Department of Physiology and Biophysics and Center for Excellence in Cardiovascular-Renal Research, University of Mississippi Medical Center, Jackson, Mississippi 39216
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ABSTRACT |
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Gender
differences in vascular reactivity have been suggested; however, the
cellular mechanisms involved are unclear. We tested the hypothesis that
the gender differences in vascular reactivity reflect gender-related,
possibly estrogen-mediated, distinctions in the expression and activity
of specific protein kinase C (PKC) isoforms in vascular smooth muscle.
Aortic strips were isolated from intact and gonadectomized male and
female Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats
(SHR). Isometric contraction was measured in endothelium-denuded aortic
strips. PKC activity was measured in the cytosolic and
particulate fractions, and the amount of PKC was measured using Western
blots and isoform-specific anti-PKC antibodies. In intact male WKY
rats, phenylephrine (Phe, 105 M) and phorbol
12,13-dibutyrate (PDBu, 10
6 M) stimulated contraction to
0.37 ± 0.02 and 0.42 ± 0.02 g/mg tissue wt, respectively.
The basal particulate/cytosolic PKC activity ratio was 0.86 ± 0.06, and Western blots revealed
-,
-, and
-PKC isoforms. Phe
and PDBu increased PKC activity and caused significant translocation of
- and
-PKC from the cytosolic to particulate fraction. In intact
female WKY rats, basal PKC activity, the amount of
-,
-, and
-PKC, the Phe- and PDBu-induced contraction, and PKC activity and
translocation of
- and
-PKC were significantly reduced compared
with intact male WKY rats. The basal PKC activity, the amount of
-,
-, and
-PKC, the Phe and PDBu contraction, and PKC activity and
- and
-PKC translocation were greater in SHR than WKY rats. The
reduction in Phe and PDBu contraction and PKC activity in intact
females compared with intact males was greater in SHR (~30%) than
WKY rats (~20%). Phe and PDBu contraction and PKC activity were not
significantly different between castrated males and intact males but
were greater in ovariectomized (OVX) females than intact females.
Treatment of OVX females or castrated males with 17
-estradiol, but
not 17
-estradiol, subcutaneous implants caused significant reduction
in Phe and PDBu contraction and PKC activity that was greater in SHR
than WKY rats. Phe and PDBu contraction and PKC activity in OVX females
or castrated males treated with 17
-estradiol plus the estrogen
receptor antagonist ICI-182,780 were not significantly different from
untreated OVX females or castrated males. Thus a gender-related
reduction in vascular smooth muscle contraction in female WKY rats with
intact gonads compared with males is associated with reduction in the expression and activity of vascular
-,
-, and
-PKC. The gender differences in vascular smooth muscle contraction and PKC activity are
augmented in the SHR and are possibly mediated by estrogen.
contraction; protein kinase C; sex hormones
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INTRODUCTION |
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CARDIOVASCULAR DISEASES REPRESENT a major health problem in the industrialized world. The greater incidence of cardiovascular diseases in men and postmenopausal women compared with premenopausal women and the potential vascular benefits of estrogen replacement therapy in postmenopausal women have suggested gender-related, possibly estrogen-mediated, vascular protective effects (9, 11). The putative beneficial vascular effects of estrogen have been ascribed to a variety of factors, including endothelium-dependent (14) and endothelium-independent vascular relaxation (5, 13, 17). The estrogen-induced relaxation in deendothelialized vascular strips (5, 17) has suggested other mechanisms in addition to the classic genomic pathway of steroid action (27), possibly involving effects on the cellular mechanisms of vascular smooth muscle contraction.
It is widely accepted that vascular smooth muscle contraction is
triggered by increases in intracellular free Ca2+
concentration ([Ca2+]i; see Refs.
15, 25, 36, 39).
Also, activation of several protein kinases such as myosin light chain
(MLC) kinase, Rho kinase, and mitogen-activated protein kinase as well
as inhibition of protein phosphatases such MLC phosphatase have been
suggested to contribute to smooth muscle contraction (15,
39). Additionally, in numerous cell types, including vascular
smooth muscle, the interaction of an -adrenergic agonist such as
phenylephrine (Phe) with its receptor not only increases
[Ca2+]i but is also coupled to increased
breakdown of plasma membrane phospholipids and increased production of
diacylglycerol (DAG; see Refs. 20 and 34). DAG binds to
and activates protein kinase C (PKC). PKC is mainly cytosolic under
resting conditions and undergoes translocation from the cytosolic to
the particulate fraction when it is activated by endogenous DAG or
exogenous phorbol esters (20, 34). Also, direct activation
of PKC by phorbol esters such as phorbol 12,13-dibutyrate (PDBu) causes
sustained contraction of vascular smooth muscle (26) with
no significant change in [Ca2+]i
(16), suggesting a role for PKC in regulating vascular
smooth muscle contraction. PKC is now known to be a family of several isoforms that have different enzyme properties, substrates, and functions and exhibit different subcellular distributions in the same
blood vessel from different species and in different vessels from the
same species (20, 23, 29, 34).
The putative vascular protective effects of estrogen in females with intact gonadal function and their proposed absence in males or in females with reduced gonadal function (9, 11) suggest that vascular smooth muscle contraction may be modified by gender and by the presence or absence of functional female gonads. Although several studies have shown gender differences in vascular reactivity to a variety of agonists (21, 40, 42), little is known about the effect of gender and the status of the gonads on the various cellular mechanisms of vascular smooth muscle contraction. Because [Ca2+]i is a major determinant of vascular smooth muscle contraction (15, 25, 36, 39), several previous studies have focused on the effect of gender and sex hormones on [Ca2+]i and the Ca2+ mobilization mechanisms of vascular reactivity (5, 6, 31, 32, 35, 44). However, the effects of gender and sex hormones on the expression and activity of other protein kinases and phosphatases that contribute to smooth muscle contraction are less clear. Specifically, although the changes in PKC activity have been well characterized in blood vessels of male rats and ferrets (23, 29), no information is available on the effect of gender and the status of the gonads on the expression and activity of the specific PKC isoforms in vascular smooth muscle. Also, because the gender differences in vascular reactivity could be small in animal models with normal vascular reactivity, such as the Wistar-Kyoto (WKY) rat, any gender differences in vascular reactivity or PKC activity are predicted to be more prominent in animal models that exhibit enhanced vascular reactivity, such as the spontaneously hypertensive rat (SHR; see Refs. 8, 10, 30). Although several studies have shown that the enhanced vascular reactivity in the SHR is associated with increased vascular PKC activity (2, 41), it is not clear whether the effects of gender and the status of the gonads on the expression and activity of vascular PKC isoforms are augmented in the SHR compared with the WKY rat.
The purpose of this study was to test the hypothesis that the gender differences in vascular reactivity reflect gender-related, possibly estrogen-mediated, distinctions in the expression and activity of PKC isoforms in vascular smooth muscle. We investigated 1) whether PKC-mediated vascular smooth muscle contraction is modified by gender and by the presence or absence of the gonads; 2) whether the gender-specific differences in vascular reactivity are associated with changes in the amount and/or activity of specific PKC isoforms in vascular smooth muscle; 3) whether the gender-specific differences in vascular reactivity and PKC activity are augmented in animal models with enhanced vascular reactivity such as the SHR compared with animal models with normal vascular reactivity such as the WKY rat; and 4) whether the gender-related distinctions in vascular smooth muscle contraction and PKC activity are possibly estrogen mediated by investigating the effects of long-term exposure to exogenous estrogen in gonadectomized female and male rats in vivo.
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METHODS |
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Animals.
WKY and SHR (12 wk; Harlan, Indianapolis, IN) were divided into the
following four groups: intact males (n = 16), intact
females (n = 16), castrated males (n = 16), and ovariectomized (OVX) females (n = 16).
Gonadectomy was performed by the vendor at 8 wk of age. Other OVX
female WKY (n = 8) and SHR (n = 8) and
castrated male WKY (n = 8) and SHR (n = 8) were given a subcutaneous timed-release 17-estradiol implant
(30-day release, 0.125 mg/pellet; Innovative Research of America,
Sarasota, FL) 3 days after gonadectomy and were studied 4 wk later.
Some OVX female WKY (n = 6) and castrated male WKY
(n = 6) rats were given subcutaneous timed-release
17
-estradiol implant (30-day release, 0.125 mg/pellet, Innovative
Research of America) 3 days after gonadectomy and were studied 4 wk
later. Other OVX female WKY (n = 6) and castrated male
WKY (n = 6) rats were simultaneously given subcutaneous
timed-release 17
-estradiol implants plus daily subcutaneous
injection of the estrogen receptor antagonist ICI-182,780 (1 mg · kg
1 · day
1 in corn
oil; Tocris, Ballwin, MO) 3 days after ovariectomy and studied 4 wk
later (43). All procedures followed 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, rats were anesthetized by inhalation of
isoflurane, and blood was collected for measurement of plasma
17-estradiol using an RIA kit (ICN Biomedicals, Costa Mesa, CA; see
Table 1).
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Isometric contraction.
The thoracic aorta was excised, placed in oxygenated Krebs solution,
cleaned of connective tissue, and cut into 3-mm-wide strips. The
putative vascular protective effects of estrogen have been ascribed to
several factors, including endothelium-dependent and
endothelium-independent vascular relaxation (5, 13, 14, 17). Thus measurement of the contractile response and the amount and activity of PKC isoforms in endothelium-intact aortic strips would
provide the lumped effects of gender and estrogen on both the vascular
endothelium and the vascular smooth muscle cells. To specifically
measure the endothelium-independent, gender-related, and
estrogen-mediated differences in the contractile response and the
expression and activity of PKC isoforms in vascular smooth muscle and
to avoid the possible contribution of endothelium-mediated effects of
estrogen on the contractile response and on the expression and activity
of PKC isoforms in endothelial cells, the present study was performed
in endothelium-denuded aortic strips. The endothelium was removed by
rubbing the vessel interior with forceps. One end of the strip was
attached to a glass hook, and the other end was connected to a Grass
force transducer (FT03E; Astro-Med, West Warwick, RI). Aortic strips
were stretched to optimal length (1.5 the initial unloaded length) and
allowed to equilibrate for 1 h in oxygenated Krebs solution at
37°C. The changes in isometric contraction were recorded on a Grass
polygraph model 7D (Astro-Med). Removal of the endothelium was verified
routinely by the absence of ACh (106 M)-induced
relaxation in aortic strips precontracted with Phe (3 × 10
7 M).
Tissue fractions.
Aortic strips were homogenized and centrifuged at 10,000 g
for 2 min, and the supernatant was used as the whole tissue fraction. Other strips were stimulated with Phe (105 M) or PDBu
(10
6 M) for 30 min, the time at which the contractile
response reaches steady state. In other experiments, the tissues were
pretreated with staurosporine (10
6 M) or calphostin C
(10
6 M) for 30 min and then were stimulated with Phe
(10
5 M) or PDBu (10
6 M) for 30 min. The
tissues were rapidly transferred to ice-cold equilibrating buffer
A (see composition under Solutions, drugs, and
chemicals), homogenized in homogenizing buffer B (see
composition under Solution, drugs, and chemicals), and
centrifuged at 100,000 rpm for 20 min, and the supernatant was used as
the cytosolic fraction. The pellet was resuspended in homogenizing
buffer B containing 1% Triton X-100 and centrifuged at
100,000 rpm for 20 min, and the supernatant was used as the particulate
fraction (19, 18). Protein concentrations were determined
using a protein assay kit (Bio-Rad, Hercules, CA).
PKC activity.
The cytosolic and particulate fractions were applied to DEAE-cellulose
columns (Bio-Rad), and the protein was eluted with 0.1 M NaCl. PKC
activity in the aliquots was determined by measuring the incorporation
of 32P from [-32P]ATP (ICN) into histone
III-S. The assay mixture contained 25 mM Tris · HCl (pH 7.5),
10 mM MgCl2, 200 µg/ml histone III-S, 80 µg/ml
phosphatidylserine, 30 µg/ml diolein, 1-3 × 105
counts · min
1 · nmol
1
[
-32P]ATP, and 0.5-3 µg protein. After 5 min of
incubation at 30°C, the reaction was stopped by spotting 25 µl of
the assay mixture on phosphocellulose discs. The discs were washed
3 × 5 min with 5% TCA and were placed in 4 ml Ecolite (ICN), and
the radioactivity was measured in a scintillation counter. The PKC
assay was performed in tissues both untreated or pretreated with the
PKC inhibitor staurosporine (10
6 M) or calphostin C
(10
6 M) for 30 min to confirm the specificity of the
assay (18, 19).
Immunoblotting.
Protein-matched samples of the whole tissue, cytosolic, and particulate
fractions were subjected to electrophoresis on 8% SDS-PAGE and then
were transferred to nitrocellulose membranes. The membranes were
incubated in 5% dry milk in PBS-Tween for 1 h and then were
incubated in the primary anti-PKC antibody solution at 4°C overnight.
Experiments were performed primarily with polyclonal antibodies to
-,
-,
-,
-,
-,
-, and
-PKC isoforms from GIBCO
(Grand Island, NY; 1:500). The specificity of each antibody has
previously been shown by the absence of cross-reactivity with other
endogenous forms of PKC or with fractions of cells transfected with other PKC sequences (23, 37). The specificity of each antibody was verified routinely by demonstrating that it was blocked by
the synthetic peptide to which the antibody was raised and not with
other sequences of the PKC molecule, a more stringent control than
simple elimination of the primary antibody. To confirm the results with
the GIBCO polyclonal antibodies, we also used polyclonal anti-PKC
antibodies from Sigma (St. Louis, MO; 1:500), polyclonal anti-PKC
antibodies from Chemicon (Temecula, CA; 1:100), and monoclonal anti-PKC
antibodies from Seikagaku America (Ijamsville, MD; 1:100) and obtained
similar results. We used the same titer of PKC antibodies and the same
amount of protein (10 µg) in all samples from the different rats.
These PKC antibody titers and protein concentration produced
significant immunoreactive bands while remaining on the linear portion
of the titration curve. The nitrocellulose membranes were washed 5 × 15 min and then were incubated in horseradish peroxidase-conjugated
anti-mouse or anti-rabbit IgG for 1.5 h (18, 19). The
blots were washed 5 × 15 min and were visualized with the
enhanced chemiluminescence detection system (Amersham, Arlington
Heights, IL). The reactive bands corresponding to PKC isoforms were
analyzed using an optical densitometer (GS-700; Bio-Rad).
Solutions, drugs, and chemicals.
Normal Krebs solution contained (in mM) 120 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 1.2 NaH2PO4, and 11.5 dextrose, pH 7.4. The high
KCl (96 mM) depolarizing solution had the same composition as normal
Krebs with equimolar substitution of NaCl with KCl. Equilibrating
buffer A contained (in mM) 25 Tris · HCl (pH 7.5), 5 EGTA, 0.02 leupeptin, 0.2 phenylmethylsulfonyl fluoride, and 1 dithiothreitol. Homogenizing buffer B had the same
composition as buffer A plus 250 mM sucrose. Phe (Sigma) was
prepared in distilled water. PDBu, 4- PDBu (Alexis Laboratories, San
Diego, CA), staurosporine, and calphostin C (Kamiya Laboratories,
Seattle, WA) were dissolved in DMSO to form a stock solution of
10
3 M. The final concentration of DMSO in solution was
0.1%. All other chemicals were of reagent grade or better.
Statistical analysis. All data were normalized to the tissue weight (in mg) or the amount of protein (in µg). Data were presented as means ± SE and were compared using the nonparametric Mann-Whitney test. Differences were considered statistically significant at P < 0.05.
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RESULTS |
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Gender differences in Phe- and PDBu-induced contraction.
In aortic strips of all groups of rats incubated in normal Krebs
solution, Phe caused concentration-dependent contractions that reached
a maximum at 105 M concentration. Also, the PKC activator
PDBu caused concentration-dependent contractions that reached a maximum
at 10
6 M. No significant contraction was observed in
tissues treated with the inactive 4-
PDBu. In intact male WKY rats,
the ED50 for Phe and PDBu were 0.27 ± 0.03 × 10
6 and 0.29 ± 0.04 × 10
7 M,
respectively. The sensitivity to Phe or PDBu was not significantly different between intact male WKY and other WKY rats of different gender or status of the gonads (Table 1).
The sensitivity to Phe and PDBu was enhanced significantly in all
groups of SHR compared with WKY rats (Table 1). However, no significant
differences in the ED50 of Phe or PDBu were observed
between SHR of different gender or status of the gonads (Table 1).
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Gender differences in basal and Phe- and PDBu-stimulated PKC
activity.
We investigated whether the gender differences in vascular smooth
muscle contraction reflect differences in vascular PKC activity. In
aortic strips of intact male WKY rats, the basal PKC activity was
greater in the cytosolic fraction than the particulate fraction (Fig.
2A), and the
particulate/cytosolic (P/C) PKC activity ratio was 0.86 ± 0.06 (Fig. 2B). Phe (105 M) and PDBu
(10
6 M) caused a significant increase in PKC activity in
the particulate fraction and a concomitant decrease in the cytosolic
fraction (Fig. 2, C and E) and increased the P/C
PKC activity ratio to 1.55 ± 0.08 and 1.75 ± 0.07, respectively (Fig. 2, D and F). The Phe- and
PDBu-stimulated PKC activity was inhibited completely in aortic strips
pretreated with the PKC inhibitor staurosporine (10
6 M)
or calphostin C (10
6 M) for 30 min (Table 2). In intact
female WKY rats, the basal and Phe- and PDBu-stimulated PKC activity
was significantly reduced compared with intact male WKY rats (Fig. 2,
B, D, and F). The basal, Phe- and
PDBu-stimulated PKC activities were not significantly different between
castrated males and intact males but were significantly greater in OVX
females than intact females (Fig. 2, B, D, and F). In SHR, the basal and Phe- and PDBu-induced PKC activity
was significantly greater than that in WKY in all groups of rats (Fig. 2, B, D, and F). The percent reduction
in basal and Phe- and PDBu-stimulated PKC activity in intact female SHR
compared with intact male SHR (29.6, 31.3, and 33.3%, respectively)
was greater than that observed in intact female WKY compared with
intact male WKY rats (20.9, 19.4, and 21.1%, respectively; Table 3).
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Gender differences in the amount and distribution of PKC isoforms.
Immunoblot analysis using the whole tissue homogenate showed
significant immunoreactive bands at ~80, ~80, and ~70 kDa with specific antisera to -,
-, and
-PKC, respectively (Fig.
3). The specificity of the
-,
-,
and
-PKC reactive bands was confirmed by the loss of immunoreactive
signal when the primary antibody was omitted from the experimental
protocol or when the tissue samples were incubated in the primary
antibody solution plus the synthetic peptide to which the antibody was
raised. No significant immunoreactive bands were detected with
antibodies to
-,
-,
-, or
-PKC. In intact male WKY rats,
the amount of
-,
-, and
-PKC, as indicated by the optical
density, was 1.4 ± 0.1, 1.3 ± 0.1, and 1.9 ± 0.1, respectively (Fig. 3). In intact female WKY rats, the amount of
-,
-, and
-PKC was significantly reduced compared with intact male
WKY rats (Fig. 3). The amount of
-,
-, and
-PKC was not
significantly different between castrated males and intact males but
was significantly greater in OVX females than intact females (Fig. 3).
The amount of
-,
-, and
-PKC was greater in SHR than WKY in
all groups of rats (Fig. 3). The percent reduction in the amount of
whole tissue
-,
-, and
-PKC in intact female SHR compared
with intact male SHR (30.0, 31.6, and 32.1%, respectively) was greater
than that observed in intact female WKY compared with intact male WKY
rats (21.4, 23.1, and 21.1%, respectively; Table 3).
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Vascular reactivity and PKC activity in gonadectomized rats
with estrogen implants.
The Phe and PDBu contraction and the basal and Phe- and
PDBu-stimulated PKC activity were not significantly different between WKY OVX females with 17-estradiol implants and untreated OVX females
(Table 4). In contrast, significant
reductions in contraction and PKC activity were observed in OVX female
WKY rats with 17
-estradiol implants compared with untreated OVX
females (Table 4). In OVX females simultaneously treated with
17
-estradiol implants plus the estrogen receptor antagonist
ICI-182,780, the Phe and PDBu contraction and the basal and Phe- and
PDBu-stimulated PKC activity were not significantly different from
untreated OVX females (Table 4). Similarly, significant
reductions in contraction and PKC activity were observed in castrated
male WKY rats with 17
-estradiol but not 17
-estradiol implants
compared with untreated castrated males (Table 4). In castrated males
simultaneously treated with 17
-estradiol implants plus
ICI-182,780, the Phe and PDBu contraction and the basal and Phe- and
PDBu-stimulated PKC activity were not significantly different from
untreated castrated males (Table 4). The percent reduction in Phe and
PDBu contraction and the basal and Phe- and PDBu-stimulated PKC
activity in OVX females with 17
-estradiol implants compared with
untreated OVX females was greater in SHR than WKY rats (Table
5).
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DISCUSSION |
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Gender differences in agonist-induced vascular smooth muscle contraction. The present study showed that maximal Phe contraction was greater in intact males than intact females in both WKY and SHR. These results are consistent with reports that norepinephrine contraction in the aorta of WKY and SHR is ~1.7-fold greater in males than females (42), that the maximal Phe contraction in the aorta of male Sprague-Dawley rats is almost two times that in females (40), and that the contraction to norepinephrine and thromboxane A2 analogs in canine renal and coronary vessels is greater in males than in females (21). However, based on these data, we do not wish to generalize that the gender difference in vascular reactivity is a uniform phenomenon that applies to all types of contractile agonists. Interestingly, vasopressin-induced contraction in rat aorta exhibits sexual dimorphism opposite to that observed with Phe, i.e., vasopressin contraction in females is almost two times that in males (40). The cause of the difference between the contractile response to Phe and vasopressin is not clear but could be related to the following: 1) in contrast to the present experiments in endothelium-denuded aortic strips, the vasopressin experiments were performed in endothelium-intact strips, raising the possibility of the contribution of endothelium-derived vasoactive factors; 2) vasopressin may exhibit a tachyphylactic response in isolated vessels that is different from its effects when administered in vivo. This is supported by reports that the pressor response to vasopressin infusion in vivo is two- to threefold greater in male than female rats (7); and 3) the differences between Phe and vasopressin responses may be related to differences in their signaling mechanisms downstream from receptor activation.
Gender differences in PKC activity of vascular smooth muscle.
The lack of gender differences in the ED50 of Phe suggests
that the differences in vascular reactivity may not be related to the
sensitivity of -adrenergic receptors to Phe. On the other hand, the
observed gender differences in the maximal Phe contraction suggest
differences in the postreceptor signaling mechanisms of smooth muscle
contraction. We investigated whether the gender differences in vascular
reactivity reflect differences in the PKC-mediated pathway of smooth
muscle contraction. Phorbol esters are known to strongly bind to and
activate PKC (20, 34). Although no gender differences in
the ED50 of PDBu were observed, the maximal PDBu
contraction was greater in intact males than in intact females. PDBu
also significantly increased PKC activity, and both PDBu-induced contraction and PKC activity were inhibited by the PKC inhibitors staurosporine and calphostin C. Phe also caused a significant increase
in PKC activity that was inhibited by staurosporine and calphostin C. These results are consistent with other reports and suggest that PKC is
involved in Phe-induced contraction of rat aortic smooth muscle
(23, 29). The observation that the Phe and PDBu
contraction and PKC activity were greater in intact males than intact
females suggested gender differences in the PKC-mediated pathway of
smooth muscle contraction. The gender differences in vascular PKC
activity could be related to the amount of PKC expressed in vascular
smooth muscle and/or the sensitivity of the PKC pathway to endogenous
sex hormones.
Gender differences in the amount and distribution of PKC isoforms.
We investigated whether the gender differences in vascular PKC activity
are related to the amount and distribution of specific PKC isoforms in
vascular smooth muscle. The immunoblot analysis showed significant
amounts of - and
-PKC, and both Phe and PDBu caused significant
redistribution of
- and
-PKC in aortic smooth muscle of
intact male WKY rats. These results are consistent with reports that
both
- and
-PKC are expressed in the aorta of male Sprague-Dawley
rats and that they undergo translocation during Phe- and PDBu-induced
contraction in this tissue (29). The observations that the
amount of
- and
-PKC and the basal and Phe- and PDBu-induced redistribution of
- and
-PKC from the cytosolic to the
particulate fraction were reduced in intact females compared with
intact males suggest that the gender differences in vascular reactivity
are related, in part, to underlying changes in the amount and activity of
- and
-PKC.
Effects of gonadectomy and estrogen replacement on vascular
reactivity and PKC activity.
The Phe and PDBu contraction and PKC activity were not different
between castrated males and intact males but were significantly greater
in OVX females than intact females, suggesting that the gender
differences in vascular reactivity and PKC activity are less likely
related to androgens but are more likely related to estrogen. This is
supported by the observation that insertion of 17-estradiol implants
in OVX females and castrated males was associated with significant
reduction in vascular reactivity and PKC activity. These data suggest
that the gender differences in vascular reactivity and PKC activity may
be related, in part, to the plasma level of estrogen.
Gender differences in PKC activity in an animal model with enhanced
vascular reactivity.
It has been reported that PKC activity is greatest in the particulate
fractions relative to the soluble fractions and similar in aortic
smooth muscle of SHR and WKY rats (38). We found that the
Phe- and PDBu-induced vascular reactivity and PKC activity were greater
in SHR than WKY rats in all groups of rats. Our results are consistent
with reports that vascular tone is enhanced in SHR (8, 10,
30) and that vascular PKC activity is augmented in the SHR and
other rat models with enhanced vascular reactivity (2, 29,
41). We also found that the reduction in vascular reactivity and
PKC activity in intact females compared with intact males was greater
in SHR than WKY rats. Our first prediction was that the greater
reduction in vascular reactivity and PKC activity in intact female SHR
compared with WKY rats is related to differences in their plasma
estrogen levels. Although the plasma estrogen levels appeared to be
higher in intact female SHR than intact female WKY rats (Table 1), the
difference did not reach statistical significance, suggesting that the
greater reduction in vascular reactivity and PKC activity in intact
female SHR compared with WKY rats is less likely related to the plasma
levels of estrogen per se. We can only suggest that the greater
reduction in vascular reactivity and PKC activity in intact female SHR
compared with WKY rats could be related, among other factors, to
inherent differences in both the amount of vascular PKC isoforms
expressed in vascular smooth muscle and the sensitivity of the PKC
pathway to the effects of estrogen. This is supported by the
observations that 1) the amounts of PKC isoforms were
consistently greater in SHR than WKY rats and 2) the
reduction in PKC activity in OVX females with 17-estradiol implants
compared with untreated OVX females was greater in SHR than WKY rats
although the plasma 17
-estradiol was restored to similar levels in
both SHR and WKY rats.
Other potential mechanisms of gender differences in vascular reactivity. Although the present results provide evidence that the gender differences in vascular reactivity may be related to differences in vascular PKC activity, these results should be interpreted with caution. In addition to PKC, other protein kinases, such as MLC kinase, Rho kinase, and tyrosine kinase, as well as protein phosphatases, such as MLC phosphatase, have been suggested to regulate vascular smooth muscle contraction (15, 20, 39). Whether the expression and activity of these protein kinases and phosphatases differ with gender is unclear and should be examined in future investigations. In relation to this question, we and others have shown that the vascular reactivity to membrane depolarization by high KCl, which mainly stimulates Ca2+ entry from the extracellular space (25, 26), is greater in SHR than WKY rats (3, 6, 32). We have also reported that the KCl-induced contraction is reduced in intact female compared with intact male or OVX female WKY or SHR (6, 32), suggesting that Ca2+ entry is modified by gender and is reduced in the presence of estrogen. This is supported by reports that estrogen inhibits Ca2+ entry in isolated porcine coronary artery (5) and blocks Ca2+ channels in cultured rat aortic smooth muscle cells (33, 44). Although the gender differences in vascular PKC activity and Ca2+ entry may represent two independent signaling events, we cannot rule out the possibility that they are related. This is supported by reports that Ca2+ entry in vascular smooth muscle is modified during PKC activation by phorbol esters (12, 26).
Finally, although the observed gender differences in vascular reactivity could be explained, in part, by possible effects of estrogen on the signaling mechanisms of vascular smooth muscle contraction, the effects of estrogen on other vascular cell types such as the endothelium cannot be trivialized and should raise several questions. For example, what happens to this pattern of observations if the endothelium is left intact? Also, with an intact endothelium, is the impact of gender on the contractile response and PKC activity still observed? Additionally, are the changes in the contractile response and PKC activity responsive to estrogen replacement in the OVX animals? Previous studies in endothelium-intact aortic strips of Sprague-Dawley rats have shown gender differences in the contractile response to Phe and vasopressin (40). Other studies have also shown that endothelium-dependent vascular relaxation in endothelium-intact aortic strips is greater in female compared with male SHR (22). Thus gender differences in the contractile response are observed not only in endothelium-denuded aortic strips but also in endothelium-intact aortic strips. However, the question remains whether gender differences in PKC activity also occur in endothelial cells. This question can be studied by comparing PKC activity in endothelium-intact and endothelium-denuded vascular strips of intact and gonadectomized animals or by studying the effects of estrogen on a uniform population of endothelial cells such as cultured endothelial cells from intact and gonadectomized animals and should represent important areas for future investigations. In conclusion, a gender-specific reduction in vascular smooth muscle reactivity in female rats with intact gonadal function compared with males is associated with a reduction in the expression and activity of vascular ![]() |
ACKNOWLEDGEMENTS |
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This work was supported by grants from the American Heart Association (Grant-in-Aid, Mississippi Affiliate) and by National Heart, Lung, and Blood Institute Grants HL-52696 and HL-51971.
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FOOTNOTES |
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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 (E-mail: rkhalil{at}physiology.umsmed.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 22 June 2000; accepted in final form 23 August 2000.
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