Khromov A, Somlyo A V, Somlyo A P
Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22906-0011, USA.
Biophys J. 1998 Oct;75(4):1926-34. doi: 10.1016/S0006-3495(98)77633-7.
Tonic rabbit femoral artery and phasic rabbit ileum smooth muscles permeabilized with Triton X-100 were activated either by increasing [Ca2+] from pCa > 8.0 to pCa 6.0 (calcium-ascending protocol) or contracted at pCa 6.0 before lowering [Ca2+] (calcium-descending protocol). The effects of, respectively, high [MgATP]/low [MgADP] [10 mM MgATP + creatine phosphate (CP) + creatine kinase (CK)] or low [MgATP]/[MgADP] (2 mM MgATP, 0 CP, 0 CK) on the "force-[Ca]" relationships were determined. In femoral artery at low, but not at high, [MgATP]/[MgADP] the force and the ratio of stiffness/force at pCa 7.2 were significantly higher under the calcium-descending than calcium-ascending protocols (54% vs. 3% of Po, the force at pCa 6.0) (force hysteresis); the levels of regulatory myosin light chain (MLC20) phosphorylation (9 +/- 2% vs. 10 +/- 2%) and the velocities of unloaded shortening V0 (0.02 +/- 0.004 l/s with both protocols) were not significantly different. No significant force hysteresis was detected in rabbit ileum under either of these experimental conditions. [MgADP], measured in extracts of permeabilized femoral artery strips by two methods, was 130-140 microM during maintained force under the calcium-descending protocol. Exogenous CP (10 mM) applied during the descending protocol reduced endogenous [MgADP] to 46 +/- 10 microM and abolished force hysteresis: residual force at low [Ca2+] was 17 +/- 5% of maximal force. We conclude that the proportion of force-generating nonphosphorylated (AMdp) relative to phosphorylated cross-bridges is higher on the Ca2+-descending than on the Ca2+-ascending force curve in tonic smooth muscle, that this population of positively strained dephosphorylated cross-bridges has a high affinity for MgADP, and that the dephosphorylated AMdp . MgADP state makes a significant contribution to force maintenance at low levels of MLC20 phosphorylation.
用Triton X - 100通透处理的强直性兔股动脉和平滑性兔回肠平滑肌，通过将[Ca2+]从pCa > 8.0增加到pCa 6.0（钙上升方案）或在pCa 6.0收缩后再降低[Ca2+]（钙下降方案）来激活。分别测定了高[MgATP]/低[MgADP] [10 mM MgATP + 磷酸肌酸（CP）+ 肌酸激酶（CK）]或低[MgATP]/[MgADP]（2 mM MgATP，0 CP，0 CK）对“力 - [Ca]”关系的影响。在股动脉中，低[MgATP]/[MgADP]（而非高[MgATP]/[MgADP]）时，在钙下降方案下，pCa 7.2时的力和刚度/力比值显著高于钙上升方案（分别为pCa 6.0时力Po的54% 对3%）（力滞后）；调节性肌球蛋白轻链（MLC20）磷酸化水平（9 ± 2% 对10 ± 2%）和无负荷缩短速度V0（两种方案均为0.02 ± 0.004 l/s）无显著差异。在这两种实验条件下，兔回肠均未检测到明显的力滞后。用两种方法在通透处理的股动脉条提取物中测得的[MgADP]，在钙下降方案维持力期间为130 - 140 μM。在下降方案期间施加外源性CP（10 mM）可将内源性[MgADP]降至46 ± 10 μM并消除力滞后：低[Ca2+]时的残余力为最大力的17 ± 5%。我们得出结论，在强直性平滑肌中，相对于磷酸化横桥，产生力的非磷酸化（AMdp）横桥在钙下降力曲线上的比例高于钙上升力曲线，这种正应变的去磷酸化横桥群体对MgADP具有高亲和力，并且去磷酸化的AMdp·MgADP状态在低水平MLC20磷酸化时对力的维持有显著贡献。
