DIFFERENTIAL ACTIVATING AND DEACTIVATING EFFECTS OF NATURAL RYANODINECONGENERS ON THE CALCIUM-RELEASE CHANNEL OF SARCOPLASMIC-RETICULUM - EVIDENCE FOR SEPARATION OF EFFECTS AT FUNCTIONALLY DISTINCT SITES

Citation
Ra. Humerickhouse et al., DIFFERENTIAL ACTIVATING AND DEACTIVATING EFFECTS OF NATURAL RYANODINECONGENERS ON THE CALCIUM-RELEASE CHANNEL OF SARCOPLASMIC-RETICULUM - EVIDENCE FOR SEPARATION OF EFFECTS AT FUNCTIONALLY DISTINCT SITES, Molecular pharmacology, 44(2), 1993, pp. 412-421
Citations number
43
Categorie Soggetti
Pharmacology & Pharmacy",Biology
Journal title
ISSN journal
0026895X
Volume
44
Issue
2
Year of publication
1993
Pages
412 - 421
Database
ISI
SICI code
0026-895X(1993)44:2<412:DAADEO>2.0.ZU;2-9
Abstract
Two novel natural ryanoids from extracts of the wood of Ryania specios a Vahl were evaluated with sarcoplasmic reticulum (SR) vesicles for th eir binding affinities and their activating and deactivating effects o n Ca2+ release channels. The new ryanoids, which are more polar than t he known Ryania constituents ryanodine and didehydro-(9,21)-ryanodine, were purified using silica gel column chromatography and reverse phas e high performance liquid chromatography. The new ryanoids were design ated ester E and ester F, in keeping with nomenclature previously used in the literature. These compounds were identified by NMR spectroscop y and mass spectroscopy as C9ax-hydroxyryanodine and C8ax-hydroxy-C-10 -epi-dehydroryanodine, respectively. Binding of esters E and F to the high affinity (nanomolar K(d)) site on SR Ca2+ release channels was de termined from relative binding affinity assays using 6.7 nm [H-3]ryano dine. Apparent K(d) values of ryanodine, ester E, and ester F for bind ing to this domain on the skeletal muscle ryanodine receptor/SR Ca2+ r elease channel were 4.4 +/- 0.8, 65 +/- 10, and 257 +/- 53 nM, respect ively (mean +/- standard deviation, four or more experiments). Apparen t K(d) values for cardiac muscle receptors were 0.51 +/- 0.01, 12 +/- 0.4, and 57 nm, respectively. As a functional indication of the effect s of the ryanoids, channel-opening (activator) and channel-closing (de activator) actions were assessed from the ability of the ryanoids to a lter the rate of Ca2+ efflux from passively loaded skeletal muscle jun ctional sarcoplasmic reticular vesicles (JSRV). Activator actions amon g the ryanoids were similar, in that they exhibited apparently paralle l 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 muM for ryanodine, ester E, and es ter F, respectively. Maximal channel opening by ester E was significan tly less than that produced by the other ryanoids. The deactivator act ions 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-depe ndent Ca2+ accumulation by cardiac JSRV provided a second means to eva luate deactivator actions of the ryanoids. Results from cardiac JSRV a ssays were in general similar to those from skeletal JSRV assays. CCRs among the natural ryanodine analogs were obviously disparate, providi ng strong evidence for separation of ryanoid effectiveness at function ally distinct domains on (or states of) the ryanodine receptor/SR Ca2 release channel. In support of this conclusion, results of studies wi th the parent molecule ryanodine also revealed a variant CCR between h alf-maximally deactivating and half-maximally activating concentration s. In the presence of the allosteric channel activator adenosine-5'-be ta,gamma-methylenetriphosphate, ryanodine exhibited an activation curv e that lay >3 orders of magnitude to the left of its deactivation curv e. The CCR of ryanodine in the absence of this adenine nucleotide was only 2% of that in its presence. The observation that activation is no t invariantly concentration-coupled to deactivation suggests that the molecular features responsible for the activator actions of ryanoids o n Ca2+ release channels are different from those inducing deactivation of the channels. The present results constitute the first evidence th at the activator effects of ryanoids are functionally separable from t heir deactivator effects.