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Circulation Research. 2000;87:e44-e52

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(Circulation Research. 2000;87:e44.)
© 2000 American Heart Association, Inc.

UltraRapid Communication

Estrogen Receptor {alpha} and Endothelial Nitric Oxide Synthase Are Organized Into a Functional Signaling Module in Caveolae

Ken L. Chambliss, Ivan S. Yuhanna, Chieko Mineo, Pingsheng Liu, Zohre German, Todd S. Sherman, Michael E. Mendelsohn, Richard G. W. Anderson, Philip W. Shaul

From the Departments of Pediatrics (K.L.C., I.S.Y., C.M., Z.G., T.S.S., P.W.S.) and Cell Biology (P.L., R.G.W.A.), University of Texas Southwestern Medical Center, Dallas, Tex; Molecular Cardiology Research Institute (M.E.M.), New England Medical Center, Tufts University School of Medicine, Boston, Mass.

Correspondence to Philip W. Shaul, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75235-9063. E-mail pshaul{at}mednet.swmed.edu


*    Abstract
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 *Abstract
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 down arrowMaterials and Methods
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 down arrowDiscussion
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Abstract—Estrogen causes nitric oxide (NO)-dependent vasodilation due to estrogen receptor (ER) {alpha}-mediated, nongenomic activation of endothelial NO synthase (eNOS). The subcellular site of interaction between ER{alpha} and eNOS was determined in studies of isolated endothelial cell plasma membranes. Estradiol (E2, 10–8 mol/L) caused an increase in eNOS activity in plasma membranes in the absence of added calcium, calmodulin, or eNOS cofactors, which was blocked by ICI 182,780 and ER{alpha} antibody. Immunoidentification studies detected the same 67-kDa protein in endothelial cell nucleus, cytosol, and plasma membrane. Plasma membranes from COS-7 cells expressing eNOS and ER{alpha} displayed ER-mediated eNOS stimulation, whereas membranes from cells expressing eNOS alone or ER{alpha} plus a myristoylation-deficient mutant eNOS were insensitive. Fractionation of endothelial cell plasma membranes revealed ER{alpha} protein in caveolae, and E2 caused stimulation of eNOS in isolated caveolae that was ER-dependent; noncaveolae membranes were insensitive. Acetylcholine and bradykinin also activated eNOS in isolated caveolae. Furthermore, the effect of E2 on eNOS in caveolae was prevented by calcium chelation. Thus, a subpopulation of ER{alpha} is localized to endothelial cell caveolae where they are coupled to eNOS in a functional signaling module that may regulate the local calcium environment. The full text of this article is available at http://www.circresaha.org.


Key Words: acetylcholine • bradykinin • caveolin • cell membrane • endothelium • estrogens


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The hormone estrogen classically exerts its effects by modifying gene expression through the activation of estrogen receptors (ERs), which serve as transcription factors.1 2 3 However, there are also rapid, presumably nongenomic effects of estrogen in a variety of tissues including the vasculature.4 5 6 Estrogen has important atheroprotective properties that are at least partially related to its capacity to enhance the bioavailability of nitric oxide (NO).5 6 7 NO is a potent regulator of blood pressure, platelet aggregation, leukocyte adhesion, and vascular smooth muscle mitogenesis that is produced in the vascular wall primarily by the endothelial isoform of NO synthase (eNOS) on the conversion of the substrate Ld-arginine to Ld-citrulline.8 The function of the Ld-arginine/eNOS system is altered in a variety of vascular disorders.9

We have previously shown that estrogen rapidly stimulates eNOS activity in endothelial cells, that the response is attenuated by ER antagonism but not by inhibiting gene transcription, and that ER{alpha} is expressed in endothelium.10 11 We have also shown that the overexpression of ER{alpha} in endothelial cells causes enhancement of the acute response to estradiol (E2) that is blocked by ER antagonism, specific to E2 versus other agonists, and dependent on the ER{alpha} hormone binding domain. In addition, the acute stimulation of eNOS by E2 can be reconstituted in COS-7 cells cotransfected with wild-type ER{alpha} and eNOS.11 Thus, the short-term effects of estrogen on eNOS that are central to cardiovascular physiology are mediated by ER{alpha} functioning in a novel, nongenomic manner. However, the subcellular site of interaction between ER{alpha} and eNOS is unknown.

Although it is not firmly established, studies using immunoidentification or conjugated estrogen suggest that a subpopulation of ERs may be associated with the cell surface in certain cell types.12 13 There is strong evidence that eNOS is targeted to the endothelial plasma membrane, particularly to caveolae, which are specialized, cholesterol-rich domains that compartmentalize signal transduction.14 15 16 We therefore designed experiments to localize the interaction between endothelial cell ER{alpha} and eNOS, raising the hypothesis that a subpopulation of ER{alpha} and eNOS are colocalized and functionally coupled on the endothelial plasma membrane. To evaluate the presence of additional molecules required for eNOS activation, 3H-Ld-arginine conversion to 3H-Ld-citrulline was measured in isolated membranes in the absence of added calcium, calmodulin, or eNOS cofactors. We also assessed whether ER{alpha} and eNOS are further colocalized and functionally linked in plasmalemmal caveolae, whether the caveolae scaffolding protein caveolin-1 plays a role in the membrane association of ER{alpha}, whether other agonists whose receptors are found in caveolae activate eNOS directly in this microdomain, and whether the localization of ER{alpha}-eNOS interaction may clarify the role of calcium in the process.


*    Materials and Methods
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 *Materials and Methods
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Cell Culture
Immortalized ovine pulmonary artery endothelial cells (iPAECs) and COS-7 cells were propagated as previously described.11 17 Near-confluent iPAECs were studied at passages 16 to 26.

Subcellular Fractionation
Highly purified plasma membrane and cytosolic fractions were prepared using modifications of a detergent-free method.14 18 Additional studies were performed with caveolae membranes isolated from iPAEC plasma membranes using a method that takes advantage of the unique buoyant density of caveolae.14 18 Plasma membrane purity and the exclusion of other cellular components were confirmed with measurements of alkaline phosphatase (plasma membrane), galactosyl transferase (Golgi), and NADPH cytochrome c reductase (endoplasmic reticulum) activity.19 All fractionation steps were done in the absence of exogenous calcium. Successful separation of caveolae from noncaveolae plasma membrane was confirmed by immunoblot analyses for the caveolae marker protein caveolin-1 and the noncaveolae protein RACK-1.20 The protein contents of all samples were determined with the method of Bradford.21

NOS Activation
For determinations of NOS activation, purified whole-plasma membranes or noncaveolae or caveolae subfractions of plasma membrane were reconstituted in 50 mmol/L Tris HCl buffer (pH 7.4) with 0.1 mmol/L EDTA, 10 µg/mL pepstatin A, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 10 µg/mL N{alpha}-p-tosyl-Ld-lysine chloromethyl ketone, 10 nmol/L phenylmethylsulfonyl fluoride, 3 mmol/L dithiothreitol, and 10 mmol/L 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Membranes (10 µg plasma membrane or 2 µg noncaveolae or caveolae membrane protein in 100 µL volume) were exposed to 2.0 µCi/mL of 3H-Ld-arginine and 2 µmol/L cold Ld-arginine, and citrulline generation was evaluated during 15- to 60-minute incubations at 37°C. Studies were performed in the absence of added calcium, calmodulin, or eNOS cofactors to assess the presence of these molecules, which are required for eNOS activation. The assay was terminated by the addition of 400 µL of 40 mmol/L HEPES buffer, pH 5.5, with 2 mmol/L EDTA and 2 mmol/L EGTA. 3H-Ld-citrulline was purified with 1 mL Dowex AG50WX-8 columns and quantified by liquid scintillation spectroscopy. In selected studies, NOS activation was also determined in cytosolic fractions. NOS activity in all samples was inhibited by the addition of 2 mmol/L nitro-Ld-arginine methyl ester. To assess maximal eNOS activity, studies were done in the presence of 2 mmol/L ß-NADPH, 20 µmol/L tetrahydrobiopterin, 10 µmol/L flavin adenine dinucleotide, 10 µmol/L flavin mononucleotide, 0.5 mmol/L CaCl2 in excess of EDTA, and 15 nmol/L calmodulin. To reveal the role of membrane-associated ERs in E2 responses, studies were done in the absence or presence of the ER antagonist ICI 182,780 or the mouse monoclonal antibody TE111 directed against the ligand binding domain (amino acids 302 to 553) of human ER{alpha} (4 µg/100 µL, Neomarkers, Inc) or unrelated IgG (4 µg/100 µL). The role of membrane-associated calcium was assessed in studies performed in the absence or presence of added calcium (2.5 mmol/L CaCl2) or in the absence of added calcium with 2.5 mmol/L EGTA added.

The capacities of E2 (10–8 mol/L), acetylcholine (10–6 mol/L),22 and bradykinin (10–6 mol/L)23 to stimulate eNOS in intact endothelial cells were compared using previously described methods.10 In addition, the abilities of the agents to activate eNOS in isolated membranes were evaluated as outlined above.

Immunoprecipitation
Immunoprecipitation was done using methods modified from those previously described.24 Plasma membrane fractions (200 µg protein) were diluted with an equal volume of 2x buffer (pH 7.4) containing 50 mmol/L Tris HCl, 150 mmol/L NaCl, 1% NP-40, 0.25% Na deoxycholate, 1 mmol/L EDTA, and protease inhibitors and incubated with 2 µg of mouse monoclonal antibody directed against amino acids 495 to 595 of human ER{alpha} (AER320, Neomarkers, Inc) or antiserum to caveolin-1 (Transduction Laboratories) at 4°C overnight. The sample was further incubated for 2 hours with 25 µL of protein A/G agarose beads (Calbiochem), and washed twice with ice-cold 25 mmol/L Tris HCl buffer (pH 7.4) containing 150 mmol/L NaCl, 5 mmol/L EDTA, and 1% Triton X-100 and twice with 10 mmol/L Tris HCl buffer (pH 7.5) with 5 mmol/L EDTA. Immunoprecipitated proteins were eluted from the beads by boiling for 3 minutes in SDS sample buffer and separated by SDS-polyacrylamide gel electrophoresis.

Immunoblot Analyses
Immunoblot analyses were performed to evaluate the distribution of ER{alpha}, eNOS, and caveolin-1.14 Equivalence of protein loads was confirmed by amido black staining (Sigma Chemical Co). The analyses used mouse monoclonal antibodies directed against either amino acids 495 to 595 (AER320, 2.5 µg/mL), 302 to 553 (TE111, Neomarkers, Inc, 1 µg/mL), or 120 to 170 of human ER{alpha} (AER304, Neomarkers, Inc, 1 µg/mL), antiserum to eNOS (1:2000, a gift of Dr Thomas Michel, Harvard Medical School), or antiserum to caveolin-1 (0.05 µg/mL).

Cell Transfection Experiments
COS-7 cells were transiently transfected with a C-terminal c-myc–tagged human ER{alpha} construct (ER{alpha}-myc) using Lipofectamine Plus (Life Technologies, Inc) as previously reported.11 pCMV3-ER{alpha} was first constructed by cloning full-length human ER{alpha} cDNA into the EcoRI site of pCDNA3.1 (Invitrogen Corp).11 25 ER{alpha}-myc was created by excision of the ER{alpha} insert of pCMV3-ER{alpha} using BamHI and XhoI followed by ligation of the insert into the same sites of the pCMV-Tag1 vector (Stratagene Cloning Systems). The stop codon was removed and the ER{alpha} sequence was placed in-frame with the c-myc epitope tag by polymerase chain reaction. The construct was confirmed by DNA sequencing. Membranes were studied from COS-7 cells transiently cotransfected with eNOS cDNA and either ER{alpha} cDNA or sham plasmid 72 hours earlier.11 Additional studies were done with membranes from cells cotransfected with a myristoylation-deficient mutant of eNOS14 and ER{alpha} cDNA. Coimmunofluorescence experiments revealed that transfection efficiency was {approx}20% for either ER{alpha} or eNOS, and that at least one-half to two-thirds of transfected cells expressed both ER{alpha} and eNOS.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
eNOS Activation by E2
To evaluate plasma membrane eNOS stimulation by E2, NOS activity was measured in plasma membranes isolated from iPAECs. In the absence of added calcium, calmodulin, or cofactors, NOS activity was detectable in quiescent membranes, yielding 2.4±0.1 pmol citrulline/mg protein per minute (n=4). This basal level of NOS activity was inhibited by calcium chelation (data not shown). In the absence of added calcium, calmodulin, or cofactors, 10–8 mol/L E2 (15 minutes) caused a 92% increase in NOS activity compared with basal levels (Figure 1ADown). Neither basal nor E2-stimulated NOS activity was detectable in the cytosolic fraction, and in contrast to 17ß-estradiol (E2), 17{alpha}-estradiol had no effect on NOS activity in isolated plasma membranes (data not shown). To assess maximal NOS activity, E2 was replaced with a mixture of calcium, calmodulin, and cofactors, yielding a 170% rise in activity compared with no additives; E2 (10–8 mol/L) did not enhance this activity. Time course experiments in the presence of E2 alone revealed a progressive, linear increase in NOS activity during the first 30 minutes of incubation, followed by a plateau (Figure 1BDown). This contrasts with NOS activity with added calcium, calmodulin, and cofactors, which displayed linearity with time for at least 120 minutes (data not shown). In dose-response studies, NOS stimulation was evident at hormone concentrations of 10–8 mol/L and greater (Figure 1CDown).



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  		Figure 1. Figure 1Up. Activation of eNOS in endothelial cell plasma membranes. A, Effect of E2 on NOS activity. 3H-Ld-arginine conversion to 3H-Ld-citrulline was measured in isolated plasma membranes over 15 minutes in the absence (basal) or presence of 10–8 mol/L E2 and in the absence or presence of exogenous eNOS cofactors, calcium, and calmodulin (CM). B, Time course of the effect of E2 on NOS activity. Membrane incubations were performed without added eNOS cofactors, calcium, and calmodulin, in the absence (basal) or presence of 10–8 mol/L E2 for 15 to 60 minutes. C, Dose-response effect of E2 on NOS activity. Membrane incubations were performed without added eNOS cofactors, calcium, or calmodulin, in the absence (basal) or presence of 10–10 to 10–6 mol/L E2 for 15 minutes. Values are mean±SEM, n=4 to 6. *P<0.05 vs basal; {dagger}P<0.05 vs no added cofactors, calcium, or calmodulin.

Role of ER
The role of ERs in E2 activation of plasma membrane eNOS was examined using the ER antagonist ICI 182,780 (Figure 2ADown). E2-stimulated NOS activity was prevented by ICI 182,780. In addition, antibody to the ligand binding domain of ER{alpha} (TE111) blocked E2-stimulated NOS activation, whereas unrelated IgG had no effect (Figure 2BDown). Immunoblot analyses detected ER{alpha} protein (67 kDa) in whole-cell lysates and plasma membranes (Figure 2CDown). As anticipated, eNOS protein was also readily detected in whole-cell lysates and plasma membrane.



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  		Figure 2. Figure 2Up. Role of ER{alpha} in eNOS activation by E2 in endothelial cell plasma membranes. A, Effect of ER antagonism on response to E2. 3H-Ld-arginine conversion to 3H-Ld-citrulline was measured over 15 minutes in isolated plasma membranes without added eNOS cofactors, calcium, and calmodulin, in the absence (basal) or presence of 10–8 mol/L E2 with or without 10–5 mol/L ICI 182,780 added. B, Effect of ER{alpha} antibody on response to E2. Membrane incubations were performed over 15 minutes without added eNOS cofactors, calcium, and calmodulin, in the absence (basal) or presence of 10–8 mol/L E2 with or without antibody to ER{alpha} (TE111) or unrelated IgG added. C, Immunoblot analysis for ER{alpha} and eNOS in endothelial whole-cell lysates (WC) and plasma membranes (PM). Signals for ER{alpha} and eNOS were evident at 67 and 135 kDa, respectively. Values are mean±SEM, n=4 to 6. *P<0.05 vs basal; {dagger}P<0.05 vs E2 alone. Results are representative of 3 independent experiments.

The identity of plasma membrane–associated ERs was further evaluated by comparison with cytosolic and nuclear ERs in immunoblot analyses using antibodies directed against 3 different ER{alpha} epitopes. Antibodies directed against amino acids 495 to 595 (AER320), 302 to 553 (TE111), or 120 to 170 of human ER{alpha} (AER304) all detected a single 67-kDa protein species in endothelial cell plasma membranes that was identical in size to the protein detected in nuclear and cytosolic fractions (Figure 3ADown). To confirm these observations, we determined whether epitope-tagged ER{alpha} introduced into COS-7 cells is targeted to plasma membrane (Figure 3BDown). Whereas antibody to ER{alpha} revealed no signal in sham-transfected cells, there was a positive signal for ER{alpha} of comparable size in nucleus, cytosol, and plasma membrane from cells transfected with ER{alpha}-myc. In parallel, immunoblot analysis with antibody to the myc tag revealed no signal in sham-transfected cells, but a similarly sized protein was detected in the nucleus, cytosol, and plasma membranes of cells expressing the tagged receptor. To determine whether ER{alpha} is recruited to the plasma membrane on E2 stimulation, immunoprecipitations for ER{alpha} were performed on plasma membranes isolated from iPAECs or COS-7 cells transfected with ER{alpha} cDNA, at baseline and after 20 minutes of E2 (10–8 mol/L) exposure. In immunoblots for plasma membrane ER{alpha}, there was no discernible increase in the abundance of the protein with E2 treatment (Figure 3CDown).



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  		Figure 3. Figure 3Up. Characterization of plasma membrane–associated ERs. A, Immunoblot analysis for ER{alpha} in endothelial cell nucleus (Nuc), cytosol (Cyt), and plasma membrane (PM). The monoclonal antibodies used were directed against amino acids 495 to 595 (AER320), 302 to 553 (TE111), or 120 to 170 (AER304) of human ER{alpha}. B, Targeting of epitope-tagged ER{alpha} to plasma membranes. After transient transfection of COS-7 cells with myc-tagged ER{alpha}, immunoblot analysis was performed for ER{alpha} and myc on cell fractions. Whole-cell lysate from a prior COS-7 cell transfection was used as a positive control (Con). C, Effect of E2 on ER{alpha} abundance in plasma membrane. iPAECs or COS-7 cells transfected with ER{alpha} cDNA were incubated in the absence (-) or presence (+) of E2 (10–8 mol/L) for 20 minutes, and plasma membranes were isolated and immunoprecipitated with anti-ER{alpha} antibody. Immunoprecipitated proteins were analyzed by immunoblot analysis for ER{alpha}. Results are representative of 3 independent experiments.

Requirement for Colocalization on the Plasma Membrane
Transient transfection of COS-7 cells was also used to determine the importance of plasma membrane colocalization for ER{alpha}-stimulated eNOS activity. In plasma membranes from COS-7 cells transfected with eNOS alone, E2 had no effect on NOS activity (Figure 4ADown). However, in plasma membranes from cells transfected with both eNOS and ER{alpha}, E2 caused a >150% increase in NOS activity that was ER-dependent (Figure 4BDown). To determine whether normal plasma membrane targeting of eNOS is required for ER-mediated activation of the enzyme, membranes from COS-7 cells cotransfected with myristoylation-deficient mutant eNOS and ER{alpha} were studied. Myristoylation-deficient mutant eNOS is minimally targeted to plasma membrane compared with wild-type eNOS.14 Measurements of NOS activity in cell lysates revealed that cells transfected with wild-type or mutant eNOS expressed similar amounts of the enzyme, with mutant eNOS activity being 98±3% of wild-type (n=4). However, as anticipated, basal NOS activity in plasma membranes determined in the absence of added calcium, calmodulin, and cofactors was markedly diminished for myristoylation-deficient mutant eNOS compared with wild-type (1.07±0.08 versus 17.9±2.0 pmol citrulline/mg protein per minute, respectively, P<0.05, n=4). In contrast to the findings for ER-mediated stimulation of wild-type enzyme (Figure 4BDown), E2 caused a decline in NOS activity in plasma membranes from cells transfected with mutant eNOS plus ER{alpha} (Figure 4CDown), and this effect was partially reversed by ICI 182,780.



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  		Figure 4. Figure 4Up. Reconstitution of E2 response in plasma membranes from COS-7 cells. Transfections were performed with human eNOS cDNA and either sham plasmid (A) or ER{alpha} cDNA (B), and NOS activation was determined 72 hours later in isolated plasma membranes over 15 minutes. Membrane incubations were done without added eNOS cofactors, calcium, and calmodulin, in the absence (basal) or presence of 10–8 mol/L E2, with or without 10–5 mol/L ICI 182,780 added. Additional studies were performed in plasma membranes from cells transfected with myristoylation-deficient mutant eNOS and wild-type ER{alpha} (C). Values are mean±SEM, n=4 to 6. *P<0.05 vs basal; {dagger}P<0.05 vs E2 alone.

Localization of ER-eNOS Interaction to Caveolae
Because plasma membrane eNOS is exclusively in caveolae,14 experiments were done to determine whether ER{alpha} protein is also associated with this subfraction of endothelial cell plasma membranes. Immunoblot analyses for caveolin-1 confirmed separation of caveolae and noncaveolae fractions (Figure 5ADown). ER{alpha} protein was detected in caveolae, and it was also detected, but to a lesser extent, in the noncaveolae fraction.



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  		Figure 5. Figure 5Up. Localization of ER-eNOS interaction to caveolae. A, Immunoblot analysis for ER{alpha} and caveolin-1 in noncaveolae membranes (NCM) and caveolae membranes (CAV) obtained from endothelial cell whole-plasma membranes. Signals for ER{alpha} and caveolin-1 were evident at 67 and 22 kDa, respectively. Results are representative of 3 independent experiments. B, E2-mediated activation of eNOS in endothelial cell caveolae membranes. 3H-Ld-arginine conversion to 3H-Ld-citrulline was measured in noncaveolae and caveolae membranes obtained from endothelial cell plasma membranes. Membrane incubations were performed over 60 minutes without added eNOS cofactors, calcium, and calmodulin, in the absence (basal, B) or presence of 10–8 mol/L E2 with or without 10–5 mol/L ICI 182,780 added. NOS activity was undetectable in noncaveolae fractions in all groups, and it was also not detected in caveolae under basal conditions. Values are mean±SEM, n=4 to 6. *P<0.05 vs basal; {dagger}P<0.05 vs E2 alone. C, Role of caveolin-1 in ER{alpha} association with plasma membrane. COS-7 cells transfected with eNOS and ER{alpha} cDNAs were incubated in the absence (lane 1) or presence (lane 2) of E2 (10–8 mol/L) for 20 minutes, and plasma membranes were isolated and immunoprecipitated with anti–caveolin-1 antibody (left). A negative control without plasma membranes is shown in lane 3. Immunoprecipitated proteins were analyzed by Western blot analyses (WB) for eNOS, ER{alpha}, or caveolin-1. IgG heavy chain (IgG HC) was detectable at 55 to 60 kDa. Blots of whole-cell lysate revealed equivalent eNOS and ER{alpha} expression between study groups (right). Results are representative of 3 independent experiments.

Experiments were then performed to evaluate the capacity of E2 to activate eNOS in caveolae and noncaveolae fractions (Figure 5BUp). In the absence of added calcium, calmodulin, or cofactors, there was no measurable NOS activity in the noncaveolae fraction under basal conditions or with E2 added. Basal NOS activity was also below detection limits in caveolae membranes. However, 10–8 mol/L E2 caused robust activation of NOS in caveolae membranes, and this effect was prevented by ICI 182,780.

To begin to explore the mechanisms responsible for ER{alpha} localization to plasmalemmal caveolae, the potential role of caveolin was assessed in immunoprecipitation experiments with caveolin-1 antibody using plasma membranes isolated from COS-7 cells transfected with ER{alpha} and eNOS. In plasma membranes from quiescent cells, eNOS was coimmunoprecipitated with caveolin-1 (Figure 5CUp); this association was decreased after 20 minutes of E2 stimulation. In contrast, no association was detected between caveolin-1 and ER{alpha} in plasma membranes under either basal or stimulated conditions. Immunoblots of whole-cell lysates confirmed comparable eNOS and ER{alpha} expression in both study groups. Similar to the findings in plasma membrane, no association was observed between caveolin-1 and ER{alpha} in coimmunoprecipitations performed on whole-cell lysates (data not shown).

To generalize the observation of eNOS activation in caveolae to other agonists whose receptors are known residents of the microdomain, responses to E2 and acetylcholine were compared. Experiments in intact endothelial cells revealed similar eNOS stimulation by the two agents (Figure 6ADown). In isolated membranes studied in the absence of added calcium, calmodulin, or cofactors, there was no measurable NOS activity in the noncaveolae fraction under basal conditions, nor with E2 or acetylcholine added (Figure 6BDown). Basal NOS activity was also nondetectable in caveolae membranes. However, 10–8 mol/L E2 and 10–6 mol/L acetylcholine caused equivalent robust eNOS activation in caveolae membranes. Similarly, bradykinin (10–6 mol/L) activated eNOS to a level of 87±7 pmol citrulline/mg protein per minute (n=4) in the caveolae fraction but had no effect in noncaveolae membranes.



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  		Figure 6. Figure 6Up. Comparison of eNOS activation by E2 and acetylcholine in intact cells and isolated caveolae. A, E2- and acetylcholine-mediated stimulation of eNOS in intact endothelial cells. 3H-Ld-arginine conversion to 3H-Ld-citrulline was measured over 15 minutes in the absence (basal, B) or presence of 10–8 mol/L E2 or 10–6 mol/L acetylcholine (Ach). Results are expressed as percent of basal NOS activity. B, E2- and acetylcholine-mediated activation of eNOS in endothelial cell caveolae membranes. 3H-Ld-arginine conversion to 3H-Ld-citrulline was measured in noncaveolae and caveolae membranes obtained from endothelial cell plasma membranes. Membrane incubations were performed over 60 minutes without added eNOS cofactors, calcium, and calmodulin, in the absence (basal, B) or presence of 10–8 mol/L E2 or 10-6 mol/L acetylcholine. NOS activity was undetectable in noncaveolae fractions in all groups, and it was also not detected in caveolae under basal conditions. Values are mean±SEM, n=4 to 6. *P<0.05 vs basal.

Role of Calcium
The role of calcium in E2-mediated activation of eNOS has been controversial.10 26 27 We therefore determined the effect of exogenous calcium on the response to E2 in isolated plasma membranes. The level of eNOS activity with E2 stimulation in the absence of added calcium was comparable to that obtained by adding calcium alone, and there was no additional increase with E2 plus calcium (Figure 7ADown). We also examined the effect of calcium chelation in isolated plasma membranes and caveolae studied in the absence of exogenous calcium. E2-stimulated eNOS activity in plasma membranes was completely blocked by EGTA (Figure 7BDown). Similarly, calcium chelation fully prevented eNOS activation by the hormone in isolated caveolae membranes (Figure 7CDown).



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  		Figure 7. Figure 7Up. Role of calcium in eNOS activation by E2 in endothelial cell plasma membranes and caveolae. A, Effect of exogenous calcium on eNOS activation in isolated plasma membranes. 3H-Ld-arginine conversion to 3H-Ld-citrulline was measured over 15 minutes in isolated plasma membranes without added eNOS cofactors, in the absence of E2 or exogenous calcium (basal), with E2 (10–8 mol/L) or calcium (2.5 mmol/L CaCl2) added, or with both E2 and calcium added. B, Effect of calcium chelation on eNOS activation in isolated plasma membranes. Membranes were incubated (15 minutes) in the absence (basal) or presence of 10–8 mol/L E2 with or without 2.5 mmol/L EGTA added. C, Effect of calcium chelation on eNOS activation in isolated caveolae. 3H-Ld-arginine conversion to 3H-Ld-citrulline was measured in noncaveolae and caveolae membranes obtained from endothelial cell plasma membranes. Membrane incubations were performed over 60 minutes without added eNOS cofactors, calcium, and calmodulin, in the absence (basal, B) or presence of 10–8 mol/L E2 with or without 2.5 mmol/L EGTA added. Values are mean±SEM, n=4 to 6. *P<0.05 vs basal; {dagger}P<0.05 vs E2 alone.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
In the present study, we have shown that E2 causes acute, ER-dependent activation of eNOS in plasma membranes isolated from endothelial cells. These findings indicate that the ER-mediated stimulation of the enzyme previously observed in intact endothelium occurs directly at the cell surface, and, therefore, that ERs have roles that are quite distinct from their classical action as nuclear transcription factors.1 2 3 To our knowledge, this is the first demonstration of ER function in isolated plasma membranes from any source.

Physiological concentrations of E2 (10–8 mol/L) caused eNOS activation in endothelial plasma membranes within 15 minutes of exposure to the hormone, which is consistent with the nongenomic nature of the response in arteries and intact endothelial cells.5 6 10 11 The response in plasma membranes was elicited without the addition of calcium, calmodulin, or eNOS cofactors during membrane preparation or the incubations for NOS activity. Furthermore, although both ER{alpha} and eNOS are found in endothelial cell cytosol,14 their functional coupling was not evident in cytosolic fractions. These observations indicate that all of the signal transduction machinery necessary for eNOS stimulation by E2 is associated with the plasma membrane. E2 alone activated the enzyme to approximately half of maximal levels, indicating that the response is quite robust. eNOS activation with E2 alone plateaued after 30 minutes but did not plateau in the presence of added calcium, calmodulin, and cofactors (data not shown), suggesting that the availability of one or more of these molecules is limited in the isolated membranes. In intact cells, there are most likely mechanisms that replenish these factors in the locale of the plasma membrane.

Having demonstrated that E2 activates eNOS in the plasma membrane, the role of estrogen receptors was evaluated. E2-mediated stimulation of the enzyme was prevented by concomitant membrane treatment with ICI 182,780 or antiserum to the ligand binding domain of ER{alpha}. In addition, ER{alpha} protein was detected in endothelial cell plasma membranes along with eNOS, and 3 antibodies directed to different ER{alpha} epitopes revealed an identical, single 67-kDa protein species in endothelial cell plasma membranes, cytosol, and nucleus. Epitope-tagged ER{alpha} introduced into COS-7 cells was also present in the plasma membrane. Studies of plasma membrane–associated ER{alpha} before and after E2 exposure did not reveal recruitment to the cell surface on agonist stimulation. These cumulative findings indicate that E2-stimulated eNOS activity is mediated by a subpopulation of ER{alpha} that is associated with the plasma membrane before receptor activation.

The requirement for plasma membrane colocalization for ER{alpha}-stimulated eNOS activity was evaluated by reconstituting ER{alpha}-eNOS interactions in COS-7 cells. Plasma membranes from cells expressing eNOS and ER{alpha} displayed rapid ER-mediated NOS stimulation, whereas membranes from cells expressing eNOS alone or ER{alpha} plus myristoylation-deficient mutant eNOS were insensitive. In fact, membranes from cells expressing myristoylation-deficient mutant eNOS and ER{alpha} displayed a decline in NOS activity with E2 that was partially reversed by ICI 182,780. Because myristoylation-deficient mutant eNOS is minimally directed to the plasma membrane but unaltered in enzymatic activity,14 these findings indicate that both normal plasma membrane targeting of eNOS and localization of ERs to that site are required for eNOS activation by E2.

Although it currently remains a topic of controversy, there is evidence for cell surface ER{alpha} in certain cell types including immunoidentification studies of GH3/B6/F10 pituitary cells and MCF-7 human breast cancer cells using multiple ER{alpha} antibodies.12 13 28 29 However, immunolocalization does not reveal whether the cell surface protein being recognized is functional. To evaluate such a possibility, impeded ligands such as E2 conjugated to BSA (E2-BSA) have been used, including in a recent report in human umbilical vein endothelial cells.12 13 However, it is important to note that freshly prepared solutions of E2-BSA contain free immunoassayable E2, E2-BSA does not bind to ER, and certain E2-BSA preparations are of very high molecular weight. This suggests extreme protein crosslinking,30 indicating that E2-BSA does not mimic unconjugated E2. The present work with isolated endothelial plasma membranes directly demonstrates for the first time that there is a subpopulation of functional ER{alpha} associated with the plasma membrane in cells expressing a constitutive level of the receptor. Confounding nuclear effects of E2 are uniquely avoided by the use of isolated plasma membranes. Furthermore, identical findings were obtained with plasma membranes from COS-7 cells expressing ER{alpha} and eNOS.

Further experiments were done to determine whether ER{alpha} coupling to eNOS occurs in plasmalemmal caveolae, which are cholesterol-rich membrane domains that serve to compartmentalize numerous signal transduction molecules including eNOS.16 ER{alpha} protein was found in endothelial cell caveolae, and it was also detected, but at lower levels, in the noncaveolae fraction. In the absence of added calcium, calmodulin, and eNOS cofactors, E2 caused a dramatic activation of eNOS in caveolae membranes that was ER-dependent, whereas noncaveolae membranes were insensitive. The findings in caveolae are consistent with a recent report that was limited solely to immunoblot analysis of the caveolae fraction.31 Interestingly, we have previously shown in intact cells that eNOS stimulation by E2 involves activation of the tyrosine kinase–mitogen-activated protein kinase pathway,11 which is a signaling cascade that has been localized to caveolae in human fibroblasts.32 The present data strongly indicate that ER{alpha} and all of the additional molecular machinery necessary for E2-mediated activation of eNOS exist in a functional signaling module in endothelial caveolae. Because ER{alpha} was found in both caveolae and noncaveolae fractions and eNOS is solely in caveolae,14 the specificity of ER{alpha} coupling to eNOS to caveolae is evidently due to the localization of the effector, and not the receptor, in this microdomain.

Studies were performed to begin the exploration of the mechanisms underlying ER{alpha} localization to plasmalemmal caveolae in a system in which ER{alpha} coupling to eNOS is demonstrable. Coimmunoprecipitation experiments were done using plasma membranes from COS-7 cells transfected with ER{alpha} and eNOS, which express endogenous caveolin-1. The previously described association between caveolin-1 and eNOS, which diminishes with activation, was evident,27 serving as a positive control for protein interaction with caveolin-1 on the plasma membrane. In contrast, caveolin-1–ER{alpha} association was not apparent in plasma membranes under any conditions, nor was it evident in coimmunoprecipitations done on whole-cell lysates (data not shown). Our findings differ from those of Schlegel et al,33 who demonstrated coimmunoprecipitation of ER{alpha} and caveolin-1 in cell lysates of 293T cells transfected with both cDNAs. In overexpression paradigms, these investigators have also shown that caveolin-1 is a positive regulator of ER{alpha} nuclear translocation and function. The disparate findings may be explained by differences in methodology or the cell types used or perhaps more likely by the study of endogenous versus overexpressed levels of caveolin-1. Because ER{alpha} coupling to eNOS function was readily apparent in plasma membranes in the present work in the absence of evidence of ER{alpha} interaction with membrane-associated caveolin-1, alternative mechanisms of ER{alpha} targeting should also be considered. Along with revealing a lack of known caveolin interaction motifs, sequence analysis of ER{alpha} yields no known acylation or prenylation motifs that would target the receptor protein to caveolae lipids.34 35 36 Detailed mutagenesis studies are now warranted to reveal the specific processes by which a subpopulation of ER{alpha} is uniquely targeted to and functional in caveolae.

The effects of other agonists whose receptors are found in caveolae were also determined. Both acetylcholine and bradykinin caused eNOS stimulation in isolated caveolae. To date, the mere localization of molecules involved in receptor-mediated eNOS activation in caveolae has been presumed to imply functional interactions that have never been demonstrated. The present studies are the first to show that signal coupling occurs in caveolae leading to eNOS stimulation, indicating that all required molecules are present and mechanistically linked in the domain. For acetylcholine, bradykinin, and a variety of other agonists, the signaling partners include caveolae-associated G proteins.16 37 There is also evidence in an overexpression paradigm that G proteins may be involved in ER{alpha}-initiated, membrane-associated signaling events.38 However, considerable additional work will be required to determine the potential role of G proteins in ER signaling under physiological conditions.

The role of calcium in E2-mediated activation of eNOS has been controversial. Certain previous investigations have indicated that the process is calcium-dependent, whereas others have suggested calcium independence because cytosolic free calcium concentrations were unaltered.10 26 27 In the present experiments, the addition of E2 alone to isolated plasma membranes yielded eNOS activity comparable to that observed with the provision of exogenous calcium, and E2 plus calcium was not additive. Furthermore, in the absence of added calcium, calcium chelation completely blocked E2-stimulated eNOS activity in both isolated plasma membranes and caveolae. Previous studies using potassium oxalate precipitation have shown that calcium is highly localized to the cell surface and in particular to caveolae in certain cell types.39 The present observations suggest that there is a pool of caveolae-associated calcium that is released upon E2 stimulation, leading to a localized increase in calcium that causes eNOS activation without requiring a global rise in cytosolic free calcium. The trace amounts of calcium that may be present in the incubation buffer are most likely not the source underlying eNOS activation with E2 in caveolae because eNOS activity is not detectable in the buffer in the absence of agonist. Isolated endothelial cell caveolae should provide a unique modeling system in which the mechanisms regulating the calcium environment and other signal transduction events at the plasma membrane can be elucidated.

The observation that physiologically relevant activation of eNOS occurs within a signaling module in plasma membrane caveolae has important implications on endothelial cell biology. We have recently shown that oxidized LDL depletes endothelial caveolae of cholesterol, resulting in the displacement of eNOS from caveolae and impaired eNOS activation.19 Furthermore, HDL maintains the concentration of caveolae-associated cholesterol, thereby preventing the negative impact of oxidized LDL on eNOS targeting and activation.40 The present findings indicate that caveolar colocalization of eNOS with partner signaling molecules, such as ER{alpha}, is required for activation of the enzyme by extracellular stimuli such as E2. Changes in membrane cholesterol balance may disrupt this signaling module, resulting in impaired endothelial cell NO production and the onset of the pathogenetic cascade characteristic of atherosclerosis.41

The localization of an intact, functional eNOS signaling module to caveolae is further evidence that this membrane domain compartmentalizes signal transduction at the cell surface.16 A signaling module consists of transducers, effectors, adaptors, and scaffolds that are connected together to form a functional signaling pathway.42 Our results strongly indicate that the functionality of the eNOS module is dependent on its localization to caveolae. Future studies of the signaling circuitry within this module will provide valuable new clues about the role of compartmentalization in signal transduction.


*    Acknowledgments
 

This work was supported by National Institutes of Health grants HL58888, HL53546, and HD30276 (to P.W.S.), GM52016 (to R.G.W.A.), and HL56069 and HL59953 (to M.E.M.). This project was supported in part by the Lowe Foundation and the Perot Family Foundation. We are indebted to Marilyn Dixon for preparing this manuscript.

Received September 19, 2000; revision received October 23, 2000; accepted October 24, 2000.


*    References
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 *References
 

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