Pregnancy and sex steroid hormones enhance circulating calcitonin gene-related peptide concentrations in rats

P.R.R. Gangula1, S.J. Wimalawansa2 and C. Yallampalli1,3

1 Departments of Obstetrics and Gynecology and 2 General Medicine, University of Texas Medical Branch, Galveston, Texas, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Calcitonin-gene-related peptide (CGRP) is a 37 amino acid neuropeptide synthesized primarily in dorsal root ganglia (DRG) and distributed widely in the perivascular nerves, suggesting that this peptide may play a role in the regulation of peripheral vascular tone. Since female sex steroid hormones have been implicated in the regulation of peripheral vascular tone during pregnancy, we postulated that they may alter the concentration of CGRP in the circulation and thus modulate the increased blood flow observed during pregnancy. In the present study, we measured changes in plasma concentrations of CGRP in non-pregnant, pregnant, and post-partum rats. Groups of ovariectomized rats were treated s.c. for 3 days either with 17ß-oestradiol (2.5 µg per injection twice daily), progesterone (2 mg per injection twice daily), or vehicle. Another group of adult, non-pregnant rats at dioestrus stage of the oestrous cycle was also used in this study. Plasma concentrations of CGRP were higher (P < 0.05) in rats on day 19 of pregnancy (22.0 ± 3.0 pmol/l) compared to that during delivery (5.0 ± 2.0), post-partum day 2 (2.0 ± 0.7) or in non-pregnant (4.9 ± 1.6) state. Furthermore, in adult ovariectomized (6.0 ± 0.6) rats, plasma CGRP concentrations were increased significantly (P < 0.05) by oestradiol (10.0 ± 1.0), progesterone (9.5 ± 1.0) and oestradiol + progesterone (14.0 ± 1.0). Thus, circulating concentrations of CGRP are elevated during pregnancy and by oestrogen and progesterone, suggesting that the elevated concentrations of CGRP may play an important role in vascular adaptations that occur during pregnancy.

Key words: calcitonin gene-related peptide/oestrogen/pregnancy/progesterone/vascular system


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pregnancy is associated with profound changes in maternal haemodynamics, including increases in plasma volume and decreases in blood pressure and peripheral vascular tone in women (Duvekot et al., 1993; Mabie et al., 1994Go; Magness et al., 1994Go), sheep (Magness and Rosenfeld, 1988Go; Magness and Zheng, 1996Go; Magness, 1998Go), rats (Slangen et al., 1996Go, 1997Go), and in numerous other species (Magness, 1998Go). The mechanisms responsible for these cardiovascular changes during normal gestation are not well understood. Increased production of nitric oxide (NO), or prostacyclin (PGI2) has been proposed as possible mechanisms for vascular changes during pregnancy and for reduced sensitivity of the maternal vascular system to angiotensin II (Walsh, 1985Go; Magness, 1991Go). Furthermore, female sex steroid hormones are implicated in the regulation of vascular tone during pregnancy (Jaffe, 1983Go). These hormonal influences appear to be mediated through changes in the production of vasoactive agents including NO (Sladek et al., 1997Go; Vagnoni et al., 1998Go), and prostaglandins (Lockitch, 1997Go) and modify the effects of calcitonin gene-related peptide (CGRP) (Yallampalli et al., 1996Go; Gangula et al., 1997aGo,bGo; Dong et al., 1998Go).

CGRP is a 37 amino acid molecule derived from alternative processing of the primary transcript of the calcitonin gene (Rosenfeld et al., 1983Go), and is the most potent endogenous vasodilator known (DiPette and Wimalawansa, 1995Go; Wimalawansa, 1996Go). CGRP is primarily synthesized in dorsal root ganglia (DRG) and distributed throughout the central and peripheral nervous systems and is located in areas involved in cardiovascular function (McEvan et al., 1989Go; DiPette and Wimalawansa, 1995Go). Immunohistochemical studies showed CGRP-containing nerve fibres throughout the cardiovascular system (Mulderry et al., 1985Go), uterus (Shew et al., 1993Go) and ovary (Calka et al., 1988Go). On the basis of these potent vasodilator effects and the perivascular localization of CGRP, we postulate that this peptide plays a role in the regulation of blood pressure (BP) and regional organ blood flows both under normal physiological conditions and in the pathophysiology of hypertension.

Two separate studies in humans have shown that circulatory immunoreactive CGRP (i-CGRP) concentration increases during pregnancy and decreases in the post-partum period (Stevenson et al., 1986Go; Saggese et al., 1990Go). Moreover, plasma concentrations of i-CGRP were significantly higher in females than in males and women on contraceptive pills had significantly higher i-CGRP concentrations (Valdemarsson et al., 1990Go) suggesting that female sex hormones may influence the plasma concentrations of CGRP. However, it is unclear if CGRP concentrations in the circulation are altered consistent with its vasodilator role during pregnancy and in steroid hormone-treated animals. Furthermore, CGRP is also a potent uterine relaxant and thus it could modulate the uterine contractility during pregnancy and labour (Chan et al., 1997Go; Dong et al., 1998Go). Therefore, the objectives of the present study were to determine whether: (i) circulatory concentrations of i-CGRP in the rat increase during pregnancy and decrease during labour at term and (ii) CGRP concentrations in the circulation are altered by exogenous sex steroid hormones in ovariectomized rats.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
Rats
Adult non-pregnant (170–200 g body weight) or timed pregnant (300–325 g body weight) rats were purchased from Harlan Sprague Dawley (Houston, TX, USA). Pregnant animals were received in the Animal Care Facilities on day 16 of pregnancy (day 1 is day of positive sperm smear). All animals were given free access to food and water. Non-pregnant animals, regardless of stage of oestrous cycle, were bilaterally ovariectomized while they were under halothane anaesthesia and after 7 days of surgery groups of animals were treated with various agents as described below. All procedures were approved by the Animal Care and Use Committee of the University of Texas Medical Branch.

Groups of 8–10 non-pregnant rats were treated s.c. either with 17ß-oestradiol (2.5 µg per injection, twice daily for 3 days in 0.2 ml sesame oil), progesterone (2 mg per injection; twice daily for 3 days in 0.2 ml of sesame oil), combination of both oestradiol and progesterone (2.5 mg and progesterone; 2 mg per injection; twice daily for 3 days in 0.2 ml of sesame oil) or vehicle (sesame oil) (Sigma Chemical Company, St Louis, MO, USA). Blood was collected into heparinized tubes from the vena cava immediately following exposure to CO2 in an inhalation chamber. Blood was then centrifuged at 1600 g for 15 min at 4°C, and plasma collected and stored at –70°C until used. In a similar manner, plasma was also collected from groups of pregnant rats on day 19 of gestation, during labour and post-partum day 2 and from non-pregnant rats at dioestrus stage of the oestrous cycle.

Extraction of CGRP from plasma and CGRP radioimmunoassay
Plasma samples were acidified with 10% of trifluoroacetic acid (TFA) (0.5 ml plasma with 50 µl TFA) and incubated on ice for 20 min. Acidified samples were spun at 6000 g for 15 min at 4°C and the supernatant was retained. The pellets were then treated with 300 µl of 10% TFA, resuspended and respun as above. Supernatants were pooled and passed (3x) through Sep-Pack C18 cartridges (Water Associates, Milford, MA, USA) primed with 100% acetonitrile. After washing with 0.5% TFA, the peptide was eluted in a solution containing 60% acetonitrile and TFA (0.5% v/v). The eluates were vacuum-dried and stored at –70°C for measurement of i-CGRP by radioimmunoassay using CGRP RIA kit (Phoenix, Belmont, CA, USA). The dried eluate was reconstituted in a small amount (|~15 µl) of 0.1% TFA over a period of 30 min at 4°C. 300 µl of radioimmunoassay buffer was added to each tube and the assay was performed in duplicate aliquots for each of these samples. CGRP was measured by radioimmunoassay utilizing a rabbit antiserum raised against synthetic rat {alpha}-CGRP conjugated to bovine albumin. The sensitivity was 32 pg/tube and the intra-assay and inter-assay variations were 5% and 10% respectively and the cross-reactivity for the antibody used was 100% and 35.5% for rat {alpha}-CGRP and human {alpha}-CGRP respectively.

Statistical analysis
Results were expressed as means ± SEM. Data were analysed for statistical differences using an analysis of variance (ANOVA) followed by Dunnet's t-test. Differences were considered significant if P < 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Figure 1Go illustrates the concentrations of i-CGRP in the circulation of non-pregnant rats at dioestrus, pregnancy on day 19 of gestation, term labour and post-partum day 2 rats. Concentrations of i-CGRP in plasma were elevated (P < 0.05) on day 19 of gestation (22.0 ± 3.0 pmol/l) compared to non-pregnant (4.9 ± 1.6 pmol/l) rats. Moreover, compared to pregnant (day 19) rats, concentrations of i-CGRP in circulation were significantly decreased in rats during spontaneous labour at term (5.0 ± 2.0 pmol/l, P < 0.05) or on post-partum day 2 (2.0 ± 0.7 pmol/l, P < 0.05).



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Figure 1. Plasma immunoreactive calcitonin gene-related peptide (i-CGRP) concentrations in rats either non-pregnant at dioestrus (Non pregnant), pregnant on day 19 of gestation (pregnant), during labour (Labour) and post-partum day 2 (Postpartum). Values are means ± SEM of the concentrations of CGRP in circulation of four to six animals in each group. *P < 0.05 compared with all other groups.

 
We examined the effects of 17ß-oestradiol and progesterone either alone or in combination on the production of i-CGRP in non-pregnant ovariectomized rats. Plasma concentrations of i-CGRP were significantly (P < 0.05) elevated in oestradiol-treated (10 ± 1.0 pmol/l) and progesterone-treated (9.5 ± 1.0 pmol/l) rats compared to the vehicle-treated (6.0 ± 0.6 pmol/l) ovariectomized rats (Figure 2Go). Furthermore, plasma i-CGRP concentrations were substantially elevated in animals treated with both oestradiol and progesterone (14.0 ± 1.0 pmol/l) compared to the vehicle-treated ovariectomized rats (Figure 2Go). Plasma concentrations of i-CGRP in rats treated with both oestradiol and progesterone were significantly (P < 0.05) higher compared to that of animals treated with either progesterone or oestradiol.



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Figure 2. Effect of 17ß-oestradiol and progesterone either alone or in combination on the plasma immunoreactive calcitonin gene-related peptide (i-CGRP) concentrations in non-pregnant ovariectomized rats. Non-pregnant ovariectomized rats were treated for 3 days, s.c., with either 17ß-oestradiol (2.5 µg per injection twice daily), progesterone (2 mg per injection twice daily) or combination of both oestradiol and progesterone in sesame oil or sesame oil alone (Vehicle). Data are means ± SEM for eight to 10 animals in each group. *P < 0.05 compared to vehicle-treated ovariectomized group. E = oestradiol-17ß|, P = progesterone.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the current study, we demonstrated that concentrations of i-CGRP in the circulation were elevated during pregnancy and decreased at term in rats. Furthermore, plasma i-CGRP concentrations in non-pregnant ovariectomized rats were elevated following treatment with oestrogen and progesterone compared to that of the vehicle administration. These studies suggest that increased concentrations of circulatory i-CGRP during pregnancy may be involved in regulating vasodilation (Gangula et al., 1997bGo) as well as uterine relaxation (Dong et al., 1998Go) and that these changes may be in part steroid hormone dependent.

In the present study, in rats, circulatory concentrations of i-CGRP were significantly increased during pregnancy, but they decreased at term and post-partum. These studies are in agreement with the studies of Stevenson et al. (1986) and Sagesse et al. (1990) demonstrating that plasma i-CGRP concentrations were elevated in pregnant women and decreased during post-partum. Furthermore, plasma concentrations of i-CGRP were higher in females than in males and women on contraceptive pills had significantly higher CGRP concentrations (Valdemarsson et al., 1990Go). Collectively, these data suggest that concentrations of i-CGRP during pregnancy are elevated and that these elevated CGRP concentrations may play a role in the vascular adaptations that occur during pregnancy and possibly in the uterine quiescence required for successful pregnancy.

Since, the circulatory concentrations of i-CGRP were increased during normal pregnancy, we postulated that this increase in i-CGRP concentrations may be sex steroid hormone dependent. In the present study, 3 day treatment either with 17ß-oestradiol or progesterone, either alone or in combination, significantly elevated the plasma i-CGRP compared to the vehicle (sesame oil)-administered ovariectomized rats. Furthermore, the circulatory concentrations of i-CGRP in the combined oestrogen + progesterone-treated group were significantly greater compared to either oestradiol, or progesterone-administered rats. The doses of oestrogen and progsterone utilized in these studies are similar to those that mimic the concentrations achieved during pregnancy (Kennedy et al., 1983aGo, bGo), and these concentrations are substantially higher compared with non-pregnant, ovary-intact animals. We recently reported that sensitivity to the vasodilator effects of exogenous CGRP were significantly elevated in the presence of similar doses of either oestradiol or progesterone (Gangula et al., 1999Go). Furthermore, in post-menopausal women, circulatory CGRP concentrations were elevated in the hormone-treated group compared to controls (Valentini et al., 1996Go; Spinetti et al., 1997Go). These studies and the report from Valdemarsson et al. (1990) showing higher i-CGRP concentrations in women with contraceptive pills, together with our current findings strongly suggest that both oestrogen and progesterone may increase the synthesis and/or release of CGRP into the circulation.

The predominant site of CGRP synthesis is the DRG which contain the cell bodies of primary afferent neurons, which extend CGRP-containing nerves to peripheral sites such as blood vessels and to central spinal cord sites known to be involved in blood pressure regulation (Gibson et al., 1984Go; Marti et al., 1987Go; McEvan et al., 1989Go; DiPette and Wimalawansa, 1995Go). Several reports suggest that the synthesis and/or release of CGRP from the nerve endings is influenced by nerve growth factor (NGF) bradykinin, prostaglandins (Andreeva and Rang, 1993Go; Vasko et al., 1994Go) and catecholamines (Supowit et al., 1995Go) in rats. It is plausible that even in females, one or more of these factors may stimulate the synthesis or release of CGRP into the circulation during pregnancy and thus regulate the blood flow and peripheral vascular tone. Our studies indicate a role for oestradiol and progesterone in these processes. Furthermore, oestradiol and progesterone have been implicated in the regulation of cardiovascular functions and may play a role in pathophysiology of hypertension and coronary heart disease in humans and in several animal species (Suzanne, 1999Go). Oestrogens have vasodilatory and as well as anti-atherogenic functions, while the role of progesterone is not well understood. Recent reports suggest that prolonged treatment with 17ß-oestradiol increases reproductive and non-reproductive organ blood flows in sheep (Magness et al., 1998Go). These studies together with our current findings suggest that oestrogen and progesterone elevate the circulatory concentrations of CGRP and thus maintain vascular adaptations during pregnancy and perhaps in post-menopausal women.

Since perivascular nerve terminals contain neuropeptides, such as CGRP, it was suggested that the primary mode of action of these peptides in regulating vascular tone is via the paracrine system. It is thought that circulatory CGRP is largely derived from perivascular nerve terminals and represents a spillover phenomenon related to the release of the peptide to promote vasodilation or other functions. Our previous studies show that systemic administration of CGRP produces vasodilation and that the vasodilatory effects of CGRP are enhanced with the treatment of steroid hormones and during pregnancy (Gangula et al., 1999Go). Our current data on the increases in circulatory concentrations of CGRP in the presence of female sex steroid hormones and during pregnancy in rats suggest that fluctuations in circulatory concentrations of CGRP may also play a role in regulating vascular tone (Gangula et al., 1999Go) and uterine smooth muscle relaxation (Dong et al., 1998Go) during pregnancy.

Oestradiol and progesterone both stimulate the renin–angiotensin system (Oel Kers, 1996). Angiotensin II is known to increase both prostaglandins (McGiff et al., 1970Go) and catecholamines (Corwin et al., 1985Go). Since both these types of hormones have been shown to increase the circulating concentrations of CGRP (Vasko et al., 1994Go; Supowit et al., 1995Go), it seems possible that these could be some of the mechanisms utilized in this system. Furthermore, it is also known that in pregnancy the vascular system is desensitized to the vasoconstrictor effects of angiotensin II (Novak et al., 1997Go). Therefore, increasing production of CGRP in response to prostaglandins would have a greater vasodilatory effect in the pregnant as opposed to non-pregnant state. However, this does not appear to explain why the CGRP concentrations fall at term when prostaglandins are increased, when the concentrations of oestradiol would still be high. Further detailed studies are required to address some of these mechanisms.

At present, we do not know whether the increase of i-CGRP results from an increase in CGRP mRNA accumulation during pregnancy or by oestradiol and progesterone. However, previous studies in rats have demonstrated that agents which act via the protein kinase C (PMA) and dibutyryl cAMP signalling pathways significantly increase CGRP mRNA content and i-CGRP secretion in primary cultures of DRG neurons (Supowit et al., 1995Go). It also appears that the stimulatory effects of PMA and dibutyryl cAMP on i-CGRP synthesis are more pronounced in the DRG neurons when compared to the vagal neurons. In addition, several lines of studies support the hypothesis that CGRP synthesis and/or release from DRG is mediated by nerve growth factor (NGF) (Lindsay and Harmar, 1989Go). Therefore, it is possible that oestrogen and progesterone either directly or indirectly through other factor(s), such as the NGF system, may increase the CGRP mRNA and i-CGRP content in DRG and thus release into the circulation. Previous observations demonstrate that oestrogen receptor mRNA and protein was present in DRG of non-pregnant normal (cycling) and ovariectomized rats (Toran-Allerand., 1994Go). These studies further showed that NGF receptor mRNA such as trkA was up-regulated in sensory neurons during pro-oestrus, as compared to the ovariectomized condition. In the present study, we have shown that supplementation of both oestradiol and progesterone significantly elevated the i-CGRP concentrations in ovariectomized rats and these concentrations were further increased with combined treatment of oestrogen + progesterone, suggesting that oestrogen and progesterone may up-regulate the NGF or its receptors or some other factors in DRG, and thus stimulating the synthesis/or release of CGRP into the circulation.

Pregnancy is a complex process in which several vascular changes occur in order to maintain blood supply to the growing fetuses. Reports in humans and sheep indicate that blood pressure will decrease during the course of gestation compared to the non-pregnant state (Fregly, 1957Go; MacGillivray et al., 1969Go; Magness, 1998Go). Furthermore, it has been shown that blood flow to the reproductive organs during pregnancy was elevated compared to the non-pregnant state in rats (Bruce, 1976Go; Buelke-Sam et al., 1982Go). Several locally produced factors such as NO and prostacyclin are known to play a major role in reducing vascular tone during pregnancy in sheep (Magness, 1998Go). It has been reported that the sensitivity to CGRP in human uterine arteries (Nelson et al., 1993Go) and myometrial tissues (Chan et al., 1997Go) was increased from non-pregnant to pregnant state. Previous studies from our laboratory demonstrated that CGRP significantly decreased the blood pressure in L-NAME-treated hypertensive rats only during pregnancy, but not during post-partum or in hormone-depleted (ovariectomized) non-pregnant rats (Yallampalli et al., 1996Go). We further showed that progesterone significantly reduced the blood pressure to control levels only in the CGRP-administered group in both non-pregnant and post-partum rats, suggesting that oestrogen and progesterone regulate the vascular tone through CGRP receptors at least in this animal model. The current study clearly demonstrates that i-CGRP concentrations were significantly elevated during pregnancy and in the presence of oestrogen and progesterone. Taken together, we postulate that the elevated concentrations of i-CGRP in the circulation may play a role in regulating blood pressure as well as relaxing uterine smooth muscle during pregnancy. However, the circulating CGRP concentrations in pregnant rats were greater than those achieved with oestrogen and progesterone replacement, indicating that other factors, besides oestrogen and progesterone, might play a significant role in the control of plasma CGRP concentrations. Further studies are required to fully assess the factor(s) that are responsible for increasing the CGRP expression and i-CGRP content in DRG during pregnancy.

In summary, circulatory i-CGRP concentrations were significantly elevated during pregnancy and were dramatically decreased at term and post-partum in rats. These data suggest that the elevated concentrations of i-CGRP may participate in both uterine and vascular relaxation during pregnancy. Both oestrogen and progesterone treatment in ovariectomized rats increased i-CGRP concentrations, suggesting that the vasoprotective effects in females may involve increased CGRP synthesis. In our previous studies we demonstrated that CGRP may be involved in vascular adaptations that occur during pregnancy (Yallampalli et al., 1996Go; Gangula et al., 1997aGo,bGo) and in uterine quiescence during pregnancy (Dong et al., 1998Go). Together, our present studies suggest an important role for elevated CGRP during pregnancy in modulating vascular and perhaps uterine relaxations.


    Acknowledgments
 
This work was supported by grants from the National Institutes of Health to C.Y.: HD 30273, HL 58144.


    Notes
 
3 To whom correspondence should be addressed at: Department of Ob/Gyn, 301 University Blvd Rt 1062, Medical Research Building Rm 11.138, Galveston, TX 77555-1062, USA Back


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 Abstract
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 Materials and methods
 Results
 Discussion
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Submitted on September 6, 1999; accepted on December 23, 1999.