Published online before print October 25, 2001, doi: 10.1161/hh2301.100812
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© 2001 American Heart Association, Inc.
Integrative Physiology |
Elevated Sympathetic Nervous Activity in Mice Deficient in 
CGRP
From the Department of Cardiovascular Medicine (Y.O., T.S., Y.K., T.I., Y.W., H.M., Y.I., Y.H., R.N.), Department of Urology (H.N.), Department of Cardiothoracic Surgery (Y.S.), Graduate School of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan; Department of Physiology (Y.K., T.K.), School of Medicine, Chiba University, Inohana, Chiba, Japan; The Hospital International Medical Center of Japan (Y.Y.), Tokyo, Japan; and Division of Integrative Cell Biology (H.K.), Department of Embryogenesis, Institute of Molecular Embryology and Genetics, Kumamoto University, Honjo, Kumamoto, Japan.
Correspondence to Hiroki Kurihara, Division of Integrative Cell Biology, Department of Embryogenesis, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan. E-mail kurihara{at}kaiju.medic.kumamoto-u.ac.jp
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Abstract |
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-Calcitonin gene-related peptide (CGRP) is a pleiotropic neuropeptide implicated in a variety of physiological processes. To better understand the biological functions of CGRP, we developed an CGRP-null mouse model using a gene targeting approach. Recordings of mean arterial pressure (MAP) and heart rate (HR) showed that basal MAP and HR were significantly higher in both anesthetized and conscious, unrestrained CGRP-null mice than in corresponding wild-type mice. The elevated MAP in CGRP-null mice was shown to be the result of elevated peripheral vascular resistance by -adrenergic blockade with prazosin and by transthoracic echocardiogram, which revealed no significant differences between CGRP-null and wild-type mice in the stroke volume, fractional shortening, and ejection fraction. Moreover, evaluation of autonomic nervous activity by measuring HR after pretreatment of atropine and/or atenolol and by analyzing arterial baroreceptor reflexes showed sympathetic nervous activity to be significantly elevated in CGRP-null mice; elevated levels of urinary catecholamine metabolites and decreased HR variability in mutant mice were also consistent with that finding. These findings suggest that CGRP contributes to the regulation of cardiovascular function through inhibitory modulation of sympathetic nervous activity.
Key Words: calcitonin gene-related peptide • gene targeting • blood pressure • autonomic nervous system • hypertension
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Calcitonin gene-related peptide (CGRP) is a 37-amino acid vasoactive neuropeptide produced by tissue-specific alternative splicing of the primary transcript of the calcitonin/
CGRP gene.1 While calcitonin (CT), which controls calcium homeostasis, is expressed almost exclusively in the C cells of the thyroid gland, CGRP is widely distributed in the central and peripheral nervous systems in mammals. In addition, a second CGRP isoform, ßCGRP, is encoded by a different gene locus and is expressed almost exclusively in specific neuronal sites.2 These two CGRP isoforms— and ß in rat and I and II in humans—exhibit overlapping biological activities in most vascular beds.3
Within the nervous system, CGRP immunoreactivity has been detected in spinal cord motor neurons, dorsal root ganglia, and motor nerve endings.4 Other neuropeptides, the tachykinins, which include substance P (SP) and neurokinin A (NKA), exhibit similar expression patterns to CGRP in several regions of the nervous system. CGRP and tachykinins coexist in primary afferent neurons, forming the part of the so-called nonadrenergic, noncholinergic (NANC) nervous system,5 and their release from sensory pain fibers has been implicated in the perception of pain.
NANC neurons containing CGRP are also widely distributed among autonomic fibers innervating the vasculature; for example, they are found in blood vessels at the junction of the adventitia and the media passing into the muscle layer.5 Moreover, CGRP is a potent vasodilator,6 and several investigators have claimed that CGRP may play a key role in regulating peripheral vascular tone and regional blood flow under both physiological and pathophysiological conditions.7,8 Immunoreactive CGRP has also been identified in the lower brain stem, which is known to be involved in the control of both arterial baroreceptor and respiratory reflex functions.9–11
Although many biological actions have been attributed to CGRP,7,12 its physiological and pathophysiological functions are not precisely known. During the period in which this study was ongoing, four groups reported the development of CGRP-deficient mutant mice using gene-targeting strategies.13–16 One group of CT/CGRP mutants exhibited elevated arterial blood pressure compared with wild-type counterparts,13 but no such difference was noted in another group.14 The other reports dealt only with the perception of pain and did not address the cardiovascular phenotype of CGRP-null mice.15,16 Thus, despite analysis of specific, gene-targeted models, the role of CGRP in the regulation of cardiovascular function remains controversial. In the present study, we have focused on the proposed role of CGRP in the regulation of cardiovascular function in our mice that have a permanent deletion of the CGRP gene. We discuss our findings in the context of those earlier studies using similar mouse models.
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Materials and Methods |
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Generation of
CGRP-Null MiceThe mouse CT/
CGRP genomic DNA was cloned from a BALB/c mouse genomic library in EMBL3 using synthetic oligonucleotide probes derived from the mouse CT/CGRP cDNA sequence. A 7.0-kb fragment containing exons 3 to 5 of the mouse CT/CGRP gene was subcloned into pBluescript (Stratagene). A targeting vector was constructed by replacing the 1.6-kb XbaI-XbaI fragment encompassing exon 5, which is specific for CGRP, with the neomycin resistance gene and flanking the thymidine kinase gene. This plasmid was linearized with NotI and introduced into 129/Sv-derived SM-1 embryonic stem (ES) cells by electroporation, after which the cells were selected in medium containing G418 (300 µg/mL) and ganciclovir (2 µmol/L). Homologous recombinants were identified by PCR and Southern blot analysis. Targeted ES cell clones were injected into C57BL/6 mouse blastocysts to generate chimeric mice. Male chimeras were then crossbred with C57BL/6 females and germline transmission was achieved. Littermates obtained by breeding heterozygotes with the genetic background of the 129/SvxC57BL/6 hybrid were used for phenotypic analysis. Only males were used in this study. All experiments were performed in accordance with the Declaration of Helsinki and were approved by the University of Tokyo Ethics Committee for Animal Experiments.
RT-PCR
Total RNA was prepared from brain stem, spinal cord, lung, and thyroid gland using RNAzol (BIOTEX) and then reverse-transcribed. PCR was performed on the cDNA samples using a GeneAmp PCR kit (Perkin Elmer Cetus), and primer sets were chosen as shown in Figure 1A, top: (a) for CGRP, the sense primer (5'-GCATGGCCACTCTCAGTGAAG-3') was chosen from within exon 3 and the antisense primer (5'-TTATCTGTTCAAGCCTG-AAGG-3') from within exon 5; (b) for CT, the sense primer (5'-GCATGGCCACTCTCAGTGAAG-3') was chosen from within exon 3 and the antisense primer (5'-CTTTGCCTCAGGAAA-GCAACC-3') from within exon 4. Thirty-five cycles (95°C for 1 minute, 61°C for 1 minute, 72°C for 1 minute) were used to amplify the products, which were then subjected to electrophoresis.
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Immunohistochemistry
Spinal cords and thyroid glands were fixed in 10% phosphate-buffered formalin (pH 7.4), embedded in OCT compound, and cut into 8-µm frozen sections on a cryostat. Slide-mounted tissue sections were washed with PBS, treated with 0.3% H2O2 in methanol, preincubated with goat nonimmune serum, and incubated with a rabbit anti-rat CGRP antibody (Peninsula Laboratories, Inc, at a dilution of 1:100) or a rabbit anti-human CT antibody (Progen Biotechnik GMBH, at a dilution of 1:25) for 16 hours at 4°C. The sections were then incubated with biotinylated goat anti-rabbit IgG for 60 minutes at 37°C. After washing, the sections were treated with avidin-biotinylated horseradish peroxidase complex (Vectastain ABC kit, Vector Labs) and developed with 0.004% H2O2 and 0.02% diaminobenzidine tetrahydrochloride. As a negative control, some samples were incubated with preimmune serum instead of the primary anti-CGRP or anti-CT antibody. The amino acid sequence of rat CGRP is completely identical to that of mouse CGRP, enabling the rat antibody to cross-react with the mouse protein. Moreover, this antibody also cross-reacts with rat ßCGRP. The sequence of human CT differs from that of mouse at two amino acid residues, but the human antibody was nonetheless expected to sufficiently cross-react with the mouse protein. We used hematoxylin as the counterstaining.
Serum and Urine Laboratory Analysis
Mice were anesthetized with pentobarbital (1 mg/10 g BW, IP), after which 21-gauge needles were inserted into the inferior vena cava and blood samples were drawn for measurement of urea nitrogen, creatinine, electrolytes, inorganic phosphate, calcium, and calcitonin. Sampling was always carried out between 4:00 and 6:00 PM. To determine the urinary levels of catecholamines and their metabolites, animals were housed in individual metabolic cages. After 3 days, a small amount of 6 N HCl was added to the beaker placed in the cage, and the acidic urine was collected for the next 24 hours and analyzed.
Measurement of Blood Pressure and Heart Rate
Male mice (8 to 12 weeks old) were anesthetized by urethane injection (1.5 mg/g, IP). Polyethylene tubing (SP31, Natsume Seisakusyo; inner diameter [ID] 0.50 mm, outer diameter [OD] 0.80 mm), heat-elongated so that the OD was reduced to 
0.4 mm, was inserted into the right femoral artery, so that the tip lay in the abdominal aorta. The other end of the cannula was connected to a pressure transducer (TP-400T, Nihon-Kohden) via a 3-way stopcock; the third port was used for drug injection. The ECG (ECG, lead II) was also monitored (JB210J, Nihon-Kohden) throughout the experiment. Heart rate (HR) was computed using a tachometer (AT-601G, Nihon-Kohden). Arterial pressure (AP), HR, and ECG signals were fed into and stored in a computer using an analog-to-digital (A/D) converter (MacLab/16s, ADInstruments) at the sampling rate of 200, 200, and 4000 Hz, respectively. Mean AP (MAP) and average values of HR and MAP for selected periods were calculated using the digital waveform analyzing software (Chart, ADInstruments). To examine basal MAP under conscious, unrestrained conditions, some of the mice were used again for measurement of MAP on the third day after the cannulation.
To assess peripheral vascular resistance in another series of experiments, mice were sequentially treated with intraperitoneal prazosin hydrochloride (1 µg/g), an 1-adrenergic receptor antagonist, and hexamethonium chloride (40 µg/g), an autonomic ganglionic blocker, on the third day after the cannulation. The MAP and HR were measured before and after the administration of each drug under conscious, unrestrained conditions.
Telemetric Monitoring of ECG
To assess HR in conscious, unrestrained animals, a radio transmitter (TA10ETA-F20, Data Sciences) was implanted in the abdominal cavities of some animals under halothane anesthesia. On the seventh day after the operation, each unrestrained mouse was placed on top of a radio receiver (RLA1020, Data Sciences) in its home cage, and the ECG was continuously monitored for 24 hours. The data were processed using a computer-assisted data acquisition system (Dataquest IV, Data Sciences) that yielded a time series of R waves and values for HR every 30 seconds. To achieve time domain analysis of heart rate variability, SDNN index (mean of the standard deviations of all normal-to-normal intervals for all 30-second segments of the entire 24-hour recording) was calculated.17
Moreover, to investigate the cardiac autonomic nervous activity, we performed several drug administration studies using autonomic nerve blockers after evaluating the basal HR. Atropine (5 µg/g), which blocks the muscarinic acetylcholine receptors, atenolol (5 µg/g), which blocks the ß-adrenergic receptors, and then both drugs were sequentially injected into the abdominal cavity. Drugs were administered at intervals of more than 24 hours during the afternoon in a random order. The HR was measured before and after the administration of atropine and/or atenolol.
Echocardiographic Analysis
Animals (16 to 20 weeks old) anesthetized with pentobarbital (1 mg/10 g BW, IP) were observed with a 2D-guided M-mode echocardiographic system equipped with a 10-MHz linear transducer (LOGIQ 500; GE Yokokawa Systems Ltd, Tokyo, Japan). Left ventricular (LV) diastolic diameter (LVDD) and LV systolic diameter (LVSD) were measured. Percent fractional shortening (%FS) and LV ejection fraction (EF) were automatically calculated on a cardiac ultrasound machine. LV end-diastolic volume (LVEDV) and LV end-systolic volume (LVESV) were calculated by the cubed method as (LVDD)3 
/3 and (LVSD)3 /3, respectively. Stroke volume (SV) was calculated as LVEDV-LVESV. Three beats were averaged for each measurement.
Arterial Baroreceptor Reflex
Arterial baroreceptor reflexes (MAP-HR relations) were evoked as follows. MAP was altered between 40 and 200 mm Hg by an intra-arterial bolus injection of sodium nitroprusside (SNP; 1 to 100 µg/100 µL), a vasodilator, or phenylephrine (PE; 1 to 30 µg/100 µL), an 1-adrenergic receptor agonist. Peak HR values were correlated with those of MAP. The data obtained from each urethane-anesthetized animal were fitted to a logistic curve using a graphics-assisted fitting program (Delta Graph, SPSS Inc) to obtain MAP-HR relations.18 The curve was expressed as y=p4+p1/(1+exp[p2(x-p3)]), where y is HR (bpm), and x is MAP (mm Hg). The four parameters in the equation are defined as follows: p1, the range of y (ie, maximum minus minimum values of y), which represents recruitment of efferent nerve activity; p2, coefficient of curvature, which is related to the central gain of the reflex; p3, median MAP (MAP50), or MAP corresponding to the midpoint over the range of y; and p4, the minimum value of y. These parameters were used to reconstruct single logistic curves representing each group, enabling comparison of groups receiving no autonomic nerve blockers, intraperitoneal atropine, or intraperitoneal atenolol.
Statistical Analysis
Quantitative values are expressed as mean±SEM. Student’s t tests were used to determine significant differences. Values of P<0.05 were considered significant.
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Results |
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Establishment and Characterization of
CGRP-Null MiceThe CT/
CGRP gene encodes two distinct prohormones via alternative RNA splicing. CT derives from exons 1 to 4, whereas CGRP derives from exons 1 to 3 and 5 to 6. A targeting DNA construct was designed to replace exon 5, which encodes an CGRP-specific region, with the neomycin resistance gene (Figure 1A). The targeting vector was introduced into 129/Sv-derived ES cells by electroporation, and targeted ES clones were then injected into C57BL/6 blastocysts. By crossbreeding heterozygous for CGRP mutant, we were able to obtain live CGRP-null mice. When the genomic DNA was digested by EcoRI or SacI, 5.7-kb or 7.0-kb recombinant bands were distinguishable from the 7.0-kb or 10.0-kb authentic gene bands, respectively, by Southern blotting using the indicated probes in ES cells (Figure 1B) and mice (Figure 1C).
By using RT-PCR primer set a, which spanned a region from exons 3 to 5, we were unable to detect amplification of 286-bp bands representing CGRP in CGRP-/- mice (Figure 2A). In contrast, using the primer set b, which spanned a region from exons 3 to 4, the 413-bp bands representing CT were detected in CGRP-/- mice (Figure 2B). Immunohistochemistry for CGRP revealed no positive staining in the spinal cords of CGRP-/- mice, whereas intense labeling was detected in the dorsal horns of CGRP+/+ mice (Figure 2C). Although we did not specifically examine the expression of mouse ßCGRP, which has yet to be identified, negative staining by the antibody for both CGRP and ßCGRP in CGRP-/- mice suggested that the lack of CGRP did not cause a compensatory increase of ßCGRP synthesis. We wondered whether CT would be overexpressed in the absence of the CGRP-specific gene locus as a consequence of predominant RNA splicing, but same as RT-PCR, immunostaining analysis showed the CT content to be the same in CGRP-/- and CGRP+/+ mice (Figure 2D).
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Laboratory Analysis
Basal levels of serum urea nitrogen, creatinine, electrolytes, inorganic phosphate, and calcium did not significantly differ in CGRP-/- and CGRP+/+ mice (data not shown), nor did serum CT levels (Figure 3A). To assess sympathetic nerve activity, we measured urinary levels of catecholamines and their metabolites. Although water intake and urine volume were not different in either genotype (data not shown), urinary metanephrine excretion was significantly higher in CGRP-/- mice than CGRP+/+ mice (Figure 3B), suggesting that sympathetic input to the adrenal medulla was increased in the mutant mice. Urinary HVA (homovanillic acid) was also elevated (Figure 3D), suggesting that central dopaminergic neurons were more active in the mutant mice. Urinary levels of normetanephrine (Figure 3C), VMA (vanillylmandelic acid), adrenaline, noradrenaline, and dopamine (data not shown) were not significantly increased, but tended to be higher in CGRP-/- than CGRP+/+ mice. These data collectively suggest that some, although not all, of the sympathetic nerve activities are augmented in CGRP-/- mice.
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Basal Cardiovascular Hemodynamics
To evaluate the role of CGRP in the regulation of cardiovascular function, basal MAPs and HRs of both genotypes were first measured under urethane anesthesia. We found MAP to be significantly elevated in CGRP-/- mice (107.7±2.2 versus 99.3±2.2 mm Hg) (Figure 4A), which also exhibited higher HRs (681.1±6.3 versus 622.3±8.3 bpm) (Figure 4B). When MAP was then measured in conscious, unrestrained mice, both genotypes exhibited MAPs that were 5 to 10 mm Hg higher than in anesthetized animals; still MAP remained significantly higher in CGRP-/- than CGRP+/+ mice (119.0±2.9 versus 104.6±5.7 mm Hg) (Figure 4C). Unrestrained CGRP-/- mice also displayed significantly higher HRs, as assessed by telemetric monitoring (554.4±26.4 versus 475.0±23.5 bpm) (Figure 4D). Thus, both basal MAPs and HRs were markedly higher in CGRP-/- mice than in CGRP+/+ mice under both anesthetized and conscious conditions. HR variability, which was calculated as SDNN index (n=7 for both genotypes), was smaller in CGRP-/- (5.8±0.8 ms) than in CGRP+/+ (9.4±1.1 ms, P<0.05) indicating higher sympathetic tone or lower parasympathetic tone in the former.
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) and mutant mice (
). The fractional increase in HR was significantly higher in wild-type mice (n=7 for both +/+ and -/-). F, Intraperitoneal atenolol significantly decreases HR in both genotypes with the fractional reduction in HR being significantly higher in mutant mice. G, Injection of both atropine and atenolol elicited significant decreases in HR only in mutant mice. Changes in HR in wild-type mice did not significantly differ. *P<0.05; **P<0.01.
Autonomic Nervous Activity in Circulatory Regulation
Having observed significantly elevated hemodynamic parameters and the indication of sympathetic or parasympathetic alteration in CGRP-/- mice, we assessed the activity of the cardiac autonomic nervous systems more directly with the pharmacological method. For that purpose, HR was measured by telemetric monitoring after pretreatment with intraperitoneal atropine, which blocks muscarinic neurotransmission in the parasympathetic nervous system, or atenolol, which blocks the ß-adrenergic sympathetic neurotransmission. Thus, changes in HR were examined under the inhibition of parasympathetic and/or sympathetic nervous activity.
Administration of atropine resulted in a significant increase in HR in both genotypes (Figure 4E). The fractional change in HR was significantly higher in CGRP+/+ mice (25.5±4.2% in CGRP-/- versus 51.0±6.1% in CGRP+/+). Cardiac sympathetic nervous blockade by intraperitoneal injection of atenolol produced significant decreases in HR in both genotypes (Figure 4F). The fractional reduction in HR was significantly higher in CGRP-/- mice (22.1±1.7 versus 12.9±2.6%). These results suggested overactivation of basal sympathetic control of HR and relatively diminished vagal control. When both atropine and atenolol were administered, CGRP-/- mice exhibited a significant reduction in HR, whereas CGRP+/+ mice showed no significant change (Figure 4G). Absolute values of the resultant HR did not differ between CGRP+/+ and CGRP-/- mice, indicating that intrinsic pacemaking activity of the heart was not different between the 2 genotypes.
Assessment of Peripheral Vascular Resistance
To investigate the involvement of peripheral vascular resistance in the elevated MAP in CGRP-/- mice, MAP and HR were measured after treatment with intraperitoneal prazosin, a peripheral 1-adrenergic sympathetic blocker, and hexamethonium, an autonomic ganglionic blocker, under conscious, unrestrained conditions (Figure 5). With HR being still significantly higher in CGRP-/- mice than in CGRP+/+ mice after prazosin injection, MAP was not significantly different between CGRP+/+ and CGRP-/- mice. Resultant MAP and HR after ganglionic blockade by hexamethonium showed no difference between CGRP+/+ and CGRP-/- mice.
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On the other hand, to evaluate the stroke volume (SV) and cardiac function of the mice, transthoracic echocardiography was performed. Despite the possibility of underestimation of SV due to the calculation of LVEDV and LVESV by the cubed method, basal contractile functions were unchanged (Table 1); LVDD, LVSD, LVEDV, LVESV, SV, %FS, and EF were not significantly different between the 2 groups.
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Thus, it is suggested that the elevated MAP in CGRP-/- mice is a result of elevated peripheral vascular resistance rather than a function of increased HR.
Arterial Baroreceptor Reflex
To further examine cardiac autonomic nervous responsiveness, PE, a vasopressor, or SNP, a vasodepressor, was administered via the right femoral artery under urethane anesthesia, and peak HRs were correlated with MAP to assess the arterial baroreceptor reflex. Figure 6 shows the MAP-HR relationships in CGRP-/- and CGRP+/+ mice, with and without autonomic nerve blockers. Atropine or atenolol was used to evaluate the cardiac autonomic nervous responsiveness under the inhibition of either parasympathetic or sympathetic nervous activity, respectively. Under basal conditions, baroreceptor reflex control of HR in CGRP-/- mice was very similar to that in CGRP+/+ mice; in fact no parameters differed significantly (Figure 6A, Table 2). By contrast, when mice were pretreated with intraperitoneal atropine, there was a clear difference between CGRP-/- and CGRP+/+ mice. Namely, the MAP50 was significantly elevated in CGRP-/- mice (Table 2). The minimum HR was significantly higher in both genotypes and was not different between CGRP-/- and CGRP+/+ mice. Parasympathetic nervous blockade by atropine thus elicited a rightward shift in the MAP-HR relationship in CGRP-/- mice. When mice were pretreated with atenolol, maximum HRs obtained from the MAP-HR relationship were significantly reduced in the both genotypes, but no other parameters were significantly affected (Figure 6B, Table 2).
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All of these results, together with the fact that basal MAP and HR were higher in CGRP-/- mice than CGRP+/+ mice, suggest that sympathetic nervous activity is significantly elevated in CGRP-/- mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we have obtained and characterized mutant mice in which the ability to express
CGRP has been selectively ablated. The goal of this study was to clarify the role of CGRP in the regulation of cardiovascular function in physiological and pathophysiological conditions, making comparisons with the previously reported findings on CGRP-deficient mice.13–16
Although much evidence suggests that CGRP participates in the regulation of cardiovascular function under both physiological and pathophysiological conditions,7,8 its precise role is still unclear. In the present study, we demonstrated that MAP and HR were significantly elevated in both anesthetized and conscious, unrestrained CGRP-null mice, which is consistent with data from CGRP/CT-null mice recently reported by Gangula et al.13 By contrast, Lu et al,14 who, like us, generated mutant mice in which CGRP was selectively deleted, failed to detect elevations in blood pressure (BP) or HR in their mice. That our results are more similar to those of Gangula et al indicates that the discrepancy between the two earlier studies does not reflect differences in the integrity of the CT gene. The genetic background used for phenotypic analysis in our study is 129/SvxC57BL/6 hybrid, whereas the mice used by Lu et al were back-bred onto a Black Swiss line. In addition, we inserted the cannula for measurement of blood pressure by transfemoral approach to keep the carotid sinus intact, whereas Lu et al accessed the left carotid artery. Similar discrepancy on blood pressure between femoral and carotid cannulation has been reported in angiotensin II type 2 receptor knockout mice.19,20 Moreover, Lu et al also cannulated the right jugular vein, which meant bilateral operation of the neck, which might have affected the autonomic nervous responsiveness. These differences in genetic background or experimental approach may account for the discrepancy observed between Lu et al and us. Nevertheless, our results refer not only to the basal hemodynamics but also to the peripheral vascular resistance and the autonomic nervous activity and clearly indicate that CGRP plays a significant role in the regulation of basal BP and HR under normal physiological conditions.
Another major finding of the present study is that basal sympathetic nervous activity is significantly higher in CGRP-null mice than wild-type mice. Having observed that MAP and HR were significantly elevated in the CGRP-null mutants, we applied atropine and/or atenolol and assessed autonomic nervous system responsiveness as reflected by the arterial baroreceptor reflex. Administration of atenolol markedly reduced HR in both genotypes with the fractional change being significantly larger in CGRP-null mice, whereas administration of atropine resulted in the significantly smaller increase rate of HR in CGRP-null mice, suggesting overactivation of basal sympathetic control of HR and relatively diminished vagal control. Note that, after administration of both atropine and atenolol, resultant HRs were similar in CGRP-null and wild-type mice, indicating that intrinsic pacemaking activity of the heart was independent from the existence of CGRP. Moreover, parasympathetic nervous blockade by atropine, which resulted in HR being determined solely by cardiac sympathetic nerve activity, induced a rightward shift in the MAP-HR curve in CGRP-null mice, whereas no difference in the baroreceptor reflex control of HR was noted among atenolol-treated mice of either genotype. Elevated levels of urinary catecholamine metabolites and decreased SDNN index of the HR variability in CGRP-null mice are also consistent with increased sympathetic nerve activity. In addition, although there was still significantly higher HR in CGRP-null mice after prazosin administration, similar resultant MAP in the two genotypes was observed. Echocardiographic analysis displayed no differences in the stroke volume and contractile parameters such as fractional shortening and ejection fraction. These results suggest elevated peripheral vascular resistance in CGRP-null mice caused by increased peripheral sympathetic activity. It thus appears that CGRP has an inhibitory effect on the sympathetic regulation of cardiovascular function.
CGRP is widely distributed in the central nervous system of mammalian species,9 with the highest density of CGRP-immunoreactive neurons being found in the lower brain stem. These CGRP-containing areas include the nucleus tractus solitarius (NTS), the nucleus ambiguus, the dorsal motor nucleus of the vagus, and the ventrolateral medulla (VLM), all of which are involved in the control of cardiovascular function, including arterial baroreceptor reflexes. Primary afferent fibers originating from baroreceptors send neural input signals to neurons in the NTS.10 The NTS in turn projects to neurons of the nucleus ambiguus and caudal ventrolateral medulla (cVLM). The former excites efferent activity in the cardiac vagus, thereby eliciting bradycardia,21 whereas the latter tonically inhibits the spontaneous activity of rostral ventrolateral medulla (rVLM) neurons,10 which has been shown to excite sympathetic preganglionic neurons in the spinal cord.22 Intracisternal administration of human CGRP (hCGRP) reduces baroreflex-mediated tachycardia evoked by sodium nitroprusside, but not the bradycardia evoked by phenylephrine. Conversely, intracisternal hCGRP-(8-37), an hCGRP antagonist, increases nitroprusside-induced tachycardia and inhibits the suppressive effects of hCGRP. This suggests that CGRP released from spontaneously active rVLM neurons may feed back to suppress the discharge, thereby decreasing sympathetic activity and reducing baroreflex-mediated tachycardia.23 On the other hand, intracerebroventricularly administered (exogenous) CGRP has been shown to enhance sympathetic nerve activity and to increase HR and arterial BP.24 Nonetheless, our findings, as well as those of Gangula et al,13 strongly suggest that CGRP acts as a depressor, but not a pressor, in the regulation of systemic cardiovascular hemodynamics. Thus, it is suggested that CGRP contributes to the regulation of cardiovascular function through inhibitory modulation of sympathetic nervous activity, affecting in particular the arterial baroreceptor reflex pathway.
Limitations
Although we have shown that CGRP influences cardiovascular function by augmentation of sympathetic nervous activity in the mutant mice, it is still unclear whether this effect mainly occurs in the central nervous system, peripheral nervous system, or at both sites. To clarify this point, a conditional knockout study may be needed.
In conclusion, we have characterized CGRP-deficient mutant mice established using a gene targeting approach. Our major finding is that permanent deletion of CGRP significantly elevates sympathetic nervous system effects on hemodynamic parameters and arterial baroreceptor reflexes. This suggests that CGRP acts to inhibit sympathetic effects on cardiovascular function. The CGRP-null mouse may thus represent a useful new animal model with which to clarify the pathogenesis of cardiovascular diseases.
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Acknowledgments |
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This work was supported by JSPS (Japan Society for the Promotion of Science) Research for the Future Program, Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan, the Research Grant for Cardiovascular Diseases (11C-1) from the Ministry of Health and Welfare and the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Drug ADR Relief, R&D Promotion and Product Review of Japan (to H.K.), and by grants from the Naito Foundation and Shimadzu Science Foundation (to T.K.).
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Footnotes |
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*Y.O. and T.S. contributed equally to this work;
T.K. and H.K. are equal contributors as last authors. 
Received March 27, 2001; revision received October 15, 2001; accepted October 15, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1. Rosenfeld MG, Mermod JJ, Amara SG, Swanson LW, Sawchenko PE, Rivier J, Vale WW, Evans RM. Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature.. 1983; 304: 129–135.
2. Amara SG, Arriza JL, Leff SE, Swanson LW, Evans RM, Rosenfeld MG. Expression in brain of a messenger RNA encoding a novel neuropeptide homologous to calcitonin gene-related peptide. Science.. 1985; 229: 1094–1097.
3. Wimalawansa SJ, Morris HR, Etienne A, Blench I, Panico M, MacIntyre I. Isolation, purification and characterization of ß-hCGRP from human spinal cord. Biochem Biophys Res Commun.. 1990; 167: 993–1000.
4. Gibson SJ, Polak JM, Giaid A, Hamid QA, Kar S, Jones PM, Denny P, Legon S, Amara SG, Craig RK. Calcitonin gene-related peptide messenger RNA is expressed in sensory neurones of the dorsal root ganglia and also in spinal motoneurones in man and rat. Neurosci Lett.. 1988; 91: 283–288.
5. Holzer P. Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides. Neuroscience.. 1988; 24: 739–768.
6. Brain SD, Williams TJ, Tippins JR, Morris HR, MacIntyre I. Calcitonin gene-related peptide is a potent vasodilator. Nature.. 1985; 313: 54–56.
7. Wimalawansa SJ. Calcitonin gene-related peptide and its receptors: molecular genetics, physiology, pathophysiology, and therapeutic potentials. Endocr Rev.. 1996; 17: 533–585.
8. DiPette DJ, Schwarzenberger K, Kerr N, Holland OB. Dose-dependent systemic and regional hemodynamic effects of calcitonin gene-related peptide. Am J Med Sci.. 1989; 297: 65–70.
9. Skofitsch G, Jacobowitz DM. Calcitonin gene-related peptide: detailed immunohistochemical distribution in the central nervous system. Peptides.. 1985; 6: 721–745.
10. Kumada M, Terui N, Kuwaki T. Arterial baroreceptor reflex: its central and peripheral neural mechanisms. Prog Neurobiol.. 1990; 35: 331–361.
11. Spyer KM. Central nervous mechanisms contributing to cardiovascular control. J Physiol.. 1994; 474: 1–19.
12. Goodman EC, Iversen LL. Calcitonin gene-related peptide: novel neuropeptide. Life Sci.. 1986; 38: 2169–2178.
13.
Gangula PR, Zhao H, Supowit SC, Wimalawansa SJ, Dipette DJ, Westlund KN, Gagel RF, Yallampalli C. Increased blood pressure in -calcitonin gene-related peptide/calcitonin gene knockout mice. Hypertension.. 2000; 35: 470–475.
14.
Lu JT, Son YJ, Lee J, Jetton TL, Shiota M, Moscoso L, Niswender KD, Loewy AD, Magnuson MA, Sanes JR, Emeson RB. Mice lacking -calcitonin gene-related peptide exhibit normal cardiovascular regulation and neuromuscular development. Mol Cell Neurosci.. 1999; 14: 99–120.
15.
Salmon AM, Damaj I, Sekine S, Picciotto MR, Marubio L, Changeux JP. Modulation of morphine analgesia in CGRP mutant mice. Neuroreport.. 1999; 10: 849–854.
16.
Zhang L, Hoff AO, Wimalawansa SJ, Cote GJ, Gagel RF, Westlund KN. Arthritic calcitonin/ calcitonin gene-related peptide knockout mice have reduced nociceptive hypersensitivity. Pain.. 2001; 89: 265–273.
17. Task Force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology. Heart rate variability: standards of measurement, physiological interpretation, and clinical use. Circulation.. 1996; 93: 1043–1065.
18. Ling GY, Cao WH, Onodera M, Ju KH, Kurihara H, Kurihara Y, Yazaki Y, Kumada M, Fukuda Y, Kuwaki T. Renal sympathetic nerve activity in mice: comparison between mice and rats and between normal and endothelin-1 deficient mice. Brain Res.. 1998; 808: 238–249.
19. Ichiki T, Labosky PA, Shiota C, Okuyama S, Imagawa Y, Fogo A, Niimura F, Ichikawa I, Hogan BL, Inagami T. Effects on blood pressure and exploratory behaviour of mice lacking angiotensin II type-2 receptor. Nature.. 1995; 377: 748–750.
20. Hein L, Barsh GS, Pratt RE, Dzau VJ, Kobilka BK. Behavioural and cardiovascular effects of disrupting the angiotensin II type-2 receptor in mice. Nature.. 1995; 377: 744–747.
21. Stuesse SL, Fish SE. Projections to the cardioinhibitory region of the nucleus ambiguus of rat. J Comp Neurol.. 1984; 229: 271–278.
22. Ross CA, Ruggiero DA, Park DH, Joh TH, Sved AF, Fernandez-Pardal J, Saavedra JM, Reis DJ. Tonic vasomotor control by the rostral ventrolateral medulla: effect of electrical or chemical stimulation of the area containing C1 adrenaline neurons on arterial pressure, heart rate, and plasma catecholamines and vasopressin. J Neurosci.. 1984; 4: 474–494.
23. Kim S, Ouchi Y, Sekiguchi H, Fujikawa H, Shimada K, Yagi K. Endogenous calcitonin gene-related peptide modulates tachycardiac but not bradycardiac baroreflex in rats. Am J Physiol.. 1998; 274: H1489–H1494.
24. Fisher LA, Kikkawa DO, Rivier JE, Amara SG, Evans RM, Rosenfeld MG, Vale WW, Brown MR. Stimulation of noradrenergic sympathetic outflow by calcitonin gene-related peptide. Nature.. 1983; 305: 534–536.
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