Differential membrane potential and ion current responses to different types of shear stress in vascular endothelial cells

Deborah K. Lieu,1 Pamela A. Pappone,2 and Abdul I. Barakat1

1Department of Mechanical and Aeronautical Engineering and 2Section of Neurobiology, Physiology and Behavior, University of California, Davis, California 95616

Submitted 11 June 2003 ; accepted in final form 30 January 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Vascular endothelial cells (ECs) distinguish among and respond differently to different types of fluid mechanical shear stress. Elucidating the mechanisms governing this differential responsiveness is the key to understanding why early atherosclerotic lesions localize preferentially in arterial regions exposed to low and/or oscillatory flow. An early and very rapid endothelial response to flow is the activation of flow-sensitive K+ and Cl channels that respectively hyperpolarize and depolarize the cell membrane and regulate several important endothelial responses to flow. We have used whole cell current- and voltage-clamp techniques to demonstrate that flow-sensitive hyperpolarizing and depolarizing currents respond differently to different types of shear stress in cultured bovine aortic ECs. A steady shear stress level of 10 dyn/cm2 activated both currents leading to rapid membrane hyperpolarization that was subsequently reversed to depolarization. In contrast, a steady shear stress of 1 dyn/cm2 only activated the hyperpolarizing current. A purely oscillatory shear stress of 0 ± 10 dyn/cm2 with an oscillation frequency of either 1 or 0.2 Hz activated the hyperpolarizing current but only minimally the depolarizing current, whereas a 5-Hz oscillation activated neither current. These results demonstrate for the first time that flow-activated ion currents exhibit different sensitivities to shear stress magnitude and oscillation frequency. We propose that flow-sensitive ion channels constitute components of an integrated mechanosensing system that, through the aggregate effect of ion channel activation on cell membrane potential, enables ECs to distinguish among different types of flow.

ion channels; atherosclerosis; mechanotransduction


IN LARGE ARTERIES, the combined effects of arterial geometry, blood flow pulsatility, and vascular wall compliance lead to a complex and highly dynamic flow field that is characterized by a wide range of shear stresses, and, in certain arterial regions, by periodic changes in flow direction (1, 4, 18, 24). This diversity of arterial flow patterns is particularly significant in light of the preferential localization of early atherosclerotic lesions at arterial regions exposed to low and/or oscillatory shear stress (1, 18, 24, 27). Although the biological basis of this correlation remains to be elucidated, shear stress-mediated endothelial dysfunction is likely centrally involved. The local hemodynamic environment in an artery is sensed by the vascular endothelium (34). This is accomplished via an intricate and coordinated sequence of shear stress-induced humoral, metabolic, and structural responses in endothelial cells (ECs) (2, 10, 12, 21, 27, 39). The mechanisms by which ECs sense fluid mechanical forces and subsequently transduce these forces into biochemical signals remain incompletely understood and are under intense investigation.

Of more direct relevance to shear stress involvement in atherogenesis is the fact that ECs, beyond being responsive to shear stress, distinguish among and respond differently to different types of shear stress. For instance, in vivo, ECs at branchings and/or bifurcations where blood flow is disturbed are cuboidal in shape, whereas cells away from branches and within relatively undisturbed flow fields have a more elongated and fusiform morphology (19, 31). In vitro, whereas both steady and nonreversing pulsatile flow induce EC elongation and mobilize intracellular Ca2+, purely oscillatory flow does not elicit either of these responses (14, 15). Steady and oscillatory flow also impact endothelial gene expression and protein synthesis differently (7, 20, 38). Turbulent flow has a considerably different impact on EC gene expression, morphology, and turnover rates from laminar flow with the same time-averaged shear stress (11, 12). Nothing is known about how ECs distinguish among different types of flow.

To discriminate among different types of shear stress, ECs must be able to sense and resolve the different components of different flow waveforms. One of the most rapid EC responses to flow is the activation of flow-sensitive ion channels; therefore, these channels have been proposed as possible flow sensors (2, 3, 10). To date, two types of flow-sensitive ion currents have been reported in ECs: a hyperpolarizing current that is principally carried by inward-rectifying K+ channels (16, 17, 32) and a depolarizing current that is at least partly due to activation of outward-rectifying Cl channels (5, 26, 35). Flow-sensitive K+ channels, which are rapidly activated on flow initiation, lead to a current that initiates at a shear stress of 0.1 dyn/cm2, reaches half-maximal activation at 0.7 dyn/cm2, and saturates >15 dyn/cm2 (16, 32). These channels desensitize slowly with sustained flow but rapidly regain sensitivity after deactivation. Typical of inward-rectifying K+ channels, flow-sensitive K+ channels are entirely blocked by external Ba2+ or Cs+. Activation of these channels hyperpolarizes the cell membrane by 2–6 mV in response to shear stresses of 1–5 dyn/cm2 (5, 17, 25). Shear stress-sensitive Cl channels are activated independently of the K+ channels and lead to cell membrane depolarization following the initial K+-mediated hyperpolarization (5, 26). The dependence of the Cl current on shear stress magnitude remains to be determined.

If flow-sensitive ion channels participate in flow sensing, then the resulting changes in cell membrane potential might be expected to play an important role in regulating the ability of vascular ECs to distinguish among different types of flow. We hypothesized that different types of flow elicit different membrane potential responses in ECs. In this study, we have used whole cell current- and voltage-clamp recordings to demonstrate for the first time that EC membrane currents are differentially sensitive to different shear stress magnitudes and pulsatile flow frequencies.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Bovine aortic ECs (BAECs) (CSC, Kirkland, WA) in passages 39 were cultured by standard procedures. To provide a system within which the flow field is well characterized while simultaneously permitting patch-clamp recording, BAECs were cultured under static (no flow) conditions inside attachment factor-treated (CSC) square cross-section (1 x 1 mm) borosilicate glass capillary tubes (VitroCom, Mountain Lakes, NJ), as described previously (32, 37, 40). Standard parallel-plate flow chambers or cone-and-plate viscometers are not appropriate for the present experiments because they do not permit patch-clamp recording. The capillary tube system used here permits patch pipette access to cells near the ends of the tube. For a given flow rate, the shear stress to which cells in the capillary tube are exposed is determined analytically (40) and is relatively flat (a variation of <10%) within 200 µm of the tube centerline. Therefore, all patch-clamp recordings were limited to cells within 200 µm of the tube centerline. Cells were grown in a 37°C, 5% CO2 humidified incubator in CSC medium supplemented with CSC growth factors (CSC) and 10% heat-inactivated calf serum (Hyclone Laboratories, Logan, UT) and were used within 1 or 2 days of plating.

Flow exposure protocol. BAECs in the capillary tubes were exposed to either steady or purely oscillatory flow (zero net flow rate). In the steady flow experiments, flow corresponding to a shear stress at the tube centerline of either 1 or 10 dyn/cm2 was infused through the capillary tube with the use of a syringe pump. For oscillatory flow, the syringe pump was programmed to cyclically infuse and withdraw to create flow with a peak shear stress of 10 dyn/cm2 (i.e., 0 ± 10 dyn/cm2) and oscillation frequencies of 0.2, 1, or 5 Hz. In all cases, the cells were exposed to a continuous-flow period of 3 min. The 3-min flow period was initiated after a stable no-flow baseline had been established for at least 1 min.

Patch-clamp protocol. Whole cell voltage-clamp recordings on single (subconfluent) BAECs and current-clamp recordings on confluent BAEC monolayers were performed as previously described (5). Briefly, borosilicate microelectrode pipettes with resistances of 2–6 M{Omega} were filled with a patch pipette solution consisting of the following (in mM): 100 K-aspartate, 35 KCl, 10 K2EGTA, 1 CaCl2, and 10 MOPS at pH 7.2 and 300 mosM. The extracellular Ringer solution consisted of (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 MOPS, and 5.55 glucose at pH 7.4 and 300 mosM. Recordings were made using a patch-clamp amplifier (Axopatch 200A, Axon Instruments, Union City, CA), and data were collected using Pulse+Pulsefit software (HEKA Electronik, Nova Scotia, Canada).

The current-clamp protocol used to record whole cell membrane potential consisted of clamping whole cell currents at 0 pA and recording the membrane potential every 65.5 ms, generating a trace of membrane potential behavior over time. The voltage-clamp protocol consisted of a step from a holding potential of –70 to +60 mV for 40 ms, followed by a ramp to –160 mV over 400 ms and finally a return to the holding potential. Voltage ramps were executed every 1 s during the recording period, yielding 60 current-voltage (I-V) traces per minute. Whole cell conductance as a function of time was calculated using Igor Pro software (Wavemetrics, Lake Oswego, OR) by taking the derivative of a least-squares straight line fit to the I-V plots for two different voltage ranges within which the I-V behavior was approximately linear: –20 to +30 mV (denoted as V+) and –130 to –90 mV (denoted as V–). To separate the two known types of flow-sensitive ion currents from one another, the voltage-clamp recordings were performed in the presence of either the K+ channel blocker Ba2+ (100 µM) or the Cl channel blocker 5-nitro-2-(3-phenopropylamino)benzoic acid (NPPB; 100 µM). Net flow-induced current was obtained by subtracting the preflow I-V trace from the I-V trace exhibiting the largest current during the flow period. All measurements were made at room temperature.

Statistical analysis. Data are presented as means ± SE. Statistical comparison of means was performed with the use of one-way ANOVA, followed by Dunnett's posttest. P < 0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Membrane potential responses to flow depend on type of flow imposed. The involvement of flow-sensitive ion channels in overall endothelial mechanotransduction likely occurs through the direct effect of activation of these channels on EC membrane potential. We previously reported that flow induces initial EC membrane hyperpolarization that is subsequently reversed to depolarization within 35–160 s of flow initiation (5). We performed current-clamp recordings on BAEC monolayers to investigate BAEC membrane potential responses to a steady shear stress of 10 dyn/cm2 and to a purely oscillatory shear stress of 0 ± 10 dyn/cm2 having different oscillation frequencies. Before flow initiation, BAECs had a resting membrane potential of –59.0 ± 1.2 mV (n = 45). A steady shear stress of 10 dyn/cm2 elicited initial cell membrane hyperpolarization that was subsequently reversed to depolarization (Fig. 1), similarly to our previous studies (5). Over the ensemble of cells studied (n = 20), steady shear stress of 10 dyn/cm2 induced a hyperpolarization of 2.1 ± 0.3 mV, followed by a depolarization of 12.1 ± 2.3 mV.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 1. Membrane potential responses to a steady shear stress of 10 dyn/cm2. Shear stress elicited membrane hyperpolarization, followed by depolarization. The bar denotes the period of flow application.

 
Flow-sensitive inward-rectifying K+ channels and outward-rectifying Cl channels were previously implicated in regulating EC membrane potential responses to steady flow (5, 32). To probe whether our membrane potential results (Fig. 1) were consistent with this notion, we performed a limited number of current-clamp measurements in the presence of either the K+ channel blocker Ba2+ or the Cl channel blocker NPPB. These pharmacological agents were previously shown to effectively block flow-sensitive K+ and Cl currents (5, 32, 38). The presence of NPPB (100 µM) resulted in the absence of the depolarization so that flow led only to sustained membrane hyperpolarization (Fig. 2A), whereas recordings made in the presence of Ba2+ (100 µM) resulted in flow-induced depolarization with no hyperpolarization (Fig. 2B). It should be noted that in the presence of Ba2+, the background inward-rectifying K+ channels were blocked so that the preflow baseline membrane potential was more positive than that in the absence of Ba2+ (range –47 to –55 mV, n = 3). However, because this membrane potential was more negative than the Cl reversal potential (ECl) (–36 mV), depolarization toward ECl was observed. These results are consistent with previous findings suggesting that flow-sensitive K+ and Cl channels contribute to overall membrane potential responses to flow.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 2. Membrane potential responses to steady flow in the presence of the Cl channel blocker 5-nitro-2-(phenopropylamino)benzoic acid (NPPB; 100 µM) (A) and the K+ channel blocker Ba2+ (100 µM) (B). Depolarization was absent in the presence of NPPB, whereas hyperpolarization was absent in the presence of Ba2+. The bars denote the period of flow application.

 
Subjecting BAECs to a 1-Hz oscillatory shear stress of 0 ± 10 dyn/cm2 (Fig. 3A) elicited hyperpolarization in all cells studied but led to depolarization in only 5 of 9 cells. The extent of flow-induced hyperpolarization (1.8 ± 0.6 mV; n = 9) was not significantly different (P > 0.05) from that in response to a steady shear stress of 10 dyn/cm2. On the other hand, the depolarization, when it occurred, was very small (1.1 ± 0.9 mV for the five cells that depolarized and 0.6 ± 0.4 mV for the ensemble of nine cells tested), significantly smaller than that in response to steady shear stress (P < 0.05). A 0.2-Hz oscillation (Fig. 3B) elicited full hyperpolarization (2.8 ± 0.3 mV; n = 6) in all cells, no depolarization in two of six cells, and only limited depolarization in four of six cells (1.7 ± 1.6 mV for the four cells that depolarized and 1.1 ± 0.8 mV for the ensemble of six cells tested). Finally, a 5-Hz oscillation (Fig. 3C) elicited neither hyperpolarization nor depolarization in any of the cells studied (n = 6). As shown in Fig. 3, AC, exposing the cells to a steady shear stress of 10 dyn/cm2 after the oscillatory flow elicited the standard hyperpolarization, followed by robust depolarization. These results directly demonstrate differences in membrane potential responses to steady and oscillatory flow and to different frequencies of oscillatory flow.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 3. Membrane potential responses to a purely oscillatory shear stress of 0 ± 10 dyn/cm2 at oscillation frequencies of 1 Hz (A), 0.2 Hz (B), and 5 Hz (C). In each case, a steady shear stress of 10 dyn/cm2 after the oscillatory flow episode induced membrane hyperpolarization, followed by depolarization. The bars denote the period of flow application.

 
Membrane potential responses to steady flow depend on shear stress amplitude. The peak shear stress in all the steady and oscillatory flow data shown thus far was 10 dyn/cm2. To probe possible differential membrane potential responsiveness to different shear stress levels, we performed current-clamp recordings on BAECs exposed to a steady shear stress of 1 dyn/cm2. In contrast to 10 dyn/cm2, 1 dyn/cm2 elicited BAEC membrane hyperpolarization with no significant depolarization (Fig. 4). The magnitude of flow-induced hyperpolarization (3.2 ± 1.6 mV; n = 4) was not significantly different from that in response to 10 dyn/cm2 (P > 0.05); however, depolarization (1 mV) occurred in only one of four cells. These results demonstrate that the hyperpolarizing current is responsive to lower levels of steady shear stress than the depolarizing current. As also shown in Fig. 4, exposing BAECs to a steady shear stress of 10 dyn/cm2 after the 1 dyn/cm2 flow episode elicited hyperpolarization, followed by full depolarization (similar to Fig. 1). This suggests that the capacity for full depolarization is preserved in cells that had been exposed to shear stress levels that hyperpolarize the cells but fall below the threshold for flow-induced depolarization.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4. Membrane potential responses to a steady shear stress of 1 dyn/cm2. The shear stress elicited membrane hyperpolarization with no depolarization. Subsequent exposure of the same cell to a steady shear stress of 10 dyn/cm2 induced hyperpolarization, followed by depolarization. The bars denote the period of flow application.

 
Figure 5 provides a quantitative comparison of the impact of the various flow conditions on BAEC membrane potential. Similar levels of hyperpolarization were induced by steady shear stresses of 1 and 10 dyn/cm2 as well as by an oscillatory shear stress of 0 ± 10 dyn/cm2 at frequencies of 0.2 or 1 Hz. On the other hand, a steady shear stress of 10 dyn/cm2 induced considerably larger depolarization than a steady shear stress of 1 dyn/cm2 or an oscillatory shear stress of 0 ± 10 dyn/cm2 at frequencies of 0.2 or 1 Hz. A 5-Hz oscillatory flow induced neither hyperpolarization nor depolarization. These results provide further support to the notion that the two types of flow-sensitive ion currents exhibit different responses to different types of imposed flow.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 5. Comparison of bovine aortic endothelial cell (BAEC) membrane potential responses to the different types of flows studied. Data are means ± SE. BAECs subjected to steady shear stress of 1 dyn/cm2 or to oscillatory shear stress of 0 ± 10 dyn/cm2 at frequencies of 0.2 or 1 Hz exhibited similar hyperpolarization but significantly smaller depolarization than cells exposed to a steady shear stress of 10 dyn/cm2. BAECs exposed to 5-Hz oscillatory shear stress of 0 ± 10 dyn/cm2 neither hyperpolarized nor depolarized (shown as bars of zero amplitude). The depolarization data are averages for the ensemble of cells studied for each experimental condition and assume zero depolarization for cells within the ensemble that did not depolarize. *P < 0.05, statistically significant difference relative to steady shear stress of 10 dyn/cm2.

 
Hyperpolarizing and depolarizing flow-sensitive currents are differentially responsive to different types of flow. Previous work in our laboratory and by others has shown that BAECs have at least two flow-sensitive currents: an inwardly rectifying K+ current that is activated at membrane potentials negative to the K+ reversal potential (EK) and that can be completely blocked by Ba2+ and an outwardly rectifying Cl current that can be blocked by NPPB (5, 16, 17, 26, 32, 38). To probe whether the membrane potential responses described above are consistent with these currents, whole cell voltage-clamp recordings were performed on BAECs in the presence of either NPPB or Ba2+. Figures 6 and 7 show the net flow-induced current (as defined in MATERIALS AND METHODS) in individual whole cell patch-clamped cells in capillary tubes in the presence of these blockers. Under these conditions, zero current potentials of unstimulated cells are more positive than the resting potentials measured in current clamp for cells forming part of a syncytium (see below), presumably due to changes in resting conductances caused by the replacement of cytosolic contents by pipette solution and/or significant contribution from seal leak. Currents induced by a steady shear stress of 10 dyn/cm2 and by a purely oscillatory shear stress of 0 ± 10 dyn/cm2 having different oscillation frequencies were studied, as in the current-clamp experiments.



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 6. Response of the net flow-activated hyperpolarizing current to steady (10 dyn/cm2) and purely oscillatory (0 ± 10 dyn/cm2) shear stress. Measurements were made in the presence of NPPB (100 µM) to block the flow-activated Cl current. The bars in B, D, F, and G denote the period of flow application. A and B: current-voltage (I-V) behavior (A) and whole cell conductance plot (B) for BAEC exposed to steady shear stress. C and D: I-V behavior (C) and conductance plot (D) for BAEC exposed to 0.2-Hz oscillatory shear stress. E and F: I-V behavior (E) and conductance plot (F) for BAEC exposed to 1-Hz oscillatory shear stress. G and H: I-V behavior (G) and conductance plot (H) for BAEC subjected to 5-Hz oscillatory shear stress. The conductance values were derived from the derivative of a straight line fit within the V– range (see MATERIALS AND METHODS). Significant hyperpolarizing currents were elicited by all flow types except for 5-Hz oscillatory flow. Time points 1 and 2 in B, D, F, and H correspond to the two values used for determining the net current.

 


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 7. Response of the net flow-activated depolarizing current to steady (10 dyn/cm2) and purely oscillatory (0 ± 10 dyn/cm2) shear stress. Measurements were made in the presence of Ba2+ (100 µM) to block the flow-activated K+ current. The bars in B, D, F, and G denote the period of flow application. A and B: I-V behavior (A) and whole cell conductance plot (B) for BAEC exposed to steady shear stress. C and D: I-V behavior (C) and conductance plot (D) for BAEC exposed to 0.2-Hz oscillatory shear stress. E and F: I-V behavior (E) and conductance plot (F) for BAEC exposed to 1-Hz oscillatory shear stress. G and H: I-V behavior (G) and conductance plot (H) for BAEC subjected to 5-Hz oscillatory shear stress. The conductance values were derived from the derivative of a straight line fit within the V+ range (see MATERIALS AND METHODS). Significant depolarizing currents were only elicited by steady flow. Time points 1 and 2 in panels B, D, F, and H correspond to the two values used for determining the net current.

 
In the presence of the Cl channel blocker NPPB, a steady shear stress of 10 dyn/cm2 induced a robust hyperpolarizing current at voltages negative to –50 mV (Fig. 6A). For the ensemble of cells studied (n = 9), the average reversal potential of this current was –60 ± 8 mV, more positive than EK (–80 mV). Because this concentration of NPPB is known to block the Cl current, this suggests that the steady flow stimulus may be activating an additional current (possibly a nonspecific cation current) in addition to the flow-sensitive K+ current previously reported (17, 32). Activation of the hyperpolarizing current by a steady shear stress of 10 dyn/cm2 is further illustrated in Fig. 6B by the flow-induced increase in whole cell conductance in the V– voltage range (see MATERIALS AND METHODS). The ratio of peak conductance during flow to preflow baseline conductance averaged 1.18 ± 0.07 (n = 9). The higher conductance level often persisted for several minutes after cessation of flow, suggesting that deactivation of the flow-sensitive hyperpolarizing current after removal of the flow stimulus was slow.

A purely oscillatory shear stress of 0 ± 10 dyn/cm2 with a physiological frequency of oscillation of 1 Hz also activated the flow-sensitive hyperpolarizing current (Fig. 6, E and F). In this case, the reversal potential of the net current was –62 ± 4 mV and the ratio of maximum whole cell conductance during flow to preflow baseline conductance in the V– range was 1.18 ± 0.03 (n = 5), not significantly different from the conductance increase induced by steady shear stress (P > 0.05). To test whether the extent of hyperpolarizing current activation by oscillatory flow was sensitive to the frequency of flow oscillation, recordings were made at a subphysiological frequency of 0.2 Hz (Fig. 6, C and D) and a superphysiological frequency of 5 Hz (Fig. 6, G and H). A 0.2-Hz oscillation activated the hyperpolarizing current (reversal potential of –53 ± 6 mV), leading to a conductance increase of 1.16 ± 0.07 (n = 7), which was not significantly different from that induced by steady flow (P > 0.05). On the other hand, a 5-Hz oscillation did not activate the hyperpolarizing current and hence failed to elicit any change in whole cell conductance in any of the cells studied (n = 6). These results are consistent with the hypothesis that the flow-sensitive hyperpolarizing current is responsive to oscillatory flow but that this response depends on the frequency of oscillation. The current is equally responsive to 0.2- and 1-Hz oscillatory flow because it is to steady flow but is nonresponsive to a 5-Hz oscillation.

To test the responses of the flow-sensitive depolarizing current to steady flow and to oscillatory flow of different frequencies, voltage-clamp recordings were performed on BAECs in the presence of the K+ channel blocker Ba2+. A steady shear stress of 10 dyn/cm2 elicited a significant current in the positive voltage range with limited rectification at negative voltages (Fig. 7A) and with a reversal voltage of –32 ± 6 mV (n = 6). This value is near the Cl reversal potential (ECl = –36 mV) and is consistent with the activation by steady flow of outward-rectifying flow-sensitive Cl channels, as we have demonstrated previously (5). The possible presence of other depolarizing currents cannot, however, be excluded at this point. The resulting increase in whole cell conductance is depicted in Fig. 7B. For the ensemble of cells studied, the ratio of peak conductance during flow to preflow baseline conductance in the V+ range (see MATERIALS AND METHODS) averaged 1.33 ± 0.10 (n = 6).

In contrast to steady flow, an oscillatory shear stress of 0 ± 10 dyn/cm2 at all frequencies tested (1, 0.2, and 5 Hz) elicited minimal depolarizing current in BAECs (Fig. 7, CH). The conductance increases in response to 0.2-, 1-, and 5-Hz oscillation were 1.04 ± 0.04 (n = 5), 1.04 ± 0.04 (n = 5), and 1.04 ± 0.02 (n = 5), all significantly smaller than the increase induced by steady shear stress (P < 0.05). For the cases where these small depolarizing currents were observed, the reversal potentials were close to ECl. These results demonstrate that the flow-sensitive depolarizing current, unlike the flow-sensitive hyperpolarizing current, responds minimally to oscillatory flow.

Figure 8 summarizes the impact of the different types of flow studied on the ratio of maximum whole cell conductance during flow to the preflow baseline conductance in the V– and V+ voltage ranges. In the V– range, steady shear stress and 0.2- and 1-Hz oscillatory shear stress induce sizable and similar increases in conductance, whereas 5-Hz oscillatory shear stress has no significant impact on conductance. In the V+ range, steady shear stress induces a significant increase in conductance, whereas oscillatory shear stress elicits significantly smaller increases in conductance at all of the oscillation frequencies tested.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 8. Ratio of maximum whole cell conductance during flow to the preflow baseline conductance for the different types of flows studied and for the voltage ranges V– and V+ (see MATERIALS AND METHODS). Data are means ± SE. For the V– range, the conductance increase was comparable for a steady shear stress of 10 dyn/cm2 (n = 9) to that for an oscillatory shear stress of 0 ± 10 dyn/cm2 with oscillatory frequencies of either 0.2 Hz (n = 7) or 1 Hz (n = 5). A 5-Hz oscillatory frequency (n = 6) did not induce any increase in conductance (shown as a bar of zero amplitude). For the V+ range, the conductance increase induced by 0.2- (n = 5), 1- (n = 5) or 5-Hz (n = 5) oscillatory shear stress of 0 ± 10 dyn/cm2 was significantly smaller than that elicited by a steady shear stress of 10 dyn/cm2 (n = 6). *P < 0.05, statistically significant difference relative to steady shear stress of 10 dyn/cm2.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Beyond being responsive to fluid mechanical shear stress, vascular ECs distinguish among and respond differently to different types of shear stress (7, 11, 12, 14, 15, 20, 31, 38). However, the mechanisms by which ECs resolve differences among different flow waveforms remain completely unknown. Activation of flow-sensitive K+ and Cl currents that, respectively, hyperpolarize and depolarize the cell membrane is one of the most rapid EC responses to flow; therefore, these ion currents have been implicated in flow sensing (2, 3, 10). The present study tested the specific hypothesis that flow-sensitive ion currents respond differently to different types of imposed flow.

We have demonstrated that flow-sensitive hyperpolarizing and depolarizing ion currents exhibit different sensitivities to shear stress magnitude and oscillatory flow frequency. Our steady flow results have revealed that while the flow-activated hyperpolarizing current is equally responsive to low (1 dyn/cm2) and relatively high shear stress (10 dyn/cm2), the depolarizing current is significantly less responsive to 1 dyn/cm2 than to 10 dyn/cm2. Because the hyperpolarizing current in response to steady flow is carried at least in part by K+ and the depolarizing current is carried at least partly by Cl, these results are consistent with the notion that flow-sensitive K+ channels have a lower shear stress threshold for activation than Cl channels and raises the intriguing possibility that K+ channels might primarily participate in sensing changes in shear stress within a range of "low" shear stresses, whereas Cl channels may be involved in sensing shear stress changes at relatively "high" shear stresses. This construct is consistent with previous data indicating that K+ channel activation occurs at very low shear stresses (0.1 dyn/cm2) with half-maximal activation attained at a shear stress as low as 0.7 dyn/cm2 (32). This issue merits further investigation, however, especially in light of the observation that the flow-induced hyperpolarizing current reverses at a voltage more positive than EK, which suggests there is an NPPB-insensitive current activated by low flow in addition to the K+ current.

Our oscillatory flow results have demonstrated that the flow-activated hyperpolarizing current responds to pulsatile frequencies smaller than or equal to a physiological frequency of 1 Hz but is nonresponsive to a 5-Hz oscillation. On the other hand, the depolarizing current is only minimally responsive to a 0.2- or 1-Hz oscillation. If this frequency dependence occurs in vivo, then our results suggest that it is the hyperpolarizing current that endows ECs with the needed sensitivity to detect changes in pulsatile frequencies within the physiological range (1–2 Hz).

We propose that the present oscillatory flow results reflect differences in the dynamics of activation between the flow-sensitive hyperpolarizing and depolarizing currents. At an oscillatory frequency of 1 Hz, flow-sensitive ion channels are periodically exposed to shear stress in each direction for 500 ms before flow-directional reversal. The integrated mechanical stimulus during that period is sufficient for activating the hyperpolarizing current but not the depolarizing current, suggesting that the activation dynamics of the hyperpolarizing current are more rapid than those of the depolarizing current. This assertion is supported by the fact that when both types of ion currents are activated by a steady flow stimulus (see Fig. 1), the cell membrane first hyperpolarizes in spite of the fact that the electrochemical driving force is larger for Cl than for K+. The eventual reversal of membrane potential to depolarization occurs only after the slower Cl channel activation has been fully realized. Indeed, the ensemble of our oscillatory flow data suggests that whereas the time constant associated with activation of the hyperpolarizing current is in the range of 100–500 ms, the time constant for activation of the depolarizing current is of the order of 2.5–5 s. The basis for the differences in activation dynamics between the two types of flow-sensitive ion currents remains to be determined but may relate to inherent structural differences between the ion channels carrying these currents or to differences in how these flow-sensitive ion channels interact with the cell membrane or with intracellular structures.

We speculate that the different responses of flow-activated hyperpolarizing and depolarizing currents to different types of shear stress may be interpreted within the context that flow-sensitive ion channels constitute components of an integrated mechanosensory system that allows ECs to discriminate among different types of flow waveforms. Within this system, channels responsible for flow-induced membrane hyperpolarization (most notably flow-sensitive K+ channels) participate in sensing changes in low shear stress magnitudes as well as changes in pulsatile frequency while channels responsible for membrane depolarization (most notably Cl channels) are involved in resolving changes in high shear stress magnitudes.

Although the physiological relevance of ion channel activation by flow remains incompletely understood, these channels have previously been shown to interact with EC volume regulatory processes (36) and to regulate various endothelial flow responses including increased production of nitric oxide, downregulation of endothelin-1 transcript levels, upregulation of transforming growth factor-{beta}1 mRNA, release of cGMP, and increased expression of the Na-K-Cl cotransport protein (9, 21, 29, 30, 38). One pathway by which this regulation may occur is through the effect of membrane potential changes mediated by these ion channels on the transport of Ca2+, a fast and sensitive second messenger, across the EC membrane (28). In this study, we have demonstrated that a steady shear stress of 10 dyn/cm2 ultimately depolarizes the cell membrane by ~12 mV after the initial transient hyperpolarization. In rat megakaryocytes, a nonexcitable cell type like ECs, depolarization stimulates Ca2+ release from intracellular stores, causing intracellular Ca2+ oscillations (22). Moreover, the fact that 1-Hz oscillatory flow fails to either depolarize the EC membrane or to increase intracellular Ca2+ levels (14) suggests that these responses may share common pathways. Another intriguing link is suggested by recent data demonstrating that flow regulates the expression of ATP-sensitive K+ channels, which in turn are implicated in the generation of endothelial reactive oxygen species, increased intracellular Ca2+, and activation of nitric oxide synthase (8). The relationship between changes in membrane potential and mobilization of intracellular Ca2+ needs to be explored further.

In vivo, most ECs in arteries are subjected to nonreversing pulsatile flow, and a recent study (35) using membrane potential-sensitive fluorescent dyes has suggested that pulsatile flow stimulates coronary EC hyperpolarization to a larger extent than steady flow. In the present study, we have focused on differences between steady and purely oscillatory flow. Oscillatory flow occurs within regions of arterial flow separation and recirculation that periodically appear and disappear with blood flow pulsatility. These regions, which are also associated with low time-averaged wall shear stress, are especially prone to the development of early atherosclerotic lesions (1, 18, 27). It is interesting that our present results predict that low and oscillatory shear stress lead to largely similar EC membrane potential profiles that are in turn different from those of ECs exposed to relatively high shear stress. An additional consideration in this regard, however, is that whereas our present experiments subjected ECs to flow acutely, ECs in vivo are exposed to flow chronically. It is likely that subjecting ECs to a specific type of flow stimulus chronically will ultimately result in desensitization of flow-sensitive ion channels and/or will elicit adaptive mechanisms that restore membrane potential to its baseline levels. Therefore, our results are expected to have their most direct relevance to acute changes in shear stress that may occur as a result of alterations in stress or activity levels.

Our findings suggest that ECs subjected acutely to low and/or oscillatory flow would only undergo membrane hyperpolarization, whereas cells exposed to relatively high and unidirectional shear stress would undergo hyperpolarization, followed by depolarization. Although membrane potential in ECs and other nonexcitable cells modulates numerous cellular functions and has been suggested as an indicator of general EC health (23), there is conflicting evidence on the physiological and pathological implications of changes in EC membrane potential. Recent data demonstrate that relatively high doses of hydrogen peroxide, which has been implicated in atherosclerosis through induction of oxidative stress, lead to EC membrane hyperpolarization (6). On the other hand, the membrane potential of coronary ECs in hypertensive rats is more depolarized than that of normal rats (13), and oxidative stress created by carbonylcyanide p-trifluoromethoxyphenylhydrazone, a mitochondrial uncoupler that disrupts production of ATP, leads to BAEC membrane depolarization (33). EC hyperpolarization also correlates with suppressed production of superoxide, a molecule that disturbs EC metabolism and facilitates low-density lipoprotein oxidation (23). Whether these various responses are direct or indirect consequences of membrane potential changes and the role of flow-sensitive ion channels in mediating these effects remain to be established.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was supported by a Biomedical Engineering Research Grant from The Whitaker Foundation, an Atorvastatin Research Award from Pfizer/Parke-Davis, and Philip Morris USA.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. I. Barakat, Dept. of Mechanical and Aeronautical Engineering, Univ. of California, 1 Shields Ave., Davis, CA 95616 (E-mail: abarakat{at}ucdavis.edu).

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


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Asakura T and Karino T. Flow patterns and spatial distribution of atherosclerotic lesions in human coronary arteries. Circ Res 66: 1045–1066, 1990.[Abstract]

2. Barakat AI. Responsiveness of vascular endothelium to shear stress: potential role of ion channels and cellular cytoskeleton. Int J Mol Med 4: 323–332, 1999.[ISI][Medline]

3. Barakat AI and Davies PF. Mechanisms of shear stress transmission and transduction in endothelial cells. Chest 114: 58S-63S, 1998.[Free Full Text]

4. Barakat AI, Karino T, and Colton CK. Microcinematographic studies of the flow patterns in the excised rabbit aorta and its major branches. Biorheology 34: 195–221, 1997.[CrossRef][ISI][Medline]

5. Barakat AI, Leaver EV, Pappone PA, and Davies PF. A flow-activated chloride-selective membrane current in vascular endothelial cells. Circ Res 85: 820–828, 1999.[Abstract/Free Full Text]

6. Bychkov R, Pieper K, Ried C, Milosheva M, Bychkov E, Luft FC, and Haller H. Hydrogen peroxide, potassium currents, and membrane potential in human endothelial cells. Circulation 13: 1719–1725, 1999.

7. Chappell DC, Varner SE, Nerem RM, Medford RM, and Alexander RW. Oscillatory shear stress stimulates adhesion molecule expression in cultured human endothelium. Circ Res 82: 532–539, 1998.[Abstract/Free Full Text]

8. Chatterjee S, Al-Mehdi A, Levitan I, Stevens T, and Fisher AB. Shear stress increases expression of a KATP channel in rat and bovine pulmonary vascular endothelial cells. Am J Physiol Cell Physiol 285: C959–C967, 2003.[Abstract/Free Full Text]

9. Cooke JP, Rossitch E Jr, Andon NA, Loscalzo J, and Dzau VJ. Flow activates an endothelial potassium channel to release an endogenous nitrovasodilator. J Clin Invest 88: 1663–1671, 1991.[ISI][Medline]

10. Davies PF. Flow-mediated endothelial mechanotransduction. Physiol Rev 75: 519–560, 1995.[Abstract/Free Full Text]

11. Davies PF, Remuzzi A, Gordon ES, Dewey CF Jr, and Gimbrone MA Jr. Turbulent fluid shear stress induces vascular endothelial turnover in vitro. Proc Natl Acad Sci USA 83: 2114–2117, 1986.[Abstract]

12. Garcia-Cardena G, Comander J, Anderson KR, Blackman BR, and Gimbrone MA Jr. Biomechanical activation of vascular endothelium as a determinant of its functional phenotype. Proc Natl Acad Sci USA 98: 4478–4485, 2001.[Abstract/Free Full Text]

13. Gauthier K and Rusch NJ. Rat coronary endothelial cell membrane potential responses during hypertension. Hypertension 37: 66–71, 2001.[Abstract/Free Full Text]

14. Helmlinger G, Berk BC, and Nerem RM. Calcium responses of endothelial cell monolayers subjected to pulsatile and steady laminar flow differ. Am J Physiol Cell Physiol 269: C367–C375, 1995.[Abstract/Free Full Text]

15. Helmlinger G, Geiger RV, Schreck S, and Nerem RM. Effects of pulsatile flow on cultured vascular endothelial cell morphology. J Biomech Eng 113: 123–131, 1991.[ISI][Medline]

16. Hoger JH, Ilyin VI, Forsyth S, and Hoger A. Shear stress regulates the endothelial Kir2.1 ion channel. Proc Natl Acad Sci USA 99: 7780–7785, 2002.[Abstract/Free Full Text]

17. Jacobs ER, Cheliakine C, Gebremedhin D, Birks EK, Davies PF, and Harder DR. Shear activated channels in cell-attached patches of cultured bovine aortic endothelial cells. Pflügers Arch 431: 129–131, 1995.[ISI][Medline]

18. Ku DN, Giddens DP, Zarins CK, and Glagov S. Pulsatile flow and atherosclerosis in the human carotid bifurcation–positive correlation between plaque location and low and oscillating shear stress. Arteriosclerosis 5: 293–302, 1985.[Abstract]

19. Langille BL, Graham JJ, Kim D, and Gotlieb AI. Dynamics of shear-induced redistribution of F-actin in endothelial cells in vivo. Arterioscler Thromb 11: 1814–1820, 1991.[Abstract]

20. Lum RM, Wiley LM, and Barakat AI. Influence of different forms of shear stress on vascular endothelial TGF-{beta}1 mRNA expression. Int J Mol Med 5: 635–641, 2000.[ISI][Medline]

21. Malek AM and Izumo S. Molecular aspects of signal transduction of shear stress in the endothelial cell. J Hypertens 12: 989–999, 1994.[ISI][Medline]

22. Mason MJ, Hussain JF, and Mahaut-Smith MP. A novel role for membrane potential in the modulation of intracellular Ca2+ oscillations in rat megakaryocytes. J Physiol 524: 437–446, 2000.[Abstract/Free Full Text]

23. McCarty MF. Endothelial membrane potential regulates production of both nitric oxide and superoxide–a fundamental determinant of vascular health. Med Hypotheses 53: 277–289, 1999.[CrossRef][ISI][Medline]

24. Moore JE Jr, Xu C, Glagov S, Zarins CK, and Ku DN. Fluid wall shear stress measurements in a model of the human abdominal aorta: oscillatory behavior and relationship to atherosclerosis. Atherosclerosis 110: 225–240, 1994.[ISI][Medline]

25. Nakache M and Gaub HE. Hydrodynamic hyperpolarization of endothelial cells. Proc Natl Acad Sci USA 85: 1841–1843, 1988.[Abstract]

26. Nakao M, Ono K, Fujisawa S, and Iijima T. Mechanical stress-induced Ca2+ entry and Cl current in cultured human aortic endothelial cells. Am J Physiol Cell Physiol 276: C238–C249, 1999.[Abstract/Free Full Text]

27. Nerem RM. Vascular fluid mechanics, the arterial wall, and atherosclerosis. J Biomech Eng 114: 274–282, 1992.[ISI][Medline]

28. Nilius B, Viana F, and Droogmans G. Ion channels in vascular endothelium. Annu Rev Physiol 59: 145–170, 1997.[CrossRef][ISI][Medline]

29. Ohno M, Cooke JP, Dzau VJ, and Gibbons GH. Fluid shear stress induces endothelial TGF-{beta}1 transcription and production: modulation by potassium channel blockade. J Clin Invest 95: 1363–1369, 1995.[ISI][Medline]

30. Ohno M, Gibbons GH, Dzau VJ, and Cooke JP. Shear stress elevates endothelial cGMP: role of a potassium channel and G protein coupling. Circulation 88: 193–197, 1993.[Abstract]

31. Okano M and Yoshida Y. Endothelial cell morphometry of atherosclerotic lesions and flow profiles at aortic bifurcations in cholesterol fed rabbits. J Biomech Eng 114: 301–308, 1992.[ISI][Medline]

32. Olesen SP, Clapham DE, and Davies PF. Hemodynamic shear stress activates a K+ current in vascular endothelial cells. Nature 331: 168–170, 1988.[CrossRef][ISI][Medline]

33. Park K, Jo I, Pak YK, Bae S, Rhim H, Suh S, Park SJ, Zhu MH, So I, and Kim KW. FCCP depolarizes plasma membrane potential by activating proton and Na+ currents in bovine aortic endothelial cells. Pflügers Arch 443: 344–352, 2002.[CrossRef][ISI][Medline]

34. Pohl U, Holtz J, Busse R, and Bassenge E. Crucial role of endothelium in the vasodilator response to increased flow in vivo. Hypertension 8: 37–44, 1986.[Abstract]

35. Qiu WP, Hu Q, Paolocci N, Ziegelstein RC, and Kass DA. Differential effects of pulsatile versus steady flow on coronary endothelial membrane potential. Am J Physiol Heart Circ Physiol 285: H341–H346, 2003.[Abstract/Free Full Text]

36. Romanenko VG, Davies PF, and Levitan I. Dual effect of fluid shear stress on volume-regulated anion current in bovine aortic endothelial cells. Am J Physiol Cell Physiol 282: C708–C718, 2002.[Abstract/Free Full Text]

37. Soghomonians A, Barakat AI, Thirkill TL, Blankenship TN, and Douglas GC. Effect of shear stress on migration and integrin expression in macaque trophoblast cells. Biochim Biophys Acta 1589: 233–246, 2002.[CrossRef][ISI][Medline]

38. Suvatne J, Barakat AI, and O'Donnell ME. Flow-induced expression of endothelial Na-K-Cl cotransport: dependence on K+ and Cl channels. Am J Physiol Cell Physiol 280: C216–C227, 2001.[Abstract/Free Full Text]

39. Traub O and Berk BC. Laminar shear stress–mechanisms by which endothelial cells transduce an atheroprotective force. Arterioscler Thromb Vasc Biol 18: 677–685, 1998.[Abstract/Free Full Text]

40. Wiesner TF, Berk BC, and Nerem RM. A mathematical model of the cytosolic-free calcium response in endothelial cells to fluid shear stress. Proc Natl Acad Sci USA 94: 3726–3731, 1997.[Abstract/Free Full Text]