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(Circulation Research. 2004;95:523.)
© 2004 American Heart Association, Inc.

Integrative Physiology

Renovascular Hypertension in Mice With Brain-Selective Overexpression of AT_1a Receptors Is Buffered by Increased Nitric Oxide Production in the Periphery

Eric Lazartigues^*, Andrew J. Lawrence^*, Fred S. Lamb, Robin L. Davisson

From the Departments of Anatomy & Cell Biology (E.L., R.L.D.) and Pediatrics (F.S.L.), Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City; Howard Florey Institute (A.J.L.), University of Melbourne, Victoria, Australia.

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

	Abstract

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We recently established a new transgenic mouse model with brain-restricted overexpression of angiotensin II (Ang II) type 1a receptors (NSE-AT_1a) to unmask the role of the brain renin-angiotensin system in hypertension. To test the hypothesis that these mice would exhibit an early exacerbation of renovascular hypertension, NSE-AT_1a and nontransgenic (NT) mice underwent 2-kidney-1-clip (2K1C) surgery and blood pressure (BP) and heart rate (HR) were recorded continuously by radiotelemetry for 28 days. Results show that NSE-AT_1a mice developed hypertension much more rapidly than NT, and this was not attributable to genotype-related differences in plasma or brain Ang II levels. A marked bradycardia accompanied this early increase in BP in NSE-AT_1a mice, as did a substantial cardiovascular region-specific downregulation of AT₁ receptor binding in brain but not in kidney. As BP reached its plateau in NT (1 week after clip), hypertension began to abate and eventually stabilized at significantly lower levels in NSE-AT_1a mice despite marked elevations in Ang II levels in brain stem and hypothalamus at these later time points. This hypertension reversal and the bradycardia were prevented by chronic infusion of the nitric oxide synthase (NOS) blocker L-NAME. These data, along with evidence showing enhanced NOS expression and NO-mediated compensatory responses in 2K1C NSE-AT_1a peripheral arteries during this later phase, suggest that activation of endogenous NO systems plays an important role in buffering the maintenance of hypertension caused by overexpression of AT_1a receptors in the brain.

Key Words: renovascular hypertension • nitric oxide • brain • transgenic mice

	Introduction

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Overactivity of the renin-angiotensin system (RAS) is postulated to participate in various forms of hypertension development. However, therapeutic effects of angiotensin-converting enzyme inhibitors or AT₁ receptor antagonists have been difficult to explain when angiotensin II (Ang II) plasma levels are not elevated.¹ Because of this observation, involvement of individual tissue RAS located in heart, kidneys, adrenals, vessels, and brain has been suggested in the pathogenesis of hypertension.²

There is now considerable evidence that the brain RAS is important in the development and maintenance of several experimental and genetic forms of hypertension.^3–5 However, these studies have not been conclusive because of the difficulty in separating peripheral and central systems. Traditional techniques such as lesioning⁶ or the use of antagonists⁷ cannot fully address the relative roles of peripheral and brain RAS in long-term regulation of blood pressure (BP) because of their lack of selectivity, short-term actions, and the complexity of the system. For example, in the Goldblatt 2-kidney-1-clip (2K1C) model, involvement of the brain RAS in either the early phase of 2K1C hypertension, when plasma Ang II are elevated, or the late phase, when plasma Ang II levels are back to normal, remains unclear.⁸

However, transgenic technology allows selective deletion or amplification of RAS gene expression, and a number of transgenic models targeting this system have already been generated.⁹ However, even some of these models do not have the level of precision in gene manipulation necessary for dissecting the RAS. For example, transgenic animals overexpressing angiotensin-converting enzyme or angiotensinogen present the disadvantage of involving multiple systems (bradykinin and RAS) or various receptors (AT_1a, AT_1b, and AT₂).^2,10 Furthermore, in models in which RAS genes have been globally manipulated,¹¹ it is difficult to unravel the role of individual tissue RAS in cardiovascular regulation.

To circumvent these problems in directly addressing the role of the brain RAS, we recently generated a new transgenic mouse with selective overexpression of AT_1a receptors in neurons of the brain (NSE-AT_1a).¹² Not surprisingly, these mice are normotensive under basal conditions when Ang II levels are normal. However, on activation of the receptors with acute central administration of Ang II, NSE-AT_1a respond with exaggerated pressor and bradycardic responses compared with their nontransgenic counterparts (NT).¹² These findings confirm the transgene functional overexpression and suggest that the model is ideally suited to investigate the role of brain AT_1a receptors in chronic Ang II-dependent hypertension.

The specific objective of this study was to test the hypothesis that pathophysiological elevation of endogenous Ang II levels would lead to exacerbated hypertension in NSE-AT_1a mice. Using the 2K1C intervention to activate the endogenous RAS in these mice,¹³ in conjunction with continuous telemetric recording of BP and heart rate (HR), our results demonstrate that brain AT_1a receptor-mediated signaling plays a significant role in the initiation of Ang II-dependent hypertension. Furthermore, our studies suggest that nitric oxide (NO) is involved in buffering this brain-dependent hypertension.

	Materials and Methods

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What follows is a brief summary of the experimental strategy. A detailed description of the experimental protocols appears in the online data supplement available at http://circres.ahajournals.org.

Radiotelemetric Recording of BP and HR
Adult NSE-AT_1a and NT mice were anesthetized and surgically instrumented with radiotelemeters. After recovery and baseline recordings, mice were re-anesthetized and 2K1C (NSE-AT_1a, n=9; NT, n=7) or sham surgery (NSE-AT_1a, n=8; NT, n=9) was performed. Cardiovascular parameters were then continuously recorded over 28 days.

Plasma and Brain Ang II Assay
Blood samples were collected before and 3, 7, and 28 days after 2K1C or sham surgery (NSE-AT_1a, n=6 to 14; NT, n=6 to 18 per group). For brain Ang II levels, NSE-AT_1a and NT (n=5 to 10 per group) were euthanized before and 3, 7, and 28 days after 2K1C surgery, and the hypothalamus, brain stem, and cerebellum were isolated. Ang II extraction and assay were performed using C18 SEP columns and enzyme-linked immunosorbent assay kit according to the manufacturer’s instructions.

Autoradiography for AT₁ Receptors
To determine AT₁ receptor binding, coronal brain/longitudinal kidney sections (20 µm) from NSE-AT_1a (2K1C: n=8 [brain], n=3 [kidney]; sham: n=4 [brain], n=3 [kidney]) and NT (2K1C: n=9 [brain]; n=4 [kidney]; sham: n=4 [brain], n=4 [kidney]) were analyzed. Binding to AT₂ receptors was prevented by coincubation with PD123319 (10 µmol/L, kind gift from Parke-Davis), whereas nonspecific binding was defined with 10 µmol/L Ang II.

Chronic Nitric Oxide Synthase Blockade During 2K1C Hypertension
To investigate the role of NO in buffering 2K1C hypertension, separate NSE-AT_1a (n=4) and NT (n=5) mice were implanted with radiotelemeters and subjected to 2K1C or sham surgery as described. At the time of clipping, mice were implanted with osmotic minipumps for subcutaneous infusion of the nitric oxide synthase (NOS) inhibitor L-NAME (300 µg/d) over 28 days.

Vascular Reactivity in 2K1C Hypertension
Twenty-eight days after 2K1C or sham surgery, NSE-AT_1a (5 to 6 per group) and NT (n=5 per group) were euthanized and right carotid arteries were dissected and mounted in wire myographs. After incubation with L-NNA (10^–4 M) or vehicle and precontraction with U46619 (10^–7 M), relaxation to acetylcholine (ACh, 10^–9 to 3x10^–6 M) and contraction to phenylephrine (Phe, 3x10^–10 to 3x10^–5 M) were assessed. Similar experiments were also performed in second-order mesenteric arteries (n=4 per group). Results were expressed as maximal response (carotids) or ED₅₀ (mesenterics).

Immunohistochemical Detection of NOS in Thoracic Aortas
Thoracic aortas from 2K1C and sham NSE-AT_1a and NT (n=3 per group) were collected, fixed, and sectioned as described.¹² Sections (5 µm) were incubated with primary antibodies to detect either the endothelial (eNOS) or the neuronal (nNOS) isoform of NOS and visualized using DAB/H₂O₂.

Western Blot Analysis of NOS in Mesenteric Arteries
Mesenteric arteries were isolated from 2K1C and sham NSE-AT_1a and NT (n=4 per group) and protein was extracted. Western blot analysis was performed using 25 µg of protein incubated with eNOS or nNOS antibodies. Quantification of the blots was performed using Image J software.

Statistics
Data are expressed as mean±SEM. Data were analyzed by Student t test or ANOVA (after Bartlett test of homogeneity of variance), followed by Newman-Keuls correction for multiple comparisons between means. Statistical comparisons were performed using Prism.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Early Onset of Renovascular Hypertension in NSE-AT_1a Mice
To determine whether chronic activation of the endogenous RAS would lead to exacerbated hypertension in mice with brain-selective overexpression of AT_1a receptors, renal artery stenosis was induced in NSE-AT_1a and NT mice and cardiovascular parameters were continuously recorded for 28 days. Before 2K1C surgery, resting mean arterial pressure (MAP) and HR values were similar in NSE-AT_1a and NT (MAP, 110±3 versus 109±2 mm Hg; HR, 569±5 versus 590±12 bpm; P>0.05). This is in agreement with our earlier studies using indwelling fluid-filled catheters for BP and HR measurements.¹² Immediately after clipping, BP increased dramatically in NSE-AT_1a. Within 24 hours, MAP had increased nearly 35 mm Hg, and by 3 days after clip, it peaked at 160±8 mm Hg (P<0.01), whereas it remained unchanged in NT (Figure 1). In fact, it was not until 6 days after clipping that MAP in NT increased to anywhere near the levels observed in NSE-AT_1a, and it took them nearly 2 weeks to reach the same hypertensive levels observed initially in NSE-AT_1a. The early accelerated increase in MAP in NSE-AT_1a after clipping was accompanied by an immediate and marked bradycardia (200 bpm) that, unlike the MAP response, remained stable throughout the experimental period. This was in contrast to the lack of HR changes in 2K1C NT, as well as the unaltered HR levels in both sham groups (Figure 1).

View larger version (30K): [in this window] [in a new window]   Figure 1. Altered development of renovascular hypertension in NSE-AT1a mice. Summary of MAP (mm Hg) and HR (bpm) recorded by radiotelemetry before and for 28 days after 2K1C or sham surgery in conscious freely moving NT and NSE-AT1a mice. Data are expressed as mean ±SEM. *P<0.05, 2K1C NSE-AT1a vs preclip and/or shams. P<0.05, 2K1C NT vs preclip and/or shams. P<0.05, 2K1C NSE-AT1a vs 2K1C NT. Sham and 2K1C surgeries were performed at day 0.

Surprisingly, just as BP began to increase in NT, it started to progressively decline in NSE-AT_1a mice (Figure 1). In fact, the MAP curves for these 2 groups crossed 1 week after clipping, resulting in significantly greater MAP values at the plateau (between 2 and 4 weeks) in NT (158±4 mm Hg) versus NSE-AT_1a (125±7 mm Hg; P<0.05). Although not a complete reversal of hypertension, BP in NSE-AT_1a eventually stabilized 35 mm Hg lower than the early postclip period. It should be noted that MAP remained unchanged over the course of the experiment in both sham groups. Taken together, these results demonstrate that central overexpression of AT_1a receptors leads to an accelerated 2K1C-induced hypertension. The subsequent sustained lowering of BP in NSE-AT_1a suggests that increased central AT_1a signaling leads to the activation of mechanisms involved in buffering this hypertensive response.

2K1C-Induced Modulation of Central and Peripheral RAS Components
To track the 2K1C-induced activation of the endogenous RAS, Ang II and AT₁ receptor levels were measured in NSE-AT_1a and NT before and after clipping. For brain Ang II assays, we focused on the brain stem and hypothalamus because of their known links to neurogenic hypertension.^6,14,15 No differences in basal circulating Ang II levels were detected between NSE-AT_1a and NT (67±11 versus 79±6 pg/mL; P>0.05) (Figure 2A). Similarly, these groups had equivalent resting Ang II levels in the brain stem (NSE-AT_1a 73±8 versus NT 70±6 fg/mg tissue; P>0.05) and hypothalamus (NSE-AT_1a 74±9 versus NT 71±7 fg/mg tissue; P>0.05) before 2K1C surgery. Importantly, these findings demonstrate that brain-selective overexpression of AT_1a receptors does not alter baseline brain or plasma Ang II levels.

View larger version (34K): [in this window] [in a new window]   Figure 2. 2K1C-induced modulation of central and peripheral RAS components. A, Plasma, brain stem, and hypothalamus Ang II levels before (day 0) and 3, 7, and 28 days after induction of 2K1C hypertension in NT and NSE-AT1a. *P<0.05, vs day 0. B, Quantification of AT1 receptor density in brain regions (NTS indicates nucleus of tractus solitarii; RVLM, rostral ventrolateral medulla; PVN, paraventricular nucleus; VP, ventral pallidum) of NSE-AT1a before (day 0) and 3 and 28 days after clip. C, Summary of AT1 receptor binding in renal cortex and medulla 3 days after clip. Data are expressed as mean±SEM.

Overexpression of AT_1a receptors in the brain also did not appear to differentially affect the 2K1C-induced changes in Ang II production. Although there were marked alterations in plasma and brain Ang II levels at the various postclip time points, changes were comparable between NSE-AT_1a and NT (Figure 2A). By 3 days, plasma and brain Ang II levels were similarly elevated in NT and NSE-AT_1a, and these changes were maintained through the first week after 2K1C. After 1 month, plasma Ang II levels had returned to baseline values in both groups, whereas brain stem and hypothalamic levels were even more dramatically elevated than they were previously. In fact, hypothalamic Ang II levels were 9-fold higher than at baseline, and there was >3-fold increase in brain stem. However, again, it should be emphasized that Ang II levels in these brain regions were increased to a similar extent in NSE-AT_1a and NT. Sham animals did not exhibit any changes in plasma (eg, at day 28, NSE-AT_1a 70±13 versus NT 66±2 pg/mL; P>0.05) or brain Ang II levels over this time period (eg, at day 28 in hypothalamus NSE-AT_1a 63±2 versus NT 70±7 pg/mL; P>0.05). Taken together, these results demonstrate that in the early phase of renovascular hypertension, plasma and brain Ang II levels increase to a similar extent in both NT and NSE-AT_1a. Moreover, during the later phase, maintenance of hypertension in both groups is associated with an even greater activation of the brain RAS but a normalization of plasma Ang II levels.

As previously reported, AT₁ levels in NSE-AT_1a brain are markedly elevated compared with NT.¹² To characterize the effect of 2K1C on central AT₁ receptors in NSE-AT_1a, quantitative receptor autoradiography was performed. Consistent with our previous findings, AT₁ receptor binding density was dramatically upregulated in NSE-AT_1a brains compared with those of NT. In fact, receptor density was so high in NSE-AT_1a, with films being saturated as early as 6 hours of exposure, that we could not make side-by-side comparisons with NT brains using the same film exposures. However, in NSE-AT_1a, we did observe significant 2K1C-induced changes in AT₁ binding in select cardiovascular regulatory nuclei (Figure 2B). By 3 days after clip, when both BP and Ang II levels were significantly elevated, there was a marked downregulation of AT₁ receptor density in the nucleus tractus solitarius, rostral ventrolateral medulla, and paraventricular nucleus compared with preclip values. No other effects of clipping were observed in any other brain region analyzed, including noncardiovascular control nuclei such as the ventral pallidum. Interestingly, by 4 weeks after 2K1C, the density of AT₁ receptors in nucleus tractus solitarius, rostral ventrolateral medulla, and paraventricular nucleus had returned to preclip levels in NSE-AT_1a.

Receptor autoradiography was also used to determine the effects of clipping on AT₁ binding in both kidneys. Three days after surgery, when central AT₁ receptor density was decreased in NSE-AT_1a, there were no significant changes in renal AT₁ levels in any group (Figure 2C). A similar observation was made at 28 days for AT₁ density in renal cortex in clipped (NT 52±2 versus NSE 53±4 dpm/mm²; P>0.05) and unclipped (NT 47±2 versus NSE 39±4 dpm/mm²; P>0.05) kidneys. Although there was a significant increase in receptor binding in renal medulla of clipped (right side) (NT, 46±4; NSE, 57±6 dpm/mm²) versus unclipped kidney (NT, 33±2; NSE, 29±5 dpm/mm²; P<0.05), there was no genotype effect in this response.

Prevention of Renovascular Hypertension Reversal in NSE-AT_1a Mice With Chronic Blockade of NOS
To determine whether augmented NO production could account for the late-phase buffering of hypertension in NSE-AT_1a, 2K1C was performed in separate cohort of NT (n=5) and NSE-AT_1a (n=4) infused with L-NAME. As before, there were no differences in baseline MAP (NT 105±8 versus NSE-AT_1a 102±5 mm Hg; P>0.05) or HR (NT 609±21 versus NSE-AT_1a 566±14 bpm; P>0.05) before surgery and drug infusion. L-NAME had no effect on the early phase of renovascular hypertension in NSE-AT_1a because the magnitude and time course of the BP increase during the first week was similar to that of nontreated NSE-AT_1a (Figure 3 versus Figure 1). In contrast, NOS inhibition had a marked effect on the later phase of hypertension in this group. L-NAME-treated NSE-AT_1a did not exhibit the progressive decline in BP (Figure 3, weeks 2 to 4) observed in untreated transgenic mice (Figure 1). Furthermore, L-NAME prevented the 2K1C-induced bradycardic responses observed in untreated NSE-AT_1a (Figure 3 versus Figure 1). Although it did not further enhance BP level in NT, NOS blockade caused a major acceleration in the development of 2K1C-induced hypertension in these animals compared with untreated NT (24 hours versus 4 days) (Figure 3 versus Figure 1).

View larger version (28K): [in this window] [in a new window]   Figure 3. NO buffers renovascular hypertension in NSE-AT1a mice. Changes in MAP and HR recorded by radiotelemetry before and for 28 days after clipping in conscious freely moving NT and NSE-AT1a mice chronically infused with L-NAME (300 µg/d) starting on the day of 2K1C surgery. Data are expressed as mean±SEM. *P<0.05, vs NSE-AT1a; P<0.05, vs baseline.

2K1C-Induced Changes in NO-Mediated Vasorelaxation and NOS Levels in NSE-AT_1a and NT
To address the hypothesis that altered peripheral vasoactivity may account for the differential effects on BP in 2K1C NSE-AT_1a, we examined carotid and mesenteric artery responses to ACh and Phe in the presence or absence of L-NNA in sham-operated and 2K1C mice 4 weeks after surgery.

In carotid arteries from sham mice, maximal relaxation to ACh was not different between NT (90±2%) and NSE-AT_1a (92±2%), and it was similarly inhibited by L-NNA in both groups (NSE-AT_1a 46±4 versus NT 42±11%; P>0.05) (Figure 4A). Maximal contraction to Phe was significantly increased in NSE-AT_1a compared with NT (48±8% versus 26±8%; P<0.05) (Figure 4C), and this difference was abolished by L-NNA (NSE-AT_1a, 86±6%; NT, 96±10%). This suggests that basal NO bioavailability may be decreased in this particular vessel in NSE-AT_1a, which could be related to the enhanced local sympathetic tone observed in preliminary studies in these mice.¹⁶

View larger version (30K): [in this window] [in a new window]   Figure 4. Enhanced NO-mediated vasorelaxation in carotid rings from 2K1C NSE-AT1a mice. Summary of ACh-induced relaxation and Phe-induced contraction in L-NNA-incubated or vehicle-incubated carotid arteries isolated from NT and NSE-AT1a before (A and C) or 28 days after clip (B and D). Data are expressed as mean±SEM. *P<0.05, vs NT; P<0.05, vs L-NNA.

Four weeks after 2K1C, maximal relaxation to ACh in carotid rings was significantly impaired in 2K1C NT (78±6%) (Figure 4B) compared with sham NT (90±2%; P<0.05) (Figure 4A). Accordingly, immunohistochemical data revealed decreased eNOS in 2K1C NT aortas (Figure 5C). Although ACh-induced vasorelaxation in NSE-AT_1a carotid arteries was not altered by 2K1C (sham, 92±2%; 2K1C, 96±2%; P>0.05) (Figure 4B), contraction to Phe was significantly reduced in these vessels by 2K1C in both NT (11±3%) and NSE-AT_1a (21±3%) compared with shams (NT, 26±8%; NSE-AT_1a, 48±8%; P<0.05) (Figure 4D). NOS blockade improved contraction in 2K1C NT (49±10%, P<0.05), but to a similar extent as in NT, ie, a 4-fold increase (Figure 4D). In contrast, L-NNA caused a dramatic augmentation in Phe-induced contractile response in 2K1C NSE-AT_1a carotid arteries (143±14%, P<0.05) that far exceeded that observed in NSE-AT_1a (Figure 4D). Coupled with the lack of diminished vasorelaxation in 2K1C NSE-AT_1a, these data suggest NOS may be upregulated in NSE-AT_1a during the late phase of renovascular hypertension. These observations were supported by immunohistochemistry showing a lack of eNOS downregulation (Figure 5G) but enhanced nNOS expression (Figure 5H) in 2K1C NSE-AT_1a aortas.

View larger version (83K): [in this window] [in a new window]   Figure 5. NOS levels are elevated in thoracic aortas from 2K1C NSE-AT1a mice. Representative immunohistochemical staining for eNOS (A through D) and nNOS (E through H) in NT (A, C, E, and G) and NSE-AT1a (B, D, F, and H) thoracic aortas. Immunostaining was performed on aortic sections 28 days after sham (A, B, E, and F) or 2K1C (C, D, G, and H) surgery. Examples of NOS staining are indicated by arrow heads.

To determine whether a similar vasoactivity profile is observed in resistance vessels, the same experiments were repeated in second-order mesenteric arteries. In shams, ACh and Phe ED₅₀ values were not different in NT (1.3±0.4 and 0.29±0.06 µmol/L, respectively) and NSE-AT_1a (2.1±0.9 and 0.37±0.07 µmol/L, respectively; P>0.05), and were similarly inhibited by L-NNA (NSE-AT_1a 3.4±0.7 and 0.15±0.02 versus NT 3.6±1.2 and 0.17±0.04 µmol/L, respectively; P>0.05) (Figure 6A and 6C). By 4 weeks after clip, vasorelaxation to ACh in these vessels was significantly improved in 2K1C NSE-AT_1a (0.11±0.05 µmol/L, P<0.05), whereas it was not changed in 2K1C NT (1.61±0.85 µmol/L; P>0.05) (Figure 6B). This difference was abolished by L-NNA (NT 2.0±0.5 versus NSE-AT_1a 1.2±0.6 µmol/L), suggesting that 2K1C induces enhanced NO-mediated vasorelaxation in NSE-AT_1a. Additionally, contraction to Phe was not altered in 2K1C NT mesenterics (0.42±0.09 µmol/L) but was significantly impaired in 2K1C NSE-AT_1a (1.10±0.31 µmol/L, P<0.05) (Figure 6D). As with the carotid vessels, NOS blockade improved vasoconstriction in 2K1C NSE-AT_1a (0.61±0.15 µmol/L) to a similar extent as in 2K1C NT (0.22±0.04 µmol/L), ie, a 2-fold decrease (Figure 6D).

View larger version (52K): [in this window] [in a new window]   Figure 6. Increased NOS levels and NO-mediated relaxation in mesenteric arteries from 2K1C NSE-AT1a mice. Summary of ACh-induced relaxation and phenylephrine-induced contraction in L-NNA-treated or vehicle-treated mesenteric arteries isolated from NT and NSE-AT1a before (A and C) or 28 days after clip (B and D). *P<0.05, vs NT; P<0.05, vs L-NNA. Representative Western blots and summary of NOS levels (E and F) in mesenteric arteries from NSE-AT1a and NT before and after (28 days) clipping. *P<0.05, vs NSE-AT1a 2K1C. Data are expressed as mean±SEM.

To provide further support of these findings, we quantified eNOS and nNOS levels in mesenteric resistance arteries using Western blot analysis (Figure 6E and 6F). Similar to the immunohistochemical findings in aortic vessels, these data show that there is no significant difference in either NOS isoform between sham-operated NT and NSE-AT_1a. However, 2K1C hypertension induced a marked increase in both eNOS and nNOS levels in NSE-AT_1a but not in NT.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
An overactive brain RAS has been implicated in the pathogenesis of genetic and experimental hypertension, but a definitive role for the brain versus peripheral tissues has not been established because of the difficulty in experimentally separating these RAS. We postulated that NSE-AT_1a mice, with brain-restricted overexpression of AT_1a receptors, would provide an ideal opportunity for unmasking the brain role in the development of chronic Ang II-dependent hypertension. Normotensive under basal conditions, these mice exhibit enhanced pressor and bradycardic responses after central administration of Ang II.¹² Using 2K1C as a model for chronic activation of endogenous Ang II production, our results show that hypertension develops in NSE-AT_1a much more rapidly than NT, and this was not attributable to genotype-related differences in plasma or brain Ang II levels. A marked bradycardia accompanied this early increase in BP in NSE-AT_1a, as did a substantial cardiovascular region-specific downregulation of AT₁ receptor binding in brain but not in kidney. Just as BP reached its plateau in NT (1 week after clip), hypertension began to abate and eventually stabilized at significantly lower levels in NSE-AT_1a despite marked elevations in Ang II levels in brain stem and hypothalamus. This hypertension reversal, like the bradycardia, was prevented by chronic infusion of L-NAME. These data, along with evidence showing enhanced NOS expression and NO-mediated compensatory responses in NSE-AT_1a peripheral arteries during this later phase, suggest that activation of endogenous NO systems plays an important role in buffering the maintenance of hypertension caused by overexpression of AT_1a receptors in the brain.

The time course and magnitude of BP change and Ang II levels observed in NT are consistent with what many investigators have observed with unilateral renovascular hypertension in various species.^13,17,18 Hypertension developed by 1 week and was sustained for 1 month after 2K1C surgery. The chronic phase of hypertension occurred in the presence of normal plasma Ang II levels, but marked increases in levels of Ang II in brain stem and hypothalamus. This suggests an important role for local activation of the brain RAS in the maintenance of hypertension in this model and is consistent with other reports showing renovascular hypertension-induced increases in angiotensinogen mRNA levels in these brain regions during the chronic phase,⁴ as well as evidence that 2K1C hypertension is ameliorated at this stage with central infusion of AT₁ receptor antisense.^8,18 Increases in brain stem and hypothalamic Ang II levels at 3 and 7 days after clip most likely reflect plasma-derived Ang II reaching the brain through structures lacking a blood-brain barrier, so-called circumventricular organs, located in each of these brain areas. However, the 4- to 6-fold increases in Ang II in each of these regions at 28 days, along with normal plasma Ang II levels at this time, leave little doubt that Ang II is centrally derived during the chronic phase.

In contrast to the 1-week delay for 2K1C hypertension to develop in NT, NSE-AT_1a exhibited marked increases in BP within 24 hours after clip, with maximum hypertension being reached by 3 days. To the best of our knowledge, this is the first report of such a rapid BP effect after 2K1C intervention, so it is important to note that the surgical procedure and clip size are the same as that reported by other investigators in mice.^13,19 These findings suggest the unmasking of an important role for the brain RAS early on in this model, and we speculate that increased plasma Ang II binding to AT_1a receptors in circumventricular organs plays a key role in the acceleration of hypertension in NSE-AT_1a. These regions have been shown to be important in the development of 2K1C hypertension in rats,^6,20 and NSE-AT_1a exhibit very high levels of AT_1a transgene expression in these regions.¹² Although plasma Ang II levels are elevated to the same level in NSE-AT_1a and NT at these early time points, there are much greater numbers of AT_1a receptors for Ang II to bind in these brain regions in NSE-AT_1a. Given the well-established connections between these sites and brain sympatho-modulatory centers,²¹ along with considerable evidence that increased Ang II signaling in the brain potentiates hypertension through modulation of sympathetic activity,²⁰ we surmise that the early rapid increase in BP in these mice is attributable to exaggerated plasma Ang II/AT_1a-mediated effects on central sympathetic outflow. Locally generated Ang II in brain stem and hypothalamus activating increased numbers of AT_1a receptors in NSE-AT_1a may also be important, although as discussed, we cannot rule out the possibility that significant Ang II elevations observed in these regions derive from the plasma. Regardless of the source of Ang II, it is clear that the rapid increase in BP is mediated by increased expression of AT_1a receptors selectively in the brain because this is the only difference between NSE-AT_1a and NT.¹² The importance of these central AT₁ receptors in the development of 2K1C hypertension is further suggested by their subsequent decline in brain (but not in kidney), which as discussed, parallels the onset of BP lowering in these animals. We believe it is likely that the immediate dramatic increase in BP over several days in NSE-AT_1a triggered the subsequent downregulation in central AT₁ binding density observed 3 days after clip.

Just as intriguing as the acceleration of hypertension in the NSE-AT_1a is its subsequent diminution in these animals. Although not a complete reversal of hypertension, BP in NSE-AT_1a stabilized at a significantly lower level than in NT. There are several potential mechanisms that could account for this, such as hypersensitivity of 2K1C animals to certain concentrations of Ang II in the brain leading to decreased BP and HR in this model.²² Additionally, augmented NO production seems to play a prominent role. First, NSE-AT_1a exhibited a dramatic and sustained 2K1C-induced bradycardia that was completely abolished by chronic treatment with L-NAME. Given the important role of NO in modulating autonomic control of HR through its actions in central cardiovascular sites,²³ along with evidence that peripherally administered L-NAME crosses the blood-brain barrier,²⁴ it is interesting to speculate that activation of endogenous NO mechanisms in the brain is involved in this response and that this could be blocked by L-NAME infusion. Wang et al recently reported increased nNOS expression in 2K1C rats brain stem, which was reduced by peripheral administration of L-NAME,²⁵ and the potent bradycardic effects of NO in this and other brain regions are now established.²³ It is also well-known that Ang II and renovascular hypertension triggers NO release in the periphery,^26–28 and evidence is accumulating suggesting a similar situation in central cardiovascular sites.^29,30 Thus, 2K1C-induced increases in Ang II/AT_1a signaling in NSE-AT_1a brain, leading to increased NO-mediated bradycardia, is 1 possible scenario. Interestingly, the nucleus ambiguus²³ and dorsal motor nucleus of the vagus³¹ are important brain stem sites for NO-mediated bradycardia, and these are among the regions with the highest levels of AT_1a expression in the NSE-AT_1a model.¹² Investigations are ongoing using brain site-selective targeting of genetic inhibitors of NOS to address this possibility.

Although central NO-mediated bradycardia may have some compensatory effects on renovascular hypertension in NSE-AT_1a, we were puzzled by the mismatch in time course between the bradycardia and the BP decline. That is, whereas bradycardia was immediate and sustained, the BP decrease was gradual. To determine whether NO-mediated effects on peripheral vasculature could account for at least part of the hypertension reversal, we studied the effects of NOS blockade on BP and vascular reactivity in NSE-AT_1a. Similar to its effects on the bradycardia, L-NAME prevented the late-phase BP decline. Furthermore, in both mesenteric and carotid arteries isolated from late-phase 2K1C NSE-AT_1a, there was evidence of increased NO-mediated dilation. Coupled with immunohistochemical evidence and Western blot analysis showing increased NOS levels in both types of vessels compared with 2K1C NT, the data suggest a robust activation of compensatory NO synthesis in peripheral arteries to modulate 2K1C hypertension in NSE-AT_1a. It is known that renovascular hypertension is associated with a compensatory increase in vasodilator function,²⁸ particularly in the late phase,³² and stretch-mediated increases in nNOS expression in vascular smooth muscle cells parallels the development of hypertension.^33,34 Interestingly, numerous hemodynamic compensatory responses are reported with rapid Ang II-induced pressor responses but not with more gradual Ang II-dependent BP increases.³⁵ Furthermore, Ang II activation of AT₁ receptors has been linked to NOS upregulation.³⁶ As such, we hypothesize that the initial rapid increase in BP in 2K1C NSE-AT_1a triggers the activation of NO production and compensatory responses in the peripheral vasculature. The precise mechanisms linking increased central AT₁ activation and peripheral NOS upregulation remain to be identified.

In conclusion, these studies reveal an important role for the brain RAS in the pathogenesis of renovascular hypertension. Dysregulation of AT_1a receptor expression in the brain causes an early exacerbation of BP increases but also provokes powerful compensatory responses that involve augmentation of endogenous NO mechanisms.

	Acknowledgments

 
This work was supported by grants to R.L.D. from the National Institutes of Health (HL 14388 and HL 63887) and the American Heart Association (0030017N). E.L. was funded by postdoctoral fellowships from the American Heart Association (20572Z) and American Physiological Society (Postdoctoral Fellowship in Physiological Genomics). A.J.L. was the Helen C. Levitt Visiting Professor at the University of Iowa. F.S.L. was supported by National Institutes of Health grant HL 062483. The authors thank Dr Haralambos Gavras and Conrado Johns for help in implementing the 2K1C procedure, Drs Nancy Kanagy, William Talman, and Martine Dunnwald for invaluable discussions and Dennis Dunnwald and Paul Reimann for their expert assistance with the figures.

	Footnotes

 
*Both authors contributed equally to this work.

Original received March 15, 2004; revision received June 24, 2004; accepted July 20, 2004.

	References

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