The mononuclear cell population in rat leg muscle: its contribution to the lysosomal enzyme activities of whole muscle extracts.

Abstract

The mononuclear cell fraction of rat hind-limb muscle was obtained by digestion with clostridial collagenase in the presence of calcium ions, filtration through nylon screens and washing to remove the enzyme. Final traces of contaminant myofibrillar debris were separated by isopycnic centrifugation in a Percoll density gradient. Whole muscle, washed cells and Percoll-fractionated cells were extracted in the presence of non-ionic detergent and the supernatants assayed for the lysosomal enzymes cathepsins B + L, N-acetyl-beta-glucosaminidase, beta-glucuronidase and protein. The enzyme levels were highest in the muscle from young rats, but the percentage of recovered activity in the mononuclear cell fraction was little altered by the age of the animal. The values obtained were: cathepsin B + L, 2.4-4.0%; N-acetyl-beta-glucuronidase, 4.3-7.6%; and beta-glucuronidase, 6.3-10.3%. Because of unavoidable losses in preparation these are minimal values and the actual levels of activity from the mononuclear cell fraction in muscle would be higher. The specific activity values of the cell lysates were raised after isopycnic centrifugation and were nearly constant over the age range 65-180 days. Substantially higher specific activity values were obtained for the cells from rats of 38 days. When grown in culture the mononuclear cell fractions were seen to contain mainly fibroblasts and myoblasts with only few leucocytes. The cultures reached confluence by the second week, at which time numerous myotubes had formed. In addition there were groups of large, circular cells with a prominent centrosphere. The origin of these latter cells is uncertain. It was concluded that although total lysosomal enzyme activity was higher in young rats there was little effect of age on the distribution of activity between muscle fibres and mononuclear cells in the muscle.