Fluid Shear Stress Activates Ca2+ Influx Into Human Endothelial Cells via P2X4 Purinoceptors
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.
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-β1. 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-dependent4 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
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 (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 (γ, s−1) and shear stress (τ, dyne/cm2) to the cells were calculated as follows: γ=6Q/ab2, τ=μγ, 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
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.
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.
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⇓).
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
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.
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⇓).
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.
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.
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 (τ) was calculated as τ=μγ, where μ is the viscosity of perfusate, and γ is shear rate. If the flow-induced Ca2+ influx is caused by shear stress, a certain τ induces the same magnitude of Ca2+ influx regardless of the values of μ and γ. If it is caused by the diffusional accumulation of ATP to the cell surface, a certain γ induces the same Ca2+ influx regardless of τ, 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 γ. 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 γ and that Ca2+ influx correlated well with τ 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.
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.
- © 2000 American Heart Association, Inc.
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