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Am J Physiol Heart Circ Physiol 293: H1955-H1961, 2007. First published July 20, 2007; doi:10.1152/ajpheart.00306.2007
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Estrogen increases smooth muscle expression of {alpha}2C-adrenoceptors and cold-induced constriction of cutaneous arteries

A. H. Eid,1 K. Maiti,1 S. Mitra,1 M. A. Chotani,1 S. Flavahan,2 S. R. Bailey,3 C. S. Thompson-Torgerson,2 and N. A. Flavahan2

1Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio; 2Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University, Baltimore, Maryland; and 3Faculty of Veterinary Science, University of Melbourne, Parkville, Victoria, Australia

Submitted 12 March 2007 ; accepted in final form 17 July 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Raynaud's phenomenon, which is characterized by intense cold-induced constriction of cutaneous arteries, is more common in women compared with men. Cold-induced constriction is mediated in part by enhanced activity of {alpha}2C-adrenoceptors ({alpha}2C-ARs) located on vascular smooth muscle cells (VSMs). Experiments were therefore performed to determine whether 17beta-estradiol regulates {alpha}2C-AR expression and function in cutaneous VSMs. 17beta-Estradiol (0.01–10 nmol/l) increased expression of the {alpha}2C-AR protein and the activity of the {alpha}2C-AR gene promoter in human cultured dermal VSMs, which was assessed following transient transfection of the cells with a promoter-reporter construct. The effect of 17beta-estradiol was associated with increased accumulation of cAMP and activation of the cAMP-responsive Rap2 GTP-binding protein. Transient transfection of VSMs with a dominant-negative mutant of Rap2 inhibited the 17beta-estradiol-induced activation of the {alpha}2C-AR gene promoter, whereas a constitutively active mutant of Rap2 increased {alpha}2C-AR promoter activity. The effects of 17beta-estradiol were inhibited by the estrogen receptor (ER) antagonist, ICI-182780 (1 µmol/l), and were mimicked by a cell-impermeable form of the hormone (estrogen:BSA) or by the selective ER-{alpha} receptor agonist 4,4',4'''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)tris-phenol (PPT; 10 nmol/l) or the selective ER-beta receptor agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; 10 nmol/l). Therefore, 17beta-estradiol increased expression of {alpha}2C-ARs by interacting with cell surface receptors to cause a cAMP/Rap2-dependent increase in {alpha}2C-AR transcription. In mouse tail arteries, 17beta-estradiol (10 nmol/l) increased {alpha}2C-AR expression and selectively increased the cold-induced amplification of {alpha}2-AR constriction, which is mediated by {alpha}2C-ARs. An estrogen-dependent increase in expression of cold-sensitive {alpha}2C-ARs may contribute to the increased activity of cold-induced vasoconstriction under estrogen-replete conditions.

Raynaud's phenomenon; Rap1; Rap2; adenosine 3',5'-cyclic monophosphate; estrogen receptors

COLD-INDUCED VASOCONSTRICTION in the cutaneous circulation is a protective physiological response that acts to reduce heat loss (49). The constriction results from a reflex increase in sympathetic activity and a direct local effect of cold to increase vasoconstriction to nerve-released norepinephrine (49). This latter effect is mediated by a cold-induced, selective increase in {alpha}2-adrenoceptor ({alpha}2-AR) reactivity on vascular smooth muscle cells (VSMs) (19, 20, 23). Although {alpha}2-ARs comprise three subtypes ({alpha}2A, {alpha}2B, and {alpha}2C) (41), only {alpha}2C-ARs appear to be regulated by cold (11, 30). Moderate cooling causes redistribution of {alpha}2C-ARs from the trans-Golgi, where they are normally retained, to the cell surface where they can respond to agonist stimulation (2, 3, 30). The local cold sensitivity of the cutaneous vascular system is increased in individuals with Raynaud's phenomenon and scleroderma, resulting in cold-induced peripheral vasospasm, which can be prevented by {alpha}2-AR blockade (25).

Primary Raynaud's phenomenon is more common in women compared with men, and in women the severity of complaints is most pronounced in the period between menarche and menopause (6, 50). In postmenopausal women, the prevalence of Raynaud's phenomenon increased significantly in individuals receiving estrogen replacement therapy (24). These studies suggest that female sex hormones, in particular estrogen, may increase susceptibility to cold-induced vasopasm. In healthy individuals, cutaneous vasoconstriction evoked by local cooling of the hand is more intense and more prolonged in women compared with men (5, 8, 14). Furthermore, the increased cold-induced vasoconstriction in women is most prominent under estrogen-replete conditions and is prevented by {alpha}2-AR but not by {alpha}1-AR blockade (5, 8, 26). Indeed, the mRNA level for {alpha}2C-ARs, but not {alpha}2B-ARs or {alpha}2A-ARs, is higher in VSMs of female compared with male cutaneous arteries (38). The present experiments were therefore performed to determine whether estrogen might increase expression of {alpha}2C-ARs in cutaneous VSMs and, if so, to identify the signaling pathway responsible for this effect.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Actinomycin D, 17beta-estradiol, beta-estradiol 6-(O-carboxy-methyl)oxime:BSA, 3-isobutyl-1-methyxanthine (IBMX), SQ-22536, and UK-14304 were purchased from Sigma (St. Louis, MO). 2,3-Bis(4-hydroxyphenyl)-propionitrile (DPN; ICI-182780) and 4,4',4'''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)tris-phenol (PPT) were purchased from Tocris (Ellisville, MO).

Cell culture. Cutaneous VSMs were cultured from human dermal arterioles (male and female donors) and used between passages 9–12, as previously described (2, 12, 13). VSMs were grown in Ham's growth medium (DMEM: F12, 50:50) supplemented with 10% heat inactivated FBS, L-glutamine, and an antibiotic/antimycotic cocktail (at 37°C). At least 3 days before the addition of 17beta-estradiol, cells were washed (3 times) and made quiescent in phenol red-free medium (DMEM: F12, 50:50, supplemented with L-glutamine and an antibiotic/antimycotic cocktail) without serum or containing 0.5% of charcoal-stripped serum. Responses to 17beta-estradiol were similar in cultured VSMs derived from male and female donors.

Western blot analysis. Cells were rinsed twice with phosphate-buffered saline (PBS) and scraped into lysis buffer (2% SDS and 60 mM Tris, pH 6.8). After sonication, the lysate was centrifuged at 5,000 g for 10 min, and the supernatant was analyzed for protein concentration (bicinchoninic acid, Pierce; Rockford, IL). Equal protein amounts of cell lysates (25 µg) were separated using 10% SDS-PAGE, transferred to polyvinyldifluoride membrane (Millipore), and incubated with a specific {alpha}2C-AR affinity-purified polyclonal antibody (1:1,000 dilution, 1 h, room temperature), which specifically recognizes the {alpha}2C-AR on Western blots (2, 12, 13). {alpha}2C-ARs have a molecular mass of ~70 kDa (2, 12, 13, 30). After being extensively washed, the membrane was incubated with a secondary anti-rabbit antibody (1:2,000 dilution, 1 h, room temperature). Blots were then developed using enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ) and quantified by densitometry (Personal Densitometer, Molecular Dynamics).

Measurement of intracellular cAMP levels. Cells were treated with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 0.225 mM) for 30 min before and during exposure to estrogen or BSA-conjugated estrogen (12, 30). At selected times, cells were placed on ice, washed with ice-cold PBS, and then lysed with 10 mM HCl in absolute ethanol (30 min at –20°C). Cells were scraped into the acidified ethanol and centrifuged at 5,000 g for 10 min at 4°C, and cAMP levels in the supernatant were measured using an 125I radioimmunoassay kit (Biomedical Technologies, Stoughton, MA), as previously described (12, 30).

Plasmids. The constitutively active mutant of Rap2GTPase (Rap2V12; Rap2-CA) and the corresponding empty murine stem cell virus vector (pMSCV) were a generous gift from Dr. Michael Gold (University of British Columbia, Vancouver, Canada) (37). The dominant-negative mutant of Rap2 GTPase (Rap-DN) and the corresponding empty (pRK5) plasmids were kindly provided by Dr. Martina Schmidt (Institute für Pharmakologie, Universitätsklinikum Essen, Germany) (44). The {alpha}2C-AR promoter/reporter plasmid spanning the full-length promoter sequence (–1,915/+892, relative to the transcription start site +1) was a kind gift from Dr. Herve Paris and is engineered as described by Schaak et al. (43). The {alpha}2A-AR full-length (–1,066/+928) promoter/reporter construct has been described previously (13). Renilla luciferase plasmid (pRL)-cytomegalovirus (CMV) (Renilla luciferase gene driven by CMV promoter/enhancer) was purchased from Promega (Madison, WI).

Transient transfections. Cells were transiently transfected by nucleofection with the Amaxa Nucleofector (Amaxa Biosystems, Gaithersburg, MD) according to the manufacturer's instructions. Transfection was optimized to achieve an ~80% transfection efficiency, with minimal toxicity, achieved with nucleofection of 400,000 cells with 4 µg of nucleic acids. The total amount of transfected DNA was kept constant throughout the study by using the appropriate empty plasmids. When we used reporter constructs, pRL-CMV was used as an internal control to normalize the firefly luciferase units (12, 13). After transfection, cells were allowed to recover overnight in Ham's growth medium. After aspiration of the growth medium, cells were washed thoroughly and maintained in serum-free phenol red-free media for 72 h before the addition of estrogen. For luciferase analysis, cells were washed, lysed in luciferase lysis buffer (Promega), snap frozen, and then thawed at room temperature. Cell lysates were centrifuged at 9,300 g for 10 min, and luciferase activity in the supernatant was determined.

Rap pull-down assay. Activation of RapGTPases was assessed using a pull-down assay (48), with an activation-specific probe corresponding to 97 amino acids of human Ral GDS-rap-binding domain, according to the manufacturer's instructions (Upstate, Lake Placid, NY). The pull-down assay was performed using 200 µg of VSM lysate, whereas total Rap expression was determined using 30 µg of the same cell lysate. Western blot analysis for activated, as well as total, Rap GTPases was assessed using antibodies specific for Rap1 (1:500 dilution, Upstate) or Rap2 (1:2,500 dilution, BD Transduction, Lexington, KY) for 1 h at room temperature.

Vasomotor activity of isolated arterioles. Male and female mice (C57BL6) were euthanized by CO2 asphyxiation, and segments of tail artery were rapidly removed and placed in cold Krebs-Ringer bicarbonate solution containing (in mM) 118.3 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, 25.0 NaHCO3, and 11.1 glucose (control solution). In some experiments, the responses of tail arteries from male and female mice were analyzed immediately. In others, paired arterial segments were incubated in the absence and presence of 17beta-estradiol (10 nmol/l, 24 h) in serum-free, phenol red-free DMEM (37°C, 5% CO2-balance air). The arteries were then rinsed in control solution and prepared for Western blot (see Western blot analysis) or vasomotor analyses.

For vasomotor analysis, arteries were cannulated at both ends with glass micropipettes in a microvascular chamber (Living Systems, Burlington, VT). The arteries were maintained at a constant transmural pressure of 60 mmHg in the absence of flow, and vasoconstrictor responses were analyzed as previously described (2, 11). The chamber was superfused with control solution and maintained at 37°C (pH 7.4) and gassed with 16% O2-5% CO2-balance N2. The chamber was placed on the stage of an inverted microscope (x20, Nikon TMS-F) connected to a video camera (Panasonic, CCTV camera). The vessel image was projected onto a video monitor, and the internal diameter was continuously determined by a video dimension analyzer (Living Systems Instrumentation) and monitored using a Biopac (Santa Barbara, CA) data acquisition system. Concentration-effect curves to the selective {alpha}1-AR agonist, phenylephrine, or the selective {alpha}2-AR agonist, UK-14304, were generated by increasing the concentration of the agonists in half-log increments, once the constriction to the previous concentration had stabilized (2, 11). After the completion of the concentration-effect curve, the influence of the agonists was terminated by repeatedly exchanging the buffer solution and allowing the artery to return to its stable baseline level. When the influence of cold was analyzed, the temperature of the superfusate was decreased to 28°C for 30 min before assessing {alpha}2-AR vasoconstriction (2, 11). Concentration-effect curves were analyzed by comparing the agonist concentration causing 20% constriction (CC20), determined by regression analysis.

Statistical analyses. Statistical evaluation of the data was performed by Student's t-test for either paired or unpaired observations. When more than two means were compared, ANOVA was used: either a one-way ANOVA with Dunnett's post hoc test or a two-way ANOVA with Tukey-Kramer's post hoc test (GraphPad Software, San Diego, CA). Data are presented as means ± SE, where n equals the number of different cell culture experiments or the number of animals from which arteries were studied.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Estrogen increases expression of {alpha}2C-ARs. 17beta-Estradiol (0.01–10 nmol/l, 24 to 48 h) caused a concentration-dependent increase in {alpha}2C-AR protein expression, which was maximal at 0.1 nmol/l with a 4.4 ± 0.7-fold increase (Fig. 1, n = 6, P < 0.01).


Figure 1
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  		Fig. 1. The effect of 17beta-estradiol (17betaE; 0.01–10 nmol/l) on protein expression of {alpha}2C-adrenceptors ({alpha}2C-ARs) in human cutaneous vascular smooth muscle cells (VSMs). Similar results were obtained after 24 or 48 h of incubation with the hormone, and the results were therefore combined. {alpha}2C-AR expression was quantified by SDS-PAGE/Western blot analysis of cell lysates, and the effect of 17betaE is presented as the fold increase in the basal, unstimulated level of {alpha}2C-ARs. Top: data from a representative experiment. The molecular mass of {alpha}2C-ARs is ~70 kDa (see Refs. 2, 12, 13, and 30). Data are presented as means ± SE for n = 6. *P < 0.05 and **P < 0.01 compared with control (C).

 
Estrogen increases {alpha}2C-AR transcription. Actinomycin D (1.6 µmol/l), an inhibitor of DNA-dependent RNA synthesis, prevented the increase in {alpha}2C-AR expression in response to 17beta-estradiol (0.1 nmol/l) [1.2 ± 0.1-fold increase in {alpha}2C-ARs, n = 4, P = not significant (NS)]. To directly assess the effect of the hormone on the promoter activity of {alpha}2-ARs, VSMs were transiently transfected with {alpha}2C-AR or {alpha}2A-AR promoter: luciferase reporter constructs. 17beta-Estradiol (0.1 nmol/l) increased the transcriptional activity of the {alpha}2C-AR reporter by 3.1 ± 0.3-fold (n = 3, P < 0.05) but did not affect the activity of the {alpha}2A-AR reporter (0.97 ± 0.02-fold, n = 3, P = NS).

Estrogen increases cAMP accumulation. In a number of cell types, estrogen regulates the activity of adenylyl cyclase and the intracellular levels of cAMP (21). In human cutaneous VSMs, 17beta-estradiol (0.01–1 nmol/l) increased cAMP accumulation, reaching a maximal effect at 0.1 nmol/l, which increased cAMP from 0.31 ± 0.04 to 1.05 ± 0.09 pmol/105 cells (n = 4, P < 0.005) (Fig. 2A). At this concentration, the increase in cAMP peaked within 15 min of exposure to the hormone (Fig. 2B). The increase in cAMP evoked by 17beta-estradiol (0.1 nmol/l) was significantly inhibited by the adenylyl cyclase inhibitor SQ-22536 (400 µmol/l). Under control conditions, 17beta-estradiol (0.1 nmol/l, 15 min) increased cAMP accumulation from 0.38 ± 0.09 to 0.94 ± 0.14 pmol/105 cells (n = 3, P < 0.01). SQ-22536 (400 µmol/l) did not significantly affect the basal level of cAMP (0.24 ± 0.02 pmol/105 cells, n = 3, P = NS) but markedly reduced the cAMP levels attained after 17beta-estradiol (0.1 nmol/l) (to 0.50 ± 0.07 pmol/105 cells, n = 3, P < 0.01). Indeed, after SQ-22536, 17beta-estradiol no longer significantly increased cAMP accumulation above basal levels.


Figure 2
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  		Fig. 2. The effect of 17betaE on cAMP accumulation in human cutaneous VSMs. The influence of increasing concentrations (A: 0.01–1 nmol/l, 15 min) and increasing times of incubation (B: 5–30 min, 0.1 nmol/l) with the hormone was determined. Intracellular cAMP is presented as picomoles of cAMP per 105 cells, and data are presented as means ± SE for n = 3 to 4. *P < 0.05 and **P < 0.01 compared with control.

 
17beta-Estradiol modulates the activity of Rap GTPases. Elevations in cAMP can increase {alpha}2C-AR expression in cutaneous VSMs by activating a Rap GTPase pathway (12). Experiments were therefore performed to determine whether 17beta-estradiol altered Rap GTPase activity in these cells. Indeed, 17beta-estradiol (0.1 nmol/l) increased the activity of Rap2 GTPase (2.7 ± 0.3-fold, n = 3, P < 0.05) but decreased the activity of Rap1 GTPase (0.4 ± 0.1-fold, n = 3, P < 0.05) (Fig. 3A). After inhibition of adenylyl cyclase with SQ-22536 (400 µmol/l), 17beta-estradiol (0.1 nmol/l) failed to increase the activity of Rap2 GTPase (Fig. 3B).


Figure 3
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  		Fig. 3. The effect of 17betaE on the activity of Rap GTPases in cutaneous VSMs. Rap activity was determined by a pull-down assay, as described in METHODS. Active Rap was normalized to total Rap in the sample and then expressed as a fold increase in the basal, unstimulated activity. A: influence of 17betaE (0.1 nmol/l, 30 min) on the activity of Rap1 and Rap2. Data are presented as means ± SE for n = 3. *P < 0.05 compared with untreated cells (–). Inset: time course for the effect of 17betaE on Rap1 and Rap2 activity. B: effects of the adenylyl cyclase inhibitor SQ-22536 (400 µmol/l) on the 17betaE-induced stimulation of Rap2 GTPase. Cells were incubated with SQ-22536 for 60 min before and during administration of 17betaE (0.1 nmol/l, 30 min). Cell lysates were then analyzed for Rap2 activity. Data are presented as means ± SE for n = 4. a,bP < 0.05, values with the same letter are significantly different.

 
Activation of Rap GTPase modulates {alpha}2C-AR expression. To determine whether activation of Rap2 GTPase was responsible for the increased {alpha}2C-AR expression in response to 17beta-estradiol, transient transfections were employed in cutaneous VSMs. Transfection with a constitutively active mutant of Rap2 GTPase [Rap2-CA (37)] caused a significant increase in the promoter activity of the cotransfected {alpha}2C-AR promoter/reporter construct (3.8 ± 0.6-fold increase; n = 4; P < 0.05). Furthermore, transfection of microvascular cells with a dominant-negative mutant of Rap2 [Rap2-DN (44)] abolished the effect of 17beta-estradiol (0.1 nmol/l) to increase the activity of the {alpha}2C-AR promoter. Under control conditions (after transfection with control vector), 17beta-estradiol (0.1 nmol/l) increased the {alpha}2C-AR promoter activity by 3.9 ± 0.5-fold (n = 4, P < 0.01). Rap2-DN did not significantly affect the basal activity of the {alpha}2C-AR promoter (1.5 ± 0.2-fold, n = 4, P = NS) but abolished the increased activity following 17beta-estradiol (1.3 ± 0.2-fold, n = 4; P = NS compared with basal activity, and P < 0.001 compared with the control response to the hormone).

Role of estrogen receptors. The estrogen receptor (ER) antagonist ICI-182780 (1 µmol/l) abolished the increase in {alpha}2C-AR expression in response to 17beta-estradiol (0.1 nmol/l) (1.2 ± 0.3-fold increase in {alpha}2C-ARs, n = 3, P = NS), suggesting that the effects of 17beta-estradiol were mediated by binding to its receptors, ER-{alpha} and/or ER-beta. To test whether activation of ER-{alpha} or ER-beta could mimic the observed effects of estrogen, we used the following subtype-selective agonists: PPT for ER-{alpha} and DPN for ER-beta. PPT (10 nmol/l) increased {alpha}2C-AR expression by 3.9 ± 1.0-fold (n = 4; P < 0.005), and DPN (10 nmol/l) increased expression by 2.9 ± 0.5-fold (n = 7, P < 0.05). Although nuclear ER-{alpha} and/or ER-beta can activate gene transcription, the increase in cAMP, which mediated the increased expression of {alpha}2C-ARs to 17beta-estradiol, was too rapid to involve transcriptional regulation. In addition to nuclear localization, estrogen receptors are also present at the plasma membrane. To assess the role of a plasma membrane receptor, we analyzed the response of cutaneous VSMs to a cell-impermeable form of estrogen, 17beta-estradiol 6-(O-carboxy-methyl)oxime:BSA (17beta-estradiol:BSA). As observed with 17beta-estradiol, 17beta-estradiol:BSA (0.1 to 10 nmol/l) increased cAMP accumulation in a concentration-dependent manner [from a basal level of 0.34 ± 0.07 pmol/105 cells to 0.72 ± 0.12 at 0.1 nmol/l (P < 0.01), 0.94 ± 0.09 at 1 nmol/l (P < 0.01), and 0.98 ± 0.08 pmol/105 cells at 10 nmol/l 17beta-estradiol:BSA (P < 0.01), n = 3]. According to the manufacturer, ~3% of 17beta-estradiol:BSA may be present in the unbound form. However, conjugated 17beta-estradiol was only 10-fold less potent than free 17beta-estradiol (Fig. 3), which is consistent with the activity of the conjugated form of the hormone (47).

Cold-induced vasoconstriction. In mouse tail arteries, {alpha}2-AR constriction at 37°C is mediated by {alpha}2A-ARs, whereas the heightened constriction observed during cooling (e.g., to 28°C) is mediated by {alpha}2C-ARs (11). At 37°C, constriction to the {alpha}2-AR agonist UK-14304 was similar between tail arteries of male and female mice (at 1 nmol/l, UK-14304 caused constriction of 9.0 ± 1.2% and 8.7 ± 0.8% in male and female arteries, respectively, n = 6 or 7, P = NS). However, during exposure to 28°C, the increased constriction to {alpha}2-AR activation by UK-14304 was further increased in female compared with male arteries (at 1 nmol/l, UK-14304 caused constriction of 19.1 ± 2.2% and 26.5 ± 1.6% in male and female arteries, respectively, n = 6 or 7, P ≤ 0.02).

To determine the effects of 17beta-estradiol, tail arteries from male arteries were incubated with the hormone for 24 h (at 37°C). 17beta-Estradiol (10 nmol/l, 24 h) increased the expression of {alpha}2C-ARs in tail arteries by 4.1 ± 0.8-fold (n = 4, P ≤ 0.001, Fig. 4). When subsequently assessed at 37°C, 17beta-estradiol (10 nmol/l, 24 h) did not significantly affect constriction evoked by activation of {alpha}1-ARs with phenylephrine (0.01 to 3 µmol/l) (data not shown) or activation of {alpha}2-ARs with UK-14304 (Fig. 4). However, cooling to 28°C augmented constriction to the {alpha}2-AR agonist, which was significantly further increased in 17beta-estradiol-treated arteries (Fig. 4). Therefore, although the hormone did not significantly affect constriction to UK-14304 at 37°C, it increased constriction to the {alpha}2-AR agonist during cold exposure [log CC20 values of –8.36 ± 0.12 and –8.91 ± 0.07 for control and 17beta-estradiol-treated arteries, respectively; log shift of 0.55 ± 0.08 (3.5-fold shift), n = 5, P < 0.005, Fig. 4].


Figure 4
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  		Fig. 4. Effect of 17betaE (10 nmol/l, 24 h) on the expression and function of {alpha}2-ARs in mouse isolated tail arteries. Inset: representative Western blot demonstrating the effect of 17betaE (10 nmol/l, 24 h) to increase the expression of {alpha}2C-ARs (by 4.1 ± 0.8-fold, n = 4, P ≤ 0.001). The molecular mass of {alpha}2C-ARs is ~70 kDa (2, 12, 13, 30). Vasoconstriction to the {alpha}2-AR agonist, UK-14304 (1–100 nmol/l) was assessed at 37° and 28°C. Responses to the agonist were expressed as a percentage of the stable baseline diameter and are presented as means ± SE (n = 5).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, 17beta-estradiol increased the expression of {alpha}2C-ARs in cutaneous VSMs by activating the {alpha}2C-AR gene promoter. The effect of 17beta-estradiol was mediated by a novel signaling cascade, namely activation of plasma membrane estrogen receptors, which resulted in a rapid increase in cAMP levels and a subsequent activation of the Rap2 GTP-binding protein. Indeed, 17beta-estradiol-induced activation of the {alpha}2C-AR gene promoter was inhibited by transient transfection with a Rap2-DN and mimicked by transfection with a constitutively active mutant of the GTP binding protein. Therefore, activation of Rap2 was necessary and sufficient for the transcriptional activation of the {alpha}2C-AR gene. In isolated cutaneous arteries, 17beta-estradiol did not alter {alpha}2-AR vasoconstriction at warm temperatures, which is mediated by {alpha}2A-ARs, but increased cold-induced amplification of the {alpha}2-AR response, which is mediated by {alpha}2C-ARs. Therefore, increased expression of {alpha}2C-ARs in response to 17beta-estradiol might contribute to the increased activity of cold-induced vasoconstriction occurring in estrogen-replete conditions.

Cutaneous arterial VSMs express {alpha}2A- and {alpha}2C- but not {alpha}2B-ARs (13, 38). In the present study, physiological levels of 17beta-estradiol increased the activity of the {alpha}2C-AR full-length promoter, resulting in increased expression of {alpha}2C-ARs in cutaneous VSMs. The hormone-induced increase in protein expression was also abolished by actinomycin D, confirming that the effect was mediated by transcriptional activation of the {alpha}2C-AR gene rather than by posttranscriptional regulation. The effect of 17beta-estradiol was selective for {alpha}2C-ARs, and the hormone did not increase activity of the {alpha}2A-AR promoter. A previous report demonstrated that VSM expression of {alpha}2C-ARs, but not {alpha}2A-ARs, was increased in cutaneous tail arteries of female compared with male rats (38). The results of the present study suggest that this sex-dependent difference in {alpha}2-AR subtype expression in cutaneous VSMs is mediated by estrogen-dependent activation of {alpha}2C-AR transcription.

{alpha}2C-AR expression in cutaneous VSMs is regulated in a complex fashion by cAMP signaling pathways (12). In addition to the prototypic A-kinase signaling cascade, cAMP can activate other effectors including exchange protein activated by cAMP (EPAC), a guanine nucleotide exchange factor for the Ras-like GTPases Rap1 and Rap2 (18, 33). cAMP has positive and negative effects on the {alpha}2C-AR promoter activity mediated through these distinct pathways: A-kinase-mediated inhibition and an EPAC/Rap-mediated activation (12). cAMP elevation increases the expression of {alpha}2C-AR in human cutaneous VSMs, indicating that the positive cAMP/EPAC/Rap pathway dominates the inhibitory cAMP/A-kinase cascade (12). Physiological concentrations of estrogen activate adenylyl cyclase and increase cAMP levels in different cell types, including rat VSMs (21). We therefore investigated the effect of estrogen on cAMP/Rap signaling in human cutaneous VSMs. Indeed, 17beta-estradiol, at physiological concentrations, increased intracellular accumulation of cAMP in these cells. 17beta-Estradiol also increased the activity of Rap2 but inhibited the activity of Rap1. Inhibition of the increase in cAMP by SQ-22536, an adenylyl cyclase inhibitor, prevented the 17beta-estradiol-induced activation of Rap2, indicating that it reflected cAMP/EPAC signaling. Transient transfection of cutaneous VSMs with a Rap2-DN abolished the hormone-induced activation of the {alpha}2C-AR promoter/reporter, indicating that this signaling pathway was essential for the induction of {alpha}2C-ARs in response to the hormone. Furthermore, transient transfection using a Rap2-CA demonstrated that Rap2 activity was sufficient for the 17beta-estradiol-mediated activation of the {alpha}2C-AR promoter. Taken together, these data indicate that Rap2 is a crucial mediator for estrogen to increase expression of {alpha}2C-ARs in human cutaneous VSMs.

The 17beta-estradiol-induced activation of the cAMP/Rap2 signaling pathway was rapid (<30 min), which is not consistent with a genomic mechanism involving classical cytoplasmic/nuclear estrogen receptors. Plasma membrane ER-{alpha} and/or ER-beta receptors have been demonstrated in a number of cell types (21, 40). Other reports have demonstrated the presence of a distinct membrane receptor, which is resistant to inhibition by the ER-{alpha}/ER-beta antagonist ICI-182780 (16, 22, 27, 28, 31). In the present study, the effect of 17beta-estradiol to increase {alpha}2C-AR expression was inhibited by ICI-182780 and mimicked by the selective ER-{alpha} agonist PPT or the selective ER-beta agonist DPN. Likewise, the rapid increase in cAMP levels in cutaneous VSMs evoked by 17beta-estradiol was mimicked by a cell-impermeable analog of the hormone, 17beta-estradiol:BSA (9, 47). Therefore, these results suggest that the effects of 17beta-estradiol were mediated by ER-{alpha} and/or ER-beta located in the plasma membrane.

This is the first demonstration that 17beta-estradiol can regulate Rap activity. Previous studies have proposed that estrogen can inhibit EPAC-dependent signaling in dorsal root ganglia neurons (29) but increase EPAC-dependent signaling in neuroendocrine cells (46). The present results indicate that the divergence may reside at the level of the Rap GTPases, with estrogen causing stimulation of Rap2 but inhibition of Rap1. Interestingly, adenylyl cyclase, EPAC, Rap GTPases, and estrogen receptors have been reported to reside in caveolae microdomains (1, 7, 17). Restriction of cAMP signaling within caveolae has been shown to play a role in the selective activation of certain signaling pathways (15, 45). Therefore, compartmentalization and interaction between these signaling components might account for a selective activation of Rap2, but decreased Rap1 activity, in response to 17beta-estradiol. Indeed, stimulation of GS-coupled receptors has been reported to activate extracellular-signal regulated kinase (ERK)1/2 in a cAMP- and Rap2-dependent, but Rap1-independent, mechanism (34). Interestingly, ERK1/2 can activate cytoplasmic/nuclear estrogen receptors by phosphorylating a critical Ser118 needed for activation (32). Therefore, although increased expression of {alpha}2C-ARs appears to be initiated by activation of membrane estrogen receptors and cAMP/Rap2 signaling, this pathway may act in part by enhancing activation of cytoplasmic/nuclear estrogen receptors. Indeed, the {alpha}2C-AR promoter encompasses many SP1 sites, one AP1 site, and a half estrogen response element site, transcription from all of which can be modulated by estrogen receptors (4, 39, 42). Further studies are needed to define whether these additional mechanisms may contribute to the increased expression of {alpha}2C-ARs by 17beta-estradiol.

Cold-induced vasoconstriction of cutaneous blood vessels is mediated by a reflex increase in sympathetic activity and a direct sensitizing effect of cold to increase constriction to the sympathetic neurotransmitter norepinephrine. This latter effect results from a selective increase in {alpha}2C-AR constrictor activity, which is mediated by cold-induced mobilization of {alpha}2C-ARs from the Golgi compartment, where they are normally retained, to the plasma membrane (2, 3, 11, 30). Indeed, {alpha}2-AR constriction at 37°C is mediated predominantly by {alpha}2A-ARs, whereas the heightened cold-induced constriction is mediated by {alpha}2C-ARs (11). As was observed in cultured VSMs, 17beta-estradiol also increased expression of {alpha}2C-ARs in cutaneous arteries. Interestingly, 17beta-estradiol did not alter constriction to {alpha}2-AR activation at warm temperatures but significantly increased {alpha}2-AR constriction during cold exposure. These results suggest that increased expression of {alpha}2C-ARs augments the pool of receptors available for cold-induced mobilization to the cell surface, resulting in a selective increase in cold-induced amplification of sympathetic constriction.

The present study describes a novel signaling pathway whereby estrogen can increase the expression of {alpha}2C-ARs, which may contribute to increased cold-induced vasoconstriction and to increased prevalence of Raynaud's phenomenon in estrogen-replete women (6, 24, 50). In fact, the increased cold-induced constriction in healthy women and in those suffering from Raynaud's phenomenon is prevented by {alpha}2-AR blockade (5, 8, 26). In addition to increasing expression of constrictor adrenergic receptors, estrogen also increases the expression of endothelial nitric oxide (NO) synthase and endothelial production of NO (36). Indeed, the elevated estrogen level occurring in the late follicular phase of the menstrual cycle is associated with increased vasodilatation to the endothelium-dependent agonist, bradykinin, as well as increased constriction to cold exposure or to norepinephrine (10, 26). Furthermore, cutaneous vasodilatation evoked by local warming, which is mediated in part by NO (35), is increased in women compared with men (5, 14). Therefore, although estrogen appears to have antagonistic effects on the cutaneous vascular system, it appears to generate a system that is especially sensitive to changes in temperature.


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This work was funded by National Institutes of Health Grants HL-080119 and OH-008531 (to N. A. Flavahan) and a grant from the Scleroderma Research Foundation (to N. A. Flavahan).


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. A. Flavahan, Dept. of Anesthesiology and Critical Care Medicine, Johns Hopkins Univ., Ross Research Bldg., R 370/372, 720 Rutland Ave., Baltimore, MD 21205 (e-mail: nflavah1{at}jhmi.edu)

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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