Enhanced Vascular Responses to Adrenomedullin in Mice Overexpressing Receptor-ActivityeCModifying Protein 2
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《循环研究杂志》
the Cardiovascular Division (C.W.T., N.C.C., J.E.C., Z.L., A.D.G., R.P., I.Mc.F., M.S.M., S.D.B.) and Division of Reproduction, Endocrinology and Diabetes (G.D., D.S., I.P.), King’s College London, United Kingdom
Research Laboratory for Calcium Metabolism (K.H., L.M.I., J.A.F., W.B.), Department of Orthopedic Surgery, University of Zurich, Balgrist University Hospital, Switzerland
Division of Psychiatry Research (J.G.), University of Zeich, Switzerland.
Abstract
Adrenomedullin (AM) levels are elevated in cardiovascular disease, but little is known of the role of specific receptor components. AM acts via the calcitonin receptor-like receptor (CLR) interacting with a receptor-activityeCmodifying protein (RAMP). The AM1 receptor is composed of CLR and RAMP2, and the calcitonin geneeCrelated peptide (CGRP) receptor of CLR and RAMP1, as determined by molecular and cell-based analysis. This study examines the relevance of RAMP2 in vivo. Transgenic (TG) mice that overexpress RAMP2 in smooth muscle were generated. The role of RAMP2 in the regulation of blood pressure and in vascular function was investigated. Basal blood pressure, acute angiotensin IIeCraised blood pressure, and cardiovascular properties were similar in wild-type (WT) and TG mice. However, the hypotensive effect of IV AM, unlike CGRP, was enhanced in TG mice (P<0.05), whereas a negative inotropic action was excluded by left-ventricular pressureeCvolume analysis. In aorta relaxation studies, TG vessels responded in a more sensitive manner to AM (EC50, 8.0±1.5 nmol/L) than WT (EC50, 17.9±3.6 nmol/L). These responses were attenuated by the AM receptor antagonist, AM22-52, such that residual responses were identical in all mice. Remaining relaxations were further inhibited by CGRP receptor antagonists, although neither affected AM responses when given alone. Mesenteric and cutaneous resistance vessels were also more sensitive to AM in TG than WT mice. Thus RAMP2 plays a key role in the sensitivity and potency of AM-induced hypotensive responses via the AM1 receptor, providing evidence that this receptor is a selective target for novel therapeutic approaches.
Key Words: adrenomedullin blood pressure CGRP receptor-activityeCmodifying protein RAMP2 transgenic mouse
Introduction
Adrenomedullin (AM), discovered in 1993,1 is considered to play a pivotal role in cardiovascular disease. AM levels are upregulated in vascular tissue in response to factors including inflammatory cytokines and hypoxia.2,3 Plasma concentrations are raised in several cardiovascular conditions including heart failure and sepsis.4eC6 Despite the possible importance of AM in cardiovascular biology, little is known of the receptor components involved. Their influence in vivo has only been inferred from molecular and cellular studies.7,8
AM is a member of the calcitonin family of peptides and acts via unique G proteineClinked receptors, composed of a common calcitonin receptoreClike receptor (CLR) associated with 1 of 3 receptor-activityeCmodifying proteins (RAMPs). The complex of CLR with RAMP1 defines a CGRP receptor, whereas CLR/RAMP2 is an AM1 receptor and the CLR/RAMP3 heterodimer is known as an AM2 receptor.9,10 Studies in a range of species show that CGRP is more potent as a vasodilator than AM and that AM is an agonist at both AM and CGRP receptors, whereas CGRP predominantly acts via CGRP receptors. Both receptors are primarily linked to cAMP synthesis,8,11 but vascular relaxation can also occur via endothelial-derived NO-dependent mechanisms.12 Experiments in mice have demonstrated the importance of AM in vascular development as homozygous AM knockout mice exhibit a fatal phenotype.13,14 However, AM transgenics and heterozygote knockouts show that AM plays a protective role in sepsis,15 ischemia,16 and cardiovascular damage.17 Thus upregulation of the peptide correlates with important cardiovascular activities. Recently, a novel form of AM, AM2, has been identified that possesses vasodilator activity.18 Currently, little is known of the functional importance of receptor components and the relative importance of CGRP and AM receptors in AM responses in vivo.11
The murine RAMPs have been cloned and shown to interact with CLR as described above in single-cell systems.19,20 Of potential functional relevance, decreased RAMP2 mRNA expression has been reported in the umbilical artery of patients with pregnancy-induced hypertension.21 In addition, mRNA for RAMP2 is upregulated in rodent models of cardiovascular disease.22,23,24 Furthermore, increased gene expression of RAMP2 has been detected in left-ventricular hypertrophy and, to a greater degree, after the transition to heart failure.25 Thus increased expression of RAMP proteins may be related to the onset and progression of cardiovascular disease. To probe the functional relevance of the AM1 receptor in vivo, transgenic (TG) mice have been developed that overexpress RAMP2 primarily in smooth muscle containing tissues. We demonstrate for the first time that overexpression of RAMP2 leads to an enhanced selective vascular responsiveness to AM, but not CGRP, through functionally active AM1 receptors.
Materials and Methods
Generation of RAMP2 TG Mice
The transgene consisted of an -actin promoter fragment fused to DNA encoding the signal sequence of the CD33 protein, a myc epitope-tag and mouse (m) RAMP2 lacking the signal sequence (Figure 1). A DNA fragment encoding myc-mRAMP2 was excised from a pcDNA3 expression construct20 and cloned into a pSMP8-CAT vector,26 replacing the chloramphenicol acetyltransferase (CAT) coding sequence, a SV40 small t intron and a SV40 polyadenylation signal downstream of the -actin promoter. The transgene was excised from the vector before pronuclear injection of B6D2F1 X B6D2F1 oocytes, as previously described.27 The genotype was determined by PCR analysis of genomic DNA extracted from tail biopsies. Transgene-specific primers 5'-AGCAAAAGCTC- ATTTCTGAAGAGG-3' and 5'-GTGGGAAGGATGAGAGTAA-GTGG-3' were used (in house). PCR products with a predicted size of 578 bp were analyzed by agarose (1.8% wt/vol) gel electrophoresis in TBE buffer (45 mmol/L)/EDTA (1 mmol/L) (Figure 1). Hemizygous TG mice were mated with wild-type (WT) B6D2F1 mice. TG hemizygous and non-TG littermates were used.
Western Blot Analysis and Immunohistochemistry
Expression of myc-mRAMP2 was examined by Western blot on tissue extracts and by immunohistochemistry on frozen mouse aorta sections. Tissues from TG mice and controls were sonicated in 125 mmol/L Tris/HCl, pH 6.8, 20% (vol/vol) glycerol, 4.5% (wt/vol) sodium dodecyl sulfate (SDS), and 500 mmol/L -mercaptoethanol (Ultrasonic, Olainview, NY) and then rotated at 4°C for 2 hours. Tissue fragments were removed by centrifugation. Fifty micrograms of the extracted proteins were separated by 12% SDS-PAGE. The proteins were transferred to a nitrocellulose membrane, which was blocked with 5% low fat milk in 50 mmol/L Tris/HCl, pH 7.5, and 150 mmol/L NaCl (TBS) at 4°C for 2 hours. Myc-tagged mRAMP2 was detected with antigen-purified rabbit antibodies to myc (1:6000; Bethyl Laboratories Inc, Montgomery, Tex) and with secondary alkaline phosphatase conjugated goat anti-rabbit antibodies (Sigma, St Louis, Mo), 1:30 000 in TBS+0.1% Tween 20 and 1% low fat milk. The proteins were visualized with Immun-Star-AP Chemiluminescent reagent (Bio-Rad, Hercules, Calif). Ten-micrometer frozen sections of aorta from TG and control mice were fixed with 2% formaldehyde (Sigma). Myc-mRAMP2 was immunostained with antigen-purified goat antibodies to myc (1:100; Bethyl Laboratories Inc). Bound antibodies were visualized with biotinylated rabbit anti-goat antibodies from the Vectastain Elite ABC detection kit (Vector Laboratories, Burlingame, Calif) and with Enhanced DBA (Pierce Biotechnology Inc, Rockford, Ill).
Measurement of Blood Pressure and Cardiac Function
Experiments were performed under the Animals (Scientific Procedures) Act, 1986, UK. Physical characteristics of the mice are shown in Table 1. Age- and sex-matched adult mice were used to measure blood pressure in the conscious mouse using tail cuff plethysmography (Coda 6; Kent Scientific, Torrington, Conn). Alternatively, mice were anesthetized with urethane (2.5 mg/g, IP; Baxter Healthcare, Berkshire, UK). The left carotid artery was cannulated (external diameter <0.7 mm) and connected to a transducer and data acquisition systems (Disposable BP Transducer and PowerLab, ADInstruments, Oxfordshire, UK). Agents were administered IV. To measure cardiac performance, mice were isoflurane (1.5%) anesthetized and artificially ventilated (MiniVent Type 845; Hugo Sachs Elektronik, March-Hugstetten, Germany). Left-ventricular pressure and volume were measured using a microconductance catheter (SPR-839; Millar Instruments, Houston, Tex) inserted into the left ventricle via an apical puncture. PressureeCvolume loops were acquired under normal conditions and with varying preload, at baseline and 2 minutes following IV administration of vehicle or AM (12 nmol/kg). Data were analyzed using PVAN 2.9 Millar Instruments software.
Vascular Relaxant Activity
Mice were killed by CO2 exposure and cervical dislocation. The thoracic aortae were dissected, cut transversely into rings 2 to 4 mm wide, and mounted in organ baths containing Krebs solution (composition in mmol/L: 120 NaCl, 4.7 KCl, 25 NaHCO3, 0.5 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, and 11 glucose) gassed with 95% O2/5% CO2 at 37°C. Tissues were equilibrated for 60 minutes. Responses were measured isometrically using force displacement transducers (Lectromed UK Ltd, Hertfordshire, UK) and recorded via a MacLab (ADInstruments). Aortic rings were precontracted with noradrenaline (NA) (100 nmol/L; >0.2 g tension). Increasing concentrations of CGRP or AM were added cumulatively. Tissues were preincubated with antagonist for 20 minutes before addition of agonist. The response to acetylcholine (1 eol/L) was assessed to ensure an intact endothelium (>50% relaxation). Endothelium was removed when required. Results are expressed as percentage relaxation of the NA response. Second-order branches of the mesenteric arcade were dissected and mounted on a MulvanyeCHalpern small vessel wire myograph (model 610D; DMT, Aarhus, Denmark) containing Krebs solution gassed as above, at 37°C. Vessels (0.2 to 0.3 mm at 100 mm Hg pressure) were normalized28 and viability assessed. After 15 minutes of equilibration, arteries were submaximally constricted with NA (80% of maximum), and studies with agonists performed as above in endothelium intact vessels.
Responses in Cutaneous Microvasculature
The assay is based on the ability of microvascular vasodilators (eg, CGRP and AM) to potentiate plasma extravasation induced by substance P (SP), as described previously.29,30 The assay allows indirect quantitative measurement of the vasodilator effects of AM and CGRP, as the direct measurement of blood flow is difficult in murine dorsal skin. Urethane anesthetized mice (2.5 mg/g, IP) were administered with 125I-labeled BSA (125I-BSA) (30 to 60 kBq IV). SP±AM or CGRP were injected intradermally (ID) in Tyrode’s solution (composition in mmol/L: 136.9 NaCl, 2.68 KCl, 0.42 NaH2PO4, 11.9 NaHCO3, 1.05 MgCl2, and 5.55 glucose). Accumulation of extravasated 125I-BSA was assessed 30 minutes after ID injection; plasma was obtained following cardiac puncture and dorsal skin sites (8 mm diameter) were removed and weighed. 125I-BSA radioactivity was assessed in skin and plasma samples (Wallac 1260 Multigamma II; Buckinghamshire, UK).
Histological Analysis
Thoracic aorta and mesenteric tissue were placed in 10% formalin. Aortae were cut transversely into 3 sections. The tissues were processed for histology of paraffin sections. Slices (4 e) were stained with hematoxylin/eosin. Aortic wall width was measured at 10 sites in each of the three sections and an average value obtained for each tissue. Mesenteric vessel diameter was measured at 2 sites in ten arterioles and the mean vessel wall width determined from readings at 4 sites in each. The average value was taken for each of 3 tissues.
Ligands
Human -CGRP (CGRP) was purchased from Phoenix Pharmaceuticals Ltd. Stock solutions were made in 0.01% BSA, as were dilutions. AM1-52 (human), AM22-52, and CGRP8-37 were purchased from Bachem (Merseyside, UK) and BIBN4096BS31 was a gift from Boehringer Ingelheim (Ingelheim, Germany).
Expression of Results and Statistical Analysis
Results are as mean±SEM For blood pressure analysis; data were analyzed using unpaired t tests. Otherwise, statistical analysis was by ANOVA plus Bonferroni’s modified t test.
Results
Myc-mRAMP2 TG Mice
Eight myc-mRAMP2 founder animals were obtained of which 2 were propagated to obtain lines. The TG mice developed normally and were fertile and healthy. Mating of hemizygous TG mice with WT animals produced 50% TG offspring with an equal sex distribution. Expression of myc-mRAMP2 was predominant in tissues rich in smooth muscle cells, including the stomach and urinary bladder (Figure 2a). Expression was also found in the smooth muscle cell layer of the aorta. Relatively low or negligible levels of expression were observed in liver, kidney, and brain. Of the 2 lines, 1 was selected for detailed analysis of which physical characteristics are shown in Table 1. The morphology of the aorta and mesenteric microvessels in WT and TG mice was histologically indistinguishable (Figure 2b).
Blood Pressure in WT and TG Mice and Enhanced Response to AM
Baseline mean arterial pressure (MAP) and heart rate (HR), was measured in conscious (WT: 117±8 mm Hg, 714±19 bpm; TG: 118±5 mm Hg, 708±23 bpm; n=12) and urethane anesthetized mice (WT: 80±8.5 mm Hg, 420±44 bpm; TG: 85±2 mm Hg, 404±30 bpm; n=4) and was indistinguishable between WT and TG groups. IV administration of submaximal doses of AM (5.2 nmol/kg in conscious, 12 nmol/kg in anesthetized mice) and CGRP (0.4 nmol/kg) produced decreases in blood pressure (Figure 3). Whereas AM-induced depressor effects were significantly enhanced in TG mice, CGRP-induced depressor effects were unaltered in conscious and urethane anesthetized mice (Figure 3). This indicates that the TG mice show an increased sensitivity to AM following increased expression of RAMP2 and in turn an increase in functional AM1 (CLR/RAMP2) receptors. Table 2 summarizes cardiac function from readings obtained at baseline and following administration of AM. WT and TG mice were indistinguishable under basal conditions. In WT mice, AM (12 nmol/kg) produced a significant increase in HR, with no other cardiac effects. In TG mice, AM had a comparable effect on HR, whereas stroke volume was significantly increased and end-systolic pressure reduced. These changes likely reflect the enhanced systemic vasodilation with AM on the TG background because there were no significant changes in the independent indices preload adjusted maximal power and preload recruitable stroke work.
The role of AM receptors in conditions of raised blood pressure was also examined. IV injections of the vasoconstrictor agent angiotensin II increased blood pressure in anesthetized mice, which was indistinguishable between WT and TG groups (ED50: WT, 3.3±0.4 nmol/kg; TG, 4.0±1.9 nmol/kg; Figure 3e).
Relaxation to AM in Isolated Aortic Tissues of WT and TG Mice
The maximum contractile responses to NA in both WT and TG tissues was comparable (0.29±0.01 g and 0.28±0.01 g, respectively). The cumulative addition of AM to aortae precontracted with NA revealed a concentration-dependent relaxation with increased sensitivity from TG as compared with WT mice; EC50 values for AM in WT and TG mice were 17.9±3.6 nmol/L and 7.9±1.5 nmol/L (P<0.01), respectively. The relaxant effect of AM at doses up to 10 nmol/L was inhibited by the AM antagonist AM22-52, in both WT and RAMP2 TG mice (Figure 4). The residual response observed in the presence of AM22-52 was of a similar magnitude in WT and TG mice. These findings suggest that at low concentrations, the effects of AM are mediated through the AM1 (CLR/RAMP2) receptor (potentiated in TG mice), whereas at higher concentrations (>30 nmol/L), the peptide acts through a distinct receptor, possibly the CGRP receptor (see below).
The role of the endothelium in the relaxant response to AM was assessed using endothelium intact and denuded vessels, determined by response to acetylcholine (Figure 4c). The responses to AM in both WT and TG mice were the same in the presence and absence of endothelium (Figure 4d). These data suggest that the response to AM is not endothelium dependent and thus may be stimulating vascular relaxation via receptors in vascular smooth muscle cells in both TG and WT mice.
The possibility that the residual responses to AM observed in the presence of the antagonist AM22-52 were attributable to AM acting via the CGRP (CLR/RAMP1) receptor was tested by examining the effect of the CGRP antagonists CGRP8-37 and BIBN4096BS. Neither antagonist caused significant inhibition of AM responses when administered individually (Figure 5), at concentrations that significantly inhibited CGRP responses. Interestingly, both CGRP antagonists, in the presence of AM22-52, caused a significant reduction (P<0.01) in the residual response to AM, providing evidence that AM can act via CGRP receptors, in addition to AM receptors, to mediate relaxation.
Relaxation to AM in Resistance Vessels
The maximum response to NA induced tone in second-order mesenteric arteries was similar in both WT and TG mesenteric vessels (1.86±0.15 mN/mm and 1.75±0.12 mN/mm, respectively). Arteries from TG mice exhibited an enhanced relaxant response to AM at 30 and 300 nmol/L compared with WT (P<0.05; Figure 6a and 6b). In contrast, responses to CGRP were similar.
Previous studies demonstrate the ability of AM and CGRP to act as vasodilators and potentiate plasma extravasation in the cutaneous microvasculature.29 Here AM potentiated the effects of SP in both WT and TG mice (Figure 6c through 6f). A significant potentiating effect of SP was observed at 30 pmol/site of AM (P<0.05) in the TG mice but not the WT littermates. By comparison, CGRP (0.03 pmol/site) potentiated edema formation in WT mice, but not in TG littermates. Thus in the TG mice an increase in the sensitivity to AM and a concomitant decrease in sensitivity to CGRP is shown.
Discussion
This study, through use of RAMP2 TG mice, displaying normal growth and physical characteristics, has demonstrated for the first time the functional importance of RAMP2 in influencing AM responses in vivo. The results provide direct evidence that upregulation of RAMP2 in vascular smooth muscle cells leads to an increased sensitivity to AM, in terms of vasoactive responses. These effects are unlikely to be compounded by a direct action on the heart because the changes observed were compensatory and exaggerated in RAMP2 TG mice. Thus AM produced an enhanced hypotensive vasodilatory effect as demonstrated by direct measurement of blood pressure in vivo, tone in isolated aortae, and mesenteric and skin microvessels. These results provide functional correlates to in vitro molecular studies describing the activity and the pharmacology of the RAMPs subsequent to the discovery that signaling of these members of the CGRP family of peptides occurs via the formation of a CLR/RAMP complex.9
Similar basal blood pressure was observed in WT and TG animals, suggesting basal concentrations of AM are too low to influence blood pressure in the normal mouse. This is in keeping with studies of conscious genetically modified C57Bl6 mice with reduced AM concentrations (AM+/eC).17 However, a small but significant increase in basal blood pressure in AM+/eC compared with WT mice has also been reported.14 Overall, the results from the present experiments support the theory that basal concentrations of AM do not play a major role in regulating blood pressure. However, IV administration of AM, in both the anesthetized and conscious mouse, revealed an enhanced hypotensive effect attributable to increased vascular relaxation in TG when compared with WT mice. This is a critical result in determining the influence of RAMP2 on biological activity. The vascular wall is a major source of AM2 and circulating AM is raised in a number of cardiovascular disease conditions.11 By comparison, the response of the more potent, structurally related peptide CGRP, that is considered to mediate vasoactive effects via the CGRP (CLR/RAMP1) receptor, was similar in both WT and TG mice. This is evidence that the presence of excess RAMP2 does not influence the level of functional CGRP receptors in the blood vessels.
Examination of responses in isolated aorta allowed a detailed investigation of the receptors involved and of the pharmacological phenotype of the RAMP2 TG at the vascular level. A major finding from the comparative study of isolated aortic and mesenteric microvessels is the increased responsiveness to AM in TG compared with WT tissue. Again, CGRP induced similar responses in both WT and TG mice. Detailed pharmacological analysis was performed in the aorta. The AM receptor antagonist AM22-5232 is of relatively low affinity but the only available inhibitor. It is a more selective antagonist at the AM1 (CLR/RAMP2) than at the AM2 (CLR/RAMP3) receptor.33 By comparison, the CGRP antagonists CGRP8-3734 and BIBN4096BS31 are both established as selective CGRP (CLR/RAMP1) antagonists. BIBN4096BS is considered to possess greater selectivity for the CGRP receptor than CGRP8-37 from cell-based studies.33,35 BIBN4096BS, although extremely effective against the human CGRP receptor, is approximately 200-fold less active but selective against the rodent CGRP receptor.30,31,36 Here, the experiments reveal the importance of both AM and CGRP receptors in mediating the AM response. A striking observation is that AM22-52 acted to inhibit the AM-induced responses such that the residual response was similar in both WT and TG mice. This provides crucial evidence that RAMP2 was able to complex in a functional manner with CLR on the cell surface as an AM1 receptor in RAMP2 TG mice and proves the phenotype of the RAMP2 TG mice from a pharmacological viewpoint. Alone, the CGRP antagonists had no effect on AM responses but inhibited each response to a similar extent when given in the presence of AM22-52. CGRP8-37, but not BIBN4096BS, is considered to also have antagonist activity at the AM2 (CLR/RAMP3) receptor.37 The similar inhibitory profile of CGRP8-37 (selective at CGRP and AM2 receptors) and BIBN4096BS (selective at the CGRP receptor) in the presence of CGRP indicates a lack of functional involvement of the AM2 receptor, of which least is known. Interestingly, the AM1 receptor appears to mediate the sensitivity of the tissue to AM as observed by a shift in the dose response curve to the right in the presence of AM22-52. The inhibitory activity of the CGRP antagonists was only revealed once the AM1 receptor had been blocked. This suggests redundancy in that the interaction of the CGRP receptor with AM at high nanomolar concentrations is only revealed under situations where the AM1 receptor is blocked. Both CGRP receptor antagonists acted similarly, on the vasoactive response to AM, such that the residual responses to AM in the presence of AM22-52 with either CGRP8-37 or BIBN4096BS were attenuated.
CGRP and AM are established potent vascular relaxants in the mouse aorta.37eC39 Interestingly, studies with AM TG mice, where the AM has been targeted to endothelial cells, indicate that the endothelial cell and NO play critical roles in the relaxant response to AM.39 Partial (20%) inhibition of AM responses has been observed in endothelial-denuded vessels37 or endothelial NO synthase knockout mice.39 By comparison, the present results, with overexpression of AM1 (CLR/RAMP2) receptors in smooth muscle cells, suggest that endothelial cells, and thus NO, play a minor role in AM-evoked vascular relaxation. However, there are acknowledged species and tissue differences,11 with respect to the relative involvement of the endothelial-dependent mechanisms as compared with smooth muscle mechanisms involving increased cAMP.
The CGRP receptor has been considered essential for mediating both AM and CGRP responses in the cutaneous microvasculature40,41 as effects are inhibited by CGRP antagonists in several species including mice.30 The present results provide evidence that the AM receptor can become involved in mediating AM responses, at low agonist concentrations. An increase in sensitivity to AM and a decrease in sensitivity to CGRP in TG mice is observed. It has been previously shown in rat osteoblast-like cells that competition between RAMP1 and RAMP2 for the CLR exists.42 It is possible that at low agonist concentrations in the cutaneous microvasculature, the usually predominant RAMP1 is no longer able to compete with the overexpressed RAMP2 for CLR, and thus an increase in potency of AM in TG mice is observed.
In conclusion, there are now several publications in which the effect of increased levels of AM have been studied or detected in animal models and human cardiovascular disease,43 but this is the first time the AM1 receptor, rather than the peptide, has been targeted for study in vivo. Through use of RAMP2 TG mice, we provide direct evidence for a functional relevance of RAMP2 in influencing AM vasoactive responses. When the in vivo and isolated tissue studies are combined, they provide compelling evidence for the primary importance of AM1 receptor in vasorelaxant responses to AM. Thus AM receptor agonists, or strategies that enhance AM receptor activity, have potential value in enhancing vascular hypotensive states and possibly in enhancing a range of cardiovascular protective effects.
Acknowledgments
This work was supported by the British Heart Foundation (UK), King’s College London, the British Biotechnology Scientific Research Council, Tommy’s the Baby Charity, the Swiss National Research Foundation, The Wellcome Trust, and the Schweizerische Verein Balgrist. Z.L. was supported by an Eotvos Hungarian Postdoctoral Fellowship. We thank D. Schuppli for excellent technical expertise in generating the RAMP2 transgenic mice, T. Clemens for providing the pSMP8-CAT vector, and Dr M. Curtis and Prof M. Avkiran for discussion.
These authors contributed equally to this study.
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Tam C, Brain SD. The assessment of vasoactive properties of CGRP and adrenomedullin in the microvasculature: a study using in vivo and in vitro assays in the mouse. J Mol Neurosci. 2004; 22: 117eC124.
Grant AD, Tam CW, Lazar Z, Shih MK, Brain SD. The calcitonin gene-related peptide (CGRP) receptor antagonist BIBN4096BS blocks CGRP and adrenomedullin vasoactive responses in the microvasculature. Br J Pharmacol. 2004; 142: 1091eC1098.
Doods H, Hallermayer G, Wu D, Entzeroth M, Rudolf K, Engel W, Eberlein W. Pharmacological profile of BIBN4096BS, the first selective small molecule CGRP antagonist. Br J Pharmacol. 2000; 129: 420eC423.
Eguchi S, Hirata Y, Iwasaki H, Sato K, Watanabe TX, Inui T, Nakajima K, Sakakibara S, Marumo F. Structure-activity relationship of adrenomedullin, a novel vasodilatory peptide, in cultured rat vascular smooth muscle cells. Endocrinology. 1994; 135: 2454eC2458.
Hay DL, Howitt SG, Conner AC, Schindler M, Smith DM, Poyner DR. CL/RAMP2 and CL/RAMP3 produce pharmacologically distinct adrenomedullin receptors: a comparison of effects of adrenomedullin22eC52, CGRP8eC37 and BIBN4096BS. Br J Pharmacol. 2003; 140: 477eC486.
Chiba T, Yamaguchi A, Yamatani T, Nakamura A, Morishita T, Inui T, Fukase M, Noda T, Fujita T. Calcitonin gene-related peptide receptor antagonist human CGRP-(8eC37). Am J Physiol. 1989; 256: E331eCE335.
Hay DL, Howitt SG, Conner AC, Doods H, Schindler M, Poyner DR. A comparison of the actions of BIBN4096BS and CGRP(8eC37) on CGRP and adrenomedullin receptors expressed on SK-N-MC, L6, Col 29 and Rat 2 cells. Br J Pharmacol. 2002; 137: 80eC86.
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Chu DQ, Legon S, Smith DM, Costa SK, Cuttitta F, Brain SD. The calcitonin gene-related peptide (CGRP) antagonist CGRP(8eC37) blocks vasodilatation in inflamed rat skin: involvement of adrenomedullin in addition to CGRP. Neurosci Lett. 2001; 310: 169eC172.
Buhlmann N, Leuthauser K, Muff R, Fischer JA, Born W. A receptor activity modifying protein (RAMP)2-dependent adrenomedullin receptor is a calcitonin gene-related peptide receptor when coexpressed with human RAMP1. Endocrinology. 1999; 140: 2883eC2890.
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Research Laboratory for Calcium Metabolism (K.H., L.M.I., J.A.F., W.B.), Department of Orthopedic Surgery, University of Zurich, Balgrist University Hospital, Switzerland
Division of Psychiatry Research (J.G.), University of Zeich, Switzerland.
Abstract
Adrenomedullin (AM) levels are elevated in cardiovascular disease, but little is known of the role of specific receptor components. AM acts via the calcitonin receptor-like receptor (CLR) interacting with a receptor-activityeCmodifying protein (RAMP). The AM1 receptor is composed of CLR and RAMP2, and the calcitonin geneeCrelated peptide (CGRP) receptor of CLR and RAMP1, as determined by molecular and cell-based analysis. This study examines the relevance of RAMP2 in vivo. Transgenic (TG) mice that overexpress RAMP2 in smooth muscle were generated. The role of RAMP2 in the regulation of blood pressure and in vascular function was investigated. Basal blood pressure, acute angiotensin IIeCraised blood pressure, and cardiovascular properties were similar in wild-type (WT) and TG mice. However, the hypotensive effect of IV AM, unlike CGRP, was enhanced in TG mice (P<0.05), whereas a negative inotropic action was excluded by left-ventricular pressureeCvolume analysis. In aorta relaxation studies, TG vessels responded in a more sensitive manner to AM (EC50, 8.0±1.5 nmol/L) than WT (EC50, 17.9±3.6 nmol/L). These responses were attenuated by the AM receptor antagonist, AM22-52, such that residual responses were identical in all mice. Remaining relaxations were further inhibited by CGRP receptor antagonists, although neither affected AM responses when given alone. Mesenteric and cutaneous resistance vessels were also more sensitive to AM in TG than WT mice. Thus RAMP2 plays a key role in the sensitivity and potency of AM-induced hypotensive responses via the AM1 receptor, providing evidence that this receptor is a selective target for novel therapeutic approaches.
Key Words: adrenomedullin blood pressure CGRP receptor-activityeCmodifying protein RAMP2 transgenic mouse
Introduction
Adrenomedullin (AM), discovered in 1993,1 is considered to play a pivotal role in cardiovascular disease. AM levels are upregulated in vascular tissue in response to factors including inflammatory cytokines and hypoxia.2,3 Plasma concentrations are raised in several cardiovascular conditions including heart failure and sepsis.4eC6 Despite the possible importance of AM in cardiovascular biology, little is known of the receptor components involved. Their influence in vivo has only been inferred from molecular and cellular studies.7,8
AM is a member of the calcitonin family of peptides and acts via unique G proteineClinked receptors, composed of a common calcitonin receptoreClike receptor (CLR) associated with 1 of 3 receptor-activityeCmodifying proteins (RAMPs). The complex of CLR with RAMP1 defines a CGRP receptor, whereas CLR/RAMP2 is an AM1 receptor and the CLR/RAMP3 heterodimer is known as an AM2 receptor.9,10 Studies in a range of species show that CGRP is more potent as a vasodilator than AM and that AM is an agonist at both AM and CGRP receptors, whereas CGRP predominantly acts via CGRP receptors. Both receptors are primarily linked to cAMP synthesis,8,11 but vascular relaxation can also occur via endothelial-derived NO-dependent mechanisms.12 Experiments in mice have demonstrated the importance of AM in vascular development as homozygous AM knockout mice exhibit a fatal phenotype.13,14 However, AM transgenics and heterozygote knockouts show that AM plays a protective role in sepsis,15 ischemia,16 and cardiovascular damage.17 Thus upregulation of the peptide correlates with important cardiovascular activities. Recently, a novel form of AM, AM2, has been identified that possesses vasodilator activity.18 Currently, little is known of the functional importance of receptor components and the relative importance of CGRP and AM receptors in AM responses in vivo.11
The murine RAMPs have been cloned and shown to interact with CLR as described above in single-cell systems.19,20 Of potential functional relevance, decreased RAMP2 mRNA expression has been reported in the umbilical artery of patients with pregnancy-induced hypertension.21 In addition, mRNA for RAMP2 is upregulated in rodent models of cardiovascular disease.22,23,24 Furthermore, increased gene expression of RAMP2 has been detected in left-ventricular hypertrophy and, to a greater degree, after the transition to heart failure.25 Thus increased expression of RAMP proteins may be related to the onset and progression of cardiovascular disease. To probe the functional relevance of the AM1 receptor in vivo, transgenic (TG) mice have been developed that overexpress RAMP2 primarily in smooth muscle containing tissues. We demonstrate for the first time that overexpression of RAMP2 leads to an enhanced selective vascular responsiveness to AM, but not CGRP, through functionally active AM1 receptors.
Materials and Methods
Generation of RAMP2 TG Mice
The transgene consisted of an -actin promoter fragment fused to DNA encoding the signal sequence of the CD33 protein, a myc epitope-tag and mouse (m) RAMP2 lacking the signal sequence (Figure 1). A DNA fragment encoding myc-mRAMP2 was excised from a pcDNA3 expression construct20 and cloned into a pSMP8-CAT vector,26 replacing the chloramphenicol acetyltransferase (CAT) coding sequence, a SV40 small t intron and a SV40 polyadenylation signal downstream of the -actin promoter. The transgene was excised from the vector before pronuclear injection of B6D2F1 X B6D2F1 oocytes, as previously described.27 The genotype was determined by PCR analysis of genomic DNA extracted from tail biopsies. Transgene-specific primers 5'-AGCAAAAGCTC- ATTTCTGAAGAGG-3' and 5'-GTGGGAAGGATGAGAGTAA-GTGG-3' were used (in house). PCR products with a predicted size of 578 bp were analyzed by agarose (1.8% wt/vol) gel electrophoresis in TBE buffer (45 mmol/L)/EDTA (1 mmol/L) (Figure 1). Hemizygous TG mice were mated with wild-type (WT) B6D2F1 mice. TG hemizygous and non-TG littermates were used.
Western Blot Analysis and Immunohistochemistry
Expression of myc-mRAMP2 was examined by Western blot on tissue extracts and by immunohistochemistry on frozen mouse aorta sections. Tissues from TG mice and controls were sonicated in 125 mmol/L Tris/HCl, pH 6.8, 20% (vol/vol) glycerol, 4.5% (wt/vol) sodium dodecyl sulfate (SDS), and 500 mmol/L -mercaptoethanol (Ultrasonic, Olainview, NY) and then rotated at 4°C for 2 hours. Tissue fragments were removed by centrifugation. Fifty micrograms of the extracted proteins were separated by 12% SDS-PAGE. The proteins were transferred to a nitrocellulose membrane, which was blocked with 5% low fat milk in 50 mmol/L Tris/HCl, pH 7.5, and 150 mmol/L NaCl (TBS) at 4°C for 2 hours. Myc-tagged mRAMP2 was detected with antigen-purified rabbit antibodies to myc (1:6000; Bethyl Laboratories Inc, Montgomery, Tex) and with secondary alkaline phosphatase conjugated goat anti-rabbit antibodies (Sigma, St Louis, Mo), 1:30 000 in TBS+0.1% Tween 20 and 1% low fat milk. The proteins were visualized with Immun-Star-AP Chemiluminescent reagent (Bio-Rad, Hercules, Calif). Ten-micrometer frozen sections of aorta from TG and control mice were fixed with 2% formaldehyde (Sigma). Myc-mRAMP2 was immunostained with antigen-purified goat antibodies to myc (1:100; Bethyl Laboratories Inc). Bound antibodies were visualized with biotinylated rabbit anti-goat antibodies from the Vectastain Elite ABC detection kit (Vector Laboratories, Burlingame, Calif) and with Enhanced DBA (Pierce Biotechnology Inc, Rockford, Ill).
Measurement of Blood Pressure and Cardiac Function
Experiments were performed under the Animals (Scientific Procedures) Act, 1986, UK. Physical characteristics of the mice are shown in Table 1. Age- and sex-matched adult mice were used to measure blood pressure in the conscious mouse using tail cuff plethysmography (Coda 6; Kent Scientific, Torrington, Conn). Alternatively, mice were anesthetized with urethane (2.5 mg/g, IP; Baxter Healthcare, Berkshire, UK). The left carotid artery was cannulated (external diameter <0.7 mm) and connected to a transducer and data acquisition systems (Disposable BP Transducer and PowerLab, ADInstruments, Oxfordshire, UK). Agents were administered IV. To measure cardiac performance, mice were isoflurane (1.5%) anesthetized and artificially ventilated (MiniVent Type 845; Hugo Sachs Elektronik, March-Hugstetten, Germany). Left-ventricular pressure and volume were measured using a microconductance catheter (SPR-839; Millar Instruments, Houston, Tex) inserted into the left ventricle via an apical puncture. PressureeCvolume loops were acquired under normal conditions and with varying preload, at baseline and 2 minutes following IV administration of vehicle or AM (12 nmol/kg). Data were analyzed using PVAN 2.9 Millar Instruments software.
Vascular Relaxant Activity
Mice were killed by CO2 exposure and cervical dislocation. The thoracic aortae were dissected, cut transversely into rings 2 to 4 mm wide, and mounted in organ baths containing Krebs solution (composition in mmol/L: 120 NaCl, 4.7 KCl, 25 NaHCO3, 0.5 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, and 11 glucose) gassed with 95% O2/5% CO2 at 37°C. Tissues were equilibrated for 60 minutes. Responses were measured isometrically using force displacement transducers (Lectromed UK Ltd, Hertfordshire, UK) and recorded via a MacLab (ADInstruments). Aortic rings were precontracted with noradrenaline (NA) (100 nmol/L; >0.2 g tension). Increasing concentrations of CGRP or AM were added cumulatively. Tissues were preincubated with antagonist for 20 minutes before addition of agonist. The response to acetylcholine (1 eol/L) was assessed to ensure an intact endothelium (>50% relaxation). Endothelium was removed when required. Results are expressed as percentage relaxation of the NA response. Second-order branches of the mesenteric arcade were dissected and mounted on a MulvanyeCHalpern small vessel wire myograph (model 610D; DMT, Aarhus, Denmark) containing Krebs solution gassed as above, at 37°C. Vessels (0.2 to 0.3 mm at 100 mm Hg pressure) were normalized28 and viability assessed. After 15 minutes of equilibration, arteries were submaximally constricted with NA (80% of maximum), and studies with agonists performed as above in endothelium intact vessels.
Responses in Cutaneous Microvasculature
The assay is based on the ability of microvascular vasodilators (eg, CGRP and AM) to potentiate plasma extravasation induced by substance P (SP), as described previously.29,30 The assay allows indirect quantitative measurement of the vasodilator effects of AM and CGRP, as the direct measurement of blood flow is difficult in murine dorsal skin. Urethane anesthetized mice (2.5 mg/g, IP) were administered with 125I-labeled BSA (125I-BSA) (30 to 60 kBq IV). SP±AM or CGRP were injected intradermally (ID) in Tyrode’s solution (composition in mmol/L: 136.9 NaCl, 2.68 KCl, 0.42 NaH2PO4, 11.9 NaHCO3, 1.05 MgCl2, and 5.55 glucose). Accumulation of extravasated 125I-BSA was assessed 30 minutes after ID injection; plasma was obtained following cardiac puncture and dorsal skin sites (8 mm diameter) were removed and weighed. 125I-BSA radioactivity was assessed in skin and plasma samples (Wallac 1260 Multigamma II; Buckinghamshire, UK).
Histological Analysis
Thoracic aorta and mesenteric tissue were placed in 10% formalin. Aortae were cut transversely into 3 sections. The tissues were processed for histology of paraffin sections. Slices (4 e) were stained with hematoxylin/eosin. Aortic wall width was measured at 10 sites in each of the three sections and an average value obtained for each tissue. Mesenteric vessel diameter was measured at 2 sites in ten arterioles and the mean vessel wall width determined from readings at 4 sites in each. The average value was taken for each of 3 tissues.
Ligands
Human -CGRP (CGRP) was purchased from Phoenix Pharmaceuticals Ltd. Stock solutions were made in 0.01% BSA, as were dilutions. AM1-52 (human), AM22-52, and CGRP8-37 were purchased from Bachem (Merseyside, UK) and BIBN4096BS31 was a gift from Boehringer Ingelheim (Ingelheim, Germany).
Expression of Results and Statistical Analysis
Results are as mean±SEM For blood pressure analysis; data were analyzed using unpaired t tests. Otherwise, statistical analysis was by ANOVA plus Bonferroni’s modified t test.
Results
Myc-mRAMP2 TG Mice
Eight myc-mRAMP2 founder animals were obtained of which 2 were propagated to obtain lines. The TG mice developed normally and were fertile and healthy. Mating of hemizygous TG mice with WT animals produced 50% TG offspring with an equal sex distribution. Expression of myc-mRAMP2 was predominant in tissues rich in smooth muscle cells, including the stomach and urinary bladder (Figure 2a). Expression was also found in the smooth muscle cell layer of the aorta. Relatively low or negligible levels of expression were observed in liver, kidney, and brain. Of the 2 lines, 1 was selected for detailed analysis of which physical characteristics are shown in Table 1. The morphology of the aorta and mesenteric microvessels in WT and TG mice was histologically indistinguishable (Figure 2b).
Blood Pressure in WT and TG Mice and Enhanced Response to AM
Baseline mean arterial pressure (MAP) and heart rate (HR), was measured in conscious (WT: 117±8 mm Hg, 714±19 bpm; TG: 118±5 mm Hg, 708±23 bpm; n=12) and urethane anesthetized mice (WT: 80±8.5 mm Hg, 420±44 bpm; TG: 85±2 mm Hg, 404±30 bpm; n=4) and was indistinguishable between WT and TG groups. IV administration of submaximal doses of AM (5.2 nmol/kg in conscious, 12 nmol/kg in anesthetized mice) and CGRP (0.4 nmol/kg) produced decreases in blood pressure (Figure 3). Whereas AM-induced depressor effects were significantly enhanced in TG mice, CGRP-induced depressor effects were unaltered in conscious and urethane anesthetized mice (Figure 3). This indicates that the TG mice show an increased sensitivity to AM following increased expression of RAMP2 and in turn an increase in functional AM1 (CLR/RAMP2) receptors. Table 2 summarizes cardiac function from readings obtained at baseline and following administration of AM. WT and TG mice were indistinguishable under basal conditions. In WT mice, AM (12 nmol/kg) produced a significant increase in HR, with no other cardiac effects. In TG mice, AM had a comparable effect on HR, whereas stroke volume was significantly increased and end-systolic pressure reduced. These changes likely reflect the enhanced systemic vasodilation with AM on the TG background because there were no significant changes in the independent indices preload adjusted maximal power and preload recruitable stroke work.
The role of AM receptors in conditions of raised blood pressure was also examined. IV injections of the vasoconstrictor agent angiotensin II increased blood pressure in anesthetized mice, which was indistinguishable between WT and TG groups (ED50: WT, 3.3±0.4 nmol/kg; TG, 4.0±1.9 nmol/kg; Figure 3e).
Relaxation to AM in Isolated Aortic Tissues of WT and TG Mice
The maximum contractile responses to NA in both WT and TG tissues was comparable (0.29±0.01 g and 0.28±0.01 g, respectively). The cumulative addition of AM to aortae precontracted with NA revealed a concentration-dependent relaxation with increased sensitivity from TG as compared with WT mice; EC50 values for AM in WT and TG mice were 17.9±3.6 nmol/L and 7.9±1.5 nmol/L (P<0.01), respectively. The relaxant effect of AM at doses up to 10 nmol/L was inhibited by the AM antagonist AM22-52, in both WT and RAMP2 TG mice (Figure 4). The residual response observed in the presence of AM22-52 was of a similar magnitude in WT and TG mice. These findings suggest that at low concentrations, the effects of AM are mediated through the AM1 (CLR/RAMP2) receptor (potentiated in TG mice), whereas at higher concentrations (>30 nmol/L), the peptide acts through a distinct receptor, possibly the CGRP receptor (see below).
The role of the endothelium in the relaxant response to AM was assessed using endothelium intact and denuded vessels, determined by response to acetylcholine (Figure 4c). The responses to AM in both WT and TG mice were the same in the presence and absence of endothelium (Figure 4d). These data suggest that the response to AM is not endothelium dependent and thus may be stimulating vascular relaxation via receptors in vascular smooth muscle cells in both TG and WT mice.
The possibility that the residual responses to AM observed in the presence of the antagonist AM22-52 were attributable to AM acting via the CGRP (CLR/RAMP1) receptor was tested by examining the effect of the CGRP antagonists CGRP8-37 and BIBN4096BS. Neither antagonist caused significant inhibition of AM responses when administered individually (Figure 5), at concentrations that significantly inhibited CGRP responses. Interestingly, both CGRP antagonists, in the presence of AM22-52, caused a significant reduction (P<0.01) in the residual response to AM, providing evidence that AM can act via CGRP receptors, in addition to AM receptors, to mediate relaxation.
Relaxation to AM in Resistance Vessels
The maximum response to NA induced tone in second-order mesenteric arteries was similar in both WT and TG mesenteric vessels (1.86±0.15 mN/mm and 1.75±0.12 mN/mm, respectively). Arteries from TG mice exhibited an enhanced relaxant response to AM at 30 and 300 nmol/L compared with WT (P<0.05; Figure 6a and 6b). In contrast, responses to CGRP were similar.
Previous studies demonstrate the ability of AM and CGRP to act as vasodilators and potentiate plasma extravasation in the cutaneous microvasculature.29 Here AM potentiated the effects of SP in both WT and TG mice (Figure 6c through 6f). A significant potentiating effect of SP was observed at 30 pmol/site of AM (P<0.05) in the TG mice but not the WT littermates. By comparison, CGRP (0.03 pmol/site) potentiated edema formation in WT mice, but not in TG littermates. Thus in the TG mice an increase in the sensitivity to AM and a concomitant decrease in sensitivity to CGRP is shown.
Discussion
This study, through use of RAMP2 TG mice, displaying normal growth and physical characteristics, has demonstrated for the first time the functional importance of RAMP2 in influencing AM responses in vivo. The results provide direct evidence that upregulation of RAMP2 in vascular smooth muscle cells leads to an increased sensitivity to AM, in terms of vasoactive responses. These effects are unlikely to be compounded by a direct action on the heart because the changes observed were compensatory and exaggerated in RAMP2 TG mice. Thus AM produced an enhanced hypotensive vasodilatory effect as demonstrated by direct measurement of blood pressure in vivo, tone in isolated aortae, and mesenteric and skin microvessels. These results provide functional correlates to in vitro molecular studies describing the activity and the pharmacology of the RAMPs subsequent to the discovery that signaling of these members of the CGRP family of peptides occurs via the formation of a CLR/RAMP complex.9
Similar basal blood pressure was observed in WT and TG animals, suggesting basal concentrations of AM are too low to influence blood pressure in the normal mouse. This is in keeping with studies of conscious genetically modified C57Bl6 mice with reduced AM concentrations (AM+/eC).17 However, a small but significant increase in basal blood pressure in AM+/eC compared with WT mice has also been reported.14 Overall, the results from the present experiments support the theory that basal concentrations of AM do not play a major role in regulating blood pressure. However, IV administration of AM, in both the anesthetized and conscious mouse, revealed an enhanced hypotensive effect attributable to increased vascular relaxation in TG when compared with WT mice. This is a critical result in determining the influence of RAMP2 on biological activity. The vascular wall is a major source of AM2 and circulating AM is raised in a number of cardiovascular disease conditions.11 By comparison, the response of the more potent, structurally related peptide CGRP, that is considered to mediate vasoactive effects via the CGRP (CLR/RAMP1) receptor, was similar in both WT and TG mice. This is evidence that the presence of excess RAMP2 does not influence the level of functional CGRP receptors in the blood vessels.
Examination of responses in isolated aorta allowed a detailed investigation of the receptors involved and of the pharmacological phenotype of the RAMP2 TG at the vascular level. A major finding from the comparative study of isolated aortic and mesenteric microvessels is the increased responsiveness to AM in TG compared with WT tissue. Again, CGRP induced similar responses in both WT and TG mice. Detailed pharmacological analysis was performed in the aorta. The AM receptor antagonist AM22-5232 is of relatively low affinity but the only available inhibitor. It is a more selective antagonist at the AM1 (CLR/RAMP2) than at the AM2 (CLR/RAMP3) receptor.33 By comparison, the CGRP antagonists CGRP8-3734 and BIBN4096BS31 are both established as selective CGRP (CLR/RAMP1) antagonists. BIBN4096BS is considered to possess greater selectivity for the CGRP receptor than CGRP8-37 from cell-based studies.33,35 BIBN4096BS, although extremely effective against the human CGRP receptor, is approximately 200-fold less active but selective against the rodent CGRP receptor.30,31,36 Here, the experiments reveal the importance of both AM and CGRP receptors in mediating the AM response. A striking observation is that AM22-52 acted to inhibit the AM-induced responses such that the residual response was similar in both WT and TG mice. This provides crucial evidence that RAMP2 was able to complex in a functional manner with CLR on the cell surface as an AM1 receptor in RAMP2 TG mice and proves the phenotype of the RAMP2 TG mice from a pharmacological viewpoint. Alone, the CGRP antagonists had no effect on AM responses but inhibited each response to a similar extent when given in the presence of AM22-52. CGRP8-37, but not BIBN4096BS, is considered to also have antagonist activity at the AM2 (CLR/RAMP3) receptor.37 The similar inhibitory profile of CGRP8-37 (selective at CGRP and AM2 receptors) and BIBN4096BS (selective at the CGRP receptor) in the presence of CGRP indicates a lack of functional involvement of the AM2 receptor, of which least is known. Interestingly, the AM1 receptor appears to mediate the sensitivity of the tissue to AM as observed by a shift in the dose response curve to the right in the presence of AM22-52. The inhibitory activity of the CGRP antagonists was only revealed once the AM1 receptor had been blocked. This suggests redundancy in that the interaction of the CGRP receptor with AM at high nanomolar concentrations is only revealed under situations where the AM1 receptor is blocked. Both CGRP receptor antagonists acted similarly, on the vasoactive response to AM, such that the residual responses to AM in the presence of AM22-52 with either CGRP8-37 or BIBN4096BS were attenuated.
CGRP and AM are established potent vascular relaxants in the mouse aorta.37eC39 Interestingly, studies with AM TG mice, where the AM has been targeted to endothelial cells, indicate that the endothelial cell and NO play critical roles in the relaxant response to AM.39 Partial (20%) inhibition of AM responses has been observed in endothelial-denuded vessels37 or endothelial NO synthase knockout mice.39 By comparison, the present results, with overexpression of AM1 (CLR/RAMP2) receptors in smooth muscle cells, suggest that endothelial cells, and thus NO, play a minor role in AM-evoked vascular relaxation. However, there are acknowledged species and tissue differences,11 with respect to the relative involvement of the endothelial-dependent mechanisms as compared with smooth muscle mechanisms involving increased cAMP.
The CGRP receptor has been considered essential for mediating both AM and CGRP responses in the cutaneous microvasculature40,41 as effects are inhibited by CGRP antagonists in several species including mice.30 The present results provide evidence that the AM receptor can become involved in mediating AM responses, at low agonist concentrations. An increase in sensitivity to AM and a decrease in sensitivity to CGRP in TG mice is observed. It has been previously shown in rat osteoblast-like cells that competition between RAMP1 and RAMP2 for the CLR exists.42 It is possible that at low agonist concentrations in the cutaneous microvasculature, the usually predominant RAMP1 is no longer able to compete with the overexpressed RAMP2 for CLR, and thus an increase in potency of AM in TG mice is observed.
In conclusion, there are now several publications in which the effect of increased levels of AM have been studied or detected in animal models and human cardiovascular disease,43 but this is the first time the AM1 receptor, rather than the peptide, has been targeted for study in vivo. Through use of RAMP2 TG mice, we provide direct evidence for a functional relevance of RAMP2 in influencing AM vasoactive responses. When the in vivo and isolated tissue studies are combined, they provide compelling evidence for the primary importance of AM1 receptor in vasorelaxant responses to AM. Thus AM receptor agonists, or strategies that enhance AM receptor activity, have potential value in enhancing vascular hypotensive states and possibly in enhancing a range of cardiovascular protective effects.
Acknowledgments
This work was supported by the British Heart Foundation (UK), King’s College London, the British Biotechnology Scientific Research Council, Tommy’s the Baby Charity, the Swiss National Research Foundation, The Wellcome Trust, and the Schweizerische Verein Balgrist. Z.L. was supported by an Eotvos Hungarian Postdoctoral Fellowship. We thank D. Schuppli for excellent technical expertise in generating the RAMP2 transgenic mice, T. Clemens for providing the pSMP8-CAT vector, and Dr M. Curtis and Prof M. Avkiran for discussion.
These authors contributed equally to this study.
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