Abstract

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 [³H]ryanodine displacement binding affinity assays, TTR and 3′-TTR exhibited IC₅₀ values of 17.3 ± 1.3 μM (K_d = 6.7 ± 0.4 μM) and 6.6 ± 1.4 μM (K_d = 2.4 ± 0.2 μM), respectively, for fast skeletal muscle RyR (RyR1) compared with an IC₅₀ 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) [IC₅₀ value of 5.2 ± 0.6 μM (K_d = 0.95 ± 0.3 μM)] 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 ⁴⁵Ca²⁺ from JSRVs in a concentration-dependent manner (EC_50act of 750 μM). 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 μM. 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 [³H]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.