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Am J Physiol Heart Circ Physiol 293: H1646-H1653, 2007. First published June 1, 2007; doi:10.1152/ajpheart.01385.2006
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Involvement of cell surface ATP synthase in flow-induced ATP release by vascular endothelial cells

Kimiko Yamamoto,1,2 Nobutaka Shimizu,1 Syotaro Obi,1 Shinichiro Kumagaya,1 Yutaka Taketani,3 Akira Kamiya,1 and Joji Ando1

1Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, Tokyo; 2Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama; and 3Department of Clinical Nutrition, Institution of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan

Submitted 20 December 2006 ; accepted in final form 29 May 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Endothelial cells (ECs) release ATP in response to shear stress, a mechanical force generated by blood flow, and the ATP released modulates EC functions through activation of purinoceptors. The molecular mechanism of the shear stress-induced ATP release, however, has not been fully elucidated. In this study, we have demonstrated that cell surface ATP synthase is involved in shear stress-induced ATP release. Immunofluorescence staining of human pulmonary arterial ECs (HPAECs) showed that cell surface ATP synthase is distributed in lipid rafts and co-localized with caveolin-1, a marker protein of caveolae. Immunoprecipitation indicated that cell surface ATP synthase and caveolin-1 are physically associated. Measurement of the extracellular metabolism of [3H]ADP confirmed that cell surface ATP synthase is active in ATP generation. When exposed to shear stress, HPAECs released ATP in a dose-dependent manner, and the ATP release was markedly suppressed by the membrane-impermeable ATP synthase inhibitors angiostatin and piceatannol and by an anti-ATP synthase antibody. Depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin (MbetaCD) disrupted lipid rafts and abolished co-localization of ATP synthase with caveolin-1, which resulted in a marked reduction in shear stress-induced ATP release. Pretreatment of the cells with cholesterol prevented these effects of MbetaCD. Downregulation of caveolin-1 expression by transfection of caveolin-1 siRNA also markedly suppressed ATP-releasing responses to shear stress. Neither MbetaCD, MbetaCD plus cholesterol, nor caveolin-1 siRNA had any effect on the amount of cell surface ATP synthase. These results suggest that the localization and targeting of ATP synthase to caveolae/lipid rafts is critical for shear stress-induced ATP release by HPAECs.

THE VASCULAR ENDOTHELIAL CELLS (ECs) that line vessels are constantly exposed to shear stress, a mechanical force generated by flowing blood. ECs recognize shear stress and transmit the signal into the interior of the cell, where it triggers cell responses that involve changes in a variety of cell functions (10). For example, in response to shear stress, ECs increase production of vasodilators, such as nitric oxide (NO) and prostacyclin, and cell surface expression of an anti-thrombotic molecule, thrombomodulin. Many of the alterations of EC functions by shear stress are accompanied by changes in expression of related genes (2). These EC responses to shear stress are thought to play important roles in blood flow-dependent phenomena, such as angiogenesis, vascular remodeling, and atherogenesis, as well as in the homeostasis of circulatory functions. At present, however, shear stress signal transduction is not fully understood.

Ca2+ signaling plays an important role in shear stress signal transduction. Our previous studies demonstrated that a shear stress-dependent Ca2+ influx occurs in human pulmonary arterial ECs (HPAECs) when exposed to flow, and that the ATP-gated P2X4 ion channel is the major contributor to flow-induced Ca2+ influx (29, 30). Mice lacking P2X4 channels do not show normal EC responses to flow, such as Ca2+ influx and subsequent production of NO, and as a result they exhibit impaired flow-dependent control of vascular tone and remodeling (31). We have also shown that flow-induced activation of P2X4 requires ATP, which is supplied in the form of endogenous ATP released by ECs (32). The molecular mechanism of the shear stress-dependent ATP release by ECs, however, remains unclear.

F1Fo ATP synthase (referred to as ATP synthase below) was thought to be located exclusively in the mitochondria, but it has recently been identified on the cell surface of many types of cells, including human tumor cells, hepatocytes, keratinocytes, adipocytes, and ECs (7, 9). However, we are only beginning to understand the functions of cell surface ATP synthase. ATP generated by cell surface ATP synthase may be transported into the cells and provide an additional energy source, or it may act through the P2X/P2Y purinoceptors, thereby activating Ca2+-dependent signaling cascades that modulate cell functions. Since ATP synthase has a proton-transporting function that regulates intracellular pH, cell surface ATP synthase may play important roles in cell homeostasis and pathological phenomena. Cell surface ATP synthase has been found to be enzymatically active in the synthesis of ATP in human umbilical vein ECs (HUVECs) and to be involved in sustaining cell proliferation (3, 19, 20), and inhibition of cell surface ATP synthase activity with the ATP synthase inhibitor angiostatin reduced proliferation by HUVECs (19, 20). Our previous study (32) showed that angiostatin suppresses flow-induced ATP release by HPAECs, and based on that finding, we hypothesized that cell surface ATP synthase plays a role in the process of ATP release. To test our hypothesis, we investigated whether HPAECs possess cell surface ATP synthase that is active in ATP synthesis and, if so, the role of the cell surface ATP synthase in flow-induced ATP release.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Cell culture. HPAECs were obtained from Clonetics and grown on a 1% gelatin-coated tissue culture flask in M199 supplemented with 15% FBS, 2 mmol/l L-glutamine (Gibco), 50 µg/ml heparin, and 30 µg/ml EC growth factor (Becton Dickinson). The cells used in the present experiments were in the 7th and 10th passages.

Flow loading experiments. A parallel plate-type apparatus was used to apply laminar shear stress to the cells, as described previously (30). Briefly, one side of the flow chamber consisted of a 1% gelatin-coated glass plate on which the cultured HPAECs rested, and the other side consisted of a polycarbonate plate. Their flat surfaces were held 200 µm apart with a Teflon gasket. The chamber was provided with an entrance and an exit for the fluid, and the entrance was connected to an upper reservoir with a silicone tube. The exit was open to a lower reservoir. The flow was driven by gravitational force. The fluid (Hanks' balanced salt solution) passed from the upper reservoir through the flow chamber into the lower reservoir. The flow rate was monitored with an ultrasonic transit time flow meter (HT107, Transonic Systems) placed at the entrance, and it was controlled by changing the difference in height between the upper reservoir and the exit of the flow chamber. The intensity of the shear stress ({tau}, dynes/cm2) acting on the EC layer was calculated by using the formula {tau} = 6µQ/a2b, where µ is the viscosity of the perfusate (poise), Q is the flow volume (ml/s), and a and b are the cross-sectional dimensions of the flow path (cm).

Quantification of extracellular ATP. The ATP released by the HPAECs was measured by means of a luciferin-luciferase assay. The cells were exposed to laminar flow in a parallel plate flow chamber at 37°C. The intensity of shear stress was increased every minute in a stepwise manner from 0 to 3, 8, and 15 dynes/cm2 by increasing the volumetric flow rate from 0 to 2.2, 5.8, and 10.9 ml/min, respectively. Since the perfusion was performed in an open circuit, there was no ATP accumulation in the system. The effluent that accumulated in the lower reservoir was collected every minute, and 100 µl of the fluid were applied to the ATP assay system (Toyo, Tokyo, Japan). The ATP assay mixture (luciferase, D-luciferin, and bovine serum albumin) was injected into each sample, and its relative light intensity was recorded for 10 s in a Lumat LB 9501 luminometer (Berthold) at room temperature. A calibration curve for ATP concentrations was obtained for each experiment by using the same batch of luciferin-luciferase reagents. Since the perfusion had a dilution effect on ATP, the ATP concentrations were calculated accordingly.

Measurement of the extracellular metabolism of 3H-labeled adenine nucleotide. HPAECs were seeded onto gelatin-coated 24-well tissue culture plates at a density of 5 x 104 cells per well. Twenty-four hours later, confluent HPAECs were rinsed twice with RPMI 1640 medium and then incubated at 37°C with gentle orbital rotation in a starting volume of 0.25 ml of RPMI 1640 containing 50 µM [3H]ADP as initial substrates. Aliquots of the mixture were periodically applied to an Alugram TLC sheet (~5 x 104 dpm per spot), and adenine nucleotides and adenosine were separated with an appropriate solvent system as described previously (34). Radioactive areas that co-migrated with respective nucleotide/nucleoside standards were scraped into scintillation vials, extracted from silica with 0.1 N HCl, and quantified by scintillation counting with a Wallac-1409 beta-spectrometer.

Immunohistochemistry. HPAECs on the coverslip were fixed with 4% paraformaldehyde (Sigma) and maintained in 1% normal bovine serum albumin (Sigma) to block nonspecific protein-binding sites. The cells were incubated with antibodies (5 µg/ml) against the beta-subunit of ATP synthase (Molecular Probes), caveolin-1 (Transduction Laboratories), or Alexa Fluor 594-conjugated cholera toxin subunit B (Molecular Probes). After a washing, they were incubated with Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 594 goat anti-rabbit IgG (Molecular Probes) at a dilution of 1:500. Stained cells were photographed through a confocal fluorescence microscope (Leica), and all images were imported into Adobe Photoshop as JPEGs for contrast manipulation and figure assembly.

Purification of caveola fractions. Caveola membranes were prepared by the detergent-free method as described previously (24). Briefly, cells were homogenized with the Teflon homogenizer (Wheaton) and centrifuged at 1,000 g for 10 min. The postnuclear supernatant fraction (PNS) was collected, and the plasma membrane fraction was isolated by 30% Percoll-gradient fractionation (84,000 g for 30 min) and sonicated six times for 5 s each. Caveola membranes were separated by the OptiPrep density gradients, a continuous 23% to 10% gradient followed by centrifugation (52,000 g for 90 min) against a discontinuous gradient to concentrate the lightest material. Proteins from each fraction were analyzed by Western blot.

Immunoprecipitation and Western blot analysis. Immunoprecipitation of caveolin-associated proteins was performed as previously described (27). Briefly, equal amounts of protein in the caveolae-enriched fraction were diluted with RIPA buffer and precleared by incubation for 2 h with protein G-Sepharose beads (Amersham). Protein G-Sepharose beads were incubated with polyclonal anti-caveolin-1 antibody (BD Transduction Laboratories) for 2 h, and, after the beads were washed with RIPA buffer, precleared supernatants were incubated with the beads overnight at 4°C with gentle mixing. The immune complexes were collected by centrifugation at 2,000 g for 5 min, washed, and eluted in SDS-PAGE sample buffer.

Western blot analysis was performed as previously described (32). Briefly, cells were dissolved in RIPA buffer and centrifuged at 26,000 g for 30 min. A 30-µg sample of total cell lysate was resolved in an SDS-PAGE gel and then transferred to an Immobilon membrane (Millipore) and incubated for 1 h. The membrane was probed with the anti-ATP synthase beta-subunit antibody (4 µg/ml) or anti-caveolin-1 antibody (3 µg/ml) and then incubated with anti-rabbit IgG horseradish peroxidase-conjugated antibody. The same membrane was reprobed with monoclonal anti-beta-actin antibody (Abcam). The densitometry values of the ATP synthase blots and caveolin-1 blots were standardized to those of the beta-actin blots.

Small interfering RNA preparation and transfection. Targeted small interfering RNA (siRNA) was used to knock down caveolin-1 expression in HPAECs as described previously (13). A DNA fragment flanked by the BamH I and HindIII sites containing the sense target sequence corresponding to bases 167–186 from the open reading frame of the human caveolin-1 (GenBank accession no. NM001753) (5'-CTAAACACCTCAACGATGA-3'), the hairpin loop sequence (5'-CTGTGAAGCCACAGATGGG-3'), and the antisense target sequence (5'-TCATCGTTGAGGTGTTTAG-3') were synthesized and inserted between the human U6 promoter and the terminator sequences of pBAsi-hU6 (Takara) to generate a stem-loop type of siRNA in transfected cells. The randomized sequence 5'-TAACATGAACCACGA CTAC-3' was used to construct the vector for a negative control. HPAECs were treated with the construct using Lipofectamine 2000 (Invitrogen) as the transfection reagent, and experiments were conducted 48 h after transfection.

Statistical analysis. All results are expressed as means ± SD. Statistical significance was evaluated by an ANOVA and a Bonferonni adjustment applied to the results of a t-test performed with SPSS software (SPSS). P values <0.01 were regarded as statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cell surface ATP synthase is distributed in lipid rafts and co-localized with caveolin-1. HPAECs were immunostained with anti-ATP synthase beta-subunit antibody to determine whether they possess cell surface ATP synthase. It should be noted that the cells were not permeabilized for immunofluorescence. Photomicrographs showed that the ATP synthase beta-subunit was distributed on the surface of the HPAECs and was concentrated at localized regions at the cell edges (Fig. 1A). Similar findings were obtained in the cells immunostained with anti-ATP synthase {alpha}-subunit antibody (data not shown). Since the punctate staining pattern of ATP synthase resembles that of caveolae/lipid rafts, HPAECs were immunostained with an antibody against cholera toxin subunit B, a ganglioside-binding protein used as lipid raft landmarker, or with an antibody against caveolin-1, a key constituent protein of caveolae. The photomicrographs showed that ATP synthase was co-localized with caveolin-1 and cholera toxin, suggesting that cell surface ATP synthase is localized in caveolae/lipid rafts.


Figure 1
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  		Fig. 1. Surface localization of ATP synthase, caveolin-1, and lipid rafts on human pulmonary arterial endothelial cells (HPAECs). A: immunofluorescence photomicrograph. Nonpermeabilized HPAECs were fixed and stained with antibodies against ATP synthase beta-subunit, caveolin-1, or a lipid raft marker, cholera toxin subunit B. Co-localization of ATP synthase with caveolin-1 and cholera toxin is visible in the merged images as yellow areas. The confocal microscope images of single cells at a higher magnification also show co-localization of ATP synthase with caveolin-1. Representative images are shown; n = 22. B: Western blot (WB) analysis. Five fractions were used in the immunoblot assay: postnuclear supernatant (PNS), cytosol (Cyt), plasma membrane (PM), caveolae-rich membrane (CM), and noncaveola membrane (NCM). A 25-µg protein sample from each fraction was separated by SDS-PAGE and immunoblotted with antibody against ATP synthase beta-subunit or caveolin-1. The CM fraction, but not the NCM fraction, contained both ATP synthase and caveolin-1, indicating co-localization of ATP synthase and caveolin-1. The results of 3 independent experiments were similar. C: co-immunoprecipitation of ATP synthase with caveolin-1. The CM fraction was incubated with [immunoprecipitation (IP) fraction] or without (control) polyclonal anti-caveolin-1 antibody (caveolin-1 pAb) overnight at 4°C. The immunocomplexes were assessed by gel electrophoresis and Western blot (WB) analysis against monoclonal anti-ATP synthase beta-subunit and anti-caveolin-1 antibodies. Experiments were repeated 3 times, with similar results.

 
To confirm the localization of ATP synthase in caveolae/lipid rafts, detergent-resistant caveolae-rich membranes were isolated and analyzed by immunoblotting with anti-ATP synthase beta-subunit antibody and anti-caveolin-1 antibody. The caveolae-rich membrane fraction contained both ATP synthase beta-subunit and caveolin-1, whereas the noncaveola fraction contained neither (Fig. 1B). This means that ATP synthase is localized in the caveolae.

To determine whether ATP synthase in HPAECs is physically associated with caveolin-1, we performed co-immunoprecipitation experiments. The caveolae-rich fraction was incubated with a polyclonal anti-caveolin-1 antibody to precipitate caveolin-1 and its associated proteins. The immune complexes were collected with protein G beads and analyzed by immunoblotting against monoclonal anti-ATP synthase and anti-caveolin-1 antibodies. These results indicated that the ATP synthase in the caveolae-rich fraction was co-precipitated with caveolin-1, suggesting that cell surface ATP synthase and caveolin-1 are physically associated (Fig. 1C).

Cell surface ATP synthase is active in extracellular ATP generation. To explore the function of cell surface ATP synthase, HPAECs were incubated with [3H]ADP and analyzed for subsequent ATP production by TLC. ATP generation was detected within 1 min after incubation, peaked at 5 min, and then declined (Fig. 2, A and B). Angiostatin, a membrane-impermeable ATP synthase inhibitor, significantly inhibited the production of [3H]ATP. No labeled product was detected within the cell pellets, confirming that the ATP had been synthesized on the cell surface (Fig. 2C). These findings indicate that the cell surface ATP synthase of HPAECs functions to synthesize ATP.


Figure 2
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  		Fig. 2. Extracellular ATP generation by cell surface ATP synthase. A: TLC assay. HPAECs were incubated with [3H]ADP, and, after incubation for the periods indicated, aliquots of medium were spotted on Alugram TLC sheets and analyzed by TLC. Autoradiography showed [3H]ATP formation on the cell surface (control). [3H]ATP formation was markedly suppressed by angiostatin (1 µM), a membrane-impermeable ATP synthase inhibitor, indicating that cell surface ATP synthase is active in ATP synthesis. The first lane shows the radiochemical purity of [3H]ADP after a 10-min incubation in the absence of HPAECs. B: quantitative densitometry analysis. Graph shows relative amounts of [3H]ATP in the absence (open bars) and presence (solid bars) of angiostatin. All values are means ± SD of 4 separate experiments. *P < 0.01 vs. control. C: radioactivity inside or outside the cells. Aliquots of the medium or cell lysate were collected during incubation of the cells with [3H]ADP, and their radioactivity (dpm) was quantified with a scintillation counter. No 3H radioactivity was detected in the cell lysate, indicating that [3H]ATP was produced on the cell surface of HPAECs.

 
Cell surface ATP synthase is involved in flow-induced ATP release. HPAECs were exposed to flow, and the concentration of ATP in the perfusate was measured by a bioluminescence assay. The ATP concentration increased in response to flow and became increasingly greater with the intensity of the shear stress (Fig. 3). ATP synthase inhibitors, such as angiostatin and piceatannol (36), or anti-ATP synthase beta-subunit antibody markedly suppressed the shear stress-dependent ATP release by the HPAECs, but none of these inhibitors had any effect on the intracellular ATP concentration, suggesting that none of these inhibitors crossed the cell membrane and inhibited mitochondrial ATP synthase. These findings indicate that cell surface ATP synthase plays a critical role in the shear stress-dependent ATP release by HPAECs.


Figure 3
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  		Fig. 3. Extracellular ATP release in response to flow. The intensity of shear stress was increased every minute in a stepwise manner, and the effluent that accumulated every minute was applied to ATP measurement. ATP concentrations in the perfusate (Hanks' balanced salt solution) were measured by luminometry. When HPAECs were exposed to shear stress, ATP release increased in a dose-dependent manner (control). Angiostatin (1 µM), piceatannol (20 µM), or anti-ATP synthase beta-subunit antibody (250 ng/ml) significantly suppressed the shear stress-dependent ATP release. All values are means ± SD of 5 different experiments. *P < 0.01 vs. control. Inset: intracellular ATP concentration. Angiostatin, piceatannol, or anti-ATP synthase beta-subunit antibody did not change the intracellular ATP concentration. Exposure to shear stress (15 dynes/cm2) for 3 min had no effect on the intracellular ATP concentration. Static, cells cultured under static conditions. Results are shown as means ± SD of 5 separate samples.

 
Depletion of plasma membrane cholesterol inhibits flow-induced ATP release. Next, we investigated whether the localization of ATP synthase in caveolae/lipid rafts is critical for flow-induced ATP release to occur. After treating HPAECs with methyl-beta-cyclodextrin (MbetaCD), which disrupts lipid rafts by depleting plasma membrane cholesterol, we examined the cells for changes in the distribution of ATP synthase, cholera toxin, and caveolin-1 by confocal immunofluorescence microscopy. Treatment with MbetaCD abolished the number and size of the lipid rafts and resulted in redistribution of ATP synthase and caveolin-1 (Fig. 4A). ATP synthase and caveolin-1 were distributed throughout the cells in a diffuse pattern, and an outline-like accumulation of caveolin-1 was visible some distance away from the cell edge. These MbetaCD effects were prevented by cholesterol pretreatment. In HPAECs incubated with cholesterol for 24 h before MbetaCD treatment, lipid rafts appeared, and both ATP synthase and caveolin-1 were distributed in the same localized regions at the cell edge. Western blotting revealed that neither MbetaCD nor MbetaCD plus cholesterol changed the amount of ATP synthase or caveolin-1 in either the cells as a whole or their plasma membrane (Fig. 4B). MbetaCD markedly suppressed the flow-induced ATP release by HPAECs, and it was almost completely prevented by cholesterol pretreatment (Fig. 4C). Co-immunoprecipitation showed that MbetaCD abolished the physical association between ATP synthase and caveolin-1, whereas shear stress had no effect (Fig. 4D). These results suggest that the localization of ATP synthase in caveolae/lipid rafts and its association with caveolin-1 are required for shear stress-dependent ATP release by HPAECs.


Figure 4
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  		Fig. 4. Effect of membrane cholesterol depletion on the localization of ATP synthase, lipid rafts, and caveolin-1 and on flow-induced ATP release. A: immunofluorescence photomicrograph. HPAECs were treated with 10 mM methyl-beta-cyclodextrin (MbetaCD) or pretreated with 1.3 mM cholesterol and then with 10 mM MbetaCD. Cells were then washed in PBS, fixed, and immunostained with antibodies against ATP synthase beta-subunit, cholera toxin subunit B, or caveolin-1. MbetaCD disrupted the lipid rafts of the HPAECs and abolished the co-localization of ATP synthase with caveolin-1. Cholesterol pretreatment prevented these effects of MbetaCD. Representative images are shown; n = 16. B: Western blot analysis. Neither MbetaCD nor MbetaCD plus cholesterol changed the amount of ATP synthase or caveolin-1 proteins in the PNS fraction or in the PM fraction. The cytoskeletal protein beta-actin serves as a protein loading control. C: flow-induced ATP release. The intensity of shear stress was increased every minute in a stepwise manner, and the effluent that accumulated every minute was applied to ATP measurement. MbetaCD markedly suppressed the shear stress-dependent ATP release, but the suppression was prevented by cholesterol pretreatment. Results are shown as means ± SD of 6 separate samples. *P < 0.01 vs. control. D: co-immunoprecipitation of ATP synthase and caveolin-1. HPAECs were cultured under static conditions (control), treated with 10 mM MbetaCD, or exposed to shear stress (15 dynes/cm2) for 3 min. Three fractions were used in the immunoblot assay: PNS, PM, and CM (for abbreviations, see legend to Fig. 1). MbetaCD abolished the association between ATP synthase and caveolin-1, but shear stress had no effect. Experiments were repeated 3 times, with similar results.

 
Downregulation of caveolin-1 expression inhibits flow-induced ATP release. To further examine the role of caveolin-1 in the cell surface ATP synthase-mediated ATP release, we used siRNA to specifically knock down expression of caveolin-1. Transfection of HPAECs with caveolin-1 siRNA resulted in a significant and specific reduction in caveolin-1 protein expression, as judged by both immunofluorescent staining and Western blotting (Fig. 5, A and B). The cells transfected with caveolin-1 siRNA showed a clear loss of staining intensity compared with control cells (subjected to transfection conditions alone). Western blotting showed a marked reduction of caveolin-1, but not of ATP synthase, in both the PNS and plasma membrane fractions, and the level of caveolin-1 protein expression declined to nearly 15% of the control at 48 h posttransfection (Fig. 5C). Downregulation of caveolin-1 markedly suppressed the shear stress-dependent ATP release by HPAECs (Fig. 5D). These findings suggest that the association of ATP synthase with caveolin-1 is critical for flow-induced ATP release by HPAECs.


Figure 5
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  		Fig. 5. Effect of caveolin-1 knockdown on the localization of ATP synthase and caveolin-1 and on flow-induced ATP release. A: immunofluorescence photomicrograph. Nonpermeabilized HPAECs transfected with caveolin-1 siRNA were fixed and stained with antibodies against ATP synthase beta-subunit or caveolin-1. Caveolin-1 siRNA markedly decreased cell surface caveolin-1. Transfection of disordered siRNA with a scrambled nucleotide sequence had no effect on caveolin-1 expression (data not shown). B: Western blot analysis. Control cells were treated with the transfection reagent alone without siRNA (control). Caveolin-1 siRNA transfection significantly decreased caveolin-1 protein in the PNS and PM fractions. The cytoskeletal protein beta-actin serves as a protein loading control. C: quantitative densitometry analysis. Results are shown as means ± SD of 6 separate samples. *P < 0.01 vs. control. D: flow-induced ATP release. The intensity of shear stress was increased every minute in a stepwise manner, and the effluent that accumulated every minute was applied to ATP measurement. Caveolin-1 siRNA transfection markedly suppressed the shear stress-dependent ATP release. Experiments were performed 48 h after transfection. Results are shown as means ± SD of 6 separate samples. *P < 0.01 vs. control.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of this study demonstrated that HPAECs express cell surface ATP synthase that is active in extracellular ATP synthesis. Inhibition of cell surface ATP synthase with angiostatin, piceatannol, or anti-ATP synthase antibody markedly reduced flow-induced ATP release, and since none of these inhibitors crosses the plasma membrane, they have no effect on mitochondrial ATP synthase activity. Thus our results suggest that cell surface ATP synthase is involved in flow-induced ATP release by HPAECs. The cell surface ATP synthase was found to be localized in the caveolae/lipid rafts of HPAECs, consistent with the findings obtained in HUVECs, hepatocytes, and adipocytes (4, 17, 21, 26). Immunoprecipitation results indicated that the cell surface ATP synthase and caveolin-1 are physically associated. Depletion of plasma membrane cholesterol with MbetaCD disrupted lipid rafts and abolished the association of ATP synthase with caveolin-1, which led to marked suppression of flow-induced ATP release. Downregulation of expression of caveolin-1 by siRNA also markedly inhibited flow-induced ATP release. Taken together, these findings show that the localization of ATP synthase in caveolae/lipid rafts and its association with caveolin-1 are critical for shear stress-induced ATP release by HPAECs.

A variety of types of cells, including epithelial cells, fibroblasts, and ECs, release ATP in response to mechanical stresses (5, 12, 14, 28). However, the molecular mechanisms of mechanical stress-induced ATP release have not been fully elucidated (15). One possibility is that cells release ATP through a process called exocytosis in which intracellular vesicles containing ATP fuse with plasma membranes and release ATP into the extracellular space. Bodin and Burnstock (6) demonstrated that shear stress-induced ATP release by HUVECs is vesicular, because ATP release was significantly reduced by monensin, an inhibitor of vesicle formation at the level of the Golgi apparatus, and by N-ethylmaleimide, an inhibitor of vesicle fusion to the plasma membrane. Another possible mechanism is ATP-binding cassette (ABC) protein-mediated ATP export across the plasma membrane. ABC proteins, such as the cystic fibrosis transmembrane conductance regulator (a cAMP-activated ATP-dependent Cl– channel), P-glycoprotein (a channel transporting both Cl– and ATP out of the cell), and the ATP-sensitive K+ channel, have been identified in ECs (8, 16, 25). Although the present study did not address these two mechanisms, vesicular exocytosis or ABC transporters may be involved in flow-induced ATP release by HPAECs. The results of the present study clearly demonstrate a novel mechanism for flow-induced ATP release mediated by cell surface ATP synthase. It remains unknown, however, whether cell surface ATP synthase interacts with ATP-releasing mechanisms via vesicular transport or ABC proteins.

Vascular ECs express a variety of ecto-enzymes that contribute to the metabolism and/or interconversion of extracellular adenine nucleotides. Thus the ATP concentration of the perfusate is thought to represent the net change that occurs as a result of the release of ATP, conversion of nucleotides to ATP, and degradation of ATP, not just the release of ATP. Our flow experiments were done in an open circulation system, and the perfusate was not recirculated. Since flow quickly removes ATP from the cell surface, it seems difficult to evaluate the degree to which ecto-enzymes degrade endogenously released ATP. Yegutkin et al. (33) demonstrated that stimulation of HUVECs by shear stress induced concomitant release of endogenous ATP and soluble ecto-enzymes that degrade both ATP (ATPase) and AMP (5'-nucleotidase). Thus soluble ecto-enzymes that were released into the effluent may have affected the ATP concentration in the effluent. Although the present study did not extend to the kinetics of ATP generation and degradation, quantitative understanding of the kinetics would provide better insight into the role of cell surface ATP synthase in shear stress-induced ATP release by ECs.

Lipid rafts are specialized membrane microdomains that are very rich in glycosphingolipids and cholesterol, unlike other membrane regions, and caveolae are a subset of lipid rafts characterized by the presence of the protein caveolin-1 and plasma membrane invaginations (1). Caveolae/lipid rafts are endowed with signaling functions because various receptors and mediators for signal transduction are clustered in them (23). There is accumulating evidence in support of the concept that caveolae/lipid rafts have the general function of cell surface mechanotransduction sites within the plasma membrane. Caveolae/lipid rafts must be intact for shear stress signal transduction to occur (35). Disruption of caveolae/lipid rafts by MbetaCD blocks the activation of extracellular signal-regulated kinase (ERK) induced by shear stress in bovine aortic ECs (BAECs) (22). Cyclosporine A decreases the cholesterol content of the plasma membrane and also suppresses shear stress-mediated activation of endothelial NO synthase (eNOS) in BAECs (18). Depletion of plasma membrane cholesterol in human fetal osteoblasts by MbetaCD significantly dampens hydrostatic pressure- and shear stress-induced mechanotransduction, including protein tyrosine phosphorylation, activation of ERK, and enhanced expression of c-fos (11). In the present study, we found that MbetaCD markedly inhibited shear stress-dependent ATP release by HPAECs. The inhibitory effect was prevented by the pretreatment of the cells with cholesterol. These findings suggest that intact caveolae/lipid rafts are required for ATP synthase-mediated ATP release in response to shear stress. Thus the cell surface ATP synthase localized at caveolae/lipid rafts may be involved in shear stress signal transduction through activation of P2X/P2Y purinoceptors rather than directly contributing to ATP release by producing ATP on the cell surface. Further study is needed to clarify the true role of cell surface ATP synthase in shear stress-induced ATP release by ECs.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by Grants-in-Aid for Scientific Research on Priority Areas and from the Ministry of Education, Culture, Sports, Science and Technology and by a research grant for cardiovascular diseases from the Japanese Ministry of Health, Labor and Welfare.


   ACKNOWLEDGMENTS
 
We thank Yuko Sawada for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Ando, Dept. of Biomedical Engineering, Graduate School of Medicine, Univ. of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan (e-mail: joji{at}m.u-tokyo.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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