The antiapoptotic effect of guanosine is mediated by the activation of the PI 3-kinase/AKT/PKB pathway in cultured rat astrocytes.
Guanosine has many trophic effects in the CNS, including the stimulation of neurotrophic factor synthesis and release by astrocytes, which protect neurons against excitotoxic death. Therefore, we questioned whether guanosine protected astrocytes against apoptosis induced by staurosporine. We evaluated apoptosis in cultured rat brain astrocytes, following exposure (3 h) to 100 nM staurosporine by acridine orange staining or by oligonucleosome, or caspase-3 ELISA assays. Staurosporine promoted apoptosis rapidly, reaching its maximal effect (approximately 10-fold over basal apoptotic values) in 18-24 h after its administration to astrocytes. Guanosine, added to the culture medium for 4 h, starting from 1 h prior to staurosporine, reduced the proportion of apoptotic cells in a concentration-dependent manner. The IC50 value for the inhibitory effect of guanosine is 7.5 x 10(-5) M. The protective effect of guanosine was not affected by inhibiting the nucleoside transporters by propentophylline, or by the selective antagonists of the adenosine A1 or A2 receptors (DPCPX or DMPX), or by an antagonist of the P2X and P2Y purine receptors (suramin). In contrast, pretreatment of astrocytes with pertussis toxin, which uncouples Gi-proteins from their receptors, abolished the antiapoptotic effect of guanosine. The protective effect of guanosine was also reduced by pretreatment of astrocytes with inhibitors of the phosphoinositide 3-kinase (PI3K; LY294002, 30 microM) or the MAPK pathway (PD98059, 10 microM). Addition of guanosine caused a rapid phosphorylation of Akt/PKB, and glycogen synthase kinase-3beta (GSK-3beta) and induced an upregulation of Bcl-2 mRNA and protein expression. These data demonstrate that guanosine protects astrocytes against staurosporine-induced apoptosis by activating multiple pathways, and these are mediated by a Gi-protein-coupled putative guanosine receptor.
Di Iorio
Patrizia
P
Department of Biomedical Sciences, School of Medicine, University of Chieti, Chieti, Italy.
Ballerini
Patrizia
P
Traversa
Ugo
U
Nicoletti
Ferdinando
F
D'Alimonte
Iolanda
I
Kleywegt
Sonya
S
Werstiuk
Eva S
ES
Rathbone
Michel P
MP
Caciagli
Francesco
F
Ciccarelli
Renata
R
D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006151:Guanosine / Q000378:metabolism* / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D058914:Purinergic Antagonists; D058915:Purinergic P1 Receptor Antagonists; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D011983:Receptors, Purinergic / Q000378:metabolism; D018047:Receptors, Purinergic P1 / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics
D053148:Caspase 3; D020169:Caspases; D004791:Enzyme Inhibitors; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D006151:Guanosine; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2; D058914:Purinergic Antagonists; D058915:Purinergic P1 Receptor Antagonists; D012333:RNA, Messenger; D011983:Receptors, Purinergic; D018047:Receptors, Purinergic P1; C494920:Akt1 protein, rat; C000605508:Gsk3b protein, rat; C505410:Casp3 protein, rat