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(Hypertension. 2000;35:470.)
© 2000 American Heart Association, Inc.

Scientific Contributions

Increased Blood Pressure in -Calcitonin Gene–Related Peptide/Calcitonin Gene Knockout Mice

Pandu R. R. Gangula; Huwai Zhao; Scott C. Supowit; Sunil J. Wimalawansa; Donald J. Dipette; Karin N. Westlund; Robert F. Gagel; Chandra Yallampalli

From the Departments of Obstetrics and Gynecology (P.R.R.G, C.Y.), Internal Medicine (H.Z., S.C.S., S.J.W., D.J.D.), and Anatomy and Neurosciences (K.N.W.), The University of Texas Medical Branch, Galveston, Tex; and the Division of Endocrinology (R.F.G.), MD Anderson Cancer Center, Houston, Tex.

Correspondence to Chandra Yallampalli, DVM, PhD, Department of Obstetrics and Gynecology, 301 University Blvd, Medical Research Bldg, Room 11.138, Galveston, TX 77555-1062. E-mail chyallam{at}utmb.edu

	Abstract

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Abstract—Nerves that contain calcitonin gene–related peptide (CGRP) are components of the sensory nervous system. Although these afferent nerves have traditionally been thought to sense stimuli in the periphery and transmit the information centrally, they also have an efferent vasodilator function. Acute administration of a CGRP receptor antagonist increases the blood pressure (BP) in several models of hypertension, which indicates that this potent vasodilator plays a counterregulatory role to attenuate the BP increase in these settings. To determine the role of this peptide in the long-term regulation of cardiovascular function, including hypertension, we obtained mice that have a deletion of the -calcitonin gene–related peptide (-CGRP) gene. Although the ß-calcitonin gene–related peptide (ß-CGRP) gene is intact in these mice, -CGRP is by far the predominant species of CGRP produced in dorsal root ganglia (DRG) sensory neurons. Initially, we examined the effect of deletion of the -CGRP on baseline BP and ß-CGRP and substance P mRNA expression. Systolic BP was significantly higher in the knockout mice (n=7) compared with wild-type in both male (160±6.1 vs 125±4.8 mm Hg) and female (163±4.8 vs 135±33 mm Hg) mice. Next, groups (n=7) of knockout and wild-type mice had catheters surgically placed in the right carotid artery for mean arterial pressure recording. With the animals fully awake and unrestrained, the knockout mice displayed an elevated mean arterial pressure compared with wild-type in both male (139±4.9 vs 118±4.9 mm Hg) and female (121±3.4 vs 107±3.1 mm Hg) mice. Northern blot analysis of DRG RNA samples confirmed the absence of -CGRP mRNA in the knockout mice. Substance P mRNA content in DRG was unchanged between the 2 groups; however, ß-CGRP mRNA levels were reduced 2-fold in the knockout mice. These results indicate for the first time that -CGRP may be involved in the long-term regulation of resting BP and suggest that these mice are particularly sensitive to challenges to BP homeostasis because of the loss of a compensatory vasodilator mechanism.

Key Words: peptides • blood pressure • mice • genes

	Introduction

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Calcitonin gene–related peptide (CGRP) is a 37–amino acid vasoactive neuropeptide that is widely distributed in the central and peripheral nervous systems in mammals.^{1 2} -CGRP is produced by the tissue-specific alternative splicing of the primary transcript of the calcitonin/-CGRP gene and is synthesized almost exclusively in neuronal tissues.^{3 4} There is a second CGRP gene, ß-CGRP, which does not produce calcitonin.⁵ Again, expression of ß-CGRP is limited almost exclusively to specific neuronal sites.^{4 5} The 2 CGRP genes, and ß in the rat and I and II in humans, differ in their protein sequences by 1 and 3 amino acids, respectively, and their biological activities are quite similar in most vascular beds.^{6 7}

The dorsal root ganglia (DRG) is a prominent site of CGRP synthesis that contains the cell bodies of primary afferent neurons that terminate peripherally on blood vessels and other tissues innervated by the sensory nervous system and centrally in laminae I/II of the dorsal horn of the spinal cord.^{8 9} A comparison of -CGRP and ß-CGRP content in rat DRG indicates that -CGRP levels are at least 6-fold higher in these neurons. Nerve fibers containing CGRP are widely distributed in the cardiovascular system. These nerves are found in blood vessels at the junction of the adventitia and the media passing into the muscle layer.¹⁰ In these nerves CGRP is often colocalized with substance P (SP) and other tachykinins.¹¹

Calcitonin gene–related peptide is a very potent vasodilator, 100 to 1000 times more potent than other vasodilators such as adenosine, SP, or acetylcholine.^{12 13} CGRP has been shown to dilate multiple vascular beds, with the coronary vasculature being a particularly sensitive target.^{12 13} Systemic administration of CGRP decreases blood pressure (BP) in a dose-dependent manner in both normotensive animals and humans, as well as the spontaneously hypertensive rat.^{1 2} The primary mechanism for this BP reduction is peripheral arterial dilation.^{2 13 14} These findings suggest that CGRP may play a significant role in regulating peripheral vascular tone and regional organ blood flows, both under normal physiological conditions and in the pathophysiology of hypertension.

A direct role for CGRP in experimental hypertension has now been established. Earlier reports demonstrated that CGRP can modulate chronic hypoxic pulmonary hypertension.¹⁵ Through the use of chronic infusion (by osmotic minipumps) of CGRP, its antibody, or the CGRP receptor antagonist, it was found that endogenous CGRP plays an important role in pulmonary pressure homeostasis during hypoxia. CGRP directly dilates the pulmonary vasculature, thus alleviating the development of chronic hypoxic pulmonary hypertension in rats.¹⁵ In addition, we have demonstrated for the first time that CGRP acts as a compensatory depressor mechanism to partially attenuate the systemic BP increase in 3 models of experimental hypertension: DOC-salt,¹⁶ SN-salt,¹⁷ and L-NAME–induced hypertension during pregnancy.^{18 19 20} Furthermore, this antihypertensive effect of CGRP appears to be mediated either by an upregulation of neuronal (DRG) CGRP synthesis and release (DOC-salt model,^{16 17} ) or through an enhanced sensitivity of the vasculature to the vasodilator effects of CGRP (SN-salt and L-NAME models).^{19 20 21} Because these studies were done acutely, there is still a question regarding the long-term participation of CGRP in the pathophysiology of hypertension. Moreover, it is not known whether this peptide plays a significant role in the regulation of systemic BP under normal physiological conditions. To begin to address these questions, we have obtained mice that have a permanent deletion of the -CGRP gene. For this initial study, we compared baseline systolic BPs and mean arterial pressures (MAPs) in the -CGRP knockout and wild-type (WT) mice of either sex and examined the expression of ß-CGRP and SP mRNA species in DRG from these animals.

	Methods

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Animals
Mice deficient in both calcium and -CGRP were created by targeted mutagenesis with the use of standard techniques.²² The targeting vector replaced the protein encoding exons 2 through 5 of the mouse calcitonin 1 gene with PGK neoBPA. Homologous recombination was confirmed by Southern analysis with 5' and 3' probes. The homozygous (-/-) breeding pairs were derived from an inbred strain on a 129/c57 genetic background. The control WT mice were also 129/c57 (CGRP+/+). The knockout mice were generated by one of the authors (R.F. Gagel), and details were recently reported.²³ All procedures were approved by the Animal Care and Use Committee of the University of Texas Medical Branch.

BP Determination
Systolic BP
Groups (7 per group) of either WT or -CGRP knockout (-/-) mice (25 to 30 g) of either sex were used in our study. Systolic BP was measured from each animal by the tail-cuff method with a RTBP1001 Rat Tail Blood Pressure System (Kent Scientific). Each mouse was trained for these studies for 3 days. Final BP values were obtained for each mouse by taking 4 to 6 readings daily and averaging the values obtained for 5 days. All systolic BP measurements were performed between 9 and 11 AM by a single investigator in a blinded fashion.

Mean Arterial BP
On the day MAPs were taken, mice were anesthetized with ketamine (80 mg/kg body wt; Fort Dodge Laboratory) and xylazine (4 mg/kg body wt; Burns Veterinary Supply). A catheter (PE-10) was inserted into the left carotid artery to continuously measure MAP with a Gould pressure transducer coupled to a Gould recorder. Mean arterial BP determinations were made 3 hours after surgery with the mice fully awake and unrestrained. At the end of experiment, the animals were killed, and thoracic and lumbar DRG from each mouse were immediately dissected and frozen in liquid nitrogen for subsequent analysis of -CGRP, ß-CGRP, and SP mRNA.

Hybridization Probes, RNA Isolation, and Analysis
The -CGRP hybridization probe was a 1.4-kb Sau3A rat genomic restriction fragment containing CGRP exons 5 and 6,³ and the ß-CGRP hybridization probe was a 0.22 kb HpaII-AluI restriction fragment from the 3' noncoding region of the ß-CGRP gene. The 18S rRNA probe was 1.15-kb BamHI-EcoRI restriction fragment of the mouse 18S rRNA gene.²⁴ The DNA inserts were purified by agarose-gel electrophoresis and subsequently labeled with (-³²p) dCTP with the use of a random hexanucleotide DNA labeling kit (Amersham). Total cellular RNA was isolated from the DRG tissue by the guanidine-isothiocyanate method.²⁵ The RNA samples were fractionated by electrophoresis on denaturing formaldehyde-agarose gels and transferred to nylon membranes. The membranes were initially hybridized with ³²p-labeled -CGRP DNA probe. The -CGRP probe was removed from the membranes that were then rehybridized with the ß-CGRP probe and subsequently with the SP probe. As an internal control, the membrane was hybridized with the 18S rDNA probe after removal of the SP probe from the membrane. After hybridization, the membranes were washed and placed on a phosphor screen. The exposed screen was then placed in a PhosphorImager (Molecular Dynamics), which generates an image of the hybridized RNA and quantifies the radioactivity in each hybridization signal.

Immunocytochemistry
Animals were anesthetized and perfused transcardially with 1% heparin in saline and 4% paraformaldehyde in PBS. Animals were dissected, and their spinal cords were removed rapidly. The tissues were postfixed in the same fixative for an additional 2 hours. Tissues were then placed in 30% sucrose in PBS for 24 hours. Spinal cord sections, 16 mm in thickness, were cut transversely on a freezing microtome (IEC Minotome Plus, International Equipment Co), and tissue sections were mounted onto chrome gelative coated slides. The spinal tissue sections were processed for localization of CGRP-like immunoreactivity (CGRP-LI) with the ABC method (Vector). The polyclonal primary antibodies were used specifically directed against -CGRP (rabbit anti-hCGRP, Peninsula, used at a dilution of 1:25 000). Spinal cord tissue was taken from both the -CGRP (-/-) and -CGRP (+/+) groups.

Statistical Analysis
Data are presented as mean±SEM. We used 2-factor ANOVA with pairwise comparisons by Tukey’s test. The acceptable value of significance was P<0.05.

	Results

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Effect of Deletion of the -CGRP gene on Systolic BP
To evaluate the role of -CGRP in BP regulation, we first determined the tail-cuff BP of -CGRP-/- or WT mice of either sex (Figure 1). Systolic BP was significantly (P<0.05) elevated in -CGRP-/- male mice (160±6.1 mm Hg, n=7) compared with WT mice (125±4.8 mm Hg, n=7). Similarly, -CGRP–deficient female mice exhibited higher (P<0.01) BP (163±4.8 mm Hg, n=7) compared with their WT (135±3.3 mm Hg, n=7) counterparts.

View larger version (31K): [in this window] [in a new window]   Figure 1. Systolic BP measurements in -CGRP-/- (-CGRP-KO) and WT mice of either sex, with the tail-cuff method. BP values were obtained for each animal by taking 4 to 6 readings daily for 5 days. Mean±SEM for each group (n=7) are presented for both male and female mice. Asterisk indicates significant differences from WT (P<0.05).

Analysis of MAP in WT and Mutant Mice
Having observed that the systolic BP was significantly elevated in the knockout mice, it was necessary to determine whether this was reflected in the MAP. After surgical placement of an indwelling arterial catheter, continuous MAP measurements were made with the mice fully awake and unrestrained (3 hours after surgery). Each MAP value was determined by the average of 3 separate measurements (5 minutes each) and the variability between measurements within each animal was <5%. Figure 2 shows that in the -CGRP-/- male mice, the MAP was significantly (P<0.05) elevated (139±4.5 mm Hg, n=7) compared with WT controls (118±4.9 mm Hg, n=7). Similarly, WT female mice displayed a lower MAP (107±3.1 mm Hg, n=7), compared with the (-/-) female mice (121±3.4 mm Hg, n=6) (P<0.05). When the data were analyzed for main effect differences between male and female mice, the MAP was significantly (P<0.01) higher in male than female -CGRP -/- mice but not in WT mice.

View larger version (25K): [in this window] [in a new window]   Figure 2. MAP determinations made with an indwelling carotid arterial catheter in -CGRP-/-(-CGRP-KO) and WT mice of either sex in a fully awake and unrestrained state. Mean±SEM for each group (n=7) are presented for both male and female mice. Asterisk indicates significant differences from WT (P<0.05).

To address the question of whether the knockout mice would have an enhanced sensitivity to exogenous CGRP, studies were performed in which the mice were instrumented as described above except the jugular vein was also cannulated for administration of saline or CGRP. With the mice in a fully awake and unrestrained state, administration of saline (0.1 mL) produced a negligible increase in the MAP in both the knockout (n=4) and WT (n=4) mice. Administration of a bolus dose of human -CGRP (1.5 ng/g body wt) resulted in a rapid 16.3±4.3 mm Hg decrease in the MAP in the WT mice and a 27.5±4.3 mm Hg reduction in the knockout male mice. This occurred within 1 minute after administration of human -CGRP. On the other hand, this dose of -CGRP produced 19.8±2.3 mm Hg and 25.9±3.8 mm Hg decrease in MAP in the WT and knockout female mice.

Analysis of -CGRP, ß-CGRP, and SP mRNA Species in (-/-) Mice
DRG tissue was then processed from the 2 groups of mice to evaluate -CGRP, ß-CGRP, and SP mRNA levels. Figure 3 (A, B) clearly shows the absence of the -CGRP mRNA species in the knockout mice after Northern blot analysis, whereas the 18S rRNA can be seen in the DRG RNA samples from both groups of mice. Because the -CGRP probe used in this experiment hybridizes to both CGRP mRNA species, the CGRP hybridization signal shown in this figure is from a 5-hour exposure of the phosphor screen to the membrane. Because -CGRP is by far the predominant CGRP species produced in DRG, there is a very strong -CGRP hybridization signal in the RNA samples from the WT mice, whereas none can be observed in the knockout mice with this exposure time. If the phosphor screen is exposed to the membrane for a much longer time (24 hours), the ß-CGRP signal can be detected in the RNA samples from the knockout mice (not shown). To confirm the lack of -CGRP peptide in the sensory neurons, immunostaining for immunoreactive CGRP was performed in the spinal cords of both knockout and WT mice. Figure 4A shows the intense staining of CGRP in laminae I/II of the dorsal horn of the spinal cord in the WT male mice. Even though the antibody used in this experiment recognizes both -CGRP and ß-CGRP, no staining is detected in the cords of the knockout mice (Figure 4B) because of the absence of -CGRP expression and the very low levels of ß-CGRP that are produced in DRG neurons.

View larger version (38K): [in this window] [in a new window]   Figure 3. Northern blot analysis of mRNA from DRG from WT and -CGRP-/-(-CGRP-KO) male (A) and female (B) mice. Total cellular RNA samples isolated from DRG taken from WT and -CGRP-/- mice were fractionated on a denaturing formaldehyde-agarose gel and transferred to a nylon membrane. The membrane was hybridized with the 32P-labeled -CGRP genomic DNA insert. The -CGRP was removed from the membrane, which was subsequently hybridized with 32P-labeled 18S rDNA probe (bottom). After hybridization with each probe, the membrane was washed and placed on a phosphor screen, and the image was generated from phosphorimager analyses of the exposed screen. Note the lack of -CGRP mRNA in the -CGRP-KO group.

View larger version (89K): [in this window] [in a new window]   Figure 4. Light photomicrograph showing the location of immunoreactive -CGRP in the spinal cord. The -CGRP staining is localized in laminae I and II of the dorsal horn of the spinal cord in the WT (A) but not in the -CGRP-/- (-CGRP-KO; B) male mice.

The same membrane was stripped and then hybridized sequentially with the ß-CGRP–specific (Figures 5A and 5B) and SP (Figures 6A and 6B) hybridization probes. As expected, the mRNA species for ß-CGRP (24-hour exposure) and SP (7-hour exposure) was present in both groups. The hybridization signals in Figure 5 were normalized to those for the 18S rRNA internal control. The relative changes in ß-CGRP expression, calculated as a ratio of ß-CGRP to 18S rRNA, are 0.05+0.01 and 0.13+0.02 in knockout and WT female mice and 0.02+0.01 and 0.04+0.01 in knockout and WT male mice, respectively. Densitometric analysis of Figure 6 revealed no significant changes in SP mRNA expression between the 2 groups in both male and female mice.

View larger version (34K): [in this window] [in a new window]   Figure 5. Northern blot analysis of mRNA from DRG of WT and -CGRP-/-(-CGRP-KO) male (A) and female (B) mice. The membrane was hybridized initially with the 32P-labeled ß-CGRP genomic DNA insert and subsequently with 32P-labeled 18S rDNA probe (bottom). (See legend for Figure 2 for more details.)

View larger version (42K): [in this window] [in a new window]   Figure 6. Northern blot analysis of mRNA from DRG of WT and (-CGRP-/-(-CGRP-KO) male (A) and female (B) mice. The membrane was hybridized initially with the 32P-labeled SP genomic DNA insert and subsequently with 32P-labeled 18S rDNA probe (bottom). See legend for Figure 3 for more details.

	Discussion

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The primary goal of this study was to determine whether permanent deletion of the -CGRP gene in the mouse significantly alters baseline BP in the absence of a challenge to BP homeostasis. Systolic BPs, as determined by the indirect tail-cuff method, were markedly higher in both the male and female knockout mice compared with the WT controls. Although the WT female mice displayed a higher systolic BP than the WT males, these values were not significantly different. Likewise, the direct recording of BP through the use of an indwelling arterial catheter clearly demonstrated that the MAPs were significantly higher (20 mm Hg) in both the male and female knockout mice compared with their normal counterparts. The mean BPs tended to be lower in the WT female mice compared with male mice, and the differences between the sexes were significant (P<0.01) when -CGRP was depleted. It should be noted that because the -CGRP gene is part of the calcitonin/-CGRP gene locus, deletion of the -CGRP gene inactivates the calcitonin gene as well as katacalcin. Katacalcin is derived from the carboxy-terminal end of the calcitonin peptide precursor during posttranslational processing of this peptide.^{1 4} Although this inactivation of the calcitonin gene could present a problem in the interpretation of these results, it is well documented that endogenous calcitonin or katacalcin do not participate in the regulation of cardiovascular function.^{1 2 26} Furthermore, administration of exogenous calcitonin does not influence BP or vascular function except at very high pharmacological doses (1000 times physiological levels).^{1 2 26} Administration of pharmacological doses of katacalcin also does not alter cardiovascular function.^{1 2 4} Therefore the results of the present study suggest, for the first time, that -CGRP plays a role in the regulation of resting BP under normal physiological conditions. We are, however, well aware of the shortcomings that are inherent when using knockout mice. It is difficult to determine the importance of a single factor on the regulation of BP by simply removing the factor and making a measurement. Permanent deletion of a gene may lead to the alteration of a number of physiological functions, some of which may influence BP homeostasis. Therefore it is possible that the increase in BP in the knockout mice is an indirect effect of the loss of -CGRP function. This could occur through the increased activity of a pressor system and/or the decreased activity of a depressor system.

The rather substantial increase in baseline BP in the knockout mice was somewhat unexpected, based on our earlier studies in rat models of experimental hypertension. As described previously, acute administration of the CGRP receptor antagonist CGRP_8-37 in studies with 3 different models of acquired hypertension resulted in a significant increase in MAPs in the hypertensive rats but not the normotensive controls, which suggests that CGRP acts as a partial counterregulatory mechanism to attenuate the BP increase in these settings. The inability of the CGRP antagonist to alter BP in the control rats implied that this peptide is not involved in the regulation of systemic BP in the normotensive state; rather, its primary cardiovascular role under these conditions is to regulate blood flow to various organs by its potent vasodilator actions. For example, CGRP participates in the regulation of regional organ blood flow in the gut,²⁷ and it has been reported that in studies in which CGRP_8-37 was used in normal rats, CGRP was responsible for 30% of basal coronary blood flow.²⁸ Although we do not yet know the reason for this discrepancy, there are several possibilities. First, whereas the studies with the CGRP receptor antagonist were done acutely, the physiological effects of deletion of the -CGRP gene obviously are ongoing throughout the entire life of the animal. It may be that in the complete absence of such a potent vasodilator, the vasoconstrictor actions of the various pressor systems are more pronounced. The second possibility relates to the CGRP receptor antagonist CGRP_8-37. Although a large number of in vivo and in vitro studies confirm that this antagonist is an effective competitive inhibitor of CGRP binding, it does have its limitations. CGRP_8-37 is a peptide that in practice is best suited for acute studies when used in vivo.^{1 29 30} Because it is a peptide, it is rapidly degraded in the circulation and the pressor effects that are observed when it is administered to the hypertensive rats described previously are short lived.^{17 31 32} In addition, it is well documented that at higher doses, CGRP_8-37 acts as an agonist.^{1 29 30} Because the normotensive rats are less sensitive to the pressor effects of this antagonist when compared with the hypertensive animals, it appears that the dose of CGRP_8-37 required to significantly alter the mean BP in the normal rats is in the range where this peptide starts to exert its agonist effects. Therefore it is difficult to answer this question until the development of a long-acting nonpeptide CGRP receptor antagonist that does not have agonist properties.

As anticipated, the knockout mice displayed an enhanced sensitivity to the hypotensive actions of exogenously administered CGRP. This result suggests that the relative lack of CGRP in the knockout mice increases the responsiveness of the vasculature to the vasodilator activity probably through a CGRP receptor–mediated mechanism. A similar increase in vascular sensitivity to exogenous CGRP has been reported in the spontaneously hypertensive rat, which has markedly lower levels of circulating CGRP and CGRP content in DRG compared with Wistar-Kyoto controls.¹

Both Northern blot analysis of DRG neurons and immunohistochemical localization studies in laminae I and II of the dorsal horn of the spinal cord confirmed the absence of -CGRP gene and protein, respectively, in the knockout but not in the WT mice. In this study, we also confirmed that -CGRP was the predominant form of CGRP produced in DRG neurons, and Northern blot analysis was used to begin to characterize ß-CGRP and SP mRNA synthesis in the knockout and WT mice. Surprisingly, ß-CGRP mRNA content was reduced 2-fold in the knockout mice. In light of the very low levels of ß-CGRP in DRG, it is probably not likely that the decrease in this species of CGRP contributes to the BP elevation observed in the knockout mice but this remains to be determined. The mechanism by which ß-CGRP mRNA production in DRG is decreased is also unknown, but it may be related to some type of positive feedback loop that is modulated either directly or indirectly by -CGRP. We also observed that SP mRNA production was unchanged between the 2 groups of mice. This was done was to determine whether SP production was enhanced as a compensatory response to the elevated BP, since Kohlmann et al³³ have reported that SP acts as a partial counterregulatory mechanism to counteract the BP increase in some types of experimental hypertension. Experiments to directly test for the participation of SP in BP control in this model have not yet been done.

In summary, the key observation made in the study is that permanent deletion of the -CGRP gene results in a significant increase in baseline BP in both male and female mice. This is the first demonstration that the sensory nervous system, through the vasodilator effects of CGRP, participates in the regulation of resting BP under normal physiological conditions. These studies also predict that these -CGRP–deficient mice will be particularly sensitive to challenges to BP homeostasis because of the loss of a potent compensatory vasodilator system.

Note Added in Proof
While this manuscript was in press, a manuscript by Lu et al, titled "Mice Lacking -Calcitonin Gene-Related Peptide Exhibit Normal Cardiovascular Regulation and Neuromuscular Development" was published in Molecular and Cellular Neuroscience (1999;14:99–120). In this study, -CGRP null mutation was achieved, without disrupting calcitonin. These authors failed to detect elevations in blood pressure in these animals, an observation different from ours. More detailed studies are required to assess the role of -CGRP in cardiovascular hemodynamics.

	Acknowledgments

 
This study was supported in part by grants from the National Institutes of Health, HD-30273 and HL-58144, to Dr Yallampalli.

Received September 14, 1999; first decision October 20, 1999; accepted November 11, 1999.

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