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(Hypertension. 1997;29:945-950.)
© 1997 American Heart Association, Inc.

Articles

Calcitonin Gene–Related Peptide Is a Depressor of Deoxycorticosterone-Salt Hypertension in the Rat

Scott C. Supowit; Huawei Zhao; Diane M. Hallman; ; Donald J. DiPette

From the Departments of Internal Medicine (Division of General Internal Medicine) and Human Biological Chemistry and Genetics, The University of Texas (Galveston) Medical Branch.

Correspondence to Scott C. Supowit, PhD, The University of Texas Medical Branch, 8.104 Medical Research Building, Galveston, TX 77555-1065.

	Abstract

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Abstract Calcitonin gene–related peptide (CGRP) is a potent vasodilator neuropeptide. We previously demonstrated that neuronal CGRP expression is significantly increased in deoxycorticosterone (DOC)–salt hypertensive rats. To determine the hemodynamic role of CGRP in this setting, we used CGRP_8-37, a specific CGRP receptor antagonist. DOC-salt hypertension was induced in Sprague-Dawley rats. To control for DOC pellet implantation, left nephrectomy, and/or saline drinking water, we also studied four normotensive groups. Four weeks after the initiation of each protocol, all rats had intravenous (for drug administration) and arterial (for continuous mean arterial pressure monitoring) catheters surgically placed and were studied in the conscious, unrestrained state. Baseline mean arterial pressure was higher in the DOC-salt than normotensive rats (175±5 versus 119±4 mm Hg, P<.001). Vehicle administration did not alter mean arterial pressure in any group, and CGRP_8-37 administration (bolus doses of 3.2x10⁴ or 6.4x10⁴ pmol/L) did not change mean arterial pressure in the four normotensive groups. However, CGRP_8-37 administration to the DOC-salt rats rapidly and significantly increased mean arterial pressure at both the lower dose (9±1 mm Hg, P<.001) and higher dose (14±1 mm Hg, P<.001). In addition, the increase in mean arterial pressure between the two CGRP_8-37 doses was also significant (P<.01), indicating a dose-dependent response. We conclude that the increase in neuronal CGRP expression in DOC-salt hypertension plays a compensatory vasodilator role to attenuate the elevated blood pressure. These results provide the first conclusive evidence that CGRP plays a direct role in DOC-salt hypertension.

Key Words: calcitonin gene–related peptide • blood pressure • hypertension, experimental • mineralocorticoids • neuropeptides

	Introduction

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Calcitonin gene–related peptide (CGRP) is produced by the tissue-specific alternative splicing of the primary transcript of the calcitonin/CGRP gene.¹ This peptide is distributed throughout the central and peripheral nervous systems and is located in areas involved in cardiovascular function.^{2 3} A prominent site of CGRP synthesis is the DRG. DRG contain the cell bodies of primary afferent neurons that extend CGRP-containing nerves to peripheral sites such as blood vessels and the central spinal cord.^{4 5} A dense perivascular CGRP neural network is seen around the blood vessels in virtually all vascular beds.² CGRP has been shown to dilate multiple vascular beds, with the coronary vasculature being a particularly sensitive target.^{6 7} Systemic administration of CGRP decreases BP in a dose-dependent manner in both normotensive animals and humans as well as in SHR.^{2 3 7} The primary mechanism responsible for this BP reduction is peripheral arterial dilation.⁷ Therefore, it has been postulated that CGRP plays a role in the modulation of BP and/or regional organ blood flows both under normal physiological conditions and in the pathophysiology of hypertension.

The role that CGRP plays in hypertension is not known. Data concerning circulating levels of immunoreactive CGRP (iCGRP) in hypertensive humans and experimental rodent models of hypertension have been conflicting.^{2 8 9 10} Such results have been attributed to the heterogeneous nature of hypertension or simply to differences in assays.² We previously reported that the neuronal expression of CGRP is differentially regulated in two different models of hypertension. In the SHR, a genetic normal-renin, sodium-independent model, iCGRP content was decreased in laminae I and II of the dorsal horn of the spinal cord, and CGRP mRNA levels were reduced in DRG compared with levels in normotensive Wistar-Kyoto control rats.^{11 12} In contrast, in the DOC-salt–induced hypertensive rat, an acquired low-renin, sodium-dependent model, iCGRP levels were elevated in the spinal cord and CGRP mRNA production was increased in DRG compared with normotensive controls.¹³ This latter finding is in agreement with an earlier study involving hypertensive humans that showed an increase in circulating CGRP levels both in individuals with primary aldosteronism and in subjects placed on high versus low salt diets.¹⁴ Therefore, these results suggest that a decrease in CGRP expression, as observed in the SHR, could contribute to the high BP by the relative reduction of vasodilator activity, whereas an increase in CGRP, as seen in DOC-salt hypertension, could attenuate the high BP by the compensatory augmentation of vasodilator activity.

To determine the hemodynamic role of the enhanced CGRP expression in DOC-salt hypertension, we used CGRP_8-37, a potent and specific CGRP receptor antagonist. High-affinity vascular CGRP receptors have been demonstrated,^{2 3 15} and it has been reported that CGRP_8-37 can inhibit vasodilation of the rat mesenteric arterial bed induced by periarterial nerve stimulation and the hemodynamic actions of intravenously administered -CGRP in the conscious rat.^{16 17} In other in vivo studies, it was shown that the CGRP antagonist could significantly inhibit the hypotensive effects of intravenously administered CGRP but not the hypotensive effects of other vasodilators, such as bradykinin, histamine, and substance P.¹⁸ Furthermore, in studies designed to investigate the CGRP-evoked increase in skin blood flow (via vasodilation), CGRP_8-37 was able to block the increased blood flow induced by CGRP administration but had no effect on the vasodilator response produced by vasoactive intestinal peptide or prostaglandin E₁.¹⁹ Importantly, CGRP_8-37 was also able to inhibit the increase in blood flow in response to capsaicin, an agent that stimulates CGRP release from sensory nerve terminals. This indicates that the CGRP antagonist can block the vasodilation induced by endogenously released CGRP. Therefore, if the upregulation of CGRP expression in DOC-salt hypertension is a compensatory vasodilator mechanism to attenuate the elevated BP, then CGRP_8-37 administration to these rats should further increase the already high BP and have considerably less effect on BP in normotensive controls.

	Methods

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Animals
All protocols were approved by the institutional Animal Care and Use Committee. For all surgical procedures, rats were anesthetized with ketamine and xylazine (80 and 4 mg/kg IP, respectively). Male Sprague-Dawley rats (Harlan), initially weighing 175 g, were studied. DOC-salt hypertension (n=7) was induced by a left nephrectomy and implantation in the nape of deoxycorticosterone acetate (150 mg pellet, Innovative Research of America) and the drinking of water containing 0.9% NaCl/0.2% KCl (Table, group A). All control rats had a placebo pellet similarly implanted. Seven rats had a sham left nephrectomy and were given tap water to drink (group B). Five rats underwent a left nephrectomy and were given tap water to drink (group C). Six rats underwent a sham nephrectomy and were given NaCl/KCl drinking water (group D), and an additional six rats underwent a left nephrectomy and were given NaCl/KCl drinking water (group E).

View this table: [in this window] [in a new window]   Table 1. Summary of Deoxycorticosterone-Salt (Group A) and Normotensive Control Groups (Groups B Through E) Studied

Hemodynamic Determinations
Human -CGRP was obtained from Phoenix Laboratories. Human -CGRP_8-37 was synthesized by standard solid-phase T-BOC chemistry. The peptide was deprotected and cleaved with hydrofluoric acid. Amino acid analysis after acid hydrolysis gave the proper molar ratios. Both CGRP and CGRP_8-37 were dissolved in saline. Each rat was anesthetized as described previously. The left carotid artery was cannulated for continuous measurement of MAP with a pressure transducer linked to a recorder (Gould Instruments). The right jugular vein was also cannulated for administration of either vehicle (saline), CGRP, or CGRP_8-37. Hemodynamic studies were performed approximately 3 hours after surgery with rats fully awake and unrestrained.

Cell Culture
We prepared DRG neurons following a modified protocol initially described by Lindsay.²⁰ DRG (cervical, thoracic, and lumbar; 40 to 45 per rat) were dissected from 150- to 175-g male Sprague-Dawley rats and collected in Ham's F-12 medium supplemented with 10% horse serum (growth medium). Ganglia, freed of roots, were dissociated in 0.125% collagenase with a constant flow of 5% CO₂/95% O₂, washed, and then treated with 0.25% trypsin. After another wash, the ganglia were transferred to growth medium containing DNase (80 µg/mL) and soybean trypsin inhibitor (100 µg/mL). Single cell suspensions were obtained by trituration of enzymatically softened ganglia. After additional washes, the dissociated neurons were plated in six-well culture dishes coated with polyornithine and maintained in growth medium at 37°C in 5% CO₂. After 48 hours, the cells were placed in serum-free conditions. The medium was Ham's F-12 supplemented with insulin (5 µg/mL), transferrin (100 µg/mL), progesterone (20 nmol/L), selenium (30 nmol/L), and putrescine (100 µmol/L, N2 supplement, GIBCO-BRL). The yield of neurons was approximately 1.5x10⁵ to 2.0x10⁵ from 40 to 45 ganglia. For these studies, the dissociated DRG cells were plated at a density of 20 000 to 30 000 neurons per well.

Hybridization Probes, RNA Isolation and Analysis, and Radioimmunoassay
The -CGRP hybridization probe was a 1.4-kb Sau3A rat genomic restriction fragment containing CGRP exons 5 (0.2 kb) and 6 (0.46 kb).¹ The 18S rRNA hybridization probe was a 1.15-kb BamHI-EcoRI restriction fragment of the mouse 18S rRNA gene.²¹ The DNA inserts were purified by agarose gel electrophoresis and subsequently labeled with [-³²P]dCTP using a random hexanucleotide DNA labeling kit (Amersham). After dissociation and plating (72 hours), the cultured neurons were treated with either DOC (10^-6 mol/L) or vehicle. Total cellular RNA was isolated by the guanidine isothiocyanate method and analyzed by Northern blot hybridization.^{22 23} The membranes were initially hybridized with the ³²P-labeled CGRP DNA probe. As a control, the CGRP probe was removed from the membrane, which was then rehybridized with the 18S rDNA probe. After hybridization, the membranes were washed and exposed to x-ray film at -70°C with an intensifying screen. The relative levels of CGRP mRNA and 18S rRNA were quantified by computerized scanning laser densitometry.

To measure released iCGRP in the medium from control and treated DRG neurons, we used a commercially available rabbit anti-rat CGRP radioimmunoassay kit (Phoenix Pharmaceuticals).²³ All assays were performed under conditions recommended by the supplier. The total protein content in each sample was determined by the Bradford method (Bio-Rad).

Statistical Analysis
Statistical significance was determined by Student's t test or, where appropriate, by ANOVA followed by the Tukey-Kramer multiple comparisons test. The acceptable level of significance was set at a value of P<.05. Data in the figures are presented as mean±SE.

	Results

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Validation of the CGRP Receptor Antagonist
For the initial studies, it was necessary to verify that our CGRP_8-37 preparation could block the hypotensive effects of exogenously administered -CGRP in normal rats. Sprague-Dawley rats (300 g, n=3) were anesthetized, and the left carotid artery was cannulated for continuous MAP recording. The right jugular vein was also cannulated for administration of either vehicle, CGRP, or CGRP_8-37. After the rats were fully awake and in the unrestrained state, bolus doses of CGRP (100 and 500 pmol/L in 0.1 mL saline) were administered intravenously. The CGRP doses of 100 and 500 pmol/L produced MAP decreases of 18±6 (Fig 1A) and 35±10 mm Hg (Fig 1B), respectively. To assess the inhibitory effects of CGRP_8-37, we initially showed that a bolus injection of the indicated dose of CGRP_8-37 by itself had no effect on MAP (Fig 1C). Similar results were observed after administration of CGRP_8-37 doses of either 3.2x10⁴ or 6.4x10⁴ pmol/L. When the indicated CGRP and CGRP_8-37 doses were given sequentially (either CGRP first or CGRP_8-37 first), the antagonist completely inhibited any reduction in MAP (Fig 1C). Likewise, both 3.2x10⁴ and 6.4x10⁴ pmol/L CGRP_8-37 completely inhibited the MAP decrease secondary to the administration of 500 pmol/L CGRP. These results confirm that CGRP_8-37 blocks the hypotensive actions of exogenously administered CGRP.

View larger version (18K): [in this window] [in a new window]   Figure 1. CGRP8-37 inhibits the hypotensive effect of exogenously administered -CGRP in normal rats. Panels display representative MAP tracings. A and B, Administration of the indicated doses of CGRP; C, administration of either CGRP8-37 alone or the sequential administration of CGRP8-37 and CGRP.

Hemodynamic Effects of CGRP_8-37 in DOC-Salt Hypertensive and Normotensive Control Rats
DOC-salt hypertension was induced in Sprague-Dawley rats. Four other groups were studied to control for the pellet implantation, left nephrectomy, and/or salt administration (Table). As described previously, in an earlier study using identical groups of DOC-salt and normotensive control rats, we observed enhanced neuronal CGRP expression only in the DOC-salt hypertensive rats. Four weeks after the initiation of each protocol, the rats were instrumented for continuous MAP recording and intravenous drug administration as previously described. The DOC-salt rats had a significantly higher baseline MAP than each of the four control groups (Table; P<.001, DOC-salt versus each of the four control groups). As expected, the two control groups that received a left nephrectomy tended to have a slightly higher MAP than the other two normotensive groups; however, there were no statistically significant differences in MAP among any of the four control groups.

Administration of vehicle (0.1 mL IV) did not significantly increase MAP in any of the five groups studied (DOC-salt, 2±1 mm Hg versus average of the four control groups, 2±1 mm Hg). Similarly, CGRP_8-37 administration at either indicated dose did not significantly increase MAP in any of the four normotensive groups (Fig 2). However, CGRP_8-37 administration to the DOC-salt hypertensive rats rapidly (the MAP increase began approximately 15 to 20 seconds after antagonist administration) induced a further increase of the already elevated MAP at both the lower (9±1 mm Hg, P<.001) and higher (14±1 mm Hg, P<.001) dose. Furthermore, the increase in MAP between the two CGRP_8-37 doses was also significant (P<.01), indicating a dose-dependent response. The duration of the pressor activity of CGRP_8-37 was relatively short (approximately 60 seconds for the lower dose and 90 seconds for the higher dose). This transient effect of CGRP_8-37 has been observed by other investigators who have used this antagonist in vivo and most likely reflects the rapid proteolysis of this peptide in the circulation.^{16 17} During these experiments, we also measured heart rate, which was not significantly changed by the CGRP antagonist in any of the five groups studied.

View larger version (20K): [in this window] [in a new window]   Figure 2. CGRP8-37 increases MAP in DOC-salt hypertensive rats but not normotensive controls. Rats were instrumented for continuous MAP recording and CGRP8-37 administration as described in the text. With the rats fully awake and unrestrained, bolus doses of the indicated amounts of CGRP8-37 were given. **P<.001, DOC-salt (group A) vs each of the four control groups at both CGRP8-37 doses; *P<.01, higher vs lower dose of CGRP8-37 in DOC-salt rats, MAP values are reported as mean±SE.

Effect of DOC on CGRP Expression In Vitro
To determine whether DOC could directly stimulate neuronal CGRP expression, we used primary cultures of rat DRG neurons. We recently published data demonstrating that NGF or activators of the protein kinase A and C signal transduction pathways significantly stimulate CGRP mRNA production and iCGRP release in cultured DRG neurons, whereas the glucocorticoid dexamethasone attenuates the stimulatory effects of NGF on CGRP expression.²³ Therefore, for this study, primary DRG neurons were treated (24 hours) with either DOC (10^-6 mol/L) or vehicle, and CGRP mRNA content was determined by Northern hybridization analysis and iCGRP release by a specific CGRP radioimmunoassay. As expected, no significant differences in either CGRP mRNA production (ratio of CGRP mRNA to 18S rRNA: DOC, 0.93±0.09 versus control, 0.83±0.15; n=4) or iCGRP release (DOC, 1.10±0.14 pg iCGRP/µg total protein per 0.1 mL versus control, 1.41±0.31; n=4) were observed.

	Discussion

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The results of the present study demonstrate that the pressor effect of CGRP_8-37 occurs only in the DOC-salt hypertensive rats, in which neuronal CGRP expression is markedly increased compared with the four normotensive groups. Importantly, these data support the hypothesis that in DOC-salt hypertension, CGRP is acting as a compensatory depressor to attenuate the elevated BP. CGRP_8-37 administration presumably blocks vascular CGRP receptors and further increases BP. It should be noted that these experiments were performed toward the end of the onset phase of DOC-salt hypertension.^{2 24} We do not know whether the compensatory vasodilator role of CGRP is maintained during the established phase of DOC-salt hypertension. Thus, further studies are required to determine whether CGRP can modulate the long-term regulation of BP in this model. The inability of CGRP_8-37 to alter BP in the control rats implies that CGRP does not play a major role in the regulation of systemic BP in the normotensive state but does not rule out a role for CGRP in the modulation of regional organ blood flows in this setting. In a recent report from other investigators,²⁵ CGRP_8-37 was used in studies with normal rats to show that CGRP is responsible for approximately 30% of basal coronary blood flow.

The question then arises as to how afferent neurons, whose primary function is the transmission of sensory information from peripheral tissues to the spinal cord, are able to regulate BP. DRG neurons give rise to afferent axons that terminate on virtually all peripheral tissues, including blood vessels, and centrally in the spinal cord where CGRP-containing nerve terminals innervate laminae I and II in the dorsal horn as well as the intermediolateral cell column, which contains the sympathetic preganglionic neurons.^{3 4 5} This connection could influence the activity of the sympathetic nervous system and thus vascular tone. In peripheral tissues, considerable evidence demonstrates the efferent release of neuropeptides (CGRP, substance P) from primary afferent nerve terminals.^{26 27 28 29 30 31} Local factors such as NGF,³⁰ vascular wall tension,^{28 31} and bradykinin/prostaglandins^{26 32} as well as interactions with the sympathetic nervous system³³ have been shown to modulate the release of CGRP and other neuropeptides. Moreover, using primary cultures of adult DRG neurons, we have demonstrated that NGF²³ or bradykinin/prostaglandins³⁴ can upregulate CGRP synthesis and release, whereas glucocorticoids²³ or ₂-adrenoceptor agonists can attenuate the stimulatory effects of NGF on CGRP (unpublished data, 1996). Thus, alterations in these factors, some of which are known to occur in hypertension, may mediate any changes seen in neuronal CGRP expression. If basal CGRP synthesis is increased or decreased, then these local factors could be expected to release more or less CGRP, respectively, resulting in a greater or lesser degree of vasodilation. Therefore, CGRP could modulate vascular tone via both efferent and afferent neuronal activities. Given the rapid onset of the hypertensive effect of CGRP_8-37 in the DOC-salt rats and because the antagonist probably does not penetrate the central nervous system, it is likely that most of the pressor activity of CGRP_8-37 seen in the present experiments results from a direct interaction of the antagonist with peripheral vascular CGRP receptors.

Both in vivo and in vitro studies demonstrate the existence of subclasses of CGRP receptors, and the target-organ distribution of these receptors is consistent with the known biological actions of this neuropeptide.^{2 3 15} In a number of systems, CGRP acts through increases in intracellular cAMP.^{35 36} There is also evidence for other mechanisms of action. In vascular smooth muscle, CGRP is reported to gate ATP-sensitive K⁺ channels,³⁷ but other reports indicate that the opening of this K⁺ channel is not involved in vascular relaxation.³⁵ However, there is considerable evidence that the vasodilator response evoked by CGRP is mediated in part by nitric oxide release and that various vascular beds differ in their degree of dependence on the presence of endothelium for the vasodilator effects of CGRP.³⁵ Thus, the depressor effects of CGRP appear to be partially mediated by endothelium-derived nitric oxide and may also involve a direct relaxation of arteries by increasing cAMP.

Regarding the mechanism or mechanisms responsible for the enhancement of neuronal CGRP expression in DOC-salt hypertension, CGRP could be stimulated simply by the elevated BP, or alterations in other factors may be required. In a previous study from our laboratory,³⁸ normal rats were chronically treated with the potent vasoconstrictor angiotensin II. As expected, these rats exhibited a marked increase in BP; however, CGRP mRNA content in DRG was not significantly altered. This result was confirmed by in vitro studies which showed that angiotensin II treatment of cultured DRG neurons had little effect on CGRP mRNA production or iCGRP release. Thus, these data indicate that an increase in BP by itself does not change CGRP expression and support the hypothesis that specific alterations in local and/or circulating factors (neuronal, hormonal, autocrine/paracrine) mediate the enhanced expression of CGRP in DOC-salt hypertension.

Another possibility is that DOC directly stimulates CGRP expression. This is unlikely because DOC itself has no effect on CGRP mRNA content or iCGRP release in primary cultures of adult rat DRG neurons. Although unlikely, we cannot rule out possible effects of DOC metabolites. It is also possible that DOC could regulate CGRP expression through an indirect mechanism by either stimulating or inhibiting factors that directly modulate neuronal CGRP expression. Alternatively, DOC-salt hypertension is characterized by significant alterations in calcium homeostasis, including decreased serum ionized calcium and increased dihydroxyvitamin D₃ and parathyroid hormone levels.²⁴ Because CGRP is a product of the calcitonin gene, which is intricately involved in calcium metabolism, and these changes in calcium homeostasis occur, it would be logical that these factors mediate the observed increase in CGRP. However, the SHR, which has decreased levels of neuronal CGRP expression, also displays alterations in serum ionized calcium and calcitropic hormones similar to those seen in DOC-salt hypertension.³⁹ Therefore, it appears that these factors alone do not totally explain the differential regulation of CGRP between the two models of hypertension. Thus, further studies are required to resolve this question.

In summary, these results indicate that the enhanced synthesis, and presumably release, of CGRP in DOC-salt hypertension is a compensatory depressor response intended to lower the elevated BP. This is the first demonstration that CGRP plays a significant and direct role in DOC-salt hypertension in the rat.

	Selected Abbreviations and Acronyms

 

BP = blood pressure CGRP = calcitonin gene–related peptide DOC = deoxycorticosterone DRG = dorsal root ganglia MAP = mean arterial pressure NGF = nerve growth factor SHR = spontaneously hypertensive rat(s)

	Acknowledgments

 
These studies were supported by National Institutes of Health grant HL-44277-01A1 and American Heart Association, Texas Affiliate, grant 90G-663. We thank Lagaya Boyd and Wilma Frye for the excellent secretarial assistance and Drs Richard Bukoski, Donna Wang, and Sunil Wimalawansa for a critical reading of the manuscript.

Received March 15, 1996; first decision July 19, 1996; accepted October 29, 1996.

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