INORGANIC LEAD MAY INHIBIT NEURITE DEVELOPMENT IN CULTURED RAT HIPPOCAMPAL-NEURONS THROUGH HYPERPHOSPHORYLATION

Inorganic lead inhibits neurite initiation in cultured rat hippocampal neurons at concentrations as low as 100 nM. Conflicting reports suggest that Pb2+ may stimulate or inhibit protein kinase C, adenylyl cyclase, phosphodiesterase, and calmodulin, or increase intracellular free Ca2+ concentrations. Therefore, Pb2+ may alter the activities of Ca2+/calmodulin-dependent protein kinase (CaM kinase) or protein kinases C or A. We cultured rat hippocampal neurons in 100 nM PbCl2 alone or in combination with kinase or calmodulin inhibitors. Inhibiting protein kinase C with calphostin C exacerbated the inhibition of neurite initiation caused by PbCl2, but inhibiting protein kinase A with KT5720, CaM kinase with KN62, or calmodulin with calmidazolium completely reversed the effects of PbCl2. These results indicate that Pb2+ may inhibit neurite initiation by inappropriately stimulating protein phosphorylation by CaM kinase or cyclic AMP-dependent protein kinase (PKA), possibly by stimulating calmodulin, This hypothesis is supported by findings that other treatments that should increase protein phosphorylation (okadaic acid, a protein phosphatase inhibitor, and S-p-cAMPS, a PKA activator) also reduced neurite initiation. Whole-cell intracellular free Ca2+ ion concentrations were not significantly altered by 100 nM PbCl2 at 4, 12, 24, or 48 hr, Therefore, the hypothesized stimulatory effects of Pb2+ exposure on calmodulin, CaM kinase, or PKA are probably not caused by increases in whole-cell intracellular free Ca2+, but may be attributable either to intracellular Pb2+ or to localized increases in [Ca2+](i), that are not reflected in whole-cell measurements. (C) 1995 Academic Press, Inc.