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Am J Physiol Heart Circ Physiol 281: H1498-H1507, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 4, H1498-H1507, October 2001

Progesterone regulation of vascular thromboxane A2 receptors in rhesus monkeys

Richard D. Minshall1, Dusan Pavcnik2, Perry V. Halushka4, and Kent Hermsmeyer1,3

1 Division of Reproductive Sciences, Oregon Regional Primate Research Center, Beaverton 97006; 2 Dotter Interventional Institute and 3 Departments of Medicine and Cell and Developmental Biology, Oregon Health Sciences University, Portland, Oregon 97201; and 4 Division of Clinical Pharmacology, Department of Medicine, and Departments of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, South Carolina 29425


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We hypothesized that progesterone regulates thromboxane A2 receptor (TxA2R) density in primate vascular muscle and that TxA2R density correlates with coronary reactivity in vivo and in vitro. Reactivity to serotonin + U-46619 was determined by angiography in surgically postmenopausal [ovariectomized (Ovx)] rhesus monkeys without progesterone replacement and after 2-wk progesterone treatment (1-2 ng/ml). In untreated Ovx animals, 100 µmol/l serotonin + 1 µmol/l U-46619 (syringe concentrations) provoked vasospasm-like constrictions in six of six monkeys; zero of six progesterone-treated monkeys developed vasospasms. Sustained Ca2+ responses in vascular muscle cells isolated from Ovx coronaries (208 ± 63% of basal 20 min after stimulation) treated with serotonin + U-46619 contrasted with transient Ca2+ responses (143 ± 18% of basal and decreasing 5 min after stimulation) in progesterone-treated monkeys. The maximum density of [1S-(1I,2J(5Z),3I(1E,3R*),4I)]-7-[3-(3-hydroxy-4-(4'-[125I]iodophenoxy)- 1-butenyl)-7-oxabicyclo[2.2.1]heptan-2-yl]-5-heptenoic acid ([125I]-BOP) binding was greater (P < 0.01) in carotid arteries and aortic membranes from Ovx (109 ± 11 fmol/mg) compared with progesterone-treated (43 ± 15 fmol/mg) monkeys. TxA2R immunolabeling revealed greater coronary TxA2R labeling in Ovx compared with progesterone-treated monkeys. The results suggest that progesterone can decrease arterial TxA2R in Ovx monkeys.

postmenopausal; vascular reactivity; coronary; ischemic heart disease


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ISCHEMIC HEART DISEASE is thought to be the result of occlusion of coronary arteries by plaques, acute thrombus formation, and profound vasoconstriction (43). However, severe prolonged reduction in coronary artery diameter (vasospasm) constitutes another possible pathophysiological mechanism, as detected by angiography (25). While atherosclerosis is generally thought to account for the pathology occurring in patients with coronary artery disease (1, 13, 43), structural mechanisms or plaques do not appear to explain angina episodes associated with Prinzmetal's (variant) angina, Syndrome X, and other cases where angiographic evidence of organic (structural) coronary occlusion is absent or minimal (7, 10, 34).

Postmenopausal women have an increased incidence of ischemic heart disease that can, under favorable circumstances, be significantly reduced by hormone replacement therapy (5). The potential mechanisms by which estrogen protects against heart disease in postmenopausal women include lowering circulating levels of lipids and lipoproteins, inhibition of lipoprotein oxidation, an antiatherosclerotic effect, augmenting endothelium-dependent responses, vasodilation by endothelium-independent mechanisms, and increasing aortic compliance and cardiac inotropy (2, 3, 9, 14, 16, 20, 23, 36a, 38, 49). Although considerable attention has been given to antiatherosclerotic effects of estrogen, it is estimated that only one-quarter of the benefits of estrogen may be attributable to lowering lipids and preventing lipoprotein oxidation (4, 36a). The addition of progesterone is desirable for balanced hormone replacement therapy and to counter the increased risk of endometrial and breast cancers associated with unopposed estrogen (6, 40); however, little is known about the effects of progesterone per se on coronary and cardiac functions.

Recently, the Heart and Estrogen/Progestin Replacement Study (19) found no significant differences between hormone replacement therapy and untreated groups. In fact, the initial year of treatment was adverse rather than beneficial for coronary arteries (19), indicating a need to reexamine the strategy for optimal hormone replacement therapy. Our finding that progesterone is protective against coronary vasospasm but that medroxyprogesterone acetate (MPA; the progestin used in aforementioned trial) predisposes toward coronary hyperreactivity and vasospasm is particularly relevant to this issue (29, 31). Thus, whereas MPA tends to negate the benefits of estrogen on coronary reactivity, natural progesterone does not. In fact, progesterone appears to reduce coronary muscle cell reactivity independent of estrogen (29, 30).

Excessive coronary artery vasoconstriction (and, ultimately, vasospasm) can occur due to vascular muscle cell hyperreactivity to vasoconstrictor stimuli (17, 21, 22-25, 29-32). Although elusive and often difficult to study separately from atherosclerosis, vasospasm can be provoked by a variety of physiological and hormonal stimuli, including physiologically relevant concentrations of serotonin and U-46619 [a thromboxane A2 (TxA2) mimetic] (12). The combination of serotonin and TxA2 is hypothesized to represent the transient release of stored platelet products from a thrombus (17).

Coronary artery vasoconstrictor responses have been demonstrated by both angiography and exaggerated vascular muscle cell (VMC) Ca2+ and protein kinase C (PKC) responses to serotonin and U-46619 in nonatherosclerotic, surgically postmenopausal rhesus monkeys (17, 32). Reactivity can be restored to normal by treatment for 4-8 wk with 17beta -estradiol (E2beta ) or by treatment for 2 wk with progesterone after a 2-wk priming period with E2beta (29, 31). This return to normal reactivity is thought to occur via restoration of normal VMC constrictor responsiveness (17, 21, 22, 30). Other studies have shown that 1) testosterone increases guinea pig coronary artery reactivity (42) and TxA2 receptor density (also known as the thromboxane-prostanoid receptor) (26-28) and 2) E2beta replacement decreases the contractility of U-46619-challenged guinea pig coronary arteries (48).

Consistent with the hypothesis of steroid hormone modulation of vascular muscle reactivity, the goal of the present study was to determine if the level of expression of vascular muscle TxA2 receptors and the contraction signaling mechanism activated would be augmented in menopausal primates and restored to normal by replacement of progesterone to physiological levels. Specifically, we hypothesized that treatment of ovariectomized (Ovx) rhesus monkeys for 2 wk with 1-2 ng/ml progesterone (lower end of the physiological range) would decrease serotonin + U-46619-stimulated coronary artery reactivity in vivo, freshly isolated vascular VMC Ca2+ responses in vitro, and TxA2 receptor density in homogenized arteries from the same animals.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal Model

The adult rhesus monkeys used in these experiments (age, 12.4 ± 0.8 yr; weight, 6.1 ± 0.5 kg) underwent the ovariectomy at least 3 mo before the study. The monkeys were then either 1) left untreated (Ovx monkeys, n = 6) or 2) treated with a 10-mm subdermal implant of silastic tubing containing ~66 mg of dry progesterone for 2 wk (n = 6). E2beta and progesterone levels were measured by radioimmunoassay as described previously (18). The lower limit of detection of E2beta was 2 pg/ml and that of progesterone was 0.05 ng/ml; these values were used in the calculations above when the steroid was not detected. Surgically postmenopausal 12-yr-old monkeys were chosen to closely approximate postmenopausal women not treated with hormone replacement therapy, who are known to have a higher incidence of ischemic coronary events than occurs before menopause (7, 39). Surgically postmenopausal rhesus monkey comparisons with low-dose parenteral progesterone, but no exogenous estrogen, further previous studies (17, 29-32) of physiological or lower concentrations of natural and synthetic progestins on coronary artery reactivity.

Provocation of Coronary Vasospasm

Monkeys were subjected to coronary artery stimulus provocation challenges in a catheterization laboratory as previously described (17, 29, 31). All procedures in the experimental protocol were approved by the Oregon Regional Primate Research Center Animal Care and Use Committee following the NIH Guide for the Care and Use of Laboratory Animals (ISBN 0-309-05377-3, Revised 1996). The injection protocol employed tested the ability of intracoronary injections of endogenous vasoactive substances to initiate prolonged (>5 min), severe (to <33% of control diameter) vasoconstriction that appears to mimic human coronary artery vasospasm. The interventional drugs, diluted in saline, were slowly and continuously injected directly into the coronary artery as a 1-ml inflow over a 30-s interval. It is estimated that this results in an immediate 15-fold dilution of the syringe concentration (17). Thus the physiologically relevant concentrations achieved for serotonin, U-46619, and ACh at the time of injection were ~6.7 µmol/l, 67 nmol/l, and 67 nmol/l, respectively. Briefly, the protocol was as follows: Rhesus monkeys were sedated with 10 mg/kg ketamine at the radiology catheterization suite. Before the arterial and venous catheterization began, 1-1.5% isoflurane in 100% O2 was continuously administered by inhalation to induce and maintain a surgical plane of anesthesia. Blood pressure, electrocardiogram, heart rate, temperature, end-tidal CO2, and percent O2 saturation of the blood were continuously monitored and recorded. Before coronary catheterization, 1,000 units of heparin were administered intravenously. Control angiograms were acquired to establish baseline diameter and the pattern of the epicardial coronary arteries using a 1- to 2-ml injection of Hexabrix, a radioopaque contrast media. Angiograms were acquired immediately (within 15 s from the end of the stimulus infusion), 3 min after the first injection, and at later times (up to 15 min) when it was apparent that a persistent severe vasoconstriction warranted additional images.

The first part of the protocol tested endothelium-dependent vasodilation. Endothelial integrity was tested by vasodilation (vs. constriction) with 100 µmol/l ACh. Seven minutes later, intracoronary injection of 100 µmol/l serotonin was used to differentiate between 1) an intact endothelium (by either lack of constriction or dilation of the large coronary arteries) and 2) endothelial defects (vasoconstriction instead) if there was a site of artery damage (17). After a minimum interval of 7 min and return to control heart rate and blood pressure ± 15% (which were the interinjection criteria used throughout this protocol), the coronary arteries were then stimulated with 1 µmol/l U-46619, a stable TxA2 mimetic.

The next five injections constituted the main vasospasm challenges: three separate injections of combined serotonin (100 µmol/l) and U-46619 (1 µmol/l), followed by the triple combination of serotonin (100 µmol/l) and U-46619 (1 µmol/l) together with endothelin-1 (1 nmol/l), and finally serotonin (100 µmol/l) with a higher concentration of U-46619 (3 µmol/l). The 7-min interval and return to <15% change of control heart rate and blood pressure with stable conditions were observed throughout these challenges in all monkeys. If focal constrictions that meet the vasospasm criteria had not developed by the time cardiogenic shock was evident (diastolic blood pressure <30 mmHg), the animal was termed "protected." Upon completion of the in vivo protocol, ketamine analgesia and tranquilization were reinstated, and the monkeys were taken to necropsy, where they were given an overdose of pentobarbital sodium. The hearts were then immediately removed for gross and histological examination of the coronary arteries to determine if there were indications of coronary artery disease.

Measurements of coronary artery diameters were made from angiographic images using computer enhancement and edge detection by an observer blinded as to the treatment group, as in our previous studies (17, 29, 31). Diameters were measured in large epicardial coronary arteries at or above the first branch point (circumflex or left anterior descending coronary artery) >5 mm beyond the catheter tip under control conditions (before any injection except the Hexabrix radioopaque medium) and at exactly the same point at the instant of minimum (or maximum) diameter. Responses for serotonin + U-46619 were selected at the point of minimum diameter, which occurred between the first and third challenges. chi 2-analysis was used to test the significance (P < 0.05) of observing the defined <= 33% of control diameter for >5-min vasospasm criteria.

VMC Reactivity

VMC preparation. Coronary artery VMC were isolated from the left anterior descending, circumflex, and right coronary arteries as described elsewhere (29). Coronary artery pieces were dissociated by treatment for 15-30 min in Ca2+- and Mg2+-free PBS containing 0.37 mg/ml type II collagenase, 0.37 mg/ml bacterial protease type XXIX, 0.67 mg/ml BSA, and 1.33 mg/ml trypsin inhibitor. The dispersed cells were collected by centrifugation for 1 min at 200 g, resuspended in PBS, and held at 4°C until used.

VMC Ca2+ and PKC responses. To assess VMC reactivity, Ca2+ and PKC responses were determined in one or more freshly isolated VMC from each monkey. Ca2+ and PKC responses were studied in the same VMC using double labeling with different wavelength fluophores and multiple-fluorescence filter sets as specified previously (25). Briefly, VMC seeded onto glass-bottomed flow chambers were equilibrated for 15 min in isotonic solution for mammals (ISM), second generation (ISM2), containing (in mmol/l) 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. After equilibration, VMC were loaded at room temperature with 30 µl of 10-50 µmol/l fluo 3 for 15 min (total volume of ~300 µl). To determine PKC content, localization, translocation, and response to vasoconstrictor stimuli, VMC were labeled with 100-500 nmol/l hypericin (29). Hypericin was loaded for 5 min, the excess indicator was washed out for 5 min (coinciding with the fluo 3 washout), and a time 0 (control) image was acquired. To the buffer directly above VMC, 30 µl of 100 µmol/l serotonin + 1 µmol/l U-46619 was added. After 15 s under no-flow conditions, thus exposing the VMC to a calculated final bath concentration of 10 µmol/l serotonin + 100 nmol/l U-46619, a continuous flow of ISM2 at 1 ml/min was reinstated, and fluorescent Ca2+ and PKC images were acquired at 1, 2, 5, 10, 15, 20, and 30 min and 3, 4, 9, 16, 21, and 31 min, respectively, using a Zeiss Axiovert microscope with a C-Apochromat ×40/1.2 numerical aperture (NA) "confocal design" water immersion objective. Illumination was limited to eight exposures of 5-s duration to minimize fading. Fluorescent fluo 3 Ca2+ images (excitation filter, 487 nm; dichroic mirror, 505 nm; emission filter, 530 nm) and hypericin PKC images (excitation filter, 535 nm; dichroic mirror, 560 nm; long-pass emission filter, 570 nm) for the whole cell thicknesses are expressed relative to the predrug baseline (percent control).

The fluorescent Ca2+ and PKC images, which provide contraction signals that reflect vascular reactivity, were made with very low illumination levels using multiple layers of filtering and an ultra-high sensitivity (photon counting) microchannel plate camera (32). Data acquired with the Hamamatsu VIM camera were controlled and processed with Image Pro software customized for our studies with Visual Basic. Statistical comparisons were made using ANOVA with repeated measures; significance was set at P < 0.05.

TxA2 Receptor Binding

The radioligand binding assays for TxA2 receptors were carried out on isolated arterial membranes using [1S-(1I,2J(5Z),3I(1E,3R*),4I)]-7-[3-(3-hydroxy-4-(4'-[125I]iodophenoxy)-1-butenyl)-7-oxabicyclo[2.2.1]heptan-2-yl]-5-heptenoic acid ([125I]BOP), a TxA2 receptor agonist (26), as described previously (21). These methods are based on the high-affinity binding of [125I]BOP and the unlabeled compound, which was synthesized as previously described (26).

Carotid arteries and aortas were removed from the animals during necropsy, placed in ice-cold buffer (containing 25 mmol/l HEPES, 150 mmol/l NaCl, 10 µmol/l indomethacin, and 75 µg/ml phenylmethylsulfonyl fluoride; pH 7.4), cleaned of connective tissue, perfused with buffer, and quick-frozen in liquid nitrogen. The whole arteries were kept at -80°C until the day of use. Crude homogenates were prepared using a modification of a previously described method (44). The protein content of the final membrane preparation was adjusted to ~250 µg/ml, and the sample was immediately used in the radioligand-binding assay.

Incubations (30 min at 30°C) were performed in silanized glass tubes by adding 100 µl of the artery membranes, 20 µl of [125I]BOP (0.01 nmol/l-1 µmol/l), or buffer (see above) containing 20 mmol/l MgCl2 and 80 µl [125I]BOP (40,000 cpm). The final pH of the incubation mixture was 7.4. The binding reactions were terminated by addition of 4 ml of ice-cold buffer, followed by vacuum filtration through glass fiber filters and three subsequent buffer washes. The filters were then counted for bound radioactivity. Specific binding was defined as the amount of total bound radioactivity minus the bound counts observed in the presence of L-657,925 (10 µmol/l), a TxA2 receptor antagonist (Merck; Frost, Canada). Nonspecific binding was 23.5 ± 3.4% (n = 8) for carotid artery and aortic membranes. The dissociation constant (Kd) and maximum receptor density (Bmax) were calculated from Scatchard-transformed binding data with the iterative, mass action law-based curve-fitting program LIGAND. Statistical comparisons were made using Student's t-test, with P < 0.05 being significant.

TxA2 Receptor Immunolabeling

To determine whether the differences in TxA2 receptor density measured in the aorta and carotid artery were also present in the coronary artery, the relative expression of TxA2 receptors was determined using immunocytochemical techniques. TxA2 receptor immunostaining was performed on coronary artery and aorta cross sections from Ovx and progesterone-treated groups. Segments of the left anterior descending, right, and/or circumflex coronary arteries and aorta were immediately fixed in 10% neutral buffered formalin and embedded in paraffin. Sections (5 µm) were incubated overnight at 4°C with a 1:100 dilution of "PH3" or "PH4," anti-peptide antibodies made against amino acids 88-97 (QHAALFEWHAC) and 221-231 (TLCHVYHGQEC) of the human TxA2 receptor, respectively (35). Secondary antibody labeling was for 60 min at room temperature with rhodamine-conjugated goat-anti rabbit IgG (1:100 dilution). Control experiments lacking exposure to the primary antibody showed no detectable secondary fluorescence, indicating the specificity of the fluorescence label. Fluorescent images were collected with a Leica TCS 4D laser scanning confocal microscope. The artery cross sections were scanned with 568-nm (red) and 488-nm (green) excitation lasers, averaging four scans from each 0.5-µm plane, using a ×40/1.25 NA oil immersion objective. The 488-nm laser line was used to visualize autofluorescent connective tissue proteins. TxA2 receptor images were collected in one experiment that is representative of the five experiments performed. All images were processed under identical conditions to allow direct comparison of the fluorescence intensity.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The data support the hypothesis that the density of TxA2 receptors measured by two independent criteria (immunolocalization and affinity binding) correspond with the dynamic functional responses (hyperreactivity to serotonin + U-46619 stimuli) as determined in both 1) intact coronary arteries imaged in the catheterization laboratory and 2) isolated coronary artery VMC Ca2+ responses.

Coronary Vasodilation and Vasoconstriction

Epicardial coronary arteries vasodilated in response to intracoronary injections of 100 µmol/l ACh during angiography revealed a dilator action in all monkeys tested (Fig. 1, B and E). On the other hand, the epicardial coronary arteries showed hyperreactive responses to TxA2 receptor stimulation by U-46619 (Fig. 1, C and F), with progressive diameter reductions that culminated in severe coronary constrictions that met the criteria of coronary artery vasospasm only in the Ovx group (Fig. 1C). Vasospasm-like constrictions occurred in the steroid-deficient Ovx group after a cumulative total of 0.7-1.05 µg U-46619 (after the second or third serotonin + U-46619 challenge, n = 6). In monkeys exposed to physiological (or less) levels (1-4 ng/ml) of progesterone for 2 wk (Fig. 1F), coronary angiography revealed only transient vasoconstriction of epicardial coronary arteries, and thus the vasospasm criteria were not met even after an accumulated intracoronary dose of over 2.35 µg U-46619. These data contrast the reactivity to TxA2 receptor stimulation in surgically postmenopausal with that in low-dose progesterone-treated primates. Untreated postmenopausal monkeys showed hyperreactive coronary artery responses to serotonin + U-46619 even in the absence of vascular injury or endothelial dysfunction. Replacement of a basal level of progesterone via silastic implants, which delivered a low physiological concentration of 2.4 ± 1.4 ng/ml (n = 6) of progesterone, significantly reduced coronary artery reactivity, as evidenced by the absence of vasospasm. Progesterone in the untreated Ovx group monkeys was 0.07 ± 0.04 ng/ml (n = 6) compared with 2.97 ± 1.42 ng/ml reported in normally cycling luteal-phase monkeys in our previous report (22).


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  		Fig. 1.   Coronary artery angiograms of ovariectomized (Ovx) monkeys without (Ovx group; A-C) and with progesterone treatment (P-treated group; D-F). Representative agonist-stimulated epicardial coronary artery responses to 100 µmol/l ACh (B and E) and 1 µmol/l U-46619 (C and F) in monkey epicardial coronary arteries are shown 3 min after drug injection compared with the vehicle control (A and D). Vasodilator responses to ACh were similar between the Ovx and P-treated groups. However, the epicardial coronary arteries in the Ovx group showed hyperreactive responses to the vasoconstrictor U-46619 (C) 3 min after the challenge. Vasoconstriction to the point of severe (>70%) and prolonged (>5 min) coronary artery focal constrictions (see arrows in C) met the criteria for coronary artery vasospasm in monkeys from the Ovx group only. A similar vasoconstrictor challenge in a progesterone-treated Ovx monkey (F) showed only transient (<5 min) decreased diameter. These data are representative of responses observed in 6 monkeys in each group.

Table 1 summarizes the average changes in coronary artery diameter in response to 1-ml constant intracoronary injections (over 30 s) of 100 µmol/l ACh, 0.1 mmol/l serotonin, 1 µmol/l U-46619, and a combination of 0.1 mmol/l serotonin + 1 µmol/l U-46619 in surgically postmenopausal untreated and progesterone-treated monkeys. The steroid levels shown were determined from blood serum collected the day before the angiography. Control diameters of the large epicardial coronary arteries (main, left anterior descending, right, and circumflex coronary arteries) where vasospasms were typically identified ranged from 1.2 to 1.8 mm for both monkey groups. Dilation (4.4 vs. 8.7%) and a transient decrease in heart rate (2-5 missed heart beats and a decrease to 65 ± 5 beats/min) in response to ACh were the same in the untreated and progesterone-treated monkeys, indicating normal endothelial and pacemaker function. A transient dilation (0.07 vs. 9.4%) and increase in heart rate up to 190 beats/min were observed in the Ovx and progesterone-treated groups after intracoronary injection of serotonin, respectively (no difference between groups). A more pronounced and longer lasting vasoconstriction was observed in response to U-46619 alone in the Ovx group, resulting in focal constrictions to <= 33% of the control diameter (>67% occlusion) in one of six Ovx monkeys (Fig. 1c). The combination of 100 µmol/l serotonin + 1 µmol/l U-46619, however, resulted in severe focal vasospasm-type constrictions that occurred at one or more sites in the large epicardial arteries in all monkeys tested in the Ovx group but in none of the progesterone-treated group. Vasospasm-like constrictions reducing diameter by >= 67% with little or no blood flow for >5 min were evident after the second or third challenge with serotonin + U-46619 in the Ovx group. In monkeys treated with progesterone, transient (3 min or less) constrictions were observed after serotonin + U-46619 injections, resulting in a 25-75% decrease in artery diameter. No additional decrease in artery diameter was observed upon the addition of 1 nmol/l endothelin-1 to the serotonin + U-46619 challenge. A further increase in concentration of U-46619 to 3 µmol/l (not allowing for dilution) in progesterone-treated monkeys resulted in a more pronounced constriction, rapidly decreasing diameter by up to 60-80%, followed by a threatening fall in blood pressure to <40/25 mmHg. However, in progesterone-treated monkeys, the constriction reversed within 3 min, and thus the combined diameter and duration criteria for vasospasm were not met. There was no evidence of clotting or increase in coagulation of blood by U-46619, even at the highest dose, in either Ovx or progesterone-treated monkeys.

                              
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  		Table 1.   Effect of low-dose progesterone on coronary artery diameter in vivo

VMC Intracellular Ca2+ and PKC Signals

Coronary artery VMC from the Ovx group showed corresponding hyperreactive intracellular Ca2+ (Fig. 2A) responses (with an elevated Ca2+ concentration progressing beyond 15 min) to the combined stimuli of 10 µmol/l serotonin and 100 nmol/l U-46619. In comparison, VMC from the progesterone-treated group showed relatively small transient responses. In VMC from postmenopausal monkeys, Ca2+ levels (whole cell averages) increased to 208% of their prestimulation levels at 20 min after stimulation (P < 0.05 vs. 148% in VSM from the progesterone-treated group) (Fig. 2A). In VMC from the progesterone-treated group, Ca2+ levels increased only at the earlier time points (minutes 1, 2, and 5) (Fig. 2A), declined after the 5-min peak, and returned to prestimulation values (99% of basal) by 20 min after stimulation.


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  		Fig. 2.   Ca2+ fluorescence (A) and protein kinase C (PKC; B) in freshly dispersed coronary artery vascular muscle cells (VMC) as a function of steroid hormone treatment. Whole cell Ca2+, as indicated by fluo 3 fluorescence intensity, was measured from images recorded immediately before (time 0) and 1, 2, 5, 10, 15, 20, and 30 min after a 15-s stimulation with 10 µM serotonin + 100 nM U-46619 (S + U). The data are means ± SE of the fluo 3 fluorescence intensity from experiments on 8 VMC from each group, calculated as the percent change from the control (time 0) fluorescence for each individual VMC. Fluo 3 fluorescence in VMC from the Ovx group showed amplified Ca2+ responses with sustained time courses (with increases persisting beyond 30 min). There were no such prolonged Ca2+ signals in VMC from the P-treated group. PKC responses measured by hypericin fluorescence intensity at time 0 and 3, 4, 9, 16, 21, and 31 min after stimulation in the same Ovx and progesterone-treated monkey VMC shown in A were less at the 21-min time point in the P-treated group than in the Ovx group. *P < 0.05, P-treated group vs. Ovx group.

PKC levels (Fig. 2B) in VMC from the Ovx group, as indicated by hypericin fluorescence intensity, increased in response to vasoconstrictor stimulation to 122 ± 7% at 21 min (n = 8) of the prestimulated baseline. As with Ca2+, the increase in fluorescence intensity was most evident at times of 21 min or later and maintained for up to 31 min, whereas VMC from the progesterone-treated group coronary arteries showed no change or a slight decrease in PKC fluorescence intensity through the 31-min time course (Fig. 2B). Thus treatment of Ovx monkeys in vivo with low concentrations of progesterone appeared to eliminate late phase responses and tended to restore the "normal" Ca2+ and PKC responses of VMC.

TxA2 Receptor Immunolabeling

To determine whether the relative level of TxA2 receptor expression correlated with the in vivo coronary artery responses, TxA2 receptor immunostaining of coronary artery and aorta cross sections were visualized by laser scanning confocal microscopy. Fluorescent labeling of vascular TxA2 receptors with anti-peptide antibodies to the human TxA2 receptor revealed that the predominant cell type expressing the receptor was the VMC in both the left anterior descending coronary artery and aorta, with relatively less labeling of the endothelial lining. TxA2 receptors in coronary vessels from a monkey with levels of 4 pg/ml E2beta and 0.07 ng/ml progesterone (Fig. 3A) showed relatively greater fluorescent labeling than arteries from a monkey from the progesterone-treated group (5 pg/ml E2beta , 2 ng/ml progesterone) (Fig. 3B). Thus treatment of Ovx monkeys in vivo with progesterone for 2 wk via silastic implant appeared to reduce the amount of immunolabeling of coronary arterial muscle cells. Cross sections of aortas from Ovx and progesterone-treated groups labeled with TxA2 receptor antibodies are also shown in Fig. 3. In these images, the green autofluorescence of elastin differentiates VMC from the connective tissue in the vessel wall. The relative amount of TxA2 receptor immunolabeling of muscle cells in the aorta of a monkey from the Ovx group (Fig. 3C) was greater than that from a monkey from the progesterone-treated group (Fig. 3D). No specific fluorescence was detected when sections were incubated with incubation buffer alone, followed by rhodamine-conjugated secondary antibody (no primary antibody negative control). Labeling profiles using two different TxA2 receptor antipeptide antibodies were similar (no detectable differences) when comparing the monkey aorta with the coronary artery. Differences noted between Ovx and progesterone-treated group coronary arteries were indistinguishable from differences between Ovx and progesterone-treated group aortas.


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  		Fig. 3.   Thromboxane A2 receptor (TxA2R) immunolabeling of monkey coronary artery and aorta cross sections. TxA2R were labeled in the left anterior descending (LAD) coronary artery and aorta from surgically postmenopausal (Ovx; A and C) and progesterone-treated Ovx (B and D) monkeys. Paraffin-embedded LAD sections (A and B) were incubated with PH4, an anti-peptide antibody to TxA2R, followed by fluorescent labeling with a rhodamine-conjugated secondary antibody. Aortic sections (C and D) were labeled with the anti-peptide antibody PH3. Images were collected with a Leica TCS 4D confocal laser scanning microscope using a ×40/1.25 numerical aperture oil immersion objective. The red fluorescent labeling is thought to represent membrane-associated TxA2R and, to a lesser extent, TxA2R found in the cytosol or associated with organelles. Green autofluorescence of the connective tissue excited by the 488-nm laser was observed in the aortic cross sections, as shown in C. Note the localization of TxA2R primarily in VMC and, at a lesser density, in endothelial cells and the upregulation of coronary artery TxA2R in the Ovx group compared with the P-treated group. These data (representative of 5 identical experiments comparing Ovx and P-treated groups) show increased arterial TxA2R expression in primates after removal of the ovaries and the inverse suppression by physiological (or less) concentrations of progesterone.

TxA2 Receptor Radioligand Binding

To independently and more accurately quantify the effect of progesterone treatment on the density of arterial TxA2 receptors and to characterize the relative affinity of agonist binding, [125I]BOP binding was measured in crude membranes from carotid arteries and aortas. These arteries were used instead of coronary arteries because adequate quantities of the later could not be obtained for the binding assay. A comparison of the Bmax is shown in Fig. 4A. Aortic and carotid artery membranes prepared from Ovx group monkeys had 111 ± 5 fmol TxA2 receptor/mg protein (n = 4). Treatment of Ovx monkeys in vivo with progesterone significantly decreased the expression of TxA2 receptors to 43 ± 15 fmol TxA2 receptor/mg protein (P = 0.01, n = 4), as measured by [125I]BOP binding. There was no significant difference (P = 0.205) in the Kd values for the Ovx group (1.50 ± 0.74 nmol/l, n = 4) compared with the progesterone-treated group (0.31 ± 0.16 nmol/l, n = 4) (Fig. 4B).


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  		Fig. 4.   Maximum TxA2R density (Bmax; A) and affinity [dissociation constant (Kd); B] in rhesus monkey carotid artery and aorta crude membranes. Density of [1S-(1I,2J(5Z),3I(1E,3R*),4I)]-7-[3-(3-hydroxy-4-(4'-[125I]iodophenoxy)-1-butenyl)-7-oxabicyclo[2.2.1]heptan-2-yl]-5-heptenoic acid ([125I]BOP) binding sites (A) was significantly greater (P < 0.01) in membranes prepared from the Ovx group (n = 4) compared with the P-treated group (n = 4). Relative affinity (Kd) of [125I]BOP binding (B) was not significantly different (P = 0.205) between samples from the P-treated and Ovx groups, although there was a tendency for higher affinity after progesterone treatment. These data demonstrate that primate coronary arteries expressed increased TxA2R density in the Ovx group compared with that of the P-treated group even with low physiological plasma levels of progesterone. NS, not significant. **Significant difference between P and Ovx (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present investigation, five independent methods were used to evaluate coronary artery reactivity in untreated and progesterone-treated Ovx rhesus monkeys. The data generated support a hypothesis that incorporates the five variables, which are as follows: 1) coronary artery reactivity (incidence of serotonin + U-46619-stimulated coronary artery vasospasm-like constrictions), 2) single coronary artery VMC Ca2+ and PKC responses to serotonin + U-46619, 3) the relative level of coronary artery and aorta TxA2 receptor antibody immunostaining, 4) aorta and carotid artery TxA2 receptor density ([125I]BOP binding), and 5) circulating progesterone concentration. In the near absence of progesterone, the monkeys showed significantly exaggerated coronary artery and isolated VMC reactivity to concentrations of serotonin and U-46619, a vasoconstrictor combination used to simulate platelet release products. Administration of natural progesterone via a subdermal silastic implant for 2 wk virtually eliminated vasospasm-like constrictions, greatly reduced single cell Ca2+ responses, and significantly reduced TxA2 receptor density compared with the progesterone-deficient state.

While TxA2 receptors have not been previously demonstrated in primate coronary arteries, pharmacological evidence (pronounced increases in coronary reactivity) suggests the likelihood of their presence (17, 29-32). In this study, we were able to demonstrate TxA2 receptors using immunocytochemical techniques. Of particular importance was the observation that the expression of TxA2 receptors was significantly reduced in progesterone-treated monkeys compared with surgically menopausal monkeys without hormone replacement. We were not able to get sufficient quantities of coronary arteries to be able to biochemically quantify the density of TxA2 receptors using radioligand binding assays. Thus we prepared and pooled crude membranes from the carotid artery and aorta. In both arteries, progesterone treatment resulted in a significant decrease in TxA2 receptor density compared with the untreated Ovx group. These observations are consistent with the hypothesis that progesterone can decrease expression of TxA2 receptors in aortas and coronary and carotid arteries.

The levels of progesterone that proved to be protective were in the low physiological range of normally cycling rhesus monkeys, which therefore might be expected to minimize side effects when used in hormone replacement therapy. Data from the vasospasm provocation studies and single cell Ca2+ experiments indicate that serum progesterone levels of <4 ng/ml are sufficient to restore the protected state to both rhesus monkey coronary arteries and isolated VMC. Previously, we (29) reported that 5-7 ng/ml progesterone treatment for 4 wk after a 2-wk E2beta priming period was cardioprotective. Here, exogenous progesterone, independent of added estrogen (levels below 10 pg/ml) and at lower progesterone concentrations (2.4 ng/ml average, with 1 ng/ml sufficient for effect), restored the protected state. It is not clear whether residual (5-10 pg/ml) E2beta in the plasma of surgically postmenopausal monkeys might have contributed indirectly to the protective effect of progesterone, e.g., by maintaining a minimum level of progesterone receptor expression. We (30) previously demonstrated VMC progesterone receptor expression in coronary artery sections from Ovx and E2beta  + progesterone- or E2beta  + MPA-treated Ovx monkeys; treatment with E2beta in vivo appeared to enhance or stabilize progesterone receptor expression. These data are in stark contrast to what we observed in monkeys treated with the synthetic progestin MPA, which tended to negate the benefits of estrogen (31). Indeed, VMC from Ovx group coronary arteries showed hyperreactivity in the catheterization laboratory and exaggerated Ca2+ signals beyond 15 min (in response to serotonin + U-46619 stimuli), which could be abolished by treatment of VMC in vitro with 1-3 ng/ml progesterone for 24-48 h (30), whereas MPA tended to further potentiate or have no effect on the prolonged Ca2+ responses (unpublished observations).

Mice lacking thromboxane-prostanoid receptors via gene targeting showed insensitivity to TxA2 receptor stimulation by U-46619 or arachidonic acid that would have caused cardiovascular collapse and death in wild-type mice (47). Unexplained TxA2 receptor downregulation has been reported in a subpopulation of male rabbits that showed virtually no contractile response to U-46619 (8, 37). Nonresponder rabbits had significantly reduced densities of VMC TxA2 receptors compared with outbred male New Zealand White rabbits. However, testosterone, corticosterone, and E2beta levels did not differ between the responder and nonresponder rabbits. Progesterone was not measured in these rabbit genetic model experiments but should be explored, because the data presented in this report indicate that progesterone suppresses the expression of VMC TxA2 receptors in surgically postmenopausal rhesus monkeys.

Our studies did not measure whether VMC serotonin receptor expression differed between Ovx and progesterone-treated groups, although the combination of U-46619 and serotonin was more likely to produce coronary vasospasm in the untreated Ovx group monkeys compared with those treated with U-46619 alone. Recently, Bethea et al. (15, 36) demonstrated that progesterone regulates the expression of brain serotonin receptors in rhesus monkeys. Thus, in addition to downregulating TxA2 receptor density, as we are proposing, progesterone may also downregulate serotonin receptor expression in the coronary artery. Alternatively, it may be that combined activation of both TxA2 receptors and serotonin receptors is synergistic. Because both receptors are known to couple to the G protein subfamily Gq and phospholipase C activation, perhaps a signaling intermediate activated by serotonin receptor stimulation may potentiate the thromboxane response directly at the level of the TxA2 receptor, via redistribution of G proteins (10, 11), or by potentiation of Ca2+ and PKC signaling.

Coronary artery VMC Ca2+ responses from the low-dose progesterone-treated postmenopausal monkeys (without E2beta treatment) were significantly decreased compared with VMC from untreated Ovx monkeys, consistent with a lower incidence of coronary artery vasospasm. Brief 15-s stimulation with 10 µmol/l serotonin + 100 nmol/l U-46619 resulted in a sustained increase in intracellular Ca2+ in Ovx group VMC that reached two times the basal value >= 20 min after stimulation. In contrast, Ca2+ responses in progesterone-treated group VMC increased only transiently (3-5 min). Kuga et al. (24) also concluded that coronary hyperreactivity after balloon injury in miniature pigs was due to VMC mechanisms. These data support the in vivo findings and our earlier report (32) suggesting that hyperreactivity to serotonin + U-46619 (the putative stimulus for coronary vasospasm) may be explained by an abnormal sustained increase in intracellular Ca2+ in VMC. The mechanism by which VMC can maintain elevated intracellular Ca2+ for 20-30 min may involve enhanced activation/translocation of PKC (29, 30, 32).

In summary, growing evidence indicates that progesterone beneficially regulates coronary artery reactivity in monkeys and humans (17, 30-32, 39, 41, 45, 50). The mechanism may involve regulation (repression) of TxA2 receptor expression by progesterone. If the regulation of coronary TxA2 receptor by progesterone were found to be similarly significant in women, this abnormal hyperreactivity manifested as the tendency for prolonged, severe, vasospastic constriction in progesterone deficiency states (such as the postmenopausal years) would represent one readily preventable etiology of coronary ischemia.


    ACKNOWLEDGEMENTS

The technical assistance of Linda Walker is gratefully acknowledged.


    FOOTNOTES

This research was supported by National Institutes of Health Grants HL-51723, HL-51750, and HD-18185.

Address for reprint requests and other correspondence: K. Hermsmeyer, Dimera LLC, 2525 NW Lovejoy, Suite 401, Portland, OR 97210 (E-mail: rkh{at}dimera.net).

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 16 December 2000; accepted in final form 14 June 2001.


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