Ppads

Keratinocytes (courtesy of Leena Bruckner-Tuderman, Department of Dermatology, University Hospital Freiburg) isolated from human foreskin were transformed with the human papillomavirus oncogenes E6 and E7 for immortalization (Kaur and McDougall, 1988 (link)). Cells were kept in Keratinocyte Serum-Free Medium (KSFM; Invitrogen, Carlsbad, CA) supplemented with 0.3 ng/ml recombinant EGF and 30 ng/ml bovine pituitary extract and 100 U/ml penicillin/streptomycin (culture medium). The experiment medium contained an additional 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Sigma-Aldrich, St. Louis, MO). For pH experiments, the medium was adjusted to a pH of 7.45 by 2.5 N NaOH or to 6.6 by 10% HCl. The inhibitors CK-666 (Sigma-Aldrich), NSC-23766 (Tocris, Bristol, UK), ZCL-278 (Tocris), PIK-90 (Selleck Chemicals, Houston, TX), AG-1478 (Cayman Chemicals, Ann Arbor, MI), Y-27632 (Merck-Millipore, Billerica, MA), (–)-blebbistatin (Sigma-Aldrich), suramin (Tocris), pertussis toxin (Enzo Scientific, Farmingdale, NY), gallein (Tocris), telenzepine (Tocris), PPADS (Tocris), and CK-689 (Merck-Millipore) were used at the indicated concentrations.

The propagation of spontaneous Ca²⁺ transients via gap junctions in iCx26GJCs was carried out using methods modified from a previous study (Schutz et al., 2010 (link)). Samples were incubated for 20 min at 37°C in high-calcium Dulbecco’s PBS (DPBS) containing 2 mM CaCl₂ (Nacalai Tesque) in DPBS (Gibco) supplemented with 5 μM fluo-4 AM (Dojindo), 0.01% (w/v) pluronic F-127 (Invitrogen), and 250 μM sulfinpyrazone (Sigma) as loading medium. To record spontaneous Ca²⁺ transients, we replaced the loading medium with low-calcium DPBS, which contained 20 μM CaCl₂.
The pharmacological study of iCx26GJCs was carried out using methods modified from previous studies (Schutz et al., 2010 (link), Tritsch et al., 2007 (link)). The spontaneous Ca²⁺ activity was recorded for 3 min, then the cells were superfused with PPADS (50 μM; Tocris) or FFA (50 μM; Sigma) for 5 min, followed by washout of the drug and superfusion with low-Ca²⁺ DPBS for 8 min. Sequential fluorescence images were acquired using a Nipkow disc confocal system (CSU22, Yokogawa) and AQUACOSMOS software (Hamamatsu Photonics). Live-cell imaging experiments were performed at near-physiological temperature (32°–35°C) or room temperature (24°–26°C). Signals were measured as relative changes of fluorescence intensity (Δf/f₀), where f₀ is the minimum fluorescence and f is the recorded fluorescence, and Δf = f − f₀.