Visualization of flow-induced ATP release and triggering of Ca2+ waves at caveolae in vascular endothelial cells
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Research Article 3477
Visualization of flow-induced ATP release and
triggering of Ca2+ waves at caveolae in vascular
endothelial cells
Kimiko Yamamoto1, Kishio Furuya2, Makiko Nakamura3, Eiry Kobatake3, Masahiro Sokabe4 and Joji Ando5,*
1
Laboratory of System Physiology, Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan
2
FIRST Research Center for Innovative Nanobiodevice, Nagoya University, Nagoya 466-8550, Japan
3
Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan
4
Department of Physiology, Nagoya University School of Medicine, Nagoya 466-8550, Japan
5
Laboratory of Biomedical Engineering, School of Medicine, Dokkyo Medical University, 880 Kita-kobayashi, Mibu, Tochigi 321-0293, Japan
*Author for correspondence (jo-ji@umin.ac.jp)
Accepted 23 May 2011
Journal of Cell Science 124, 3477–3483
ß 2011. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.087221
Summary
Endothelial cells (ECs) release ATP in response to shear stress, a fluid mechanical force generated by flowing blood but, although its
release has a crucial role in controlling a variety of vascular functions by activating purinergic receptors, the mechanism of ATP release
has never been established. To analyze the dynamics of ATP release, we developed a novel chemiluminescence imaging method by
Journal of Cell Science
using cell-surface-attached firefly luciferase and a CCD camera. Upon stimulation of shear stress, cultured human pulmonary artery ECs
simultaneously released ATP in two different manners, a highly concentrated, localized manner and a less concentrated, diffuse manner.
The localized ATP release occurred at caveolin-1-rich regions of the cell membrane, and was blocked by caveolin-1 knockdown with
siRNA and the depletion of plasma membrane cholesterol with methyl-b-cyclodexrin, indicating involvement of caveolae in localized
ATP release. Ca2+ imaging with Fluo-4 combined with ATP imaging revealed that shear stress evoked an increase in intracellular Ca2+
concentration and the subsequent Ca2+ wave that originated from the same sites as the localized ATP release. These findings suggest that
localized ATP release at caveolae triggers shear-stress-dependent Ca2+ signaling in ECs.
Key words: Endothelial cells, Shear stress, ATP release, Calcium (Ca2+) signalling, Caveolae
Introduction previous studies have shown that ATP-mediated Ca2+ signaling
Adenosine 59-triphosphate (ATP) is the source of the energy that plays an important role in shear stress mechanotransduction.
drives virtually all cell functions; it also functions as an autocrine Shear stress increases intracellular Ca2+ concentration dose-
and paracrine regulatory signaling molecule. It is generally dependently by causing an influx of extracellular Ca2+ through a
acknowledged that many different cell types release ATP in subtype of P2X purinoceptors, the P2X4 receptors (Yamamoto
response to mechanical or biochemical stimulation, and that the et al., 2000a; Yamamoto et al., 2000b). Activation of P2X4
released ATP modulates cell function by activating nearby receptors requires ATP, which is supplied in the form of
purinoceptors, such as ion channel P2X receptors and G-protein- endogenous ATP released by ECs (Yamamoto et al., 2003). In
coupled P2Y receptors (Khakh and Burnstock, 2009; Milner et addition, our recent study in P2X4-knockout mice revealed that
al., 1990; Yegutkin, 2008). However, it remains unclear how the Ca2+ signaling triggered by shear stress has a crucial role in
cells release endogenous ATP into the extracellular space and, so vascular physiology and pathophysiology, because P2X4-
far, a main hurdle in ATP research has been the difficulty of knockout mice were found to exhibit impaired vasodilator
directly visualizing ATP release by living cells. responses to acute increases in blood flow and to have higher
The vascular endothelial cells (ECs) that line the inner surface blood pressure than wild-type mice, both of which were
of blood vessels are exposed to shear stress – a biomechanical attributable to reduced endothelial nitric oxide (NO) production
force generated by flowing blood – and they alter their (Yamamoto et al., 2006). Adaptive vascular structural remodeling
morphology, function and gene expression in response to in response to a chronic decrease in blood flow was also impaired
changes in shear stress (Ando and Yamamoto, 2009). These EC in the knockout mice (Yamamoto et al., 2006). The mechanism
responses to shear stress play important roles not only in the of ATP release as an early response to shear stress, however, is
homeostasis of the circulatory system but in blood-flow- still unknown, and whether ATP release occurs at a specific
dependent phenomena, such as angiogenesis, vascular cellular location and whether it is sufficient to activate nearby
remodeling, aneurysm formation and atherosclerosis. Numerous purinoceptors remain unclear.
studies have been undertaken to clarify how ECs sense shear To analyze the dynamics of ATP release, we developed a novel
stress and transmit the signal into the cell interior, but the chemiluminescence imaging method by utilizing a biotin–
mechanism has only partially been revealed (Davies, 1995). Our luciferase chimera protein that can be stably immobilized on a
3478 Journal of Cell Science 124 (20)
biotinylated cell surface with streptavidin, and an intensified
charge-coupled device (CCD) camera. This method allowed us to
visualize ATP release at the cell surface in real time and at high
resolution. Since caveolae have been implicated as plasma
membrane microdomains that sense or transduce altered shear
stress into biochemical signals, thereby regulating EC function
(Yu et al., 2006), we investigated the relationship between ATP
release and caveolae by immunostaining with an antibody against
caveolin-1, the primary structural protein of caveolae. In
addition, to investigate the role of ATP release in shear stress
Ca2+ signaling we performed Ca2+ imaging by using a fluorescent
probe Fluo-4 in the same cells as examined by ATP imaging.
Results
Distribution and ATP sensitivity of cell-surface-
attached luciferase
The distribution of cell-surface-attached luciferase on cells
immunostained with a FITC-labeled antibody against luciferase
was examined with a confocal laser scanning microscope. En-
face images showed an even and dense distribution of biotin–
luciferase over the entire cell surface, and longitudinally cut
images showed that the biotin–luciferase was localized uniformly
on the apical cell membrane (Fig. 1A). Application of shear
Journal of Cell Science
stress caused no significant change in the distribution of cell-
surface-attached luciferase, indicating that this method can be
used even under flow conditions.
Fig. 1. Distribution and ATP sensitivity of luciferase attached to the cell
To assess the ability of cell-surface-attached luciferase to surface. (A) Confocal laser scanning photomicrographs of luciferase-labeled
detect ATP, various concentrations of ATP were added to HPAECs immunostained with a FITC-labeled antibody to luciferase. En-face
luciferase-labeled human pulmonary artery ECs (HPAECs), and images of the apical cell membrane show an even and dense distribution of
luminescence emitted as a result of the ATP-triggered luciferase– biotin–luciferase over the entire cell surface under static conditions (Static),
luciferin reaction was measured with a CCD camera. The and longitudinal images cut at the dashed lines depict the biotin–lucifease
intensity of the luminescence in response to the addition of ATP localized to the apical cell membrane. Application of shear stress (15 dynes/
increased in a concentration-dependent manner, and there was a cm2) for 10 minutes caused no change in the distribution of luciferase.
(B) ATP sensitivity of cell-surface-attached luciferase. Increasing
clear correlation between the luminescence intensity and ATP
concentrations of ATP were added to luciferase-labeled HPAECs and the
concentration (Fig. 1B). These findings indicate that this method luminescence emitted was measured with a CCD camera. Luminescence
of ATP detection allows to determine quantitatively the increased as the ATP concentration increased. The inset shows a correlation
extracellular ATP concentrations at the cell surface. between luminescence intensity and the ATP concentration. Values are
means¡s.d. of data obtained from three separate HPAEC cultures.
Visualization of shear-stress-induced ATP release by ECs
Luminescent ATP signals at the cell surface were monitored with ATP release that was almost spatiotemporally identical to that of
a CCD camera before and after shear stress application, and the the initial stimulation (data not shown).
signals were transformed into pseudo-color images. As soon as
the cells were exposed to shear stress, ATP was released from the Localized ATP release occurs in caveolin-1-rich regions of
entire surface of the cell membrane that was monitored, and the cell edge
the ATP signals were particularly strong at localized regions at To examine the relationship between the regions of localized
the edge of the cell (Fig. 2A), thereby indicating the existence of ATP release and caveolae (cholesterol-rich plasma membrane
two distinct manners of ATP release, a diffuse manner and a microdomains) after visualizing ATP release, cells were
highly concentrated, localized manner. immunostained with an antibody to caveolin-1, a marker
The temporal changes in ATP signals were quantified in the protein for caveolae. Caveolin-1 was unevenly distributed over
regions of diffuse ATP release and the regions of localized ATP the cell surface and was concentrated at specific parts of the cell
release (Fig. 2B). ATP release began simultaneously in both edge. Comparison between the sites of ATP release and caveolin-
regions, but the peak of the ATP signal in both regions was 1 distribution revealed that the localized ATP release occurred in
markedly different. The ATP concentration estimated from the the caveolin-1-rich cell edge regions (Fig. 3).
luminescence intensity reached more than 10 mM in the regions To investigate the role of caveolae in shear-stress-induced ATP
of localized ATP release, but it remained below 1 mM in the release, we used small interfering RNA (siRNA) in order to
regions of diffuse ATP release (Fig. 2C). The ATP signal in both specifically knockdown the expression of caveolin-1. In clear
regions increased further when the shear stress was raised from contrast to the control cells, which had been subjected to
10 dynes/cm2 to 40 dynes/cm2, indicating that the amount of transfection conditions alone, marked suppression of localized
ATP release is shear-stress dependent (Note: 1 dyne510 mN). ATP release was observed in the HPAECs transfected with
Secondary application of shear stress to the same cells induced an caveolin-1 siRNA (Fig. 4A,B). Caveolin-1 siRNA had no
Visualization of endothelial ATP release 3479
Journal of Cell Science
Fig. 2. Visualization of ATP release in response to shear stress. (A) Sequential pseudo-color images of shear-stress-induced ATP release. Luciferase-labeled
HPAECs were exposed to shear stress (10 dynes/cm2) for 3 seconds and changes in the ATP signal at the cell surface were recorded in real time. Each pseudo-
color image was created by integrating the luminescence for 5 seconds. Relatively low concentrations of ATP were released diffusely from the entire surface of
the cells; at the same time highly concentrated ATP release occurred at localized regions at the edge of the cell. (B) Comparison between localized ATP release
and diffuse ATP release. The temporal changes in ATP signal were quantified by placing regions of interest on a region of localized ATP release (L) and a region
of diffuse ATP release (D). The start of ATP release after shear stress application was identical in both regions, but the peak ATP signal in the localized release
region was substantially larger than in the diffuse release region. It should be noted that luminescence decays slowly because the PicaGene reagent contains
coenzyme A as a substrate of luciferase in addition to luciferin. (C) Shear-stress-dependency of ATP release. The amount of ATP that was released increased
further when shear stress was increased from 10 dynes/cm2 to 40 dynes/cm2. The ATP concentration was determined from the correlation with the intensity of
luminescence as shown in Fig.1B. Values are means¡s.d. of 30 cells in three separate experiments. *P,0.01 10 dynes/cm2 vs 40 dynes/cm2.
significant effect on diffuse ATP release. Next, we treated localized ATP release coincided exactly with the initiation sites
HPAECs with methyl-b-cyclodextrin (MbCD), which disrupts of Ca2+ waves. Comparison between the start of localized ATP
caveolae and lipid rafts by depleting plasma-membrane release and increase in [Ca2+]i showed that ATP release always
cholesterol. Treatment with MbCD significantly inhibited preceded the increase in [Ca2+]i. Treatment of HPAECs with
shear-stress-induced localized ATP release but did not have a angiostatin, a known blocker of ATP release, almost completely
significant effect on diffuse ATP release (Fig. 4A,B). The abolished both the shear-stress-induced ATP release and the
inhibitory effect of MbCD on localized ATP release was increase in [Ca2+]i (Fig. 5B). These results suggest that the
partially prevented by pretreatment with cholesterol. These localized release of ATP at caveolae triggers the increase in
findings suggest that caveolae are involved in shear-stress- [Ca2+]i by activating nearby purinoceptors.
induced, localized ATP release.
Discussion
Colocalization of localized ATP release and the The novel ATP imaging method described here clearly
subsequent initiation of Ca2+ waves demonstrated that HPAECs release ATP in response to shear
Luciferase-labeled HPAECs were exposed to shear stress and stress in two distinct manners; i.e. a highly concentrated,
examined for changes in intracellular Ca2+ concentration localized manner and a diffuse manner. A variety of methods
([Ca2+]i) by using the Ca2+ indicator Fluo-4 and a fluorescence can be used to detect ATP that is released at the surface of living
microscope. Shear stress evoked a rapid increase in [Ca2+]i that cells, including biosensor techniques (Bell et al., 2003; De Proost
started at a single site in the cell and propagated throughout the et al., 2009; Hayashi et al., 2004; Hazama et al., 1998; Llaudet et
entire cell in the form of a Ca2+ wave (Fig. 5A). The Ca2+ wave al., 2005; Schneider et al., 1999) and methods that induce
also propagated into the cell nucleus. After Ca2+ imaging, ATP luminescence or measure fluorescence by using ATP-sensitive
imaging was performed on the same cells. The regions of proteins added to the extracellular space (Arcuino et al., 2002;
3480 Journal of Cell Science 124 (20)
the protein marker caveolin-1 and are known to play crucial roles
in multiple signal transduction events at the surface of various
cell types (Shaul and Anderson, 1998). Recent studies have
demonstrated that caveolae sense or transduce shear stress into
biochemical signals that regulate EC functions. Shear stress has
been found to activate extracellular-signal-regulated kinase
(ERK) in bovine aortic ECs that was blocked by the delivery
of polyclonal caveolin-1 antibody into the cells (Park et al.,
2000). The production of a potent vasodilator, nitric oxide (NO),
by ECs increases in response to shear stress, and one of the
mechanisms of shear-stress-induced NO production is
dissociation of endothelial NO synthase (eNOS) from caveolae
followed by activation of eNOS (Rizzo et al., 1998). A more
recent study comparing caveolin-1 knockout mice with wild-type
mice showed that lack of caveolin-1 impaired blood-flow-
dependent eNOS activation, vasodilation, and vascular
remodeling and that these abnormalities were rescued by
reconstituting caveolin-1 into the vascular endothelium of the
knockout mice (Yu et al., 2006). The present study revealed that
localized ATP release induced by exposing HPAECs to shear
stress occurs in caveolin-1-rich regions of the plasma membrane.
The localized ATP release was markedly suppressed by
knockdown of caveolin-1 expression with siRNA and by
depletion of membrane cholesterol with MbCD, which disrupts
Journal of Cell Science
caveolae/lipid rafts. Although the possibility remains that the
inhibition of the localized ATP release by caveolin-1 siRNA and
MbCD is attributable to secondary rather than primary effects,
these findings suggest that caveolae are involved in the localized
ATP release that occurs in response to shear stress.
It remains unclear why the localized ATP release occurs
Fig. 3. Comparison between the regions of localized ATP release and
caveolin-1 distribution. ATP image, pseudo-color images of shear-stress-
preferentially at caveolin-1-rich regions of the cell membrane.
induced ATP release. Each image was created by integrating luminescence Since shear stress causes microscale deformation/displacement of
for 5 seconds. Caveolin-1, fluorescent photomicrographs of caveolin-1. DIC, cell surface membrane proteins, the lipid bilayer itself, and the
differential interference contrast (DIC) images. Broken lines represent the cell cytoskeleton and connected proteins, it seems likely that the sites
outlines obtained from the DIC images. Six pairs of images demonstrate that of shear-stress-induced deformation are linked to the localization
the regions of localized ATP release coincide with the caveolin-1-rich regions of ATP release. Caveolae/lipid raft microdomains are
at the cell edge. These six sets of images were selected because they are characterized by a unique lipid composition that contains high
representative of dozens of images, all of which showed similar results. concentrations of cholesterol and sphingolipids in places where
the lipid bilayer is more rigid and lipid movement is more
Corriden et al., 2007; Wang et al., 2000) or targeted to the plasma restricted than in other parts of the membrane (Gaus et al., 2003).
membrane (Beigi et al., 1999; Joseph et al., 2003; Okada et al., These differences in the physical properties of the membrane
2006; Pellegatti et al., 2005). However, some of these methods may be responsible for the localization of ATP release. On the
provide only semi-quantitative information on ATP release, other hand, caveolae/lipid rafts are closely associated with
others are unsuitable for the visualization of ATP release, mainly cytoskeleton filaments, and shear stress may affect the caveolae
because of weak signals. In our study, however, we were able to and associated molecules through cytoskeleton networks. Helmke
visualize ATP release of ECs by using a cell-surface-targeting et al. showed that strain in the intermediate filament was
luciferase and a high-resolution CCD camera. To obtain stronger heterogeneous, with small regions of high-strain concentrations
signals, we generated a biotin–luciferase fusion protein that can located at the cell periphery and at several sites in the cell
attach to biotinylated cell surfaces through interaction with interior, and that the hot spots of strain concentration were
streptavidin (Nakamura et al., 2006). Since various plasma repositioned by shear stress (Helmke et al., 2003). These hot
membrane proteins can bind to many biotins and one streptavidin spots of cytoskeletal strain may coincide with the locations of
molecule has four biotin-binding sites, a large amount of ATP release. It requires further research to clarify whether the
luciferase can be bound to the cell surface. In addition, by localization of ATP release is a plasma membrane domain
using the PicaGene reagent, which emits several times more phenomenon or influenced by interference with cytoskeleton
luminescence than other luciferase–luciferin reaction reagents, it function or both.
became possible to obtain luminescence strong enough for high- Depending on the cell type and extrinsic stimulus, ATP is
resolution imaging of ATP release. This imaging method should released into the extracellular space by ATP-permeable
prove useful for studying ATP release mechanisms and the membrane channels, including connexin hemichannels (Stout et
functional roles of ATP in various cell types. al., 2002) and volume-regulated anion channels (Sabirov and
Caveolae are small flask-shaped invaginations of cholesterol- Okada, 2004), by diffusion facilitated by ATP-binding cassette
rich cell membranes. They are characterized by the presence of transporters, such as the cystic fibrosis transmembrane
Visualization of endothelial ATP release 3481
Fig. 4. Involvement of caveolae in shear-stress-induced ATP release. (A) Pseudo-color images of ATP release obtained 5–10 seconds after application of shear
stress (10 dynes/cm2 for 3 seconds). Each image was created by integrating luminescence for 5 seconds. Control, control luciferase-labeled HPAECs. Caveolin-1
siRNA, cells transfected with caveolin-1 siRNA. MbCD, the cells treated with 10 mM methyl-b-cyclodextrin. MbCD+Cholesterol, the cells pretreated with
1.3 mM cholesterol and then with 10 mM MbCD. Caveolin-1 knockdown with siRNA and disruption of caveolae and lipid rafts with MbCD markedly suppressed
Journal of Cell Science
the shear-stress-induced localized ATP release. Pretreatment with cholesterol partially prevented the effect of MbCD. (B) Quantitative analysis of shear-stress-
induced ATP release. Luminescence intensity was measured separately in the region of diffuse ATP release and the region of localized ATP release by setting an
region of interest over the cell nucleus and over the site in the cell periphery where the intensity was highest. The data are expressed as means¡s.d. (n530).
*P,0.01 vs control. Localized ATP release was significantly suppressed by caveolin-1 siRNA and MbCD, but diffuse ATP release was not, suggesting that
caveolae are involved in the shear-stress-induced localized ATP release.
conductance regulator (Schwiebert et al., 1999), or by vesicular adhesion molecule-1, the cytoskeleton, the glycocalyx, and primary
transport and exocytotic secretion (Bodin and Burnstock, 2001). cilia (Ando and Yamamoto, 2009; Davies, 1995). However, it is
Our previous study revealed that ATP synthase is localized in unclear how these molecules and microdomains enable ECs to sense
caveolae and involved in shear-stress-induced ATP release by shear stress and transmit the signal to downstream effectors to allow
HPAECs (Yamamoto et al., 2007). However, it remained unclear cells to respond. Our previous studies in ECs demonstrated that the
whether caveolar ATP synthase is involved in any of the above- P2X4 receptor contributes to shear stress mechanotransduction
mentioned ATP-releasing pathways or in an unknown pathway. through Ca2+ signaling, which plays a crucial role in the control of
In view of the discovery of the two different manners of vascular function in vivo (Yamamoto et al., 2000a; Yamamoto et al.,
ATP release in this study, HPAECs may use dual pathways to 2003). Clarification of the mechanisms of ATP release as an early
release ATP in response to shear stress. Further study will be response to shear stress should lead to a better understanding of the
needed to identify the pathway responsible for each manner of mechanotransduction of shear stress.
ATP release.
In the present study we performed intracellular Ca2+ imaging and Materials and Methods
Cell culture
ATP imaging of the same cells. Shear stress evoked an increase in Human pulmonary artery endothelial cells (HPAECs) were obtained from
[Ca2+]i that originated at a specific site and propagated throughout Clonetics and grown on a 1% gelatin-coated tissue culture flask in M199
the entire cell in the form of a Ca2+ wave in a manner that resembled supplemented with 15% FBS, 2 mM L-glutamine (Gibco), 50 mg/ml heparin, and
to the Ca2+ responses previously observed in bovine fetal aortic ECs 30 mg/ml EC growth factor (Becton Dickinson). The cells used in the present
experiments were in the 7th and 10th passage.
(Isshiki et al., 1998). The sites where the increase in [Ca2+]i
originated coincided with the sites of localized ATP release. Production and purification of biotin–luciferase
Comparisons between the start of the [Ca2+]i increase and ATP Biotin–luciferase protein was produced and purified as previously reported
release revealed that ATP release always preceded the Ca2+ (Nakamura et al., 2006). Briefly, biotin acceptor peptide (BAP) was fused to
thermostabilized firefly luciferase, and the biotin–luciferase gene fusion plasmid,
increase. The ATP concentration at the sites of localized ATP pET-NHis-BAP-Luc, was constructed. For purification, six repeats of the histidine
release reached more than 10 mM, which is sufficient to activate sequence (His-tag) were coded to the N terminus of the fusion gene. The pET-
purinoceptors. Thus, it seems that shear stress first triggers ATP NHis-BAP-Luc plasmid was transduced into Escherichia coli BL21 (DE3)
competent cells and the cells were grown and lyzed by sonication. The soluble
release, which activates nearby P2X and/or P2Y receptors, and, in fraction of the bacterial lysates was subjected to metal-ion affinity chromatography
turn, leads to Ca2+ responses. Many studies have been devoted to in order to purify biotin–luciferase protein by using the His-tag.
investigation of shear stress mechanotransduction and have
demonstrated the involvement of various membrane molecules Cell-surface labeling with biotin–luciferase
and cellular microdomains in its mechanisms, including ion HPAECs cultured on coverslips were incubated with 250 mg/ml EZ-Link2 Sulfo-
NHS-Biotin (Thermo Scientific Pierce, Rockford, IL) for 10 minutes at room
channels, growth factor receptors, G proteins, caveolae, adhesion temperature. After washing with HBSS, cells were treated with 2 mM streptavidin
proteins such as integrin, VE-cadherin, and platelet endothelial cell (Wako) for 30 minutes at 37 ˚C and then, after another HBSS wash, incubated with3482 Journal of Cell Science 124 (20)
Journal of Cell Science
Fig. 5. Colocalization of sites of localized ATP release and the subsequent initiation of Ca2+ waves. (A) Pseudo-color images of shear-stress-induced ATP
release and Ca2+ responses. Ca2+ imaging with Fluo-4 showed that shear stress evoked an increase in intracellular Ca2+ concentrations ([Ca2+]i) that started at a single
site and propagated throughout the entire cell in the form of a Ca2+ wave. The first Ca2+ image was obtained 1 second after application of shear stress, the rest of the
images shown were captured at intervals of 140 mseconds. ATP imaging was performed after the Ca2+ imaging of the same cells. Each ATP image was created by
integrating luminescence for 1 second after the application of shear stress. Broken lines represent the cell outlines obtained from the DIC images. Five pairs of images
demonstrate colocalization of the localized ATP release and sites of initiation of the Ca2+ waves. Quantitative analysis of the rising phase of ATP release and [Ca2+]i
increase showed that ATP release always preceded the [Ca2+]i increase. Values of [Ca2+]i are expressed as a ratio of Fluo-4 fluorescence (DF:F0) to the control before
application of shear stress. Open rectangles indicate the duration of shear stress (10 dynes/cm2 for 3 seconds), and the left end of each rectangle corresponds to time
zero. These five sets of images were selected because they are representative of dozens of images, all of which showed similar results. (B) Role of ATP release in the
shear-stress-induced [Ca2+]i increase. Treatment of cells with angiostatin, a known blocker of ATP release, abolished the ATP release and [Ca2+]i increase, and both
were restored by removing the angiostatin (by flushing). This suggests that the ATP release triggered the Ca2+ increase. Values are means¡s.d. of data obtained from
24 cells in three separate experiments. *P,0.01 angiostatin vs after flushing.
1 mg/ml biotin–luciferase for 30 minutes at room temperature. To observe the Shear-stress stimulation and ATP imaging
distribution of biotin–luciferase on the cell surface, cells were immunostained with A coverslip on which luciferase-labeled cells had been cultured was placed in a
FITC-conjugated anti-luciferase antibody (10 mg/ml, Rockland). parallel-plate flow chamber whose temperature can be controlled (FCS2,Visualization of endothelial ATP release 3483
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We acknowledge Akira Kamiya for his invaluable support in our
Stout, C. E., Costantin, J. L., Naus, C. C. and Charles, A. C. (2002). Intercellular
work and thank Yuko Sawada for technical assistance. calcium signaling in astrocytes via ATP release through connexin hemichannels. J.
Biol. Chem. 277, 10482-10488.
Funding Wang, Z., Haydon, P. G. and Yeung, E. S. (2000). Direct observation of calcium-
independent intercellular ATP signaling in astrocytes. Anal. Chem. 72, 2001-2007.
This work was partly supported by Grants-in-Aid for Scientific Yamamoto, K., Korenaga, R., Kamiya, A. and Ando, J. (2000a). Fluid shear stress
Research (Grant numbers S21220011 and B22300150) from the activates Ca2+ influx into human endothelial cells via P2X4 purinoceptors. Circ. Res.
Ministry of Education, Culture, Sports, Science and Technology to 87, 385-391.
J.A. and K.Y, and Grant-in-Aid from the Japan Science and Yamamoto, K., Korenaga, R., Kamiya, A., Qi, Z., Sokabe, M. and Ando, J. (2000b).
P2X4 receptors mediate ATP-induced calcium influx in human vascular endothelial
Technology Agency to K.Y. (Grant number 7815). cells. Am. J. Physiol. Heart Circ. Physiol. 279, H285-H292.
Yamamoto, K., Sokabe, T., Ohura, N., Nakatsuka, H., Kamiya, A. and Ando, J.
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