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Neurofilament phosphorylation and [125I]Calmodulin binding by Ca2+/calmodulin-dependent protein kinase in the brain subcellular fractions of diisopropyl phosphorofluoridate (DFP)-treated hen
Authors:Ram P Gupta  Mohamed B Abou-Donia
Institution:(1) Department of Pharmacology, Duke University Medical Center, P.O. Box 3813, 27710-0001 Durham, North Carolina
Abstract:Diisopropyl phosphorofluoridate (DFP) produces organophosphorus ester-induced delayed neurotoxicity (OPIDN) in humans and sensitive animal species, e.g., adult chicken. The chickens were sacrificed 18 days after a single dose of DFP (1.7 mg/kg, sc.), which produced severe ataxia or paralysis in 10–14 days. We studied Ca2+/calmodulin-dependent in vitro neurofilament phosphorylation by the brain subcellular fractions of control and DFP-treated hens. There was enhanced phosphorylation of all three NF subunits by the brain supernatant of treated hens. This was accompanied by enhanced autophosphorylation of both Ca2+/CaM-dependent protein kinase II (CaM-kinase II) subunits and increased calmodulin binding using either125I-CaM or biotinylated calmodulin to only agr subunit without concomitant increase in the amount of this enzyme. This enhanced phosphorylation of neurofilament subunits was completely and partially inhibited by mastoparan and KN-62, respectively. There was no alteration in the distribution of CaM-kinase II activity in treated hens and the activity was not related to its concentration in different subcellular fractions. The difference in125I-CaM binding to CaM-kinase II agr subunit in the brain supernatants of control and DFP-treated hens was not altered by its phosphorylation or dephosphorylation. The increased CaM-kinase II activity in the soluble fraction of DFP-treated hen brain may be involved in the aberrant phosphorylation of axonal neurofilaments, and thus play a role in OPIDN.Abbreviations CaM calmodulin - CaM-kinase II Ca2+/calmodulin-dependent protein kinase II - DFP diisopropyl phosphorofluoridate - ECL enhanced chemiluminescence - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(beta-aminoethyl ether)N,N,Nprime,Nprime-tetraacetic acid - MAP-2 microtubule-associated protein-2 - MBP myelin basic protein - OPIDN organophosphorus ester-induced delayed neurotoxicity - PIPES 1,4-piperazinediethanesulfonic acid - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid
Keywords:Neurofilament phosphorylation  Ca2+/calmodulin-dependent protein kinase  calmodulin-binding  hen  diisopropyl phosphorufluoridate  brain
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