Date of Award
Doctor of Philosophy (PhD)
Edwin E. Daniel
Ryanodine, a neutral alkaloid, is a widely used pharmacological tool in the studies
of muscle excitation-contraction coupling. The specific binding sites for ryanodine have
been identified to exist on sarcoplasmic reticulum (SR) in both skeletal muscle and
cardiac muscle. The ryanodine receptor has been purified from different tissue types. The purified ryanodine receptor from skeletal muscle was found to be identical with foot
structure, a protein spanning the gap between t-tubule and SR membrane. The ryanodine
receptor was also found to have Ca²⁺ channel activity, a Ca²⁺-induced Ca²⁺ release channel.
In functional studies with isolated SR vesicle or single channel, ryanodine was found to
have dual effects on Ca²⁺ channel. At lower concentrations, ryanodine locked Ca²⁺ channel
in the open state, but fully closed the channel at higher concentrations. In smooth muscle,
ryanodine was also found to affect intracellular Ca²⁺ movement in the functional studies
using intact tissue. However, direct evidence was not available for the presence of
ryanodine receptor in smooth muscle before this research was carried out.
In the present study, a high affinity binding site was found located on SR
membranes of rat vas deferens (RVD) smooth muscle (Kd=5.6 nM, Bmax=435 fmol/mg).
The ryanodine receptor in smooth muscle shared many similarities with that of skeletal
muscle, but was not identical. The time required for [³H]ryanodine binding to microsomal
fraction was about two hours to reach a steady state, and [³HJryanodine could be dissociated from its binding site by 20 fold dilution. However, the dissociation was very
slow and incomplete when initiated by excess ryanodine. The [³H]ryanodine binding in
smooth muscle was Ca²⁺ dependant. The affinity was increased with increased Ca²⁺
concentrations, but the Bmax was unchanged. The [³H]ryanodine binding also increased
with higher ionic strength and higher osmolarity, but later has less effect. Many factors
that affect Ca²⁺-induced Ca²⁺ release (CICR) channel activity were also found to affect [³H]ryanodine binding in my study; e.g., both Mg²⁺ and ruthenium red inhibited binding
and caffeine potentiated it, especially in the presence of a low Ca²⁺ concentration. In the
present study, I also showed that varied levels of [³H]ryanodine binding site existed among different smooth muscles. This variation was not correlated with the density of innervation or the SR content of different smooth muscles.
In dog mesentery artery smooth muscle, a low affinity binding site (Kd=269 nM) was also identified, in addition to the high affinity binding site.
In the present study, I also tried to show functional effect of ryanodine on the
CICR channel using subcellular membrane vesicles from vas deferens. Ryanodine, at
higher concentrations, inhibited oxalate-stimulated Ca²⁺ uptake, an effect which was observed as early as 5 minutes after uptake was initiated. This inhibitory effect was partially additive to that of cyclopiazonic acid (CPA), a potent SR Ca²⁺ pump inhibitor,
when this agents were used at submaximal concentrations. However, when CPA was used at a maximal concentration, ryanodine had no additional effect. Ryanodine at concentrations of 10⁻⁹ to 5x10⁻⁴ M, did not significantly change the Ca²⁺ release rate. In Ca²⁺ release experiments, no functional Ca²⁺ channel was observed, but the data are consistent with an inhibition of the SR Ca²⁺ pump at high ryanodine concentrations.
My study provided the first direct evidence ofthe existence ofryanodine receptors located on smooth muscle SR which may represent a CICR channels. Since another type of Ca²⁺ channel, IP₃-induced Ca²⁺ release channel (IICR), has also been identified in different smooth muscles, the varied levels of ryanodine binding site observed in the present study may be correlated to the ratio of occurrences of the two types of Ca²⁺ channel. This suggests that different excitation-contraction coupling mechanisms may exist in different tjpes of smooth muscle depending on different expression of these two channels. The present study also suggests that ryanodine, at higher concentrations, may inhibit the Ca²⁺ pumps located on SR. Suitable conditions for membrane isolation and Ca²⁺ transport experiment must be defmed to restore a functional Ca²⁺ release channel in smooth muscle, in order to study the functional effect of ryanodine on this channel.
Zhang, Zhen-Du, "Characterization of A Ca²⁺ release channel in smooth muscle" (1994). Open Access Dissertations and Theses. Paper 3965.