Activation of protein kinase C inhibits ATP‐induced [Ca2+]i elevation in rat osteoblastic cells: Selective effects on P2Y and P2U signaling pathways

Abstract

Extracellular ATP elicits transient elevation of cytosolic free Ca2+ concentration ([Ca2+]i) in osteoblasts through interaction with more than one subtype of cell surface P2-purinoceptor. Elevation of [Ca2+]i arises, at least in part, by release of Ca2+ from intracellular stores. In the present study, we investigated the possible roles of protein kinase C (PKC) in regulating these signaling pathways. [Ca2+]i of indo-1-loaded UMR-106 osteoblastic cells was monitored by spectrofluorimetry. In the absence of extracellular Ca2+, ATP (100 microM) induced transient elevation of [Ca2+]i to a peak 57 +/- 7 nM above basal levels (31 +/- 2 nM, means +/- S.E.M., n = 25). Exposure of cells to the PKC activator 12-O-tetradecanoyl-beta-phorbol 13-acetate (TPA, 100 nM) for 2 min significantly reduced the amplitude of the ATP response to 13 +/- 4 nM (n = 11), without altering basal [Ca2+]i. Inhibition was half-maximal at approximately 1 nM TPA. The Ca2+ response to ATP was also inhibited by the PKC activators 1,2-dioctanoyl-sn-glycerol or 4 beta-phorbol 12,13-dibutyrate, but not by the control compounds 4 alpha-phorbol or 4 alpha-phorbol 12,13-didecanoate. Furthermore, exposure of cells to the protein kinase inhibitors H-7 or staurosporine for 10 min significantly attenuated the inhibitory effect of TPA. However, these protein kinase inhibitors did not prolong the [Ca2+]i response to ATP alone, indicating that activation of PKC does not account for the transient nature of this response. When the effects of other nucleotides were examined, TPA was found to cause significantly greater inhibition of the response to the P2Y-receptor agonists, ADP and 2-methylthioATP, than the response to the P2U-receptor agonist, UTP. These data indicate that activation of PKC selectively inhibits the P2Y signaling pathway in osteoblastic cells. In vivo, endocrine or paracrine factors, acting through PKC, may regulate the responsiveness of osteoblasts to extracellular nucleotides.