1 GI Research Unit, Department of Physiology, and Tumor Biology Program, Mayo
Clinic, Rochester, MN 55905, USA
2 Division of Cardiovascular Disease, and Molecular Medicine Program, Mayo
Clinic, Rochester, MN 55905, USA
* Author for correspondence (e-mail: shah.vijay{at}mayo.edu)
Accepted 12 May 2003
![]() |
Summary |
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Key words: Nitric oxide, Nitric oxide synthase, Bradykinin, Dynamin-2
![]() |
Introduction |
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BK is a soluble peptide that acts via NO to promote vasodilation and
capillary permeability. Soluble BK binds the membrane-bound BK2 receptor with
downstream activation of eNOS through divergent and convergent signaling
pathways that include Hsp 90, MAP kinase, receptor tyrosine kinases, and eNOS
dephosphorylation (Bernier et al.,
2000; Harris et al.,
2001
; Thuringer et al.,
2002
; Venema et al.,
1996
). In addition to these complimentary signaling pathways, it
has been observed that treatment of cells with BK is associated with changes
in the subcellular distribution of eNOS
(Prabhakar et al., 1998
;
Thuringer et al., 2002
;
Venema et al., 1996
). Owing to
the prominent influence of GTPases, particularly the large GTPase, dynamin-2,
on internalization of plasmalemmal vesicles and downstream signaling pathways
such as that described for the extracellular signal-regulated kinase cascade
(Pierce et al., 1999
), we
sought to determine the influence of perturbation of dynamin-2 and GTP
hydrolysis on cellular eNOS localization and NO production. We hypothesized
that the specific targeting of eNOS, as a passive cargo within cellular
vesicles, may influence the ability of the enzyme to produce and release NO.
We demonstrate, using two complimentary cell systems, that BK-stimulated NO
production is associated with a redistribution of the plasmalemmal pool of
eNOS protein, with enrichment within Triton X-100 insoluble, low buoyant
density cell fractions. Furthermore, we demonstrate that disruption of
dynaminand GTP-dependent endocytosis, but not clathrin-dependent endocytosis,
abrogates the BK-mediated redistribution of eNOS within cells. Also,
disruption of dynamin GTP hydrolysis attenuates BK-dependent cellular NO
production. These studies implicate a role for GTPase-dependent,
clathrin-independent, trafficking of eNOS-containing vesicles in the mechanism
of BK-mediated NO generation.
![]() |
Materials and Methods |
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Cell culture
Complimentary techniques were performed in both bovine aortic endothelial
cells (BAEC), which express eNOS endogenously, and in ECV-304 cells stably
transfected with eNOS-GFP (eNOS-GFP ECV 304 cells)
(Sowa et al., 1999). Although
the latter cells are probably derived from a human bladder tumor
(Brown et al., 2000
), their
phenotype has been documented to partially overlap with that of endothelial
cells; and owing to their stable and uniform expression of eNOS-GFP at
relatively high levels (Paxinou et al.,
2001
; Sowa et al.,
1999
), these cells have served as a useful model in which to study
eNOS trafficking. BAEC were from Clonetics (San Diego, CA) and used between P2
and P5, while eNOS-GFP-ECV 304 cells were a generous gift from Dr William
Sessa (Sowa et al., 1999
).
Both cell types were cultured in Dulbecco's modified Eagle's medium (DMEM),
supplemented with 10% fetal bovine serum; 1 mM L-glutamine, and 100 IU/ml
penicillin. eNOS-GFP ECV 304 cells were additionally supplemented with 400
µg/ml G418. For transfection experiments, eNOS-GFP ECV 304 cells were
plated on glass coverslips in 12- or 24-well plates, or in 100 mm culture
dishes and transfected using a calcium phosphate method with pcDNA3-dyn 2
K44A, pCMV-myc AP 180, or appropriate control vectors. Twenty-four hours after
transfection of eNOS-GFP ECV 304 or 24 hours after plating of BAEC, the cells
were incubated with 10 µM BK or vehicle for 10 minutes at 37°C. After
BK stimulation, cells were prepared for various subcellular fractionation
procedures, microscopic analyses, or NO measurements as individually described
below. For permeabilization experiments, cells were pretreated with either 1
µM digitonin, 10 µM GTP-
-s, 1 µM digitonin plus 10 µM
GTP-
-s, or vehicle (PBS) for 5 minutes, then washed with PBS prior to
BK stimulation. In some experiments 10 µM ATP-
-s was used in place
of GTP-
-s and in some experiments, cells were treated with 15 µM
chlorpromazine (CP), an agent that blocks clathrin-dependent endocytosis, for
10 minutes prior to BK stimulation (Petris
et al., 2002
). To assess transferrin uptake in response to AP 180
transfection (Ford et al.,
2001
), cells were incubated in low serum medium (0.2% BSA) for 30
minutes at 37°C and Texas Red-conjugated transferrin was added from a
stock of 5 mg/ml to the medium at a ratio of 1:1000. Cells were washed with
PBS after 10 minutes incubation at 37°C. Cells were washed further with
low serum medium (pH 3.5) to reduce background signals and fixed with 2%
formaldehyde solution. Uptake of transferrin was assessed by confocal laser
scanning microscopy.
Confocal laser scanning microscopy
To study eNOS-GFP localization in living cells in response to BK
stimulation, a cell culture system with stable temperature control (POC-R;
Zeiss) was mounted to the laser scanning confocal microscope (Pasqual LSM 5;
Zeiss). Briefly, eNOS-GFP ECV 304 cells plated and cultured on 32 mm
coverslips were mounted in fresh medium in the 37°C POC-R chamber on the
microscope. BK was added to the chamber and the cells were scanned and single
confocal images were captured at 4-minute intervals using a 63x
C-apochromat lens. Micrographs of live cells were digitized and exported to
Adobe Photoshop, version 5.0, for calculation of the intensity of the
fluorescence in the perinuclear and plasmalemmal regions at each time
interval, using data pooled from three independent experiments. For
immunofluorescence microscopic analysis, BAEC were fixed in acetone/methanol
1:1 for 3 minutes at -20°C after BK stimulation (10 minutes), while ECV
304 cells were fixed with 2% paraformaldehyde. Fixed cells were incubated with
the appropriate dilution of indicated Ab or blocking serum for 2 hours at room
temperature, and primary Ab was detected with an FITC- or Texas Red-conjugated
secondary antibody and mounted in Anti-fade (Molecular Probes, Oregon). Cells
were visualized using a confocal laser scanning microscope with a 63x
lens. Owing to the previously noted heterogeneity in subcellular distribution
of eNOS between neighboring cells
(Prabhakar et al., 1998;
Sowa et al., 1999
), we
classified cells into two groups for the purpose of quantifying changes in NOS
distribution in association with BK: (1) plasma membrane pattern (PM:
cells with distinct plasma membrane and perinuclear localization of eNOS-GFP)
and (2) non-plasma membrane pattern (Non PM: cells with perinuclear
localization of eNOS-GFP in the absence of plasmalemmal staining).
Quantification of eNOS redistribution in fixed cells was performed in a blind
fashion from 200 individual cells in each group from two independent
experimental preparations.
Measurement of cellular NO production
NO production was measured using the complimentary fluorescent NO indicator
probes, DAF 2DA and DAF-2, the former detects intracellular NO accumulation
and the latter detects NO production by assessing NO released from cells
(Fulton et al., 2002;
Goetz et al., 1999
;
Kojima et al., 1998
), as well
as NO-specific chemiluminescence (Shah et
al., 1997
). For the DAF-2DA assay, BAEC were cultured on glass
coverslips in 12-well plates. After the permeabilization procedure described
above, cells from each of the experimental groups were washed twice with PBS,
preincubated with 0.1 mM NOS substrate, L-arginine at 37°C, or 1.0 mM of
the NOS inhibitor, L-NAME for 10 minutes, then loaded with DAF-2DA (10 µM).
After 10 minutes, BK or vehicle was added to the cells. Intracellular DAF-2DA
fluorescence was visualized using a conventional fluorescence microscope
(5100TV, Zeiss, Germany) and fluorescence was quantified from ten selected
regions of the digitized micrographs, each taken at 1 minute time intervals,
using Adobe Photoshop 5.0 software. Specificity of DAF-2DA fluorescence was
established in preliminary experiments, which demonstrated a linear
relationship between DAF-2DA fluorescence intensity in response to the
NO-stimulating calcium ionophore A23187 and parallel control experiments using
DAF-4, a nonfluorescent analog (data not shown). To measure cellular NO
release using DAF-2, eNOS-GFP ECV304 cells were transfected as described above
and 24 hours later cells were incubated in a solution containing 1 µM DAF-2
with either 0.1 mM L-arginine or 1 mM L-NAME at 37°C. After 30 minutes, BK
(10 µM) was added to the cells for an additional 10 minutes at which point
the cell supernatant was transferred to microcuvettes for fluorescence
quantification with excitation of 485 nm and emission of 538 nm using a
fluorimeter (VersaFluor Flurometer, BioRad). Fluorimetry was performed with
duplicate readings from duplicate wells and run along side a standard curve
generated using known amounts of sodium nitroprusside (0-500 nM) and
normalized for cellular protein.
NO-specific chemiluminescence was performed using a Seivers NOA,
essentially as previously described (Shah
et al., 1997), except that glacial acetic acid containing sodium
iodine was substituted for vanadium/hydrochloric acid as the refluxing agent.
A standard curve was generated using known nitrite standards. Samples of
medium were measured in duplicate and normalized for protein
concentration.
Subcellular fractionation and western blotting
For separation of cell membranes from cytosol, BAEC or eNOS-GFP ECV 304
cells were scraped in a buffer containing 50 mM Tris-HCl pH 7.4, 0.1 mM EDTA,
0.1 mM EGTA, plus a protease inhibitor cocktail (Roche Diagnostics, Germany),
and sonicated on ice at 200 W/cm2 for 3 cycles with 10 seconds
intervals, then centrifuged at 100,000 g for 1 hour at
4°C. The pellet constituted the membrane fraction and the supernatant was
utilized as the cytosolic fraction. Equal amounts of protein from the membrane
and cytosol fractions were boiled in Laemmli buffer for 5 minutes and
subjected to SDS-PAGE and western blotting. In experiments examining eNOS
segregation in response to Triton X-100, the membrane pellet was resuspended
in 0.5% Triton X-100 in 50 mM Tris-HCl buffer for 15 minutes at 4°C and
centrifuged at 10,000 g for 10 minutes. The resulting pellet
constituted the Triton insoluble fraction (TIF) while the supernatant
represented the Triton soluble fraction (TS). The TIF membrane pellet was
homogenized with 5 cycles of 20 strokes in a Dounce homogenizer. Protein
concentrations of these fractions were determined by BCA assay and equal
aliquots were boiled in Laemmli buffer and subjected to SDS-PAGE and western
blotting. Cellular proteins were also fractionated by buoyant density, using a
well established protocol (Smart et al.,
1995) that we have slightly modified as we previously described
(Peterson et al., 1999
). For
separation of low buoyant density membranes, four 150 mm dishes of confluent
eNOS-GFP ECV 304 cells were washed and scraped in 5 ml of fractionation buffer
(0.25 M sucrose, 6 mM EDTA, 120 mM Tricine, pH 7.8) and pelleted by
centrifugation (1400 g). Cells were resuspended in 1 ml of the
identical buffer and homogenized with 20 strokes of a Dounce homogenizer. A
postnuclear supernatant was collected by centrifugation for 10 minutes at 1000
g. The supernatant was layered on 30% Percoll in fractionation
buffer and centrifuged at 84,000 g for 30 minutes. A distinct
visible band comprising plasma membranes was collected and sonicated three
times (200 W/cm2). Sonicated samples were mixed with Optiprep to a
final concentration of 23%, in a Sorvall Ultrafuge tube. Two additional layers
of 20% and 10% Optiprep were added prior to centrifugation at 52,000
g for 90 minutes. Eight gradient fractions were collected and
30 µl of each fraction were boiled in Laemmli buffer for 5 minutes and used
for SDS-PAGE. After cell fractionation and SDS-PAGE, gels were prepared for
Coommassie staining or alternatively for transfer to nitrocellulose membranes
for western blotting. For western blotting, membranes were blocked with 5% dry
milk for 1 hour at room temperature prior to incubation with antibodies
specific for eNOS, caveolin, ß-COP, dynamin-2, myc and V5 epitope as
previously described (Shah et al.,
1999
).
Statistical analysis
Where appropriate, analysis of variance (two-tailed, paired values) was
used to evaluate the differences in the levels of parameters studied, using
Statgraff, Version 3. A P value of 0.05 was considered statistically
significant.
![]() |
Results |
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|
To examine the influence of BK on eNOS subcellular distribution, we
performed biochemical and microscopic analyses. First, eNOS-GFP ECV 304 cells
were stimulated with BK (10 µM) or vehicle for 10 minutes, and cell lysates
were separated into cytosolic and membrane fractions. Western blot analysis of
these fractions did not demonstrate differences in distribution of eNOS
between these two fractions in cells treated with BK as compared to vehicle,
as previously reported (Liu et al.,
1995). However, further separation of the membrane pellet by
Triton X-100 solubility revealed an enrichment of eNOS protein within the
Triton-insoluble fraction of cells treated with BK as compared to vehicle
(Fig. 2, right).
|
We next assessed the influence of BK on eNOS localization by performing
studies in live eNOS-GFP ECV 304 cells using a cell culture system adapted for
a laser scanning confocal microscope. Previous studies in these cells have
established that tracking of GFP accurately assesses, and does not confound,
the subcellular localization of the fused eNOS protein
(Sowa et al., 1999). Treatment
of eNOS-GFP ECV 304 cells with BK promoted the time-dependent internalization
of eNOS from the plasmalemmal eNOS location that is characteristic of these
cells when grown to confluence (Fig.
3A, bottom panel micrographs). Temporal quantification of
fluorescence intensity of both Golgi and plasmalemmal pools of eNOS
demonstrate that Golgi distribution of eNOS did not decrease in parallel with
the plasmalemmal pool of eNOS, reducing the likelihood of a photobleaching
effect (Fig. 3A, graph in
bottom panel). Cells treated with vehicle showed no change in the fluorescence
pattern of eNOS-GFP over a similar time period indicating that eNOS
localization was not influenced by the cell culture conditions within the
experimental system (Fig. 3A,
top panel, micrographs and graph). The graphs were generated by compiling
cumulative data points from 3 cells from each group (mean ±
s.e.m.).
|
Next, to further demonstrate that eNOS does indeed leave the plasma
membrane in response to BK, immuno-localization studies of plasma membrane and
Golgi marker proteins were performed in conjunction with eNOS localization in
fixed cells after BK stimulation. As seen in
Fig. 3B (top panels),
Na+-K+ ATPase immuno-localization is detected on the
plasma membrane of cells (top left panel), and substantive pools of eNOS are
situated on the plasma membrane as well (top middle panel). The merged image
(top right panel) indicates that pools of the two proteins co-distribute on
discrete regions of plasma membrane (arrows, indicated in yellow). As seen in
Fig. 3B (lower panels), after
stimulation of cells with BK, Na+-K+ ATPase distribution
(Mobasheri et al., 1997)
remains largely unchanged (bottom left panel), while plasma membrane pools of
eNOS are no longer prominent (bottom middle panel). Subsequently,
colocalization of the two proteins on plasma membrane is no longer detected
(bottom right panel). Next, the Golgi membrane pool of eNOS was examined in an
analogous manner (Fig. 3C).
Under basal conditions, substantive pools of eNOS (top middle panel; arrows)
reside within a perinuclear pattern reminiscent of the location of the Golgi
marker, 58K protein (top left panel). Indeed, merging of these images (top
right panel, yellow) demonstrates colocalization of eNOS-GFP with 58K protein.
After stimulation of cells with BK, neither 58K protein (bottom left panel) or
eNOS-GFP (bottom middle panel) redistribute from the Golgi membrane, as
further substantiated by persistent colocalization of eNOS-GFP with Golgi 58K
protein in a perinuclear distribution (bottom right panel, yellow). These
observations suggest that BK stimulation of cells promotes the selective
dissociation of eNOS from plasma membrane but does not promote dissociation of
eNOS from Golgi.
Inhibition of GTP-dependent endocytosis, but not clathrin-dependent
endocytosis, influences the subcellular redistribution of eNOS in response to
BK
To test the role of dynamin and retrograde vesicle trafficking on
BK-mediated internalization of eNOS, we first interrupted GTP hydrolysis
within eNOS-GFP ECV 304 cells using cell permeabilization and GTP--S.
Cells were preincubated with either the cell permeant, digitonin (1 µM for
5 minutes); digitonin with the nonhydrolyzed GTP analog, GTP-
-S; or
digitonin with the non-hydrolyzed ATP analog, ATP-
-S. Cells were
stimulated with BK or vehicle and prepared for confocal imaging. The left
micrograph in Fig. 4A
demonstrates the plasmalemmal and perinuclear distribution of eNOS observed in
cells treated with digitonin alone, which was similar to that observed in the
absence of digitonin (seen in Fig.
3C, top middle panel). BK stimulation (10 µM) was associated
with a decrease in the fluorescence intensity of plasmalemmal eNOS (middle
micrograph), consistent with the analyses in
Fig. 3. Interestingly, the
influence of BK on plasmalemmal eNOS was abrogated by 5 minutes preincubation
of cells with GTP-
-s (right micrograph), though not so prominently by
ATP-
-s (not shown). Similar results were obtained in fixed BAEC, and
this data was utilized for quantification of imaging as depicted in the graph
in Fig. 4B. For this analysis,
quantification was performed in a blind manner by assessing the percentage of
cells with and without plasmalemmal eNOS after the various experimental
treatments (n=200 BAEC per group in two independent experiments).
Consistent with that observed qualitatively in the representative micrographs
from ECV304 cells in Fig. 4A,
BK stimulation of BAEC was associated with a reduction in the number of cells
that expressed plasmalemmal eNOS (open bars), and this effect was abrogated by
preincubation of cells with GTP-
-s
(Fig. 4B). These studies
indicate that inhibition of GTP hydrolysis abrogates eNOS internalization
after BK stimulation.
|
GTP--s may inhibit both clathrin and caveolae vesicle-mediated
endocytosis (Oh et al., 1998
).
However, as plasmalemmal eNOS resides largely within caveolae
(Shaul et al., 1996
), we
anticipated that the inhibitory influence of GTP-
-s on BK-mediated eNOS
redistribution from the plasma membrane was largely mediated through a
caveolae-dependent process rather than via a clathrin-dependent process. To
further examine this, we used complimentary pharmacological and molecular
approaches, to examine a role for clathrin-dependent endocytosis in this
process. First, CP, a cationic amphiphilic drug that inhibits the assembly of
the clathrin adapter protein AP2 on clathrin-coated pits
(Petris et al., 2002
), was
incubated with eNOS-GFP ECV 304 cells prior to BK stimulation. As seen in
Fig. 4C, in cells preincubated
with CP, internalization of eNOS-GFP is still detected in response to BK
(image c; compare with cells in the absence of BK and CP in image a, and cells
in the presence of BK and absence of CP in image b). Consistent with this
observation, inhibition of clathrin-mediated endocytosis via the
overexpression of AP 180 protein, which limits the size and distribution of
clathrin cages (Ford et al.,
2001
), also did not prevent BK-mediated eNOS internalization
(image d; compare to cells in the absence of BK in image a, and cells in
presence of BK and absence of AP 180 in image b). However, AP 180 did reduce
the uptake of Texas Red-conjugated transferrin from transfected cells by
greater than 50% compared to cells transfected with the empty vector,
indicating that this reagent was indeed functional under our experimental
conditions (n=250 cells per group, data not shown). These studies
suggest that the inhibitory influence of GTP-
-s on BK-mediated eNOS
internalization is likely a caveolae-dependent process rather than a
clathrin-dependent process.
We next dissected the role of the specific cellular GTPase, dynamin-2, as
this GTPase is essential to retrograde vesicle trafficking events from the
plasma membrane (Dessy et al.,
2000; Henley et al.,
1998
; Oh et al.,
1998
). We tested the role of dynamin in this process by
overexpressing a dominant negative dynamin-2 K44A construct in eNOS-GFP ECV
304 cells, and measuring eNOS distribution within the varying buoyant density
membrane fractions after BK stimulation. This construct contains a point
mutation in the dynamin GTPase domain that impairs the ability of dynamin to
hydrolyze GTP (Cao et al.,
2000
). Fig. 5A
demonstrates the validity of the density gradient used in these experiments,
to distinguish low and high buoyant density proteins prepared from eNOS-GFP
ECV 304 cells as shown by the enrichment of the 22 kDa protein, caveolin, in
the low buoyant density fractions (fractions 1-4), and the converse enrichment
of the Golgi and trans-Golgi compartment marker protein, ß-COP, in the
high buoyant density fractions (fractions 6-7), consistent with prior studies
by us and others using this technique
(Peterson et al., 1999
;
Smart et al., 1995
). After BK
stimulation of eNOS-GFP ECV 304 cells transfected with empty vector, eNOS was
enriched within low buoyant density fractions
[Fig. 5B; rectangular
box, see eNOS signal in lanes labeled E in fractions #1-4; (-) vs (+)],
while in cells overexpressing K44A, BK-mediated enrichment of eNOS in low
buoyant density fractions was not detected
[Fig. 5B; rectangular box, see
eNOS signal in lanes labeled K in fractions #1-4; (-) vs (+)]. Control
experiments confirm overexpression of K44A as assessed by immunodetection of
the V5 epitope tag (see V5 western blot band in
Fig. 5B) and similar level of
total proteins within each fraction as assessed by Coommassie Blue staining of
gels (not shown). These studies indicate that the biochemical redistribution
of eNOS in response to BK stimulation requires the GTPase function of
dynamin-2.
|
Perturbation of GTP hydrolysis inhibits cellular NO production
We next sought to determine whether inhibition of dynamin-2-dependent
vesicle trafficking might influence cellular NO production as well. First, to
examine the influence of GTP--s on the cellular production of NO in
cells that express eNOS endogenously, BAEC were permeabilized with digitonin
and pretreated with GTP-
-s (10 µM) or vehicle. Cells were then
loaded with the NO indicator dye, DAF-2DA, and BK-stimulated NO production was
assessed by fluorescence microscopy. As seen in
Fig. 6A (top panel
micrographs), there was a time-dependent increase in cellular fluorescence
intensity in response to BK. However, BK-induced NO production was markedly
abrogated in cells pretreated with GTP-
-s (middle panel micrographs in
Fig. 6A), and further
diminished in the presence of the NOS inhibitor, L-NAME, as well (bottom panel
micrographs in Fig. 6A).
Quantification of the fluorescent intensity in cells from digitized
micrographs revealed a 60% decrease in NO production by GTP-
-s in
comparison to digitonin alone, in response to BK stimulation (lower graph in
Fig. 6A). Next, experiments
were performed to assess the effect of K44A overexpression on cellular NO
production. eNOS-GFP ECV cells were transfected with empty pcDNA vector or
K44A-V5 epitope-tagged vector, and cellular NO release was measured from the
media using the fluorescent NO probe, DAF-2. Transfected cells were treated
with either L-arginine or L-NAME and stimulated with BK or vehicle. As seen in
Fig. 6B, overexpression of K44A
markedly inhibited the cellular production of NO in response to BK. L-NAME
inhibited NO production in all groups, indicating specificity of effect.
Western blot control experiments from cell lysates, confirmed the expression
of V5 epitope and overexpression of dyn-2 in cells transfected with the vector
encoding K44A-V5 as compared to cells transfected with the empty vector, as
well as similar eNOS protein levels throughout the groups. To further confirm
the inhibitory influence of K44A on cellular NO production, we performed
complimentary studies using NO-specific chemiluminescence, which assesses
levels of the NO metabolite, nitrite. As seen in
Fig. 6C, L-NAME-inhibited,
BK-stimulated nitrite accumulation was reduced in cells transfected with K44A
as compared to empty vector. Thus, studies using complimentary NO measurement,
indicate that inhibition of cellular GTPases, particularly dynamin-2, abrogate
BK-mediated NO production.
|
![]() |
Discussion |
---|
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---|
The precise role of nucleotide hydrolysis and dynamin-2 in the molecular
steps of membrane scission, internalization, and ensuing retrograde vesicle
trafficking remain an area of active investigation
(Henley et al., 1998;
McNiven, 1998
;
Oh et al., 1998
;
Schnitzer et al., 1996
;
Schekman and Orci, 1996
). In
the present studies, we chose to exploit the energy requirement of vesicle
trafficking to study the mechanism by which perturbation of vesicle
trafficking might influence eNOS distribution and NO production by using both
pharmacological and molecular approaches, in conjunction with complimentary
techniques, to measure cellular NO and assess NOS localization. Indeed, a
similar approach was recently utilized to dissect the role of vesicle
trafficking in the desensitization of G protein-coupled receptor signaling and
eNOS-caveolin complex stability in cardiac myocytes
(Dessy et al., 2000
). We find
that inhibition of GTP hydrolysis by GTP-
-S impairs BK-dependent eNOS
redistribution and NO production. Furthermore, perturbation of cells with
dominant negative form of dynamin-2 mimic the inhibitory effects of
GTP-
-S on NO generation, indicating a particular importance of
dynamin-2 in NOS trafficking and function. Dynamin-2 is of particular
relevance as this protein colocalizes with caveolin and eNOS within caveolae
and Golgi vesicles, and is essential in the scission and internalization of
plasmalemmal vesicles (Cao et al.,
2001
; Henley et al.,
1998
; Oh et al.,
1998
). Our current observations are consistent with several prior
studies, which have demonstrated that inhibition of dynamin GTPase function
prevents internalization of caveolin-coated membrane vesicles
(Henley et al., 1998
;
Oh et al., 1998
;
Pierce et al., 1999
), and add
to these studies by demonstrating that this process influences downstream
signaling pathways, in this case relating to NOS activation. However, it is
important to recognize that the influence of GTP-
-S on eNOS trafficking
may reflect inhibition of cellular GTPases in addition to dyn-2, which are
essential to the processes of vesicle trafficking, such as Rab 3, Rab 5 and
Rab 7 (Chavrier and Goud,
1999
). Several converging and diverging BK-mediated signaling
pathways that culminate in NOS activation have been well delineated (Bernier
et al., 275; Harris et al.,
2001
; Thuringer et al.,
2002
; Venema et al.,
1996
), and the interplay of these pathways with dynamin-dependent,
BK-mediated eNOS internalization will require further elucidation.
We have previously demonstrated that dynamin-2 binds in a specific manner
with eNOS and promotes the catalytic activity of recombinant eNOS protein in
vitro (Cao et al., 2001). In
the current studies, we anticipate that K44A dynamin is likely influencing NO
production through effects on vesicle internalization rather than through
direct binding actions. This concept is supported by the observation that the
eNOS binding domain within dynamin, resides within the carboxy-terminal
proline-rich domain of dynamin-2, rather than the amino-terminal GTPase
domain, wherein lies the K44A mutation (Cao
et al., 2003
). Furthermore, colocalization of eNOS and dynamin in
cells is most prominent in the Golgi membranes
(Cao et al., 2001
), where a
significant and bioactive pool of both proteins reside
(Fulton et al., 2002
), rather
than within plasma membrane, where the influence of K44A appears to be most
prominent. It is possible that direct binding regulation between eNOS and
dynamin occurs in the Golgi membranes while dynamin-dependent endocytosis
selectively influences the plasmalemmal pool of eNOS.
In conclusion, these studies, using complimentary cell lines and methodologies, demonstrate that perturbation of specific vesicle trafficking pathways has the capability to influence cellular eNOS distribution and the ensuing NO production. These studies add to the current understanding of eNOS biology by demonstrating that BK-dependent eNOS trafficking is a GTPase-dependent process, with particular importance of dynamin-2 GTPase, and that perturbation of this process impairs the cellular production of NO. Additionally, they provide further evidence that dynamin-dependent endocytosis is intimately linked to downstream cell signaling events.
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Acknowledgments |
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