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
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ABSTRACT |
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ion channels; atherosclerosis; mechanotransduction
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 26 mV in response to shear stresses of 15 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.
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MATERIALS AND METHODS |
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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 26 M 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.
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RESULTS |
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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.
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DISCUSSION |
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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 (12 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 100500 ms, the time constant for activation of the depolarizing current is of the order of 2.55 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-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.
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GRANTS |
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FOOTNOTES |
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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.
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