Assessment of connexin hemichannel involvement in air-stimulated ATP release.

<p>Uptake of the connexin permeable dye lucifer yellow (LY) was measured to confirm that connexin hemichannels open in response to air exposure and are blocked by the same drugs that prevent ATP release. Cells were pre-incubated with ATP release inhibitors using the same procedure as for the pharmacological analysis assays. LY (500 µM final concentration) was added in the media after air exposure or a change of half the media (control). Sixty minutes later, cultures were rinsed twice with media containing 1-octanol (2 mM) to remove residual LY, but minimize leakage of absorbed LY. LY levels in the cell lysates were then measured. Air exposure significantly increased LY uptake compared to control and the same drugs that inhibited air-stimulated ATP release prevented this increase in LY uptake. This indicates that connexin hemichannels are open during air-stimulated ATP release. Values represent 8 or 9 wells per group +/− SEM, * <i>p</i><0.05 vs. control.</p

Primers used for qPCR analyses.

<p>Primers used for qPCR analyses.</p

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

<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

Assessment of MDR1 transporter involvement in air-stimulated ATP release.

<p>Exclusion of a radiolabeled substrate of the MDR1 ABC transporter was measured to determine whether activity of this transporter is increased during air-stimulated ATP release. Note that this transporter acts to exclude substrates, so an increase in [³H]-digoxin uptake reflects an inhibition of transporter activity. Confluent NHEK cultures were pre-incubated with ATP release inhibitors using the same protocol as for the pharmacological analysis assays. [³H]-digoxin (50 nM final concentration) was added in the media after air exposure or a change of half the media (control). After 7 minutes, the time of peak air-stimulated ATP release, [³H]-digoxin levels in cell lysates were measured. Air exposure significantly increased [³H]-digoxin uptake, as did the ABC transporter inhibitors glibenclamide and verapamil. The connexin blocker 1-octanol also significantly inhibited uptake, while the other connexin blocker carbenoxolone was without effect. The inhibition of MDR1 following air exposure, combined with the disparate effects of ATP release inhibitors, indicates that this transporter is not involved in air-stimulated ATP release. Values represent 6 wells per group +/− SEM, * <i>p</i><0.05 vs. control.</p

Time course and extent of intracellular ATP release. A. Time course of air-stimulated ATP release.

<p>Extracellular ATP levels were quantified following a 15 second air exposure (Air) or an exchange of half the media (Control) in proliferating (Prolif) and calcium differentiated (Diff) NHEK cultures. Air-stimulated ATP release was significantly higher in differentiated cultures at the five minute time point, but remained significantly elevated for 25 minutes in both cultures. Values represent the mean +/− SEM for 13 air-exposed and 2 control dishes per group shown as % baseline. * <i>p</i><0.05 vs. 0 minute Prolif, + <i>p</i><0.05 vs. 0 minute Diff, # <i>p</i><0.05 Diff vs. Prolif. <b>B. Intracellular ATP following air exposure.</b> To determine the extent of intracellular ATP reduction after air exposure, levels of ATP in cell lysates of air-exposed and control confluent NHEK cultures were measured. Air-exposed cultures showed a significant reduction in intracellular ATP at the time of peak ATP release (7 minutes after exposure). This reduction was no longer significant by 60 minutes after air exposure. Values represent averages +/− SEM of 6 dishes per group, * <i>p</i><0.05 vs. control 7 minutes.</p