HIGH-AFFINITY C-10-O-EQ ESTER DERIVATIVES OF RYANODINE - ACTIVATOR-SELECTIVE AGONISTS OF THE SARCOPLASMIC-RETICULUM CALCIUM-RELEASE CHANNEL

The plant alkaloids ryanodine and dehydroryanodine are specific and po tent modulators of the sarcoplasmic reticulum calcium release channel. In the present study, acidic, basic, and neutral side chains esters o f these diterpene compounds were prepared and their pharmacologic acti vities were assessed. Binding affinities of the novel C10-Oeq ester de rivatives for the sarcoplasmic reticulum Ca2+ release channel were eva luated with sarcoplasmic reticular vesicles prepared from rabbit skele tal muscle. K-d values of the derivatives varied 500-fold, ranging fro m 0.5 to 244 nM. In comparison, K-d values for ryanodine and dehydrory anodine were 4.4 nM and 5.4 nM, respectively. Basic substituents at th e C-10-O-eq side chain terminus produced the highest affinity derivati ves (K-d values from 0.5 to 1.3 nM). Neutral and/or hydrophobic side c hain derivatives exhibited intermediate affinities for the high affini ty ryanodine receptor site (K-d values from 2.5 to 39 nM), whereas a d erivative with a terminal acidic group had the lowest affinity (K-d va lue >100 nM). Certain of the higher affinity C-10-O-eq derivatives wer e evaluated more extensively for their pharmacologic activity on the s arcoplasmic reticular Ca2+ release channel. Both channel activating (o pening) and deactivating (closing) actions were assessed from the abil ity of the ryanoids to alter Ca2+ efflux rates from skeletal junctiona l sarcoplasmic reticular Vesicles that had been passively loaded with Ca2+. The natural Ryania secondary metabolites ryanodine, dehydroryano dine and esters E and F, all exhibit antithetical concentration-effect curves, indicating both activator and deactivator actions. In contras t, the semi-synthetic C-10-O-eq esters selectively activate the Ca2+ r elease channel. Half-maximal concentrations for such activation (EC(50 act) ranged from 0.87 mu M to 4.2 mu M, compared with an EC(50 act) o f 1.3 mu M for ryanodine. These derivatives were also evaluated for th eir ability to augment ATP-dependent Ca2+ accumulation by cardiac junc tional sarcoplasmic reticular vesicles, an effect that results from de activation of the Ca2+ release channels. None of the derivatives teste d was able to significantly augment Ca2+ accumulation, further substan tiating their inability to deactivate the sarcoplasmic reticular Ca2release channel. Additionally, these derivatives functionally antagoni zed the action of ryanodine to close the Ca2+ release channel. The res ults presented demonstrate that these C-10-O-eq ester derivatives of r yanodine 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 r yanodine at its low affinity (deactivator) site(s).