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Am J Physiol Cell Physiol 282: C708-C718, 2002. First published November 21, 2001; doi:10.1152/ajpcell.00247.2001
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Vol. 282, Issue 4, C708-C718, April 2002

Dual effect of fluid shear stress on volume-regulated anion current in bovine aortic endothelial cells

Victor G. Romanenko, Peter F. Davies, and Irena Levitan

Institute for Medicine and Engineering, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The key mechanism responsible for maintaining cell volume homeostasis is activation of volume-regulated anion current (VRAC). The role of hemodynamic shear stress in the regulation of VRAC in bovine aortic endothelial cells was investigated. We showed that acute changes in shear stress have a biphasic effect on the development of VRAC. A shear stress step from a background flow (0.1 dyn/cm2) to 1 dyn/cm2 enhanced VRAC activation induced by an osmotic challenge. Flow alone, in the absence of osmotic stress, did not induce VRAC activation. Increasing the shear stress to 3 dyn/cm2, however, resulted in only a transient increase of VRAC activity followed by an inhibitory phase during which VRAC was gradually suppressed. When shear stress was increased further (5-10 dyn/cm2), the current was immediately strongly suppressed. Suppression of VRAC was observed both in cells challenged osmotically and in cells that developed spontaneous VRAC under isotonic conditions. Our findings suggest that shear stress is an important factor in regulating the ability of vascular endothelial cells to maintain volume homeostasis.

chloride channels; hemodynamic environment; vascular endothelial cells


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SWELLING OF VASCULAR ENDOTHELIAL cells is observed during ischemia and may result in severe pathological consequences, such as narrowing of the capillary luminal space, impairment of blood flow, lowered integrity of the endothelial permeability barrier, and edema formation (11, 27, 28). During the onset of reperfusion, swollen cells cause further damage to the vascular system by restricting blood flow, increasing capillary hydraulic resistance, and enhancing edema formation. Thus flow environment changes considerably and may become an important component of the volume-regulatory mechanisms of vascular endothelial cells in ischemia and subsequent reperfusion.

The key mechanism responsible for preventing pathological cell swelling is activation of volume-regulated anion current (VRAC). Activation of VRAC allows Cl- and small organic osmolytes (taurine, aspartate, glutamate) to flow out of the cell, reducing the intracellular osmolarity and restoring cell volume back to normal (reviewed in Refs. 17 and 52). Several lines of evidence suggest that changes in the hemodynamic environment may be an important factor in the regulation of VRAC in vascular endothelial cells. First, exposure to shear stress activates a large array of signaling molecules that are known to regulate VRAC activity. These include small G proteins (24, 38, 53), tyrosine kinase (18, 26, 46, 48), and mitogen-activated protein kinases extracellular signal-regulated kinase (ERK)-1 and ERK-2 (5, 44, 54). Second, it is well known that both fluid shear stress (9, 14, 56) and osmotic stress (19, 58) induce dramatic rearrangement of the cytoskeleton network. VRAC sensitivity to an osmotic stress is enhanced by the disruption of F-actin (19, 30). Finally, it was shown recently that exposure of endothelial cells to fluid shear stress, in the absence of an osmotic stimulus, can activate a small Cl--selective current that may be identical to VRAC (2, 32). The flow field, however, was not well defined in these studies because they were conducted in open surface flow chambers, and, therefore, the quantitative relationship between the current and the level of shear stress could not be evaluated.

To expose cells to a defined flow field simultaneously with electrophysiological recording, we (23) developed a minimally invasive flow (MIF) device. The MIF device is a novel modification of a parallel-plate flow chamber based on the principle that a narrow opening can be cut in the upper plate of the parallel-plate chamber without creating a fluid overflow if the surface tension forces at the opening are sufficient to counteract the hydrostatic pressure generated in the chamber. We showed previously (23) that the flow streamlines are only minimally disturbed by lowering the tip of a micropipette into the MIF chamber and that the cells inside the chamber are exposed to well-defined unidirectional flow with a parabolic profile. Here, using the MIF device, we show that there is a strong interaction between the osmotic and the shear stress-dependent pathways in the regulation of VRAC and that there are at least two mechanisms by which the two pathways interact. Our results also imply that different regimes of flow may affect the degree of endothelial cell swelling during reperfusion injury.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Bovine aortic endothelial cells (BAECs) between passages 10 and 15 were grown in Dulbecco's modified Eagle's medium (DMEM; Cell Grow, Washington, DC) supplemented with 10% bovine serum (GIBCO BRL, Grand Island, NY). Cell cultures were maintained in a humidified incubator at 37°C with 5% CO2. The cells were fed and split every 3-4 days.

Exposure of cells to flow: MIF device. To record membrane currents under well-defined flow conditions, cells grown on thin coverslips (0.1-mm thickness) were mounted in the MIF device as previously described (23). Briefly, the MIF device consists of three connecting flow chambers: 1) an upper chamber that serves to spread fluid evenly across the inlet width to the flow chamber, 2) a parallel plate flow chamber (chamber height 1 mm) with a series of narrow longitudinal slits cut into its upper plate (cells are grown on the lower surface of this chamber), and 3) an open lower chamber from which the perfusion medium is removed by vacuum suction. A recording micropipette accesses the flow chamber through the open slits in the top plate and reaches the cells grown on the lower surface of the chamber. The device permits unidirectional laminar flow of up to 15 dyn/cm2 to be applied to the cells. The flow was driven by gravitational force. The fluid (extracellular recording solution) was passed from a reservoir through the MIF device, from which it was suctioned to a flask for return to the fluid reservoir. The flow rate was set by adjusting the height difference between the fluid reservoir and the microscope stage/MIF device and was measured by collecting the perfusion fluid in a vacuum flask. The shear stress (tau ) was calculated from tau  = 6µQ/wh2 (3), where µ is fluid viscosity (0.010 g · cm-1 · s-1), Q is the flow rate (ml/s), w is the width (2 cm), and h is the height (0.1 cm) of the chamber. The flow rates were measured simultaneously with VRAC recording in every experiment. All cells were continuously exposed to a background level of shear stress (0.05-0.1 dyn/cm2) generated by a slow perfusion of the chamber (1-2 ml/min) maintained for the duration of the experiment. This slow perfusion is necessary to maintain the osmolarity of the extracellular solution and to prevent the accumulation of chemicals that may be released from the cells.

Electrophysiological recording. The external recording solution contained (in mM) 150 NaCl, 1 EGTA, 2 CaCl2, and 10 HEPES, pH 7.2. Internal solutions contained (in mM) 140 Cs-glutamate, 10 HEPES, and 4 ATP, pH 7.2 (CsOH) with free Ca2+ concentration ([Ca2+]) of 15-16 nM (0.1 CaCl2, 1.1 EGTA). Free [Ca2+] was calculated with MAXC software (4). Chemicals were obtained from Fisher Scientific (Fairlawn, NJ) or Sigma (St. Louis, MO). The osmolarities of all solutions were determined immediately before recording with a vapor pressure osmometer (Wescor, Logan, UT) and were adjusted by the addition of sucrose as required. Current was monitored by 500-ms linear voltage ramps from -60 to +60 mV at an interpulse interval of 5 s. The holding potential between the ramps was -60 mV. Ionic currents were measured with the whole cell configuration of the standard patch-clamp technique (13). Pipettes (SG10 glass; Richland Glass, Richland, NJ) were pulled to give a final resistance of 2-6 MOmega with the above-described recording solutions. Pipettes were coated with Sylgard (Dow Corning) to decrease electrical capacitance. These pipettes generated high-resistance seals without fire polishing. A saturated salt agar bridge was used as reference electrode. Currents were recorded with an EPC9 amplifier (HEKA Electronik, Lambrecht, Germany) and accompanying acquisition and analysis software (Pulse and PulseFit; HEKA Electronik) running on a PowerCenter 150 (Mac OS) computer. Pipette and whole cell capacitance was automatically compensated. Whole cell capacitance and series resistance were monitored throughout each recording. Series resistance was compensated for in all experiments (75-95%).

Volume measurements. BAECs were loaded with 20 mM 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) in serum-free medium overnight. Cells were then washed three times with Cl--free perfusion medium [in mM: 111 NaNO3, 4 KNO3, 1.6 Ca(NO3)2, 0.6 Mg(NO3)2, 50 HEPES, 5 glucose, and 28.5 mM gluconic acid, pH 7.2] and incubated in the same medium for 2 h at 25°C. Fluorescence was measured with a microplate fluorometer (Fluoroscan Ascent FL; Labsystems) adapted for use with the MIF device. Excitation and emission filters were 355 and 460 nm, respectively.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Enhancement of VRAC activation by shear stress (1 dyn/cm2). The sensitivity of membrane anion conductance to fluid shear stress was compared in BAECs that were challenged with a transmembrane osmotic gradient (extracellular-to-intracellular osmolarity ratio of 0.8) or maintained in an isosmotic environment. The osmolarity of the extracellular solution in all experiments was maintained constant at 300 ± 10 mosM. The osmolarity of the intracellular solution in osmotically challenged cells was 370 ± 10 mosM, whereas in cells that were exposed to an isotonic environment it was maintained at 300 ± 10 mosM. Challenging the cells with a transmembrane osmotic gradient resulted in a gradual VRAC development over the period of 15-20 min followed by a steady-state plateau, as described previously (20).

Imposition of a shear stress step from a background level to 1 dyn/cm2 during the activation phase of VRAC resulted in an immediate increase in VRAC activation rate (Fig. 1). Typical examples are shown in Fig. 1A,b and in Fig. 1B (top curve). The same effect was observed in the presence of 5 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) in the intracellular recording solution (Fig. 1B, inset), indicating that an increase in VRAC activation rate is not due to an increase in cytosolic Ca2+ level. The reversal potential of the current was unaffected, indicating that the selectivity of the shear stress-induced component of the current is identical to the selectivity of the swelling-induced current. The values of the reversal potential during the exposure to flow and under no-flow conditions were -46 ± 3 and -44 ± 2 mV respectively. Glutamate--to-Cl- permeability ratios calculated from these reversal potentials with the Goldman-Hodgkin-Katz equation were 0.20 and 0.22, respectively, similar to amino acid-to-Cl- permeability ratios reported previously for glutamate, aspartate, and taurine (15, 22, 45). In symmetrical Cl- conditions the reversal potential of the current became -1.5 ± 4 mV, as expected. In contrast to osmotically challenged cells, application of shear stress to cells maintained under isotonic conditions did not result in the development of VRAC in a consistent and repeatable way (Fig. 1A,a; Fig. 1B, bottom). Similarly, no current development was observed in cells exposed to hypertonic extracellular medium (not shown).


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  		Fig. 1.   Typical current-voltage relationships and time-courses of volume-regulated anion current (VRAC) development in individual bovine aortic endothelial cells (BAECs) exposed simultaneously to osmotic gradient and to a shear stress step of 1 dyn/cm2. A: representative current-voltage relationships recorded from 2 individual cells, a cell challenged with a mild osmotic gradient (extracellular-to-intracellular osmotic ratio 0.8) and a cell at isosmotic conditions. Currents were elicited by 500-ms linear voltage ramps from a holding potential of -60 mV to +60 mV. The ramps were delivered with time intervals of 5 s for the duration of the experiment. The traces shown were recorded 50, 100, and 150 s before (a) and after (b) application of fluid shear stress (in isosmotic conditions the traces overlap). B: time courses of VRAC development in the same two cells. The top curve shows VRAC development in the cell challenged with an osmotic gradient, and the bottom curve shows the current in the cell maintained under isosmotic conditions. Each point represents current amplitude of an individual voltage ramp at +60 mV. During the entire experiment cells were slowly perfused (1 ml/min, 0.05 dyn/cm2) with external recording solution, except when the fluid flow was increased to 22 ml/min (bar), generating shear stress of 1.1 dyn/cm2. Inset, the time course of VRAC development recorded with 5 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) in the pipette solution.

To calculate the average time course of VRAC development before and after application of shear stress, the currents in the individual cells were normalized to the value of the current in the same cell recorded 2.5 min before the application of the shear. As expected from the increase in VRAC activation rate in individual cells, the average slope of VRAC development increased approximately threefold on introduction of the shear stress step (Fig. 2). We compared the rates of current development before and during the application of the flow in the same cells. No statistically significant change in current activity was observed in cells maintained in an isosmotic environment (Fig. 2B).


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  		Fig. 2.   An increase in VRAC activation rate by a shear stress step of 1 dyn/cm2. A: an average time course of VRAC development before, during, and after the application of a shear stress step of 0.99 ± 0.1 dyn/cm2 (n = 8). For every cell, VRAC amplitudes elicited by individual ramps for the duration of the experiment were normalized to the VRAC amplitude recorded in the same cell 2.5 min before the application of the shear stress. The points show means ± SE (vertical deflections). B: VRAC activation rates before and on application of shear stress in cells at hyposmotic and isosmotic conditions. The rates were calculated as slopes of linear regression for the linear segments of the time courses. The VRAC rates before and during application of the flow were measured from the same cells. All values are means ± SE (n = 5-8). * P < 0.05 vs. no flow.

An enhancement of VRAC activity by the application of shear stress was observed not only during the activation phase of the current but also after the current reached steady-state plateau levels (Fig. 3A,b; Fig. 3B, top). In these experiments, the currents induced by an osmotic challenge were allowed to develop to the steady-state level (15-20 min) as described above. Application of the shear stress step (1 dyn/cm2) induced a gradual increase in VRAC activity, and termination of the flow resulted in a slow and sometimes delayed decrease of VRAC. The response to shear stress was attenuated after repetitive applications of shear. Figure 3B (top) demonstrates that the VRAC response to repetitive applications of the same shear step to the same cell was blunted. This observation was further confirmed by calculating the average responses of the normalized VRAC after the first (Fig. 4A) and after the third (Fig. 4B) applications of the same shear stress step to the same cells. To determine whether an increase in the current amplitude is statistically significant, maximal current amplitudes measured during the application of the flow in each individual cell were normalized to the current amplitudes measured in the same cells before the application of the flow. The ratio between the maximal current density during the exposure to flow (first exposure) and the plateau current amplitude before the application of flow is 1.2 ± 0.03 (P < 0.05, flow vs. no flow). A similar effect was observed in the presence of 5 mM BAPTA (current ratio 1.16 ± 0.04; P < 0.05, flow vs. no flow). No significant changes in maximal current density were observed after repetitive flow applications. Figure 4, C and D, shows the rate of VRAC change before and after the application of shear. Before shear, the slope of the VRAC change is zero because the current is at its plateau level. Immediately after the introduction of the first shear stress step the slope becomes strongly positive (an increase in VRAC activity), and after the termination of the step the slope becomes negative (a decrease in VRAC; Fig. 4C). In contrast, after repetitive step applications (a third application) there are no statistically significant changes in VRAC activity (Fig. 4D).


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  		Fig. 3.   An increase in maximal VRAC amplitude on the application of 1 dyn/cm2 shear stress. In this experiment, shear stress steps were introduced only after VRAC amplitude reached its maximal steady-state value (15-25 min after the cells were challenged osmotically). A: current-voltage relationships recorded 50, 100, and 150 s before the application of flow (a; note that the traces completely overlap) and 150 s after the application of shear stress (b). The top traces were recorded in a cell challenged with a 0.8 osmotic gradient, and the bottom traces were recorded in a cell maintained under isosmotic conditions. B: time courses of the shear stress-induced enhancement of VRAC in the 2 cells described in A. VRAC responses to the 1st and 4th applications of the shear stress step in the same cells are shown (note the break in the time axis).



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  		Fig. 4.   Sensitivity to shear stress runs down with repetitive stimulation. A and B: average time courses of the shear stress-induced enhancement of VRAC induced by the 1st application of the shear stress step (1.1 ± 0.08 dyn/cm2; A) and by the subsequent (3rd and 4th) applications of the same shear stress step to the same cells (B). The points show means ± SE (vertical deflections). C and D: VRAC activation rates calculated as described in Fig. 2. All values are means ± SE (n = 5-8). * P < 0.01, significant difference vs. cell currents before shear stress challenge.

Biphasic effect of shear stress on VRAC activation (3 dyn/cm2). Increasing the shear stress level from 1 to 3 dyn/cm2 did not increase the facilitatory effect of the shear stress on VRAC activation (Table 1). To investigate further the effect of a 3 dyn/cm2 shear stress step on VRAC, flow was introduced during the steady-state phase of VRAC (Fig. 5). In this experiment, the introduction of shear stress induced a transient increase in VRAC activity, followed by a slower inhibitory phase, an average response calculated from four cells (Fig. 5A). The increase in maximal current amplitude in cells exposed to 3 dyn/cm2 was similar to that in cells exposed to 1 dyn/cm2. An increase in current amplitude shown in Fig. 5A is ~15%. This, however, is an underestimation because maximal effect of shear stress does not occur exactly at the same time in all cells and, therefore, averaging the time courses of different cells diminishes the effect. The ratio between maximal current amplitude calculated for each individual cell and that normalized for the current amplitude before the application of the flow in the same cell was 1.2 ± 0.06 (P < 0.05 flow vs. no flow).

                              
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  		Table 1.   VRAC activation rates



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  		Fig. 5.   Biphasic effect of shear stress on VRAC activation (3 dyn/cm2). A: average time course of VRAC response to a 3 dyn/cm2 shear stress step (3.0 ± 0.4 dyn/cm2). The points show means ± SE (vertical deflections). B: average VRAC activation rates before, during, and after the termination of flow. The 1st and 4th bars are VRAC activation rates before and after application of shear stress, respectively. The 2nd and 3rd bars represent the VRAC currents during application of shear stress calculated for the period before and after the peak of the current, respectively. All values are means ± SE (n = 4-5). * P < 0.05, significant difference vs. cell currents before shear stress challenge.

The transient nature of the enhancement was also manifested by a decrease in time to peak, defined as the length of time from the application of the shear step to the maximum amplitude of the current. Specifically, the time to peak in cells exposed to 3 dyn/cm2 was 34 ± 16 s, whereas the time to peak in cells exposed to 1 dyn/cm2 was 162 ± 25 s (P < 0.01). The rates of VRAC change before and after the introduction of flow are shown in Fig. 5B.

Suppression of VRAC activation by higher shear stress levels (5-10 dyn/cm2). A further increase in the magnitude of the shear stress from control to 5-10 dyn/cm2 caused pronounced suppression of VRAC activity in 12 of 16 cells, whereas a facilitatory effect was observed only in 2 cells. When a high-shear stress step was applied during the activation phase of VRAC development, the current was strongly suppressed in a steplike manner (4 of 6 tested cells; Fig. 6). Furthermore, in those cells the amplitude of the current after termination of the flow was significantly larger than its amplitude before the application of the shear stress step. This overshoot suggests that there is a component(s) of VRAC activated by osmotic stress that continues to develop in the presence of the (suppressive) shear stress, so that when the shear stress is removed the current rises to a higher value. With repetitive applications of shear the suppression becomes noticeably slower (compare the 1st and 3rd applications of the step), and, eventually, the response runs down completely. To test whether the rundown of VRAC responsiveness to flow is due to the repetitive flow applications or to the loss of the current sensitivity after prolonged cell swelling, the cells were maintained under hypotonic conditions for 30-35 min before the first application of flow. Figure 6B, inset, shows that cells maintained their sensitivity to flow after the prolonged swelling if they were not exposed to flow but lost their responsiveness to flow after repetitive exposures to flow. This experiment strongly supports the hypothesis that the rundown of the responsiveness of VRAC to the shear stress stimulus is caused by repetitive applications of flow.


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  		Fig. 6.   Inhibitory effect of higher levels of shear stress (5 dyn/cm2) on osmotically induced VRAC development. A: typical current-voltage relationships recorded at hyposmotic and isosmotic conditions. The currents shown were recorded before application of shear stress (a), immediately after the application of shear stress (b), and immediately after the termination of the flow (c). B: time courses of shear stress-induced inhibitory effect on VRAC development (top curve). Bottom curve, currents recorded from a control cell maintained in isosmotic conditions. The bar indicates the time of the application of the shear stress. Inset, an example of an experiment in which the 1st exposure to flow was applied 33 min after challenging the cells osmotically. The bars indicate the times and durations of the exposures to flow. The experiment was repeated in 3 cells.

Application of the high-shear stress step at the steady-state plateau phase of VRAC also resulted in a gradual inhibition of VRAC (Fig. 7A; n = 10). The normalized average decrease in current amplitude during the flow exposure was 0.73 ± 0.05 (P < 0.01 flow vs. no flow). Under isosmotic conditions, cells that did not develop anionic currents before the application of the shear stress had no response to the shear step (n = 4). Cells that did develop a small anionic current, however, showed some decrease in the current amplitude (n = 4). This variability between cells is reflected in the SE, and the overall effect of the shear stress on cells maintained in isosmotic environment was not statistically significant (Fig. 7B). We conclude, therefore, that application of 5-10 dyn/cm2 of shear stress has a significant inhibitory effect on the activity of VRAC.


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  		Fig. 7.   Inhibitory effect of shear stress on maximal VRAC amplitude after the current reaches its steady-state level (>5 dyn/cm2). A: average time course of VRAC response to a 6 dyn/cm2 shear stress step. The points show means ± SE (vertical deflections). B: VRAC activation rates before, on, and after application of shear stress in the cells at hyposmotic and isosmotic conditions. All values are means ± SE (n = 8-10). * P < 0.05 vs. cell currents before shear stress challenge.

Shear stress does not affect voltage-dependent inactivation and rectification of Cl- conductance. Voltage- and time-dependent decay of Cl- conductance at depolarized voltages (greater than +80 mV) is the hallmark of VRAC in a variety of cell types (reviewed in Refs. 34 and 39) including vascular endothelial cells (20, 35). These inactivation properties are unique to VRAC and can be used to distinguish between VRAC and other Cl- conductances such as Ca2+-dependent Cl- conductance (36). A two-pulse voltage protocol with a conditioning pulse ranging from -60 to +160 mV followed by a test pulse to +100 mV was used to determine the inactivation properties of the current under flow and no-flow conditions. Voltage-dependent inactivation of the current recorded under flow (1 dyn/cm2) is apparent from the accelerated decay of the current amplitude shown in Fig. 8A. The inactivation ratio was defined as the ratio between the amplitude of the test pulse delivered after a preconditioning pulse and the amplitude of a control test pulse delivered directly from the holding potential. Dependence of the inactivation ratio on the voltage of the preconditioning pulse provides a measure of the voltage sensitivity of the inactivation and the amount of charge that has to move for a channel to change its conformation from an open to an inactivated state. Figure 8B shows that the inactivation curves measured before and during the application of the flow are very similar.


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  		Fig. 8.   Voltage-dependent inactivation and rectification of VRAC on the application of 1 dyn/cm2 shear stress. A: VRAC current recorded in response to a family of voltage steps from -60 to +160 mV (inset). The traces show 2 families of currents recorded from the same cell before (a) and during (b) the application of shear stress of 1 dyn/cm2. B: voltage dependences of inactivation of currents before () and during (black-triangle) application of shear stress of 1.1 ± 0.1 dyn/cm2. R, normalized ratio of the current amplitude in response of a test voltage pulse (+100 mV) after a conditioning voltage pulse (-60 mV to +160 mV). All values are means ± SE (n = 4-5). C: current-voltage relationships of VRAC before and during application of flow under symmetrical Cl- conditions.

Another typical feature of VRAC is an outward rectification in symmetrical ionic conditions (reviewed in Refs. 34 and 39). Figure 8C shows the currents recorded from the same cell before and during the application of the flow in symmetrical Cl- conditions. The rectification ratio, defined as the ratio between the slope of the VRAC current-voltage relationship at +60 mV and the slope of the current-voltage relationship at -60 mV, was 1.67 ± 0.04 during the exposure to flow and 1.65 ± 0.07 in no-flow conditions. The similarities between the inactivation curves and the rectification ratios measured at the two experimental conditions strongly suggest that the shear stress-sensitive Cl- current is VRAC.

Shear stress has no effect on BAEC cell volume. To test whether exposure of BAECs to shear stress results in changes in cell swelling or shrinking, cell volume was measured by loading the cells with SPQ, a fluorescent dye that is sensitive to cell volume (49, 50). The principle of measuring cell volume with SPQ is that the dye is quenched by intracellular osmolytes, presumably by the intracellular anions. When cells swell, the intracellular concentrations of both the SPQ and the quencher are decreased, lowering the probability of interaction between the two, and the fluorescence intensity increases. However, SPQ is quenched by Cl-, and therefore it is necessary to deplete the intracellular Cl- and to substitute it with NO<UP><SUB>3</SUB><SUP>−</SUP></UP>, which has little effect on SPQ fluorescence (42). Indeed, after the anion substitution SPQ fluorescence stabilized, producing a stable baseline with no detectable variation for the duration of 30 min. The substitution of Cl- by NO<UP><SUB>3</SUB><SUP>−</SUP></UP> did not have a significant effect on shear stress-induced facilitation of VRAC; the ratio between the maximal current density during the exposure to flow and the plateau current amplitude before the application of flow is 1.15 ± 0.05 (P < 0.05, flow vs. no flow).

The effect of shear stress on BAEC cell volume was tested under both isosmotic and hyposmotic conditions (Fig. 9). When cells were exposed to flow of 1 dyn/cm2 while being maintained in an isosmotic environment and then challenged with a strong osmotic gradient (50%), there was no change in SPQ fluorescence in response to the application of flow (Fig. 9A). Challenging the cells osmotically resulted in a significant increase in SPQ fluorescence, as expected. Application of flow after the cells were challenged osmotically also had no effect on SPQ fluorescence (Fig. 9B). In the latter experiment, the cells were challenged with a mild osmotic gradient (20%) to simulate more closely the conditions of the electrophysiological experiments.


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  		Fig. 9.   Cell volume is not affected by shear stress. A: normalized 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) fluorescence of BAECs during the application of shear stress under isosmotic conditions and during subsequent osmotic shock. The cells were continuously perfused with isotonic or hypotonic (50%) medium. The rate of perfusion during "no flow" was maintained at 0.05 dyn/cm2. B: normalized SPQ fluorescence of BAECs exposed to flow of 1.0 dyn/cm2 after they were challenged with a mild osmotic shock (20%). The shear stress step of 1.0 dyn/cm2 was applied from the basal perfusion level of 0.05 dyn/cm2. Cell volumes were normalized to the values at the beginning of the recordings. All values are means ± SE (n = 3). The experiments were performed in the minimally invasive flow (MIF) device.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The role of hemodynamic mechanical forces in the regulation of cell volume homeostasis in vascular endothelial cells was studied by determining the effect of fluid shear stress on the activation of VRAC, the key mechanism protecting the cells against pathological swelling (reviewed in Refs. 17 and 52). It has been suggested that, in vascular endothelial cells, the physiological role of VRAC is not only to regulate cell volume but also to sense changes in the hemodynamic environment (33, 35). This is the first study to investigate the sensitivity of VRAC to shear stress in a controlled flow environment. Our results demonstrate that VRAC is regulated by acute changes in shear stress, indicating that both osmotic and shear stress stimuli contribute to VRAC regulation.

Two lines of evidence suggest that the shear stress-sensitive Cl- current in BAECs is VRAC and not a Ca2+-dependent Cl- current that is also expressed in vascular endothelial cells (36). First, stress-induced increase in Cl- conductance was observed in cells in which VRAC was activated by an osmotic stress but not in cells maintained in isosmotic or hypertonic conditions, and there was a strong correlation between the ability of cells to develop VRAC and their ability to respond to shear stress, as discussed in more detail below. Second, the voltage-dependent properties of shear stress-sensitive Cl- current observed in our study are similar to those of VRAC and not Ca2+-dependent Cl- current. Specifically, the currents recorded under flow exhibited slow voltage-dependent decay at depolarizing potentials (greater than +80 mV), a feature that is typical for VRAC (see, e.g., Refs. 21 and 35). In contrast, Ca2+-dependent Cl- current is known to exhibit voltage-dependent activation, the opposite trend at these potentials (31, 36). VRAC and Ca2+-dependent Cl- current also exhibit opposite trends in their voltage-dependent properties at negative membrane potentials. Ca2+-dependent Cl- current rapidly inactivates, whereas VRAC has no voltage dependency at these potentials. The behavior of the Cl- current recorded under flow was identical to that of VRAC. In addition, the rectification properties of the current were also similar to those of VRAC and not to those of Ca2+-dependent Cl- current, whose rectification is significantly stronger than that of VRAC (31, 36). We conclude, therefore, that the increase in Cl- current on the application of shear stress is due to potentiation of VRAC and not to activation of Ca2+-dependent Cl- current.

The pattern of VRAC regulation by shear stress is complex. Exposing the cells to shear stress in a range of 1-10 dyn/cm2 has a biphasic effect on the swelling-induced development of VRAC: a facilitatory phase at low levels of shear stress (1 dyn/cm2) and an inhibitory phase at the higher levels of shear (5-10 dyn/cm2). Importantly, the two phases coexist at a medium level of shear stress (3 dyn/cm2). In the latter case, a facilitatory phase develops immediately after the introduction of the shear step and an inhibitory phase develops more slowly and becomes dominant after a delay of ~1 min, an observation consistent with the concept that acute changes in shear stress and steady levels of shear may have differential effects on the physiology of endothelial cells (6, 8). To exclude the possibility that the observed effects are due to variations in extracellular osmolarity that may occur during the experiment because of evaporation, the cells were continuously exposed to a background shear stress level of 0.05-0.1 dyn/cm2. A step from no flow to the background shear stress level in the beginning of an experiment did not induce any overt response. Because of the high Cl- concentration in the extracellular solution, we can exclude washout of Cl- from the vicinity of the cell membranes as an explanation of the observed effects of flow. We also show that application of shear stress does not result in changes in cell volume of BAECs, suggesting that the sensitivity of VRAC to shear stress is not due to shear stress-induced changes in cell volume. The biphasic effect of shear stress on VRAC suggests that there are at least two mechanisms by which swelling-induced and shear stress-induced transduction pathways interact with each other in the regulation of the current.

These observations provide a possible explanation for the earlier contradictory reports on the ability of flow to activate endothelial Cl- current. Although low-amplitude flow-induced Cl- currents were observed in vascular endothelial cells (2), no flow-sensitive Cl- currents were observed under similar flow conditions (41). It is well known that establishment of a whole cell configuration may induce "spontaneous" VRAC under isosmotic conditions (see, e.g., Ref. 57), an effect that is attributed to a Donnan equilibrium between the cytosol and the pipette (57). We suggest, therefore, that in previous reports flow-induced Cl- current was observed in cells that may have developed spontaneous VRAC before the flow exposure. This hypothesis is supported in our study, in which there was a strong correlation between the ability of cells to develop VRAC in response to osmotic stress and their ability to respond to shear stress. Specifically, cells that failed to develop VRAC in response to osmotic stress, typically 20-30% of the cell population (21, 47), also had no response to shear stress (not shown). The source of the heterogeneity in swelling-induced and spontaneous VRAC development is not known, but the correlation between the ability of cells to respond to osmotic and shear stimuli, observed in our study, provides further evidence that the two effects are coupled.

Several mechanisms may underlie the interactions between osmotic and shear stress stimuli. The nonadditive nature of the interaction between the stimuli suggests that parallel pathways may be involved in VRAC regulation. Rho A-induced reorganization of the cytoskeleton (37, 38, 53, 55) and protein tyrosine kinase-induced phosphorylation cascade (18, 46, 48, 55) are required for VRAC activation (reviewed in Ref. 33). Both signaling pathways are also regulated by shear stress. Specifically, shear stress induces the translocation of Rho A from the cytosol to the membrane (25), a step that is known to activate the Rho-induced signaling pathway (12). Translocation of Rho A is required for the shear stress-induced reorganization of the stress fibers (25). Protein phosphorylation cascades are also regulated by shear stress in a complex biphasic manner (1, 40, 54). The dual effect of shear stress on VRAC, therefore, may be mediated by a shift in the equilibrium between phosphorylation/dephosphorylation cascades and/or by reorganization and detachment of cytoskeleton from the membrane.

The levels of shear stress examined in this study are in the low physiological range (1-10 dyn/cm2). Similar levels of shear stress are known to induce a variety of endothelial responses, including activation of flow-sensitive K+ currents (1-15 dyn/cm2; Refs. 16 and 41), reorganization of the cytoskeleton (9), translocation of Rho A (25), and activation of protein tyrosine kinase (40). Typical average values of shear stress in the major human arteries during basal conditions are 2-20 dyn/cm2 with local transients to 30-100 dyn/cm2 (7). The basal levels of shear in small coronary arteries and arterioles, measured in canine vascular bed, average 10 and 19 dyn/cm2, respectively (51). High levels of shear stress (>10 dyn/cm2) were not tested in our experiments because it was impossible to maintain a stable seal between the recording pipette and cellular membrane.

Regulation of VRAC by shear stress may have important clinical implications. Swelling of microvascular endothelial cells observed during low-flow ischemia (10, 27, 29) can reduce the capillary diameters below the diameter of the blood cells, resulting in further impairment of blood flow. The narrowing of capillary luminal space persists on the start of reperfusion so that full blood supply is not reestablished (10, 28). This condition of "no reflow" is considered to be the major postischemic dysfunction of capillaries, and it has been suggested that therapeutic strategies should be aimed to prevent endothelial swelling. Although there are some differences between ischemia-induced cell swelling and swelling induced by an acute osmotic shock, VRAC activation plays a major role in protecting the cells against excessive swelling in both conditions (reviewed in Ref. 43). In summary, our study suggests that the level of shear stress during the onset of reperfusion may significantly affect cell volume homeostasis of endothelial cells: a step to low shear stress is advantageous because it enhances protection against cell swelling, whereas a step to high shear stress may be detrimental because it suppresses the protection.


    ACKNOWLEDGEMENTS

We thank Drs. Aron Fisher, Yefim Manevich, and Kevin Foskett of the University of Pennsylvania for critical reading of this manuscript. We also thank Nadeene Harbeck and Alan Sun for superb technical assistance.


    FOOTNOTES

This work was supported by American Heart Association Grant 0060197U and American Heart Association Scientist Development Grant 0130254N to I. Levitan and by National Heart, Lung, and Blood Institute Grants HL-62250 and HL-64388-01 to P. F. Davies.

Address for reprint requests and other correspondence: I. Levitan, Univ. of Pennsylvania, IME, 1160 Vagelos Research Laboratories, Philadelphia, PA 19104 (E-mail: ilevitan{at}mail.med.upenn.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpcell.00247.2001

Received 14 June 2001; accepted in final form 14 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 282(4):C708-C718
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