Al-Jarallah Aishah, Khan Islam, Oriowo Mabayoje A
Department of Biochemistry, Faculty of Medicine, Kuwait University, Kuwait.
Eur J Pharmacol. 2008 Jan 28;579(1-3):365-73. doi: 10.1016/j.ejphar.2007.10.069. Epub 2007 Nov 1.
Inflammatory bowel disease is associated with reduced colonic smooth muscle contractility. However the underlying mechanism responsible for the decrease in contractility is not fully understood. In this study we investigated the role of Ca(2+)-sensitization in reduced carbachol-induced contraction of colonic segments from rats treated with trinitrobenzenesulphonic acid (TNBS). Functional alterations in RhoA/Rho-kinase and protein kinase C (PKC) pathways were examined using specific antagonists, Y-27632 and GF-109203X respectively. In this study, TNBS-induced colitis was associated with a decrease in the maximum response but not sensitivity to carbachol. Permeabilized inflamed colonic segments showed greater sensitivity to Ca(2+) as compared to controls, indicating greater Ca(2+)-sensitivity of the myofilaments. In contrast, carbachol-induced increase in Ca(2+)-sensitization was reduced in these tissues suggesting that the reduced carbachol-induced contraction could be due to decreased Ca(2+)-sensitization. Y-27632, a Rho-kinase inhibitor, induced significantly greater relaxation in colon strips from TNBS-treated rats indicating higher basal tone in these tissues. This is consistent with increased expression of Rho-kinase in the inflamed colon. Y-27632 concentration-dependently inhibited carbachol-induced contractions in control and TNBS-treated rats. However its effect was not significantly different between the two groups. GF-109203X, a PKC antagonist, produced concentration-dependent reduction in carbachol-induced contractions in control and TNBS-treated rats. GF-109203X was less effective in reducing carbachol-induced contractions of colonic segments from TNBS-treated rats suggesting a defect in PKC activation. Western blotting analysis showed reduced expression of total PKC in inflamed colonic smooth muscle. Carbachol-induced phosphorylation of CPI-17 was also reduced in colonic segments from TNBS-treated rats. These findings suggest that Ca(2+)-sensitization in rat colon involves both the PKC and the Rho-kinase pathways and that the reduced carbachol-induced contraction in colitis was due to inflammation-induced changes in Ca(2+)-sensitization involving a defect in the PKC pathway.
炎症性肠病与结肠平滑肌收缩性降低有关。然而，导致收缩性降低的潜在机制尚未完全明确。在本研究中，我们调查了Ca(2+)敏化在三硝基苯磺酸（TNBS）处理的大鼠结肠段中卡巴胆碱诱导的收缩降低中的作用。分别使用特异性拮抗剂Y-27632和GF-109203X检测RhoA/Rho激酶和蛋白激酶C（PKC）途径的功能改变。在本研究中，TNBS诱导的结肠炎与最大反应降低有关，但与对卡巴胆碱的敏感性无关。与对照组相比，通透化的炎症结肠段对Ca(2+)表现出更高的敏感性，表明肌丝对Ca(2+)的敏感性更高。相反，这些组织中卡巴胆碱诱导的Ca(2+)敏化增加减少，提示卡巴胆碱诱导的收缩降低可能是由于Ca(2+)敏化降低所致。Rho激酶抑制剂Y-27632在TNBS处理的大鼠结肠条中诱导出明显更大的舒张，表明这些组织中的基础张力更高。这与炎症结肠中Rho激酶表达增加一致。Y-27632在对照组和TNBS处理的大鼠中浓度依赖性地抑制卡巴胆碱诱导的收缩。然而，两组之间其作用无显著差异。PKC拮抗剂GF-109203X在对照组和TNBS处理的大鼠中浓度依赖性地降低卡巴胆碱诱导的收缩。GF-109203X在降低TNBS处理的大鼠结肠段卡巴胆碱诱导的收缩方面效果较差，提示PKC激活存在缺陷。蛋白质印迹分析显示炎症结肠平滑肌中总PKC表达降低。TNBS处理的大鼠结肠段中卡巴胆碱诱导的CPI-17磷酸化也降低。这些发现提示大鼠结肠中的Ca(2+)敏化涉及PKC和Rho激酶途径，且结肠炎中卡巴胆碱诱导的收缩降低是由于炎症诱导的Ca(2+)敏化改变，涉及PKC途径的缺陷。
