Protein Kinase C (PKC) Mediates Purinergic Receptor Induced Contraction in MC3T3‐E1 Osteoblasts

P2X7 receptor activation mediates the load‐induced increase in bone formation, in vivo. We find that activation of this receptor produces a rapid contraction of the osteoblast that we hypothesize is modulated by two distinct pathways; RhoA GTPase and PKC. To test this hypothesis, we measured MC3T3‐E1 preosteoblast contractions using the Zeiss 5LIVE rapid confocal microscope during activation of the P2X7 receptor in the presence or absence of specific inhibitors of RhoA GTPase and PKC pathways. BzATP, a known agonist of the P2X7 receptor, was added to the cells and changes in cell area following BzATP stimulation were quantified using Differential Interphase Contrast (DIC) microscopy. Addition of 0.5mM BzATP to MC3T3‐E1 cells resulted in a 34.14% reduction in cell area. Similar results were seen using a PKC activator, PMA. Non‐specific inhibition of PKC with, GF109203X, significantly attenuated the BzATP‐induced contraction. Specific inhibition of PKCá, a Ca2+ dependent isoform of PKC, amplified the contraction in response to BzATP, suggesting that PKCá is not responsible for the P2X7 induced contraction. Inhibition of myosin light chain kinase and Rho kinase (ROCK) also failed to block BzATP‐induced contractions. These studies suggest that PKC mediates the response of P2X7 receptor activation that may be important in skeletal remodeling. (Supported by INBRE2 P20 RR016472‐08 and NIH/NIDDK R01 DK058246)