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(Hypertension. 1995;25:1333-1338.)
© 1995 American Heart Association, Inc.
Articles |
Enhanced Neuronal Expression of Calcitonin Gene–Related Peptide in Mineralocorticoid-Salt Hypertension
From the Departments of Internal Medicine (Division of General Internal Medicine and Hypertension Section), Human Biological Chemistry and Genetics, and the Marine Biomedical Institute, University of Texas Medical Branch, Galveston.
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Abstract |
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Abstract Dorsal root ganglia neuronal cell bodies synthesize the vasodilator neuropeptide calcitonin gene–related peptide and innervate the blood vessels and spinal cord sites (laminae I and II) involved in blood pressure regulation. We previously demonstrated that calcitonin gene–related peptide mRNA content is significantly decreased in dorsal root ganglia and that immunoreactive calcitonin gene–related peptide levels are reduced in laminae I and II of the dorsal horn of the spinal cord in the spontaneously hypertensive rat compared with Wistar-Kyoto control rats. To determine whether neuronal calcitonin gene–related peptide expression is also altered in mineralocorticoid-salt hypertension, we quantified calcitonin gene–related peptide mRNA levels in dorsal root ganglia and protein content in laminae I and II of the spinal cord in rats with mineralocorticoid-salt–induced hypertension. To control for pellet implantation, saline drinking water, and/or uninephrectomy, four normotensive groups were similarly studied. By Northern hybridization analysis, the ratio of calcitonin gene–related peptide mRNA to 18S rRNA was increased approximately fivefold in hypertensive rats (33±7) compared with each of the four normotensive control groups (average of the four groups, 6±0.5; P<.01, mineralocorticoid-salt group versus each group). The density of the peptide, quantified by computer-assisted image analysis, in laminae I and II in the hypertensive rats was also increased (66±1 versus average of the four groups, 46±2 arbitrary units; P<.001, mineralocorticoid-salt group versus each group). In conclusion, neuronal levels of calcitonin gene–related peptide mRNA and protein are increased in mineralocorticoid-salt hypertension. Therefore, increased neuronal synthesis and available stores of this potent vasodilator may be compensatory responses to and thus attenuate the blood pressure elevation in this experimental model of hypertension.
Key Words: calcitonin gene–related peptide • blood pressure • hypertension, experimental • mineralocorticoids • genes • neuropeptides • immunohistochemistry • RNA
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Introduction |
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Calcitonin gene–related peptide (CGRP) is a potent vasodilator neuropeptide produced by alternative processing of the primary transcript of the calcitonin-CGRP gene.1 Altered calcium homeostasis has been reported in both human essential hypertension and experimental animal models of hypertension such as the spontaneously hypertensive rat (SHR) and mineralocorticoid (DOC)-salt–induced hypertension.2 3 4 Such alterations included decreased serum ionized calcium levels and increased serum parathyroid hormone (PTH) and 1,25 dihydroxyvitamin D3 levels. These changes appear to be particularly prominent in low-renin hypertension.2 Because CGRP is a product of the calcitonin gene and these changes in calcium metabolism are observed, it is logical to speculate that alterations in CGRP may also be present in hypertension.
CGRP has pronounced cardiovascular effects, including vasodilation and positive chronotropic and inotropic effects.5 6 7 The systemic administration of CGRP decreases blood pressure by peripheral vasodilation, and the coronary circulation appears to be particularly sensitive to the vasodilator effects of CGRP.6 7 Immunocytochemical and radioimmunoassay techniques have identified CGRP-containing nerve fibers throughout the cardiovascular system, particularly in association with blood vessels.8 We previously demonstrated in Sprague-Dawley rats that altered dietary calcium intake directly changes the neuronal content of immunoreactive CGRP (iCGRP) in laminae I and II of the dorsal horn of the spinal cord: low dietary calcium decreased serum ionized calcium and iCGRP content; high dietary calcium increased serum ionized calcium and iCGRP content.9 Laminae I and II of the dorsal horn of the spinal cord are the site of termination for incoming primary afferents rich in CGRP. The cell bodies for these afferent axons are found in the dorsal root ganglia (DRG). These neurons produce abundant levels of CGRP and project axons and fibers not only into the spinal cord but also to all peripheral tissues, including blood vessels.10 11 12
Our laboratory also demonstrated that the SHR had decreased serum-ionized calcium levels compared with Wistar-Kyoto control animals. Furthermore, there were significant reductions in DRG CGRP mRNA and iCGRP in laminae I and II of the spinal cord.13 14 Support for these results is provided by reports that demonstrated an age-related decrease in iCGRP and CGRP vasodilator activity in perivascular nerves associated with mesenteric vascular beds isolated from SHR compared with those isolated from age-matched Wistar-Kyoto control rats.15 Other reports indicate that CGRP-containing nerves counteract adrenergic vasoconstriction in peripheral resistance vessels.16 17 Therefore, it has been suggested that in the SHR the observed decrease in neuronal CGRP synthesis and/or release results in reduced vasodilation and may facilitate adrenergic vasoconstriction. These effects, in turn, could contribute to the elevated peripheral resistance observed in this experimented model of hypertension.13 14 15 16 17 Because DOC-salt hypertension is also characterized by marked abnormalities of calcium homeostasis similar to the SHR, the present study was undertaken to quantify CGRP mRNA accumulation in DRG and iCGRP levels in laminae I and II of the spinal cord in DOC-salt hypertensive rats and normotensive controls.
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Methods |
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Animals
A total of 59 male Sprague-Dawley rats (Harlan), initially weighing between 150 and 175 g, were studied. All protocols were approved by the institutional Animal Care and Use Committee. All rats were anesthetized with ketamine-xylazine (4:1 solution at 0.1 mL/100 g body weight IP) for surgical procedures and pellet implantation. DOC-salt hypertension (n=13) was induced by a left nephrectomy and implantation in the nape of the neck of deoxycorticosterone acetate (150-mg pellet, Innovative Research of America) and 0.9% NaCl–0.2% KCl drinking water ad libitum (the Table, group A). Other groups of rats were studied to control for the nephrectomy, salt administration, and pellet implantation. All control rats had a placebo pellet similarly implanted. A total of 16 rats had a sham left nephrectomy and were given tap water ad libitum. (the Table, group B). The 10 rats that underwent a left nephrectomy also were given tap water ad libitum (the Table, group C). In addition, 10 rats underwent a sham nephrectomy and were given a similar NaCl-KCl drinking water ad libitum (the Table, group D), and another 10 rats underwent a left nephrectomy and were given NaCl-KCl drinking water ad libitum (the Table, group E). Body weights and systolic blood pressures of all rats were recorded 3 to 4 weeks after the initiation of each protocol, the latter by indirect tail cuff in the nonheated state with a photoelectric sensor (IITC). An indicated number of animals were killed 2 to 3 days after blood pressures were determined, and the thoracic and lumbar DRG from each animal were immediately dissected and frozen in liquid nitrogen for subsequent RNA analysis.
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Hybridization Probes
The 
-CGRP hybridization probe was a 1.4-kb Sau3A
rat genomic restriction fragment containing CGRP exons 5 and 6; the 18S
rRNA hybridization probe was a 1.15-kb
BamH1-EcoR1 restriction fragment of the mouse 18S
rDNA gene.18 19 The DNA inserts were excised from the
plasmid vectors with the appropriate restriction endonucleases and
purified by agarose gel electrophoresis. The hybridization probes were
subsequently labeled with (-32P) deoxycytidine
triphosphate (dCTP) by random hexanucleotide DNA labeling
(Amersham).
RNA Analysis
Total cellular RNA was isolated from the DRG tissue by the
guanidinium–cesium chloride method.20 The RNA samples
were subjected to electrophoresis on denaturing formaldehyde agarose
gels.21 The fractionated RNAs were transferred to nylon
membranes and hybridized with the 32P-labeled CGRP DNA
probe that hybridizes to both the - and ß-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.
Immunocytochemical Analysis
The remaining animals from each of the five groups of rats were
deeply anesthetized with sodium pentobarbital intraperitoneally and
perfused intracardially with warm saline (100 to 200 mL) followed by
4% paraformaldehyde (1 L) in phosphate buffer (4°C, pH 7.4) for 45
to 60 minutes. The spinal cord (T-2 through L-4) was dissected and put
in sucrose (30% in phosphate buffer) overnight. Spinal cords were cut
frozen (30 µm) on a sliding microtome and stained
immunocytochemically for CGRP with the peroxidase-antiperoxidase method
in partitioned screen-bottomed trays that allowed simultaneous exposure
of tissues from all animals to each reagent. The CGRP antibody was
rabbit anti-human (1:2000, Peninsula). Positive and negative
immunocytochemical controls demonstrated specificity.9 13
Immunocytochemically stained spinal cords were analyzed from a light
table linked through a video recorder to a Quantex computer-assisted
image processing system. Readings were taken with a point counting
program in laminae I and II of the dorsal horn of the thoracic and the
lumbar spinal cord. We took 7 to 18 radiance readings from laminae I
and II and 4 to 6 readings from white matter as background levels from
each of five sections to be averaged for each animal. The average
values recorded from laminae I and II were subtracted from the
background to yield a corrected radiance or density reading for each
rat in both groups (ie, background equals zero).
Statistical Analysis
All data are expressed as mean±SEM. ANOVA followed by the
Scheffé test were used to determine statistical significance
where appropriate. A value of P<.05 was considered
statistically significant.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Blood Pressure Analysis
The Table summarizes the treatments received by the experimental (group A) and control (groups B through E) groups described in the "Methods" section and gives the final systolic tail-cuff blood pressures attained by each group. After 3 to 4 weeks of each treatment, the DOC-salt rats had a highly significant increase in blood pressure compared with each of the four control groups (P<.001, DOC-salt rats versus each control group). The two control groups that received salt in their drinking water displayed slightly higher blood pressures compared with the other two normotensive control groups. Only the blood pressure increase for group E achieved statistical significance compared with group B (P<.05); however, this is not surprising because these animals underwent a uninephrectomy in addition to receiving salt in the drinking water.
RNA Analysis
In a previous study in which we quantified relative DRG CGRP mRNA
levels between SHRs and Wistar-Kyoto rats using Northern hybridization
analysis, we established that this assay was specific for CGRP mRNA
and that the intensity of the hybridization signal was proportional to
the amount of RNA in each sample.14 We also observed that
this was true for the 18S rRNA used as an internal control for each RNA
sample. This technique was therefore used to quantify DRG CGRP mRNA
content in the DOC-salt and normotensive control animals.
In the first series of experiments, six DOC-salt (group A) rats were
studied. To control for the possible effects of the pellet
implantations, nephrectomy, and/or salt administration, it was
necessary to also examine DRG CGRP mRNA expression in four groups of
control rats (groups B through E). Fig 1 is a
representative Northern blot demonstrating the levels of both
the 1.2-kb CGRP mRNA species (both and ß) and 18S rRNA
present in DRG RNA samples from three animals in the DOC-salt group
and three normotensive control rats from group B. Fig 2
is a representative blot that demonstrates the levels of CGRP
mRNA and 18S rRNA present in DRG RNA samples from three (group B)
or four (groups C through E) rats in each of the four control groups.
DRG RNA samples from each animal in the five groups were analyzed
similarly. Laser densitometric analysis revealed that the DOC-salt
rats had significantly higher levels of CGRP mRNA than did animals from
the four control groups. As an internal control for possible
differences in loading of RNA samples between the groups, 18S rRNA
levels were similarly determined. There was no significant difference
between the DOC-salt rats and the control rats. As Fig 3
shows, when the values for the CGRP mRNA levels were normalized to
those for 18S rRNA, the ratio of CGRP mRNA to 18S rRNA was
significantly greater in the DOC-salt rats compared with the four
control groups (33±7 versus the average of 6±0.5, respectively;
P<.01, DOC-rats versus each group). These data clearly
demonstrate that CGRP mRNA accumulation is significantly altered only
in the DOC-salt rats. Thus, pellet implantation, uninephrectomy, and
salt administration cannot account for the increase seen in the
DOC-salt rats.
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Immunocytochemical Analysis
For the next set of experiments, quantitative immunocytochemical
techniques were used to evaluate changes in iCGRP content in laminae I
and II of the dorsal horn of the spinal cord in the DOC-salt
hypertensive rats and the four normotensive control
groups.9 13 Fig 4 shows four
representative sections (two lumbar, two thoracic) of the
spinal cords (laminae I and II) from the DOC-salt and control (group B)
animals. Immunocytochemical staining of the spinal cord sections
revealed a visually perceptible increase in the density of iCGRP
staining in laminae I and II of the DOC-salt rats compared with the
normotensive control animals. Similar results were observed when spinal
cord sections from the DOC-salt rats were compared with those from the
other three control groups (S.C.S., D.J.D., unpublished data, 1994). As
Fig 5 shows, computer-assisted image processing
confirmed that the density of iCGRP in laminae I and II from the
DOC-salt rats was significantly increased compared with each of the
four control groups (66±1 versus the average 46±2 arbitrary units,
respectively; P<.001 versus each group).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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For these studies, we have quantified relative levels of DRG CGRP mRNA and iCGRP content in laminae I and II of the dorsal horn of the spinal cord in DOC-salt hypertensive rats and normotensive control rats. DRG were chosen for study because they contain the neuronal cell bodies that produce abundant CGRP and give rise to the afferent axons and fibers that terminate peripherally on blood vessels and centrally in laminae I and II of the spinal cord. The CGRP-containing nerve terminals innervate laminae I and II and the intermediolateral cell column of the spinal cord, which contains the sympathetic preganglionic neurons.12 This connection could influence the activity of the sympathetic nervous system and thus vascular tone. In peripheral tissues (ie, blood vessels) innervated by these afferent neurons, there is considerable evidence demonstrating the efferent release of neuropeptides (substance P, CGRP) from these primary afferent nerve terminals.22 23 Local factors such as vascular wall tension, bradykinin, prostaglandins, cytokines, and interactions with the sympathetic nervous system have been shown to modulate CGRP release.16 17 24 25 26 If basal neuronal CGRP content is increased or decreased, then these local factors would be expected to release more or less CGRP, respectively, resulting in a greater or lesser degree of vasodilation. Therefore, CGRP could modulate vascular tone through both efferent and afferent neuronal activity.
On the basis of both the dietary calcium and SHR studies and the observation that DOC-salt–induced hypertension in the rat is accompanied by a decrease in serum-ionized calcium levels, we anticipated that this model of experimental hypertension might also exhibit a reduction in neuronal CGRP expression. In contrast to our expectations, DRG CGRP mRNA levels were increased approximately 500%, and iCGRP content in laminae I and II of the spinal cord was elevated approximately 50% over that in the control normotensive rats. Furthermore, these increases were specific for DOC-salt–induced hypertension because they were not seen in the other groups of normotensive rats receiving the same NaCl-KCl drinking water, nephrectomy alone, or a combination of both. Rather than being implicated in the pathogenesis and/or maintenance of the elevation in blood pressure, as has been suggested for the SHR, it may be that the CGRP mRNA and iCGRP levels are increased as a compensatory vasodilator response in DOC-salt hypertension.
Because the CGRP hybridization probe used in these studies hybridizes
to both - and ß-CGRP mRNA species, both of which are synthesized
in the DRG, we do not yet know if the increase in neuronal CGRP
expression results from the enhanced production of one or both CGRP
gene products. Although differential expression of the and ß
genes has been reported in neuronal tissues, its significance is not
clear because in the rat and in humans the and ß protein
sequences differ by only one and three amino acids, respectively, and
there are no significant differences in the biological activities of
the two peptides.18 27 We also observed a quantitative
difference between the increase in CGRP mRNA accumulation in the DRG
and the elevated iCGRP levels observed in laminae I and II of the
spinal cord in the DOC-salt rats. One possible explanation for these
results is that neuronal CGRP expression has been shown to be regulated
at the translational and posttranslational levels. In this regard, it
was reported that in a rat model of experimental diabetes, CGRP and
substance P content in the sciatic nerve was significantly reduced in
the absence of any alterations in velocity of axonal transport compared
with normal control rats.28 There were, however, no
detectable changes in CGRP or substance P mRNAs in the lumbar DRG.
Other possibilities include altered rates of axonal transport of CGRP
in the DOC-salt rats or asymmetrical axonal transport of CGRP between
central and peripheral sensory nerve terminals.29
It is intriguing that two different models of hypertension, the SHR and DOC-salt–induced hypertension, have opposite effects on neuronal CGRP expression. One possible explanation is that in the DOC-salt rats, alterations in unidentified neuronal, hormonal, and autocrine or paracrine factors increase CGRP expression as a compensatory response to high blood pressure. Using primary cultures of adult rat DRG neurons, we and others demonstrated that nerve growth factor and agents that activate the protein kinase A and C signal transduction pathways can significantly upregulate CGRP mRNA accumulation and release of iCGRP.30 31 In the SHR it may be that changes or defects in receptors and/or intracellular signaling mechanisms that regulate CGRP synthesis are responsible for the significant decrease in neuronal CGRP expression observed in this genetic model of hypertension. Alterations in intracellular signaling mechanisms, specifically at the level of the GTP-binding regulatory proteins, have been described in vascular tissues in rodent models of genetic hypertension.32 33 It is not known, however, whether functional differences related to CGRP regulation exist in DRG neurons that are unique to the SHR. Another possible explanation is that DOC directly enhances neuronal CGRP expression. We think this is unlikely because DOC has no effect on CGRP mRNA content or iCGRP release in primary cultures of DRG neurons, although an effect of DOC metabolites has not been ruled out (unpublished data, 1994). Because both SHRs and DOC-salt hypertensive rats have been shown to have decreased serum-ionized calcium and increased PTH levels, it appears that these factors alone do not totally explain the differential regulation of CGRP expression between the two hypertensive models. Thus, if there is a relation between CGRP expression and hypertension, the role of CGRP could differ, depending on the physiological defects responsible for the development and maintenance of high blood pressure.
In summary, we have demonstrated that DOC-salt–induced hypertension in the rat is associated with a significant increase in neuronal CGRP mRNA and peptide levels. Thus, an increase in the neuronal production and release of CGRP, a potent vasodilator, may be compensatory responses to the blood pressure elevation in this experimental model of hypertension. Future studies should delineate the factor(s) responsible for this enhanced CGRP gene expression. Alterations in CGRP, either increased in DOC-salt hypertension or decreased in the SHR, may play a role in the pathophysiology of the blood pressure elevation and regional blood flows in these settings.
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Acknowledgments |
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We wish to thank Vicki Isaacks and Michelle McCall for the excellent secretarial assistance and Jay Carson, Scott Richardson, and Charlotte Barker for excellent technical assistance. These studies were supported by NIH grants NS-11255, NS-28064, and R01HL-44277-01A1 and American Heart Association, Texas Affiliate grants 87R-654 and 90G-663. Dr DiPette is supported by an Established Investigator Award from the American Heart Association, and Dr Westlund is supported by a Research Career Development Award, NS01445.
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Footnotes |
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Reprint requests to Scott C. Supowit, PhD, 8.104 Medical Research Bldg 1065, University of Texas Medical Branch, Galveston, TX 77555-1065.
Received November 8, 1994; first decision December 7, 1994; accepted January 23, 1995.
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 P.R.R. Gangula, P. Lanlua, S. Wimalawansa, S. Supowit, D. DiPette, and C. Yallampalli Regulation of Calcitonin Gene-Related Peptide Expression in Dorsal Root Ganglia of Rats by Female Sex Steroid Hormones Biol Reprod, April 1, 2000; 62(4): 1033 - 1039. [Abstract] [Full Text]
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P. R. R. Gangula, H. Zhao, S. C. Supowit, S. J. Wimalawansa, D. J. Dipette, K. N. Westlund, R. F. Gagel, and C. Yallampalli Increased Blood Pressure in {alpha}-Calcitonin Gene-Related Peptide/Calcitonin Gene Knockout Mice Hypertension, January 1, 2000; 35(1): 470 - 475. [Abstract] [Full Text] [PDF]
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D. H. Wang, J. Li, and J. Qiu Rapid Communication: Salt-Sensitive Hypertension Induced by Sensory Denervation : Introduction of a New Model Hypertension, October 1, 1998; 32(4): 649 - 653. [Abstract] [Full Text] [PDF]
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S. C. Supowit, H. Zhao, D. M. Hallman, and D. J. DiPette Calcitonin Gene-Related Peptide Is a Depressor in Subtotal Nephrectomy Hypertension Hypertension, January 1, 1998; 31(1): 391 - 396. [Abstract] [Full Text] [PDF]
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S. C. Supowit, H. Zhao, D. M. Hallman, and D. J. DiPette Calcitonin Gene–Related Peptide Is a Depressor of Deoxycorticosterone-Salt Hypertension in the Rat Hypertension, April 1, 1997; 29(4): 945 - 950. [Abstract] [Full Text]
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P. R. R. Gangula, S. C. Supowit, S. J. Wimalawansa, H. Zhao, D. M. Hallman, D. J. DiPette, and C. Yallampalli Calcitonin Gene-Related Peptide Is a Depressor in NG-Nitro-L-Arginine Methyl Ester-Induced Hypertension During Pregnancy Hypertension, January 1, 1997; 29(1): 248 - 253. [Abstract] [Full Text] [PDF]
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S. C. Supowit, H. Zhao, D. H. Wang, and D. J. DiPette Regulation of Neuronal Calcitonin Gene–Related Peptide Expression : Role of Increased Blood Pressure Hypertension, December 1, 1995; 26(6): 1177 - 1180. [Abstract] [Full Text]
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