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Flow-activated Chloride Channels in Vascular Endothelium Flow-activated Chloride Channels in Vascular Endothelium
SHEAR STRESS SENSITIVITY, DESENSITIZATION DYNAMICS,
AND PHYSIOLOGICAL IMPLICATIONS
*
Received for publication, June 19, 2006, and in revised form, September 6, 2006
Published, JBC Papers in Press, September 13, 2006, DOI 10.1074/jbc.M605866200
Mamta Gautam , Yue Shen , Twanda L. Thirkill
§
, Gordon C. Douglas
§
, and Abdul I. Barakat
1
From the Department of Mechanical and Aeronautical Engineering and the
§
Department of Cell Biology and Human Anatomy,
University of California, Davis, California 95616
Although activation of outward rectifying Cl channels is one
of the fastest responses of endothelial cells (ECs) to shear stress,
little is known about these channels. In this study, we used
whole-cell patch clamp recordings to characterize the flow-ac-
tivated Cl current in bovine aortic ECs (BAECs). Application of
shear stress induced rapid development of a Cl
current that
was effectively blocked by the Cl channel antagonist 5-nitro-2-
(3-phenopropylamino)benzoic acid (100
M
). The current initi-
ated at a shear stress as low as 0.3 dyne/cm
2
, attained its peak
within minutes of flow onset, and saturated above 3.5 dynes/cm
2
( 2.53.5-fold increase over pre-flow levels). The Cl current
desensitized slowly in response to sustained flow, and step
increases in shear stress elicited increased current only if the
shear stress levels were below the 3.5 dynes/cm
2
saturation level.
Oscillatory flow with a physiological oscillation frequency of 1
Hz, as occurs in disturbed flow zones prone to atherosclerosis,
failed to elicit the Cl
current, whereas lower oscillation fre-
quencies led to partial recovery of the current. Nonreversing
pulsatile flow, generally considered protective of atheroscle-
rosis, was as effective in eliciting the current as steady flow.
Measurements using fluids of different viscosities indicated
that the Cl current is responsive to shear stress rather than
shear rate. Blocking the flow-activated Cl current abolished
flow-induced Akt phosphorylation in BAECs, whereas block-
ing flow-sensitive K currents had no effect, suggesting that
flow-activated Cl
channels play an important role in regu-
lating EC flow signaling.
The responsiveness of vascular endothelial cells (ECs)
2
to
fluid mechanical shear forces is essential for vasoregulation and
arterial wall remodeling (1, 2). Furthermore, the fact that ECs
respond differently to different types of shear stress is likely the
basis for the localization of early atherosclerotic lesions in arte-
rial regions exposed to low and/or oscillatory shear stress (3, 4).
Although much has been learned about endothelial signaling
pathways activated by flow (57), the early events involved in
shear stress sensing and initiation of flow-mediated signaling
remain incompletely understood.
Activation of flow-sensitive ion channels is one of the most
rapid endothelial responses to shear stress; therefore, these
channels have been proposed as candidate flow sensors (5, 8).
Sudden exposure of ECs to shear stress immediately activates
inward-rectifying K
channels, leading to cell membrane
hyperpolarization (9, 10). Simultaneously, flow stimulates out-
ward-rectifying Cl channels whose activation antagonizes the
K
channel-mediated hyperpolarization and leads to mem-
brane depolarization (11, 12). Although characteristics of flow-
sensitive K
channels, including their unitary conductance,
dependence on the magnitude of applied shear stress, and reg-
ulation, have been reported in previous studies (9, 13, 14), little
is known about flow-activated Cl channels.
Shear stress rapidly phosphorylates the serine/threonine
kinase Akt (also called protein kinase B or Rac kinase) (15, 16).
Akt phosphorylation regulates endothelial nitric-oxide syn-
thase activation, integrin-mediated signaling, matrix adhesion,
and suppression of cell apoptosis (1719). The mechanisms
governing Akt activation by shear stress are not known.
In this study, we used the whole-cell patch clamp technique
in bovine aortic ECs (BAECs) to establish the sensitivity of the
flow-activated Cl
current to the amplitude of applied shear
stress, probe the desensitization characteristics of the current,
investigate the responsiveness of the current to sudden changes
in shear stress, and demonstrate that the current is responsive
to shear stress rather than shear rate. We also show that the Cl
current regulates shear stress-induced Akt phosphorylation.
EXPERIMENTAL PROCEDURES
Cell Culture and Flow Experiments
BAECs procured from
Cell Systems Corp. (Kirkland, WA) were cultured in Dulbeccos
modified Eagles medium/F-12 nutrient media supplemented
with antibiotics (penicillin/streptomycin) and 10% fetal bovine
serum (Gemini Bio-Products, Woodland, CA) and used in pas-
sage 4 7. For patch clamp experiments, cells were plated in 1
1-mm square cross-section borosilicate glass capillary tubes
(Vitrocom, Mountain Lakes, NJ). As detailed elsewhere (20),
this flow system allows determination of the shear stress to
which BAECs are exposed during recording. Cells located near
one end of the capillary tube were used for patch clamp record-
ings, whereas the other end was connected to a syringe pump
(Cole Parmer) capable of generating either steady or purely
*
This work was supported in part by an Atorvastatin Research Award from
Pfizer/Parke-Davis and by Philip Morris (to A. I. B.). The costs of publication
of this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
To whom correspondence should be addressed: Dept. of Mechanical and
Aeronautical Engineering, University of California, One Shields Ave., Davis,
CA 95616. Tel.: 530-754-9295; Fax: 530-752-4158; E-mail: abarakat@
ucdavis.edu.
2
The abbreviations used are: EC, endothelial cell; BAEC, bovine aortic endo-
thelial cell; cP, centipoise; NPPB, 5-nitro-2-(3-phenopropylamino)benzoic
acid.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 48, pp. 3649236500, December 1, 2006
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.
Printed in the U.S.A.
36492
JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 281 NUMBER 48 DECEMBER 1, 2006
at University of California, Davis on December 6, 2006
www.jbc.org
Downloaded from oscillatory flow (zero net flow rate) at prescribed flow rates (and
hence known wall shear stresses) and frequencies. Nonrevers-
ing pulsatile flow was generated by combining the outputs of a
syringe pump supplying steady flow with another syringe pump
supplying oscillatory flow.
Western Blot Analysis for Akt Phosphorylation
For Akt
studies, BAECs were plated on tissue culture-treated plastic
slides (Permanox, Nalge Nunc International, Naperville, IL)
and starved for 12 h prior to experiments in Dulbeccos modi-
fied Eagles medium/F-12 containing 0.1% fatty acid-free
bovine serum albumin (Gemini Bio-Products, Woodland, CA)
and 1% penicillin (10,000 IU/ml) and streptomycin (10,000
g/ml). Steady and oscillatory flow Akt studies were performed
in a parallel plate flow chamber as described elsewhere (21).
Flow periods ranged from 5 to 60 min. After removal from the
flow chamber, the cells were washed twice in ice-cold phos-
phate-buffered saline. The cells were then disrupted with lysis
buffer containing 20 m
M
Tris-HCl, pH 7.6, 150 m
M
NaCl, 1 m
M
EDTA, 1 m
M
EGTA, 1% Triton X-100, 2.5 m
M
sodium pyro-
phosphate, 1 m
M
-glycerophosphate (MP Biomedicals Inc.,
Aurora, OH), 1 m
M
sodium vanadate, 0.1% protease inhibitor
mixture, 1 m
M
phenylmethylsulfonyl fluoride, and 1
M
micro-
cystin (MP Biomedicals Inc.). The cell lysates were then centri-
fuged at 14,000
g
for 15 min at 4 °C. The protein concentra-
tion of each sample supernatant was determined by the
modified Lowrys method (22). Equal amounts of protein were
resolved in 8% SDS-PAGE and blot-
ted onto nitrocellulose membranes
(Bio-Rad). After blocking with 5%
bovine serum albumin, the mem-
branes were incubated (1 h at room
temperature) with phospho-specific
polyclonal antibodies that recognize
the phosphorylated site Ser(P)
473
in
Akt (Cell Signaling Technology,
Beverly, MA). After incubation
with secondary antibody (horse-
radish peroxidase-conjugated goat
anti-rabbit IgG), the blots were
incubated with SuperSignal West
Pico chemiluminescence substrate
(Pierce) and exposed to x-ray film.
The membranes were then stripped
with Restore
TM
Western blot strip-
ping buffer (Pierce) and re-probed
with total Akt antibody (Cell Signal-
ing Technology) as an internal con-
trol. Densities of the