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Biology of Reproduction 65, 1601-1605 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Gestational Changes in Calcitonin Gene-Related Peptide, Nerve Growth Factor, and Its Receptors in Rat Dorsal Root Ganglia1

P. Lanluaa,b, P.R.R. Gangulaa, G. Taglialatelab, and C. Yallampalli2,,a,b

a Departments of Obstetrics and Gynecology and b Anatomy and Neuroscience, The University of Texas Medical Branch, Galveston, Texas 77555

ABSTRACT

In dorsal root ganglia (DRG) cell cultures, levels of calcitonin gene-related peptide (CGRP) are increased in the presence of ovarian hormones and nerve growth factor (NGF). In addition, injection of ovariectomized rats with ovarian hormones led to an increase in levels of two NGF receptors, TrkA and p75NTR, in DRG. Thus, we hypothesized that increased levels of ovarian hormones during pregnancy may elevate the synthesis of CGRP and NGF receptors in the DRG. DRG harvested from rats on specific days of pregnancy, on Day 2 postpartum, and after ovariectomy were subjected to radioimmunoassay, Western blot analysis, and NGF immunoassay to determine levels of CGRP, TrkA and p75NTR, and NGF, respectively. CGRP levels in rat DRG were significantly higher during pregnancy than at Day 2 postpartum or in ovariectomized rats. Levels of both TrkA and p75NTR in DRG increased during pregnancy and remained elevated at Day 2 postpartum, but CGRP levels declined. Levels of NGF reached a statistically significant peak at Day 18 of gestation, and were not significantly reduced at Day 2 postpartum. Increased levels of ovarian steroid hormones during pregnancy may be involved in the synthesis of CGRP, however, the postpartum decreases in CGRP synthesis appear to be unrelated to NGF and its receptors.

estradiol, growth factors, pregnancy, progesterone

INTRODUCTION

Although it is known that increased blood volume, elevated cardiac output, and decreased total peripheral resistance occur during pregnancy [1–3], the precise mechanisms of these maternal hemodynamic changes are not well understood. The ovarian hormones, estrogen and progesterone, may be partially responsible for regulating vascular tone during pregnancy [4] via nitric oxide [5], prostaglandins [6], and calcitonin gene-related peptide (CGRP) [7–10]. CGRP is a neuropeptide of 37 amino acids that is synthesized primarily in the dorsal root ganglia (DRG), and is released into peripheral circulation [11] to lower blood pressure via peripheral vasodilation [12, 13]. However, no reports are available on how CGRP synthesis is regulated. When ovariectomized rats were injected with the female steroid hormones estradiol-17ß (E2) and progesterone (P4) either alone or together [14–17], CGRP levels became elevated in both the peripheral circulation and the DRG. Further, when these hormones increased during pregnancy, plasma CGRP levels were also shown to increase, and to then decrease at term, when the steroid hormone levels declined. However, it has remained unclear whether these changes in plasma CGRP were due to variations in CGRP synthesis in the DRG. Nerve growth factor (NGF) [18–21] was also reported to stimulate CGRP synthesis in the DRG. We observed that CGRP synthesis could not be achieved in DRG cell cultures by the addition of E2 or P4 unless NGF was also present [17].

NGF, a secretory protein from the neurotrophin family, is released from organs that are innervated by responsive neurons. NGF is important in the development and trophic maintenance of peripheral and central nervous systems [22, 23]. After its release, NGF binds to two types of receptors, a high-affinity receptor (TrkA) and a low-affinity receptor (p75NTR) [24, 25]. Female steroid hormones have been implicated in the regulation of NGF receptors in the DRG. The number of NGF binding sites in the DRG is greater in nonpregnant, intact female rats and in female rats injected with E2 at birth compared with the number of sites found in males [26]. With short-term E2 injections of ovariectomized rats and rats at the proestrus stage of the estrous cycle, levels of TrkA mRNA and protein in the DRG are increased [27–29]. Moreover, TrkA levels are decreased when ovarian steroid hormones are depleted by ovariectomy [27]. Therefore, the up-regulation of TrkA in the DRG may depend on high levels of female sex steroid hormones. However, the effects of ovarian hormones on p75NTR are not consistent. In a study by Sohrabji and coworkers [28], p75NTR mRNA in the DRG was shown to be increased in proestrus rats, but when ovariectomized rats were injected with E2, decreases in the receptor mRNA in the DRG were noticed. Recently, we have shown that p75NTR protein in the DRG was elevated with injections of P4 and a mixture of E2 and P4 in ovariectomized rats; E2 injection alone did not have a significant effect on p75NTR protein [27]. Thus, it appears that female sex steroid hormones in nonpregnant female rats regulate both TrkA and p75NTR in the DRG.

Because both E2 and P4 were increased during pregnancy, we hypothesized that CGRP and NGF receptor levels in the DRG are elevated during pregnancy. Therefore, in this study we assessed the changes in CGRP, TrkA, and p75NTR in the DRG during pregnancy and in the postpartum period. We also examined NGF levels in the DRG to determine whether NGF levels fluctuated during pregnancy and the postpartum period.

MATERIALS AND METHODS

Animals

All procedures were approved by the Animal Care and Use Committee at the University of Texas Medical Branch and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Adult male rats, adult female nonpregnant (8–9 wk old) rats, and Day 14 pregnant Sprague-Dawley rats were purchased from Harlan Sprague-Dawley (Houston, TX). After arrival at the animal care facility, all rats were maintained in the colony room at constant temperature with a 12L:12D cycle and allowed free access to water and rodent chow.

Groups of nonpregnant female rats were killed after ovariectomy or at various days of gestation after mating with male rats. For ovariectomy, female nonpregnant rats were deeply anesthetized with an i.p. injection of a ketamine (50 mg/kg) and xylazine (8 mg/kg) mixture, and the ovaries were removed bilaterally. The ovariectomized rats were killed a week after the surgery. For early pregnancy, rats were mated (presence of sperm in the vaginal flush was recorded as Day 1) and maintained in the animal care facility until they were killed at Days 5 and 10 of gestation. In addition, groups of pregnant rats were killed on Days 16, 18, 20, and 22, and postpartum Day 2 (PP2). All fetuses and pups were killed by halothane inhalation. All adult animals were killed with CO2 inhalation, and the DRG were collected carefully from both sides of the spinal cord from cervical 1 to lumbar 5 levels. All DRGs were kept at -80°C. Each parameter was measured in the pooled DRGs from a single animal, and three to five animals were used per treatment.

Radioimmunoassay for CGRP

CGRP levels in DRGs were determined with the use of a CGRP radioimmunoassay (RIA) kit (Phoenix Pharmaceuticals, Inc., Belmont, CA) according to the manufacturer's instructions. Briefly, DRGs were homogenized with an RIA buffer, and the homogenates were centrifuged at 5000 x g at 4°C for 10 min. The supernatant was separated and used for RIA reactions in duplicate, according to the manufacturer's instructions. CGRP was measured by RIA using a rabbit antiserum raised against synthetic rat {alpha}-CGRP conjugated to bovine albumin. The sensitivity was 32 pg/tube; the intraassay and interassay variations were 5% and 10%, respectively; and the cross-reactivities for the antibody used were 100% and 35.5% for rat {alpha}-CGRP and human {alpha}-CGRP, respectively.

Western Immunoblotting

DRGs used for Western blot analysis of both TrkA and p75NTR came from the same groups of animals. Briefly, DRGs were homogenized in lysis buffer (50 mM TBS, 1 mM EDTA, 5% SDS, and 50 mM PMSF), then the homogenate was centrifuged at 4000 x g for 20 min at 4°C. Aliquots containing 50 µg of protein were electrophoresed on 7.5% SDS-polyacrylamide denaturing gels (Bio-Rad, Hercules, CA), and each gel was blotted to a polyvinylidene fluoride membrane (Millipore, Bedford, MA). The membranes were placed in blocking buffer (5% powdered milk in wash buffer; Tris-buffered saline with 0.1% Tween 20) for 1 h at room temperature, and the blots were then incubated with primary antibodies (TrkA, 0.1 µg/ml; p75NTR, 0.1 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature. Next, the blots were incubated with horseradish peroxidase conjugated to either goat anti-rabbit (for TrkA receptor, 1:5000; Bio-Rad) or mouse anti-goat (for p75NTR receptor, 1:10 000; Santa Cruz Biotechnology) antibodies. The membranes were rinsed with wash buffer, and the enhanced chemiluminescence reagent (ECL kit; Amersham, Piscataway, NJ) was added and incubated for 1 min at room temperature. The blots were exposed to Hyperfilm ECL, and the intensity of specific immunoreactive bands was quantified by a densitometric scanning program (AlphaEase; Alpha Innotech Corporation, San Leandro, CA). Densitometric units of specific protein bands were measured for each animal. Specimens for each animal to be compared for each parameter were run together, and all blots were probed with antibody at the same time. Blots from all groups were exposed to one film, and the arbitrary units were compared among groups. For easy appreciation of the relative changes among groups, data are plotted as percentage change from controls. The signals obtained for both TrkA and p75NTR proteins from DRG were in linear range (data not shown). Because TrkA proteins with this antibody consistently showed two bands [27], we quantitated both these bands in our analysis.

NGF Immunoassay

The procedures used to measure NGF were based on the manufacturer's instructions for the NGF immunoassay system kit (Promega, Madison, WI). Briefly, DRGs were homogenized in a lysis buffer (20 mM Tris pH 8.0, 137 mM NaCl, 1% NP-40, 10% glycerol, 1 mM PMSF, 10 µg/ml aprotinin, 1 µg/ml leupeptin, and 0.5 mM sodium metavanadate), and the homogenate was then centrifuged at 1500 x g at 4°C for 20 min. The supernatant was diluted in DPBS (2.68 mM KCl, 136.9 mM NaCl, 1.47 mM KH2PO4, 8.09 mM Na2HPO4, 0.9 mM CaCl2·2H2O, and 0.49 mM MgCl2·6H2O pH 7.35). Acid treatment was used to break the 7 S NGF to the 2.5 S NGF form and to release NGF from its receptors and binding proteins. After the samples were diluted with DPBS, 1 N HCl (pH 2.6) was added to the samples for 15 min at room temperature. Next, the samples were neutralized with 1 N NaOH (pH 7.6). All diluted samples were kept at -80°C until the procedure was performed. The NGF immunoassay kit consisted of polyclonal antibodies against murine NGF, which was shown to react with rat NGF. The sensitivity was 5 pg/well, and the intraassay and interassay variations were 5.5% and 6.5%, respectively.

Statistical Analysis

The number of animals in each group varied from three to five. Results are presented as the mean ± SEM. Statistical differences between means in the pregnant groups and the control group were compared by ANOVA, followed by the Bonferroni test. A value of P < 0.05 was considered statistically significant. Densitometric arbitrary units for protein bands from each animal were used to assess the differences among various groups.

RESULTS

CGRP Levels in DRG Neurons During Pregnancy and Postpartum

CGRP levels in the DRG of rats on Days 5–22 of gestation, postpartum Day 2, and after ovariectomy were measured by RIA (Fig. 1). CGRP levels in the DRG of Day 5 pregnant rats did not vary significantly from those of ovariectomized rats. Furthermore, CGRP levels in the DRG on Day 10 to Day 22 of pregnancy were significantly increased, and the levels were significantly reduced at postpartum Day 2.



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  		FIG. 1. Immunoreactive CGRP in the DRG of pregnant rats at various days of gestation (Days 5–22), Day 2 postpartum (PP2), and in ovariectomized rats. The amount of CGRP in the DRG is normalized to the total protein content in the DRG. Values are mean ± SEM from four animals. *P < 0.05 compared with ovariectomized rats (ANOVA)

NGF Receptors in the DRG During Pregnancy and Postpartum

The NGF receptors, TrkA and p75NTR, in the DRG were measured by Western blot analysis during Day 5 to Day 22 of gestation, on postpartum Day 2, and in ovariectomized rats (Figs. 2 and 3). The reported sizes of TrkA and p75NTR are 140 and 75 kDa, respectively. Two bands at 140 and 150 kDa immunoreacted with the TrkA antibody. Moreover, the 150-kDa band did not appear to be due to phosphotyrosine on TrkA, because a phosphotyrosine antibody (Amersham Life Science Inc., Arlington Heights, IL) did not react with this band (unpublished data). The molecular weight of the band that immunoreacted with the p75NTR antibody in the DRG homogenate was 75 kDa, which is similar to what was previously reported [27, 30, 31]. Figures 2A and 3A show TrkA and p75NTR immunoreactive bands in the DRGs of pregnant, postpartum Day 2, and ovariectomized rats. Densitometric analysis of the bands showed that TrkA and p75NTR in the DRG were elevated during pregnancy and the postpartum period (Figs. 2B and 3B). Similar to CGRP levels in DRG, both TrkA and p75NTR levels in DRG were increased during pregnancy. The increases in p75NTR levels were significant from Day 5 of gestation; however, increases in TrkA levels attained significance on Day 20 of gestation. Although both TrkA and p75NTR levels remained high in the postpartum period, levels of CGRP in the DRG decreased.



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  		FIG. 2. Change in TrkA levels in the DRG of pregnant rats at various days of gestation (Days 5–22), Day 2 postpartum (PP2), and in ovariectomized rats. A) Western blots using TrkA antibody (n = 3–4). The numbers on the right side are molecular weight markers. B) A plot of the percentage change in the density of bands relative to ovariectomized animals (mean ± SEM from three to four rats). *P < 0.05, **P < 0.01 compared with ovariectomized rats (ANOVA)

NGF Levels in the DRG During Pregnancy and Postpartum

NGF levels in the DRG were measured to determine whether changes in CGRP levels were due to alterations in NGF levels. The NGF contents in the DRG of rats that were ovariectomized, pregnant, and on postpartum Day 2 were measured by an NGF immunoassay (Fig. 4). NGF levels on Day 18 of gestation were significantly elevated compared with those of nonpregnant ovariectomized rats. NGF content in the DRG from rats on Days 5–16 and 20–22 of gestation and at postpartum remained low, and were similar to those of ovariectomized rats.



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  		FIG. 4. NGF content in the DRG of pregnant rats at various days of gestation (Days 5–22), Day 2 postpartum (PP2), and in ovariectomized rats. The amount of NGF in the DRG is normalized to the total protein content in the DRG. Values are mean ± SEM from four animals. *P < 0.05 compared with ovariectomized rats (ANOVA)

DISCUSSION

In this study we showed that CGRP levels in the DRG were elevated during pregnancy and decreased at postpartum. Moreover, the NGF receptors, TrkA and p75NTR, were up-regulated during pregnancy, but did not decrease significantly at postpartum. Because it is known that CGRP synthesis is dependent upon both NGF receptors and NGF levels in the DRG, we measured the NGF contents in these specimens to explain the decreased CGRP levels at Day 2 postpartum. We found that NGF levels were significantly elevated at gestational Day 18, whereas CGRP levels were highest between Days 10 and 22. Levels of CGRP were significantly decreased at Day 2 postpartum, but NGF levels did not change. Therefore, we conclude that fluctuations in levels of female steroid hormones during pregnancy and postpartum may regulate CGRP synthesis in the DRG, but that these changes are not entirely dependent on levels of NGF and its receptors in the neurons.

Evidence of elevated CGRP synthesis in the DRG of ovariectomized rats after female steroid hormone injection [17] supported our hypothesis that CGRP synthesis in the DRG was dependent on ovarian steroid hormone levels. Another recent report demonstrated that estrogen receptors were coexpressed with CGRP in DRG neurons [32]. Together, these reports suggest that steroid hormones may regulate CGRP synthesis. Other published studies have reported that NGF stimulated CGRP synthesis in the DRG [18–21], and that TrkA and p75NTR, the NGF receptors, are coexpressed with CGRP in DRG neurons [33–35]. Furthermore, we and others [26–28] have shown that more NGF receptors are found in the DRG when ovarian hormones are elevated; we have shown this in animals at the proestrus stage of the estrous cycle and in ovariectomized rats after female sex steroid hormone treatments. In this study, we showed that CGRP levels in the DRG increased during pregnancy but decreased at postpartum. NGF receptor levels increased during early gestation and remained high throughout pregnancy and into the postpartum period. We also noted that the elevation of NGF receptors paralleled the increase in CGRP synthesis during pregnancy, however, the postpartum decreases in CGRP levels occurred despite the lack of a decrease in the levels of NGF receptors. Consequently, we examined the changes in NGF levels in the DRG during pregnancy and the postpartum period to determine whether the alterations in NGF levels reflected changes in CGRP synthesis. We found that the levels of NGF in the DRG were not entirely correlated with CGRP levels through pregnancy and postpartum.

Because our data indicate that NGF levels are not solely responsible for CGRP synthesis, we suggest that other contributing factors may be involved in CGRP synthesis during pregnancy. Others have shown that the promoter of the calcitonin/CGRP gene in the DRG contains response elements to NGF and cAMP [36, 37]. The TrkA signaling pathway also involves the transcription factor cAMP response element-binding protein (CREB) [38]. After NGF binds to its receptors, CREB is generated and activates CGRP synthesis in the DRG. Thus, it is possible that the changes in cAMP/CREB generation or binding to promoter regions may also contribute to CGRP gene transcription. Some of these additional regulation sites may be involved in NGF-induced CGRP synthesis, and thus may help to explain a lack of correlation between NGF, NGF receptor levels, and CGRP synthesis during pregnancy and postpartum.

The NGF mRNA has not been detected in the DRG; therefore, NGF in the DRG may primarily reflect the retrograde transport from the target organs—the female reproductive tract, the submaxillary gland, and the vasculature [39]. The transport of NGF to the DRG requires NGF receptors as well. The lack of substantial changes in NGF levels in the DRG suggests that there were no changes at the sites of NGF synthesis, which means that the available NGF could be transported to the DRG at postpartum via the NGF receptors. Zapf-Colby and Olefsky [40] reported that 40% of 125I-NGF is bound to TrkA receptors at pH 6.0; thus the homogenates were treated with acid at pH 2.6 to dissociate the NGF from its receptors and other binding proteins. Our unpublished data show that NGF levels in the DRG without acid treatment were several times lower than those with acid treatment. In this manuscript, we reported total NGF levels in the DRG and further report that these levels did not decrease at postpartum, when CGRP levels declined.

In summary, CGRP levels in the DRG were elevated during pregnancy, and these elevations occurred when NGF receptor levels were increased in the DRG. Nevertheless, CGRP levels in the DRG declined at postpartum, but there was not a concomitant decrease in levels of NGF or its receptors. Thus, CGRP synthesis may depend not only on NGF and its receptors, but perhaps on other factors as well, such as alterations in the second messenger systems involved in CGRP synthesis. The changes in ovarian steroid hormones during pregnancy and postpartum may regulate some of these mechanisms along with NGF receptors in the DRG. More detailed studies are required to delineate the mechanisms involved in CGRP synthesis during pregnancy and the role of female steroid hormones in this process.



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  		FIG. 3. Change in p75NTR levels in the DRG of pregnant rats at various days of gestation (Days 5–22), Day 2 postpartum (PP2), and in ovariectomized rats. A) Western blots using p75NTR antibody (n = 3–4). The numbers on the right-hand side are molecular weight markers. B) A plot of the percentage change in the density of bands relative to ovariectomized rats (mean ± SEM from three to four animals). *P < 0.05, **P < 0.01, ***P < 0.001 compared with ovariectomized rats (ANOVA)

ACKNOWLEDGMENTS

We thank Ms. P. Necessary for editorial comments.

FOOTNOTES

First decision: 22 May 2001.

1 Supported in part by the National Institutes of Health through grants HL 58144 and HD 30273. Back

2 Correspondence: Chandrasekhar Yallampalli, Department of Obstetrics and Gynecology, The University of Texas Medical Branch, 301 University Boulevard, Route 1062, Galveston, TX 77555-1062. FAX: 409 747 0475; chyallam{at}utmb.edu Back

Accepted: July 5, 2001.

Received: April 30, 2001.

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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.
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