Dokhac L, D'Albis A, Janmot C, Harbon S
J Muscle Res Cell Motil. 1986 Jun;7(3):259-68. doi: 10.1007/BF01753559.
Myometrial strips from oestrogen-primed rat uterus were exposed to various treatments, isometric contraction was measured, and the extent of myosin light chain phosphorylation determined after rapid freezing in liquid nitrogen. Two-dimensional electrophoresis revealed five spots having the same molecular weight as the light chain, with isoelectric points comprised between 5.15 and 4.95. Two of these spots (pI 5.09 and 5.00) were not present in pure uterine myosin, whether prepared from incubated or nonincubated tissue; they do not represent light chain isoforms or electrophoresis artefacts but rather degradation products appearing during the treatment. Two spots (pI 5.15 and 5.06) were identified as the nonphosphorylated and the phosphorylated forms of the light chain. The fifth minor spot (pI 4.95) may represent a diphosphorylated myosin species. Strips incubated in a normal Ca2+-medium 0.8 mM) exhibited basal contractions and an incorporation of 0.2 mol phosphate per mol light chain. Removal of Ca2+ resulted in almost complete dephosphorylation, coincident with a total relaxation of the muscle. Exposure of the myometrium to carbachol caused tetanic contractions with an increase to 0.5 mol phosphate per mol light chain. Isoproterenol, a beta-adrenergic agonist elevated intracellular cyclic AMP and induced uterine relaxation. Addition of isoproternol to a resting myometrium caused a slight but significant decrease in phosphorylation; its addition prior to carbachol markedly prevented the increase in myosin phosphorylation normally induced by the cholinergic effector. Forskolin (1 microM) increased intracellular cyclic AMP, caused relaxation and a concomitant decrease in basal myosin phosphorylation. Prostaglandin E2-induced elevation in intracellular cyclic AMP was however accompanied by an increase in contraction together with an increase in light chain phosphorylation. The data imply that light chain phosphorylation-dephosphorylation, regulated by Ca2+-dependent mechanisms, is essential for both uterine contraction and relaxation but question the role of cyclic AMP in exclusively mediating relaxation and myosin dephosphorylation in intact myometrium.
将来自经雌激素预处理的大鼠子宫的子宫肌条进行各种处理，测量等长收缩，并在液氮中快速冷冻后测定肌球蛋白轻链磷酸化程度。二维电泳显示有五个斑点，其分子量与轻链相同，等电点在5.15至4.95之间。其中两个斑点（pI 5.09和5.00）在纯子宫肌球蛋白中不存在，无论该肌球蛋白是由孵育组织还是未孵育组织制备的；它们既不代表轻链同工型，也不是电泳假象，而是在处理过程中出现的降解产物。两个斑点（pI 5.15和5.06）被鉴定为轻链的非磷酸化形式和磷酸化形式。第五个小斑点（pI 4.95）可能代表双磷酸化的肌球蛋白种类。在正常的0.8 mM Ca²⁺ 培养基中孵育的肌条表现出基础收缩，每摩尔轻链掺入0.2摩尔磷酸盐。去除Ca²⁺ 导致几乎完全去磷酸化，同时肌肉完全松弛。将子宫肌层暴露于卡巴胆碱会引起强直性收缩，每摩尔轻链的磷酸盐增加至0.5摩尔。异丙肾上腺素，一种β-肾上腺素能激动剂，可提高细胞内环磷酸腺苷水平并诱导子宫松弛。将异丙肾上腺素添加到静息的子宫肌层中会导致磷酸化略有但显著降低；在卡巴胆碱之前添加它可显著阻止通常由胆碱能效应器诱导的肌球蛋白磷酸化增加。福斯可林（1 microM）可增加细胞内环磷酸腺苷水平，引起松弛并伴随基础肌球蛋白磷酸化的降低。然而，前列腺素E2诱导的细胞内环磷酸腺苷水平升高伴随着收缩增加以及轻链磷酸化增加。这些数据表明，由Ca²⁺ 依赖性机制调节的轻链磷酸化-去磷酸化对于子宫收缩和松弛都是必不可少的，但质疑环磷酸腺苷在完整子宫肌层中仅介导松弛和肌球蛋白去磷酸化的作用。
