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BOR - Papers in Press, published online ahead of print July 30, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.031369
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BIOLOGY OF REPRODUCTION 71, 1739–1745 (2004)
DOI: 10.1095/biolreprod.104.031369
© 2004 by the Society for the Study of Reproduction, Inc.

Pregnancy

Mesenteric Arterial Relaxation to Calcitonin Gene-Related Peptide Is Increased During Pregnancy and by Sex Steroid Hormones1

P.R.R. Gangula4, P. Lanlua4,5, R.D. Bukoski3,7, S.J. Wimalawansa6, and C. Yallampalli2,4,5

Departments of Obstetrics & Gynecology,4 Anatomy & Neuroscience,5 Internal Medicine,6 The University of Texas Medical Branch, Galveston, Texas 77555 Cardiovascular Disease Research Program,7 Julius L. Chambers Biomedical Biotechnology Research Institute, North Carolina Central University, Durham, North Carolina 27707


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The present study investigated whether pregnancy and circulatory ovarian hormones increase the sensitivity of the mesenteric artery to calcitonin gene-related peptide (CGRP)-induced relaxation and possible mechanisms involved in this process. Mesenteric arteries from young adult male rats or female rats (during estrous cycle, after ovariectomy, at Day 20 of gestation, or Postpartum Day 2) were isolated, and the responsiveness of the vessels to CGRP was examined with a small vessel myograph. The CGRP (10–10 to 10–7 M) produced a concentration-dependent relaxation of norepinephrine-induced contractions in mesenteric arteries of all groups. Arterial relaxation sensitivity to CGRP was significantly (P < 0.05) greater in female rats compared with male rats. Pregnancy increased the sensitivity to CGRP significantly (P < 0.05) compared to ovariectomized and Postpartum Day 2 rats. In pregnant rats, CGRP-receptor antagonist, CGRP8–37, inhibited the relaxation responses produced by CGRP. The CGRP-induced relaxation was not affected by NG-nitro-L-arginine methyl ester (nitric oxide inhibitor, 10–4 M) but was significantly (P < 0.05) attenuated by an inhibitor of guanylate cyclase (1H-[1 , 2 , 4 ]oxadizaolo[4 , 3 -a]quinoxalin-1-one, 10–5 M). Relaxation responses of CGRP on mesenteric arteries were blocked (P < 0.05) by a cAMP-dependent protein kinase A inhibitor, Rp-cAMPs (10–5 M). The CGRP-induced vasorelaxation was significantly (P < 0.05) attenuated by calcium-dependent (tetraethylammonium, 10–3 M), but not ATP-sensitive (glybenclamide, 10–5 M), potassium channel blocker. Therefore, the results of the present study suggest that mesenteric vascular sensitivity to CGRP is higher during pregnancy and that cAMP, cGMP, and calcium-dependent potassium channels appear to be involved. Therefore, we propose that CGRP-mediated vasodilation may be important to maintain vascular adaptations during pregnancy.

pregnancy, steroid hormones


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pregnancy is a complex process in which several vascular changes occur to maintain blood supply to growing fetuses. Reports in humans and other species indicate that during gestation, blood pressure and vascular resistance decrease, whereas blood volume, cardiac output, and heart rate increase [1]. The mesenteric circulation makes a major contribution to peripheral resistance in rats [2]. Calcitonin gene-related peptide (CGRP) is a potent vasodilator that lowers blood pressure via peripheral vasodilation [3–5]. The CGRP is synthesized primarily in the sensory neurons of dorsal root ganglia that extend axons centrally to the spinal cord and peripherally to various organs, including blood vessels [3, 6–8].

The CGRP-induced relaxation in the mesenteric artery is greater than in the vein [9]. In addition, CGRP has more pronounced dilator effects on small, resistance arteries, such as basilar, gastroepiploic, and mesenteric, than on large, conduit arteries [5, 10–12]. True resistance arteries, including those in the mesenteric circulation, regulate gastrointestinal vascular resistance and regional blood flow and contribute to blood pressure regulation [13]. In addition, evidence from several reports indicates that the effects of CGRP on the mesenteric artery are endothelium-independent [14–17].

Men and postmenopausal women are at higher risk for cardiovascular diseases, including hypertension [18, 19]. Although a recent women's health initiative report [20] questioned the value of hormone replacement therapy (HRT) in postmenopausal women, several previous reports showed decreases in the incidence of cardiovascular problems, such as hypertension, coronary heart disease, and stroke [21–24]. In view of these findings, we suggest that vasodilators sensitive to CGRP may be lower in males compared to females when sex steroid hormone levels are elevated. In the present study, we examined the vascular relaxation effects of CGRP in male and female rat mesenteric artery segments.

During pregnancy, a generalized decrease in blood pressure results from reduced vascular resistance, and blood pressure returns to nonpregnant levels in the postpartum period [25]. Our previous studies [26] have shown that CGRP reduced the NG-nitro-L-arginine methyl ester (L-NAME)-induced hypertension in pregnant rats. A CGRP-induced decrease in the mean arterial pressure was greater during gestation and with the injection of ovarian hormone to young adult ovariectomized (ovx) rats [27, 28]. Coronary, mesenteric, and renal vasculature appeared to be more sensitive to CGRP-induced vasodilation in pregnant and as well as in young adult ovx rats treated with female sex steroid hormones [29]. Moreover, infusion of CGRP8–37, a receptor antagonist for CGRP, increases blood pressure and fetal mortality and reduces fetal growth in pregnant rats [30]. In view of these findings, we hypothesized that increased vasodilatory responses to CGRP during pregnancy result from increased relaxation sensitivity of resistance arteries to CGRP. Several second-messenger pathways, including cAMP, nitric oxide (NO), as well as ATP-sensitive and Ca2+-dependent potassium channels (K+), have been suggested to mediate CGRP-induced relaxation in different blood vessels [7]. However, it is unknown as to whether these mechanisms are involved in CGRP-induced mesenteric artery relaxation in pregnant rats. Therefore, we examined involvement of second-messengers systems, such as cAMP, ATP-activated and calcium-activated potassium channels, and the NO system, in CGRP-mediated mesenteric relaxation in pregnant rats.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

All procedures were approved by the Animal Care and Use Committee at the University of Texas Medical Branch in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Adult male, female nonpregnant (age, 8–9 wk), and Day 14 pregnant (body weight, 275–300 g) 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 with fixed photoperiod of 12L:12D. Animals were allowed free access to water and rodent chow.

Groups of nonpregnant female rats were killed at each stage of the estrous cycle and after ovariectomy. The stage of estrous cycle was assessed by examining cells in a vaginal flush using light microscopy [31]. Pregnant rats were killed on Day 20 and on Postpartum Day 2 (PP2). All fetuses and pups were killed by halothane inhalation.

Isometric Force Wire Myography

Tissue preparation The procedure for using the isometric force wire myograph (Kent Scientific, Litchfield, CT) is similar to that described by Wang and Bukoski [32]. Young adult males; females during proestrous, estrous, and diestrous stages of the estrous cycle; ovx females; and pregnant rats on Day 20 of gestation were killed by exsanguination under deep anesthesia induced by i.p. injection of ketamine (50 mg/kg) and xylazine (8 mg/kg). The small intestine, including the blood supply, was cut and placed in physiological salt solution (PSS) and kept on ice. The PSS contained 114 mM NaCl, 4.7 mM KCl, 1.15 mM KH2PO4, 1.10 mM Na2HPO4, 1.18 mM MgSO4·7H2O, 15 mM NaHCO3, 1.50 mM CaCl2, and 5.0 mM glucose. Secondary branches of the mesenteric artery were then isolated and cleaned of fat and connective tissue. The arterial segments (length, ~2 mm) were then mounted on a wire myograph using tungsten wires and incubated for 15 min in PSS at 37°C, which was gassed with 95% air and 5% CO2 to maintain pH 7.4. The segment was then stretched to a length that was equivalent to a diameter of 200–225 µm and incubated for an additional 15 min. Finally, the segment was induced to contract by the addition of 5 µM norepinephrine (NE) until reproducible responses were obtained. The response to CGRP was assessed by cumulative addition of the peptide from 10–10 to 10–7 M to vessels that were precontracted with the ED70 (the dose of NE that induced 70% of the maximal response) concentration of NE determined for that vessel.

Mechanisms for the CGRP-induced relaxation in pregnant rat mesenteric artery The involvement of receptors, various intracellular messengers, and ion channels in CGRP-induced vascular relaxation was investigated using selective inhibitors. In these studies, we first assessed vascular reactivity of mesenteric artery from rats on Day 20 of pregnancy to varying doses of CGRP (10–10 to 10–7 M) with NE precontraction using wire myography. Subsequently, CGRP was washed out with PSS, and the mesenteric arterial segments were incubated for 30 min in fresh PSS with either CGRP8–37 (10–4 M, a receptor antagonist for CGRP), the inhibitors of cAMP-dependent protein kinase A (Rp-cAMPs, 10–5 M), guanylate cyclase (1H-[1, 2, 4]oxadizaolo[4, 3-a]quinoxalin-1-one [ODQ], 10–5 M), ATP-sensitive (glybenclamide, 10–5 M) or calcium-dependent (tetraethylammonium, 10–3 M) potassium (K+) channels, or NO (L-NAME, 10–4 M). After the incubation period, relaxation responses to cumulative doses of CGRP (10–10 to 10–7 M) were repeated in NE precontracted arterial segments. For each segment, the CGRP-induced vasorelaxations were calculated as the percent of precontracted tension induced by norepinephrine (ED70).

Statistical Analysis

Data are presented as the mean ± SEM. Relaxation to CGRP was expressed as a percentage of the initial precontraction to NE. The Emax (maximal increase in tension) and pD2 (–logEC50, or concentration of the agent that inhibited 50% of the maximal contraction) were calculated using a nonlinear regression curve (Prism GraphPad Software, Inc., San Diego, CA) from the individual concentration-response relationships. Two arteries were used from each animal, and means were calculated. Raw data for individual concentration-response curves were also compared by two-way repeated-measurement ANOVA. The Bonferroni/Dunn post-hoc test was used for determining significant differences between factors. The Student unpaired t-test was used for statistical comparison for logEC50 values. A P value of less than 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CGRP-Induced Mesenteric Vascular Reactivity in Pregnant, Postpartum, and Young Adult ovx Rats

Levels of the female sex steroids during pregnancy are elevated, but these levels are decreased during the postpartum period. To study the effects of these hormones on the vascular response to CGRP, the relaxation response of mesenteric artery segments from rats at Day 20 of pregnancy and at PP2 as well as from nonpregnant ovx rats were compared (Fig. 1). CGRP-induced a concentration-dependent relaxation of the mesenteric artery in pregnant, postpartum, and ovx rats. The overall relaxant response (P < 0.05, two-way ANOVA) to CGRP was significantly enhanced in arteries of pregnant rats compared with arteries of ovx and PP2 rats. The pD2 values were significantly (P < 0.05) lower in ovx (7.39 ± 0.21) and postpartum (6.70 ± 0.41) animals compared to pregnant animals (8.51 ± 0.21).



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  		FIG. 1. Vascular relaxation response to cumulative dose of CGRP (10–10 to 10–7 M) in the mesenteric artery of rats at Day 20 of pregnancy (D20), PP2, and young adult ovx rats (NP-ovx). The percentage of initial tension of each CGRP dose was calculated as a percentage of precontraction by NE (100%). The comparison was made by repeated-measurement ANOVA (mean ± SEM, n = 6 animals/group). **P < 0.01, ***P < 0.005

CGRP-Induced Mesenteric Vascular Reactivity in Young Adult Male and Female Rats

It is possible that the enhanced sensitivity of the isolated arteries to CGRP during pregnancy might be the result of elevated levels of ovarian hormones. To assess the influence of ovarian hormones on CGRP-induced relaxation, mesenteric arteries of male and female rats at the proestrous, estrous, and diestrous stages of the estrous cycle were examined (Fig. 2). Cumulative addition of CGRP to the mesenteric arteries of intact male and female rats induced concentration-dependent relaxation. Sensitivity to CGRP was greater for mesenteric arteries of female rats than for vessels from male rats (Fig. 2A and Table 1). Further analysis was performed to determine how the stage of the estrous cycle in these female rats influences the sensitivity to CGRP. The CGRP-induced vasorelaxation of mesenteric arteries isolated from rats at the diestrous, proestrous, and estrous stages was significantly (P < 0.05) greater than arteries from male animals (Fig. 2B), although the differences among various stages of the estrous cycle in the female rats were not significant.



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  		FIG. 2. Cumulative dose of CGRP-induced vasorelaxation in the mesenteric artery of male (M) and female (F) rats (A) and at different stages of the estrous cycle (B): diestrus (D), proestrus (P), and estrus (E). The percentage of the initial tension of each CGRP dose was calculated as a percentage of precontraction by NE (100%). The percentage of initial tension was compared by repeated-measurement ANOVA (mean ± SEM; M, n = 7; F, n = 21; D, n = 9; P, n = 7; E, n = 5). *P < 0.05 vs. M


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  		TABLE 1. CGRP concentrations causing 50% relaxation (pD2) of mes enteric artery in male and female rats at various stages of estrous cycle or during Day 20 of pregnancy.a

Assessment of Receptor and/or Postreceptor Mechanisms Involved in CGRP-Induced Mesenteric Artery Relaxation in Pregnant Rats

In all of these studies, rats at Day 20 of pregnancy were utilized to assess the mechanisms of action of CGRP; comparisons were made to the mesenteric artery relaxation responses to CGRP (control) obtained before and after the addition of various inhibitors. To examine whether CGRP-induced mesenteric artery relaxation is mediated by its receptors, we used the antagonist of CGRP, CGRP8–37, before the addition of cumulative doses of CGRP. As shown in Figure 3, blockade of the CGRP receptor by CGRP8–37 (pD2, 6.43 ± 0.61) substantially inhibited the CGRP-induced relaxation of the pregnant rat mesenteric artery (pD2, 8.20 ± 0.30; P < 0.05).



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  		FIG. 3. Effect of CGRP8–37 on vasodilator activity of CGRP in isolated mesenteric artery of rats at Day 20 of pregnancy. Graphs show the concentration-response relationship for CGRP-induced relaxation in the absence (control) and the presence of CGRP8–37 (10–4 M). The percentage of the initial tension of each CGRP dose was calculated as a percentage of precontraction by NE (100%). The comparison was made by repeated-measurement ANOVA. Values are presented as the mean ± SEM (n = 3). *p < 0.05

To investigate whether NO and cGMP pathways are involved in CGRP-induced relaxation effects, mesenteric artery segments were incubated for 30 min in the presence of either L-NAME (NO inhibitor, 10–4 M) or ODQ (inhibitor of guanylate cyclase, 10–5 M). As shown in Figure 4A, L-NAME appeared to inhibit the vasodilator effects of CGRP in pregnant rat mesenteric artery, but these effects did not reach statistical significance (P = 0.08). However, the relaxation responses to CGRP were significantly (P < 0.05) attenuated by the inhibitor of guanylate cyclase (ODQ; pD2, 6.7 ± 0.38) compared to the control group (7.99 ± 0.11) (Fig. 4B).



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  		FIG. 4. Effect of inhibition of NO synthase (L-NAME; A) or guanylate cyclase (ODQ; B) on relaxation response of mesenteric artery from rats at Day 20 of pregnancy induced by cumulative concentrations of CGRP. Graphs show the concentration-response relationship for CGRP-induced relaxation in the absence (control) and the presence of L-NAME (10–4 M) or ODQ (10–5 M). The percentage of initial tension of each CGRP dose was calculated as a percentage of precontraction by NE (100%). The comparison was made by repeated-measurement ANOVA. Values are presented as the mean ± SEM (n = 3). *P < 0.05

Furthermore, we evaluated whether the cAMP pathway is involved in CGRP-induced vascular relaxation in pregnant rats. The effects of CGRP were assessed in the presence of 10–5 M Rp-cAMPs, an inhibitor of a cAMP-dependent protein kinase A. As shown in Figure 5, the sensitivity and the maximal response to CGRP were reduced (P < 0.05) with preincubation of the mesenteric artery with Rp-cAMPs (pD2: control, 8.07 ± 0.27; Rp-cAMPs, 6.70 ± 0.27).



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  		FIG. 5. Effect of Rp-cAMPs, a cyclic AMP-dependent protein kinase A inhibitor, on CGRP-induced vasorelaxation in the mesenteric artery of rats at Day 20 of pregnancy. Graphs show the concentration-response relationship for CGRP-induced relaxation in the absence (control) and the presence of Rp-cAMPs (10–5 M). The percentage of the initial tension of each CGRP dose was calculated as a percentage of precontraction by NE (100%). The percentage of initial tension was compared by repeated-measurement ANOVA. Values are presented as the mean ± SEM (n = 3 animals). *P < 0.05

The CGRP-induced vasorelaxation was significantly (P < 0.05) attenuated by calcium-dependent (tetraethylammonium, 10–3 M) K+-channel blocker (Fig. 6A). The pD2 was in the presence of tetraethylammonium was 6.94 ± 0.51, compared with 8.10 ± 0.10 in the control. However, the ATP-dependent K+-channel blocker, glybenclamide, only had minimal effects (Fig. 6B) on CGRP-induced vascular relaxation (pD2: control, 8.13 ± 0.09; glybenclamide, 7.67 ± 0.28).



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  		FIG. 6. Influence of potassium-channel blockers, A) tetraethylammonium (TEA; calcium-activated channel, 10–3 M), or B) glybenclamide (Glib; ATP-activated channel, 10–5 M) on vascular relaxation response to cumulative doses of CGRP (control) in mesenteric artery segments of rats at Day 20 of pregnancy. Graphs show the concentration-response relationship for CGRP-induced relaxation in the absence (control) and the presence of inhibitors of potassium channel blockers. The percentage of the initial tension of each CGRP dose was calculated as a percentage of precontraction by NE (100%). The percentage of initial tension was compared by repeated-measurement of ANOVA. Values are presented as the mean ± SEM (n = 3). *P < 0.05


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The major findings of the present study are as follows: 1) The relaxation response to CGRP of the mesenteric branch arteries was significantly greater in pregnant rats compared with young adult ovx and postpartum animals; 2) the mesenteric branch arteries of female rats were more sensitive to CGRP-induced vasorelaxations at stages when steroid hormones were elevated compared to male rats; 3) CGRP8–37, a receptor antagonist for CGRP, significantly inhibited the vasorelaxation to CGRP in pregnant rat mesenteric artery segments; and 4) several second-messenger pathways were involved in CGRP-induced relaxation in mesenteric arteries from pregnant rats, including cAMP, cGMP, and calcium-sensitive as well as, perhaps, ATP-sensitive K+ channels. These results indicate that the vascular relaxation sensitivity to CGRP of the resistance vessels is enhanced in female rats compared to male rats as well as during pregnancy. Our previous studies have shown hypertension in both male and female CGRP-knockout mice, demonstrating the importance of CGRP in maintaining normal blood pressure [33]. Current studies provide further insights regarding our previous findings that CGRP may play a role in the decreased vascular resistance during pregnancy [27, 28].

In the present study, the mesenteric arterial relaxation sensitivity to CGRP appeared to be decreased in young adult ovx and postpartum rats compared to pregnant rats (Fig. 1). These results are in agreement with previous observations that CGRP-induced mesenteric vascular relaxation responses are greater on Day 10 of gestation compared to nonpregnant rats [34]. During pregnancy, the maternal cardiovascular system undergoes several adaptations, including increased cardiac output, decreased blood pressure, and decreased systemic vascular resistance [35–37]. This decreased systemic vascular resistance during pregnancy may result from increased sensitivity of the resistance vessels to vasodilators, such as acetylcholine [38], and/or from decreased resistance arterial responses to vasoconstrictors, such as NE and phenylephrine [39, 40]. Recent studies from our laboratory provide evidence that increased vasodilation to CGRP during pregnancy could result from decreased total vascular resistance, particularly to coronary, mesenteric, and renal vascular beds, and that these effects are sex steroid hormone-dependent [27–29]. The present study provides direct evidence of increased mesenteric vascular relaxation to CGRP, which could be contributing for lowered blood pressure during pregnancy. Previous studies from our laboratory [26] demonstrated that CGRP administration cannot decrease blood pressure in L-NAME-infused rats during the postpartum period unless progesterone is coinjected, indicating the requirement of female steroid hormones for elucidation of the effect of CGRP. Thus, we suggest that increased relaxation sensitivity to CGRP of both resistance and uterine arteries, as reported earlier [41], may be important to maintain vascular relaxation and to increase uterine blood flow during pregnancy.

Because the data obtained with the pregnant animals suggest that ovarian hormones modulate the vasorelaxant responses to CGRP, we assessed the responses of arteries taken from animals at different stages of the estrous cycle and in male rats. The sensitivity of male rat arteries to CGRP is significantly lower than that of female rat arteries at all stages of the estrous cycle (Fig. 2A). Furthermore, the sensitivity of the relaxation responses to CGRP was substantially greater in nonpregnant rats at the proestrous stage with higher ovarian steroid hormone levels (Fig. 2B and Table 1). The vascular responses of the mesenteric artery to vasoconstrictors, such as noradrenaline and potassium chloride, are reduced in female compared to male and ovx rats [42]. That female steroid hormones may increase the arterial sensitivity to vasodilators, such as CGRP, as found in the present study, together with the decrease in the sensitivity to vasoconstrictors, as reported elsewhere [42], could provide protection against cardiovascular diseases. These changes in sensitivity to CGRP may play a role in vascular adaptations not only during pregnancy [25] but also in ovx rats injected with female steroid hormones [27, 28] and in postmenopausal women with HRT [21–24]. The lack of significant differences in the CGRP-related vasorelaxation among various stages of estrous cycle in the females may result from the relatively short duration of each stage for manifesting significant effects by steroid hormones.

The present study further demonstrated that preincubation of mesenteric artery segments with CGRP8–37 significantly (P < 0.05) inhibited CGRP-induced relaxation (Fig. 3). Recent studies from our laboratory postulated that the vasodilatory effects of CGRP was inhibited by CGRP8–37 in pregnant uterine artery segments [41]. Furthermore, we have reported that CGRP-receptor components, calcitonin receptor like-receptor, and receptor activity-modifying protein 1 (RAMP1) are elevated during pregnancy and by treatment with female sex steroid hormones in mesenteric arteries [34, 43]. Collectively, these studies suggest that CGRP receptors may play a role in mediating vasodilator effects of CGRP in both mesenteric and uterine blood vessels.

To address the mechanisms of CGRP-induced vasorelaxation, pregnant mesenteric artery rings were preincubated for 30 min either with L-NAME, an inhibitor of NO synthase, or with ODQ, inhibitor of guanylate cyclase, and dose-dependent effects of CGRP were measured in NE precontracted vessels. Results show that ODQ significantly (Fig. 4B), and L-NAME (Fig. 4A) partially, inhibited the CGRP-induced vascular relaxation effects in pregnant rat mesenteric artery. Furthermore, our studies show that Rp-cAMPs, a cAMP-dependent protein kinase A inhibitor, significantly inhibited CGRP-induced vascular relaxant effects in pregnant rat mesenteric artery. Previous studies demonstrated that the vasodilator effects of CGRP are NO-dependent in rat aortas [44], whereas they are independent of NO in mouse uteri [45]. Furthermore, the relaxation effects of CGRP appear to be mediated through the CGRP inhibition of type III phosphodiesterase and subsequent accumulation of cAMP in smooth muscle cells [46]. Thus, CGRP-induced mesenteric vasodilator effects appear to be partially NO-dependent, and both cAMP and cGMP may be involved in this process.

In the present study, tetraethylammonium, an inhibitor of calcium-activated K+ channels, reduced the vascular relaxation responses to CGRP in pregnant rat mesenteric artery. Although incubation with glybenclamide, an ATP-sensitive K+-channel blocker, inhibited CGRP-induced mesenteric arterial relaxation, this was not statistically significant. Previous studies have shown that at least part of the CGRP-induced relaxation in mesenteric arteries is mediated by the opening of ATP-sensitive K+ channels [47]. The CGRP-mediated vasodilatory effects were antagonized by glybenclamide [48] in pulmonary vascular beds of the cat. In contrast, studies by Gao et al. [49] demonstrated that glybenclamide did not alter the CGRP-induced mesenteric artery relaxation in either stroke-prone spontaneously hypertensive or age-matched Wistar-Kyoto rats. Our findings that tetraethylammonium significantly inhibited the CGRP-induced mesenteric artery relaxation suggest that Ca2+-activated K+ channels exist in pregnant rat mesenteric arteries and that these channels may be involved in CGRP-induced mesenteric relaxation during pregnancy.

Thus, data from the present study suggest that cAMP, cGMP, and potassium channels appear to be involved in CGRP-mediated mesenteric vascular relaxation in pregnant rats. The CGRP has been shown to relax vascular smooth muscle cells via both endothelium-dependent and endothelium-independent signal transduction pathways [50–54]. Both pathways can be activated by adenylate cyclase and production of cAMP [55–57]. In many vascular beds, the endothelium-dependent pathway begins with CGRP binding to receptors in the endothelial cells, leading to activation of adenylcyclase. The resultant increased levels of cAMP may then activate the NO synthase, leading to increased levels of NO. However, CGRP may also elevate NO directly, without the involvement of adenylcyclase. Then, NO acts on smooth muscle cells by activating soluble guanylate cyclase and production of cGMP, which, in turn, leads to smooth muscle relaxation. In the endothelium-independent pathway, CGRP directly binds to CGRP receptors located on smooth muscle cells, activating adenylcyclase, which, in turn, produces cAMP, leading to vascular relaxation. In the present study, we have shown that both cAMP and cGMP are involved in CGRP-induced vascular relaxation in pregnant rat mesenteric artery. Moreover, reports indicate that the cAMP pathway is also involved in the CGRP-induced K+-channel activation, a transient decrease in the [Ca2+]I, and a long-lasting Ca2+ desensitization [58] in vascular smooth muscle [59, 60]. However, a decrease of intracellular Ca2+ in the vascular smooth muscle cells could be the final common pathway of vascular relaxation effects of CGRP. At present, it is unclear whether these signal-transduction pathways involved in CGRP-induced vascular relaxation are independent or interdependent. Additional studies, including assessment of the generation of cAMP and cGMP in mesenteric vascular tissues in response to CGRP, are required to fully understand the signaling "cross-talk" in CGRP-induced mesenteric artery relaxation.

In summary, our data provide evidence that vascular relaxation sensitivity to CGRP during pregnancy is increased and that this may be the underlying mechanism for the lowered blood pressure in pregnant rats. The CGRP-induced vascular relaxation effects are greater in female rats compared to male rats, suggesting that female sex steroid hormones are involved in this process. The effects of CGRP on mesenteric artery relaxation are mediated by CGRP receptors. Cyclic AMP, cGMP and Ca2+-activated K+ channels appear to be the primary mediators involved in CGRP-induced mesenteric artery relaxation in pregnant rats. These results suggest that elevated levels of female sex steroid hormones may play a critical role in maintaining vascular relaxant effects of CGRP in resistance blood vessels and, therefore, regulate vascular adaptations during pregnancy.


   ACKNOWLEDGMENTS
 
We thank Ms. K. Barrett for editorial comments, Mr. J. Helms for his skillful graphic designs, Dr. N. Ishioka for instruction in the myograph technique, and Ms. K. Mitchell and Cheryl Welch for typing the manuscript. This paper is dedicated in honor of R.D. Bukoski, coauthor, who recently passed away. Dr. Bukoski provided his expertise in guiding us to establish myograph techniques in our laboratory as well as in preparing this manuscript. He will be sadly missed.


   FOOTNOTES
 
1 Supported by the following National Institutes of Health (NIH) grants: HL 58144, HD 40828, and HL 72650. Back

2 Correspondence: Chandrasekhar Yallampalli, Department of Obstetrics & 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

3 Deceased Back

Received: 26 April 2004.

First decision: 2 June 2004.

Accepted: 9 July 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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