Inorganic Lead May Inhibit Neurite Development in Cultured Rat Hippocampal Neurons through Hyperphosphorylation

Abstract

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 SpcAMPS, 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+]in that are not reflected in whole-cell measurements.