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The localized elevation of cathepsins B and L in rat gastrocnemius muscle following tenotomy.

Tenotomy of the rat gastrocnemius muscle is commonly employed as a model of accelerated protein breakdown or as a system in which to follow the hypertrophy of the ipsilateral plantaris or soleus muscles [l]. We have studied changes in the activities of cathepsins B and L in rat gastrocnemius muscle over the period following tenotomy when the muscle atrophies. Individually caged rats (160 g) were tenotomized under ether anaesthesia with a sham operation on the contralateral leg. Rats were killed 1 to 9 days thereafter and the gastrocnemius muscle or muscle segment was homogenized in 19 volumes of ice-cold phosphate buffered saline, pH 6.0 containing 2 mM-EDTA and 2 mwcysteine, using a Polytron homogenizer at 30% of full speed for 15 s. The homogenate was further diluted and assayed for cathepsins B and L with Z-Phe-Arg-NMec [2]. A time course of cathepsin B and L specific activities in the whole tenotomized gastrocnemius showed a maximum at day S (Fig. la). However, this increase in activity was not evenly distributed throughout the muscle. When the gastrocnemius was divided into three segments along its length, the changes in cathepsin activities were confined to the distal portion of the muscle, i.e. adjacent to the severed tendon (Fig. 1 b; 3 days after tenotomy). The activity in the distal segment at day 2 was significantly greater than that in the contralateral control segment (data not shown). In contrast, catheptic activity in the proximal segment was similar to that in the comparable segment of the contralateral, shamoperated gastrocnemius at days 1, 2 or 3. The medial segment showed a significant increase in activity over its contralateral control segment only on day 3 (Fig. 1 h ) . Previously, such changes in enzyme activities have been ascribed to the muscle tissue alone, particularly when a whole muscle has been the source of a preparation. However, the differential changes in cathepsin activities following tenotomy (Fig. 1 h ) suggest either a localized response within the muscle fibres, a concept difficult to rationalize with the highly ordered structure of muscle, or a contribution from non-muscle cells. This latter view receives some support from the literature. Sano et al. (31 showed by immunolocalization that cathepsins B, H and L were scarcely detectable in normal mouse muscle but all enzymes were readily demonstrated in macrophages resident in that muscle. Cathepsins B, H and L were present in all tissues of the rat but leucocytes and macrophages (from peripheral blood) also had appreciable levels of activity [4]. Significantly, when such macrophages were activated, the cathepsin B activity increased almost 10fold [4]. That non-muscle cells show a metabolic response to tenotomy was demonstrated by Jablecki et al. [S] in a study of soleus hypertrophy following tenotomy of the synergistic gastrocnemius and plantaris muscles. Autoradiographic detection of RNA synthesis at 12, 24 and 48 h showed that all the activity was associated with the connective tissues (capillaries and fibroblasts) while muscle tissue showed no response. Macrophages and other cells involved in the inflammatory response are likely to be present at the distal end of the gastrocnemius following surgical tenotomy. Armstrong et a/. [6] and Kelso et al. [7], using surgical ablation of the gastrocnemius and soleus in a study of hypertrophy of the synergistic plantaris, showed a short-term response of cell -