INTRODUCTION

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PROGESTINS ARE GENERALLY PRESCRIBED together with estrogen to postmenopausal women for hormone-replacement therapy. This regimen is intended to provide cardioprotection and at the same time limit the unwanted effects of unopposed estrogen on the endometrium and breast. Although the cardioprotective effects of estrogen are now widely appreciated, there are divergent effects of bioidentical progesterone as contrasted with synthetic progestins on the cardiovascular system (14, 18, 27).

Classical nuclear estrogen receptors (ER-) and progesterone receptors (PR) are expressed in vascular muscle cells (VMCs) (8-13, 14, 21). The function of ovarian steroid hormone receptors in vascular muscle has recently received greater interest, in part, due to the heightened awareness of the cardioprotective effects of hormone-replacement therapy for postmenopausal women (20).

Rhesus monkey coronary artery muscle cells identified as being positive for smooth muscle myosin heavy chain showed nuclear immunolabeling with PR and ER- antibodies (14). Pretreatment of these cells with 0.3-3 ng/ml progesterone or 3-100 pg/ml 17-estradiol for >24 h decreased the amplitude and duration of serotonin + U-46619 (5-HT+U)-stimulated intracellular Ca²⁺ and protein kinase C (PKC) responses (14). Modulation of VMC excitation-contraction signaling by estrogen and progesterone was blocked by ER- and PR antagonists ICI-182780 and RU-486, respectively. This suggests that physiologically relevant concentrations of estrogen and progesterone reduce VMC reactivity via nuclear ER and PR signaling.

The mechanism by which estrogen and progesterone modulate VMC Ca²⁺ and PKC responses to 5-HT+U is not known but may involve modulation of thromboxane A₂-receptor expression on the VMC membrane (15). In addition, the relative degree of PR expression is decreased in the absence of 17-estradiol or presence of medroxyprogesterone acetate (MPA) (16). MPA is a synthetic progestin commonly used in oral contraceptives and hormone-replacement regimens shown to negate the benefits of 17-estradiol, whereas bioidentical progesterone does not (16, 18, 19, 27). Replacement of progesterone and/or 17-estradiol in ovariectomized (OVX) monkeys to levels observed before surgical menopause was shown to provide cardioprotection against 5-HT+U-induced coronary artery vasospasm (5, 6, 16, 18), suggesting that ovarian steroid hormones modulate coronary artery vasoconstrictor reactivity through classical nuclear steroid receptor signaling.

Chronic progesterone treatment reduces systemic blood pressure in humans (24), and a membrane progesterone binding site was recently cloned from porcine coronary artery muscle cells (4), providing a possible basis for progesterone to mediate rapid effects on blood vessels. Thus, in the present study, we first examine the hypothesis that a 100-ng bolus of progesterone, administered by direct intracoronary injection during coronary ischemia, is capable of coronary vasodilation powerful enough to counteract vasospasm induced by a putative vasospasm stimulus combination, even in the presence of MPA. Second, we test whether estrogen or progesterone added directly to an agonist-stimulated isolated VMC in a form attached to albumin, which is believed to not rapidly cross the cell membrane and thus act extracellulary, would affect intracellular Ca²⁺ and PKC signals.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animal model. OVX rhesus macaques (n = 14; average age = 13.3 ± 0.7 yr; average weight = 6.5 ± 0.4 kg) were either not treated with steroids (control; n = 6) or treated with 4 wk of estrogen in combination with a progestin for the last 2 wk, i.e., 17-estradiol and MPA (EM; n = 6), 17-estradiol, progesterone, and MPA (EPM; n = 1), or 17-estradiol and norethindrone (EN; n = 1) via Silastic implants to achieve physiological levels of estrogen (60-120 pg/ml), progesterone (1-4 ng/ml), and therapeutic levels of MPA or norethindrone (0.5-2 ng/ml) (see Ref. 16 for further details). Monkeys reported herein are part of a larger study on the mechanism of coronary vasospasm and vascular muscle hyperreactivity. The epicardial coronary arteries in these monkeys showed hyperreactive responses to the vasoconstrictor challenges, which culminated in severe coronary constrictions that met the criteria for vasospasm. By definition, the criterion for coronary artery vasospasm is focal areas of severe constriction to less than one-third of the control diameter, followed by downstream dilation that persists for >5 min (6). OVX monkeys treated with estrogen, progesterone, or estrogen + progesterone, in previous studies, were not hyperreactive to the same coronary vasoconstrictor challenges and, with rare exception, did not meet the vasospasm criteria (16, 18). Therefore, this report deals with a subset of monkeys in which we were able to evoke severe vasospasms, to which we applied progesterone or mibefradil and monitored the coronary diameter.

Coronary vasospasm provocation. The injection protocol employed for initiation of vasoconstriction, which appeared analogous to coronary vasospasm in humans, has been described previously (6, 16, 18). The interventional challenge drugs, diluted in saline, were injected directly into the coronary artery as a 1-ml bolus over a 30-s interval. It is estimated that this results in an immediate 15-fold dilution of the syringe concentration in nonconstricted arteries (6). In brief, the protocol is as follows. Rhesus monkeys were sedated with an intramuscular injection of ketamine (10 mg/kg) at the radiology catheterization suite. For artery and venous catheterizations, 1-1.5% isoflurane in 70% O₂ and 30% nitrous oxide were continuously administered by inhalation to maintain a surgical plane of anesthesia. Blood pressure, electrocardiogram, heart rate, temperature, and percent O₂ saturation were continuously monitored and recorded. Before coronary catheterization, 1,000 units of heparin were administered intravenously. Control angiograms were acquired to document the anatomy of the epicardial coronary arteries by using a 1- to 2-ml injection of Hexabrix, a radiopaque contrast media. Angiograms were acquired immediately (within 15 s), at 3 min after each injection, and at later times when it was apparent that a persistent effect warranted additional images. Endothelial integrity was demonstrated by vasodilation with 1 µM ACh, and intracoronary injection of 100 µM serotonin (5-HT) was then used to show a lack of constriction in the large coronary arteries, due to an intact endothelium, or would detect endothelial defects as vasoconstriction if there was a site of artery damage. Next, coronary arteries were stimulated with 1 µM U-46619 (U), a stable thromboxane A₂ mimetic. The next five injections constitute the main vasospasm challenges: three separate injections of combined 5-HT (100 µM) and U (1 µM), followed by the triple combination of 5-HT (100 µM) and U (1 µM) together with endothelin-1 (1 nM), and finally 5-HT (100 µM) with a higher concentration of U (3 µM). The vasospasm-like constrictions occurred after a cumulative total of 1-2 ng of U (5-HT+U challenges 2 and 3 in most cases). If focal constrictions that meet the vasospasm criterion had not developed by the time cardiogenic shock was evident (diastolic blood pressure of <30 mmHg), the animal was termed "protected." We have shown that protection against vasospasm is strongly correlated with and probably is dependent on physiological levels of circulating estrogen and/or progesterone (16, 19).

VMC preparation. OVX rhesus monkeys (n = 6) were subjected to coronary artery vasospasm provocation challenges in a catheterization laboratory as described above. The epicardial coronary arteries in these groups showed hyperreactive responses to the vasoconstrictor challenges, which culminated in severe coronary constrictions that met the criteria for vasospasm in all six untreated OVX monkeys.

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 (14-16, 19). Ca²⁺ and PKC were studied in the same VMC by using double labeling with different wavelength fluophores and two fluorescence filter sets. The fluorescent Ca²⁺ indicator fluo 3 acetoxymethyl ester (fluo 3; Molecular Probes) was used to sense Ca²⁺. Live cell measurements of PKC were made by using hypericin (LC Laboratories). The concentration range used to detect PKC in our experiments was 30-300 nM, which is 10-100 times less than the IC₅₀ (3.4 µM) and below the threshold concentration for inhibition of purified PKC (400 nM) (26).

Ca²+ fluorescence. VMC on small glass coverslips were placed in a 300-µl laminar flow chamber (7). Isotonic solution for mammals, second generation (ISM2), which contains (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, VMCs were loaded at room temperature by adding 30 µl of 1-10 µM fluo3 for 15 min (into a total volume of ~300 µl). VMCs were then washed for 5 min, and a time 0 (control) image was acquired. To the buffer directly above a VMC, 30 µl of 10 µM 5-HT plus 100 nM U was added. After 15 s under no-flow conditions, thus exposing the VMC to a final bath concentration of 1 µM 5-HT + 10 nM U, continuous flow of ISM2 at 1 ml/min was reinstated, the stimuli were washed out [half time (t_1/2) = 30 s], and fluorescent Ca²⁺ images were acquired at 1, 2, 5, 10, 15, 20, and 30 min by using a Zeiss Axiovert microscope with a C-Apochromat ×40, 1.2 numerical aperture confocal-design water-immersion objective. After the 30-min image was acquired, at which time the agonist-stimulated Ca²⁺ responses were maximal, BSA-conjugated progesterone (P-BSA; final concentration = 3 ng/ml) or BSA-conjugated 17-estradiol (E₂-BSA; 333 pg/ml) was added. After 1 min, flow of 1 ml/min was reinstated, and Ca²⁺ images were acquired 3, 5, 10, 15, 20, 25, and 30 min later. Fluorescent Ca²⁺ images (excitation filter, 487 nm; dichroic mirror, 505 nm; emission filter, 530 nm) for the whole cell thickness are expressed relative to the prestimulated basal level (percentage of control).

PKC. Hypericin was loaded for 5 min, and the excess indicator was washed out for 5 min (coinciding with the fluo 3 washout). By using the end of the 5-min washout as time 0, fluorescent PKC images were acquired immediately before and 3, 4, 9, 16, 21, and 31 min after stimulation with 5-HT+U. Additional PKC images were collected 5, 10, 15, 20, and 30 min after adding P-BSA or E₂-BSA. Hypericin 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. The whole cell fluorescence intensity was taken as an indication of the relative level of PKC in the VMC.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Coronary artery angiograms in hyperreactive female rhesus monkeys showed focal constrictions with an intracoronary injection of 1 ml of 100 µM 5-HT + 1 µM U. These focal constrictions were relieved by intracoronary progesterone injection. A rapid and therefore putatively nongenomic action of progesterone on primate coronary arteries was observed. Progesterone actions were seen within minutes of intracoronary injection of 100 ng of the bioidentical hormone applied to severely constricted coronary arteries. Relaxation in response to progesterone was observed within about the same time frame as vasorelaxation by mibefradil, a selective T-type Ca²⁺-channel blocker. Thus progesterone may act as a direct vasodilator to relieve even severe focal coronary artery constrictions in non-human primates.

Figure 1, a-d, shows example angiograms of the epicardial coronary arteries of an OVX monkey (Fig. 1a) in which focal constrictions of hourglass shape, as found in human coronary artery vasospasms, were induced and sustained at 5, 10, and 20 min (Fig. 1, b-d, respectively). Spontaneous relaxation of focal constrictions was observed only rarely, including in this example, as shown in Fig. 1e. Rather, coronary vasospasms in monkeys characteristically persisted for >20 min in arteries in which no vasodilator was given. Focal constrictions were reinitiated at the same anatomic point with a subsequent injection of 5-HT+U (Fig. 1f). Constrictions to less than one-third of control average diameter () in a specific segment of an epicardial artery were dilated by direct injection of 100 ng progesterone (1 ml injected over 30 s, which would be diluted ~15-fold by the coronary circulation if and when blood flow was reinstated; Fig. 1, g and h). Progesterone relieved vasospasm-like constrictions in six of six monkeys tested; the monkeys tested in this group were untreated OVX (n = 3) or OVX treated for 2 wk with EM (n = 2) or EPM (n = 1). This response began in the most rapid instances as early as the first measurement after progesterone administration (15 s later) when the radiopaque contrast media was injected. By 3 min after injection of progesterone,  at the site of focal constrictions increased dramatically and significantly. Average  preconstriction vs. 3 min after injection of progesterone showed a return to control, i.e., diameters were not significantly different (Fig. 2A). Completely restored  were observed in monkeys over times ranging between 1 and 20 min after progesterone injection (means ± SE = 8.2 ± 2.6 min).

View larger version (98K): [in this window] [in a new window]   Fig. 1.   Coronary artery angiograms in an ovariectomized (OVX) rhesus monkey show focal constrictions (similar in form and function, particularly ovarian steroid responsiveness, and thus relevant to human coronary vasospasm) on stimulation with 1 ml of 1 µM U-46619 (U), a thromboxane A2 mimetic, with or after 100 µM serotonin (S). a-d: Prestimulated left anterior descending coronary artery (a), followed by angiograms taken 5 (b), 10 (c), and 20 min (d) after intracoronary injection of 1 µM U. Arrows in b, c, and d point to the site of focal constriction. Spontaneous relaxation, which occurred 30 min after initiation of the vasospasm (e), allowed a second stimulation, this time with 100 µM S + 1 µM U (S+U), which reinduced the focal constriction at the same anatomic point (f; see arrow). Direct injection of 100 ng of progesterone (1 ml/30 s) dilated the artery within 1 min (g) and completely restored the prestimulated diameter by 20 min (h).

View larger version (51K): [in this window] [in a new window]   Fig. 2.   Average diameter () of coronary arteries measured during a S+U-induced spasm (focal constriction to <30% of control diameter that persisted for more than 5 min; S+U constriction) was significantly reduced compared with the  of arteries measured during a control Hexabrix injection (control). A: administration of 100 ng of progesterone (P; 1 ml injected over 30 s) resulted in rapid vasorelaxation at the site of the focal constriction, as shown by  measured 3 min after injection of P (P 3' ). After an average of 8.2 ± 2.6 min after injection of P (n = 6), the focal constrictions had fully relaxed to their preconstriction diameters (P max ). B: administration of 1 µM mibefradil (Ro40) injected over 30 s resulted in rapid vasorelaxation at the site of the focal constriction, as shown by  measured 3 min after injection (Ro40 3' ). After an average of 3.3 ± 0.6 min after injection of mibefradil (n = 6), the focal constrictions had fully relaxed to their preconstriction diameters (Ro40 max ). Values are means ± SE. NS, not different from control. *P < 0.05 vs. other groups.

Intracoronary injection of the Ca²⁺ antagonist mibefradil (1 µM) during an ongoing coronary artery vasospasm in six hyperreactive rhesus monkeys rapidly relieved the focal constrictions in every case (Fig. 2B); the monkeys tested in this group were untreated OVX (n = 1) or OVX treated for 2 wk with EM (n = 4) or EN (n = 1). The average time required to relieve spasms with mibefradil was 3.3 ± 0.6 min, which tended to be more rapid but was not statistically different than that observed with progesterone injections. Example angiograms, showing a similar time course of vasospasm relief by mibefradil and progesterone, are shown in Fig. 3. In Fig. 3, a-d, 5-HT+U caused a focal constriction (vasospasm), as defined by the hourglass-shaped reduction in coronary artery diameter, to 25% (3,b and c) of the control diameter (3a) in an untreated OVX monkey. Mibefradil (Ro-40-5967, 1 µM), injected at 1 ml/30 s, restored the diameter to 87.5% of the control diameter (Fig. 3d) by 3 min after the intracoronary injection. Similarly, coronary artery angiograms in an EPM-treated OVX rhesus monkey showed focal constrictions on stimulation with 5-HT+U and relief by progesterone. Figure 3, d-g, shows the control (prestimulated) angiogram of the left anterior descending coronary artery (Fig. 3e), followed by angiograms taken 3 (Fig. 3f) and 15 min (Fig. 3g) after intracoronary injection of 5-HT+U. The arrows in Fig. 3, f and g, point to the site of focal constriction at which the diameter of the artery decreased from 2.0 to 0.1 mm. Direct injection of 100 ng of progesterone relieved the vasospasm beginning within 3 min (diameter = 1.75 mm; Fig. 3h) and completely restored the prestimulated diameter (2.0 mm) by 5 min (not shown). Similar results were observed when nifedipine, an L-type Ca²⁺-channel blocker, or nitroglycerin, a source of nitric oxide, were injected during vasospasm (data not shown). Electrocardiogram correlates of the severe vasoconstrictions manifested themselves as 1- to 3-mm elevations in ST segments and enlarged or inverted T waves. Intracoronary progesterone or mibefradil infusion during the severe, persistent focal coronary constrictions led to normalization of electrocardiogram abnormalities with approximately the same time course as the increase in coronary artery diameter.

View larger version (92K): [in this window] [in a new window]   Fig. 3.   Relief of coronary artery vasospasm with mibefradil vs. progesterone. a-d: intracoronary injection of 1 µM mibefradil (Ro-40-5967), a selective T-type Ca2+-channel blocker with only weaker blocking action on L-type Ca2+ channels, 18 min after initiation of coronary vasospasm with S+U in an OVX rhesus monkey. The control diameter (2.0 mm; a) at the site of the focal constriction caused by S+U stimulation (0.5 mm; b and c) and the diameter after intracoronary injection of mibefradil (1.75 mm; d) are shown. Coronary artery angiograms in an 17-estradiol + progesterone + medroxyprogesterone-treated OVX rhesus monkey also showed focal constrictions (similar in form and function to human coronary vasospasm) on stimulation with S+U. e-h: angiograms of the control (prestimulated) left anterior descending coronary artery (e), followed by intracoronary injection of S+U over 30 s and the vasoconstrictions 3 (f) and 15 min (g) after. Arrows in f and g point to the site of focal constriction at which the diameter of the artery decreased from 2.0 to 0.1 mm. Direct intracoronary injection of 100 ng of progesterone almost completely relieved the vasospasm within 3 min (h) (diameter = 1.75 mm).

As a separate in vitro correlate, we tested whether physiologically relevant levels of membrane-impermeant albumin-conjugated ovarian steroid hormones would modulate 5-HT+U-stimulated VMC Ca²⁺ and PKC responses. Maximum Ca²⁺ levels were achieved 30 min after agonist stimulation in hyperreactive monkey VMC, at which time a final concentration of 333 pg/ml E₂-BSA or 3 ng/ml P-BSA, both physiologically relevant concentrations, was applied to VMC. After 1 min, laminar flow of ISM2 at a rate of 1 ml/min was reinstated and fluorescent Ca²⁺ and PKC images were acquired for an additional 30 min.

Figure 4 shows the effect of P-BSA and E₂-BSA on 10 µM 5-HT + 100 nM U stimulated intracellular Ca²⁺ responses. Ca²⁺ levels in VMC from OVX monkeys increased to 203 ± 32% (n = 5) and 250 ± 34% (n = 5) of the baseline fluorescent Ca²⁺ signal 30 min after stimulation in the E₂-BSA and P-BSA groups, which did not differ significantly. With the addition of 3 ng/ml of P-BSA, the intracellular Ca²⁺ signal indicated by fluo 3 fluorescence immediately began to decrease and continued to decrease until reaching the basal Ca²⁺ level (113 ± 17% of basal) 25-30 min after P-BSA was added. The time required for the Ca²⁺ signal to decay to one-half of the maximum value was 7 min (Fig. 4B). A significant change in the intracellular Ca²⁺ signal was not observed within 30 min after adding 333 pg/ml E₂-BSA to a 5-HT+U stimulated VMC (t_1/2 > 30 min; Fig. 4A).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Nongenomic Ca2+ lowering effect of progesterone-BSA contrasted with lack of effect of estrogen-BSA. Vascular muscle cells isolated from OVX monkey coronary arteries were cultured on glass coverslips in medium containing 1% HS, loaded with fluo 3-AM, and stimulated with 10 µM S + 100 nM U. At the peak of the hyperreactive Ca2+ transient (A: 203 ± 32%; B: 250 ± 34%), 17-estradiol-conjugated BSA (E2-BSA; 333 pg/ml; A) or progesterone-conjugated BSA (P-BSA; 3 ng/ml; B) was added and fluo 3 fluorescence intensity continuously monitored. Values are means ± SE (n = 5) change over basal fluo 3 fluorescence intensity.

PKC, which is thought to facilitate sustained increases in intracellular Ca²⁺ in hyperreactive VMC (19), was observed simultaneously with Ca²⁺ by using the indicator hypericin. The relative abundance of PKC was represented by the whole cell average of the hypericin fluorescence (14). In the primary cultured VMC from OVX monkeys, PKC levels increased to 133 ± 18% and 144 ± 21% of the prestimulated basal level 31 min after adding 5-HT+U in two separate studies. With the addition of P-BSA, whole cell PKC average tended to increase, although the maximum level achieved (152 ± 37%) was not significantly different from the PKC fluorescence intensity just before the addition of P-BSA (n = 5). After the addition of E₂-BSA, whole cell PKC fluorescence appeared to decrease to ~110-120% of the basal value but, as with P-BSA treatment, this difference did not achieve statistical significance at any time point after the addition of the steroid. Thus we observed no effect of either P-BSA or E₂-BSA (brief stimulation with physiological concentrations) on 5-HT+U-activated PKC in monkey coronary artery muscle cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The data reported herein suggest a rapid beneficial vasodilatory action of progesterone that has not previously been recognized as important in the coronary circulation. Direct vasorelaxation of constricted coronary arteries by the intracoronary administration of progesterone (100 ng) may be critical to understanding the cardiovascular benefits of ovarian steroid hormones used in hormone-replacement regimens.

In our experimental model, the phenomenon of ovarian steroid modulation of vascular reactivity characterized in nonhuman primates has been attributed (until now) to a genomic mechanism of action (5, 6, 14-16, 18, 19). In this model, coronary artery focal constrictions with the characteristics of vasospasms are reliably induced pharmacologically in 12-yr-old nonatherosclerotic hypoestrogenic monkeys with 5-HT+U. These focal constrictions are prevented when 17-estradiol, progesterone, or a combination of estradiol and progesterone are replaced in these OVX rhesus monkeys for 1, 2, or 4 wk. The in vivo protective effect of estrogen, progesterone, and the combination on coronary VMC reactivity in OVX rhesus monkeys correlates with 1) circulating ovarian steroid hormone levels, 2) intracellular Ca²⁺ and PKC responses of VMCs dissociated from coronary arteries, and 3) expression of receptors for estrogen and progesterone in coronary artery cross sections taken from the same animals (16). Treatment of OVX monkeys for 4 wk with physiological levels of progesterone (in the absence of added exogenous estrogen) also effectively eliminated the hyperreactivity of coronary arteries in vivo and the exaggerated and prolonged Ca²⁺ responses of isolated VMC in vitro by reducing thromboxane A₂-receptor density in arteries (15, 16)

That a physiologically relevant level of progesterone can also reverse coronary vasospasm suggests a second, more rapid effect on the vessel wall. A small transient peripheral vasodilatory effect was observed in four of the six animals in which progesterone was injected into the coronary artery. However, vasodilatory actions for both the radiopaque contrast medium (Hexabrix) and the anesthetic agent employed (isoflurane) may have caused underestimation of the peripheral dilation by progesterone. Nevertheless, replacement of progesterone appears to prevent ovariectomy-induced coronary artery hyperreactivity by a genomic mechanism and to relax focal constrictions by a nongenomic mechanism. This benefit does not appear to be true for treatment with MPA, a commonly used synthetic progestin, which we and others have shown consistently negates the protective effects of estrogen on coronary reactivity in vivo (16, 18, 19, 27).

These data strongly suggest the existence of an independent surface membrane progesterone receptor distinct from the classical nuclear progesterone receptor that is part of the transcription-activating superfamily. Rapid vasodilation by progesterone poses powerful evidence of the existence of such a second progesterone mechanism. This study did not examine the effect of promiscuous progesterone-receptor blocker RU-486, as in vitro studies revealed this compound, by itself, affected intracellular Ca²⁺ in VMC (Minshall and Hermsmeyer, unpublished observations). Our earlier studies have shown that, when progesterone or MPA are coexistent, MPA dominates, causing hyperreactivity despite the presence of physiological concentrations of estrogen and progesterone (16, 18, 19). Surprisingly, a modest dose of progesterone injected into coronary arteries produced dilation, even in monkeys treated with MPA. In contrast, a prolonged vasodilatory action of progesterone was not observed in monkeys treated for 2 or more weeks with EPM (16, 18), i.e., studies designed to test the transcriptional actions of 17-estradiol, progesterone, and MPA. Thus the directly observed angiographic dilation, and this action by progesterone superceding the hyperactive response expected in the MPA-treatment group, argues in favor of a separate mechanism of action.

Both immunohistochemical and physiological data suggest that the main site of action of progesterone is the VMC, with a lesser role for the endothelial cell. Thus we suspect that nonhuman primate coronary artery VMCs express a traditional ligand-inducible transcription factor, as well as a plasma membrane progesterone binding site (4). Our results suggest that membrane binding of progesterone rapidly modulates coronary artery diameter and can relieve focal constrictions (vasospasms) with nearly the same time course as the T-type Ca²⁺-channel blocker mibefradil, which is known to act on T-type Ca²⁺ channels in the VMC membrane (17). Our in vitro data reported herein also support the hypothesis that primary cultured coronary artery VMCs contain membrane progesterone binding sites capable of transducing a Ca²⁺-lowering effect within minutes.

Several reports of rapid actions of progesterone in a variety of tissue types indicate the existence of a membrane receptor. Progesterone acts on the plasma membrane of sperm, but in this case it increases intracellular Ca²⁺, which is thought to initiate the acrosome reaction (3). On human intestinal smooth muscle cells, Bielefeldt et al. (2) found that progesterone inhibits Ca²⁺ signals consistent with blocking the L-type Ca²⁺ channel. Both of these effects occurred very rapidly (within 1 min), were observed with supraphysiological levels of progesterone (0.1-50 µg/ml), and were not blocked by antiprogestins, which prevent genomic actions of progesterone and other progestins (2, 3). Whether the present observation of coronary vasodilation and VMC Ca²⁺ suppression by progesterone can also be explained by this mechanism (progesterone blockade of L-type Ca²⁺ channels) is not clear. Recently, a progesterone binding protein was isolated and cloned from porcine VMCs (4). The 1.9-kb transcripts shown by Northern analysis suggest that the protein is expressed in the liver > kidney > lung > cerebellum  heart (4). It is interesting to speculate that this membrane progesterone binding site leads to inhibition of Ca²⁺ conductance via the L-type Ca²⁺ channel.

Synthetic progestins, such as MPA, do not appear to act at this nongenomic membrane binding site (3). Thus it appears that a unique membrane progesterone binding site exists on sperm cells, intestinal cells, and VMCs. In each case, the functional significance is only beginning to be recognized. However, the evidence reported here suggests the rationale for more extensive investigation of this new-found beneficial role for progesterone. Taken together, the transcriptional actions on thromboxane A₂-receptor expression (15) and the direct relaxant action on coronary arteries and isolated coronary VMCs appear to justify new interest in cardiovascular actions of progesterone. The rapid, nongenomic relaxant effect of progesterone suggested by both our in vivo and in vitro nonhuman primate analog of coronary artery reactivity provides evidence for a cardiovascular benefit of progesterone independent of classical genomic actions.