A438079

Starting at 2 wk of age, mdx mice were treated daily by intraperitoneal injection for 4 wk with 125 mg/kg body weight Coomassie Brilliant Blue G 250 (CBB) (Sigma-Aldrich) or for 4 wk with 8.4 mg/kg body weight oxidized ATP (ox-ATP) (Tocris Bioscience) or for 2 wk with 50 mg/kg body weight A-438079 (Tocris Bioscience). Dosage was based on previous studies [9 (link),23 (link),24 (link)]. Age-matched control mice received the same volume of sterile saline (for CBB and ox-ATP) or saline with 20% v/v DMSO solution (for A-438079).

Each inhibitor was added 20 min before stimulation with LL-37 or CRAMP. BAPTA (inhibitor of intracellular calcium release); U-73122 (phospholipase C inhibitor); GF109203X (Protein kinase C inhibitor); SB203580 (p38-MAPK inhibitor), MG101 (calpain inhibitor); wortmannin (phosphoinositide 3-kinase inhibitor); Boc-MLF (FPR1-receptor inhibitor); WRW4 (FPR2-receptor inhibitor); and A438079 (P2X7 receptor inhibitor) were all obtained from Tocris (Bristol, UK). Pertussis toxin and cholera toxin (inhibitors of G-protein signaling) and Tirofiban (GPIIb/IIIa inhibitor) were from Sigma. Abciximab was from Elly Lilly (USA). Dasatinib (inhibitor of Src-family kinases), R406 (Syk Inhibitor), and Stattic (STAT3-Inhibitor) were from Selleckchem (Houston, USA). Anti-mouse GPVI antibody was from Emfret (clone JAQ1, Cat No. M011-0). The inhibitory monoclonal antibody HGP5C4 directed against human GPVI and the respective isotype control antibody RmC7H8 were generated by immunization of Lou/C rats with an adenovirally expressed human GPVI-Fc fusion protein. The latter represents a soluble form of GPVI with the extracellular domain of human GPVI fused to the human Fc domain. 4C9 and 5C4 monoclonal antibodies (immunoglobulin G1 subtype) specifically bound to GPVI-Fc but not control Fc^{65 (link)}. RmC7H8, raised in rats against an irrelevant human antigen, served as control monoclonal antibody (mAb).

Mice were anesthetized with ketamine (75 mg/kg) and xylazine (3 mg/kg) intraperitoneally and the muscles overlying the T5 to T8 vertebrae were dissected, followed by laminectomy to expose the spinal cord. To induce SCI, extradural compression of the spinal cord at the level of the T6-T7 vertebra was conducted via placement of an aneurysm clip with a closing force for 1 min, as described by Paterniti et al. [17 (link)]. The aneurysm clip is with a closing force of 30 g based on previous study [18 (link)]. Mice undergoing laminectomy alone served as the sham group.
Mice in treatment groups received a daily single-dose intraperitoneal injection of BAY 11-7082 (Tocris Bioscience, Bristol, UK) at 20 mg/kg or A438079 (Tocris Bioscience) at 80 mg/kg in 0.1 ml vehicle (DMSO and 0.9% NaCl, 1:3) immediately after injury and following 2 days. Sham and SCI groups received daily 0.1 ml vehicle immediately post injury and following 2 days intraperitoneally. Mice underwent manual bladder expression twice a day until recovery of reflex bladder emptying. The timing and dose of BAY 11-7082 [19 (link), 20 (link)] and A438079 [21 (link)] were based on previous studies.

A438079 (P2X7 antagonist), Go6983 (PKC inhibitor), or BAPTA (intracellular Ca²⁺ chelator) was purchased from Tocris Bioscience (Bristol, UK). ATP, BzATP, Phorbol 12-myristate 13-acetate (PMA, PKC activator), apocynin (NADPH oxidase inhibitor), Trolox (ROS scavenger), or apyrase were purchased from Sigma Aldrich (St. Louis, MO, USA). ATP (5~50 mg/kg), BzATP (5 mg/kg), apyrase (0.2 U/kg) were administered intravenously in 0.3 mL PBS, and apocynin (2.5 mg/kg) or Trolox (2.5 mg/kg) was dissolved in DMSO and administered intravenously in 0.3 mL PBS after 9 h of pMCAO.

Electrophysiological recording was performed using a patch/whole cell clamp amplifier (Axopatch 200B) [39 (link)]. A micropipette was filled with an internal solution (in mM) containing KCl (140), MgCl₂ (2), HEPES (10), EGTA (11), and ATP (5); the solution’s osmolarity was adjusted to 340 mosmol/kg with sucrose and its pH was adjusted to 7.4 with KOH. The external solution (in mM) contained NaCl (150), KCl (5), CaCl₂ (2.5), MgCl₂ (1), HEPES (10), and D-glucose (10); the solution’s osmolarity was adjusted to 340 mosmol/kg with sucrose, and its pH was adjusted to 7.4 with NaOH. The resistance of the recording electrodes was in the range of 1 to 4 MΩ, with 3 MΩ being optimal. A small patch of membrane underneath the tip of the pipette was aspirated to form a seal (1–10 GΩ), and then more negative pressure was applied to rupture it to establish a whole-cell mode. The holding potential (HP) was set to −60 mV. A-438079 (a selective antagonist of P2X₇ receptor, Tocris Bioscience, Ellis-ville, MO), BzATP, and ATP (Sigma Company) were dissolved in the external solution and delivered by gravity flow from an array of tubules (500 μm O.D., 200 μm I.D.) connected to a series of independent reservoirs. The distance from the tubule mouth to the examined cell was approximately 100 μm. Rapid solution exchange was achieved by shifting the tubules horizontally with a micromanipulator.

The immortalized rat enteric glial cell (EGC) line PK060399egfr (ATCC CRL-2690, VA, United States), which has been shown to exhibit similar morphology and functional properties to primary enteric glial cells (37 (link)), was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) and supplemented with 10% fetal bovine serum, 1% antibiotics (100 μg/mL penicillin and 100 μg/mL streptomycin, Gibco) and 1 mM sodium pyruvate (Gibco) at 37°C in a humidified incubator under 5% CO2 for no more than 16 passages. For all experiments, EGCs were released using 0.05% trypsin-EDTA for 5 min. Cells were incubated with 10 and 50 µM 10Panx trifluoroacetate (Panx1 inhibitor, Sigma-Aldrich SML2152) or 300 µM A438079 (P2X7R antagonist, Tocris 2972) 1 h before incubation with TcdA (50 ng/mL) or TcdB (1 ng/mL). All drug concentrations used were based on MTT assay results (Figure S1). Purified TcdA and TcdB (TechLab, VA, United States) produced by C. difficile strain VPI10463 were used in this study.