Brozovich F V, Yamakawa M
Bockus Research Institute, Graduate Hospital, Philadelphia, Pennsylvania 19146.
Am J Physiol. 1995 Jan;268(1 Pt 1):C237-42. doi: 10.1152/ajpcell.1995.268.1.C237.
To investigate thin filament regulation of force activation in smooth muscle, we recorded force and stiffness of alpha-toxin-permeabilized single smooth muscle cells. At pCa 9, the rigor state was characterized by high in-phase stiffness, low force, and low quadrature stiffness, suggesting that the attachment of rigor cross bridges does not depend on either Ca2+ or myosin light chain (MLC) phosphorylation, and cross bridges can enter a rigor state without producing force. At pCa 4, 20 microM ATP increased force, in-phase stiffness, and quadrature stiffness, while 20 microM CTP did not increase any of these parameters, suggesting that although MLC phosphorylation is not required for the formation of rigor cross bridges, MLC phosphorylation is required for detached cross bridges to attach to actin and undergo a force-producing isomerization. These results also suggest that for smooth muscle, force activation is regulated by myosin light-chain kinase. From rigor, 20 microM ATP (pCa 9) increased force and quadrature without changing in-phase stiffness. This force increase could be explained if in rigor solution both actomyosin (AM) and AM.ADP cross bridges exist (2, 32), and ATP-induced detachment of AM cross bridges is accompanied by AM.ADP cross bridges undergoing a force-producing isomerization in combination with cooperative cross-bridge reattachment (36). Thus results of our experiments suggest that thin filament-based regulation of force activation is not essential in smooth muscle, and a population of cross bridges must begin in an attached state for force to be produced in the absence of MLC phosphorylation.
为了研究平滑肌中细丝对力激活的调节作用，我们记录了经α-毒素通透处理的单个平滑肌细胞的力和刚度。在pCa 9时，强直状态的特征是同相刚度高、力低和正交刚度低，这表明强直横桥的附着不依赖于Ca2+或肌球蛋白轻链（MLC）磷酸化，并且横桥可以在不产生力的情况下进入强直状态。在pCa 4时，20μM ATP增加了力、同相刚度和正交刚度，而20μM CTP没有增加这些参数中的任何一个，这表明虽然MLC磷酸化对于强直横桥的形成不是必需的，但MLC磷酸化是分离的横桥附着于肌动蛋白并经历产生力的异构化所必需的。这些结果还表明，对于平滑肌，力激活受肌球蛋白轻链激酶调节。从强直状态开始，20μM ATP（pCa 9）增加了力和正交度，而同相刚度没有变化。如果在强直溶液中同时存在肌动球蛋白（AM）和AM.ADP横桥（2, 32），并且ATP诱导的AM横桥分离伴随着AM.ADP横桥与协同横桥重新附着一起经历产生力的异构化（36），那么这种力的增加就可以得到解释。因此，我们的实验结果表明，细丝对力激活的调节在平滑肌中不是必需的，并且一定数量的横桥必须以附着状态开始，才能在没有MLC磷酸化的情况下产生力。
