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 of P2 ATP receptors prevent cell death evoked by hypoglycaemia, chemical hypoxia, mitochondria dysfunction, as well as glutamate-dependent excitotoxicity and low potassium-induced apoptosis. Experiments are now designed to examine which biological pathway contributes to cell death/survival under glucose starvation. We show here that, consequently to hypoglycaemic insults, cerebellar granule neurones undergo a combination of apoptosis and necrosis both inhibited by the P2 receptor antagonist basilen blue. This is demonstrated by morphological and biochemical features, such as TdT-mediated dUTP-biotin nick end-labelling, fluorescent staining of nuclear chromatin using Hoechst 33258, direct counting of intact viable nuclei and extracellular releasing 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 dissect the role played by P2 receptor antagonism in sustaining neuroprotection against metabolic stresses.
Cavaliere
F
F
Fondazione Santa Lucia, Via Ardeatina 306, 00179, Rome, Italy.
D'Ambrosi
N
N
Sancesario
G
G
Bernardi
G
G
Volonté
C
C
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015415:Biomarkers; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects*; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004396:Coloring Agents / Q000494:pharmacology; D005947:Glucose / Q000172:deficiency / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D007003:Hypoglycemia / Q000378:metabolism*; D020287:In Situ Nick-End Labeling; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D058919:Purinergic P2 Receptor Antagonists; D051381:Rats; D017208:Rats, Wistar; D014227:Triazines / Q000494:pharmacology*
D015415:Biomarkers; D004396:Coloring Agents; D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; D018696:Neuroprotective Agents; D019253:Proto-Oncogene Proteins c-bcl-2; D058919:Purinergic P2 Receptor Antagonists; D014227:Triazines; C018551:glucose-regulated proteins; C012842:Cibacron Blue F 3GA; D007770:L-Lactate Dehydrogenase; D020169:Caspases; D005947:Glucose