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(Circulation Research. 2000;87:385.)
© 2000 American Heart Association, Inc.

Cellular Biology

Fluid Shear Stress Activates Ca²⁺ Influx Into Human Endothelial Cells via P2X4 Purinoceptors

Kimiko Yamamoto, Risa Korenaga, Akira Kamiya, Joji Ando

From the Department of Biomedical Engineering (K.Y., R.K., J.A.), Graduate School of Medicine, University of Tokyo, Tokyo, Japan; and Interdisciplinary Science Center (A.K.), Nihon University, Tokyo, Japan.

Correspondence to Dr Joji Ando, Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 Japan. E-mail joji{at}m.u-tokyo.ac.jp

	Abstract

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Abstract—Ca²⁺ signaling plays an important role in endothelial cell (EC) responses to shear stress generated by blood flow. Our previous studies demonstrated that bovine fetal aortic ECs showed a shear stress–dependent Ca²⁺ influx when exposed to flow in the presence of extracellular ATP. However, the molecular mechanisms of this process, including the ion channels responsible for the Ca²⁺ response, have not been clarified. Here, we demonstrate that P2X4 purinoceptors, a subtype of ATP-operated cation channels, are involved in the shear stress–mediated Ca²⁺ influx. Human umbilical vein ECs loaded with the Ca²⁺ indicator Indo-1/AM were exposed to laminar flow of Hanks’ balanced salt solution at various concentrations of ATP, and changes in [Ca²⁺]_i were monitored with confocal laser scanning microscopy. A stepwise increase in shear stress elicited a corresponding stepwise increase in [Ca²⁺]_i at 250 nmol/L ATP. The shear stress–dependent increase in [Ca²⁺]_i was not affected by phospholipase C inhibitor (U-73122) but disappeared after the chelation of extracellular Ca²⁺ with EGTA, indicating that the Ca²⁺ increase was due to Ca²⁺ influx. Antisense oligonucleotides designed to knockout P2X4 expression abolished the shear stress–dependent Ca²⁺ influx seen at 250 nmol/L ATP in human umbilical vein ECs. Human embryonic kidney 293 cells showed no Ca²⁺ response to flow at 2 µmol/L ATP, but when transfected with P2X4 cDNA, they began to express P2X4 purinoceptors and to show shear stress–dependent Ca²⁺ influx. P2X4 purinoceptors may have a "shear-transducer" property through which shear stress is perceived directly or indirectly and transmitted into the cell interior via Ca²⁺ signaling.

Key Words: endothelium • shear stress • purinergic receptors/purinoceptors • Ca²⁺ • adenosine triphosphate

	Introduction

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Endothelial cells (ECs) covering the inner surface of blood vessels are constantly exposed to shear stress generated by blood flow. Shear stress, a mechanical stimulus, modulates not only the morphology but also various functions of ECs.¹ For example, shear stress increases the production of vasodilating substances such as NO, prostacyclin, C-type natriuretic peptide, and adrenomedulin and growth factors, including platelet-derived growth factor and transforming growth factor-ß₁. Shear stress also alters the expression of antithrombotic molecules on the cell surface, such as thrombomodulin and heparan sulfate. Shear stress affects gene expression in ECs. It has become apparent that shear stress regulates gene expression at the transcriptional or posttranscriptional level and that some transcription factors and their binding sites on gene promoters, shear stress–responsive elements, play an important role in transcriptional regulation.^{2 3}

A number of recent studies have shown that shear stress activates many signal transduction pathways in which a variety of molecules, including Ca²⁺, K⁺ channels, GTP-binding protein, adenylate cyclase, integrin, and protein kinases such as protein kinase C, mitogen-activated protein kinase, and tyrosine kinase, are involved.¹ It remains unclear, however, which pathway is primary or secondary or whether a specific feature of shear stress is the simultaneous activation of many signal transduction pathways. Ca²⁺ signaling has been thought to play a role in shear stress signal transduction. The application of flow to ECs causes an increase in [Ca²⁺]_i in an ATP-dependent^{4 5 6} or -independent manner.^{7 8} Our previous studies demonstrated that at a certain level of extracellular ATP, cultured bovine fetal aortic ECs showed an increase in [Ca²⁺]_i in response to flow stimulation and that this increase in [Ca²⁺]_i was shear stress, rather than shear rate, dependent.⁹ The increase in [Ca²⁺]_i was thought to be mainly due to the influx of extracellular Ca²⁺ across the cell membrane, because the Ca²⁺ response disappeared after the removal of extracellular Ca²⁺. These findings suggest the presence of Ca²⁺-permeable channels responsible for the shear stress–dependent Ca²⁺ influx in ECs.

To date, it has been indicated that ECs have a variety of Ca²⁺-permeable channels, including receptor-mediated channels, Ca²⁺-leak channels, stretch-activated cation channels, and channels involved in internal Na⁺-dependent Ca²⁺ entry and Ca²⁺ release–activated Ca²⁺ entry.¹⁰ Most of these channels, however, have not yet been characterized at the molecular level, with the exception of some receptor-mediated channels, including acetylcholine receptors and 5-hydroxytryptamine receptors. Recently, we found that P2X4 purinoceptors, a subtype of ATP-operated cation channels, are predominantly expressed in ECs cultured from human umbilical vein, pulmonary artery, aorta, and skin microvessels and are involved in the Ca²⁺ response to ATP.¹¹ Thus, in the present study, we investigated the role of P2X4 purinoceptors in flow-induced Ca²⁺ signaling in human umbilical vein ECs (HUVECs). The effect of antisense oligonucleotides against P2X4 on flow-induced Ca²⁺ response was examined, and we established human embryonic kidney (HEK) 293 cell lines that stably express P2X4 purinoceptors and analyzed their Ca²⁺ responses to flow.

	Materials and Methods

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Cell Culture
HUVECs were cultured in M199 supplemented with 15% FBS, 2 mmol/L L-glutamine, 50 µg/mL heparin, and 30 µg/mL EC growth factor as previously described.^{11 12} HEK 293 cells (ATCC) were cultured in DMEM supplemented with 10% FBS. Cells were used for the present experiments in the 4th and 10th passages.

Reverse Transcription–Polymerase Chain Reaction Analysis
Reverse transcription (RT)-polymerase chain reaction (PCR) was performed as previously described.^{11 12} PCR was performed with sense and antisense primer pairs for each P2X4 (5'-AACTGCTCATCCTGGCCTAC-3' and 5'-ACGTGTGTG-TCATCCTCCAC-3') and GAPDH (5'-ACATCATCCCTG-CCTCTACTGG-3' and 5'-AGTGGGTGTCGCTGTTGAAGTC-3'). Each temperature cycle consisted of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute. The PCR products were separated with 5% polyacrylamide gel electrophoresis and analyzed with a GS363 Molecular Imager System (Bio-Rad).

Western Blot Analysis
Western blot analysis was performed as previously described.¹¹ Briefly, cells were dissolved in RIPA buffer and centrifuged at 26 000g for 30 minutes. The supernatants were immunoprecipitated with the anti-P2X4 purinoceptor antiserum, which was prebound to protein A–Sepharose beads. SDS-polyacrylamide gels were transferred to Immobilon membranes (Millipore) and incubated for 1 hour with the anti-P2X4 antiserum (3 µg/mL). Membranes were then incubated with anti-rabbit IgG horseradish peroxidase–conjugated antibody.

Antisense Oligonucleotides
Antisense oligonucleotides (AS-oligos) targeted to the P2X4 purinoceptor and scrambled control oligonucleotides (S-oligos) were designed and synthesized at Biognostik GmbH.¹¹ The sequences of phosphorothiorate AS-oligos and S-oligos were 5'-CCTGAAATTGTAGCC-3' and 5'-TAATCGCTTCAGACG-3', respectively, and these were FITC labeled at the 5'-end. AS-oligos or S-oligos were transfected into cells with LipofectAMINE PLUS (GIBCO). Cellular uptake of AS-oligos was checked through the observation of FITC with a fluorescence microscope.

Stable Expression of P2X4 Purinoceptor in HEK 293 Cells
P2X4 cDNA was subcloned into a pcDNA3.1(+) expression vector. The plasmids were transfected into HEK 293 cells with LipofectAMINE PLUS, and clones that stably expressed P2X4 purinoceptor were selected through treatment with geneticin. pcDNA3.1(+) alone was also transfected into HEK 293 cells as a control.

Flow Stimulation and [Ca²⁺]_i Measurement
HUVECs or HEK 293 cells were loaded with 5 µmol/L Indo-1/AM. The coverslip was placed in a parallel plate-type flow chamber. The flow chamber was set on the stage of an inverted microscope. One end of the chamber was connected to a reservoir filled with Hanks’ balanced salt solution (HBSS) via a silicon tube. By changing the height of the reservoir and monitoring flow rate at the outflow tube with a flow sensor, we exposed the cells to a stepwise increase in flow rate. Intensity of shear rate (, s^-1) and shear stress (, dyne/cm²) to the cells were calculated as follows: =6Q/ab², =µ, where Q is volume flow (mL/s), a and b are cross-sectional dimensions (cm) of the flow path, and µ is the viscosity of perfusate (10² mPa · s), respectively. Changes in [Ca²⁺]_i were measured with a confocal laser scanning system equipped with an ultraviolet argon ion laser, as previously described.¹³

	Results

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Shear Stress–Dependent Ca²⁺ Influx Occurs in HUVECs at 250 nmol/L Extracellular ATP
HUVECs were exposed to stepped rises in shear stress of 3, 8, and 15 dyne/cm² at various concentrations of extracellular ATP, and changes in [Ca²⁺]_i were monitored (Figure 1, top). At 100 nmol/L ATP, [Ca²⁺]_i showed only a very weak response to flow. At 250 nmol/L ATP, [Ca²⁺]_i increased stepwise with increasing shear stress, and when the flow was stopped, it soon returned to the basal level. At 500 nmol/L or 2 µmol/L ATP, [Ca²⁺]_i increased in response to flow but not in a shear stress–dependent manner. When extracellular Ca²⁺ was eliminated with EGTA (Figure 1, bottom), the flow-induced Ca²⁺ response completely disappeared at 100 or 250 nmol/L ATP, whereas there was only an early transient spike increase in [Ca²⁺]_i at 500 nmol/L or 2 µmol/L ATP.

View larger version (30K): [in this window] [in a new window]   Figure 1. Flow-induced Ca2+ responses in HUVECs at various concentrations of extracellular ATP. HUVECs loaded with Indo-1/AM were exposed to serial stepped rises in shear stress of 3, 8, and 15 dyne/cm2 in the presence of 100, 250, or 500 nmol/L or 2 µmol/L ATP (top). Similar experiments were also performed after a 1-minute incubation of cells with 1 mmol/L EGTA (bottom). Six groups of 8 to 10 cells were chosen with a 40x objective and placed into 6 regions of interest in the measuring field of a photometric fluorescence microscope. Cells were excited with light of 351-nm wavelength, and the emitted light was divided into 480 nm (F480) and 405 nm (F405) with a beam splitter. The ratio of these 2 values (F405/F480), which reflects changes in [Ca2+]i, was monitored. Each graph represents 6 Ca2+ responses of a group of 8 to 10 cells. Before the perfusion of HBSS, a hydrostatic pressure load in the absence of flow was applied by elevating the position of the reservoir so that the pressure in the chamber might increase to that (2.5, 4.0, and 9.5 mm Hg) during the subsequent flow. The pressure alone had no effect on [Ca2+]i (data not shown). Note that shear stress–dependent increase in [Ca2+]i occurred at 250 nmol/L ATP but not at 100 nmol/L, 500 nmol/L, or 2 µmol/L. The Ca2+ response seen at 250 nmol/L ATP disappeared in the absence of extracellular Ca2+. Similar results were obtained in 3 different experiments.

To clearly differentiate Ca²⁺ release from Ca²⁺ influx, HUVECs were treated with the phospholipase C inhibitor U-73122 (2 µmol/L) or its inactive analog U-73343 (2 µmol/L), and Ca²⁺ responses to flow were examined. Neither U-73122 nor U-73343 influenced the stepwise increase in [Ca²⁺]_i seen at 250 nmol/L ATP, whereas U-71322, but not U-73343, abolished the early spike increase in [Ca²⁺]_i at 500 nmol/L or 2 µmol/L ATP (data not shown) (Figure 2). These results indicate that the shear stress–dependent increase in [Ca²⁺]_i seen at 250 nmol/L ATP was due to the influx of extracellular Ca²⁺ across the cell membrane and that the early spike increase in [Ca²⁺]_i at 500 nmol/L or 2 µmol/L ATP was due to Ca²⁺ release from intracellular Ca²⁺ stores.

View larger version (32K): [in this window] [in a new window]   Figure 2. Effects of phospholipase C inhibitor on the flow-induced Ca2+ responses. HUVECs loaded with Indo-1/AM were exposed to shear stress in the presence of the phospholipase C inhibitor U-73122 (2 µmol/L) or its inactive analog U-73343 (2 µmol/L), and changes in [Ca2+]i were measured. Neither U-73122 nor U-73343 affected the shear stress–dependent increase in [Ca2+]i seen at 250 nmol/L ATP in the absence of EGTA (top). U-73122, but not U-73343, abolished the early spike increase in [Ca2+]i at 500 nmol/L ATP in the presence of EGTA (bottom). These data indicate that the shear stress–dependent increase at 250 nmol/L ATP was due to the influx of extracellular Ca2+ and that the early spike increase at 500 nmol/L ATP was due to Ca2+ release.

Quantitative analysis showed a good correlation between [Ca²⁺]_i (F₄₀₅/F₄₈₀) and shear stress (dyne/cm²) at an ATP level of 250 nmol/L, which was lost in the presence of EGTA but was not affected by U-73122 (Figure 3).

View larger version (23K): [in this window] [in a new window]   Figure 3. Relationship between flow-induced changes in [Ca2+]i (F405/F480) and shear stress at 250 nmol/L ATP. F405/F480 values at the center of each step of the Ca2+ response are given as the mean±SD of 6 groups of 8 to 10 cells. A linear regression analysis showed a good correlation between F405/F480 and shear stress (Control, r=0.9955, P<0.01). The relationship disappeared in the absence of extracellular Ca2+ [EGTA (+), 1 mmol/L] but was not affected by the phospholipase C inhibitor [U-73122 (+), 2 µmol/L].

P2X4 Purinoceptors Mediate Shear Stress–Dependent Ca²⁺ Influx
To investigate the role of P2X4 purinoceptors in the shear stress–dependent Ca²⁺ influx, HUVECs were treated with AS-oligos targeted against P2X4 purinoceptors and their Ca²⁺ responses to flow were determined. AS-oligos decreased the P2X4 mRNA and protein levels, reaching a minimum (approximately one fourth of the control) at 3 days after the transfection (Figure 4). S-oligos did not affect the P2X4 mRNA or protein levels. The AS-oligos had no effect on expression of P2X1, P2X3, P2X5, P2X7, P2Y1, or P2Y2 mRNA or on the Ca²⁺ responses induced by agonists other than ATP, such as histamine or bradykinin (data not shown), indicating that the AS-oligos specifically knockout the expression of P2X4 purinoceptor.¹¹

View larger version (25K): [in this window] [in a new window]   Figure 4. P2X4 mRNA and protein levels in HUVECs treated with AS-oligos to P2X4. P2X4 mRNA and protein levels were determined with RT-PCR (left) and Western blot analysis (right), respectively. Control indicates nontreated; S-oligos, S-oligo–treated HUVECs; and AS-oligos, AS-oligo–treated HUVECs. Top, Representative signal bands. Bottom, Quantitative analyses with densitometry. The vertical axis represents the percentage of control. Data are mean±SD of 5 separate samples. Both P2X4 mRNA and protein levels in AS-oligo–treated HUVECs were decreased to 25% of the control levels.

Ca²⁺ measurements were made 3 days after the transfection of AS-oligos or S-oligos. A shear stress–dependent Ca²⁺ response was seen in HUVECs treated with S-oligos but not in those treated with AS-oligos at 250 nmol/L ATP (Figure 5), suggesting that P2X4 purinoceptors play a crucial role in the shear stress–dependent Ca²⁺ influx in HUVECs.

View larger version (20K): [in this window] [in a new window]   Figure 5. Flow-induced Ca2+ responses in HUVECs treated with AS-oligos to P2X4. Cells were exposed to stepped increases in shear stress of 3, 8, and 15 dyne/cm2 at 250 nmol/L ATP. Each graph represents 6 average [Ca2+]i responses of a group of 8 to 10 cells. [Ca2+]i increased stepwise with increasing shear stress in HUVECs treated with S-oligos but not with AS-oligos. Similar results were obtained in 3 separate experiments.

Transfection of P2X4 cDNA Leads to Shear Stress–Dependent Ca²⁺ Influx in HEK Cells
To further examine the role of P2X4 purinoceptors in flow-induced Ca²⁺ signaling, HEK 293 cell lines stably expressing various levels of P2X4 purinoceptors (+P2X4) were established through the transfection of P2X4 cDNA, and their Ca²⁺ responses to flow were examined. Control HEK cells expressed little P2X4, but in +P2X4, the P2X4 mRNA and protein levels were significantly increased (Figure 6).

View larger version (26K): [in this window] [in a new window]   Figure 6. P2X4 mRNA and protein levels in HEK 293 cells stably expressing P2X4 purinoceptors. P2X4 mRNA and protein levels were determined with RT-PCR (left) and Western blot analysis (right), respectively. Control indicates HEK 293 cells transfected with vector alone; +P2X4, 3 clones of HEK 293 cells stably expressing various levels of P2X4 (low, medium, or high). Top, Representative signal bands. Bottom, Quantitative analyses with densitometry. Northern and Western blotting showed that the level of P2X4 expression in +P2X4 (high) was 1.5-fold that in HUVECs (data not shown). The vertical axis represents the percentage of control. Data are mean±SD of 5 separate samples.

The control HEK cells did not show any significant increase in [Ca²⁺]_i when exposed to flow at an ATP level of 2 µmol/L (Figure 7). However, +P2X4 showed stepwise increases in [Ca²⁺]_i in response to stepwise rises in shear stress. The Ca²⁺ response showed a dose-response relationship with the level of the P2X4 expression (ie, the Ca²⁺ response became greater as the level of P2X4 expression increased). The flow-induced stepwise increase in [Ca²⁺]_i completely disappeared in the absence of extracellular Ca²⁺. These findings suggest that the ectopic expression of P2X4 purinoceptors made HEK cells sensitive to flow, facilitating a shear stress–dependent Ca²⁺ influx.

View larger version (18K): [in this window] [in a new window]   Figure 7. Flow-induced Ca2+ responses in HEK 293 cells stably expressing P2X4 purinoceptors. Cells were exposed to stepped increases in shear stress of 3, 8, and 15 dyne/cm2 at 2 µmol/L ATP. Control indicates HEK 293 cells transfected with vector alone; +P2X4, 3 clones of HEK 293 cells transfected with P2X4 cDNA and stably expressing various levels of P2X4 (low, medium, or high); EGTA (+), +P2X4 (high) incubated with 1 mmol/L EGTA for 1 minute. Each graph represents 6 average [Ca2+]i responses of a group of 8 to 10 cells. The control did not show any significant Ca2+ response to flow, but +P2X4 showed shear stress–dependent Ca2+ influx. The shear stress–dependent Ca2+ response became larger as the level of the P2X4 expression increased. The Ca2+ response in P2X4 (high) disappeared in the absence of extracellular Ca2+.

Flow-Induced Ca²⁺ Influx via P2X4 Purinoceptors Is Shear Stress, Rather Than Shear Rate, Dependent
To examine whether the flow-induced Ca²⁺ influx via P2X4 purinoceptors is dependent on shear stress or shear rate, HUVECs or +P2X4 (high) were exposed to the flow of 2 perfusates with different viscosities at 250 nmol/L or 2 µmol/L ATP, respectively, and changes in [Ca²⁺]_i were measured. A stepwise elevation of shear rate induced stepwise increases in [Ca²⁺]_i but to a greater extent with the high-viscosity perfusate (HBSS+5% dextran) in HUVECs (Figure 8A) or +P2X4 (data not shown). This tendency was quantitatively confirmed by plotting the percent increases in [Ca²⁺]_i against shear rate (Figure 8B, left). When plotted against shear stress, they formed an almost single curve, indicating that the percent increases in [Ca²⁺]_i are well correlated with shear stress regardless of viscosity (Figure 8B, right). These results indicate that flow-induced Ca²⁺ influx via P2X4 purinoceptors is shear stress, rather than shear rate, dependent.

View larger version (29K): [in this window] [in a new window]   Figure 8. Shear stress dependency of flow-induced Ca2+ influx via P2X4 purinoceptors. A, Ca2+ responses to flow of 2 perfusates with different viscosities in HUVECs. Cells were exposed to stepped increases in shear rate of 440, 990, and 1990 seconds at 250 nmol/L ATP. Each value of shear stress was shown in the parentheses. Left, Ca2+ response of a group of 8 to 10 cells to flow of low-viscosity perfusate (HBSS). Right, Response to flow of high-viscosity perfusate (HBSS with 5% dextran [MW 162 000]). The viscosity, specific gravity, and osmotic pressure of each solution were 0.765 and 3.372 mPa · s, 1.005 and 1.025, and 289 and 292 mOsm/kg H2O, respectively. The flow-induced increases in [Ca2+]i were always higher at high viscosity than at low viscosity at any shear rate. B, Percentage increases in [Ca2+]i plotted against shear rate (left) or shear stress (right). Top, HUVECs. Bottom, HEK cells stably expressing P2X4. •, Low viscosity; , high viscosity. The lines were drawn by curvilinear regression with Microsoft Excel. The ratio test of the composite hypothesis of Neyman and Pearson indicated that the low- and high-viscosity groups should be regarded as separate groups with P<0.01.9 Data are mean±SD for 6 groups of 8 to 10 cells. Note that data plotted against shear stress form almost a single line, indicating that flow-induced increases in [Ca2+]i were shear stress, rather than shear rate, dependent.

	Discussion

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The results of the present study demonstrated that the exposure of HUVECs to fluid flow increased Ca²⁺ influx in a shear stress–dependent manner at 250 nmol/L extracellular ATP. These findings are consistent with our previous results obtained in fetal bovine aortic ECs.⁹ The shear stress–dependent Ca²⁺ influx seen at 250 nmol/L ATP was markedly suppressed in HUVECs treated with AS-oligos targeted against P2X4 purinoceptors. Furthermore, the transfection of P2X4 cDNA made HEK 293 cells sensitive to flow, facilitating a shear stress–dependent Ca²⁺ influx. These results indicated that P2X4 purinoceptors play a key role in the shear stress–dependent Ca²⁺ influx.

Pattern of flow-induced Ca²⁺ responses in HUVECs varied according to extracellular ATP concentrations. At ATP levels of >500 nmol/L, both Ca²⁺ release and influx occurred in response to flow, but at ATP levels of <250 nmol/L, Ca²⁺ influx alone occurred. It is possible that at higher ATP levels, flow activates not only P2X4 but also G protein–coupled P2Y purinoceptors. The activation of P2Y purinoceptors increases inositol triphosphate (IP₃) via phospholipase C. Then, the IP₃ binds to its receptors on intracellular Ca²⁺ stores such as endoplasmic reticulum and releases Ca²⁺. At relatively low ATP levels of <250 nmol/L, flow may activate P2X4, but not P2Y, purinoceptors. This may be due to the difference in the expression level or sensitivity to ATP between P2X4 and P2Y. Competitive PCR showed that P2Y1 and P2Y2 mRNA levels were 30% and 15% of the P2X4 mRNA levels, respectively.¹¹ P2X4 purinoceptors are most sensitive to ATP, whereas P2Y1 purinoceptors are generally more sensitive to ADP.¹⁴ Shear stress–dependent Ca²⁺ influx was clearly seen at relatively low ATP levels but not at higher ATP levels. Once Ca²⁺ release from intracellular Ca²⁺ stores occurred at higher ATP levels, Ca²⁺ release–activated Ca²⁺ entry may become predominant and make the shear stress dependency of P2X4-mediated Ca²⁺ influx indistinct.

HUVECs showed shear stress–dependent Ca²⁺ influx at 250 nmol/L ATP, but HEK cells required 2 µmol/L ATP to show similar responses. Although the exact reason for this discrepancy is not clear, these 2 cell lines may express distinct types of P2X4 receptors with different sensitivity to ATP (eg, the HEK cells transfected with P2X4 cDNA express a P2X4 homomer, whereas the native receptor in HUVECs may be a heteromer of P2X4 and other P2X subtypes). It has been shown that ATP-gated channels can be formed through a heteropolymerization of P2X receptor subunits.¹⁵ On the other hand, there is the possibility that the amount of cell surface ecto-ATPases that degrade ATP or of endogenous ATP released from cells¹⁶ may differ between the 2 cell lines, which would modify the ATP level required for shear stress–dependent Ca²⁺ responses.

Flow exerts 2 effects on cells. One effect relates to mass transport (ie, the increase in flow rate or shear rate decreases the thickness of the diffusion boundary layer of ATP, which increases the amount of ATP that reaches the cell surface and further stimulates cells). The other effect is due to the flow-derived mechanical force, shear stress. To discriminate these 2 effects, flow-loading experiments with 2 perfusates with different viscosities were performed. As shown in Materials and Methods, shear stress () was calculated as =µ, where µ is the viscosity of perfusate, and is shear rate. If the flow-induced Ca²⁺ influx is caused by shear stress, a certain induces the same magnitude of Ca²⁺ influx regardless of the values of µ and . If it is caused by the diffusional accumulation of ATP to the cell surface, a certain induces the same Ca²⁺ influx regardless of , or because higher viscosity decreases the diffusion of ATP, the flow of high-µ perfusate should induce smaller Ca²⁺ responses compared with those of low-µ perfusate at the same . The results showed that the magnitude of Ca²⁺ influx with the high-µ perfusate were always larger than those with the low-µ perfusate at any and that Ca²⁺ influx correlated well with regardless of µ. This means that the flow-induced Ca²⁺ influx was shear stress, rather than shear rate, dependent.

The mechanisms for the shear stress–dependent Ca²⁺ influx, however, remain unclear. Shear stress may modulate P2X4 purinoceptor functions indirectly or directly. Receptors other than P2X4 may recognize shear stress and transduce the signal into changes in some second messengers that affect P2X4 function. It has been shown that shear stress can cause hyperpolarization of EC membrane by increasing the probability of opening of potassium ion channels.¹⁷ Membrane hyperpolarization is thought to function as a driving force for Ca²⁺ influx. It has been also shown that shear stress can be perceived by GTP-binding protein¹⁸ or adhesion molecules such as integrin¹⁹ and platelet-endothelial cell adhesion molecule-1²⁰ and transmitted into the cell interior. These signal transduction events may play a role in the activation of P2X4 purinoceptors. Shear stress also stimulates ECs to release ATP.¹⁶ In addition, it has been reported that endogenous ATP released by shear stress induces [Ca²⁺]_i transient in mouse fibroblasts in an autocrine manner.²¹ Thus, these endogenous ATP may modulate the shear stress–induced Ca²⁺ response in ECs. On the other hand, P2X4 function may be directly modulated by shear stress. Interestingly, the molecular structure of P2X4, which has 2 transmembrane domains,²² resembles that of mechanosensitive ion channels such as amiloride-sensitive epithelial Na⁺ channels²³ and large conductance mechanosensitive ion channels.²⁴ However, it is still not known whether shear stress induces any conformational change in the P2X4 purinoceptor or how shear stress alters P2X4 functions.

ECs respond to shear stress in a dose-dependent rather than an all-or-none manner. To understand shear stress signal transduction, it is important to find pathways that show a shear stress dependency. P2X4-mediated Ca²⁺ signaling would be one candidate shear stress–dependent pathway. Although Ca²⁺ signaling is involved in the shear stress–mediated production of NO, prostacyclin, and endothelin and the activation of some transcription factors,¹ the physiological significance of the occurrence of shear stress–dependent Ca²⁺ signaling at relatively low levels of ATP remains unclear. The exact concentration of ATP in blood vessels has not yet been determined, but according to some reports, it would be <2 µmol/L, which may be an overestimation.²⁵ With the assumption that the concentration would be around several hundred nanomoles per liter, it seems likely that the mechanism by which the intensity of shear stress is quantitatively transduced into changes in [Ca²⁺]_i works in vivo. Further studies with neutralizing antibody or P2X4 knockout mice are needed to clarify the physiological role of P2X4-mediated Ca²⁺ signaling in vivo.

	Acknowledgments

 
This work was supported in part by grants-in-aid for scientific research and for scientific research on priority areas from the Japanese Ministry of Education, Science and Culture; a research grant for cardiovascular diseases from the Japanese Ministry of Health and Welfare; Special Coordination Funds for Promoting Science and Technology; and research funds from the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Drug ADR Relief, R&D Promotion and Product Review of Japan. We sincerely thank Dr M. Sokabe (Nagoya University) and Dr Y. Takada (Asahi Chemical Industry Company) for advice.

Received March 9, 2000; revision received June 28, 2000; accepted July 11, 2000.

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