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INTERNALIZATION OF eNOS VIA CAVEOLAE REGULATES PAF-INDUCED

INFLAMMATORY HYPERPERMEABILITY TO MACROMOLECULES

Fabiola A. Sánchez, David D. Kim, Ricardo G. Durán, Cynthia J. Meininger1, and Walter N.

Durán

From Program in Vascular Biology, Department of Pharmacology and Physiology, UMDNJ -

New Jersey Medical School, 185 South Orange Ave, Newark, NJ 07101-1709; 1Department of

Systems Biology and Translational Medicine, Texas A & M Health Science Center, Temple, TX

76504

Running Title: eNOS endocytosis and hyperpermeability

Address correspondence to:

Fabiola A. Sánchez, PhD

185 S. Orange Ave., MSB H-638

Newark, NJ 07101-1709

Phone: 973-972-3981

Fax: 973-972-7950

Email: sanchefa@umdnj.edu

Articles in PresS. Am J Physiol Heart Circ Physiol (August 15, 2008). doi:10.1152/ajpheart.00629.2008

Copyright © 2008 by the American Physiological Society.

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ABSTRACT

Endothelial nitric oxide synthase (eNOS) is thought to regulate microvascular permeability via

NO production. We tested the hypotheses that expression of eNOS and eNOS endocytosis by

caveolae are fundamental for appropriate signaling mechanisms in inflammatory endothelial

permeability to macromolecules. We used bovine coronary postcapillary venular endothelial

cells (CVEC) because these cells are derived from the microvascular segment responsible for

transport of macromolecules in inflammation. We stimulated CVEC with platelet-activating

factor (PAF) at 100 nM and measured eNOS phosphorylation, NO production and CVEC

monolayer permeability to FITC-dextran-70. PAF translocated eNOS from plasma membrane to

cytosol, induced changes in the phosphorylation state of the enzyme and increased NO

production from 4.3 ± 3.8 to 467 ± 22.6 nM. PAF elevated CVEC monolayer permeability to

FITC-dextran-70 from 3.4 ± 0.3 x 10-6

cm/s to 8.5 ± 0.4 x 10-6

cm/s. Depletion of endogenous

eNOS with siRNA abolished PAF-induced hyperpermeability demonstrating that expression of

eNOS is required for inflammatory hyperpermeability responses. Inhibition of the caveolar

internalization by blocking caveolar scission using transfection of dyn2K44A (dynamin

dominant negative mutant) inhibited PAF-induced hyperpermeability to FITC-dextran-70. We

interpret these data as evidence that 1) eNOS is required for hyperpermeability to

macromolecules, and 2) internalization of eNOS via caveolae is an important mechanism in the

regulation of endothelial permeability. We advance the novel concept that eNOS internalization

to cytosol is a signaling mechanism for onset of microvascular hyperpermeability in

inflammation.

Key Words: endothelial nitric oxide; endothelial cells; microvascular permeability; eNOS

translocation; acetylcholine; platelet-activating factor

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INTRODUCTION

Increased microvascular permeability (hyperpermeability) is a hallmark of inflammation.

Hyperpermeability to macromolecules is a process that occurs in vivo mainly at postcapillary

venules. While the significance of endothelial nitric oxide synthase (eNOS)-derived nitric oxide

(NO) in the regulation of microvascular transport across postcapillary venules is an

experimentally supported emerging concept (16, 27, 34, 36), the mechanisms by which eNOS

controls microvascular permeability are poorly understood.

Pro-inflammatory agonists that increase permeability - such as vascular endothelial growth factor

(VEGF), bradykinin (BK), and platelet activating factor (PAF) - stimulate signaling cascades that

converge on eNOS (20), activate the enzyme and cause NO production (9). The activation of

eNOS proceeds mainly through phosphorylation of the enzyme at serine 1177 (Ser1177

); (7, 12),

and de-phosphorylation at threonine 495 (Thr495

); (8). Interestingly, acetylcholine (ACh), an

agent that causes vasodilation but does not alter microvascular permeability (27), induces exactly

the same changes in phosphorylation of eNOS (30). These observations suggest that

phosphorylation of eNOS per se is not a determinant of the functional consequences of eNOS-

derived NO.

We reasoned that location of eNOS may contribute to determine the functional significance of

eNOS-derived NO. eNOS is found in plasma membrane (mostly in caveolae) and Golgi in

control endothelial cells (EC). It is established that eNOS can produce NO regardless of its

location, even though its efficiency may vary (5, 37). Also, eNOS can translocate, upon

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stimulation, from plasma membrane to intracellular compartments (10, 23, 30). This movement

has been associated with depalmitoylation of eNOS (35), and more recently, it has been

suggested that caveolae may serve as a vehicle for eNOS translocation (4).

The functional significance of eNOS internalization or traffic, if any, has not been previously

investigated. Work from Michel at al shows eNOS movement to the cytosol after VEGF

application, an agonist that induces hyperpermeability (10) and we demonstrated that PAF-

induced eNOS preferential internalization to cytosol was associated with hyperpermeability to

macromolecules (30). Based on these and the above-mentioned observations, we hypothesized

that eNOS expression is required for development of hyperpermeability and that eNOS

internalization to NO-receptors serves to determine the functional significance of eNOS-derived

NO in response to PAF, a pro-inflammatory autacoid. The experiments reported herein were

designed to test these hypotheses in bovine coronary endothelial cells derived from postcapillary

venules (CVEC; (31). We confirm in endothelial cells that eNOS expression is required for an

increase in permeability and propose, for the first time, that eNOS internalization via caveolae

plays a functional role in determining PAF-induced hyperpermeability to macromolecules.

MATERIALS AND METHODS

Antibodies. We used mouse anti-human eNOS, mouse anti-phospho-human eNOS(Ser1177

),

mouse anti-phospho-human eNOS(Thr495

) and rabbit anti-caveolin antibodies from BD

Biosciences (San Jose, CA). All the above-mentioned mouse antibodies recognize the

corresponding eNOS epitopes in the bovine CVEC.

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Cell culture and transfection. We grew CVEC in Dulbeccco’s modified Eagle’s Medium

supplemented with 10% fetal bovine serum; 20 units/ml sodium heparin, 50 ug/ml penicillin, 50

µg/ml streptomycin and 10 µg/ml neomycin. All the experiments were performed using passage

3-7 CVEC. Using electroporation, we transfected CVEC with cDNA for the dominant negative

mutant of dynamin 2 coupled to green fluorescent protein (dyn2K44A; kindly provided by Dr.

Mark McNiven, Mayo Clinic College of Medicine, Rochester, MN). Briefly, we grew cells to

70-80% confluence, trypsinized them and re-suspended them in 100 µl of nucleofection solution.

We, then, added 1 µg of dyn2K44A cDNA to the cells. We electroporated CVEC using a basic

nucleofector kit for primary mammalian endothelial cells. We applied program T23 from Amaxa

Biosystems (Gaithersburg, MD). We electroporated CVEC with nucleofection solution and with

the empty vector as a control. The experiments were carried out 72 h after transfection. We

verified the expression of transfected dyn2K44A by the cellular fluorescence.

eNOS siRNA transfection. We used specific siRNA (Ambion, Austin, TX), as designed by Zhang

et al (37), to deplete eNOS. We transfected CVEC with siRNA using lipofectin and following

the protocol recommended by the manufacturer (Invitrogen, Carlsbad, CA). We lysed the cells at

pre-determined times and performed western blotting against eNOS to confirm depletion of the

protein.

NO measurements. We measured NO production using NO-sensitive recessed-tip

microelectrodes (1). We used 100% nitrogen and 400 and 800 parts per million NO in nitrogen to

establish a calibration range for NO of 0-600-1,200 nM (in saline). We placed coverslips

containing confluent CVEC in a perfusion chamber. We superfused the cells with media

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supplemented with L-arginine at a rate of 1 ml/min. We administered PAF (Sigma Chemicals, St.

Louis, MO) through a side-port in the perfusion tubing to achieve a PAF concentration of 100

nM in the chamber.

Imunofluorescence microscopy. We fixed confluent monolayers of CVEC grown on glass

coverslips with 3% paraformaldehyde for 15 minutes, permeabilized them with 0.5% PBS-Triton

for 5 minutes, blocked them in 1% PBS-BSA for 30 minutes and then incubated them with anti-

eNOS antibodies and Alexa Fluor secondary antibodies. We examined the CVEC with an

inverted fluorescence microscope (Axiovert 200 M; Zeiss).

Detergent free purification of caveolae enriched membrane fractions. We grew CVEC in 100

mm tissue culture dishes (2 plates per treatment). After stimulation with 100 nM PAF, we

homogenized and sonicated the cells according to described protocols (21). We placed cell

lysates adjusted to 45% sucrose in a volume of 1.5 ml adding buffer 2 (90% sucrose/ 25 mM

MES, pH 6.5/ 150 mM NaCl, and inhibitors of proteases and phosphatases) at the bottom of a

4.5 ml centrifuge tube. We layered 1.5 ml of each buffer 3 (35% sucrose/ 250 mM sodium

carbonate, pH 11/ 25 mM MES/ 150 mM NaCl) and buffer 4 (5% sucrose/ 250 mM sodium

carbonate, pH 11/ 25 mM MES/ 150 mM NaCl) on top of it, and centrifuged the samples at

44,000 rpm for 18 h in a Beckman L8-70M Ultracentrifuge equipped with a SW60Ti rotor. We

collected 12 fractions from the top to the bottom of each tube. An equal volume of each

fraction

was used for Western blot analysis and probed for eNOS and caveolin-1.

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Western blot analysis. We grew CVEC to confluence in 100mm plates. Immunoblot analyses of

protein expression and phosphorylation were assessed as we described previously (2, 30).

Densitometric analyses of Western blots were performed using the NIH Image J program.

Measurement of monolayer permeability. We determined control and PAF-stimulated

permeability to fluorescein isothiocyanate dextran 70 KDa (FITC-Dx-70, a macromolecule that

mimics albumin) across confluent CVEC monolayers using an established method (2, 30). We

obtained samples for baseline permeability every 15 minutes from the abluminal chamber for a

period of 60 minutes. After addition of PAF to both sides of the chamber (final concentration =

100 nM), we collected samples for additional 60 minutes.

Statistical analysis. Data are presented as mean ± S.E.M. Groups were analyzed for differences

by one-way ANOVA followed by Tukey-Kramer’s test. Significance was accepted at p < 0.05.

RESULTS

PAF stimulates eNOS phosphorylation and NO production in CVEC. Because we used

endothelial cells derived from postcapillary venules, our first step was to confirm that CVEC

express eNOS and that the enzyme can be activated to produce NO. We verified the expression

of eNOS in CVEC by immunofluorescence. Figure 1A shows immunofluorescent localization of

eNOS preferentially in the plasma membrane and Golgi, in agreement with the typical

distribution of eNOS described in endothelial cells (13, 14, 30). We corroborated eNOS

activation in response to PAF by measuring NO production and eNOS phosphorylation.

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Application of 100 nM PAF rapidly increased NO production by CVEC from 4.3 ± 3.8 nM to

467.0 ± 22.6 nM (mean ± SEM; p < 0.05; Figure 1B). After quantification of eNOS

phosphorylation, we calculated the ratio of phosphorylated eNOS to total eNOS at control (time

= 0), 0.5, 1.0 and 3.0 minutes after PAF. Figure 1C (upper panel) shows that PAF significantly

increased phosphorylation of eNOS at Ser1177

as early as 0.5 minutes. The phosphorylation at

Ser1177

remained elevated up to minute 3.0. PAF-induced de-phosphorylation of eNOS at Thr495

was significant 1 minute after application of the autacoid and showed a trend for returning

towards baseline levels at 3 minutes. Figure 1C (lower panel) displays western blots illustrating

changes in eNOS phosphorylation at Ser1177

and Thr495

. Interestingly, NO production reached a

maximum approximately 3 minutes after the application of PAF, indicating a close temporal

correlation between eNOS phosphorylation and NO production in CVEC.

PAF-stimulated hyperpermeability is regulated by eNOS-derived NO in CVEC. To correlate

molecular signals with functional end-points, we investigated PAF-induced hyperpermeability to

FITC-Dx-70 in CVEC monolayers. PAF significantly increased permeability to FITC-Dx-70

from 3.4 ± 0.3 x 10-6

cm/s to 8.5 ± 0.4 x 10-6

cm/s (Fig. 2A). This change was rapid inasmuch as

a change in the slope of flux of FITC-Dx-70 was detected in the first sample taken at 5 minutes

after PAF application. To test that the increase in permeability is mediated by eNOS, we

depleted eNOS from CVEC using specific siRNA (37). Figure 2B shows that eNOS expression

decreases as early as 16 hours after transfection of CVEC with eNOS siRNA and illustrates that

maximal depletion of eNOS occurred at 72 hours using 30 nM eNOS siRNA. We chose this

maximal depletion time-point and siRNA concentration to perform our measurements of CVEC

monolayer permeability to FITC-Dx-70. Scrambled siRNA, used as control, does not decrease

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the normal expression of eNOS in CVEC. Figure 2C shows that the basal permeability is normal

in eNOS-depleted CVEC. However, the hyperpermeability response to PAF is completely absent

in eNOS-depleted CVEC (Fig. 2C).

PAF induces eNOS trafficking in CVEC. Having demonstrated that eNOS is required for

development of an increase in CVEC monolayer permeability to macromolecules, we

investigated whether or not translocation of eNOS is also required for PAF-induced

hyperpermeability. As a first approach, we verified eNOS translocation in CVEC using

immunofluorescence microscopy and purification of caveolae enriched membrane fractions.

Figure 3A shows eNOS is located in the plasma membrane and Golgi in control CVEC, and

illustrates that PAF stimulates eNOS translocation from plasma membrane. Since caveolin is a

widely used marker for caveolae, we confirmed the information provided by microscopy by

isolating caveolin-containing lipid rafts fractions and probing for eNOS and caveolin by western

blotting. Figure 3B shows that, under baseline conditions, eNOS is distributed in fractions 7-12,

and is greatly enriched in fractions 7 and 8. Baseline caveolin is enriched in the same fractions.

Three minutes after PAF application, eNOS is found mainly, if not only, in heavier fractions

(particularly in fraction 12). Taken together, the microscopy and lipid rafts data indicate that

PAF induces eNOS translocation in CVEC.

eNOS internalization via caveolae and PAF-induced hyperpermeability. Because PAF-induced

eNOS preferential translocation to cytosol is associated with PAF-induced hyperpermeability

(30), we hypothesized that stimulated trafficking of eNOS-containing caveolae may serve to

deliver eNOS to subcellular effectors which determine the functional outcome of the initial

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stimulus. To test this hypothesis, we inhibited eNOS internalization by transfecting CVEC with

dyn2K44A, a dominant negative mutant of dynamin-2 (18, 24, 25). Figure 4A shows the

expression of dyn2K44A in CVEC. The efficiency of transfection, assessed by cell counting

(fluorescent versus total cells), was 60%. Transfection of CVEC with dyn2K44A completely

prevented PAF-induced hyperpermeability, whereas CVEC transfected with the empty vector

(used to deliver dyn2K44A) developed a hyperpermeability response to PAF (Figure 4B),

showing that transfection of CVEC with the empty vector did not inhibit PAF-induced

hyperpermeability.

To further verify the efficacy of dyn2K44A in preventing scission of caveolae from plasma

membrane in CVEC, we collected, at the end of the experiment, the snapwell inserts containing

the CVEC monolayers used to measure transport of FITC-DX-70 and examined the location of

eNOS by indirect immunofluorescence microscopy. Figure 5 displays the images obtained from

the experimental snapwells. Control CVEC show eNOS located in the cell membrane and Golgi.

Non-transfected cells show that 100 nM PAF causes translocation of eNOS to subcellular

locations, as eNOS appears diffuse in the cytosol. Importantly, CVEC transfected with

dyn2K44A demonstrate that eNOS is located at the cell membrane and Golgi after application of

100 nM PAF, indicating that dyn2K44A is highly efficacious in preventing movement of eNOS

from the cell membrane (caveolae) to cytosol.

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DISCUSSION

Our results demonstrate 1) eNOS expression is required for robust hyperpermeability response to

PAF, a pro-inflammatory agent, 2) internalization of eNOS is an important element of the signals

determining the permeability-enhancing function of eNOS-derived NO, and 3) traffic of eNOS

via caveolae is associated with the onset of hyperpermeability.

Translocation of eNOS from plasma membrane to subcellular compartments in response to

agonists has been reported to occur via enzyme depalmitoylation (29, 35) and/or in association

with internalization via caveolae (4) in endothelial cells derived from large vessels. A few

reports have advanced speculations concerning functional consequences of eNOS translocation

(15, 37). Our work is the first to suggest a close association between eNOS translocation via

caveolae and increase in endothelial monolayer permeability to macromolecules. In addition, our

data strongly indicate that eNOS translocation is required to stimulate the onset of PAF-induced

hyperpermeability.

While our results are strengthened by the fact that we obtained them in endothelial cells derived

from the microvascular segment normally involved in enhancing transport of macromolecules in

inflammation, preferential translocation of eNOS to the cytosol by agents that cause

hyperpermeability appears to be a property shared by other endothelial cells in culture. VEGF

(10) and BK (23), two well characterized hyperpermeability enhancing agents, cause eNOS

translocation to cytosol. In contrast, acetylcholine – an agent that causes vasodilation via eNOS-

derived NO but does not alter permeability - induces preferential translocation of eNOS to the

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Golgi region (30). The consistency of eNOS translocation to defined subcellular locations in

response to agents that induce different functional end-points argues against the interpretation

that eNOS movement reflects regular protein traffic, and argues in favor of a response to specific

external stimuli or agonists.

Pharmacologic inhibition of eNOS blocks hyperpermeability induced by PAF, VEGF and

histamine in vivo and in cultured EC (2, 20, 27, 33, 36). In mice, deletion of the gene encoding

for eNOS leads to loss of microvascular hyperpermeability responses to PAF (16) and to VEGF

(11). We confirm in CVEC monolayers that eNOS expression is an absolute requirement for

development of increased endothelial permeability to macromolecules in response to PAF.

Interestingly, loss or depletion of eNOS does not influence baseline permeability in mice and in

CVEC. It seems that baseline permeability is maintained at a set-point by a number of redundant

mechanisms while the ability to respond to pro-inflammatory agents (PAF, VEGF) is exquisitely

sensitive to or dependent on mechanisms based on functional eNOS.

Based on our results, we propose eNOS internalization via caveolae as a novel mechanism in

endothelial regulation of microvascular permeability to macromolecules in response to PAF, a

well known pro-inflammatory agent. Our data support the concept that eNOS-derived NO serves

as an onset signal for hyperpermeability. This assessment is based on the close temporal

correlation among the changes induced by PAF on eNOS activation (Ser1177

phosphorylation at

0.5 – 1.0 minutes; Thr495

de-phosphorylation at 1.0 minute), NO production (peaking at about 1-

3 minutes) and increases in permeability to FITC-Dx-70 detected shortly after application of

PAF.

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The concept that eNOS internalization to subcellular compartment plays a role in determining

stimulus-initiated function serves to complement the fund of knowledge demonstrating activation

of eNOS through phosphorylation or de-phosphorylation at several sites (7, 8, 12). While

phosphorylation is clearly important as a mechanism to activate eNOS, it has been difficult to

ascribe functional specificity to it inasmuch as agents that stimulate different functional

outcomes do phosphorylate eNOS at the same sites, as is the case for example for PAF and ACh

(30). Internalization of eNOS may serve as a mechanism to bring the enzyme in close contact

with soluble guanylyl cyclase (sGC) – its main effector – or an as yet unidentified subcellular

effector.

Our data support the concept that eNOS translocation and its association with elevation in

permeability involves internalization of eNOS via caveolae. This idea is based on the ability of

dyn2K44A to block the scission of caveolae from the plasma membrane, an event intimately

linked to inhibition of PAF-induced hyperpermeability to macromolecules. Even though we

cannot completely rule out the contributions of endocytosis via coated-pit vesicles, it is normally

accepted that caveolae cover about 85% of endothelial cell membranes (26). Thus, we interpret

our results with dyn2K44A as an inhibition of caveolar internalization. It is known, mainly

through in vitro experiments, that dynamin2 interacts with eNOS (3). For this reason, it could be

argued that dyn2K44A blocks eNOS directly. However, it has been pointed out that dyamin2 and

eNOS co-localize mainly in Golgi and that the eNOS binding domain with dynamin resides at a

location different from the K44 mutation (3, 4). Thus, it appears safe to conclude that relevant

action of dyn2K44A is to prevent the separation of caveolae from the plasma membrane. Our

data (Figure 5) supports this conclusion as it shows that eNOS stays at the plasma membrane

after PAF challenge in dyn2K44A-transfected CVEC. However, a recent article reports that

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dyn2K44A can inhibit caveolar endocytosis and NO production, in response to gp60 (22). This

article did not address the mechanism by which dyn2K44A inhibits NO production nor did they

address the discrepancy that the K44A mutation site should not alter NO production. Thus, it is

plausible that dyn2K44A inhibits PAF-induced permeability simultaneously through anchoring

caveolae to plasma membrane and inhibiting caveolae-associated NO production.

Why is eNOS internalization via caveolae necessary? There are no evidence-based answers to

these questions yet. Given that NO is a highly diffusible gas, one would anticipate that location

of the source would not be essential. We and others have shown that the hyperpermeability

response is impaired in the presence of NOS inhibitors such as L-NAME and L-NMMA (2, 20,

27, 33, 36). Furthermore, in eNOS knockout mice, NO produced by other NOS-isozymes does

not restore the ability of striated muscle microvasculature to produce a robust hyperpermeability

in response to PAF (16). Thus, not only eNOS expression is needed for adequate microvascular

function but also its location is important. In regards to traffic via caveolae, we speculate that

caveolae may possess a necessary target recognizing molecule that allows eNOS to efficaciously

promote the appropriate protein-protein signaling interactions in the intracellular environment.

Another speculation is that internalization via caveolae may serve to protect eNOS from S-

nitrosylation, which inactivates the enzyme (8). In addition, we may speculate that this traffic

may serve to deliver the appropriate NO concentration to achieve the correct stimulation of sGC,

the main NO-receptor, a cytosolic protein. As it is established sGC and its product (cyclic GMP)

play a necessary role in the development of hyperpermeability (32). The mechanisms by which

sGC-cGMP activate hyperpermeability is unknown; however, cGMP may activate

phosphodiesterase 2 and induce degradation of cAMP (17). In turn, degradation of cAMP may

15

reduce the barrier properties of the microvascular EC, and thus increase permeability to

macromolecules (6, 19, 28).

In conclusion, we report and propose for the first time a novel mechanism in the regulation of

microvascular endothelial permeability. Our data strongly indicate that internalization of eNOS

via caveolae is required for PAF-induced hyperpermeability. Moderate increases in permeability

are helpful to allow the exchange of macromolecules needed for wound healing and tissue

remodeling, but a highly elevated vascular permeability may have deleterious effects. Inhibition

of caveolar endocytosis may be help to control excessive hyperpermeability in inflammation.

ACKNOWLEDGMENTS

This work was supported by NIH grant 5RO1 HL070634 and by Institutional grants from the

Department of Pharmacology and Physiology, the Dean’s Biomedical Research Support (New

Jersey Medical School) and the Foundation of the UMDNJ.

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LEGENDS TO FIGURES

Fig. 1: PAF (at 100 nM) induces eNOS phosphorylation and NO production in CVEC. A)

Immunofluorescence showing eNOS expression in cell membrane and Golgi in control CVEC.

B) PAF significantly stimulated a robust increase in pericellular NO concentration. The induced

response was rapid, peaked at about 3 minutes and returned to basal levels approximately 10

minutes after PAF application (mean ± SEM, n = 6, p < 0.05). C) PAF significantly increased

phosphorylation of eNOS at Ser1177

and decreased eNOS phosphorylation at Thr495

as a function

ot time, as indicated by the ratio of phosphorylated (p-eNOS) to total eNOS (*: p<0.05, n=3).

The western blots illustrate typical examples of changes in eNOS phosphorylation at Ser1177

and

Thr495

.

Fig. 2: Depletion of eNOS inhibits PAF-induced hyperpermeability. A) 100 nM PAF

increases permeability to FITC-Dx-70 in CVEC. Data are expressed as permeability coefficients

(mean S.E.M). *: p<0.05, n = 5. B) Western blots showing depletion of eNOS as a function of

siRNA concentration and time. C) Depletion of endogenous eNOS in CVEC abrogates the

development of PAF-induced hyperpermeability. CVEC transfected with scrambled siRNA

served as a control. The increase in permeability elicited by 100 nM PAF is significant compared

to all other interventions (mean S.E.M; *: p<0.05, n = 5).

Fig. 3: PAF induces eNOS translocation in CVEC. A) Immunofluorescence images of CVEC

were obtained in control cells and after 100 nM PAF treatment. The image shows that PAF

induces the disappearance of eNOS from plasma membrane and its appearance in a diffuse

22

fashion in cytosol. The images are representative of 3 independent experiments. B) Western blots

of isolated lipid rafts in control and PAF treated cells (Fraction 1 = lightest; fraction 12 =

heaviest). Fractions were probed against eNOS and caveolin.

Fig. 4: Inhibition of caveolar internalization decreases PAF-induced hyperpermeability in CVEC.

A) Image showing expression of dyn2K44A in transfected CVEC. B) The panel shows the

impact of CVEC transfection with dyn2K44A on permeability to FITC-DX-70. CVEC

transfected with the corresponding empty vector served as control. PAF induced a robust

hyperpermeability in the control CVEC. Transfection of CVEC monolayers with dyn2K44A

significantly inhibited the PAF-induced hyperpermeability to FITC-DX-70. (*: p<0.05 compared

to control and interventions, n=5).

Fig. 5: Dyn2K44A inhibits eNOS traffic in CVEC. The figure displays eNOS

inmunofluorescence images taken from CVEC monolayers at the end of the measurement of

permeability. In the control (baseline) CVEC, eNOS is distributed in the cell membrane and in

the Golgi area. Non-transfected CVEC show eNOS translocation to cytosol (diffuse material)

after challenge with PAF (center panel). In contrast, eNOS is efficaciously retained in the

plasma membrane in the dyn2K44A transfected CVEC after administration of 100 nM PAF.

Figure 1

23

Figure 2

24

Figure 3

25

Figure 4

26

27

Figure 5