Miyakoda G, Yoshida A, Takisawa H, Nakamura T
Department of Biology, Faculty of Science, Osaka University.
J Biochem. 1987 Jul;102(1):211-24. doi: 10.1093/oxfordjournals.jbchem.a122034.
Spontaneously beating heart myocytes were prepared from adult rat ventricular tissues to study the correlation between beta-adrenergic receptor-stimulated changes in contractile performance and protein phosphorylation in vitro. The plasma membrane of isolated myocardial cells was permeabilized by saponin in the presence of EGTA and Mg-ATP. The permeabilized myocytes, which formed a homogeneous cell population, retained the rod-cell morphology of heart cells in situ and showed spontaneous cyclic contractions. Their contractile activity in response to extracellularly added cAMP mimicked the effects caused by beta-adrenergic stimulation of the whole heart: both the frequency and longitudinal velocity of free contraction and relaxation of the cells increased. Similar increases were observed when beta-agonist, isoproterenol, and GTP were added to suspending medium. In addition, isoproterenol maximally enhanced the adenylate cyclase activity of the cells in the presence of GTP. Both of these effects of isoproterenol were completely blocked by the beta-antagonist propranolol. cAMP-mediated phosphorylation of proteins in the permeabilized myocytes was investigated under conditions in which the beating frequency increased. cAMP elevated the phosphorylation level of five proteins; three of them with apparent molecular masses of 24, 15, and 12 kDa were membrane proteins and the other two with apparent molecular masses of 150 and 28 kDa were myofibrillar proteins. The 24-kDa phosphoprotein dissociated into 12-kDa molecules when boiled in sodium dodecyl sulfate, suggesting that these proteins are oligomeric and monomeric forms of phospholamban. The phosphorylation of these five proteins was stimulated by isoproterenol. The effect of isoproterenol was enhanced by GTP but completely blocked by propranolol. The time course of their phosphorylation correlated well with that of the increase in the beating frequency of the cells; both were measured after the administration of isoproterenol and GTP. When propranolol was added after the start of the stimulation by isoproterenol, only phospholamban and the 15-kDa protein were rapidly dephosphorylated in close correlation with the decrease of the beating frequency. These results demonstrate for the first time that the permeabilized myocytes retain the functional beta-adrenergic receptor and cellular responses to beta-adrenergic stimulation. They also suggest that cAMP-mediated phosphorylation of proteins, possibly phospholamban and/or the 15-kDa protein, is involved in the increased contractile activity of permeabilized heart cells.
从成年大鼠心室组织中制备自发搏动的心肌细胞，以研究β-肾上腺素能受体刺激引起的收缩性能变化与体外蛋白质磷酸化之间的相关性。在EGTA和Mg-ATP存在的情况下，用皂素使分离的心肌细胞质膜通透化。通透化的心肌细胞形成了均匀的细胞群体，保留了原位心脏细胞的杆状细胞形态，并表现出自发的周期性收缩。它们对细胞外添加的cAMP的收缩活性模拟了β-肾上腺素能刺激整个心脏所产生的效应：细胞自由收缩和舒张的频率以及纵向速度均增加。当向悬浮培养基中添加β-激动剂异丙肾上腺素和GTP时，也观察到了类似的增加。此外，在GTP存在的情况下，异丙肾上腺素最大程度地增强了细胞的腺苷酸环化酶活性。异丙肾上腺素的这两种效应均被β-拮抗剂普萘洛尔完全阻断。在搏动频率增加的条件下，研究了通透化心肌细胞中cAMP介导的蛋白质磷酸化。cAMP提高了五种蛋白质的磷酸化水平；其中三种表观分子量为24、15和12 kDa的是膜蛋白，另外两种表观分子量为150和28 kDa的是肌原纤维蛋白。24 kDa的磷蛋白在十二烷基硫酸钠中煮沸时解离成12 kDa的分子，这表明这些蛋白质是受磷蛋白的寡聚体和单体形式。异丙肾上腺素刺激了这五种蛋白质的磷酸化。GTP增强了异丙肾上腺素的作用，但被普萘洛尔完全阻断。它们磷酸化的时间进程与细胞搏动频率增加的时间进程密切相关；两者都是在给予异丙肾上腺素和GTP后测量的。当在异丙肾上腺素开始刺激后添加普萘洛尔时，只有受磷蛋白和15 kDa的蛋白质迅速去磷酸化，与搏动频率的降低密切相关。这些结果首次证明通透化的心肌细胞保留了功能性β-肾上腺素能受体以及对β-肾上腺素能刺激的细胞反应。它们还表明，cAMP介导的蛋白质磷酸化，可能是受磷蛋白和/或15 kDa的蛋白质，参与了通透化心脏细胞收缩活性的增加。
