Bidasee K R, Maxwell A, Reynolds W F, Patel V, Besch H R
Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis 46202-5120, USA.
J Pharmacol Exp Ther. 2000 Jun;293(3):1074-83.
The isoflavones tectoridin (TTR) and 3'-hydroxy TTR (3'-TTR) were isolated from an Ayurvedic herbal preparation Vacä and evaluated for their affinity and effect on ryanodine receptors (RyR) using junctional sarcoplasmic reticulum vesicles (JSRVs). In [(3)H]ryanodine displacement binding affinity assays, TTR and 3'-TTR exhibited IC(50) values of 17.3 +/- 1.3 microM (K(d) = 6.7 +/- 0.4 microM) and 6.6 +/- 1.4 microM (K(d) = 2.4 +/- 0.2 microM), respectively, for fast skeletal muscle RyR (RyR1) compared with an IC(50) value for ryanodine of 6.2 +/- 0.4 nM (K(d) = 2.4 nM). TTR demonstrated a 3-fold higher affinity for cardiac RyR (RyR2) [IC(50) value of 5.2 +/- 0.6 microM (K(d) = 0.95 +/- 0.3 microM)] than for RyR1. The displacement isotherms for both TTRs paralleled that for ryanodine, consistent with the notion that all three are likely binding to similar site(s) on the receptors. Calcium efflux from and calcium influx into JSRVs were used to measure function effects of TTRs on binding to RyR. In calcium efflux assays, TTR (up to 1 mM) enhanced the release of (45)Ca(2+) from JSRVs in a concentration-dependent manner (EC(50act) of 750 microM). Higher concentrations deactivated (partially closed) RyR1. 3'-TTR had similar effects, but was approximately 2-fold more potent, exhibiting an EC(50act) value of 480 microM. Using passive calcium influx assays, TTR activated and deactivated RyR1 in a time- and concentration-dependent manner. The aglycone tectorigenin also was effective in displacing [(3)H]ryanodine from RyR1 but not from RyR2. These results demonstrate that TTRs are capable of interacting at ryanodine binding sites to differentially modulate fast skeletal and cardiac calcium-release channels.
从阿育吠陀草药制剂瓦察（Vacä）中分离出异黄酮染料木苷（TTR）和3'-羟基TTR（3'-TTR），并使用连接肌浆网囊泡（JSRV）评估它们对兰尼碱受体（RyR）的亲和力和作用。在[³H]兰尼碱置换结合亲和力测定中，与兰尼碱的IC50值6.2±0.4 nM（Kd = 2.4 nM）相比，TTR和3'-TTR对快速骨骼肌RyR（RyR1）的IC50值分别为17.3±1.3 μM（Kd = 6.7±0.4 μM）和6.6±1.4 μM（Kd = 2.4±0.2 μM）。TTR对心脏RyR（RyR2）的亲和力比对RyR1高3倍[IC50值为5.2±0.6 μM（Kd = 0.95±0.3 μM）]。两种TTR的置换等温线与兰尼碱的等温线平行，这与三者可能都结合在受体上相似位点的观点一致。利用JSRV的钙外流和钙内流来测量TTR对与RyR结合的功能影响。在钙外流测定中，TTR（高达1 mM）以浓度依赖性方式增强了JSRV中⁴⁵Ca²⁺的释放（EC50act为750 μM）。更高浓度会使RyR1失活（部分关闭）。3'-TTR有类似作用，但效力约高2倍，EC50act值为480 μM。使用被动钙内流测定，TTR以时间和浓度依赖性方式激活和失活RyR1。糖苷配基黄豆黄素也能有效从RyR1而非RyR2上置换[³H]兰尼碱。这些结果表明，TTR能够在兰尼碱结合位点相互作用，以差异方式调节快速骨骼肌和心脏钙释放通道。
