Fast muscles of the rat hind limb were stimulated continuously at 10 or 20 Hz for periods of 55-61 days by means of an implantable neuromuscular stimulator. Gel electrophoresis clearly demonstrated the presence in stimulated muscles of slow myosin light and heavy chains, although fast isoforms were still present in all cases. Thus, contrary to previous reports, induction of slow myosin isofonns does occur in this, as in other, mammalian species. The time course of the response to stimulation appears to be more extended than that seen in the rabbit.

Chronic electrical stimulation of skeletal muscle at 10 Hz induces fast-to-slow fiber type transformation. Does a lower aggregate amount of activity lead to a less complete transformation, or does it produce the same transformation over a longer time course? We examined this question by subjecting adult rabbit tibialis anterior and extensor digitorum longus muscles to continuous stimulation at 2.5 Hz for 2-12 wk. Most of the fibers acquired the histochemical and immunocytochemical characteristics of type 2A, not type 1, fibers. There was a corresponding rise in oxidative activity, but this was accompanied by a marked decline in anaerobic glycolysis. The activities of hexokinase and 3-oxoacid CoA-transferase stopped increasing after 2 wk, glutamate oxaloacetate transaminase after 4 wk, and beta-hydroxyacyl-CoA dehydrogenase after 6 wk of stimulation. Succinate dehydrogenase, citrate synthase, lactate dehydrogenase, and creatine phosphokinase continued to change up to 12 wk of stimulation. Changes in enzyme activity were not as rapid or as marked as those observed for stimulation at 10 Hz, and none showed the typical two-phase response of oxidative enzyme activities to stimulation at 10 Hz. The latter may therefore be dependent on induction of type 1 myosin isoforms.

We studied changes in the mechanical properties and myosin isoform composition of rabbit tibialis anterior muscles that were subjected to continuous stimulation at 2.5 Hz for up to 12 wk. The effects of stimulation at 2.5 Hz were less profound than those observed for the same duration of stimulation at 10 Hz (12). Stimulation at 10 Hz for 12 wk induced complete transformation to a slow-contracting muscle homogeneous in slow myosin isoforms; stimulation for the same period at 2.5 Hz resulted in moderate changes in contractile speed and a very small increase in the synthesis of slow myosin isoforms. On the other hand, the fatigue resistance of muscles stimulated at 2.5 Hz was as great, in both isometric and dynamic fatigue tests, as that of the muscles stimulated at 10 Hz. Thus entire fast skeletal muscles can be transformed to a state in which fast myosin isoforms continue to be synthesized, but the oxidative capacity is sufficient to support sustained working at a higher power output than that associated with slow muscle.

ATP released from damaged cells or by controlled secretion could be an important factor in the formation or remodeling of bone. In a variety of other tissues ATP has been shown to control cellular processes by acting on P2-purinoceptors and activating the calcium signaling pathway. Here we demonstrate for the first time that extracellular ATP increases the intracellular free calcium [Ca2+]i concentration in normal human osteoblasts and in SaOS-2 cells, a human osteosarcoma-derived cell line, but not in ROS 17/2.8 cells. The ATP-induced increase in [Ca2+]i was dose dependent, and the concentrations of ATP required were similar to those reported to regulate cellular functions in other cell types. Although ATP is metabolized rapidly by bone cells, the effects on [Ca2+]i appeared to be mediated directly by ATP rather than one of its metabolites. Adenosine 3-thiotriphosphate, a nonhydrolyzable analog of ATP, induced similar changes in [Ca2+]i. This indicates that P2-purinoceptors are present on osteoblast-like cells and that extracellular ATP from various sources might be an important factor in the regulation of osteoblast functions.

Immunogold labelling was used to study the distribution of newly synthesized slow muscle myosin (SM) at the ultrastructural level as it replaced fast muscle myosin (FM) in rabbit muscles undergoing stimulation-induced type transformation. Control fast muscle was labelled strongly with antibody to FM and control slow muscle with antibody to SM; label was confined to the A-band. Well-defined differences in the distribution of label within the A-band suggested that the monoclonal antibodies used corresponded to epitopes on different parts of the myosin molecule; this was confirmed by Western blots of subfragments prepared from FM and SM. After 4 weeks of continuous stimulation at 10 Hz, fibres of the tibialis anterior muscle reacted with antibodies to both isoforms; after 6 weeks, labelling was obtained only with antibody to SM. After a 7-week period of stimulation and 3 further weeks of recovery, fibres again reacted with both antibodies. In all positively-labelled sections, the distribution of gold particles was characteristic of the antibody and independent of the origin or history of the fibres. This observation supports the conclusion that newly synthesized myosin is capable of being incorporated throughout the length and cross-section of the A-band.