Insulin receptor autophosphorylation in BC3H-1 whole cell assays is inhibited by the specific calmodulin inhibitors calmidazolium and W-7

Recent reports suggest the involvement of Ca++ and the Ca++ binding protein calmodulin in the insulin stimulated receptor tyrosine kinase activity in cell free (adipocyte) phosphorylation systems. Working with the insulin-responsive well characterized muscle cell line BC3H-1, they have investigated the effects of calmodulin antagonists on insulin receptor phosphorylation in cultured intact cells. BC3H-1 myocytes were grown to confluency (10-12 days) then exposed to media containing 32P-orthophosphate for 24 hours (100 mCi/ml). Insulin treatment stimulated the phosphorylation of a 95K protein which is immunoprecipitable with antireceptor antibodies indicating insulin-induced phosphorylation of the insulin receptor beta subunit in vivo. This phosphorylation occurs rapidly within 30 minutes at physiologic insulin concentrations at 370C. Phosphorylation can also be stimulated by the B-10 anti-insulin receptor antibody (1:500). Pretreatment of cells for 30 min with 1uM calmidazolium (R24571) and 10nM W-7 (n-[6-AminoHexyl]-s-chloro-1-naphalenesulfonamide) each significantly inhibited insulin-stimulated phosphorylation. This would suggest that calmodulin may play a role in mediation of the insulin receptor tyrosine kinase activity in the BC3H-1 myocyte