SR 57227A 2.5, 5, 10, 20, 40 mg o.d. and 20 mg b.i.d. were administered during 7 consecutive days

Mouse neuroblastoma x rat glioma hybrid cells NG 108-15 were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 40 mM sodium bicarbonate, 1.8 mM L-glutamine, 10% inactivated foetal calf serum (Gibco) and HAT (100mkM hypoxanthine, 1 mkM aminopterin, 16 mkM thymine) (Gibco) and subcultured every 2 days. Cells were grown for 2 days in 35-mm culture dishes in 3 ml growth medium. Before the experiment was started the cell layer was washed twice with 1.5 ml buffer A (145 mM NaC1, 5.4 mM KCI, 1.8 mM CaCl2, 1.0 mM MgCl 2, 2.0 mM Na2HPO4, 20 mM glucose and 20 mM HEPES, the pH being adjusted to 7.4 with NaOH) The incubation (10 min at 37°C) was then performed in 1 ml buffer B (135 mM NaCI 4.5 mM KCI, 1.8 mM CaCl2, 1.0 mM MgCl2, 2.0 mM Na2HPO4, 20 mM glucose, 20 mM HEPES, pH 7.4 with NaOH), supplemented with 10 mM guanidinium chloride, 200-250 nCi (7.40-9.25 kBq) [14C]guanidinium, 10 mkM substance P and the SR-57227A. The incubation was stopped by aspiration of the medium, and the cell layer was washed 3 times with 1.5 ml ice-cold buffer C (same composition as buffer A except that NaCl was replaced by choline chloride). The cells were then dissolved in 0.5 ml of 0.4 N NaOH and transferred to scintillation vials. The culture dishes were rinsed with 0.5 ml 1 N HCI and 0.5 ml 0.4 N NaOH, which was mixed with the first extract for determination of radioactivity in the presence of 10 ml Aquasol (NEN).