Substance P Regulation in Cultured DRG Cells

The isolated DRG cells from adult Wistar rats (6–9 weeks of age) were cultured at 37°C in a water-saturated atmosphere with 5% CO₂ for 5 days before the initiation of experiments [3 (link)]. Next, the DRG cells (5–6 DRGs/35 mm dish) were separately exposed to Dkk1 (R&D Systems, Minneapolis, MN 55413, USA) or NS-398 (Sigma Chemical Co., St Louis, MO, USA) in serum-free DMEM containing peptidase inhibitors (1 μM phosphoramidon, 4 μg/ml bacitracin and 1 μM captopril; Sigma) for 15 min at 37°C in a water-saturated atmosphere with 5% CO₂. Thereafter, the cells pretreated with various drugs were continuously stimulated with TWS119 (Cayman Chemical, Ann Arbor, MI) or Wnt-3a (R&D Systems) for 1, 6, 24 or 48 h in DMEM at 37°C. The DRG cells treated by peptidase inhibitors alone were used as a control. The substance P content in the cultured rat DRG cells was measured using a highly sensitive radioimmunoassay as previously described [13 (link)].