Clostridium difficile toxin B inhibits carbachol-induced force and myosin light chain phosphorylation in guinea-pig smooth muscle:: role of Rho proteins
Clostridium difficile toxin B inhibits carbachol-induced force and myosin light chain phosphorylation in guinea-pig smooth muscle:: role of Rho proteins
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DOI：
10.1111/j.1469-7793.1998.083bx.x
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发表时间：
1998-01-01
影响因子：
5.5
通讯作者：
Pfitzer, G
中科院分区：
文献类型：
--
作者：
Lucius, C;Arner, A;Pfitzer, G
1.艰难梭菌毒素B使Rho亚家族的Ras相关低分子量GTP酶葡糖基化，从而使其失活。本研究以毒素B为工具，检测Rho蛋白是否参与了卡巴胆碱诱导的完整肠平滑肌细胞力钙敏感性和肌球蛋白轻链（MLC）磷酸化的增加.将豚鼠小肠纵肌的小条在毒素B（40 ng/ml）中孵育过夜。测定卡巴胆碱诱导的力和细胞内[Ca 2 +]，并在单独的系列中测定力和MLC磷酸化.卡巴胆碱诱导双相收缩：力的初始快速增加（峰1），随后部分松弛和力的第二次延迟增加（峰2）。用Fura-2测量的C2+信号的峰值先于力的峰值1，然后下降到较低的基底上稳态水平。峰2与[Ca 2 +]的显著增加无关。毒素B几乎完全抑制峰2，而峰1未被显著抑制。毒素B对Ca 2+瞬变无影响.在对照条中，峰2处的MLC磷酸化为27.7%，其显著高于静息值（18.6%）。抑制毒素B诱导的第二次延迟性力上升与完全抑制MLC磷酸化增加相关。静息MLC磷酸化与对照条无显著差异.暴露于卡巴胆碱后3秒测定的MLC磷酸化的初始增加在对照条中为54%。毒素B也抑制了该初始磷酸化峰，尽管事实上毒素B不抑制Ca 2+瞬变和力的初始增加。这表明Rho蛋白在设定MLC磷酸化和去磷酸化反应之间的平衡中起重要作用，即使在高水平的细胞内Ca 2+下。这些研究结果是一致的假设，延迟上升的力量引起的卡巴胆碱是由于增加的Ca 2+的敏感性，由Rho蛋白介导的MLC磷酸化。
1. Clostridium difficile toxin B glucosylates the Ras-related low molecular mass GTPases of the Rho subfamily thereby inactivating them. In the present report, toxin B was applied as a tool to test whether Rho proteins participate in the carbachol-induced increase in the Ca2+ sensitivity of force and myosin light chain (MLC) phosphorylation in intact intestinal smooth muscle.2. Small strips of the longitudinal muscle of guinea-pig small intestine were incubated in toxin B (40 ng ml(-1)) overnight. Carbachol-induced force and intracellular [Ca2+], and, in a separate series, force and MLC phosphorylation, were determined.3. Carbachol induced a biphasic contraction: an initial rapid increase in force (peak 1) followed by a partial relaxation and a second delayed increase in force (peak 2). The peak of the C2+ signal measured with fura-2 preceded peak 1 of force and then declined to a lower suprabasal steady-state level. Peak 2 was not associated with a significant increase in [Ca2+]. Toxin B nearly completely inhibited peak 2 while peak 1 was not significantly inhibited. Toxin B had no effect on the Ca2+ transient.4. In control strips, MLC phosphorylation at peak 2 was 27.7 %, which was significantly higher than the resting value (18.6%). The inhibition of the second, delayed, rise in force induced by toxin B was associated with complete inhibition of the increase in MLC phosphorylation. The resting MLC phosphorylation was not significantly different from that of the control strips.5. The initial increase in MLC phosphorylation determined 3 s after exposure to carbachol was 54 % in the control strips. Toxin B also inhibited this initial phosphorylation peak despite the fact that the Ca2+ transient and the initial increase in force were not inhibited by toxin B. This suggests that Rho proteins play an important role in setting the balance between MLC phosphorylation and dephosphorylation reactions even at high levels of intracellular Ca2+.6. These findings are consistent with the hypothesis that the delayed rise in force elicited by carbachol is due to an increase in the Ca2+ sensitivity; of MLC phosphorylation mediated by Rho proteins.