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. 2012 Jan;227(1):172-82.
doi: 10.1002/jcp.22715.

Mechanisms of ATP release by human trabecular meshwork cells, the enabling step in purinergic regulation of aqueous humor outflow

Ang Li  1 , Chi Ting Leung, Kim Peterson-Yantorno, W Daniel Stamer, Claire H Mitchell, Mortimer M Civan
Affiliations

Mechanisms of ATP release by human trabecular meshwork cells, the enabling step in purinergic regulation of aqueous humor outflow

Ang Li et al. J Cell Physiol. 2012 Jan.

Abstract

Our guiding hypothesis is that ecto-enzymatic conversion of extracellular ATP to adenosine activates A(1) adenosine receptors, reducing resistance to aqueous humor outflow and intraocular pressure. The initial step in this purinergic regulation is ATP release from outflow-pathway cells by mechanisms unknown. We measured similar ATP release from human explant-derived primary trabecular meshwork (TM) cells (HTM) and a human TM cell line (TM5). Responses to 21 inhibitors indicated that pannexin-1 (PX1) and connexin (Cx) hemichannels and P2X(7) receptors (P2RX(7) ) were comparably important in modulating ATP release induced by hypotonic swelling, whereas vesicular release was insignificant. Consistent with prior studies of PX1 activity in certain other cells, ATP release was lowered by the reducing agent dithiothreitol. Overexpressing PX1 in HEK293T cells promoted, while partial knockdown (KD) in both HEK293T and TM5 cells inhibited hypotonicity-activated ATP release. Additionally, KD reduced the pharmacologically defined contribution of PX1 and enhanced those of Cx and P2RX(7) . ATP release was also triggered by raising intracellular Ca(2+) activity with ionomycin after a prolonged lag time and was unaffected by the PX1 blocker probenecid, but nearly abolished by P2RX(7) antagonists. We conclude that swelling-stimulated ATP release from human TM cells is physiologically mediated by PX1 and Cx hemichannels and P2X(7) receptors, but not by vesicular release. PX1 appears not to be stimulated by intracellular Ca(2+) in TM cells, but can be modulated by oxidation-reduction state. The P2RX(7) -dependent component of swelling-activated release may be mediated by PX1 hemichannels or reflect apoptotic magnification of ATP release, either through itself and/or hemichannels.

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Figures

Fig. 1
Fig. 1. Purinergic regulation of aqueous humor outflow
The present aim was to identify which of the mechanisms thought to subserve ATP release in other preparations might underlie release by TM cells. The current results indicate that PX1, Cxs and P2RX7 play major roles, but not CFTR, VSOR, the maxi-anion channel or vesicular release. Following release, ATP can directly activate multiple P2X and P2Y receptors to initiate downstream purinergic signaling, or can be ecto-enzymatically degraded into adenosine, stimulating A1AR-mediate matrix metalloproteinase 2 (MMP-2) secretion. The MMP-2 lowers outflow resistance, thereby reducing intraocular pressure.
Fig. 2
Fig. 2. Time courses of ATP concentration in solutions bathing HEK293T (A) and TM5 cells (B)
Fig. 2A illustrates how the parameters Ccon, Cmax and Cexp were quantified in order to calculate percentage inhibition with Eq. 1 (Materials and Methods). Baseline isotonic ATP release was stable throughout the 60-min period of measurement. Ccon was quantified as the mean isotonic ATP concentration during the bracketed period 10–40 min after beginning measurement, and averaged over the wells in a single experiment (here, n=18). Cmax (arrow) was the mean of the peak ATP concentrations measured in drug-untreated hypotonic solution of the wells of the single experiment. Cexp (arrow) was the peak ATP concentration released by cells exposed to inhibitor in hypotonic solution in a single well. These values of Ccon, Cmax and Cexp were inserted in Eq. 1 to calculate the percentage inhibition observed in a single well. In this experiment, Ccon, Cmax and Cexp, and percentage inhibition were 7.8 nM, 41.5 nM and 24.4 nM, respectively. Inserting these values in Eq. 1, Inhibition(%)=100%•[(41.5−24.4)/(41.5−7.8)]=50.7% The inhibitions calculated for the single wells were pooled and averaged to generate the final percentage inhibitions presented in the figures and tables. Fig. 2B illustrates the similar time courses of ATP release by TM5 cells in isotonic solution, hypotonic solution and hypotonic solution containing 1 mM heptanol. The data are the means ± SE of measurements obtained with 40–77 wells from five experiments.
Fig. 3
Fig. 3. Linear correlation between inhibition of ATP release from a normal human TM cell line (TM5) and human explant-derived primary TM Cells (HTM)
Filled squares (■) reflect measurements after hypotonic exposure in the presence of 0.1 mM PRO, 1 mM HEP, 1 μM KN-62, 2 μM BAF, 50 μM Gd3+, 1 μM LAC, 100 μM GLY, or 30 μM CBX + 1 mM HEP + 1 μM KN-62. Open circles (○) refer to data obtained after exposure to ionomycin together with 0.1 mM PRO, 1 mM HEP, 1 μM KN-62, or 1 μM LAC. The linear regression line relates inhibition of HTM cells (y) to inhibition of the TM5 cell line (x) by the same drug at the identical concentration after the same stimulation: y = (−2.57 ± 1.53) + (1.04 ± 0.03)· x, with a correlation coefficient (R) of 0.99 (P<0.0001).
Fig. 4
Fig. 4. Molecular identification of multiple ATP-permeable conduits from two preparations of human TM cells
RT-PCR with subsequent DNA sequencing verified that both TM5 and HTM cells express PX1, Cx26, Cx31, Cx43, P2RX7, and CFTR. No product was detectable upon omitting reverse-transcriptase [RTase (−)], confirming that the cDNA derived from total RNA was free of genomic DNA contamination (A). The relative expressions of PX1, Cx43, P2RX7, and beta-actin genes in the two cell preparations were quantified by real-time PCR (N=5–7 independent experiments), using GAPDH as the internal control (B). The expression of PX1 protein in TM5 and HTM cells was further verified by western-blotting, taking HEK293T cells as the positive control (C).
Fig. 5
Fig. 5. Heterologous expression of PX1 in HEK293T cells
Western-blotting confirmed the expression of human PX1 proteins tagged with myc-6×His, using anti-myc and anti-6×His monoclonal antibodies. No band was visible in either non-treated (NT) or mock control (MC, transfected with blank vector) cells (A). Confocal microscopic images demonstrated the localization of the PX1 close to or within the plasma membrane. No such staining was observed in NT or MC cells (B).
Fig. 6
Fig. 6. PX1 knock-down (KD) in TM5 cells by lentivirus-mediated RNA interference
The successful KD of PX1 mRNA and protein in TM5 cells was verified by real-time PCR (A) and semi-quantitative immunoblotting (B), respectively. The stable PX1 KD in the TM5 cell line lasted over 2 months after transduction (A). N refers to numbers of independent experiments performed. *P< 0.01 compared with mock controls (MC) by Student’s t-test.
Fig. 7
Fig. 7. Alteration of pharmacologic profile of ATP release from TM5 cells after PX1 knockdown (KD)
The decrease of the inhibitory effect of PRO after Px1 KD indicated the involvement of PX1, consistent with the accordingly enhanced efficacies of HEP and KN-62. In contrast, KN-62 abolished ionomycin-induced ATP release in both mock control (MC) and KD, suggesting that PX1-independent P2RX7 predominated in the Ca2+ triggered release. Numbers along the abscissa are numbers of wells measured. *P<0.01 compared with MC controls by Student’s t-test.
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