Regulation of Neuronal Calcitonin Gene–Related Peptide Expression
Role of Increased Blood Pressure
Abstract Calcitonin gene–related peptide (CGRP) is a potent vasodilator neuropeptide. We have previously demonstrated that CGRP mRNA levels are increased in dorsal root ganglia, and immunoreactive CGRP content is elevated in the spinal cord in mineralocorticoid-salt hypertension. Dorsal root ganglia neuronal cell bodies synthesize CGRP and send axons peripherally to blood vessels and centrally to spinal cord sites involved in blood pressure regulation. This increased synthesis of a potent vasodilator is a compensatory response to attenuate the increase in blood pressure; however, it is not known if neuronal CGRP is regulated simply by the elevated blood pressure or by changes in other parameters. To determine if elevation of blood pressure in normal rats induced by the administration of a potent vasoconstrictor can increase neuronal CGRP mRNA, 7-week-old male Sprague-Dawley rats were treated for 2 weeks with either angiotensin II (n=6) or vehicle (n=6) by using implanted osmotic minipumps. After the treatment period, the angiotensin II–treated rats displayed a marked increase in systolic blood pressure (angiotensin II, 217±18 versus control, 131±3 mm Hg, P<.001), and decrease in plasma renin activity (angiotensin II, 3.7±3.5 versus control, 35.9±14.2 ng · mL−1 · h−1, P<.05). However, dorsal root ganglia CGRP mRNA content did not significantly differ between the two groups of rats. These results demonstrate that a marked increase in blood pressure, by itself, does not increase neuronal CGRP mRNA accumulation. We propose that in mineralocorticoid-salt hypertension there are specific changes in as yet unidentified factor(s) that modulate neuronal synthesis and release of CGRP independent of blood pressure elevation.
Calcitonin gene–related peptide, a 37–amino-acid neuropeptide, is produced by the tissue-specific alternative splicing of the primary transcript from the calcitonin/CGRP gene.1 2 This peptide is distributed throughout the central and peripheral nervous systems and is located in areas involved in cardiovascular function.3 4 A prominent site of CGRP synthesis is the DRG. The DRG contain the cell bodies of primary afferent neurons, which extend CGRP-containing nerves to peripheral sites such as blood vessels and to central spinal cord sites known to be involved in BP regulation.3 4 5 6
CGRP is a potent vasodilator, approximately 1000 times more potent than acetylcholine or substance P.7 8 CGRP has significant and selective regional hemodynamic effects and has been shown to increase blood flows and/or decrease vascular resistance in the coronary, common carotid, renal, mesenteric, and hindquarter vascular beds.8 9 10 The coronary vasculature has been demonstrated to be a particularly sensitive target.7 8 Systemic administration of CGRP decreases BP in a dose-dependent manner in both normotensive animals and humans and in the spontaneously hypertensive rat.3 10 The primary mechanism responsible for this BP reduction is peripheral arterial dilation.8 9 10 On the basis of these potent vasodilator effects and the perivascular localization of CGRP, it has been postulated that this peptide plays a role in the regulation of BP and regional organ blood flows both under normal physiological conditions and in the pathophysiology of hypertension.3 4
The role that CGRP plays in hypertension is unclear. The data concerning circulating levels of iCGRP in hypertensive humans have been conflicting, with investigators reporting increased,11 unchanged,12 or decreased13 levels. Such results have been attributed to the heterogeneous nature of hypertension or due to differences in assays.3 Similarly, contradictory results concerning circulating levels of iCGRP in experiments with rodent models of hypertension have also been reported.3 4 In a previous study we demonstrated that the neuronal expression of CGRP is enhanced in DOC-salt hypertensive rats.14 In this acquired low-renin, sodium-dependent model of hypertension, iCGRP levels were elevated in laminae I and II of the dorsal horn of the spinal cord and, correspondingly, CGRP mRNA accumulation was increased in DRG compared with normotensive control rats. In a related study, CGRP8-37, a potent and specific CGRP receptor antagonist, was intravenously administered to DOC-salt hypertensive rats and normotensive controls.15 Administration of the antagonist had little effect on MAP in the normotensive rats whereas in the DOC-salt hypertensive rats CGRP8-37 significantly increased the already elevated MAP in a dose-dependent manner. These results demonstrate that the enhanced neuronal expression of CGRP in DOC-salt hypertension is a compensatory vasodilator mechanism to attenuate the elevated BP. However, it is not known if neuronal CGRP mRNA production is upregulated simply by the elevated BP or whether changes in other factors are required. Therefore, the purpose of the present study was to determine if the elevation of BP alone, via long-term administration of the potent vasoconstrictor Ang II would increase the neuronal levels of CGRP mRNA.
All protocols were approved by the institutional Animal Care and Use Committee. For pump implantation, the rats were anesthetized with ketamine/xylazine (4:1 solution at 0.1 mL/100 g body wt IP). Male Sprague-Dawley rats (7 weeks old; Harlan, Houston, Tex) were treated for 2 weeks with either Ang II (200 ng/kg per day, n=6) or vehicle (n=6) using implanted osmotic minipumps. Systolic BPs were determined in the nonheated state by the indirect tail-cuff method by using a photoelectric sensor. At the end of the treatment period, the animals were euthanatized by decapitation, and the thoracic and lumbar DRG were immediately dissected and frozen in liquid nitrogen for later RNA analysis. Blood samples were taken to determine PRA using a PRA radioimmunoassay kit (Incstar).
Hybridization Probes and RNA Analysis
The α-CGRP hybridization probe was a 1.4-kb Sau3A rat genomic restriction fragment containing CGRP exons 5 and 6, and the 18S rRNA hybridization probe was a 1.15-kb BamHI/EcoRI restriction fragment of the mouse 18S rDNA gene.1 16 The DNA inserts were excised from the plasmid vectors by using the appropriate restriction endonucleases and were purified by agarose gel electrophoresis.17 The hybridization probes were subsequently labeled with [α-32P]dCTP by random hexanucleotide DNA labeling (Amersham).
Total cellular RNA was isolated from the DRG tissue by the guanidinium–cesium chloride method.18 The RNA samples were subjected to electrophoresis on denaturing formaldehyde-agarose gels.17 The fractionated RNAs were transferred to nylon membranes and hybridized with the [32P]-labeled CGRP DNA probe, which hybridizes to both the α- and β-CGRP mRNA species. As a control, the CGRP probe was removed from the membrane, which was then hybridized with the 18S rDNA probe. After hybridization, the membranes were washed and exposed to Kodak X-Omat x-ray film (Eastman Kodak Co) at −70°C with an intensifying screen. After autoradiography, the relative levels of CGRP mRNA and 18S rRNA were quantified by computerized scanning laser densitometry.
Student’s t test was employed to determine statistical significance.
Long-term administration of Ang II to normal rats produced a rapid and significant increase in BP that was maintained throughout the treatment period. At the end of the 2-week treatment period, the final systolic BP attained by the rats receiving Ang II was 217±18 mm Hg compared with the vehicle-treated control rats (137±3 mm Hg, P<.001). Moreover, there was no significant difference in final body weight between the two groups (Ang II, 286±7 versus control, 270±7 g).
In addition to its hypertensive effects, long-term Ang II treatment would be expected to markedly decrease PRA. At the end of the 2-week treatment period, the Ang II–treated rats had approximately a 10-fold lower PRA compared with the vehicle-treated controls (Ang II, 3.7±3.5 versus control, 35.9±14.2 ng generated angiotensin I/mL per hour, P<.05).
To determine if the elevated BP in the Ang II–treated rats would result in an increase in neuronal CGRP mRNA production, as we previously observed in DOC-salt hypertensive rats, we used Northern hybridization analysis to compare relative DRG CGRP mRNA content between the two groups of animals. We have previously employed this technique to quantify CGRP mRNA levels both in intact DRG and primary cultures of adult DRG neurons.15 19 Fig 1⇓ demonstrates the levels of the 1.2-kb CGRP mRNA species (both α and β) present in DRG RNA samples from the six Ang II–treated and six control rats. As an internal control for possible differences in loading of RNA samples between the groups, 18S rRNA levels were similarly determined. As can be seen, there was no apparent difference in CGRP mRNA levels between the Ang II–treated and control rats. To confirm this observation, ratios of CGRP mRNA to 18S rRNA were determined by computer-assisted laser densitometric analysis of the autoradiogram (Fig 2⇓). There was no statistically significant difference in CGRP mRNA/18S rRNA ratios between the two groups of rats (Ang II, 1.16±0.18 versus control, 0.96±0.17). In addition, similar results were obtained when the values for CGRP mRNA were normalized to those for the glyceraldehyde 3-phosphate dehydrogenase mRNA species (Ang II, 0.41±0.06 versus control, 0.39±0.09). These results clearly demonstrate that the elevation of BP alone is not sufficient to stimulate neuronal CGRP mRNA accumulation.
As expected, long-term administration of the potent vasoconstrictor Ang II to normal rats markedly increased BP and decreased PRA. Although the increase in BP was similar to that observed in DOC-salt hypertensive rats, there was no significant difference in DRG CGRP mRNA content between the Ang II–treated and normotensive control rats. These data demonstrate that a marked increase in BP (secondary to Ang II) by itself does not alter neuronal CGRP mRNA production. Because we did not measure DRG iCGRP levels in this study, we cannot rule out the possibility that an increase in BP could modulate iCGRP levels through a regulatory mechanism at the translational level. However, in previously published studies both in vivo14 and in vitro in primary cultures of adult DRG neurons,19 we have always observed a direct correlation between changes in CGRP mRNA content and iCGRP levels. Taken together, these results support the hypothesis that specific alterations in local and/or circulating factors (neuronal, hormonal, autocrine/paracrine) may mediate the enhanced expression of CGRP in DOC-salt hypertension. As described previously, we recently reported that the upregulation of CGRP in DOC-salt hypertensive rats is a compensatory vasodilator mechanism to attenuate the elevated BP.
Another important finding to come from this study is that Ang II does not regulate neuronal CGRP mRNA production either directly or indirectly. In support of these data we observed that in primary cultures of adult DRG neurons,19 which have been shown to have functional Ang II receptors,20 Ang II did not significantly alter either CGRP mRNA content (CGRP mRNA/18S rRNA; Ang II, 1.82±0.03 versus control, 1.96±0.18; n=3) or iCGRP release (pg iCGRP/μg total protein/0.1 mL; Ang II, 0.72±0.11 versus control, 0.69±0.08). Therefore, an increase in BP and/or alterations in Ang II do not appear to play a role in the stimulation of neuronal CGRP mRNA accumulation in DOC-salt hypertension.
Another possibility is that DOC directly upregulates CGRP expression. This is unlikely since we have shown that DOC itself has no effect on CGRP mRNA content or iCGRP release in primary cultures of adult rat DRG neurons.14 Another potential explanation is that DOC-salt hypertension is characterized by significant alterations in calcium homeostasis, and that changes in these parameters mediate the increase in the neuronal expression of CGRP that we previously observed. Serum ionized calcium levels are significantly decreased while vitamin D3 and parathyroid hormone levels have been reported to be increased in DOC-salt hypertension.21 Because CGRP is a product of the calcitonin gene, which is intricately involved in calcium homeostasis, and these changes in calcium metabolism are observed, it would be logical to speculate that these factors might play a role in the regulation of CGRP in DOC-salt hypertension. However, the spontaneously hypertensive rat, which we have previously demonstrated to have significantly lower levels of neuronal CGRP expression, also displays alterations in serum ionized calcium and calcitropic hormones similar to these observed in DOC-salt hypertension.22 23 24 Therefore, it appears that these parameters of calcium homeostasis alone do not totally explain the differential regulation of CGRP between the two hypertensive models. Although we have not yet identified the factor or factors that mediate the upregulation of neuronal CGRP expression in DOC-salt hypertension, other possible candidates include nerve growth factor and other neurotrophic factors, bradykinin/prostaglandins, and the sympathetic nervous system.
In conclusion, the results of this study demonstrate that the elevation of BP alone does not lead to changes in neuronal CGRP mRNA production. Future studies are warranted to determine specific factor(s) involved in the regulation of CGRP that also may be altered in hypertension and/or play a role in the pathophysiology of hypertension.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|CGRP||=||calcitonin gene–related peptide|
|DRG||=||dorsal root ganglia|
|MAP||=||mean arterial pressure|
|PRA||=||plasma renin activity|
These studies were supported by NIH grant HL44277-0141 and American Heart Association Texas Affiliate grant 90G-663. Donald J. DiPette is supported by an Established Investigator Award of the American Heart Association. We thank Rhoda Thompson for the excellent secretarial assistance.
Reprint requests to Scott C. Supowit, PhD, 8.104 Medical Research Bldg 1065, University of Texas Medical Branch, Galveston, TX 77555-1065.
- Received June 18, 1995.
- Revision received August 18, 1995.
- Accepted September 7, 1995.
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