engleski

Phorbol 12-myristate 13-acetate Mediated Signalling in Murine Bone Marrow Cells

Phorbol 12-myristate 13-acetate (PMA)-mediated signalling was investigated in relation to the ability of murine (CBA) bone marrow cells to form colonies in vitro. Treatment of marrow cells with PMA did not influence 1,2-diacylglycerol and cAMP concentration, Ca^2^+_i and phospholipase D activity. PMA increased particulate phospholipase PLA_2 (PLA_2) activity, and the formation of lyso-PtdCho and arachidonic acid relelase from bone marrow cells and these effects were abolished when cells were pretreated with putative PLA_2 inhibitors heparin and mepacrine. While indomethacin and NDGA inhibited either cyclo-oxigenase and lipoxygenase pathway of arachidonic acid metabolism, as measured by their products PGE_2 and LTB_4, they did not influence PMA-mediated PLA_2 activation or translocation of protein kinase C (PKC) from soluble to particulate fraction. Treatment of cells with PMA increased the amount of membrane bound alpha, beta, delta, epsilon and zeta isoforms of PKC in bone marrow cells. Pretreatment of cells with PLA_2 inhibitors reduced the amount of membrane bound PKC-zeta in unstimulated cells and diminished PMA-induced translocation of PKC-zeta to membranes without affecting other PKC isoforms. Their effect could be overcome by exogenoud addition of arachidonic acid suggesting that PKC-zeta may operate downstream of activated PLA_2. On the other hand, wortmannin, an inhibitor of phosphatidylinositol-3-kinase, did not influence the amount of PKC-zeta associated with particulate fraction in control cells and could not abolish its PMA-mediated translocation. Short exposure (45 min) of bone marrow cells to PMA, phorbol 12,13-dibutyrate or arachidonic acid increased the number of colonies formed over seven days in methylcellulose-based culture in vitro. The effect of PMA but not arachidonic acid could be prevented by puitative PLA_2 inhibitors, suggesting that PMA-mediated activation of cPKCs and nPKCs leads to PLA_2 activation which, by releasing the arachidonic acid from phospholipids, activates PKC-zeta, and that this signalling pathway appears to be mitogenic for bone marrow cells.

phorbol 12-myristate 13-acetate; signalling; murine bone marrow cells

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