Paradoxical potentiation by low extracellular Ca2+ of acute chemical anoxic neuronal injury in cerebellar granule cell culture

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 >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 <10% of control. Removal of Cat 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 Cat chelator Fura-2 did not influence *Author to whom all correspondence and reprint requests should be addressed. Molecular and Chemical Neuropathology 217Vol. 15, 1991 218 Verity, Torres, and Sarafian IAA-induced cytotoxicity in control or low Cat media. Addition of 50 µM glutamate had a minimal cytotoxic effect over 3 h and the combined addition of 0.2 mM IAA plus 50 µM glutamate did not potentiate IAA-induced injury. The glutamate receptor antagonists, D-2-amino -5-phosphonovaleric acid (APV) or kynurenate did not block IAA-injuced injury in control medium but inhibited the potentiation of toxicity seen in the low Ca t 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 Ca t+ medium. The low Cat potentiated component was sensitive to glutamate/NMDA receptor antagonists and associated with reduction of intracellular GSH.

Extracellular HIV-1 viral protein R affects astrocytic glyceraldehyde 3-phosphate dehydrogenase activity and neuronal survival

“…The interplay between ATP and GSH metabolites is particularly important, as different compounds might independently reduce the levels of ATP or GSH or both: for example, treatment with iodoacetate reduces ATP but not GSH (Verity et al , 1991). The role of GAPDH in OS has recently been evaluated and could be due to its nuclear translocation (Dastoor and Dreyer, 2001), thus disabling its specific role in glycolysis and inducing apoptosis.…”

Extracellular human immunodeficiency virus type 1 viral protein R causes reductions in astrocytic ATP and glutathione levels compromising the antioxidant reservoir

“…Indeed, impairment of glyceraldehyde 3-phosphate dehydrogenase activity and glycolysis would lead to decreased levels of ATP and an increased accumulation of ROS, which may not be disposed of in a timely manner, thereby inducing a decrease in GSH concentrations. Another chemotherapeutic compound (iodoacetate) has been shown to decrease ATP but not GSH (Verity et al, 1991), pointing to a drug-dependent mechanism of action insofar as ATP and GSH reductions are concerned. While, our studies within the U-87 MG astroglioma cell line could represent an in vitro limitation with respect to our findings although in line with preliminary data in primary human fetal astrocytes, further studies are currently under way to determine the intricate relationship between the reductions in ATP and GSH in order to determine which of the two aforementioned scenarios are caused by recombinant Vpr.…”