Am J Physiol Heart Circ Physiol
281: H2105–H2112, 2001.
Chronic estrogen depletion alters adenosine diphosphateinduced pial arteriolar dilation in female rats
H. L. XU, R. A. SANTIZO, H. M. KOENIG, AND D. A. PELLIGRINO
Neuroanesthesia Research Laboratory, University of Illinois at Chicago, Chicago, Illinois 60607
Received 27 February 2001; accepted in final form 27 July 2001
sion (15, 21), while negatively affecting the expression
of the endogenous eNOS inhibitor caveolin-1 (21).
In the present study, using a closed cranial window
system and intravital microscopy, we sought to corroborate the aforementioned estrogen influence on cerebrovascular eNOS function by monitoring pial arteriolar reactivity to another purported eNOS-dependent
vasodilator ADP (14, 22). Intact females, Ovx females,
and 17␤-estradiol (E2)-treated Ovx females were studied. The eNOS-dependent portion of the ADP response
was ascertained by comparing ADP-induced pial arteriolar dilations before and after suffusion of a NOS
inhibitor, N␻-nitro-L-arginine (L-NNA). With the use of
a series of established pharmacological inhibitors, additional experiments were performed to compare, in
intact and OVX rats, potential contributions from: 1)
prostanoids [indomethacin (Indo) applications], 2)
Ca2⫹-dependent K⫹ (KCa) channels [iberiotoxin (IbTX)],
3) ATP-regulated K⫹ (KATP) channels (glibenclamide),
4) the KCa-regulating cytochrome P-450 epoxygenase
pathway (miconazole), and 5) by virtue of possible
ectonucleotidase actions toward ADP, adenosine receptor activation [8-sulfophenyltheophylline (8-SPT)].
METHODS
WE RECENTLY REPORTED (21) that estrogen depletion via
ovariectomy in rats was accompanied by a virtually
complete loss of pial arteriolar responses to the endothelial nitric oxide (NO) synthase (eNOS)-dependent
vasodilator acetylcholine (ACh). Those responses were
restored in ovariectomized (Ovx) rats given chronic
estrogen replacement. The apparent estrogen-associated changes in eNOS function probably can be attributed, at least in part, to the reported ability of estrogen
in cerebral vessels to positively influence eNOS expres-
The study protocol was approved by the Institutional Animal Care and Use Committee. In initial experiments, three
groups of Sprague-Dawley rats (250–350 g) were used: intact
female, ovariectomized (Ovx), and E2-treated (0.1 mg 䡠 kg⫺1 䡠
day⫺1 ip for 1–2 wk) Ovx rats (Ovx ⫹ E2). The vendor
(Charles River) performed the ovariectomies 1 wk before the
rats were shipped. The Ovx rats were then used at 4–6 wk
postovariectomy. As shown in a previous report from our
laboratory (21), the 0.1 mg 䡠 kg⫺1 䡠 day⫺1 dose results in an
average daily plasma E2 concentration that falls between the
peak and nadir levels observed over the normal rat estrous
cycle. Further details regarding animal treatments are provided in earlier publications from our laboratory (21, 23, 26).
Anesthesia was induced with halothane. The rats were then
paralyzed with curare, tracheotomized, and mechanically
ventilated. For surgery, anesthesia was maintained with
0.8% halothane and 70% N2O-30% O2. Catheters were placed
in the femoral artery to measure systemic arterial pressure
and to obtain arterial blood samples, and in the femoral vein
for infusion of drugs. Rectal temperature was maintained at
37°C with a servo-controlled heating pad. The rats were then
Address for reprint requests and other correspondence: D. A.
Pelligrino, Neuroanesthesia Research Laboratory, Univ. of Illinois at
Chicago, MBRB (M/C 513), 900 S. Ashland Ave., Chicago, IL 60607
(E-mail: [email protected]).
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.
ATP-sensitive K⫹ channel; Ca2⫹-dependent K⫹ channel; endothelium-derived hyperpolarizing factor; miconazole; nitric
oxide
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0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society
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Xu, H. L., 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 281: H2105–H2112, 2001.—
We examined pial arteriolar reactivity to a partially endothelial nitric oxide synthase (eNOS)-dependent vasodilator ADP
as a function of chronic estrogen status. The eNOS-dependent portion of the ADP response was ascertained by comparing ADP-induced pial arteriolar dilations before and after
suffusion of a NOS inhibitor, N␻-nitro-L-arginine (L-NNA; 1
mM) in intact, ovariectomized (Ovx), and 17␤-estradiol (E2)treated Ovx females. We also examined whether ovariectomy
altered the participation of other factors in the ADP response. Those factors were the following: 1) the prostanoid
indomethacin (Indo); 2) the Ca2⫹-dependent K⫹ (KCa) channel, iberiotoxin (IbTX); 3) the ATP-regulated K⫹ (KATP) channel glibenclamide (Glib); 4) the KCa-regulating epoxygenase
pathway miconazole (Mic); and 5) the adenosine receptor
8-sulfophenyltheophylline (8-SPT). In intact females, the
eNOS-dependent (L-NNA sensitive) portion of the ADP response represented ⬃50% of the total. The ADP response was
retained in the Ovx rats but L-NNA sensitivity disappeared.
On E2 replacement, the initial pattern was restored. ADP
reactivity was unaffected by Indo, Glib, Mic, and 8-SPT. IbTX
was associated with 50–80% reductions in the response to
ADP in the intact group that was nonadditive with L-NNA,
and 60–100% reductions in the Ovx group. The present
findings suggest that estrogen influences the mechanisms
responsible for ADP-induced vasodilation. The continued
sensitivity to IbTX in Ovx rats, despite the loss of a NO
contribution, is suggestive of a conversion to a hyperpolarizing factor dependency in the absence of E2.
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any inhibitor additions. No changes from the initial ADP or
SNAP response were observed (data not shown). Mean arterial blood pressure (MABP) was continuously monitored and
arterial blood samples were taken at 30-min intervals for
measurement of PO2, PCO2, and pH by using a blood gas/pH
analyzer (model ABL 520, Radiometer; Copenhagen, Denmark). The rats were euthanized with a halothane overdose
at the termination of the experiment.
Statistical comparisons of pial arterioles diameter values
within groups were made using one-way analysis of variance
combined with a post hoc Tukey’s test. A level of P ⬍ 0.05 was
considered significant in all statistical tests. Values are presented as means ⫾ SE.
ADP, L-NNA, Indo, IbTX, miconazole, and 8-SPT were
purchased from Sigma (St. Louis, MO), glibenclamide was
obtained from Research Biochemicals (Natick, MA). ADP,
L-NNA, and miconazole were prepared in aCSF. Indo was
dissolved by sonication in a Na2CO3 solution. IbTX was
dissolved in ethanol and diluted in aCSF before suffusion.
The stock glibenclamide was made by initially dissolving this
agent in a small amount of dimethyl sulfoxide and the balance in ethanol. This vehicle was then diluted 1:1,000 in
aCSF to make the working solution. All concentrations were
expressed as the final molar concentration suffused under
the cranial window.
RESULTS
In animals from both experimental series, the arteriolar PCO2, pH, and MABP did not show any significant variations over the length of the study. Thus,
initial and final pH, PCO2, and MABP values were (in
mmHg) 7.38 ⫾ 0.01 and 7.37 ⫾ 0.01, 38.8 ⫾ 1.2 and
34.6 ⫾ 1.7, and 125 ⫾ 3 and 132 ⫾ 2, respectively. PO2
was maintained above 100 mmHg during all experiments.
No significant changes in pial arteriolar diameters
were observed in the presence of 1 ␮M ADP. Thus, in
the results described below, only the findings obtained
during suffusions of 10 and 100 ␮M ADP are reported.
The results of series 1 experiments are shown in Fig. 1.
The initial ADP response evaluations revealed similar
dose-dependent vasodilations (at 10 and 100 ␮M ADP)
in the intact (17 ⫾ 2 and 28 ⫾ 2%, respectively), Ovx
(14 ⫾ 1 and 22 ⫾ 2%, respectively), and Ovx ⫹ E2 (15 ⫾
2 and 28 ⫾ 3%, respectively) groups. In all groups, pial
arteriolar diameters reverted to pre-ADP levels within
10 min after return to control aCSF. In intact females,
topical application of the NOS inhibitor L-NNA (1 mM)
was accompanied by significant 60 and 41% reductions
in pial arteriolar dilations at 10 and 100 ␮M ADP,
respectively. No reductions in the ADP response at any
dose were observed in the Ovx group. However, in the
E2-treated Ovx females, the L-NNA sensitivity was
restored, to the extent that the response was reduced
by 81 and 50% at 10 and 100 ␮M ADP, respectively.
Before ADP suffusion, application of L-NNA for 60 min
was associated with virtually no change in pial arteriolar diameters (data not shown).
The results of series 2 experiments are summarized
in Figs. 2–7. In the cyclooxygenase inhibition experiments (Fig. 2), before administration of Indo, ADP
elicited statistically similar dose-dependent responses
in intact and Ovx females (18 ⫾ 1 vs. 14 ⫾ 2%, at 10
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prepared for placement of a closed cranial window, according
to established procedures (21, 23). Briefly, the surface of the
skull was exposed after a midline scalp incision. A craniotomy (10-mm diameter) was performed over the midline of the
skull, and the dura was carefully removed, keeping the sagittal sinus intact. An acrylic cranial window equipped with
three ports (inflow, outflow, and intracranial pressure monitoring) was fixed to the skull with cyaroacrylate gel. After
the surgery, the halothane was discontinued and a loading
dose of intravenous fentanyl (10 ␮g/kg) was given. The rat
was maintained on 70% N2O-30% O2 and fentanyl (25
␮g 䡠 kg⫺1 䡠 h⫺1 iv) thereafter. The space under the window was
filled with artificial cerebrospinal fluid (aCSF) that was
equilibrated with 10% O2-5% CO2, with a balance of N2 (9).
The aCSF was suffused at 0.5 ml/min and was maintained at
a temperature of 37°C. Cerebral arterioles were observed
using a microscope equipped with a video camera. Magnifications of ⫻800 were displayed on a video monitor. Measurements of arteriole diameters were made using a calibrated
video microscaler.
In all experiments, initial diameter measurements were
made after a 40-min period of cortical suffusion with drugfree aCSF (at 1 h after halothane). Hypercapnia (PaCO2, ⬃65
mmHg) was then imposed for 3 min, and the arterioles
displaying an adequate response to CO2 (pial arteriolar “reactivity” ⬎1.0% diameter increase/mmHg CO2 change) were
used in each experiment. In the first series of experiments,
ADP was suffused at concentrations of 1, 10, and 100 ␮M (10
min at each level). Baseline diameters were then restored via
10- to 15-min suffusion of drug-free aCSF. At this time, the
nonselective NOS inhibitor, N␻-nitro-L-arginine (L-NNA, 1
mM), was introduced into the suffusate and applied for 60
min. The ADP suffusion sequence was then repeated.
In the second series of experiments, only intact and Ovx
females were evaluated. Series 2 rats were further divided
into five subgroups, according to drug treatment: 1) cyclooxygenase inhibitor, 2) KCa channel blocker, 3) KATP channel
blocker, 4) cytochrome P-450 epoxygenase inhibitor, and 5)
adenosine receptor antagonist. After suffusions of ADP (1,
10, and 100 ␮M), baseline diameters were restored with
suffusion of drug-free aCSF for 10 min. At this point, experimental protocols in the different groups diverged. In group
2.1, the effect of indomethacin on the pial arteriolar response
to ADP was examined before and 20 min after an intravenous
injection of indomethacin (10 mg/kg). After return to baseline, L-NNA (1 mM) was added to the suffusate, and the ADP
response was evaluated after 45–60 min. In group 2.2, before
ADP infusion, IbTX (0.1 ␮M) was suffused for 30 min. After
the return to IbTX suffusion alone, L-NNA (1 mM) was added,
and the ADP response was evaluated 45–60 min later. In
one-half of the animals, the pial arteriolar responses to suffusions of the NO donor S-nitroso-N-acetylpenicillamine
(SNAP; 0.1 and 1.0 ␮M) were measured. SNAP was administered before ADP both in the absence and then in the
presence of IbTX. In group 2.3, glibenclamide (5 ␮M) was
suffused for 10 min before cosuffusion with ADP. In group
2.4, miconazole (10 ␮M) was added to the suffusate, and ADP
cosuffusion was initiated 20 min later. In group 2.5, a slight
modification in the initial vasodilator application sequence
was introduced. Thus, adenosine (100 ␮M) was suffused for 5
min, and then after 10 min of drug-free aCSF, the ADP
application was initiated. The general adenosine receptor
antagonist 8-SPT (10 ␮M) was then suffused for 15 min
before repeating the additions of adenosine and ADP. Several
intact and Ovx females were utilized as time controls, where
ADP and SNAP responses were evaluated in the absence of
ESTROGEN AND CEREBRAL VASODILATION
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Fig. 1. Pial arteriolar diameter increases (expressed as a percentage
of the baseline diameters) during suffusions of increasing concentrations of ADP, both in the absence (initial) and in the presence of
topically applied N␻-nitro-L-arginine (L-NNA; 1 mM). Results for
intact (A), ovariectomized (B), and 17␤-estradiol (E2)-treated ovariectomized (C) females are presented. Baseline diameters were 36.7 ⫾
5.9, 37.5 ⫾ 1.9, and 37.9 ⫾ 2.1 ␮m for the intact, ovariectomized, and
E2-treated ovariectomized rats, respectively. Values are means ⫾
SE. *P ⬍ 0.05 vs. initial; n ⫽ 4–5 rats per group.
␮M; and 37 ⫾ 3 vs. 24 ⫾ 1% at 100 ␮M, respectively).
Treatment with Indo had no effect on baseline diameters (data not shown), and did not alter responses of
pial arterioles to ADP in either group. When L-NNA
was subsequently added to the suffusate, baseline diameters were not altered but the ADP response in the
intact females was reduced to an extent (68 and 51% at
10 and 100 ␮M, respectively) similar to that seen in the
data summarized in Fig. 1. The addition of L-NNA had
no effect on the ADP response in Ovx females. In all
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Fig. 2. ADP-induced pial arteriolar diameter increases in intact (A)
and ovariectomized (B) females before (initial) and after indomethacin (Indo; 10 mg/kg iv), and then Indo plus topically applied L-NNA
(1 mM). Baseline diameters were 38.8 ⫾ 4.4 and 37.5 ⫾ 7.5 ␮m for
the intact and ovariectomized rats, respectively. Values are means ⫾
SE. *P ⬍ 0.05 vs. initial and Indo; n ⫽ 4 rats per group.
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cases, pre-ADP diameters were rapidly restored on
cessation of ADP suffusions.
In experiments examining the roles of KCa and KATP
channels in ADP-induced vasodilation in intact and
Ovx rats, contrasting findings were obtained. The results of the experiments examining KCa channel contributions are given in Fig. 3. Consistent with the
results described above, no differences in the initial
reactivities to 10 and 100 ␮M ADP were seen in the
intact versus Ovx females (17 ⫾ 2 vs. 18 ⫾ 2% at 10
␮M, and 32 ⫾ 3 vs. 35 ⫾ 5% at 100 ␮M, respectively).
IbTX alone did not significantly alter baseline diameters, although modest (2–6%) reductions were observed (not shown). However, IbTX was found to have
a significant effect on ADP reactivities in both groups.
Thus, at 10 ␮M ADP, the diameter increases were
reduced by 76% and 97% in the intact and Ovx females,
respectively. At 100 ␮M ADP, the diameter increases
were reduced by 45% and 59%, respectively. After ADP
withdrawal, and in the presence of continued IbTX
suffusion, baseline diameters were restored. At this
point, a cosuffusion of IbTX and L-NNA was initiated
and ADP reactivities were subsequently retested. No
further changes in ADP reactivities were observed,
compared with those measured in the presence of IbTX
alone. The most important information revealed by
this experimental manipulation (coupled to the results
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ESTROGEN AND CEREBRAL VASODILATION
provided in Fig. 1) is that the NO- and KCa-dependent
portions of the ADP response in intact females completely overlap. Ostensibly, these findings might be
explained by a mechanism whereby NO elicits pial
arteriolar dilations in rats via a KCa-dependent process
(19). However, recent findings (25) from our laboratory
indicated that SNAP-induced pial arteriolar relaxation
in male rats was unaffected by IbTX. Therefore, we
examined the effects of IbTX on SNAP-induced pial
arteriolar dilations in intact and Ovx females. Suffusions of SNAP at 0.1 and 1.0 ␮M produced identical
pial arteriolar responses in the intact and Ovx females
(Fig. 4). Furthermore, those initial responses were significantly reduced (by ⬃50% at 0.1 ␮M SNAP and by
30–40% at 1.0 ␮M SNAP) in both groups in the presence of IbTX.
A rather different result was obtained in experiments investigating the role of KATP channels (Fig. 5).
Before the addition of glibenclamide, ADP, at 10 and
100 ␮M, elicited nearly identical diameter increases in
both groups (16 ⫾ 2 vs. 14 ⫾ 2% at 10 ␮M and 29 ⫾ 3
vs. 31 ⫾ 3% at 100 ␮M, in the intact vs. Ovx females,
respectively). Glibenclamide by itself elicited no
changes in baseline diameters (not shown) and did not
affect ADP-induced dilations in either group.
To examine whether the KCa dependency of the ADP
response in Ovx females (and, for comparison, intact
females) relates in any way to the generation of cytochrome P-450 epoxygenase products, which have been
AJP-Heart Circ Physiol • VOL
Fig. 4. Pial arteriolar diameter increases elicited by increasing suffusate concentrations of the nitric oxide donor, S-nitroso-N-acetyl
penicillamine (SNAP), in intact (A) and ovariectomized (B) rats, in
the absence (initial) and in the presence of topically applied IbTX (0.1
␮M). Baseline diameters were 40.4 ⫾ 2.0 and 33.8 ⫾ 0.6 ␮m for
intact and ovariectomized rats, respectively. Values are means ⫾ SE.
*P ⬍ 0.05 vs. initial; n ⫽ 4 rats per group.
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Fig. 3. Pial arteriolar diameter increases elicited by increasing suffusate concentrations of ADP, in intact (A) and ovariectomized (B)
rats, in the absence (initial) and in the presence of topically applied
iberiotoxin (IbTX; 0.1 ␮M), followed by combined suffusion of IbTX
and L-NNA (1 mM). Baseline diameters were 38.9 ⫾ 2.1 and 36.0 ⫾
1.1 ␮m for the intact and ovariectomized rats, respectively. Values
are means ⫾ SE. *P ⬍ 0.05 vs. initial; n ⫽ 8 rats per group.
linked to KCa channel activation (7), we tested whether
the epoxygenase inhibitor miconazole (10 ␮M) had any
effect on ADP reactivities. At 10 ␮M, miconazole has
been shown to be a selective epoxygenase inhibitor,
with no effects on NOS activity (1). Before miconazole
was given, and identical to the findings given in Figs.
1–5, statistically similar ADP responses were seen in
the intact and Ovx females (17 ⫾ 2 vs. 13 ⫾ 3% at 10
␮M and 27 ⫾ 2 vs. 25 ⫾ 3% at 100 ␮M, respectively).
Miconazole had no significant effect on baseline diameters (not shown) and did not alter ADP-induced vasodilations in either intact or Ovx female rats (Fig. 6),
suggesting that the KCa dependency revealed by the
results summarized in Fig. 3 was not related to generation of P-450 epoxygenase products.
Finally, to establish whether ADP-induced pial arteriolar dilations were in any way related to nucleotidase-influenced adenosine production, experiments
were conducted in the absence and presence of suffusions of the adenosine receptor antagonist 8-SPT (10
␮M). As in all of the other experiments, ADP reactivities before inhibitor addition (Fig. 7) were statistically
similar in intact and Ovx females (13 ⫾ 2 vs. 12 ⫾ 1%
at 10 ␮M, and 29 ⫾ 2 vs. 29 ⫾ 3% at 100 ␮M, respectively). Also, similar responses to topically applied
adenosine were observed in the two rat groups. In the
ESTROGEN AND CEREBRAL VASODILATION
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Fig. 5. ADP-induced pial arteriolar diameter increases in intact (A)
and ovariectomized (B) females before (initial) and after topically
applied glibenclamide (Glib; 5 ␮M). The baseline diameters were
34.4 ⫾ 3.5 and 39.7 ⫾ 2.5 ␮m for the intact and ovariectomized rats,
respectively. Values are means ⫾ SE; n ⫽ 4 rats per group.
presence of 8-SPT, no changes in baseline diameters
were observed (not shown). However, in both groups,
the adenosine response was completely inhibited, indicating the effectiveness of the adenosine receptor
blockade. Despite this, ADP-induced dilations were not
reduced, demonstrating that ADP does not act via
adenosine receptors.
DISCUSSION
The principal findings in this investigation can be
summarized as follows. First, although the magnitude
of ADP-induced pial arteriolar dilation is essentially
unaffected by chronic estrogen depletion and subsequent repletion, the NO dependency of the ADP response is lost in ovariectomized females, but restored
to the levels observed in intact females on chronic
estrogen replacement. Second, in both intact and Ovx
female rats, the ADP responses were substantially
reduced in the presence of the large-conductance KCa
channel (big KCa) channel blocker, IbTX. Moreover, in
the intact females, the effects of the NOS and big KCa
channel inhibitors completely overlapped. However,
the implication of these findings, that the NO- and
KCa-dependent portions of the ADP response involve a
common pathway, was only partly supported on further study. Third, cytochrome P-450 epoxygenase products are not involved in the KCa channel-dependency of
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Fig. 6. Pial arteriolar diameter increases elicited by increasing suffusate concentrations of ADP, in intact (A) and ovariectomized (B)
rats, in the absence (initial) and in the presence of topically applied
miconazole (Mic; 10 ␮M). Baseline diameters were 41.5 ⫾ 2.4 and
36.7 ⫾ 6.9 ␮m for the intact and ovariectomized rats, respectively.
Values are means ⫾ SE; n ⫽ 4 rats per group.
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the ADP response. Fourth, irrespective of chronic estrogen status, vasodilator prostanoids, KATP channels,
and adenosine receptors, do not participate in ADPinduced pial arteriolar dilations.
The present findings would indicate that the mechanism or mechanisms through which ADP promotes
cerebral vasodilation can be substantially altered by
estrogen. Thus in the face of chronic estrogen depletion, the KCa channel and NO-dependent portion of the
ADP-induced vasodilating process is essentially replaced by a mechanism that continues to rely on KCa
channel activation, but not NO.
Evidence obtained in studies (10, 14, 20, 22) on
rodent pial arterioles in vivo clearly supports at least a
partial NO contribution to the ADP-induced vasodilating response; although some disagreement exists as to
the precise percentage of the ADP response affected by
NOS inhibition. That is, the ⬃50% reduction in ADP
reactivity seen in the intact females of the present
study is less than the 65–90% reductions observed in
our previous studies (10, 20). Because the experiments
were performed on Sprague-Dawley rats of the same
age, exposed to similar surgical and anesthetic conditions and NOS inhibitor doses, gender represents the
only major model difference, when comparing the
present (female) and previous (male) studies from our
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ESTROGEN AND CEREBRAL VASODILATION
laboratory. Thus the possibility exists that the NOdependent portion of the ADP response is greater in
male versus female rats. Results to date also suggest
that the source of that NO is eNOS. The latter can be
inferred from studies in isolated rat cerebral arteries,
or murine pial arterioles in vivo, where ADP-induced
relaxation was repressed on endothelial removal (28)
or endothelial injury (22), respectively; and from preliminary data obtained in our laboratory (unpublished
communications) showing that ADP-induced pial arteriolar dilation in vivo was not altered in the presence of
a neuronal NOS-selective inhibitor.
The current findings would appear to be in line with
the concept that ADP-induced cerebral arteriolar dilation can occur via more than one pathway. Thus, if one
pathway is blocked, a “compensatory switch” takes
place, thereby maintaining ADP reactivity, but via an
alternative route. The pathway utilized, at least in
female animals, is determined by chronic estrogen status. Our present results bear some resemblance to
recent findings by Golding and Kepler (6), which
showed that ATP-induced dilations in cerebral arteries
isolated from intact and estrogen-treated Ovx female
rats were much more sensitive to NOS inhibition than
vessels obtained from untreated Ovx females (6). Moreover, like the present study, KCa channel (but not
cyclooxygenase) blockade substantially reduced the response in the estrogen-depleted group. One plausible
explanation for the retention of agonist-induced vasodilating function relates to the production of another
endothelium-derived relaxing factor (EDRF): the endothelium-derived hyperpolarizing factor (EDHF). Previous studies involving both cerebral (2) and peripheral
(13, 18) vessels have suggested that NO may prevent
agonist-induced increases in EDHF generation. Thus,
only when NO synthesis is blocked, will an increase in
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Fig. 7. Adenosine (Ado; 100 ␮M) and ADP (10 and 100 ␮M) induced
pial arteriolar diameter increases in intact and ovariectomized (Ovx)
females, before (initial), and after topically applied 8-sulfophenyltheophylline (8-SPT, 10 ␮M). Baseline diameters were 38.8 ⫾ 3.4
and 39.5 ⫾ 3.2 ␮m for the intact and ovariectomized rats, respectively. Values are means ⫾ SE. *P ⬍ 0.05 vs. initial; n ⫽ 4 rats per
group.
EDHF be revealed. However, the results from other
laboratories do not support this view (4). Nevertheless,
in the absence of any measure of vascular smooth
muscle membrane potential, the contribution of an
EDHF to agonist-induced vasodilation is often identified by the sensitivity of the vessel to KCa channel
blockade combined with its insensitivity to NOS and
cylooxygenase blockade. Those criteria were met in the
Ovx rats of the present study, as well as in the study by
Golding and Kepler (6).
Of some relevance to the present model of chronic
reduction in eNOS function (i.e., estrogen depletion)
are the findings of Meng et al. (16), who reported a
fairly normal pial arteriolar ACh reactivity in eNOS
knockout mice. The data obtained in that study indicated that the retention of ACh reactivity was partly
attributable to a compensatory increase in the role of
nNOS, although the authors did suggest the possibility
of an increase in the contribution from an EDHF.
Recent findings from our laboratory showed that
eNOS-dependent vasodilating function (ACh reactivity) in pial arterioles in vivo is almost completely lost in
Ovx females but restored with chronic estrogen replacement (21). Those findings, in principal, agree with
the present results showing a complete loss of the
NO-sensitive component of ADP-induced dilations in
estrogen-depleted females. On the other hand, in Ovx
rats, the absence of any ACh reactivity, as opposed to a
“normal” ADP-induced vasodilating response would
seem to indicate that the signal transduction pathways
activated by endothelial muscarinic and ADP-sensitive
P2Y1 purinoceptor stimulation are different. Although
specific differences have not as yet been identified, one
possible (but perhaps oversimplified) speculation
would be that in rat pial arterioles, P2Y1 receptor
activation is capable of activating both eNOS and an
“EDHF generator,” whereas muscarinic agonists stimulate only eNOS. In the former case, the NO produced
would prevent EDHF release.
As appealing as the above model is in explaining the
data obtained in the present study, it remains only
speculative, both because of published evidence that
appears to oppose features of this model and because of
limitations in our experimental approach. Regarding
the former, relaxation of mesenteric arteries harvested
from female versus male rats (27), or aortas from
female rats with pregnancy-induced chronic elevations
in circulating estrogen (3), displayed a greater dependence on a non-NO/nonprostanoid hyperpolarizing factor when acetylcholine was applied. These findings,
suggesting a direct relationship between chronic estrogen status and the relative EDHF contribution to an
endothelium-mediated vasodilating stimulus, would
seem to contradict the results obtained for cerebral
vessels (Ref. 6 and present results). However, the possibility that estrogen negatively influences EDHF activity only in cerebral vessels is unlikely: in another
study (8) that compared mesenteric arteries from male
and female rats, the apparent EDHF component was
greater in the “estrogen-deprived” males. Although
methodological factors (i.e., the use of different rat
ESTROGEN AND CEREBRAL VASODILATION
AJP-Heart Circ Physiol • VOL
ide (11). The first has received the greatest attention.
However, because miconazole did not affect ADP-induced relaxation in the current study, it is unlikely
that an epoxide played any role in the responses observed in the Ovx rats. Whether anandamide, carbon
monoxide, or some other EDHF is involved will require
additional experimentation.
Studies to date consistently indicate that, irrespective of the factor involved, ADP-induced cerebrovascular dilation is strongly endothelium dependent (see
METHODS). This also includes pial arterioles studied in
vivo, at least in the mouse (22). Nevertheless, because
the endothelium dependence of the ADP response under current experimental conditions was not evaluated, we cannot eliminate the prospect that this nonNO, nonprostanoid but KCa-dependent mechanism,
which is switched on in the face of estrogen depletion,
may indeed involve a paracrine factor that is nonendothelial in origin. Moreover, lack of information as to the
extent of endothelial participation also does not permit
us to ignore the possibility that ADP-induced pial arteriolar relaxation may occur via direct interactions
with P2Y1 receptors on vascular smooth muscle. Such
a process merits consideration in light of recent findings (24) showing a KCa channel-dependent hyperpolarization in isolated pig aortic smooth muscle cells in
the presence of ADP and other P2Y1 agonists.
In conclusion, present findings suggest that chronic
estrogen depletion, while not influencing the magnitude of ADP-induced dilations, is associated with a
change in the mechanism mediating the vasorelaxant
response. That is, the partial NO dependence of the
ADP action is completely lost in ovariectomized females but is restored on chronic estrogen replacement.
The NO-independent component was strongly influenced by KCa channel blockade only in the estrogendepleted group. Also, the ADP response was unaffected
by blockade of prostanoid synthesis, KATP channels, or
adenosine receptors. Taken together, these results suggest the appearance of an EDHF-like factor as an
important participant in the pial arteriolar response to
ADP when estrogen is chronically depleted.
The authors thank Susan Anderson and Dennis Riley for expert
technical assistance.
This study was supported by National Heart, Lung, and Blood
Institute Grants HL-56162 and HL-52594.
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