Gender-related distinctions in protein kinase C activity in rat vascular smooth muscle

Celia A. Kanashiro 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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, 10-5 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 alpha -, delta -, and zeta -PKC isoforms. Phe and PDBu increased PKC activity and caused significant translocation of alpha - and delta -PKC from the cytosolic to particulate fraction. In intact female WKY rats, basal PKC activity, the amount of alpha -, delta -, and zeta -PKC, the Phe- and PDBu-induced contraction, and PKC activity and translocation of alpha - and delta -PKC were significantly reduced compared with intact male WKY rats. The basal PKC activity, the amount of alpha -, delta -, and zeta -PKC, the Phe and PDBu contraction, and PKC activity and alpha - and delta -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 17beta -estradiol, but not 17alpha -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 17beta -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 alpha -, delta -, and zeta -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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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.


    METHODS
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INTRODUCTION
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 17beta -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 17alpha -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 17beta -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 17beta -estradiol using an RIA kit (ICN Biomedicals, Costa Mesa, CA; see Table 1).

                              
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Table 1.   Plasma 17beta -estradiol level and ED50 for Phe and PDBu in aortic strips of intact and gonadectomized male and female WKY rats and SHR untreated or chronically treated with 17beta -estradiol implants

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 (10-6 M)-induced relaxation in aortic strips precontracted with Phe (3 × 10-7 M).

The effects of the alpha -adrenergic agonist Phe, as a more physiological activator of PKC (24, 23, 29), were compared with direct activation of PKC by PDBu. After three control 96 mM KCl contractions followed by washing with normal Krebs solution, the aortic strips were stimulated with different concentrations of Phe or PDBu, and both the maximal contractile responses and the ED50 were measured. Other tissues were stimulated with Phe (10-5 M) or PDBu (10-6 M) until the contraction reached steady state (~30 min) and were treated with the PKC inhibitors staurosporine (10-6 M) or calphostin C (10-6 M), and the inhibition of contraction was measured when it reached steady state. In other experiments, the tissues were pretreated with staurosporine or calphostin C for 30 min and stimulated with Phe (10-5 M) or PDBu (10-6 M), and the contractile responses were measured after 30 min of stimulation.

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 (10-5 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 [gamma -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 [gamma -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 alpha -, beta -, gamma -, delta -, epsilon -, eta -, and zeta -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-alpha 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.


    RESULTS
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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
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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 10-5 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-alpha 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).

In aortic strips of all groups of rats, the contractions to maximal concentrations of Phe (10-5 M) and PDBu (10-6 M) reached a plateau in ~30 min and were maintained for at least 1 h. The Phe- and PDBu-induced contractions were sensitive to the PKC inhibitors staurosporine and calphostin C (Fig. 1A and Table 2). Staurosporine (10-6 M) caused significant inhibition of Phe and PDBu contraction that reached steady state in ~30 min. Calphostin C (10-6 M) also significantly inhibited Phe and PDBu contraction (Fig. 1A) but was slower in onset than staurosporine and reached steady state in ~1 h. Pretreatment of the aortic strips with staurosporine or calphostin C for 30 min significantly inhibited the Phe and PDBu contractions to levels not significantly different from those observed when Phe or PDBu was added first and then staurosporine or calphostin C. 


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Fig. 1.   A: effect of the protein kinase C (PKC) inhibitors staurosporine (10-6 M) and calphostin C (10-6 M) on phenylephrine (Phe; 10-5 M) and phorbol 12,13-dibutyrate (PDBu; 10-6)-induced contraction in aortic strips of intact male Wistar-Kyoto (WKY) rats. B and C: vascular smooth muscle contraction in response to Phe (10-5 M; B) or PDBu (10-6 M; C) in aortic strips of intact male, intact female, castrated male, and ovariectomized (OVX) female WKY rats and spontaneously hypertensive rats (SHR). Bars represent means ± SE of measurements in 12-16 individual aortic strips from 6-8 rats of each group. P < 0.05: *SHR significantly different from WKY rats; #intact female WKY rats significantly different from other groups of WKY rats; dagger intact female SHR significantly different from other groups of SHR.


                              
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Table 2.   Effect of staurosporine and calphostin C on Phe- and PDBu-induced contraction and P/C PKC activity in aortic strips of intact and gonadectomized male and female WKY rats and SHR

We compared the maximal Phe- and PDBu-induced contraction in the different groups of rats. In intact male WKY rats, the Phe (10-5 M)- and PDBu (10-6 M)-induced contraction was 0.37 ± 0.02 and 0.42 ± 0.02 g/mg tissue wt, respectively (Fig. 1). In intact female WKY rats, the Phe and PDBu contraction was reduced significantly compared with intact male WKY rats (Fig. 1, B and C). The Phe- and PDBu-induced contractions were not significantly different between castrated males and intact males but were significantly greater in OVX females than intact females (Fig. 1, B and C). In SHR, the Phe and PDBu contractions were significantly greater than those of WKY in all groups of rats (Fig. 1, B and C). The percent reduction in Phe- and PDBu-induced contraction in intact female SHR compared with intact male SHR (29.5 and 27.3%, respectively) was greater than that in intact female WKY compared with intact male WKY rats (21.6 and 19.1%, respectively; Table 3).

                              
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Table 3.   Contraction, P/C PKC activity, and the amount in whole tissue and P/C distribution of PKC isoforms under basal conditions and during stimulation with Phe or PDBu in aortic strips of intact male and intact female WKY rats and SHR

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 (10-5 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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Fig. 2.   Basal (A and B) and Phe (10-5 M; C and D)- and PDBu (10-6 M; E and F)-induced changes in PKC activity in the cytosolic (Cyt) and particulate (Part) fraction (A, C, and E) and the particulate/cytosolic (P/C) PKC activity ratio (B, D, and F) in aortic strips of intact male, intact female, castrated male, and OVX female WKY rats and SHR. Data represent means ± SE of measurements from 18 experiments on tissue samples from 6 rats in each group. P < 0.05: *SHR significantly different from WKY rats; #intact female WKY rats significantly different from other groups of WKY rats; dagger intact female SHR significantly different from other groups of SHR.

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 alpha -, delta -, and zeta -PKC, respectively (Fig. 3). The specificity of the alpha -, delta -, and zeta -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 beta -, gamma -, epsilon -, or eta -PKC. In intact male WKY rats, the amount of alpha -, delta -, and zeta -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 alpha -, delta -, and zeta -PKC was significantly reduced compared with intact male WKY rats (Fig. 3). The amount of alpha -, delta -, and zeta -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 alpha -, delta -, and zeta -PKC was greater in SHR than WKY in all groups of rats (Fig. 3). The percent reduction in the amount of whole tissue alpha -, delta -, and zeta -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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Fig. 3.   Expression of alpha -, delta -, and zeta -PKC in aortic strips of intact male, intact female, castrated male, and OVX female WKY rats and SHR. The whole tissue homogenates of aortic strips were prepared for Western blot analysis using anti-alpha -PKC (Seikagaku; 1:100; A), anti-delta -PKC (B), and anti-zeta -PKC (C) antibodies (GIBCO; 1:500). Position of the molecular mass marker is shown on right. Autoradiographs were scanned by optical densitometry, and the amounts of alpha -, delta -, and zeta -PKC were expressed as optical density. Bars represent means ± SE of measurements in 12 experiments on tissue samples from 6 rats in each group. P < 0.05: *SHR significantly different from WKY rats; #intact female WKY rats significantly different from other groups of WKY rats; dagger intact female SHR significantly different from other groups of SHR.

Immunoblot analysis of the cytosolic and particulate fractions showed that, in resting aortic strips of intact male WKY rats, the distribution of alpha - and delta -PKC was greater in the cytosolic than particulate fraction, with a basal P/C distribution ratio of 0.87 ± 0.06 and 0.86 ± 0.05, respectively (Table 3). Phe and PDBu caused a significant increase in the distribution of alpha - and delta -PKC in the particulate fraction, a concomitant decrease in the cytosolic fraction, and significantly increased the P/C distribution ratio (Table 3). In intact female WKY rats, the basal and Phe- and PDBu-induced P/C distribution of alpha - and delta -PKC was reduced significantly compared with intact male WKY rats (Table 3). The basal and Phe- and PDBu-induced P/C distribution of alpha - and delta -PKC was greater in SHR than WKY rats (Table 3). The percent reduction in the P/C distribution of alpha - and delta -PKC in intact females compared with intact males was greater in SHR than WKY rats (Table 3). zeta -PKC was equally distributed in the cytosolic and particulate fraction both under basal conditions and during stimulation with Phe or PDBu, and no significant differences in the P/C distribution of zeta -PKC were observed between intact male and intact female WKY or SHR (Table 3).

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 17alpha -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 17beta -estradiol implants compared with untreated OVX females (Table 4). In OVX females simultaneously treated with 17beta -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 17beta -estradiol but not 17alpha -estradiol implants compared with untreated castrated males (Table 4). In castrated males simultaneously treated with 17beta -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 17beta -estradiol implants compared with untreated OVX females was greater in SHR than WKY rats (Table 5).

                              
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Table 4.   Contraction and PKC activity under basal conditions and during stimulation with Phe or PDBu in aortic strips of OVX female and castrated male WKY rats and SHR untreated or chronically treated with 17alpha -estradiol, 17beta -estradiol, or 17beta -estradiol + the estrogen receptor antagonist ICI-182,780


                              
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Table 5.   Contraction and PKC activity under basal conditions and during stimulation with Phe or PDBu in aortic strips of OVX female WKY rats and SHR untreated or treated with 17beta -estradiol implants


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha - and delta -PKC, and both Phe and PDBu caused significant redistribution of alpha - and delta -PKC in aortic smooth muscle of intact male WKY rats. These results are consistent with reports that both alpha - and delta -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 alpha - and delta -PKC and the basal and Phe- and PDBu-induced redistribution of alpha - and delta -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 alpha - and delta -PKC.

The observation that zeta -PKC did not show redistribution with Phe or PDBu is consistent with reports that it lacks the DAG/phorbol ester binding site (20, 34) and thus provided a control experiment and increased the level of confidence in the immunoblot analysis. The absence of zeta -PKC translocation during Phe and PDBu contraction suggests that it may not be involved in the gender differences in vascular reactivity. However, the significant reduction in the amount of zeta -PKC in intact females compared with intact males suggests that it may play a role in other possible gender-related vascular changes, e.g., vascular smooth muscle growth and proliferation. This is supported by reports that vascular zeta -PKC is localized in the vicinity of the nucleus (23).

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 17beta -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.

The findings that the vascular reactivity and PKC activity in OVX females and castrated males simultaneously treated with 17beta -estradiol implants and estrogen receptor antagonist were not significantly different from that in untreated OVX females and castrated males suggest possible involvement of estrogen receptors. Because the expression of estrogen receptors in arterial smooth muscle may vary depending on the gender and the status of the gonads (4), the observed gender differences in vascular reactivity and PKC activity may also be related to the relative abundance of estrogen receptors. This is supported by reports that estrogen receptors have been identified in rat aorta (1, 28) and that females have more estrogen receptors in their arteries than males (4).

The question arises as to how the exposure to estrogen might affect vascular PKC activity. The effects of estrogen have been classically thought of as resulting from genomic actions mediated through interaction with cytoplasmic receptors and translocation of the hormone-receptor complex to the nucleus (27). Thus a genomic action of estrogen on the expression of PKC isoforms in vascular smooth muscle might well underlie the reduction in vascular reactivity and PKC activity observed in intact females compared with intact males. However, additional nongenomic effects of estrogen in vascular smooth muscle have been suggested (5, 9, 17), and direct effects of estrogen on the PKC molecule or its lipid cofactors or other protein kinases upstream from PKC or perhaps secondary vascular effects due to effects of estrogen on other control mechanisms of the systemic hemodynamics cannot be excluded under the present chronic experimental conditions. These questions could be further clarified by studying the acute effects of estrogen on vascular PKC activity and should represent important areas for future investigations.

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 17beta -estradiol implants compared with untreated OVX females was greater in SHR than WKY rats although the plasma 17beta -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 alpha -, delta -, and zeta -PKC isoforms. The gender-specific differences in vascular reactivity and PKC activity are possibly related to endogenous estrogen. The gender-related distinctions in vascular reactivity and PKC activity are augmented in animal models with enhanced vascular reactivity such as the SHR.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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