Hypoglycaemia-induced cell death: features of neuroprotection by the P2 receptor antagonist basilen blue

Our previous work in neuronal cultures has shown that several antagonists o f P2 ATP receptors prevent cell death evoked by hypoglycaemia, chemical hyp oxia, mitochondria dysfunction, as well as glutamate-dependent excitotoxici ty and low potassium-induced apoptosis. Experiments are now designed to exa mine which biological pathway contributes to cell death/survival under gluc ose starvation. We show here that, consequently to hypoglycaemic insults, c erebellar granule neurones undergo a combination of apoptosis and necrosis both inhibited by the P2 receptor antagonist basilen blue. This is demonstr ated by morphological and biochemical features, such as TdT-mediated dUTP-b iotin nick end-labelling, fluorescent staining of nuclear chromatin using H oechst 33258, direct counting of intact viable nuclei and extracellular rel easing of the cytosolic enzyme LDH. Furthermore, we show that hypoglycaemia induces outflow of cytochrome c from mitochondria and it up-regulates heat -shock proteins HSP70, but not HSP90, glucose-regulated proteins GRP75 and GRP78, as well as expression and activity of the enzyme caspase-2. Basilen blue can modulate only some of these effects. Our data contribute to dissec t the role played by P2 receptor antagonism in sustaining neuroprotection a gainst metabolic stresses. (C) 2001 Elsevier Science Ltd. All rights reserv ed.