In vitro modulation of primate coronary vascular muscle cell reactivity by ovarian steroid hormones

^a Division of Reproductive Sciences, Oregon Regional Primate Research Center, Beaverton, Oregon 97006, USA
^b Department of Endocrinology, Women's Health Research Institute, Wyeth-Ayerst Research, Radnor, Pennsylvania 19084, USA
^c Departments of Medicine, Oregon Health Sciences University, Portland, Oregon 97201, USA
^d Cell and Developmental Biology, Oregon Health Sciences University, Portland, Oregon 97201, USA
^e Obstetrics and Gynecology, Oregon Health Sciences University, Portland, Oregon 97201, USA

	ABSTRACT

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Susceptibility to drug-induced coronary vasospasm in rhesus monkeys increases after removal of the ovaries and can be normalized by adding back physiological levels of estradiol-17ß (E₂) and/or natural progesterone (P) in vivo as reported recently by our group. Furthermore, the reactivity status (Ca²⁺ and protein kinase C responses) of freshly isolated and primary culture coronary artery vascular muscle cells (VMC) mimic the intact coronary artery responses to 5-HT + U46619. Since coronary reactivity is maintained in the isolated VMC, we hypothesized that the reactivity state inherent in the VMC was modulated directly by ovarian steroids in vitro as in the whole animal. To test this hypothesis, we treated hyperreactive VMC from ovariectomized (ovx) monkeys in vitro with E₂ or P and measured VMC reactivity to combined stimulation with 5-HT and U46619, as determined by the amplitude and especially the duration of intracellular Ca²⁺ signals, as well as protein kinase C (PKC) activation/translocation. VMC were treated for 12–96 h with 3–100 pg/ml E₂ (10–365 pM) and/or 0.3–3 ng/ml P (0.95–9.5 nM). Hyperreactive responses to the combination of 5-HT and U46619 in untreated VMC were significantly and dose-dependently reduced by treatment in vitro with physiological levels of either E₂ or P for at least 24 h. Both the early transient and late sustained increases in intracellular Ca²⁺ and PKC translocation were blunted, and the effects of 0.2 nM E₂ and 3.2 nM P were specifically antagonized by the receptor blockers ICI 182,780 (200 nM) and RU486 (15 nM), respectively. Antibodies to the estrogen receptor and progesterone receptor labeled nuclei in VMC, which were also positively labeled by a smooth muscle myosin heavy chain monoclonal antibody. These data indicate that natural ovarian steroids directly reduce hyperreactive 5-HT and thromboxane A₂-stimulated Ca²⁺ and PKC responses of coronary artery VMC from surgically menopausal rhesus macaques. We hypothesize that vascular hyperreactivity, which may be a critical factor involved in the increased incidence of coronary artery vasospasm and ischemic heart disease in postmenopausal women, can be normalized by E₂ and/or P through direct actions on coronary artery vascular muscle cells.—Minshall, R. D., Miyagawa, K., Chadwick, C. C., Novy,M. J., Hermsmeyer, K. In vitro modulation of primate coronary vascular muscle cell reactivity by ovarian steroid hormones. FASEB J. 12, 1419–1429 (1998)

Key Words: estrogen • progesterone • coronary artery reactivity • ischemic heart disease

	INTRODUCTION

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VASCULAR HYPERREACTIVITY (1–7) is a phenomenon in which local sites in coronary arteries become hyperreactive to a variety of vasoconstrictor substances, including thromboxane A₂ (TxA₂)² and 5- hydroxytryptamine (5-HT; serotonin) (3, 5, 8, 9). Such severe and sustained coronary occlusions are the hallmark of Prinzmetal's angina but are also thought to lead to sudden cardiac death (10, 11), which is more prevalent among postmenopausal women than any other segment of the female population (12, 13). Estrogen replacement therapy significantly reduces the risk of developing coronary heart disease (14) and decreases overall mortality in postmenopausal women (15).

Recently, we established that coronary artery vasospasms can be drug induced in ovariectomized nonhuman primates (rhesus macaques) by administering intracoronary injections of 5-HT and U46619 (a TxA₂ mimetic) (3). Susceptibility to coronary vasospasm increases after removal of the ovaries, which reduces the plasma estrogen levels to less than 10 pg/ml and progesterone levels to 0.1 ng/ml (7). Vascular hyperreactivity and the ability to provoke coronary vasospasm can be normalized by adding back physiological levels of estradiol-17ß (E₂, 60–120 pg/ml serum) and/or natural progesterone (P, 5–7 ng/ml serum) in vivo (3–7). The pathophysiological drug combination of 5-HT and TxA₂ was chosen because it represents perhaps the most important vasoconstrictors released by activated platelets and is thus thought to be involved in initiating coronary artery cyclic flow variations and coronary artery vasospasms (8, 16, 17). The absence of balloon injury of the endothelium and underlying vascular muscle, or cholesterol feeding, makes this model strictly dependent on vascular muscle cell (VMC) reactivity.

Work from this lab has also shown that the reactivity status of freshly isolated rhesus monkey coronary artery muscle cells mimics the coronary artery responses measured in vivo in the catheterization lab during vasospasm provocation (i.e., in monkeys in which coronary vasospasm-like 15 min prolonged responses could be provoked, the VMC from the coronary arteries showed amplified and prolonged Ca²⁺ and protein kinase C (PKC) responses to the same stimulus). Since coronary reactivity is maintained when the isolated VMC are studied as either freshly dispersed cells (7) or primary cell cultures (6), we hypothesized that the reactivity state inherent in the VMC is modulated directly by ovarian steroids, both in vitro and in the whole animal. To test this hypothesis, the present study was designed to determine the effects of physiological levels of the ovarian steroid hormones E₂ and P on hyperreactive monkey coronary artery VMC responses. Our objectives were to determine whether in vitro treatment of hyperreactive VMC from ovariectomized (ovx) monkeys with ovarian steroids would decrease reactivity, as determined by the amplitude and especially the duration of intracellular Ca²⁺ signals, as well as PKC activation/translocation in response to combined stimulation with 5-HT and U46619. Furthermore, the expression of specific E₂ and P receptors in monkey coronary artery VMC was assessed to identify the signaling pathway of direct effects of E₂ and P on coronary arteries.

	MATERIALS AND METHODS

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VMC preparation
VMC from the left anterior descending, circumflex, and right coronary arteries were prepared as primary cell cultures (6, 18) from ovx adult rhesus monkeys and used in experiments from day 3 to day 30 after isolation. These cells were never subcultured and maintained the characteristics of the source tissue, including contraction, relaxation, receptor integrity, and membrane electrical properties (19). The cells were dissociated with collagenase and protease enzymes in a potassium glutamate solution (KG) that prevents loading with Na⁺, Ca²⁺, or Cl^- and results in a high proportion of viable, contracting cells (20, 21). [KG solution consists of (in mM): 140 K- glutamate, 16 NaHCO₃, 0.5 NaH₂PO₄, 30 HEPES, and 16.5 dextrose at pH 7.3]. VMC were seeded at low density (small drop on each coverslip containing 10,000 cells/ml) in cardiovascular cell culture medium, fifth generation (CV5M), on 9 x 22 mm or 20 x 45 mm glass coverslips. After 72 h, the cells were placed in a maintenance medium (CV5MM), which was replaced weekly. CV5M contains MEM + 10% HS, 1% L-glutamine, and 100 µg/L ciprofloxicin. In CV5MM, the concentration of horse serum is reduced to 1.5%. Phenol red was excluded from all solutions because of known actions on estrogen receptors (22). There was no detectable estradiol activity and less than 10 pg P was detected per milliliter of horse serum.

We studied isolated primary culture monkey coronary artery VMC in two groups of experiments: VMC Ca²⁺ and PKC fluorescence microscopy, and immunofluorescent staining and confocal image analysis of estrogen and progesterone receptors in VMC. For live cell analysis of agonist-stimulated Ca²⁺ and PKC signals, VMC cultures from ovx monkeys were used in an attempt to restore the reactivity to that observed in VMC from intact monkeys. The treatments were as follows: 1) no in vitro treatment of ovx VMC, 2) 3 to 100 pg/ml E₂, 3) 60 pg/ml E₂ + 200 nM ICI 182,780, 4) 0.3 to 3 ng/ml P, and 5) 1 ng/ml P + 15 nM RU 486. The concentrations were chosen to mimic the E₂ and P plasma levels measured in monkeys (6, 7) or were determined empirically to be the minimum concentrations required. Initial time course experiments were conducted in which VMC were treated in vitro for 12 h to 7 days. Subsequently, it was determined that the optimum duration of steroid hormone treatment was 48–72 h. Exogenous P added to the culture medium was stable for at least 7 days, and E₂ levels decreased less than 20% in 7 days, as measured by specific radioimmunoassay (23–25).

VMC reactivity
To assess vascular reactivity, measurements of intracellular free Ca²⁺ and PKC fluorescence intensity and mobilization/translocation were made on monkey coronary artery VMC as previously described (6). Ca²⁺ and PKC were studied in the same VMC using double labeling with different wavelength fluophores and multiple fluorescence filter sets. The fluorescent Ca²⁺ probe fluo3-acetoxymethylester (Molecular Probes, Inc., Eugene, Oreg.) was used to sense Ca²⁺. Fluo3 is a fluorescent indicator of wide dynamic range ( 200x) that reports kinetics and sources of Ca²⁺ signals. The Ca²⁺ sensing characteristics of fluo3 measured in our laboratory indicate that an intracellular free Ca²⁺ concentration in the range of 15 nM to 1 µM can be reliably measured, which was determined using a CaEGTA buffering system (Molecular Probes). The K_d of fluo3 for Ca²⁺ was determined to be 0.3 µM, similar to that reported by the manufacturer (Molecular Probes, MP 108).

Live cell measurements of PKC were made using hypericin (LC Laboratories), a naturally fluorescent, high-affinity aromatic polycyclic dione isolated from Hypericum erectum plants (26). The concentration range used to detect PKC in our experiments was 30–100 nM, which is less than the IC₅₀ and below the threshold concentration for inhibition of purified PKC (400 nM) (26). Hypericin inhibits PKC (IC₅₀ = 3.4 µM) by interacting with the regulatory domain, similar in action to calphostin C (26). The advantages of using hypericin as the fluorescent PKC indicator rather than, for example, BODIPY 12'-phorbol ester-13ß- acetate (PBA) (27, 28), include 1) bright red fluorescence emission and exceptional photostability of hypericin, 2) activation of PKC translocation with PBA per se (29), and 3) preliminary studies in our lab show increased hypericin fluorescence intensity and translocation of fluorescence in VMC stimulated with 1,2 dioctanoyl-sn-glycerol, a known activator of PKC that mimics the endogenous PKC activator.

The fluorescent Ca²⁺ and PKC images, which provide sensitive measures of vascular reactivity, were made with very low light levels, using multiple layers of filtering and an ultra-high sensitivity (photon counting) microchannel plate camera (6). Data acquired with the Hamamatsu VIM camera were controlled and processed with Image Pro software customized for our studies with Visual Basic using a Pentium PC and an Imagraph A-D acquisition card with PCI local bus. Live cell images acquired during 5 s exposures over a 30 min protocol were analyzed to determine Ca²⁺ and PKC fluorescence intensity and distribution, and to provide statistical analysis and look up table color mapping.

Ca²⁺ and PKC fluorescence
VMC on small glass coverslips were placed in a 300 µl laminar flow chamber (30). Large coverslips were sealed to the bottom of flow chambers with Apiezon grease (GEC Alsthom, Manchester, U.K.). Isotonic solution for mammals, second generation (ISM2), containing (in mM) 100 NaCl, 4 NaHCO₃, 0.5 NaH₂PO₄, 4.7 KCl, 1.8 CaCl₂, 0.41 MgCl₂, 0.41 MgSO₄, 50 HEPES (pH 7.37 at 22°C), and 5.5 dextrose was continuously washed over the cells (at 1 ml/min) to facilitate equilibration and washout of stimuli. After a 15 min equilibration period in ISM2, VMC were loaded at room temperature by adding 30 µl of 10 to 50 µM fluo3 for 15 min (into a total volume of approximately 300 µl). The VMC were then washed for 5 min (extracellular fluo3 was 75% removed within 1 min) and a time 0 (control) image was acquired. Hypericin was loaded for 5 min and the excess indicator was washed out for 5 min (coinciding with the fluo3 washout). To the buffer directly above a VMC, 30 µl of 100 µM 5-HT plus 1 µM U46619 was added. After 15 s under no- flow conditions, thus exposing the VMC to a final bath concentration of 10 µM 5-HT + 100 nM U46619, continuous flow of ISM2 at 1 ml/min was reinstated, washing away the stimuli (t_1/2=30 s). Fluorescent Ca²⁺ images were acquired at 1, 2, 5, 10, 15, 20, and 30 min, and fluorescent PKC images at 3, 4, 9, 16, 21, and 31 min after stimulation with 5-HT and U46619 using a Zeiss Axiovert microscope with a C-Apochromat 40X/1.2 NA `confocal design' water immersion objective. The fluorescent images were obtained with 5 s duration shuttered exposures with the following filters: 535 nm excitation, 560 nm dichroic mirror, and 570 nm long pass emission filter for PKC; and excitation filter, 487 nm; dichroic mirror, 505 nm; emission filter, 530 nm for Ca²⁺. Fluorescence intensity averaged for the whole cell thickness is expressed relative to the predrug baseline (% of control). The whole cell fluorescence intensity was taken as an indication of the relative level of PKC activity in the VMC. The ratio of a 1 µm band at the cell perimeter to the remaining central fluorescence (peripheral/central ratio) was calculated to determine whether the PKC fluorescence signal moved from central to peripheral regions (decreasing ratio) after agonist stimulation. Statistical analysis was performed using two-factor analysis of variance with one-factor repeated measures. Post-hoc testing of individual time points against controls was done using Dunnet's test.

Immunocytochemical identification of estrogen and progesterone receptors (ER and PR)
The presence of ER and PR in isolated monkey coronary artery VMC was examined using immunocytochemical staining techniques as described (7, 31). In brief, ovx and intact monkey VMC grown on 12 mm round coverslips were treated with vehicle or 60 pg/ml E₂ ± 200 nM ICI 182,780 for 24 h. The coverslips were then rinsed and microwave stabilized with an Amana Radarrange Touchmatic (Amana, Iowa) on ice for 3 s, fixed in 0.2% picric acid-2% paraformaldehyde in phosphate-buffered saline (PBS) + 1.5% polyvinyl-pyrrolidine (PVP) for 10 min at room temperature, rinsed twice for 2 min at 4°C in 85% ethanol in H₂O+ 1.5% PVP, three times for 3 min in PBS + 1.5% PVP, twice for 7 min in 0.37% glycine in PBS + 1.5% PVP, 3x for 3 min in PBS + 1.5% PVP, and 3x for 3 min in PBS + 1.5% PVP with 0.1% gelatin. The VMC were then treated for 20 min with PBS + 1.5% PVP + 0.1% gelatin + 0.2% bovine serum albumin (BSA) + 5% goat serum and incubated overnight at 4°C with 0.5 to 2 µg/ml ER-21, a polyclonal rabbit antibody raised against human ER, or 1 µg/ml JZB39, a rat anti-PR monoclonal antibody (32). JZB39 and ER-21 were generously provided by Dr. Geoffrey Greene, University of Chicago. In addition, some of the coverslips were coincubated with a 1:100 dilution of monoclonal antibody against smooth muscle myosin heavy chain (clone 9A9 G4-G9 to isoforms SM-1 and SM-2) (33) provided by Dr. Gary Owens, University of Virginia Medical Center, Charlottesville. Nonspecific PR staining was determined using the same concentrations of an unrelated, nonspecific monoclonal antibody (rat anti-Timothy) as primary antibody. Specific ER-21 immunostaining was determined by preabsorbing the antibody with an equivalent amount (weight/weight) of the immunizing peptide. After the primary antibody incubation, the coverslips were rinsed three times for 3 min in PBS + 0.1% gelatin and 0.075% BRIJ 35 solution (a polyoxyethylene ether used to facilitate antibody penetration), and again blocked for 20 min. The coverslips were then incubated for 90 min at room temperature with goat anti-rat or anti-rabbit IgG coupled to rhodamine to detect PR and ER and with goat anti-mouse IgG coupled to fluorescein isothyocyanate to detect smooth muscle myosin heavy chain. Finally, the VMC on coverslips were rinsed three times for 3 min with PBS + 0.1% gelatin and mounted in 1 drop Fluoromount G.

Confocal microscopy of immunolabeled VMC
Fluorescent images of ER and PR, in the presence or absence of smooth muscle myosin heavy chain labeling, were collected with a Leica TCS 4D confocal laser scanning microscope. The fluorescent cells were either individually or simultaneously scanned with 488 (green) and 568 nm (red) excitation lasers in 16 planes, averaging 4 scans from each plane, using a 40X 1.25 NA oil immersion objective. The digital images from each serial section were stored and later reconstructed (overlayed) and processed with Adobe Photoshop 4.0 digital image processing software.

	RESULTS

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These studies show direct modulation of monkey coronary artery VMC reactivity by estradiol-17ß (E₂) or progesterone (P) treatment in vitro. The modulation of VMC reactivity was assessed using responsiveness (intracellular Ca²⁺ and PKC responses) to a brief application of putative pathophysiological concentrations of U46619, a thromboxane A₂ mimetic, together with 5-HT. Adult (10 to 18 year old) rhesus macaques were killed after vasospasm provocation testing in vivo, and coronary artery VMC were isolated and maintained in primary culture for up to 30 days. Our primary findings are as follows. 1) Coronary artery VMC derived from ovx monkeys show hyperreactive responses to 5-HT + U46619, whereas VMC derived from intact female monkeys show only small, transient Ca²⁺ and PKC responses. 2) Short-term treatment of VMC from ovx monkeys in vitro with physiological levels of E₂ or P decreased the responsiveness (Ca²⁺ and PKC responses) to 5-HT + U46619. 3) The effects of E₂ and P were specifically antagonized by ICI 182,780 and RU486, respectively. 4) High-resolution images obtained with a confocal laser scanning microscope indicate the presence of nuclear ER and PR immunostaining in monkey coronary artery VMC, which were also identified as smooth muscle myosin heavy chain positive cells with a specific monoclonal antibody.

Effects of E₂ and P on hyperreactive VMC Ca²⁺ and PKC responses
Primary culture VMC derived from ovx rhesus monkey coronary arteries, as compared to VMC from intact (non-ovx) monkeys, showed hyperreactive Ca²⁺ and PKC responses to the pathophysiological combination of 5-HT and U46619. The whole cell average intracellular Ca²⁺ in VMC from intact monkeys is not significantly increased after 5-HT and U46619 stimulation, although transient responses were observed in most cells 1 min after stimulation. Measurements made at earlier time points (e.g., 15, 30, and 45 s) may more effectively capture the Ca²⁺ transient. In contrast, VMC from ovx monkeys showed biphasic increases in whole cell average intracellular Ca²⁺ that reached a maximum level of 181±37% of baseline fluorescence intensity at min 30 (n=11 cells; P<0.05 vs. time 0 fluo3 fluorescence intensity). The increase in intracellular Ca²⁺ in response to 5-HT + U46619 stimulation in ovx monkey VMC was significantly greater than the relative level of intracellular Ca²⁺ observed in intact monkey VMC (86±8%; n=15) at the 20 and 30 min time points.

Treatment of VMC in vitro with 3 to 100 pg/ml E₂ (10–365 pM) significantly reduced the agonist-stimulated Ca²⁺ responses ( Fig. 1). Hyperreactive VMC exposed to 30–100 pg/ml E₂ for greater than 48 h showed Ca²⁺ responses to 5-HT + U46619 that were significantly decreased compared to untreated ovx monkey VMC as early as 1 min after stimulation; these responses continued to decrease below the baseline intracellular Ca²⁺ level through minute 30 after stimulation. The effect of E₂ was dose dependent; with 3 pg/ml E₂, the Ca²⁺ response to 5-HT + U46619 was similar to that observed in VMC derived from ovx and intact female monkeys for the first 10 min after stimulation, but then showed statistically significant decreases in Ca²⁺ 15–30 min after stimulation. Treatment of VMC with concentrations of E₂ between 30 and 100 pg/ml yielded statistically indistinguishable Ca²⁺ responses. The reactivity lowering effect of 60 pg/ml E₂ on VMC Ca²⁺ responses was completely blocked at each time point measured ( Fig. 1) when the cultures were pretreated for 30 min with 200 nM ICI 182,780 (n=6), an estrogen receptor antagonist that blocks E₂ actions on both the and ß ER subtypes. The Ca²⁺ responses in VMC treated with E₂ + ICI 182,780 were not different from the responses of untreated ovx monkey VMC.

View larger version (31K): [in this window] [in a new window]   Figure 1. Agonist-stimulated changes in intracellular Ca2+ in coronary artery VMC as a function of E2 treatment in vitro. Whole cell Ca2+, as indicated by fluo3 fluorescence intensity, was measured from images recorded immediately before (time 0) and 1, 2, 5, 10, 15, 20, and 30 min after 15 s stimulation with 10 µM serotonin + 100 nM U46619. The data are the mean ±SEM of the fluo3 fluorescence intensity from experiments on 5–11 VMC from each group, which were calculated as the percent change from the control (time 0) fluorescence image of each individual VMC. Fluo3 fluorescence in VMC from ovx (n=11; filled circles) monkeys showed amplified Ca2+ responses with sustained time courses (with increases persisting beyond 30 min) that differed significantly (P<0.05) from intact monkey VMC. Physiological E2 levels (30 pg/ml, open circle; n=7; 60 pg/ml, open square, n=5; 100 pg/ml, open diamond, n=6) significantly reduced the Ca2+ response to 5-HT+U46619 at every time point. No change in the Ca2+ response was noted with 3 pg/ml E2 (open triangle; n=6) until 15–30 min after stimulation. The effect of 60 pg/ml E2 was completely abolished by 200 nM ICI 182,780 (n=6; filled squares), an estrogen receptor antagonist. *,**,***Data points below the asterisk are significantly different (P<0.05, 0.01, 0.001) compared to untreated ovx VMC.

Treatment of hyperreactive ovx monkey VMC with 0.3–3 ng/ml P (0.95 or 9.5 nM) in culture dose-dependently and significantly reduced 5-HT + U46619-stimulated increases in intracellular Ca²⁺ ( Fig. 2). An effect was not observed with 0.3 ng/ml P treatment until 20–30 min after agonist stimulation, similar to that observed in VMC from intact `normal' monkeys. In vitro treatment of ovx VMC with 1 or 3 ng/ml P for greater than 48 h significantly reduced the 5- HT + U46619-stimulated whole cell average intracellular Ca²⁺ signal measured 2–30 min after stimulation. The maximum decrease in the Ca²⁺ signal was observed after 15 min. Ovx monkey VMC pretreated for 30 min with the progesterone receptor antagonist RU486 (15 nM) before treatment with 1 ng/ml P for greater than 48 h significantly blocked the protective effect of P. In the presence of RU486, the 5-HT + U46619-stimulated Ca²⁺ response did not differ from untreated ovx monkey VMC Ca²⁺ responses ( Fig. 2), but was significantly higher than the Ca²⁺ response in VMC treated with 1 ng/ml P. Thus, physiologically relevant concentrations of P dose-dependently protect VMC from abnormal (hyperreactive) agonist-stimulated increases in intracellular Ca²⁺. The protective effect of P was blocked by RU486, suggesting that the phenomenon is mediated by PR.

View larger version (29K): [in this window] [in a new window]   Figure 2. Agonist-stimulated changes in intracellular Ca2+ in coronary artery VMC as a function of P treatment in vitro. Treatment of VMC primary cultures from ovx monkeys with 1 ng/ml P (open squares; n=6) or 3 ng/ml P (open circles; n=4) decreased Ca2+ responses compared to the untreated ovx monkey VMC (filled circles; n=11). Significantly reduced Ca2+ responses of VMC treated with 0.3 ng/ml P (open diamonds; n=8) were observed 20–30 min after stimulation. The effect of 1 ng/ml P was blocked by cotreatment of the cells with 15 nM RU486, a progesterone receptor antagonist (n=10; filled squares). Data points below *, **, or ***P < 0.05, 0.01, and 0.001 vs. untreated ovx VMC.

Measurement of PKC in parallel with intracellular Ca²⁺ revealed amplified PKC responses in ovx VMC that were coincident with the late phase increase in intracellular Ca²⁺ ( Fig. 3). Hyperreactive PKC responses were negated by 30–100 pg/ml E₂, but relatively unaffected by 3 pg/ml E₂ (not shown). The reduction of the PKC response by 60 pg/ml E₂ was not blocked by ICI 182,780 (not shown), perhaps indicating that the effect of E₂ on isolated monkey coronary artery VMC Ca²⁺ and PKC responses can occur through separate mechanisms. Hyperreactive PKC response amplitudes observed in ovx VMC were also decreased in the presence of 0.3 to 3 pg/ml P. However, this effect of P was not antagonized by RU486.

View larger version (27K): [in this window] [in a new window]   Figure 3. Protein kinase C (PKC) fluorescence in coronary artery VMC as a function of sex steroid hormone treatment. Whole cell PKC, as indicated by hypericin fluorescence intensity, was measured in coronary artery VMC (concurrently with Ca2+, shown in Figs. 1 and 2) immediately before (time 0) and 3, 4, 9, 16, 21, and 31 min after stimulation with 10 µM serotonin and 100 nM U46619. Note the sustained increase in PKC in VMC from ovx monkeys (n=15; filled circles), as compared to VMC from intact monkeys (not shown) and those treated with 30–100 pg/ml E2 (n=24; open squares) or 0.3–1 ng/ml P (n=21; open diamonds). Unlike the effect of E2 and P on Ca2+, however, the selective receptor antagonists ICI 182,780 and RU 486 did not reverse the decrease in PKC fluorescence (not shown). *P < 0.05 vs. E2-treated VMC; #P < 0.05 vs. P-treated VMC at the same time point.

Estimated measurements of PKC translocation in VMC from ovx monkeys untreated in vitro or treated with 30–100 pg/ml E₂ or 0.3–3 ng/ml P are shown in Fig. 4. The relative level of fluorescence measured from a 1 µm band of the cell perimeter divided by the remaining fluorescence of the cell was used as an indication of fluorescence movement over time. The peripheral to central ratio decreased from 0.55 ± 0.04 to 0.49 ± 0.02 (mean ±SEM; n=15) in ovx VMC by min 4, and then tended to return toward the baseline value during the remainder of the time course. PKC responses in VMC treated in vitro with E₂ or P did not change during the 31 min after agonist stimulation, suggesting minimal or no significant translocation of PKC in these cells.

View larger version (30K): [in this window] [in a new window]   Figure 4. Protein kinase C (PKC) translocation in coronary artery VMC as a function of sex steroid hormone treatment in vitro. The peripheral to central ratio of hypericin fluorescence, an indicator of PKC translocation, was calculated from the PKC fluorescence signals and summarized in Fig. 4. The ratio of fluorescence decreased in untreated VMC from ovx monkeys, whereas the ratio did not significantly change in VMC treated in vitro with 30–100 pg/ml E2 or 0.3–3 ng/ml P.

Representative fluorescent Ca²⁺ and PKC images from untreated hyperreactive ovx monkey VMC and VMC treated with 60 pg/ml E₂ and 1 ng/ml P for 48 h are shown in Fig. 5. Intracellular Ca²⁺ in the ovx monkey VMC was still increased 30 min after stimulation, whereas the Ca²⁺ observed in the VMC after treatment in vitro with E₂ + P significantly decreased below baseline fluorescence at 30 min. Both the rapid transient intracellular Ca²⁺ signal and the sustained elevation in intracellular Ca²⁺ were significantly increased in VMC from ovx monkeys compared to VMC treated in vitro with at least 30 pg/ml E₂ or 1 ng/ml P in six experiments for each group for which this figure is representative. PKC fluorescence intensity increased continuously to the 31 min termination of the protocol in ovx ( Fig. 5A), but did not remain greater than control in the E₂ + P treated VMC ( Fig. 5B).

View larger version (92K): [in this window] [in a new window]   Figure 5. Representative Ca2+ and PKC fluorescent images from monkey coronary artery VMC. Whole cell Ca2+ (fluo3 fluorescence) and PKC images (hypericin fluorescence) from monkey coronary artery VMC contrasting the hyperreactive state (ovariectomized monkey VMC) with the protected state after E2 and P treatment in vitro are shown. In the far left column, the bright-field Nomarski image of each cell is shown, followed by time 0 and 30 min Ca2+ images and time 0 and 31 min PKC images. The color bar shows increasing Ca2+ and PKC levels from violet (lowest) to red (highest). Note the dramatically increased Ca2+ signal 30 min after stimulation by 5-HT + U46619 in VMC from an ovx monkey (A), which is decreased significantly in the VMC treated in vitro with physiological levels of E2 (60 pg/ml) and P (1 ng/ml) (B) for 48 h. The fluorescence intensity of the PKC indicator was also increased 31 after min stimulation in the ovx derived VMC (A) but not the E2+P treated VMC (B). The original magnification was 10,000x for all images (40x/1.2 W objective).

Coronary artery VMC ER and PR immunostaining
Immunolabeling of ER and PR was performed with the rabbit polyclonal anti-ER IgG, ER-21 ( Fig. 6 a, b, red fluorescence), and rat monoclonal anti-PR antibody, JZB39 ( Fig. 6d, e, red fluorescence), respectively. Colabeling (green fluorescence) with mouse monoclonal smooth muscle myosin heavy chain antibody (1:100; Fig. 6b, e) reveals positive nuclear ER and PR steroid receptor immunolabeling in cells that also stain positively for smooth muscle myosin heavy chain (SM-1/SM-2).

View larger version (133K): [in this window] [in a new window]   Figure 6. Confocal images of ER and PR immunostaining in smooth muscle myosin heavy chain (SM-1/SM-2) positive VMC. Immunolabeling of ER;ha and PR (red fluorescence) in monkey coronary artery VMC was performed with ER-21 (a–c) and JZB39 (d–f), respectively. Colabeling (green fluorescence) was with mouse monoclonal smooth muscle myosin heavy chain antibody (b, e), revealing steroid receptor labeling of cells that also stain positively for smooth muscle myosin heavy chain. ER;ha was localized in VMC in nuclear/perinuclear regions (a, b) with ER-21. As shown in panel c, preincubation of ER-21 with an equivalent amount (wt/wt) of the immunizing peptide eliminated both the nuclear and cytosolic staining. Note that the SM-1/SM-2 negative cells (b), which are presumably fibroblasts, also show positive immunostaining for ER-21 in and around the nucleus. A perinuclear/nuclear staining pattern was also observed for PR (d, e) that was not detected when an irrelevant rat monoclonal antibody (anti-Timothy) was used in place of JZB39 (f). Modestly stained nuclear PR was observed in SM-1/SM-2 immunopositive cells (e).

Nuclear and perinuclear staining with the antibody ER-21 to ER was observed in VMC ( Fig. 6a, b), which was substantially eliminated by preincubating the antibody with an equivalent amount (wt/wt) of the immunizing peptide ( Fig. 6c). Many cells on the coverslips were immunopositive for SM-1/SM-2 smooth muscle myosin heavy chain ( Fig. 6b). These data, together with the thick, refractile morphology of the cells, ability to contract in response to 5-HT and U46619, and sustained Ca²⁺ signals, further imply that they are representative of vascular muscle. The SM-1/SM-2 negative cells, as shown in Fig. 6b, are presumably fibroblasts, which notably show positive immunostaining for ER in and around the nucleus.

A perinuclear/nuclear staining pattern was also observed for PR ( Fig. 6d, e), which was not detected when an irrelevant rat monoclonal antibody (anti-Timothy) was used in place of JZB39 ( Fig. 6f). The modestly stained nuclear PR was observed in SM-1/SM-2 immunopositive cells ( Fig. 6e).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this series of experiments, we have shown that application of physiological levels of either E₂ or P to isolated monkey coronary artery VMC in vitro reduces the responsiveness to 5-HT and U46619. These data provide evidence that has been difficult to obtain, i.e., that physiological levels of ovarian steroids have direct vascular protective actions. Previous reports of direct actions of ovarian steroids on vascular muscle and on endothelial cell biochemical and physiological mechanisms have relied on pharmacological doses of E₂ and P (between 10 nM and 10 µM) to achieve an effect (34–41). In the present study, however, 30–60 pg/ml E₂ and 0.3–1 ng/ml P restored Ca²⁺ responses to 5-HT + U46619 in VMC from OVX monkeys to that observed in VMC from intact monkeys. In our in vivo study (7), we showed that the circulating blood levels of E₂ and P measured in intact adult rhesus monkeys in luteal (n=4) and follicular (n=7) phase of the menstrual cycle were 33 ± 10 and 80 ± 34 pg/ml E₂ and 2.97 ± 1.42 and 0.29 ± 0.07 ng/ml P, respectively. By comparison, the E₂ and P levels in ovariectomized monkeys were 6.4 ± 3.4 pg/ml and 0.1 ± 0.04 ng/ml (n=7), respectively. Thus, restoring the levels of E₂ or P to that measured in vivo (both alone or in combination) directly onto the isolated coronary artery VMC normalized hyperreactive Ca²⁺ and PKC responses relative to the Ca²⁺ and PKC responses observed in VMC from intact monkey coronary arteries. These observations provide compelling evidence supporting the hypothesis that one of the cardioprotective mechanisms of E₂ and P is through regulation of VMC reactivity to circulating vasoconstrictor hormones (3).

Similar to the results obtained in our study, Bhalla and co-workers (42) showed that in vitro application of 50 pg/ml E₂ for 4 days to guinea pig coronary artery VMC reduced bradykinin-stimulated intracellular Ca²⁺ transients (both the peak effect and time to peak). In support of the Bhalla study and the results obtained in the present investigation, exposure of MCF-7 human breast cancer cells to E₂ (1 nM) for more than 5 min inhibited phosphatidylinositol 4,5-bisphosphate phospholipase C (PIP₂-PLC) activity (43), which would result in decreased inositol 1,4,5-trisphosphate (IP₃) -stimulated Ca²⁺ signals.

Stimulation of VMC with agonists that activate PLC only transiently elevated intracellular Ca²⁺ and PKC under normal E₂ conditions, but Ca²⁺ and PKC responses were dramatically enhanced and sustained in VMC derived from monkeys in a hypoestrogenic state (ovariectomized) or after treatment in vivo with medroxyprogesterone acetate (MPA) (6, 7). Although several proteins involved in Ca²⁺ signaling may be altered after the loss of E₂ or when exposed to MPA, leading to hyperreactive and potentially spastic coronary arteries, these data suggest a focus on the signal transduction pathway initiated by TxA₂ and 5-HT receptors, presumably PLC activation and, ultimately, IP₃ and PKC signals in the coronary artery VMC. Since PKC is known to regulate Ca²⁺ signals (44, 45), this enzyme may be a key protein regulated by E₂ and P ( Figs. 3 and 4). However, the effect of E₂ and P on agonist-stimulated PKC activity was not blocked by the ER and PR antagonists ICI 182,780 and RU486, whereas steroid hormone modulation of Ca²⁺ responses were blocked. Nongenomic actions of P like that observed on sperm are seen within 1 min with BSA-conjugated-P, a form that is unable to cross the plasma membrane (46). Protein kinase C activity appears necessary for P to initiate the [Ca²⁺]_i rise and acrosome reaction in sperm, but not the depolarizing effects of this steroid (47). Since BSA-conjugated-P does not have access to the cytosolic PR, effects mediated by the membrane binding site pathway are not blocked by antiprogestins. A similar observation made by Bielefeldt et al. (48) suggests that P can directly inhibit Ca²⁺ currents in a human intestinal smooth muscle cell line, albeit the concentration of P was 1000-fold the physiological level.

Alternatively, there may exist a delicate balance of modulation by E₂ and P that regulates vascular muscle contractile responses by changing the sensitivity of the vessel wall to several vasoactive agents. Drastic changes in that balance could, for example, alter the expression of steroid hormone receptors, TxA₂, and/or 5-HT receptors or downstream signaling mechanisms, which would change the physiological responses of the VMC to vasoconstrictor stimuli.

The heart and great vessels have for some time been considered to be targets for steroid hormones (for a review, see ref 49). Specific binding of [³H]estradiol has been observed in subcultured rat coronary artery (50) and aortic VMC (51), and ER mRNA has been confirmed in VMC from the rat aorta by reverse transcriptase-polymerase chain reaction (38). Furthermore, Grohé et al. (52) showed that cultured rat cardiac myocytes and fibroblasts contain functional ER and that E₂ regulates the expression of specific cardiac genes. Subcultured VMC derived from human left internal mammary artery stain positive for ER when using the rabbit polyclonal anti-human ER antibody ER-21 (53), and the human VMC ER functionally responds to estrogen (54). Similarly, nuclear PR protein and/or message has been identified in the cell nuclei of the tunica media and neointima of human saphenous vein biopsy samples (55), human thoracic ascending aorta, internal carotid, coronary artery, left atrial appendage (56), and cytosolic fractions of canine aorta (57). In fresh-frozen rhesus monkey coronary artery sections, similar expression profiles were observed when using specific antibody labeling techniques; ER was present primarily in coronary artery endothelial cells, interstitial cells, and in relatively fewer VMC, whereas PR was primarily located in the vascular muscle layer (7). These data, together with the epidemiological findings that postmenopausal hormone users have reduced coronary heart disease, suggest direct beneficial actions of natural ovarian steroid hormones on the vascular muscle cells of coronary arteries and on cardiac muscle and connective tissue.

The loss of E₂ that occurs after menopause may shift a delicate balance of factors that regulate vascular tone and reactivity (58–60; for a review, see ref 61). Several mechanisms may explain similar reactivity changes that occur in coronary arteries of both postmenopausal women and ovx monkeys. Although E₂ potentiates the release of prostacyclin and nitric oxide, it also inhibits the production of TxA₂, endothelin, and superoxide ions by endothelial cells (59). Thus, altered vascular reactivity may be an important factor independent of atherosclerosis or blood pressure (3). The results of the present study suggest that cardioprotective effects of sex steroid hormones can occur independent of changes in cholesterol and vasodilator actions via prostacyclin and nitric oxide, prompting further investigations into possibly multiple mechanism(s) by which E₂ and P can reduce agonist-stimulated intracellular Ca²⁺ signals.

Vasodilator actions have been shown for the acute administration of both E₂ (62) and P (63). In the present study, we also show that physiological levels of either E₂ or P alone decrease agonist-stimulated VMC Ca²⁺ and PKC responses. A potent inhibitory action of physiologically relevant concentrations of E₂ and P on VMC proliferation in vitro has also been demonstrated (42, 64–66). These studies thus indicate that P by itself, compared to E₂ alone, may be equally effective at providing cardioprotection. If substantiated, hormone replacement therapy with natural P could reduce or perhaps eliminate the estrogenic component in selective patients, which may be an attractive alternative to the increased risk of breast or uterine cancer observed with estrogen (14).

In summary, this report and previous studies from this lab have shown that the response of rhesus monkey coronary artery VMC to vasoconstrictor agonists is modulated by physiological levels of E₂ and P (6, 7). Coronary artery reactivity to the platelet release products 5-HT and TxA₂ (as U46619) is increased after removal of the ovaries in rhesus monkeys and is restored by replacement of E₂ and/or P both in vivo (5, 7) and in vitro (present study). These findings demonstrate that E₂ and P directly modulate VMC Ca²⁺ and PKC hyperreactivity to 5-HT and U46619. The effects of E₂ and P are suggested to be via a genomic mechanism of transcription factor activation because specific nuclear receptor sites were shown by confocal microscopy, the time course of the E₂ and P effect (inhibitory) on Ca²⁺ signaling was slow (optimal after treatment for 48 h), and the E₂ and P effects were blocked by the specific nuclear ER and PR receptor antagonists, ICI 182,780 and RU486.

	ACKNOWLEDGMENTS

 
This research was supported by the Women's Health Research Institute, Wyeth-Ayerst Research, and National Institutes of Health grants HL 51723, HD18185, and HD06159.

	FOOTNOTES

 
¹ Correspondence: Oregon Regional Primate Research Center, 505 N.W. 185th Ave., Beaverton, OR 97006, USA. E-mail: rkh@compuserve.com

² Abbreviations: 5-HT, 5-hydroxytryptamine (serotonin); VMC, vascular muscle cell(s); ovx, ovariectomized; KG, potassium glutamate solution; PBA, 12'-phorbol ester-13'- acetate; PBS, phosphate-buffered saline; E₂, estradiol-17ß; TxA₂, thromboxane A₂; P, progesterone; ER, estrogen receptors; PR, progesterone receptors; PVP, polyvinyl-pyrrolidine; PLC, phospholipase C; IP₃, inositol 1,4,5-trisphosphate; MPA, medroxyprogesterone acetate; BSA, bovine serum albumin.

Received for publication March 11, 1998. Accepted for publication May 11, 1998.

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