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VASCULAR BIOLOGY

Effects of estradiol on phenylephrine contractility associated with intracellular calcium release in rat aorta

Sección de Estudios de Posgrado e Investigación de la Escuela Superior de Medicina, Instituto Politécnico Nacional, Mexico Distrito Federal, Mexico

Submitted 31 October 2005 ; accepted in final form 21 July 2006

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The ability of estradiol to affect phenylephrine-induced contraction and the subsequent increase in resting tone, associated with capacitative Ca²⁺ entry across the plasma membrane, was evaluated in rat aortic rings incubated in Ca²⁺-free solution. The incubation with estradiol (1–100 nM, 5 min) inhibited both the phenylephrine-induced contraction and the IRT. Neither cycloheximide (1 µM; inhibitor of protein synthesis) nor tamoxifen (1 µM; blocker of estrogenic receptors) modified the effects of estradiol. Estradiol (100 µM) also blocked the contractile response to serotonin (10 µM) but not to caffeine (10 mM). In addition, estradiol (100 µM) inhibited the contractile responses to cyclopiazonic acid (1 µM; selective Ca²⁺-ATPase inhibitor) associated with capacitative Ca²⁺ influx through non-L-type Ca²⁺ channels. Finally, estradiol inhibited the Ca²⁺-induced increases in intracellular free Ca²⁺ (after pretreatment with phenylephrine) in cultured rat aorta smooth muscle cells incubated in Ca²⁺-free solution. In conclusion, estradiol interfered in a concentration-dependent manner with Ca²⁺-dependent contractile effects mediated by the stimuli of ₁-adrenergic and serotonergic receptors and inhibited the capacitative Ca²⁺ influx through both L-type and non-L-type Ca²⁺ channels. Such effects are in essence nongenomic and not mediated by the intracellular estrogenic receptor.

estrogen; ₁-adrenergic agonists

On the other hand, sex hormones are among the endogenous compounds with vasodilator properties. The female sex hormones estradiol and progesterone, as well as the androgenic hormone testosterone, have shown vasodilator activity (3, 8, 9, 17, 26). Some of the vascular effects of estrogen are of nongenomic origin and are mediated by estrogen receptors localized into caveolae in endothelial cells (24). These receptors activate the endothelial nitric oxide synthase and enhance nitric oxide availability (1). Moreover, the vasodilator activity of estradiol seems to be at least partially explained by hormone-induced inhibition of voltage-dependent Ca²⁺ channels in vascular smooth muscle cells (12, 27). Thus there is evidence about the ability of estradiol to interfere with the entry of extracellular Ca²⁺, but there is not enough information related to the capacity of this hormone to affect contractile processes dependent on the release of Ca²⁺ from intracellular stores.

The ability of estradiol to induce acute vasodilator effects has been demonstrated using several approaches, including arterial preparations precontracted with different agents, including ₁-adrenergic agonists, but there is not enough information about the ability of this hormone to interfere, specifically, with the mechanism of action of each of the contractile agents. As previously mentioned, there is particularly scarce information about the ability of estradiol to interfere with the contractile processes associated with the activity of ₁-adrenergic agonists on intracellular Ca²⁺ stores.

With the aim to provide additional information about this matter and using endothelium-denuded rat aortic rings incubated in Ca²⁺-free solution, we evaluated the effects of estradiol on both the contractile effect of phenylephrine and the subsequent increase in resting tone (IRT) associated with capacitative Ca²⁺ entry across the plasma membrane. In addition, we also evaluated the effects of estradiol on the increases in intracellular Ca²⁺ concentration elicited by phenylephrine (1 µM) as well as by the addition of Ca²⁺ (after pretreatment with phenylephrine) in cultured rat aorta smooth muscle cells incubated in the absence of Ca²⁺.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
All procedures were approved by institutional research and ethics committees. We used male Wistar rats (250–300 g) maintained at a 12:12-h light-dark cycle in a special room at constant temperature (22 ± 2°C) with food and water freely available. The rational of using male rats was to exclude the effects of changes in sex hormones during the estrous cycle in female rats.

Preparation of aortic rings. The animals were anesthetized with pentobarbital sodium (63 mg/kg ip), and the thoracic aortas were immediately excised, placed in cold buffer, cleaned, and freed from surrounding connective tissue. The isolated arteries were cut into rings (4–5 mm long) and placed in 10-ml tissue chambers filled with Krebs-bicarbonate solution of the following composition (in mM): 118 NaCl, 4.7 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄·7H₂O, 2.5 CaCl₂·2H2O, 25 NaHCO₃, 11.7 dextrose, and 0.026 calcium disodium-EDTA. In the Ca²⁺-free solution, CaCl₂ was omitted and EGTA (2 mM) was added. Tissue baths were maintained at 37°C, pH 7.4, and bubbled with a mixture of 95% O₂ and 5% CO₂. Experiments with caffeine were performed at 25 and 37°C, because it has been reported that the contractile effect of this xanthine is more evident at 25°C (16). Rings were mounted on two stainless steel hooks to fix them to the bottom of the chamber and to a Grass FTO3 force displacement transducer connected with a 7D Grass polygraph (Grass Instruments, Quincy, MA) to record the isometric tension developed by aortic rings. The rings were given 2 g of initial tension and allowed to equilibrate for 2 h. Thirty minutes after the organ bath was set up, tissues were first contracted with phenylephrine (1 µM) to test their contractile responses, and then they were rinsed three times with Krebs solution to restore tension to the precontracted level. Optimal tension was selected from preliminary experiments in which the rings were stretched so that the greatest response to phenylephrine (1 µM) could be obtained. We used endothelium-denuded aortic rings to evaluate of the direct effects of drugs on the vascular smooth muscle. Endothelial integrity was pharmacologically assessed using acetylcholine-induced vasodilation (1 µM); segments showing no relaxation were considered to be endothelium denuded.

Experimental protocol. After the equilibration period, a submaximal concentration of phenylephrine (1 µM) was administered to contract aortic rings that were incubated in Ca²⁺-free solution during 30 min; then the arterial preparations were washed, and the administration of phenylephrine was repeated two more times, at the end of which Ca²⁺ (2.5 mM) was added to the medium. The repeated administration of phenylephrine leads to depletion of the Ca²⁺ intracellular pools, and this phenomenon, in turn, enables the capacitative entry of Ca²⁺ from the extracellular space when this ion is added to the incubation medium, leading to an IRT (16, 18, 19). Once the IRT due to the addition of Ca²⁺ was observed, the rings were washed with Ca²⁺-containing solution, remaining in it for an hour (reequilibration period) before further experimentation. In some experiments, serotonin (10 µM) or caffeine (10 mM) was employed as a contractile agent, following the same protocol as with phenylephrine.

Effects of estradiol, progesterone, and testosterone on the contractile response to phenylephrine in aortic rings incubated in Ca²⁺-free solution. After the reequilibration period, the solution in the incubation medium was replaced by Ca²⁺-free solution, the rings were incubated in this solution for 30 min, and then estradiol (1–100 nM), progesterone, or testosterone (10–100 µM) was added 5 min before the administration of either phenylephrine or Ca²⁺. In other experiments, diltiazem (10 µM) (26) was administered to the incubation medium 15 min before the addition of Ca²⁺, to assess whether the entry of extracellular Ca²⁺ related to intracellular Ca²⁺ store depletion occurs through the L-type calcium channel.

Mechanisms of estradiol effects. To assess the participation of the estrogenic receptors in the inhibitory effects of estradiol, we followed the protocol as described previously except that the aorta rings were pretreated with tamoxifen (estrogenic antagonist; 1 µM) (22) or ICI 182,780 (specific pure antiestrogen, 1 µM) (24) 30 min before estradiol. To evaluate the genomic nature of estradiol effects, in addition to the short incubation with estradiol (2 and 5 min) the aortic rings were pretreated with cycloheximide (inhibitor of protein synthesis; 1 µM) 60 min (45 min in the presence of Ca²⁺ and 15 min in its absence) (5) before estradiol was added.

To assess estradiol direct effects on the sarcoplasmic reticulum, we added caffeine (an intracellular Ca²⁺ releaser; 10 mM) (1, 20) instead of phenylephrine, to contract aortic rings incubated in Ca²⁺-free solution. It has been shown that cyclopiazonic acid induces capacitative Ca²⁺ entry through non-L-type Ca²⁺ channels in rat aorta (16); hence, the ability of estradiol to interfere with such Ca²⁺ entry was assessed by studying the effects of the hormone (pretreatment for 5 min) on the contractile response to cyclopiazonic acid (10 µM) of aortic rings incubated in Ca²⁺-containing solution. To evaluate whether estradiol activates Ca²⁺-dependent K⁺ channels in vascular smooth muscle, we incubated the rings with tetraethylammonium (10 mM) (13) 15 min before estradiol.

In all the experiments, each ring served as its own control. One preparation was run in parallel with the experimental tissues, receiving no treatment (except the contractile agent) and was used to determine any time-dependent changes in agonist sensitivity. When any special solvent was used, a control was run in parallel to determine the effect of the solvent on a given tissue. The vehicles had no effect on either the basal resting tension or the contractile responses to agonists. Data are presented as means ± SE of six experiments on eight aortic rings from different animals. Each aortic ring was its own control and also was compared with parallel rings.

Cell culture. Vascular smooth muscle cells were isolated from aorta by enzymatic digestion as previously described (2). In brief, aortas were incubated for 10 min in a dissecting solution containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 0.1% bovine serum albumin, penicillin (100 U/ml), streptomycin (100 mg/ml), and collagenase (300 U/ml). The dispersed smooth muscle cells were plated in 90-mm petri dishes containing DMEM supplemented with 20% fetal calf serum (FCS) plus penicillin (100 U/ml) and streptomycin (100 µg /ml). The cells were allowed to grow in a humidified incubator (37°C) under a 5% CO₂ atmosphere until confluence. Vascular smooth muscle cells were identified as cell expressing -actin immunoreactivity; more than 99% of cells expressed -actin.

Experimental set up. Cells were trypsinized from a stock culture and resuspended in culture medium for a final concentration of 1 x 10⁴ cells/ml. A droplet of 25 µl was placed onto coverslip dishes no. 1 (glued to a perforated plastic petri dish). After the cells began to attach, additional medium was added. At 48–72 h before the experiment, the cells were serum deprived, and the incubation medium was replaced with culture medium containing Pen-Strep and 0.2% FCS.

Ca²⁺ measurement and experimental protocol. The cells were washed three times with HEPES-buffered Hanks' 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, 2.5 mM CaCl₂, 2 mM Na-pyruvate, and 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 Ca²⁺-free solution had the same composition except that CaCl₂ was omitted and EGTA (2 mM) was added. The cells were washed three times with buffer and post-incubated with the same buffer for an additional hour. The experimental chambers were placed on an inverted microscope (dual-wavelength fluorescence imaging system InCytIm2; Intracellular Imaging, Cincinnati, OH).

After the solution in the incubation medium was replaced by the Ca²⁺-free solution, a submaximal concentration of phenylephrine (1 µM) was administered to release Ca²⁺ from intracellular stores of the cultured aortic cells. The cells were then washed, and the administration of phenylephrine and washing was repeated two more times, at the end of which Ca²⁺ (2.5 mM) was added to the medium. The repeated administration of phenylephrine leads to the depletion of the intracellular pools of Ca²⁺, and this phenomenon in turn enables the entry of Ca²⁺ from the extracellular space when this ion is added to the incubation medium, causing an increase in the intracellular free-Ca²⁺ concentration that corresponds to the IRT observed in aortic rings (16, 18, 19). We evaluated changes in the intracellular Ca²⁺ concentration associated with either the first administration of phenylephrine or the addition of Ca²⁺. In some cases, before the administration of either phenylephrine or Ca²⁺, cultured aortic cells were pretreated for 5 min with either estradiol (100 nM), testosterone (100 µM) or progesterone (100 µM) to evaluate their ability to interfere with the release of intracellular Ca²⁺ elicited by the application of phenylephrine or the entry of extracellular Ca²⁺ dependent on the depletion of the intracellular Ca²⁺ stores. The fura-2 fluorescence response to the intracellular Ca²⁺ concentration was calibrated as previously described (20).

Drugs. All drugs and tissue culture media were purchased from Sigma Chemical (St. Louis, MO) except for cycloheximide (ICN, Costamesa, CA), ICI 182,780 (Ellisville, MO), and pentobarbitone sodium (Anestesal, SmithKline Beecham). Estradiol, testosterone, progesterone, and cycloheximide were dissolved in ethanol (1%). Cyclopiazonic acid was dissolved in DMSO (0.1%). The rest of the drugs were prepared and diluted in distilled water. All the subsequent drug dilutions were made in assay buffer and are expressed as the final molar concentration in the organ chamber.

Statistical analysis. Data are presented as means ± SE. In all experiments, n equals the number of rats from which vessel segments were obtained in six experiments on eight aortic rings from different animals. Each aortic ring was its own control and also was compared with parallel rings.

Statistical comparisons were performed using Student's t-test for paired observations. When necessary, ANOVA was used to determine the statistical significance of differences in obtained data, followed by a post hoc test. In all cases, a P value <0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Phenylephrine (1 µM) and caffeine (25 or 37°C; 10 mM) elicited transient contractile effects on endothelium-denuded aortic rings incubated in Ca²⁺-free solution (maximal effects were 0.40 ± 0.04 and 0.36 ± 0.01, respectively). The contractile effect almost disappeared when each of the drugs was administered for the third time (drugs were washed out with Ca²⁺-free solution after each administration). The addition of Ca²⁺ (2.5 mM) led to an IRT in the case of phenylephrine (see typical trace in Fig. 1; maximal effects were 1.08 g ± 0.05), but no change in resting tone was observed when caffeine was added or in time control arterial preparations, which were also incubated in Ca²⁺-free solution but not exposed to contractile agents. It was noted that the Ca²⁺ compartment sensitive to caffeine was at least partially shared by phenylephrine and serotonin, because the previous administration of each of these drugs almost eliminated the contractile effect of the others (data not shown).

View larger version (7K): [in this window] [in a new window]   Fig. 1. Typical traces obtained in aortic rings incubated in Ca2+-free solution showing the contractile effect of phenylephrine (Phen, 1 µM) associated with the release of intracellular Ca2+ (left) and the consequent increase in resting tone (IRT) due to capacitative Ca2+ entry when this ion (2.5 mM) was added to the incubation medium (right).

View larger version (13K): [in this window] [in a new window]   Fig. 2. Inhibitory effect of estradiol (10–100 nM), testosterone (100 µM), and progesterone (100 µM) on the contractile response to phenylephrine (1 µM; control) of aortic rings incubated in Ca2+-free solution. Effects are expressed in grams, and each column represents the mean ± SE of 6 different experiments. P < 0.05 vs. control.

View larger version (9K): [in this window] [in a new window]   Fig. 3. Concentration-response curve to estradiol (1–100 nM) and testosterone (1–100 µM) in isolated aorta rings without endothelium and precontracted with phenylephrine (1 µM). Effects are expressed as percentages of the initial contraction. Each point represents the mean ± SE of 6 experiments.

Effects of sex hormones on IRT associated with the reincorporation of Ca²⁺ in the incubation medium. Pretreatment with each of the three sex hormones (5 min) reduced in a concentration-dependent manner the IRT observed in aortic rings previously depleted of Ca²⁺ stores when Ca²⁺ (2.5 mM) was added to the incubation medium. Once again, estradiol (1–100 nM) was the most potent, whereas testosterone and progesterone (each at 10–100 µM) were equipotent. For clarity, only the effect of the highest concentration of the three sexual hormones tested (100 nM in the case of estradiol and 100 µM for the other 2 drugs) is shown in Fig. 4. Similarly, pretreatment (5 min) with the voltage-dependent Ca²⁺ channel blocker diltiazem (10 µM) significantly inhibited the IRT induced by the addition of Ca²⁺ in the aortic rings (0.92 ± 0.14 g without and 0.14 ± 0.02 g with diltiazem, P = 0.02; not shown; n = 6 in each case).

View larger version (8K): [in this window] [in a new window]   Fig. 4. Inhibitory effect of estradiol (100 nM), testosterone (100 µM), and progesterone (100 µM) on the IRT induced by the addition of Ca2+ (2.5 mM; control) into the incubation medium after the intracellular Ca2+ stores had been depleted by phenylephrine. Effects are expressed in grams, and each column represents the mean ± SE of 6 different experiments. *P < 0.05 vs. control.

View larger version (8K): [in this window] [in a new window]   Fig. 5. Effects of estradiol (100 nM) on the contractile response to phenylephrine (1 µM; control) of aortic rings incubated in Ca2+-free solution in the absence and presence of cycloheximide (1 µM; A) or tamoxifen (1 µM; B). Effects are expressed in grams, and each column represents the mean ± SE of 6 different experiments. P < 0.05, control vs. estradiol.

View larger version (10K): [in this window] [in a new window]   Fig. 6. Effect of estradiol (1 nM) on the contractile response to caffeine (10 mM) of aortic rings incubated in Ca2+-free solution at 25 or 37°C. Effects are expressed in grams, and each column represents the mean ± SE of 6 different experiments. P > 0.05.

Measurement of intracellular free Ca²⁺. Using the fura-2 Ca²⁺ measurement technique and in the absence of extracellular Ca²⁺, the effect of either phenylephrine (1 µM) or the addition of Ca²⁺ (after pretreatment with phenylephrine to induce capacitative Ca²⁺ entry) in the incubation medium on total intracellular free Ca²⁺ in cultured vascular smooth muscle cells is shown in Fig. 7. As shown, phenylephrine elicited a transient increment in the intracellular Ca²⁺ concentration (Fig. 7A). On the other hand, the addition of Ca²⁺ (2.5 mM, after pretreatment with phenylephrine) to the incubation medium also induced an increment in the intracellular Ca²⁺ concentration with a longer lasting plateau (Fig. 7B). As shown in Fig. 7, C and D, estradiol (100 nM) administered 5 min before phenylephrine or Ca²⁺ inhibited, in both cases, the increase in the intracellular Ca²⁺ concentration. Testosterone and progesterone also inhibited the increase in the intracellular Ca²⁺ concentration associated with either the administration of phenylephrine or the addition of Ca²⁺, but higher concentrations of these hormones were needed (100 µM in both cases).

View larger version (28K): [in this window] [in a new window]   Fig. 7. Representative tracings of effects of phenylephrine (1 µM; A) or Ca2+ (2.5 mM; B) after pretreatment with phenylephrine on intracellular Ca2+ concentration in cultured aortic smooth muscle cells incubated in Ca2+-free solution in the absence or presence (C and D) of estradiol (100 nM). Effects are expressed as the ratio 340/380 of fluorescence excitation as an indirect measure of intracellular Ca2+ concentration changes. E: mean values of estradiol (E2, 100 nM), testosterone (T, 100 µM), and progesterone (P, 100 µM) effects on phenylephrine (PE)- and CaCl2-induced increases in intracellular Ca2+ concentration. Each bar represents 5 experiments on 10,000 cells. *P < 0.05, phenylephrine vs. phenylephrine + each hormone. **P < 0.05, CaCl2 vs. CaCl2 + each hormone.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Interestingly, the inhibitory effects of the sex hormones were observed after only 2 min of exposure. Therefore, they seem to be of nongenomic origin. Moreover, we observed that an inhibitor of protein synthesis, cycloheximide, did not modify the inhibition of the transient contractile response elicited by estradiol.

On the other hand, such inhibitory effects of estradiol are not mediated by the intracellular estrogenic receptor, because they are not modified by tamoxifen, a selective antagonist of this receptor (10), or ICI 182,780 (specific pure antiestrogen) (24). However, the fact that the effects are observed with relatively low concentrations of estradiol (1–100 nM) suggests that they are a consequence of the interaction of this steroid with a specific receptor, perhaps an estrogenic receptor insensitive to tamoxifen or ICI 182,780 but with high affinity for estradiol. The estradiol-induced effects, particularly those found with 100 nM, may not have clinical relevance, because they are to high compared with the physiological serum concentration; however, at the microenvironmental level, higher concentrations of estradiol may be reached. More work is necessary to clarify this issue.

The activation of ₁-adrenoceptors causes phospholipase C-mediated hydrolysis of the membrane phospholipid, phosphatidylinositol 4,5-bisphosphate, yielding two second messengers, inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (6, 21, 23). IP₃ binds to receptors located on the sarcoplasmic reticulum and releases stored Ca²⁺ (11, 23), which is responsible for the transient contractile response to phenylephrine that was observed in rat aortic rings incubated in Ca²⁺-free solution. Therefore, the inhibition of this contractile effect elicited by the sex hormones may be a consequence of interference at some point of the ₁-adrenoceptor signal transduction pathway. However, at least in the case of estradiol, it is unlikely that a direct action of the sex hormone on the sarcoplasmic reticulum could explain its inhibitory effect, because it did not modify the contractile effect of caffeine on aortic rings incubated in Ca²⁺-free solution [the caffeine transient contractile response is attributed to the release of stored Ca²⁺ from a compartment shared by phenylephrine in the sarcoplasmic reticulum (7, 16)]. Therefore, the inhibition of estradiol on the intracellular Ca²⁺ store-dependent contractile effect of phenylephrine may be related to an effect on the ₁-adrenoceptor signal transduction pathway produced at a different level from the sarcoplasmic reticulum, probably at the plasma membrane, and might be related to PLC activity.

As mentioned above, estradiol also inhibited the IRT induced by Ca²⁺ addition to the medium after the intracellular Ca²⁺ stores had been depleted with phenylephrine. It has been established that the depletion of intracellular Ca²⁺ stores produced by the administration of ₁-adrenergic agonists leads to the opening of Ca²⁺ channels at the cellular membrane, which permits the influx of extracellular Ca²⁺ and, consequently, the IRT (16). Hence, the internal Ca²⁺ stores have a crucial role in the contractile response of vascular smooth muscle cells, because the depleted Ca²⁺ stores are the signal for the so-called capacitative or store-operated entry of extracellular Ca²⁺ (18, 19). In agreement with Noguera et al. (14, 15), and because the effect was blocked by diltiazem (10 µM), the present results support that the IRT produced by the addition of Ca²⁺ under depletion of intracellular Ca²⁺ stores is, at least partially, due to capacitative Ca²⁺ influx through voltage-dependent Ca²⁺ channels. Since estradiol also inhibited such a process, it may have blocker properties on voltage-dependent Ca²⁺ channels. This is not a novel proposal, because the rapid vasorelaxing effects of estrogens have been attributed to the inhibition of Ca²⁺ influx through L-type Ca²⁺ channels in the vascular smooth muscle cell (8, 27). Moreover, it has been demonstrated that activation of Ca²⁺-dependent K⁺ channels account for endothelium-independent vascular relaxation in porcine coronary arteries (25). Indeed, tetraethylammonium, a selective antagonist of Ca²⁺-dependent K⁺ channel (13), inhibited estradiol effects. Therefore, hyperpolarization of vascular smooth muscle cells and the subsequent closing of Ca²⁺ channels cannot be excluded in this study.

On the other hand, at higher concentration (100 µM) estradiol also inhibited the contractile effect elicited by cyclopiazonic acid on aortic rings incubated in Ca²⁺-containing solution. The contractile effect of this drug in rat aorta seems to be a consequence of the capacitative influx of extracellular Ca²⁺ through non-L-type Ca²⁺ channels stimulated by the prior depletion of intracellular Ca²⁺ stores (16). Estradiol, in addition to blocking L-type Ca²⁺ channels, may also block non-L-type Ca²⁺ channels or nonselective cation channels that are responsible for the contractile effect of cyclopiazonic acid. However, since a higher concentration of estradiol was required to block the non-L-type Ca²⁺ channels, a nonspecific effect is suggested.

On the other hand, direct evidence of the ability of estradiol to affect the intracellular Ca²⁺ increments was obtained by measuring the intracellular concentration of this ion, in cultured aortic smooth muscle cells incubated in Ca²⁺-free solution, using the fura-2 Ca²⁺ measurement technique. Estradiol inhibited both the transient increment in the intracellular Ca²⁺ concentration elicited by phenylephrine as well as the longer lasting increment in the intracellular concentration of Ca²⁺ induced by the addition of this ion to the incubation medium after the intracellular pools of Ca²⁺ had been depleted with phenylephrine.

Together, the results lead us to believe that the sex hormones, because of their ability to intercalate into the plasma membrane, could alter in a nonspecific way some molecules included in the vascular smooth muscle cell membrane. Those molecules include L-type and non-L-type Ca²⁺ channels as well as elements of the signal transduction pathway of G_q11-coupled receptors, including the hormone receptors themselves, G proteins, and phospholipase C. In agreement with this relatively nonspecific effect are the high concentrations of testosterone and progesterone (10–100 µM in both cases) needed to inhibit in general the intracellular Ca²⁺-dependent events evaluated in this work, as well as the high concentrations of estradiol (10–100 µM) needed to specifically inhibit the contractile effect of cyclopiazonic acid. However, a specific effect is suggested by the low concentrations of estradiol (1 nM) required to inhibit both the intracellular Ca²⁺-dependent contractile effect of phenylephrine and the increment in resting tone consequent to the depletion of the intracellular Ca²⁺ stores induced by this amine.

In conclusion, we have demonstrated that estradiol inhibits contractile effects associated with both the release of intracellular Ca²⁺ produced by stimulation of G_q11-coupled receptors and with the capacitative influx of Ca²⁺ through L-type and non-L-type Ca²⁺ channels.

	FOOTNOTES

 

Address for reprint requests and other correspondence: C. Castillo, Escuela Superior de Medicina del I.P.N., Plan de San Luis y Díaz Mirón, Col. Casco de Santo Tomás, México D.F., México C.P. 11340 (e-mail: drcarloscastillo{at}esmas.com)

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.

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