Altered Calcium Dynamics Do Not Account for Attenuation of Endothelium-Derived Hyperpolarizing Factor–Mediated Dilations in the Female Middle Cerebral Artery
Background and Purpose— The contribution of endothelium-derived hyperpolarizing factor (EDHF) to ATP-mediated dilations is significantly attenuated in the rat middle cerebral artery of intact and estrogen-treated ovariectomized (OVX) females compared with males and vehicle-treated OVX females. Since an increase in endothelial calcium appears to be a critical prerequisite in the EDHF response, we tested the hypothesis that endothelial cell intracellular calcium ([Ca2+]i) fails to reach sufficient levels to elicit robust EDHF-mediated dilations in females and that this effect is mediated by estrogen.
Methods— Vascular diameter and [Ca2+]i were measured concomitantly in perfused middle cerebral artery segments with the use of videomicroscopy and fura 2 fluorescence, respectively.
Results— In the presence of NG-nitro-l-arginine methyl ester and indomethacin, the dilation to 10−5 mol/L ATP was significantly reduced (P<0.05) in intact females (42±8%; n=6) and estrogen-treated OVX females (25±6%; n=9) compared with intact males (89±5%; n=6) and vehicle-treated OVX females (92±2%; n=7). Contrary to our initial hypothesis, endothelial cell [Ca2+]i increased to comparable levels in intact females (461±116 nmol/L), estrogen-treated OVX females (417±50 nmol/L), intact males (421±77 nmol/L), and vehicle-treated OVX females (530±92 nmol/L). In response to luminal ATP (10−5 mol/L), smooth muscle cell [Ca2+]i decreased to a greater degree in males (37±4%; n=8) compared with females (21±5%; n=7) and in vehicle-treated OVX females (18±7%; n=7) compared with estrogen-treated OVX females (3±5%; n=9).
Conclusions— Our data suggest that loss of a factor coupling EDHF to reduction of ionized smooth muscle cell [Ca2+]i accounts for the attenuated EDHF-mediated dilations in the female middle cerebral artery.
An emerging concept is being developed in the cerebral microcirculation that exposes a central role for endothelium-derived hyperpolarizing factor (EDHF) in the modulation of vascular tone.1 While many laboratories are currently working toward pinpointing the actual identity of EDHF, we know that the initial stimulus for EDHF-mediated dilations involves calcium influx into the endothelium either directly by virtue of calcium ionophores2 or indirectly via stimulation of endothelial receptors.3 Such endothelial calcium mobilization culminates in hyperpolarization of the vascular smooth muscle with ensuing relaxation of the artery.
Sex-specific differences in vascular reactivity have recently been described both in the periphery4 and in the cerebral circulation.5 In particular, nitric oxide (NO)6,7⇓ and prostacyclin8 appear to be upregulated in females. We have recently shown that in the female middle cerebral artery (MCA), EDHF-mediated dilations are significantly attenuated compared with their male counterparts.9 These dilations in females were enhanced to levels similar to that of an intact male after ovariectomy and subsequently were lost after chronic estrogen replacement, suggesting that this effect is mediated by estrogen. In contrast to our findings in cerebral vessels, estrogen appears to potentiate EDHF-mediated dilations in peripheral vessels.10,11⇓ This adds to the accumulating data supporting the notion that the mechanism for the EDHF response is distinct in the periphery and the cerebrovasculature.12
The specific mechanisms through which estrogen acts to reduce EDHF-mediated dilations have not yet been identified. This task is confounded by the fact that the identity of EDHF in the brain still remains unclear.12 Nevertheless, it is well established that the primary stimulus for the production and/or release of EDHF is a rise in endothelial cell (EC) intracellular calcium ([Ca2+]i).13,14⇓ Moreover, the magnitude of the [Ca2+]i increase appears to distinguish an NO-mediated dilation from an EDHF-mediated dilation, with the latter requiring a slightly higher increase in [Ca2+]i.3 Given the critical role of EC [Ca2+]i in the EDHF pathway, it is reasonable to speculate that alterations in endothelial calcium regulation may account for the attenuated EDHF-mediated dilations in the female MCA.
The present study has tested the hypothesis that in the female MCA, EC [Ca2+]i fails to reach critical threshold levels to elicit a robust EDHF response and that this effect is mediated by estrogen. We demonstrate herein that EDHF-mediated increases in EC [Ca2+]i were sufficiently elevated to potentially elicit an EDHF-mediated dilation in intact females and estrogen-treated ovariectomized (OVX) females. However, the EDHF-associated reduction in smooth muscle cell (SMC) [Ca2+]i was markedly attenuated. Our results suggest that estrogen acts to uncouple the EDHF response at a point beyond the change in EC [Ca2+]i. This uncoupling prevents the reduction of SMC [Ca2+]i and resultant dilatation.
Materials and Methods
Experiments were performed in strict accordance with National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the Animal Protocol Review Committee at Baylor College of Medicine. Rats were housed under a 12-hour/12-hour light/dark cycle with unrestricted access to food and water. Experiments were performed on age-matched (aged 70 to 90 days) male (weight, 275 to 324 g) and female (weight, 200 to 224 g) Long-Evans rats. Four groups of rats were used in the present study: (1) intact males (n=14); (2) intact females (n=13); (3) vehicle-treated OVX females (n=14); and (4) estrogen-treated OVX females (n=18).
Estrogen Depletion and Repletion
All surgical procedures were undertaken under aseptic conditions. Animals were secured to a nose cone and allowed to breathe spontaneously (2% isoflurane). Rectal temperature was maintained at 37°C with the use of a heating pad and a temperature controller (Harvard Apparatus Inc). A bilateral ovariectomy was then performed by accessing the ovaries through 2 lateral abdominal incisions.15 A dorsal incision was made, and an osmotic minipump (model 2004, Alza Corporation) was subcutaneously implanted to deliver either 17β-estradiol or 50% dimethyl sulfoxide/0.045% NaCl (vehicle). 17β-Estradiol was administered at a physiological dose of 4 μg/kg per day. Validation of estrogen depletion and repletion comprised monitoring body weight and measuring plasma estradiol concentration. For the latter, trunk blood (3 mL) was obtained from all animals immediately after decapitation. The blood was centrifuged for 3 minutes at 8000 rpm, and the resulting plasma was frozen at −20°C. 17β-Estradiol was measured at a later time with the use of an ultrasensitive radioimmunoassay (Diagnostic Systems Laboratory). After completion of surgery, wounds were sutured, and the animals were returned to the holding facility for 2 weeks.
Harvesting and Mounting Cerebral Vessels
Animals were placed in an anesthetic chamber, allowed to breathe isoflurane spontaneously, and then decapitated. The brain was removed from the cranium and placed in ice-cold physiological salt solution (PSS). The MCA was excised, cleaned of surrounding connective tissue, and cannulated with micropipettes in a custom-made vessel chamber. PSS was circulated abluminally and perfused luminally. Monitoring of intraluminal pressure was performed with in-line transducers, which were connected to 2 strain gauge panel meters (Omega). Once mounted, vessels were tested for leaks by clamping the proximal and distal tubing and monitoring intraluminal pressure. Vessels that did not maintain a steady pressure were discarded. The vessel chamber was mounted on the stage of an inverted fluorescence microscope. Transmural pressure was set at 85 mm Hg with a flow of 100 μL/min through the lumen, and the vessels were allowed to equilibrate for 1 hour. During this time they developed spontaneous tone by constricting from their fully dilated diameter at initial pressurization. After the development of tone, the experiment was initiated (see EDHF-Mediated Dilations).
Measurement of Vascular Diameter and Calcium
Vascular diameter and intracellular calcium were measured simultaneously as recently described in detail16 with the use of a charge-coupled device video system and photomultiplier tube, respectively (Intracellular Imaging). Frequency of acquisition was 8 Hz for vascular diameter and 4 and 5 Hz for EC and SMC [Ca2+]i, respectively. For the measurement of EC or SMC [Ca2+]i, the Ca2+-sensitive indicator fura 2 was selectively loaded into either the EC or SMC, respectively (see below). Fura 2 was excited at 340 and 380 nm, and the emission light was sampled at 510 nm. The fluorescence ratio (340/380 nm) was calculated after subtraction of the background fluorescence.
Selective loading of the endothelium was achieved by perfusing fura 2-AM (0.67 μmol/L) through the lumen for a period of 4 minutes. The fura 2-AM containing buffer was subsequently washed out, and an additional period of 15 minutes was allowed for complete de-esterification of the dye. Smooth muscle loading was achieved from the adventitial side by replacing the abluminal PSS with MOPS buffer containing fura 2-AM (1 μmol/L). Loading was continued for 5 minutes at room temperature followed by a washout period with fresh PSS at 37°C. The vessel was allowed 30 minutes for complete de-esterification of fura 2-AM before SMC [Ca2+]i measurements were acquired. Previous studies have confirmed the selectivity of loading the ECs and SMCs with the use of this paradigm.16
EDHF-mediated dilations were assessed by luminal application of ATP, a P2Y2 purinoceptor agonist, in the presence of NG-nitro-l-arginine methyl ester (L-NAME) and indomethacin.17 After the development of spontaneous tone, L-NAME (3×10−5 mol/L) and indomethacin (10−5 mol/L) were added to the luminal and abluminal baths to remove the NO synthase and cyclooxygenase contributions, respectively. A concentration-response curve to luminal application of ATP (10−7 to 10−5 mol/L) was determined in all 4 groups. Vascular diameter and either EC or SMC [Ca2+]i were assessed in parallel (see Measurement of Vascular Diameter and Calcium). In some experiments, a concentration-response curve to luminal application of the calcium ionophore A23187 (10−7 to 10−4 mol/L) was determined. To assess whether female MCAs were responsive to SMC hyperpolarization, 15 mmol/L KCl was added to the abluminal bath. This concentration of KCl has been demonstrated to produce inwardly rectifying potassium channel–dependent dilations in cerebral arteries.18 Experiments were terminated by replacing PSS with calcium-free PSS containing 1 mmol/L EGTA to obtain the maximum dilation of the vessel.
Reagents and Buffers
All chemicals were purchased from Sigma with the exception of fura 2-AM and Pluronic F-127 (TefLabs). The ionic composition of PSS contained the following (mmol/L): NaCl 119, NaHCO3 21, KCl 4.7, KH2PO4 1.18, MgSO4 1.17, CaCl2 1.6, glucose 5.5, and EDTA 0.026. MOPS buffer consisted of the following (mmol/L): NaCl 145, NaH2PO4 1.2, KCl 4.7, MgSO4 1.17, CaCl2 1.6, glucose 5, pyruvate 2, EDTA 0.02, and MOPS 3. The MOPS buffer was adjusted to pH 7.4 at room temperature. Stock solutions of ATP (10−2 mol/L) and L-NAME (3×10−2 mol/L) were prepared in distilled water, aliquoted, and then frozen. A stock solution of indomethacin (10−2 mol/L) was prepared in a solution of Na2CO3 and distilled water. Fura 2-AM was mixed with 50 μL dimethyl sulfoxide and 25 μL Pluronic F-127 in dimethyl sulfoxide.
Data Analysis and Calculations
All data are presented as mean±SEM. Both diameter and [Ca2+]i measurements were averaged over a 2-minute period immediately after luminal exposure to ATP. Changes in vascular diameter are presented as a percentage of the maximum diameter of the MCAs, calculated as follows equation
where DATP is the diameter of the MCA after luminal administration of ATP, Dbase is the baseline diameter of the MCA before addition of ATP, and Dmax is the maximal diameter of the MCA in the presence of calcium-free PSS. EC and SMC [Ca2+]i were determined as follows equation
where [Ca2+]i is EC or SMC calcium concentration, β is the ratio of 380 unbound/380 bound, Kd is the dissociation constant for fura 2 to Ca2+, R is the ratio of the 340/380 emission, Rmin is the ratio in calcium-free conditions, and Rmax is the ratio in calcium-saturating conditions. In situ calibration16 was performed on both intact male MCAs (n=8) and intact female MCAs (n=5). The calibration curves did not differ statistically and were therefore averaged to yield the following values: 2.017 (Rmax), 0.1365 (Rmin), and 5.218 (β). The in situ Kd was previously determined by Knot and Nelson19 to be 282 nmol/L.
Statistical comparisons of body weight changes and plasma estradiol concentration were performed with a 1-way ANOVA followed by a Bonferroni t test for multiple comparisons. Statistical comparisons of the concentration-response curves to ATP were performed with a 2-way ANOVA with repeated measures, and multiple comparisons were made with a Student-Newman-Keuls test. Comparisons of baseline [Ca2+]i were made with a 1-way ANOVA followed by a Bonferroni t test. Differences were considered significant at error probabilities <0.05 (P<0.05).
At 2 weeks after OVX and pump insertion, plasma estradiol levels were significantly elevated in estrogen-treated OVX females (36±7 pg/mL) compared with vehicle-treated OVX females (11±2 pg/mL; P<0.05, 1-way ANOVA). Estrogen-treated OVX females had also gained significantly less weight compared with vehicle-treated OVX females (1.9±5% versus 47±3%; P<0.05, 1-way ANOVA).
Endothelial Calcium Changes to EDHF- Mediated Dilations
Previous studies in our laboratory have shown that EDHF-mediated dilations are attenuated in the intact female and estrogen-treated OVX rat MCA.9 To determine whether this could be attributed to an insufficient elevation in EC [Ca2+]i, changes in vascular diameter and EC [Ca2+]i were measured simultaneously.
After the development of tone, resting MCA diameters were similar between groups: 241±9 μm (intact males), 230±3 μm (intact females), 231±15 μm (vehicle-treated OVX females), and 212±3 μm (estrogen-treated OVX females). Exposure to L-NAME and indomethacin resulted in the MCAs constricting by a similar amount between groups: 17±3% (intact males), 25±3% (intact females), 24±2% (vehicle-treated OVX females), and 19±3% (estrogen-treated OVX females).
Changes in diameter and EC [Ca2+]i in response to luminal delivery of ATP were then assessed in intact males, intact females (Figure 1), and vehicle-treated and estrogen-treated OVX females (Figure 3). ATP-induced dilations were significantly reduced in intact females and estrogen-treated OVX females compared with intact males and vehicle-treated OVX females (P<0.05, repeated-measures ANOVA) (Figures 1A and 3⇓A, respectively). Representative dynamic responses of MCAs isolated from intact males and females are shown in Figure 2A and 2B, respectively. The diameter of the pressurized MCAs was not affected by loading the endothelium with fura 2-AM. In the presence of L-NAME and indomethacin, resting EC [Ca2+]i was significantly greater in intact females (174±13 nmol/L) compared with intact males (126±10 nmol/L) and in estrogen-treated OVX females (113±15 nmol/L) compared with vehicle-treated OVX females (62±5 nmol/L) (P<0.05, 1-way ANOVA). However, in response to 10−5 mol/L ATP, EC [Ca2+]i increased to similar levels in intact males (421±77 nmol/L), intact females (461±116 nmol/L), vehicle-treated OVX females (530±92 nmol/L), and estrogen-treated OVX females (417±50 nmol/L) (P=NS, 1-way ANOVA) (Figures 1B and 3⇓B, respectively).
To rule out the possibility that there is modulation of calcium sensitivity at the level of the endothelium, MCAs were luminally exposed to the calcium ionophore A23187 in the presence of L-NAME and indomethacin. A23187 increases [Ca2+]i by directly facilitating Ca2+ influx into the cell. As shown in Figure 4, A23187-induced dilations were significantly attenuated in the intact female MCA compared with the intact male MCA.
Smooth Muscle Calcium Changes to EDHF-Mediated Dilations
To further dissect out the mechanisms associated with the effect of estrogen on EDHF-mediated dilations, changes in SMC [Ca2+]i in response to luminal application of ATP were assessed. The diameter of the pressurized MCAs was not affected by loading the SMCs with fura 2-AM. In the presence of L-NAME and indomethacin, resting SMC [Ca2+]i was comparable in intact males (264±6 nmol/L), intact females (237±12 nmol/L), vehicle-treated OVX females (221±10 nmol/L), and estrogen-treated OVX females (237±11 nmol/L) (Figures 5 and 6⇓). In response to luminal application of 10−5 mol/L ATP, SMC [Ca2+]i decreased in both intact males (170±9 nmol/L) and vehicle-treated OVX females (176±17 nmol/L), while it increased in intact females (244±15 nmol/L) and estrogen-treated OVX females (264±17 nmol/L). The increase in SMC [Ca2+]i reflects the fact that these vessels dilated transiently, followed by constriction. We therefore calculated the minimum value that SMC [Ca2+]i reached in response to 10−5 mol/L ATP and verified that SMC [Ca2+]i decreased to a greater degree in males (37±4%) compared with females (21±5%) and in vehicle-treated OVX females (18±7%) compared with and estrogen-treated OVX females (3±5%).
SMC hyperpolarization induced by 15 mmol/L KCl caused a comparable dilation in intact males (71±9%) and intact females (64±4%), with a similar corresponding decrease in SMC [Ca2+]i by 22±6% (intact males) and 24±3% (intact females). Comparable dilations were also observed in estrogen-treated (69±5%) and vehicle-treated OVX females (73±6%). The concomitant decrease in SMC [Ca2+]i was also comparable between estrogen-treated OVX females (29±2%) and vehicle-treated OVX females (30±4%).
The results of the present study suggest that a factor or mechanism coupling EDHF to reduction of ionized SMC [Ca2+]i accounts for the attenuated EDHF-mediated dilations in the female MCA. This conclusion is supported by 3 lines of evidence. First, EC [Ca2+]i reached sufficient levels to potentially elicit an EDHF response in all experimental groups. However, this was not accompanied by a robust dilation in intact females and estrogen-treated OVX females. Second, imposed increases in EC [Ca2+]i using the calcium ionophore A23187 failed to elicit a substantial EDHF-mediated dilation. Third, SMC [Ca2+]i decreased to a greater degree in intact males and vehicle-treated OVX females compared with intact females and estrogen-treated OVX females.
Endothelial Calcium Changes to EDHF- Mediated Dilations
In the presence of L-NAME and indomethacin, resting but not ATP-stimulated EC [Ca2+]i was significantly elevated in MCAs isolated from intact females and estrogen-treated OVX compared with intact males and vehicle-treated OVX. Our findings agree with those of Knot and colleagues,20 who found that in coronary arteries, basal EC [Ca2+]i but not acetylcholine-stimulated [Ca2+]i was significantly elevated in females compared with their male counterparts. Although they agree, the latter studies do not reflect an EDHF-mediated dilation. The acetylcholine response represented a purely NO-mediated dilation, while in the present study NO was inhibited and we are therefore observing an EDHF-mediated dilation. To substantiate this, we have previously shown that the ATP-induced dilation resistant to L-NAME and indomethacin in intact males and OVX females can be abolished either by denudation or inhibition of Ca2+-sensitive potassium channels (charybdotoxin).9
Our finding that basal EC [Ca2+]i is elevated in the MCA isolated from intact females and estrogen-treated OVX females deserves some attention. An elevated EC [Ca2+]i could suggest a greater driving force for calcium, perhaps instigated by greater hyperpolarization.21 Furthermore, the notion that female ECs are more hyperpolarized may offer an explanation for the attenuated EDHF-mediated dilations. The amplitude of the ATP-induced hyperpolarization depends on the resting membrane potential, as shown in cells with a more negative membrane potential, where the hyperpolarization in response to endothelium-dependent vasodilators was much smaller in magnitude.21–23⇓⇓ In other words, the magnitude of the agonist-induced hyperpolarization may be a result of the difference in resting membrane potential between male and female MCAs.
While EDHF-mediated increases in EC [Ca2+]i were sufficiently elevated to potentially elicit an EDHF-mediated dilation, a robust dilation was not observed in intact females and estrogen-treated OVX females. One could reason that a reduced calcium sensitivity at the level of the endothelium may account for this phenomenon. To address this possibility, the calcium ionophore A23187 was delivered to the endothelium in the presence of L-NAME and indomethacin. If there was a Ca2+ sensitivity issue at the level of the endothelium, one would expect that imposed increases in EC Ca2+ would elicit robust EDHF-mediated dilations. However, A23187 was also ineffective in eliciting a robust EDHF relaxation in intact females compared with intact males (Figure 4). Taken together, our data suggest that at the level of the endothelium, neither insufficient agonist-induced increases in [Ca2+]i nor calcium sensitivity can account for the attenuated EDHF-mediated dilations in the female rat MCA.
Smooth Muscle Calcium Changes to EDHF-Mediated Dilations
Resting SMC [Ca2+]i was comparable in MCAs isolated from intact males, intact females, vehicle-treated OVX females, and estrogen-treated OVX females (Figures 5 and 6⇑). However, in response to luminal ATP (10−5 mol/L), SMC [Ca2+]i decreased to a greater degree in males (37±4%) and vehicle-treated OVX females (18±7%) compared with females (21±5%) and estrogen-treated OVX females (3±5%). Previous studies have reported that basal levels of [Ca2+]i are reduced in aortic SMCs isolated from intact females and estrogen-treated OVX females.24 The disparity in the 2 studies is most likely a reflection of differences in vascular bed (MCA versus aorta), strain (Long-Evans versus Wistar-Kyoto rats), and experimental paradigm (pressurized versus nonpressurized vessels).
Since dilation to EDHF in the male MCA is mediated by hyperpolarization of the SMC,1,25⇓ we investigated the possibility that the attenuation of the EDHF-mediated dilation in females is attributed to lack of hyperpolarization of the SMC. However, exposure to 15 mmol/L KCl elicited decreases in SMC [Ca2+]i and a concomitant dilation, suggesting that females can respond appropriately to hyperpolarization of the SMC. Although this is indirect evidence, it suggests that hyperpolarization of the SMC is inadequate in the EDHF-mediated pathway to produce a dilation in intact female and estrogen-treated OVX female MCAs.
In conclusion, the results of the present study suggest that a factor or mechanism coupling EDHF to reduction of ionized SMC [Ca2+]i accounts for the attenuated EDHF-mediated dilations in the female MCA. Our results support the idea that there is inadequate hyperpolarization of the SMC in the female MCA to elicit a robust EDHF-mediated dilation. This study underscores the fact that sex-related differences in vascular reactivity exist and promotes new perspectives for clinical research.
This work was supported by American Heart Association National Scientist Development grant 0130250N.
- Received April 10, 2002.
- Revision received June 25, 2002.
- Accepted June 26, 2002.
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- ↵Golding EM, Kepler TE. Role of estrogen in modulating EDHF-mediated dilations in the female rat middle cerebral artery. Am J Physiol. 2001; 280: H2417–H2423.
- ↵White RM, Rivera CO, Davison CA. Nitric oxide-dependent and -independent mechanisms account for gender differences in vasodilation to acetylcholine. J Pharmacol Exp Ther. 2000; 292: 375–380.
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