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Endogenously released ATP mediates shear stress-induced Ca²⁺ influx into pulmonary artery endothelial cells

¹Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo; and ²Interdisciplinary Science Center, Nihon University, Tokyo 113-0033, Japan

Submitted 31 December 2002 ; accepted in final form 22 April 2003

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
The mechanisms by which flow-imposed shear stress elevates intracellular Ca²⁺ in cultured endothelial cells (ECs) are not fully understood. Here we report finding that endogenously released ATP contributes to shear stress-induced Ca²⁺ responses. Application of flow of Hanks' balanced solution to human pulmonary artery ECs (HPAECs) elicited shear stress-dependent increases in Ca²⁺ concentrations. Chelation of extracellular Ca²⁺ with EGTA completely abolished the Ca²⁺ responses, whereas the phospholipase C inhibitor U-73122 or the Ca²⁺-ATPase inhibitor thapsigargin had no effect, which thereby indicates that the response was due to the influx of extracellular Ca²⁺. The Ca²⁺ influx was significantly suppressed by apyrase, which degrades ATP, or antisense oligonucleotide targeted to P2X₄ purinoceptors. A luciferase luminometric assay showed that shear stress induced dose-dependent release of ATP. When the ATP release was inhibited by the ATP synthase inhibitors angiostatin or oligomycin, the Ca²⁺ influx was markedly suppressed but was restored by removal of these inhibitors or addition of extracellular ATP. These results suggest that shear stress stimulates HPAECs to release ATP, which activates Ca²⁺ influx via P2X₄ receptors.

hemodynamic force; mechanotransduction; ATP synthase; purinoceptors; and P2X₄ receptor

Shear stress has been demonstrated to activate a variety of molecules known to function in signal transduction including ion channels, G proteins, inositol trisphosphate, and many protein kinases (8), which suggests that multiple pathways are involved in shear stress signal transduction. However, it remains unclear which pathways are primary and which are secondary, and the initial sensor that recognizes shear stress has yet to be identified. Shear stress may activate several pathways simultaneously.

Ca²⁺ signaling has been demonstrated to be involved in shear stress signal transduction. ECs show an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in response to shear stress (1, 4), and, at a certain extracellular ATP concentration, shear stress induces a dose-dependent influx of extracellular Ca²⁺ across the cell membrane (3). A subtype of ATP-gated cation channel, the P2X₄ purinoceptor, has even been shown to mediate the shear stress-dependent Ca²⁺ influx (26, 27). Nevertheless, reports show that Ca²⁺ responses to shear stress occur even in the absence of extracellular ATP (11, 23), and the role of ATP in shear stress-induced Ca²⁺ responses is a matter of controversy (9, 18).

ECs are known to synthesize, store, and release a variety of vasoactive substances in response to shear stress. ATP has been shown to be rapidly released by cultured ECs and vascular beds in response to increases in shear stress (6, 13, 17). Thus endogenously released ATP may modify the flow-induced Ca²⁺ responses, but this issue has not yet been fully investigated. Here we report for the first time that endogenously released ATP mediates shear stress-induced Ca²⁺ responses in human pulmonary artery ECs (HPAECs).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

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

Flow stimulation and Ca²⁺ measurement. HPAECs were seeded at a density of 1.5 x 10⁴ cells/cm² on a coverslip with a surface area of 3.7 cm² and were cultured for 3 days to form a confluent monolayer that reached a density of 4.5 x 10⁴ cells/cm². The cells were loaded with 5 µmol/l indo 1-acetoxymethyl ester (indo 1-AM; Dojindo), the coverslip was placed in a parallel plate-type flow chamber (FSC2, Bioptechs), and the flow chamber was set on the stage of an inverted microscope (Diaphot 300, Nikon). One end of the chamber was connected to a reservoir filled with Hanks' balanced salt solution (HBSS) via a silicon tube, and the cells were exposed to a stepwise increase in flow rate at 37°C by changing the height of the reservoir and monitoring the flow rate at the outflow tube with a flow sensor (Transonic Systems). The intensity of shear rate (, measured in s^–1) and shear stress (, in dyn/cm²) to the cells were calculated as follows: = 6/ab² and = µ, where is volume flow (in ml/s), a and b are cross-sectional dimensions (in cm) of the flow path, and µ is the viscosity of the perfusate (in Poise). Changes in [Ca²⁺]_i were measured with a confocal laser scanning system (MRC-1000 UV, Bio-Rad) equipped with an ultraviolet argon ion laser as previously described (14). Briefly, light from the laser at a 351-nm wavelength was used to excite the cells through a x20 objective, and the emitted light was divided into 480- and 405-nm wavelengths (by a beam splitter) and counted with photomultipliers. The time course of the 405-to-480-nm fluorescence ratio (F₄₀₅/F₄₈₀) in cells of interest was monitored with the accessory time-course software of the Bio-Rad 1000 UV system.

ATP measurement. ATP released from HPAECs was measured by a luciferin-luciferase assay. The cells were exposed to a stepwise increase in flow rate under the same conditions of Ca²⁺ measurement at 37°C, and, after the perfusate was collected every 20 s, 100-µl aliquots were applied to the ATP assay system (Toyo Ink; Tokyo, Japan). The ATP assay mixture (luciferase, D-luciferin, and BSA) was injected into each sample, and its relative light intensity was recorded for 10 s in a Lumat LB 9501 luminometer (Berthold; Wildbad, Germany) at room temperature. A calibration curve for ATP concentrations was obtained for each experiment by using the same batch of luciferin-luciferase reagents, and the total moles of ATP released per second per 10⁶ cells were calculated.

Western blot analysis. Western blot analysis was performed as previously described (27). Briefly, cells were dissolved in RIPA buffer (1% Nonidet P-40, 20 mM Tris · HCl, 0.15 M NaCl, 0.5% sodium deoxycholate, 2 mM EDTA, 2 mM EGTA, 0.1% SDS, 0.2 mM Na₂MoO₄, 10 mM NaF, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml antipain, 5 µg/ml pepstatin A, and 0.2 U/ml aprotinin; pH 7.4) and centrifuged at 26,000 g for 30 min. A 30-µg sample of total cell lysate was resolved in SDS sample buffer (0.2 M Tris · HCl, 18% glycerol, 4% SDS, 0.01% bromophenol blue, and 10% -mercaptoethanol; pH 8.8) for SDS-PAGE and transferred to an Immobilon membrane (Millipore), where it was incubated for 1 h. The membrane was probed with the anti-P2X₄ antiserum (3 µg/ml) and then incubated with anti-rabbit IgG horseradish peroxidase-conjugated antibody. The same membrane was reprobed with monoclonal anti--actin antibody (Abcan). The densitometry values of the P2X₄ blots were standardized to those of the -actin blots. An antiserum against human P2X₄ receptor protein was generated in rabbits injected with a synthetic peptide (NH₂-RLYYREKKYKYVEDYC-COOH) comprising amino acid residues 364–378 of the sequences for the intracellular COOH-terminal domains of the cloned human P2X₄ receptor. The peptide was covalently linked to keyhole limpet hemocyanin, and rabbits were immunized by injection with the conjugated peptide every 2 wk for 8 wk. The anti-P2X₄ receptor antiserum was then affinity purified with synthetic peptide (P2X_{4 364–378}) immobilized on Sepharose 4B (Asahi Techno Glass).

Competitive PCR. Both sense and antisense primers were synthesized according to the gene-specific sequence (Table 1), and the RNA competitor fragments were obtained as previously described (27). With the use of these primers, DNA competitor fragments were generated and then transcribed into RNA fragments using SP6 RNA polymerase. Competitive PCR was performed by adding 2 µl of the mRNA samples obtained from HPAECs to 2 µl of different 10-fold dilutions of the RNA competitor fragments that ranged from 0.00008 to 800 attomol/µl. After reverse transcription, the PCR reactions were carried out using each target gene-specific primer in a solution that contained ExTaq DNA polymerase (Takara) and [-³²P]dCTP. A second series of competitive PCR assays was then carried out with consecutive 1:2 dilutions of competitors mixed with a constant amount of each target mRNA. The PCR products were separated by electrophoresis in a 5% polyacrylamide gel. The radioactivity of both target mRNA bands and competitor bands (known concentrations) was measured with a GS363 Molecular Imager System. The logarithm of the ratio of target to competitor bands was plotted as a function of the logarithm of the known amount of competitor. The concentration of target mRNA molecules present in cells corresponds to that of competitor at the competition equivalence point [log (target/competitor) = 0]. The -actin gene, which is a housekeeping gene, was used as a control for variation in the quality and quantity of RNA.

Real-time PCR analysis. Total RNA samples were prepared from HPAECs with Isogen (Nippon Gene; Tokyo). First-strand cDNAs were generated using Moloney murine leukemia virus reverse transcriptase (MMLV-RT; GIBCO-BRL) and RNA primed with oligo(dT) primer. After reverse transcription of RNA into cDNA, real-time PCR was used to monitor gene expression with a Smart Cycler (Cepheid) according to the standard procedure. PCR was performed with a TaKaRa ExTaq R-PCR version (Takara), SYBR green I (BioWhittaker), and the primer pairs (Table 1). The temperature profile included initial denaturation for 30 s at 95°C, followed by 35 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 15 s, elongation at 72°C, and fluorescence monitoring at 85°C. The specificity of the amplification reaction was determined by performing a melting-curve analysis. Standard curves for expression of each gene were generated by serial dilutions of known quantities of the respective cDNA gene template. Relative quantification of the signals was achieved by normalizing the signals of the different genes to GAPDH.

Antisense oligonucleotides. Antisense oligonucleotides (AS-oligos) targeted to the P2X₄ receptor and control scrambled oligos (S-oligos) were designed and synthesized by Biognostik (Göttingen, Germany; Ref. 26). The sequences of the phosphorothiorate AS- and S-oligos were 5'-CCTGAAATTGTAGCC-3' and 5'-TAATCGCTTCAGACG-3', respectively, and they were FITC labeled at the 5'-end. The AS- or S-oligos were transfected into cells with LipofectAMINE PLUS (GIBCO-BRL).

Statistical analysis. All results are expressed as means ± SD. Statistical significance was evaluated by an ANOVA and a Bonferonni's adjustment applied to the results of a t-test performed with SPSS software. P values <0.01 were regarded as statistically significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Flow induces a shear stress-dependent Ca²⁺ influx into HPAECs. HPAECs were exposed to flow of HBSS, and changes in [Ca²⁺]_i were monitored. [Ca²⁺]_i increased stepwise in tandem with the increases in shear stress, and, when flow was terminated, [Ca²⁺]_i returned to its basal level (Fig. 1A). Chelation of extracellular Ca²⁺ with EGTA completely abolished the flow-induced Ca²⁺ responses, whereas the phospholipase C inhibitor U-73122 or the Ca²⁺-ATPase inhibitor thapsigargin that induces Ca²⁺-store depletion had no effect (Fig. 1, B–D). When the [Ca²⁺]_i F₄₀₅/F₄₈₀ was plotted against shear stress (in dyn/cm²), a linear relationship between the two was observed in the control, but this relationship was absent in the presence of EGTA and was unaffected by U-73122 or thapsigargin. These results indicate that the shear stress-dependent increase in [Ca²⁺]_i was due to the influx of extracellular Ca²⁺ across the cell membrane.

View larger version (31K): [in this window] [in a new window]   Fig. 1. Flow-induced Ca2+ responses in human pulmonary artery endothelial cells (HPAECs). HPAECs loaded with indo 1-acetoxymethyl ester (indo 1-AM) were exposed to stepwise increases in shear stress to 3, 8, and 15 dyn/cm2, and the 405-to-480-nm fluorescence ratio (F405/F480), which reflects changes in intracellular Ca2+concentration ([Ca2+]i), was monitored. Each graph represents 12 Ca2+ responses by a group of 8–10 cells. A: stepwise increases in [Ca2+]i were observed in response to stepwise increments in shear stress. B: when extracellular Ca2+ was eliminated with EGTA (1 mmol/l), the Ca2+ response completely disappeared. C: phospholipase C inhibitor U-73122 (2 µmol/l) had no effect on the shear stress-dependent increases in [Ca2+]i. D: Ca2+-ATPase inhibitor thapsigargin (1 µmol/l) induces Ca2+ store depletion but had no effect on the shear stress-dependent increases in [Ca2+]i. Relationships between [Ca2+]i (F405/F480) and shear stress are shown (insets). F405/F480 values at the center of each step of the Ca2+ responses are given as means ± SD of 12 groups of 8–10 cells each. A linear regression analysis showed a good correlation between F405/F480 and shear stress. This relationship disappeared in the absence of extracellular Ca2+ [EGTA (+)] but was unaffected by U-73122 or thapsigargin.

Shear stress-induced Ca²⁺ influx is ATP dependent. HPAECs were exposed to apyrase (Enzyme Commission No. 3.6.1.5 [EC] ; Sigma grade III), which degrades ATP and ADP, and their Ca²⁺ responses to flow were examined. Apyrase markedly inhibited the shear stress-dependent Ca²⁺ influx in a dose-dependent manner (Fig. 2), which indicates that ATP and ADP are involved in the flow-induced Ca²⁺ influx in HPAECs. Because no extracellular ATP is present in HBSS, endogenous ATP and ADP appeared to play a role.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Effects of apyrase on the flow-induced Ca2+ responses in HPAECs. HPAECs were exposed to shear stress in the absence (control) or presence of apyrase, which degrades ATP, and were examined for changes in [Ca2+]i. Shear stress-dependent increase in [Ca2+]i that is seen in the control was inhibited by apyrase in a dose-dependent manner.

HPAECs predominantly express ATP-operated cation channel P2X₄. Using a competitive PCR method, we compared the mRNA expression of the P2X subtypes P2X₁, P2X₂, P2X₃, P2X₄, P2X₅, P2X₆, and P2X₇. HPAECs had markedly elevated expression of P2X₄, whereas expression of P2X₁, P2X₂, P2X₃, P2X₅, P2X₆, and P2X₇ was very low or almost undetectable (Fig. 3). ATP-operated G protein-coupled receptor P2Y₁ and P2Y₂ expression was also very low compared to P2X₄ expression.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Competitive PCR analysis of P2X purinoceptor subtypes in HPAECs. Expression of mRNA level of each subtype is shown as a percentage of P2X4 mRNA levels. Results are presented as means ± SD of three separate samples.

P2X₄ purinoceptors mediate shear stress-induced Ca²⁺ influx in HPAECs. AS-oligos targeted to the P2X₄ receptor were used to examine whether P2X₄ receptors mediate the shear stress-induced Ca²⁺ influx. The AS-oligos markedly decreased the P2X₄ mRNA and protein levels in the HPAECs, whereas control S-oligos had no effect (Fig. 4A). The shear stress-dependent Ca²⁺ influx was seen in HPAECs exposed to control S-oligos but not in those exposed to AS-oligos (Fig. 4B). These findings suggest that P2X₄ receptors play a central role in the shear stress-dependent Ca²⁺ influx in HPAECs, although the possibility of some involvement by other purinergic receptors besides P2X₄ cannot be excluded.

View larger version (31K): [in this window] [in a new window]   Fig. 4. Effects of antisense oligonucleotides (AS-oligos) to P2X4 on flow-induced Ca2+ responses in HPAECs. A: P2X4 mRNA and protein levels in HPAECs treated with AS-oligos to P2X4. P2X4 mRNA and protein levels were determined by real-time PCR (left) and Western blot (right), respectively. Western blot analysis was repeated three times, and representative signal bands are shown (top). A single band with molecular size consistent with that of P2X4 appeared on the membrane of a Western blot loaded with whole cell lysate, which suggests that the anti-P2X4antibody used has no cross-reactivity with other proteins. Same membrane was reprobed by a monoclonal anti--actin antibody to standardize the P2X4 protein levels. Results of quantitative analyses by densitometry are shown. Vertical axis represents percentages of the control values. Control, nontreated; S-oligos, scrambled-oligonucleotide treated; AS-oligos, AS-oligo-treated HPAECs. Data are means ± SD of three separate samples. Both the P2X4 mRNA and protein levels in the AS-oligo-treated HPAECs decreased to 20% of the control levels. *P < 0.001 vs. control. B: flow-induced Ca2+ responses in HPAECs treated with AS-oligos to P2X4. Cells were exposed to stepped increases in shear stress to 3, 8, and 15 dyn/cm2. Each graph represents 12 Ca2+ responses by a group of 8–10 cells. [Ca2+]i increased stepwise with increasing shear stress in HPAECs treated with S-oligos but not in those treated with AS-oligos. Similar results were obtained in three separate experiments.

Shear stress stimulates HPAECs to release ATP. To examine whether HPAECs release ATP, the amount of ATP in perfusate was determined by a luciferase luminometric assay. The cells released a very small amount of ATP basally, but when exposed to flow, the amount of ATP released increased in a stepwise manner in response to the stepwise increments in shear stress (Fig. 5).

View larger version (25K): [in this window] [in a new window]   Fig. 5. Flow-induced ATP release by HPAECs. ATP concentrations in the perfusate obtained every 15 s from HPAECs cultured under static conditions or exposed to shear stress (3, 8, and 15 dyn/cm2) were measured by luminometry. Under basal conditions, HPAECs released a very small amount of ATP. When exposed to stepwise increments in shear stress, ATP release increased in a stepwise manner. Results are presented as means ± SD of five separate samples.

Flow-induced Ca²⁺ influx and ATP release are shear stress rather than shear rate dependent. To examine whether the flow-induced Ca²⁺ influx and ATP release are dependent on shear stress or shear rate, flow-loading experiments were conducted with two perfusates of different viscosities. Stepwise elevation of the shear rate induced increases in [Ca²⁺]_i but to a greater extent with the high-viscosity perfusate (HBSS + 5% dextran; Fig. 6A). This tendency was quantitatively confirmed by plotting the percent increases in [Ca²⁺]_i against shear rate: the different viscosities produced two distinct curves (Fig. 6B, left). By contrast, when plotted against shear stress, they almost formed a single curve (Fig. 6B, right), which indicates that the percent increases in [Ca²⁺]_i are well correlated with shear stress regardless of viscosity. A similar tendency was observed in flow-induced ATP release (Fig. 6C). These findings indicate that the flow-induced Ca²⁺ influx and ATP release are dependent on shear stress rather than on shear rate.

View larger version (25K): [in this window] [in a new window]   Fig. 6. Shear stress dependency of the flow-induced Ca2+ responses and ATP release. A: Ca2+ responses to flow of two perfusates with different viscosities. HPAECs were exposed to stepwise increases in shear rate to 440, 990, and 1,990 s–1. Shear stress values are shown in parentheses. Twelve Ca2+ responses of a group of 8–10 cells to flow of the low-viscosity perfusate (HBSS) are shown (left) next to the response to flow of the high-viscosity perfusate (HBSS with 5% dextran, mol wt 162,000; right). Viscosity, specific gravity, and osmotic pressure values for the low-viscosity perfusate were 0.765 mPas · s, 1.005, and 289 mosmol/kgH2O, respectively, and those of the high-viscosity perfusate were 3.372 mPas · s, 1.025, and 292 mosmol/kgH2O, respectively. Flow-induced increases in [Ca2+]i were always higher in the high-viscosity solution than in the low-viscosity solution regardless of the shear rate. B: percent increases in [Ca2+]i plotted against shear rate (left) and shear stress (right). C: percent increases in ATP concentrations plotted against shear rate (left) and shear stress (right). Lines were drawn by curvilinear regression with Microsoft Excel software. Data are means ± SD of five separate samples. Note that the data plotted against shear rate yielded two separate curves. 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 (4). Data plotted against shear stress formed almost a single line, on the other hand, which indicates that the flow-induced increases in [Ca2+]i and ATP release were shear stress rather than shear rate dependent.

Endogenously released ATP mediates flow-induced Ca²⁺ influx. To investigate whether endogenously released ATP is involved in flow-induced Ca²⁺ influx, ATP release was suppressed with angiostatin or oligomycin, which are inhibitors of ATP synthase (19, 20), and changes in the Ca²⁺ responses were examined. The ATP release was reduced by angiostatin and oligomycin in a dose-dependent manner (Fig. 7), and the flow-induced Ca²⁺ responses were also strongly suppressed by angiostatin and oligomycin (Fig. 8). When these inhibitors were removed by flushing, the ATP release and the Ca²⁺ responses were recovered (Fig. 8). Addition of extracellular ATP also resulted in recovery of the Ca²⁺ responses in HPAECs treated with angiostatin or oligomycin (Fig. 9). These findings suggest that endogenously released ATP actually mediates the shear stress-induced Ca²⁺ influx via P2X₄ receptors in HPAECs.

View larger version (22K): [in this window] [in a new window]   Fig. 7. Effects of angiostatin and oligomycin on flow-induced ATP release. HPAECs that had been incubated with angiostatin or oligomycin for 1 h were exposed to flow, and the amount of ATP released was measured. Both of the inhibitors were tested for effects on the sensitivity of the luciferin-luciferase reaction for ATP measurement, but neither had any effect on its sensitivity. Shear stress-dependent ATP release was inhibited by angiostatin (left) and oligomycin (right) in a dose-dependent manner. ATP release recovered after inhibitors were washed off for 5 min. Results are presented as means ± SD of six separate samples. *P < 0.01 vs. control.

View larger version (34K): [in this window] [in a new window]   Fig. 8. Effects of angiostatin (hp) and oligomycin (bottom) on flow-induced Ca2+ responses. HPAECs were exposed to flow in the presence of angiostatin or oligomycin and examined for changes in [Ca2+]i. Each graph represents 12 [Ca2+]i responses of a group of 8–10 cells. Angiostatin and oligomycin markedly suppressed the flow-induced Ca2+ responses. Flow-induced Ca2+ responses recovered after inhibitors were washed away for 5 min, which indicates that endogenously released ATP mediates the Ca2+ responses. Similar results were obtained in four separate experiments.

View larger version (33K): [in this window] [in a new window]   Fig. 9. Effects of extracellularly applied ATP on the flow-induced Ca2+ responses in HPAECs exposed to angiostatin or oligomycin. HPAECs were exposed to flow in the absence or presence of extracellular ATP and angiostatin or oligomycin and examined for changes in [Ca2+]i. Each graph represents 12 [Ca2+]i responses of a group of 8–10 cells. Ca2+ responses that were inhibited by angiostatin or oligomycin recovered after the addition of extracellular ATP. Similar results were obtained in three separate experiments.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
The present study demonstrates that a shear stress-dependent increase in [Ca²⁺]_i occurs in HPAECs in response to flow of HBSS and that the increase in [Ca²⁺]_i is due to the influx of extracellular Ca²⁺ via P2X₄ receptors. HPAECs also release ATP in a shear stress-dependent manner in response to flow. When the release of ATP was suppressed with ATP synthase inhibitors, the flow-induced Ca²⁺ influx became much weaker but was restored by washing away the inhibitors or adding extracellular ATP. Thus it appears that the ATP release occurs in response to flow, and the endogenously released ATP activates P2X₄ receptors, thereby facilitating shear stress-dependent Ca²⁺ influx in HPAECs. An increase in [Ca²⁺]_i is known to enhance the production of a variety of factors involved in the control of vascular tone such as nitric oxide and prostacyclin (21). It was demonstrated (13) in the rat pulmonary circulation perfused with blood-free Krebs solution that an increase in flow induces ATP release from ECs, which leads to pulmonary vasodilation. Increases in pulmonary blood flow caused by rapid constriction of the ductus arteriosus in fetal lambs are also shown (15) to cause pulmonary vasodilation that leads to a decrease in pulmonary vascular resistance. Thus the [Ca²⁺]_i-increasing mechanism to which ATP contributes may play a role in the pulmonary vascular response to the increase in blood flow that occurs, e.g., during acute exercise and in the fetal lung at birth.

Under the present experimental conditions, flow induced Ca²⁺ influx but not Ca²⁺ release in HPAECs, which suggests that endogenously released ATP activates P2X₄ but not G protein-coupled P2Y receptors. The activation of P2Y receptors increases inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃], which binds to Ins(1,4,5)P₃ receptors on intracellular Ca²⁺ stores (such as the endoplasmic reticulum) and releases Ca²⁺. This may be due to the difference between P2X₄ and P2Y in expression level or sensitivity to ATP. Competitive PCR showed that the P2Y₁ and P2Y₂ mRNA levels were only 5% of the P2X₄ mRNA levels in HPAECs. P2X₄ receptors are most sensitive to ATP, whereas P2Y₁ receptors are more sensitive to ADP.

Flow has two effects on ECs. The first involves flow-induced changes in mass transport. If perfusates contain any bioactive substances capable of stimulating ECs, the mass transport of the substance increases as the flow rate or shear rate increases and thereby further stimulates ECs to release ATP or to increase [Ca²⁺]_i. The second effect is shear stress, which mechanically deforms the ECs. To separate these two effects, we conducted flow-loading experiments with two perfusates that have different viscosities, which allowed us to apply different levels of shear stress at the same flow rate. The results clearly demonstrate that the flow-induced ATP release and Ca²⁺ influx are dependent on shear stress as a mechanical force rather than on flow rate-dependent mass transport.

Apyrase and AS-oligos to P2X₄ receptors inhibited the flow-induced Ca²⁺ responses in HPAECs, which indicates that P2X₄ and its ligands ATP and ADP are involved in the responses. Our previous study showed that human umbilical vein ECs (HUVECs) required exogenous ATP to exhibit Ca²⁺ responses to flow (3, 27). Thus the conditions required for Ca²⁺ responses seem to differ between HPAECs and HUVECs. This may be explained by the difference in the amount of ATP released in response to shear stress. HPAECs release more ATP in response to shear stress than HUVECs (data not shown). Because of their relatively poor ability to release ATP, HUVECs may require exogenously applied ATP to activate P2X₄ receptors. P2X receptors are multisubunit receptors that exist in the form of homomers or heteromers of different subtypes. Our competitive PCR analysis shows that both HPAECs and HUVECs express the P2X subtypes P2X₄, P2X₅, and P2X₇, but P2X₄ was by far the most strongly expressed isoform. A recent study demonstrated abundant expression of P2X₄ and P2X₅ in human EC primary cultures derived from multiple blood vessels (22). Thus it is possible that HPAECs and HUVECs express distinct P2X homomers or heteromers with different affinities for ATP, and this may be related to the different conditions for Ca²⁺ responses between the two cell lines.

Abundant ecto-enzymes that degrade ATP are present on the EC surfaces, and these ecto-ATPases modify purinoceptor-mediated signaling by exerting an influence on the local concentration of ATP released by ECs. It was recently demonstrated that soluble ATPase is released from ECs in response to shear stress (28). It was also reported that ATP is generated on the surface of ECs by phosphotransfer reactions of ecto-nucleotide kinase (29) and by F₀-F₁ ATP synthase. Thus the P2X₄-mediated Ca²⁺ response to shear stress seems to be dependent on correlations between two opposing mechanisms: an ATP-increasing mechanism and an ATP-decreasing mechanism. The extent to which each mechanism plays a role in ATP-mediated Ca²⁺ responses to shear stress, however, remains unclear.

Although the precise mechanism of shear stress-induced ATP release remains uncertain, possible mechanisms include cell lysis, exocytosis of secretary vesicles that contain ATP, ATP release via ATP channels or transporters, or ATP generation on the cell surface. Cell lysis, however, seems unlikely, because none was detected when shear stressed cells were examined microscopically, and cell lysis was quantitated by Trypan blue exclusion tests and lactate dehydrogenase assays (data not shown). Mechanical stimuli can modulate membrane traffic, such as exocytosis and endocytosis (5), and shear stress has been shown to enhance exocytosis of von Willebrand factor (vWF) from the vWF storage organelles (Weibel-Palade bodies), thereby increasing release of vWF from HUVECs (10). Evidence was recently obtained that ATP release by ECs during increased shear stress probably occurs via vesicular exocytosis (7). Inhibitors of vesicular transport such as monensin and N-ethylmaleimide significantly reduced the release of ATP from ECs in response to shear stress. This mechanism appears most possible, but other mechanisms should also be considered. Erythrocytes are reported to release ATP in response to mechanical deformation; this requires the activity of the cystic fibrosis transmembrane conductance regulator, which is a member of the ATP binding cassette (24). However, controversy remains as to whether the cystic fibrosis transmembrane conductance regulator actually mediates ATP release, because there are conflicting reports on its role in the regulation of ATP release (12, 25). Although additional studies are needed to clarify the mechanism of shear stress-dependent ATP release, elucidation of this problem would provide new insight into our understanding of shear stress signal transduction in ECs.

	DISCLOSURES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
This work was partly supported by Grants-in-Aid for Scientific Research (A); for Scientific Research on Priority Areas; for Young scientists (B); and for Special Coordination Funds for Promoting Science and Technology from the Japanese Ministry of Education, Culture, Sports, Science, and Technology; and a research grant for cardiovascular diseases from the Japanese Ministry of Health and Welfare.

	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.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 

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