Introduction

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Epidemiological studies have established the role of female gender as a protective factor in the development of various cardiovascular diseases, including atherosclerosis and hypertension (Drizd et al., 1986; Barrett-Connor and Bush, 1991). It has been suggested that one of the mechanisms underlying this "cardioprotection" of females is enhanced vascular production of nitric oxide (NO). In women, NO-dependent vasodilation to the endothelium-dependent dilator acetylcholine (ACh) is greater than in men (Celermajer et al., 1994; Algotsson et al., 1995). The aorta, as well as coronary, cerebral, and skeletal muscle arteries from female rats appear to produce a greater amount of NO compared with those from males (Kauser and Rubanyi, 1994; Wellman et al., 1996; Huang et al., 1997, 1998; Rahimian et al., 1997; Skarsgard et al., 1997). The importance of this enhanced NO production in females has not been fully elucidated, but NO is known to inhibit platelet aggregation and adhesion (Radmoski et al., 1990), prevent neointimal plaque progression, and mediate vasodilation. Thus, an enhanced function of the NO system in females may contribute to a lower incidence of vascular disease.

Endothelial cell production of NO is regulated by a variety of factors, including nitric oxide synthase (NOS) substrate levels (i.e., arginine), cofactors (tetrahydrobiopterin, NADPH), and intracellular calcium concentrations. It has been demonstrated that the agonist-induced release of endothelium-derived relaxing factor (presumably NO) is, in part, dependent on activation of endothelial calcium-activated potassium (K_Ca) channels (Demirel et al., 1994). The resulting hyperpolarization is thought to provide an electrochemical gradient that favors calcium influx, resulting in elevated cytoplasmic calcium levels that stimulate NO release and subsequent relaxation. To date, no study has addressed the contribution of endothelial potassium channels to gender differences in NO-mediated, endothelium-dependent vasodilation. Thus, the purpose of the current study was to pharmacologically determine: 1) the extent to which NO contributes to ACh-induced vasodilation in arteries from male and female rats and 2) if gender differences in endothelial potassium channel activity mediate differences in NO-mediated vascular relaxation.

	Materials and Methods

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Animals

Sexually mature male and female Sprague-Dawley rats (13 weeks of age) were used in these studies (Taconic Farms, Inc., Germantown, NY). All rats were housed in groups of two or three in temperature-controlled, light-cycled (6:00 AM-5:00 PM) quarters with ad libitum access to standard rat chow (Ralston Purina, St. Louis, MO) and water. Stage of the estrous cycle was determined by daily vaginal lavage, and approximately equal numbers of females from each stage were used in the experimental groups. All procedures involving the use of animals were approved by the Institutional Animal Care and Use Committee and conform to federal, state, and institutional guidelines.

Preparation of Isolated Arteries

After at least one or two full estrous cycles in females, or at least 1 week of institutional housing for males, rats were weighed and then euthanized with sodium pentobarbital (120 mg/kg i.p). Rats were sacrificed between 8:00 AM and 9:00 AM. A segment of small intestine was removed and placed in cold physiological salt solution (PSS) of the following composition: 130 mM NaCl, 4.7 mM KCl, 1.17 mM MgSO₄(7H₂O), 1.18 mM KH₂PO₄, 14.9 mM NaHCO₃, 5.5 mM dextrose, 0.03 mM NaCa₂ EDTA, and 1.6 mM CaCl₂(2H₂O). Under a dissecting microscope, two third- or fourth-order branches of the superior mesenteric artery were dissected free of fat and adhering connective tissue. Each artery was placed in a single chamber of a dual-chamber pressure arteriograph (Living Systems Instrumentation, Burlington, VT), and cannulated at one end with a glass microcannula. The lumen was gently flushed with PSS to remove any blood and the distal end of the artery was then cannulated. Arteries were secured to the cannulae with silk sutures. The vessels were pressurized to 60 mm Hg and pressure was controlled with an automatic servo device (Living Systems Instrumentation). There was no flow through the lumen of the vessels.

Each artery was bathed with warmed (37°C), gassed (95% O₂/5% CO₂) PSS, pH 7.35, at a flow rate of 20 ml/min. All vessels were allowed to equilibrate for 30 to 60 min before the beginning of experiments. The arteriograph chamber was placed on the stage of an inverted microscope, and vessel outer diameter was continuously monitored via computer image analysis consisting of a Framegrabber card (PCVision Plus) and appropriate software (Microscience, Seattle, WA). After equilibration, the arteries were stimulated with 10⁶ M phenylephrine. Once the contraction reached a stable level, ACh (10⁵ M) was applied to determine viability of the endothelium. Resting vessel diameters were as follows: males, 246 ± 14 µm (n = 21); females, 260 ± 10 µm (n = 37).

Vascular Reactivity Studies

Pharmacologic Analysis of ACh-Induced Relaxation. In each artery, a concentration-response curve to the -adrenergic agonist phenylephrine was performed (1.0 × 10⁹-1.0 × 10⁵ M). From this curve, the concentration of phenylephrine causing 80% of the maximal constriction (EC₈₀) was determined. The vessels were then rinsed with fresh PSS and allowed to re-equilibrate for 20 to 30 min. The arteries were then constricted with the EC₈₀ concentration of phenylephrine and allowed to reach a plateau. A cumulative concentration-response curve to ACh was then performed (1.0 × 10⁹-1.0 × 10⁶ M). In some experiments, the artery was pretreated with one of the following for 20 min before the start of the ACh concentration-response curve: 1) N-nitro-L-arginine (LNA; 100 µM), an inhibitor of NOS; 2) apamin (30 nM), an inhibitor of the small conductance K_Ca channel; 3) a combination of LNA and apamin; and 4) indomethacin (INDO; 10 µM), a cyclooxygenase inhibitor. In addition, some arteries were constricted with phenylephrine in the presence of 40 mM KCl (isoosmotic substitution for NaCl in the buffer), and ACh concentration-response curves were performed. In the presence of elevated potassium, the concentration of phenylephrine was reduced as necessary to maintain the same degree of vasoconstriction as obtained at the phenylephrine EC₈₀ in the absence of high potassium.

Relaxation to Spermine-NO complex (SPERNO). Arteries were preconstricted with the EC₈₀ concentration of phenylephrine as determined above and were preincubated with 100 µM LNA to eliminate endogenous NO production. Concentration-response curves to SPERNO (1.0 × 10⁸-1.0 × 10⁵ M) were performed in the absence and presence of the K_Ca channel inhibitor apamin (30 nM). At the conclusion of all concentration-response curves, arteries were rinsed with calcium-free PSS containing 1 mM EGTA to determine diameter at maximal relaxation.

Source of Materials

Phenylephrine, ACh, LNA, INDO, and apamin were obtained from Sigma Chemical Co. (St. Louis, MO). SPERNO was obtained from Research Biochemicals (Natick, MA).

Statistical Analysis

All values are expressed as means ± S.E. Dilator responses were expressed as the percentage of the phenylephrine-induced constriction remaining with the following formula: % of phenylephrine constriction remaining = [(D_max  D_conc)/(D_max  D_PE)] × 100, where D_max = diameter in calcium-free PSS, D_conc = diameter at a given concentration of vasodilator, and D_PE = diameter after phenylephrine constriction. Thus, all diameters were normalized to the diameter at full relaxation induced by the calcium-free PSS.

Relaxations between groups were compared with a repeated-measures two-way ANOVA (factor 1, gender; factor 2, presence or absence of each inhibitor). When the F values were significant, individual comparisons among groups were made with the Newman-Keuls test. The EC₅₀ (the concentration of agonist that produced 50% of the maximal response) was calculated with nonlinear regression of the sigmoidal dose-response curve (GraphPad PRISM, version 2.01). The negative logs of the EC₅₀ values were compared with either Student's t test or ANOVA. A P value of <.05 was considered statistically significant.

	Results

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Relaxation to ACh in mesenteric arteries from male and female rats is shown in Fig. 1. Over the concentration range of 4.0 × 10⁸ to 2.5 × 10⁷ M, relaxation in arteries from females was significantly greater than that in males. Sensitivity, as measured by the EC₅₀ value, was significantly greater in arteries from females compared with males (log EC₅₀: male = 6.74 ± 0.06; female = 6.96 ± 0.06; P = .037). Maximal relaxation to ACh in arteries from male and female rats was not significantly different.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Line graph represents ACh concentration-response curves in mesenteric arteries from male (, ) and female (, ) rats in the absence (, ) or presence (, ) of LNA (100 µM). Arteries were preconstricted with an EC80 concentration of phenylephrine. Relaxation in arteries from females was significantly greater than that in males at the concentrations indicated by an asterisk. LNA significantly inhibited relaxation and eliminated the gender difference over the concentration range of 4.0 × 108 to 4.0 × 107 M. In the presence of LNA, relaxation in females remained significantly greater than that in males at the concentrations indicated by a double asterisks. Numbers in parentheses represent n values.

The NOS inhibitor LNA significantly blunted relaxation to ACh in arteries from both male and female rats (Fig. 1). Over the ACh concentration range of 4.0 × 10⁸ to 4.0 × 10⁷ M, LNA eliminated the difference in relaxation between male and female rats. In the presence of LNA, relaxation at the two highest concentrations of ACh examined was significantly greater in arteries from females compared with males (percentage of phenylephrine constriction remaining at 1 µM ACh in the presence of LNA was 62 ± 9% in males and 44 ± 6% in females; P < .05; Fig. 1). Thus, over the ACh concentration range in which females demonstrate enhanced relaxation compared with males, LNA eliminated the gender difference in relaxation. However, at the higher end of the ACh concentration range studied, a non-LNA-sensitive mechanism of relaxation appears to be enhanced in females.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Line graph represents ACh concentration-response curves in mesenteric arteries from male (, ) and female (, ) rats in the presence (, ) or absence (, ) of apamin (30 nM). Numbers in parentheses represent n values. Arteries were preconstricted with an EC80 concentration of phenylephrine. Apamin significantly inhibited relaxation and eliminated the gender difference over the entire concentration range examined.

The small conductance K_Ca channel inhibitor apamin inhibited relaxation to ACh in arteries from both males and females over the entire ACh concentration range tested (Fig. 2). Similar to LNA, apamin eliminated the gender difference in ACh vasodilation over the range of concentrations in which relaxation is greater in females (4.0 × 10⁸-2.5 × 10⁷ M). The extent to which apamin and LNA inhibited ACh relaxation was not significantly different over this range. In contrast to LNA, apamin inhibited relaxation to maximal concentrations of ACh equally in both males and females. In arteries from males, the effects of apamin alone and LNA alone on ACh responses were similar. However, in arteries from females, at the maximal ACh concentrations tested (2.5 × 10⁷-1.0 × 10⁶ M), the inhibitory effect of apamin was greater than that of LNA.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Line graph represents ACh concentration-response curves in mesenteric arteries from male and female rats in the presence or absence of LNA (100 µM) and apamin (30 nM). Numbers in parentheses represent n values. Arteries were preconstricted with an EC80 concentration of phenylephrine. The combination of LNA and apamin significantly inhibited relaxation and eliminated the gender difference over the entire concentration range examined.

To determine whether the inhibition of ACh-induced relaxation by LNA and apamin was additive, experiments using both inhibitors combined were performed, as shown in Fig. 3. The combination of apamin and LNA significantly inhibited relaxation to ACh in both males and females. Over the concentration range of 4.0 × 10⁸ to 2.5 × 10⁷ M, the extent of inhibition with the combination of LNA and apamin was similar to that found with either inhibitor alone. Thus, the effects of apamin and LNA were not additive over the ACh concentration range in which relaxation is greater in females. Similar to the results with apamin alone, there was no gender difference in maximal relaxation after the combination of LNA and apamin.

Figure 4 summarizes the effects of LNA alone, apamin alone, and the combination of LNA + apamin on the vasodilator response to the maximal concentration of ACh used in our experiments (1 µM). The bar graph shows that in males, the percentage of inhibition of the response to 1 µM ACh was roughly equivalent across the three interventions (LNA, 52%; apamin, 60%; apamin + LNA, 63%). In contrast, in females the percentage of inhibition produced by apamin was twice that produced by LNA (LNA, 35%; apamin, 74%; apamin + LNA, 54%).

View larger version (58K): [in this window] [in a new window]   Fig. 4.   Bar graph represents a summary of the average percentage of inhibition of the vasodilator response to 106 M ACh produced by LNA, apamin, and the combination of apamin + LNA in arteries from females () and males (). These data are derived from the full concentration-response curves are found in Figs. 1, 2, and 3. At 106 M ACh, LNA had a larger inhibitory effect in the males (see Fig. 1 for statistical comparison), whereas the effects of apamin and apamin + LNA were similar in males and females (see Figs. 2 and 3 for statistical comparisons).

The effect of INDO (10 µM) on ACh concentration-response curves in male and female rats is shown in Fig. 5. In the presence of INDO, ACh curves were shifted significantly to the right in arteries from both males (P = .003) and females (P = .01, two-way repeated-measures ANOVA). The gender difference in ACh relaxation persisted in the presence of INDO (Fig. 5). The maximum response to ACh was not altered by INDO in arteries from either sex. Preconstriction of arteries with phenylephrine in the presence of 40 mM KCl totally abolished the vasodilator response to ACh in arteries from both males and females (data not shown). In preliminary experiments, we found that this concentration of KCl had a slight effect to decrease sensitivity to the NO donor SPERNO, but did not alter the maximal vasodilation to this agent. Thus, under the conditions of our experiments, 40 mM KCl appears to be relatively selective in blocking a non-NO-mediated response.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Line graph represents ACh concentration-response curves in mesenteric arteries from male (, ) and female (, ) rats in the presence (, ) or absence (, ) of INDO (10 µM). Numbers in parentheses represent n values. Arteries were preconstricted with an EC80 concentration of phenylephrine. INDO decreased the sensitivity to ACh in arteries from males and females, but did not eliminate the gender difference in vasodilator response.

There was no gender difference in sensitivity to relaxation with the NO donor SPERNO (Fig. 6; log EC₅₀, male = 5.44 ± 0.14 and female = 5.58 ± 0.13; P = .28; ANOVA and Newman-Keuls test). Apamin did not inhibit relaxation to SPERNO in arteries from either males or females (Fig. 6, log EC₅₀, male + apamin = 5.25 ± 0.07 and female + apamin = 5.33 ± 0.14). These data suggest that smooth muscle apamin-sensitive K_Ca channels are not involved in the vasodilator effects of NO.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Line graph represents SPERNO concentration-response curves in mesenteric arteries from male (, ) and female (, ) rats in the presence (, ) or absence (, ) of apamin (30 nM). Numbers in parentheses represent n values. Arteries were preconstricted with an EC80 concentration of phenylephrine. All concentration-response curves were performed in the presence of LNA (100 µM) to eliminate endogenous NO production. There was no difference in relaxation between males and females. Apamin did not significantly alter the relaxation to SPERNO in either males or females.

	Discussion

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This study was designed to explore the mechanisms of the gender difference in vasodilation to the endothelium-dependent vasodilator ACh. Our results confirm those of several other studies that demonstrate enhanced endothelium-dependent relaxation in females compared with males. In women, infusion of ACh into the forearm circulation leads to a greater vasodilation compared with that observed in males (Celermajer et al., 1994; Algotsson et al., 1995). Similarly, in the rat, ACh-induced vascular relaxation is greater in females (Rahimian et al., 1997), and may, in part, be due to a greater basal NO release (Wellman et al., 1996; Huang et al., 1997). Our data further contribute to the understanding of sexual dimorphism in artery function by providing evidence of enhanced agonist-stimulated NO and endothelium-derived hyperpolarizing factor (EDHF) release from the endothelium of arteries from female rats compared with those from males.

Inhibition of NOS with LNA eliminated the gender difference in ACh-induced relaxation over the midrange concentrations at which females exhibit enhanced relaxation compared with males. These data suggest that the contribution of NO to ACh-induced vasodilation is greater in females than in males and contributes to the gender difference in dilation to this agonist. We also found that the maximal relaxation to ACh, although not different between males and females, appears to be mediated by different endothelial factors. We concluded this based on the findings that: 1) in the presence of LNA, maximal relaxation to ACh was greater in arteries from females compared with males, 2) apamin eliminated this gender difference in maximal relaxation, and 3) INDO did not effect the maximal response to ACh. Consistent with other reports in the literature (Garland and McPherson, 1992; Hwa et al., 1994; Shimokawa et al., 1996; McCulloch et al., 1997), our results indicate that mesenteric arteries from both males and females elaborate a non-NO, noncyclooxygenase EDHF in response to ACh. This EDHF appears to act, at least in part, on small conductance K_Ca channels that are blocked by apamin (Murphy and Brayden, 1995; Chen and Cheung, 1997). Our experiments demonstrating that 40 mM KCl abolishes the relaxation to ACh in males and females further emphasize the importance of potassium channel activation in the vasodilator response to ACh, and also implicate EDHF as a mechanism of action of ACh. Our findings of a non-NO, noncyclooxygenase component of ACH vasodilation that is greater in females suggest that differences in production or action of EDHF also may contribute to gender differences in vasodilation.

Potential mechanisms for enhanced NO contribution to vasodilation in females include: 1) increased release of NO from endothelium of females, or 2) increased sensitivity of smooth muscle from females to NO. To assess smooth muscle sensitivity to NO in arteries from male and female rats, we performed concentration-response curves to the NO donor SPERNO. SPERNO is an NO donor that has been shown to cause endothelium-independent relaxation of arteries. The degree of vasorelaxation induced by nucleophile-NO compounds such as SPERNO is highly correlated with their ability to release NO, and is associated with increases in cGMP (Morley et al., 1993). In the current studies, we used SPERNO as an NO donor that directly relaxes vascular smooth muscle cells. SPERNO responses were performed in the presence of LNA to eliminate the interference of endogenous NO production with the relaxant actions of the exogenous NO donor. We found no difference in SPERNO-mediated relaxation between arteries from male and female rats. Thus, it is unlikely that differences in smooth muscle sensitivity to NO explain the enhanced LNA-sensitive relaxation to ACh in arteries from female rats. Our data support the postulate that enhanced ACh-induced relaxation in mesenteric arteries from females is due to increased agonist-stimulated NO production from the vascular endothelial cells of female rats compared with male rats.

The concept of enhanced NO release from the endothelium of female rats is supported by our results with apamin, an inhibitor of the small conductance K_Ca channel (Brayden, 1996). Apamin, like LNA, eliminated the gender difference in relaxation to ACh over the midrange concentrations at which females exhibit greater relaxation than males. The extent of inhibition by apamin and LNA was similar over this range. Furthermore, the effects of apamin and LNA were not additive; relaxation to ACh in the presence of LNA and apamin combined was not significantly different from each of these inhibitors separately over the ACh concentration range of the gender difference. Collectively, these data suggest that LNA and apamin act on a common pathway to inhibit relaxation to ACh over the concentration range of 4.0 × 10⁸ to 2.5 × 10⁷ M.

Apamin may interact with the NO system at the level of either the endothelium or the smooth muscle, and there is evidence in the literature of apamin-sensitive potassium channels on both of these cell types (Khan et al., 1993; Demirel et al., 1994; Murphy and Brayden, 1995; Brayden, 1996). We found that apamin did not inhibit relaxation to the NO donor SPERNO, making it unlikely that NO causes relaxation of rat mesenteric arteries by opening of smooth muscle small-conductance K_Ca channels. Thus, our data suggest that both apamin and LNA act at the endothelium to decrease NO production. Recent patch-clamp studies have demonstrated the presence of an apamin-sensitive potassium channel on aortic endothelium that mediated hyperpolarization of the endothelium when stimulated by ACh (Marchenko and Sage, 1996). Studies in the cat (Champion and Kadowitz, 1997) and the rabbit (Demirel et al., 1994) have suggested that activation of tetraethylammonium-sensitive K_Ca channels on endothelium, with resulting hyperpolarization of the endothelial cell, is involved in stimulating endothelium-derived relaxation factor (presumably NO) release. Thus, our findings are consistent with the mechanism that apamin inhibits endothelial cell small conductance K_Ca channels, which inhibits endothelial cell hyperpolarization and subsequent NO release.

The mechanisms by which stimulation of endothelial potassium channels lead to release of NO remain somewhat speculative. However, it is known that agonists such as ACh induce a biphasic peak in endothelial cytoplasmic calcium concentrations, with the initial peak due to inositol 1,4,5-triphosphate (IP₃) production and the second, sustained phase requiring extracellular calcium and a negative membrane potential. Although the ion channels regulating calcium influx into endothelial cells have been shown to be voltage insensitive, negative endothelial cell membrane potential will electrochemically favor the movement of calcium ions from the extracellular environment into the cytoplasm. In rabbit aortic strips, ACh induces a rise in intracellular calcium concentration at the endothelial surface, an effect that is inhibited by the large-conductance K_Ca channel blocker tetraethylammonium (Demirel et al., 1994). In addition, Sakai (1990) examined the effects of blocking the IP₃ receptor with heparin on the outward current induced by ACh in rabbit aortic endothelial cells and concluded that ACh activates a calcium-dependent potassium channel via the release of calcium from IP₃-sensitive intracellular stores. Collectively, available evidence suggests that ACh leads to an increase in intracellular calcium via the IP₃ pathway; this increased calcium then activates endothelial cell K_Ca channels, causing endothelial cell hyperpolarization and subsequently increasing the driving gradient for calcium to move into the endothelium. This sustained elevation of calcium may then maintain activity of the calcium-dependent NOS. Although there are no literature reports of an estrogen response element in the promoter region of the K_Ca gene, or of gender differences in the resting membrane potential of endothelial cells, it has been shown that endothelial cells from females have a higher intracellular calcium level than endothelial cells from males (Rahimian et al., 1998; Knot et al., 1999). Knot et al. (1999) predict that the observed gender differences in endothelial cell calcium levels would lead to a nearly 3-fold greater NO production in arteries from females than from males. These reported gender differences in endothelial cell calcium levels support our interpretation of our pharmacological analyses, and support the postulate that enhanced NO-dependent relaxation in arteries from females is due to an increased contribution of endothelial calcium-activated potassium channels.

In summary, we found that arteries from females are more sensitive to ACh-induced dilation than those from males. The augmented relaxation in females is associated with: 1) an enhanced NO component of the relaxation, and 2) an increased contribution of a non-NO, noncyclooxygenase EDHF. The increased NO-mediated relaxation in females is related to heightened activity of an endothelial apamin-sensitive potassium channel. Because potassium channels have been postulated to be involved in maintenance of NO production from the vascular endothelium, our studies provide evidence that enhanced vasodilation in arteries from female rats may be due to a greater degree of agonist-induced cellular hyperpolarization compared with males, leading to augmented NO release from the vascular endothelium.