Net synthesis of the fast-type sarcoplasmic reticulum (SR) Ca2(+)-ATPase was studied in the muscle cell line L6AM using an immunochemical assay (e.l.i.s.a.). In addition, Ca2+ uptake by SR was monitored in muscle cell homogenates by a method employing the fluorescent Ca2+ indicator fura-2. Measurements were done both in differentiating myoblasts and in myotubes. Ca2(+)-ATPase levels were low (1 pmol/mg of protein) in undifferentiated myoblasts (controls) and only doubled over a period of 8 days in the absence of thyroid hormone (L-triiodothyronine; T3). This corresponded to a similar increase in Ca2+ uptake activity. Only half of the myoblasts fused under these conditions. Fusion was not increased in the presence of T3 (5 nM), but Ca2(+)-ATPase levels increased 4-fold and the Ca2+ uptake activity doubled compared with controls. In contrast, insulin-like growth factor-I (IGF-I) induced almost complete myotube formation (greater than 90% fusion), but only slightly stimulated (50%) net Ca2(+)-ATPase synthesis above control levels. However, the doubling of the Ca2+ uptake stimulation by IGF-I was comparable with that caused by T3. The effects of T3 plus IGF-I on Ca2(+)-ATPase levels and Ca2+ uptake activity were more than additive. Furthermore, the temporal relationship between the induction of Ca2(+)-ATPase net synthesis and Ca2+ uptake activity was identical with the two hormones. Qualitatively similar results were obtained when T3 and IGF-I were added to maximally fused cell cultures. The enhanced effect of T3 on Ca2(+)-ATPase net synthesis and Ca2+ uptake activity in the presence of IGF-I cannot therefore be explained by an increased myotube formation stimulated by the latter. In both differentiating myoblasts and myotubes the effect of T3 was more prominent on Ca2(+)-ATPase net synthesis than on Ca2+ uptake activity, whereas in myotubes the opposite was observed for IGF-I. This could imply complementary actions of the two agents in the development of a functional SR.
