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Endocrinology Vol. 144, No. 10 4315-4324
Copyright © 2003 by The Endocrine Society

Short-Term Activation by Low 17ß-Estradiol Concentrations of the Na+/H+ Exchanger in Rat Aortic Smooth Muscle Cells: Physiopathological Implications

Sandra Incerpi, Silvia D’Arezzo, Maria Marino, Roberto Musanti, Valentina Pallottini, Andrea Pascolini and Anna Trentalance

Department of Biology (S.I., S.D., M.M., V.P., A.P., A.T.), University of Rome "Roma Tre," 00146 Rome, Italy; and Newron Pharmaceuticals (R.M.), 21040 Gerenzano, Varese, Italy

Address all correspondence and requests for reprints to: Professor Sandra Incerpi, Department of Biology, University of Rome ‘Roma Tre,’ Viale Marconi, 446, 00146 Roma, Italy. E-mail: incerpi{at}uniroma3.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Low physiological concentrations of 17ß-estradiol increased the intracellular pH of rat aortic smooth muscle cells by a rapid nongenomic mechanism. This effect was due to stimulation of the Na+/H+ exchanger activity, measured using the intracellular pH-sensitive fluorescent probe 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein. The 17ß-estradiol gave rise to a bell-shaped dose response, with a maximum at 10-12 M and no significant effect at 10-9 M. The specificity of the effect was verified by the use of the Na+/H+ exchanger inhibitor 5-(ethyl-N-isopropyl)amiloride and the lack of effect of the isomer 17{alpha}-estradiol. Inhibitors of the nuclear estrogen receptors, tamoxifen and ICI 182,780, completely prevented activation of the exchanger by 17ß-estradiol. The effect of low estrogen concentrations on the intracellular pH was mimicked by both norepinephrine and phenylephrine, suggesting a connection between the increase of intracellular pH and the muscle contraction process. The transduction mechanism for this nongenomic effect of estrogens did not involve modulation of the cAMP content, whereas inositol 1,4,5-trisphosphate, protein kinase C and MAPK pathways appear to play a role, as indicated by both pharmacological approaches and immunoblot experiments on protein kinase C translocation and ERK phosphorylation. These results for the first time provide evidence for a nongenomic effect of low physiological concentrations of 17ß-estradiol on intracellular pH that, together with other factors, may contribute to the development of hypertension and atherosclerosis in men and postmenopausal women and increase the risk of cardiovascular disease. Paradoxically, the lack of stimulation at high physiological estradiol levels could explain the protective effects found in premenopausal women.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGENS MODULATE A WIDE variety of cellular effects, acting both at the intracellular level and on the plasma membrane. In the classical model, estrogens bind to the nuclear estrogen receptors (ERs){alpha} and -ß whose molecular characteristics have been well established, acting as ligand-dependent transcription factors to regulate gene expression and protein synthesis (1). The membrane actions of estrogens are believed to be triggered after hormone binding to plasma membrane receptors, giving rise to nongenomic effects characterized by a rapid onset of action, within seconds to minutes (2, 3), but the nature of the putative membrane ER is still debated. It is not yet clear whether this membrane receptor is similar or even identical with the nuclear ERs or whether it has a completely different structure (4, 5); the presence of a membrane receptor unrelated to both ER{alpha} and ERß has been postulated and defined as {gamma}-adrenergic receptor (6).

The estradiol-induced nongenomic responses observed seem to depend on the type of cell and tissue, and the signaling mechanisms involved appear to be similar to those evoked by the most common growth factors and hormones acting through membrane receptors: intracellular [Ca2+], protein kinase C (PKC), phospholipase C (PLC), cAMP, the MAPK pathway, and intracellular pH (7, 8, 9). At present it is completely unknown which genomic or nongenomic mechanisms are responsible for the vascular response to estrogen influence. It is generally accepted that estrogens have direct acute vasodilatory effects on vascular tone, brought about through both endothelium-independent (10) and endothelium-dependent mechanisms (11) and that this is part of the cardioprotective action of estrogens in premenopausal women (12). Some specific functions of vascular smooth muscle cells such as contraction, proliferation, production, and composition of the extracellular matrix have been shown to be affected by estrogens. A decreased contractile response of rat tail artery to norepinephrine, arginine-vasopressin and KCl (13) and isolated aorta to {alpha}-adrenergic stimulation has been observed after exposure to high concentrations of estradiol (14). The estradiol effect in the physiological to supraphysiological concentration range on smooth muscle contraction has been ascribed to different mechanisms: the blocking of Ca2+ channels and inhibition of Ca2+ influx, the activation of calcium- or voltage-activated K+-channels, and changes in cAMP and cGMP levels (15). On the other hand, the estrogen-induced endothelium-dependent relaxation was commonly associated with the stimulation of nitric oxide production from the vascular endothelium (16). Although many data are available on the high estradiol concentration effects, the lower concentrations corresponding to the postmenopausal or masculine levels have been less investigated. Epidemiological studies have shown significant differences between males and females in the onset of osteoporosis or cardiovascular disease. In the males the risk is constantly growing with the age, but premenopausal women show a lower occurrence attributed to the presence of estrogens; in postmenopausal women, when the estrogens level is low, the risk become comparable with that of men.

It could therefore be of high physiological relevance to know the mechanisms taking place when estrogens fall to postmenopausal levels, and the protective effect is lost in cells that are strongly affected by estrogens such as aortic smooth muscle cells. Taking into account that the physiological concentration range of estrogens can be very wide, going from pregnancy (10-9 to 10-8 M) to postmenopause (10-12 to 10-10 M), we will call the first range high physiological and the second range low physiological throughout this article (17).

A quite complex relationship has been reported in smooth muscle between intracellular pH and the contraction/relaxation process. Changes in intracellular pH have been found to produce marked effects on the contraction of smooth muscles. In general, muscle tension increases as intracellular pH increases, even though some smooth muscles can produce more force during an intracellular acidification as it happens in the pulmonary artery (18). The intracellular pH is regulated by the Na+/H+ antiport, a plasma membrane protein exchanging Na+ and H+ ions according to the concentration gradient; besides its housekeeping function of regulation of intracellular pH and cell volume, this protein is currently considered also to play a more regulatory role in the cell (19). In particular, an increase of intracellular pH because of activation of the Na+/H+ exchanger by different kinases can represent the first response of the cell to a wide range of physiological (hormones and growth factors) (20, 21, 22) and pathological signals (oxidative stress, ischemia, inflammatory cytokines) (23, 24, 25, 26, 27).

Thyroid hormones are best known for exerting their physiological action through intracellular receptors and genomic long-term mechanism, in analogy with estrogens, they can also give rise to short-term nongenomic effects with a time course of seconds to minutes. Among these effects is a fast increase in intracellular pH because of activation of the Na+/H+ antiport in L-6 myoblasts from rat skeletal muscle (21). Nongenomic thyroid hormone stimulation of the Na+/H+ exchanger and the amino acid transport, system A, was recently shown in chick embryo hepatocytes and was found to be mediated by a signal transduction pathway involving PKC, the MAPK pathway, and phosphatidylinositol-3 kinase (22). However, short-term effects on the Na+/H+ exchanger have also been reported for aldosterone by a rapid nongenomic mechanism in human mononuclear cells and vascular smooth muscle cells (2). Furthermore, 17ß-estradiol (10-8 M) is able to activate, by a mechanism not sensitive to cycloheximide, a regulatory factor for the Na+/H+ exchanger (NHE-RF), involved in the inhibition of the Na+/H+ exchanger by the protein kinase A (PKA)-cAMP-dependent mechanism in the renal tubule (28). These findings suggest that both estrogens and thyroid hormones may give rise to nongenomic effects through modulation of the exchanger activity. Taking into account the role played by intracellular pH in the contraction/relaxation process of smooth muscle, we have studied whether estrogens modulate the activity of the exchanger in rat aortic smooth muscle (RASM) cells. Our data show that 17ß-estradiol, in a low physiological concentration range (10-12 to 10-10 M) increases intracellular pH in RASM cells, by activation of the Na+/H+ exchanger, with a nongenomic mechanism involving both PKC and MAPK pathways. We discuss the potential physiopathological implications of the observed effects at these low hormone concentrations.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
RASM cells were prepared from explants of thoracic aorta of 15-wk-old male and female Wistar rats by the method of Ross (29). Cells were cultured in 25-cm2 plastic tissue flasks and grown in DMEM, supplemented with 10% fetal calf serum, 100 µg/ml streptomycin, and 100 U/ml penicillin, in an atmosphere of 5% CO2 at 37 C. RASM cells were harvested once a week by treatment with 0.25% trypsin and refed every 2 d. For the experiments cells were used at passages 3–10 as confluent monolayers after 6–8 d from seeding.

Cell growth was measured on cells seeded in 60 x 15-mm petri dishes 1.0–1.5 x 105 cells/well and grown in DMEM supplemented as reported above, in the presence and absence of different concentrations of hormones. The medium with or without hormones was changed every 24 h. Cells were harvested after mild trypsinization and counted in a Neubauer chamber. These studies were conducted in accordance with the directives of the European Community (86/609/EEC) on the care and use of laboratory animals.

Determination of intracellular pH
For the experiments of fluorescence assays, cells were grown in chamber slides (Lab-Tek, Nunc, Naperville, IL) and used at confluency. Before the experiment cells were rendered quiescent by serum deprivation for 5 h.

Intracellular pH was measured by the fluorescent intracellular pH indicator 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein tetra-acetoxymethyl ester (BCECF/AM). To rule out the contribution of HCO3-dependent transport mechanisms (30), all experiments were carried out in bicarbonate-free buffer with the following composition (mM): 135 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, 20 HEPES (pH 7.3). This buffer (henceforth called Na+ buffer) was used for the incubation with the fluorescent probe and the determination of intracellular pH unless otherwise stated; the cells incubated in this buffer were considered virtually depleted of bicarbonate.

Incubation with the fluorescent dye was carried out as follows: Cells were washed twice with Na+ buffer and were thus considered bicarbonate free. Cells were then incubated in Na+ buffer with the fluorescent dye (1 mg/ml in dimethyl sulfoxide) at the final concentration of 1 µg/106 cells, for 10 min at 37 C in the dark. Then the medium containing the dye was eliminated, and the cells were washed twice with the Na+ buffer.

Routinely at the end of each experiment, calibration of fluorescence vs. pH was carried out by an established method (31) using the K+-H+ ionophore nigericin (5 µM) added to cells suspended in a potassium solution having the same composition as Na+ buffer but with NaCl substituted by equimolar concentrations of KCl. Under these conditions, intracellular and extracellular pH are equilibrated (pHi = pHo). The extracellular pH was changed with 10-µl aliquots of 1 M 2-[N-morpholino] ethanesulfonic acid or 1 M Tris(hydroxymethyl)aminomethane (Tris) and determined with a glass electrode inserted directly into the cuvette. Intracellular fluorescence was determined and plotted vs. extracellular pH. The calibration curve was linear in the pH range 6.5–7.8 (not shown).

Fluorescence was measured under continuous magnetic stirring at a controlled temperature (37 C) in a LS-50B luminescence spectrometer (Perkin-Elmer, Norwalk, CT) equipped with a fast filter accessory for the dual-excitation single-emission ratio technique. Excitation wavelengths were set at 498 nm (pH-dependent component) and 450 nm (pH-independent component) with emission at 530 nm, using 5- and 10-nm slits, respectively, for the two light paths. This allowed measurements of intracellular pH that were not depending on cell concentration and dye loading (32).

Inositol polyphosphate production
Subconfluent cells were exposed for 24 h to 2-[3H]myoinositol (1 µCi/ml; specific activity, 20 Ci/mmol). The medium was discarded and, after three washes with PBS, substituted with serum-free fresh medium containing 17ß-estradiol (10-8 M) or vehicle (dimethyl sulfoxide/PBS 1/100 vol/vol). In some experiments ICI 182,780 (1 µM) was added 15 min before estradiol treatment.

At the end of the incubation cells were rinsed with PBS and scraped with 1 ml trichloroacetic acid (TCA) 10% containing 2 mM EDTA. The TCA-soluble fraction was washed with diethyl ether and analyzed by anionic exchange chromatography on Dowex 1x-8 resin, formate form. Free inositol, inositol monophosphate, inositol bisphosphate, inositol trisphosphate, and inositol tetrakisphosphate were eluted from small glass columns with water, 0.2, 0.4, 0.8, and 1.0 M ammonium formate in formic acid (0.1 M), respectively (33), and the radioactivity was measured by a liquid scintillation counter (Packard, Downers Grove, IL). Under these conditions, a [3H]inositol 1,4,5-trisphosphate (IP3) standard was completely eluted from columns with 15 ml 0.8 M ammonium formate in formic acid (0.1 M). The TCA-insoluble fraction was dissolved in 1 M NaOH, and the protein content was measured (34). IP3 and cAMP levels in the cells were determined by radioligand assay kits.

Western blot analysis
Subconfluent cells after hormone treatment were washed twice in ice-cold PBS and then scraped into ice-cold 10 mM Tris-HCl (pH 7.4) containing 1 mM CaCl2, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml leupeptin. After sonication, soluble and particulate fractions were obtained by centrifuging samples at 100,000 x g for 30 min. Total cell lysate were obtained as already described (9, 33). Proteins were solubilized in 0.125 M Tris-HCl (pH 6.8) containing 10% sodium dodecyl sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, and boiled for 2 min. Equal amounts (20 µg) of cytosol, membrane, and total proteins were separated by 7% SDS-PAGE (PKC-{alpha}) or 10% SDS-PAGE (ERK and ERK-P) at 40 mA for 1 h and then transferred to nitrocellulose filters. Filters were saturated with 1% BSA and probed at room temperature for 1 h with monoclonal anti-PKC-{alpha} or anti-ERK or anti-ERK-P antibodies (1 µg/ml). Bound antibodies were visualized using enhanced chemiluminescence detection (CDP-Star, NEN Life Science Products, Boston, MA). Protein content was quantified by densitometry analysis of Western blots (Fluor-S, Bio-Rad, Hercules, CA).

Solutions
BCECF/AM (1 mg/ml), 5-(N-ethyl-N-isopropyl)amiloride (EIPA; 10 mM), genistein, U 73122, H-7, and calphostin C were dissolved in dimethyl sulfoxide, which did not affect the fluorescence signal. Nigericin (10 mM) and 17ß-estradiol 3-benzoate [1,3,5 (10)-estratriene-3,17ß-diol 3-benzoate; 1 mM], 17{alpha}-estradiol [1,3,5 (10)-estratriene-3,17{alpha}-diol], tamoxifen, ICI 182,780, and neomycin were dissolved in ethanol. Norepinephrine and phenylephrine were aqueous solutions.

Materials
DMEM, antibiotics, and sterile plastic ware for cell culture were from Flow Laboratory (Irvine, UK). Fetal bovine serum was from Gibco (Grand Island, NY). BCECF/AM was obtained from Molecular Probes (Eugene, OR). [3H]-cAMP and [3H]IP3 assay kits were from Amersham (Freiburg, Germany). Nigericin, HEPES, 2-[N-morpholino]ethanesulfonic acid, Tris, 17{alpha}-estradiol, 17ß-estradiol, genistein, H-7, neomycin, norepinephrine, phenylephrine, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), and Dowex 1x-8 resin were from Sigma (St. Louis, MO). EIPA was obtained from Research Biochemicals International (Natick, MA). PD 98059, U 0126 were from Alexis Biochemicals (Laufelfingen, Switzerland). Tamoxifen and ICI 182,780 were from Tocris Cookson (Bristol, UK). U 73122 and RO 31–8220 were obtained from Calbiochem (La Jolla, CA). Calphostin C was from ICN Pharmaceuticals (Costa Mesa, CA). Monoclonal anti PKC-{alpha} or anti-ERK or anti-ERK-P antibodies were provided from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals were of the purest grade available from Merck (Darmstadt, Germany).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of 17ß-estradiol on cell proliferation
To verify the inhibitory effect of estrogens on cell growth, considered to be part of the cardioprotective actions of these hormones in premenopausal women, the dose response of 17ß-estradiol on RASM cell proliferation was determined over a wide concentration range (Fig. 1Go). The hormone showed antiproliferative properties after 72 h, corresponding to the most active period of exponential cell growth. The effect was significant only in the 10-10 to 10-8 M concentration range, indicating that the antiproliferative properties, which were previously observed with high physiological and pharmacological concentrations in the same cells (35, 36, 37), are not present at low physiological concentrations of the hormone.



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  		FIG. 1. Dose response of 17ß-estradiol on the proliferation of RASM cells. Results are reported as number of cells (%) with respect to control cells without hormone treatment and are the mean ± SD of at least four to six different experiments. The antiproliferative effect was significant in the physiological concentration range (10-10 to 10-8 M). *, P < 0.01 as from a t test with respect to control.

 
Effect of 17ß-estradiol on the intracellular pH at the steady state
In the standard bicarbonate-free Na+ buffer solution, the mean steady-state pHi of rat aortic smooth muscle cells in culture was 7.00 ± 0.04 (mean ± SD; n = 20), in agreement with earlier reported data (24).

Addition of very low concentrations of 17ß-estradiol, 10-12 to 10-10 M, resulted in a lasting increase in the intracellular pH because of the stimulation of the Na+/H+ exchanger. The maximum increase at equilibrium was 0.18 ± 0.04 {Delta} pH/40 min over the basal value (mean ± SD; n = 12) and was found at 10-12 M 17ß-estradiol, whereas lower effects were obtained at higher and lower hormone concentration, as reflected by the bell-shaped dose-response curve (Fig. 2Go, upper panel). The time course of the effect of 10-12 M estradiol showed that the intracellular pH started to increase immediately after hormone addition and the maximum was reached within about 30–40 min, a slightly longer time than that normally required for most hormones or growth factors to activate the Na+/H+ exchanger (Fig. 2Go, lower panel). The effect was specific because it was inhibited by 10 µM EIPA, a derivative of amiloride and a specific inhibitor of this transport system (Fig. 2Go, lower panel).



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  		FIG. 2. Effect of 17ß-estradiol on the steady-state intracellular pH in RASM cells. Upper panel, Dose response of hormone effect on intracellular pH at steady state. Results are given as {Delta}pHi/40 min over basal value and are the mean ± SD of at least 10 different experiments. *, P < 0.01 starting from 10-10 M, with respect to baseline pH, as from a t test. Lower panel, Time course of the effect of 17ß-estradiol (10-12 M), on the intracellular pH. The effect of the inhibitor EIPA (10 µM) is shown. EIPA by itself did not affect the fluorescent signal. The graphs are representative of at least 10 similar experiments. The arrow indicates the addition of hormone, with or without EIPA

 
The stereospecificity of the stimulation obtained with 17ß-estradiol was evaluated by the use of 17{alpha}-estradiol. This isomer was tested over a wide range of concentrations, but no significant effect was observed on the intracellular pH; the result obtained with one representative concentration (10-12 M) is reported in Fig. 3Go. The same figure shows that treatment with the most common inhibitors of the nuclear estrogen receptor, ICI 182,780 and tamoxifen, completely prevented the effect of 17ß-estradiol on the intracellular pH. Taken together these results confirm the presence of an estrogen receptor with similar binding site topology as the nuclear receptor types.



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  		FIG. 3. Effect of nuclear ER inhibitors and of the stereoisomer 17{alpha}-estradiol on the intracellular pH at the steady state. Tamoxifen (10-7 M) and ICI 182,780 (10-8 M) were given to the cells together with 17ß-estradiol (10-12 M), and none of them affected the fluorescent signal. The effect of the stereoisomer 17{alpha}-estradiol (10-12 M) alone is also reported. Results are shown as {Delta}pHi/40 min over basal value and are mean ± SD of at least 3–10 different experiments.

 
Norepinephrine and phenylephrine mimic the nongenomic activation of the Na+/H+ exchanger by 17ß-estradiol
It is known that changes of pH can alter the force development in smooth muscle affecting various aspects of muscle contraction such as the rate of myosin ATPase activity, the sensitivity of the contractile units to Ca2+ and many others (18). The sensitivity of myofilaments to Ca2+ is pH dependent, and in general tension falls when pH falls. Estrogens in the range 10-6–10-7 M give rise to vasodilation of smooth muscle by a rapid nongenomic mechanism involving membrane-based phenomena, such as alteration of membrane ionic permeability and activation of membrane-bound enzymes, even though the classical genomic effects must be also taken into account (38, 39). To find a correlation between the increase of intracellular pH by 17ß-estradiol and vascular tone, experiments were carried out with {alpha}-adrenergic agonists for smooth muscle.

Norepinephrine and phenylephrine increased intracellular pH by activating the Na+/H+ exchanger with a time-course similar to that showed by 17ß-estradiol, and again the effect was blocked by EIPA, indicating the specificity of these agonists (Fig. 4Go). The addition of an {alpha}-adrenergic agonist together with 17ß-estradiol did not give rise to any additive response or inhibition of the intracellular pH increase, suggesting the possibility of a common signal transduction pathway (not shown). All experiments involving the {alpha}-adrenergic agonists to be reported in the following were carried out with both norepinephrine and phenylephrine, but only the data obtained with phenylephrine will be shown for the sake of brevity.



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  		FIG. 4. Time course of the effect of norepinephrine and phenylephrine on the steady-state intracellular pH. Norepinephrine (upper panel; NE) and phenylephrine (lower panel; PHE) were tested at 10 µM. The effect of the inhibitor EIPA (10 µM) is also reported in the presence of each agonist. Results are representative of at least 6–10 similar experiments. The arrow indicates the addition of hormone/agonist, with or without EIPA.

 
Transduction pathway for the short-term effect of 17ß-estradiol and phenylephrine on intracellular pH
To identify the transduction pathway involved in the activation of the Na+/H+ exchanger by 17ß-estradiol in RASM cells, we assessed the production of second messengers as well as the involvement of kinases and phospholipases, by means of a pharmacological approach and using the Western blotting technique. The tyrosine kinase inhibitor genistein, the PLC inhibitors neomycin and U 73122, the PKC inhibitors H-7 and calphostin C, and the MAPK pathway inhibitors PD 98059 and U 0126 were all able to prevent the activation of the Na+/H+ exchanger by estrogens (Fig. 5Go). These results suggest a transduction mechanism involving all these enzymes. To verify whether activation of the Na+/H+ exchanger by {alpha}-adrenergic agonists used similar pathways, the involvement of PKC and MAPK was probed by the use of inhibitors. All compounds used were effective in the inhibition of the Na+/H+ exchanger activity by phenylephrine, too (Fig. 6Go).



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  		FIG. 5. Effect of inhibitors of tyrosine kinase, PLC, PKC, and the MAPK pathway on the increase of intracellular pH by 17ß-estradiol. The inhibitor concentration was 10-4 M for neomycin and H-7 and 10-7 M for U 0126, U 73122 and genistein; light-activated calphostin C was 1 µM, and PD 98059 was 10 µM. All inhibitors except calphostin C were given to the cells 5 min before hormone addition, whereas the samples containing calphostin C were light activated with 30-min dark and 30-min light preincubation before hormone treatment. Results are reported as {Delta}pHi/40 min over basal value and are mean ± SD of at least three different experiments.

 


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  		FIG. 6. Effect of inhibitors of PKC and the MAPK pathway on the increase of intracellular pH by phenylephrine. A, The PKC inhibitor H-7 (100 µM) was given to the cells together with the hormone, whereas calphostin C (1 µM) was light activated (see Fig. 5Go). B, Inhibitors of the MAPK pathway PD 98059 (10 µM) and U 0126 (100 nM) were given to the cells 5 min before hormone treatment and at the concentrations employed did not affect the fluorescent signal. Results are reported as {Delta}pHi/40 min over basal value and are mean ± SD of at least six to seven different experiments.

 
Inhibitors of signal transduction pathways often turn out to be less specific than assumed, and studies on activation mechanisms should not be based only on the effects observed with inhibitors. Further experiments were therefore made to verify the role of key compounds of the phosphoinositide and MAPK pathways. 17ß-Estradiol stimulated the production of IP3, the second messenger derived from PLC-catalyzed hydrolysis, as reflected in the higher steady-state level of IP3 (Fig. 7AGo). The increase in IP3 production, measured as incorporation of 3H-myoinositol was rapid, detectable after 1 min, well in agreement with previously published results (40) and with a maximum at the hormone concentration of 10-10 M (Fig. 7BGo). Inhibition of the IP3 increase by ICI 182,780 and U 73122 indicated an ER-dependent involvement in PLC activation (Fig. 7CGo).



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  		FIG. 7. Effect of 17ß-estradiol on IP3 level and production. A, Dose response of 17ß-estradiol in a wide concentration range on IP3 levels. Results are reported as pmol/mg protein and are the mean ± SD of three different experiments. *, P < 0.05, at least, as from a t test with respect to control. B, Time course of IP3 production in RASM cells with ({blacksquare}) or without ({circ}) 17ß-estradiol (10-10 M). Results are reported as fmol/mg protein. C, Effect of ICI 182,780 (1 µM) and U 73122 (10-7 M) on IP3 production. Results are reported as fmol/mg protein and are the mean ± SD of four to six different experiments. P < 0.001 as from a t test with respect to control (*) or 17ß-estradiol alone (°).

 
The sharp increase in IP3 production prompted us to evaluate the role of intracellular calcium in the mediation of nongenomic effects of estrogens on the intracellular pH. For that purpose we used BAPTA, a selective chelator of intracellular Ca2+ (41). Experiments were carried out on RASM cells preincubated with BAPTA/AM (25 µM), a hydrophobic form of BAPTA, that enters the cell and then becomes deesterified to BAPTA and trapped within. BAPTA/AM pretreatment completely prevented the effect of both estrogens and {alpha}-adrenergic agonists (Fig. 8Go). The same result was found by chelating extracellular calcium with 5 µM EGTA (not shown).



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  		FIG. 8. Effect of the Ca2+ chelator BAPTA (25 µM) on the responses to 17ß-estradiol (10-12 M) and phenylephrine (10-5 M) of the intracellular pH at the steady state. Results are the mean ± SD of seven to eight different experiments and are reported as {Delta}pHi/40 min. BAPTA/AM at the concentration used did not affect the fluorescent signal.

 
The dose-response of estrogen effect on the translocation of PKC-{alpha} from the cytosol to the membrane showed a major effect in the concentration range of 10-12 to 10-10 M (Fig. 9AGo). ICI 182,780 and U 73122 (Fig. 9BGo) fully inhibited the translocation, even at the most effective estrogen concentration (10-10 M), confirming a receptor-dependent involvement of PLC activation. As to the MAPK pathway, our data showed that the level of phosphorylation of ERK2 induced by estrogen was increased in the range 10-14 to 10-8 M, indicating the ability of 17ß-estradiol to activate this enzyme (Fig. 10AGo). The same figure also shows the effect of inhibitors ICI 182,780, RO 31–8220, and U 0126 on the activation of the MAPK pathway by 17ß-estradiol. The ICI inhibition and the ineffectiveness of the PKC inhibitor, RO 31–8220, on ERK2 phosphorylation supports the involvement of an estrogen receptor and the IP3/PKC-{alpha} independence of this activation (Fig. 10BGo).



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  		FIG. 9. Effect of 17ß-estradiol on PKC{alpha} translocation. A, Dose response of 17ß-estradiol (E) on the PKC{alpha} translocation from cytosol to the membrane. B, Effect of the nuclear ER inhibitor ICI 182,780 (10-8 M) and the PLC inhibitor U 73122 (1 µM) on the translocation of PKC{alpha} from cytosol (empty bars) to the membrane (filled bars) treated with and without 17ß-estradiol (10-10 M). Data reported on the right side of both panels show the translocation of PKC{alpha} determined by densitometric scanning and are the mean ± SD of three different experiments of Western Blotting. P < 0.001 as from a t test with respect to its own control (C, *) or estrogen-treated cells (E, °). The ß-actin was used to verify iso-loading of the samples.

 


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  		FIG. 10. Effect of 17ß-estradiol on the phosphorylation and content of the ERK2. A, Dose response of 17ß-estradiol (E) on the levels and phosphorylated ERK2 (ERK2-P). B, Effects of the nuclear ER inhibitor ICI 182,780 (10-8 M), the PKC inhibitor RO 31–8220 (1 µM), and the inhibitors of ERK phosphorylation U 0126 (100 nM) in RASM cells treated 15 min with 17ß-estradiol (10-12 M). The right side of both panels shows the densitometric scanning and are the mean ± SD of at least three different experiments of Western blotting. P < 0.001 as from a t test with respect to its own control (C, *) or estrogen-treated RASM cells (E, °). The ß-actin was used to verify the iso-loading of the samples.

 
It has been reported that estrogens regulate the expression of NHE-RF, which is involved in the inhibition of the Na+/H+ exchanger in the renal tubule through a nongenomic mechanism depending on cAMP and PKA activation (28). In our cells, 17ß-estradiol did not give rise to any modulation of the cAMP levels, thus ruling out the contribution of cAMP to the activity of the Na+/H+ exchanger by estrogens in this experimental system (Fig. 11Go).



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  		FIG. 11. Dose response of 17ß-estradiol on cAMP levels. The cAMP levels was measured after 15 min incubation with the hormone. Results are given as pmol/mg protein and are the mean ± SD of four to six different experiments.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this article we report for the first time that 17ß-estradiol, in the low physiological concentration range (10-12 to 10-10 M) close to that found in men or postmenopausal women (42), increases intracellular pH of RASM cells in culture. The effect on intracellular pH is due to the specific activation of the Na+/H+ exchanger because it is prevented by the inhibitor EIPA and because the stereoisomer 17{alpha}-estradiol is totally ineffective. The rapid onset of the intracellular pH response suggests that this effect cannot be explained by a classical genomic mechanism and must be considered nongenomic. The hormone dose response in a wide concentration range displays a bell-shaped curve, as is already known for other hormones that typically interact with plasma membrane receptors (i.e. insulin, atrial natriuretic factor) and shows a maximum effect in the low physiological range. The lack of effect at higher concentrations could be considered the expression of a desensitization or down-regulation phenomenon, by which the cells protect themselves against high hormone levels (24, 43).

To understand the possible involvement of the classical nuclear ER, we used well-known inhibitors: tamoxifen that is a partial ER antagonist (12) and ICI 182,780 that is a pure ER antagonist (44). 17ß-Estradiol action in RASM cells was completely prevented by cell treatment with low concentrations of either of these inhibitors. The nongenomic effects of estradiol in some cases are prevented by the cell treatment with tamoxifen or ICI 182,780 (45, 46) and in some cases are not (47, 48). In these cell lines the expression of both {alpha} and ß ERs isoforms have been reported, although their absolute level in the vascular tissue is not yet well characterized (42). Nadal et al. (6) reported nongenomic estrogen actions in the endocrine pancreas that are not prevented by ICI 182,780, ascribing the estradiol effects to a new {gamma}-adrenergic receptor (6). Our data suggest that the involved receptors, a putative membrane receptor, that following the Mannheim classification of nongenomic steroid actions, may be called a classical ER, similar in structure to or at least shares some analogy with the nuclear receptor (8). For sake of simplicity, we considered the receptor involved in the nongenomic response to be a plasma membrane receptor, in analogy with the situation found for other hormones. However, it should be remembered that this receptor may just as well be located in the cell interior; it could even be a nuclear receptor operating through a mechanism different from transcription factor activation (8).

As to the transduction mechanism, it might be useful to recall at this point that the Na+/H+ exchanger is activated by kinase phosphorylation or modulation of intracellular [Ca2+] or both (19). Our data show that the activation of the Na+/H+ exchanger by estrogens is mediated by at least two parallel pathways and that their relative importance depends on the hormone concentration: the IP3 and PKC pathway is functioning mainly at 10-10 M, as assessed by the use of inhibitors of PKC and immunoblot experiments of translocation of PKC from the cytosol to the membrane. The activation of PKC{alpha} and the increase of both levels and production of IP3 within 1 min has been reported for the nongenomic effects of estrogens in different cells (33, 49). At 10-12 M, there appears to be a major involvement of the MAPK pathway, confirmed by the use of inhibitors of MAPK pathway, and through immunoblotting experiments. The activation of these pathways was prevented by cell treatment with ICI 182,780.

The participation of the MAPK pathway has recently been reported in a wide variety of genomic and nongenomic effects of estrogens (50, 51, 52, 53, 54, 55), even though the physiological response appears to be very much dependent on the cellular context. In addition, recent results suggest that thyroid hormones and estrogens, despite their different structures and biological effects, show similarities in the mechanism of their nongenomic actions, even though the physiological significance of this coincidence is not yet known (54, 55, 56).

The relationship between pH and vascular tone has been known since the article by Gaskell (57), and more than a century later the molecular mechanisms of the pH effects on vascular smooth muscle are still poorly understood. Intracellular pH affects the degree of contraction and relaxation of the smooth muscle and, in general, a direct relationship exists between alkalinization and muscle contraction. Intracellular acidification decreases the affinity of calcium ions for the myofibrils, thus decreasing the vascular tone. On the other hand, following alkalinization, the increased affinity of calcium ions for the myofibrils leads to an increased vascular tone. Changes of pH can alter such things as the force development in smooth muscle affecting the rate of myosin ATPase activity, the sensitivity of the contractile units to Ca2+, the fluxes of Ca2+ into the cell, the transmitter release, and the competition at Ca2+-binding sites (18). In an attempt to understand the physiological meaning of the increase in pHi in a low range of hormone concentrations, we examined the effects of two {alpha}-adrenergic agonists on RASM cells, norepinephrine and phenylephrine. Our data show that these two vasoconstrictors caused an increase in intracellular pH superimposable to that of estrogen, with an identical time course and no potentiation of the effects when the 17ß-estradiol was given together with the {alpha}-adrenergic agonists.

It has been reported that the activity of the nuclear estrogen receptor may be stimulated also in the absence of an estrogen ligand (58). Multiple signaling pathways initiated by growth factors (59) or hormones (60) stimulate nuclear receptor activities. We suggest that the observed effects of estradiol on intracellular pH of RASM cells represent another example of the reported cross-talk between different steroid receptors as well as between different signaling pathways (6).

A simplified scheme for the smooth muscle relaxation indicates that estrogen can modify the expression of adrenergic receptors that in turn could give rise to an increase in cAMP, PKA activation, and myosin light chain kinase phosphorylation with consequent muscle relaxation (15). In general, an increase in cAMP and activation of PKA in mammalian cells inhibits the activity of the Na+/H+ exchanger (61). RASM cells after a short-term treatment with estrogen did not show any increase in intracellular cAMP; this could be a factor that at the low estrogen concentrations allows the increase of intracellular pH and muscle contraction.

In conclusion the increase of intracellular pH because of 17ß-estradiol is the consequence of three different factors: the activation of PKC, the increase of [Ca2+]i caused by IP3, and the activation of the MAPK pathway.

There are many potential targets for PKC in vascular smooth muscle that could play a role in the modulation of the contractile process. First, PKC can phosphorylate the myosin light chain on sites different from those of myosin light chain kinase, contributing to contraction (62). Second, PKC can phosphorylate other proteins such as calponin and caldesmon, which may depress the interaction between actin and phosphorylated myosin. In particular, caldesmon can be phosphorylated also by MAPK pathway, and PKC can activate an early step of this kinase cascade (62). On the other hand, the role of IP3 in the release of intracellular calcium and in smooth muscle contraction is well known. The role of MAPK in contractile smooth muscle is not well understood, even though in the last few years there has been much interest on MAPK in this field (63). A relationship between MAPK pathway and smooth muscle contraction has been reported because the specific inhibitor of MAPKK PD 98059 significantly inhibited the contraction to phenylephrine (64). A similar result has recently been obtained for ovine uterine artery (65).

We hypothesize that the increase of intracellular pH in response to low concentrations of estrogens may give a contribution to the development of hypertension and atherosclerosis. An article from one of us (26) has shown that an increase of intracellular pH in human vascular smooth muscle cells can trigger cell proliferation in response to mitogens and affect cell locomotion, key events in atherogenesis. All together these data suggest that the lower estrogen concentrations found in postmenopausal women might remove the hormone protective effects typical of premenopausal women that, among other factors, regulate the pace of the Na+/H+ exchanger activity.


   Footnotes
 
This work was supported by a grant ("Sviluppo 2000") from the University of Rome "Roma Tre" (to A.T.) and grants from the Ministry of University and Scientific Technological Research "ex60%" 1998, 1999, and "Cofinanziamento 2000" (to S.I.).

Abbreviations: BAPTA/AM, 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester; BCECF/AM, 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein tetra-acetoxymethyl ester; EIPA, 5-(N-ethyl-N-isopropyl)amiloride; ER, estrogen receptor; IP3, inositol 1,4,5-trisphosphate; NHE-RF, regulatory factor for the Na+/H+ exchanger; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; RASM, rat aortic smooth muscle; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid.

Received April 18, 2003.

Accepted for publication June 24, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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