Fax. Schanne et al., LEAD-INDUCED RISE IN INTRACELLULAR FREE CALCIUM IS MEDIATED THROUGH ACTIVATION OF PROTEIN-KINASE-C IN OSTEOBLASTIC BONE-CELLS, Biochimica et biophysica acta. Molecular basis of disease, 1360(3), 1997, pp. 247-254
Lead characteristically perturbs processes linked to the calcium messe
nger system. This study was undertaken to determine the role of PKC in
the Pb2+ induced rise of [Ca2+](i). [Ca2+](i) was measured using the
divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy) ethane N,N
,N',N'-tetraacetic acid (5F-BAPTA) and F-19-NMR in the osteoblast cell
line, ROS 17/2.8. Treatment of cells with Pb2+ at 1 and 5 mu M produc
ed a rise in [Ca2+](i) from a basal level of 125 nM to 170 nM and 230
nM, respectively, while treatment with phorbol 12-myristate 13-acetate
(PMA) (10 mu M), an activator of PKC, produced a rise in [Ca2+](i) to
210 nM. Pretreatment with calphostin C, a potent and highly selective
inhibitor of PKC activation failed to produce a change in basal [Ca2](i) and prevented any rise in [Ca2+](i) in response to Pb2+. To deter
mine whether Pb2+ acts directly on PKC, we measured the Pb2+-dependent
activation of phosphatidylserine/diolein-dependent incorporation of P
-32 from ATP into histone and endogenous TCA precipitable proteins in
the 100 000 X g supernatant from homogenized ROS 17/2.8 cells. The fre
e concentrations of Pb2+ and Ca2+ were set using 5F-BAPTA; and [Ca2+]
and [Pb2+] in the PKC reaction mixtures were confirmed by F-19-NMR. We
found that Pb2+ activates PKC in the range of 10(-11)-10(-7) M, with
an activation constant of 1.1 X 10(-10) M, whereas Ca2+ activates PKC
in the range from 10(-8) to 10(-3) M, with an activation constant of 3
.6 X 10(-7) M. These data suggest that Pb2+ activates PKC in ROS 17/2.
8 cells and that Pb2+ activation of PKC mediates the documented rise i
n [Ca2+](i) and, perhaps, other toxic effects of Pb2+.