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

• Published in: Journal of cellular physiology Vol. 227; no. 1; pp. 172 - 182
• Main Authors: Li, Ang, Leung, Chi Ting, Peterson-Yantorno, Kim, Stamer, W. Daniel, Mitchell, Claire H., Civan, Mortimer M.
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01.01.2012
• Subjects: Adenosine - metabolism; Adenosine Triphosphate - metabolism; Aqueous Humor - metabolism; Blotting, Western; Connexins - metabolism; Glaucoma - metabolism; Glaucoma - physiopathology; HEK293 Cells; Humans; Intraocular Pressure - physiology; Luminescent Measurements; Microscopy, Confocal; Nerve Tissue Proteins - metabolism; Real-Time Polymerase Chain Reaction; Receptors, Purinergic P2X7 - metabolism; Reverse Transcriptase Polymerase Chain Reaction; Trabecular Meshwork - cytology; Trabecular Meshwork - metabolism
• ISSN: 0021-9541; 1097-4652
• DOI: 10.1002/jcp.22715

Our guiding hypothesis is that ecto‐enzymatic conversion of extracellular ATP to adenosine activates A1 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 P2X7 receptors (P2RX7) 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 P2RX7. ATP release was also triggered by raising intracellular Ca2+ activity with ionomycin after a prolonged lag time and was unaffected by the PX1 blocker probenecid, but nearly abolished by P2RX7 antagonists. We conclude that swelling‐stimulated ATP release from human TM cells is physiologically mediated by PX1 and Cx hemichannels and P2X7 receptors, but not by vesicular release. PX1 appears not to be stimulated by intracellular Ca2+ in TM cells, but can be modulated by oxidation‐reduction state. The P2RX7‐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. J. Cell. Physiol. 227: 172–182, 2012. © 2011 Wiley Periodicals, Inc.