Neurotoxicity of free-radical-mediated serotonin neurotoxin in cultured embryonic chick brain neurons.

Exposure of serotonin (5-HT) to oxygen-derived free-radical-generating system, xanthine oxidase-hypoxanthine or to a Fenton reaction results in the formation of the neurotoxin, tryptamine-4,5-dione. In cultured embryonic chick brain neurons, incubation of tryptamine-4,5-dione or its ethyl carbonate derivative resulted in a dose-dependent neurotoxicity (1-100 microM). The addition of sulfhydryl compound, glutathione at 2 or 10 microM significantly enhanced the toxicity induced by 10 microM tryptamine-4,5-dione. On the contrary, glutathione at 10 microM decreased the neurotoxic effect caused by 10 microM 5,6- and 5,7-dihydroxytryptamine in the cultured neurons. The toxicity resulted from 5,6- and 5,7-dihydroxytryptamine could be fully prevented by a 5-HT uptake inhibitor, fluoxetine. However, the toxicity caused by tryptamine-4,5-dione and glutathione conjugate could not be blocked by fluoxetine (10 or 100 microM) or by a glutathione transferase inhibitor, boric acid/serine. The results indicate a different molecular mechanism among 5-HT derived neurotoxins and suggest that tryptamine-4,5-dione and/or its glutathione conjugate would cause neuronal damage, if they are formed in vivo.

Identification of a protein kinase as an intrinsic component of rat liver coated vesicles.

Purified rat liver coated vesicles phosphorylate two peptides, Mr 53 000 and Mr 51 000, in the presence of [gamma-32P]ATP. Incorporation of phosphate into these peptides is not stimulated by cAMP, Ca2+, or Ca2+ plus calmodulin and occurs principally on a threonine residue. Mild conditions that result in removal of coat proteins from coated vesicles remove most of the protein kinase activity, suggesting the enzyme(s) is (are) not an integral membrane protein. Photolabeling of coated vesicles with 8-azido-[alpha-32P]ATP results in specific labeling of only the Mr 53 000 and Mr 51 000 peptides. Preincubation with 10 mM N-ethylmaleimide inhibits kinase activity and concomitantly reduces photolabeling of the two peptides. Thus, the data are consistent with the hypothesis that protein kinase activity resides with these two coated vesicle proteins and that they are catalyzing an autophosphorylation reaction.

Chick embryo spinal cord neurons synthesize a transferrin-like myotrophic protein.

Highly enriched cultures of chick embryo spinal cord neurons synthesize and secrete a protein which is immunoprecipitable by anti-ovotransferrin. Ovotransferrin, an iron-binding glycoprotein of Mr 80 000, is also shown to stimulate in vitro myogenesis of cultured chick embryo myotubes as measured by saturable dose-dependent increase in acetylcholine receptors. This effect is probably dependent on ovotransferrin's ability to donate iron to the cells. In many respects ovotransferrin is similar to 'sciatin', a myotrophic protein isolated from chicken sciatic nerves.

Coated vesicles from rat liver and calf brain contain cryptic mannose 6-phosphate receptors.

Highly purified clathrin-coated vesicles, isolated from rat liver and calf brain, contain mannose 6-phosphate receptors. The coated vesicle receptors appear to have the same subunit molecular weight and similar binding affinity as the receptor previously purified from bovine liver and rat chondrosarcoma microsomes (Sahagian, G. G., Distler, J. J., and Jourdian, G. W. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4289-4293 and Steiner, A. W., and Rome, L. H. (1982) Arch. Biochem. Biophys. 214, 681-687). There is a considerable (greater than 60-fold) enrichment of receptors in liver coated vesicles as compared to liver microsomes. Experiments carried out with intact and detergent-disrupted coated vesicles indicated that the receptors face toward the inside of the coated vesicles. The data suggest that coated vesicles are involved in the intracellular transport of the mannose 6-phosphate receptor.