Study On Actions Of Substance P On Colonic Dysmotility

Part I The potential effects of chronic stress on the expression of substance P and its related receptors in rat colonAim To investigate the effects of chronic stress on the expression of substance P and its related receptors in rat colon.Methods Male Wistar rats were exposed to water-avoidance stress(WAS)or sham WAS (SWAS) (1 h/day) for up to 10 consecutive days. The spontaneous contractile activities of longitudinal muscle (LM) strip and circular muscle (CM) strip from rat colon were studied in an organ bath system. Enzyme Immunoassay Kit was applied to detect the SP serum level and the presence of SP in the colonic mucosa. The distribution and expression of neurokinin-1 receptor (NK1R) and neurokinin-2 receptor (NK2R) were investigated by Immunohistochemistry and Western blotting.Results The numbers of fecal pellets expelled per hour by the WAS rats at days 1, 3,5,7 and 10 were significantly higher than those in the SWAS rats (7.3±0.2 vs. 2.6±0.1,7.1±0.4 vs.2.4±0.3.6.4±0.2 vs.2.0±0.2; 6.0±0.3 vs.2.0±0.2 and 5.4±0.4 vs. 2.3±0.2. respectively, P<0.01). The spontaneous contraction of both LM and CM strips from the WAS rats were significantly increased than those in the SWAS rats (1.14±0.05 g vs.0.94±0.04g and 0.36±0.02 g vs.0.25±0.02g, P<0.01). The serum levels of SP and the expression of SP in the colonic mucosa were elevated following WAS. Immunohistochemistry proved the expression of NK1R and NK2R in mucosa, muscularis and myenteric plexus. Western blotting demonstrated stress-induced up-regulation of NK1R in colon devoid of mucosa and submucosa, but not NK2R.Conclusion Chronic stress enhanced the contraction of muscle strips from rat colon. SP-NK1R signal system might be involved in this pathological process.PartⅡ The regulation effect of substance P and the selective antagonist of NK1R receptor on chronic stress-induced hypermotilityAim To investigate the regulation effect of substance P (SP) and the selective antagonist of NK1R receptor on chronic stress-induced hypermotility.Methods Wistar rats were exposed to daily 1-hour water avoidance stress (WAS) or sham WAS (SWAS) for 10 consecutive days. The spontaneous contractile activities of longitudinal muscle (LM) strip and circular muscle (CM) strip from rat colon were studied in an organ bath system to observe the effects of SP on spontaneous contractile activity of muscle strips. The effects of SP on muscle strips were also observed when these strips were pretreated with selective NK1R antagonist L-703,606 oxalate salt hydrate (L-703).Results The contraction of LM from both SWAS and WAS rats were increased after addition of SP (0.1μM), but the R values between the two had no significance. (P>0.05); In both the SWAS and WAS rats, SP (1 μM) significantly increased the spontaneous contraction of the LM strips. The R values of the LM strips from the two groups increased from 1 (baseline) to 1.39±0.12 and 1.67±0.08, respectively (P<0.01). Moreover, the R values of the WAS group were significantly higher than those of the SWAS group (P<0.05). SP (0.1 and 1 μM) dose-dependently increased the spontaneous contractile amplitude of the CM strips from the SWAS and WAS rats. At the concentration of 0.1 μM, SP significantly increased the R values of the two groups from 1 (baseline) to 5.35±0.29 and 9.65±0.85, respectively (P<0.01), and the R values of the WAS group were significantly higher than those of the SWAS group (P<0.01). At the concentration of 1 μM, the R values of the CM strips from the two groups increased from 1 (baseline) to 7.31±1.45 and 14.49±2.75, respectively (P<0.01). Moreover, the R values of the WAS group were significantly higher than those of the SWAS group (P<0.05)Conclusion Repeated WAS increased the contractile activities of longitudinal muscle and circular muscle strips induced by SP and this effect was reversed by the selective NK1R antagonist L-703.PartⅢ The effect of substance P on L-type calcium channel current in smooth muscle cells from both WAS and SWAS rat colonAim To investigate the effect of substance P on L-type calcium channel current in smooth muscle cells from both WAS and SWAS rat colon.Methods Wistar rats were exposed to daily 1-hour water avoidance stress (WAS) or sham WAS (SWAS) for 10 consecutive days. L-type calcium channel currents (ICa,L) of smooth muscle cells (SMCs) were recorded by whole-cell patch-clamp technique.Results Repeated stress did not change the shape of the averaged steady-state current-voltage (Ⅰ-Ⅴ) relationships of ICa,L and the ICa,L current density between the two groups did not differ significantly (P>0.05). Bath application of SP (1 μM) significantly increased the ICa,L of SMCs in both the SWAS and WAS rats, the current density at 0 mV increased from -3.93±0.38 pA/pF and -3.49±0.17 pA/pF to -4.54±0.39 pA/pF and-4.89±0.31 pA/pF, respectively (P<0.05). Furthermore, SP caused a significant 129±3% increase in the control peak current density measured compared with that measured before SP application in the WAS rats, and this increase was significantly higher than that of the SWAS group (P<0.05).Conclusion Chronic stress has no effect on the expression or numbers of L-type calcium channels in smooth muscle cells; however, increased NK1R expression in the muscularis may contribute to this effect.