The use of lanthanum and cytochalasin B to study calcium effects on skeletal muscle differentiation in vitro.

Abstract

AbstractA dual effect of external Ca2+ on creatine kinase (CPK) accumulation during myogenesis has recently been demonstrated (Morris and Cole, '79). Ca2+ inhibits muscle‐specific CPK accumulation at intermediate (50–100 μ) concentrations compared with both lower (no added Ca2+) and higher (2–3 μ) concentrations. Myoblast fusion, however, requires high Ca2+ and is inhibited at both low and intermediate Ca2+ levels.These effects are now investigated further by studying the effects of lanthanum ion (La3+), which interferes with Ca2+‐binding to membranes and Ca2+‐transport, and cytochalasin B, which affects the cell membrane and prevents cell fusion without inhibiting CPK accumulation.The results show that low concentrations (10–100 μ) of La3+ inhibit the appearance of the muscle‐specific (MM) CPK isoenzyme during myogenesis without significantly affecting cell fusion or intracellular cyclic AMP levels. Three further observations are consistent with the existence of myotube‐specific membrane‐binding sites for Ca2+, which are involved in the stimulation of CPK accumulation on increasing external Ca2+ from intermediate to high concentrations.(1) CPK levels are not affected by La3+ at 0–50 μ external Ca2+.(2) CPK levels in cytochalasin B treated myoblasts are hardly affected by La3+ at any Ca2+ concentration.(3) In cytochalasin B treated cultures, CPK levels are not increased by raising external Ca2+ from intermediate to high levels.In contrast, the stimulation of CPK accumulation on decreasing external Ca2+ from intermediate to very low concentrations is not affected by either La3+ or cytochalasin B.Some alternative interpretations of the data are also considered, including direct disruption of a membrane Ca2+‐binding site by cytochalasin B.