Abstract
Purpose: :
To determine the relative functional contributions to ATP release by large channels we have identified in human trabecular meshwork (TM) cells.
Methods: :
ATP release was measured with a luminometer by the luciferin-luciferase reaction 24-48hrs after plating cells into 96-well plates. Cultured human TM (hTM5) cells were pre-incubated isotonically with/without drugs for 1hr, and stimulated by 50% hypotonicity or by raising intracellular Ca2+ with 10µM ionomycin. ATP concentrations were calibrated in isotonic and hypotonic solutions with/without drugs.
Results: :
Control ATP concentration in the isotonic bath was 14.0+0.2nM (mean+SE, n=839 wells). Hypotonicity increased ATP concentration 4-fold to 60.9+0.5nM (n=751, P<0.01), and ionomycin 9-fold to 134.7+3.8nM (n=138, P<0.01). The selective pannexin-1 hemichannel (PANX1) blockers carbenoxolone (3µM, CBX), mefloquine (30nM) and probenecid (100µM) inhibited hypotonicity-triggered ATP release by 43+5% (n=24), 42+3% (n=36) and 21+3% (n=46), respectively. The relatively specific connexin (Cx) inhibitor heptanol (1mM) also reduced release by 43%+1% (n=53). Antagonists of the P2X7 ionotropic receptor, 1µM KN62 and 0.5µM brilliant blue G decreased hypotonicity-induced ATP release by 36+5% (n=36) and 33+2% (n=54), respectively. Blockers of the maxi-Cl- channel (1mM SITS or 50µM Gd3+), of CFTR (100µM glybenclamide), of CaMKII (1µM Lavendustin C), and of vesicular release (2µM bafilomycin or 100µM monensin) had no effect. Contrary to their effects on hypotonicity-elicited ATP release, KN62 totally abolished ionomycin-triggered release, while CBX had no effect.
Conclusions: :
PANX1, Cxs and P2X7, directly or via PANX1, contribute to swelling-activated ATP release, whereas the maxi-Cl- channel, CFTR and vesicular release are not involved. KN62, but not CBX, abolished cytosolic Ca2+-evoked ATP release, suggesting that PANX1-independent P2X7 pathways may dominate following ionomycin stimulation. The results reveal that homogenous populations of TM cells functionally express multiple ATP conduits, whose relative importance depends on the stimulus applied.
Keywords: trabecular meshwork • ion channels • receptors: pharmacology/physiology