Adenosine is a neuromodulator acting via four (A1, A2A, A2B and A3) receptors (Cunha, 2001). Adenosine is released in situations of metabolic stress and is considered a neuroprotective agent based on the ability of A1 receptor activation to inhibit glutamate release, hyperpolarize neurons and decrease cellular metabolism (Cunha, 2001). Recent observations have highlighted the neuroprotective impact of A2A receptor blockade in CNS stressful situations (Fredholm et al. 2003). However, it is unknown if the impact of the A2A receptor on neuronal viability results from a direct action on neurons. Thus, we now used cultured neurons to test the effect of adenosine receptor agonists and antagonists on the outcome of neuronal viability.
Cerebral cortices were obtained from rat embryos (E15-16) and primary neurons were cultured for 6 days (Silva et al. 2001). They were then subjected to an excitotoxic stimulus (100 µM kainate plus 30 µM cyclothiazide) for 24 h, after which cell viability was assessed using syto-13 and propidium iodide (Silva et al. 2001). Adenosine receptor agonists and antagonists were added at least 30 min before the excitotoxic stimulus. All animals were humanely killed. The values are presented as means ± S.E.M. Statistical analysis was performed using Student’s paired t test.
The excitotoxic stimulus led to a 42.7 ± 4.5 % (n = 13) neuronal death. Adenosine deaminase (2 U ml-1, that converts adenosine into its inactive metabolite – inosine) reduced neuronal death to 33.5 ± 2.9 % (n = 4, P < 0.05, Student’s t test). The A1 receptor agonist, CPA (100 nM), had no effects on neuronal survival but the A1 receptor antagonist, DPCPX (50 nM), decreased neuronal cell death to 30.7 ± 4.9 % (n = 4, P < 0.05). The A2A receptor agonist, CGS 21680 (30 nM) reduced death to 25.8 ± 4.7 % (n = 6, P < 0.05) and this effect was prevented by the A2A receptor antagonists, ZM 241385 (50 nM, n = 4) and SCH 58261 (50 nM, n = 3), which alone had no effect on neuronal viability. Finally, neither the A3 receptor agonist, Cl– IBMECA (100 nM, n = 3), nor the A3 receptor antagonist, MRS 1191 (5 µM, n = 3), had any significant effect on neuronal survival.
These results indicate that in isolated neurons in culture adenosine has a neuroprotective effect, which is essentially due to removal of a noxious activation of A1 receptors. Futhermore the activation rather than the blockade of A2A receptors also protected neurons in accordance with what was shown in cerebral ischaemia in pups (Aden et al. 2003). This unexpected neuroprotective pattern of adenosine receptors in cultured cortical neurons might result from the different sensitivity to intracellular calcium of neurons from newborn rats (Turner et al. 2003), which indicates that neuronal cultures might not be adequate models to study neurotoxic phenomena in the adult brain.
This work was supported by FCT.