Spontaneous relaxation of isolated smooth muscle cells shortened with muscarinic receptor stimulation

当收缩激动剂被移除时，收缩的平滑肌自发地松弛。在这项研究中，我们首次提供证据表明，在ACh诱导的细胞缩短过程中，M受体介导的PKC激活对于随后的细胞缩短的松弛是必要的。本研究以链溶素-O通透性的单个平滑肌细胞为研究对象，在无任何张力负荷的条件下，通过测量细胞缩短的方法，发现PKC依赖的松弛机制对M受体的收缩刺激是特异的。此外，在ACh诱导的细胞缩短过程中，受体介导的PKC的激活是随后缩短的细胞松弛所必需的。因此，随后的松弛很可能需要依赖PKC的促进阶段，这种松弛是由收缩刺激剂ACh的去除和游离Ca~(2+)的下降所触发的。相反，钙离子诱导的细胞缩短的松弛作用不依赖于PKC。Th…PKC依赖的更多差异可能是由于M受体介导的代谢性信号的激活。目前尚不清楚PKC依赖促进松弛的机制。然而，在激动剂诱导的收缩过程中，最有可能的是依赖于PKC激活调节机制。据报道，与音调和闩锁状态的维持有关的几种可能的调节机制是依赖于PKC激活的。我们专注于这些机制之一，细胞骨架的重组。我们已经获得了一些证据表明，PKC对收缩过程中肌动蛋白细胞骨架重组的调节与随后的激动剂诱导的收缩的松弛有关。在张力负荷条件下，收缩的平滑肌组织很容易被洗涤松弛，即使ACh在蛋白激酶抑制剂的存在下诱导收缩也是如此。这些结果表明，在张力负荷的整个组织制备过程中，PKC依赖的松弛机制可能被张力相关松弛所掩盖，而PKC依赖的松弛机制积极地参与了激动剂缩短的单个平滑肌细胞的松弛。提示在激动剂诱导的收缩过程中，依赖于PKC的肌动蛋白细胞骨架的重组促进了自发的松弛。较少

Contracted smooth muscle relaxs spontaneously when contractile agonist is removed. In this study, we have provide first evidence that muscarinic receptor-mediated activation of PKC in the process of ACh-induced cell shortening is necessary for the subsequent relaxation of the shortened cells. This study was done with streptolysin-O-permeabilized single smooth muscle cells by measuring cell shortening under the condition without any tension load.PKC-dependent relaxation mechanism was found to be specific for the contractile stimulation of muscarinic receptors. Moreover, the receptor-mediated activation of PKC in the process of ACh-induced cell shortening was required for the subsequent relaxation of the shortened cells. Thus, it is likely that PKC-dependent promotion stage is required for the subsequent relaxation which is triggered by removal of contractile stimulant ACh and fall of free Ca^<2+>. In contrast, the relaxation from Ca^<2+>-induced cell shortening was independent on PKC.Th … More e differences in PKC-dependence could be due to the activation of muscarinic receptor-mediated metabotropic signaling.It is unclear what is the mechanism underlying PKC-dependent promotion of relaxation. It is, however, most likely that PKC-dependent activation of regulatory mechanisms during agonist-induced contractions could be involved. Several possible regulatory mechanisms which are related to maintenance of tone and latch state are reported to be activated PKC-dependently. We focused one of these mechanisms, the reorganization of cytoskeleton. We have obtained several evidences that PKC regulation of actin cytoskeletal reorganization during contraction is responsible for subsequent relaxation from agonist-induced contraction.Contracted smooth muscle tissues under the condition with tension-load were readily relaxd by a wash even if the contraction was induced by ACh in the presence of a protein kinase inhibitor. These results arise the possibility that the PKC-dependent relaxation mechanism, which actively participates in the relaxation of agonist-shortened individual smooth muscle cells, is apparently concealed by the tension-related relaxation in the preparation of tension-loaded whole tissues.In conclusion, we demonstrated a novel mechanism of smooth muscle relaxation. It is suggested that PKC-dependent reorganization of actin cytoskeleton during agonist-induced contraction promotes spontaneous relaxation. Less