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C. T. Leung, A. Li, K. Peterson-Yantorno, M. M. Civan; Molecular Identification of ATP-Permeable Channels in Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1483.
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To determine whether channels putatively regarded as conduits for ATP release are expressed in human trabecular meshwork cells (hTM5). Release of ATP is the enabling first step in purinergic signaling and regulation of aqueous humor dynamics.
Total RNA was extracted from the cultured human trabecular meshwork cell line hTM5. Channel mRNA was detected by the reverse transcribed-polymerase chain reaction, and verified by DNA sequencing. Channel protein distribution was confirmed by immunocytochemistry. Functional expression of the purinergic receptor P2X7 was tested by monitoring intracellular Ca2+ concentration with fura-2.
Connexins Cx26, Cx31 and Cx43, cystic fibrosis transmembrane conductance regulator (CFTR), pannexin-1 (PANX1) and P2X7 were detected in hTM5 cells, as were all other P2X receptors except P2X2. Both ATP and the P2X7 agonist BzATP strongly increased cytosolic Ca2+. Removal of extracellular Ca2+ almost completely abolished BzATP- but not ATP-elicited elevations. Ca2+ increase was reversibly-inhibited by both P2X7 antagonists (KN62, brilliant blue G) and PANX1 antagonists (mefloquine, probenecid), but not by the connexin antagonist heptanol.
Multiple ATP release conduits as well as five other P2X receptors were expressed in hTM5 cells. The intracellular Ca2+ changes in response to P2X7 agonists and antagonists substantiated its functional expression. Mefloquine and probenecid, antagonists of PANX1 but not P2X7, also inhibited BzATP-triggered increases in calcium, indicating that P2X7 modulates Ca2+ entry either directly or by stimulating PANX1 hemichannels.
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