Pharmacological analysis of the air-stimulated ATP release mechanism. A. propidium iodide (PI) labeling.

Abstract

<p>PI labeling was measured to ensure that air exposure and drugs used in inhibition experiments did not compromise cell membranes. Only pretreatment with carbenoxolone significantly increased PI labeling and this remained below 3% of cells. All values represent averages +/− SEM of 6 dishes per group, * <i>p</i><0.05 vs. control. <b>To determine the mechanism of air-stimulated ATP release</b>, keratinocytes were pre-incubated for 10 minutes with known inhibitors of ATP release pathways before air exposure. ATP levels in the media were assayed just prior to air exposure to determine any effects of the drugs themselves on baseline ATP release (<b>B</b>, pre-air) and 7 minutes after air exposure (<b>C</b>, post-air) to measure the ability of drugs to inhibit air-stimulated ATP release. <b>B</b>. Pre-air ATP levels were significantly lower in differentiated cultures, but similar for most drug treatment groups. <b>C</b>. In both proliferating and differentiated cultures, pre-incubation with the connexin hemichannel blockers 1-octanol and carbenoxolone largely abolished air-stimulated ATP release. Glibenclamide and verapamil, drugs traditionally used as ABC transporter blockers, also significantly inhibited air-stimulated ATP release. Post-air ATP levels are expressed as percent of air alone. Values represent 12 (drug treatment) or 24 (air alone) dishes. Error bars are +/− SEM, *,⁺<i>p</i><0.05 vs. vehicle or air alone for proliferating and differentiated cells, respectively. <b>Inset. Schematic showing potential keratinocyte ATP release pathways and the major targets of inhibitors used.</b> Secondary targets discussed in the text are also shown in parentheses. Red dots and arrows represent regulatory ATP binding sites and routes of release, respectively. <b>Release Pathways.</b> P2X7: P2X7 ATP receptors, Px1/2: pannexins-1 & 2, Cx43: connexin-43, VRAC: volume-regulated anion channels (unknown molecular identity) and the ATP binding cassette (ABC) transporters CFTR, MDR1 and MRP1.</p