Humerickhouse R A, Bidasee K R, Gerzon K, Emmick J T, Kwon S, Sutko J L, Ruest L, Besch H R
Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis 46202.
J Biol Chem. 1994 Dec 2;269(48):30243-53.
The plant alkaloids ryanodine and dehydroryanodine are specific and potent modulators of the sarcoplasmic reticulum calcium release channel. In the present study, acidic, basic, and neutral side chains esters of these diterpene compounds were prepared and their pharmacologic activities were assessed. Binding affinities of the novel C10-Oeq ester derivatives for the sarcoplasmic reticulum Ca2+ release channel were evaluated with sarcoplasmic reticular vesicles prepared from rabbit skeletal muscle. Kd values of the derivatives varied 500-fold, ranging from 0.5 to 244 nM. In comparison, Kd values for ryanodine and dehydroryanodine were 4.4 nM and 5.4 nM, respectively. Basic substituents at the C10-Oeq side chain terminus produced the highest affinity derivatives (Kd values from 0.5 to 1.3 nM). Neutral and/or hydrophobic side chain derivatives exhibited intermediate affinities for the high affinity ryanodine receptor site (Kd values from 2.5 to 39 nM), whereas a derivative with a terminal acidic group had the lowest affinity (Kd value > 100 nM). Certain of the higher affinity C10-Oeq derivatives were evaluated more extensively for their pharmacologic activity on the sarcoplasmic reticular Ca2+ release channel. Both channel activating (opening) and deactivating (closing) actions were assessed from the ability of the ryanoids to alter Ca2+ efflux rates from skeletal junctional sarcoplasmic reticular vesicles that had been passively loaded with Ca2+. The natural Ryania secondary metabolites ryanodine, dehydroryanodine and esters E and F, all exhibit antithetical concentration-effect curves, indicating both activator and deactivator actions. In contrast, the semi-synthetic C10-Oeq esters selectively activate the Ca2+ release channel. Half-maximal concentrations for such activation (EC50 act) ranged from 0.87 microM to 4.2 microM, compared with an EC50 act of 1.3 microM for ryanodine. These derivatives were also evaluated for their ability to augment ATP-dependent CA2+ accumulation by cardiac junctional sarcoplasmic reticular vesicles, an effect that results from deactivation of the Ca2+ release channels. None of the derivatives tested was able to significantly augment Ca2+ accumulation, further substantiating their inability to deactivate the sarcoplasmic reticular Ca2+ release channel. Additionally, these derivatives functionally antagonized the action of ryanodine to close the Ca2+ release channel. The results presented demonstrate that these C10-Oeq ester derivatives of ryanodine and dehydroryanodine bind specifically to the SR Ca2+ release channel, selectively activate the channel, and, although they fail to effect channel closure, they nevertheless functionally compete with ryanodine at its low affinity (deactivator) site(s).
植物生物碱莱克多巴胺和去氢莱克多巴胺是肌浆网钙释放通道的特异性强效调节剂。在本研究中，制备了这些二萜类化合物的酸性、碱性和中性侧链酯，并评估了它们的药理活性。用从兔骨骼肌制备的肌浆网囊泡评估了新型C10 - Oeq酯衍生物对肌浆网Ca2 +释放通道的结合亲和力。衍生物的Kd值变化了500倍，范围从0.5到244 nM。相比之下，莱克多巴胺和去氢莱克多巴胺的Kd值分别为4.4 nM和5.4 nM。C10 - Oeq侧链末端的碱性取代基产生了亲和力最高的衍生物（Kd值从0.5到1.3 nM）。中性和/或疏水性侧链衍生物对高亲和力莱克多巴胺受体位点表现出中等亲和力（Kd值从2.5到39 nM），而具有末端酸性基团的衍生物亲和力最低（Kd值> 100 nM）。对某些亲和力较高的C10 - Oeq衍生物在肌浆网Ca2 +释放通道上的药理活性进行了更广泛的评估。从莱克多巴胺类化合物改变被动加载Ca2 +的骨骼肌连接肌浆网囊泡中Ca2 +流出速率的能力评估了通道激活（开放）和失活（关闭）作用。天然的雷公藤次生代谢产物莱克多巴胺、去氢莱克多巴胺以及酯E和F，均呈现相反的浓度 - 效应曲线，表明既有激活剂作用又有失活剂作用。相比之下，半合成的C10 - Oeq酯选择性地激活Ca2 +释放通道。这种激活的半数最大浓度（EC50 act）范围从0.87 microM到4.2 microM，而莱克多巴胺的EC50 act为1.3 microM。还评估了这些衍生物增强心脏连接肌浆网囊泡ATP依赖性Ca2 +积累的能力，这种效应是由Ca2 +释放通道失活引起的。所测试的衍生物均不能显著增强Ca2 +积累，进一步证实了它们无法使肌浆网Ca2 +释放通道失活。此外，这些衍生物在功能上拮抗莱克多巴胺关闭Ca2 +释放通道的作用。给出的结果表明，莱克多巴胺和去氢莱克多巴胺的这些C10 - Oeq酯衍生物特异性结合到SR Ca2 +释放通道，选择性地激活该通道，并且，尽管它们不能导致通道关闭，但它们在其低亲和力（失活剂）位点上与莱克多巴胺在功能上存在竞争。
