From the Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5120
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ABSTRACT |
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Ryanodine derivatives are differentially effective on the two limbs of the ryanodine concentration-effect curve. This study comparing ryanodine, ryanodol, and pyridyl ryanodine and nine C10Oeq esters of them focuses on structure-function relations underlying their differential effectiveness. Ryanodol and pyridyl ryanodine had significantly lower affinities than ryanodine, but their EC50act values (concentration of ryanoid that induces one-half of full efficacy), potencies, and efficacies were not diminished in like fashion. Ryanodine and ryanodol were partial agonists, whereas pyridyl ryanodine was a full agonist, having a diminished deactivation potency. C10Oeq esterifications enhanced affinities and efficacies of the base ryanoids. The C10-Oeq ester derivatives of ryanodine and pyridyl ryanodine, but not those of ryanodol, lost their capacity to deactivate RyR1s. Thus, affinity differences among ryanoids clearly do not predicate functional differences as regards activation of Ca2+ release channels. The pyrrole carboxylate on the C3 of ryanodine is dispensable to ryanoid activation of Ca2+ release channels. Ryanodol lacks this ring, but it nevertheless effects substantial activation. Moreover, its C10-Oeq esters display full efficacy. The increased ability of all the C10-Oeq derivatives to release Ca2+ from the vesicles strengthens their role in directly impeding deactivation of RyR1, perhaps by interaction with some component within the transmembrane ionic flux pathway.
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INTRODUCTION |
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Significant progress has been made recently in delineating the role of the sarcoplasmic reticulum (SR)1 in the excitation-contraction (E-C) coupling processes of muscles (1-6). In major part, pharmacologic dissection of the pivotal role of the Ca2+ pump of SR in E-C coupling has been achieved with aid of the microbial metabolites cyclopiazonic acid (7) and thapsigargin (8), which are potent inhibitors of Ca2+,Mg2+ATPases (sarco(endo)plasmic reticulum Ca2+-ATPase) (9). Furthermore, knock-out genetic mutants (10) have solidified the importance of the regulator protein phospholamban (2) on physiologic function of the sarcoplasmic reticulum. Especially contributory to our current understanding of SR function has been studies with the complex plant alkaloid ryanodine (C3-O-(pyrrole-2-carbonyl) ryanodol), a potent and specific modulator of a class of intracellular Ca2+ release channels (11-15).
For over a decade it has been appreciated that Ca2+ release channels (both on sarcoplasmic reticulum and endoplasmic reticulum) exhibit a complex response to ryanodine. At the single channel level, the effects of ryanodine are now appreciated to be quantal, occurring in a minimum of two discrete steps. Submicromolar concentrations induce an abrupt change from a flickery, predominantly closed (zero conductance) state to a sustained, higher (fractional, i.e. less than full) conductance state. "Locked" in this fractional conductance state and hovering about half-way between closed and fully open, the Ca2+ release channel modified by ryanodine is less restive, displaying fewer opening events than in the absence of ryanodine (16, 17). Rarely at submicromolar but more readily at micromolar concentrations, ryanodine may induce a second step change in the ionic conductance of the Ca2+ release channel to a persistently closed state displaying opening events only rarely. For convenience of discussion, we adopt the convention describing the locked, fractional conductance state as "modified" (18) and the persistently closed, zero conductance state produced at high ryanodine concentrations as "shut."
Assays at the multi-channel level with muscle microsomes reveal graded
responses to ryanodine, reflecting the ensemble of the many individual
quantal events. Passive Ca2+ efflux assays evaluating
ensemble effects typically employ sarcoplasmic reticulum membrane
vesicles prepared from fast skeletal muscles (19-21). Low
concentrations of ryanodine (60 µM) activate or open the RyR1s of such preparations, whereas higher concentrations deactivate, that is they close them. The resultant, biphasic response curves reflect the aggregate of the step quantal changes occurring in
the multitude of individual Ca2+ release channels of such
microsomes. We have recently shown that the behavior of single channels
incorporated into lipid bilayers (18) is consistent with their
aggregate behavior judged from flux studies with sarcoplasmic reticulum
vesicles. Calcium sparks seem to demonstrate a similar phenomenon
(22).
Details of the molecular processes that trigger RyRs to respond to ryanodine in a biphasic manner remain obscure. A robust strategy to approach understanding of the modulation of RyR1 could be assembled from structure-activity relationships that would identify chemical functionalities on the molecule that prove to be integral for high affinity interactions and comparison of these with topological features found essential for ryanoid channel modulating characteristics. The present study is a step in the latter direction.
It is generally recognized that the C3 substituent on the A-ring of ryanodine is important to its affinity for ryanodine-binding sites on RyR1 (23-25). However, few studies have been conducted to evaluate whether the chemical nature of C3 substituents are especially important for the efficacy of ryanoids at opening, on the one hand, or closing, on the other, the Ca2+ release channel to ionic flux. This paucity results from chemical synthetic limitations. Although de-esterification of the C3 ester is facile, re-esterification with original stereochemistry is not. In fact, it is presently unattainable. Thus, comparisons of efficacy of C3-modified ryanoids have been limited for the most part to comparisons between ryanodine and ryanodol, the latter having no C3 ester. The present study adds pyridyl ryanodine to the mix. Also termed ryanodyl-3-nicotinate in the literature, the affinity but not the functional consequences of pyridyl ryanodine have been investigated. Jefferies et al. (23) first identified this ryanoid as a naturally occurring minor secondary metabolite from Ryania wood. They found pyridyl ryanodine to be "essentially inactive compared with ryanodine for insecticidal activity, for toxicity to mice, and for competition with [3H]ryanodine at the Ca2+ ryanodine receptor complex of skeletal muscle." For use in the present study, we purified to >98% a sufficient quantity of pyridyl ryanodine to permit a more extensive functional evaluation of this natural ryanoid.
Among the numerous derivatives of ryanodine thus far reported, only a few C10-Oeq esters increased RyR1 affinity (26-28). Such effective ester substituents were incorporated into pyridyl ryanodine and compared in the present study with ryanodine and ryanodol and certain other of their esters. The results show clearly that affinity consequences of C3 ester substituents do not predict their channel activating character. Furthermore, anchoring substituents at the C10-Oeq position effect no greater changes in activation potency of weak ryanoids than they do of highly effective ryanodines. Apparently the topologic features determinant for RyR1 activation and deactivation are not congruent with those responsible for apparent affinity for binding to RyR1.
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EXPERIMENTAL PROCEDURES |
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Materials
Ryanodine
(C3-O-(pyrrole-2-carbonyl) ryanodol) and pyridyl
ryanodine (C3-O-(pyridyl-3-carbonyl) ryanodol)
used in this study were obtained from Integrated Biotechnology Corp.
(Carmel, IN) or were isolated and purified to 98% single compounds
from chipped Ryania speciosa wood supplied by them,
using procedures previously described (21, 29).
[3H]Ryanodine (specific activity 87 Ci/mmol) and
45CaCl2 (specific activity 2.7 Ci/mmol) were
purchased from NEN Life Science Products. Brevital® sodium was
obtained from Lilly. All other reagents and solvents used were of the
highest purity available commercially.
Skeletal Muscle Junctional Sarcoplasmic Reticular Membrane Vesicles (JSRV)
Crude (mixtures of junctional and longitudinal) sarcoplasmic
reticular membrane vesicles (CVs) were prepared from fast twitch skeletal muscles of rabbits. The CVs were then subfractionated using a
discontinuous sucrose gradient as described previously (21, 26-28,
30). The interface between 1.2 and 1.5 M sucrose which
typically contains the highest percentage of ryanodine receptors was
collected by aspiration and resuspended in freshly prepared isolation
buffer at concentrations of 5-10 mg/ml, quickly frozen, and stored at
70 °C.
Chemical Methods
Preparation of Ryanodol The C3 hydroxyl substituent was removed from ryanodine using the alkaline de-esterification procedure described by Kelley et al. (31) and ryanodol the product of interest, was further purified by column chromatography giving a yield of 85%. Detection of ryanodol on thin layer chromatographic (silica gel) plates was achieved by spraying with 10% ceric ammonium nitrate in 85% phosphoric acid and heating for 5 min. Ryanodol was identified as a reddish-brown spot. The product was characterized by mass spectroscopy and 1H NMR.
Semi-synthesis of C10-Oeq Ester
Derivatives of Ryanodol, Ryanodine, and Pyridyl Ryanodine
Esterifications of the accessible C10 secondary
hydroxyl of the base ryanoids (Fig. 1)
with Cbz--alanine,
-alanine, and guanidino propionate were
conducted using procedures described (26-28) as outlined in
Scheme 1.
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Preparation of C10-Oeq Cbz--alanyl
Ester Derivatives of Ryanodol, Ryanodine, and Pyridyl
Ryanodine--
Each of the base ryanoids (0.5 mM in 20 ml
of dried CH2Cl2) were derivatized and purified
by column chromatography as described. The products of interest were
eluted with 4-6% MeOH in CH2Cl2. Yields
averaged
80% after freeze-drying from dioxane. The structures of the
derivatives were confirmed using 1H NMR and mass
spectroscopy.
Deprotection of C10-Oeq -Alanyl Ester
Derivatives of the Base Ryanoids--
Hydrogenolysis of the
C10Oeq Cbz-
-alanyl ryanoid esters was
performed as described (26-28). The products of interest eluted with
12-15% MeOH in CH2Cl2 and on rotary
evaporation and freeze-drying from dioxane afforded yields of at least
85%. The structures of the C10-Oeq
-alanyl
ryanoids were characterized by 1H NMR and mass
spectroscopy.
Preparation of C10-Oeq Guanidino-propionyl Ester Derivatives of the Base Ryanoids-- C10Oeq guanidino acyl ester derivatives of these ryanoids were prepared from their C10-Oeq aminoacyl esters as described previously (26-28) or by the procedure of Bernatowicz et al. (32). The structures of the derived compounds were confirmed by 1H NMR and mass spectroscopy.
Biochemical and Pharmacological Properties of the Base Ryanoids and Selected C10-Oeq Ester Derivatives
Relative Binding Affinities of the Base Ryanoids for RyR1s
Relative binding affinities of ryanodol, ryanodine, and pyridyl
ryanodine, as well as their C10-Oeq
Cbz--alanyl,
-alanyl, and guanidino-propionyl ester derivatives
were assessed using equilibrium displacement binding affinity assays
based on rabbit skeletal muscle JSRV as described (18, 21, 24-30, 33).
Briefly, JSRV (0.1 mg/ml) were incubated in binding buffer (500 mM KCl, 20 mM Tris·HCl, 0.2 mM
CaCl2, pH 7.4 at 37 °C) containing 6.7 nM
[3H]ryanodine and varying concentrations of the ryanoids
(up to 50 µM) for 2 h at 37 °C. At the end of the
incubation, the vesicles were filtered through Whatman GF/C filters
(0.45 µm) using a cell harvester (Brandel model M-24R), washed 3 times with 3 ml of ice-cold binding buffer (pH 7.4 at 0 °C),
quantitated by liquid scintillation counting. Nonspecific binding was
determined simultaneously by incubating JSRV with a concentration of
the respective unlabeled ryanoid 10 × higher than the highest
concentration used in the binding assay. Displacement curves and
IC50 and Kd values were calculated using
Microsoft Excel®, CA-Cricket Graph (version 1.1), the coupled binding
analysis program EBDA/Ligand (34), and GraphPad Prism 2.0 (San Diego,
CA).
Channel Modulating Characteristics of the Ryanoids Ryanoid-induced changes in Ca2+ efflux were assessed by measuring their abilities to alter passive Ca2+ efflux from JSRV previously loaded with 45Ca2+ as described (19-21, 26-30). In this assay, JSRV (3.5 mg/ml) were incubated in Ca2+ loading buffer (140 mM NaCl, 20 mM HEPES, 1.1 mM Ca2+ (spiked with 0.25 µM 45Ca2+), 0.1 mM EGTA, and 1 mM MgCl2 (pH 7.0 at 22 °C)), in the presence of varying concentrations of the ryanoids (up to 5 mM) for 2 h at 22 °C. The Ca2+ load achieved was 20-30 nmol/mg JSRV protein. Passive Ca2+ (45Ca2+) efflux through RyR1 pathways was determined by diluting the vesicles (5 µl) 100-fold into an efflux buffer (140 mM NaCl, 20 mM HEPES, 0.2 mM Ca2+, and 1 mM EGTA, pH 7.0 at 22 °C). After 3 s, efflux was stopped by further diluting the vesicles 6-fold into an ice-cold stop solution (140 mM NaCl, 20 mM HEPES, 0.1 mM EGTA, 5 mM MgCl2, and 0.01 mM ruthenium red) and rapidly filtering. The vesicles on the filters were then washed 3 times with 3 ml of rinse solution (identical to stop solution except without ruthenium red), and the 45Ca2+ remaining inside the vesicles was determined by liquid scintillation counting.
Since Ca2+ efflux may be different from different vesicle populations (reflecting, for example, different densities of RyR1s), the measurements can be taken to represent only an average over all vesicles in the preparation. We use herein the term ensemble functional patency to describe the effect of a given ryanoid-induced opening toward net Ca2+ efflux. The lower the amount of 45Ca2+ remaining in the vesicles the greater the ensemble functional patency of RyRs to Ca2+ efflux and vice versa. It is not clear whether one or more than one channel on a given vesicle may be adequate to empty Ca2+ vesicular content in 3 s. In addition, as discussed below, efficacy for channel opening varies among ryanoids and must be considered when evaluating efficacy consequences of chemical modifications of ryanodine.Full Efficacy of Ryanoid Actions
To compare the ability of the various ryanoids to open the Ca2+ release channels fully, it was necessary to institute a protocol that would establish it for each JSRV population. In previously published studies with activation selective ryanodine derivatives (26-38), we found guanidino-propionyl ryanodine to consistently produce maximal Ca2+ efflux at 30 µM (in the absence of 1 mM AMP-PCP) and at 300 nM in its presence. Concentration-effect curves with several dozen ryanodine derivatives never revealed efflux any greater than this combination (data not shown). Thus we used as an operational definition of full efficacy that which occurs with 1 µM guanidino-propionyl ryanodine in the presence of 1 mM AMP-PCP. It was 88 ± 5% (mean ± S.D.) for all nine preparations used in the present studies. The residual 12% could be released within 2 s by 1 µM A23187 (data not shown).
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RESULTS |
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Relative Binding Affinities of the Base Ryanoids for Rabbit Skeletal Muscle RyR1
Binding affinities were determined from the ability of the
compounds to compete with 6.7 nM
[3H]ryanodine for binding to receptor sites on RyR1,
using displacement binding affinity assays. As shown in Fig.
2a, the model ryanoids exhibit
substantial differences in apparent receptor affinity. Ryanodol ()
was weakest in this regard, exhibiting an IC50 value of
8000 ± 50.9 nM (Kd = 2970 ± 49.6 nM) for RyR1. Derivatives varying at the
C3 secondary hydroxyl of ryanodol, containing either pyridyl-3-carbonate (pyridyl ryanodine) or pyrrole-2-carbonate (ryanodine), showed significantly enhanced affinity for RyR1
(IC50 value of 700 ± 10.2 nM
(Kd = 260 ± 12.2 nM), for pyridyl ryanodine and IC50 value of 6.2 ± 0.2 nM
(Kd = 2.4 ± 0.2 nM for ryanodine,
respectively (Fig. 2a,
and
)). All three displacement
binding affinity isotherms display apparent saturation binding kinetics
and are parallel to each other, as anticipated from their similarities
in chemical structure.
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These results are compatible with those from previous studies showing that an ester substituent on the C3 carbon enhances high affinity ryanoid interactions with RyR1 from rabbit skeletal muscles. The present studies suggest that, in addition, the chemical nature of the C3 ester plays a substantial role in the high affinity interaction of the ryanoids with RyR1 (see also Table I, data column 2). High affinity interactions of ryanoids with RyR1 apparently depend to some degree on an appropriately sized substituent on the C3 carbon. These results confirm and extend reports from prior investigations (23-25, 35).
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It should be noted, however, that there is a substantial potential for distortion of affinity assays by minor variations in purity of the applied ryanoids. To minimize this confounding variable in the present study, we re-assessed ryanoids' affinities using exactly the same ryanoid preparations subsequently applied to evaluate channel modulating characteristics in determinations of passive Ca2+ efflux effects.
Relative Binding Affinities of C10-Oeq
Derivatives of the Base Ryanoids with Cbz--Alanyl,
-Alanyl, and
Guanidino-propionyl Esters
Ryanodol--
The relative binding affinities of ryanodol
derivatives, including C10-Oeq Cbz--alanyl
ryanodol, C10-Oeq
-alanyl ryanodol, and
C10-Oeq guanidino-propionyl ryanodol were
compared with ryanodol using displacement binding affinity assays as
described above. As shown in Fig. 2b, increasing the
basicity of the C10-Oeq ester substituent
terminus, from Cbz-
-alanyl to
-alanyl, and then to
guanidino-propionyl progressively enhanced the affinity of these
ryanoids for binding sites on RyR1 (see also Table I, data column 3).
These values demonstrate that C10-Oeq anchoring
by the esterifications restored affinity toward that of ryanodine by
2-, 7-, and 19-fold, with Cbz-
-alanyl,
-alanyl, and
guanidino-propionyl side chains, respectively (compared with parent
ryanodol).
Ryanodine--
Esterification of the C10 equatorial
secondary hydroxyl of ryanodine to produce the Cbz--alanyl,
-alanyl, and guanidino-propionyl derivatives also progressively
enhances their affinities for RyR1 (Fig. 2c). Notably, the
degree of leftward shift by each of the ryanodine esters in the series
was not as large as that of the corresponding ryanodol ester in the
same series (Table I, data column 3). These displacement binding
isotherms are all parallel, consistent with the notion that all four
ryanoids are interacting with the same site(s) on RyR1. The higher
affinities of these semi-synthetic ester derivatives of ryanodine are
apparently due to the anchoring influence of the C10 ester
substituents on RyR1, but they are able to add only a lesser
stabilizing influence to the predominant binding strength afforded by
the C3 pyrrole-2-carbonyl (and the remaining polycyclic,
polyhydroxylic backbone of the parent ryanodine molecule).
Pyridyl Ryanodine--
As for ryanodol and ryanodine,
esterification of the C10 secondary hydroxyl group of
pyridyl ryanodine with basic Cbz--alanine,
-alanine, and
guanidino-propionate side chains produced derivatives that exhibited
enhanced affinities for RyR1 (Fig. 2d; Table I, data column
3). Notably, the affinity enhancements by anchoring esterifications of
pyridyl ryanodine were greater than those for either ryanodol or
ryanodine. The guanidino-propionyl derivative of pyridyl ryanodine
demonstrated an apparent affinity of about 20 nM, a value
not substantially different from that of ryanodine.
Efficacy Consequences of the C3-O Modifications: Comparisons among the Base Ryanoids and Their C10-Oeq Ester Derivatives
To relate affinity consequences of ryanoid structural changes with consequent functional changes, we evaluated their ability to enhance (and to diminish) Ca2+ efflux from calcium-loaded junctional sarcoplasmic reticulum vesicles (Fig. 3). Like other investigators we have expressed in previous communications concentration-dependent effects of ryanoids on Ca2+ efflux as a percent of initial total vesicular content, the latter being determined in the absence of any ryanoid (19-21, 26-30). However, this presents an incomplete picture, in that it leaves unaccounted the variable degree of contamination of each preparation of junctional vesicles with vesicles bearing no functional Ca2+ release channels. This can distort determination of EC50 values, because the 100% effect has been tacitly assumed as the maximal opening effect on the Ca2+ release channels. Previous reports establish that substantial differences exist among ryanoids in their efficacy, as judged from their ability to maximally open RyR1 to Ca2+ efflux. For extended structure-activity evaluations it seemed necessary to establish a robust means to ascertain maximal ensemble Ca2+ channel opening (full efficacy, see "Experimental Procedures").
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Channel Modulating Characteristics of the Base Ryanoids on Rabbit Skeletal Muscle RyR1-- The ability of the base ryanoids to modulate Ca2+ efflux via RyR1 was assessed with passive efflux assays using rabbit skeletal muscle JSRV (Fig. 3a). In a dose-dependent manner, ryanodine (dashed lines) at concentrations up to 60 µM increased Ca2+ efflux from the vesicles by opening RyR1s (EC50act = 3.4 µM) (where EC50act is the concentration of ryanoid that induces one-half of full efficacy; full efficacy is the ability to induce 100% of maximum Ca2+ releasable from JSRV, as established in control experiments using the combination of 1 µM guanidino-propionyl ryanodine plus 1 mM AMP-PCP). At its optimal concentration, ryanodine (60 µM) released a maximum of 81.5% of the Ca2+ load from the JSRV. Thus ryanodine functioned as a partial agonist, having less than full efficacy for channel opening. At higher concentrations, ryanodine impeded Ca2+ efflux from the vesicles in a manner predicted by the usual biphasic quasi-bi-sigmoidal relationship (EC50deact = 260 µM) (where EC50deact is the concentration of ryanoid that reduces the Ca2+ release by one-half of full efficacy). The ratio between the midpoints of the two limbs, that is the concentration coupling ratio previously defined (21) as the ratio EC50deact/EC50act, was 76.
The channel modulating profile of ryanodol (Fig. 3a,Relative Efficacies of C10-Oeq Derivatives
of Ryanodol: Cbz--alanyl Ryanodol and
-Alanyl
Ryanodol--
C10-Oeq ester derivatives of
ryanodol, like ryanodol itself, also activated and at least partially
deactivated RyR1, with only small changes in potencies (Fig.
3b, Table I). However, these semi-synthetic derivatives
demonstrated enhanced activating efficacies. Up to concentrations of 30 µM, these ryanoids were unable to alter significantly the
ensemble Ca2+ efflux. Thereafter, progressively increasing
the concentrations of C10-Oeq Cbz-
-alanyl
ryanodol and C10-Oeq
-alanyl ryanodol up to
3 mM resulted in activating efficacies greater than that of
ryanodol and perhaps even of ryanodine (Fig. 3b (
) and
(
)). Whereas C10-Oeq Cbz-
-alanyl ryanodol
(3 mM) approached full agonist activity,
C10-Oeq
-alanyl ryanodol (3 mM)
released a maximum of only 84.5%. Their EC50act values
were similar, at about 300 µM. Thus, the latter compounds
increased the ensemble functional patency of RyR1 to Ca2+
efflux well beyond the maximum affordable by parent ryanodol.
Relative Efficacies of C10-Oeq Derivatives
of Ryanodine--
As previously reported,
C10-Oeq derivatives of ryanodine (Fig.
3c) induced Ca2+ release from vesicles, with
greater efficacies than ryanodine. All three became full agonists or
nearly so. C10-Oeq Cbz--alanyl ryanodine
demonstrated an EC50act of 6.9 µM, releasing
a maximum of 87.5% of the intravesicular Ca2+ at a
concentration of 60 µM, and
C10-Oeq
-alanyl ryanodine exhibited an
EC50act of 2.3 µM, releasing a maximum of
98.5% of the Ca2+ from JSRV at a concentration of 30 µM, and C10-Oeq
guanidino-propionyl ryanodine released a maximum of 94.5% at a
concentration of 30 µM (EC50act = 2.9 µM). Clearly, the channel activating profiles of
C10-Oeq
-alanyl ryanodine and
C10-Oeq guanidino-propionyl ryanodine did not
mimic that of ryanodine. At the low end of the concentration-effect
curve (concentrations less than about 1 µM), their
channel activating profiles are approximately superimposable on that of
ryanodine. Higher concentrations of these derivatives resulted in
greater Ca2+ efflux than ryanodine.
C10-Oeq Cbz-
-alanyl ryanodine appears to be
less potent than ryanodine, or either of the two other derivatives in
ability to activate RyR1, i.e. its concentration-effect
curve was shifted slightly to the right.
Relative Efficacy of C10-Oeq -Alanyl
Pyridyl Ryanodine--
Like the other ryanoid derivatives,
C10-Oeq
-alanyl pyridyl ryanodine was
assessed for its channel modulating characteristics using passive
Ca2+ efflux assays. As is shown in Fig. 3d
(
), it too activated the RyR1 in a
concentration-dependent manner (EC50act = 3.9 µM) and exhibited full agonist action. At a concentration
of 300 µM, 98% of the intravesicular Ca2+
was released. In fact, up to a concentration of 30 µM the
channel activating effects of pyridyl ryanodine and its
C10-Oeq
-alanyl derivative were essentially
superimposable. Concentrations of pyridyl ryanodine above these
appeared to be slightly more efficacious than
C10-Oeq
-alanyl pyridyl ryanodine in ability
to induce Ca2+ loss from the vesicles. In preliminary
passive Ca2+ efflux assays, we found that
C10-Oeq Cbz-
-alanyl pyridyl ryanodine and
C10-Oeq guanidino-propionyl pyridyl ryanodine
were also activator-selective ryanoids and that their channel
activating profiles were superimposable on that of parent pyridyl
ryanodine. Further experimentation is currently ongoing to confirm or
refute these latter observations.
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DISCUSSION |
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A principal result of the present study is that pyridyl ryanodine, a natural secondary metabolite from R. speciosa, shows for the first time a definitive separation of activation potency from high affinity for the ryanodine receptor. Pyridyl ryanodine had only 1% of the affinity of ryanodine for RyR1-binding sites but showed 100% of ryanodine's activation potency and even greater efficacy than ryanodine. The 100-fold loss in apparent affinity was associated with a 10-fold loss in deactivation potency.
We previously suggested that the effects of ryanoids on Ca2+ efflux might usefully be rationalized as the algebraic sum of individual activation and deactivation concentration-effect curves for the ryanoids (21). A straightforward means to characterize the degree of overlap of the deactivation limb on the activation limb of ryanoid effects is to compare the mean values for their midpoints. For convenience of discussion, we have termed the ratio of EC50deact to EC50act, the concentration coupling ratio, abbreviated as the CCR (21). Recently Meissner et al. (37) coined the term "Ca2+ window of receptor activation" to describe a similar phenomenon, namely anionic influences that separate the limbs of the pCa-ryanodine binding curve.
The CCR for ryanodine in the present study was approximately 76, but it increased >8-fold to 680 for pyridyl ryanodine. The CCR for ryanodol, on the other hand, was only 0.68. Strong overlap of the deactivation curve on the activation curve rationalizes the poor efficacy of ryanodol, and the lack of overlap rationalizes the full channel opening character of pyridyl ryanodine. The wide berth between the EC50act and the EC50deact for pyridyl ryanodine was caused entirely by a loss of potency for deactivation; activation potency for pyridyl ryanodine was equal to that of ryanodine. Pyridyl ryanodine thus activated the channel equally as well as ryanodine but deactivated it only as poorly as ryanodol.
Taken together, these results strongly imply that sites at which ryanoids enlist functionally differential effects on RyR1 are physically separate as well. Both activation and deactivation appear to arise from initially high affinity interactions. If such suggested sites actually exist, they are kinetically indistinguishable, so far, by binding competition studies using 6.7 nM [3H]ryanodine.
Displacement binding affinity assays are most discriminant of subsites when the concentration of the radiolabeled ligand used is less than the lowest concentration of unlabeled ligand against which it is allowed to compete. To approach this ideal, in an earlier study we prepared a radioiodinated derivative of ryanodine, namely C10-Oeq N-(4-azido-5-125I-iodosalicyloyl) glycyl ryanodine, having a specific activity (1400 Ci/mmol) 12-fold higher than that of [3H]ryanodine (120 Ci/mmol). By using 0.5 nM of this radioligand in displacement binding affinity assays, we reported a pseudo-sigmoidal displacement curve that had a distinct shoulder at about 6 nM ryanodine (28). This could represent two sequential ryanodine binding isotherms within the nanomolar concentration range. Applied to the present results, the higher affinity site would have an IC50 of 0.5 nM (Kd = 0.26 ± 0.02 nM) while the lower (high) affinity site would demonstrate an IC50 of about 15 nM (Kd = 11.7 ± 1.2 nM).
An alternate explanation for those results is that the intense radiation from the 125I label produced an autodegradation product as a minor contaminant in the binding displacement medium. Although this seems unlikely, we are currently investigating this possibility. If such an artifact can be ruled out, those findings would complement the present data, suggesting that the biphasic effects of ryanoids on RyRs may arise from sequential interactions at functionally antagonistic binding sites (38) that both apparently have high affinity constants, at least initially (39). If one of these (the deactivating sites?) were multicomponent and displayed negative cooperativity, Scatchard plots based on [3H]ryanodine could be misconstrued to represent only a single high and one (or a few) low affinity sites (40, 41). This possibility is under current investigation using a more selective derivative, aryldiazirine ryanodine (42).
Prior studies with two other minor secondary Ryania metabolites, namely ester E (C9ax-hydroxy-ryanodine) and ester F (C8ax-hydroxy-C10-epi-dehydroryanodine) provided evidence that the topologic features responsible for activator actions of ryanoids are not coincident with those inducing deactivation of the channels (21). However, those natural ryanodines lost activation potency by about as much as they lost deactivation potency. Furthermore, affinities of ester E and ester F were much greater than those of the present studies. Pyridyl ryanodine is the first (natural or semi-synthetic) ryanoid to possess full ability to activate the Ca2+ release channel while losing, by over 100-fold, the ability to compete for binding at the high affinity ryanodine-binding sites.
A second major conclusion from the present results is that the C3 ester substituent on ryanodines plays only a secondary role in their potency and efficacy for activation of Ca2+ release channels. This was an unexpected finding in light of the apparently primary role of the pyrrole substituent in affinity (36). As noted above in the discussion of our prior results with a radioiodinated ryanoid, the present results suggest that if receptor affinity may be correlated to potency of unidirectional effects on RyR1, then such a correlation appears more consistent with the deactivating than the activating limb of the biphasic concentration-effect curve.
In any case, the present results show that a C3 ester is dispensable for activation of the Ca2+ release channel. Ryanodol lacks an ester substituent at C3 but nevertheless is able to open the Ca2+ release channel to Ca2+ flux. The potency and efficacy of ryanodol, however, are limited. Its half-activation of Ca2+ efflux (EC50act) was 412-fold higher than that of ryanodine. This was not simply consequent to the diminished affinity of ryanodol for RyR1, as expressed in its IC50 value, because the latter was 1290-fold less than that of ryanodine. Thus, while ryanodol binds weakly to RyR1, it nevertheless has fairly strong efficacy for opening the Ca2+ release channel.
The chemical nature of the C3 substituent appears determinant of potency for activation of RyR1 by the base ryanoids of the present study. Pyrrole- and pyridyl-carbonate derivatives of ryanodol were approximately equally potent at opening the channel. This was not further enhanced by substituent esters, despite the fact that such C10-Oeq substituents anchored them to the receptor (as suggested by their up to 35-fold increase in affinity on esterification). The same was true for ryanodol. What C10-Oeq esters with basic termini provide is an increased efficacy for channel opening. Anchoring substituent esters at the C10-Oeq position of the base ryanoids enhanced affinity and especially efficacy of the derived ryanoids but produced little consequence as regards potency.
Two direct and an additional indirect experimental paradigms are generally used to evaluate structure-activity effects on ryanodine receptor/calcium release channels as follows: flux measurements, lipid bilayer measurements, and [3H]ryanodine binding studies. The first and third provide data averaged over many Ca2+ channels, with time courses over hours. The second reflects single (or a few) channels, with time courses over minutes and individual gating events in the millisecond range. In previously published studies we have used all three methods. The present results rely on direct flux for functional measurements because these permit a wide range of experimental manipulations. Furthermore, the present results confirm that, at least under some conditions, ryanoid bind affinity does not go hand-in-hand with activity.
Qualitatively, results with single channel measurements nevertheless are in general consistent with efficacy results with vesicle population measurements. Certain ryanoids possessing full efficacy in vesicles lack ability to produce the shut state in single channel studies (18). This translates as full efficacy in the alternate experimental paradigm. Interestingly, however, at the higher resolution possible with bilayer measurements, a new feature of ryanoid actions emerges (18). Each ryanoid in our series produced a characteristic modified partial conductance state of the channel. The resultant slope conductances were not related to efficacy as judged from ensemble Ca2+ efflux data.
Specifically, ryanodol induces the largest modified conductance state of any ryanoid examined thus far. Yet ryanodol produces the least efficacy. Furthermore, C10-Oeq derived ryanodines produce even smaller modified conductance states, in direct proportion to their enhanced affinities, but exhibit full efficacy for ensemble Ca2+ efflux.
Apparently the determinant feature of full agonists is not the residual size of the intermediate subconductance state, but rather the probability of inducing the terminal shut state. Fully efficacious C10-Oeq derivatives seem to lock the channel into a state where Po approximates 1. Not only do they not close the channel, they prevent ryanodine from closing it, even when ryanodine is elevated to 1 mM (18, 27). Ryanodol, on the other hand, is able to produce the shut channel state at submillimolar concentrations. (We have not yet completed studies with pyridyl ryanodine at the single channel level. They may prove particularly instructive.)
These results are compatible with the notion that basic termini of C10-Oeq side chains interact with the ryanodine receptor in such a manner as to interfere physically with ionic flux through the pore region of the transmembrane conduction pathway, regardless of the experimental paradigm. By extension, the C10-hydroxyl of ryanodine may also interact within the transmembrane conduction pathway.
Almost a decade after the total synthesis of ryanodol (43), the complete synthesis of ryanodine per se has yet to be achieved. Ruest and Deslongchamps (44) reincorporated the pyrrole-2-carbonate onto the C3 secondary hydroxyl of anhydroryanodol using the mild reaction conditions of Neises and Steglich (45) that we adapted for C10-Oeq esterification of ryanoids (26-30). However, owing to stereochemical hindrances, Ruest and Deslongchamps were able to produce only C3 epi-ryanodine. It has so far proved impossible to vary chemically the ester substituent on the C3 carbon while maintaining the original stereochemical orientation found in ryanodine. The present studies with the naturally oriented C3-pyridyl substituent of ryanodine were permitted by our isolation of sufficient quantities of the natural secondary metabolite pyridyl ryanodine from Ryania wood.
C3 epi-ryanodine had an affinity 100-fold less than ryanodine. In collaboration with Ruest and Deslongchamps (44), Welch et al. (36) confirmed the low affinity of C3 epi-ryanodine and further showed that esterification of the C10-Oeq position of ryanodol with the basic side chain pyrrole-2-carbonate partially restored the affinity of ryanodol for RyR1, to within 7-fold that of parent ryanodine. We interpret their data to indicate that a C10-Oeq pyrrole carbonate is able to anchor the ryanoid polycyclic backbone structure to the ryanodine receptor, much as does other basic termini such as the guanidino-propionyl moiety. They concluded differently. However, in their binding paradigm, both the C9 and C10-Oeq positions were systematically excluded from their elegant comparative molecular field analysis relating topologic ryanoid features to ryanoid affinity. They modeled both to be in solvent space. Whereas this exclusion may well be justified for C9 substituents, it seems less likely for C10-Oeq esters, since the latter can considerably increase the binding affinity of all ryanoids, including ryanodine itself (24, 26-30, 33 and present results). If effective, basic C10-Oeq ryanoids had been included in the Welch et al. (36) basis set, the interpretation that certain pyrrole derived ryanoids might be "reoriented in the binding site ... such that the pyrrole [when present] always occupies the same subsite" likely could not have emerged as a viable hypothesis. In fact, if a substituent pyrrole-2-carbonate predicated the binding orientation of any ryanoid containing it, then pyrrole-2-carbonate itself logically should compete with [3H]ryanodine for high affinity binding sites. Having none of the steric hindrances associated with the bulk polycyclic backbone of ryanodine and none of its numerous polar groups, pyrrole-2-carbonate should be an especially effective competitor for [3H]ryanodine binding. It is not. Up to 1 mM, pyrrole-2-carbonate has absolutely no effect on [3H]ryanodine binding (data not shown).
Numerous other studies of function-activity relationships among ryanoids support the proposition that the polycyclic backbone of ryanoids and its multiple polar moieties figure prominently in affinity and effectiveness of ryanodine derivatives. For example, Casida and co-workers (46) showed that partial oxidation of the C10 secondary hydroxy to a ketone reduced the affinity of this ryanoid for RyR1. These investigators also showed that conversion of the C10 secondary hydroxyl to its axial isomer or other axially oriented functionalities (e.g. amino) decreased the affinity of ryanoids for RyR1 (47). Systematic evaluation of ryanoid-structure activity relationships among many ryanoids have demonstrated affinity consequences of various polar regions of the ryanodine molecule (48). Jefferies et al. (49) examined 22 derived ryanoids prepared by nucleophilic additions to 4,12-seco- and 4,12-dioxoryanodines and extended such studies to two additional series of derivatives. Additionally, Welch and co-workers (24, 33, 36) sought to identify structural components of ryanodine responsible for modulation of sarcoplasmic reticulum Ca2+ channel function by examining dozens of ryanoids.
For the most part, however, prior studies have focused on changes in affinities of ryanoids consequent to imposed structural variations. In a recent review, Sutko and colleagues (50) provided an excellent summary of ryanoid structure-activity studies to date. Prior studies also have shown that the affinities of natural ryanoids for their receptor sites may be altered by modifications and derivatizations. We have recently shown that esterification of the C10 secondary hydroxyl of ryanodine and 9,21-didehydroryanodine with substituents having basic termini significantly enhances their affinities for the receptor sites by as much as 6-8-fold. The potencies and efficacies of these molecules were not correspondingly shifted.
Taken collectively with the present data, the composite results permit assignment of the ester on the C3 secondary hydroxyl to less than a central role for high affinity interactions of ryanoids with RyR1s.
As regards the anchoring substituents, for each of the base ryanoids of the present study, C10-Oeq esterification with side chains bearing basic termini advanced their affinities approximately equally. For each, the most basic terminus, guanidino-propionyl, produced the highest affinity interaction. Whereas apparent affinity for binding was substantially augmented by basic termini, potency for activation was only marginally advanced, if it was moved at all. C10-Oeq substituents anchoring the ryanoids to ryanodine receptor site(s) were found to affect primarily efficacy for channel opening, with little effect on potency. Thus increased binding affinity clearly desegregated from increased potency.
Unlike ryanodine that demonstrates a biphasic effect on RyRs, most of the semi-synthetic C10-Oeq derived ryanoid molecules showed only activator-selective actions. Assuming that the biphasic effects of ryanoids on RyRs are due to their interactions with (at least) two functionally antagonistic receptor sites, it still remained uncertain whether the activator selective actions of certain ryanoids resulted from their inability to interact with a putative lower affinity binding site or whether they might be antagonists at such a site. Our functional competition studies with JSRV favored the latter interpretation, and this conclusion has recently been strengthened by our single channel experiments. The present communication provides further evidence in support of our working hypothesis that the activator selective actions of these C10-Oeq ester derivatives of ryanoids result from their physical interference with deactivation.
In summary, the present results show that affinity consequences of ryanoid chemical modifications experimentally segregate from their consequences on potency and efficacy. Pyridyl ryanodine was poorly effective in its ability to compete with ryanodine for binding to high affinity sites on the ryanodine receptor but nevertheless was fully effective in its capacity to open the channels to Ca2+ efflux. In fact, pyridyl ryanodine opened the Ca2+ release channels fully at a concentration some 100-fold lower than its minimal concentration for initiating channel closure. Ryanoid anchoring via C10-Oeq side chains with basic termini also desegregated affinity consequences from potency consequences; affinities were increased but potencies remained essentially unchanged.
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ACKNOWLEDGEMENTS |
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We thank Luc Ruest for some of the confirmatory 1H NMR spectrographic analyses. We are grateful to Phil Wilson and Lydia Gerbig for valuable help with the illustrations; Kurt Besch, Sangyeol Kwon, and Bruce Henry for technical assistance; and Lisa Cunningham for untiring secretarial aid.
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FOOTNOTES |
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* This work was supported in part by the Showalter Trust Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence and reprint requests should be sent: Dept.
of Pharmacology and Toxicology, Indiana University School of Medicine,
635 Barnhill Dr., Indianapolis, IN 46202-5120. Tel.: 317-274-7844; Fax:
317-274-7714; E-mail: besch{at}indiana.edu.
1
The abbreviations used are: SR, sarcoplasmic
reticulum; E-C, excitation-contraction; RyR1, sarcoplasmic reticulum
Ca2+-release channels from rabbit fast skeletal muscle;
JSRV, junctional sarcoplasmic reticular vesicles; ryanodine,
C3-O-(pyrrole-2-carbonyl) ryanodol; pyridyl
ryanodine, C3-O-(pyridyl-3-carbonyl) ryanodol; Cbz, benzyloxycarbonyl; AMP-PCP, ,
-methyleneadenosine
5'-triphosphate; CCR, concentration coupling ratio, the ratio of
EC50deact to EC50act.
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REFERENCES |
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