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Am J Physiol Cell Physiol 278: C834-C844, 2000;
0363-6143/00 $5.00
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Vol. 278, Issue 4, C834-C844, April 2000

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 17beta -estradiol implants and intact females. Exogenous application of 17beta -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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, 17beta -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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 17beta -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 17beta -estradiol by RIA using a 17beta -estradiol kit (ICN Biomedicals, Costa Mesa, CA). In WKY rats, the plasma 17beta -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 17beta -estradiol implants. The plasma 17beta -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 alpha -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)
[Ca<SUP>2+</SUP>]<SUB>i</SUB> = <IT>K</IT><SUB>d</SUB>(Sf<SUB>2</SUB>/Sb<SUB>2</SUB>)[(R − R<SUB>min</SUB>)/(R<SUB>max</SUB> − R)]
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 17beta -estradiol (2,3,5[10]-estratriene-3,17beta -diol; Sigma) was prepared as 5 × 10-2 M in 100% ethanol. 17alpha -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-{[17beta -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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 17beta -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.


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  		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 17beta -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 17beta -estradiol implants are significantly different from other groups of WKY. dagger  Intact female SHR and OVX female SHR with 17beta -estradiol implants are significantly different from other groups of SHR.



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  		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 17beta -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 17beta -estradiol implants are significantly different from other groups of WKY. dagger  Intact female SHR and OVX female SHR with 17beta -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 17beta -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 17beta -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 17beta -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.


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  		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 17beta -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.



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  		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 17beta -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 17beta -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).


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  		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 17beta -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 17beta -estradiol implants are significantly different from other groups of WKY. dagger  Intact female SHR and OVX female SHR with 17beta -estradiol implants are significantly different from other groups of SHR.

Effect of 17beta -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 17beta -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 17beta -estradiol on Phe- or KCl-induced cell contraction or [Ca2+]i could not be identified. In OVX female WKY rats, application of 17beta -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 17beta -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 17beta -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 17beta -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 17beta -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 17beta -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 17beta -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 17beta -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). 17beta -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.


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  		Fig. 6.   Effect of 17beta -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 17beta -estradiol.



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  		Fig. 7.   Effect of 17beta -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 17beta -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 17beta -estradiol is significantly different from that in absence of the hormone. dagger  Decrease in cell contraction and [Ca2+]i in cells of OVX female SHR in presence of 17beta -estradiol is significantly different from that in absence of the hormone.

Pretreatment of cells from OVX female WKY rats with 17alpha -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 17alpha -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 17beta -estradiol. Phe contraction in the presence of 17beta -estradiol plus tamoxifen (31 ± 2%, n = 15) or 17beta -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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 17beta -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 alpha -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 17beta -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 17beta -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.


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DISCUSSION
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