Different protein kinase C isozymes could modulate bradykinin-induced extracellular calcium-dependent and -independent increases in osteoblast-like MC3T3-E1 cells

Abstract

Effects of protein kinase C (PKC) on bradykinin (BK)-induced intracellular calcium mobilization, consisting of rapid Ca 2+ release from internal stores and a subsequent sustained Ca 2+ inflow, were examined in Fura-2-loaded osteoblast-like MC3T3-E1 cells. The sustained Ca 2+ inflow as inferred with Mn 2+ quench method was blocked by Ni 2+ and a receptor-operated Ca 2+ channel blocker SK&F 96365, but not by nifedipine. The short-term pretreatment with phorbol 12-myristate 13-acetate (PMA), inhibited BK-stimulated Ca 2+ inflow, and the prior treatment with PKC inhibitors, H-7 or staurosporine, enhanced the initial internal release and reversed the PMA effect. Moreover, 6 h pretreatment with PMA caused similar effect on the BK-induced inflow to that obtained with PKC inhibitors, whereas 24 h pretreatment was necessary to affect the internal release. On the other hand, the translocation and down-regulation of PKC isozymes were examined after PMA treatment of MC3T3-E1 cells by immunoblot analyses of PKCs with the isozyme-specific antibodies. 6 h treatment with PMA induced down-regulation of PKCβ, whereas longer treatment was needed for down-regulation of PKCα. Taken together, it was suggested that the BK-induced initial Ca 2+ peak and the sustained Ca 2+ inflow through the activation of a receptor-operated Ca 2+ channel, are differentially regulated by PKC isozymes α and β, respectively, in osteoblast-like MC3T3-E1 cells.