Liepe B A, Burnside B
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
J Gen Physiol. 1993 Jul;102(1):75-98. doi: 10.1085/jgp.102.1.75.
Retinal rod photoreceptors of teleost fish elongate in the light and shorten in the dark. Rod cell elongation and shortening are both mediated by actin-dependent mechanisms that occur in the inner segment myoid and ellipsoid. The intracellular signaling pathways by which light and dark regulate the actin cytoskeleton in the inner segment are unknown. To investigate the intracellular signals that regulate teleost rod motility, we have been using mechanically isolated rod inner/outer segments (RIS-ROS) obtained from the retinas of green sunfish, Lepomis cyanellus. In culture, RIS-ROS retain the ability to elongate in response to light; myoids elongate 15-20 microns in length during 45 min of light culture. A pharmacological approach was taken to investigate the role of cyclic nucleotides, cyclic nucleotide-dependent kinases, and protein phosphatases in the regulation of RIS-ROS motility. Millimolar concentrations of cAMP and cGMP analogues were both found to inhibit light-induced myoid elongation and two cyclic nucleotide analogues, SpCAMPS and 8BrcGMP, promoted myoid shortening after RIS-ROS had elongated in response to light. The cyclic nucleotide-dependent kinase inhibitor, H8, mimicked light by promoting myoid elongation in the dark. The effects of H8 were dose dependent, with maximal elongation occurring at concentrations of approximately 100 microM. Similar to the effects of cyclic nucleotide analogues, the phosphatase inhibitor, okadaic acid (0.1-10 microM), inhibited light-induced elongation and promoted shortening. The results presented here suggest that RIS-ROS motility is regulated by protein phosphorylation: phosphorylation in the dark by cyclic nucleotide-dependent protein kinases promotes rod shortening, while dephosphorylation in the light promotes rod elongation.
硬骨鱼的视网膜视杆光感受器在光照下伸长，在黑暗中缩短。视杆细胞的伸长和缩短均由肌动蛋白依赖性机制介导，这些机制发生在内段的肌样和椭球体中。光照和黑暗调节内段肌动蛋白细胞骨架的细胞内信号通路尚不清楚。为了研究调节硬骨鱼视杆运动的细胞内信号，我们一直在使用从绿太阳鱼（Lepomis cyanellus）视网膜中获得的机械分离的视杆内/外段（RIS-ROS）。在培养过程中，RIS-ROS保留了对光作出反应而伸长的能力；在光照培养45分钟期间，肌样长度可伸长15-20微米。我们采用药理学方法来研究环核苷酸、环核苷酸依赖性激酶和蛋白磷酸酶在调节RIS-ROS运动中的作用。发现毫摩尔浓度的cAMP和cGMP类似物均抑制光诱导的肌样伸长，并且两种环核苷酸类似物SpCAMPS和8BrcGMP在RIS-ROS对光作出反应伸长后促进肌样缩短。环核苷酸依赖性激酶抑制剂H8通过在黑暗中促进肌样伸长来模拟光照。H8的作用是剂量依赖性的，在浓度约为100 microM时出现最大伸长。与环核苷酸类似物的作用相似，磷酸酶抑制剂冈田酸（0.1-至10 microM）抑制光诱导的伸长并促进缩短。此处给出的结果表明，RIS-ROS的运动受蛋白质磷酸化调节：在黑暗中由环核苷酸依赖性蛋白激酶进行的磷酸化促进视杆缩短，而在光照下的去磷酸化促进视杆伸长。
