1 Department of Human Physiology, School of Medicine, and 2 Department of Mechanical and Aeronautical Engineering, University of California, Davis, California 95616
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ABSTRACT |
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Steady laminar
shear stress has been shown previously to markedly increase Na-K-Cl
cotransporter mRNA and protein in human umbilical vein endothelial
cells and also to rapidly increase endothelial K+ and
Cl channel conductances. The present study was done to
evaluate the effects of shear stress on Na-K-Cl cotransporter activity and protein expression in bovine aortic endothelial cells (BAEC) and to
determine whether changes in cotransporter expression may be dependent
on early changes in K+ and Cl
channel
conductances. Confluent BAEC monolayers were exposed in a
parallel-plate flow chamber to either steady shear stress (19 dyn/cm2) or purely oscillatory shear stress (0 ± 19 dyn/cm2) for 6-48 h. After shearing, BAEC monolayers
were assessed for Na-K-Cl cotransporter activity or were subjected to
Western blot analysis of cotransporter protein. Steady shear stress led
to a 2- to 4-fold increase in BAEC cotransporter protein levels and a
1.5- to 1.8-fold increase in cotransporter activity, increases that
were sustained over the longest time periods studied. Oscillatory flow,
in contrast, had no effect on cotransporter protein levels. In the
presence of flow-sensitive K+ and Cl
channel
pharmacological blockers, the steady shear stress-induced increase in
cotransporter protein was virtually abolished. These results suggest
that shear stress modulates the expression of the BAEC Na-K-Cl
cotransporter by mechanisms that are dependent on flow-activated ion channels.
endothelium; mechanotransduction; ion channels
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INTRODUCTION |
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AN ELECTRONEUTRAL Na-K-Cl cotransport system is present in the membranes of most mammalian cells. In vascular endothelial cells, this system is highly active and is regulated by various vasoactive substances, including bradykinin and vasopressin, as well as by important signaling molecules, including calcium and protein kinase C (6, 39, 40). This suggests that the Na-K-Cl cotransport system is an important component of endothelial cell function (40). In general, this system has been shown to participate in the vectorial transport of ions across polarized epithelial cells (19, 43) and in the regulation of intracellular volume in various cell types, including vascular endothelium (15, 26, 40). Previous studies have shown that intracellular volume of endothelial cells is a determinant of the endothelial barrier permeability (25, 41). It has also been suggested that, in the arterial system, abnormalities in the ability of the endothelium to regulate permeability likely play a role in the development of early atherosclerotic lesions (4, 11, 36).
In addition to its responsiveness to various vasoactive hormones, the
Na-K-Cl cotransport system in vascular endothelial cells responds to
the biophysical stimulus of osmotic stress, with cell shrinkage
stimulating and cell swelling inhibiting activity of the cotransporter
(40). Cell shrinkage-induced stimulation of the
endothelial cotransporter allows the cells to restore intracellular volume to preset levels even in the continued presence of elevated extracellular osmolarity (40). More recently, steady
laminar fluid mechanical shear stress has been reported to upregulate Na-K-Cl cotransport mRNA and protein expression in human umbilical vein
endothelial cells in vitro (54). Shear stress, in fact, elicits a wide range of humoral, metabolic, and structural responses in
endothelial cells (1, 2, 10, 49, 55). These responses include activation of flow-sensitive K+ and
Cl ion channels (3, 23, 35, 46) and G
proteins (17, 18), oscillations in intracellular
Ca2+ concentration (13, 16, 50), changes in
expression of various gene products (49), and extensive
cytoskeletal reorganization leading to cellular elongation and
alignment in the flow direction (12, 14, 37). The precise
mechanisms by which endothelial cells sense shear stress and transmit
it to the nucleus to alter gene expression remain poorly understood. It
has been suggested that this may occur through a sequence of events
involving direct interaction of the fluid mechanical stimulus with
cell-surface structures, which act as primary flow sensors, and
subsequent transmission of the mechanical signal, possibly via
cytoskeletal elements, to the nucleus (1, 2, 10).
Candidate flow-sensing systems to date include flow-activated ion
channels, G protein-coupled receptors, mitogen-activated receptors, and
focal adhesion complexes (10).
The increased expression of Na-K-Cl cotransport mRNA observed in response to steady laminar shear stress does not occur when the cells are exposed to turbulent shear stress of the same time-averaged magnitude (54). This finding suggests that endothelial cell Na-K-Cl cotransport expression responds differentially to different forms of shear stress. Differential endothelial responses to different types of flow have also been reported for other flow-induced responses, including intracellular Ca2+ oscillation (21), cytoskeletal reorganization (22), and transcriptional changes of various genes (8, 30, 56). This finding may be especially important in light of the fact that early atherosclerotic lesions localize preferentially in arterial regions exposed to disturbed and multidirectional shear stresses, while regions subjected to unidirectional flow remain largely spared of early lesions (2, 28, 36).
The present study was conducted to quantitate the effects of shear
stress on aortic endothelial cell Na-K-Cl cotransporter protein and
activity and to examine the time course of effects. Because increases
in flow-sensitive K+ and Cl channel
conductances are early events triggered by exposure of endothelial
cells to steady shear stress, in the present study we also investigated
the possibility that changes in cotransporter protein and activity may
be secondary to changes in K+ and/or Cl
channel conductances. We report here that steady laminar shear stress
upregulates Na-K-Cl cotransport protein levels and activity in cultured
aortic endothelial cells, whereas oscillatory shear stress has no
effect on the cotransporter. Moreover, we have demonstrated that the
steady shear stress-induced Na-K-Cl cotransport upregulation occurs
through mechanisms that are at least partially dependent on
flow-sensitive K+ and Cl
channels.
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MATERIALS AND METHODS |
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Cell culture. Bovine aortic endothelial cells (BAEC) were isolated and cultured by using standard procedures in 75-cm2 collagen-coated tissue culture flasks as described previously (40). The cells were grown in Eagle's minimal essential medium (MEM; GIBCO BRL, Grand Island, NY) supplemented with 7% heat-inactivated fetal bovine serum (FBS) (Gemini BioProducts, Calabasas, CA), L-glutamine, and penicillin/streptomycin. Cells were used between the 5th and 15th passages. After brief trypsinization, the cell suspension was plated at subconfluent density onto standard (75 × 25 mm) cell culture plastic slides (Permanox; Miles Scientific, Napierville, IL) coated with type I rat tail collagen (Collaborative Research, Bedford, MA). Flow experiments were performed on cells within 3-4 days after they reached confluence.
Exposure of cells to shear stress. BAEC monolayers cultured on Permanox slides were subjected to shear stress with the use of a parallel-plate flow chamber similar to those described elsewhere (22, 29). The mechanical stress vector within the parallel-plate flow chamber used in the present study has both tangential (shear stress) and normal (pressure) components. Thus, in this study, the term "shear stress" refers to laminar fluid flow through the parallel-plate flow chamber. In all experiments, the flow medium was Dulbecco's modified Eagle's medium (DMEM, high glucose; GIBCO BRL) containing 25 mM HEPES, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 5% heat-inactivated FBS (Gemini BioProducts).
Two different flow setups were used, one for steady flow and the other for oscillatory flow. The steady flow experiments were intended to simulate conditions that occur within arterial regions characterized by largely undisturbed flow, while the oscillatory flow studies simulated disturbed flow regimes that might occur in the vicinity of arterial branches and bifurcations (36). Although endothelial cells in undisturbed flow regions in vivo are not subjected to steady flow but, rather, to nonreversing pulsatile flow, the impact of nonreversing pulsatile flow on various aspects of endothelial cell structure and function appears to resemble that of steady flow (21, 22, 30). In the steady flow experiments, the flow chamber was inserted into a recirculating flow loop. Components of the loop were connected by small-diameter Silastic tubing (Cole-Parmer, Chicago, IL). A reservoir of culture medium (125 ml) was maintained at 37°C, and DMEM, gently gassed with 5% CO2-95% air, was drawn through the system by a peristaltic flow pump (Cole-Parmer). To dampen pulsatility, we inserted two buffer reservoirs between the pump and the flow chamber. In all the steady flow experiments, the cells were subjected to a flow rate of 45 ml/min, which generated a shear stress of 19 dyn/cm2. This shear stress value was selected because it approximates the mean shear stress value (averaged over the course of the cardiac cycle) to which macrovascular endothelial cells in regions of largely undisturbed flow are exposed (27). In the oscillatory flow experiments, the flow chamber was connected via Silastic tubing to a syringe pump (Cole-Parmer) capable of generating purely oscillatory flow (i.e., zero net flow rate) of a given pulsatile frequency by alternating between infusion and withdrawal modes at specified rates. The infusion/withdrawal rates were selected to generate an oscillatory shear stress of 0 ± 19 dyn/cm2, and the oscillation rate was adjusted to give a frequency of oscillation of 1 Hz. A small, steady flow component of 0.1 ml/min (corresponding to a shear stress of 0.04 dyn/cm2) was superimposed on the oscillatory flow to replenish the cell culture medium during the flow period.Western blot analysis of cotransporter protein.
BAEC monolayers on Permanox slides were removed from the flow chamber
after being subjected to steady or oscillatory flow, and then they were
rinsed with ice-cold phosphate-buffered saline with 2 mM EDTA
(PBS-EDTA, pH 7.4) containing protease inhibitors. The cells were then
scraped into PBS-EDTA-protease inhibitor solution plus 1% SDS. With
the use of a 26-gauge needle, the extract was mixed well and
transferred to centrifuge tubes. Samples of each supernatant were
analyzed for protein content by using the bicinchoninic acid (BCA)
method (51) to ensure equal loading of cell protein into
each gel lane. Supernatant samples (500-µl aliquots) and prestained
molecular weight markers (Bio-Rad, Hercules, CA) were denatured in SDS
reducing buffer and then either used immediately for gel
electrophoresis or stored at 80°C until use. Protein samples were
then electrophoretically separated on 8% SDS gels (Bio-Rad
Mini-PROTEAN II), and the resolved proteins were electrophoretically transferred to nitrocellulose with a Bio-Rad Trans-Blot apparatus. The
blots were incubated in 5% nonfat dry milk-containing Tris-buffered saline (TBS) for 2 h at room temperature. Subsequently, blots were
incubated with T4 monoclonal antibody (which recognizes Na-K-Cl cotransport protein) (32), rinsed five times with TBS, and
then exposed to secondary antibody (horseradish peroxidase-conjugated goat anti-mouse IgG). After five washes to remove unbound secondary antibody, bound antibody was visualized by using the enhanced chemiluminescence assay (ECL; Amersham Pharmacia Biotech, Piscataway, NJ). Densities of bands in Western blots were analyzed using an IS-1000
Digital Imaging System (Innotech Scientific, San Leandro, CA). Data are
expressed as relative abundance of cotransport protein (i.e.,
experimental value relative to control value for each blot).
Assay of Na-K-Cl cotransport activity.
BAEC monolayers on Permanox slides were exposed to shear stress or no
shear stress (control) plus the various conditions described in Figs.
1-5. At the end of the shear period, the slides were
rapidly removed from the flow chamber and carefully cut into 12 pieces each. Slide segments were then placed (1 each) into wells of a 24-well
cluster plate containing prewarmed isotonic (290 mosM) or hypertonic
(390 mosM, by addition of NaCl) assay pretreatment medium
(HEPES-buffered MEM with 0 or 10 µM bumetanide). After a 9-min
pretreatment period at 37°C on a gyratory water bath, the medium was
replaced with assay medium (identical to the pretreatment medium but
containing 86Rb), and the cells were incubated for 9 min.
To terminate the assay, slide segments were rinsed four times with 3 ml
of ice-cold TBS. The slide segments were air-dried and then extracted
in 1% SDS for protein (51) and radioactivity
determination (Tri-Carb 2500 TR liquid scintillation counter).
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Cell Cl content determination.
Cell Cl
content was determined by the 36Cl
equilibration method, as described previously (48).
Briefly, BAEC monolayers grown in 24-well cluster plates were
preequilibrated for 30 min in HEPES-buffered MEM at 37°C in an air
atmosphere, followed by a 60-min incubation in HEPES-buffered MEM
containing varying extracellular Cl
concentrations
([Cl
]o) and also
36Cl
(0.2 µCi/ml). The concentration of
Cl
in medium varied from 0 to 137 mM, with methane
sulfonate used as the replacement anion. To terminate the assay,
monolayers were rinsed with isotonic ice-cold 0.1 M MgCl2,
and then SDS extracts of the cells were prepared to determine
radioactivity and protein content of each well. Specific activities
(cpm/µmol) of 36Cl
were determined for each
assay condition and used to calculate intracellular Cl
content (expressed as µmol/g protein.
Intracellular volume determination. Intracellular volume of BAEC was determined by radioisotopic evaluation of BAEC intracellular water space by using [14C]urea and [14C]sucrose as markers of total and extracellular space, respectively. Details of this methodology have been described previously by O'Donnell (40). BAEC monolayers on 24-well plates were preequilibrated for 30 min in HEPES-buffered MEM at 37°C in an air atmosphere. The cells were then treated for 30 min with HEPES-buffered MEM containing varying [Cl]o (10, 30, 70, 100, or 136 mM), followed by a 30-min incubation in the same media containing either [14C]urea or [14C]sucrose (both at 1 µCi/ml). To terminate the assay, we rinsed monolayers with isotonic ice-cold 0.1 M MgCl2 and then extracted with SDS. Radioactivity of the SDS extracts was determined by liquid scintillation, and the protein content of each extract was assessed using the BCA method (51). The amounts of radioactivity in assay media containing [14C]urea and [14C]sucrose (cpm/ml) were used to calculate water space associated with the radioactive markers (expressed as µl/mg protein). Intracellular volume was calculated as the difference between water space determined for [14C]urea (a marker for intracellular plus trapped extracellular space) and [14C]sucrose (a marker for trapped extracellular space).
Materials. BaCl2, tetraethylammonium (TEA), DIDS, and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) were purchased from Sigma (St. Louis, MO), and diphenylamine-2-carboxylate (DPC) was purchased from Aldrich (Milwaukee, WI). Bumetanide was purchased from ICN Pharmaceuticals (Costa Mesa, CA). 86Rb was from Dupont NEN (Boston, MA).
Statistical analysis. All data are presented as means ± SE. Means were statistically compared with a Student's t-test. Statistical significance was set at 5% level (P < 0.05).
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RESULTS |
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Steady laminar shear stress increases Na-K-Cl cotransport protein expression and activity. BAEC were exposed in the parallel-plate flow chamber to a steady laminar shear stress of 19 dyn/cm2 for periods of 6, 12, 24, and 48 h. As has been shown elsewhere (12, 14, 37), vascular endothelial cells subjected to a steady shear stress greater than ~5 dyn/cm2 undergo extensive cytoskeletal reorganization, which leads to progressive cellular elongation and alignment in the direction of flow. Figure 1 is an example of this flow-induced elongation in BAEC observed after 6 and 24 h of steady shear stress in our flow chamber.
Figure 2 shows the results of Western blot and densitometric analyses of the time course of changes in BAEC Na-K-Cl cotransport protein expression in response to a steady shear stress of 19 dyn/cm2. The representative Western blot shown in Fig. 2A reveals that steady shear stress led to a considerable and sustained increase in Na-K-Cl cotransport protein expression at 6, 12, 24, and 48 h of exposure. For BAEC exposed to 6 h of steady shear stress, a doublet of bands was detected by the T4 antibody, with an upper band of ~170 kDa and a lower band of ~135 kDa, compared with a single band of ~170 kDa in the control cells. These sizes are respectively consistent with the mature, glycosylated form of the cotransporter and the immature, nonglycosylated form of the cotransporter, as reported previously (32, 42). With longer times of steady shear stress exposure, the lower band decreased in density as the upper band increased in density, consistent with maturation of the cotransporter protein over time. By 48 h of shear stress, most of the cotransporter protein appeared in a single band of ~170 kDa. When we analyzed cotransporter protein levels from multiple experiments by densitometry, we found that, at all time points studied, cotransport protein levels were significantly higher than the no-flow static controls, as shown in Fig. 2B. The peak increase in cotransport protein (3.7 fold) occurred after 12 h of flow. Although the relative abundance of cotransporter protein was less after 24 h of shear stress than after 12 h, it was nevertheless significantly greater than in no-flow static controls. In two experiments examining the effects of 48 h of shear stress, we observed increases in cotransporter protein of 3.0- and 3.7-fold above control (data not shown). To determine whether the Na-K-Cl cotransport protein induced by steady laminar shear stress actively participates in the transport of ions across the endothelial cell membrane, we studied the effect of shear stress on Na-K-Cl cotransporter activity, evaluated as bumetanide-sensitive K influx (40-42). Figure 3 demonstrates that, in an isotonic extracellular solution (osmolarity of 290 mosM), steady shear stress imposed for periods of either 6 or 24 h did indeed increase Na-K-Cl cotransport protein activity. However, this increase was smaller than the flow-induced increase in cotransport protein levels. For instance, while 6 and 24 h of flow resulted in protein levels that were more than double those of static no-flow controls (Fig. 2), cotransporter protein activity increased only 1.5- to 1.8-fold. We had previously demonstrated that elevation of extracellular tonicity stimulates BAEC Na-K-Cl cotransport activity (40). To determine whether shear stress-induced cotransport protein that was not rendered active by shear stress alone could be stimulated into activity by changes in tonicity, BAEC were challenged for 5 min with a hypertonic (390 mosM) extracellular solution following the 6- or 24-h exposure to steady shear stress. As shown in Fig. 3, exposure to hypertonic medium caused a significant increase in Na-K-Cl cotransporter activity in both control and sheared endothelial cells. In this case, the cotransporter activity of BAEC sheared for 24 h and assayed in hypertonic medium was increased 2.6-fold compared with that of the isotonic, nonsheared control.Oscillatory shear stress has no effect on Na-K-Cl cotransport protein expression. Vascular endothelial cells have been shown to respond differentially to different types of imposed shear stress. For instance, while steady laminar flow induces intracellular Ca2+ oscillations and extensive cytoskeletal reorganization, purely oscillatory flow does not elicit either response (21, 22). Steady and oscillatory flows have also been shown to affect endothelin-1 and endothelial constitutive nitric oxide synthase mRNA differently (8). To establish whether oscillatory flow affects endothelial Na-K-Cl cotransport differently from steady laminar flow, we quantitated cotransporter protein levels in BAEC monolayers that were exposed in the parallel-plate flow chamber to a purely oscillatory flow of 0 ± 19 dyn/cm2 (zero net flow rate) with a pulsatile frequency of 1 Hz for periods of 6 or 24 h. Figure 4A demonstrates that BAEC exposed to 6 h of purely oscillatory flow in our chamber did not elongate but remained rounded like cells in static culture. These results are consistent with those published elsewhere (22, 30). Figure 4B is a representative Western blot showing BAEC Na-K-Cl cotransport protein levels occurring in response to 6 h of oscillatory shear stress. Densitometric analysis of four separate experiments revealed that the relative abundance of cotransporter protein in cells exposed to oscillatory shear was 1.13 ± 0.1 compared with 1.0 for control, nonsheared cells. Thus, in contrast to steady shear stress, oscillatory flow is without effect on the Na-K-Cl cotransporter of BAEC.
Steady laminar shear stress-induced increase in cotransporter
protein is dependent on flow-sensitive ion channels.
One of the fastest known endothelial responses to shear stress is the
activation of flow-sensitive K+ and Cl ion
channels (3, 23, 35, 46). Therefore, these ion channels are thought to play an important role in shear stress sensing and
transduction in the vascular endothelium (1, 10). We hypothesized that the increase in Na-K-Cl cotransport protein induced
by steady shear stress depends on the activation of shear stress-sensitive ion channels. We tested this hypothesis by
investigating the effect of steady shear stress on BAEC cotransport
protein in the presence of pharmacological blockers of flow-activated K+ and Cl
ion channels. For these
experiments, we evaluated the effects of Ba2+ (1 mM) and
TEA (3 mM), K+ channel blockers (9, 44, 46).
We also evaluated the effects of DPC (1 mM), DIDS (1 mM), and NPPB (100 µM), agents that block Cl
channels (3, 7, 52,
53). Of these blockers, TEA has been shown to markedly attenuate
several flow-induced responses, including the increase in intracellular
cGMP (45) and the upregulation of transforming growth
factor (TGF)-
1 mRNA (44). Also, DPC and DIDS have been
shown by Barakat and coworkers (3) recently to block
flow-activated Cl currents.
Changes in intracellular Cl concentration modulate
Na-K-Cl cotransport protein expression and activity.
One possible explanation for our finding that the responsiveness of
endothelial Na-K-Cl cotransport protein to shear stress appears to be
mediated by flow-activated K+ and Cl
ion
channels is that the intracellular concentrations of these ions
may modulate Na-K-Cl cotransport protein levels and activity. Consistent with this are previous studies that have shown intracellular Cl
concentration ([Cl
]i) to
be a potent regulator of the Na-K-Cl cotransporter such that elevated
[Cl
]i inhibits, whereas reduced
[Cl
]i stimulates, cotransporter activity
(31, 48). Thus it is possible that shear stress activation
of Cl
channels reduces [Cl
]i,
which in turn causes increased cotransporter activity. As a first step
to investigating possible mechanisms by which flow-sensitive ion
channels may modulate cotransporter abundance, we investigated the
effects of altering [Cl
]i on BAEC
cotransporter activity and cotransporter protein expression. To vary
[Cl
]i, we incubated BAEC in media
containing decreasing [Cl
]o. As shown in
Fig. 6A,
this resulted in a linear decrease in [Cl
], as
determined by 36Cl equilibration methods (48).
Figure 6B shows that when BAEC were incubated for 60 min in
reduced [Cl
]o to decrease
[Cl
]i, activity of the Na-K-Cl
cotransporter was stimulated, with an approximately threefold increase
observed for cells pretreated with 10 mM
[Cl
]o compared with cells pretreated with
140 mM [Cl
]o. Similarly, a 30-min
pretreatment of cells in media containing 0 mM Cl
resulted in a 2.76 ± 0.33-fold stimulation of cotransporter
activity (n = 4 experiments; data not shown). We showed
previously that Na-K-Cl cotransporter activity of BAEC is stimulated by
cell shrinkage (40). To evaluate the possibility that the
reduced [Cl
]o media, which contain methane
sulfonate as the replacement anion, simply cause the cells to shrink
and thereby stimulate cotransporter activity, we tested the effect of
these media on BAEC intracellular volume. As shown in Fig.
6C, a 60-min exposure of the cells to reduced
[Cl]o media (containing Na-methane sulfonate in place of
NaCl) did not cause a significant reduction of intracellular volume.
Thus, using the same reduced [Cl]o pretreatment
conditions (with respect to both time of pretreatment and
[Cl]o), we found that BAEC intracellular volume does not
change, yet cotransporter activity is significantly stimulated.
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DISCUSSION |
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Vascular endothelial cells exhibit a very active electroneutral
Na-K-Cl cotransport system that is regulated by various vasoactive substances and that participates in the modulation of endothelial cell
volume (6, 39, 40). The results of the present study demonstrate that Na-K-Cl cotransport protein expression and activity in
cultured aortic endothelial cells are also regulated by fluid mechanical shear stress. BAEC exposed to a steady laminar shear stress
of 19 dyn/cm2 exhibited a significant increase in Na-K-Cl
cotransport protein levels, and this increase was sustained for flow
periods as long as 48 h. At their peak 12 h after the onset
of flow, Na-K-Cl cotransport protein levels reached more than 3.5-fold
the levels of no-flow static controls. In contrast, exposing BAEC to
6 h of a purely oscillatory flow (0 ± 19 dyn/cm2) with a pulsatile frequency of 1 Hz resulted in no
significant change in the amount of Na-K-Cl cotransporter protein in
the cells. It should be noted that Lum and coworkers (30)
recently showed that both steady and oscillatory shear stress induce
expression of TGF-1 in BAEC; however, the response to oscillatory
shear stress was not observed until 24 h after the start of shear
stress. Thus it is possible that a longer exposure to oscillatory shear stress could have an effect on cotransporter expression. In any case,
it is clear that the response of the BAEC with respect to cotransporter
protein expression differs between steady and oscillatory flow. In
addition to the observed shear stress effects on cotransporter protein
levels, BAEC Na-K-Cl cotransporter activity, assessed as
bumetanide-sensitive K influx, also increased in response to steady
laminar shear stress (evaluated 30-60 min after flow was stopped).
The magnitude of this increase in cotransporter activity, however, was
considerably smaller than that of the increase in protein, suggesting
that only a fraction of the flow-induced protein was actively
participating in ion transport at the plasma membrane. Na-K-Cl
cotransport activity of BAEC exposed to steady shear stress was
markedly stimulated by hypertonic medium, previously shown to cause
cell shrinkage in BAEC and to thereby stimulate cotransporter activity
(40). Thus it appears that, although the shear
stress-induced increase in cotransporter protein does not result in an
increase in cotransporter activity of corresponding magnitude, the
sheared BAEC exhibit an increased capacity for cotransporter activity, which in our studies was revealed by stimulating the cells with hypertonic medium.
Activation of flow-sensitive K+ and Cl ion
channels is one of the earliest responses to shear stress observed in
vascular endothelial cells (3, 23, 35, 46). These ion
channels, along with other shear stress-responsive membrane-associated
structures, have been implicated in both the sensing and transduction
of shear stress (1, 10). In the present study, we have
established that when pharmacological blockers of K+ and
Cl
ion channels are present during shear stress, the
Na-K-Cl cotransporter protein upregulation induced by shear stress is
greatly attenuated or even abolished. This suggests that the BAEC
Na-K-Cl cotransport protein response to flow occurs by mechanisms that
are dependent either specifically on shear stress-sensitive ion
channels or more generally on endothelial cell membrane potential.
Blocking of flow-sensitive K+ channels has previously been
shown to considerably attenuate a number of endothelial responses to
flow, including release of an endogenous nitrovasodilator
(9), upregulation of transforming growth factor-
1 mRNA
levels (44), and downregulation of endothelin-1 gene
expression (33). The present findings suggest that
upregulation of Na-K-Cl cotransporter protein is another endothelial
response triggered by shear stress activation of K+
channels. In addition, our finding that three different agents known to
block Cl
channels, i.e., DPC, DIDS, and NPPB, all abolish
the shear stress-induced increase in cotransporter protein suggests
that shear-sensitive Cl
channels also participate in
shear stress effects on BAEC cotransporter protein abundance. It is
important to note that while these channel blockers reduced or
abolished the shear stress-induced increase in cotransporter protein,
they had no effect on the amount of cotransporter protein in control,
nonsheared cells.
The mechanism whereby shear stress activation of K+ and
Cl channels causes increased cotransporter protein in
BAEC is as yet unclear and will require further investigation. Our
present studies suggest that the increase in cotransporter protein is
not simply driven by a reduction of [Cl
]i,
which is expected to occur during Cl
channel activation.
We have shown here that reduction of [Cl
]i
causes an increase in BAEC Na-K-Cl cotransporter activity. We also
report here, for the first time, that cotransporter protein expression
can be increased by exposure of the BAEC to media containing reduced
[Cl
]o. Despite this, the elevation in
cotransporter protein caused by a 6-h treatment with reduced
[Cl
]o is considerably smaller than the
increase observed following 6-h of laminar shear stress. Thus even a
sustained reduction of [Cl
]i cannot alone
account for the shear stress-induced increase in cotransporter protein.
It is also important to note that the shear stress activation of
flow-sensitive K+ and Cl
channels is expected
to be relatively short-lived due to channel desensitization and/or the
activation of mechanisms that restore resting membrane potential. Thus
one would predict that if the shear stress-induced increase in
cotransporter protein is induced by a drop in
[Cl
]i, then a transient fall in
[Cl
]i should be sufficient to trigger the
increased expression of cotransporter protein. Previous studies of the
effects of reduced [Cl
]i on cotransporter
activity in other cells have provided evidence for
Cl
-sensitive intracellular signaling pathways that appear
to involve changes in kinase and/or phosphatase activities
(31). In this regard, it is possible that shear stress
activation of BAEC Cl
channels may trigger
Cl
-sensitive signaling pathways that result in altered
expression of the Na-K-Cl cotransporter. It should be noted that the
reduction of shear stress-induced cotransporter protein observed with
K+ channel blockers is consistent with this scenario,
because shear stress activation of K+ channels with the
subsequent reduction of [K+]i would be
expected to cause a concomitant decrease in
[Cl
]i because the electrochemical driving
forces favor Cl
efflux under these conditions. These
observations notwithstanding, it is clear that clarifying the role of
shear stress-activated K+ and Cl
channels and
the subsequent signaling pathways that lead to increase in Na-K-Cl
cotransport expression will require further investigation.
To our knowledge, the only other report of the influence of fluid mechanical forces on Na-K-Cl cotransport in endothelium is that of Topper et al. (54), who demonstrated that a steady laminar shear stress of 10 dyn/cm2 upregulates Na-K-Cl cotransport mRNA and protein in cultured human umbilical vein endothelial cells. Interestingly, turbulent shear stress of the same average magnitude (10 dyn/cm2) did not induce the Na-K-Cl response, and steady laminar shear stress of 4 dyn/cm2 induced only a minimal change in cotransport gene expression (54). It is noteworthy that while a steady shear stress of 10 dyn/cm2 leads to extensive endothelial cytoskeletal reorganization and consequent cellular elongation and alignment in the direction of flow, a shear stress of 4 dyn/cm2 may be below the threshold needed for inducing these cell shape changes (12). Similarly, it has been demonstrated that a 10 dyn/cm2 turbulent shear stress fails to produce endothelial cell morphological changes (11). These results suggest the possibility that cytoskeletal regulation of cotransporter gene transcription and/or translation occurs in response to shear stress. Our results with laminar oscillatory flow, which also does not lead to cellular elongation (22), are consistent with possible cytoskeletal regulation of cotransporter expression. Many examples of cytoskeletal regulation of ion flux pathways have been documented (20, 34). Microfilaments have been implicated in regulation of ion channel activity, as well as various transporters (20, 34). Recent studies of the T84 human intestinal epithelial cell line and the mouse medullary thick ascending limb (mTAL) cell line have shown that agents that stimulate activity of the Na-K-Cl cotransporter also cause a redistribution of F-actin in the cells and that the F-actin stabilizer phalloidin blocks this process (20). In other studies in which T84 cells and Ehrlich ascites tumor cells were used, it was shown that the microfilament-disrupting cytochalasins cause stimulation of Na-K-Cl cotransporter activity in a manner blocked by phalloidin (20). It has been proposed that formation of "short" actin filaments by cytochalasin causes the stimulation of cotransporter activity as well as activation of other ion flux pathways such as epithelial Na+ channels (20, 47). However, the mechanism by which rearrangement of actin filaments may regulate activity of the Na-K-Cl cotransporter has yet to be elucidated.
A number of membrane-associated structures, including ion channels, G
protein-coupled receptors, and focal adhesion complexes, have been
shown to respond rapidly to shear stress and have therefore been
hypothesized to be candidate flow sensors in endothelial cells
(10). As an integral membrane protein that
responds to shear stress, the Na-K-Cl cotransporter must be considered
to possibly serve as a flow sensor. Whether the cotransporter does in
fact respond to flow with sufficient rapidity to allow it to serve as a
sensor is unclear. It is difficult to assess the immediate response of
the cotransporter to flow because, unlike activation of flow-sensitive
ion channels, which can be detected within milliseconds of an imposed
flow, the electrically silent Na-K-Cl cotransport system must be
assessed by considerably slower radioisotopic methods, performed after
the cells are removed from the flow apparatus. Despite this, our
finding that the flow-induced increase in cotransporter protein
expression can be abolished by K+ and Cl
channel blockers suggests that changes in cotransporter protein and/or
activity occur downstream of flow-sensitive K+ and
Cl
channel activation.
Clarification of the role of the cotransporter in the integrated
endothelial cell response to steady laminar shear stress remains to be
determined. One possibility is that activation of the cotransport
system plays a role in maintaining endothelial ionic balance following
flow-induced activation of ion channels. Consistent with this are our
present findings that the BAEC Na-K-Cl cotransporter is quite sensitive
to changes in [Cl]i, with cotransporter
activity increasing as [Cl
]i falls and
decreasing as [Cl
]i rises. Thus, by sensing
changes in [Cl
]i, the cotransporter may
function to regulate [Cl
]i in these cells,
as has been proposed for other cells (20). A second
possibility relates to the role that the Na-K-Cl cotransport system
plays in the regulation of endothelial cell volume (39). There is evidence suggesting that the shear stress-induced
Cl
channels recently demonstrated in endothelial cells
(3) may be identical to volume-regulated Cl
channels previously described in these cells (38, 52).
This raises the interesting possibility that shear stress may induce changes in endothelial intracellular volume, thus leading to direct activation of the Na-K-Cl cotransport system. Because intracellular volume appears to be a determinant of endothelial barrier permeability (25), this sequence of events may have important
implications in the regulation of normal vascular function. A third
possibility relates to the observation that early atherosclerotic
lesions localize preferentially in arterial regions exposed to
disturbed and/or oscillatory flow (36). Previous studies
suggest that the endothelial phenotypes induced by laminar and
turbulent shear stresses differ (54). These findings,
together with the present observation that cotransport protein is
induced by steady laminar but not by oscillatory shear stress, suggest
the possibility that the cotransporter plays a role in atheroprotective
mechanisms and is hence downregulated as part of a lesion-prone
phenotype at arterial sites exposed to disturbed and/or oscillatory flow.
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ACKNOWLEDGEMENTS |
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This work was supported by University of California, Davis, Research Initiation Funds (to A. I. Barakat) and the American Heart Association (to M. E. O'Donnell).
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. E. O'Donnell, Dept. of Human Physiology, School of Medicine, Univ. of California, Davis, CA 95616 (E-mail: meodonnell{at}ucdavis.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.
Received 8 December 1999; accepted in final form 29 August 2000.
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