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Biology of Reproduction 64, 331-338 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Female Steroid Hormones Modulate Receptors for Nerve Growth Factor in Rat Dorsal Root Ganglia1

P. Lanluaa,b, F. Decortia, P.R.R. Gangulaa, K. Chungb, G. Taglialatelab, and C. Yallampalli2,a

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

ABSTRACT

Calcitonin gene-related peptide (CGRP) is a vasodilatory peptide, and it is primarily synthesized in dorsal root ganglia (DRG). Plasma CGRP levels increase during pregnancy and with steroid hormones, and nerve growth factor (NGF) stimulates calcitonin/CGRP promoter and CGRP synthesis in DRG. We previously showed that CGRP levels in DRG were stimulated with steroid hormone treatments in vivo but not in vitro. Thus, the stimulation of CGRP by these hormones may be indirect through the upregulation of NGF effects. We hypothesized that the female sex steroid hormones upregulate NGF receptors, trkA and p75NTR, in DRG. We examined the effects of 17ß-estradiol (E2) and progesterone (P4) on NGF receptors in DRG obtained from ovariectomized (ovx) rats. Groups of 4 ovx rats were injected s.c. with 5 µg E2, 4 mg P4, or 5 µg E2 + 4 mg P4 in 0.2 ml sesame oil or injected with oil only and were killed at 6, 24, and 48 h. In addition, ovx rats were also injected s.c. with varying doses (0.2, 1.0, 5.0, 25 µg) of E2 (0.5, 1.5, 4, 10 mg) P4, and (5 µg) E2 + (0.5, 1.5, 4.0, 10 mg) P4 in 0.2 ml sesame oil, or vehicle, and killed at 6 (for E2) or 24 (for P4 and E2 + P4) h. Furthermore, groups of ovx rats were also killed at 12 and 24 h; 3 and 7 days; 2, 4, and 6 wk after ovariectomy. The DRGs were collected from all groups and then processed for Western immunoblotting to examine both trkA and p75NTR levels. Estradiol increased trkA at 6 h but not p75NTR. Progesterone caused upregulation of trkA and p75NTR at 6 and 24 h. 17ß-Estradiol + P4 increased trkA at 6 and 24 h and p75NTR at all time points examined. One microgram of E2 increased trkA but did not affect p75NTR levels. Progesterone at 4 and 10 mg upregulated trkA but only 10 mg P4 increased p75NTR. Five micrograms of E2 coinjected with P4 at 1.5 and 4 mg increased trkA, while p75NTR receptor was upregulated when coinjected with P4 at 1.5 to 10 mg. The ovariectomy caused a decrease in trkA receptors compared to proestrus rats, and these decreases were significant by 6 wk, but surprisingly p75NTR increased at 2 wk after ovariectomy. 17ß-Estradiol increased trkA but not p75NTR receptors in DRG, whereas P4 caused increases in both trkA and p75NTR in DRG. In addition, the combination of these steroid hormones had more effect on both receptors than either hormone alone. Thus, we concluded that high levels of female steroid hormones such as those due to pregnancy or hormonal replacement therapy could increase NGF receptor expression in DRG that carry more NGF to elevate the CGRP synthesis in these groups. We suggested that the regulation of NGF receptors by ovarian steroids may underlie steroidal regulation of other factors such as CGRP.

estradiol, growth factors, hormone action, polypeptide receptors, progesterone

INTRODUCTION

Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide encoded from the same gene as calcitonin [1–3]. The primary site of CGRP synthesis occurs in the sensory neurons of the dorsal root ganglia (DRG) [4] that send efferent sensory nerve fibers around blood vessels and release CGRP at the nerve terminals [5, 6]. Released CGRP binds to specific receptors on the vasculature [2, 7] and decreases blood pressure via peripheral vasodilation [8, 9]. Mechanisms in the regulation of CGRP synthesis in the DRG are not well known. However, evidence indicates that nerve growth factor (NGF) can stimulate the calcitonin/CGRP promoter [10] and CGRP synthesis in DRG [11–14]. Nerve growth factor is a neurotrophic factor that is important in the development and trophic maintenance of peripheral and central nervous systems [15, 16]. Nerve growth factor binds to two classes of receptors: trkA, a member of the family of tyrosine kinase receptors and p75NTR, a member of the tumor-necrosis factor (TNF) receptor superfamily [17–20]. Moreover, CGRP is coexpressed with trkA and p75NTR in DRGs [21–23].

It has been suggested that like NGF, female sex steroid hormones stimulate CGRP synthesis. Plasma CGRP levels are upregulated when female steroid hormone levels are elevated such as in women on oral contraceptives [24], pregnancy in humans and rats [25–27], or hormonal replacement therapy (HRT) in postmenopausal women [28] and ovariectomized (ovx) rats injected with the female steroid hormones [27]. Female steroid hormones, estradiol and progesterone, are suggested to modulate vascular tone during pregnancy [29] and to reduce the risks and conditions of cardiovascular diseases such as hypertension in postmenopausal women [30–32]. Therefore, elevated female steroid hormones such as during pregnancy and HRT can increase synthesis of CGRP and cause lower blood pressure [33, 34]. In addition, our previous studies showed that female steroid hormones administrated to ovx rats increased CGRP mRNA and protein levels in DRG. However, these hormones failed to increase CGRP levels in isolated DRG neurons in culture except when NGF is present [35]. Thus, it appears that steroid hormone-induced increases in CGRP synthesis seem to be dependent upon NGF. Therefore, we hypothesized that female sex steroid hormones upregulate NGF receptors; trkA and p75NTR in DRG. Increases of NGF receptors bind to NGF and then elevate CGRP in DRG. In the present study, we examined the effects of estradiol, progesterone, and the combination of these two steroid hormones on the trkA and p75NTR receptors in DRG in female ovx rats.

MATERIALS AND METHODS

Animals

Female nonpregnant Sprague-Dawley rats (150–170 g body weight, 8–10 wk old) were purchased from Harlan Sprague-Dawley (Houston, TX). Animals were allowed free access to food and water. To assess the stage of estrous cycle in these rats, the vaginas were flushed with normal saline solution, and the cell types in the flush were observed under light microscope. One group of three rats in proestrus stage was identified by the presence of mostly nucleated epithelial cells, a few cornified squamous epithelial cells, but no leukocytes [36]. All other rats were bilaterally ovx under ketamine (50 mg/kg)/xylazine (8 mg/kg) anesthesia. All animals were killed by carbon dioxide inhalation and then the skin, the connective tissue, and muscles on the back of the animals were cut and removed until the vertebral laminae of cervical 1 to lumbar 3 were exposed. After removal of the vertebral laminae, DRGs were carefully collected from both sides of the spinal cord, frozen in liquid nitrogen, and stored at -80°C until further analyses were performed. All procedures were approved by the Animal Care and Use Committee at University of Texas Medical Branch and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Immunocytochemistry

Female rats were killed by i.p. injection of ketamine (50 mg/kg) and xylazine (8 mg/kg), perfused through the ascending aorta with 200 ml heparinized saline (1000 IU heparin and 1 ml of a 1% solution of NaNO2 in 5000 ml saline). Then, the perfusion was followed by 500 ml of 4% paraformaldehyde and 0.1% picric acid in 0.1 M phosphate buffer (PB). The right atrium was cut to drain blood perfusate. The DRGs were collected at T11–L6 and immersed in the same fresh fixative overnight. Next, the ganglia were put into 30% sucrose in 0.1 M PB overnight and followed by freezing in OCT medium. Five- to seven-micron-thick (cryostat sectioning) sections were incubated in 3% blocking buffer (3% normal goat serum [NGS] for trkA or normal rabbit serum [NRS] for p75NTR, 1% BSA, 0.1 M PB, 0.3% Triton X-100) for 1 h at room temperature (RT). Then, the sections were immersed into the primary antibodies or primary antibodies preadsorbed with the respective proteins prior to incubation (trkA, 1:1000; p75NTR, 1:500; protein for trkA, 1:100; protein for p75NTR, 1:50; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at RT. The sections were then incubated with biotinylated secondary antibodies (goat anti-rabbit for trkA, 1:1000 and rabbit anti-goat for p75NTR, 1:250; Vector Laboratories Inc., Burlingame, CA) for 1 h at RT. Next, the sections were incubated with Vectastain Elite ABC kit (Vector Laboratories) for 2 h at RT and washed with 0.1 M PB. The slides were incubated with DAB Reagent set (Zymed Laboratories Inc., South San Francisco, CA) for 3 min and then washed with 0.1 M PB. The slides were allowed to air dry overnight, dehydrated by 70% to 100% ethanol, xylene, and then coverslipped and observed under light microscope.

Female Steroid Hormone Treatments

Study 1: time course One week after ovariectomy, groups of four rats were injected s.c. with a single dose of 17ß-estradiol (E2, 5 µg in 0.2 ml of sesame oil), progesterone (P4, 4 mg in 0.2 ml sesame oil), or a combination of E2 (5 µg) together with P4 (4 mg) or vehicle (0.2 ml sesame oil). Both hormones were purchased from Sigma (St. Louis, MO). Groups of four rats were killed at different times after injections of hormones (6, 24, and 48 h) and DRGs were collected.

Study 2: dose response One week after ovariectomy, groups of three rats were injected s.c. with a single dose of E2 at 0.2, 1, 5, or 25 µg in 0.2 ml sesame oil and then killed at 6 h after injection; P4 at 0.5, 1.5, 4, or 10 mg in 0.2 ml of sesame oil and killed at 24 h after the injection; E2 (5 µg) together with P4 at 0.5, 1.5, 4, or 10 mg in 0.2 ml sesame oil or vehicle (0.2 ml sesame oil) killed at 24 h. The DRGs were then collected.

Study 3: long-term ovariectomy Groups of three rats per group were killed at 12, 24 h, 3, 7 days, 2, 4, or 6 wk after ovariectomy to assess the time course effect of depletion of steroid hormones on NGF receptors in DRGs. A group of three rats at the proestrus stage (high levels of both E2 and P4) served as a control for determining the effects of long-term depletion of steroid hormones.

Western Immunoblotting

The Western blotting procedure was similar to the protocols previously used [37, 38]. The DRGs from same groups of animals were used for Western blot analysis of both trkA and p75NTR. Briefly, DRGs were homogenized in lysis buffer (50 mM Tris-buffered saline/1 mM EDTA, 5% SDS, 50 mM PMSF), then centrifuged at 4000 x g for 20 min at 4°C. The supernatants were collected and the protein levels measured (BCA kit; Pierce, Rockford, IL). Aliquots containing 50 µg proteins were diluted in 4x SDS loading buffer (100 mM Tris-HCl, 8% SDS, 0.02% bromophenol blue, 40% glycerol, 500 mM ß-mercaptoethanol) and electrophoresed on 7.5% SDS-polyacrylamide denaturing gels (Bio-Rad, Hercules, CA). After electrophoresis, the gels were blotted to a polyvinylidene fluoride membrane (Millipore, Bedford, MA). The blots were placed in blocking buffer (5% powdered milk in wash buffer; Tris-buffered saline-0.1% Tween 20) for 1 h at RT, and then incubated with specific primary antibodies (trkA, 1:1000; p75NTR, 1:500, Santa Cruz Biotechnology) for 1 h at room temperature. After washing three times for 10 min each with wash buffer, the blots were incubated with horseradish peroxidase-conjugated goat-antirabbit (1:5000, for trkA; Bio-Rad) or mouse-antigoat (1:10 000, for p75NTR receptor; Santa Cruz Biotechnology) antibodies diluted in 2.5% powdered milk in wash buffer. The membranes were rinsed with wash buffer three times for 30 min and the enhanced chemiluminesence reagent (ECL kit; Amersham, Piscataway, NJ) was added and incubated for 1 min at RT. The blots were exposed to hyperfilm ECL (high performance chemiluminescence film; Amersham), and the intensity of specific immunoreactive bands was quantified by densitometric scanning program (AlphaEase). Densitometric units of specific protein bands were expressed relative to the values from the control animals.

Statistical Analysis

Results were presented as means ± SEM. The statistical differences between means of the experimental groups and the control group were compared by ANOVA followed by the Bonferroni t-test. A value of P < 0.05 was considered significant.

RESULTS

Immunohistochemical localization of trkA and p75NTR was performed in DRGs from female rats, to assess the cell types that express these receptors. The data presented in Figure 1 demonstrated that both trkA and p75NTR were expressed primarily in small- and medium-sized neurons in DRG.



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  		FIG. 1. Immunohistochemical localization of trkA and p75NTR in female rat DRGs. The sections were stained as per procedures described in Materials and Methods. The positive staining for trkA and p75NTR are shown in A and B, respectively. The antibody controls using antibodies preadsorbed with the respective proteins prior to incubations for trkA (C) and p75NTR (D) were performed (x20)

Effects of E2 and P4 on NGF Receptors in DRG of Ovariectomized Rats

Study 1: time course The reported sizes of trkA and p75NTR are 140 and 75 kDa, respectively. We observed two bands (140 and 150 kDa) that immunoreacted with the trkA antibody. The 150-kDa band did not appear to be due to phosphotyrosine on trkA, because a phosphotyrosine antibody did not react with this band (unpublished data). Therefore, both bands were used for statistical analysis. The molecular weight of the band that immunoreacted with the p75NTR antibody in the DRG homogenates was 75 kDa, similar to that previously reported [19, 20]. Densitometric analysis of the bands revealed that trkA was increased in DRG (Fig. 2B). The effect of E2 was significant at 6 h, but the levels of trkA returned to the control levels by 48 h after E2 injection. Similarly, both P4 alone and E2 + P4 injections increased trkA expression in DRG by 6 h, and the levels returned to control levels by 48 h. Furthermore, the magnitude of this increase in trkA levels seemed higher in the E2 + P4 group compared to either hormone alone.



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  		FIG. 2. Effect of E2 and P4 on trkA protein in DRG. Ovariectomized rats were injected 1 wk postovariectomy with a single dose of E2 (5 µg), P4 (4 mg), the combination of E2 and P4 (E2 + P4), or oil only (veh). Dorsal root ganglia were collected 6, 24, and 48 h after injection. A) Western blots using trkA antibody (n = 4 per group). The numbers on the lefthand side are molecular weight markers. B) Percentage of change in the density of bands compared to veh (means ± SEM from four animals). *P < 0.05; **P < 0.01 compared to veh (ANOVA)

The p75NTR protein levels in DRG were significantly elevated in rats treated with P4 and E2 + P4 (Fig. 3). The effects of both P4 and E2 + P4 were significant at 6 h and remained elevated for longer periods (24–48 h). On the other hand, the effects of E2 on p75NTR did not attain significance at any given time point. These data indicated that E2 and P4 increased trkA and p75NTR levels in DRG from ovx rats in a time-dependent manner. In addition, female steroid hormone effects on p75NTR protein were relatively smaller as compared to the changes of trkA.



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  		FIG. 3. Effect of E2 and P4 on p75NTR protein in DRG. Ovariectomized rats were injected 1 wk postovariectomy with a single dose of E2 (5 µg) and P4 (4 mg), the combination of E2 and P4 (E2 + P4), or oil only (veh). Dorsal root ganglia were collected 6, 24, and 48 h after injection. A) Western blots using p75NTR antibody (n = 4 per group). The numbers on the lefthand side are molecular weight markers. B) Percentage of change in the density of bands compared to veh (means ± SEM from four animals). *P < 0.05; **P < 0.01; ***P < 0.001 compared to veh (ANOVA)

Study 2: dose response Figure 4 shows trkA levels in rat DRGs after treatment with varying doses of E2, P4, and E2 + P4. The density of trkA in DRG was significantly increased by 1 µg E2, whereas lower (0.2 µg) or higher (5 or 25 µg) doses were not effective. Only high doses of P4 (4 and 10 mg) increased trkA levels. In the combined treatment group, animals treated with 1.5 or 4.0 mg P4 along with 5 µg E2 showed increases in trkA levels in DRG while the highest dose of P4 (10 mg) was ineffective. In contrast, significant increases in p75NTR protein levels in DRG were not observed with E2 or P4 treatments (Fig. 5). Only the highest dose of P4 had a modest effect on p75NTR levels in DRG. When 0.5 mg of P4 was combined with 5 µg E2, there was no effect on p75NTR levels, whereas higher doses of P4 (1.5, 4.0, and 10 mg) combined with 5 µg E2 significantly increased p75NTR levels in DRG. These data suggested that E2 and P4 amplified trkA and p75NTR in a dose-dependent manner. As observed with the time course study, hormone treatments on p75NTR induced a smaller change than on trkA.



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  		FIG. 4. Effect of E2 and P4 on trkA protein in DRG. Ovariectomized rats were injected 1 wk postovariectomy with a single dose of E2; (0.2, 1, 5, 25 µg) and P4 (0.5, 1.5, 4, 10 mg), the combination of E2 and P4 (E2 + P4), or oil only (veh), and DRG were collected 6 (for E2) and 24 (for P4 and E2 + P4) h after injection. A) Western blots using trkA antibody (n = 3 per group). The numbers on the lefthand side are molecular weight markers. B) Percentage of change in the density of bands compared to veh (means ± SEM from three animals). *P < 0.05; **P < 0.01; ***P < 0.001 compared to veh (ANOVA)



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  		FIG. 5. Effect of E2 and P4 on p75NTR protein in DRG. Ovariectomized rats were injected 1 wk postovariectomy with a single dose of E2 (0.2, 1, 5, 25 µg) and P4 (0.5, 1.5, 4, 10 mg), the combination of E2 and P4 (E2 + P4), or oil only (veh). Dorsal root ganglia were collected 6 (for E2) and 24 (for P4 and E2 + P4) h after injection. A) Western blots using p75NTR antibody (n = 3 per group). The numbers on the lefthand side are molecular weight markers. B) Percentage of change in the density of bands compared to veh (means ± SEM from three animals). *P < 0.05 compared to veh (ANOVA)

Effects of Female Steroid Hormone Depletion on NGF Receptors in DRG of Rats

Study 3: long-term ovariectomy To assess the effects of steroid hormone depletion, the density of trkA protein levels in the DRG of proestrus rats with elevated levels of both estrogen and progesterone was compared to that at various times after ovariectomy (Fig. 6). The trkA levels in DRG of ovx rats decreased with time after ovariectomy, reaching statistical significance as compared to proestrus rats at 6 wk after ovariectomy. The p75NTR levels were also measured in the ovx rats at different times after ovariectomy (Fig. 7). At variance with trkA, the p75NTR levels increased with time after ovariectomy and reached statistical significance at 2 wk after ovariectomy (P < 0.001) before decreasing by 4 wk. The result of p75NTR in DRG of both proestrus and ovx rats for 2 wk was confirmed by adding more animals in Figure 8.



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  		FIG. 6. Effect of the hormonal deficiency on trkA protein in DRG. Rats were ovariectomized and killed at 12, 24 h, 3, 7 days, 2, 4, and 6 wk after ovariectomy. Rats in the proestrus (PE) stage of the cycle were used as the control group. A) Western blots using trkA antibody (n = 3 per group). The numbers on the righthand side are molecular weight markers. B) Percentage of change in the density of bands compared to PE animals (means ± SEM from three animals). *P < 0.05 compared to PE rats (ANOVA)



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  		FIG. 7. Effect of the hormonal deficiency on p75NTR in DRG. Rats were ovariectomized and killed in 12, 24 h, 3, 7 days, 2, 4, and 6 wk after ovariectomy. Rats in the proestrus (PE) stage of the cycle were used as control group. A) Western blots using p75NTR antibody from three rats in each group. The numbers on the righthand side are molecular weight markers. B) Percentage of change in the density of bands compared to PE animals (means ± SEM from three animals). ***P < 0.001 compared to PE group (ANOVA)



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  		FIG. 8. Effect of the hormonal depletion on p75NTR in DRG. Rats were ovariectomized and killed at 2 wk after ovariectomy. Rats in the proestrus (PE) stage of the estrous cycle were used as a control group. A) Western blot using p75NTR antibody from six rats in each group. The numbers on the lefthand side are molecular weight markers. B) Percentage of change in the density of bands compared to PE animals (mean ± SEM from six animals). **P < 0.02 compared to PE rats (Student's t-test)

DISCUSSION

The present results indicate that steroid hormones stimulate the expression of NGF receptors in DRG. Both trkA and p75NTR were localized primarily to the small to medium-sized neurons in the DRG. The effects of E2 and P4 on trkA receptor protein in DRG, measured by densitometric analysis of the immunoreactive bands, were time dependent between 6–24 h after single injection of the steroid. The steroid hormone effects on trkA were also dose dependent. Furthermore, the combined E2 and P4 treatments appeared to be more effective on trkA than either hormone alone. On the other hand, only P4 injections with or without E2 increased the p75NTR receptors in DRGs in time- and dose-dependent manners, while E2 administration alone had no significant effects. Depletion of female sex steroid hormones for a prolonged period (ovariectomy) caused decreases in trkA at 6 wk but surprisingly increased p75NTR in DRG as early as 2 wk after ovariectomy. These findings show that female steroid hormone, especially P4 with or without E2 upregulated NGF receptors. Moreover the hormonal depletion decreased only trkA but not p75NTR.

Data on the effects of female steroid hormones on NGF receptors in DRG are limited. It has been shown that E2 injection (10 µg) in ovx animals downregulated p75NTR mRNA in DRG temporally at 4 h and then increased to the same level as the control ovx group at 52 h. On the other hand, E2 injection increased trkA mRNA in DRG [39, 40]. Our results also showed that trkA in DRG increased, while p75NTR did not change after E2 injection. Moreover, our results showed that the effects of P4 or the combination of E2 and P4 injections appeared to be greater on both trkA and p75NTR in DRG than that with E2 only injections. This could be related to the well-known estrogen-induced increased expression of the progesterone receptor (PR) gene at the transcriptional level [41, 42] in the combination injections. In addition, the estrogen receptor was expressed in neurons in DRG [40] and Schwann cells [43], whereas PR distribution is not well understood at the present time. It has been shown that trkA was found in neurons of DRG but not in the Schwann cells [44–46]; however, p75NTR receptors were expressed in not only neurons but also Schwann cells [47–49]. Therefore, the steroids can bind to their receptors in these cells and thus increase expression of NGF receptors.

Depletion of female sex steroid hormones for a prolonged period (ovariectomy) mimicked the condition in the postmenopausal women. The long-term ovariectomy caused decreases in trkA at 6 wk but increased p75NTR in DRG at 2 wk after ovariectomy. The mechanisms seemed to be more complicated in p75NTR receptor regulation in the hormonal depletion model. Several functions for p75NTR have been suggested such as increasing trkA affinity for NGF, transporting NGF retrogradely and providing the substrates for trkA in cell signaling [18, 50, 51]. In cells expressing both trkA and p75NTR, the ratio of binding to NGF of p75NTR to trkA is normally about 10:1. When the p75NTR receptor is not present, trkA-binding affinity to NGF is decreased. However, when p75NTR is present, the effects of NGF through the trkA binding cascade are amplified [19, 20, 51]. Decreased trkA after long-term ovariectomy could result in less NGF being carried to DRG. To maintain these functions, increased p75NTR may help transport and concentrate more NGF to the DRG. The other possibility is that in some situations trkA and p75NTR inhibited each other biochemically and functionally. For example, high activity of p75NTR with low activity of trkA may cause cell apoptosis and vice versa [51–55]. Therefore, the hormonal depletion may have differential effects on the DRG levels of trkA and p75NTR receptors, and these changes could result in altered NGF responsiveness.

Several studies showed that NGF induces CGRP synthesis in DRG [10–14]. Nerve growth factor is a secretory protein and binds to two types of receptors: trkA and p75NTR [17–20]. Our immunocytochemistry study demonstrated that the neurons immunoreactive to trkA and p75NTR were small to medium in size [21]. About 37% of neurons in DRG express CGRP and 84% to 92% of these neurons express trkA mRNA also [21, 22, 56]. The percentage of coexpression of CGRP with p75NTR is 46%, which is lower than the coexpression with trkA [23]. About 93% of neurons in DRG coexpress both trkA and p75NTR [57]. Therefore, NGF could stimulate CGRP synthesis by specifically binding to NGF receptors in DRG neurons. Other investigators and we have demonstrated that plasma CGRP is elevated during pregnancy in either humans or rats [25–27], in ovx rats injected with E2 and P4 [27] with HRT in postmenopausal women [28]. We further reported that steroid hormone-induced stimulation of CGRP synthesis in cultured DRG is contingent upon the presence of NGF [35]. The present study, together with previous observations [25–28, 35] suggest that steroid hormones stimulate expression of CGRP synthesis in DRG via the upregulation of NGF receptors. Therefore, mechanisms of female steroid hormones modulating vascular tone during pregnancy [29] and helping alleviate high blood pressure in HRT [30–32] work at least in part through upregulation of CGRP synthesis via increase of NGF receptors.

In conclusion, NGF receptors, trkA and p75NTR, were upregulated directly by female steroid hormones in a time- and dose-dependent manners. Progesterone and the combination of estradiol and progesterone treatments appeared to be more effective in stimulating NGF receptors in DRG than the estradiol injection alone. The hormonal depletion decreased trkA levels but had more complicated effects on p75NTR. These studies together with our previous studies [27, 35] suggest that steroid hormones upregulate NGF receptor expression, and then increase CGRP synthesis in DRG. We suggest that the regulation of NGF receptors by ovarian steroids may underlie steroidal regulation of other factors such as CGRP.

FOOTNOTES

First decision: 10 May 2000.

1 Supported in part by NIH through grants HL58144, HD30273, and AG13945. 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: September 5, 2000.

Received: April 4, 2000.

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