Dual effect of fluid shear stress on volume-regulated anion
current in bovine aortic endothelial cells
Victor G.
Romanenko,
Peter
F.
Davies, and
Irena
Levitan
Institute for Medicine and Engineering, Department of
Pathology and Laboratory Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania 19104
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ABSTRACT |
The key mechanism responsible for
maintaining cell volume homeostasis is activation of
volume-regulated anion current (VRAC). The role of hemodynamic
shear stress in the regulation of VRAC in bovine aortic endothelial
cells was investigated. We showed that acute changes in shear stress
have a biphasic effect on the development of VRAC. A shear stress step
from a background flow (0.1 dyn/cm2) to 1 dyn/cm2 enhanced VRAC activation induced by an osmotic
challenge. Flow alone, in the absence of osmotic stress, did not induce
VRAC activation. Increasing the shear stress to 3 dyn/cm2,
however, resulted in only a transient increase of VRAC activity followed by an inhibitory phase during which VRAC was gradually suppressed. When shear stress was increased further (5-10
dyn/cm2), the current was immediately strongly suppressed.
Suppression of VRAC was observed both in cells challenged osmotically
and in cells that developed spontaneous VRAC under isotonic conditions. Our findings suggest that shear stress is an important factor in
regulating the ability of vascular endothelial cells to maintain volume homeostasis.
chloride channels; hemodynamic environment; vascular endothelial
cells
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INTRODUCTION |
SWELLING OF
VASCULAR ENDOTHELIAL cells is observed during ischemia
and may result in severe pathological consequences, such as narrowing
of the capillary luminal space, impairment of blood flow, lowered
integrity of the endothelial permeability barrier, and edema formation
(11, 27, 28). During the onset of reperfusion, swollen
cells cause further damage to the vascular system by restricting blood
flow, increasing capillary hydraulic resistance, and enhancing edema
formation. Thus flow environment changes considerably and may become an
important component of the volume-regulatory mechanisms of vascular
endothelial cells in ischemia and subsequent reperfusion.
The key mechanism responsible for preventing pathological cell swelling
is activation of volume-regulated anion current (VRAC). Activation of
VRAC allows Cl
and small organic osmolytes (taurine,
aspartate, glutamate) to flow out of the cell, reducing the
intracellular osmolarity and restoring cell volume back to normal
(reviewed in Refs. 17 and 52). Several lines of evidence
suggest that changes in the hemodynamic environment may be an important
factor in the regulation of VRAC in vascular endothelial cells. First,
exposure to shear stress activates a large array of signaling molecules
that are known to regulate VRAC activity. These include small G
proteins (24, 38, 53), tyrosine kinase (18, 26, 46,
48), and mitogen-activated protein kinases extracellular
signal-regulated kinase (ERK)-1 and ERK-2 (5, 44, 54).
Second, it is well known that both fluid shear stress (9, 14,
56) and osmotic stress (19, 58) induce dramatic
rearrangement of the cytoskeleton network. VRAC sensitivity to an
osmotic stress is enhanced by the disruption of F-actin (19,
30). Finally, it was shown recently that exposure of endothelial
cells to fluid shear stress, in the absence of an osmotic stimulus, can
activate a small Cl
-selective current that may be
identical to VRAC (2, 32). The flow field, however, was
not well defined in these studies because they were conducted in open
surface flow chambers, and, therefore, the quantitative relationship
between the current and the level of shear stress could not be evaluated.
To expose cells to a defined flow field simultaneously with
electrophysiological recording, we (23) developed a
minimally invasive flow (MIF) device. The MIF device is a novel
modification of a parallel-plate flow chamber based on the principle
that a narrow opening can be cut in the upper plate of the
parallel-plate chamber without creating a fluid overflow if the surface
tension forces at the opening are sufficient to counteract the
hydrostatic pressure generated in the chamber. We showed previously
(23) that the flow streamlines are only minimally
disturbed by lowering the tip of a micropipette into the MIF chamber
and that the cells inside the chamber are exposed to well-defined
unidirectional flow with a parabolic profile. Here, using the MIF
device, we show that there is a strong interaction between the osmotic
and the shear stress-dependent pathways in the regulation of VRAC and
that there are at least two mechanisms by which the two pathways interact. Our results also imply that different regimes of flow may
affect the degree of endothelial cell swelling during reperfusion injury.
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METHODS |
Cell culture.
Bovine aortic endothelial cells (BAECs) between passages 10 and 15 were grown in Dulbecco's modified Eagle's medium
(DMEM; Cell Grow, Washington, DC) supplemented with 10% bovine serum (GIBCO BRL, Grand Island, NY). Cell cultures were maintained in a
humidified incubator at 37°C with 5% CO2. The cells were
fed and split every 3-4 days.
Exposure of cells to flow: MIF device.
To record membrane currents under well-defined flow conditions, cells
grown on thin coverslips (0.1-mm thickness) were mounted in the MIF
device as previously described (23). Briefly, the MIF
device consists of three connecting flow chambers: 1) an
upper chamber that serves to spread fluid evenly across the inlet width to the flow chamber, 2) a parallel plate flow chamber
(chamber height 1 mm) with a series of narrow longitudinal slits cut
into its upper plate (cells are grown on the lower surface of this chamber), and 3) an open lower chamber from which the
perfusion medium is removed by vacuum suction. A recording micropipette accesses the flow chamber through the open slits in the top plate and
reaches the cells grown on the lower surface of the chamber. The device
permits unidirectional laminar flow of up to 15 dyn/cm2 to
be applied to the cells. The flow was driven by gravitational force.
The fluid (extracellular recording solution) was passed from a
reservoir through the MIF device, from which it was suctioned to a
flask for return to the fluid reservoir. The flow rate was set by
adjusting the height difference between the fluid reservoir and the
microscope stage/MIF device and was measured by collecting the
perfusion fluid in a vacuum flask. The shear stress (
) was calculated from
= 6µQ/wh2
(3), where µ is fluid viscosity (0.010 g · cm
1 · s
1), Q is the
flow rate (ml/s), w is the width (2 cm), and h is the height (0.1 cm) of the chamber. The flow rates were measured simultaneously with VRAC recording in every experiment. All cells were
continuously exposed to a background level of shear stress (0.05-0.1 dyn/cm2) generated by a slow perfusion of
the chamber (1-2 ml/min) maintained for the duration of the
experiment. This slow perfusion is necessary to maintain the osmolarity
of the extracellular solution and to prevent the accumulation of
chemicals that may be released from the cells.
Electrophysiological recording.
The external recording solution contained (in mM) 150 NaCl, 1 EGTA, 2 CaCl2, and 10 HEPES, pH 7.2. Internal solutions contained (in mM) 140 Cs-glutamate, 10 HEPES, and 4 ATP, pH 7.2 (CsOH) with free
Ca2+ concentration ([Ca2+]) of 15-16 nM
(0.1 CaCl2, 1.1 EGTA). Free [Ca2+] was
calculated with MAXC software (4). Chemicals were obtained from Fisher Scientific (Fairlawn, NJ) or Sigma (St. Louis, MO). The
osmolarities of all solutions were determined immediately before
recording with a vapor pressure osmometer (Wescor, Logan, UT) and were
adjusted by the addition of sucrose as required. Current was monitored
by 500-ms linear voltage ramps from
60 to +60 mV at an interpulse
interval of 5 s. The holding potential between the ramps was
60
mV. Ionic currents were measured with the whole cell configuration of
the standard patch-clamp technique (13). Pipettes (SG10
glass; Richland Glass, Richland, NJ) were pulled to give a final
resistance of 2-6 M
with the above-described recording
solutions. Pipettes were coated with Sylgard (Dow Corning) to decrease
electrical capacitance. These pipettes generated high-resistance seals
without fire polishing. A saturated salt agar bridge was used as
reference electrode. Currents were recorded with an EPC9 amplifier
(HEKA Electronik, Lambrecht, Germany) and accompanying acquisition and
analysis software (Pulse and PulseFit; HEKA Electronik) running on a
PowerCenter 150 (Mac OS) computer. Pipette and whole cell capacitance
was automatically compensated. Whole cell capacitance and series
resistance were monitored throughout each recording. Series resistance
was compensated for in all experiments (75-95%).
Volume measurements.
BAECs were loaded with 20 mM 6-methoxy-N-(3-sulfopropyl)
quinolinium (SPQ) in serum-free medium overnight. Cells were then washed three times with Cl
-free perfusion medium [in mM:
111 NaNO3, 4 KNO3, 1.6 Ca(NO3)2, 0.6 Mg(NO3)2,
50 HEPES, 5 glucose, and 28.5 mM gluconic acid, pH 7.2] and incubated
in the same medium for 2 h at 25°C. Fluorescence was measured
with a microplate fluorometer (Fluoroscan Ascent FL; Labsystems)
adapted for use with the MIF device. Excitation and emission filters
were 355 and 460 nm, respectively.
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RESULTS |
Enhancement of VRAC activation by shear stress (1 dyn/cm2).
The sensitivity of membrane anion conductance to fluid shear stress was
compared in BAECs that were challenged with a transmembrane osmotic
gradient (extracellular-to-intracellular osmolarity ratio of 0.8) or
maintained in an isosmotic environment. The osmolarity of the
extracellular solution in all experiments was maintained constant at
300 ± 10 mosM. The osmolarity of the intracellular solution in osmotically challenged cells was 370 ± 10 mosM,
whereas in cells that were exposed to an isotonic environment it was
maintained at 300 ± 10 mosM. Challenging the cells with a
transmembrane osmotic gradient resulted in a gradual VRAC development
over the period of 15-20 min followed by a steady-state plateau,
as described previously (20).
Imposition of a shear stress step from a background level to 1 dyn/cm2 during the activation phase of VRAC resulted in an
immediate increase in VRAC activation rate (Fig.
1). Typical examples are shown in Fig.
1A,b and in Fig. 1B (top curve). The
same effect was observed in the presence of 5 mM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA) in the intracellular recording solution (Fig. 1B, inset), indicating that an increase in VRAC activation rate
is not due to an increase in cytosolic Ca2+ level. The
reversal potential of the current was unaffected, indicating that the
selectivity of the shear stress-induced component of the current is
identical to the selectivity of the swelling-induced current. The
values of the reversal potential during the exposure to flow and under
no-flow conditions were
46 ± 3 and
44 ± 2 mV
respectively. Glutamate
-to-Cl
permeability
ratios calculated from these reversal potentials with the
Goldman-Hodgkin-Katz equation were 0.20 and 0.22, respectively, similar
to amino acid-to-Cl
permeability ratios reported
previously for glutamate, aspartate, and taurine (15, 22,
45). In symmetrical Cl
conditions the reversal
potential of the current became
1.5 ± 4 mV, as expected. In
contrast to osmotically challenged cells, application of shear stress
to cells maintained under isotonic conditions did not result in the
development of VRAC in a consistent and repeatable way (Fig.
1A,a; Fig. 1B, bottom).
Similarly, no current development was observed in cells exposed to
hypertonic extracellular medium (not shown).

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Fig. 1.
Typical current-voltage relationships and
time-courses of volume-regulated anion current (VRAC) development in
individual bovine aortic endothelial cells (BAECs) exposed
simultaneously to osmotic gradient and to a shear stress step of 1 dyn/cm2. A: representative current-voltage
relationships recorded from 2 individual cells, a cell challenged with
a mild osmotic gradient (extracellular-to-intracellular osmotic ratio
0.8) and a cell at isosmotic conditions. Currents were elicited by
500-ms linear voltage ramps from a holding potential of 60 mV to +60
mV. The ramps were delivered with time intervals of 5 s for the
duration of the experiment. The traces shown were recorded 50, 100, and
150 s before (a) and after (b) application
of fluid shear stress (in isosmotic conditions the traces overlap).
B: time courses of VRAC development in the same two cells.
The top curve shows VRAC development in the cell challenged
with an osmotic gradient, and the bottom curve shows the
current in the cell maintained under isosmotic conditions. Each point
represents current amplitude of an individual voltage ramp at +60 mV.
During the entire experiment cells were slowly perfused (1 ml/min, 0.05 dyn/cm2) with external recording solution, except when the
fluid flow was increased to 22 ml/min (bar), generating shear stress of
1.1 dyn/cm2. Inset, the time course of
VRAC development recorded with 5 mM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
(BAPTA) in the pipette solution.
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To calculate the average time course of VRAC development before and
after application of shear stress, the currents in the individual cells
were normalized to the value of the current in the same cell recorded
2.5 min before the application of the shear. As expected from the
increase in VRAC activation rate in individual cells, the average slope
of VRAC development increased approximately threefold on introduction
of the shear stress step (Fig. 2). We compared the rates of current development before and during the application of the flow in the same cells. No statistically significant change in current activity was observed in cells maintained in an
isosmotic environment (Fig. 2B).

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Fig. 2.
An increase in VRAC activation rate by a shear stress
step of 1 dyn/cm2. A: an average time course of
VRAC development before, during, and after the application of a shear
stress step of 0.99 ± 0.1 dyn/cm2 (n = 8). For every cell, VRAC amplitudes elicited by individual ramps for
the duration of the experiment were normalized to the VRAC amplitude
recorded in the same cell 2.5 min before the application of the shear
stress. The points show means ± SE (vertical deflections).
B: VRAC activation rates before and on application of shear
stress in cells at hyposmotic and isosmotic conditions. The rates were
calculated as slopes of linear regression for the linear segments of
the time courses. The VRAC rates before and during application of the
flow were measured from the same cells. All values are means ± SE
(n = 5-8). * P < 0.05 vs. no
flow.
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An enhancement of VRAC activity by the application of shear stress was
observed not only during the activation phase of the current but also
after the current reached steady-state plateau levels (Fig.
3A,b; Fig. 3B,
top). In these experiments,
the currents induced by an osmotic challenge were allowed to develop to
the steady-state level (15-20 min) as described above. Application of the shear stress step (1 dyn/cm2) induced a gradual
increase in VRAC activity, and termination of the flow resulted in a
slow and sometimes delayed decrease of VRAC. The response to shear
stress was attenuated after repetitive applications of shear. Figure
3B (top) demonstrates that the VRAC response to
repetitive applications of the same shear step to the same cell was
blunted. This observation was further confirmed by calculating the
average responses of the normalized VRAC after the first (Fig.
4A) and after the third (Fig.
4B) applications of the same shear stress step to the same
cells. To determine whether an increase in the current amplitude is
statistically significant, maximal current amplitudes measured during
the application of the flow in each individual cell were normalized to
the current amplitudes measured in the same cells before the
application of the flow. The ratio between the maximal current density
during the exposure to flow (first exposure) and the plateau current amplitude before the application of flow is 1.2 ± 0.03 (P < 0.05, flow vs. no flow). A similar effect was
observed in the presence of 5 mM BAPTA (current ratio 1.16 ± 0.04; P < 0.05, flow vs. no flow). No significant
changes in maximal current density were observed after repetitive flow
applications. Figure 4, C and D, shows the rate
of VRAC change before and after the application of shear. Before shear,
the slope of the VRAC change is zero because the current is at its
plateau level. Immediately after the introduction of the first shear
stress step the slope becomes strongly positive (an increase in VRAC
activity), and after the termination of the step the slope becomes
negative (a decrease in VRAC; Fig. 4C). In contrast, after
repetitive step applications (a third application) there are no
statistically significant changes in VRAC activity (Fig.
4D).

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Fig. 3.
An increase in maximal
VRAC amplitude on the application of 1 dyn/cm2 shear
stress. In this experiment, shear stress steps were introduced
only after VRAC amplitude reached its maximal steady-state value
(15-25 min after the cells were challenged osmotically).
A: current-voltage relationships recorded 50, 100, and
150 s before the application of flow (a; note
that the traces completely overlap) and 150 s after the
application of shear stress (b). The top traces
were recorded in a cell challenged with a 0.8 osmotic gradient, and the
bottom traces were recorded in a cell maintained under
isosmotic conditions. B: time courses of the shear
stress-induced enhancement of VRAC in the 2 cells described in
A. VRAC responses to the 1st and 4th applications of
the shear stress step in the same cells are shown (note the break in
the time axis).
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Fig. 4.
Sensitivity to shear stress
runs down with repetitive stimulation. A and B:
average time courses of the shear stress-induced enhancement of VRAC
induced by the 1st application of the shear stress step (1.1 ± 0.08 dyn/cm2; A) and by the subsequent (3rd and
4th) applications of the same shear stress step to the same cells
(B). The points show means ± SE (vertical
deflections). C and D: VRAC activation rates
calculated as described in Fig. 2. All values are means ± SE
(n = 5-8). * P < 0.01, significant difference vs. cell currents before shear stress
challenge.
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Biphasic effect of shear stress on VRAC activation (3 dyn/cm2).
Increasing the shear stress level from 1 to 3 dyn/cm2 did
not increase the facilitatory effect of the shear stress on VRAC activation (Table 1). To investigate
further the effect of a 3 dyn/cm2 shear stress step on
VRAC, flow was introduced during the steady-state phase of VRAC (Fig.
5). In this experiment, the introduction
of shear stress induced a transient increase in VRAC activity, followed by a slower inhibitory phase, an average response calculated from four
cells (Fig. 5A). The increase in maximal current amplitude in cells exposed to 3 dyn/cm2 was similar to that in cells
exposed to 1 dyn/cm2. An increase in current amplitude
shown in Fig. 5A is ~15%. This, however, is an
underestimation because maximal effect of shear stress does not occur
exactly at the same time in all cells and, therefore, averaging the
time courses of different cells diminishes the effect. The ratio
between maximal current amplitude calculated for each individual cell
and that normalized for the current amplitude before the application of
the flow in the same cell was 1.2 ± 0.06 (P < 0.05 flow vs. no flow).

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Fig. 5.
Biphasic effect of shear stress on VRAC activation (3 dyn/cm2). A: average time course of VRAC
response to a 3 dyn/cm2 shear stress step (3.0 ± 0.4 dyn/cm2). The points show means ± SE (vertical
deflections). B: average VRAC activation rates before,
during, and after the termination of flow. The 1st and 4th bars are
VRAC activation rates before and after application of shear stress,
respectively. The 2nd and 3rd bars represent the VRAC currents during
application of shear stress calculated for the period before and after
the peak of the current, respectively. All values are means ± SE
(n = 4-5). * P < 0.05, significant difference vs. cell currents before shear stress
challenge.
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The transient nature of the enhancement was also manifested by a
decrease in time to peak, defined as the length of time from the
application of the shear step to the maximum amplitude of the current.
Specifically, the time to peak in cells exposed to 3 dyn/cm2 was 34 ± 16 s, whereas the time to peak
in cells exposed to 1 dyn/cm2 was 162 ± 25 s
(P < 0.01). The rates of VRAC change before and after
the introduction of flow are shown in Fig. 5B.
Suppression of VRAC activation by higher shear stress levels
(5-10 dyn/cm2).
A further increase in the magnitude of the shear stress from control to
5-10 dyn/cm2 caused pronounced suppression of VRAC
activity in 12 of 16 cells, whereas a facilitatory effect was observed
only in 2 cells. When a high-shear stress step was applied during the
activation phase of VRAC development, the current was strongly
suppressed in a steplike manner (4 of 6 tested cells; Fig.
6). Furthermore, in those cells the
amplitude of the current after termination of the flow was
significantly larger than its amplitude before the application of the
shear stress step. This overshoot suggests that there is a component(s)
of VRAC activated by osmotic stress that continues to develop in the
presence of the (suppressive) shear stress, so that when the shear
stress is removed the current rises to a higher value. With repetitive
applications of shear the suppression becomes noticeably slower
(compare the 1st and 3rd applications of the step), and, eventually,
the response runs down completely. To test whether the rundown of VRAC
responsiveness to flow is due to the repetitive flow applications or to
the loss of the current sensitivity after prolonged cell swelling, the cells were maintained under hypotonic conditions for 30-35 min before the first application of flow. Figure 6B,
inset, shows that cells maintained their sensitivity to flow
after the prolonged swelling if they were not exposed to flow but lost
their responsiveness to flow after repetitive exposures to flow. This
experiment strongly supports the hypothesis that the rundown of the
responsiveness of VRAC to the shear stress stimulus is caused by
repetitive applications of flow.

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Fig. 6.
Inhibitory effect of
higher levels of shear stress (5 dyn/cm2) on osmotically
induced VRAC development. A: typical current-voltage
relationships recorded at hyposmotic and isosmotic conditions. The
currents shown were recorded before application of shear stress
(a), immediately after the application of shear stress
(b), and immediately after the termination of the flow
(c). B: time courses of shear stress-induced
inhibitory effect on VRAC development (top curve).
Bottom curve, currents recorded from a control cell
maintained in isosmotic conditions. The bar indicates the time of the
application of the shear stress. Inset, an example of an
experiment in which the 1st exposure to flow was applied 33 min after
challenging the cells osmotically. The bars indicate the times and
durations of the exposures to flow. The experiment was repeated in 3 cells.
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Application of the high-shear stress step at the steady-state plateau
phase of VRAC also resulted in a gradual inhibition of VRAC (Fig.
7A; n = 10).
The normalized average decrease in current amplitude during the flow
exposure was 0.73 ± 0.05 (P < 0.01 flow vs. no
flow). Under isosmotic conditions, cells that did not develop anionic
currents before the application of the shear stress had no response to
the shear step (n = 4). Cells that did develop a small
anionic current, however, showed some decrease in the current amplitude
(n = 4). This variability between cells is reflected in
the SE, and the overall effect of the shear stress on cells maintained
in isosmotic environment was not statistically significant (Fig.
7B). We conclude, therefore, that application of 5-10
dyn/cm2 of shear stress has a significant inhibitory effect
on the activity of VRAC.

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Fig. 7.
Inhibitory effect of shear stress on maximal VRAC
amplitude after the current reaches its steady-state level (>5
dyn/cm2). A: average time course of VRAC
response to a 6 dyn/cm2 shear stress step. The points show
means ± SE (vertical deflections). B: VRAC activation
rates before, on, and after application of shear stress in the cells at
hyposmotic and isosmotic conditions. All values are means ± SE
(n = 8-10). * P < 0.05 vs.
cell currents before shear stress challenge.
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Shear stress does not affect voltage-dependent inactivation and
rectification of Cl
conductance.
Voltage- and time-dependent decay of Cl
conductance at
depolarized voltages (greater than +80 mV) is the hallmark of VRAC in a
variety of cell types (reviewed in Refs. 34 and 39)
including vascular endothelial cells (20, 35). These
inactivation properties are unique to VRAC and can be used to
distinguish between VRAC and other Cl
conductances
such as Ca2+-dependent Cl
conductance
(36). A two-pulse voltage protocol with a conditioning pulse ranging from
60 to +160 mV followed by a test pulse to +100 mV
was used to determine the inactivation properties of the current under
flow and no-flow conditions. Voltage-dependent inactivation of the
current recorded under flow (1 dyn/cm2) is apparent from
the accelerated decay of the current amplitude shown in Fig.
8A. The inactivation ratio was
defined as the ratio between the amplitude of the test pulse delivered
after a preconditioning pulse and the amplitude of a control test pulse
delivered directly from the holding potential. Dependence of the
inactivation ratio on the voltage of the preconditioning pulse provides
a measure of the voltage sensitivity of the inactivation and the amount of charge that has to move for a channel to change its conformation from an open to an inactivated state. Figure 8B shows that
the inactivation curves measured before and during the application of
the flow are very similar.

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Fig. 8.
Voltage-dependent inactivation
and rectification of VRAC on the application of 1 dyn/cm2
shear stress. A: VRAC current recorded in response to a
family of voltage steps from 60 to +160 mV (inset). The
traces show 2 families of currents recorded from the same cell before
(a) and during (b) the application of shear
stress of 1 dyn/cm2. B: voltage dependences of
inactivation of currents before ( ) and during
( ) application of shear stress of 1.1 ± 0.1 dyn/cm2. R, normalized ratio of the current amplitude in
response of a test voltage pulse (+100 mV) after a conditioning voltage
pulse ( 60 mV to +160 mV). All values are means ± SE
(n = 4-5). C: current-voltage
relationships of VRAC before and during application of flow under
symmetrical Cl conditions.
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Another typical feature of VRAC is an outward rectification in
symmetrical ionic conditions (reviewed in Refs. 34 and
39). Figure 8C shows the currents recorded from the same
cell before and during the application of the flow in symmetrical
Cl
conditions. The rectification ratio, defined as the
ratio between the slope of the VRAC current-voltage relationship at +60
mV and the slope of the current-voltage relationship at
60 mV, was
1.67 ± 0.04 during the exposure to flow and 1.65 ± 0.07 in
no-flow conditions. The similarities between the inactivation curves
and the rectification ratios measured at the two experimental
conditions strongly suggest that the shear stress-sensitive
Cl
current is VRAC.
Shear stress has no effect on BAEC cell volume.
To test whether exposure of BAECs to shear stress results in changes in
cell swelling or shrinking, cell volume was measured by loading the
cells with SPQ, a fluorescent dye that is sensitive to cell volume
(49, 50). The principle of measuring cell volume with SPQ
is that the dye is quenched by intracellular osmolytes, presumably by
the intracellular anions. When cells swell, the intracellular
concentrations of both the SPQ and the quencher are decreased, lowering
the probability of interaction between the two, and the fluorescence
intensity increases. However, SPQ is quenched by Cl
, and
therefore it is necessary to deplete the intracellular Cl
and to substitute it with NO
, which has little
effect on SPQ fluorescence (42). Indeed, after the anion
substitution SPQ fluorescence stabilized, producing a stable baseline
with no detectable variation for the duration of 30 min. The
substitution of Cl
by NO
did not have
a significant effect on shear stress-induced facilitation of VRAC; the
ratio between the maximal current density during the exposure to flow and the plateau current amplitude before the application of flow is
1.15 ± 0.05 (P < 0.05, flow vs. no flow).
The effect of shear stress on BAEC cell volume was tested under both
isosmotic and hyposmotic conditions (Fig.
9). When cells were exposed to flow of 1 dyn/cm2 while being maintained in an isosmotic environment
and then challenged with a strong osmotic gradient (50%), there was no
change in SPQ fluorescence in response to the application of flow (Fig.
9A). Challenging the cells osmotically resulted in a
significant increase in SPQ fluorescence, as expected. Application of
flow after the cells were challenged osmotically also had no effect on
SPQ fluorescence (Fig. 9B). In the latter experiment, the
cells were challenged with a mild osmotic gradient (20%) to simulate
more closely the conditions of the electrophysiological experiments.

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Fig. 9.
Cell volume is not affected by shear stress.
A: normalized 6-methoxy-N-(3-sulfopropyl)
quinolinium (SPQ) fluorescence of BAECs during the application of shear
stress under isosmotic conditions and during subsequent osmotic shock.
The cells were continuously perfused with isotonic or hypotonic (50%)
medium. The rate of perfusion during "no flow" was maintained at
0.05 dyn/cm2. B: normalized SPQ fluorescence of
BAECs exposed to flow of 1.0 dyn/cm2 after they were
challenged with a mild osmotic shock (20%). The shear stress step of
1.0 dyn/cm2 was applied from the basal perfusion level of
0.05 dyn/cm2. Cell volumes were normalized to the values at
the beginning of the recordings. All values are means ± SE
(n = 3). The experiments were performed in the
minimally invasive flow (MIF) device.
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DISCUSSION |
The role of hemodynamic mechanical forces in the regulation of
cell volume homeostasis in vascular endothelial cells was studied by
determining the effect of fluid shear stress on the activation of VRAC,
the key mechanism protecting the cells against pathological swelling
(reviewed in Refs. 17 and 52). It has been suggested that,
in vascular endothelial cells, the physiological role of VRAC is not
only to regulate cell volume but also to sense changes in the
hemodynamic environment (33, 35). This is the first study
to investigate the sensitivity of VRAC to shear stress in a controlled
flow environment. Our results demonstrate that VRAC is regulated by
acute changes in shear stress, indicating that both osmotic and shear
stress stimuli contribute to VRAC regulation.
Two lines of evidence suggest that the shear stress-sensitive
Cl
current in BAECs is VRAC and not a
Ca2+-dependent Cl
current that is also
expressed in vascular endothelial cells (36). First,
stress-induced increase in Cl
conductance was observed in
cells in which VRAC was activated by an osmotic stress but not in cells
maintained in isosmotic or hypertonic conditions, and there was a
strong correlation between the ability of cells to develop VRAC and
their ability to respond to shear stress, as discussed in more detail
below. Second, the voltage-dependent properties of shear
stress-sensitive Cl
current observed in our study are
similar to those of VRAC and not Ca2+-dependent
Cl
current. Specifically, the currents recorded under
flow exhibited slow voltage-dependent decay at depolarizing potentials
(greater than +80 mV), a feature that is typical for VRAC (see, e.g.,
Refs. 21 and 35). In contrast, Ca2+-dependent
Cl
current is known to exhibit voltage-dependent
activation, the opposite trend at these potentials (31,
36). VRAC and Ca2+-dependent Cl
current also exhibit opposite trends in their voltage-dependent properties at negative membrane potentials. Ca2+-dependent
Cl
current rapidly inactivates, whereas VRAC has no
voltage dependency at these potentials. The behavior of the
Cl
current recorded under flow was identical to that of
VRAC. In addition, the rectification properties of the current were
also similar to those of VRAC and not to those of
Ca2+-dependent Cl
current, whose
rectification is significantly stronger than that of VRAC (31,
36). We conclude, therefore, that the increase in
Cl
current on the application of shear stress is due to
potentiation of VRAC and not to activation of
Ca2+-dependent Cl
current.
The pattern of VRAC regulation by shear stress is complex. Exposing the
cells to shear stress in a range of 1-10 dyn/cm2 has a
biphasic effect on the swelling-induced development of VRAC: a
facilitatory phase at low levels of shear stress (1 dyn/cm2) and an inhibitory phase at the higher levels of
shear (5-10 dyn/cm2). Importantly, the two phases
coexist at a medium level of shear stress (3 dyn/cm2). In
the latter case, a facilitatory phase develops immediately after the
introduction of the shear step and an inhibitory phase develops more
slowly and becomes dominant after a delay of ~1 min, an observation
consistent with the concept that acute changes in shear stress and
steady levels of shear may have differential effects on the physiology
of endothelial cells (6, 8). To exclude the possibility
that the observed effects are due to variations in extracellular
osmolarity that may occur during the experiment because of evaporation,
the cells were continuously exposed to a background shear stress level
of 0.05-0.1 dyn/cm2. A step from no flow to the
background shear stress level in the beginning of an experiment did not
induce any overt response. Because of the high Cl
concentration in the extracellular solution, we can exclude washout of
Cl
from the vicinity of the cell membranes as an
explanation of the observed effects of flow. We also show that
application of shear stress does not result in changes in cell volume
of BAECs, suggesting that the sensitivity of VRAC to shear stress is
not due to shear stress-induced changes in cell volume. The biphasic effect of shear stress on VRAC suggests that there are at least two
mechanisms by which swelling-induced and shear stress-induced transduction pathways interact with each other in the regulation of the current.
These observations provide a possible explanation for the earlier
contradictory reports on the ability of flow to activate endothelial
Cl
current. Although low-amplitude flow-induced
Cl
currents were observed in vascular endothelial cells
(2), no flow-sensitive Cl
currents were
observed under similar flow conditions (41). It is well
known that establishment of a whole cell configuration may induce
"spontaneous" VRAC under isosmotic conditions (see, e.g., Ref.
57), an effect that is attributed to a Donnan equilibrium between the cytosol and the pipette (57). We suggest,
therefore, that in previous reports flow-induced Cl
current was observed in cells that may have developed spontaneous VRAC
before the flow exposure. This hypothesis is supported in our study, in
which there was a strong correlation between the ability of cells to
develop VRAC in response to osmotic stress and their ability to respond
to shear stress. Specifically, cells that failed to develop VRAC in
response to osmotic stress, typically 20-30% of the cell
population (21, 47), also had no response to shear stress
(not shown). The source of the heterogeneity in swelling-induced and
spontaneous VRAC development is not known, but the correlation between
the ability of cells to respond to osmotic and shear stimuli, observed
in our study, provides further evidence that the two effects are coupled.
Several mechanisms may underlie the interactions between osmotic and
shear stress stimuli. The nonadditive nature of the interaction between
the stimuli suggests that parallel pathways may be involved in VRAC
regulation. Rho A-induced reorganization of the cytoskeleton (37,
38, 53, 55) and protein tyrosine kinase-induced phosphorylation cascade (18, 46, 48, 55) are required for VRAC activation (reviewed in Ref. 33). Both signaling pathways are also
regulated by shear stress. Specifically, shear stress induces the
translocation of Rho A from the cytosol to the membrane
(25), a step that is known to activate the Rho-induced
signaling pathway (12). Translocation of Rho A is required
for the shear stress-induced reorganization of the stress fibers
(25). Protein phosphorylation cascades are also regulated
by shear stress in a complex biphasic manner (1, 40, 54).
The dual effect of shear stress on VRAC, therefore, may be mediated by
a shift in the equilibrium between phosphorylation/dephosphorylation
cascades and/or by reorganization and detachment of cytoskeleton from
the membrane.
The levels of shear stress examined in this study are in the low
physiological range (1-10 dyn/cm2). Similar levels of
shear stress are known to induce a variety of endothelial responses,
including activation of flow-sensitive K+ currents
(1-15 dyn/cm2; Refs. 16 and 41),
reorganization of the cytoskeleton (9), translocation of
Rho A (25), and activation of protein tyrosine kinase
(40). Typical average values of shear stress in the major human arteries during basal conditions are 2-20
dyn/cm2 with local transients to 30-100
dyn/cm2 (7). The basal levels of shear in
small coronary arteries and arterioles, measured in canine vascular
bed, average 10 and 19 dyn/cm2, respectively
(51). High levels of shear stress (>10
dyn/cm2) were not tested in our experiments because it was
impossible to maintain a stable seal between the recording pipette and
cellular membrane.
Regulation of VRAC by shear stress may have important clinical
implications. Swelling of microvascular endothelial cells observed during low-flow ischemia (10, 27, 29) can reduce
the capillary diameters below the diameter of the blood cells,
resulting in further impairment of blood flow. The narrowing of
capillary luminal space persists on the start of reperfusion so that
full blood supply is not reestablished (10, 28). This
condition of "no reflow" is considered to be the major
postischemic dysfunction of capillaries, and it has been
suggested that therapeutic strategies should be aimed to prevent
endothelial swelling. Although there are some differences between
ischemia-induced cell swelling and swelling induced by an acute
osmotic shock, VRAC activation plays a major role in protecting the
cells against excessive swelling in both conditions (reviewed in Ref.
43). In summary, our study suggests that the level of
shear stress during the onset of reperfusion may significantly affect
cell volume homeostasis of endothelial cells: a step to low shear
stress is advantageous because it enhances protection against cell
swelling, whereas a step to high shear stress may be detrimental
because it suppresses the protection.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Aron Fisher, Yefim Manevich, and Kevin
Foskett of the University of Pennsylvania for critical reading of this manuscript. We also thank Nadeene Harbeck and Alan Sun for superb technical assistance.
 |
FOOTNOTES |
This work was supported by American Heart Association Grant 0060197U
and American Heart Association Scientist Development Grant 0130254N to
I. Levitan and by National Heart, Lung, and Blood Institute Grants
HL-62250 and HL-64388-01 to P. F. Davies.
Address for reprint requests and other correspondence: I. Levitan, Univ. of Pennsylvania, IME, 1160 Vagelos Research
Laboratories, Philadelphia, PA 19104 (E-mail:
ilevitan{at}mail.med.upenn.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00247.2001
Received 14 June 2001; accepted in final form 14 November 2001.
 |
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