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a Department of Obstetrics and Gynecology,
b Internal Medicine, The University of Texas Medical Branch, Galveston, Texas 77555
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CGRP is a neuropeptide produced by the alternative processing of the primary transcript of the calcitonin/CGRP gene [10, 11]. CGRP is a potent vasodilator, approximately 100–1000 times more potent than acetylcholine or substance P [12, 13]. CGRP has significant and selective hemodynamic effects and has been shown to increase blood flow and/or decrease vascular resistance in multiple vascular beds [13]. This peptide is distributed throughout the central and peripheral nervous system. A prominent site of CGRP expression is the dorsal root ganglia (DRG), where neuronal cell bodies extend CGRP-containing nerves peripherally to blood vessels and centrally to the spinal cord [14, 15]. Immunohistochemical studies showed CGRP-containing nerve fibers throughout the cardiovascular system [16, 17], uterus [18], and ovary [19].
Several lines of evidence suggest that factors such as prostaglandins, bradykinin, and nerve growth factor (NGF) stimulate CGRP synthesis and release from peripheral tissues [20–23]. During development, NGF is essential for the survival and differentiation of sympathetic neurons and the majority of neural crest-derived sensory neurons such as those found in DRG [24, 25]. In mature sensory neurons, NGF is required to maintain the fully differentiated phenotype [24, 25]. Reports indicate that both NGF and CGRP are colocalized in 40% of cells in DRG [26]. NGF can directly stimulate CGRP expression in mature sensory neurons in normal adult rats in vivo (unpublished results), as well as in primary cultures of adult DRG neurons [24, 27]. These data suggest that NGF may be one of the important factors for the synthesis and release of CGRP.
Recent studies from our group [28] and others [29, 30] suggested that circulatory levels of CGRP are significantly elevated during pregnancy and decreased at term and postpartum in rats and humans. Furthermore, our studies indicate that both estradiol-17ß and progesterone up-regulate plasma CGRP levels in adult ovariectomized rats [28], suggesting that elevated levels of female sex steroid hormones during pregnancy may be responsible for the increased CGRP levels in circulation during pregnancy to maintain vascular adaptations. We hypothesize that steroid hormones either directly or indirectly through modulation of other systems, such as the NGF system, may stimulate CGRP synthesis in DRG. To test this hypothesis, we measured the effects of estradiol-17ß and progesterone on CGRP mRNA expression and protein synthesis in DRG neurons in ovariectomized rats. Further, primary cultures of adult rat DRG neurons were used to determine whether the effects of these steroid hormones are direct or are mediated indirectly through the augmentation of the stimulatory actions of NGF on CGRP synthesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult nonpregnant (170–200 g BW) animals were purchased from Harlan Sprague Dawley (Indianapolis, IN). All animals were housed in a climate-controlled room with a 12L:12D schedule and were fed standard rat chow with water to drink ad libitum. All procedures were approved by the Animal Care and Use Committee of the University of Texas Medical Branch (Galveston, TX).
In Vivo Treatments
Adult nonpregnant rats were bilaterally ovariectomized (ovx) under ketamine (45 mg/kg BW; Fort Dodge Laboratory, Fort Dodge, IA) and xylazine (5 mg/kg BW; Burns Veterinary Supply, New York, NJ) and allowed 7 days to recover before treatment. Groups of animals received a single s.c. injection of 5 µg estradiol-17ß (E2) or 4 mg progesterone (P4) in 0.5 ml sesame oil, either alone or in combination (E2+P4), or vehicle (sesame oil) only. Groups of three to four rats were killed at 4, 8, 24, and 48 h after treatments, and DRG (cervical, thoracic, and lumbar; 40–45 per rat) were collected from both sides. The immunoreactive (i-)CGRP and CGRP mRNA levels in the pooled DRG from each rat were analyzed by RIA and Northern blot analysis, respectively.
In Vitro Treatments
Primary cultures of DRG neurons from adult nonpregnant rats regardless of estrous cycle, in duplicate, were used according to the protocol described by Lindsay [25]. DRG (cervical, thoracic, and lumbar; 40–45 per rat) were dissected and collected in Ham's F-12 medium (no phenol red) supplemented with 10% horse serum (growth medium) (Gibco-BRL, Grand Island, NY). Ganglia, freed of roots, were dissociated in 0.125% collagenase (Boehringer Mannheim, Indianapolis, IN) with a constant flow of 5% CO2 and 95% O2, washed, and then treated with 0.25% trypsin (Worthington, Freehold, NY). After another wash, the ganglia were transferred to growth medium containing DNase (80 µg/ml) and soybean trypsin inhibitor (100 µg/ml; Sigma Chemical Co., St. Louis, MO). Single-cell suspensions were obtained by trituration of enzymatically softened ganglia. After additional washes, the dissociated neurons were plated on multiwell culture dishes coated with polyornithine (Sigma) and cultured for 24 h in growth medium (no phenol red) at 37°C in 5% CO2. After 24 h, the growth medium was replaced with serum-free F-12 media (no phenol red) supplemented with N2 (Gibco-BRL) and incubated for another 24 h with or without various concentrations of E2 (0.1–10.0 nM), P4 (1.0–100 nM), or E2+P4 (1 + 10; 1 + 100 nM) (Sigma). In another study, the neurons were incubated for 24 or 48 h with E2 (10 nM), P4 (100 nM), or E2+P4 (10 nM + 100 nM) in absence or presence of NGF (20 ng/ml). In addition, we examined the effects of varying doses of E2 and P4 in the presence of NGF (20 ng/ml) on CGRP synthesis by DRG neurons in culture. In these in vitro cultures, i-CGRP in the medium and mRNA for CGRP in the neurons were measured. The yield of neurons was approximately 1.5 x 105 to 2.0 x 105 from 40 to 45 ganglia. For these studies, the dissociated DRG cells were plated at a density of 20 000–30 000 neurons per well.
Hybridization Probes, RNA Isolation and Analysis, and RIA
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-CGRP hybridization probe was a 1.4-kilobase (kb) Sau 3A rat genomic restriction fragment containing CGRP axons 5 (0.2 kb) and 6 (0.46 kb). The 18S rRNA hybridization probe was a 1.15 kb BamHI-EcoRI restriction fragment of the mouse 18S rRNA gene. The DNA inserts were purified by agarose gel electrophoresis and subsequently labeled with [-32P]dCTP using a random hexanucleotide DNA labeling kit (Amersham Pharmacia Biotech, Piscataway, NJ). Total cellular RNA was isolated by the guanidine isothiocyanate method and analyzed by Northern blot hybridization [31,32]. 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 i-CGRP in the medium from control and treated DRG neurons, we used a commercially available rabbit anti-rat CGRP RIA kit (Phoenix Pharmaceuticals, Belmont, CA). 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, Hercules, CA).
Statistical Analysis
Statistical significance was determined by one-way ANOVA followed by the Bonferonni t-test. The acceptable level of significance was P < 0.05. Data in the figures are presented as mean ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As shown in Figure 1a, CGRP mRNA levels in DRG from E2-treated animals were significantly (P < 0.05) elevated at 24 h; they returned to control levels by 48 h. On the other hand, i-CGRP levels from E2-, P4-, or E2+P4-treated rats were significantly (P < 0.05) elevated at both 24 and 48 h (Fig. 1b). Both mRNA and peptide levels were unchanged at 4 and 8 h after treatment (data not shown). These data suggest that E2 and P4 can stimulate CGRP expression (both mRNA and protein) in DRG in a time-dependent manner.
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Effects of Steroid Hormones on the Expression of CGRP in Cultures of DRG Neurons
To determine whether female sex steroid hormones alter CGRP synthesis or peptide levels either directly or indirectly, we utilized primary neuronal cultures in this study. We therefore initially examined the effects of varying doses of E2 (0.1, 1, 10 nM) or P4 (1, 10, 100 nM) on CGRP expression either alone or in combination (E2+P4; 1 + 10, or 1 + 100 nM) by treating the cultured cells for 24 h. Neither E2 nor P4 at any of the doses used was effective in modulating either CGRP mRNA content (Fig. 2) or peptide levels (data not shown) in these neurons, indicating that these steroid hormones do not have direct effects on CGRP expression in adult rat DRG. Lack of stimulatory effects of steroid hormones on CGRP synthesis was also observed in cultured cells of DRG obtained from ovx rats (data not shown).
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Steroid Hormone Modulation of NGF-Induced CGRP Expression
To assess whether E2 or P4 can modulate the stimulatory effects of NGF on CGRP expression in a time-dependent manner, primary cultures of DRG neurons from female rats were incubated with E2 (10 nM), P4 (100 nM), E2+P4 (10 nM + 100 nM), or vehicle in the presence or absence of NGF (20 ng/ml) for 24 and 48 h. A representative Northern blot for the 24-h time period is shown in Figure 3. As shown in Figures 4 and 5, NGF alone stimulated both CGRP mRNA and peptide levels both at 24 and at 48 h (P < 0.05). However, the stimulatory effects of NGF on CGRP mRNA and protein were amplified in the presence of E2, P4, and E2+P4 (Figs. 4 and 5) (P < 0.05). The effects of E2, P4, and E2+P4 in the presence of NGF were substantial compared to those with NGF alone as measured by both mRNA and peptide levels for CGRP, and these effects were time dependent (Figs. 4 and 5). Both CGRP mRNA and CGRP protein levels were substantially (P < 0.05) higher at 48 h compared to the 24-h time period, and this occurred in the E2, P4, and E2+P4 groups in the presence of NGF. These studies suggest that sex steroids stimulate both CGRP mRNA accumulation and i-CGRP release through amplifying the effects of NGF on DRG neurons.
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We then evaluated the effects of varying doses of E2 (0.01–10 nM) and P4 (0.01–1 µM), or E2+P4 (E2 0.1 nM + P4 1.0 µM), in the presence of NGF (20 ng/ml) on the expression of CGRP in DRG neurons. DRG neurons were treated for 24 h. As shown in Figure 6, E2 increased CGRP mRNA in the neurons in a dose-dependent manner with maximal effects at 1 nM of E2 in the presence of NGF (P < 0.05). However, the effects of E2 appeared to be biphasic, since the 10 nM dose was less effective than 1 nM. Similarly, P4 treatment also increased the mRNA for CGRP in a dose-dependent manner, with maximal effects at 0.1 µM in the presence of NGF (P < 0.05). Again, the effects of P4 at 1 µM were less effective than at 0.1 µM, indicating biphasic effects. These data suggest that the stimulatory effects of NGF on CGRP expression are amplified in the presence of E2 and P4.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Limited information is available on the identification of the factors and molecular mechanisms that regulate CGRP biosynthesis. We have previously reported that CGRP mRNA and i-CGRP levels were significantly decreased in DRG and laminae I/II of the dorsal horn of the spinal cord, respectively, in spontaneously hypertensive rats compared to Wistar-Kyoto controls [33, 34]. On the other hand, we observed that CGRP mRNA in DRG and i-CGRP levels in laminae I/II of the dorsal horn of the spinal cord were elevated in mineralocorticoid-salt-induced hypertensive rats compared to normotensive controls [35]. Further studies on this latter model of hypertension showed that the increase in CGRP expression was a compensatory depressor response to buffer the elevated blood pressure. Taken together, these studies suggest that in certain pathological conditions, changes in local and/or circulating factors (neuronal, hormonal, autocrine/paracrine) can significantly alter the long-term synthesis and release of CGRP and thus modulate vascular tone.
Female reproductive hormones profoundly influence the vascular system. Previous studies have shown that circulating i-CGRP levels are elevated during pregnancy [27, 29] and that they fall after delivery in humans. Recent studies from our laboratory demonstrated that in rats, plasma CGRP levels were increased during pregnancy and decreased during delivery and postpartum [28]. These studies further show that both E2 and P4, alone or in combination, elevated the CGRP levels in circulation in adult rats [28], indicating that elevated sex steroid hormones during pregnancy may increase CGRP expression in this setting. In other studies, gonadal steroids, especially estrogens, modulated the synthesis of CGRP in pituitary gland [36]. Estrogens were also shown to increase CGRP immunoreactivity in other tissues, including the periventricular preoptic nucleus and the medial preoptic nucleus [37, 38], which may dampen the activity of the sympathetic nervous system. On the other hand, dexamethasone, neurotrophic factor, and leukemia inhibitory factor are able to markedly attenuate the stimulatory effects of NGF on CGRP mRNA and i-CGRP accumulation in DRG primary cultures in male rats [27, 39]. Similarly, we previously demonstrated that 2-adrenergic receptor agonist UK 14,304 can attenuate the stimulatory effects of NGF on CGRP mRNA accumulation and i-CGRP levels in primary cultures of adult male rat DRG neurons [40]. However, activators of protein kinases A (cAMP analogues, forskolin) and C (phorbol esters), as well as the inflammatory mediators bradykinin and prostaglandins, can significantly enhance the neuropeptide release from sensory neurons and cultured embryonic [41, 42] and adult rat DRG neurons [39, 43]. Therefore, these findings suggest that several different classes of regulatory factors alter the synthesis and release of CGRP through NGF, although the mechanisms by which this occurs is not known. Data from the present studies demonstrate that in vivo administration of both E2 and P4 increased the CGRP mRNA expression at 24 h (Fig. 1a) and peptide levels at both 24 h and 48 h (Fig. 1b). These in vivo data suggest that steroid hormones stimulate the CGRP mRNA synthesis and i-CGRP in DRG and perhaps facilitate the release into the peripheral tissues and circulation and thus modulate vascular functions.
It is possible that female sex steroid hormones either directly or indirectly through other mechanisms may elevate CGRP synthesis. Therefore, to understand the mechanisms(s) involved in the regulation of CGRP synthesis, we utilized primary cultures of DRG neurons from female rats. In contrast to findings in the in vivo studies, both E2 and P4 had no effects in vitro on CGRP synthesis by DRG neurons in culture (Fig. 2). This indicated to us that the effects of steroid hormones on CGRP synthesis are not direct. Lack of steroid hormone effects was observed even when the DRG were obtained from ovx rats (data not shown), suggesting that the hormonal status of the animals at the time of DRG collection did not mask the responses in vitro to steroid hormones. Since several lines of evidence suggest that CGRP synthesis is elevated by NGF [24, 27], we incubated the DRG neurons in the presence or absence of NGF for 24-h and 48-h time periods to determine whether steroid hormones amplify the effects of NGF. In the presence of NGF, both of these hormones increased CGRP mRNA at 24 h and i-CGRP secretion at 48 h (Figs. 4a and 5b). Furthermore, the dose-dependent stimulation of CGRP synthesis in DRG with E2 and P4 occurs with in vitro cultures in the presence of NGF, suggesting that steroid hormone effects may involve up-regulation of NGF responses. We speculate that steroid hormones up-regulate NGF receptor levels in DRG or postreceptor mechanisms and thus NGF responses.
Studies are currently under way to evaluate the effects of E2 and P4 on NGF receptors in DRG neurons. NGF has two receptors, trkA and p75. It has been recently shown that estrogen receptor mRNA and protein are present in adult female rat DRG [44], and more than 80% of CGRP-immunoreactive neurons in lumbar DRG contain E2 receptors [45]. These studies [44] further showed that estrogen replacement in ovx rats resulted in a transient down-regulation of p75 mRNA and a time-dependent up-regulation of trkA mRNA in DRG neurons [44]. Moreover, ligand-binding studies demonstrated that receptors for E2 and P4 are present in DRG-Schwann cells in vitro [46]. Estrogen regulation of NGF receptor mRNA and the existence of both E2 and P4 receptors in adult ganglionic cells support the hypothesis that these hormones may be important in modulating neurotrophin actions in neuronal functions. With regard to the specificity of the DRG cells involved in steroid-induced increases in CGRP synthesis, the possibility of involvement of glia or fibroblasts in cell cultures cannot be excluded in the present studies. However, in studies on the factors that regulate CGRP synthesized in DRG, the CGRP was synthesized primarily from neurons, and NGF stimulated synthesis of CGRP in enriched neuronal cells in culture [24]. Additional studies are needed to further clarify the effects of steroids with respect to specific cell types involved in CGRP synthesis in DRG.
Previously we have demonstrated that 1) vasodilatory effects of CGRP are enhanced during pregnancy and in the presence of female sex steroid hormones [6] and 2) administration of CGRP8–37, a specific CGRP receptor antagonist, to NG-nitro-L-arginine methyl ester-induced hypertensive rats further increased the blood pressure during pregnancy [7]. These studies together with our present observations suggest that increased synthesis of CGRP in DRG neurons by female sex steroid hormones may also play a significant role in the regulation of cardiovascular functions during pregnancy.
We suggest that CGRP may not only be involved in vascular adaptations during pregnancy but also play an important mediator role in maintaining cardiovascular effects of sex hormones in females. Studies have shown that circulatory CGRP levels were elevated in postmenopausal women after hormone replacement therapy [47, 48]. Further, recent studies suggest that CGRP release from mesenteric arteries was significantly decreased in aged rats [49]. The decrease in cardiovascular protection is likely to be due to a decrease in circulating estrogens. In addition, additive factors may include reduced CGRP synthesis and/or release in peripheral tissues. However, studies in the older individual or in aging animal models are required to confirm this.
In summary, these studies show that female sex hormones stimulate CGRP mRNA and peptide synthesis in DRG neurons. Further, our studies also demonstrate that the stimulatory effects of steroid hormones on CGRP can be observed only in the presence of NGF. From these data we speculate that both E2 and P4 may increase NGF receptors in DRG neurons and consequently amplify the stimulatory effects of NGF on CGRP expression.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by grants from NIH HD 30273 and HL 58144 to C.Y. 
2 Correspondence: Chandrasekhar Yallampalli, Department of Obstetrics and Gynecology, 301 University Boulevard, Medical Research Bldg., Rm. 11.138, Galveston, TX 77555-1062. FAX: 409 747 0475; chyallam{at}utmb.edu
Accepted: November 29, 1999.
Received: June 12, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2-Adrenergic receptor activation inhibits calcitonin gene-related peptide expression in cultured dorsal root ganglia neurons. Brain Res 1998; 782:184–193.[CrossRef][Medline]This article has been cited by other articles:
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  A. M. Aukes, N. Bishop, J. Godfrey, and M. J. Cipolla The Influence of Pregnancy and Gender on Perivascular Innervation of Rat Posterior Cerebral Arteries Reproductive Sciences, April 1, 2008; 15(4): 411 - 419. [Abstract] [PDF]
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 N. Shimozawa, K. Okajima, and N. Harada Estrogen and isoflavone attenuate stress-induced gastric mucosal injury by inhibiting decreases in gastric tissue levels of CGRP in ovariectomized rats Am J Physiol Gastrointest Liver Physiol, February 1, 2007; 292(2): G615 - G619. [Abstract] [Full Text] [PDF]
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C.-L. Lu, J.-C. Hsieh, M.-L. Tsaur, Y.-H. Huang, P. S. Wang, L.-L. Wu, P.-Y. Liu, F.-Y. Chang, and S.-D. Lee Estrogen rapidly modulates mustard oil-induced visceral hypersensitivity in conscious female rats: a role of CREB phosphorylation in spinal dorsal horn neurons Am J Physiol Gastrointest Liver Physiol, January 1, 2007; 292(1): G438 - G446. [Abstract] [Full Text] [PDF]
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 N. M. Flake, T. O. Hermanstyne, and M. S. Gold Testosterone and estrogen have opposing actions on inflammation-induced plasma extravasation in the rat temporomandibular joint Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2006; 291(2): R343 - R348. [Abstract] [Full Text] [PDF]
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 P. M. Schmitt, K. Gohil, and M. P. Kaufman Spinal estrogen attenuates the exercise pressor reflex but has little effect on the expression of genes regulating neurotransmitters in the dorsal root ganglia J Appl Physiol, March 1, 2006; 100(3): 958 - 964. [Abstract] [Full Text] [PDF]
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 C.N. Mowa and R.E. Papka The Role of Sensory Neurons in Cervical Ripening: Effects of Estrogen and Neuropeptides J. Histochem. Cytochem., October 1, 2004; 52(10): 1249 - 1258. [Abstract] [Full Text] [PDF]
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 P.R.R. Gangula, S.J. Wimalawansa, and C. Yallampalli Sex Steroid Hormones Enhance Hypotensive Effects of Calcitonin Gene-Related Peptide in Aged Female Rats Biol Reprod, December 1, 2002; 67(6): 1881 - 1887. [Abstract] [Full Text] [PDF]
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P. Lanlua, R.D. Bukoski, S.J. Wimalawansa, and C. Yallampalli Effects of Pregnancy and Female Sex Steroid Hormones on Calcitonin Gene-Related Peptide Content of Mesenteric Artery in Rats Biol Reprod, November 1, 2002; 67(5): 1430 - 1434. [Abstract] [Full Text] [PDF]
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P. Lanlua, P.R.R. Gangula, G. Taglialatela, and C. Yallampalli Gestational Changes in Calcitonin Gene-Related Peptide, Nerve Growth Factor, and Its Receptors in Rat Dorsal Root Ganglia Biol Reprod, November 1, 2001; 65(5): 1601 - 1605. [Abstract] [Full Text] [PDF]
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P. Lanlua, F. Decorti, P.R.R. Gangula, K. Chung, G. Taglialatela, and C. Yallampalli Female Steroid Hormones Modulate Receptors for Nerve Growth Factor in Rat Dorsal Root Ganglia Biol Reprod, January 1, 2001; 64(1): 331 - 338. [Abstract] [Full Text]
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