GLUTAMATE-INDUCED EFFLUX OF PROTEIN, NEURON-SPECIFIC ENOLASE AND LACTATE-DEHYDROGENASE FROM A MESENCEPHALIC CELL-CULTURE

A mixed mesencephalic cell culture damaged by glutamate was used as a model to study the efflux of lactate dehydrogenase and neuron-specific enolase from neuronal cells into the culture medium. Glutamate toxici ty was induced in sister cultures by 15 min exposure to 100 mu mol/l g lutamate in a Ca2+ containing salt solution. Cell injury was monitored 24 h later by measuring the lactate dehydrogenase activity and the ne uron-specific enolase content in the cells and in the culture medium. The neuronal cell damage is reflected by an efflux of neuron-specific enolase and lactate dehydrogenase from the cells and an increase of la ctate dehydrogenase catalytic activity concentration and neuron-specif ic enolase mass concentration in the culture medium. It was found that the efflux fraction calculated from estimations of the cells was clea rly higher than the efflux fraction calculated from estimations of the amount of enzymes found in the culture medium. Calculations of the re covery of lactate dehydrogenase and neuron-specific enolase and experi ments designed to study the efflux of lactate dehydrogenase and neuron -specific enolase during incubation and washing showed that higher amo unts of neuron-specific enolase are released than lactate dehydrogenas e. A close correlation was found between the glutamate-induced changes of the neuron-specific enolase efflux fraction, based on enzyme deter minations of the cells, and the change of the microscopically counted neuron-specific enolase immunoreactive cell numbers. This indicates th at the determination of the neuron-specific enolase efflux fraction (c ells) is an accurate and sensitive marker of damaged neurons. The lact ate dehydrogenase efflux fraction seems to be less sensitive for the q uantitation of neuronal cell damage; in addition, it depends not only on the neuronal damage but also on the proportion of neurons in the ce ll culture.