Journal: PLoS ONE

Article Title: Air-Stimulated ATP Release from Keratinocytes Occurs through Connexin Hemichannels

doi: 10.1371/journal.pone.0056744

Figure Lengend Snippet: Primers used for qPCR analyses.

Article Snippet: The following inhibitors of non-vesicular ATP release pathways were purchased from Sigma Aldrich (St. Louis, MO) and used at the final concentrations indicated in parentheses: the connexin hemichannel blocker 1-octanol (2 mM), the dual connexin/pannexin hemichannel blocker carbenoxolone (100 µM), the P2X7 channel blocker brilliant blue G (BBG, 1 µM), the anion channel blockers phloretin (100 µM), 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, 100 µM) and gadolinium chloride (50 µM), the CFTR transporter inhibitor glibenclamide (200 µM), the MDR1 transporter inhibitor verapamil (10 µM) and the MRP1 transporter inhibitor probenecid (1 mM) , , , , , , , , .

Techniques:

Journal: PLoS ONE

Article Title: Air-Stimulated ATP Release from Keratinocytes Occurs through Connexin Hemichannels

doi: 10.1371/journal.pone.0056744

Figure Lengend Snippet: qPCR was used to assess potential ATP release pathways present in proliferating (white bars) and calcium differentiated (black bars) NHEK cultures. All non-vesicular ATP release pathways investigated were present in NHEK cultures. The P2X7 purinergic receptor, which is proposed to form an ATP permeable pore through an interaction with pannexins, was significantly increased in differentiated cells. The ABC transporters CFTR and MDR1 were not altered by differentiation, although the MRP1 ABC transporter showed a small, but significant increase. The hemichannel component connexin 43 (Cx43) was also increased by differentiation, while the other hemichannel components pannexin 1 and 2 (Px1 and 2) were significantly decreased. Large increases in the differentiation markers keratin 10 (K10), loricrin (Lor) and involucrin (Inv) verified the differentiation status of calcium treated cultures. The basal keratinocyte marker keratin 14 (K14) was similarly present in both proliferating and differentiated cultures. Values are shown relative to the housekeeping gene YWHAZ and represent data from 3 samples +/− SEM. Due to differences in expression levels, multiple Y axes are used. *significantly decreased in differentiated cells, + significantly increased in differentiated cells ( p <0.05).

Article Snippet: The following inhibitors of non-vesicular ATP release pathways were purchased from Sigma Aldrich (St. Louis, MO) and used at the final concentrations indicated in parentheses: the connexin hemichannel blocker 1-octanol (2 mM), the dual connexin/pannexin hemichannel blocker carbenoxolone (100 µM), the P2X7 channel blocker brilliant blue G (BBG, 1 µM), the anion channel blockers phloretin (100 µM), 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, 100 µM) and gadolinium chloride (50 µM), the CFTR transporter inhibitor glibenclamide (200 µM), the MDR1 transporter inhibitor verapamil (10 µM) and the MRP1 transporter inhibitor probenecid (1 mM) , , , , , , , , .

Techniques: Marker, Expressing

Journal: PLoS ONE

Article Title: Air-Stimulated ATP Release from Keratinocytes Occurs through Connexin Hemichannels

doi: 10.1371/journal.pone.0056744

Figure Lengend Snippet: 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, * p <0.05 vs. control. To determine the mechanism of air-stimulated ATP release , 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 , pre-air) and 7 minutes after air exposure ( C , post-air) to measure the ability of drugs to inhibit air-stimulated ATP release. B . Pre-air ATP levels were significantly lower in differentiated cultures, but similar for most drug treatment groups. C . 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, *, + p <0.05 vs. vehicle or air alone for proliferating and differentiated cells, respectively. Inset. Schematic showing potential keratinocyte ATP release pathways and the major targets of inhibitors used. 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. Release Pathways. 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.

Article Snippet: The following inhibitors of non-vesicular ATP release pathways were purchased from Sigma Aldrich (St. Louis, MO) and used at the final concentrations indicated in parentheses: the connexin hemichannel blocker 1-octanol (2 mM), the dual connexin/pannexin hemichannel blocker carbenoxolone (100 µM), the P2X7 channel blocker brilliant blue G (BBG, 1 µM), the anion channel blockers phloretin (100 µM), 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, 100 µM) and gadolinium chloride (50 µM), the CFTR transporter inhibitor glibenclamide (200 µM), the MDR1 transporter inhibitor verapamil (10 µM) and the MRP1 transporter inhibitor probenecid (1 mM) , , , , , , , , .

Techniques: Labeling, Inhibition, Incubation, Binding Assay