Even though there has been a particular effort to elucidate it, the mechanism behind steroid hormone effects at the cardiovascular level is still not clear and may include more than one process. Classic concepts of the mechanism of steroid action at target cells include hormone entry to the cell, binding to high-affinity cytoplasmic or nuclear receptors, and specific interaction of the steroid-receptor complex with DNA, which results in gene expression and production of specific proteins, that in turn affect cell function (1) However, this mechanism does not seem to be sufficient to account for all the known cellular effects of steroid hormones, including their rapid effects, which are independent of hormone interaction with the cell nucleus (2), are too fast and too transient, and are not blocked by the antagonist of intracellular receptors that blocked the genomic effects of steroids or by actinomycin D or cycloheximide, blockers of transcription and translation (3,4). These facts signal the existence of nongenomic effects of steroids (5) (independent of intracellular receptors) that are exerted at or near the cellular membrane (6,7).
In the recent past, it has been shown that 17β-estradiol can induce a fast increase in the cytosolic free [Ca2+]i in different cell types. The increase in the intracellular calcium concentration may be due to an influx of extracellular calcium, as in uterine smooth muscle cells (8), rat osteoblast (9), and hepatocytes (10); it may also be due to the mobilization of calcium from intracellular stores as in chicken granulose cells (11) and rat osteoblast. It has been proposed that estradiol enhances the formation of 1,4,5-triphosphate through phospholipase C activity (12,13).
At the cardiovascular level, the rapid onset of hormone effects suggests that specific binding sites for steroids may be present on the cell membrane and therefore suggests that different receptors mediate short-term actions (seconds to minutes) and long-term actions (hours to days) (14). Recent results from our laboratory, in the isolated and perfused rat heart, showed that testosterone induces a short-term increase in vascular resistance and blocks the effects of vasodilatory agents, even when the presence of the hormone is restricted to the coronary vessel lumen (15). These effects were of a nongenomic character and were exerted at the endothelial cell membrane (16). In this regard, two mechanisms for 17β-estradiol-induced vasodilation have been demonstrated: (a) release of nitric oxide from endothelium (17-19), and (b) a direct vasodilatory effect on smooth muscle that occurs by a competitive inhibition of Ca2+ entry through L-type calcium ion channels (20,21). Estradiol modulates endothelial cell function through several mechanisms; it has been reported that brief exposure (5-30 min) to estradiol induced increases in the intracellular calcium concentration; however, the mechanism behind this effect is still unknown and might involve nongenomic mechanisms (19,20).
The purpose of this work was to analyze the effects of briefly applied estradiol on intracellular calcium kinetics in rat aorta vascular endothelial cells in culture and to characterize their modulatory effects on agonist-induced increases in [Ca2+]i, to clarify the initial events of steroidal nongenomic effects at the cardiovascular level. This approach is based on the fact that steroid hormones are transported to their target organs through the blood, that endothelial cells are the first cellular type to be in contact with the steroids, and that their stimulation starts a series of events that in turn regulate the function of cells in close association with them, such as smooth muscle or parenchymal cells.
The endothelial cells were obtained by a previously established protocol (22,23). In brief, cells were obtained by lightly scraping the intimal surface of young adult male rat (10-12 weeks) aortas with a scalpel blade. The cells were rinsed off the scalpel into dishes containing Dulbecco's MEM supplemented with 20% fetal calf serum (FCS) plus penicillin (100 U/ml) and streptomycin (100 μg/ml). The cells were allowed to grow in an incubator under a 5% CO2 atmosphere until islands of endothelial cells appeared. Then a small number of endothelial cells were removed by the trypsin treatment using cloning rings and plated until confluence. Cells were used between passages four and ten.
Characterization of cells
Endothelial cells were identified by examination of cellular morphology by phase-contrast microscopy and characterized by the expression of factor VIII antigen (von Willebrand) using antibodies obtained in rabbit and by taking up the fluorescent probe DIL-acetylated low-density lipoprotein.
The procedure for antibody staining was as follows: Cells were fixed in 4% paraformaldehyde for 10 min, washed and treated with H2O2 in methanol, 15%, for 15 min, and washed and incubated 30 min with blocking solution (5% goat serum). Then the cells were incubated for 48 h at 4°C with von Willebrand factor (1:1,000) antibodies. After washing, the cells were incubated for 4 h at room temperature with goat anti-rabbit immunoglobulin (1:100), followed by treatment with peroxidase antiperoxidase complex (1:50). The cultures were stained for peroxidase activity with 3′3′-diaminobenzidine tetrachloride and H2O2. After washing, the cultures were mounted and observed by light microscopy. From 98 to 99% expressed the von Willebrand factor.
Cells were trypsinized from a stock culture and resuspended in culture medium at a final concentration of ∼2.4 × 105 cells/ml. A droplet of ∼25 μl was placed onto coverslip dishes no. 1 (glued to a perforated plastic Petri dish), the rest of coverslip remain dry. After the cells began to attach, additional medium was added to the well until the final volume was ∼2 ml. The cells were maintained in an incubator under a 5% CO2 atmosphere. At 48-72 h before the experiment, the cells were serum deprived, replacing the incubation medium with culture medium containing Pen-Strep and 0.2% FBS.
Calcium measurement and experimental protocol
The cells were washed 3 times with HEPES-buffered Hank's balanced salts, pH 7.4 (137 mM NaCl, 0.441 mM KH2PO4, 0.442 mM Na2HPO4, 0.885 mM MgSO4 · 7H2O, 27.7 mM glucose, 1.25 mM CaCl2, 2 mM Na-pyruvate, 20 mM HEPES), and loaded with 3 μM fura-2/AM for 2 h in the same buffer at room temperature in the dark. The cells were washed 3 times with buffer and postincubated with the same buffer for an additional 1 h. The experimental chambers were placed on an inverted microscope (Dual-wavelength fluorescence imaging system InCytIm2; Intracellular Imaging Inc., Cincinnati, OH, U.S.A.). Drugs and reagents were added directly into the experimental chamber.
Fura-2 fluorescence response to the intracellular calcium concentration was calibrated from the ratio of 340/380 nm fluorescence values after subtraction of background fluorescence, using calcium standards (Molecular Probes Kit II, Eugene, OR, U.S.A.) in the absence of cells and as described by Grynkiewicz et al. (24). The dissociation constant for the fura-2-Ca2+ complex was taken as 224 nM. The values for Rmax and Rmin were calculated from measurements using 25 μM digitonin and 4 mM EGTA, raising the pH to 8.3.
The first series of experiments was performed to have a positive control, and bradykinin (10 nM to 10 μM) was used to characterize the increases in the intracellular calcium concentration [Ca2+]i as a result of its membrane receptor stimulation. Additional series of experiments were done to evaluate the short-term effects of 17β-estradiol (E2; 0.1 nM to 10 μM) on [Ca2+]i in the endothelial cell cultures. In separate experiments, we explored the participation of the intracellular steroid receptor on the effects induced by E2, using tamoxifen (1 μM), ICI 182,780 (10 μM), and E2 covalently bound to albumin (E2-CMO-BSA).
To determine the participation of Ca2+ mobilization from intracellular stores in the E2-induced effects, two types of experiments were performed: incubation of cells with pertussis toxin (PTX; 1 μg/ml) and incubation of with U-73122 (2 μM), a phospholipase Cβ inhibitor.
We used a "water soluble" estrogen (cyclodextrin-encapsulated 17β-estradiol). Cyclodextrin was without effect on [Ca2+]i.
Tamoxifen and ICI 182,780 were dissolved in ethanol and in DMSO, respectively; the final concentration of ethanol and DMSO never exceeded 0.01%; these ethanol and DMSO concentrations were without effect on [Ca2+]i.
To eliminate the possibility that there was no free steroid or steroid-CMO in the steroid-CMO-BSA preparation, this complex was treated with charcoal to remove the noncovalently bound steroid or the steroid-CMO.
The data on the changes in [Ca2+]i (maximal concentration reached by spike-like component of curve) were analyzed by one-way analysis of variance (ANOVA). The individual contrasts between treatments were made by Dunnett's method. N represents number of experiments. Each experiment was made in ∼15 cells. Cells were randomly chosen; cell numbers were limited only by the view field of microscope objective and were representative of 6 × 103 cells per preparation. Statistically significant differences were considered at p < 0.05.
Bradykinin induced a concentration-dependent increase in [Ca2+]i (not shown). Figure 1 shows a representative tracing of the bradykinin (10 μM) effects in [Ca2+]i. This response has two components: the first one, a spike-like component due mainly to Ca2+ release from intracellular stores, mediated by IP3, and a second one that represents calcium entry to the cells (25). We considered this response a positive control.
Direct effects of 17β-estradiol on intracellular calcium
The addition of E2 to the cultures (n = 15, in each concentration assayed) resulted in an increase in [Ca2+]i. Figure 1 shows a representative tracing of the increase in [Ca2+]i induced by E2 (10 nM); the response was similar to that induced by bradykinin (10 μM). The initial spike-like response is similar (Table 1), but the second phase of the E2-induced response (a plateau) was different from the (relatively slow recovery) induced by bradykinin. Figure 2 shows the biphasic, bell-shaped concentration-response curve of the spike-like increase curve elicited by estradiol; the maximal response was obtained with 10 nM. Cyclodextrin by itself did not affect [Ca2+]i.
Participation of the intracellular receptor in the effects elicited by 17β-estradiol
The incubation of cells for 30 min with the antiestrogen tamoxifen (1 μM) did not modify the estradiol-induced increases in [Ca2+]i(Fig. 2). However, the incubation of cells for 1 h with the pure antiestrogen ICI 182,780 (10 μM), a 7α-substituted derivate of E2, blocked the estradiol-induced increase in [Ca2+]i(Fig. 2); by themselves, tamoxifen and ICI 182,780 had no effect on [Ca2+]i.
Conversely, blocking the free diffusion of estradiol into the cells by covalently binding the steroid to a macromolecule such as BSA explored the possibility that estradiol effects were elicited at the plasmalemma level. Our results show that estradiol-CMO-BSA induced effects similar to the free hormone, with concentration-dependent effects of the peak increase that were bell shaped with maximal activity at 10 nM. The only difference was that the maximal effect induced by estradiol was not reached by estradiol-CMO-BSA (Fig. 2). CMO-BSA did not induce changes in intracellular calcium (data not shown).
Effect of "calcium free" solutions on estradiol-induced increases in [Ca2+]i
In this set of experiments, we changed the loading buffer 10 min before the experiments, using a Krebs buffer with no calcium (Krebs improved solution, Ringer II). The concentration of estrogen giving a maximal [Ca2+]i response was used (10 nM; n = 15). Figure 3 shows the estradiol-induced increases in [Ca2+]i in the absence of extracellular calcium. The initial increase in the intracellular calcium concentration was not modified, and only the second component was abolished (Table 1).
Effects of pertussis toxin on [Ca2+]i response to estradiol
Cells were preincubated for 10 min with 1 μg/ml of PTX, and the concentration of estrogen giving a maximal [Ca2+]i response was used (10 nM; n = 15). PTX by itself did not modify the [Ca2+]i but induced a blockage of the estradiol-induced increase in [Ca2+]i(Table 1). Figure 4 shows a representative experiment.
Effects of U-73122, a phospholipase C inhibitor, on [Ca2+]i response to estradiol
Cells were pretreated with U-73122 (5 min; 2 μM), and the concentration of estrogen giving a maximal [Ca2+]i response was assayed (10 nM; n = 15). Estradiol-induced increases in [Ca2+]i were blocked (Table 1); this effect was not the result of intracellular calcium stores depletion because addition of caffeine (20 mM) induced a short-term increase in [Ca2+]i(Fig. 5).
Classic concepts of the mechanism of steroid action at the nuclear level do not seem to be sufficient to account for all the known cellular effects of steroid hormones, such as their rapid effects, which are independent of hormone interaction with the cell nucleus. In this work, we showed that rat aortic endothelial cells in culture are influenced by 17β-estradiol through mechanisms that are of rapid onset (milliseconds) and may be of nongenomic origin.
Bradykinin, an agonist of endothelial cell activity, which in this work was used as a positive control, induced short-term increases in [Ca2+]i by acting on membrane receptors and triggering intracellular mechanisms. This led to the activation of phosphoinositide-phospholipase C enzymatic pathway and that where the coupling between receptors and phospholipase C is mediated by G proteins (which at least in part are PTX sensitive) (9).
In regard to estradiol-induced effects, our results show very rapid (milliseconds) effects on cytosolic free calcium and phospholipid metabolism in the vascular endothelial cells in culture. The E2-induced spike-like increase in [Ca2+]i is similar to that induced by bradykinin and signals the possibility that the phospholipase C metabolic pathway could be activated by the steroid, a process that might be modulated by G proteins. However, the analysis of the E2-induced second-phase kinetics is different from that of bradykinin, which is due to the influx of extracellular calcium (25). We have no explanation for these phenomena. These effects were produced by nearly physiologic concentrations (0.1 nM); they were dose dependent in a bell-shaped manner, with a maximal activity at 10 nM.
The initial biphasic spike-like phenomena induced by 17β-estradiol persisted even in the absence of extracellular calcium, but the sustained plateau phase was abolished with solutions lacking this cation. These results agree with the results of Lantin-Hermoso (33). Conversely, U-73122, a phospholipase C inhibitor, totally blocked the initial increase in [Ca2+]i and significantly diminished the second or plateau phase. This demonstrates that E2 mainly acts by mobilizing Ca2+ from the endoplasmic reticulum. It has been reported that in these cells, the calcium discharge from intracellular pools seems to occur through the inositol 1,4,5-trisphosphate-sensitive Ca2+ release channels.
Our data are in agreement with reports showing modulation of calcium influx by 17β-estradiol in several cell types such as in rat osteoblast (9), Xenopus oocytes (16), uterine cells (8), and rat hepatocytes (26). However, in chicken and pig granulose cells, the increase in [Ca2+]i induced by estrogens is due only to calcium mobilization from intracellular stores (27), and in vascular smooth cells, E2 induces an inhibition of Ca2+ entry through L-type calcium ion channels (21,28). The differences in estrogen effects on intracellular calcium may be linked to tissue specificity and to cell specificity when cells belong to the same tissue. Some reports show the possibility that the heat-shock protein 90 system (29) and the activation of tyrosine kinases (19,20) are involved in the origin of the nongenomic stimulation of NO synthesis by estrogen. This supports the idea of interaction between those pathways and estrogens at the membrane level (30,31).
Our results show that in rat vascular endothelial cells in culture, the estrogen effects were fast and transient and were not blocked by tamoxifen, which binds to the conventional estrogen receptor and blocks genomic effects of estrogen; it agrees with the results of Moini et al. (22). However, our results showed that ICI 182,780, a pure antiestrogen, blocked the increase in [Ca2+]i, suggesting that E2-receptor α can be involved in this responses. This could be explained because the ICI 182,780 is a 7α-substituted derivate of E2, which has the same spatial arrangement in relation to the plane of the E2 nucleus; however, more work is necessary to clarify this issue.
Our data also show that estradiol immobilized by covalent linkage to BSA also increased [Ca2+]i. However, under these conditions, the maximal effect elicited by free estradiol was not reached; we do not have a feasible explanation for this result. It is clear that the bell-shaped concentration-effect curve is similar to the one obtained with the free steroid and presents the same time courses. The response was obtained with physiologic concentrations of estradiol, and it is maximal at 10 nM. We believe that the increase in [Ca2+]i induced by estradiol-BSA is not due to estradiol released from the macromolecular complex during the incubation, because the time courses of free estradiol and estradiol-BSA are identical and because we were unable to detect any free estradiol in the incubation medium after the experiment was finished (organic solvent extraction, spectrophotometric, HPLC, and RIA methods).
Recently Chen et al. (32) reported a brief (5 min) E2-mediated activation of endothelial NO synthase through stimuli of the estrogen receptor α, involving the mitogen-activated protein (MAP) kinase pathway (30), but they did not established the location of the estrogen receptor. Our results differ from theirs on latency (millisecond vs. 5 min). However, we cannot rule out any MAPk involvement in our results. The rapid onset of estradiol effects, the nonblockage by tamoxifen, but blockage by the ICI 182,780, and the capacity of E2-BSA to elicit increases in [Ca2+]i suggest that the estradiol receptor responsible for mediating intracellular calcium increases resides on the outer membrane of vascular endothelial cells. Our results agree with the results of Moini et al. (22), who showed that 17β-estradiol increases intracellular calcium concentration in HUVECs, and disagree with Caulin-Glaser et al. (34) who, using HUVECs, showed that E2 did not promote Ca2+ fluxes. We have no explanation for this difference.
Estradiol induced an increase in [Ca2+]i similar to that of bradykinin. This effect may be mediated by an increase in [IP3], which in turn is mediated through the activity of phospholipase C, leading one to speculate about the necessity of an aromatic A ring. However, it is very probable that this is not the only structural requirement for steroid action, because U-73122 inhibits the IP3-phospholipase C-induced increases in [Ca2+]i elicited by the addition of estradiol. U-73122 is an aminosteroid similar to estradiol, with an aromatic A ring and a structural modification on carbon 17 of the steroid backbone (amino hexyl-pyrrole-2,5 dione), suggesting the existence of a modulatory site for steroids in the phospholipase C molecule, where steroids (maybe through the induction of conformational changes), can block or induce the phospholipase C activity.
Conversely, the results obtained in presence of PTX suggest that the toxin uncouples the nongenomic receptor from its G protein by blocking the signal transduction that activates the phospholipase C.
All the data in this work suggest a direct interaction of steroid molecules with specific membrane steroid moieties: the rapid responses induced by estradiol in [Ca2+]i, the nonparticipation of intracellular receptors, effects obtained even with restricted free diffusion of the steroid into cells, all suggest that the putative membrane receptor may be different from the classic intracellular receptor (at least in its linkage to intracellular effectors), and that it belongs to a class of membrane receptors linked to the intracellular effector that is coupled to phospholipase C by a PTX-sensitive G protein. Another possibility is that phospholipase C, an enzyme of membrane localization, by itself is the membrane-associated acceptor of steroids. However, more work is necessary to clarify these phenomena.
Our results can be used to integrate the effects of estradiol-modulating vasodilatation processes. In the recent past, it has been proposed that estradiol induces vasodilatation, acting as a calcium channel blocker (L-type calcium channel) at the vascular smooth muscle. However, if this were the mechanism through which estradiol was acting at the endothelium level, the synthesis of nitric oxide and other vasodilators would be blocked, disrupting vasodilatation. It must be keep in mind that vascular endothelial cells are the first cell type to be in contact with the hormones circulating in the blood. Perhaps the increase in intracellular calcium, leading to the synthesis of vasodilators in this type of cells, precedes the effects at the vascular smooth muscle. These processes together can explain the vasodilator effects of estradiol in a more integrated manner.
In conclusion, our results show that in rat vascular endothelial cells in culture, 17β-estradiol behaves as an agonist on endothelial activity and increases [Ca2+]i. The steroid effects due to their rapid onset (milliseconds) are of nongenomic origin and are closely related to activation of membrane-associated phospholipase C activity.
Acknowledgment: This work was supported by the Instituto Politecnico Nacional and CONACyT 31423-M, Mexico.
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