Flow-induced expression of endothelial Na-K-Cl cotransport: dependence on K and Cl channels

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1 Am J Physiol Cell Physiol 280: C216 C227, Flow-induced expression of endothelial Na-K-Cl cotransport: dependence on K and Cl channels JIMMY SUVATNE, 1 ABDUL I. BARAKAT, 2 AND MARTHA E. O DONNELL 1 1 Department of Human Physiology, School of Medicine, and 2 Department of Mechanical and Aeronautical Engineering, University of California, Davis, California Received 8 December 1999; accepted in final form 29 August 2000 Suvatne, Jimmy, Abdul I. Barakat, and Martha E. O Donnell. Flow-induced expression of endothelial Na-K-Cl cotransport: dependence on K and Cl channels. Am J Physiol Cell Physiol 280: C216 C227, 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/cm 2 ) or purely oscillatory shear stress (0 19 dyn/cm 2 ) 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 stressinduced 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 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 Address for reprint requests and other correspondence: M. E. O Donnell, Dept. of Human Physiology, School of Medicine, Univ. of California, Davis, CA ( meodonnell@ucdavis.edu). 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 Ca 2 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 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. C /01 $5.00 Copyright 2001 the American Physiological Society

2 C217 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 Ca 2 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 flowsensitive 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 flowsensitive K and Cl channels. MATERIALS AND METHODS Cell culture. Bovine aortic endothelial cells (BAEC) were isolated and cultured by using standard procedures in 75-cm 2 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 g/ml streptomycin, and 5% heatinactivated 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% CO 2-95% 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/cm 2. 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/cm 2, 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/cm 2 ) 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- l 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

3 C218 Fig. 1. Flow-induced endothelial elongation. Micrographs show control (no flow) bovine aortic endothelial cells (BAEC) or BAEC exposed in a parallel-plate flow chamber to a steady laminar shear stress (LSS) of 19 dyn/cm 2 for 6 or 24 h. BAEC monolayers were viewed through windows in the flow chamber. Cells were photographed using a cooled charge-coupled device camera (Photometrics SenSys). 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 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 with0or10 M bumetanide). After a 9-min pretreatment Fig. 2. A: Western blot analysis of Na-K-Cl cotransport protein (NKCC) expression in cells exposed to steady shear stress. BAEC monolayers on plastic slides were exposed in the flow chamber to a steady shear stress at 19 dyn/cm 2 for 6, 12, 24, or 48 h. For each experiment, control BAEC monolayers on plastic slides were exposed to the same medium but not sheared. At the end of the perfusion/incubation period, cells were removed from slides and prepared for gel electrophoresis and Western blotting as described in MATERIALS AND METHODS. Monoclonal antibody that recognizes NKCC was used to detect cotransporter protein in the Western blots. Molecular mass markers are shown at left of each blot. The cotransporter protein appears in these images as bands of 170 and 135 kda, sizes that are consistent with the mature, glycosylated form of the protein and the immature, nonglycosylated form, respectively. Data are from a representative Western blot for cells exposed to no shear stress (control) or to LSS for 6, 12, 24, or 48 h. B: densitometric analysis of NKCC expression in BAEC exposed to steady LSS. Western blots were subjected to densitometric analysis to determine the relative abundance of NKCC in the blots. For each experiment, band size and density for the steady shear stress sample were compared with an internal control (run on the same day and treated identically except not sheared). Thus the data are expressed as relative abundance of NKCC. Data represent means SE of 9, 4, and 8 experiments, each with 2 replicate samples for 6, 12, and 24 h of steady LSS, respectively.

4 C219 period at 37 C on a gyratory water bath, the medium was replaced with assay medium (identical to the pretreatment medium but containing 86 Rb), 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). Cell Cl content determination. Cell Cl content was determined by the 36 Cl equilibration method, as described previously (48). Briefly, BAEC monolayers grown in 24-well cluster plates were preequilibrated for 30 min in HEPESbuffered 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 36 Cl (0.2 Ci/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 MgCl 2, and then SDS extracts of the cells were prepared to determine radioactivity and protein content of each well. Specific activities (cpm/ mol) of 36 Cl were determined for each assay condition and used to calculate intracellular Cl content (expressed as mol/g protein. Intracellular volume determination. Intracellular volume of BAEC was determined by radioisotopic evaluation of BAEC intracellular water space by using [ 14 C]urea and [ 14 C]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 [ 14 C]urea or [ 14 C]sucrose (both at 1 Ci/ml). To terminate the assay, we rinsed monolayers with isotonic ice-cold 0.1 M MgCl 2 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 [ 14 C]urea and [ 14 C]sucrose (cpm/ml) were used to calculate water space associated with the radioactive markers (expressed as l/mg protein). Intracellular volume was calculated as the difference between water space determined for [ 14 C]urea (a marker for intracellular plus trapped extracellular space) and [ 14 C]sucrose (a marker for trapped extracellular space). Materials. BaCl 2, 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). 86 Rb 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). RESULTS 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/cm 2 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/cm 2 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/cm 2. 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 Fig. 3. Effect of steady LSS on BAEC Na-K-Cl cotransporter activity. BAEC monolayers on plastic slides were exposed to a steady shear stress of 19 dyn/cm 2 for 6 or 24 h and then rapidly removed from the flow chamber and assayed for Na-K-Cl cotransport activity as described in MATERIALS AND METHODS. For the assay, cells were first exposed for 9 min to isotonic (290 mosm) or hypertonic (390 mosm, by addition of NaCl) medium containing 0 or 10 M bumetanide and then assayed for 9 min in identical medium also containing 86 Rb. Control cells were maintained in the shear stress medium with no flow for 6 or 24 h and then assayed for cotransporter activity at the same time the sheared cells were assayed. Data represent means SE of 3 and 4 experiments, each with 4 6 replicates for 6 and 24 h of steady laminar shear stress, respectively. Steady LSS data shown for isotonic 6- and 24-h LSS and for hypertonic 6- and 24-h LSS are all significantly different from their respective no-shear controls (P 0.01, P 0.001, P 0.05, and P , respectively; unpaired t-test).

5 C220 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 Ca 2 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 parallelplate flow chamber to a purely oscillatory flow of 0 19 dyn/cm 2 (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 compared with 1.0 for Fig. 4. Exposure of BAEC to oscillatory flow: lack of effects on elongation and NKCC abundance. A: micrograph of BAEC exposed to oscillatory flow. Micrographs show control (no flow) BAEC and BAEC exposed to a purely oscillatory flow of 0 19 dyn/cm 2 for 6 h. BAEC monolayers were photographed through flow chamber windows. B: Western blot of cells exposed to oscillatory flow. BAEC monolayers on plastic slides were exposed in the flow chamber to a purely oscillatory flow of 0 19 dyn/cm 2 at a frequency of 1 Hz for 6 h or to no flow in the same medium. At the end of the perfusion/incubation period, cells were removed from slides and subjected to Western blotting for analysis of NKCC abundance as described in MATERIALS AND METHODS. Molecular mass markers are shown at left of blot. OSS, oscillatory shear stress. Data are from a representative Western blot. Three other experiments produced the same outcome.

6 C221 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 Ba 2 (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 M), 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. Figure 5 demonstrates the effects of the ion channel blockers on the amount of Na-K-Cl cotransport protein expressed in BAEC after6hofsteady shear stress. For these studies, representative Western blots are shown in Fig. 5A and densitometric analyses of multiple experiments are shown in Fig. 5B. While steady shear stress in the absence of inhibitors produced a 2.3-fold Fig. 5. A: Western blot analysis of the effect of K and Cl channel inhibitors on NKCC abundance in BAEC exposed to6hofsteady LSS. BAEC monolayers on plastic slides were exposed in the flow chamber to a steady LSS at 19 dyn/cm 2 for 6 h with the use of perfusate that contained Ba 2 (0 or 1 mm), tetraethylammonium (TEA; 0 or 3 mm), diphenylamine-2-carboxylate (DPC; 0 or 1 mm), DIDS (0 or 1 mm), or 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB; 0 or 100 M). For each experiment, control BAEC monolayers on plastic slides were exposed to the same medium (i.e., with the various inhibitors) but not sheared. At the end of the perfusion/incubation period, cells were removed from slides and prepared for gel electrophoresis and Western blotting as described in MATERIALS AND METHODS. Data shown are representative Western blots. B: densitometric analysis of NKCC abundance in BAEC exposed to 6hofsteady LSS in the presence or absence of K and Cl channel inhibitors. Western blots were subjected to densitometric analysis to determine the relative abundance of NKCC in the blots. For each experiment, band size and density for the shear stress sample were compared with an internal control (run on the same day and treated identically except not sheared). All data are expressed as abundance of NKCC relative to the nonsheared control without inhibitors (shaded control bar). Data represent means SE of 5, 4, 5, 5, and 4 experiments for Ba 2, TEA, DPC, DIDS, and NPPB experiments, respectively. Values for 6-h LSS plus TEA, DPC, DIDS, and NPPB are significantly different from the 6-h LSS control (P 0.01 for TEA, DPC, and NPPB; P 0.05 for DIDS; unpaired Students t-test).

7 C222 increase in cotransporter protein abundance, this effect of shear stress was reduced or abolished in the presence of agents that block K or Cl channels. Thus, when cells were exposed to 6hofshear stress in the presence of TEA, no significant increase in the amount of cotransporter protein was observed (Fig. 5B, TEA, solid bar vs. control bars). Although TEA abolished the shear stress-induced increase in cotransporter protein, it had no significant effect on cotransporter protein in control, nonsheared cells (Fig. 5B, TEA vs. control, shaded bars). Similarly, in BAEC exposed to steady laminar shear stress in the presence of the K channel blocker Ba 2, the abundance of cotransporter protein was decreased relative to the sheared control cells in absence of Ba 2 (Fig. 5B, Ba 2 vs. control, solid bars). However, unlike TEA, Ba 2 also reduced the abundance of cotransporter protein in control, nonsheared cells such that the shear stress-induced increase in cotransporter protein abundance in the presence of Ba 2 was not significantly different from that in the control cells (Fig. 5B, Ba 2 vs. control, differences between shaded and solid bars). The reason for the difference in these BAEC responses to TEA vs. Ba 2 is not clear and will require further investigation. These studies also revealed that three separate agents known to block Cl channels (DPC, DIDS, and NPPB) each abolished the shear stress-induced increase in cotransporter protein. As with TEA, none of these agents had a significant effect on cotransporter protein abundance in control, nonsheared cells (6-h exposure to the inhibitors). In these studies, we also evaluated the effects of TEA and NPPB on the shear stressinduced increase in Na-K-Cl cotransporter activity. BAEC were exposed to either TEA (3 mm) or NPPB (100 M) for 6 h under no-flow conditions or in the presence of steady laminar shear stress (19 dyn/cm 2 ), as described for Fig. 3, and then assessed for Na-K-Cl cotransporter activity (in the absence of TEA and NPPB) by using methods described in Fig. 3 and MATE- RIALS AND METHODS. We found that the presence of either TEA or NPPB prevented the shear stress-induced increase in cotransporter activity from occurring in the cells. In fact, both of these channel blockers actually reduced cotransporter activity in the sheared cells compared with the control, nonsheared cells. Thus cotransporter activity of cells exposed to 6 h of TEA under no-flow vs. steady shear conditions was and mol K g protein 1 min 1, respectively. Similarly, cotransporter activity of cells exposed to6hofnppb was and mol K g protein 1 min 1 under no-flow vs. steady shear conditions, respectively. The reason that the channel blockers not only prevented the shear stressinduced increase in cotransporter activity (as shown in Fig. 3) but actually reduced cotransport activity in sheared cells below that of nonsheared cells is not clear and will require further investigation. However, it is clear that TEA and NPPB block the shear stressinduced increases in both cotransporter protein and cotransporter activity. Collectively, these observations suggest that shear stress-induced elevation of Na-K-Cl cotransport protein expression in the endothelial cells occurs through mechanisms that are at least partially dependent on flow activation of shear stress-sensitive ion channels. 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 flowactivated 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 ele- Fig. 6. A and B: effect of varying intracellular Cl content on BAEC Na-K-Cl cotransport activity. A: BAEC monolayers grown on multiwell plates were exposed to media containing varying extracellular [Cl ] ([Cl ] o ) for 60 min, and then intracellular Cl content was determined by 36 Cl equilibration as described in MATERIALS AND METHODS. [Cl ] o was varied by replacing NaCl with Na-methane sulfonate. Data are means SE of 4 replicates from a representative experiment. Similar results were obtained in 3 other experiments. B: BAEC monolayers were exposed to media with varying [Cl ] o for 60 min, and then cells were immediately assayed for 5 min in medium containing normal [Cl ] o (131 mm) to determine Na-K-Cl cotransporter activity. In these experiments, [Cl ] o in the preincubation medium was varied by replacing NaCl with Na-methane sulfonate. Data are means SE (n 8). C: intracellular volume of BAEC following pretreatment with reduced [Cl] o media. Cell volume of BAEC was evaluated by radioisotopic determination of intracellular water space as described in MATERIALS AND METHODS. Confluent BAEC monolayers were incubated in media with varying [Cl ] o for 30 min by using Na-methane sulfonate to replace NaCl. Cells were then incubated for a second 30-min period in identical media containing either [ 14 C]urea or [ 14 C]sucrose (1 Ci/ml). The cell-associated radioactivity of the monolayers was subsequently determined and used to calculate total and extracellular water space, respectively. Data are means SE of quadruplicate determinations from 4 experiments. No significant differences were found among values for cell volumes observed in 10, 30, 70, 100, and 136 mm [Cl] o media (ANOVA). D: effect of reduced [Cl ] o treatment on BAEC NKCC. BAEC monolayers grown on collagen-coated multiwell plates were exposed to media containing varying [Cl ] o for 30 min or 6 h. [Cl ] o in the media was reduced by replacing NaCl with Na-methane sulfonate. At the end of the incubation period, cells were removed from the multiwell plates and prepared for gel electrophoresis and Western blotting as described in MATERIALS AND METHODS. Data are from a representative Western blot. E: densitometric analysis of NKCC in BAEC pretreated with media containing reduced [Cl ] o. BAEC monolayers were pretreated for 6 h with media containing either 131 (control), 50, or 25 mm Cl under no-flow conditions and then evaluated for relative abundance of cotransporter protein by Western blot analysis and densitometry. Methane sulfonate was used as the replacement anion. Data are means SE of 3 experiments. Values for 50 and 25 mm [Cl ] o pretreatments are significantly different from values for 131 mm [Cl ] o pretreatment (P 0.05).

8 C223 vated [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 36 Cl 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 in-

9 C224 crease 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 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 Namethane 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. Previous studies have shown that increased extracellular tonicity, which stimulates activity of the Na- K-Cl cotransporter, can also induce increased expression of cotransporter protein in some cells (5, 24). Because we and others (31, 48) have shown that reduction of [Cl ] i is another potent stimulus of cotransporter activity, it is possible that it also causes an increased expression of cotransporter protein. To test this possibility, we examined the effect of reduced [Cl ] i on BAEC Na-K-Cl cotransporter protein abundance. BAEC monolayers grown on multiwell plates were incubated in media containing varying [Cl ] o for 30 min or 6 h. At the end of the incubation period, the cells were removed from the multiwell plates and immediately prepared for Western blotting as described in MATERIALS AND METHODS. Figure 6D demonstrates that, for an incubation time of 30 min, Na-K-Cl cotransport protein levels were largely unchanged by exposure to reduced [Cl ] o (and hence to reduced [Cl ] i ). However, after a 6-h incubation in reduced [Cl ] o, protein levels were significantly elevated for both 50 and 25 mm [Cl ] o incubation media compared with the control 131 mm [Cl ] o medium. Densitometric analysis of [Cl ] i effects on BAEC cotransporter protein revealed that 6-h incubation in 25 mm [Cl ] o caused a 1.42-fold increase in cotransporter protein over control, as shown in Fig. 6E. Thus reduced [Cl ] i not only stimulates BAEC cotransporter activity but also increases the expression of cotransporter protein. However, the elevation of cotransporter protein abundance observed with this rather large decrease in [Cl ] i is considerably less than the 2.3-fold increase in cotransporter protein observed with 6hoflaminar shear stress. This finding suggests that the shear stressinduced increase in BAEC Na-K-Cl cotransporter protein abundance, while apparently involving activation of flow-sensitive K and Cl channels, is not mediated simply by reduction of [Cl ] i. DISCUSSION 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/cm 2 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/cm 2 ) 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 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

10 C225 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 shearsensitive 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 stressinduced 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/cm 2 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/cm 2 ) did not induce the Na-K-Cl response, and steady laminar shear stress of 4 dyn/cm 2 induced only a minimal change in cotransport gene expression (54). It is noteworthy that while a steady shear stress of 10 dyn/cm 2 leads to extensive endothelial cytoskeletal reorganization and consequent cellular elongation and alignment in the direction of flow, a shear stress of 4 dyn/cm 2 may be below the threshold needed for inducing these cell shape changes (12). Similarly, it has been demonstrated that a 10 dyn/cm 2 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

11 C226 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 flowsensitive 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). 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