrolateral medulla pressor neurons from hypothalamus. Hypertension. 1999; 34: 1301–1307.[Abstract/Free Full Text]
  44. Toney GM, Porter JP. Functional role of brain AT1 and AT2 receptors in the central angiotensin II pressor response. Brain Res. 1993; 603: 57–63.[CrossRef][Medline] [Order article via Infotrieve]
  45. Meng H, Wielbo D, Gyurko R, Phillips MI. Antisense oligonucleotide to AT1 receptor mRNA inhibits central angiotensin induced thirst and vasopressin. Regul Pept. 1994; 54: 543–551.[CrossRef][Medline] [Order article via Infotrieve]
  46. Bishop VS, Hay M. Involvement of the area postrema in the regulation of sympathetic outflow to the cardiovascular system. Front Neuroendocrinol. 1993; 14: 57–75.[CrossRef][Medline] [Order article via Infotrieve]
  47. Castren E, Saavedra JM. Repeated stress increases the density of angiotensin II binding sites in rat paraventricular nucleus and subfornical organ. Endocrinology. 1988; 122: 370–372.[Abstract]
  48. Saavedra JM. Emerging features of brain angiotensin receptors. Regul Pept. 1999; 85: 31–45.[CrossRef][Medline] [Order article via Infotrieve]
  49. Johren O, Sanvitto GL, Egidy G, Saavedra JM. Angiotensin II AT1A receptor mRNA expression is induced by estrogen-progesterone in dopaminergic neurons of the female rat arcuate nucleus. J Neurosci. 1997; 17: 8283–8292.[Abstract/Free Full Text]
  50. Johren O, Saavedra JM. Expression of AT1A and AT1B angiotensin II receptor messenger RNA in forebrain of 2-wk-old rats. Am J Physiol. 1996; 271: E104–E112.[Medline] [Order article via Infotrieve]



This article has been cited by other articles:


Home page
 PhysiologyHome page
J. L. Grobe, D. Xu, and C. D. Sigmund
An Intracellular Renin-Angiotensin System in Neurons: Fact, Hypothesis, or Fantasy
Physiology, August 1, 2008; 23(4): 187 - 193.
[Abstract] [Full Text] [PDF]

Home page
 Exp PhysiolHome page
Z. Lin, Y. Chen, W. Zhang, A. F. Chen, S. Lin, and M. Morris
RNA interference shows interactions between mouse brainstem angiotensin AT1 receptors and angiotensin-converting enzyme 2
Exp Physiol, May 1, 2008; 93(5): 676 - 684.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
Y. Feng, X. Yue, H. Xia, S. M. Bindom, P. J. Hickman, C. M. Filipeanu, G. Wu, and E. Lazartigues
Angiotensin-Converting Enzyme 2 Overexpression in the Subfornical Organ Prevents the Angiotensin II-Mediated Pressor and Drinking Responses and Is Associated With Angiotensin II Type 1 Receptor Downregulation
Circ. Res., March 28, 2008; 102(6): 729 - 736.
[Abstract] [Full Text] [PDF]

Home page
 J. Appl. Physiol.Home page
T. M. Mousa, D. Liu, K. G. Cornish, and I. H. Zucker
Exercise training enhances baroreflex sensitivity by an angiotensin II-dependent mechanism in chronic heart failure
J Appl Physiol, March 1, 2008; 104(3): 616 - 624.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
M. F. Doobay, L. S. Talman, T. D. Obr, X. Tian, R. L. Davisson, and E. Lazartigues
Differential expression of neuronal ACE2 in transgenic mice with overexpression of the brain renin-angiotensin system
Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R373 - R381.
[Abstract] [Full Text] [PDF]

Home page
 Eur Heart JHome page
P. M. Kistler, P. Sanders, M. Dodic, S. J. Spence, C. S. Samuel, C. Zhao, J. A. Charles, G. A. Edwards, and J. M. Kalman
Atrial electrical and structural abnormalities in an ovine model of chronic blood pressure elevation after prenatal corticosteroid exposure: implications for development of atrial fibrillation
Eur. Heart J., December 2, 2006; 27(24): 3045 - 3056.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
I. H. Zucker
Novel Mechanisms of Sympathetic Regulation in Chronic Heart Failure
Hypertension, December 1, 2006; 48(6): 1005 - 1011.
[Full Text] [PDF]

Home page
 HypertensionHome page
R. A.L. Dampney
Angiotensin Type 1A Receptors on Glial Cells in Rostral Ventrolateral Medulla and Hypertension
Hypertension, June 1, 2006; 47(6): 1052 - 1053.
[Full Text] [PDF]

Home page
 HypertensionHome page
Y. Chen, H. Chen, A. Hoffmann, D. R. Cool, D. I. Diz, M. C. Chappell, A. Chen, and M. Morris
Adenovirus-Mediated Small-Interference RNA for In Vivo Silencing of Angiotensin AT1a Receptors in Mouse Brain
Hypertension, February 1, 2006; 47(2): 230 - 237.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
I. H. Zucker and L. Gao
The Regulation of Sympathetic Nerve Activity by Angiotensin II Involves Reactive Oxygen Species and MAPK
Circ. Res., October 14, 2005; 97(8): 737 - 739.
[Full Text] [PDF]

Home page
 Exp PhysiolHome page
Y. Chen, H. Chen, and M. Morris
Enhanced osmotic responsiveness in angiotensin AT1a receptor deficient mice: evidence for a role for AT1b receptors
Exp Physiol, September 1, 2005; 90(5): 739 - 746.
[Abstract] [Full Text] [PDF]

Home page