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. Author manuscript; available in PMC: 2011 Mar 1.
Published in final edited form as: Hypertension. 2010 Jan 25;55(3):627–635. doi: 10.1161/HYPERTENSIONAHA.109.148171

RECEPTOR ACTIVITY-MODIFYING PROTEIN-1 INCREASES BAROREFLEX SENSITIVITY AND ATTENUATES ANGIOTENSIN-INDUCED HYPERTENSION

Rasna Sabharwal 1,*, Zhongming Zhang 2,*, Yongjun Lu 1, Francois M Abboud 1,2, Andrew F Russo 2,**, Mark W Chapleau 1,2,3,**
PMCID: PMC2902886  NIHMSID: NIHMS213373  PMID: 20100989

Abstract

Calcitonin gene-related peptide (CGRP) is a powerful vasodilator that interacts with the autonomic nervous system. A subunit of the CGRP receptor complex, receptor activity-modifying protein 1 (RAMP1), is required for trafficking of the receptor to the cell surface and high-affinity binding to CGRP. We hypothesized that upregulation of RAMP1 would favorably enhance autonomic regulation and attenuate hypertension. Blood pressure, heart rate (HR) and locomotor activity were measured by radiotelemetry in transgenic mice with ubiquitous expression of human RAMP1 and littermate controls. Compared with control mice, RAMP1 mice exhibited similar mean arterial pressure (MAP), a lower mean HR, increased HR variability, reduced blood pressure variability, and increased baroreflex sensitivity (2.83±0.20 vs. 1.49±0.10 ms/mmHg in controls) (P<0.05). In control mice, infusion of angiotensin II (Ang-II) increased MAP from 118±2 mmHg to 153±4 and 174±6 mmHg after 7 and 14 days of infusion, respectively (P<0.05). In contrast, Ang-II hypertension was markedly attenuated in RAMP1 mice with corresponding values of MAP of 111±2, 119±2 and 132±3 mmHg. Ang-II induced decreases in baroreflex sensitivity and HR variability, and increases in blood pressure variability observed in control mice were also abrogated or reversed in RAMP1 mice (P<0.05). Moreover, during the Ang-II infusion, the pressor response to the CGRP receptor antagonist CGRP8-37 was significantly greater (P<0.05) in RAMP1 mice (+30±2 mmHg) than in control mice (+19±2 mmHg), confirming a significantly greater antihypertensive action of endogenous CGRP in RAMP1 mice. We conclude that RAMP1 overexpression attenuates Ang-II induced hypertension and induces a protective change in cardiovascular autonomic regulation.

Keywords: calcitonin gene-related peptide, parasympathetic nerve activity, heart rate variability, baroreflex sensitivity, blood pressure, transgenic mice

Calcitonin gene-related peptide (CGRP) is expressed predominantly in the nervous system and contributes to a variety of physiological and pathological processes including neurogenic inflammation, inhibition of cell proliferation and oxidative stress, and cardiovascular regulation (1,2). CGRP is one of the most powerful vasodilators known and sensory nerves containing CGRP provide extensive innervation of blood vessels (1,2). Furthermore, CGRP and/or CGRP receptor expression are increased in several models of hypertension, including that induced by infusion of the vasoconstrictor peptide angiotensin II (Ang-II) (3-6). While pharmacological blockade of CGRP receptors does not influence mean arterial pressure (MAP) of normotensive subjects, receptor blockade increases the severity of hypertension in several experimental models (6-8). These findings suggest that endogenous CGRP operates through a negative-feedback mechanism to oppose the development of hypertension.

Although it is reasonable to attribute antihypertensive actions of CGRP to its vasodilator activity, other mechanisms may also be involved. CGRP and CGRP receptors are expressed in sensory nerves and brain regions that powerfully influence blood pressure via their effects on parasympathetic and sympathetic nerve activity (2, 9-11). Previous studies examining autonomic responses to acute administration of CGRP have demonstrated variable responses that were dependent on site of injection and dose (12-17).

The baroreceptor reflex is a major regulator of arterial blood pressure (18). CGRP has been implicated in modulation of baroreflex sensitivity but its role and importance remain unclear (9,16,17). The baroreflex buffers fluctuations in blood pressure by evoking reflex changes in heart rate (HR) and vascular resistance. Therefore, high baroreflex sensitivity reduces blood pressure variability and increases HR variability. Decreases in baroreflex sensitivity and HR variability, and associated increases in blood pressure variability increase cardiovascular risk (19,20). Thus, a favorable influence of CGRP on autonomic regulation may provide additional protection beyond its blood pressure lowering effect in hypertension. Indeed, such a favorable action of CGRP is suggested by the finding that MAP, HR, and sympathetic tone are increased and cardiac parasympathetic tone is decreased in CGRP-deficient mice (21,22), although contradictory data have also been reported (23).

The discovery of the molecular components of the CGRP receptor complex has created new avenues for investigating the role of CGRP in blood pressure regulation. The CGRP receptor is a multimeric G-protein-coupled receptor complex consisting of calcitonin-like-receptor (CLR) and receptor activity-modifying protein-1 (RAMP1) (24). RAMP1 is a single-pass transmembrane protein that is required for high-affinity binding of CGRP and trafficking of the receptor to the cell surface (25). Studies from our laboratory (Z.Z., A.F.R.) have demonstrated that increasing expression of RAMP1 in trigeminal ganglia (26) and vascular smooth muscle (27,28) enhances functional responses to CGRP, both in vitro and in vivo. Thus, RAMP1 is rate limiting for CGRP receptor-mediated signaling.

Based on these findings, we hypothesized that transgenic upregulation of RAMP1 will enhance the antihypertensive actions of endogenous CGRP in vivo. To test this hypothesis, we generated transgenic mice with ubiquitous expression of human RAMP1 (hRAMP1) in all tissues and compared cardiovascular and autonomic phenotypes in the hRAMP1 transgenic mice and littermate controls before and during chronic infusion of a pressor dose of Ang-II.

METHODS

Animals

Experiments were performed on male mice at 10-20 weeks of age. The mice were maintained in a 12:12 hr light-dark cycle (6:00 AM to 6:00 PM), fed normal mouse chow, and had access to water ad libitum. All procedures were performed in accordance with American Physiological Society and institutional guidelines.

Transgenic mice with GFP flanked by loxP sites followed by the hRAMP1 cDNA have been described (26). Hemizygous GFP-hRAMP1 mice (strain 28412/3) were crossed with mice expressing cre recombinase under the control of the ubiquitous adenovirus EIIa promoter (B6.FVB-Tg(EIIa-cre)C5379Lmgd/J, Stock No. 003724, Jackson Laboratory) to produce double transgenic EIIa/hRAMP1 mice, referred to here as hRAMP1 mice. PCR genotyping, using primers detecting hRAMP1 (Genbank #NC_000002), SV40 (Genbank #NC001669) and cre, was performed as described (26). PCR products with a predicted size of 371 bp for hRAMP1 and 400 bp for cre were analyzed by gel electrophoresis and confirmed by sequencing.

hRAMP1 expression was confirmed in multiple tissues by RT-PCR. Tissues were also analyzed by real time quantitative PCR (qPCR) to determine the mRNA expression levels of both hRAMP1 and the endogenous mouse RAMP1 (mRAMP1) in hRAMP1 mice (n=3) and littermate controls (n=3). For a description of the methods used for detection and measurement of mRNA expression, please see http://hyper.ahajournals.org.

Cardiovascular and Autonomic Assessment before and during Ang-II Infusion

A radiotelemetry probe (Data Sciences International) was inserted into the thoracic aorta via the left common carotid artery in anesthetized mice for measurement of arterial blood pressure (29). The implant surgical procedure and care of the mice are described in the Online Supplement (see http://hyper.ahajournals.org).

Mice were allowed to recover from the transmitter implantation for 5 days before baseline measurements were made. Thereafter, diurnal variations in blood pressure, HR, and locomotor activity were measured over a minimum of 24 hrs at a sampling rate of 500 Hz for 10 s once every 5 min using the Dataquest ART Acquisition software (Version Gold 4.0, DSI) (n=7 for each group of mice). Blood pressure was continuously recorded at a higher sampling frequency (2000 Hz) for 1 hr, between 10 am and 3 pm, to collect data for assessment of baroreflex sensitivity, and blood pressure and HR variability (n=7 for each group). This period of day, when mice are relatively inactive, was chosen to minimize the influences of physical activity and arousal. Pulse interval and HR were derived from the blood pressure waveform using the DSI software. Measurements of spontaneous locomotor activity were derived from the changes in transmitter signal strength associated with movement of the mouse.

After baseline measurements were obtained, mice were anesthetized and an osmotic minipump (model 1002, Alzet) containing Ang-II was implanted subcutaneously. After recovery from surgery, 24 hr recordings of blood pressure, HR, and activity were made over the next 14 days (n=7 for each group). At the end of week 2, blood pressure was continuously recorded for one hour at 2000 Hz for assessment of baroreflex sensitivity, and HR and blood pressure variability (n=5 for each group).

In additional groups of hRAMP1 (n=4) and control (n=3) mice, blood pressure responses to acute intravenous injection of the CGRP receptor antagonist CGRP8-37 (1 μg), the Ang-II type 1 receptor (AT1R) antagonist losartan (10 μg), and the ganglionic blocker chlorisondamine (12 μg) were measured in order to estimate the contributions of endogenous CGRP, AT1R activation, and sympathetic nerve activity to the blood pressure level in Ang-II-infused mice. Drugs were injected via a chronically-implanted jugular vein catheter and blood pressure was measured by telemetry as described above. The methods are described in detail in the Online Supplement (please see http://hyper.ahajournals.org).

At the end of each experiment, mice were euthanized with pentobarbital (150 μg, IP) and the radio-telemeters were removed, cleaned and sterilized for future use.

Expression of AT1R, CGRP and RAMP1 in Neuronal and Vascular Tissues

Pro-hypertensive and anti-hypertensive actions of Ang-II and CGRP, respectively, may involve changes in expression of AT1R, CGRP, and/or the CGRP receptor. To evaluate these possibilities, we measured AT1R, CGRP, and mRAMP1 expression by qPCR in brainstem, dorsal root ganglia (DRG), aorta, and kidney of control and hRAMP1 mice with and without infusion of Ang-II. For a description of the procedures for removing tissues and performing qPCR, please see http://hyper.ahajournals.org.

Data Analysis

Diurnal variations in MAP, HR and activity were calculated as the difference between average values measured over a 6 hr night period (6pm to midnight) and average values measured over a 6 hr day period (8am to 2pm).

Baroreflex sensitivity for control of HR was calculated from spontaneous fluctuations in systolic arterial blood pressure and HR when the mice were active using the sequence technique (30). Sequences of four-or-more consecutive blood pressure pulses where the systolic arterial pressure and pulse interval are positively correlated (r2>0.85) were detected using a customized software (HemoLab, Version 2.3). Baroreflex sensitivity was calculated as the average slope of the systolic pressure-pulse interval relationships (Δms/ΔmmHg).

Blood pressure and HR variability were calculated from beat-to-beat measurements of systolic arterial pressure and pulse interval recorded for 3 min when the mice were inactive using Hemolab and Batch Processor (Version 0.5) software. The standard deviation of pulse interval measurements provided a general measure of HR variability. The root mean square of successive differences in pulse intervals (RMSSD) provided a measure of rapid, parasympathetic-mediated modulation of HR (31). The standard deviation of systolic arterial pressure measurements provided a measure of blood pressure variability.

The results are expressed as means ± SEM. Significant differences were defined at P<0.05. Statistical evaluation was performed using unpaired t-test to compare between two groups; either repeated measures ANOVA with LSD post-hoc test or paired t-test to determine effects of Ang-II, CGRP8-37, losartan and chlorisondamine; and two-factor ANOVA to determine effects of genotype and Ang-II on gene expression (StatView SAS Institute, Cary, NC).

RESULTS

hRAMP1 Transgenic Mice

The GFP-hRAMP1 mice were crossed with mice expressing cre recombinase under control of the EIIa promoter, which is active early in development and in all tissues (32) (Fig. 1A). The resultant double transgenic hRAMP1 mice expressed the hRAMP1 gene in all tissues (Fig. 1B). As expected, there was loss of GFP fluorescence in the hRAMP1 mice. In some tissues, such as the heart, there was still patchy fluorescence (Fig. 1A), which is in agreement with reported mosaic expression of the EIIa-cre transgene (32).

Figure 1. EIIa/hRAMP1 transgenic mice.

Figure 1

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A. Strategy for generating ubiquitous hRAMP1 transgenic mice. Mice with the CX1 promoter and GFP–stop–hRAMP1 cassette flanked by loxP sites (GFP–hRAMP1) were crossed with mice with EIIa promoter driving cre recombinase (EIIa–cre). CX1 drives ubiquitous expression of GFP, but not hRAMP1 due to a stop codon and polyadenylation signal following GFP. Double transgenic offspring that have both the EIIa–cre and GFP–hRAMP1 transgenes (EIIa/hRAMP1) have cre-mediated excision of the GFP–stop sequence at flanking loxP sites. The resultant hRAMP1 transgene is expressed under the control of CX1 promoter. Brightfield (left) and fluorescent (right) images of postmortem hearts from each genotype are shown.

B. hRAMP1 expression in EIIa/hRAMP1 mice. hRAMP1 RNA (228 bp RT-PCR product) was detected from double transgenic mice, but not from control GFP-hRAMP1 littermates in brain (Br), heart (H), liver (Li), stomach (St), small intestine (Si), kidney (K), aorta (Ao), carotid artery (Ca), nodose ganglia (Ng), spleen (Sp), lung (Ln), brainstem (Bs), and bladder (not shown). mRAMP1 (231 bp) and β-actin (125 bp) RT-PCR products were detected from all tissues from both genotypes. hRAMP1 RNA was not detected from the double transgenic mice when amplified in the absence of reverse transcriptase or when water was substituted for cDNA (not shown).

C. Quantitative PCR measurement of RAMP1 levels in EIIa/hRAMP1 mice (hR) and control littermates (C). The levels of hRAMP1 (h), mRAMP1 (m) and β-actin were determined by real-time PCR. The copies of RAMP1 RNAs were calculated from standard curves and normalized to 50,000 copies of β-actin mRNA (left panel). In the right panel, the fold increase of total RAMP1 level in EIIa/hRAMP1 mice relative to control mice is indicated. Data are the mean from 3 mice in each group, with the SE from 3 independent experiments.

The levels of hRAMP1 and endogenous mRAMP1 gene expression in the hRAMP1 mice were determined by qPCR using hRAMP1 and mRAMP1 specific primers (26). The combined total of hRAMP1 and mRAMP1 RNA in different tissues of the hRAMP1 mice ranged from 5- to 15-fold greater than the endogenous mRAMP1 RNA level in tissues of control littermates (Fig. 1C).

Cardiovascular/Autonomic Phenotypes under Basal Conditions

Under basal conditions, MAP and locomotor activity (24 hr avg) did not differ in control and hRAMP1 mice, while mean HR was significantly lower in hRAMP1 mice (573±14 beats/min vs. 615±12 beats/min in control mice) (Fig. 2A). Furthermore, the diurnal night-day differences in MAP, HR and locomotor activity were similar in control and hRAMP1 mice (Fig. 2B).

Figure 2. Blood pressure and autonomic regulation of conscious hRAMP1 mice compared with control littermates under basal conditions.

Figure 2

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hRAMP1 mice exhibit lower HR, less blood pressure (BP) variability, higher HR variability, and higher baroreflex sensitivity than control mice.

A. The 24 hr average values of MAP, HR, and locomotor activity (counts (cts) per min). B. Diurnal variations (night-day differences) in MAP, HR, and activity. C. Systolic arterial BP variability. D. HR variability. PI-SD = standard deviation of pulse intervals. RMSSD = root mean square of successive differences in pulse interval (indicative of parasympathetic modulation). E. Baroreflex sensitivity. Significant differences between hRAMP1 (n=7) and control mice (n=7) are indicated, * P<0.05.

We also measured short-term blood pressure and HR variability, and calculated spontaneous baroreflex sensitivity. Although MAP did not differ in the two groups of mice, systolic blood pressure variability (standard deviation, SD) was markedly reduced in hRAMP1 mice (Fig. 2C). Conversely, overall HR variability (SD of pulse intervals) and rapid beat-to-beat variability attributed to parasympathetic modulation (31) were increased significantly in hRAMP1 mice (Fig. 2D). Baroreflex sensitivity was markedly increased in hRAMP1 mice (Fig. 2E). Increased baroreflex sensitivity was evident when blood pressure was increasing (up sequences) and when it was decreasing (down sequences), and the number of baroreflex sequences per 1000 heart beats did not differ in control vs. hRAMP1 mice (Table S2, please see http://hyper.ahajournals.org).

Ang-II Hypertension and Autonomic Dysregulation are Abrogated in hRAMP1 Mice

In control mice, Ang-II increased MAP progressively over time with the rise in pressure reaching significance on days 3 through 14 (Fig. 3A). Infusion of the same dose of Ang-II in hRAMP1 mice did not increase MAP significantly until the 13th day of infusion and the severity of hypertension was modest (Fig. 3A). Mean HR and locomotor activity were not affected by Ang-II infusion (Fig. 3A). While Ang-II did not affect diurnal variations in MAP or locomotor activity in either group of mice, it essentially abolished diurnal variations in HR in control mice (Fig. 3B). The lack of diurnal HR changes during Ang-II infusion was the result of a lower HR at night (Table S3 in Online Supplement). Ang-II did not significantly reduce diurnal variations in HR in hRAMP1 mice (Fig. 3B and Table S3; please see http://hyper.ahajournals.org).

Figure 3. Transgenic hRAMP1 expression abrogates Ang-II induced hypertension and restores diurnal changes in HR.

Figure 3

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Ang-II increased MAP significantly throughout the period of infusion in control mice but caused a delayed and significantly smaller pressor response in hRAMP1 mice (ANOVA). However, mean HR and locomotor activity were not affected by Ang-II in either group.

A. The 24-hour averages (±SEM) of MAP, HR, and locomotor activity (counts (cts) per min) measured before and throughout the 14-day period of Ang-II infusion in control (n=7, dashed line) and hRAMP1 (n=7, solid line) mice. Baseline measurements were made on day 0 with the arrow indicating the beginning of Ang-II infusion (1000 ng/kg/min). * significant increase in MAP from baseline in response to Ang-II (ANOVA and LSD test, P<0.05). † significant difference in MAP in hRAMP1 vs. control mice during Ang-II infusion (ANOVA and LSD test, P<0.05).

B. The corresponding diurnal (night-day) changes (±SEM) in MAP, HR, and locomotor activity for Day 0 (before Ang-II), and for Days 7 and 14 of Ang-II infusion. Ang-II infusion essentially abolished diurnal changes in HR in control mice (* P<0.05 vs. baseline-Day 0). Diurnal changes in HR were preserved in Ang-II infused hRAMP1 mice († P<0.05, hRAMP1 vs. control mice). Data collected during the night (6pm-12 midnight) and during the day (8am-2pm) were used to calculate the diurnal changes.

Ang-II infusion increased blood pressure variability, decreased HR variability, and decreased baroreflex sensitivity in control mice (Fig. 4). The Ang-II induced increase in blood pressure variability was abolished whereas the decrease in HR variability was reversed to an increase in hRAMP1 mice (Fig. 4). The Ang-II induced decrease in baroreflex sensitivity was evident for both up and down sequences (Table S2, please see http://hyper.ahajournals.org), and was less severe in hRAMP1 mice (−32±6 %) than in control mice (−53±4 %) (P<0.05, Fig. 4).

Figure 4. Transgenic hRAMP1 expression abrogates deleterious effects of Ang-II on autonomic regulation.

Figure 4

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The Ang-II-induced increase in blood pressure (BP) variability was abolished, the decrease in HR variability was reversed to increased variability, and the decrease in baroreflex sensitivity was less pronounced in hRAMP1 mice vs. control mice. Measurements were obtained at baseline (BL) and after 2 weeks of Ang-II infusion in control (n=5) and hRAMP1 (n=5) mice.

A. Systolic BP variability (standard deviation, SD). B. HR variability (pulse interval standard deviation, PI-SD). C. HR variability reflecting parasympathetic modulation (RMSSD). D. Baroreflex sensitivity measured by the sequence technique. Responses to Ang-II infusion are expressed as a percentage of the baseline values. * significant change in response to Ang-II infusion, P<0.05. † response to Ang-II was significantly less in hRAMP1 vs. control mice, P<0.05.

Mechanisms Influencing Blood Pressure Level during Ang-II Infusion

Previous studies have demonstrated that pharmacological blockade of CGRP receptors increases MAP in several models of hypertension, while having little or no effect on MAP in normotensive animals (6-8). To estimate the contribution of endogenous CGRP to MAP in Ang-II infused mice, we measured the blood pressure response to intravenous injection of the CGRP receptor antagonist CGRP8-37. CGRP8-37 increased MAP in control mice (P<0.05), and this effect was enhanced significantly in hRAMP1 mice (Fig. 5). Thus, endogenous CGRP protects against Ang-II hypertension and the protection is potentiated by upregulation of the CGRP receptor subunit RAMP1.

Figure 5. Mechanisms contributing to blood pressure level during Ang-II infusion in control and hRAMP1 mice.

Figure 5

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A. Shown are the increases in MAP that occurred in response to intravenous injection of the CGRP receptor antagonist CGRP8-37 in control and hRAMP1 mice. The greater increase in blood pressure in hRAMP1 mice suggests that enhanced responsiveness to endogenous CGRP contributes to the lower MAP in these mice. B. Shown are the decreases in MAP that occurred in response to the AT1R blocker losartan. The small and similar response in control and hRAMP1 mice suggests that differences in AT1R signaling do not explain the differences in MAP between the groups. C. Shown are the decreases in MAP in response to the ganglionic blocker chlorisondamine. The attenuation of the depressor response in hRAMP1 mice suggests that reduced sympathetic tone contributes to the lower MAP in these mice. MAP measured during Ang-II infusion before injection of CGRP8-37, losartan, and chlorisondamine was significantly lower in hRAMP1 mice (126±1 mmHg) compared with control mice (147±3 mmHg) (P<0.05). Number of mice per group: Control, n=3; hRAMP1, n=4. * significant change in MAP after injection of CGRP8-37, losartan or chlorisondamine, P<0.05. † significant difference in the magnitude of response to drug in hRAMP1 vs. control mice, P<0.05.

The AT1R blocker losartan caused small but significant decreases in MAP in both control and hRAMP1 mice infused with Ang-II that did not differ in magnitude between the groups (Fig. 5). In contrast, injection of the ganglionic blocker chlorisondamine markedly decreased MAP in both groups of mice with the magnitude of the decrease in pressure being significantly greater in control mice (Fig. 5). These results suggest that reduced sympathetic-mediated vasoconstriction may contribute to the lower MAP in hRAMP1 mice vs. control mice.

Expression of AT1R, CGRP and RAMP1 in Control and hRAMP1 Mice

Ang-II infusion increased AT1R expression (~20 fold) and CGRP expression (~2 fold) in brainstem of control mice (Table 1, P<0.05), but did not affect expression of these genes in aorta, DRG or kidney (Table 1). In contrast, in hRAMP1 mice, Ang-II failed to increase brainstem AT1R expression and increased brainstem CGRP expression to higher levels (Table 1).

Table 1.

Effects of Ang-II infusion and transgenic expression of hRAMP1 on AT1R, CGRP, and mRAMP1 gene expression.

Gene Control Mice hRAMP1 Mice
Baseline Ang-II Baseline Ang-II
AT1R
 Aorta 0.083±0.033 0.123±0.039 0.111±0.044 0.246±0.030
 Brainstem 0.170±0.047 3.473±1.711* 0.314±0.037 0.719±0.322
 DRG 1.036±0.141 0.272±0.167 1.818±0.511† 1.534±0.078†
 Kidney 0.438±0.106 2.211±2.072 1.942±1.022 4.583±2.594
CGRP
 Aorta 0.000±0.000 0.000±0.000 0.000±0.000 0.000±0.000
 Brainstem 0.001±0.000 0.002±0.001* 0.001±0.000 0.007±0.002*
 DRG 0.797±0.097 0.422±0.358 1.066±0.083† 0.954±0.100†
 Kidney 0.000±0.000 0.000±0.000 0.160±0.159 0.165±0.110
mRAMP1
 Aorta 11.290±1.056 0.273±0.049* 16.230±4.404 0.597±0.082*
 Brainstem 14.683±3.697 35.380±7.214* 23.277±4.731† 65.551±9.157*†
 DRG 1.453±0.399 0.383±0.078* 1.059±0.229 0.722±0.107*
 Kidney 0.980±0.233 5.073±4.845 19.750±18.299 25.015±17.399
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Measurements are relative mRNA expression of target genes normalized to 18S RNA and referenced to control tissue (DRG in hRAMP1 mouse), means ± SE.

Number of mice per group:

Control-No Ang-II (baseline), n=3; Control-Ang-II, n=2.

hRAMP1-No Ang-II (baseline), n=3; hRAMP1, Ang-II, n=3.

*

Ang-II vs. Baseline, P<0.05

†

hRAMP1 vs. Control mice, P<0.05

Ang-II infusion increased mRAMP1 expression in brainstem of both control and hRAMP1 mice, but decreased mRAMP1 expression in aorta and DRG (Table 1). Furthermore, mRAMP1 expression was upregulated in brainstem of the transgenic hRAMP1 mice (1.5-2.0 fold), both at baseline and during Ang-II infusion (Table 1). CGRP and AT1R expression were increased by ~2 fold in DRG of hRAMP1 mice (P<0.05, Table 1).

DISCUSSION

We have created a mouse model that exhibits ubiquitous transgenic expression of hRAMP1 with increased RAMP1 levels in all tissues examined. We provide here the first full report of the phenotype of these mice. Under baseline conditions, the hRAMP1 mice are normotensive. The normal MAP is accompanied by striking, favorable changes in autonomic regulation including lower HR, higher HR variability, higher baroreflex sensitivity, and lower blood pressure variability. Furthermore, transgenic expression of hRAMP1 essentially abolishes Ang-II-induced hypertension and the accompanying autonomic dysregulation. The results demonstrate powerful autonomic and antihypertensive actions of RAMP1.

Cardiovascular/Autonomic Phenotypes in hRAMP1 Mice under Basal Conditions

The normal MAP observed in the hRAMP1 mice under basal conditions is consistent with the failure of peripheral administration of CGRP receptor antagonists to increase MAP in normotensive animals in previous studies (6-8). However, we did observe improved autonomic regulation in the hRAMP1 mice under basal conditions. This suggests that hRAMP1 levels are important for normal cardiovascular regulation. This conclusion is in agreement with results from CGRP (21,22) and RAMP1 (33) knockout mice that are hypertensive, although this was not seen in one CGRP−/− line (23). Our findings particularly complement the results of Oh-hashi et al. (21) who demonstrated increased sympathetic tone and decreased parasympathetic tone in hypertensive α-CGRP−/− mice. The failure of peripherally administered CGRP receptor antagonists to increase MAP in healthy animals may reflect poor penetrance of the antagonist into the central nervous system. CGRP and its receptor are expressed in central nervous system sites involved in autonomic regulation, including the nucleus tractus solitarius, hypothalamus, dorsal motor nucleus of the vagus, nucleus ambiguus, and amygdala (2,10,11). CGRP is also expressed in carotid sinus nerves (9), suggesting its presence in baroreceptor afferents.

In previous studies, CGRP microinjected into central nervous system sites has usually resulted in increases in sympathetic activity, HR and MAP (12-14), although opposite responses were sometimes observed depending on dose and site of injection (15,17). CGRP has been reported to either enhance or inhibit baroreflex sensitivity through undefined central mechanisms (16,17). Thus, our finding of high baroreflex sensitivity and HR variability, and low blood pressure variability in hRAMP1 mice under basal conditions could not have been easily predicted from the previous acute studies. More research is needed to define the mechanisms of the enhanced autonomic regulation in hRAMP1 mice.

RAMP1 in Hypertension

The results of the present study are consistent with the established role of endogenous CGRP to oppose the development of most types of hypertension (4-8). We chose the Ang-II infusion model of hypertension because the model is well characterized in a variety of species including mice (6,34-37) and Ang-II is established as an important cause of hypertension in humans (38). While direct Ang-II-induced vasoconstriction may cause the initial rise in MAP, increases in central sympathetic drive and blunting of baroreflex-mediated decreases in sympathetic activity and HR play a predominant role when Ang-II is infused chronically over several days to weeks (35-37).

While the mechanism of RAMP1's antihypertensive effect is not yet fully understood, our results along with the established strong neurogenic contribution to Ang-II hypertension suggest that high baroreflex sensitivity and suppression of central sympathetic drive are involved. The AT1R blocker losartan caused small decreases in MAP in control and hRAMP1 mice infused with Ang-II (Fig. 5B), while the ganglionic blocker chlorisondamine caused large decreases in MAP that were significantly attenuated in the hRAMP1 mice (Fig. 5C). These results are consistent with high sympathetic vasomotor tone contributing to hypertension in the Ang-II infused control mice and reduced sympathetic tone contributing to the lower MAP in hRAMP1 mice. The meager depressor response to losartan in both groups of mice may reflect desensitization of vascular AT1R during the sustained 1-2 week infusion of Ang-II and/or limited penetrance of acutely administered (IV) losartan into the brain. A progressive decrease in the magnitude of the acute depressor response to AT1R blockade and enhancement of the depressor response to ganglionic blockade during sustained Ang-II induced hypertension have been shown previously in rats (35), suggesting the predominant contribution of central AT1R to the hypertensive effect of Ang-II.

Our gene expression data support the functional results. Ang-II infusion increased AT1R mRNA expression selectively in brainstem of control mice, an effect that was abrogated in hRAMP1 mice (Table 1). Increased AT1R expression in brainstem has been associated with increased sympathetic nerve activity in heart failure (39). Peripheral AT1R expression in aorta, kidney and DRG was similar in control and hRAMP1 mice suggesting that the lower MAP in hRAMP1 mice was not caused by downregulation of peripheral AT1R.

Ang-II also increased mRAMP1 expression selectively in brainstem, and both mRAMP1 and CGRP expression in brainstem were significantly elevated in Ang-II infused hRAMP1 mice vs. control mice (Table 1). hRAMP1 expression in brainstem of the transgenic mice was confirmed (see Fig. 1B). We speculate that reduced expression of AT1R and increased expression of RAMP1 and CGRP in brainstem may have contributed to the higher baroreflex sensitivity, lower sympathetic tone, and attenuation of hypertension in Ang-II infused hRAMP1 mice. We also observed that CGRP expression was increased in DRG of hRAMP1 mice (Table 1). CGRP-positive sensory neurons in DRG provide widespread innervation of blood vessels and are a major source of circulating CGRP (1,2). Thus, CGRP-induced vasodilation may contribute to the lower MAP in hRAMP1 mice. Future studies will be required to further define the central and peripheral mechanisms by which upregulation of RAMP1 enhances autonomic regulation and reduces blood pressure. It will also be important to determine if the changes in AT1R, CGRP, and RAMP1 mRNA expression shown here translate to changes in protein expression.

While we have focused on the CGRP receptor, it is possible that interactions between RAMP1 with other G protein-coupled receptors (40) may contribute to the autonomic and cardiovascular phenotypes in hRAMP1 mice. RAMP1 interacts with the calcitonin receptor to generate an amylin receptor that can also bind CGRP (41). RAMP1/CLR receptors are also activated by intermedin, the newest member of the calcitonin/CGRP peptide family (42). Furthermore, it is possible that RAMP1 overexpression may modulate the interactions of CLR with RAMP2 or RAMP3, which generate adrenomedullin receptors (25,43). Although functional responses to upregulation of RAMP1 may involve other agonists in addition to CGRP, our finding that the pressor response to the CGRP receptor antagonist CGRP8-37 was enhanced in hRAMP1 mice suggests that CGRP contributes to the lower MAP in these mice (Fig. 5A).

It is important to note that the clinical benefits of upregulating RAMP1 expression in hypertension are likely to extend beyond its blood pressure-lowering effect. Increased blood pressure variability and low values of baroreflex sensitivity, HR variability, and diurnal HR fluctuations increase the risk of cardiovascular events and death in patients with hypertension and myocardial infarction (19,20,44). The deleterious effects of Ang-II infusion on each of these risk factors in control mice were abrogated or reversed in hRAMP1 mice.

Perspectives

We have investigated whether upregulation of a regulatory subunit of the CGRP receptor, RAMP1, may enhance the protective actions of CGRP or related peptides in the cardiovascular system. Indeed, changes in RAMP1 levels under various conditions have been reported including hypertension (4,6), heart failure (45), pregnancy (46), and ureteral obstruction (47). The magnitude of increase in RAMP1 mRNA expression in the failing heart (~3-fold) and the obstructed kidney (~13-fold) (45,47) approach the levels we observed in the hRAMP1 mice (~5-15 fold, Fig. 1). Based on these reports and our results obtained from the genetically engineered hRAMP1 mice, it seems likely that dynamic changes in RAMP1 expression will have functional implications in cardiovascular pathologies and identify RAMP1 as a therapeutic target in hypertension. The feasibility of modulating RAMP1 for therapeutic benefit has been established by the efficacy of RAMP1 antagonists for treating migraine (48,49). Analysis of the benefits and possible side effects of antagonizing and enhancing CGRP-RAMP1 signaling and development of methods to target therapies in a tissue-specific manner will be needed to optimize the therapeutic potential.

Supplementary Material

01

ACKNOWLEDGMENTS

The authors thank members of their laboratories for helpful discussions related to this research and Dr. Baoli Yang for advice and for providing the EIIa-cre mice.

SOURCE OF FUNDING

The work was supported by research grants from the American Heart Association (#0855944G), the NIH (HL14388 and DE016511), and the VA (Merit Review Award).

Footnotes

CONFLICT OF INTEREST/DISCLOSURE

None.

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