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Department of Anesthesiology, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We tested the hypothesis that
endothelium-derived hyperpolarizing factor (EDHF) plays a less dominant
role in the female cerebrovasculature. The contribution of EDHF to the
ATP-mediated dilation was determined in middle cerebral arteries (MCAs)
isolated from male and female rats. Four groups of rats were tested:
intact male (n = 12), intact female (n = 13), estrogen-treated ovariectomized female (n = 13), and vehicle-treated ovariectomized female (n = 20)
rats. Maximal dilation to ATP was similar in all groups. However, in
the presence of
N
-nitro-L-arginine methyl ester
(L-NAME, 3 × 10
5 M) and indomethacin
(105 M), the maximal dilation to ATP was significantly
reduced in intact female (24 ± 9%) and estrogen-treated
ovariectomized female (29 ± 9%) rats compared with intact male
(95 ± 4%) and vehicle-treated ovariectomized female (96 ± 2%) rats. The ATP-mediated dilation in L-NAME- and
indomethacin-treated MCAs isolated from male and ovariectomized female
rats was inhibited by charybdotoxin (107 M), an inhibitor
of large-conductance Ca2+-sensitive K+
channels. We have defined EDHF as the L-NAME- and
indomethacin-insensitive component of the ATP-mediated dilation. Our
findings indicate that EDHF-mediated dilations are negligible in the
female rat MCA; these dilations can be significantly enhanced after
ovariectomy, suggesting that this effect is mediated by estrogen.
gender; vascular
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ESTROGEN HAS RECEIVED
CONSIDERABLE attention recently as a potential therapeutic agent
in various pathophysiological states. Although the mechanisms by which
estrogen mediates its protective effects are multifactorial, the
endothelium has been shown to play a pivotal role (see Ref.
12 for review). Endothelial cells play a fundamental role
in regulating vascular resistance and blood flow through their ability
to produce vasodilators such as nitric oxide (NO) and prostacyclin. In
addition to NO and prostacyclin, a third vasodilator, known as
endothelium-derived hyperpolarizing factor (EDHF), has recently been
described in the cerebrovasculature (21) and periphery
(5). EDHF is known to hyperpolarize and relax vascular
smooth muscle predominantly through activation of
Ca2+-dependent K+ (KCa) channels.
Although its identity remains elusive, this "eternally deceptive
hyperpolarizing factor" has recently sparked much attention (7). In the present study, we have defined EDHF as the
N-nitro-L-arginine methyl ester
(L-NAME)- and indomethacin-insensitive component of the
dilation to luminal application of ATP, mediated predominantly through
activation of KCa channels.
Although estrogen has been shown to upregulate NO (11, 13) and prostacyclin (1), no studies have investigated the effect of estrogen on EDHF in the cerebrovasculature. The justification for such studies is well documented. First, there is evidence in the periphery (6) and cerebral circulation (20) that EDHF plays an important role in controlling vascular resistance. Second, EDHF acts in a compensatory manner in endothelial NO synthase (eNOS)-knockout mice, where eNOS is absent (8, 16). Third, upregulation of EDHF in the cerebrovasculature has been reported after ischemia-reperfusion (9). These studies point to the fact that an equipoise between NO and EDHF may exist, such that, in the absence of NO, EDHF may assume a compensatory role. Given that NO is upregulated in females, we tested the hypothesis that EDHF-mediated dilations play a less dominant role in the cerebrovasculature of females than in males and that this effect is mediated by estrogen.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Experiments were carried out 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.
Surgery
Experiments were performed on age-matched (70-90 days old) male (275-324 g) and female (200-224 g) Long-Evans rats. All surgical procedures were undertaken under aseptic conditions. Animals were secured to a nose cone and allowed to breathe spontaneously (2% isoflurane). A heating pad and a temperature controller (Harvard Apparatus) were used to maintain rectal temperature at 37°C. A bilateral ovariectomy was then performed (17). A dorsal incision was made, and a mini-osmotic pump (model 2004, Alza, Palo Alto, CA) was subcutaneously implanted to deliver 17
-estradiol or
50% DMSO-0.045% NaCl (vehicle). 17-Estradiol was administered at a
physiological dose of 4 µg · kg1 · day1.
Validation of our model of ovariectomy and estrogen treatment consisted
of 1) monitoring body weight, 2) measuring plasma
estradiol concentrations, and 3) obtaining vaginal smears.
After completion of surgery, the animals' ears were tagged, wounds
were sutured, and the animals were returned to the holding facility for
2 wk.
Harvesting and Mounting Cerebral Vessels
Animals were anesthetized with isoflurane and then decapitated. The brain was removed from the cranium and placed in ice-cold physiological salt solution (PSS). The middle cerebral artery (MCA) was carefully harvested, cleaned of surrounding connective tissue, and cannulated with micropipettes in an arteriograph as described elsewhere in detail (3). PSS was circulated abluminally and perfused luminally. Monitoring of intraluminal pressure was performed from in-line transducers that were connected to two strain gauge panel meters (Omega, Stamford, CT). Once mounted, vessels were tested for leaks by clamping the proximal and distal tubing, and intraluminal pressure was monitored. Vessels that did not maintain a steady pressure were discarded. Transmural pressure was set at 85 mmHg with a flow of 200 µl/min through the lumen, and the vessels were allowed to equilibrate for 1 h. During this time, the vessels develop spontaneous tone by constricting from their fully dilated diameter at initial pressurization. After the development of tone, the experiment was initiated (see Experimental Protocol).Measurement of MCA Diameter
Changes in vessel diameter were observed with an inverted microscope (Nikon) equipped with a videocamera and monitor. Outer diameters were measured directly online from the video monitor (final magnification ×600). Dynamic changes in vessel diameter were digitized (post hoc) using image-analysis software (Optimas, Bothell, WA), with an acquisition frequency of 1.1 Hz allowed.Experimental Groups
Four groups of rats were used in the present study: 1) intact male, 2) intact female, 3) estrogen-treated ovariectomized female, and 4) vehicle-treated ovariectomized female rats. Ovariectomy was performed and drug treatment was initiated 2 wk before isolation of the cerebral vessels.Measurement of Plasma Estradiol
Immediately after decapitation, trunk blood (3 ml) was obtained from all animals. The blood was centrifuged for 3 min at 8,000 rpm, and the resulting plasma was frozen at
15°C. 17-Estradiol was
measured at a later time using an ultrasensitive RIA (Diagnostic Systems Laboratory, Webster, TX).
Experimental Protocol
Experiments were conducted using isolated MCAs by luminal application of ATP, an agonist of the P2Y2 purinoceptor, which elicits a dilation in male cerebral vessels through the production of NO and EDHF (21). A concentration-response curve (CRC) to luminal application of ATP (108-104 M) was determined in all four
groups. Only one CRC to ATP was conducted for each vessel to avoid
tachyphylaxis. In some vessels, dilations to 106 M ATP
were assessed before and after removal of the endothelium (10 ml air)
to confirm that the entire response was mediated by the endothelium. At
the end of the experiment, integrity of the smooth muscle was confirmed
using 105 M
S-nitroso-N-acetylpenicillamine (SNAP). In other
vessels, the CRC to ATP was conducted in the presence of
L-NAME (3 × 105 M) and indomethacin
(105 M) to determine the non-NO, non-prostacyclin
component of the dilation. Such a dilation is consistent with an EDHF
response. In vessels exhibiting a non-NO, non-prostacyclin dilation,
charybdotoxin (107 M) was delivered abluminally to
inhibit the large-conductance KCa channels. Experiments
were terminated with Ca2+-free PSS to determine the maximum
dilation of the vessel.
Reagents and Drugs
The PSS consisted of (in mM) 119 NaCl, 24 NaHCO3, 4.7 KCl, 1.18 KH2PO4, 1.17 MgSO4, 1.6 CaCl2, 5.5 glucose, and 0.026 EDTA. It was gassed with 5% CO2-21% O2-balance N2. After the solution was gassed, the pH of the buffer was ~7.40. All drugs were purchased from Sigma (St. Louis, MO) with the exception of MAHMA NONOate, which was purchased from Alexis Biochemicals (San Diego, CA). Stock solutions of ATP (102 M), adenosine
(102 M), UTP (102 M), UDP
(102 M), L-NAME (3 × 102
M), and charybdotoxin (2 × 105 M) were prepared in
distilled water, aliquotted, and then frozen. A stock solution of
indomethacin (102 M) was prepared in a solution of
Na2CO3 and distilled water (1:1 by weight). A
stock solution of
6-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine (MAHMA NONOate) was prepared in 0.01 M NaOH.
Data Analysis and Calculations
Values are means ± SE. Absolute diameter comparisons were made using a one-way ANOVA followed by a Fisher test for multiple comparisons. For CRC to ATP, the results are presented as a percentage of the maximum diameter of the MCA, as calculated according to Eq. 1
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(1) |
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(2) |
In the presence of L-NAME and indomethacin, MCA diameter responses to ATP were analyzed using two different methods. Because these responses were very dynamic, two analysis methods provided further validation and increased confidence in our findings. Diameter changes were first analyzed by calculating a time-weighted mean from the observed maximum and minimum diameters during the first minute of drug exposure. The diameter responses were then digitized using Optimas software and integrated using Sigma Plot software during the first 5 min of drug exposure. This allowed us to take into consideration the duration, amplitude, and dynamic nature of the responses.
Statistical comparisons of body weight changes were performed using a two-way ANOVA with repeated measures. Comparisons of plasma estradiol concentration and EC50 values were made using a one-way ANOVA. Statistical comparisons of the CRC to ATP were performed using a two-way ANOVA with repeated measures. Post hoc comparisons were made using a Student-Newman-Keuls test. Differences were considered significant at P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Model Validation
Body weight.
Changes in body weight over the 2-wk period are shown in Fig.
1. Intact female and estrogen-treated
ovariectomized female rats showed a similar increase in body weight of
3 ± 1 and 4 ± 2%, respectively (not significant; 2-way
repeated-measures ANOVA). Intact male and vehicle-treated
ovariectomized female rats also demonstrated a similar increase in body
weight of 16 ± 2 and 14 ± 1%, respectively, that was
significantly greater than that of intact female and estrogen-treated
ovariectomized female rats (P < 0.05, 2-way
repeated-measures ANOVA).
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Plasma estrogen.
Plasma estrogen values of all experimental groups are shown in Table
1. Intact female and estrogen-treated
ovariectomized female rats had significantly higher plasma
17-estradiol concentrations than intact male and vehicle-treated
ovariectomized female rats (P < 0.05, 1-way ANOVA).
There was no significant difference in plasma 17-estradiol
concentration between intact female and estrogen-treated ovariectomized
female rats, suggesting that we had successfully restored physiological
levels of estrogen in ovariectomized animals. Furthermore, there was no
significant difference in plasma 17-estradiol levels in intact male
and vehicle-treated ovariectomized female rats, indicating successful
removal of physiological levels of estrogen in these latter animals.
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EDHF-Mediated Dilations
After the development of tone, the resting MCA diameters were similar among the groups: 205 ± 4, 212 ± 9, 219 ± 5, and 218 ± 7 µm in intact male, intact female, vehicle-treated ovariectomized female, and estrogen-treated ovariectomized female rats, respectively. The dilation to luminal application of ATP in all vessels is shown in Fig. 2. Vessel diameter measurements were calculated using a time-weighted mean (see Data Analysis and Calculations). Although the maximal dilation to ATP was similar in all groups (Table 2), the dilation to 106 M ATP was significantly lower in
ovariectomized female rats with estrogen and vehicle treatment
(P < 0.05 compared with intact male and intact female
rats, 2-way repeated-measures ANOVA). Therefore, the EC50
of MCAs isolated from ovariectomized female rats with both estrogen and
vehicle treatment was significantly greater than that of MCAs isolated
from intact male and female rats (P < 0.05, 1-way
ANOVA).
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To verify that the observed ATP-mediated dilations in MCAs from male
and female rats were originating from the endothelium, dilations to
106 M ATP were assessed before and after removal of the
endothelium. As shown in Fig. 3, removal
of the endothelium virtually abolished the dilation to luminal delivery
of ATP in MCAs from male and female rats. Integrity of the smooth
muscle was confirmed at the end of the experiment with
105 M SNAP. Denudation constricted MCAs from intact male
(12 ± 1%) and female (15 ± 3%) rats to a similar degree.
Therefore, removal of the endothelium, another means of eliminating NO,
confirmed our L-NAME findings suggesting that basal NO
release was similar between MCAs from intact male and female rats. The
sensitivity to NO was compared in MCAs isolated from intact male and
female rats by running CRCs (109-105
M) to the spontaneous NO donor, MAHMA NONOate, in the presence of
L-NAME (3 × 105 M) to eliminate
endogenous sources of NO. Dilations to MAHMA NONOate were similar in
MCA from male and female rats (data not shown).
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After considering the possibility that ATP could be uniquely degraded
to adenosine in the female rat MCA, we challenged these vessels to
adenosine (108-104 M CRC). The
dilation to adenosine was significantly less than that to ATP (Fig.
4). Because we observed robust dilations
to luminal application of ATP, this suggests that ATP is not degraded to adenosine in our preparation. This has been shown previously in the
male rat MCA (19).
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After the application of L-NAME and indomethacin, the MCAs
constricted by a similar amount between groups: 42 ± 2, 38 ± 3, 40 ± 1, and 39 ± 4% in intact male, intact female,
vehicle-treated ovariectomized female, and estrogen-treated
ovariectomized female rats, respectively. In the presence of
L-NAME and indomethacin, maximal dilation to ATP was
similar in intact male and vehicle-treated ovariectomized female rats
compared with dilation in the absence of L-NAME and
indomethacin. The dilation to ATP in the presence of L-NAME
and indomethacin in intact female and estrogen-treated ovariectomized
female rats was significantly attenuated (P < 0.05 compared with male and vehicle-treated ovariectomized female rats, statistical power = 100%, 2-way repeated-measures ANOVA; Fig. 5). The EC50 and maximal
dilation were not calculated in these groups, because we did not
observe a plateau in the dilation. The EC50 was
significantly shifted to the right in intact male and vehicle-treated
ovariectomized female rats (P < 0.05 compared with all groups in the absence of L-NAME and indomethacin,
1-way ANOVA).
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The dynamic response of vessels to 105 and
104 M ATP in the presence of L-NAME and
indomethacin is shown in Fig. 6. The
response can be very dynamic. For this reason, the integrated mean of
the vessel responses was then calculated (see Data Analysis and
Calculations). The integrated response to 105 and
104 M ATP was significantly lower in intact female
and estrogen-treated ovariectomized female rats
(P < 0.05 compared with male and vehicle-treated ovariectomized female rats, 2-way repeated-measures ANOVA; Fig. 7). This confirms the results from our
previous analysis in which we used a time-weighted mean. Figure
8 demonstrates that charybdotoxin, an
inhibitor of large-conductance KCa channels, abolished the non-NO component of the dilation at 105 M ATP and
significantly attenuated the dilation at 104 M ATP in
MCAs isolated from intact male and vehicle-treated ovariectomized female rats. The persistent dilation at 104 M ATP may
likely evolve from nonspecific effects of ATP at this concentration,
since this effect was observed in male and female rats.
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In supplementary studies, we used a distinct P2Y2 receptor agonist, UTP, to confirm our findings with ATP in the MCA from the intact female rat. Dilations to luminal application of UTP in the presence and absence of L-NAME and indomethacin replicated our findings with ATP (Fig. 4). Luminal exposure to UDP failed to elicit a dilation and, indeed, produced a constriction (Fig. 4). The lack of vasodilatory ability of UDP is consistent with that shown previously in the MCA from the male rat (19).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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In the present study, we have shown that EDHF-mediated dilations to ATP are, for all practical purposes, nonexistent in the female rat MCA. EDHF-mediated dilations were significantly enhanced in the ovariectomized female, however, suggesting that estrogen mediates this effect. A number of mechanisms could be mediating this observation in the MCA of the female rat, including the release of EDHF, smooth muscle sensitivity to EDHF, and changes in the half-life of EDHF(s).
This is the first study to describe gender-specific differences regarding EDHF-mediated dilations in the cerebrovasculature. In the periphery, there are two reports suggesting that the active release of EDHF to muscarinic receptor stimulation is increased in the female rat mesenteric arterial bed (10, 18). At the outset, these data may appear to be in conflict with the present data; however, it is important to realize that there are numerous indications that the cellular modes of action of EDHF show tissue and species variability (6). Although the identity of EDHF remains elusive, it has been defined as 1) being derived from the endothelium, 2) not NO, 3) not prostacyclin or a cyclooxygenase metabolite, 4) hyperpolarizing the vascular smooth muscle, and 5) activating K+ channels on the vascular smooth muscle (2, 4, 14). Possible candidates of EDHF include epoxides, anandamide, K+, and an electrical coupling mediated by myoendothelial gap junctions (7). Indeed, EDHF may not be a single entity but, rather, a number of factors or, alternatively, a biochemical process. Regardless of the identity of EDHF, its significance has recently received much attention. Upregulation of EDHF has been reported after ischemia-reperfusion (9) and head trauma (6a), suggesting a compensatory role in the absence of NO. There is evidence in the periphery (6) and cerebral circulation (20) that EDHF is a prominent relaxing factor in smaller arteries. However, more work is required to define the significance of EDHF in controlling cerebrovascular function.
It is unknown at this stage why EDHF-mediated dilations are attenuated in the female cerebrovasculature; however, estrogen has been shown to directly activate the large-conductance K+ channels (15). Because these channels play an integral role in mediating EDHF (6), it could be envisaged that estrogen acts to desensitize these channels, thereby contributing to the negligible EDHF-mediated dilations in females.
An interesting finding in the present study is that ovariectomized female rats (with estrogen and vehicle treatment) demonstrated a significantly increased EC50 compared with male and intact female rats without an effect on the maximal dilation to ATP. This suggests that MCAs isolated from ovariectomized animals have a reduced sensitivity to ATP. The reason for this finding is not known; however, one could speculate that surgical stress and/or anesthesia effects may be responsible. Alternatively, replacement with exogenous estrogen and progesterone in ovariectomized animals may prevent these differences; however, further studies are required to verify this hypothesis.
In summary, the present study has shown that EDHF-mediated dilations in the female rat MCA are negligible and that this effect is mediated by estrogen. Although estrogen most likely employs multiple signaling mechanisms to alter vascular physiology in the brain, the present report should set the stage for future studies in this area of investigation.
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ACKNOWLEDGEMENTS |
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The authors are grateful to Dr. Robert Bryan, Jr., for an invaluable critique of the manuscript and to Dr. Heather Giles (GlaxoWellcome) for advice regarding curve fitting.
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FOOTNOTES |
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This work was supported by National Institute of Neurological Disorders and Stroke Grants PO1-NS-27616 and RO1-NS-37250.
Address for reprint requests and other correspondence: E. M. Golding, Dept. of Anesthesiology, One Baylor Plaza, Suite 434D, Houston, TX 77030 (E-mail: egolding{at}bcm.tmc.edu).
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. Section 1734 solely to indicate this fact.
Received 5 September 2000; accepted in final form 3 January 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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A. Sato, H. Miura, Y. Liu, L. B. Somberg, M. F. Otterson, M. J. Demeure, W. J. Schulte, L. M. Eberhardt, F. R. Loberiza, I. Sakuma, et al. Effect of gender on endothelium-dependent dilation to bradykinin in human adipose microvessels Am J Physiol Heart Circ Physiol, September 1, 2002; 283(3): H845 - H852. [Abstract] [Full Text] [PDF]
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 E. M. Golding, S. P. Marrelli, J. You, and R. M. Bryan Jr Endothelium-Derived Hyperpolarizing Factor in the Brain: A New Regulator of Cerebral Blood Flow? Stroke, March 1, 2002; 33(3): 661 - 663. [Full Text] [PDF]
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L. A. Schildmeyer and R. M. Bryan Jr. Effect of NO on EDHF response in rat middle cerebral arteries Am J Physiol Heart Circ Physiol, February 1, 2002; 282(2): H734 - H738. [Abstract] [Full Text] [PDF]
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H. L. Xu, R. A. Santizo, H. M. Koenig, and D. A. Pelligrino Chronic estrogen depletion alters adenosine diphosphate-induced pial arteriolar dilation in female rats Am J Physiol Heart Circ Physiol, November 1, 2001; 281(5): H2105 - H2112. [Abstract] [Full Text] [PDF]
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W. B. Campbell and D. R. Harder Prologue: EDHF-what is it? Am J Physiol Heart Circ Physiol, June 1, 2001; 280(6): H2413 - H2416. [Full Text] [PDF]
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