INTRODUCTION

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CARDIOVASCULAR DISEASE-RELATED morbidity and mortality are lower in women than in men throughout middle age (10, 19). During and after menopause, this "risk advantage" can be maintained with estrogen administration (19). The mechanism for these gender differences is usually associated with estrogen; however, there are data to support a role for androgens in the differing disease development between men and women (22). One important estrogen-related mechanism involved in the lowering of cardiovascular disease risk among women is sodium and water regulation (7, 20). However, both estrogen and testosterone receptors are present in the hypothalamus of adult primates (16); therefore, both may alter control of the cardiovascular, fluid, and sodium regulation systems in the hypothalamus (24, 25, 29, 30).

Estrogen and testosterone modulate the synthesis of arginine vasopressin (AVP) in the paraventricular and supraoptice nuclei in rats (24, 25) as well as the osmotic regulation of AVP release by the pituitary in humans (26, 28). Previous studies have found greater 24-h urinary AVP (7) and greater resting and exercise plasma AVP concentration (P_[AVP]) (27) in men compared with women. Unfortunately, it is difficult to determine whether these differences are due to estrogen or testosterone effects on osmotic regulation of AVP. Moreover, no human studies have specifically addressed gender differences or how estrogen, progesterone, or testosterone may interact to alter AVP release to impact the cardiovascular, water, and sodium regulation systems.

Human studies to determine the mechanisms for sex differences in body fluid regulation are crucial to developing effective treatments for conditions such as hypertension and cardiovascular and renal disease that are equally efficacious for men and women. The purpose of this study was to determine gender differences in the P_[AVP] responses to hypertonic saline infusion (HSI) by comparing men with women in the early follicular menstrual phase (i.e., low estrogen-low progesterone) and then again in the midluteal menstrual phase (i.e., high estrogen-high progesterone). We hypothesized that greater estrogen and progesterone would enhance the AVP response to osmotic stimulation. Therefore, men would only differ from women in the midluteal phase, thereby implicating estrogen as the primary mechanism for changes in osmotic regulation of AVP. To determine the functional consequences of changes in plasma osmolality regulation of AVP, we also examined sex differences in body water and in the cardiovascular system.

	METHODS

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Study Design

Infusion Studies

After these control samples were obtained, hypertonic (3.0% NaCl) saline was infused at a rate of 0.1 ml · kg body wt¹ · min¹ for 120 min through one of the catheters. Blood was sampled at 10, 20, 30, 40, 50, 60, 75, 105, and 120 min during the infusion from the other catheter, and a urine sample was obtained at the end of the infusion. After a 30-min seated recovery period, the subject drank water (15 ml/kg body wt) during the next 30 min. Thirty minutes after the drinking period was completed, plasma volume was determined with the Evans blue dye procedure (see Blood Volume below). Blood samples were obtained at 30, 60, and 120 min after infusion, and urine was collected at 60 and 120 min after infusion. The subjects were weighed at the end of the infusion period, after the drinking period, and again at the end of the protocol.

All blood samples were analyzed for hematocrit (Hct), hemoglobin concentration ([Hb]), plasma total protein concentration, plasma osmolality, plasma creatinine concentrations, P_[AVP], plasma cortisol concentrations (P_[Cort]), and serum concentrations of sodium (S_[Na]) and potassium (S_[K]). Blood samples at baseline, at the end of the infusion, and at 60 and 120 min after the infusion were also analyzed for the plasma concentrations of atrial natriuretic peptide (P_[ANP]), aldosterone (P_[Ald]), and plasma renin activity (PRA). Baseline blood samples from the women were also analyzed for 17-estradiol and progesterone. Urine was analyzed for volume, osmolality, sodium, potassium, and creatinine concentrations.

Blood samples were separated immediately into aliquots. The first was analyzed for [Hb] and Hct. A second aliquot was transferred to a heparinized tube, and a third aliquot for the determination of S_[Na] and S_[K] was placed into a tube without anticoagulant. All other aliquots were placed in tubes containing EDTA. The tubes were centrifuged, and the plasma was taken off the heparinized sample and analyzed for sodium, potassium, osmolality, creatinine, and aldosterone. The EDTA samples were analyzed for concentrations of AVP, cortisol, ANP, and PRA.

Blood Analysis

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Blood Volume

Calculations

We used the pre-HSI [Hb] and Hct to estimate the baseline plasma volume from the Evans blue dye measurement at the end of the experiment.

Fractional excretions of water (FE_H2O) and sodium (FE_Na) were calculated from the following equations


where the subscript f is glomerular filtrate, U_v is urine flow rate, GFR is the glomerular filtration rate, U_[Na] is the urine excretion of Na concentration, and S_[Na] is in protein-free solution (meq/kgH₂O). Glomerular filtration rate was estimated from creatinine clearance.

Data Analysis

Statistics. The variables over time were analyzed by conditions (menstrual phase) and group (women vs. men) using ANOVA for repeated measures. We used separate ANOVA models to test differences between the men and women for each menstrual phase and to test the women within the menstrual phases. When significant differences were found, orthogonal contrasts tested differences between specific means related to the hypothesis of interest. Data are expressed as means ± SE. Differences were considered statistically significant at P < 0.05.

Sample size calculation. Expected P_[AVP] responses within and between groups are derived from data from our laboratory during HSI (26). In an earlier study, during HSI, P_[AVP]-plasma osmolality threshold decreased by 6 mosmol/kgH₂O with estrogen treatment. An estimate of the pooled standard deviation for the group was 3 mosmol/kgH₂O.

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	RESULTS

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Baseline (pre-HSI)

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Plasma osmolality (POsm), plasma concentration of arginine vasopressin (P[AVP]), and percent change in plasma volume at rest, in response to hypertonic saline infusion and recovery in the follicular and midluteal menstrual phases in women, and in men. *Different from the follicular phase. §Different from the midluteal phase. #Different from the men. Differences were accepted as significant at P < 0.05. Data are means ± SE.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Plasma renin activity (PRA), plasma aldosterone (P[Ald]), plasma atrial natriuretic peptide (P[ANP]), and plasma cortisol (P[Cort]) concentrations at rest, in response to hypertonic saline infusion and recovery in the follicular and midluteal menstrual phases in women, and in men. *Different from the follicular phase. §Different from the midluteal phase. #Different from the men. Differences were accepted as significant at P < 0.05. Data are means ± SE.

Baseline PRA and P_[Ald] were higher in the midluteal phase compared with the follicular phase within women and compared with the men (Fig. 2, P < 0.05). Urine flow, osmolality and sodium excretion (Fig. 3), and free water and osmotic clearances were unaffected by menstrual phase or gender, indicating that baseline hydration levels were consistent between the menstrual phases and between the men and women (Table 3). Basal heart rate was unaffected by menstrual phase or gender, but systolic, diastolic, and mean arterial pressures were greater in the men than in the women in both menstrual phases (Table 4, P < 0.05).

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Renal Na+ and cumulative Na+ excretion at rest corrected for body weight (BW) during hypertonic saline infusion and recovery in the follicular and midluteal phases in women and in men. *Different from the follicular phase. §Different from midluteal phase. Differences were accepted as significant at P < 0.05. Data are means ± SE.

Hypertonic Saline Infusion

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Mean P[AVP] responses to increases in POsm during hypertonic saline infusion in the follicular and midluteal phases in women and in men. Data are means ± SE.

P_[Cort] fell steadily during the infusion in the women in both menstrual phases but began to increase in the men at 75 min of infusion and continued to increase through the end of the infusion (Fig. 2, P < 0.05). PRA and P_[Ald] decreased during HSI in the men and the women in both menstrual phases, although P_[Ald] was greater in the midluteal phase than in the follicular phase within women (Fig. 2, P < 0.05). Although renal sodium excretion increased during HSI in the men and the women in both menstrual phases, the magnitude of increase was lower in the men compared with the women (Fig. 3 and Table 3, P < 0.05). Heart rate and blood pressure were unaffected by HSI in the women, but systolic and pulse pressures increased by the end of the infusion in the men (Table 4, P < 0.05).

Recovery

During recovery, renal function, electrolyte excretion (Fig. 3 and Table 3), and overall fluid balance (i.e., fluid intake  urine output) were not affected by phase of the menstrual cycle or by gender. Due to the greater sodium retention during the infusion in the men, cumulative sodium excretion was lower in men than in women by the end of recovery (Fig. 3, P < 0.05). Throughout recovery, blood pressure remained greater in men compared with women, and systolic blood pressure and pulse pressure were greater during recovery in men compared with their own baseline values (Table 4, P < 0.05).

	DISCUSSION

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The greater P_[AVP]-plasma osmolality slope observed in men indicated greater P_[AVP] sensitivity to changes in plasma osmolality during HSI compared with women in both phases of the menstrual cycle. This greater sensitivity occurred without differences in free water clearance, thereby suggesting lower renal sensitivity to AVP in men. In contrast, within women, the osmotic threshold was shifted to a lower plasma osmolality in the midluteal phase relative to the follicular phase of the menstrual cycle, although without a change in the P_[AVP]-plasma osmolality slope. As with the men, this shift in the osmotic threshold for AVP release occurred with no change in free water clearance or body fluid balance, suggesting a change in the osmotic operating point for fluid regulation over the menstrual cycle, most likely due to estrogen actions on AVP release (28). HSI increased P_[Cort] in men but not in women in either menstrual phase. The mechanism for the P_[Cort] response is not apparent from our data but may be due to an interaction between AVP and corticotropin-releasing hormone (CRH) in the hypothalamus that is unique to men or perhaps due to greater stress responses to the high levels of thirst induced by HSI. Finally, there were no changes in blood pressure or sodium excretion observed between the menstrual phases in women. However, HSI lowered sodium excretion and increased blood pressure in men, suggesting a hypertensive shift in the pressure natriuresis curve in men, presumably related to testosterone.

A primary difference between men and women was the greater P_[AVP]-plasma osmolality slope in men, indicating greater sensitivity of systems regulating AVP synthesis and/or release in response to changes in plasma osmolality. There was no difference in osmotic AVP sensitivity between the two menstrual phases, suggesting that androgens are a primary factor in this shift. Our study does not conclusively demonstrate that testosterone is the primary cause for the sex differences in P_[AVP]-plasma osmolality slope; however, the hypothalamus and pituitary have binding sites for testosterone (24) and testosterone has effects independent of estrogen on hypothalamic AVP stimulation in rats (8). In women, the P_[AVP]-plasma osmolality slope during dehydrating exercise is unaffected by menstrual phase or by administration of oral contraceptives containing estrogen (30). In the present study, we also found a shift to a lower osmotic threshold for AVP release in the midluteal phase within women but no difference due to gender, suggesting estrogen is the primary cause of the change in osmotic threshold but has little effect on osmotic AVP sensitivity.

Our data do not explain why estrogen and testosterone would modulate osmotic AVP regulation differently, but both estrogen and testosterone are probably having their effects within the central nervous system. Estrogen and androgens cross the blood-brain barrier, and vasopressin-producing cells in the paraventricular and supraoptic nuclei have binding sites for both testosterone and estrogen that affect osmotic regulation (1, 8, 24, 25), thereby affecting synthesis and release of AVP. Estrogen and androgens may also modulate hypothalamic AVP release indirectly through catecholaminergic neurons that bind estrogen and testosterone and project to the paraventricular and supraoptic nuclei (18) and by sex steroid modulation of cholinergic and angiotensinergic innervation of vasopressinergic cells in the paraventricular and supraoptic nuclei (29).

Another mechanism involved in the sex differences in osmotic AVP regulation may be changes in plasma volume. The volume expansion produced by HSI should have increased central venous pressure by 2.2 mmHg (15), which is a sufficient stimulus to load cardiopulmonary baroreceptors. Loading cardiopulmonary baroreceptors in itself can provide a countersignal concerning fluid status to that of the elevated plasma osmolality. In this way, cardiopulmonary baroreceptor loading can modulate the AVP response to the increased plasma osmolality. Thus a lower cardiopulmonary baroreceptor sensitivity in men relative to women would make them less sensitive to the inhibitory stimulus of plasma volume expansion and is a possible mechanism for the greater P_[AVP]-plasma osmolality slope in the men during HSI. Watenpaugh et al. (34), however, recently demonstrated that men and women have similar cardiovascular and renal responses to water immersion in which the primary stimulus is plasma volume expansion and cardiopulmonary baroreceptor loading.

Another peripheral mechanism that might have contributed to the greater P_[AVP]-plasma osmolality slope in the men could be the increase in pulse pressure during HSI, which would reflect an increase in stroke volume or a reduction in arterial compliance. An increase in stroke volume seems an unlikely mechanism because greater stroke volume increases would have attenuated, rather than augmented, the P_[AVP]-plasma osmolality slope in the men through countereffects on cardiopulmonary baroreceptors. However, a sodium load and/or vasopressin secretion can reduce arterial compliance in males. For example, in rats, the pressor response to vasopressin secretion is greater in males vs. females due to reduced total peripheral resistance in females (32), which is mediated by estrogen (31). Thus, in our study, greater arterial resistance during HSI may have augmented the pulse pressure responses in the men, which in turn increased the P_[AVP]-plasma osmolality slope during HSI.

The increase in systolic blood pressure in the men during HSI may be due to an upward shift in the pressure-natriuresis curve (22). Sodium retention was greater in the men vs. the women, which is consistent with this upward shift, already reported in spontaneously hypertensive male vs. female rats (22) and in ovariectomized female rats given testosterone (5). Moreover, there is evidence that estrogen modifies the renin-angiotensin system (21), and recent evidence in transgenic hypertensive mice indicates that estrogen may act to reduce vasoconstriction and hypertension by shifting the vasoconstrictor-vasodilation balance in the kidney (3). Finally, androgens may have direct effects on proximal sodium reabsorption in the kidney, although evidence that androgen receptors exist in the proximal tubules is still preliminary (22).

Despite the greater P_[AVP]-plasma osmolality slope in the men, the fall in free water clearance due to the osmotic load was unaffected by gender, suggesting greater renal sensitivity to AVP in women. This is consistent with data in postmenopausal women given estrogen, in whom water retention and renal concentrating response (urine osmolality/plasma osmolality) during HSI increased despite similar P_[AVP], suggesting that estrogen enhances renal free water retention (26). On the other hand, more recent data suggest lower renal sensitivity to changes in P_[AVP] in response to a water load in young women (4). Moreover, combined estrogen and progesterone administration to young women reduced the free water clearance response to increases in P_[AVP] during exercise-induced dehydration (28).

Basal P_[Cort] was similar between men and women, regardless of menstrual phase; however, P_[Cort] increased only in men during HSI. Both estrogen (11) and androgens (11) have some control over the hypothalamic synthesis and release of CRH. CRH, released by the paraventricular nucleus, is the first component of the hypothalamic-pituitary-adrenal-axis system that ultimately leads to the release of cortisol. Modulation of AVP release by the posterior pituitary is a part of the negative feedback control of ACTH secretion (23) that is modified by testosterone (2) and estrogen (16). Thus the greater P_[Cort] in men during HSI may be related to greater AVP release by the posterior pituitary.

In addition to the hypothalamic-pituitary-adrenal-axis system control, P_[Cort] can be an indication of physiological or psychological stress, so the greater P_[Cort] may be a stress response to HSI unique to the men. Men and women generally have similar basal and stress-induced blood cortisol response (9), although cortisol metabolism is lower in women (12, 33), supporting a greater P_[Cort] response in the women. Thus a greater stress response in the men to HSI is an unlikely explanation for the differences in P_[Cort] response to HSI.

Our data indicate a greater sensitivity in the P_[AVP] response to changes in plasma osmolality as the primary difference between men and women during HSI. In men, this greater sensitivity was associated with an increase in systolic blood and pulse pressures and P_[Cort] during HSI and thus may play a role in the greater cardiovascular morbidity in men compared with women. The greater blood pressure and sodium retention observed in men may also indicate a shift in the pressure-natriuresis curve, which may be due to differential sensitivity to the renin-angtiotensin system or related to direct effects on the kidney. Finally, this greater AVP sensitivity to osmotic stimulation may have also led to increases in cortisol and blood pressure during the infusion, although the functional significance of the greater adrenal stimulation is not apparent from our data.

Perspectives