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Biol Reprod 2002, 10.1095/biolreprod.102.007682
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Biology of Reproduction 67, 1881-1887 (2002)
DOI: 10.1095/biolreprod.102.007682 © 2002 Society for the Study of Reproduction, Inc.

Mechanisms of Hormone Action

Sex Steroid Hormones Enhance Hypotensive Effects of Calcitonin Gene-Related Peptide in Aged Female Rats1

P.R.R. Gangulaa, S.J. Wimalawansab, and C. Yallampalli2,a

a Department of Obstetrics/Gynecology, University of Texas Medical Branch, Galveston, Texas 77555-1062 b Department of Endocrinology, Robert Wood Johnson Medical School, New Brunswick, New Jersey 08903-0019


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aim of the present study is to investigate whether vascular protective effects of steroid hormones in aged female rats are mediated through calcitonin gene-related peptide (CGRP), a known potent vasodilator. This rat model reflects the postmenopausal state in humans. We examined whether blood pressure lowering effects of CGRP are enhanced in aged female rats when steroid hormone treatments are administered. We observed that 1) continuous infusion of CGRP lowered blood pressures in rats treated with estradiol-17ß and progesterone (P < 0.05), 2) acute hypotensive effects of CGRP were significantly (P < 0.05) greater in the presence of steroid hormones than in vehicle-treated groups, 3) blood pressure decreases in response to CGRP are lower in aged female rats than they are in young adult ovariectomized rats, and 4) age-related differences in the hypotensive effects of CGRP were nullified when animals were treated with steroid hormones. These data suggest that female sex steroid hormones may modulate arterial blood pressure by regulating the CGRP effector system in female rats regardless of age.

aging, estradiol, neuropeptides, progesterone, steroid hormones


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aging is associated with an increase in pathological processes such as hypertension, coronary artery disease, heart failure, and postural hypotension [1]. Previous studies have demonstrated that in older people, changes occur in the cardiovascular system, especially in the structure and function of the arteries [2, 3]. Further, risk of cardiovascular alterations is two to five times greater in elderly patients with hypertension than it is in normotensive individuals of the same age [4].

Several epidemiological studies have shown a gender-specific difference in hypertension morbidity in middle-aged humans (men < women) and that it increases in women after menopause [5–7] when steroid hormones are depleted. On the other hand, estrogen treatment lowers hypertension in postmenopausal women [8] and in male rats with spontaneous hypertension (SHR) [9]. In addition, reports indicate [10] that premenopausal women have a higher cardiac output and lower total peripheral resistance than do men of the same age, and potent vasodilatory effects of estrogens have been shown in several in vivo and in vitro studies [9, 11]. Studies by Armstrong [12] demonstrated a hypotensive action of progesterone (P4) in rats with renal hypertension and in humans with essential hypertension. It has been reported that circulatory levels of estradiol-17ß (E2) and P4 are lower in aged rats [13]. Collectively, these studies suggest that the age-related decrease in sex steroid hormone levels may play a role in the development of hypertension in postmenopausal women and animals. Several mechanisms such as nitric oxide (NO) [14], prostaglandins [15], serum lipids [16, 17], and calcitonin gene-related peptide (CGRP) [18–22] have been proposed for their favorable effects as steroid hormones in vascular functions.

CGRP is a potent vasodilator neuropeptide encoded in the calcitonin gene [23]. The primary site of CGRP synthesis is dorsal root ganglia (DRG) that send efferent sensory nerve fibers to the peripheral organs and around vasculature, particularly in resistance blood vessels such as mesenteric arteries [24–26]. CGRP is released from the nerve terminals, binds to its receptors on the vasculature [23, 27], and reduces the blood pressure via peripheral vasodilation [28, 29]. Systemic administration of CGRP decreases blood pressure and produces significant positive chronotropic and inotropic effects [30–32]. This peptide is believed to be involved in peripheral vascular tone regulation and regional organ blood flows through endothelium-dependent [33, 34] or endothelium-independent mechanisms [35–38].

Our previous studies in young adult female rats showed that sex steroid hormones stimulate CGRP mRNA expression in DRG neurons [39]. Circulatory CGRP levels are elevated in nonpregnant ovariectomized rats treated with steroid hormones [21] and in postmenopausal women undergoing hormone replacement therapy (HRT) [40, 41]. Moreover, plasma concentrations of immunoreactive CGRP (i-CGRP) were significantly higher in women than in men, and women who use oral contraceptives had higher i-CGRP concentrations [42], suggesting that female sex hormones may influence CGRP concentrations in plasma. Previous studies in our laboratory demonstrated that mean arterial blood pressure was lower in young adult ovariectomized rats treated with E2 and P4 than in vehicle-treated ovariectomized rats [20]. Further, hypotensive effects of systemic administration of CGRP are significantly greater with steroid hormone treatment in ovariectomized, young adult female rats [22, 43]. The effects of CGRP on greater cardiac output and lower total peripheral resistance were amplified in steroid hormone-treated rats [22]. These studies further demonstrated that coronary and mesenteric vasculature appeared to be more sensitive to CGRP in steroid hormone-treated rats. Together, these studies suggest that cardiovascular protective effects of steroid hormones appear to be mediated through the CGRP system.

The vasculature during the aging process is generally characterized by impaired vessel tone and impaired ability to adapt to circulatory insults. The age-associated deterioration in neuroendocrine regulatory function, which has been demonstrated in humans and animals [1], is believed to be one of the main reasons for age-related cardiovascular diseases. The depletion of female sex hormones in postmenopausal women and in aged female rats [5–7, 13] is one of the leading causes of blood vessel wall pathophysiology. Several reports, including ours, provide evidence that CGRP plays an important role in the pathogenesis of hypertension, and that the release, content, and innervation of CGRP neurons are lower in the central nervous system and peripheral tissues of aged humans and animals [41, 44–49]. The vasorelaxant responses induced by electric perivascular nerve-stimulated release of CGRP were also found to be lower in aged rats. Recent studies [50] reported that vasodilatory effects of CGRP are impaired in aged male and female rats. Together, these studies suggest that the decrease in steroid hormone levels in postmenopausal women results in lower CGRP synthesis and release and its effects, which lead to hypertension and greater cardiovascular problems.

Therefore, in the present study we first examined whether differences in blood pressure levels existed between young adult and aged female rats. Next, we investigated whether continuous infusion of CGRP or steroid hormone treatments either alone or in combination lowers the systolic blood pressure in aged female rats. Further, studies were focused on whether bolus injections of CGRP dose-dependently modulate the systolic, diastolic, and mean arterial blood pressures in these rats, and if they did, whether the hypotensive effects of CGRP were rescued upon steroid hormone treatment. We also assessed whether the vasodilatory effects of CGRP were more impaired in aged female rats than they were in young adults. Finally, we examined whether steroid hormone treatment can rescue the loss of CGRP-induced vasodilation in aged female rats.


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

Young adult (3 mo) nonpregnant or aged (16–18 mo) rats were purchased from Harlan Sprague-Dawley (Houston, TX) and housed in a climate controlled room with a 12L:12D schedule. All animals were fed standard rat chow and had ad libitum access to water. All procedures were approved by the Animal Care and Use Committee of the University of Texas Medical Branch (Galveston, TX).

Treatments

In the first experiment, young adult or aged female rats underwent bilateral ovariectomy after administration of ketamine (45 mg/kg body weight [BW]; Fort Dodge Laboratory, Fort Dodge, IA) or xylazine (5 mg/kg BW; Burns Veterinary Supply, New York, NY) anesthesia. After 7 days of recovery, groups (n = 6) of aged ovariectomized rats were given s.c. injections of E2 (12.5 µg/kg BW twice daily) or P4 (10 mg/kg BW twice daily) for 4 days in the presence or absence of a continuous s.c. infusion of CGRP (50 µg/day/kg BW) through osmotic minipumps (2 MLI; Alza, Palo Alto, CA). Steroid hormone doses were established on the basis of earlier studies [51]. Control groups received vehicle only (0.5 ml sesame oil). Systolic blood pressure was measured daily in all ovariectomized rats with a pneumatic tail cuff device (Narco-Biosystem, Houston, TX). Blood pressure values obtained from three consecutive measurements were averaged and recorded as the mean systolic pressure for a given rat at each point.

In our second study we used 16- to 18-mo-old ovary-intact female rats because they exhibit low sex steroid hormone levels [13], and because our present studies demonstrated no differences in blood pressure between ovary-intact and aged ovariectomized rats. Groups (n = 6) of rats at diestrus (confirmed by vaginal cytology) received 4-day steroid hormone treatments as described above. To determine age-related changes in response to CGRP in the presence or absence of steroid hormone treatments we used the data for young adult ovariectomized rats treated with steroid hormones as described above. Briefly, on the day of acute measurements, following ketamine or xylazine anesthesia, the left carotid artery was cannulated to continuously measure systolic, diastolic, and mean arterial blood pressures using a DBP001 direct blood pressure system (Kent Scientific, Litchfield, CT). The right jugular vein was also cannulated to administer either vehicle (saline) or CGRP. Animals were allowed to fully recover from the effects of anesthesia (3–4 h after surgery) before we measured systolic, diastolic, and mean arterial pressures and administered CGRP. Previous studies have shown that maximal responses to CGRP were observed within 1 min after i.v. administration [20]. Systolic, diastolic, and mean arterial blood pressures were measured immediately before and 1 min after bolus injection of either the vehicle or cumulative doses of CGRP (9–360 pmol/kg BW). The changes in arterial pressures in response to each dose of CGRP are expressed as means ± SEM for each group.

Statistical Analysis

Differences in blood pressures between groups were determined using either ANOVA followed by the Tukey-Kramer multiple comparisons test or the Student t-test. The acceptable level of significance was P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In the first experiment we investigated whether systolic and mean arterial blood pressures were elevated in aged ovary-intact or in aged ovariectomized rats, and if they were, whether steroid hormones can reverse this effect. Ovariectomy in young adult female rats causes rises in both systolic and mean arterial blood pressures [20]. Systolic blood pressure as measured by the tail cuff method was significantly (P < 0.05; n = 6 rats/group) higher in aged female rats (185 ± 5 mm Hg) than it was in adult ovariectomized rats (124 ± 3 mm Hg) (Fig. 1A). No significant change in systolic blood pressure was observed in aged rats after ovariectomy (186 ± 18 mm Hg). Treatment of aged ovariectomized rats with E2 (179 ± 4.0 mm Hg) or P4 (183 ± 9.0 mm Hg) did not decrease systolic blood pressure levels from those of controls (186 ± 18 mm Hg) (Fig. 1A). We next compared changes in mean arterial blood pressure using carotid artery indwelling catheters between young adult and aged female rats in a fully awake and free-moving state. As shown in Figure 1B, mean arterial blood pressure was higher in ovary-intact aged rats (123 ± 3.0 mm Hg) than it was in young adult ovariectomized rats (106 ± 4.0 mm Hg). Treatment with steroid hormones did not alter mean arterial blood pressure in ovary-intact aged rats (Fig. 1B).



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  		FIG. 1. Effect of sex steroid hormones on systolic blood pressure (A) and mean arterial blood pressure (B) in young adult ovariectomized and aged female rats. Groups of either ovary-intact or ovariectomized female rats 16–18 mo old received s.c. injections of E2 (12.5 µg/kg BW twice daily for 4 days), P4 (10 mg/kg BW twice daily for 4 days), or vehicle (sesame oil). Systolic blood pressure was measured in young adult ovariectomized, aged ovary-intact, and aged ovariectomized rats with the use of a pneumatic tail cuff device (A). Mean arterial blood pressure was measured in fully awake and free-moving young adult ovariectomized and ovary-intact aged rats using a carotid artery indwelling catheter. Values are means ± SEM of 6 animals per group. *P < 0.05 vs. young adult ovariectomized animals (n = 6, Student t-test)

Figure 2 illustrates the effects of a continuous infusion of CGRP on systolic blood pressure and modulation by female sex steroid hormones in aged ovariectomized rats. CGRP infusion alone did not lower systolic blood pressure in ovariectomized rats during any treatment period. On the other hand, E2 injections led to a significant drop in systolic blood pressure in CGRP-infused rats throughout the treatment period, however, P4 did not lower systolic blood pressure in the first 2 days. Nonetheless, the effects of this hormone in lowering systolic blood pressure were substantial on the third and fourth day of treatment (P < 0.05) compared with vehicle treated rats (Fig. 2).



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  		FIG. 2. Effect of CGRP on systolic blood pressure and modulation by female sex steroid hormones in aged ovariectomized rats. Groups of aged ovariectomized rats were s.c. injected with E2 (12.5 µg/kg BW twice daily), P4 (10 mg/kg BW twice daily) for 4 days in the presence or absence of a continuous s.c. infusion of CGRP (50 µg day/kg BW) through osmotic minipumps. The control group received 0.5 ml sesame oil only. Systolic blood pressure was measured daily for 4 days. Values are means ± SEM of 6 animals per group. *P < 0.05; CGRP + E2, CGRP + P4 versus vehicle

Because blood pressure was not altered by ovariectomy in aged female rats, we next examined the effects of E2 and P4 on the ability of bolus injections of CGRP to lower systolic, diastolic, and mean arterial blood pressures in ovary-intact rats. CGRP significantly (P < 0.05) lowered systolic (Fig. 3A), diastolic (Fig. 3B), and mean arterial blood pressures (Fig. 3C) in a dose-dependent manner beginning at 45 pmol/kg BW with the maximal effects achieved at 360 pmol/kg BW in aged female rats. Decreases in systolic blood pressure (measured in mm Hg) in response to CGRP at the 9 and 45 pmol/kg doses were greater with E2 (-2.2 ± 0.9 mm Hg at 9 pmol; -3.2 ± 1.41 mm Hg at 45 pmol) and P4 (-3.24 ± 1.13 mm Hg at 45 pmol) treatment than in those treated with vehicle (Fig. 3A). Decreases in diastolic blood pressure in response to varying doses of CGRP were significantly (P < 0.05) greater in rats treated with E2 (-3.04 ± 1.54 mm Hg at 9 pmol, -6.83 ± 1.49 mm Hg at 45 pmol, and -14.70 ± 5.22 mm Hg at 180 pmol) and P4 (-7.79 ± 0.96 mm Hg at 45 pmol, -10.92 ± 1.21 mm Hg at 180 pmol, and -22.09 ± 1.93 at 360 pmol) (Fig. 3B). Moreover, treatment with E2 and P4 caused larger decreases in mean arterial blood pressure in response to CGRP at 9, 45, 90, 180, and 360 pmol/kg doses than it did in ovary-intact, aged female rats (Fig. 3C).



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  		FIG. 3. Decrease in systolic (SBP) (A), diastolic (DBP) (B), and mean arterial (MAP) (C) blood pressures in response to an s.c. bolus administration of a vehicle (sesame oil) or varying doses of CGRP in intact female rats 16–18 mo old, E2 (12.5 µg/kg BW twice daily for 4 days), or P4 (10 mg/kg BW twice daily for 4 days) in 0.5 ml of sesame oil. Values are means ± SEM for 6 animals for cumulative doses of CGRP administered in each treatment group. *Significantly different from intact rats within a given dose of CGRP (P < 0.05 by ANOVA)

Further, we extended our studies to examine whether the CGRP-induced mean arterial blood pressure responses were altered in ovary-intact aged rats compared with those in young adult ovariectomized rats. As shown in Figure 4A, the decreases in mean arterial blood pressure in response to CGRP at doses of 45 and 180 pmol/kg were lower in aged rats than they were in young adult rats. We then assessed whether the effects of female sex steroid hormones on the hypotensive role of CGRP are altered with age. In aged rats treated with E2 and P4, the CGRP-induced decreases in blood pressure were substantial, and mean arterial blood pressure values following CGRP treatment in aged rats were similar to those in young adult rats, indicating that these steroid hormone can restore full responsiveness to CGRP regardless of age (Fig. 4, B and C).



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  		FIG. 4. Comparison of varying doses of CGRP-induced hypotensive responses in young adult, nonpregnant ovariectomized (3 mo old) and aged (16–18 mo old) intact female rats treated with steroid hormones or vehicle. Groups of young adult, ovariectomized, or aged-intact rats were treated with either A) sesame oil, B) E2 (12.5 µg/kg BW twice daily s.c.), or C) P4 (10 mg/kg BW twice daily s.c.) for 4 days. Values are means ± SEM for 6 animals per group. *Significantly different from intact young adult, ovariectomized rats within a given dose of CGRP (P < 0.05 by ANOVA)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study we demonstrated that in aged female rats, blood pressure was significantly higher than it was in young adult ovariectomized rats, and that the increase in blood pressure in ovary-intact aged rats was unchanged after ovariectomy. Neither continuous s.c. infusion of CGRP nor female sex steroid hormone treatments alone were able to lower the elevated blood pressure in these aged rats. Continuous infusion of CGRP maintained a lower systolic blood pressure in rats treated with E2 throughout the treatment period. A blood pressure lowering effect of infused CGRP was observed after 2 days of treatment with P4. Acute, systemic bolus administration of CGRP led to lower systolic, diastolic, and mean arterial blood pressures in a dose-dependent manner in ovary-intact aged rats, however, these responses were smaller than they were in young adult rats. The acute hypotensive effects of CGRP are substantially greater after treatment with either E2 or P4 compared with those observed in vehicle treated animals, and these changes were similar in both aged and young adult rats. These results suggest that female sex steroid hormones in aged rats can restore the hypotensive effects of CGRP.

Recent studies demonstrated no differences in mean arterial blood pressure between young adult and aged male and female anesthetized rats [50]. However, studies by Maria et al. [52] showed that in free-moving aged male rats, mean arterial blood pressure was significantly higher than it was in young adults. In the present study, we measured arterial blood pressure when animals were either not anesthetized or fully recovered from anesthesia and in a free moving state ({approx}3 h after surgery). We noticed that both systolic (Fig. 1A) and mean arterial blood pressures were significantly higher in aged female rats than in young adult rats (Fig. 1B). It is possible that the differences in mean arterial blood pressure levels between aged and young adult rats may not become apparent in animals under anesthesia. Our previous studies show that mean arterial blood pressure was significantly higher in adult rats when ovarian hormones are depleted as a result of bilateral ovariectomy [20]. Studies by Packer et al. [53] demonstrated that ovariectomy aggravates SHR, and that E2 and P4 levels are both reported to decrease with age [13]. Collectively, these data suggest that reduced steroid hormone levels in aged rats may contribute to elevated blood pressure in these animals.

Hormone replacement therapy is known to decrease blood pressure in young adult ovariectomized rats [20] and those with SHR [53], and in postmenopausal women [8]. We hypothesized that E2 or P4 supplementation would lower elevated blood pressure in aged female rats, and tested it by performing ovariectomy and then treating the animals either with s.c. hormone injections every day for 4 days. Systolic blood pressures were measured and compared with those of age-matched vehicle treated, ovariectomized rats. As shown in Figure 1A, blood pressures were unchanged in animals treated with either hormone. Further, E2 and P4 administered together were also ineffective in altering blood pressure in aged female rats (data not shown). Similarly, no changes in mean arterial blood pressure were observed in aged rats with steroid hormone treatment given alone (Fig. 1B) or together (data not shown). Recent studies in our laboratory and in those of others [20, 43] have demonstrated that E2 and P4 both enhance the blood pressure lowering effects of CGRP in young adult ovariectomized rats. We therefore hypothesized that steroid hormones may modulate CGRP effects on the cardiovascular system in aged female rats as well. Results from this study show that CGRP and steroid hormones are both required to lower blood pressure in aged rats (Fig. 2) and that neither treatment alone is effective. However, we do not know why P4 exhibits delayed effects as opposed to the rapid effect of E2 in the present study.

We next examined whether acute systemic administration of CGRP to these hormone-treated rats alters systolic, diastolic, and mean arterial blood pressures in a dose-dependent manner. Because no differences in blood pressure values were observed between ovary-intact rats at diestrus and ovariectomized aged rats, we performed further studies with ovary-intact aged rats. Data in Figure 3 show that CGRP produced dose-dependent decreases in systolic (Fig. 3A), diastolic (Fig. 3B), and mean arterial (Fig. 3C) blood pressures in ovary-intact aged rats. Further, in the present study we show that diastolic blood pressure but not systolic blood pressure drops significantly in response to an acute bolus injection of CGRP in aged hormone-treated rats. These data suggest that the effects of CGRP on blood pressure appeared to be mediated through peripheral vascular dilation in aged female rats and that this may be modulated by female sex steroid hormones. Furthermore, we speculate that lower concentrations of CGRP could be effective in lowering blood pressure in the presence of steroid hormones in aged rats.

Our present studies demonstrate that the hypotensive effects of CGRP are impaired in aged female rats, but they are not impaired in young adult ovariectomized rats (Fig. 4A). Chan et al. [50] observed similar age-related changes in the blood pressure lowering effects of CGRP in both male and female rats. Our results further show that treatment with E2 or P4 completely restored the CGRP-induced hypotensive effects in aged and young adult ovariectomized rats (Fig. 4, B and C). Several studies suggest that the vasodilatory effects of CGRP are mediated by specific CGRP receptors and do not involve adenoreceptors, nitric oxide, or prostanoids [23, 54]. Moreover, depending on the type of blood vessel, CGRP exhibits endothelium-dependent [33, 34] and endothelium-independent [35–38] vascular relaxations. We showed in previous studies that P4 modulated the vasodilator effects of CGRP in the presence of NG-nitro-L-arginine methyl ester (L-NAME, an inhibitor of nitric oxide), indicating the involvement of a nitric oxide-independent pathway. Studies in the SHR model indicate that endothelium-dependent vasodilator response to CGRP is lower in hypertensive animals [55]. Studies by Tschudi et al. [56] demonstrated that the level of nitric oxide released from endothelial cells may decrease with aging. These studies suggest that endothelium-dependent mechanisms may be impaired when sex steroid hormone levels are low in aged female rats. We speculate that E2 treatment would elevate endothelium-dependent vascular-relaxation effects of CGRP, perhaps by stimulating nitric oxide or prostacyclin, or directly by stimulating CGRP receptor levels in vascular tissues. On the other hand, P4 may modulate the vasodilator effects of CGRP through endothelium-independent mechanisms [57], including CGRP receptor levels, and greater sensitivity of the CGRP effector system in smooth muscle cells in aged female rats. Additional studies are required to address these mechanisms.

CGRP synthesis in DRG is modulated by nerve growth factor through its receptor, TrkA [58–60]. Recent studies in our laboratory have demonstrated that female sex steroid hormones stimulate TrkA receptors in DRG of young adult ovariectomized rats [61] and that prolonged depletion of these hormones (ovariectomy) decreases TrkA receptor levels in these animals. Studies in aged male rats demonstrated that TrkA receptors are lower in DRG neurons [62, 63]. Therefore, it is possible that decreases in steroid hormone levels in aged rats may reduce TrkA receptors and thus decrease CGRP synthesis in DRG, and therefore result in elevated blood pressure. These data suggest that hormone replacement therapy in postmenopausal women could increase TrkA receptor expression in DRG and may increase CGRP synthesis or its release, and thus help regulate blood pressure. In the present study we have shown that steroid hormone supplementation also increases the vasodilator effects of CGRP in these rats. Thus, we suggest that regardless of the mechanisms involved, steroid hormones enhance CGRP-related vasodilator effects in aged animals.

In summary, this study suggests that the hypotensive effects of CGRP are substantially elevated with the treatment of female sex steroid hormones in aged female rats. Blood pressure-lowering effects of CGRP are more impaired in elderly female rats than they are in adult ovariectomized rats. Furthermore, steroid hormones restored the vasodilator sensitivity effects of CGRP regardless of age. Therefore, we suggest that female sex steroid hormones may regulate arterial blood pressure through the CGRP system in aged female rats, and that decreases in these hormones concomitant with aging appears to damage or down-regulate the vascular responses to CGRP.


   ACKNOWLEDGMENTS
 
We thank Kimberly Mitchell for typing the manuscript.


   FOOTNOTES
 
1 P.R.R.G. was supported by grant AG-18999-01 from the National Institute of Aging and by the Sealy Center for Aging at the University of Texas Medical Branch, Galveston, Texas. C.Y. was supported by grant HL-58144 from the National Heart, Lung, and Blood Institute, and by grant HD-40828 from the National Institute on Child Health and Human Development. Back

2 Correspondence: Chandrasekhar Yallampalli, Department of Obstetrics and Gynecology, University of Texas Medical Branch, 301 University Boulevard, Medical Research Building, Room 11.138, Galveston, TX 77555-1062; FAX: 409 747 0475; chyallam{at}utmb.edu Back

Received: 23 May 2002.

First decision: 13 June 2002.

Accepted: 1 July 2002.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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