Taggart M J, Lee Y H, Morgan K G
Signal Transduction Group, Boston Biomedical Research Institute, Boston, Massachusets, USA.
Exp Cell Res. 1999 Aug 25;251(1):92-101. doi: 10.1006/excr.1999.4565.
Efficient receptor-coupled activation of smooth muscle requires discrete coordination of many signal transducing events from the plasma membrane to the myofilaments. Recruitment of key factors to the plasma membrane is thought to be crucial for transduction of extracellular signals leading to contractility. We investigated, therefore, for the first time in intact differentiated smooth muscle cells, the distributions of three molecules important for receptor-coupled excitation: protein kinase Calpha (PKCalpha), rhoA, and rho kinase (ROK). We also directly confirmed, by single cell force measurements, carbachol-induced Ca(2+) sensitization of contractility. Laser scanning confocal immunofluorescent microscopy of central smooth muscle cell sections determined that, at rest, PKCalpha, rhoA, and ROKalpha were distributed predominantly throughout the cytosol. Muscarinic stimulation resulted in significant redistribution of each protein to the cell membrane. By digital image analysis, peripheral:cytosolic distributions of PKCalpha, rhoA, and ROKalpha were calculated as, respectively, 1.05 +/- 0.03 (8), 1.09 +/- 0.03 (5), and 1.26 +/- 0.04 (12) at rest, increasing significantly following stimulation to 2.09 +/- 0.22 (6), 2.02 +/- 0.12 (8), and 1.93 +/- 0.05 (10). It is proposed that this receptor-coupled recruitment to the cell periphery of the downstream signaling molecules PKCalpha, rhoA, and ROKalpha contributes to the efficacy of agonist-induced contractile activation of smooth muscle.
平滑肌的高效受体偶联激活需要从质膜到肌丝的许多信号转导事件进行离散协调。关键因子募集到质膜被认为对于导致收缩性的细胞外信号转导至关重要。因此，我们首次在完整分化的平滑肌细胞中研究了三种对受体偶联兴奋重要的分子的分布：蛋白激酶Cα（PKCα）、rhoA和rho激酶（ROK）。我们还通过单细胞力测量直接证实了卡巴胆碱诱导的收缩性[Ca(2+)]i致敏。对中央平滑肌细胞切片进行激光扫描共聚焦免疫荧光显微镜检查确定，在静息状态下，PKCα、rhoA和ROKα主要分布在整个细胞质中。毒蕈碱刺激导致每种蛋白质显著重新分布到细胞膜。通过数字图像分析，PKCα、rhoA和ROKα的外周:细胞质分布在静息时分别计算为1.05±0.03(8)、1.09±0.03(5)和1.26±0.04(12)，刺激后显著增加至2.09±0.22(6)、2.02±0.12(8)和1.93±0.05(10)。有人提出，下游信号分子PKCα、rhoA和ROKα的这种受体偶联募集到细胞外周有助于激动剂诱导的平滑肌收缩激活的效力。
