Gender differences in coronary artery diameter reflect changes in both endothelial Ca²⁺ and ecNOS activity

Department of Pharmacology, University of Vermont College of Medicine, Burlington, Vermont 05405

	ABSTRACT

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Elevation of nitric oxide (NO) release from the vascular endothelium may contribute to some of the gender-associated differences in coronary artery function. The mechanisms by which gender affects NO release from the endothelium of coronary arteries are not known. In this study, endothelial function was examined in pressurized coronary arteries from female and male rats. Diameter and endothelial cell intracellular Ca²⁺ concentration ([Ca²⁺]_i) in intact arteries, as well as enzymatic activity of endothelial constitutive nitric oxide synthase (ecNOS) in arterial lysates, was measured. Elevation of intravascular pressure to 60 mmHg constricted coronary arteries from female animals less than coronary arteries from male animals (18% and 31% constriction, respectively). The increased arterial diameter of coronary arteries from females was associated with elevated endothelial [Ca²⁺]_i (female 174 nM, male 90 nM; P < 0.001). Elevation of Ca²⁺ activated ecNOS with a similar slope and half-activation constant (~160 nM) for both female and male coronary arteries. However, at [Ca²⁺] > 100 nM, ecNOS activity was significantly higher in coronary arteries from female rats compared with their male equivalents (P < 0.01). Maximal activity for ecNOS at saturating Ca²⁺ (300 nM) was 37% higher in coronary arteries from female animals compared with male animals (P < 0.05). Thus elevated [Ca²⁺]_i in the endothelium of female coronary arteries alone is predicted to increase the production of NO (by nearly 2-fold). This gender difference combined with increased ecNOS activity at a given [Ca²⁺] in females indicates that tonic NO production should be nearly threefold greater in female coronary arteries compared with male coronary arteries. We conclude that, in the regulation of endothelial Ca²⁺ and ecNOS, gender differences contribute significantly to the overall decrease in myogenic tone observed in coronary arteries of females.

coronary disease; nitric oxide; endothelium; calcium; hormones; endothelial constitutive nitric oxide synthase

	INTRODUCTION

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IT IS WIDELY HELD that the incidence of morbidity and mortality from coronary artery disease is less for young adult females compared with males or postmenopausal females [for review, see Nathan and Chaudhuri (40)]. Numerous clinical studies clearly support a role for estrogen in this gender-related cardioprotective effect (17, 58). Estrogen exerts a protective action through favorable effects on lipid profiles, decreased platelet and monocyte adhesion, and decreased vascular reactivity (2, 18, 57, 60). Although the mechanisms by which estrogen affects vascular tone are not completely understood, a change in the communication between the vascular endothelium and smooth muscle is likely an important pathway for the action of estrogen.

The endothelium exerts control over vascular tone through the generation of nitric oxide (NO), a powerful smooth muscle relaxant (43). The consensus from numerous studies is that estrogen stimulates the production of NO by the endothelium (7, 21, 27, 44, 57), including human coronary endothelium (20, 45). The mechanisms by which gender influences NO production are unclear and may involve an increase in nitric oxide synthase (NOS) expression or activity. Intracellular Ca²⁺ (combined with calmodulin) is a key physiological regulator of NO production. However, neither the levels of cytosolic free Ca²⁺ ([Ca²⁺]_i) in intact coronary endothelium (10, 53, 54) nor the sensitivity of endothelial constitutive NOS (ecNOS) to physiologically relevant Ca²⁺ concentrations is known (14).

Our previous study indicated that endogenous estrogen induces a tonic increase in NO release from the endothelium of pressurized coronary arteries, resulting in a maintained, increased diameter of arteries from female rats (57; see also Refs. 7, 15, 25, 38, 41, 50, and 51 for similar conclusions from others). The overall objective of this study was to determine endothelial [Ca²⁺]_i and ecNOS activity in female and male coronary rat arteries. We examined gender differences in endothelial Ca²⁺ regulation and ecNOS activity levels and the Ca²⁺ dependence of ecNOS. Here, we provide the first measurements of endothelial [Ca²⁺]_i in intact coronary endothelium and demonstrate a highly significant, positive correlation between endothelial [Ca²⁺]_i and the diameter of pressurized arteries. We also provide evidence that coronary arteries from female rats exhibit higher levels of both endothelial [Ca²⁺]_i and ecNOS activity than coronary arteries from male rats. In addition, we establish the Ca²⁺ dependence of ecNOS activity from coronary arteries over the physiological range of endothelial [Ca²⁺]_i. We propose that a tonic elevation of endothelial [Ca²⁺]_i provides an important contribution to the observed gender differences in coronary artery tone by amplifying the NO production in the coronary endothelium.

	METHODS

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Animals and Tissue

Mature female and male Sprague-Dawley rats (148 Rattus norvegicus, 12-14 wk old, ~228-450 g) were euthanized with pentobarbital (150 mg/kg body wt by ip injection). The hearts were removed, and isolated coronary arteries were obtained as previously described (31, 57). All procedures involving animals were conducted in accordance with the Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85-23, Revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20205].

[Ca²⁺]_i Measurements in Endothelium of Intact Pressurized Arteries

Distal septal coronary arteries were cannulated, mounted in an arteriograph, and continuously superfused with oxygenated physiological salt solution (PSS, 3-6 ml/min) at 37°C. Intravascular pressure was gradually increased from 2 to 60 mmHg as previously described (31, 57). Arterial diameter of fura 2-loaded arteries was directly measured from the video signal of the background-corrected ratio images or from videotaped experiments as previously described (30). Arteries that did not constrict to pressure were not used. The coronary artery endothelium was selectively loaded with fura 2, the Ca²⁺ indicator, as follows. After myogenic tone had developed, the lumen of the artery was filled for 5 min with PSS fura 2-AM (2 µM in PSS at 37°C) using a pressurized loading loop. The loading loop consists of a short piece of Tygon tubing connected to a 1-ml syringe barrel. This loop is connected, via two three-way stopcocks, in parallel with the pressurization line just upstream of the proximal cannula. This arrangement allows either direct connection of the pressure reservoir with the lumen of the artery or, alternately, a connection via the loop. The loop can be directly filled or backfilled with PSS or PSS containing fura 2-AM dye without disturbing intraluminal pressure. Opening the distal end of the perfusion system [i.e., distal to the artery via a small-gauge cannula (<10-µm diameter)] initiates flow of PSS or fura 2-AM into the lumen of the artery. The artery is observed under ultraviolet (UV) illumination (380 nm) during this procedure. Entry of the dye (PSS containing fura 2-AM) into the lumen causes a bright fluorescence from the lumen. After a 5-min incubation period with the distal end of the cannula closed to stop flow, the three-way valves are switched to allow PSS from the pressure reservoir to flush out the PSS or PSS-fura 2-AM solution from the lumen (by again opening the distal cannula). With the use of these loading conditions, the artery displayed a minimal drop in luminal pressure due to the high resistance of the distal opening but essentially remained pressurized. After washout of the lumen, the endothelial loading was clearly visible as a bright thin layer on the luminal side of the artery in the focal plane (see Fig. 1). After each experiment, the artery was denuded by placing an air bubble in the arterial lumen for 30 s, followed by perfusion with 1 ml distilled water while the pressurized artery was observed again under UV illumination. Total removal of the fluorescence was considered a good indication of removal of the endothelium (see Fig. 1) and of prior selective loading of the endothelial cells with fura 2-AM.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Measurement of endothelial intracellular calcium concentration ([Ca2+]i) in intact pressurized coronary arteries. A: digitized images of an intact coronary artery luminally loaded with fura 2. Top: simultaneous illumination with ultraviolet (UV) and transmitted light; bottom: illumination with UV light only. Denuded vessel is same artery after removal of the endothelium (see METHODS). B: nisoldipine added to external superfusing bath solution affects diameter of intact coronary arteries (top; maximal dilation at 100 nM). Endothelial [Ca2+]i imaging (bottom) was performed simultaneously with diameter measurement.

Ratiometric Ca²⁺ imaging was performed as described previously (30). The sampling rate in these experiments was 0.2-0.5 Hz. Several control experiments were performed to verify selective endothelial loading of the Ca²⁺ indicator (see RESULTS and Fig. 1). Successful measurements of endothelial Ca²⁺ were made in 40 of 73 arteries studied. Fourteen arteries either did not develop myogenic tone or did not hold pressure, and 19 arteries could not be used because either fura 2 loading did not occur or fura 2 loading of both endothelium and smooth muscle was observed.

Endothelial Ca²⁺ was calculated using the equation (from Ref. 19) [Ca²⁺] = K_d ×  × (R  R_min)/(R_max  R). R is the ratio between emission signals, each corrected for the background signal, when the sample is excited with 340-nm and 380-nm light; R_min is R under Ca²⁺-free conditions (5 mM EGTA); R_max is R under Ca²⁺-saturated conditions (2.4 mM Ca²⁺); and  is the ratio of the emission signal when the sample is excited with 380-nm light under Ca²⁺-free conditions to that under Ca²⁺-saturated conditions. R_min and R_max were measured from ionomycin- and nigericin-treated arteries as previously described (R_max = 2.4 ± 0.09, R_min = 0.35 ± 0.07,  = 1.98 ± 0.31; n = 22) (30, 59). For every set of experiments within a protocol, these values were pooled and used to convert the averaged ratio values (R) into a [Ca²⁺] value. A value of 282 nM was taken as the K_d of fura 2. This was experimentally determined using an in situ titration of Ca²⁺ in fura 2-loaded arteries (30).

Measurement of ecNOS Activity in Isolated Arteries

Coronary arteries were removed and immediately frozen in liquid N₂ and stored at 80°C. For experiments requiring denuded arteries, a hair containing a knot was run through the artery. Approximately 20 coronary segments (5-10 mm in length) were required per experiment. The arteries were thawed and homogenized in 600 µl of solubilization buffer [10 mM HEPES, pH 7.5, 1 µM calmodulin (CaM), 50 µM tetrahydrobiopterin (THB), 5 mM EGTA, 100 µM phenylmethylsulfonyl fluoride (PMSF), and 0.1% Tergitol NP-40]. After homogenization, samples were solubilized on ice for 30 min, then centrifuged at 12,000 g for 10 min. After centrifugation, the resulting pellets were discarded and NOS activity was measured in the supernatant (lysate). A citrulline-based assay was used to measure NOS activity (23). Reaction mixtures (250-µl final volume) contained 10 mM HEPES, pH 7.5, 1 µM CaM, 50 µM THB, 5 mM EGTA, 100 µM reduced NADP, 100 µM PMSF, 1 mM magnesium acetate, and 0-10 µM CaCl₂. Free Ca²⁺ concentrations (activity) were calculated as described by Fabiato (11). Reactions were initiated by the addition of 25 µl of arterial lysate (~50 µg protein) and L-[³H]arginine [200 µM, ~8,000 counts per minute (cpm)/nmol]. After 5 min at 37°C, reactions were terminated by the addition of 6 µl of 6 N TCA on ice. Samples were neutralized with 500 µl of 0.5 mol/l HEPES, pH 7.5, and loaded onto a 2-ml cation-exchange column [50W-X8 resin (converted to Na⁺ form) Bio-Rad Laboratories, Hercules, CA]. [³H]citrulline product was collected in the eluate by washing the column with 5 ml of H₂O and then quantified by scintillation counting. Citrulline recovery from the resin was nearly 100% as assayed using [³H]citrulline, and NOS activity was linear over the time course studied. All reactions were performed in triplicate, and cpm values were corrected by subtraction of a reaction containing no protein. Counts were normalized to protein content (Bradford assay, Bio-Rad Laboratories) and the specific activity of L-[³H]arginine and are presented as picomoles of citrulline per minute per milligram protein.

Data Analysis

Endothelial [Ca²⁺]_i values (in nM) are expressed as means ± SD from n different arteries. Diameter values (in µm) are expressed as means ± SE for n vessels. Statistical significance was tested at the 95-98% confidence level using a paired or unpaired Student's t-test or a Student-Newman-Keuls test when appropriate.

Chemicals and Buffers

Fura 2-AM was purchased from Molecular Probes (Eugene, OR). All other salts and drugs were obtained from Sigma Chemical (St. Louis, MO). A PSS was used as the bathing solution and contained (in mM) 119 NaCl, 4.7 KCl, 24.0 NaHCO₃, 1.2 KH₂PO₄, 1.6 CaCl₂, 1.2 MgSO₄, 0.023 EDTA, and 11.0 glucose (pH 7.4). This solution was continuously bubbled with 95% O₂-5% CO₂ and heated to 37°C. High external K⁺ solutions were made by isosmotic substitution of NaCl with KCl in the PSS. Drugs were added to the superfusate and thus allowed to act from the adventitial side of the artery.

	RESULTS

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Endothelial [Ca²⁺]_i in Intact Pressurized Coronary Arteries

Endothelial [Ca²⁺]_i was measured in intact pressurized coronary arteries loaded with fura 2-AM as described in METHODS. Fura 2 localization in the endothelium was supported by these observations: 1) fura 2 fluorescence disappeared with removal of the endothelium (Fig. 1A); 2) the endothelium-dependent vasodilator ACh increased the fluorescence signal (see Figs. 2 and 3); 3) membrane depolarization with external K⁺, which elevates [Ca²⁺]_i in smooth muscle and decreases [Ca²⁺]_i in endothelial cells (5), decreased fluorescence (see, e.g., Fig. 2); and 4) inhibitors of voltage-dependent Ca²⁺ channels (e.g., nisoldipine) did not affect Ca²⁺ (Fig. 1B), which is consistent with the idea that endothelial cells do not have voltage-dependent Ca²⁺ channels and therefore should not respond to blockers of such channels.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   ACh increases and elevated external K+ decreases endothelial [Ca2+]i in intact coronary arteries from female rats. A: elevated K+ causes a drop in endothelial [Ca2+]i. Original ratio recordings of endothelial [Ca2+]i plotted against a converted scale of nM Ca2+. Bars, duration of exposure to isosmotically elevated K+ in tissue bath. B: ACh elevates endothelial [Ca2+]i. Bars, duration of exposure to ACh or ACh + K+ in bath solution. C: summary of nonstimulated () and ACh-stimulated () steady-state endothelial [Ca2+]i measurements (n = 14 female rat arteries). PSS, physiological salt solution.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   ACh increases and elevated external K+ decreases endothelial [Ca2+]i in intact coronary arteries from male rats. A: elevated K+ causes a drop in endothelial [Ca2+]i. Recordings were performed as in Fig. 2. B: ACh elevates endothelial [Ca2+]i in pressurized coronary arteries from male rats. C: summary of nonstimulated () and ACh-stimulated () steady-state endothelial [Ca2+]i measurements (n = 12 male rat arteries).

The diameter of pressurized (60 mmHg) coronary arteries from female and male animals was not affected by loading the endothelium with fura 2 (174 ± 15 vs. 172 ± 14 µm, n = 14 for females; 150 ± 10 vs. 147 ± 9 µm, n = 12 for males). Lowering the intravascular pressure of coronary arteries in female rats from 60 to 10 mmHg decreased diameter from 169 ± 12 to 124 ± 7 µm (n = 4) but had no effect on endothelial [Ca²⁺]_i (187 ± 22 nM at 60 mmHg; 181 ± 18 nM at 10 mmHg). In contrast, lowering intravascular pressure reduced [Ca²⁺]_i in smooth muscle cells (30). The dihydropyridine inhibitor of voltage-dependent Ca²⁺ channels in smooth muscle cells, nisoldipine, dilated pressurized (60 mmHg) arteries to 211 ± 16 µm (n = 4) but did not significantly alter endothelial [Ca²⁺]_i (186 ± 26 nM; n = 4), further supporting the endothelial localization of fura 2 (Fig. 1B). Lowering the intravascular pressure from 60 to 10 mmHg in the presence of nisoldipine also had no effect on endothelial [Ca²⁺]_i (186 ± 26 nM before and 184 ± 18 nM after lowering pressure), although arterial diameter decreased from 211 to 127 ± 14 µm (n = 9). These results suggest that neither intravascular pressure nor smooth muscle contractility affects endothelial [Ca²⁺]_i.

Gender Differences in Regulation of Endothelial [Ca²⁺]_i

Coronary arteries from female rats. [Ca²⁺]_i in the endothelium of pressurized (60 mmHg) arteries from female rats was 174 ± 33 nM (n = 14) (Fig. 2A). Increasing external K⁺ from 6 mM to 61, 121, and 141 mM caused a graded reduction in [Ca²⁺]_i to a final concentration of 94 ± 25 nM (n = 14) (Fig. 2, A and C). High K⁺ (141 mM) also constricted these arteries from 172 µm to 64 ± 7 µm (n = 12). Stimulation of the coronary arteries from females with ACh caused a sustained rise in endothelial [Ca²⁺]_i from 174 to 337 ± 48 nM (n = 6) and caused dilation of the arteries to a final diameter of 215 ± 7 µm (n = 6). Elevating external K⁺ from 6 to 141 mM in the presence of ACh decreased endothelial [Ca²⁺]_i from 337 nM to 209 ± 15 nM (Fig. 2C) and constricted arteries to a final diameter of 66 ± 8 µm (n = 6). These results are consistent with membrane depolarization causing a decrease in endothelial Ca²⁺ entry through a reduction in the Ca²⁺ electrochemical gradient. Furthermore, these results suggest that Ca²⁺ entry in the endothelium of coronary arteries from female rats is regulated by membrane potential under both basal and ACh-stimulated conditions.

Coronary arteries from male rats. At an arterial pressure of 60 mmHg, [Ca²⁺]_i in the endothelium of coronary arteries from male rats was significantly lower than that of females [female, 174 ± 33 nM; male, 90 ± 13 nM (n = 12); P < 0.001] (Figs. 3 and 4A). Increasing external K⁺ to 61, 121, and 141 mM caused a smaller drop in endothelial Ca²⁺, to 63 ± 12 nM, in male rat arteries than in female rat arteries (n = 12) (Fig. 3, A and C). As observed in females arteries, high K⁺ (141 mM) constricted male arteries from 150 ± 10 to 66 ± 7 µm (n = 9).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Relationship between arterial endothelial [Ca2+]i and arterial diameter in coronary arteries from rats. A: basal endothelial [Ca2+]i but not ACh-stimulated [Ca2+]i is significantly higher in female compared with male coronary artery endothelium. * P < 0.001 by unpaired Student's t-test. B: elevated endothelial [Ca2+]i correlates well with arterial dilation in coronary arteries from rat. Data points represent individual arteries (n = 21 arteries).

Stimulation of coronary arteries from male rats with ACh caused a sustained rise in endothelial [Ca²⁺] from 90 to 325 ± 44 nM (n = 5), similar to the ACh-stimulated endothelial [Ca²⁺]_i in arteries from female rats (Fig. 4A). ACh dilated arteries from male rats to a final diameter of 214 ± 8 µm (n = 5). Elevation of external K⁺, in the presence of ACh, to 61, 121, and 141 mM elicited a substantial drop in endothelial [Ca²⁺]_i to 218 ± 19 nM (Fig. 3C) and a constriction to 61 ± 6 µm (n = 5). Similar to observations made in arteries from female rats, these results from male rats indicate that Ca²⁺ entry in the endothelium of arteries is regulated by membrane potential. Whereas endothelial [Ca²⁺]_i in unstimulated arteries from male rats is significantly lower than [Ca²⁺]_i in unstimulated arteries from female rats, steady-state endothelial [Ca²⁺]_i in the presence of ACh is not different between males and females (Fig. 4A).

Relationship Between Endothelial [Ca²⁺]_i and Arterial Diameter

Consistent with our previous study (57), elevating intravascular pressure from 2 to 60 mmHg constricted coronary arteries from female rats from 212 ± 15 to 174 ± 15 µm (n = 14) or by 18%, whereas the same pressure step constricted coronary arteries from male animals from 216 ± 14 to 150 ± 10 µm or by 31% (P < 0.001, n = 12). An elevation in endothelial [Ca²⁺]_i was associated with a sustained increase in arterial diameter (r = 0.92, P < 0.0001, Fig. 4B). Elevation of endothelial [Ca²⁺]_i by ACh to levels >300 nM (Fig. 4A) was associated with a maximal dilation and was similar for arteries from both female and male animals. Unstimulated coronary arteries from female rats had consistently higher endothelial [Ca²⁺]_i and were more dilated than unstimulated arteries from male rats (Fig. 4, A and B). These results suggest that the endothelial [Ca²⁺]_i level is an important regulator of coronary arterial diameter and that the higher endothelial [Ca²⁺]_i in arteries from female rats may be, in part, responsible for the observed gender differences in coronary artery tone.

Gender Differences in Ca²⁺-Dependent ecNOS Activity

To examine the Ca²⁺ dependence of NOS, the effects of Ca²⁺ on NOS activity were measured in arterial lysates from female and male coronary arteries (with and without endothelium). Elevation of Ca²⁺ increased NOS activity from female and male coronary arteries with intact endothelium (Fig. 5). In contrast, elevating Ca²⁺ to 300 nM did not significantly alter NOS activity in lysates from female and male coronary arteries previously denuded of their endothelium (n = 3). Ca²⁺-dependent NOS activity was significantly greater (~1.4-fold) in coronary arteries from female rats than their male counterparts at [Ca²⁺] >100 nM (P < 0.01) (Fig. 5). Maximal ecNOS activity from coronary arteries for both male and female rats was obtained at [Ca²⁺] (300 nM, n = 3). This level of endothelial [Ca²⁺]_i was associated with maximal dilation (Fig. 4B). The [Ca²⁺] required for half-maximal activity of ecNOS was not significantly different between arteries from males and females (K_m of 170 ± 80 nM and 150 ± 60 nM for coronary arteries from male and female rats, respectively).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Coronary arteries from female rats exhibit a higher Ca2+-sensitive, endothelium-derived nitric oxide synthase (NOS) activity than coronary arteries from male rats. Ca2+-dependent NOS activity was measured in arterial lysates derived from coronary arteries of male and female rats in presence of a range of free Ca2+ concentrations. All Ca2+ solutions were buffered with 5 mM EGTA, and 0 Ca2+ was approximated by presence of 5 mM EGTA in buffer containing no added Ca2+. Because Ca2+ had no effect on NOS activity in denuded arteries (not shown, n = 3), endothelial constitutive NOS (ecNOS) activity at different [Ca2+] was calculated as (total NOS activity  NOS activity in 0 Ca2+). Data represent 3 independent experiments performed in triplicate. Maximal velocity (Vmax) = 860 ± 121 (female) and 557 ± 115 (male) pmol citrulline · min1 · mg1. Hill coefficients (h) were 1.4 and 1.2 for Ca2+ dependence of ecNOS for extracts derived from female and male animals, respectively. ecNOS activity = Vmax/{(Km/[Ca2+])h + 1}. * P < 0.01 vs. male by Student-Newman-Keuls test.

	DISCUSSION

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This study provides the first measurements of endothelial [Ca²⁺]_i in isolated pressurized coronary arteries. It is also the first evidence that both [Ca²⁺]_i and ecNOS activity (at a given [Ca²⁺]_i) are higher in the endothelium of intact pressurized coronary arteries from female rats compared with male rats. This study suggests that a tonic elevation of endothelial [Ca²⁺]_i in female coronary arteries (Fig. 4A) leads to an increase in NO production (Fig. 5) and a maintained dilation (Fig. 4B). Our previous study (57) indicated that gender differences in myogenic tone of these coronary arteries are due to endogenous estrogen causing an increase in tonic NO release. This suggests that estrogen may be involved in the gender differences observed in endothelial [Ca²⁺]_i and NOS activity in the present study. However, our results do not exclude possible effects of other hormones (e.g., progesterone and testosterone) on endothelial [Ca²⁺]_i and NO release.

[Ca²⁺]_i in Intact Endothelium

The majority of measurements of endothelial [Ca²⁺]_i have been made using cultured cells (3, 5, 13, 37). In some instances, [Ca²⁺]_i has been measured in freshly isolated endothelial cells (9, 42) and in intact endothelium (10, 12, 26, 53). We measured [Ca²⁺]_i in intact endothelium of pressurized coronary arteries from rats. Our results indicate that endothelial [Ca²⁺]_i in intact pressurized coronary arteries ranges from 90 nM (male) to 174 nM (female) and that stimulation of the endothelium with ACh (10 µM) increases [Ca²⁺]_i to levels >300 nM. Consistent with this result is that of Usachev et al. (53), who measured endothelial [Ca²⁺]_i in intact aorta of immature rats (4-5 wk old) and found levels of 95 nM in nonstimulated aorta and 413 nM in ACh-stimulated aorta. Our observation that endothelial [Ca²⁺]_i in nonstimulated and ACh-stimulated endothelium in situ declines on depolarization with elevated K⁺ is consistent with previous studies that have shown endothelial [Ca²⁺] is regulated by the Ca²⁺ electrochemical gradient (5, 8, 34, 39). Our results do not support a contribution of stretch- or pressure-activated Ca²⁺ influx pathways in the coronary endothelium because neither pressure nor changes in arterial diameter affected endothelial [Ca²⁺]. Our finding that K⁺- or pressure-induced smooth muscle depolarization and associated increases in smooth muscle [Ca²⁺]_i do not affect endothelial [Ca²⁺] is in contrast to that of Dora et al. (10). These investigators reported that increases in smooth muscle Ca²⁺ are accompanied by an elevation in endothelial cell Ca²⁺ in hamster cheek pouch arterioles, presumably as a result of Ca²⁺ flux from smooth muscle to endothelial cells via myoendothelial junctions. One explanation for this difference is that the reported endothelial Ca²⁺ changes (10) are rapid and transient (<10-s duration). Our experimental design measured steady-state Ca²⁺ levels on a relatively slow time scale and would have missed such transient changes in endothelial Ca²⁺, should they have occurred. The presence or density of functional myoendothelial junctions may also vary with species or vascular bed, and this could also account for the contrasting results.

Gender Differences in Regulation of Endothelial [Ca²⁺]_i

Gender differences in the regulation of endothelial [Ca²⁺]_i may be due to direct or indirect effects of estrogen on the coronary endothelium because gender differences in myogenic tone of these coronary arteries appear to be due to endogenous estrogen (57). The regulation of endothelial [Ca²⁺]_i in coronary arteries is poorly understood. Steady-state [Ca²⁺]_i should be determined by the balance between Ca²⁺ influx and extrusion across the plasma membrane. Transient [Ca²⁺]_i changes could occur in response to release from internal stores. Steady-state [Ca²⁺] could be higher in the endothelium of female coronary arteries if the number or activity of Ca²⁺-permeable channels was elevated or if the driving force for Ca²⁺ entry was increased by membrane hyperpolarization caused by activation of K⁺ channels (47). Decreasing Ca²⁺ extrusion via Ca²⁺ pumps would also elevate steady-state [Ca²⁺]_i. Resolution of how gender can alter the regulation of endothelial [Ca²⁺]_i requires more detailed knowledge of the properties of Ca²⁺-permeable channels, K⁺ channels, and Ca²⁺ pumps in the intact coronary endothelium.

Ca²⁺ Dependence of ecNOS Activity

Although it is widely accepted that ecNOS activity is increased by Ca²⁺-CaM (4, 49), the relationship between Ca²⁺ and ecNOS activity is not known for the physiological range of endothelial [Ca²⁺]_i (100-400 nM) or for coronary arteries. Our results indicate that ecNOS activity is half-maximal at ~160 nM Ca²⁺ and maximal above 300 nM Ca²⁺. ACh causes a sustained elevation of [Ca²⁺]_i in the intact coronary endothelium to ~300 nM, a level that would elicit maximal ecNOS activity for both males and females. Elevation of endothelial [Ca²⁺]_i from 90 to 174 nM, which corresponds to levels in male and female coronary endothelium, respectively, would be predicted to increase ecNOS activity substantially in the intact artery.

ecNOS Activity is Elevated in Coronary Arteries From Female Rats

A number of studies indicate that NO release is elevated in arteries from females (20, 21, 25, 27, 28, 46), including coronary arteries (7, 35, 57). Estrogen appears to be responsible for the observed gender difference in NO release (7, 20, 25, 38, 41, 50, 51, 56). However, the mechanism by which estrogen elevates NO release remains elusive. Estrogen, through interaction with its receptor, may increase the transcription of ecNOS. Estrogen has been shown to increase the level of ecNOS mRNA in cultured pulmonary artery endothelial cells (36) and in uterine arteries (56). However, estrogen was not shown to increase ecNOS mRNA in cultured human myometrial or bovine aortic endothelial cells (52). A recent study by Kleinert et al. (29) provides direct evidence for a 1.8-fold increase in ecNOS protein expression by physiological levels of estrogen in cultured human endothelial cells. In addition, an estrogen-dependent increase in ecNOS activity may (22, 24) or may not (33, 55) translate to differences in protein level.

Although estrogen-independent effects and mechanisms are possible, most studies strongly implicate estrogen as a key mediator of gender differences in ecNOS activity and NO release. The effect of gender on NOS activity in coronary arteries has been reported in only one previous study, which concluded NOS activity in porcine coronary arteries from males and females is not different (1). We found that Ca²⁺-dependent ecNOS activity was 1.4-fold higher in coronary arteries from female than from male rats at several Ca²⁺ levels. The explanation for these differing results is unclear. However, this study provides the first direct evidence for elevated ecNOS activity in the endothelium of coronary arteries from female rats and supports related observations made using other endothelial preparations (36, 56). The increase in ecNOS activity, at a given [Ca²⁺], in female coronary arteries could reflect increased protein levels or increased enzymatic activity, but clarification of this issue awaits further study. ecNOS activity is also regulated by important cofactors such as tetrahydrobiopterin and CaM, and in situ the concentrations of these factors, and thus ecNOS activity, might be influenced by gender. We know of no evidence in support of such a gender effect on NOS cofactors, but this could be an important determinant of total ecNOS activity in situ. In any case, we observed a sustained gender-based elevation of intrinsic ecNOS activity in vitro. Together with the increase in endothelial [Ca²⁺]_i that we have documented, NO production in intact coronary arteries of female rats should be elevated by nearly threefold compared with arteries from male rats (see Fig. 5).

It is difficult to directly extrapolate our in vitro results to the regulation of coronary blood flow in humans because a wide variety of in vivo factors regulate coronary blood. However, several in vivo studies provide evidence that coronary blood flow is enhanced by estrogen (6, 16, 32, 41, 48). In most of these studies, this effect of estrogen on coronary blood flow has been shown to be related to increased NO-mediated coronary vasodilatation in the presence of estrogen (6, 16, 32, 41). Any gender differences in coronary artery reactivity in vivo could thus be due to effects of estrogen, but other mechanisms are certainly possible.

We propose that ecNOS activity is higher in the endothelium of coronary arteries from female compared with male rats because both endothelial [Ca²⁺]_i and intrinsic ecNOS activity are elevated. The gender differences in the regulation of Ca²⁺ and intrinsic ecNOS activity should contribute synergistically to an elevated tonic NO production, which would result in the decrease in myogenic tone observed in coronary arteries of females. These findings may promote new perspectives for basic and clinical research into diseases associated with impaired endothelial function, and stress the importance of endothelial [Ca²⁺]_i regulation.

	ACKNOWLEDGEMENTS

We thank Dr. G. Wellman for helpful comments on this manuscript.

	FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-51728 and HL-44455.

Present address of H. J. Knot: Cardiology Div., Dept. of Medicine, Given Bldg. D-014E, The Univ. of Vermont, Burlington, VT 05405-0068.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: M. T. Nelson, Dept. of Pharmacology, Univ. of Vermont College of Medicine, Given Medical Bldg., Burlington, VT 05405-0068.

Received 11 August 1998; accepted in final form 16 November 1998.

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