APE-RELATED ABNORMALITIES IN REGULATION OF THE RYANODINE RECEPTOR IN RAT FAST-TWITCH MUSCLE

The tibialis anterior (TA) muscles of 6-month-old and 24-month-old mal e Wistar rats, after being characterized, at the fast motor unit level , for twitch properties, were dissected and processed by a procedure [ Margreth A., Damiani E., Tobaldin G. Biochem Biophys Res Commun 1993; 197: 1303-1311] aimed at obtaining a representative total membrane fra ction comprising 70-80% of the total muscle content of sarcoplasmic re ticulum (SR) and transverse tubule (TT) membranes (about 20 mg protein /g). Skeletal muscle membranes were analyzed for protein composition, and the content and functional properties of specific components of th e free and junctional subcompartments of the SR and of junctional TT. Our results, while confirming a twitch prolongation in TA of old rats, do not demonstrate any associated age-related change concerning: (a) the overall number and functional properties of Ca2+ pumps, as charact erized by kinetic parameters, Ca2+-dependency, and the protein isoform specificity of SR Ca2+-ATPase; (b) the number of functional junctiona l SR Ca2+-release channels, on the basis of B-max values for high-affi nity binding of [H-3]-ryanodine to skeletal muscle membranes at optima l Ca2+; (c) the overall muscle dihydropyridine receptor/ryanodine rece ptor (RyR) ratio. We conclude from these findings, and the additional negative evidence for changes in membrane density of specific componen ts of junctional SR, including 60 kDa Ca2+-calmodulin protein kinase, that this membrane domain, like the Ca2+-pump domain of the SR, are in no way basically altered at early stages of the aging process, as inv estigated here. Because of that, we allege particular significance to the occurrence of age-related, specific abnormalities in regulation of RyR in rat TA. The main supportive evidence is as follows: (a) an inc reased sensitivity to Ca2+ of the RyR of old muscle, and, more importa ntly; (b) an increased sensitivity to caffeine of [H-3]ryanodine bindi ng to the RyR at optimal Ca2+ and also optimal for the activity of the Ca2+-release channel. The results reported here also demonstrate that there are two classes of caffeine sites in rat TA muscle, as defined by differences in EC(50) values at resting (pCa 7) and at high Ca2+ (p Ca 4-5), that sites involved in stimulation of [H-3]-ryanodine binding to the RyR are distinguished by a higher affinity (caffeine below mM) , and that only these sites undergo age-related changes. Thus, althoug h the underlying age-related abnormality of the RyR remains to be eluc idated, it appears to satisfy the requirement for being regarded as a specific change, which in itself might argue for its being fundamental ly related to the twitch prolongation of the muscle.