| The organophosphorous compounds, represented by tri-o-cresyl phosphate (TOCP), and the straight chain hexacarbons, represented by 2,5-hexanedione (2,5-HD), are two classes of central-peripheral distal axonopathic agents that produce clinically similar manifestations (hindlimb paralysis). However, they differ histopathologically in the nature of the lesions produced, internodal swellings in TOCP neuropathy (OPIDN) and paranodal neurofilament accumulation in 2,5-HD neuropathy, and in their distribution.; The rationale for this project focused primarily on the potential for antagonism between organophosphorous compounds and endogenous protein phosphorylation systems on the grounds of chemical similarity suggesting affinity for the same binding sites. Optimal conditions for in vitro phosphorylation assay were characterized for chickens which, unlike rats, are sensitive to OPIDN. Brains and spinal cords were removed and synaptosomal cytosol and "P(,2)" membranes were prepared. The assay was performed at 35(DEGREES)C, pH 6.5 with protein concentration in a final volume of 200(mu)l of 100 (mu)g for brain and 35-50 (mu)g for spinal cord cytosol and 75 (mu)g for membranes. Incubation times were 90 sec for cytosolic and 20 sec for membrane proteins. Proteins phosphorylated with ((gamma)-('32)P)ATP were separated by 10% SDS-PAGE, stained with Coomassie blue, and autoradiographed. Differences in ('32)P incorporation were quantified by microdensitometry. A striking contrast was observed between the enhancement of tubulin phosphorylation by addition of 50 (mu)M Ca('2+) in the rat (534%) and the hen (164%) brain cytosol. Other notable differences between hen and rat phosphorylatable proteins include the absence from hen spinal cord cytosol of the most phosphorylated rat spinal cord cytosolic band of M(,r) 30K and the greater phosphorylation of the 20K band in hen and the 16K band in rat spinal cord cytosol, brain membranes, and spinal cord membranes.; Paralysis of hens by oral dosing with 750 mg/kg TOCP 21 days prior to sacrifice appeared to confer Ca('2+)-dependence on their endogenous Mr 55K phosphorylation system. Enhancement of its phosphorylation fit the following criteria for involvement in OPIDN: time course, chemical selectivity, species, age, and sex selectivities and dose dependence.; In contrast, rats paralyzed by drinking 0.5% 2,5-HD for 90 days showed reduced phosphorylation of myelin basic proteins, but no significant effect on brain cytosolic proteins. These opposing phosphorylation effects may indicate different biochemical etiologies for OPIDN and hexacarbon neuropathies. |
