Lack of Ethidium Bromide Uptake during Hypotonic Stress in HEK 293 Cells that Express P2X7 Receptors

Recent reports indicate that the long known ethidium bromide uptake resulting from prolong stimulation of cell surface P2X7 receptors is due to Pannexin-1 (Panx-1) hemichannels, which are probably activated by the carboxy terminus of the receptor itself. It has also been proposed that Panx-1 is activated by exposing the cells to hypotonic solutions without the need for P2X7 receptor activation and that this maneuver results in ATP release from cells probably via Panx-1. However, other groups have previously proposed that the hypotonic-induced ATP release is via cell swelling-activated chloride channels. In this work we explored activation of endogenous Panx-1 in HEK 293 cells (untransfected or transfected with P2X7 receptors) exposed to hypotonic solutions, while simultaneously measuring whole cell chloride current and fluorescence signals resulting from ethidium bromide uptake (an index of Panx-1 activation). When HEK cells were exposed to hypotonic solutions that induced activation of cell swelling-activated chloride current (ICl,swell) no ethidium bromide uptake was detected. Treatment of the cells with the Panx-1 inhibitor mimetic peptide 10panx1 resulted in complete inhibition of ethidum bromide uptake induced by ATP activation of P2X7 receptors without affecting ICl,swell. Pretreatment with carbenoxolone (CBX), another blocker of Panx-1, inhibited ethidium bromide uptake induced by stimulation of HEK cells expressing P2X7 receptors and at the same time inhibited ICl,swell in a dose-dependent manner. We conclude that hypotonic conditions most likely induce ATP release by via activation of CBX-sensitive volume-sensitive chloride channels without participation of Panx-1 hemichannels.Supported by CONACyT grants 45895 and 79897.