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(Circulation Research. 2006;99:870.)
© 2006 American Heart Association, Inc.

Cellular Biology

Novel Mechanism of Endothelial Nitric Oxide Synthase Activation Mediated by Caveolae Internalization in Endothelial Cells

Nikolaos A. Maniatis, Viktor Brovkovych, Scott E. Allen, Theresa A. John, Ayesha N. Shajahan, Chinnaswamy Tiruppathi, Stephen M. Vogel, Randal A. Skidgel, Asrar B. Malik, Richard D. Minshall

From the Departments of Pharmacology (N.A.M., V.B., S.E.A., T.A.J., A.N.S., C.T., S.M.V., R.A.S., A.B.M., R.D.M.) and Anesthesiology (R.D.M.) and the Center for Lung and Vascular Biology (V.B., C.T., S.M.V., R.A.S., A.B.M., R.D.M.), University of Illinois College of Medicine, Chicago.

Correspondence to Richard D. Minshall, University of Illinois, Department of Pharmacology (m/c 868), 835 S Wolcott Ave, Chicago, IL 60612. E-mail rminsh{at}uic.edu

	Abstract

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Caveolin-1, the caveolae scaffolding protein, binds to and negatively regulates eNOS activity. As caveolin-1 also regulates caveolae-mediated endocytosis after activation of the 60-kDa albumin-binding glycoprotein gp60 in endothelial cells, we addressed the possibility that endothelial NO synthase (eNOS)-dependent NO production was functionally coupled to caveolae internalization. We observed that gp60-induced activation of endocytosis increased NO production within 2 minutes and up to 20 minutes. NOS inhibitor N^G-nitro-L-arginine (L-NNA) prevented the NO production. To determine the role of caveolae internalization in the mechanism of NO production, we expressed dominant-negative dynamin-2 mutant (K44A) or treated cells with methyl-ß-cyclodextrin. Both interventions inhibited caveolae-mediated endocytosis and NO generation induced by gp60. We determined the role of signaling via Src kinase in the observed coupling of endocytosis to eNOS activation. Src activation induced the phosphorylation of caveolin-1, Akt and eNOS, and promoted dissociation of eNOS from caveolin-1. Inhibitors of Src kinase and Akt also prevented NO production. In isolated perfused mouse lungs, gp60 activation induced NO-dependent vasodilation, whereas the response was attenuated in eNOS^–/– or caveolin-1^–/– lungs. Together, these results demonstrate a critical role of caveolae-mediated endocytosis in regulating eNOS activation in endothelial cells and thereby the NO-dependent vasomotor tone.

Key Words: caveolin-1 • endocytosis • vasomotor tone • albumin • transcytosis

	Introduction

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Endothelial NO synthase (eNOS) is modified by N-myristoylation and palmitoylation, which targets the enzyme to caveolae,¹ the plasma membrane cholesterol-rich microdomains.^2,3 Multiple mechanisms are involved in regulating NO production following eNOS activation. eNOS activity is regulated by Ca²⁺-calmodulin, phosphorylation activated by kinases such as Src and interactions with caveolin-1, dynamin, and heat shock protein 90 (HSP90).⁴ The effects of phosphorylation are complex. Phosphorylation of Ser116 and Thr497 negatively regulates eNOS activity, whereas phosphorylation at Ser635 and Ser1179 has the opposite effect.^5–7 Src kinase activated eNOS by inducing phosphorylation at Tyr83.⁸ Phosphorylation at Ser617 functions by affecting the phosphorylation of the above residues.⁹ Insulin, estrogen, and shear stress were shown to induce phosphorylation-dependent activation of eNOS at Ser1179 independent of increased intracellular [Ca²⁺]^10–12

eNOS in caveolae is held inactive by its association with caveolin-1,¹³ but eNOS activity can be increased by Ca²⁺/calmodulin³ and binding to HSP90 and dynamin-2.^14,15 HSP90 facilitates the phosphorylation of eNOS by forming a ternary complex with eNOS and Akt.¹⁶ Dynamin-2 regulates eNOS activity through the binding of its proline-rich domain to the FAD domain of eNOS, promoting electron transfer between the bound flavins of the reductase domain and increasing NO production.¹⁵

We have shown that activation of the albumin-binding protein gp60 in microvascular endothelial cells (ECs) induces Src activation,^17,18 resulting in phosphorylation of both dynamin-2 and caveolin-1.^18,19 This response required activation of the heterotrimeric GTP-binding protein, G_i, and release of the heterodimer G_ß.¹⁹ The G_ß-induced Src kinase activation signaled the scission of caveolae from the endothelial cell plasma membrane.^17–21 Because caveolin-1 regulates eNOS activity and caveolae-mediated endocytosis, we addressed the possibility that these are coupled processes requiring activation of similar signaling pathways. In the present study, we determined the role of caveolae-mediated endocytosis induced by caveolin-1 phosphorylation in signaling the uncoupling of eNOS from caveolin-1 and regulating NO production.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antibodies
Rabbit anti-eNOS antibody and goat anti–phospho-Ser1179-eNOS antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif); gp60 antibody was generated as described.²² Rabbit anti–phospho-Ser473-Akt antibody was purchased from Cell Signaling Technology (Danvers, Mass); mouse pTyr14-caveolin-1 antibody and rabbit anti–caveolin-1 antibody were purchased from Transduction Laboratories (Franklin Lakes, NJ).

Reagents
Fraction V BSA was purchased from Fisher Scientific (Pittsburgh, Pa), 2x recrystallized BSA from ICN Biomedicals (Solon, Ohio), acetylated BSA from Electron Microscopy Sciences (Hatfield, Pa), and 25% BSA solution in Tyrode’s buffer (used in isolated lung studies) from Sigma Chemical Co (St Louis, Mo). PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), A23187 (calcimycin), SH-6 (D-2,3-dideoxy-myo-inositol 1-[(R)-2-methoxy-3-(octadecyloxy)propyl hydrogen phosphate]), and mSIRK (myristoylated-SIRKALNILGYPDYD) were purchased from Calbiochem (La Jolla, Calif). All other reagents were purchased from Sigma unless otherwise indicated.

Cell Culture and Transfection
Rat lung microvascular endothelial cells (Vec Technologies, Rensselaer, NY) were cultured using DMEM (Invitrogen, Carlsbad, Calif) supplemented with 10% FBS, 50 U/mL penicillin, and 50 µg/mL streptomycin as described.²³ Transfection with Adv-ct-ßARK1 and Dyn-2-K44A were performed as described.^18,19 The cultures were maintained in 5% CO₂/95% room air at 37°C.

Mice
Animal studies were approved by the University of Illinois Animal Care and Use Committee. Caveolin-1 knockout (Cav1^–/–; Cav1^tm1Mls) breeding pairs were purchased from The Jackson Laboratory (Bar Harbor, Me). Adult male Black Swiss mice were purchased from Taconic (Hudson, NY) for use as strain-matched controls. Adult female C57/Black6 and age matched eNOS knockout mice (eNOS^–/–) were purchased from The Jackson Laboratory.

NO Measurement
NO produced by cells cultured on 6-well plates was measured using a porphyrinic NO electrode as described.^24,25 Briefly, the electrode is created by coating carbon fibers with a metalloporphyrinic conductive polymer and subsequently sealed with Nafion. Each electrode is calibrated using a stock solution of NO-saturated water. NO diffuses into the Nafion membrane where it is oxidized to the nitrosyl ion. The electron is transferred to the porphyrin of the conductive polymer and proceeds along the copper wire to a detector. The NO electrode is placed onto the surface of an endothelial cell monolayer and 2 additional electrodes are added to the solution to generate a 650-mV potential. The system was coupled with a FAS1 femtostat and a personal computer with electrochemical software (Gamry Instruments, Warminster, Pa). Electrode current, which is proportional to NO concentration, is measured as a function of time. The cell culture medium temperature is kept at 37°C.

Intracellular Ca²⁺ Measurements
Intracellular Ca²⁺ was measured using a ratiometric fluorescence detection method using fura-2 AM (5-oxazolecarboxylic acid, 2-(6-(bis(2-(acetyloxy)methoxy)-2-oxoethyl)amino)-5-(2-(2-(bis(2-(acetyloxy))methoxy)-2-oxoethyl)amino)-5-((methylphenoxy)ethoxy)-(2-(benzofuranyl)), (acetyloxy)methyl ester). NO production was measured in cells treated with BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid) with albumin stimulation. Cells grown on 25-mm diameter glass coverslips were washed twice with Hanks’ balanced salt solution (HBSS). Cells were loaded with either 3 µmol/L fura-2 AM alone or with 10 µmol/L BAPTA-AM for 30 minutes at 37°C. Cells were imaged using an Attofluor RatioVision digital fluorescence microscopy system (Atto Instruments, Rockville, Md) equipped with a Zeiss Axiovert S100 inverted microscope and F-Fluar x40, 1.3 NA oil immersion objective. Regions of interest in individual cells were marked and excited at 334 and 380 nm with emissions collected at 520 nm at 5-second intervals. At the end of each experiment, 10 µmol/L ionomycin was used to obtain fluorescence of Ca²⁺-saturated fura-2 (high [Ca²⁺]_i) and 10 mmol/L EGTA to obtain fluorescence of free fura-2 (low [Ca²⁺]_i). [Ca²⁺]_i was calculated based on a dissociation constant (K_d) of 225 nmol/L with a 2-point curve fit.

Immunostaining and Confocal Microscopy
Confluent RLMEVCs were washed with PBS, fixed, permeabilized, and stained with anti-eNOS polyclonal antibody (1 µg/mL) and the nuclear marker 4',6-diamidino-2-phenylindole (DAPI) (1 µg/mL), as described.¹⁹ A Zeiss LSM 510 META microscope was used for confocal microscopy. Nonconfocal DAPI images were acquired using Hg lamp excitation and UV filter set with the LSM 510. Fluorescence emission was detected in optical sections <1 µm in thickness (pinhole set to achieve 1 Airy unit).

Immunoprecipitation and Western Blotting
For Western blot analysis, cells were lysed with lysis buffer (30 minutes at 4°C in 50 mmol/L Tris-HCl, pH 7.5, containing 150 mmol/L NaCl, 1 mmol/L EDTA, 0.25% sodium deoxycholate, 1.0% Nonidet P-40, 0.1% sodium dodecyl sulfate, 1 mmol/L Na₃VO₄, 1 mmol/L NaF, 44 µg/mL phenylmethylsulfonyl fluoride, and protease inhibitor mixture) and insoluble materials were removed by centrifugation (14 000g for 15 minutes). For immunoprecipitations, the lysates were incubated with 10 µg/mL primary antibodies overnight at 4°C followed by incubation with protein A/G-agarose beads (Santa Cruz Biotechnology) for 4 hours. Proteins were run on a 5% to 20% gradient SDS-PAGE gel, transferred to a nitrocellulose membrane, and blotted with the appropriate primary antibody overnight at 4°C and with horseradish peroxidase–conjugated secondary antibody for 1 hour at room temperature. Proteins were detected using an ECL kit (Amersham, Piscataway, NJ).

Endothelial NOS and Akt Phosphorylation
Rat lung microvascular endothelial cells (RLMVECs) were grown on 60-mm dishes to 80% to 90% confluence, subsequently serum deprived for several hours in serum-free DMEM, then treated with BSA (30 mg/mL) for various time periods ranging from 30 seconds to 30 minutes. Cell lysates were used for Western blot analysis as described above. In experiments designed to assess inhibition of phosphorylation of eNOS and Akt, cells were pretreated for 20 minutes with inhibitors, BSA was added to activate gp60, and subsequently the cells were lysed and processed as above.

Mouse Lung Preparation
Mice were anesthetized with an intraperitoneal injection of a mixture of ketamine (0.3 mL/30 g of a 10 mg/mL solution) and xylazine (0.1 mL/30 g of a 2 mg/mL solution) titrated to surgical anesthesia. Heparin was administered intravenously via retroorbital injection and following tracheostomy; the lungs were ventilated with room air at a positive pressure of 15 cm H₂O. A thoracotomy was performed, PE10 tube was inserted into the pulmonary artery through the right ventricle, and the left atrium was incised. Pulmonary arterial pressure was monitored continuously through the pulmonary cannula, which was connected to a fluid-filled pressure transducer. The lungs were subsequently perfused with HEPES-buffered RPMI medium 1640 (pH=7.4) at the baseline flow of 1.8 mL/min, and pulmonary venous pressure was held constant. After a period of 15 minutes, isogravimetric conditions were achieved, defined as stable pressure and lung weight (which was also continuously monitored as described²⁶). A continuous infusion of the stable thromboxane A2 mimetic U46619 (9,11-dideoxy-11,9-epoxymethanoprostaglandin F₂) (100 nmol/L) was then initiated to induce vasoconstriction and was maintained until the end of the experiment. Endothelin-1 (400 nmol/L) was infused as a bolus for 5 minutes and then washed out for 5 minutes. Administration of these vasoactive substances resulted in an increase in pulmonary arterial pressure of 2 to 3 times the baseline. At the point of maximum vasoconstriction, the perfusate was switched to HEPES-buffered RPMI medium 1640 with 2% BSA to activate gp60 and the pressure response of the lung preparation was recorded. Pulmonary vascular resistance (PVR) (cm H₂O/mL per minute) was derived as the ratio of the pulmonary arterial pressure (P_pa) and left atrial pressure divided by the perfusate flow.

Statistical Analysis
Data are presented as mean±SEM. Means were compared using 1-way ANOVA. Dunett’s test was used for post hoc comparison of experimental groups to controls.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ca²⁺-Independent eNOS Activation
We previously showed that albumin binding to gp60 induces G_ß-activated Src kinase signaling and activation of caveolae-mediated endocytosis.^17–23 To address the role of endocytosis in regulating activation of eNOS, RLMVECs were serum deprived for 2 hours and BSA (5 mg/mL) was added while measuring NO production using the porphyrin-coated electrode. This increased NO production lasting up to 20 minutes compared with the transient response observed in cells stimulated with Ca²⁺ ionophore, A23187 (2 µmol/L) (Figure 1a). Albumin did not increase intracellular [Ca²⁺] (Figure 1b), and preincubation of RLMVEC with 1 µmol/L BAPTA-AM (intracellular Ca²⁺ chelator) had no effect on NO production compared with control (Figure 1c).

View larger version (16K): [in this window] [in a new window]   Figure 1. Calcium-independent NO production in endothelial cells induced by gp60. A, Representative tracings of current vs time show NO electrode detection method. Calcium ionophore, A23187 (2 µmol/L), or BSA (5 mg/mL) to activate gp60 were added to confluent endothelial cell monolayers, and real-time NO production was determined as described in Materials and Methods. Addition of either agent resulted in an increase in electrode current indicating increased NO concentration in the medium. The ionophore-induced response was rapid, returning to basal level in approximately 2 minutes, whereas the gp60-induced response developed more slowly and was prolonged (n=3). B, RLMVECs were loaded with fura-2 AM and stimulated with 30 mg/mL Cohn fraction V albumin to activate gp60. C, RLMVECs were pretreated with BAPTA-AM or vehicle for 30 minutes and then stimulated with 30 mg/mL BSA to activate gp60. *P<0.05 vs control; n=6 wells/group.

NO Production Induced by Gp60 Activation
We observed that albumin-activated NO production was saturable between 20 to 30 mg/mL albumin with an EC₅₀ of 5 to 7 mg/mL albumin (Figure 2A). EC₅₀ value of 100 µmol/L was identical to the concentration required for 50% activation of endocytosis shown previously²³ (Figure 2A, inset). To address whether NO production was the result of native albumin per se, we used Cohn fraction V and 2x recrystallized albumin (30 mg/mL). Both gave similar responses (Figure 2B). Albumin modified by acetylation (acet-BSA), which does not activate gp60,²¹ failed to induce NO production (Figure 2B). Activation of gp60 with a polyclonal antibody to gp60 (10 µg/mL), as described,²² induced NO release similar to albumin (Figure 2B).

View larger version (17K): [in this window] [in a new window]   Figure 2. Gp60-activated NO production in endothelial cells. A, Dose-response relationship of gp60-induced endothelial NO production. A sigmoidal relationship was observed for albumin (fraction V) from 1 to 30 mg/mL used to activate gp60. Maximal NO production was observed at physiological concentrations of plasma albumin (30 mg/mL). EC50 was 100 µmol/L albumin (6.5 mg/mL) (n=6/point). This concentration/effect relationship is indicative of a receptor-mediated event and similar to the dose-response relationship of gp60-induced endocytosis (see inset, replotted from John et al,23 with permission). B, Similarity between gp60 cross-linking and albumin-induced NO generation. Cold-alcohol precipitated and endotoxin-free Cohn fraction V BSA, double-recrystallized BSA, highly pure albumin, at 30 mg/mL, increased NO production (NO concentration present at 20 minutes is tabulated). The amount of NO produced in response to anti-gp60 antibody (Ab) (10 µg/mL), fraction V BSA (fV-BSA), or doubly recrystallized BSA (2xC-BSA) were not statistically different. A partly linearized and acetylated form of BSA (Acet-BSA) (30 mg/mL) induced significantly less NO production compared with gp60 cross-linking or native BSA, indicating the importance of charge and/or tertiary structure for the albumin-mediated effect (n=6 wells/group).

Dissociation of eNOS From Caveolin-1
We observed punctate localization of eNOS in vesicle-like structures in endothelial cells (Figure 3A). Caveolin-1 and eNOS were coimmunoprecipitated using anti-eNOS antibody (Figure 3B). The association was markedly reduced following gp60 activation used to induce caveolae internalization.²⁰ Gp60 induced the phosphorylation of caveolin-1 (Tyr14), Akt (Ser473), and eNOS (Ser1179) within 30 seconds (Figure 3C). Phospho-Akt was maximal at 15 minutes, whereas phospho-eNOS showed a biphasic response peaking at 1 minute and again at 30 minutes. Caveolin-1 phosphorylation was maximal at 1 minute.

View larger version (36K): [in this window] [in a new window]   Figure 3. Gp60 induces eNOS activation. A, The cellular localization of eNOS in serum-deprived RLMVECs was assessed by immunofluorescence staining and confocal microscopy; eNOS staining was primarily punctate. B, Caveolin-1 coimmunoprecipitation with eNOS was measured in serum-deprived RLMVECs and cells treated for 5 minutes with 30 mg/mL albumin to activate gp60. Note the decrease in eNOS coimmunoprecipitation with caveolin-1 following gp60 activation. C, Time course of eNOS, Akt, and caveolin-1 phosphorylation induced by gp60. RLMVECs were serum deprived for 2 hours, and the addition of albumin to activate gp60 resulted in phosphorylation of caveolin-1 (p-Cav1-Tyr14), peaking after 1 minute. Akt phosphorylation (p-Akt-Ser473) steadily increased for up to 15 minutes, at which time it declined. Phosphorylation of eNOS (p-eNOS-Ser1179) showed a biphasic response, increasing at 1 minute and again at 30 minutes. Results shown are representative of 3 experiments.

G_ß Dependence of NO Production
We measured NO production in endothelial cells transfected with adenoviral vector containing the C terminus of ß-adrenergic receptor kinase (ct-ßARK), a selective inhibitor of G_ß signaling.²⁷ Ct-ßARK expression reduced gp60-activated NO production by 72% indicating the important involvement of G_ß in signaling both NO production and caveolae-mediated endocytosis (Figure 4). This role of G_ß was directly addressed using mSIRK, an independent activator of G_ß-signaling.^19,28 We observed that mSIRK stimulated NO production independent of gp60 activation and the effect was prevented in endothelial cells expressing ct-ßARK (Figure 4).

View larger version (26K): [in this window] [in a new window]   Figure 4. Gß mediates gp60-activated NO production. NO production was measured in confluent monolayers of RLMVECs expressing the C-terminal domain of ß-adrenergic receptor kinase (ct-ßARK), an inhibitor Gß signaling. ct-ßARK transduction showed 72% reduction in gp60-activated NO production compared with control cells or cells transfected in empty Adv-vector (P<0.05, n=5). mSIRK, a cell permeable peptide activator of Gß heterodimers, also stimulated NO production in RLMVECs. This increase was prevented in ct-ßARK–transfected cells (*P<0.05, n=5) but not in empty vector transfected cells.

Involvement of Src Kinase and Akt in Gp60-Mediated NO Production
We addressed the roles of Src and Akt as the kinases downstream of G_ß mediating the gp60-activated NO production. Akt inhibitor SH-6 caused 80% reduction in NO production and PP2, a Src kinase inhibitor, caused 40% reduction (Figure 5A). N^G-Nitro-L-arginine (L-NNA) (2 µmol/L), an eNOS inhibitor, reduced the response by 50% (Figure 5A).

View larger version (28K): [in this window] [in a new window]   Figure 5. Gp60 activation of eNOS requires Src, PI3K, Akt, and caveolae-mediated endocytosis. A, NO production was measured on addition of albumin to activate gp60 in RLMVECs stably expressing Dyn-2 K44A, dominant-negative form of dynamin-2, or cells pretreated with cholesterol-depleting agent, methyl-ß-cyclodextrin (5 mmol/L). These interventions reduced NO production activated by endocytosis by 50%. Pretreatment of cells with 15 µmol/L PP2, a Src inhibitor, 10 µmol/L SH-6, an Akt inhibitor and 2 µmol/L L-NNA, an eNOS inhibitor, for 20 minutes before NO measurement reduced gp60-activated NO production by 40%, 80%, and 50%, respectively. *P<0.05 (n=6 to 9). B, Effect of methyl-ß-cyclodextrin, LY294002, and PP2 on gp60-induced eNOS and Akt activation. RLMVECs were pretreated with vehicle, 15 µmol/L PP2, or 10 µmol/L LY294002, a PI3K inhibitor, followed by gp60 activation with albumin for 5 minutes and assayed by Western blot to determine the amount of activated/phosphorylated Akt (p-Akt-Ser473) and eNOS (p-eNOS-Ser1179). All 3 inhibitors resulted in reduced eNOS and Akt activation, indicating that intact caveolae and caveolae scission as well as Src and PI3K signaling are required for eNOS activation. MßCD indicates methyl-ß-cyclodextrin; LY, LY294002. Blots shown are representative of 3 experiments.

Gp60-Mediated NO Production Requires Intact Caveolae and Caveolae Scission
To address the role of caveolae-mediated endocytosis in the mechanism of NO production, we examined the effects of methyl-ß-cyclodextrin, a cholesterol-binding agent that disrupts caveolae, and dynamin-2 K44A, a GTPase-defective dynamin-2 mutant that inhibits caveolae scission.^18,29 Both agents resulted in a 50% reduction in NO production (Figure 5A). Intact caveolae were also required for eNOS and Akt phosphorylation (Figure 5B). LY294002 (phosphatidylinositol 3-kinase [PI3K] inhibitor) and PP2 also decreased Akt and eNOS phosphorylation induced by gp60 activation (Figure 5B).

Impaired NO-Dependent Pulmonary Vasodilation Induced by Gp60 Activation in Caveolin-1–Null Mice
We used nonblood perfused mouse lungs, which were perfused at constant flow, to address the effects of gp60 activation on eNOS activation and NO production in the intact microcirculation. In lungs prevasoconstricted with the thromboxane A₂ mimetic U46619, infusion of albumin to activate gp60 induced pulmonary vasodilation (Figure 6A and 6B). Preconstriction with endothelin-1 also resulted in a similar response following gp60 activation (Figure 6C). However, in the absence of gp60 activation, we observed increased pulmonary vasoconstriction (Figure 6D). The effect of gp60 activation in inducing pulmonary vasodilation was markedly reduced in eNOS^–/– and caveolin-1^–/– mouse lungs. Similar results were obtained in lung preparations pretreated with the pan-NOS inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) (100 µmol/L) added to the perfusate before gp60 activation (Figure 6A).

View larger version (24K): [in this window] [in a new window]   Figure 6. Albumin induced vasorelaxation of preconstricted perfused lung preparations. A, RPMI medium 1640–perfused mouse lungs (perfused at constant flow) were treated with a continuous infusion of 100 nmol/L U46619, resulting in a 2- to 3-fold increase in pulmonary arterial pressure. Significant vasodilation was observed following addition of 2% albumin-containing RPMI medium 1640 in wild-type (WT) mouse lungs to activate gp60 (n=6). This effect was significantly reduced in lungs from eNOS knockout (n=5) and caveolin-1 knockout mice (n=4). L-NAME–pretreated lungs (100 µmol/L) showed similar responses to those observed in eNOS knockout lungs. Data are expressed as percentage of pulmonary vascular resistance (PVR) recorded during U46619 infusion (*P<0.05 vs wild type). B, Example tracing of an experiment shown in a contrasts the vasodilation seen in eNOS knockout and wild-type mouse lungs in which pulmonary vessels were preconstricted. In both cases, activation of gp60 led to initial, transient pressure increase followed by rapid vasodilation in wild-type mouse lungs; a much slower and overall attenuated response in eNOS knockout lungs. C, Gp60-induced vasodilation of endothelin-1 (40 nmol/L) preconstricted lungs (n=4). Endothelin-1 was infused for 5 minutes followed by 5 inutes washout period before introduction of 2% albumin containing perfusate to activate gp60 (red tracing). After withdrawal of BSA from the preparation and perfusion with RPMI medium 1640 alone, there was rebound of vasoconstriction. Comparison is made to endothelin-treated preparation without the addition of BSA (black tracing, n=3). D, Intact mouse lungs were perfused with RPMI medium 1640 containing 2% albumin at baseline followed by vasoconstriction with U46619 (100 nmol/L). After steady-state pulmonary arterial pressure was achieved, perfusing solution was changed to RPMI medium 1640 containing 2% IgG. This change was associated with a greater increase in U46619-induced vasoconstriction (n=3).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study shows that activation of the albumin-binding protein gp60, which signals caveolae-mediated endocytosis in pulmonary microvascular endothelial cells,¹⁷ induces eNOS activation and NO production. The gp60-mediated eNOS activation occurred by a Ca²⁺-independent mechanism. Quenching the Ca²⁺ signal with the intracellular Ca²⁺ chelator BAPTA-AM had no effect on NO production. We used 2 independent approaches to activate gp60: (1) gp60 cross-linking involving a primary rabbit anti-gp60 antibody²² and (2) ligation of gp60 by albumin addition to serum-deprived endothelial cells.²⁰ In previous studies, gp60 cross-linking was shown to promote plasmalemmal gp60 clustering.¹⁷ Gp60 activation by either means activated the Src kinase signaling pathway.^17–21 Cohn fraction V, fatty acid-free, and double-recrystallized albumin used to activate gp60 induced NO production, whereas denaturation of albumin did not result in NO production. The effect of albumin on NO production was also saturable, similar to the effect of albumin in activating gp60.²³ The sigmoid concentration-response curve showed maximal NO production between 20 to 30 mg/mL albumin, the range of the normal plasma albumin concentration to which endothelial cells are exposed. The concentration-response curve correlated precisely with the EC₅₀ of albumin-activated endocytosis,²³ suggesting that the plasma concentration bathing endothelial cells is itself an important determinant of the constitutive NO production.

We have demonstrated previously that gp60-induced phosphorylation of caveolin-1 and Src is required for the engagement of the signaling machinery responsible for caveolae-mediated endocytosis.¹⁷ As Src also phosphorylates eNOS via the PI3K/Akt pathway,³⁰ we investigated the possible relationship between caveolae-mediated endocytosis and eNOS-derived NO production. Activation of gp60 rapidly induced eNOS and Akt phosphorylation with concomitant caveolin-1 phosphorylation at Tyr14. Importantly, eNOS became uncoupled from caveolin-1 following gp60 activation, a requirement for eNOS activation.¹³ These results show that both eNOS activation and caveolae-mediated endocytosis induced by gp60 occurred together and required the Src signaling pathway.

Our previous work has identified the important role of G_ß as the downstream effector of gp60 activation, which in turn induces Src phosphorylation.¹⁸ Using endothelial cells expressing ct-ßARK to prevent ß-activation,¹⁹ we observed that inhibition of G_ß signaling greatly reduced NO production. Direct activation of ß-subunits with the synthetic peptide mSIRK induced NO production independent of gp60 and this response was abolished in ct-ßARK–transfected cells. These findings demonstrate an important role for G_ß in signaling NO production, consistent with the functional coupling of eNOS-derived NO production with caveolae-mediated endocytosis.

In a series of studies, we addressed the signaling roles of Src kinase and Akt in activating eNOS induced by gp60. Using PP2, a Src kinase inhibitor, and SH-6, an Akt inhibitor, we observed that both inhibitors significantly reduced NO production induced by gp60 activation. This was associated with reduced Akt and eNOS phosphorylation. However, the decreased NO production ranged from 40% to 80%, suggesting that other pathways are also involved in signaling eNOS activation by gp60. Additionally, the inhibitor of PI3K, LY294002, abrogated the Akt and eNOS phosphorylation induced by gp60, suggesting that Akt mediates the response downstream of PI3K.

To address the relationship of eNOS activation to endocytosis, methyl-ß-cyclodextrin, a cholesterol-depleting agent, and Dyn-2 K44A, a GTPase-defective form of dynamin-2, were used to block caveolae-mediated endocytosis. Cyclodextrin functions by disrupting the caveolae structure,^23,31 and dynamin mutant prevents scission of caveolae from the membrane.¹⁸ We have shown that both agents effectively blocked caveolae-mediated endocytosis induced by gp60.^18,23 In the present study, they resulted in 50% reduction in NO production. This finding coupled with similarities between EC₅₀ of albumin-mediated endocytosis (93 µmol/L) and EC₅₀ of gp60-mediated NO production (100 µmol/L) supports an important role of caveolae endocytosis in the mechanism of NO production. As disruption of caveolae internalization partially reduced NO production, it is probable that other mechanisms are also involved. One possibility is the recent finding that dynamin directly regulates eNOS activity independent of the scission function of dynamin.²⁹ Thus, although activation of eNOS resulting from scission of caveolae from the plasma membrane is a crucial mechanism of NO production as identified in the present study, it is clear that eNOS is also subject to regulation by other signaling pathways.

We next determined the functional significance of NO production associated with caveolae-mediated endocytosis, specifically its involvement in the regulation of vasomotor tone. We assessed the vasomotor tone of mouse lungs perfused with nonblood RPMI medium 1640–containing solution at constant flow and venous pressure to address the role of gp60-activated NO production in promoting vasodilation. In this preparation, on the basis of the Poiseuille’s equation, any change in pulmonary artery pressure directly reflects a change in pulmonary vasomotor tone. We observed that gp60 activation induced rapid and profound dilation of vessels preconstricted with either endothelin-1 or U46619. This response was independent of sex and strain, at least in the 3 mouse strains tested (CD1, Swiss Black, C57/Black6). The vasodilation was significantly reduced, but it was not prevented in lungs obtained from eNOS- or caveolin-1–null mice, suggesting that albumin likely interacts in multiple ways with the endothelium to regulate vascular tone. Other vasodilators, such as carbon monoxide³² and prostacyclin,³³ release of which have been localized to caveolae, may also be generated secondary to caveolae-mediated endocytosis. In addition, albumin may bind NO and form S-nitroso-albumin, which can induce vasodilation.^34,35 These factors help to explain why the gp60-mediated vasodilatory response was only partly attenuated in eNOS^–/– or caveolin-1^–/– mice.

Drab et al,³⁶ showed in caveolin-1^–/– mice that the vasodilator response to acetylcholine (as assessed by relaxation of preconstricted aortic rings) was enhanced. The greater relaxation induced by acetylcholine in caveolin-1^–/– aortic rings was ascribed to a higher production of NO.³⁶ Our finding that the vasodilator response to gp60-induced caveolae endocytosis was reduced in caveolin-1 null lungs seems to contradict the data found by Drab et al. We observed that impairment of caveolae endocytosis in methyl-ß-cyclodextrin–treated endothelial cells was coupled to reduced eNOS activity and NO production. Therefore, a likely explanation of our data is that caveolae-mediated endocytosis as regulated by Src-induced phosphorylation of caveolin-1 is, itself, an important signal for eNOS activation. Thus, in the absence of caveolae-mediated endocytosis as in caveolin-1^–/– lungs, there is diminished NO production and hence an impairment of gp60-activated pulmonary vasodilation. This concept is supported by evidence of a marked decrease in eNOS activation and NO production on stimulation of caveolin-1 null endothelial cells with VEGF.³⁷

In conclusion, we have uncovered a novel mechanism of eNOS activation and NO production in endothelial cells involving caveolae-mediated endocytosis induced by gp60. Inhibition of endocytosis resulted in the marked impairment of NO production. eNOS activity induced by gp60 was mediated by G_ß activation of downstream Src, Akt, and PI3K pathways. As caveolae internalization is a constitutive process in endothelial cells,^38,39 this mechanism of NO production may be important in regulating basal pulmonary vasomotor tone. Thus, strategies interfering with caveolae endocytosis may have a deleterious effect on eNOS activation and NO production and lead to pulmonary arterial hypertension.

	Acknowledgments

 
Sources of Funding

This research was supported by NIH grants T32 HL07239 (to A.B.M.), P01 HL60678 (to A.B.M., R.A.S., and R.D.M.), and R01 HL71626 (to R.D.M.).

Disclosures

None.

	Footnotes

 
Original received May 11, 2006; revision received August 9, 2006; accepted August 31, 2006.

	References

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