Porrello K, Burnside B
J Cell Biol. 1984 Jun;98(6):2230-8. doi: 10.1083/jcb.98.6.2230.
We have been using lysed cell models of teleost retinal cones to examine the mechanism of contraction in nonmuscle cells. We have previously reported that dark-adapted retinas can be lysed with the detergent Brij-58 to obtain cone motile models that undergo Ca++- and adenosine triphosphate (ATP)-dependent reactivated contraction. In this report we further dissect the roles of ATP and Ca++ in activation of contraction and force production by (a) characterizing the Ca++ and nucleotide requirements in more detail, (b) by analyzing the effects of inosine triphosphate (ITP) and the ATP analog ATP gamma S and (c) by testing effects of cyclic adenosine monophosphate (cAMP) on reactivated cone contraction. Exposing lysed cone models to differing free Ca++ concentrations produced reactivated contraction at rates proportional to the free Ca++ concentration between 3.16 X 10(-8) and 10(-6) M. A role for calmodulin (CaM) in this Ca++ regulation was suggested by the inhibition of reactivated contraction by the calmodulin inhibitors trifluoperazine and calmidazolium ( R24571 ). The results of analysis of nucleotide requirements in lysed cone models were consistent with those of smooth muscle studies suggesting a role for myosin phosphorylation in Ca++ regulation of contraction. ATP gamma S and ITP are particularly interesting in that ATP gamma S, on the one hand, can be used by kinases to phosphorylate proteins (e.g., myosin light chains) but resists cleavage by phosphatases or adenosine triphosphatases (ATPases), e.g., myosin ATPase. ITP, on the other hand, can be used by myosin ATPase but does not support Ca++/calmodulin mediated phosphorylation of myosin light chains by myosin light chain kinase. Thus, these nucleotides provide an opportunity to distinguish between the kinase and myosin ATPase requirements for ATP. When individual nucleotides were tested with cone motile models, the nucleotide requirement was highly specific for ATP; not only ITP and ATP gamma S, but also guanosine triphosphate, cytosine triphosphate, adenylyl-imidodiphosphate (AMPPNP) failed to support reactivated contraction when substituted for ATP throughout the incubation. However, if lysed cones were initially incubated with ATP gamma S and then subsequently incubated with ITP, the cones contracted to an extent that was comparable to that observed with ATP. As observed in skinned smooth muscle, adding cAMP to contraction medium strongly inhibited contraction in lysed cone models.
我们一直在使用硬骨鱼视网膜视锥细胞的裂解细胞模型来研究非肌肉细胞的收缩机制。我们之前报道过，暗适应的视网膜可以用去污剂Brij - 58裂解，以获得视锥细胞运动模型，该模型会经历依赖钙离子（Ca++）和三磷酸腺苷（ATP）的再激活收缩。在本报告中，我们通过（a）更详细地描述钙离子和核苷酸需求，（b）分析三磷酸肌苷（ITP）和ATP类似物ATPγS的作用，以及（c）测试环磷酸腺苷（cAMP）对再激活视锥细胞收缩的影响，进一步剖析了ATP和Ca++在收缩激活和力产生中的作用。将裂解的视锥细胞模型暴露于不同的游离Ca++浓度下，在3.16×10^(-8)至10^(-6) M的游离Ca++浓度范围内，产生的再激活收缩速率与游离Ca++浓度成正比。钙调蛋白（CaM）在这种Ca++调节中的作用通过钙调蛋白抑制剂三氟拉嗪和平他米松（R24571）对再激活收缩的抑制得以体现。对裂解视锥细胞模型中核苷酸需求的分析结果与平滑肌研究结果一致，表明肌球蛋白磷酸化在Ca++调节收缩中起作用。ATPγS和ITP特别有趣，一方面，ATPγS可被激酶用于使蛋白质磷酸化（如肌球蛋白轻链），但能抵抗磷酸酶或三磷酸腺苷酶（ATP酶）（如肌球蛋白ATP酶）的切割。另一方面，ITP可被肌球蛋白ATP酶利用，但不支持肌球蛋白轻链激酶介导的Ca++/钙调蛋白对肌球蛋白轻链磷酸化。因此，这些核苷酸为区分激酶和肌球蛋白ATP酶对ATP的需求提供了机会。当用单个核苷酸对视锥细胞运动模型进行测试时，核苷酸需求对ATP具有高度特异性；不仅ITP和ATPγS，而且三磷酸鸟苷、三磷酸胞苷、腺苷酰亚胺二磷酸（AMPPNP）在整个孵育过程中替代ATP时均无法支持再激活收缩。然而，如果裂解的视锥细胞最初用ATPγS孵育，随后用ITP孵育，视锥细胞的收缩程度与用ATP观察到的相当。如在去表皮平滑肌中观察到的那样，向收缩培养基中添加cAMP会强烈抑制裂解视锥细胞模型中的收缩。
