Mechanosensitive Ca²⁺ oscillations and STOC activation in endothelial cells

^a Department of Internal Medicine, University Hospital Benjamin Franklin, Free University Berlin, 12200 Berlin, Germany

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Activation of ion channels and the increase in intracellular Ca²⁺ concentration [Ca²⁺]_i play a key role in endothelial responses to hemodynamic forces and subsequent vasoregulation. In bovine aortic endothelial cells subjected to shear stress in a parallel flow chamber, we demonstrate shear stress activation of hyperpolarizing K⁺ currents that occur simultaneously with oscillating increases of [Ca²⁺]_i. Oscillating K⁺ currents, also known as spontaneous transient outward currents (STOC), were regulated in frequency and amplitude by the rate of shear stress in a range from 5 to 18 dyn/cm². Activation of STOC depended on Ca²⁺ influx; current depended on the extracellular Ca²⁺ concentration and was blocked by 50 µM Gd³⁺. Emptying of Ca²⁺ stores by BHQ abolished current responses to shear stress. STOC activation was significantly reduced by cell dialysis with ryanodine (20 µM), but not heparin (200 µg/ml). Shear stress-induced STOC activation was also observed in the intact endothelium. The endothelial response to shear stress involves oscillating [Ca²⁺]_i increase and STOC activation, which depend on Ca²⁺ influx-induced Ca²⁺ release from ryanodine-sensitive stores, demonstrating a new signaling pathway in endothelial mechanotransduction.—Hoyer, J., Köhler, R., Distler, A. Mechanosensitive Ca²⁺ oscillations and STOC activation in endothelial cells. FASEB J. 12, 359–366 (1998)

Key Words: shear stress • spontaneous transient outward currents • intracellular calcium • ryanodine • bovine aortic endothelial cells

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
MODULATION OF THE free intracellular Ca²⁺ concentration ([Ca²⁺]_i)² is essential for intracellular signal transduction in nonexcitable cells. In endothelial cells, changes in [Ca²⁺]_i play a pivotal role in the synthesis and release of vasoactive substances such as nitric oxide or prostacyclin. The release of endothelial growth factors and cytokines is also regulated by [Ca²⁺]_i. The increase in [Ca²⁺]_i after stimulation of endothelial cells by humoral factors such as acetylcholine or histamine is due to a release of Ca²⁺ from intracellular stores and an influx of extracellular Ca²⁺ ions through Ca²⁺-permeable ion channels (for a review, see ref 1). The agonist-induced release of Ca²⁺ from intracellular stores is regulated primarily by the intracellular 1,4,5 inositoltriphosphate (IP₃). Recently, ryanodine-sensitive Ca²⁺-stores have been identified in endothelial cells and may also contribute to agonist-induced increases in [Ca²⁺]_i (2).

In nonexcitable cells, intracellular Ca²⁺ signals are often organized in [Ca²⁺]_i oscillations with complex temporal and spatial patterns (3), and are due to a Ca²⁺ release from IP₃-dependent Ca²⁺ stores. In endothelial cells, Ca²⁺ oscillations have been observed after agonist stimulation (4). Spontaneous transient outward currents (STOC) originating from activation of Ca²⁺-dependent K⁺ channels (5) occur in oscillating patterns. In fact, presumably STOC directly reflect oscillating Ca²⁺ release from intracellular stores and can be used to monitor intracellular Ca²⁺ transients (5, 6).

The activation of endothelial cells by hemodynamic forces leads to an increase in [Ca²⁺]_i (7–9), presumably via the release from intracellular stores and Ca²⁺ influx into the cell. Ca²⁺ influx involves Ca²⁺-permeable, mechanosensitive ion channels (7, 10–13), but the regulation of intracellular Ca²⁺ release under mechanical stimulation by hemodynamic forces is incompletely understood. Recently, it has been demonstrated that shear stress induces oscillatory increases in [Ca²⁺]_i (14). In the present study, we characterized the regulation of endothelal Ca²⁺ homeostasis and ion channel activation by shear stress. We demonstrate that shear stress-induced [Ca²⁺]_i oscillations and STOC activation depend on Ca²⁺ influx and Ca²⁺ release from ryanodine-sensitive stores.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Bovine aortic endothelial cells (BAEC) were isolated from bovine aorta as described previously (11). Briefly, bovine aorta was incubated in phosphate-buffered saline containing 0.25% trypsin for 20 min at 37°C. Endothelial cells were harvested by gentle scraping of the luminal surface. Cells were transferred into Medium 199 containing 20% fetal calf serum, 2.2 g/l NaHCO₃, 1 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin and cultured on uncoated culture dishes at 37°C in 5% CO₂. For experiments, cells were trypsinated and transferred to the parallel flow chamber. The cells were allowed to settle down for 1 h, yielding single cells and cell clusters of two to five cells suitable for whole-cell recordings and [Ca²⁺]_i measurements.

A parallel flow chamber was modified for patch clamp experiments as described (15). Briefly, the flow channel consisted of a polycarbonate top and a coverslip as the bottom, separated by a thin gasket. The channel dimensions were given by the thickness of specific thin plastic gaskets. The patch pipette was entered from the end of the channel. Shear stress in the flow channel was calculated by the formula: = 6Q /h²b (15), where is the shear stress, Q the flow rate, the viscosity, h the channel height, and b the channel width. Pressure was held constant by a flow loop system. Laminar flow in the flow chamber was confirmed by calculating the Reynolds number (Re) with the equation: Re = Q /pb, where p is the density of the medium. A maximal Re of 281 indicated that flow was laminar.

If not otherwise stated, patch clamp experiments were performed in single BAEC in slow whole-cell recording mode with a nystatin-perforated patch. Slow whole-cell patch clamp experiments were carried out as described (16). Whole-cell currents were filtered at 200 Hz and recorded at a sample time of 5 ms with a HEKA (Heidenheim, Germany) EPC-9 patch clamp amplifier. Computer-generated voltage ramps and pulses were programmed using EPC-9 software. Data were stored on removable cartridges until analysis. Patch pipettes were pulled from borosilicate glass capillaries with wall thickness of 0.3 mm and had a tip resistance of 4–5 G in symmetrical KCl solutions. The seal resistance ranged from 2 to 4 G. Experiments were performed at 35°C.

The pipette solution for slow whole-cell recordings contained (in mmol/l): 140 KCl, 0.1 CaCl₂, 1 MgCl₂, 10 HEPES, and 150–200 µg/ml nystatin (pH 7.2). A few experiments were performed in fast whole-cell recording mode with a pipette solution containing (in mmol/l): 140 KCl, 0.0028 CaCl₂, 0.23 MgCl₂, 0.020 EGTA, 10 HEPES (pH 7.2). If not otherwise stated, the perfusion solution contained (in mmol/l): 118 NaCl, 1 NaH₂PO₄, 5 KCl, 1.4 CaCl₂, 1 MgCl₂, 20 HEPES, 5.5 D-Glucose. ATP was not added to the perfusion solution especially to avoid ATP-dependent Ca²⁺ mobilization, as described by Ando et al. (17).

[Ca²⁺]_i was measured by dual wavelength fluorescence in single BAEC loaded with the Ca²⁺-sensitive indicator fura-2 (18). Briefly, for fluorescence measurements, BAEC were loaded with fura-2-acetoxymethylester (5 µmol/l) for 15 min at 37°C. As a measure of [Ca²⁺]_i, the fluorescence emission ratio at 340/380 nm excitation wave length was calculated after subtraction of the noise signal and auto fluorescence. Analysis and calibration of fura-2 measurements were conducted as described (18) at nominally zero Ca²⁺ in the presence of 10 mM EGTA and at an excess of Ca²⁺ (5 mmol/l) for minimum and maximum fluorescence values, respectively. A quantitative estimation of the changes in [Ca²⁺]_i was made by using the equation: [Ca²⁺]_i = K_d [(R-R_min)/(R_max-R)] , where K_d is the dissociation constant of fura-2 for Ca²⁺ of 224 nmol/l, R is the measured fura-2 fluorescence ratio, and R_min and R_max are the 500–530 nm fura-2 fluorescence ratios at nominally zero Ca²⁺ and saturated Ca²⁺ concentrations. is the ratio between the fluorescence emission at 380 nm excitation in the virtual absence of Ca²⁺ and emission in the presence of an excess amount of Ca²⁺.

If not stated otherwise, data are given as mean ±SEM (n), where n refers to the number of experiments. For whole-cell current analysis, the area under curve (AUC) was determined by calculating the area of peaks by the trapezoid rule. A Mann Whitney U/Wilcoxon rank sum test was used to compare mean values within an experimental series. A P value of < 0.05 was accepted to indicate statistical significance.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
A transient increase in [Ca²⁺]_i and activation of STOC were observed in BAEC subjected to shear stress in a parallel flow chamber ( Fig. 1 A, B). [Ca²⁺] peaks occurred within 5–30 s after initiation of flow and had a duration of 5–15 s. [Ca²⁺]_i oscillations were inactivated within 15–30 s after cessation of flow ( Fig. 1A). The frequency of [Ca²⁺]_i oscillations was about 1–2 peak/min at a shear stress rate of 18 dyn/cm². The [Ca²⁺]_i increased from a basal concentration of 40 ±7 nmol/l (n=8) to a peak concentration of 350 ±49 nmol/l (n=8). The frequency and shape of Ca²⁺ oscillations closely resembled the flow-induced Ca²⁺ oscillations observed previously in single cultured endothelial cells (14).

View larger version (47K): [in this window] [in a new window]   Figure 1. Time course of shear stress-induced [Ca2+]i (A) and STOC activation (B) in two different single BAEC at shear stress rates of 18 dyn/cm2. Start and stop of shear stress are depicted by arrows, respectively. In a fura-II loaded BAEC, the increase in [Ca2+]i is denoted as a change in fura-II fluorescence ratio. Whole-cell currents were recorded at 0 mV holding potential with a KCl pipette solution and NaCl perfusion medium. Cell capacity was 14.6 pF. Simultaneous measurement of fura-II fluorescence (upper trace) and whole-cell currents (lower trace) in a single BAEC demonstrating simultaneous activation of [Ca2+]i and STOC, with similar shapes and duration of respective peaks at a fluid shear stress (FSS) of 5 and 9 dyn/cm2 (C).

STOC were activated directly after the initial rise in [Ca²⁺]_i and were observed simultaneously at the same frequency as the [Ca²⁺]_i oscillations ( Fig. 1B), since activation of STOC directly reflected the [Ca²⁺]_i oscillations. In a few combined fluorescence and patch clamp experiments (n=13), [Ca²⁺]_i and STOC were measured simultaneously, proving that the increase in [Ca²⁺]_i was followed directly by STOC activation ( Fig. 1C). In this series of experiments, stable recordings were performed over a period of 250–600 s at 5 (n=3), 9 (n=6), and 18 (n=4) dyn/cm²; between one and six peaks were observed, depending on the shear stress rate. All STOC and Ca²⁺ peaks were temporally coordinated. However, very small Ca²⁺ signals failed to activate STOC, as shown in Fig. 1C. A simultaneous activation of STOC by increases in [Ca²⁺]_i was reported for smooth muscle cells (5) and endothelial cells under agonist stimulation (6). The simultaneous activation of oscillating [Ca²⁺]_i and STOC by mechanical forces has so far not been observed. STOC characteristics are different from those reported for endothelial cells under agonist stimulation. Shear stress-induced STOC had a lower frequency, higher current amplitude, and longer spike duration than those reported for rabbit aortic endothelial cells in response to activation by caffeine (6). In other studies, an activation of K⁺ channels by shear stress was observed (19, 20). In these studies, however, only ⁸⁶Rb⁺ efflux measurements were performed; no patch clamp experiments were conducted to identify the presence Ca²⁺-dependent K⁺ channels.

The activation of STOC was completely blocked by addition to the perfusion medium of charybdotoxin (10 nmol/l), a specific blocker of Ca²⁺-dependent K⁺ channels. The activation of STOC led to a concomitant hyperpolarization of BAEC. In current clamp recordings, oscillating hyperpolarizations ranging from -30 to -65 mV were observed. Cell hyperpolarization facilitates Ca²⁺ influx by providing the electrical driving force and is an important regulator of endothelial function (21).

The activity of STOC depended on the magnitude of shear stress (frequency) and maximal current amplitude of STOC. As shown in Fig. 2A, maximal current amplitude increased from 6.6 pA/pF at 5 dyn/cm² to 16.5 pA/pF at 18 dyn/cm². The frequency of current peaks was increased at 18 dyn/cm² compared to peak frequency at 5 dyn/cm². The integrated total current of STOC over time, measured as the AUC of the whole-cell recording, increased accordingly from 69.4 pA x pF^-1 x s at 5 dyn/cm² to 241.2 pA x pF^-1 x s at 18 dyn/cm².

View larger version (147K): [in this window] [in a new window]   Figure 2. Effect of different shear stress rates on STOC activation. In representative experiments, shear stress of 5 dyn/cm2 induced STOC with lower frequency and current amplitude compared to 18 dyn/cm2 (A). Holding potential was 0 mV. Start of shear stress is depicted by arrows. Results from experiments performed at 5 dyn/cm2 (n=7), 9 dyn/cm2 (n=11), and 18 dyn/cm2 (n=13) show an increase in STOC frequency (B), maximal current amplitude (C), and total shear stress-induced current (D). Currents were standardized to cell capacity. Total shear stress-induced currents over time of single experiments were determined by computer-assisted calculation of peak areas (AUC) by the use of a trapezoid rule. Dependency of Ca2+ peak frequency on shear stress of 5 (n=8), 9 (n=13), and 18 (n=8) dyn/cm2 (E). *P<0.05, **P<0.01.

In a series of 51 patch clamp experiments, whole-cell current recordings were performed at 5 or 9 or 18 dyn/cm² over a period of 300 s, and currents were standardized to cell capacity. At 5 dyn/cm² about 50%, and at 9 dyn/cm² and at 18 dyn/cm² approximately 95% of the investigated cells, respectively, displayed a current response to shear stress. STOC frequency of responding cells increased from 0.57 ±0.13 peaks/min at 5 dyn/cm² (n=7) to 0.78 ±0.13 peaks/min at 9 dyn/cm² (n=13) and 1.09 ±0.11 peaks/min at 18 dyn/cm² (n=17; Fig. 2B). Also, the maximum current amplitude was significantly increased at 9 dyn/cm² (6.6±1.0 pA/pF; n=19) and 18 dyn/cm² (9.8±2.4 pA/pF; n=22) compared to 5 dyn/cm² (3.3±0.8 pA/pF; n=10), with no significant difference in maximum currents at 9 and 18 dyn/cm² ( Fig. 2C). Accordingly, the AUC of the whole-cell current was increased from 33 ±11 pA x pF^-1 x s (n=7) at 5 dyn/cm² to 124 ± 26 pA x pF^-1 x s (n=13) at 9 dyn/cm² and 225 ± 71 pA x pF^-1 x s (n=17) at 18 dyn/cm² ( Fig. 2D).

Corresponding to shear stress-dependent STOC frequency, Ca²⁺ peak frequency was also significantly higher at 18 dyn/cm² (n=8) compared to 9 (n=13) or 5 (n=8) dyn/cm² ( Fig. 2E). In general, [Ca²⁺]_i oscillations can be characterized as baseline or sinusoidal spikes. Baseline spikes are regulated by increases in the frequency of spikes; changes of spike amplitude also are observed only at low agonist concentrations. In contrast, sinusoidal spikes show an amplitude modulation in response to changes in agonist concentration. In the present study, the [Ca²⁺]_i oscillations are of the baseline type as judged from spike shape and the low frequency of spikes. Frequency of STOC was correspondingly low, and changes in shear stress rate resulted in modulations of STOC frequency. In response to increases from medium (9 dyn/cm²) to high shear stress (18 dyn/cm²), STOC frequency was increased but current amplitude was not significantly different. STOC current frequency and amplitude were modulated only at low shear stress.

In addition to modulation by the shear stress rate, STOC activation also depended on extracellular Ca²⁺ concentration [Ca²⁺]_o. In a series of experiments under high shear stress (18 dyn/cm²), no shear stress-induced activation of STOC was detectable at very low [Ca²⁺]_o (0.02 µmol/l). At 0.17 µmol/l [Ca²⁺]_o, low frequency of STOC and a low integrated total current were observed ( Fig. 3 A, B), whereas current amplitude was comparable to amplitudes measured at normal [Ca²⁺]_o. At medium (1.5 µmol/l) and high (1.4 mmol/l) [Ca²⁺]_o, frequency of STOC and total integrated current increased significantly ( Fig. 3A, B). This dependence of STOC activation on extracellular [Ca²⁺]_o indicates that an influx of extracellular Ca²⁺ is needed for shear stress-induced [Ca²⁺]_i oscillations. A Ca²⁺-dependent endothelial response to shear stress or stretch has been shown in studies performed on endothelial cells from human umbilical veins (11, 22) and BAEC (23). In contrast, other studies performed with BAEC showed no dependence of flow-induced increase in [Ca²⁺]_i on extracellular Ca²⁺ (24). However, these studies did not observe [Ca²⁺]_i oscillations; the experiments were performed on highly passaged BAEC, which may have undergone a down-regulation of cation channels responsible for Ca²⁺ influx. A transient increase has been observed to be induced by shear stress in BAEC (8). Ca²⁺ oscillations were observed in the presence of ATP (17). To avoid such an ATP activation of purinergic receptors and associated Ca²⁺ signaling, the present study was conducted in the absence of ATP. A study reporting [Ca²⁺]_i oscillations in response to steady and pulsatile shear stress proposes that the oscillations require a Ca²⁺ influx, according to experiments using La³⁺ to block Ca²⁺ influx pathways (14). In contrast, other studies failed to demonstrate significant shear stress-induced Ca²⁺ mobilization in the absence of additional agonists. This may be due to the use of highly passaged endothelial cells in these studies (25, 26).

View larger version (63K): [in this window] [in a new window]   Figure 3. Dependence of shear stress STOC activation on extracellular [Ca2+] in single BAEC. Experiments were performed at a shear stress rate of 18 dyn/cm2. Compared to STOC measured at extracellular [Ca2+] of 1.4 mM (n=18), STOC frequency (A) and total integrated currents measured as AUC (B) were significantly lower at extracellular [Ca2+] of 1.5µM (n=9) and 0.17 µM (n=6). *P<0.05, **P<0.01.

The Ca²⁺ entry pathway is not well characterized in endothelial cells. Ca²⁺-permeable, mechanosensitive ion channels and store depletion-activated Ca²⁺ entry pathways via small cation channels like trpl or crac channels may be involved, as described for endothelial cells (27). In experiments (n=5) using gadolinium (Gd³⁺), a trivalent lanthanoid and blocker of Ca²⁺-permeable cation channels, activation of STOC was completely blocked after addition of Gd³⁺ to the perfusion medium ( Fig. 4). This effect was not due to emptying of intracellular Ca²⁺ stores by blocking capacitative Ca²⁺ entry, indicated in these experiments by a large increase in [Ca²⁺]_i after addition of ATP (10 µmol/l). Therefore, we tested whether the activation of STOC could also be blocked by econazole, another blocker of capacitative Ca²⁺ entry (28). The addition of econazole (10 µmol/l) did not show any significant effect on shear stress-induced activation of STOC (n=5). However, more specific blockers for mechanosensitive ion channels and capacitative currents need to be used in order to identify the nature the of Ca²⁺ influx pathway under shear stress conditions.

View larger version (11K): [in this window] [in a new window]   Figure 4. Effect of the addition of Gadolinium (Gd3+) to shear stress-induced STOC at 18 dyn/cm2. Addition of Gd3+ to flow medium completely blocked within 3 min. STOC activation by ATP at the end of the experiment indicates that Gd3+ itself did not induce emptying of intracellular Ca3+ stores. Experiment was performed at 37°C and at a membrane potential of 0 mV. Cell capacity was 13.2 pF.

In a series of experiments (n=6), emptying of intracellular stores of BAEC was induced by preincubation with butylhydroquinon (BHQ), a blocker of Ca²⁺ reuptake into intracellular Ca²⁺ stores. After pretreatment with BHQ for 10 min, no shear stress-induced Ca²⁺ oscillations or STOC activation were observed, indicating the necessity of Ca²⁺ release from intracellular stores in the endothelial response to shear stress.

Agonist-induced increases in [Ca²⁺]_i are mediated by increases in intracellular IP₃ concentration and release from IP₃-sensitive stores. Also, mechanical forces have been shown to generate IP₃ via activation of phospholipase C (29). To test whether the phosphoinositol pathway and Ca²⁺ release from IP₃-sensitive stores are involved in shear stress-induced STOC activation, we performed experiments in the presence of neomycin, an inhibitor of phospholipase C (29). Preincubation of BAEC with 1 mM neomycin for 15 min failed to inhibit the activation of STOC by a shear stress of 18 dyn/cm² (n=5).

An important feature of the patch clamp technique is the opportunity to apply impermeable agents directly to their intracellular site of action by cell dialysis. We performed whole-cell recordings with pipette solutions containing heparin, a specific blocker of IP₃-sensitive Ca²⁺ stores that is impermeable to cell membranes. Internal dialysis of BAEC with a patch pipette solution containing heparin (200 µg/ml) did not affect shear stress-induced STOC activation (n=6; Fig. 5 A, B). The lack of an inhibitory effect of neomycin and heparin suggests that in contrast to agonist stimulation, IP₃-sensitive Ca²⁺ stores presumably are not involved in shear stress-induced [Ca²⁺]_i increases and subsequent STOC activation. Shear stress has been reported to increase the generation of intracellular IP₃ itself (29). However, this increase of IP₃ was observed only in the presence of agonists (ATP); simultaneous Ca²⁺ measurements have not been performed. Furthermore, the increase peaked after 30 s and remained elevated for minutes without any oscillating characteristics, and may point to a different endothelial response mechanism.

View larger version (32K): [in this window] [in a new window]   Figure 5. Original fast whole-cell recording in single BAEC. Cell dialysis with KCl pipette solution containing heparin (200 µg/ml) did not suppress STOC activation by shear stress (18 dyn/cm2). In contrast, cell dialysis with KCl pipette solution containing ryanodine (20 µM) almost completely blocked shear stress-induced STOC (A). Cell capacities were 18 and 17.5 pF, respectively. In a series of experiments, the effect of heparin (n=4) and ryanodine (n=8) was compared to control (n=7) dialyzed only with KCl solution (B). Data are given as mean ±SE. *P<0.05.

In contrast, in the presence of ryanodine, known to block Ca²⁺-induced Ca²⁺ release at concentrations in the micromolar range, STOC activation was significantly reduced. After cell dialysis with a pipette solution containing ryanodine (20 µmol/l), the total integrated current under 18 dyn/cm² was reduced to almost 20% of control currents due to a reduction in frequency as well as in current amplitude of STOC ( Fig. 5A, B). These experiments show that the increase in [Ca²⁺]_i underlying STOC activation was due mainly to the release of Ca²⁺ from ryanodine-sensitive Ca²⁺ stores and may represent a Ca²⁺-induced Ca²⁺ release.

In a series of experiments (n=4), thin tissue slices of bovine aorta with an intact endothelium were subjected to shear stress in the parallel flow chamber. To detect single-cell current responses to shear stress, endothelial cells were electrically uncoupled by preincubation with heptanol (3 mM), as described previously (30). At a shear stress rate of 9 dyn/cm², STOC could be resolved as shown in Fig. 6A, with current characteristics similar to those demonstrated in isolated BAEC. In addition, shear stress-induced STOC were observed in subconfluent BAEC (n=5) at 3 days postseeding. Due to the electrical coupling of subconfluent cells, shear stress-induced STOC showed superimposed current peaks at a higher frequency than in single cells ( Fig. 6B).

View larger version (41K): [in this window] [in a new window]   Figure 6. Representative whole-cell current recording in intact endothelium of bovine aorta (A). Cells were electrically uncoupled by addition of heptanol (3 mM) to NaCl bath medium. Shear stress was increased from 0.5 to 9 dyn/cm2, as indicated by an arrow. Shear stress induced STOC activation similar to that in isolated BAEC. B) Shear stress-induced STOC activation in a subconfluent monolayer of approximately 25 BAEC as determined by nuclei count. At a shear stress of 18 dyn/cm2, STOC from electrically coupled cells were superimposed and observed at higher frequency than in single-cell recordings. Whole-cell currents (A, B) were recorded at 0 mV holding potential with a KCl pipette solution and NaCl perfusion medium.

This study has demonstrated that intracellular Ca²⁺ oscillations and STOC activation are implicated in endothelial mechanotransduction, and a model of cellular responses to mechanical stimulation is provided. Ca²⁺ oscillations and hyperpolarizing STOC depended on Ca²⁺ influx and Ca²⁺-induced Ca²⁺ release from ryanodine-sensitive stores, and presumably not from IP₃-dependent stores. The observation of oscillating STOC in the endothelium of intact vascular tissue slices demonstrates that these endothelial responses are also present in intact endothelium and are not due to cell culture artifacts. These results show that the endothelial response to shear stress involves signaling pathways different from agonist stimulation of the endothelium. The oscillatory pattern of [Ca²⁺]_i increase and cell hyperpolarization represents a mechanism that preserves endothelial responsiveness to continuous stimulation by hemodynamic forces.

	ACKNOWLEDGMENTS

 
This study was supported by a grant from the Deutsche Forschungsgemeinschaft (Ho-1103/2–2).

	FOOTNOTES

 
¹ Correspondence: Correspondence.

² Abbreviations: BAEC, bovine aortic endothelial cells; [Ca²⁺]_i, intracellular calcium concentration; [Ca²⁺]_o, extracellular calcium concentration; BHQ, 2,5 di-tert-butylhydroquinon; IP₃, inositol 5'-triphosphate; AUC, area under curve; STOC, spontaneous transient outward currents.

Received for publication November 3, 1997. Accepted for publication November 21, 1997.

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 

1. Himmel, H. M., Whorton, A. R., and Strauss, H. C. (1993) Intracellular calcium, currents, and stimulus-response coupling in endothelial cells. Hypertension 21, 112–127[Abstract/Free Full Text]
2. Ziegelstein, R. C., Spurgeon, H. A., Pili, R., Passaniti, A., Cheng, L., Corda, S., Lakatta, E. G., and Capogrossi, M. C. (1994) A functional ryanodine-sensitive intracellular Ca²⁺ store is present in vascular endothelial cells. Circ. Res. 74, 151–156[Abstract/Free Full Text]
3. Clapham, D. E. (1995) Calcium signaling. Cell 80, 259–268[Medline]
4. Jacob, R. (1991) Calcium oscillations in endothelial cells. Cell Calcium 12, 127–134[Medline]
5. Nelson, M. T., Cheng, H., Rubart, M., Santana, L. F., Bonev, D., Knot, H. J., and Lederer, W. J. (1995) Relaxation of arterial smooth muscle by calcium sparks. Science 270, 633–637[Abstract/Free Full Text]
6. Rusko, J., Van Slooten, G., and Adams, D. J. (1995) Caffeine-evoked, calcium-sensitive membrane currents in rabbit aortic endothelial cells. Br. J. Pharmacol. 115, 133–141[Medline]
7. Nilius, B., Viana, F., and Droogmans, G. (1997) Ion channels in vascular endothelium. Annu. Rev. Physiol. 59, 145–170[Medline]
8. Shen, J., Luscinskas, Conolly, A., Dewey, C. F., and Gimbrone, M. A. (1992) Fluid shear stress modulates cytosolic free calcium in vascular endothelial cells. Am. J. Physiol. 262, C384–C390[Abstract/Free Full Text]
9. Falcone, J. C., Kuo, L., and Meininger, G. A. (1993) Endothelial cell calcium increases during flow-induced dilation in isolated arterioles. Am. J. Physiol. 264, H653–H659[Abstract/Free Full Text]
10. Lansman, J. B., Hallam, T. J., and Rink, T. J. (1987) Single stretch-activated ion channels in vascular endothelial cells as mechanotransducers. Nature (London) 325, 811–813.[Medline]
11. Naruse, K., and Sokabe, M. (1993) Involvement of stretch-activated ion channel in Ca²⁺ mobilization to mechanical stretch in endothelial cells. Am. J. Physiol. 264, C1037–C1044[Abstract/Free Full Text]
12. Hoyer, J., Distler, A., Haase, W., and Gögelein, H. (1994) Ca²⁺-influx through stretch-activated cation channel activates maxi K⁺ channels in porcine endocardial endothelium. Proc. Natl. Acad. Sci. USA 91, 2367–2371[Abstract/Free Full Text]
13. Hoyer, J., Köhler, R., and Distler, A. (1996) Up-regulation of pressure-activated Ca²⁺-permeable cation channel in intact vascular endothelium of hypertensive rats. Proc. Natl. Acad. Sci. USA 93, 11253–11258[Abstract/Free Full Text]
14. Helmlinger, G., Berk, B. C., and Nerem, R. M. (1996) Pulsatile and steady flow-induced calcium oscillations in single cultured endothelial cells. J. Vasc. Res. 33, 360–369[Medline]
15. Frangos, J. A., McIntire, L. V., and Eskin, S. G. (1988) Shear stress induced stimulation of mammalian cell metabolism. Biotech. Bioeng. 32,1053–1060
16. Horn, R., and Marty, A. (1988) Muscarinic activation of ionic currents measured by a new whole-cell recording method. J. Gen. Physiol. 92, 145–159[Abstract/Free Full Text]
17. Ando, J., Komatsuda, T., and Kamiya, A. (1988) Cytoplasmic Ca²⁺ response to fluid shear stress in cultured vascular endothelial cells. In Vitro Cell Dev. Biol. 24, 871–877[Medline]
18. Nitschke, R., Fröbe, U., and Greger, R. (1991) Antidiuretic hormone acts via V1 receptors on intracellular calcium in isolated perfused rabbit cortical thick ascending limb. Pfluegers Arch. 417, 622–632[Medline]
19. Alevriadou, B. R., Eskin, S. G., McIntire, L. V., and Schilling, W. P. (1993) Effect of shear stress on ⁸⁶Rb⁺ efflux from calf pulmonary artery endothelial cells. Ann. Biomed. Eng. 21, 1–7[Medline]
20. O'Neill, W. C. (1995) Flow-mediated NO release from endothelial cells is independent of K⁺ channel activation or intracellular Ca²⁺. Am. J. Physiol. 269, C863–C869[Abstract/Free Full Text]
21. Lückhoff, A., and Busse, R. (1990) Ca²⁺ influx into endothelial cells and formation of endothelium-derived relaxing factor is controlled by the membrane potential. Pfluegers Arch. 416, 305–311[Medline]
22. Schwarz, G., Callewaert, G., Droogmans, G., and Nilius, B. (1992) Shear stress-induced calcium transients in endothelial cells from human umbilical cord veins. J. Physiol. (London) 458, 527–538[Abstract/Free Full Text]
23. Diamond, S. L., Sachs, F., and Sigurdson, W. J. (1994) Mechanically induced calcium mobilization in cultured endothelial cells is dependent on actin and phospholipase. Arterioscler. Thromb. 14, 2000–2006[Abstract/Free Full Text]
24. Geiger, R. V., Berk, B. C., Alexander, R. W., and Nerem, R. M. (1992) Flow-induced calcium transients in single endothelial cells, spatial and temporal analysis. Am. J. Physiol. 262, C1411–C1417[Abstract/Free Full Text]
25. Mo, M., Eskin, S. G., and Schilling, W. P. (1991) Flow-induced changes in Ca²⁺ signaling of vascular endothelial cells, effect of shear stress and ATP. Am. J. Physiol. 260, H1698–H1707[Abstract/Free Full Text]
26. Dull, R. O., and Davies, P. F. (1991) Flow modulation of agonist (ATP)–response (Ca²⁺) coupling in vascular endothelial cells. Am. J. Physiol. 261, H149–H154[Abstract/Free Full Text]
27. Vaca, L., and Kunze, D. L. (1995) Depletion of intracellular Ca²⁺ stores activates a Ca²⁺-selective channel in vascular endothelium. Am. J. Physiol. 267, C920–C925
28. Franzius, D., Hoth, M., and Penner, R. (1994) Non-specific effects of calcium entry antagonists in mast cells. Pflueg. Arch. Eur. J. Physiol. 428, 433–8[Medline]
29. Nollert, M. U., Eskin, S. G., and McIntire, L. V. (1990) Shear stress increases inositol triphosphate levels in human endothelial cells. Biochem. Biophys. Res. Commun. 170, 281–287[Medline]
30. Ying, X., Minimiya, Y., Fu, C., and Battacharaya, J. (1997) Ca²⁺ waves in lung capillary endothelium. Circ. Res. 79, 898–908[Abstract/Free Full Text]