Effects of substance P on nitric oxide synthesis in HaCaT cells

Objective To observe the effects of substance P (SP), NK1 receptor antagonist and nitric oxide synthase (NOS) inhibitors on the secretion of nitric oxide (NO) and expression of inducible NOS (iNOS) by immortalized human keratinocyte line HaCaT. Methods The NO level in supernatant of cultured HaCaT cells was measured by nitrate reductase assay after treatment with different concentrations (10-9 to 10-6 mol/L) of SP, or the combination of SP (10-8 mol/L) and spantide (3×10-7 mol/L), aminoguanidine (10-7 mol/L), 7-nitroindazole (10-6 mol/L) or L-NAME (10-5 mol/L) for various durations. Reverse transcription-PCR was performed to measure the expression of iNOS mRNA in HaCaT cells incubated with SP of 10-8 mol/L for 1, 24 and 48 hours. Results The SP of 10-9 to 10-6 mol/L significantly induced the production of NO by HaCaT cells, and the highest level of NO was observed in HaCaT cells treated with SP of 10-8 mol/L. The synthesis of NO by HaCaT cells induced by SP was inhibited by Spantide of 3 × 10~7 mol/L at all time points (30 minutes, 1, 3, 6, 12, 24 hours, all P 0.05). After treatment with SP of 10-8 mol/L, the relative mRNA expression of iNOS was 0.199 ± 0.018 and 0.516 ± 0.030 at 24 and 48 hours, respectively, and there was a statistical difference between the two time points (P < 0.01). Conclusions SP can reinforce the secretion of NO by HaCaT cells via NK1 receptor activation, but iNOS is unlikely to be the primary origin of NO secreted by HaCaT cells induced by SP. Key words: Substance P;  Nitric oxide;  Nitric oxide synthase;  Cells, cultured