Abstract
Acute chemical anoxic injury was produced in primary cerebellar granule cell cultures incubated with iodoacetate (IAA) alone or IAA combined with potassium cyanide (KCN). Cytotoxicity was assessed using Trypan blue exclusion or LDH release. Four millimolars of KCN induced approx 30% neuron death at 3 h, whereas greater than 50% cell death was produced by 0.2 mM IAA. No potentiation of cytotoxicity was observed by IAA + KCN. A total of 0.2 mM IAA produced an early major reduction of intracellular ATP prior to the onset of neuron injury or reduction in intracellular glutathione (GSH). Medium Na+ replacement by choline, K+, or methylglucamine protected against IAA-induced neuronal injury, reduced the rate of decline of intracellular ATP but had no effect on intracellular GSH. Some 80% neuronal survival was obtained when Na+ was deleted from the medium even after the intracellular ATP had been reduced to less than 10% of control. Removal of Ca2+ from the medium had no effect on control culture, Trypan blue exclusion, GSH, or ATP, but potentiated the onset and magnitude of IAA-induced cytotoxicity. ATP and GSH decline. Loading of granule cells with the Ca2+ chelator Fura-2 did not influence IAA-induced cytotoxicity in control or low Ca2+ media. Addition of 50 microM glutamate had a minimal cytotoxic effect over 3 h and the combined addition of 0.2 mM IAA plus 50 microM glutamate did not potentiate IAA-induced injury. The glutamate receptor antagonists, D-2-amino-5-phosphonovaleric acid (APV) or kynurenate did not block IAA-induced injury in control medium but inhibited the potentiation of toxicity seen in the low Ca2+ medium. This study suggests the use of IAA as a chemical anoxic agent in cerebellar granule cell culture. The early, dose-dependent decline in ATP may be dissociated from GSH change. Acute IAA-induced injury is Na+/Cl- dependent but paradoxically potentiated in low Ca2+ medium. The low Ca2+ potentiated component was sensitive to glutamate/NMDA receptor antagonists and associated with reduction of intracellular GSH.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.