© 1999 American Heart Association, Inc.
Cellular Biology |
A Flow-Activated Chloride-Selective Membrane Current in Vascular Endothelial Cells
From the Department of Mechanical and Aeronautical Engineering (A.I.B.) and Section of Neurobiology, Physiology and Behavior (E.V.L., P.A.P.), University of California, Davis, Calif, and Institute for Medicine and Engineering, Department of Pathology and Laboratory Medicine, and Department of Bioengineering (P.F.D.), University of Pennsylvania, Philadelphia, Pa.
Correspondence to Abdul I. Barakat, PhD, Department of Mechanical and Aeronautical Engineering, University of California, One Shields Ave, Davis, CA 95616. E-mail abarakat{at}ucdavis.edu
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Abstract |
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Abstract—Shear stress–induced activation of endothelial ion channels, one of the earliest responses to flow, is implicated in mechano–signal transduction that results in the regulation of vascular tone. The effects of laminar flow on endothelial membrane potential were studied in vitro using both fluorescent potentiometric dye measurements and whole-cell patch-clamp recordings. The application of flow stimulated membrane hyperpolarization, which was reversed to depolarization within 35 to 160 seconds. The depolarization was caused by a Cl--selective membrane current activated by flow independently of the K+ channel-mediated hyperpolarization. Thus, flow activated both K+ and Cl- currents, with the net membrane potential being determined by the balance of the responses. Membrane potential sensitivity to flow was unchanged by flow preconditioning that elongated and aligned the cells.
Key Words: mechanotransduction • shear stress • ion channel • potentiometric dye • patch clamp
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Introduction |
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The endothelium is a biological mechanotransducer that senses shear and stretch forces associated with blood flow and converts these mechanical stimuli into biochemical signals.1 2 3 In vivo, endothelial mechanotransduction is central for both acute vasoregulation and chronic vascular remodeling.4 5 In vitro studies have demonstrated that fluid mechanical shear stress elicits acute endothelial responses that include activation of ion channels6 and G proteins,7 8 9 mobilization of intracellular calcium,10 11 and induction of protein kinase pathways.12 Subsequent transcription factor activation13 and binding to shear stress response–related sequences of several flow-responsive genes3 results in important functional and structural changes in the cells that include extensive topographic and cytoskeletal reorganization, cellular elongation, and alignment in the direction of shear stress.14 15 16
The mechanisms that initiate shear stress signaling are poorly understood. They may arise from discrete surface molecules that undergo deformation at the luminal surface or from an integrated change of intracellular tension,1 or both. An important very early response to flow, however, is the activation of a K+ current that hyperpolarizes the cell6 17 and is driven by shear stress–responsive inward-rectifying K+ channels present on the luminal surface of cultured endothelium.18 In studies using membrane potential–sensitive dyes, we noted a biphasic endothelial response to the initiation of flow in which membrane hyperpolarization was quickly attenuated and reversed to depolarization.19 In this paper, using both whole-cell patch-clamp recordings and measurements from potentiometric fluorescent dyes, we demonstrate the presence of a shear stress–induced chloride-selective current in endothelial cells that antagonizes flow-induced hyperpolarization. The membrane potential responsiveness to flow persists in flow-conditioned cells that have undergone significant topographic and cytoskeletal reorganization.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell Culture
Bovine aortic endothelial cells (BAECs) were isolated and cultured by standard procedures.20 Cells in passages 15 to 25 were used. After trypsinization, the cell suspension was plated at subconfluent density either onto standard square (22x22-mm) glass coverslips (patch-clamp experiments) or into 1x1-mm square cross-section borosilicate glass capillary flow tubes6 (VitroCom, Inc) (potentiometric dye measurements). The cells were bathed in DMEM (high sucrose; Gibco) containing 10 mmol/L HEPES, 2 mmol/mL glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% heat-inactivated calf serum (Gibco).
Reagents
The membrane potential–sensitive fluorescent dye
bis(1,3-diethylthiobarbiturate)trimethine oxonol, abbreviated to
"bisoxonol," was purchased from Molecular Probes, Inc. DIDS and
diphenylamine carboxylate (DPC) were purchased from Sigma Chemical Co.
The patch pipette fill solution consisted of (in mmol/L) potassium
aspartate 100, KCl 35, K2-EGTA 10,
CaCl2 1, MOPS 10, ATP 1, and
MgCl2 2, and pH was adjusted to 7.2 with KOH
(
6.5). ATP/MgCl2 solution (pH 7.2 with KOH)
was added to the fill solution from frozen stock at the beginning of
each day. Normal Ringer's solution contained (in mmol/L) NaCl
140, KCl 5, CaCl2 2, MgCl2
1, and MOPS 10, as well as 5.6 D-glucose, pH 7.4
with NaOH (6.5). Low-Cl- Ringer's solution
contained (in mmol/L) sodium aspartate 140, KCl 4,
CaCl2 2, MgCl2 0.5, and
MOPS 10, pH 7.4 with NaOH. Low-Na+ Ringer's
solution contained (in mmol/L)
N-methyl-D-glucamine (NMDG)
140, KCl 5, CaCl2 2, MOPS 10, and 5.6
D-glucose, pH 7.4 with HCl (130). Fill solutions had
measured osmolarities of 275 mosm. External solutions had measured
osmolarities of 265 to 310 mosm.
Patch-Clamp Recordings
All patch-clamp recordings were performed at room
temperature on BAECs cultured on coverslips which were subsequently
mounted in a Warner recording/perfusion chamber (Warner
Instrument Corp). A schematic of this chamber is illustrated in Figure 1
. Steady laminar flow was administered
via a syringe pump (Cole Parmer).
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Preliminary experiments indicated that responsiveness of cells to flow may depend on the waiting period experienced before exposure to shear stress. This possibility was not investigated in detail; instead, a uniform minimum waiting period was imposed. All data presented were from cells exposed to at least 20 minutes of no-flow conditions before and between experimental runs.
Whole-cell voltages were recorded from cells in confluent
monolayers using standard patch-electrode recording
techniques.21 Utilizing confluent cells took advantage of
the electric coupling via gap junctions known to be present between
adjacent BAECs. Currents from cells adjacent to the recording
cell reduced the significance of shunts to ground through the pipette
seal. This provided more stable and less depolarized membrane voltage
recordings in current-clamp mode compared with that obtained in
single subconfluent cells. However, this technique has 2 disadvantages,
as follows: (1) ion reversal potentials are not precisely known,
because only the recording cell is dialyzed whereas the voltage
is measured from the recording cell plus adjacent (nondialyzed)
cells, and (2) voltage clamp is not possible because of gap junctions
to other cells in the monolayer. Borosilicate pipettes had resistances
of 3 to 5 M
. The voltage offset between the patch pipette and the
bath solution was nulled immediately before seal formation. An agar
bridge containing 1 mol/L KCl was used to ground the bath solution.
Voltages were recorded from a patch-clamp amplifier (Axopatch 200A,
Axon Instruments) in current-clamp mode (I=0). Data were
collected on a Macintosh personal computer using Pulse+Pulsefit
software (HEKA Elektronik). Resting membrane voltages were defined as
average voltages over a 29- or 60-second period immediately before flow
onset.
Whole-cell currents were recorded similarly from single
(subconfluent) cells with membrane capacitances nulled through the
patch-clamp amplifier. The holding potential was -70 mV, which is
close to the resting membrane voltages observed in current-clamp mode
(-60 to -76 mV in normal Ringer's solution; n=47 traces from 23
cells) and to that reported by other investigators in similar in vitro
preparations.6 18 22 23 The voltage ramp protocol
comprised a voltage step from the holding potential to +60 mV for 40 ms
followed by a ramp to -100 mV over a period of 400 ms followed by a
return to the holding potential. During the experimental protocol,
voltage ramps were conducted once every 5 seconds for 10 minutes,
yielding 120 traces. Conductances were computed by taking the
derivative of a least-squares straight line fit to the I-V
data plots using Igor Pro software (Wavemetrics, Inc). In cases in
which the I-V plots were not linear over the entire voltage
ramp (eg, Figure 5E), computation of the whole-cell conductance
was restricted to a linear range, typically -100 to 0 mV. Data are
presented as mean±SEM.
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Membrane Potential Measurements Using Potentiometric Dyes
Experiments using the potentiometric fluorescent dye
bisoxonol were performed on single (subconfluent) cells cultured in
borosilicate glass capillary tubes.6 BAECs were washed
with PBS and then incubated in PBS containing 5x
10-7 mol/L of the bisoxonol dye for 15 minutes
at 37°C. The dye was added in ethanol stock solution, with the final
ethanol concentration in the incubated cells never exceeding 0.05%. In
some experiments, a solution made to approximate the ionic composition
of the culture medium was used. This solution consisted of (in
mmol/L) KCl 4, NaCl 150, CaCl2 1,
MgCl2 2, and HEPES 10, pH 7.4 with HCl.
Fluorimetric measurements of membrane potential were performed using a Spex ARCM-1000 microspectrofluorimeter (Spex Industries). The fluorimeter was interfaced to a Nikon Diaphot inverted microscope equipped with a Nikon Fluor objective (x40). The photometer contained a pinhole diaphragm to regulate the viewing field. The results represent measurements on single cells at room temperature located at or near the center midline of the tube. Dye-loaded cells were excited at 495 nm; emission at 515 nm was collected at 1.0-second intervals. Background fluorescence was automatically subtracted from all measurements. Similar results were obtained whether the perfusate was PBS (in most experiments) or the solution approximating the ionic composition of the culture medium.
Flow was induced by connecting the capillary tube containing dye-loaded cells to a 60-mL syringe mounted on a syringe pump (Harvard Apparatus) via a length of Silastic tubing (Masterflex, Cole Parmer). Perfusion rates were calibrated by direct measurement of collected volume for a range of pump gear ratios. Shear stress levels were calculated from the flow rates and tube geometry on the basis of the fully developed Poiseuille flow solution in a tube of circular cross section as previously described.6 Because the capillary tubes used here had square cross sections, the equivalent (or hydraulic) diameter used in the Poiseuille flow expression corresponded to the length of a side of the square tube. The perfusate contained the same concentration of bisoxonol dye as that in which the cells were incubated (5x10-7mol/L). This was necessary to avoid concentration gradient–driven dye washout from the cells.
Flow Loop
In experiments on flow-conditioned cells, BAECs were subjected
to flow by insertion of the capillary tubes into a recirculating flow
loop. Components of the loop were connected by small diameter Silastic
tubing (Cole Parmer). A reservoir of culture medium (125 mL) was
maintained at 37°C adjacent to the microscope stage that was enclosed
by a temperature-regulated Plexiglas incubator, also maintained at
37°C. Medium, gently gassed with air/5% CO2,
was drawn through the system by a peristaltic flow pump (Cole Parmer).
To dampen pulsatility, the main reservoir was vented, and 2 buffer
reservoirs were inserted between the pump and flow tube.
Filamentous Actin (F-Actin) Fluorescence Staining
Cells were fixed with 4% formaldehyde in PBS for 20 minutes at
37°C. After 3 washes with PBS, the cells were
permeabilized by 0.1% Triton X-100 in PBS for 3
minutes at room temperature and washed twice with PBS and then 3 times
with 50 mmol/L ammonium chloride (pH 7.3) for 5 minutes each wash.
After rinsing twice with PBS, F-actin was stained by addition of
NBD-phallacidin (Molecular Probes) at a dilution in PBS of 1:100
of a 3 mmol/L stock solution, for 20 minutes at room temperature
followed by 3 final washes in PBS. Cells in capillary flow tubes
containing 1:10 glycerol:PBS were viewed using a Zeiss Axiophot
fluorescence microscope equipped with epifluorescence.
Cell images were photographed on 35-mm Kodak T-Max film (ASA 400).
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Results |
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Acute Membrane Potential Shear Responses
The dependence of bisoxonol fluorescence intensity on membrane potential has previously been extensively studied17 24 25 ; membrane depolarization leads to an increase in fluorescence intensity as a result of a rise in intracellular concentration of the negatively charged bisoxonol, whereas membrane hyperpolarization has the opposite effect. We have confirmed this behavior in control experiments in which the membranes of BAECs cultured in capillary flow tubes were either depolarized with 100 mmol/L KCl or hyperpolarized with 2 µmol/L ATP (data not shown).
The variation of bisoxonol fluorescence intensity with time in
response to flow was measured in single (subconfluent) BAECs previously
unstressed by flow (n=17). Results obtained from a
representative cell are illustrated in Figure 2A. The baseline intensity was
recorded for the first 300 seconds, after which flow at a steady
rate of 0.7 mL/min (generating a wall shear stress of 1
dyne/cm2 at the midline of each face of the
square cross-section tube) was initiated and maintained for 25 minutes
(1500 seconds). This shear stress level was selected because it
approximates that required for half-maximal activation of the
flow-sensitive inward-rectifying K+ channel
initially reported by Olesen et al6 but is insufficiently
high for the induction of significant topographic and cytoskeletal
reorganization.14 The fluorescence intensity
initially sharply decreased in response to flow, indicating membrane
hyperpolarization consistent with previous
reports using patch-clamp recordings6 18 and
membrane potential–sensitive fluorescent dyes.17
With continued flow, however, the fluorescence intensity
increased above the original baseline value, reaching a plateau at a
depolarized state. Flow, therefore, induced initial cell
hyperpolarization followed by depolarization.
Membrane depolarization also occurred in a few experiments in which
flow was arrested immediately after
hyperpolarization (data not shown).
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The bisoxonol data provide a qualitative picture of BAEC membrane potential responsiveness to flow. Whole-cell patch-clamp recordings were performed to validate and quantify the bisoxonol shear experiments. Recordings in both current-clamp mode on cells in a confluent monolayer and voltage-clamp mode on single (subconfluent) cells were made. All patch-clamp experiments were performed at a steady flow rate of 3 mL/min. The corresponding shear stress to which the cells in the Warner chamber were exposed in this system is not precisely known; however, assuming a linear velocity variation in the direction orthogonal to flow, the shear stress is estimated to be in the range of 0.5 to 1 dyne/cm2.
Figure 2B
illustrates current-clamp recordings of the
response of BAEC membrane potential to the steady shear stress in
normal Ringer's solution. In these experiments, a stable resting
membrane potential (-69±0.5 mV, range -60 to -76 mV; n=23 cells)
was recorded for a period of 180 seconds before initiating flow.
Flow was then maintained for 240 seconds before being turned off. With
onset of flow, the membrane voltage rapidly hyperpolarized, reaching a
peak hyperpolarization amplitude of 1.7±0.2 mV
(range 0.4 to 6 mV) within 16±1 seconds (range 4 to 43 seconds).
Hyperpolarization was then reversed so that the
membrane potential crossed the resting voltage 64±6 seconds (range 35
to 158 seconds) after the onset of flow and then continued to increase
to a depolarization of 4.4±1.5 mV (range 0 to 36 mV). The dynamics of
the depolarization were not the same in all cells; peak depolarization
occurred at (Figure 2B), before, or after cessation of flow. As
mentioned in Materials and Methods, a no-flow waiting period of at
least 20 minutes was imposed before exposure to shear stress. In a
limited number of experiments in which a shorter waiting period was
used, the initial hyperpolarization event appeared
unaffected, but the magnitude of the subsequent depolarization was
reduced.
Although all cells in this study responded to flow with a rapid
hyperpolarization,6 18 the
depolarizing response was less consistent, occurring in 22 of
33 cells (15 of 23 current-clamp, 7 of 10 voltage-clamp cells) in
experiments in normal Ringer's solution. Some cells responded to flow
with a hyperpolarization that did not resolve to
the preflow resting potential (Figure 3A)
but instead remained stable at a more hyperpolarized level. In 4 of 5
cells (in either current- or voltage-clamp mode) that did not exhibit
flow-induced depolarization in normal Ringer's solution, it was found
that on replacement of the external solution with
low-Cl- Ringer's solution, the same flow
paradigm elicited a depolarizing response (Figure 3B). This
suggests a potential involvement of Cl- ions in
the flow-induced depolarizing response.
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Comparison of Figure 2A with Figure 2B suggests that,
whereas the bisoxonol dye system qualitatively captures the behavior of
BAEC membrane potential responsiveness to flow, the time constants and
relative magnitudes of the flow-induced changes in dye
fluorescence intensity differ from the
physiological behavior measured in the patch-clamp
experiments. The reason for this difference remains unknown but may
relate to an interaction between the flow and the dynamics of bisoxonol
partitioning between the cytoplasmic and extracellular spaces. However,
the minimal invasiveness of the dye to both the cells and the flow
field makes this technique attractive for qualitative studies of
membrane potential flow responses.
Depolarization Is Due to a Shear Stress–Induced
Cl--Selective Membrane Current
The fact that the flow-induced depolarization was maximized in
low–external Cl- Ringer's solution suggested
the involvement of a Cl--permeable conductance.
Endothelial cells express a variety of
Cl- channels.22 23 26 27 28 29 30 Cells
were therefore exposed in dye experiments to the 2 known
Cl- channel blockers DIDS (1 mmol/L) and
DPC (1 to 2 mmol/L) and subsequently subjected to a steady shear
stress of 1 dyne/cm2. In the presence of
Cl- channel inhibitors, there was a
sustained hyperpolarization without depolarization
of the cells (Figure 4), suggesting the
involvement of Cl- channels in the flow-induced
depolarization response. DIDS and DPC did not block the depolarization
in every case; DIDS was effective in 3 of 6 cells, whereas DPC blocked
flow-induced depolarization in 5 of 6 cells.
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More definitive evidence of flow activation of a
Cl--selective current was provided by whole-cell
voltage-clamp experiments. Cells were subjected to the same flow
protocol as used in the current-clamp experiments. In cells treated
with 1 mmol/L external Ba2+ to block the
flow-sensitive K+ current,6 18
whole-cell conductance was increased by 50% during flow and did not
return to resting levels until several minutes after flow had been
stopped (Figure 5A). Voltage ramps from
this experiment intersected at -18 mV (Figure 5D), indicating
that the reversal potential of the flow-activated current was
near this value and consistent with Cl-
(ECl=-36 mV) carrying a significant portion of
the current.
The reversal potential for the flow-activated current
(intersection point of voltage ramps) shifted with
ECl on replacement of external
Cl- with aspartate31 32 (current
reversal potential28 mV; ECl31 mV),
indicating that the current was largely mediated by
Cl- ions (Figure 5B). In contrast, the
reversal potential of the flow-activated current was, for the
most part, insensitive to replacement of external
Na+ with the impermeant cation NMDG (Figure 5C; current reversal potential=-11 mV;
ECl=-34 mV); the intersection point would have
shifted to a more hyperpolarized level if a
Na+-selective current were involved. These
results are consistent with the activation of an
anion-selective current.32 Figure 6 illustrates the net (leak-subtracted)
currents elicited by flow in the data of Figure 5. This figure
more clearly demonstrates the reversal potentials, and it illustrates
that the flow-induced current is altered considerably in low external
Cl- while remaining virtually unchanged in low
external Na+. The fact that the reversal
potentials obtained here do not exactly match ECl
suggests that the flow-activated Cl-
current is imperfectly selective or that other conductances are
activated. For example, a nonselective cation component, as has
been demonstrated in other Cl-
currents,31 would shift the reversal potential of the
observed current away from ECl and toward 0 mV,
as was seen in these experiments (Figure 5D through 5F).
Alternatively, the Cl- current observed here
could have some finite permeability to aspartate.33 In the
conditions presented in Figure 5, Easpartate was positive in the cases of normal
and low-Na+ Ringer's solution, whereas
Easpartate was negative in the case of
low-Cl- Ringer's solution. Again, this would be
consistent with the shift in reversal potential toward 0 mV
observed in all conditions. Lastly, the discrepancy between calculated
and observed reversal potentials could be due in part to junction
potentials in our patch-clamp setup that were not corrected for.
However, this voltage error is <7 mV for our
solutions.32
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The fact that the flow-induced Cl- current was
observed in the presence of the K+ channel
blocker Ba2+ (Figure 5) suggests that this
current is activated independently by flow and is not a
consequence of the activation of the flow-sensitive
K+ channels. Furthermore, Figures 5B and 6 reveal an additional characteristic of the
flow-activated current in low external
Cl- solution, a substantial increase in
conductance. The same cell shown in Figure 6A in normal
Ringer's solution showed a >3-fold increase in resting conductance in
low-Cl- Ringer's solution (Figure 6B).
In response to flow, conductance increased by 100% in both normal
and low-Cl- Ringer's solution. The increased
conductance in low external Cl- may be due to
the recruitment of more channels, an increase in unitary
conductance,33 34 a direct effect of aspartate (used to
replace Cl- in low-Cl-
Ringer's solution) on conductance, or a combination of these factors.
The increase in resting conductance is likely not due to an increase in
nonselective or K+-based leaks, as the
reversal potential in no-flow conditions does not shift more negatively
(compare Figure 5D trace 1 with Figure 5E trace 1 from
the same cell). More likely, this increase in conductance is
Cl--based and could be due to activation of the
volume-sensitive Cl- current known to be
present in macrovascular endothelial
cells.35 36 Osmolarity of the
low-Cl- solution was consistently 10%
to 15% less than that of the normal Ringer's solution, and this may
have been sufficient to elicit a volume-activated
Cl- current; however, because bath solution
composition did not change during shear, the fact that
Cl--based conductance did increase substantially
in response to shear in low-Cl- solution
indicates that the flow-induced conductance is not due simply to
changes in osmolarity.
Flow Preconditioning Does Not Desensitize Flow-Activated
Currents
The bisoxonol dye system was used to investigate membrane
potential responses to flow in endothelial cells
exposed to flow for periods sufficiently long to induce cell alignment.
BAECs in capillary tubes were exposed to flow at a shear stress of 13
dyne/cm2 in the flow loop for a period of at
least 24 hours. In these experiments, cells were confluent on one face
of the square cross-section capillary tube and subconfluent on the 2
adjacent faces. At the end of flow preconditioning, the F-actin in
cells within the confluent monolayer underwent extensive remodeling
(Figure 7), and the cells became
elongated and aligned in the general flow direction. Subconfluent cells
did not uniformly align but underwent significant cytoskeletal
reorganization. The cells were then taken out of the loop and
immediately washed and incubated in the bisoxonol dye in a fashion
similar to that previously described in the acute flow experiments.
Membrane potential measurements on single (subconfluent) cells in
response to a shear stress of 1 dyne/cm2 were
then recorded (n=4).
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Figure 8 illustrates the response of
flow-preconditioned BAECs to the steady shear stress. All cells
hyperpolarized and then depolarized in a fashion very similar to that
of the response recorded for previously unsheared cells. These
results indicate that endothelial cells that have
adapted to sustained flow by undergoing extensive flow-induced
topographic and cytoskeletal reorganization retain their membrane
potential responsiveness to flow.
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Discussion |
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The activation of a K+ membrane current is one of the fastest known responses to fluid mechanical shear stress in endothelium6 18 and may play a role in the sensing and transduction of fluid shear forces. In this paper, we have used a combination of patch-clamp recordings and measurements from the membrane potential-sensitive fluorescent dye bisoxonol to demonstrate that shear stress also activates a Cl- current in cultured BAECs. As a result of this current, the initial K+ channel–mediated membrane hyperpolarization is reversed to a depolarized state within 35 to 160 seconds of the onset of flow, and this depolarization often persists for several minutes after flow cessation. Evidence for the Cl--selective nature of the depolarizing current was provided by varying external Cl- concentration in the patch-clamp experiments and by using 2 Cl- channel blockers (DIDS and DPC) in the bisoxonol dye studies. Induction of the Cl- current by shear stress persisted in the presence of external Ba2+, a known blocker of the shear-sensitive K+ channel; therefore, the activation of the Cl- current is an independent shear stress–induced response and is not a consequence of the initial K+ channel–mediated hyperpolarization.
To our knowledge, this is the first demonstration of the direct induction by fluid shear stress of a Cl- current in vascular endothelium. At least 3 types of Cl- channels have been described in macrovascular endothelial cells: Ca2+-activated Cl- channels, volume-activated Cl- channels, and large-conductance Cl- channels that may be regulated by voltage or by cAMP22 23 ; however, the sensitivity of these channels to fluid mechanical forces has not been evaluated. More recently, lung-endothelial cell adhesion molecule-1 (Lu-ECAM-1), an endothelial cell surface molecule that mediates adhesion of metastatic melanoma cells to lung endothelium, has been cloned and characterized in cultured BAECs.37 Interestingly, this molecule is not a member of any of the classic families of cell-cell adhesion molecules, including the selectins, integrins, and immunoglobulins, and it is highly homologous (92% homology at the DNA level and 88% at the amino acid level) to a Ca2+-activated Cl- channel present in bovine tracheal epithelium.38 Because of this homology, it has been suggested that Lu-ECAM-1 may function both as an adhesion molecule and a Cl- channel and that this functional duality may bestow mechanosensitivity to the channel.
The only evidence to date of direct activation of
endothelial Cl- channels by
mechanical stimulation is provided by studies demonstrating that
hypotonic volume increase elicits Cl- currents
in cultured human umbilical vein and bovine pulmonary artery
endothelial cells.27 35 These
volume-activated Cl- channels, which are
different from the Ca2+-activated
Cl- channels,36 may also be
responsive to fluid mechanical shear stress (B. Nilius, personal
communication, September 1996). It is unknown whether or not the
shear stress–activated Cl- current
reported in the present study is mediated by the activation of
these volume-regulated Cl- channels; however, it
is not expected that the relatively low shear stress levels used in the
present study (1 dyne/cm2) would lead to
significant stretch of the endothelial cell membrane to
result in a direct change of cell volume.
The impact of shear stress on endothelial cell membrane ion currents has previously been studied in BAECs by whole-cell patch clamping6 39 and single-channel patch clamping18 and in bovine pulmonary artery endothelial cells by membrane potential–sensitive fluorescent dyes.17 Although all of these studies reported rapid activation by shear stress of a K+-selective membrane current, the precise character of the resulting membrane potential response to sustained flow varied from persistent hyperpolarization17 18 to ion channel desensitization and membrane potential return to the no-flow baseline6 to Ca2+-dependent spontaneous transient hyperpolarizing bursts.39 Reasons for these differences remain unclear.
It is also unclear why the Cl- current reported
here was not observed in the other studies. One possibility may be
related to our observation that in some cells the magnitude of the
flow-induced depolarization may be smaller unless there is a relatively
long no-flow waiting period (up to 20 minutes) between consecutive flow
episodes. This may suggest that the desensitization and resensitization
dynamics of the flow-sensitive K+ current differ
from those of the Cl- current. If such
differences occur in vivo, then they might be expected to have
particular relevance to endothelial cells within flow
separation regions in the vicinity of arterial bifurcations
where the time-averaged wall shear stress is very low but becomes
transiently high during exercise or stress conditions.40
Our results suggest that the length of the time period between stress
episodes (relative to the no-flow waiting period) may determine the
extent of the flow-induced Cl- current in the
endothelial cells within these flow separation zones.
It should also be noted that the whole-cell patch-clamp experiments
modified cytosolic contents and were performed at room temperature. It
is not known whether these factors impact the desensitization and
resensitization dynamics of the flow-activated channels.
Another possible reason for differences between our results and those
of others is that in the previous studies, ECl
was maintained near 0 mV, whereas an ECl of -36
mV (Figure 5D) was used in the present study; thus, a
Cl- current in the other studies could have been
interpreted as a nonspecific leak current.
It is unknown why the shear stress–induced Cl- current was not observed in all cells studied. In response to a shear stress of 5 dyne/cm2, Hoyer et al39 observed flow-induced K+ bursts in 50% of BAECs studied, whereas 95% of the cells became responsive at 9 and 18 dyne/cm2. We tested the idea that the imposed shear stress needs to exceed a critical threshold for Cl- channel activation and that this threshold is not identical in all cells. In a limited number of patch-clamp experiments (data not shown), the flow rate of the perfusate on nonresponsive cells was increased; however, the results were inconclusive, with some cells remaining nonresponsive and others exhibiting only marginal depolarization. Another possibility is that the density of the shear stress–sensitive Cl- channels on the BAEC surface is highly variable from one cell to another. Such topographic heterogeneity has been reported for K+ channels in neurons41 and for volume-activated Cl- channels in vascular endothelial cells.42 The surface density of Lu-ECAM-1 also appears to vary considerably in freshly isolated BAECs, but the extent of this variability appears to decrease with progressive passages in culture (A.D. Gruber, personal communication, April 1998). To assess the impact of cell passaging, we have performed a limited number of voltage-clamp recordings in BAECs from early passages (passages 4 to 5; data not shown) and have established that the flow-activated Cl- current reported here is consistently observed in these cells. It is interesting that in studies on epithelial cells, anion currents in response to osmotic stress have been reported to occur in only approximately two thirds of the cells studied,43 44 a fraction similar to that reported here. Cell-to-cell heterogeneity has also been observed both in vivo and in vitro in several other endothelial flow responses.45 Examples include sensitivity to shear stress of VCAM-1, ICAM-1, and [Ca2+]i.
Use of the fluorescent potentiometric dye bisoxonol for studies
of the effect of flow on endothelial cell membrane
potential is attractive because of the simplicity of application of the
dye as well as its minimal invasiveness to both the cell and the flow
field. However, the dye response time is relatively slow (a few
seconds),46 47 which prevents measurement of precise
physiological time constants associated with
membrane potential changes. At the relatively low concentrations used
in this study, dye-induced cell toxicity appears to be negligible (A.S.
Waggoner, personal communication, November 1994) and in no
instance during these studies was there any evidence of cytotoxicity.
Bisoxonol results were obtained at a shear stress of 1
dyne/cm2; we also attempted similar bisoxonol
measurements on cells subjected to larger shear stresses that are more
representative of time-average values within large
arteries in vivo (5 to 15 dyne/cm2). The results
demonstrated an immediate increase in fluorescence intensity on
onset of flow, suggesting membrane depolarization in the absence of
hyperpolarization (data not shown). J.A. Frangos
(personal communication, October 1996) has noted that there
exists a nonspecific interaction between the bisoxonol dye system and
the flow environment. This interaction becomes very pronounced at the
higher flow rates and gives an artifactual depolarization signal that
overwhelms the hyperpolarization, which is known to
occur at physiological shear
stresses.6 18 The existence of this nonspecific
interaction was confirmed by coating the inside surfaces of glass
capillary tubes with a lipid layer and applying flow to it. The simple
application of flow led to an increase in fluorescence
intensity, which resembles membrane depolarization. In combination with
the inability of the bisoxonol system to reflect
physiological time constants and magnitudes of
flow-induced membrane potential changes (Figure 2B), this
suggests that care must be exercised in the use of this dye system in
studies of flow-induced changes in membrane potential.
Endothelial cells in vivo are exposed to flow chronically and are often aligned in the flow direction.48 Our experiments demonstrate that BAEC membrane potential responsiveness to shear stress in flow-preconditioned cells that have undergone extensive topographic and cytoskeletal reorganization is similar to that in previously unsheared cells, ie, initial hyperpolarization that is subsequently reversed to depolarization. Thus, endothelial cells adapt to a chronic flow environment in a manner that maintains their membrane potential sensitivity to shear stress. The mechanisms governing such adaptive processes remain unknown but may involve association of ion channels with cytoskeletal proteins such as actin49 50 51 and annexins.36 Volume-activated Cl- channels present in macrovascular endothelial cells have recently been shown to be modulated by annexin II, a member of a family of Ca2+-dependent proteins that aggregate as cytoskeletal structures at cell membranes.36
The functional role of the flow-sensitive Cl- channel reported here remains to be elucidated. It is possible that this channel provides a mechanism for depolarization of the membrane potential after flow-induced K+ channel–mediated hyperpolarization. However, both the responsiveness of the Cl- channel to shear stress in the absence of K+ channel activation by flow (in presence of external Ba2+) and the fact that the cell ends up at a depolarized level rather than at its preflow membrane voltage suggest that this channel also serves other functions. Hoyer et al39 have reported that shear stress–induced endothelial hyperpolarization facilitates calcium influx by increasing the electric driving force; therefore, one possibility is that the activation of the Cl- current by flow acts to limit this influx. A second possibility is that the flow-induced Cl- current may be involved in volume changes that may occur as the cells prepare for the extensive cytoskeletal and morphological changes that occur in response to sustained flow. A third possibility is that the imposition of shear stress may change the metabolic demands on endothelial cells so that regulation of intracellular pH may be required, and the Cl- current may be involved in this regulation. Finally, the Cl- channel may constitute an independent candidate structure involved in mechanosensing. Given the seemingly different dynamics of flow desensitization and resensitization of the K+ and Cl- channels, an intriguing notion is that these channels may constitute components of separate systems capable of responding to shear stresses of different temporal scales. Thus, whereas the K+ channel system responds to rapid changes in shear stress, the Cl- channel may be primarily involved in responding to temporally slower fluid mechanical stimuli.
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Acknowledgments |
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This work was supported by grants from the Whitaker Foundation RG-97-0114 (to A.I.B.) and the NHLBI, HL36049 and HL15062 (to P.F.D.). E.V.L. was supported by National Science Foundation Research Training Grant 9602226. We thank Dr Debby Nelson of the University of Chicago and Dr Irena Levitan of the University of Pennsylvania for valuable discussions, Dr Sean Wilson and Deborah Lieu for assistance with patch-clamp techniques, and Richard Lum for help with cell culture.
Received October 6, 1998; accepted August 5, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Davies PF. Flow-mediated endothelial mechanotransduction. Physiol Rev. 1995;75:519–560.
2. Davies PF, Barbee KA, Volin MV, Robotewskyj A, Chen J, Joseph L, Griem M, Wernick M, Jacobs E, Polacek DC, DePaola N, Barakat AI. Spatial relationships in early signaling events of flow-mediated endothelial mechanotransduction. Annu Rev Physiol. 1997;59:527–549.[Medline] [Order article via Infotrieve]
3. Resnick N, Gimbrone MA. Hemodynamic forces: complex regulators of endothelial gene expression. FASEB J. 1995;9:874–882.[Abstract]
4.
Pohl U, Holtz J, Busse R, Bassenge E. Crucial role of
endothelium in the vasodilator response to increased
flow in vivo. Hypertension. 1986;8:37–47.
5.
Langille BL, O'Donnell F. Reductions in
arterial diameter produced by chronic decreases in blood
flow are endothelium-dependent. Science. 1986;231:405–407.
6. Olesen S-P, Clapham DE, Davies PF. Hemodynamic shear stress activates a K+ current in vascular endothelial cells. Nature. 1988;331:168–170.[Medline] [Order article via Infotrieve]
7. Berthiaume F, Frangos JA. Flow-induced prostacyclin production is mediated by a pertussis toxin-sensitive G protein. FEBS Lett. 1992;308:277–279.[Medline] [Order article via Infotrieve]
8.
Gudi S, Clark CB, Frangos JA. Fluid flow rapidly
activates G proteins in human endothelial
cells: involvement of G proteins in mechanochemical signal
transduction. Circ Res. 1996;79:834–839.
9.
Gudi S, Nolan JP, Frangos JA. Modulation of GTPase
activity of G proteins by fluid shear stress and phospholipid
composition. Proc Natl Acad Sci U S A. 1998;95:2515–2519.
10.
Shen J, Luscinskas FW, Connolly A, Dewey CF Jr,
Gimbrone MA Jr. Fluid shear stress modulates cytosolic free calcium in
vascular endothelial cells. Am J
Physiol. 1992;262:C384–C390.
11. Ando J, Ohtsuka A, Korenaga R, Kawamura T, Kamiya A. Wall shear stress rather than shear rate regulates cytoplasmic Ca2+ responses to flow in vascular endothelial cells. Biochem Biophys Res Commun. 1993;190:716–723.[Medline] [Order article via Infotrieve]
12.
Tseng H, Peterson TE, Berk BC. Fluid shear stress
stimulates mitogen-activated protein kinase in
endothelial cells. Circ Res. 1995;77:869–878.
13.
Lan Q, Mercurius KO, Davies PF. Stimulation of
transcription factors NF
B and AP1 in
endothelial cells subjected to shear stress.
Biochem Biophys Res Commun. 1994;201:950–956.[Medline]
[Order article via Infotrieve]
14. Dewey CF Jr, Bussolari SR, Gimbrone MA Jr, Davies PF. The dynamic response of vascular endothelial cells to fluid shear stress. J Biomech Eng. 1981;103:177–185.[Medline] [Order article via Infotrieve]
15. Remuzzi A, Dewey CF, Davies PF, Gimbrone MA Jr. Orientation of endothelial cells in shear fields in vitro. Biorheology. 1984;21:617–630.[Medline] [Order article via Infotrieve]
16.
Barbee KA, Davies PF, Lal R. Shear stress-induced
reorganization of the surface topography of living
endothelial cells imaged by atomic force microscopy.
Circ Res. 1994;74:163–171.
17.
Nakache M, Gaub HE. Hydrodynamic
hyperpolarization of endothelial
cells. Proc Natl Acad Sci U S A. 1988;85:1841–1843.
18. Jacobs ER, Cheliakine C, Gebremedhin D, Davies PF, Harder DR. Shear activated channels in cell attached patches of vascular endothelial cells. Pflugers Arch. 1995;431:129–131.[Medline] [Order article via Infotrieve]
19. Barakat AI, Davies PF. Effect of cytoskeletal reorganization on flow-induced membrane potential changes in endothelial cells. Ann Biomed Eng. 1996;24:207a. Abstract.
20. Schwartz SM. Selection and characterization of bovine aortic endothelial cells. In Vitro. 1980;14:966–978.
21. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981;391:85–100.[Medline] [Order article via Infotrieve]
22. Adams DJ. Ionic channels in vascular endothelial cells. Trends Cardiovasc Med. 1994;4:18–26.
23. Nilius B, Viana F, Droogmans G. Ion channels in vascular endothelium. Annu Rev Physiol. 1997;59:145–170.[Medline] [Order article via Infotrieve]
24. Wilson HA, Chused TM. Lymphocyte membrane potential and Ca2+-sensitive potassium channels described by oxonol dye fluorescence measurements. J Cell Physiol. 1985;125:72–81.[Medline] [Order article via Infotrieve]
25. Epps DE, Wolfe ML, Groppi V. Characterization of the steady-state and dynamic fluorescence properties of the potential-sensitive dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol (Dibac4(3)) in model systems and cells. Chem Phys Lipids. 1994;69:137–150.[Medline] [Order article via Infotrieve]
26. Olesen, S-P, Bundgaard M. Chloride-selective channels of large conductance in bovine aortic endothelial cells. Acta Physiol Scand. 1992;144:191–198.[Medline] [Order article via Infotrieve]
27.
Nilius B, Oike M, Zahradnik I, Droogmans G. Activation
of a Cl- channel by hypotonic volume increase in
human endothelial cells. J Gen Physiol. 1994;103:787–805.
28. Groschner K, Kukovetz WR. Voltage-sensitive chloride channels of large conductance in the membrane of pig aortic endothelial cells. Pflugers Arch. 1992;421:209–217.[Medline] [Order article via Infotrieve]
29.
Song J, Davis MJ. Chloride and cation currents
activated by bradykinin in coronary venular
endothelial cells. Am J Physiol. 1994;267:H2508–H2515.
30.
Ueda S, Lee S-L, Fanburg BL. Chloride efflux in cyclic
AMP-induced configurational change of bovine pulmonary artery
endothelial cells. Circ Res. 1990;66:957–967.
31.
Franciolini F, Nonner W. Anion and cation permeability
of a chloride channel in rat hippocampal neurons. J Gen
Physiol. 1987;90:453–478.
32.
Pappone PA, Lee SC. 
-Adrenergic stimulation
activates a calcium-sensitive chloride current in brown fat
cells. J Gen Physiol. 1995;106:231–258.
33.
Pusch M. Knocking on channel's door: the permeating
chloride ion acts as the gating charge in CIC-0. J Gen
Physiol. 1996;108:233–236.
34. Pusch M, Ludewig U, Rehfeldt A, Jentsch TJ. Gating of the voltage-dependent chloride channel CIC-0 by the permeant anion. Nature. 1995;373:527–531.[Medline] [Order article via Infotrieve]
35. Nilius B, Prenen J, Voets T, Eggermont J, Bruzik KS, Shears SB, Droogmans G. Inhibition by inositoltetrakisphosphates of calcium- and volume-activated Cl- currents in macrovascular endothelial cells. Pflugers Arch. 1998;435:637–644.[Medline] [Order article via Infotrieve]
36.
Nilius B, Gerke V, Prenen J, Szucs G, Heinke S,
Droogmans G. Annexin II modulates volume-activated chloride
currents in vascular endothelial cells. J
Biol Chem. 1996;271:30631–30636.
37.
Elble RC, Widom J, Gruber AD, Abdel-Ghany M, Levine R,
Goodwin A, Cheng H-C, Pauli BU. Cloning and characterization of
lung-endothelial cell adhesion molecule-1 suggest it is
an endothelial chloride channel. J Biol
Chem. 1997;272:27853–27861.
38.
Cunningham SA, Awayda MS, Bubien JK, Ismailov II,
Arrate MP, Berdiev BK, Benos DJ, Fuller CM. Cloning of an epithelial
chloride channel from bovine trachea. J Biol Chem. 1995;270:31016–31026.
39.
Hoyer J, Kohler R, Distler A. Mechanosensitive
Ca2+ oscillations and STOC activation
in endothelial cells. FASEB J. 1998;12:359–366.
40. Moore JE, Ku DN. Pulsatile velocity measurements in a model of the human abdominal aorta under simulated exercise and postprandial conditions. J Biomech Eng. 1994;116:107–111.[Medline] [Order article via Infotrieve]
41.
Ribera AB. Homogeneous development of
electrical excitability via heterogeneous ion channel
expression. J Neurosci. 1996;16:1123–1130.
42. Nilius B, Eggermont J, Voets T, Droogmans G. Volume-activated Cl- channels. Gen Pharmacol. 1996;27:1131–1140.[Medline] [Order article via Infotrieve]
43.
Solc CK, Wine JJ. Swelling-induced and
depolarization-induced Cl- channels in normal
and cystic fibrosis epithelial cells. Am J Physiol. 1991;261:C658–C674.
44. Levitan I, Garber SS. Voltage-dependent inactivation of volume-regulated Cl- current in human T84 colonic and B-cell myeloma cell lines. Pflugers Arch. 1995;431:297–299.[Medline] [Order article via Infotrieve]
45. Davies PF, Mundel T, Barbee KA. A mechanism for heterogeneous endothelial responses to flow in vivo and in vitro. J Biomech. 1995;28:1553–1560.[Medline] [Order article via Infotrieve]
46. Waggoner AS. Dye indicators of membrane potential. Annu Rev Biophys Bioeng. 1979;8:47–68.[Medline] [Order article via Infotrieve]
47. Smith JC. Potential-sensitive molecular probes in membranes of bioenergetic relevance. Biochim Biophys Acta. 1990;1016:1–28.[Medline] [Order article via Infotrieve]
48.
Langille BL, Adamson SL. Relationship between blood
flow direction and endothelial cell orientation at
arterial branch sites in rabbits and mice. Circ
Res. 1981;48:481–488.
49.
Undrovinas AI, Shander GS, Makielski JC. Cytoskeleton
modulates gating of voltage-dependent sodium channel in heart.
Am J Physiol. 1995;269:H203–H214.
50.
Prat AG, Xiao Y, Ausiello DA, Cantiello HF.
cAMP-independent regulation of CFTR by the actin cytoskeleton.
Am J Physiol. 1995;268:C1552–C1561.
51. Hug T, Koslowsky T, Ecke D, Greger R, Kunzelmann K. Actin-dependent activation of the ion conductances in bronchial epithelial cells. Pflugers Arch. 1995;429:682–690.[Medline] [Order article via Infotrieve]
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