The antiapoptotic effect of guanosine is mediated by the activation of the PI 3‐kinase/AKT/PKB pathway in cultured rat astrocytes

Abstract

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.

Gliopreventive effects of guanosine against glucose deprivation in vitro

“…This effect could be related, at least partially, to the release of a guanine-based purine after astrocytic hypoglycemic insults [6], its trophic properties and ability to stimulate astrocyte proliferation [84], its modulatory effects on the glutamatergic system [3], and its antioxidant properties. Additionally, guanosine promotes the activation of the PI3K signaling pathway, upstream of glycogen synthase kinase 3, which may regulate the glucose uptake, ROS levels and, thus, cell survival [13,85,86].…”

Anatomical Distribution of Nucleoside System in the Human Brain and Implications for Therapy

“…This effect appeared to be mediated by the antiapoptotic properties of Guo (Di Iorio et al 2004 ) . Guanosine was also found to modulate memory processes: its pretraining administration impaired retention of inhibitory avoidance responses in rats (Roesler et al 2000 ) .…”

Transforming growth factor alpha acts as a gliatrophin for mouse and human astrocytes

“…Astrocytes were treated with staurosporine for 3 h and death was evaluated 24 h after application (Di Iorio et al, 2004). DAPI nuclear staining revealed the presence of apoptotic bodies, which are indicative of apoptotic death (Figure 8a).…”

“…Nuclei were counterstained with DAPI (3 mM in PBS, 10 min at room temperature, Sigma, France). Staurosporine-induced astrocytes death was performed as described (Di Iorio et al, 2004), exposing astrocytes for 3 h to 100 nM staurosporine (Calbiochem, La Jolla, CA, USA). Transforming growth factor alpha (50 ng/ml), U0126 (10 mM), LY294002 (30 mM) or the corresponding vehicles were added simultaneously with staurosporine.…”