Gender-specific reduction in contractility and
[Ca2+]i in vascular smooth
muscle cells of female rat
Jason G.
Murphy and
Raouf A.
Khalil
Department of Physiology and Biophysics and Center for
Excellence in Cardiovascular-Renal Research, University of Mississippi
Medical Center, Jackson, Mississippi 39216-4505
 |
ABSTRACT |
The hypothesis
that vascular protection in females and its absence in males reflects
gender differences in [Ca2+]i and
Ca2+ mobilization mechanisms of vascular smooth muscle
contraction was tested in fura 2-loaded aortic smooth muscle cells
isolated from intact and gonadectomized male and female Wistar-Kyoto
(WKY) and spontaneously hypertensive (SHR) rats. In WKY cells incubated in Hanks' solution (1 mM Ca2+), the resting length and
[Ca2+]i were significantly
different in intact males (64.5 ± 1.2 µm and 83 ± 3 nM) than in
intact females (76.5 ± 1.5 µm and 64 ± 7 nM). In intact male WKY,
phenylephrine (Phe, 10
5 M) caused transient increase
in [Ca2+]i to 428 ± 13 nM
followed by maintained increase to 201 ± 8 nM and 32% cell
contraction. In intact female WKY, the Phe-induced [Ca2+]i transient was not
significantly different, but the maintained [Ca2+]i (159 ± 7 nM) and cell
contraction (26%) were significantly less than in intact male WKY. In
Ca2+-free (2 mM EGTA) Hanks', Phe and caffeine (10 mM)
caused transient increases in
[Ca2+]i and contraction that were
not significantly different between males and females. Membrane
depolarization by 51 mM KCl caused 31% cell contraction and increased
[Ca2+]i to 259 ± 9 nM in intact
male WKY, which were significantly greater than a 24% contraction and
214 ± 8 nM [Ca2+]i in intact
female WKY. Maintained Phe- and KCl-stimulated cell contraction and
[Ca2+]i were significantly greater
in SHR than WKY in all groups of rats. Reduction in cell contraction
and [Ca2+]i in intact females
compared with intact males was significantly greater in SHR (~30%)
than WKY (~20%). No significant differences in cell contraction or
[Ca2+]i were observed between
castrated males, ovariectomized (OVX) females, and intact males, or
between OVX females with 17
-estradiol implants and intact females.
Exogenous application of 17
-estradiol (10
8 M) to
cells from OVX females caused greater reduction in Phe- and KCl-induced
contraction and [Ca2+]i in SHR than
WKY. Thus the basal, maintained Phe- and depolarization-induced [Ca2+]i and contraction of vascular
smooth muscle triggered by Ca2+ entry from the
extracellular space exhibit differences depending on gender and the
presence or absence of female gonads. Cell contraction and
[Ca2+]i due to Ca2+
release from the intracellular stores are not affected by gender or gonadectomy. Gender-specific reduction in contractility and [Ca2+]i in vascular smooth
muscle of female rats is greater in SHR than WKY rats.
sex hormones; vascular smooth muscle; contraction; hypertension
 |
INTRODUCTION |
HYPERTENSION IS A MAJOR CARDIOVASCULAR disease in the
Western world. The higher incidence of hypertension in men and
postmenopausal women than in premenopausal women (4, 9, 12, 20) has suggested putative vascular protective effects of endogenous estrogen (1, 11, 39, 42). The potential beneficial vascular effects of estrogen
replacement therapy in postmenopausal women have further supported a
role for estrogen in protecting against hypertension (37). The
beneficial vascular effects of estrogen have been ascribed to
modification of circulating lipoproteins (11, 30), changes in blood
coagulation (3), inhibition of intravascular accumulation of collagen
(41), inhibition of vascular smooth muscle growth (5), as well as
direct vasodilation. In addition to an endothelium-dependent component
of estrogen-induced vasodilation (13, 17), estrogen causes vasodilation
in deendothelialized vessels (7, 18), suggesting an
endothelium-independent component of vascular relaxation that involves
direct action on vascular smooth muscle (9, 11, 15).
The suggested vascular protective effects of estrogen in females with
intact gonads (4, 20) and their proposed absence in males and in
females with reduced gonadal function (30, 35) imply that the
mechanisms of vascular smooth muscle contraction may be modified by
gender and by the presence or absence of functional female gonads.
However, little is known about the effect of gender and the status of
the gonads on the intracellular free Ca2+ concentration
([Ca2+]i) or the Ca2+
mobilization mechanisms of vascular smooth muscle contraction, namely
Ca2+ release from the intracellular stores and
Ca2+ entry from the extracellular space (23). Also, since
vascular smooth muscle contractility and
[Ca2+]i are often enhanced in
animal models of hypertension (8, 10, 16, 31), any gender differences
in the Ca2+ mobilization mechanisms of vascular smooth
muscle contraction are expected to be more apparent in hypertensive
than in normotensive animals. However, whether the possible effects of
gender on vascular smooth muscle contractility and
[Ca2+]i are different in
hypertensive vs. normotensive animals is unknown. Furthermore, despite
the potential vascular benefits of estrogen, most of the information
regarding the effects of estrogen on vascular smooth muscle contraction
has largely been inferred from in vitro acute studies. For example,
17
-estradiol has been shown to cause relatively rapid relaxation of
isolated vascular strips (7, 15, 18), suggesting additional mechanisms
independent of the classical genomic pathway of steroid action on gene
transcription (26), possibly mediated by alterations in the
Ca2+ mobilization mechanisms into vascular smooth muscle
and consequently [Ca2+]i. However,
whether the possible effects of estrogen on vascular smooth muscle
contraction and [Ca2+]i are
different during long-term exposure to estrogen in vivo compared with
the acute effects of estrogen in vitro remain unclear.
The purpose of this study was 1) to determine whether vascular
smooth muscle contractility exhibits differences depending on gender
and the presence or absence of gonads, 2) to determine whether
the gender-specific differences in smooth muscle contractility are
associated with changes in [Ca2+]i,
3) to determine whether the changes in
[Ca2+]i reflect changes in
Ca2+ release from the intracellular stores and/or
Ca2+ entry from the extracellular space, and
4) to determine whether the gender differences in smooth
muscle contractility and [Ca2+]i,
if any, are enhanced in animal models of hypertension. Because sex
hormones may affect various types of vascular cells, studying the
effect of gender on [Ca2+]i in a
multicellular vascular preparation could be difficult. Therefore, this
study was performed on single vascular smooth muscle cells freshly
isolated from aortic strips of intact and gonadectomized male and
female Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats.
The effects of long-term exposure to endogenous estrogen in intact
females in vivo, long-term exposure to exogenous estrogen in
ovariectomized (OVX) females in vivo, and acute exposure of vascular
smooth muscle cells to exogenous estrogen in vitro on cell contraction
and [Ca2+]i were compared.
 |
METHODS |
Animals. WKY and SHR rats (12 wk; Harlan, Indianapolis, IN)
were housed in the animal facility and maintained on ad libitum standard rat chow and tap water on a 12 h:12 h light-dark cycle. Both
WKY and SHR rats were divided into four groups: intact males (n = 16), intact females (n = 16), castrated males (n = 16), and OVX females (n = 16). Gonadectomy was performed and
verified by the vendor at 8 wk of age. Some OVX female WKY (n = 8) and SHR (n = 8) rats were given subcutaneous timed-release
17
-estradiol implants (30-day release, 0.125 mg/pellet; Innovative
Research of America, Sarasota, FL) 3 days after ovariectomy and were
studied 4 wk later. All procedures were performed in accordance with
the guidelines of the Animal Care and Use Committee at the University of Mississippi Medical Center and the American Physiological Society.
Blood samples. On the day of the experiment the rats were
anesthetized by inhalation of isoflurane. Blood was collected for measurement of plasma 17
-estradiol by RIA using a 17
-estradiol kit (ICN Biomedicals, Costa Mesa, CA). In WKY rats, the plasma 17
-estradiol was 17 ± 2 pmol/l (n = 16) in intact males,
76 ± 7 pmol/l (n = 16) in intact females, 16 ± 3 pmol/l
(n = 16) in castrated males, 18 ± 2 pmol/l (n = 16)
in OVX females, and 83 ± 9 pmol/l (n = 8) in OVX females with
17
-estradiol implants. The plasma 17
-estradiol in SHR rats was
not significantly different from that in WKY rats in each group.
Tissue preparation. The thoracic aorta was excised, placed in
oxygenated Krebs solution, cleaned of connective and adipose tissue,
and opened by cutting along its longitudinal axis. To minimize the
number of other cell types during the cell isolation procedure, the
endothelium was removed by gently rubbing the vessel interior with wet
filter paper, and the tunica media was carefully dissected from the
tunica adventitia under microscopic visualization using sharp-tipped
forceps. The tunica media was then sectioned into 2 × 2 mm
strips. Only the tunica media was used during the cell isolation procedure.
Single cell isolation. Single aortic smooth muscle cells were
freshly isolated as previously described, specifically avoiding aspiration through a pipette or centrifugation (22). Rat aortic strips
(50 mg) were placed in a siliconized flask containing a tissue
digestion mixture of collagenase type II (236 U/mg protein activity;
Worthington, Freehold, NJ), elastase grade II (3.25 U/mg protein
activity; Boehringer Mannheim, Indianapolis, IN), and trypsin inhibitor
type II soybean (10,000 U/ml; Sigma, St. Louis, MO) in 7.5 ml of
Ca2+- and Mg2+-free Hanks' solution
supplemented with 30% BSA (Sigma). The tissue was incubated three
consecutive times in the tissue digestion mixture to yield three
batches of cells. For the first batch the tissue was incubated with 5 mg collagenase, 4 mg elastase, and 147 µl trypsin inhibitor for 60 min. For batches 2 and 3 the collagenase was reduced to
2.5 mg, the trypsin inhibitor was reduced to 122 µl, and the
incubation period was reduced to 30 min. The tissue preparation was
placed in a shaking water bath at 34°C in an atmosphere of 95%
O2-5% CO2. At the end of each incubation
period, the preparation was rinsed with 12.5 ml of Hanks' with albumin
and poured over glass coverslips placed in wells and cooled to 2°C.
By using the gravitational force, the cells were allowed to settle and
adhere to the glass coverslips. Ca2+ was gradually added
back to the preparation to avoid the "Ca2+ paradox"
(34). The cell isolation procedure consistently yielded long,
spindle-shaped smooth muscle cells that consistently showed significant
contraction in response to contractile stimuli. The cells were further
characterized and consistently showed significant immunofluorescence
signal when labeled with anti-smooth muscle myosin antibody.
Contractility studies. Cell length was measured in freshly
isolated aortic smooth muscle cells viewed under the microscope using a
Nikon ×40 objective. Three different smooth muscle activators were used. The
-adrenergic agonist phenylephrine (Phe) was used to
stimulate both Ca2+ release from the intracellular stores
and Ca2+ entry from the extracellular space (23, 24).
Caffeine was used to activate the Ca2+-induced
Ca2+-release mechanism in Ca2+-free solution
(27). Membrane depolarization by high-KCl solution was used to activate
Ca2+ entry from the extracellular space (23). The changes
in cell length in response to Phe (10
5 M), caffeine
(10 mM), and high-KCl depolarizing solution (51 mM) were measured, and
the magnitude of cell contraction was expressed as
[(Li
L)/Li] × 100, where
Li is the initial cell length and L is the
final cell length. All contraction measurements were made at 22°C
as previously described (22).
Measurement of
[Ca2+]i.
[Ca2+]i was measured in fura
2-loaded single aortic smooth muscle cells using the ratio method as
previously described (21, 40). The cells were incubated in the fura
2-loading solution for 30 min at 34°C. The loading solution was
made of normal Hanks' solution supplemented with 1 µM of the cell
permeant fura 2-AM (Molecular Probes, Eugene, OR) and 0.01% Pluronic
F-127 (Sigma). The fura 2-AM was diluted from a 1 mM stock solution in
DMSO, so that the final concentration of DMSO in the loading solution was 0.1%. The fura 2-loaded cells were washed twice and further incubated in normal Hanks' solution for at least 30 min to allow complete deesterification of the dye. Nonspecific intracellular esterases hydrolyze the fura 2-AM esters and liberate the
Ca2+-sensitive indicator fura 2 (14). Due to the
photosensitivity of the fura 2 molecule, precautionary measures were
taken throughout the procedure to avoid extensive photobleaching.
The fura 2-loaded cells were viewed through a Nikon CF Fluor ×100
oil-immersion objective (numerical aperture 1.3) on an inverted Nikon
(Diaphot-300) microscope. The Ca2+ indicator was excited
alternately at 340 ± 5 and 380 ± 6 nm using a filter wheel that
alternates between the two filters at a frequency of 0.5 Hz. The SE in
the excitation wavelengths reflect the band width of the excitation
filters on both sides of the peak excitation at 340 and 380 nm. The
emitted light was collected at 510 nm to a photomultiplier tube R928
(Ludl Electronic Products, Hawthorne, NY) through a pinhole aperture 1 µm in diameter positioned 1 µm from the plasma membrane and 1 µm
from the nucleus. The fluorescent signal was digitized using a module
(Mac 2000, Ludl) and analyzed on a PC using data analysis software. The
signal-to-noise ratio was improved by averaging eight consecutive
fluorescent intensity readings collected by the photomultiplier tube.
The fluorescent signal was background subtracted. Spectral shifts that
result from binding of Ca2+ allow the fura 2 indicator to
be used ratiometrically, making the measurement of
[Ca2+]i less sensitive to changes
in cell thickness or the extent of dye loading and photobleaching. The
fluorescence ratio was calculated from the fluorescence intensity at
340 nm divided by that at 380 nm. The 340/380 ratio (R) was transformed
to the corresponding levels of
[Ca2+]i as described by Grynkiewicz
et al. (14)
where
Rmin and Rmax represent the minimal and maximal
fluorescence ratios and were measured by adding fura 2 pentapotassium salt (50 µM) into Ca2+-free (10 mM EGTA) and
Ca2+-replete (2 mM) solutions, respectively, using
Ca2+-EGTA buffers; Sf2/Sb2 is the
ratio of the 380 signal in Ca2+-free and
Ca2+-replete solutions, respectively; and
Kd is the dissociation constant of fura 2 for
Ca2+ and was established at 200 nM under these experimental
conditions as previously described (14, 21). All
[Ca2+]i measurements were made at
22°C as previously described (21). Because the basal and maintained
agonist-stimulated changes in [Ca2+]i showed significant
fluctuations, 10 consecutive measurements were averaged in each
individual cell.
Solutions. Krebs solution was used for dissecting the tissue
and contained (in mM) 120 NaCl, 5.9 KCl, 25 NaHCO3, 1.2 NaH2PO4, 11.5 dextrose, 2.5 CaCl2,
and 1.2 MgCl2 at pH to 7.4. Hanks' solution was used for
cell isolation and for performing the experiments and contained (in mM)
137 NaCl, 5.4 KCl, 0.44 KH2PO4, 0.42 Na2HPO4, 4.17 NaHCO3, 5.55 dextrose, and 10 HEPES. The solution was bubbled for 30 min with a 95%
O2-5% CO2 mixture, and the pH was adjusted to
7.4. For Ca2+- and Mg2+-containing Hanks'
solution, 1 mM CaCl2 and 1.2 mM MgCl2 were added. For Ca2+-free Hanks' solution, CaCl2
was omitted and replaced with 2 mM EGTA. The high-KCl (51 mM)
depolarizing solution had the same composition as normal Hanks'
solution, with equimolar substitution of NaCl with KCl.
Drugs and chemicals. Stock solution of Phe
(10
1 M; Sigma) was prepared in distilled water.
Caffeine (Sigma) was prepared as 10 mM in Ca2+-free (2 mM
EGTA) Hanks' solution. Stock solution of 17
-estradiol (2,3,5[10]-estratriene-3,17
-diol; Sigma) was prepared as
5 × 10
2 M in 100% ethanol. 17
-Estradiol
and tamoxifen (Sigma) and ICI-182780 (Tocris, Ballwin, MO) were
prepared as 10
2 M stock solutions in 100% ethanol.
The final concentration of the vehicle ethanol in solution was
0.001%. Neomycin sulfate and
1-(6-{[17
-3-methoxestra-1,3,5(10)-trien-17-yl]amino}hexyl)- 1H-pyrole-2,5-dione
(U-73122) were purchased from Biomol (Plymouth Meeting, PA) and
4-bromo-A-23187 was from Calbiochem (San Diego, CA). All other
chemicals were of reagent grade or better.
Statistical analysis. The data were analyzed and presented as
means ± SE. Data were compared using ANOVA with three classification criteria [strain, gender, and treatment (gonadectomized vs.
intact)]. Scheffe's F-test was used for comparison of
multiple means. Student's t-test for unpaired data was used
for comparison of two means. Differences were considered statistically
significant if P < 0.05.
 |
RESULTS |
Resting cell length and basal
[Ca2+]i.
The cell isolation procedure produced cells of variable lengths. Only
spindle-shaped cells
50 µm in length were selected for this study.
In resting cells of intact male WKY rats, the average cell length was
64.5 ± 1.2 µm (n = 25) (Fig.
1A). Basal [Ca2+]i was also measured in
resting cells. To avoid the fluctuations in basal
[Ca2+]i measurements (see Fig.
2), 10 consecutive measurements were averaged in each individual cell. In resting cells of intact male WKY,
the average basal [Ca2+]i was 83 ± 3 nM (n = 23; Fig. 1B). The average cell
length in intact female WKY rats (76.5 ± 1.5 µm, n = 25)
was 18.6% longer (Fig. 1A), and basal
[Ca2+]i (64 ± 7 nM, n = 22) was 22.9% smaller (Fig. 1B) compared with the respective
measurements in intact male WKY rats. Resting cell length and basal
[Ca2+]i in castrated male WKY were
not significantly different from that in intact male WKY. In OVX female
WKY rats, resting cell length was significantly shorter and basal
[Ca2+]i was significantly greater
than that in intact female WKY rats. On the other hand, the cell length
and [Ca2+]i in OVX female WKY rats
with 17
-estradiol implants were not significantly different from
that in intact female WKY rats (Fig. 1). In SHR rats, the average
resting cell length was shorter (Fig. 1A), and basal
[Ca2+]i (Fig. 1B) was
greater than that of WKY rats in all groups of male and female rats. In
intact female SHR rats, the average resting cell length was 27.7%
longer (Fig. 1A), and basal
[Ca2+]i was 28.5% smaller (Fig.
1B) than that in intact male SHR rats.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 1.
Resting cell length (A) and basal intracellular free
Ca2+ concentration
([Ca2+]i; B) in aortic
smooth muscle cells isolated from intact and gonadectomized, male and
female, Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats
and ovariectomized (OVX) female WKY and SHR rats with 17 -estradiol
implants and incubated in Hanks' solution (1 mM Ca2+).
Data bars represent means ± SE of measurements in 18-30 cells
from 6-10 rats of each group. * Measurement in SHR is
significantly different (P < 0.05) from WKY. # Intact
female WKY and OVX female WKY with 17 -estradiol implants are
significantly different from other groups of WKY. Intact
female SHR and OVX female SHR with 17 -estradiol implants are
significantly different from other groups of SHR.
|
|

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of phenylephrine (Phe; 10 5 M) on cell
contraction and [Ca2+]i in aortic
smooth muscle cells isolated from intact and gonadectomized, male and
female, WKY and SHR rats and OVX female WKY and SHR rats with
17 -estradiol implants and incubated in Hanks' solution (1 mM
Ca2+). Traces are representative of
[Ca2+]i measurements in cells from
WKY (A) and SHR (B). Dashed lines were drawn at
smallest basal and maintained Phe-induced increase in
[Ca2+]i recorded in cells of intact
females to facilitate comparison with other groups of rats. Horizontal
bars below traces represent time of application of Phe. Data bars
represent means ± SE of measured Phe-induced changes in cell
contraction (C), initial peak
[Ca2+]i (D), and maintained
[Ca2+]i (E) in 15-25
cells from 5-7 rats of each group. * Measurement in SHR is
significantly different (P < 0.05) from WKY. # Intact
female WKY and OVX female WKY with 17 -estradiol implants are
significantly different from other groups of WKY. Intact
female SHR and OVX female SHR with 17 -estradiol implants are
significantly different from other groups of SHR. Cast, castrated.
|
|
Effect of Phe in
Ca2+-containing medium.
Freshly isolated aortic smooth muscle cells were responsive to
contractile stimuli. In normal Hanks' (1 mM Ca2+)
solution, Phe (10
5 M) caused contraction of rat
aortic smooth muscle cells that reached a plateau at ~5 min. Phe also
caused a transient initial peak in
[Ca2+]i followed by a steady-state
increase that was maintained for at least 5 min in all groups of rats
(Fig. 2, A and B). Application of the nonselective
Ca2+ influx inhibitor NiCl2 (1 mM) on top of
the maintained Phe-induced [Ca2+]i,
decreased [Ca2+]i to basal levels,
supporting the contention that this response is mediated by
Ca2+ entry from the extracellular space. To avoid the
fluctuations in the maintained Phe-induced increase in
[Ca2+]i measurements (Fig. 2,
A and B), 10 consecutive measurements were averaged in
each individual cell. The average Phe-induced cell contraction after 5 min of stimulation (Fig. 2C), as well as the average initial
peak (Fig. 2D) and steady-state increase in
[Ca2+]i after 5 min (Fig.
2E), were compared in the different groups of rats. In cells of
intact male WKY rats, Phe caused 32 ± 1.5% (n = 25) cell contraction (Fig. 2C), an initial peak in
[Ca2+]i to 428 ± 13 nM (n = 21; Fig. 2, A and D), and a maintained increase in
[Ca2+]i to 201 ± 8 nM (n = 21; Fig. 2, A and E). The Phe-induced initial peak
[Ca2+]i was not significantly
different among the different groups of male and female WKY rats (Fig.
2, A and D). The Phe-stimulated cell contraction (Fig.
2C) and maintained [Ca2+]i
(Fig. 2, A and E) in intact female WKY were
significantly less than the respective measurements in intact male WKY
rats by 18.8% and 20.9%, respectively. The Phe-induced cell
contraction and maintained [Ca2+]i
were not significantly different between intact male and castrated male
WKY rats, but were significantly greater in OVX female WKY than in
intact female WKY rats. In OVX female WKY rats with 17
-estradiol implants, the Phe-induced cell contraction and maintained
[Ca2+]i were not significantly
different from that in intact female WKY rats. In the SHR rats,
Phe-induced cell contraction (Fig. 2C) and maintained
[Ca2+]i (Fig. 2, B and
E) were significantly greater than that of WKY in all groups of
rats. Phe-induced cell contraction and maintained [Ca2+]i in intact female SHR were
significantly less than that in intact male SHR rats by 29.4% and
28.1%, respectively. Phe-induced initial peak
[Ca2+]i was not significantly
different among the different groups of male and female SHR rats (Fig.
2, B and D).
Effect of Phe and caffeine in
Ca2+-free solution.
We investigated whether the observed gender differences in cell
contraction and [Ca2+]i reflect
changes in Ca2+ release from the intracellular stores. In
cells of intact male WKY rats incubated in Ca2+-free (2 mM
EGTA) Hanks' solution for 1 min, basal
[Ca2+]i was reduced to 33 ± 2 (n = 36), which was not significantly different from that in
cells isolated from the other groups of male and female WKY and SHR
rats. In cells of intact male WKY rats, Phe (10
5 M)
caused 10 ± 1% (n = 20) cell contraction (Fig.
3A) and a transient increase in
[Ca2+]i to 341 ± 16 (n = 16; Fig. 3, B and D), which were not significantly different from the respective measurements in WKY or SHR intact female,
castrated male, OVX female, or OVX female rats with 17
-estradiol implants (Fig. 3). Pretreatment of cells from male or female WKY or SHR
rats with the phospholipase C (PLC) inhibitor neomycin (0.5 mM) or
U-73122 (10 µM) for 30 min in Ca2+-containing Hanks'
solution, followed by brief incubation in Ca2+-free Hanks'
solution, completely abolished the Phe-induced contraction and
transient increase in [Ca2+]i.
Also, in Ca2+-free (2 mM EGTA) Hanks' solution, caffeine
(10 mM) caused 9 ± 1.5% (n = 20) cell contraction (Fig.
4A) and a transient increase in
[Ca2+]i to 441 ± 25 (n = 20; Fig. 4, B and D) in cells of intact male WKY rats,
which were not significantly different from the respective measurements
in WKY or SHR intact female, castrated male, OVX female, or OVX female
rats with 17
-estradiol implants (Fig. 4). Additionally, in cells of
intact male WKY incubated in Ca2+-free Hanks' solution,
the Ca2+ ionophore 4-bromo-A23187 (1 µM) caused a large
[Ca2+]i spike (638 ± 24 nM,
n = 11) that was not significantly different among the
different groups of rats.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 3.
Phe (10 5 M)-induced cell contraction (A) and
[Ca2+]i transient (B, C,
and D) in aortic smooth muscle cells isolated from intact and
gonadectomized, male and female, WKY and SHR rats and OVX female WKY
and SHR rats with 17 -estradiol implants and incubated in
Ca2+-free (2 mM EGTA) Hanks' solution. Data bars are means ± SE and traces are representative of
[Ca2+]i measurements in 16-25
cells from 6-7 rats of each group. Horizontal bars below traces
represent time of application of Phe.
|
|

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 4.
Caffeine (10 mM)-induced cell contraction (A) and
[Ca2+]i transient (B, C,
and D) in aortic smooth muscle cells isolated from intact and
gonadectomized, male and female, WKY and SHR rats and OVX female WKY
and SHR rats with 17 -estradiol implants and incubated in
Ca2+-free (2 mM EGTA) Hanks' solution. Data bars are means ± SE and traces are representative of
[Ca2+]i measurements in 12-25
cells from 6-7 rats of each group. Horizontal bars below traces
represent time of application of caffeine.
|
|
Effect of membrane depolarization by KCl. We investigated
whether the observed gender differences in cell contraction and [Ca2+]i reflect changes in
Ca2+ entry from the extracellular space. Membrane
depolarization by KCl is known to stimulate Ca2+ entry
through voltage-gated Ca2+ channels (23, 24). KCl caused
cell contraction that reached a plateau at ~5 min. KCl also caused an
increase in [Ca2+]i that was
maintained for at least 5 min in all groups of rats (Fig.
5). Application of the nonselective
Ca2+ influx inhibitor NiCl2 (1 mM) on top of
the KCl-induced [Ca2+]i decreased
[Ca2+]i to basal levels, supporting
the contention that this response is mediated by Ca2+ entry
from the extracellular space. To avoid fluctuations in KCl-stimulated
[Ca2+]i measurements (Fig. 5), 10 consecutive measurements were averaged in each individual cell. The
average KCl-induced cell contraction and
[Ca2+]i after 5 min of stimulation
were compared in the different groups of rats. In cells of intact male
WKY, KCl caused 31 ± 1% (n = 25) contraction (Fig.
5A) and increased [Ca2+]i
to 259 ± 9 nM (n = 22; Fig. 5, B and D). In
intact female WKY, KCl-induced cell contraction (Fig. 5A) and
[Ca2+]i (Fig. 5, B and
D) were significantly reduced by 22.6% and 17.4%, respectively, compared with intact male WKY rats. The KCl-induced responses were not significantly different between intact and castrated
male WKY but were significantly greater in OVX female WKY than in
intact female WKY rats. In OVX female WKY rats with 17
-estradiol
implants, the KCl-induced contraction and
[Ca2+]i were not significantly
different from that in intact female WKY rats (Fig. 5). In the SHR
rats, the KCl-induced cell contraction (Fig. 5A) and
[Ca2+]i (Fig. 5, C and
D) were significantly greater than that of the WKY in all
groups of rats. KCl-induced cell contraction and
[Ca2+]i in intact female SHR were
significantly reduced by 29.2% and 26.1%, respectively, compared with
intact male SHR rats (Fig. 5).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of membrane depolarization by 51 mM KCl on cell contraction
(A) and [Ca2+]i (B,
C, and D) in aortic smooth muscle cells isolated from
intact and gonadectomized, male and female, WKY and SHR rats and OVX
female WKY and SHR rats with 17 -estradiol implants. Data bars are
means ± SE and traces are representative of
[Ca2+]i measurements in 14-25
cells from 5-7 rats of each group. Dashed lines were drawn at
smallest basal and KCl-induced increase in
[Ca2+]i recorded in cells of intact
females to facilitate comparison with other groups of rats. Horizontal
bars below traces represent time of application of KCl.
* Measurement in SHR is significantly different (P < 0.05)
from WKY. # Intact female WKY and OVX female WKY with
17 -estradiol implants are significantly different from other groups
of WKY. Intact female SHR and OVX female SHR with
17 -estradiol implants are significantly different from other groups
of SHR.
|
|
Effect of 17
-estradiol on Phe- and KCl-induced cell
contraction and
[Ca2+]i.
Because the gender differences could involve a multitude of factors in
vivo, we tested the direct effect of exogenous application of
17
-estradiol on Phe- and KCl-induced cell contraction and [Ca2+]i in OVX female WKY and SHR
rats. At concentrations <10
8 M, a clear effect of
17
-estradiol on Phe- or KCl-induced cell contraction or
[Ca2+]i could not be identified. In
OVX female WKY rats, application of 17
-estradiol
(10
8 M) on top of the maintained Phe-induced
[Ca2+]i caused a rapid decrease in
[Ca2+]i (Fig.
6A). When the cells were pretreated
with 17
-estradiol (10
8 M) for 10 min, the
Phe-induced [Ca2+]i transient did
not appear to be affected, but a decrease in the maintained Phe-induced
[Ca2+]i was observed (Fig.
6A). Similarly, in cells of OVX female WKY rats, top
application or pretreatment of the cells with 17
-estradiol (10
8 M) for 10 min was associated with a decrease in
the KCl-induced increase in [Ca2+]i
(Fig. 6A). The decrease in the maintained Phe- or KCl-induced [Ca2+]i following top application
or pretreatment of the cells with 17
-estradiol
(10
8 M) for 10 min appeared to be greater in OVX
female SHR (Fig. 6B) than in OVX female WKY rats (Fig.
6A). The cumulative data from different cells showed that
pretreatment of the cells with 17
-estradiol (10
8
M) for 10 min reduced Phe- and KCl-induced cell contraction by 51.5%
and 51.6%, respectively, in OVX female WKY compared with 86.3% and
87.8%, respectively in OVX female SHR rats (Fig.
7A). Pretreatment of the cells with
17
-estradiol (10
8 M) for 10 min slightly but not
significantly decreased the basal [Ca2+]i in OVX female WKY (79 ± 6, n = 20) and SHR rats (118 ± 7, n = 21; Fig.
7B). Also, pretreatment of the cells with 17
-estradiol (10
8 M) slightly but not significantly decreased the
Phe-induced [Ca2+]i transient in
OVX female WKY (425 ± 24 nM, n = 12) and SHR rats (427 ± 16 nM, n = 12). On the other hand, pretreatment of the cells with
17
-estradiol significantly reduced the maintained Phe- and
KCl-induced [Ca2+]i by 30.4% and
34.3%, respectively, in OVX female WKY compared with 52.2% and 59.3%
reduction in OVX female SHR rats, respectively (Fig. 7B).
17
-Estradiol caused a smaller but significant inhibition of the
maintained Phe- and KCl-induced increases in
[Ca2+]i to 128 ± 5 nM (n = 12) and 204 ± 6 nM (n = 12), or 19.5% and 21.24%
reduction, respectively, in smooth muscle of intact female WKY compared
with a decrease in [Ca2+]i to 144 ± 6 nM (n = 12) and 233 ± 8 nM (n = 12), or 38.2%
and 42.2% reduction, respectively, in intact female SHR rats.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of 17 -estradiol (10 8 M) on Phe
(10 5 M)- and KCl (51 mM)-induced changes in
[Ca2+]i in aortic smooth muscle
cells isolated from OVX female WKY (A) and SHR (B)
rats. Traces are representative of measurements in 12-20 cells
from 4-5 rats of each group. Horizontal bars below traces
represent time of application of Phe, KCl, and 17 -estradiol.
|
|

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of 17 -estradiol (10 8 M) on Phe
(10 5 M)- and KCl (51 mM)-induced cell contraction
(A) and [Ca2+]i (B)
in aortic smooth muscle cells isolated from OVX female WKY and SHR
rats. Cells were pretreated with 10 8 M
17 -estradiol for 10 min then stimulated with Phe or KCl, and changes
in cell length and [Ca2+]i were
measured after 5 min of stimulation. Data bars represent means ± SE
of measurements in 12-25 cells from 4-5 rats of each group.
* SHR is significantly different (P < 0.05) from WKY.
# Decrease in cell contraction and
[Ca2+]i in cells of OVX female WKY
in presence of 17 -estradiol is significantly different from that in
absence of the hormone. Decrease in cell contraction and
[Ca2+]i in cells of OVX female SHR
in presence of 17 -estradiol is significantly different from that in
absence of the hormone.
|
|
Pretreatment of cells from OVX female WKY rats with 17
-estradiol
(10
8 M) for 10 min did not significantly inhibit
Phe-induced cell contraction. Phe (10
5 M) cell
contraction in the presence of 17
-estradiol (32 ± 2%, n = 15) was not significantly different from that in the
absence of the hormone (33 ± 1.5%, n = 20). Pretreating the
cells with the estrogen receptor antagonist tamoxifen
(10
6 M) or ICI-182780 (10
6 M) for
10 min completely abolished the inhibition of Phe-induced cell
contraction by 10
8 M 17
-estradiol. Phe
contraction in the presence of 17
-estradiol plus tamoxifen (31 ± 2%, n = 15) or 17
-estradiol plus ICI-182780 (32 ± 1.5%, n = 15) was not significantly
different from that in the absence of the hormone and the antagonist.
 |
DISCUSSION |
The main findings of the present study are as follows. 1)
Vascular smooth muscle cell contraction and
[Ca2+]i due to Ca2+
entry from the extracellular space, but not Ca2+ release
from the intracellular stores, are reduced in the presence, and
enhanced in the absence, of female gonads. 2) Estrogen
replacement in OVX female WKY rats reduces vascular smooth muscle cell
contraction and [Ca2+]i to levels
similar to those in intact female WKY rats. 3) Exogenous application of estrogen to single vascular smooth muscle cells causes
significant inhibition of cell contraction and significant reduction in
[Ca2+]i. 4) The reductions
in cell contraction and [Ca2+]i in
intact females, OVX females with estrogen implants, and in response to
exogenous application of estrogen on single vascular smooth muscle
cells of OVX females are greater in SHR than WKY rats.
Gender differences in resting cell length and basal
[Ca2+]i.
The present study showed that the average cell length in intact female
WKY was longer and basal [Ca2+]i
was smaller compared with the respective measurements in intact male
WKY rats, suggesting gender differences in the Ca2+
handling mechanisms of vascular smooth muscle even under basal conditions. The gender differences in resting cell length and basal
[Ca2+]i appear to be related to
endogenous estrogen because 1) cell length and
[Ca2+]i in castrated male WKY were
not significantly different from that in intact male WKY rats,
2) cell length was shorter and
[Ca2+]i was greater in OVX female
WKY compared with that in intact female WKY rats, and 3) cell
length and [Ca2+]i in OVX female
WKY with 17
-estradiol implants were not significantly different from
that in intact female WKY rats.
Gender differences in Phe-induced cell contraction and
[Ca2+]i.
The present study also showed that cell contraction to the
-adrenergic agonist Phe was greater in intact male than in intact female WKY rats. These results are consistent with other studies in
multicellular vascular preparations that have shown a greater contraction to Phe in aortic strips of intact male than intact female
rats (36). The observation that the Phe-induced cell contraction in
castrated males was not significantly different from that in intact
males but significantly enhanced in OVX females compared with intact
females further suggests that the gender differences are less likely
related to androgens and more likely related to estrogens. Because the
expression of sex hormone receptors in arterial smooth muscle may vary
depending on the gender and the status of the gonads (38), the observed
gender differences in cell contraction may well be related to the
relative abundance of estrogen receptors. This is supported by reports
that estrogen receptors have been identified in the rat aorta (2, 28,
33) and that females have higher levels of estrogen receptors in their arteries than males (6). However, the gender differences may also be
related to differences in the signaling mechanisms downstream from
receptor activation. We investigated whether the gender differences in
vascular smooth muscle cell contraction reflect differences in
[Ca2+]i and the mechanisms of
Ca2+ mobilization into vascular smooth muscle.
Agonist-induced smooth muscle contraction is triggered by increases in
[Ca2+]i due to initial inositol
1,4,5-trisphosphate (IP3)-induced Ca2+
mobilization from the intracellular stores in the sarcoplasmic reticulum and maintained Ca2+ influx through voltage-gated
and receptor-operated Ca2+ channels in the plasma membrane
(23, 24). The present study showed that, in cells incubated in the
presence of external Ca2+, Phe caused an initial peak in
[Ca2+]i, probably due to initial
Ca2+ release from the intracellular stores, followed by a
smaller but maintained increase in
[Ca2+]i, possibly due to
Ca2+ entry from the extracellular space. We also found that
the Phe-induced cell contraction in Ca2+-containing medium,
which is potentially due to both Ca2+ release and
Ca2+ influx, was significantly greater in male than in
female rats (Fig. 2C). On the other hand, the Phe-induced
Ca2+ transient, which is potentially due to
Ca2+ release, was not significantly different between males
and females. These results suggest that Phe-induced vascular smooth
muscle contraction is mainly due to a maintained increase in
Ca2+ influx and maintained increase in
[Ca2+]i. This is supported by the
observation that the Phe-induced maintained increase in
[Ca2+]i and cell contraction in
Ca2+-containing medium were significantly inhibited in
Ca2+-free medium.
Gender and the Ca2+ release
mechanisms.
The present study showed that pretreatment of cells from
male or female WKY or SHR rats with the PLC inhibitor neomycin or U-73122 completely abolished the Phe-induced contraction and transient increase in [Ca2+]i observed in
Ca2+-free solution. These data provide evidence that
Phe-induced contraction and [Ca2+]i
involve activation of PLC and consequently generation of
IP3. The present results also showed that Phe-induced
contraction and [Ca2+]i in
Ca2+-free solution were not significantly different among
the different groups of rats. Taken together, these results support the
contention that the IP3-mediated Ca2+ release
mechanism is less likely to be involved in the observed gender
differences in cell contraction and
[Ca2+]i. Also, caffeine, which
stimulates the Ca2+-induced Ca2+ release
mechanism (23, 27), caused a small cell contraction and a transient
increase in [Ca2+]i that were
similar in magnitude in the different groups of rats, suggesting that
the observed gender differences in cell contraction are not related to
the Ca2+-induced Ca2+-release mechanism.
However, the caffeine data should be interpreted with caution, since a
ryanodine-sensitive component of Ca2+-induced
Ca2+ release cannot be ruled out under these conditions and
should represent an important area for future investigations.
Gender differences in Ca2+
entry mechanisms.
The maintained Phe-induced
[Ca2+]i observed in cells incubated
in the presence of external Ca2+ was greater in intact male
than in intact female WKY rats, providing evidence that the gender
differences in vascular smooth muscle cell contraction could be related
to differences in Ca2+ entry mechanism from the
extracellular space. The observation that the maintained Phe-induced
increase in [Ca2+]i was not
significantly different in castrated males compared with intact males
but was significantly enhanced in OVX females compared with intact
females and was not significantly different in OVX females with
estrogen implants compared with intact females provides further
evidence that the gender differences are less likely related to
androgens and more likely related to estrogens.
Membrane depolarization by high KCl is known to mainly stimulate
Ca2+ entry from the extracellular space (23, 24). The
observation that KCl-induced cell contraction and
[Ca2+]i were greater in intact
males than in intact females further supported possible gender
differences in Ca2+ entry mechanisms. Also, the observation
that KCl-induced cell contraction and
[Ca2+]i were enhanced in OVX
females compared with intact females but were not significantly
different in OVX females with estrogen implants compared with intact
females lends support to the contention that the gender differences are
more likely related to endogenous estrogens. The causes of the gender
differences in the Ca2+ entry mechanism are not clear but
may be related, among other factors, to the plasmalemmal density and/or
the permeability of the Ca2+ entry pathways depending on
the presence or deficiency of endogenous estrogen. This is supported by
reports that the expression of the L-type Ca2+ channels in
cardiac muscle is substantially increased in estrogen receptor-deficient mice (19). However, the observed gender differences in the mechanisms of Ca2+ mobilization into vascular smooth
muscle could be due to a multitude of effects of sex hormones in vivo.
We found that exogenous application of estrogen to freshly isolated
vascular smooth muscle cells of OVX females caused significant
inhibition of high-KCl- and maintained Phe-induced increases in
[Ca2+]i. These results
are in agreement with reports that estrogen causes vascular relaxation
in preconstricted isolated strips of rabbit and porcine coronary artery
(7, 15, 18) and are consistent with the reduced cell contraction and
[Ca2+]i observed in cells of intact
female rats. Because the high-KCl-induced increases in
[Ca2+]i are mainly due to
Ca2+ entry through voltage-gated Ca2+ channels
while the Phe-induced maintained increase in
[Ca2+]i are mainly due to
Ca2+ entry through both voltage-gated and receptor-operated
Ca2+ channels, the present results suggest that both
voltage-gated Ca2+ channels and receptor-operated
Ca2+ channels are involved in the effects of
17
-estradiol on cell contraction and intracellular Ca2+
regulation. However, based on these results, we do not wish to draw
conclusions on whether estrogen inhibits Ca2+ entry by
direct or indirect action on plasmalemmal Ca2+ channels.
Other studies have shown that estrogen blocks Ca2+ channels
in cultured A7r5 and aortic smooth muscle cells (32, 43). Although the
properties of the Ca2+ channels may be different in
cultured cells, our measurements of
[Ca2+]i in freshly isolated aortic
smooth muscle cells are consistent with these reports.
Gender differences in cell contraction and
[Ca2+]i
in WKY vs. SHR rats.
In the present study, aortic strips of SHR rats showed shorter basal
cell length, greater basal [Ca2+]i,
and greater Phe- and KCl-induced cell contraction and
[Ca2+]i than those of WKY in all
groups of rats. These results are consistent with other studies that
have shown increased vascular tone in several vascular preparations
isolated from the SHR rat (8, 10, 16, 31). The underlying causes of the
observations that the reduction in cell contraction and
[Ca2+]i in intact females compared
with intact males was greater in SHR than WKY rats and that the effects
of exogenous application of estrogen on isolated cells of OVX females
were greater in SHR than WKY rats are not clear at the present time but
could be related to differences in the number of estrogen receptors or
in the number or permeability of the Ca2+ channels. This is
supported by reports that the activity of L-type Ca2+
channels is enhanced in vascular smooth muscle cells of the SHR rat
(25, 29).
Acute vs. genomic effects of estrogen. It is important to note
that exogenous estrogen caused inhibition of cell contraction and
[Ca2+]i at concentrations higher
than those observed in the plasma of intact females. Although both
exogenous application of estrogen and the presence of endogenous
estrogen were associated with reduction in cell contraction and
maintained [Ca2+]i, we do not wish
to make a definitive conclusion on whether the cellular mechanisms of
estrogen-induced inhibition of cell contraction and
[Ca2+]i observed in isolated cells
in vitro and the possible vasorelaxant effects of estrogen in vivo are
identical. The effects of estrogen on target tissues have been
classically thought of as arising from genomic actions mediated through
interaction with cytoplasmic receptors and translocation of the
hormone-receptor complex to the nucleus (26). Although a genomic action
of estrogen on the expression of the Ca2+ channels might
underlie the reduced cell contractility and
[Ca2+]i observed in aortic smooth
muscle cells of intact females, it is less likely to account for the
acute inhibitory effects of exogenous 17
-estradiol on cell
contraction and [Ca2+]i. The acute
nature of the vasorelaxant effects of exogenous estrogen may represent
additional nongenomic effects of estrogen on the mechanisms of
Ca2+ entry into vascular smooth muscle.
Finally, based on the present results in smooth muscle cells of
thoracic aorta, we cannot make a definite conclusion whether the
observed gender differences in cell contraction and
[Ca2+]i also occur in smooth muscle
of resistance vessels, which should represent an important area for
future investigations.
In conclusion, the vascular smooth muscle cell contractility and
increases in [Ca2+]i due to
maintained Ca2+ entry from the extracellular space, but not
Ca2+ release from intracellular stores, are reduced in the
presence, and enhanced in the absence, of female gonads. The
gender-specific differences in vascular smooth muscle cell contraction
and [Ca2+]i are possibly related to
endogenous estrogen. The gender-specific reduction in contractility and
[Ca2+]i in vascular smooth muscle
of female rats is greater in the SHR rat model of hypertension than the
WKY rat model.
 |
ACKNOWLEDGEMENTS |
This work was supported by a Grant-in-Aid from the American Heart
Association (Mississippi Affiliate) and by National Heart, Lung, and
Blood Institute Grants HL-52696 and HL-51971.
 |
FOOTNOTES |
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 and other correspondence: R. A. Khalil,
Dept. of Physiology and Biophysics, Univ. of Mississippi Medical
Center, 2500 North State St., Jackson, MS 39216-4505 (E-mail:
rkhalil{at}physiology.umsmed.edu).
Received 5 August 1999; accepted in final form 12 November 1999.
 |
REFERENCES |
1.
Barrett-Connor, E,
and
Bush TL.
Estrogen and coronary heart disease in women.
JAMA
265:
1861-1867,
1991[Abstract].
2.
Bayard, F,
Clamens S,
Meggetto F,
Blaes N,
Delsol G,
and
Faye JC.
Estrogen synthesis, estrogen metabolism, and functional estrogen receptors in rat arterial smooth muscle cells in culture.
Endocrinology
136:
1523-1529,
1995[Abstract].
3.
Bing, RJ,
and
Conforto A.
Reversal of acetylcholine effect on atherosclerotic coronary arteries by estrogen: pharmacologic phenomenon of clinical importance?
J Am Coll Cardiol
20:
458-459,
1992[ISI][Medline].
4.
Bush, TL,
Cowan LD,
Barrett-Connor E,
Criqui MH,
Karon JM,
Wallace RB,
Tyroler HA,
and
Rifkind BM.
Estrogen use and all-cause mortality. Preliminary results from the lipid research clinics program follow-up study.
JAMA
249:
903-906,
1983[Abstract].
5.
Clowes, AW,
Reidy MA,
and
Clowes MM.
Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in the absence of endothelium.
Lab Invest
49:
327-333,
1983[ISI][Medline].
6.
Collins, P,
Rosano GM,
Sarrel PM,
Ulrich L,
Adamopoulos S,
Beale CM,
McNeill JG,
and
Poole-Wilson PA.
17
-Estradiol attenuates acetylcholine-induced coronary arterial constriction in women but not men with coronary heart disease.
Circulation
92:
24-30,
1995[Abstract/Free Full Text].
7.
Crews, JK,
and
Khalil RA.
Antagonistic effects of 17
-estradiol, progesterone, and testosterone on Ca2+ entry mechanisms of coronary vasoconstriction.
Arterioscler Thromb Vasc Biol
19:
1034-1040,
1999[Abstract/Free Full Text].
8.
Falcone, JC,
Granger HJ,
and
Meininger GA.
Enhanced myogenic activation in skeletal muscle arterioles from spontaneously hypertensive rats.
Am J Physiol Heart Circ Physiol
265:
H1847-H1855,
1993[Abstract/Free Full Text].
9.
Farhat, MY,
Lavigne MC,
and
Ramwell PW.
The vascular protective effects of estrogen.
FASEB J
10:
615-624,
1996[Abstract/Free Full Text].
10.
Gebremedhin, D,
Fenoy FJ,
Harder DR,
and
Roman RJ.
Enhanced vascular tone in the renal vasculature of spontaneously hypertensive rats.
Hypertension
16:
648-654,
1990[Abstract].
11.
Gerhard, M,
and
Ganz P.
How do we explain the clinical benefits of estrogen? From bedside to bench.
Circulation
92:
5-8,
1995[Free Full Text].
12.
Gilligan, DM,
Badar DM,
Panza JA,
Quyyumi AA,
and
Cannon RO, III.
Acute vascular effects of estrogen in postmenopausal women.
Circulation
90:
786-791,
1994[Abstract].
13.
Gisclard, V,
Miller VM,
and
Vanhoutte PM.
Effect of 17
-estradiol on endothelium-dependent responses in the rabbit.
J Pharmacol Exp Ther
244:
19-22,
1988[Abstract].
14.
Grynkiewicz, G,
Poenie M,
and
Tsien RY.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J Biol Chem
260:
3440-3450,
1985[Abstract].
15.
Harder, DR,
and
Coulson PB.
Estrogen receptors and effects of estrogen on membrane electrical properties of coronary vascular smooth muscle.
J Cell Physiol
100:
375-382,
1979[ISI][Medline].
16.
Hayashi, K,
Epstein M,
and
Loutzenhiser R.
Pressure-induced vasoconstriction of renal microvessels in normotensive and hypertensive rats. Studies in the isolated perfused hydronephrotic kidney.
Circ Res
65:
1475-1484,
1989[Abstract].
17.
Herrington, DM,
Braden GA,
Williams JK,
and
Morgan TM.
Endothelial-dependent coronary vasomotor responsiveness in postmenopausal women with and without estrogen replacement therapy.
Am J Cardiol
73:
951-952,
1994[ISI][Medline].
18.
Jiang, CW,
Sarrel PM,
Lindsay DC,
Poole-Wilson PA,
and
Collins P.
Endothelium-independent relaxation of rabbit coronary artery by 17
-oestradiol in vitro.
Br J Pharmacol
104:
1033-1037,
1991[Abstract].
19.
Johnson, BD,
Zheng W,
Korach KS,
Scheuer T,
Catterall WA,
and
Rubanyi GA.
Increased expression of the cardiac L-type calcium channel in estrogen receptor-deficient mice.
J Gen Physiol
110:
135-140,
1997[Abstract/Free Full Text].
20.
Kannel, WB,
Hjortland MC,
McNamara PM,
and
Gordon T.
Menopause and risk of cardiovascular disease: the Framingham study.
Ann Intern Med
85:
447-452,
1976[ISI][Medline].
21.
Khalil, RA,
Lajoie C,
and
Morgan KG.
In situ determination of [Ca2+]i threshold for translocation of the
-protein kinase C isoform.
Am J Physiol Cell Physiol
266:
C1544-C1551,
1994[Abstract/Free Full Text].
22.
Khalil, RA,
and
Morgan KG.
Phenylephrine-induced translocation of protein kinase C and shortening of two types of vascular cells of the ferret.
J Physiol (Lond)
455:
585-599,
1992[Abstract].
23.
Khalil, RA,
and
van Breemen C.
Mechanisms of calcium mobilization and homeostasis in vascular smooth muscle and their relevance to hypertension.
In: Hypertension: Pathophysiology, Diagnosis, and Management, edited by Laraagh JH,
and Brenner BM.. New York: Raven, 1995, p. 523-540.
24.
Khalil, RA,
and
van Breemen C.
Sustained contraction of vascular smooth muscle: calcium influx or C-kinase activation?
J Pharmacol Exp Ther
244:
537-542,
1988[Abstract].
25.
Kubo, T,
Taguchi K,
and
Ueda M.
L-type calcium channels in vascular smooth muscle cells from spontaneously hypertensive rats: effects of calcium agonist and antagonist.
Hypertens Res
21:
33-37,
1998[Medline].
26.
Landers, JP,
and
Spelsberg TC.
New concepts in steroid hormone action: transcription factors, proto-oncogenes, and the cascade model for steroid regulation of gene expression.
Crit Rev Eukaryot Gene Expr
2:
19-63,
1992[Medline].
27.
Leijten, PA,
and
van Breemen C.
The effects of caffeine on the noradrenaline-sensitive calcium store in rabbit aorta.
J Physiol (Lond)
357:
327-339,
1984[Abstract].
28.
Lin, AL,
and
Shain SA.
Estrogen-mediated cytoplasmic and nuclear distribution of rat cardiovascular estrogen receptors.
Arteriosclerosis
5:
668-677,
1985[Abstract].
29.
Martens, JR,
and
Gelband CH.
Ion channels in vascular smooth muscle: alterations in essential hypertension.
Proc Soc Exp Biol Med
218:
192-203,
1998[Abstract].
30.
Mendoza, SG,
Zerpa A,
Carrasco H,
Colmenares O,
Rangel A,
Gartside PS,
and
Kashyap ML.
Estradiol, testosterone, apolipoproteins, lipoprotein cholesterol, and lipolytic enzymes in men with premature myocardial infarction and angiographically assessed coronary occlusion.
Artery
12:
1-23,
1983[ISI][Medline].
31.
Mulvany, MJ,
and
Nyborg N.
An increased calcium sensitivity of mesenteric resistance vessels in young and adult spontaneously hypertensive rats.
Br J Pharmacol
71:
585-596,
1980[Abstract].
32.
Nakajima, T,
Kitazawa T,
Hamada E,
Hazama H,
Omata M,
and
Kurachi Y.
17
-Estradiol inhibits the voltage-dependent L-type Ca2+ currents in aortic smooth muscle cells.
Eur J Pharmacol
294:
625-635,
1995[ISI][Medline].
33.
Nakao, J,
Chang WC,
Murota SI,
and
Orimo H.
Estradiol-binding sites in rat aortic smooth muscle cells in culture.
Atherosclerosis
38:
75-80,
1981[ISI][Medline].
34.
Nayler, WG,
Perry SE,
Elz JS,
and
Daly MJ.
Calcium, sodium, and the calcium paradox.
Circ Res
55:
227-237,
1984[Abstract].
35.
Phillips, GB,
Castelli WP,
Abbott RD,
and
McNamara PM.
Association of hyperestrogenemia and coronary heart disease in men in the Framingham cohort.
Am J Med
74:
863-869,
1983[ISI][Medline].
36.
Stallone, JN,
Crofton JT,
and
Share L.
Sexual dimorphism in vasopressin-induced contraction of rat aorta.
Am J Physiol Heart Circ Physiol
260:
H453-H458,
1991[Abstract/Free Full Text].
37.
Stampfer, MJ,
Colditz GA,
Willett WC,
Manson JE,
Rosner B,
Speizer FE,
and
Hennekens CH.
Postmenopausal estrogen therapy and cardiovascular disease. Ten-year follow-up from the nurses' health study.
N Engl J Med
325:
756-762,
1991[Abstract].
38.
Tamaya, T,
Wada K,
Nakagawa M,
Misao R,
Itoh T,
Imai A,
and
Mori H.
Sexual dimorphism of binding sites of testosterone and dihydrotestosterone in rabbit model.
Comp Biochem Physiol A Physiol
105:
745-749,
1993[ISI].
39.
Wellman, GC,
Bonev AD,
Nelson MT,
and
Brayden JE.
Gender differences in coronary artery diameter involve estrogen, nitric oxide, and Ca2+-dependent K+ channels.
Circ Res
79:
1024-1030,
1996[Abstract/Free Full Text].
40.
Williams, DA,
Becker PL,
and
Fay FS.
Regional changes in calcium underlying contraction of single smooth muscle cells.
Science
235:
1644-1648,
1987[ISI][Medline].
41.
Wolinsky, H.
Effects of estrogen and progestogen treatment on the response of the aorta of male rats to hypertension. Morphological and chemical studies.
Circ Res
30:
341-349,
1972[ISI][Medline].
42.
Wren, BG.
The effect of oestrogen on the female cardiovascular system.
Med J Aust
157:
204-208,
1992[ISI][Medline].
43.
Zhang, F,
Ram JL,
Standley PR,
and
Sowers JR.
17
-Estradiol attenuates voltage-dependent Ca2+ currents in A7r5 vascular smooth muscle cell line.
Am J Physiol Cell Physiol
266:
C975-C980,
1994[Abstract/Free Full Text].
Am J Physiol Cell Physiol 278(4):C834-C844
0363-6143/00 $5.00
Copyright © 2000 the American Physiological Society