【作者】 唐泽耀；

【导师】 林原；

【作者基本信息】 大连医科大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 已知平滑肌收缩机理可简述为:在Ca²⁺-CaM 存在的条件下,肌球蛋白轻链激酶（MLCK）使肌球蛋白轻链（MLC2）磷酸化,磷酸化MLC2与肌动蛋白（actin）相互作用而产生张力收缩。细胞内Ca²⁺ 降至静息水平及MLC2 脱磷酸化后,平滑肌仍然保持一定的张力,这一现象难以用现有的Ca²⁺ 依赖性调节理论解释,提示在平滑肌收缩过程中还存在着Ca²⁺ 依赖性调节以外途径。本研究以胃、子宫平滑肌及心肌肌球蛋白（myosin）的非Ca²⁺-CaM 依赖性磷酸化（CIPM）为研究对象,初步揭示了MLCK 使CIPM 的一些特征,期望这些研究有助于进一步揭示平滑肌、心肌张力维持的机理提供新的研究途径。 该部分研究指出: 在Ca²⁺-CaM 不存在条件下,MLCK 也可使平滑肌myosin 非Ca²⁺ 依赖性磷酸化（CIPM）。与myosin 的Ca²⁺-CaM 依赖性磷酸化（CDPM）相比,它们的主要区别如下:（1）随孵育温度升高,CIPM 未显示明显变化,而CDPM 随孵育温度升高而明显降低。（2）随孵育时间延长,CIPM 未显示明显变化;CDPM 随孵育时间延长而明显降低。（3） 随着钾离子强度从60 mmol增加至360 mmol,CIPM 未显示明显变化;然而,当钾离子强度超过120 mmol时,CDPM 不再显示双重磷酸化。（4）CDPM 可被ML-9（一种MLCK 抑制剂）显著抑制;而在同样条件下,未观察到ML-9 对CIPM 的抑制效应。（5）CIPM 的Mg2+-ATP 酶的活性比未磷酸化的myosin 活性高,比CDPM 的活性低。这些差异在统计学上均有显著性（***P < 0.001,**P < 0.01 或* P < 0.05）。更多还原

【Abstract】 The mechanism dealing with smooth muscle contraction can be simply described as that smooth muscle myosin light chains are phosphorylated by smooth muscle myosin light chain kinase (MLCK) in Ca2+-calmodulin (CaM) dependent way (CDPM), and the interaction between phosphorylated myosin and actin induces smooth muscle contraction. Since the rise of intra-cellular Ca2+ and the phsphorylation of myosin is happened in a micro-second range, the phenomena that the tension of smooth muscle may be retained as [Ca2+]i fall down to the basic level and myosin dephosphorylated are difficult to be explained by traditional hypothesis of CDPM. These phenomena suggested that other mechanisms may also be involved in the regulation of smooth muscle contraction. This study is tried to partially reveal the characterization of Ca2+-CaM independent phosphorylation way (CIPM) by MLCK in gizzard, uterus smooth muscle and cardiac muscle and factors involved in the regulation. The author hopes that these results can provide some valuable information to further study the mechanism of CIPM. Part one The characterization of CIPM by MLCK in gizzard smooth muscle Part one indicated that smooth muscle myosin may also be phosphorylated by MLCK in the absence of Ca2+-CaM. The major differences between CIPM and CDPM are as follows. (1) With the increase of incubation-temperature, the extent of CIPM by MLCK showed no apparent change. However, the extent of CDPM by MLCK apparently decreased with the increased temperature. (2) With the increase of incubation-time, the extent of CIPM by MLCK showed no apparent changes; in contrast, the extent of CDPM by MLCK was apparently decreased. (3) The increase of ionic strength from 60 to 360 mmol KCl showed no apparent effects on CIPM. However, when ionic strength was over 120 mmol/L, Ca2+-CaM dependent di-phosphorylation of myosin was not observed. (4) CDPM was significantly inhibited by ML-9 (MLCK inhibitor). By contrast, no inhibitory effects on CIPM by MLCK were observed in the same assay condition. (5) The Mg2+-ATPase activity of CIPM was higher than that of dephosphorylated myosin but lower than that of CDPM (***P <0.001,** P <0.01 or * P <0.05). Part two The characterization of CIPM by MLCK in uterus The characterization of Ca2+-CaM independent phosphorylation of uterus myosin light chains by MLCK is as following. (1) CIPM by MLCK is much lower than that of CDPM by MLCK. (2) The extent of CIPM is more stable than that of CDPM, and CIPM was less influenced by the changes of incubation-time, incubation-temperature, and increase of the concentration of KCl. (3) Both CIPM and CDPM of uterus were less influenced by achidonic acid (AA) compared to those in gizzard smooth muscle. (4) CIPM by MLCK was less efficient, less energy consumption than that of CDPM by MLCK. (5) It was not observed that CIPM in uterus was affected by oxcytocin. The results suggested that CIPM by MLCK in uterus smooth muscle share similarity to that of CIPM by MLCK in gizzard smooth muscle. Part three Characterization of cardiac CIPM by MLCK The characterizations of cardiac CIPM by MLCK and CDPM by MLCK are as follows. (1) The extent of CIPM by MLCK is lower than that of CDPM. CIPM was characterized by less efficient, fewer energy consumption comparing with that of CDPM. (2) CIPM by MLCK in cardiac muscle was more influenced by changes of ionic strength than that of CDPM by MLCK. The extent of CIPM was less influenced by the changes of AA concentration than that of CDPM and this is different from that of CIPM in smooth muscle. (3) Cardiac CIPM and CDPM were more stable than those in smooth muscle. Cardiac CIPM and CDPM by MLCK were less influenced by changes of incubation time, incubation-temperature. CIPM by MLCK in cardiac muscle can be observed only under acid assay condition and these differences are statistically significant (** P < 0.01, or * P < 0.05). Part four The effects of histamine on CIPM by MLCK in different muscle The effects of histamine in high, medium and low concentration on CIPM and CDPM in gizzard, uterus sm更多还原