Osmolarity-dependent characteristics of [3H]ryanodine binding to sarcoplasmic reticulum.
While many reports have shown that Ca2+ alone causes ryanodine binding to the heavy fraction of the sarcoplasmic reticulum (HFSR), our results demonstrate that caffeine or beta,gamma-methylene adenosine triphosphate (AMPOPCP) in addition to Ca2+ is necessary for ryanodine binding, although Ca2+ is indispensable for it. While clarifying the reasons for this discrepancy, we found that a high osmolarity of the reaction medium, but not ionic strength, is a crucial factor. In a hypertonic solution containing 1 M NaCl, Ca2+ alone causes a sizable extent of ryanodine binding. Caffeine and AMPOPCP independently stimulate it, unlike the case of 0.17 M KCl (or NaCl) medium, in which they show a potentiating interaction. Ryanodine binding in the hypertonic solution was markedly enhanced not only as to the binding rate but also the extent. The Scatchard plot was linear, indicating a single class of homogeneous binding sites. The maximum number of binding sites as well as the affinity was also increased in 1 M NaCl-medium. The presence of AMPOPCP and/or caffeine did not affect the magnitudes of them so much, especially that of the affinity, in the hypertonic medium, as in the isotonic medium. The Ca2(+)-dependence of ryanodine binding in the stimulatory range was similar to that in 0.17 M KCl- (or NaCl-) medium. However, the very weak inhibition at high Ca2+ concentrations is in striking contrast to ryanodine binding in the isotonic medium. The stimulation due to a high osmolarity is distinct, as to the mechanism, from that due to AMPOPCP, caffeine, or temperature. The dissociation of [3H]ryanodine bound was also examined under various experimental conditions.
Ogawa
Y
Y
Department of Pharmacology, Juntendo University School of Medicine, Tokyo.
Harafuji
H
H
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000470:Alkaloids / Q000378:metabolism*; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D006982:Hypertonic Solutions; D009994:Osmolar Concentration; D011892:Rana catesbeiana; D012433:Ryanodine / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D000470:Alkaloids; D006982:Hypertonic Solutions; D012433:Ryanodine; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D002110:Caffeine; D000255:Adenosine Triphosphate; C002630:alpha,beta-methyleneadenosine 5'-triphosphate