Extracellular Release of ATP Mediated by Cyclic Mechanical Stress Leads to Mobilization of AA in Trabecular Meshwork Cells

Abstract

Purpose.: To investigate the mechanisms that mediate the release of ATP induced by cyclic mechanical stress (CMS) and the role of extracellular ATP in the mobilization of arachidonic acid (AA) and prostaglandin secretion.

Methods.: Porcine trabecular meshwork (pTM) cells were subjected to CMS. Extracellular ATP was detected with a luciferin-luciferase assay in the presence or absence of transport inhibitors and a lipid raft disrupter. ATP vesicles were visualized with quinacrine. The release of AA (AA 1-14C) was measured with and without ATP, ATP inhibitors, and phospholipase-A and -C inhibitors. Prostaglandin E2 (PGE2) and viability were measured with ELISA and a lactate dehydrogenase assay, respectively.

Results.: CMS induced ATP release that was inhibited by the vesicle inhibitors N-ethylmaleimide (NEM) and monensin. Lipid raft disruption significantly increased the extracellular ATP induced by CMS. CMS induced AA release (1–4-fold increase) and its metabolic product PGE2 (3.9-fold increase). The AA mobilization induced by CMS could be mimicked by the addition of extracellular ATP and was partially inhibited by a P2 antagonist, by an ATP inhibitor, and by inhibitors of phospholipase-A2 and -C. Addition of PGE2 (10 μM) to the media exerted cytoprotective effects against long-term CMS.

Conclusions.: Extracellular release of ATP induced by CMS in TM cells is mediated by exocytosis of ATP-enriched vesicles into lipid rafts. The resulting activation of purinergic receptors leads to mobilization of AA from the plasma membrane. The subsequent release of PGE could exert protective effects by preventing TM cell loss that may result from chronic exposure to CMS.

The best characterized risk factor for primary open-angle glaucoma (POAG) is elevated intraocular pressure (IOP) ^1,2 that results from an increase in aqueous humor outflow resistance at the level of the conventional outflow pathway (trabecular meshwork [TM] and Schlemm's canal [SC]). ³ The mechanisms involved in homeostasis of normal outflow resistance, as well as those leading to abnormal levels of resistance in POAG, are still poorly understood. 

The TM is constantly subjected to mechanical forces due to transient spikes of IOP associated with systole of the cardiac cycle, blinking, and eye movement. ^4,5 These changes in IOP result in cyclic stretching and relaxation of TM cells, and the resultant cyclic mechanical stress (CMS) has been hypothesized to induce cellular responses that may have a significant role in both the maintenance of normal levels of outflow resistance and the pathologic alterations in glaucoma. ^6–9  

One response to mechanical stress frequently observed in different cell types is a regulated release of ATP into the extracellular space. The specific mechanisms involved in this release of ATP have not been fully elucidated and appear to be cell-type dependent. ^10–13 The extracellular release of ATP in response to mechanical stress has been reported in TM cells. ¹⁴ Similarly, increased hydrostatic pressure in bovine eye cups has been shown to induce an increase in extracellular ATP content of the vitreous compartment adjacent to the retina. The ATP levels correlated with the pressure and were transient at lower pressures but sustained at higher pressures. ¹¹ Increased concentrations of extracellular ATP have also been observed in the vitreous and anterior chamber in acute glaucoma. ¹⁵  

Extracellular ATP and the products generated by its digestion by ecto-ATPases are now recognized as important autocrine and paracrine signaling mediators that participate in the regulation of a broad range of cellular functions. ^16–19 Specific targets of extracellular ATP and other nucleotides include P2Y (G-coupled) and P2X (ion channel) receptors. In addition, extracellular ATP can generate adenosine, which is an agonist of the P1 receptor family. ^20,21  

A potentially important response elicited by extracellular ATP signaling in several cell types is the mobilization of AA from the plasma membrane through the activation of phospholipase. ^22–24 The regulation of AA mobilization in TM cells could be particularly important in the physiology of the outflow pathway, because AA can be metabolized by cyclooxygenases, lipoxygenases, and cytochrome P450 monooxygenase enzymes into an extensive array of biologically active products, including leukotrienes, thromboxanes, prostaglandins (PG), and endocannabinoids, ^25–27 some of which have demonstrated IOP-lowering effects. ^28–32 It is important to note that AA is also the rate-limiting substrate for prostaglandin H synthetase-2 (PGHS-2), also known as cyclooxygenase 2 (COX-2), for the production of PGs. ³³ In addition, the biosynthesis of these products has been shown to be partially inhibited by dexamethasone. ^34,35 Prostaglandins have been recently shown to exert their IOP lowering effects by increasing aqueous humor outflow, not only through the uveoscleral pathway, but through the conventional pathway as well. ^36–41  

Currently, there is little information about the specific mechanisms by which CMS mediates the extracellular release of ATP in TM cells and any possible relationship to the metabolism of AA and its derivatives. Therefore, we investigated the routes for extracellular release of ATP mediated by CMS in TM cells, the potential role of extracellular ATP signaling in the modulation of AA mobilization from the plasma membrane, and whether the mobilization of AA results in increased production of PGE2. In addition, we evaluated the effects of PGE2 on cell viability. 

Methods

Reagents

Monensin, N-ethylmaleimide (NEM), orthovanadate, piceatannol, methyl-β-cyclodextrin (MβCD), quinacrine, suramin, adenosine 5′ triphosphate disodium salt (ATP), apyrase, bromoenol lactose (BEL), O-tricyclo[5.2.1.0^2,6]dec-9-yl dithiocarbonate potassium salt (D609), methyl arachidonyl fluorophosphonate (MAFP), bromophenacyl bromide (BPB), and PGE2 were purchased from Sigma-Aldrich (St. Louis, MO). Radio-labeled AA (AA 1-14C) was purchased from Moraveck Biochemicals (Brea, CA). 

Cell Culture

Porcine (p)TM cells were obtained from fresh pig eyes. ⁴² Cell cultures were maintained at 37°C in 5% CO₂ in medium (low-glucose Dulbecco's modified Eagle's medium with l-glutamine, 110 mg/mL sodium pyruvate, 10% fetal bovine serum, 100 μM nonessential amino acids, 100 U/mL penicillin, 100 μg/mL streptomycin sulfate, and 0.25 μg/mL amphotericin B). All the reagents were obtained from Invitrogen Corp. (Carlsbad, CA). 

Cyclic Mechanical Stress

pTM cells on passage 3 or 4 were cultured on type I collagen-coated flexible silicone-bottomed plates (Flexcell, Hillsborough, NC). The culture medium was switched to serum-free DMEM 2 hours before stretching. The cells were subjected to CMS (20% stretching, 1 cycle per second) for different periods (15 minutes to 18 hours) with a computer-controlled, vacuum-operated strain unit (FX-3000 Flexercell; Flexcell). Control cells were cultured in the same conditions, but no mechanical force was applied. 

Extracellular ATP Measurements

For ATP measurements inhibitors were added 30 minutes before stretching (piceatannol 20 μM; monensin 100 μM; NEM 100 μM; MβCD 10 mM, and orthovanadate 5 mM). Extracellular ATP concentration was detected using a luciferin-luciferase bioluminescent assay (ATP Determination kit, Invitrogen, Eugene, OR) after 15 minutes of stretching. Bioluminescence was measured by a luminometer (TD-20/20; Turner Designs, Sunny Valley, CA). 

Intracellular ATP Staining

For visualization of ATP vesicles pTM cells were cultured on cover slides and incubated for 20 minutes with the fluorescent dye quinacrine (25 μM). ATP vesicles were visualized with a fluorescence microscope (X-cite Series 120; Carl Zeiss Meditec, Dublin, CA). 

AA Measurements

Confluent pTM cultures were labeled with radioactive AA (0.05 μC/mL or 5.7 μg) for 18 to 20 hours in serum-free medium. The cells were then washed three times (0.2% BSA in PBS) and kept in serum-free DMEM for 2 hours before any treatment. Inhibitors were added 30 minutes before CMS (BEL 10 μM, D609 20 μM, MAFP 5 μM, BPB 20 μM, suramin 10 μM, and apyrase 5 U/mL). ATP (100 μM) was added to complete medium. Supernatants were collected at different time points after treatments, centrifuged, and loaded on filter paper. Radioactivity was estimated after 48 hours exposure in a screen cassette (Phosphor; Molecular Dynamics, Sunnyvale, CA). Measurements were made with a molecular imager (Personal Molecular Imager FX; Bio-Rad, Hercules, CA) and analyzed (Quantity One software; Bio-Rad). 

PGE2 Assay

PGE2 was measured with a competitive binding ELISA (Parameter PGE2_; R&D Systems, Minneapolis, MN), according to the manufacturer's instructions. 

Cell Viability Assay

The effects of PGE2 on changes in cell viability induced by CMS wee evaluated by treating the cells with 10 μM PGE2 or vehicle 30 minutes before stretching. Cell viability was assayed after 18 hours of CMS by measuring the lactate dehydrogenase released to the culture medium as a result of plasma membrane damage, by using a nonradioactive cytotoxicity assay (Cito Tox 96; Promega, Madison, WI), according to the manufacturer's instructions. 

Statistical Analysis

Significance was analyzed with a nonpaired Student's t-test; a two-way ANOVA, using the variables treatment and time; and the Tukey test, when applicable. The Tukey test was used in conjunction with ANOVA to find means significantly different from one another. A probability lower than 5% was considered statistically significant. 

Results

CMS-Induced ATP Release through Vesicles

To elucidate the mechanisms of ATP transport and release during CMS, several inhibitors were used. MβCD, a disrupter of lipid rafts, significantly increased the amount of extracellular ATP. Comparison of extracellular ATP levels between cells subjected to CMS with and without MβCD showed a 98% increase in cells treated with MβCD (Fig. 1A). The vesicle inhibitors NEM and monensin clearly reduced the ATP induced by cyclic mechanical stress (Fig. 1B). The ABC cassette inhibitor, orthovanadate and the ATP synthase inhibitor piceatannol did not reduce the amount of extracellular ATP; they increased it (Fig. 1C). We also evaluated extracellular ATP synthesis (ADP+inorganic P) after incubation with and without the cell surface ATP synthase inhibitor piceatannol. Extracellular ATP was highly increased after a 10-minute incubation and then gradually declined in cells without inhibitor. Cells treated with piceatannol showed an 85% reduction in ATP synthesis (data not showed). ATP vesicles were visualized with quinacrine, a fluorescent dye used for detecting releasable ATP stores (Fig. 2). 

Figure 1.

 

Effects of transport inhibitors in the release of extracellular ATP induced by CMS. Lipid raft disruption increased the extracellular ATP induced by CMS (A). The vesicle inhibitors NEM (vehicle ethanol) and monensin (vehicle, water) significantly reduced ATP induced by CMS (B). The ABC cassette inhibitor orthovanadate (vehicle, water) and the ATP synthase inhibitor piceatannol (vehicle, ethanol) increased extracellular ATP (C). n = 4; bars represent SD; *P ≤ 0.05, t-test.

Figure 2.

 

ATP vesicles visualized with the fluorescent dye quinacrine.

CMS-Induced Release of AA Mediated by ATP

To elucidate the role of ATP as a signalling molecule in CMS, we used AA labeled with C14. We first showed that CMS induced release of AA into the extracellular medium compared with control samples (Fig. 3A) and was partially inhibited by the P2 antagonist suramin and by the ATP inhibitor apyrase (Fig. 3B). To confirm the ATP role in the release of AA, we added ATP to labeled cells, which induced a rapid and significant release in AA, mainly in the first 2 hours (Fig. 3C). 

Figure 3.

 

CMS induced release of AA is mediated by ATP. (A) CMS induced AA release in samples subjected to CMS (S) compared with nonstressed control cells (C) (n = 5; ANOVA P = 0.0041). (B) P2 antagonist (suramin), and ATP inhibitor (apyrase) partially inhibited the AA release induced by CMS. (n = 4; ANOVA for CMS/control in suramin-treated cells, P = 0.0683; ANOVA for CMS/control in apyrase-treated cells, P = 0.8887; and, ANOVA for CMS/control in nontreated cells, P < 0.0001). (C) ATP added to the media mimicked the release of AA (n = 4; ANOVA for cells treated with ATP versus nontreated, P = 0.0194 for the first three time points). *P ≤ 0.05 t-test; bars represent SD; Tukey test: &P ≤ 0.05 and &&P ≤ 0.01.

Phospholipase-A2 (PLA2) and -C (PLC) Mediated in the Mobilization of AA by CMS

To clarify whether PLA2 and/or PLC were involved in the CMS-induced release of AA from the membrane; we used the following inhibitors of phospholipases: BPB for secretory PLA2; BEL for calcium-independent PLA2; MAFP for calcium-independent and -dependent but nonsecretory PLA2; and D609 for PLC. MAFP and BEL inhibited the release of AA at almost all time points. BB and D609 had only partial inhibition; AA increased with CMS but in fewer amounts when compared with the control. Both phospholipases (A and C) participated in the CMS-induced release of AA from the membrane. AA mobilization by CMS was partially prevented by the inhibition of PLA2 by three inhibitors and of PLC by one (Table 1). 

Table 1.

 

Mobilization of AA in Nonstressed Control and CMS Samples during Inhibition of Phospholipase-A2 and-C

	30 min			1 hour			2 hours			3 hours
	Mean	SD	P	Mean	SD	P	Mean	SD	P	Mean	SD	P
BB-C	3.233	6.849		4.567	9.602		3.800	8.728		6.100	0.141
BB-S	7.767	9.421	0.052	13.067	11.347	0.047*	4.300	12.155	0.065	3.000	8.415	0.393
MAFF-C	3.033	5.036		7.433	6.529		7.333	3.765		6.050	9.018
MAFF-S	0.433	3.020	0.362	4.033	4.136	0.282	1.800	10.482	0.316	11.200	11.902	0.120
BEL-C	4.567	4.821		5.833	11.632		8.867	3.818		8.667	5.196
BEL-S	1.033	3.092	0.217	4.433	2.797	0.362	6.000	14.425	0.129	8.625	14.076	0.168
D609-C	0.400	2.281		2.500	4.842		8.775	0.010		10.500	7.202
D609-S	0.475	0.950	0.431	6.025	4.465	0.081	12.325	7.670	0.057	14.033	1.050	0.061
Control	1.275	6.485		0.933	6.357		9.450	11.255		7.633	12.632
Stressed	8.475	10.157	0.038	11.175	8.895	0.151*	23.750	15.830	0.008†	30.750	18.458	0.029†

 Mobilization of AA by CMS was partially prevented by the inhibition of phospholipase-A2 and -C. Data are the mean and SD of AA released (in arbitrary units) and the P-value (t-test) of nonstressed controls (C) and samples (S) subjected to CMS (S), with and without inhibitors of PLA2 (BB, MAFF, and BEL) and PLC (D609); n = 3, ANOVA for CMS/control was significant only for cells without inhibitors (P = 0.0055). Significance was also determined by the Tukey test.

*   P ≤ 0.05 (Tukey test).

†   P ≤ 0.01 (Tukey test).

Effect of CMS on Expression of PGE2

We investigated whether the induction of AA by CMS also resulted in an increase in metabolic products, such as prostaglandins. PGE2 was measured by ELISA assay in cells subjected to CMS or nonstressed control cells. Stressed cells showed an increase in PGE2 of 78 ± 48 pg/mL, whereas in nonstressed cells this increase was 20.5 ± 33 pg/mL after 3 hours (Fig. 4A). 

Figure 4.

 

PGE2 expression increased with CMS and conferred protection against mechanical stress. PGE2 expression increased in stressed cells after 3 hours of CMS when compared with that of nonstressed cells (A). Cells treated with PGE2 showed a small but significant increase in viability after 18 hours of CMS (B). n = 3; bars represent SD; *P ≤ 0.05, t-test.

PGE2 Protection of TM Cells against CMS

To examine whether PGE2 confers protection against CMS, we subjected pTM cells to cyclic mechanical stress for 18 hours, with and without PGE2 and analyzed cell viability. Cells treated with PGE2 showed a small but significant increase in viability (Fig. 4B). 

Discussion

The specific mechanisms that mediate ATP release in different cell types are not completely clear and may be cell-type dependent. ^10–12 Our results showed that inhibition of vesicle transport and exocytosis prevented most of the increase in extracellular ATP induced by CMS in TM cells, pointing to exocytosis of ATP-storing vesicles as the main mechanism involved in this response. Consistent with this concept, staining revealed the presence of a distinct pool of vesicles enriched in ATP in cultured pTM cells. A similar exocytic release of ATP is known to occur at synapses and in activated platelets, chromaffin cells, and mast cells. ⁴³ Exocytosis of ATP-storing vesicles is also known to contribute to the release of ATP induced by hypotonic stress in retinal pigment epithelium cells, osteoblasts, and hepatocytes. ^44–46 Inhibition of the ABC cassette (orthovanadate) and ATP synthase (piceatannol), the other two mechanisms of ATP transport tested, did not contribute to the release of ATP induced by CMS. The increase in extracellular ATP induced by these inhibitors, in our model, was probably due to its other properties; orthovanadate is a strong inhibitor of some ecto-ATPases ⁴⁷ and ATP synthases participate in both synthesis and hydrolysis of ATP. ⁴⁸ Although potential contributions of other mechanisms to the ATP release induced by mechanical stress cannot be ruled out, our results strongly support the exocytic pathway as the main route for such ATP release. 

Our results also showed that when lipid rafts were disrupted, the increase in detectable ATP in the cell culture medium was much larger than that observed in cells with intact lipid rafts. This observation suggests that the ATP liberated in response to CMS may be released into lipid rafts. One important property attributed to lipid rafts is their ability to segregate receptors and other signaling components among different domains of the cell membrane. Many components of the machinery associated with G-protein-coupled receptors have been found to localize in lipid rafts, which include P2Y and P1 receptors. Although P2X receptors are not coupled to G-proteins, there is evidence that at least some of them may also be present in lipid rafts. ⁴⁹  

In addition, the enzymes that convert ATP into ADP, AMP, and adenosine (ecto-alkaline phosphatases and ecto-5′-nucleotidases) are glycosylphosphatidylinositol-anchored proteins that are typically localized in lipid rafts. ^49,50 Therefore, the preferential release of ATP into lipid rafts observed during CMS could serve to activate the purinergic and P1 receptors by concentrating ATP and its derivatives. In our model, ATP induced by CMS should reach concentrations large enough to activate some of these receptors, since the use of ATP and purinoreceptors inhibitors (apyrase and suramin) inhibited the release of AA induced by CMS. The effects of apyrase and suramin on the release of AA strongly support the role of ATP release and subsequent activation of purinergic receptors in the mobilization of AA during CMS. In addition, administration of ATP in the absence of CMS also resulted in increased AA mobilization. TM cells in culture are known to release AA ^34,35 in the absence of any additional stressor, and that effect reflected in the levels of AA observed in our nontreated control cultures. Our results showed that these basal levels of AA release were significantly elevated by both CMS and ATP administration. This effect is expected to fade away as ATP is exhausted from the media consistently with the loss of effect observed at 3 hours. It is generally accepted that the mobilization of AA from the plasma membrane after purinergic receptor activation is regulated by PLA2. PLC is also known to be involved in this process, either through activation of PLA2 or through PLA2-independent mechanisms. ^22,51 Our results showed that inhibition of both PLC and PLA2 may be involved in AA mobilization after CMS, suggesting that, similar to what has been observed in other cell types, this response was mediated by these two phospholipases. Given the strong effects on AA release associated with inhibition of PLA2 compared with those of PLC inhibition, our results also suggest that PLC may induce AA release through activation of PLA2 rather than through other mechanisms. 

TM cells have been demonstrated to transform AA in a variety of products, including leukotrienes, hydroxyeicosatetraenoic acids, and PGE2. ^34,35 Consistent with this observation, our results showed that the increased mobilization of AA induced by CMS in TM cells was indeed associated with a significant increase in the concentration of PGE2 in the culture medium. Several of the metabolic derivatives of AA, including prostaglandins and endocannabinoids, have been demonstrated to increase outflow facility. ^52,53 Although the IOP-lowering effects of some of these molecules may be mediated through an increase in uveoscleral outflow facility, PGs have also been observed to increase the outflow facility at the levels of the TM/SC pathway. ³⁷ Therefore, one expected effect of the mobilization of AA induced by CMS would be the increase in outflow facility mediated by AA derivatives such as PGE2. This possibility is consistent with the effects of purinergic agonists on trabecular outflow facility in perfused bovine ocular anterior segments. ⁵⁴  

However, the effects of mechanical stress are likely to be complex and may also contribute to pathologic changes in the TM similar to those observed in other tissues, such as blood vessels. ⁵⁵ In this regard, it is important to note that some end products of AA metabolism are also known to have profibrotic properties and induce the synthesis of several collagen types and fibronectin. ⁵⁶ The profibrotic and inflammatory actions of some of these AA derivatives is believed to contribute to the progression of several pathologic conditions including cardiac fibrosis, Alzheimer's disease, and Parkinson's disease. ^26,57,58 In contrast, a potential mechanism that could compensate for some of the potentially pathologic effects of CMS in the TM could involve cytoprotective effects of PGs. ⁵⁹ For example, it has recently been reported that, in addition to their effects on aqueous humor outflow, PGs protect TM cells against oxidative stress damage ⁶⁰ and our own data indicate that PGs could help prevent the cell loss induced by chronic exposure to CMS. 

In conclusion, our results demonstrated that the extracellular release of ATP-induced by CMS in TM cells is mediated by exocytosis of ATP-enriched vesicles into lipid rafts. The resulting activation of purinergic receptors leads to mobilization of AA from the plasma membrane. The subsequent increase in PGE could exert protective effects by preventing the TM cell loss that may result from chronic exposure to CMS. Given the well-documented IOP-lowering effects of some AA derivatives such as prostaglandins, the observed mobilization of AA induced by CMS could also be relevant in understanding how cellular responses to CMS influences the levels of aqueous outflow facility and IOP. 

Footnotes

 Supported by National Eye Institute Grants EY01894, EY016228, and EY05722 and Research to Prevent Blindness.

Footnotes

 Disclosure: C. Luna, None; G. Li, None; J. Qiu, None; P. Challa, None; D.L. Epstein, None; P. Gonzalez, None

Footnotes

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