Differential activating and deactivating effects of natural ryanodine congeners on the calcium release channel of sarcoplasmic reticulum: evidence for separation of effects at functionally distinct sites

RA Humerickhouse, HR Besch , K Gerzon, L Ruest, JL Sutko and JT Emmick

Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis 46202.

Two novel natural ryanoids from extracts of the wood of Ryania speciosa Vahl were evaluated with sarcoplasmic reticulum (SR) vesicles for their binding affinities and their activating and deactivating effects on Ca2+ release channels. The new ryanoids, which are more polar than the known Ryania constituents ryanodine and didehydro-(9,21)-ryanodine, were purified using silica gel column chromatography and reverse phase high performance liquid chromatography. The new ryanoids were designated ester E and ester F, in keeping with nomenclature previously used in the literature. These compounds were identified by NMR spectroscopy and mass spectroscopy as C9ax-hydroxyryanodine and C8ax- hydroxy-C10-epi-dehydroryanodine, respectively. Binding of esters E and F to the high affinity (nanomolar Kd) site on SR Ca2+ release channels was determined from relative binding affinity assays using 6.7 nM [3H]ryanodine. Apparent Kd values of ryanodine, ester E, and ester F for binding to this domain on the skeletal muscle ryanodine receptor/SR Ca2+ release channel were 4.4 +/- 0.8, 65 +/- 10, and 257 +/- 53 nM, respectively (mean +/- standard deviation, four or more experiments). Apparent Kd values for cardiac muscle receptors were 0.51 +/- 0.01, 12 +/- 0.4, and 57 nM, respectively. As a functional indication of the effects of the ryanoids, channel-opening (activator) and channel- closing (deactivator) actions were assessed from the ability of the ryanoids to alter the rate of Ca2+ efflux from passively loaded skeletal muscle junctional sarcoplasmic reticular vesicles (JSRV). Activator actions among the ryanoids were similar, in that they exhibited apparently parallel concentration-effect curves, having a slope of 40% Ca2+ loss/decade increment in ryanoid concentration. Half- maximal values for activation (EC50 values) were 2.5, 63, and 43 microM for ryanodine, ester E, and ester F, respectively. Maximal channel opening by ester E was significantly less than that produced by the other ryanoids. The deactivator actions of the compounds on skeletal JSRV were dissimilar, in that their concentration-effect curves appeared not to be parallel. The quotient of the EC50 for deactivation and that for activation was taken as the concentration-coupling ratio (CCR). The CCR for ryanodine was 114 and that for ester F was 72, but the CCR for ester E was only 21. ATP-dependent Ca2+ accumulation by cardiac JSRV provided a second means to evaluate deactivator actions of the ryanoids. Results from cardiac JSRV assays were in general similar to those from skeletal JSRV assays.(ABSTRACT TRUNCATED AT 400 WORDS)

Volume 44, Issue 2, pp. 412-421, 08/01/1993
Copyright © 1993 by American Society for Pharmacology and Experimental Therapeutics

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