(Received for publication, May 18, 1995; and in revised form, June 7, 1995)
From the
The particulate enzyme, endothelial nitric oxide synthase
(eNOS), produces nitric oxide to maintain normal vasodilator tone in
blood vessels. In this study, we demonstrate that eNOS is a
Golgi-associated protein in cultured endothelial cells and intact blood
vessels. Using a heterologous expression system in HEK 293 cells, we
show that wild-type myristoylated and palmitoylated eNOS, but not
mutant, non-acylated eNOS targets to the Golgi. More importantly, HEK
293 cells expressing wild-type eNOS release substantially more NO than
cells expressing the mutant, non-acylated enzyme. Thus, eNOS is a novel
Golgi-associated protein, and Golgi compartmentalization is necessary
for the enzyme to respond to intracellular signals and produce NO. Under physiological conditions, nitric oxide (NO)
For
immunofluorescence microscopy, BAEC or HUVEC were plated on Permanox
4-chamber slides. The cells were fixed in 1.5% formaldehyde, 0.1 M sodium phosphate (pH 7.4) for 10 min at 25 °C and
permeabilized with 0.1% Triton X-100 in PBS/0.1% BSA for 5 min. Cells
were incubated with the appropriate primary antibodies for 2 h (eNOS
mAb, H32, IgG For light microscopy, frozen sections (10 µm)
of lymph nodes were air-dried, acetone-fixed for 10 min, and incubated
overnight with tissue culture supernatant mAb H32 (56 µg/ml, 1:300)
in PBS/0.1% BSA. Sections were washed and incubated with
peroxidase-conjugated rabbit anti-mouse antibody (Dako Corp.) for 45
min at room temperature and visualized with DAB reagent for 10 min.
Controls with non-immune IgG For electron microscopy, BAEC were grown as confluent
monolayers onto tissue culture slides with chambers (Tissue-Tek) and
fixed in situ at room temperature for 15 min in 3%
paraformaldehyde/0.1% glutaraldehyde, buffered to pH 7.4 in 0.1 M phosphate buffer. Cells were washed with 0.1%
BSA/phosphate-buffered saline (PBS-BSA, 30 min) and permeabilized with
0.1% Triton X-100 in PBS (5 min) and then washed again with PBS-BSA.
Cells were then incubated overnight at 4 °C with primary eNOS
antibody (H32, protein A-purified ascites fluid, 910 µg/ml) at a
dilution of 1:500. An irrelevant IgG
For measurement of NO Localization of eNOS in BAEC and HUVEC by indirect
immunofluorescence using an eNOS monoclonal antibody, H32, revealed
specific, crescent-shaped, perinuclear staining and additional diffuse,
peripheral staining of the antigen (Fig. 1, A and B). In both subconfluent and confluent EC monolayers, no
significant plasma membrane staining was observed. Due to the
difficulty in visualizing the spatial orientation of proteins in flat
endothelial monolayers that line large blood vessels, we stained human
lymph node microvessels from frozen biopsy samples that contain
cuboidal shaped, high endothelial venules. As seen in Fig. 1, C and D, staining of lymphatic tissue demonstrated an
intense, perinuclear peroxidase product in high endothelial venules, a
pattern identical to that seen in cultured BAEC and HUVEC. More
interestingly, the staining appeared to be polarized toward the vessel
lumen. This polarized orientation suggests that activation of eNOS by
shear forces and local factors (bradykinin, ATP) would favor NO to be
released lumenally to influence lymphocyte trafficking or
leukocyte-platelet adhesion to the vascular endothelium.
Figure 1:
eNOS is localized in the
perinuclear region of cultured endothelial cells and in intact human
blood vessels. BAEC (A) and HUVEC (B) were fixed,
permeabilized, and labeled with the H32 anti-eNOS mAb. Both endothelial
cell lines showed intense perinuclear staining in a Golgi-like
distribution. In C and D lymphatic tissues from
biopsy specimens were stained with H32, followed by peroxidase labeling
and counterstained with eosin (C) or hematoxylin and eosin (D). Notice the perinuclear lumenal (L) staining
pattern and polarized localization of eNOS in high endothelial
venules.
Next, we
colocalized eNOS in BAEC with various markers for cellular organelles
by confocal microscopy. eNOS did not colocalize with
dihydrotetramethylrosamine, a redox-sensitive dye for mitochondria, or
with calnexin, a resident glycoprotein chaperone of the endoplasmic
reticulum. Additionally, eNOS did not colocalize with iron-loaded
transferrin, suggesting an association of eNOS with structures other
than those associated with recycling plasmalemma-derived endocytic
vesicles.
Figure 2:
Colocalization of eNOS with mannosidase
II, a Golgi marker, and electron microscopic localization of eNOS on
Golgi membranes. In A, BAEC were double labeled by
simultaneous incubations of cells with mannosidase II (ManII) and H32 eNOS mAb antibodies and processed as above. B, transmission electron micrograph of Golgi stacks in
cultured BAEC after immunoperoxidase labeling with eNOS mAb. The
electron-dense peroxidase reaction product is concentrated on Golgi
cisternae. Bar = 0.5 µm; N =
nucleus; PM = plasma membrane. Arrows show
areas of intense peroxidase product on Golgi
membranes.
To characterize if recombinantly expressed eNOS targets to the Golgi
region as seen in endothelial cells, we generated stable cell lines
that expressed either wild-type (WT) or mutant (glycine to alanine
mutation, G2A) eNOS. G2A eNOS is not myristoylated or palmitoylated and
is a soluble protein that behaves identically to membrane-associated,
wild-type eNOS in direct measurements of NOS
activity(6, 7) . Immunolocalization of eNOS reveals a
majority of the WT enzyme in a perinuclear crescent that colocalizes
with mannosidase II, whereas the G2A enzyme exhibits a diffuse
cytoplasmic stain that does not overlap with the Golgi marker (Fig. 3, middle and lower panels).
Non-transfected HEK 293 cells do not stain for eNOS but stain for
mannosidase II (Fig. 3, top panel). Measurements of
eNOS specific activities (assessed by
nitro-L-arginine-inhibitable conversion of
Figure 3:
Wild-type eNOS colocalizes with
mannosidase II in stably transfected HEK 293 cells, whereas G2A mutant
eNOS does not. Non-transfected HEK 293 cells (NT) and WT and
G2A eNOS stably transfected HEK 293 cells were labeled with mannosidase
II (Man II) and eNOS antibodies and processed as
described.
Figure 4:
Ionomycin stimulates the release of more
NO
To examine the
functional significance of eNOS localized to the Golgi, we then
challenged both WT and G2A-transfected cells with the
calcium-mobilizing ionophore, ionomycin, and measured NO The mechanism of how eNOS
or other fatty acylated membrane proteins get to their final
destination is a subject of intense investigation in many
laboratories(12) . For myristoylated eNOS, there are two
possible fates; either it can be targeted to the Golgi where it is
palmitoylated by a Golgi membrane-associated palmitoyltransferase (13, 14) or it can be palmitoylated in the cytosol by
a cytoplasmic palmitoyltransferase and then targeted to the Golgi. For
plasma membrane proteins such as p21 The presence of eNOS on the Golgi is functionally
important for the activation of eNOS and the release of NO as
demonstrated by the ability of cells expressing Golgi-localized eNOS to
release more NO
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)produced by the vascular endothelium exerts a major
inhibitory influence on the contractile properties of vascular smooth
muscle and contributes to the non-thrombogenic surface of the
endothelium. NO is produced via the metabolism of one of the chemically
equivalent guanidino nitrogens of L-arginine by the enzyme
endothelial nitric oxide synthase (eNOS)(1, 2) . eNOS
is localized in membrane fractions (>90% of the total protein) of
freshly isolated and serially passaged endothelial cells and of
recombinant cells expressing the eNOS cDNA(3, 4) .
eNOS is the only N-myristoylated protein in the NOS family,
and N-myristoylation is necessary for the particulate
localization of the enzyme as demonstrated by the inability of
non-myristoylated mutants to efficiently bind to biological
membranes(5, 6) . Moreover, N-myristoylation
is necessary for palmitoylation of eNOS(7) ; however, the
site(s) of palmitoylation and the contribution of this
post-translational modification to the membrane association are not
known. Since the precise biological membrane(s) where eNOS resides and
the functional significance of such localization are not well
described(8, 9) , we set out to examine the
localization of eNOS in bovine aortic endothelial cells (BAEC) and
human umbilical vein endothelial cells (HUVEC).
Cell Culture and Immunofluorescent, Light, and Electron
Microscopy
Endothelial cells were isolated from BAEC or HUVEC
and were isolated and cultured as described
previously(5, 10) . Cells were grown in plastic tissue
culture flasks and used at passages 2-3. isotype, 56 µg/ml tissue culture
supernatant, 1:500 dilution(8) , and mannosidase II polyclonal
antisera, 1:500 dilution(11) ) followed by washing and
incubation for 1 h with fluorescein isothiocyanate- and/or Texas
Red-labeled second antibodies (Jackson Laboratories, 1:400 dilutions).
After washing in PBS/BSA, slides were mounted with Slowfade (Molecular
Probes) and observed with a Bio-Rad MRC 600 confocal inverted
microscope. Controls using non-immune at equivalent dilutions as immune
antibody or secondary antibody alone were negative. Identical
perinuclear localization was detected with a commercially available
eNOS mAb (1:300 dilution, Transduction Laboratories) and a rabbit
polyclonal eNOS Ab directed against a glutathione S-transferase-eNOS fusion protein (amino acids 134-579
of bovine eNOS).
and secondary antibody alone
were negative.
(1 mg/ml), at the
same dilution, served as a negative control. Sites of antibody binding
were visualized by the avidin/biotin peroxidase technique (Vectastain
ABC kit, Vector Laboratories). After the staining reaction with
diaminobenzidine/hydrogen peroxide, the cells were refixed with
half-strength Karnovsky's glutaraldehyde/paraformaldehyde
fixative, followed by 1% OsO
. Cells were dehydrated in
increasing concentrations of ethanol and embedded in epoxy resin.
Ultrathin serial sections, cut parallel to the original surface, were
left unstained or were double stained with uranyl acetate and lead
citrate and viewed in a Zeiss 109 transmission electron microscope,
operated at an accelerating voltage of 80 kV.
Generation of Stable Cell Lines and Measurement of NO
Release
Wild-type and G2A mutant (in which glycine-2 has been
mutated to alanine) eNOS cDNAs were constructed (6) and
subcloned into the mammalian expression vector, pcDNA3 (containing the
neomycin-resistant gene). Human embryonic kidney cells (HEK293, ATCC)
were transfected with wild-type or G2A eNOS cDNAs in pcDNA3 (15 µg
of plasmid DNA/1 10
cells in 100-mm dishes)
according to the standard calcium phosphate precipitation method.
Transfected cells were selected for growth in Dulbecco's modified
Eagle's medium (DMEM) containing penicillin (100 IU/ml),
streptomycin (100 µg/ml), and 10% (v/v) fetal calf serum (complete
DMEM) in the presence of G418 (800 µg/ml). After 14 days,
G418-resistant colonies were isolated and maintained in DMEM containing
G418 (250 µg/ml). Lines WT.HEK5 and G2A.HEK6 were used for this
study. Western blot analysis was performed as described
previously(5) .
(NO
and
NO
) release from stably transfected
cells, HEK-293 cells were grown in 35-mm
dishes. The medium
was changed to Hanks' balanced salt solution supplemented with L-arginine (100 µM) and CaCl
(1.3
mM), and cells were equilibrated for 30 min at 37 °C. In
some experiments cells were treated with N
-monomethyl-L-arginine (300
µM) in the absence or in the presence of additional L-arginine (2 mM). To stimulate NO release, ionomycin
(3 µM) was added for 1 h, and the supernatant was
collected for analysis of NO
by chemiluminescence. Samples
(100 µl) containing NO
were refluxed in 0.1 M vanadium(III) chloride in 2 M HCl. Under these
conditions, both NO
and
NO
are quantitatively reduced to NO,
which was quantified by a chemiluminescence detector after reaction
with ozone. Cells were scraped and lysed with 1 N NaOH for
total protein determination using the Lowry assay. Net NO
per mg of protein was calculated after subtracting unstimulated
basal release from the control wells.
(
)However, a majority of the eNOS
staining did colocalize with mannosidase II (1:500 dilution of rabbit
polyclonal antibody(11) , a lumenal Golgi protein (Fig. 2A). Immunoperoxidase labeling/electron
microscopy shows high concentrations of eNOS at the cytoplasmic face of
Golgi stacks (Fig. 2B) and some staining in adjacent
vesicles. There was no obvious reaction product in endoplasmic
reticulum or plasma membrane of various cells in multiple experiments.
H-labeled L-arginine to
H-labeled L-citrulline) were similar in lysates from both cell lines (89
and 92 pmol/mg/min in WT and G2A eNOS-transfected HEK 293 cells,
respectively) as were the amounts of immunoreactive NOS protein (Fig. 4A). Pulse-chase analysis of
S-labeled eNOS demonstrated identical turnovers for both
WT and G2A eNOS (18-20 h). These data suggest that myristoylation
and/or palmitoylation of eNOS are necessary for Golgi targeting of the
protein in a heterologous expression system.
from wild-type eNOS-transfected HEK 293 cells than G2A
mutant eNOS-transfected cells. In A, eNOS protein in total
lysates (60 µg) or proportional amounts of membrane (m) or
cytosolic (c) proteins from WT and G2A-transfected cells were
Western blotted with eNOS mAb H32. NT, lysate from
non-transfected HEK 293 cells. In B, transfected cells were
stimulated with ionomycin (3 µM) as described, and
NO
release was measured by chemiluminescence. Bars represent means; cappedverticallines represent S.E. (n = 5). L-NMMA, N
-monomethyl-L-arginine.
release (the sum of all nitrogen oxides). Agonist-dependent
increases in cytoplasmic calcium facilitate
calcium/calmodulin-dependent activation of endothelial nitric oxide
synthase and cause the subsequent release of
NO(1, 3) . Ionomycin (3 µM) induced the
release of NO
from both WT and G2A-transfected HEK 293
cells; however, 3-4 times more NO
was released from
WT eNOS cells (Fig. 4B). The release of NO
from both WT and G2A cells was L-arginine-dependent,
attenuated by the NOS inhibitor, N
-monomethyl-L-arginine and partially
reversed by L-arginine (L-Arg). These data suggest
that compartmentalization of WT eNOS on the Golgi is necessary for the
enzyme to respond to intracellular signals (calcium/calmodulin) and to
efficiently utilize the cellular cofactors (O
, NADPH, FMN,
FAD, and tetrahydrobiopterin) required for the stoichiometric
conversion of L-arginine to NO.
or
Src-related proteins, palmitoylation is necessary for membrane binding
and localization(15, 16) . However, for the
Golgi-associated enzyme, glutamic acid decarboxylase (GAD 65),
palmitoylation is not required for membrane association or
localization(17, 18) . Whether palmitoylation of eNOS
is necessary for Golgi localization is not known and is presently being
investigated.
than mutant cells. Presently, we cannot
rule out the possibility that the diffuse cytoplasmic staining in
endothelial cells represents eNOS in non-endosomal, post-Golgi vesicles
destined for another cellular compartment. However, preliminary data
demonstrate that stimulation of early passage BAEC with bradykinin or
HUVECs with histamine does not influence the localization of eNOS,
suggesting that the enzyme is a resident Golgi protein (data not
shown). Quantitative imaging of intracellular calcium has shown high
resting levels of calcium in the perinuclear and Golgi regions of
cells(19, 20) , and upon activation of surface
receptors, calcium accumulation occurs primarily in these same
regions(21, 22) . In endothelial cells, laminar fluid
shear stress, a stimulus for NO release, also increases perinuclear
calcium accumulation(23) . The perinuclear, Golgi localization
of eNOS would promote a productive interaction between calcium,
calmodulin, and the enzyme. In addition to the optimal spatial
orientation of the enzyme with its primary activator, calcium, the
Golgi localization of eNOS suggests that NO
may exert a
more generalized role in cellular processes such as the secretion,
nitration, and ADP-ribosylation of proteins.
We thank Dr. Kelley Moreman for the generous supply of
mannosidase II antibody, Dr. Ari Helenius for calnexin antibody, Drs.
Louis Ignarro and David Harrison for vanadium reflux/NO
chemiluminescence methodology, Dr. Mary Gerritsen for helpful
suggestions during the early phases of this project, and Rong Zhang for
expert technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.