Lithium chloride effects on the growth of L₆ myoblasts in culture : implication for involvement of phosphoinositide metabolism in myoblast proliferation

Abstract

The L6 myogenic cell line was utilized in a series of experiments designed to investigate the possible regulatory role of the phosphoinositide signal transduction system in the proliferation of myoblasts in vitro. The growth of the L6 myogenic cell line was assayed in the presence of increasing concentrations of LiCl, an inhibitor of phosphatidylinositol synthesis. Cultures were plated in DMEM + 10% FBS containing 0 (control), 5,10 or 20 mM LiCl and cell number, protein content and 3H-thymidine incorporation determined at 24-h intervals. Control cells exhibited a rapid rate of proliferation (7.8-fold increase in cell number by 96 h in culture) and the addition of 5 mM LiCl did not effect (P > .05) the rate or extent of proliferation. In contrast, the addition of 10 or 20 mM LiCl resulted in a 34.1 and 85.7% decrease (P < .05) in cell number by 96 h in culture. The inhibition of proliferation did not result from affects on cell viability, because cells treated with LiCl exhibited a similar (P > .05) ability to exclude trypan blue, and returned to proliferation upon removal of LiCl from the medium. The inhibition of myoblast proliferation was supported by a measurement of DNA synthesis, with a 24 h pretreatment of cultures with 10 or 20 mM LiCl resulting in a 16.9 and 64.7% decrease (P < .05) in the incorporation of 3H-thymidine. In addition, the effect of LiCl treatment could be overcome (P < .05) by the addition of the active phorbol ester, phorbol-12-myristate-13-acetate (PMA). Treatment of L6 myoblasts, prelabelled with 3H-inositol, with LiG resulted in a dose-dependent increase (P < .05) in the accumulation of label within the inositol monophosphates (402.2,770.3 and 867.7% increase vs control for the 5,10 and 20 mM LiG treatments, respectively). Treatment of cells with 10 or 20 mM LiCl also resulted in 37.1 and 90.1% increases (P < .05) vs control in label recovered as inositol bisphosphate. In contrast, LiG treatment did not substantially effect the amount of label recovered as inositol trisphosphate. Due to the ability of LiG to modulate the metabolism of the inositol phosphates, the effect of LiG on the incorporation of 3Hinositol into the phosphoinositides was investigated...