Adenosine receptor ligands protect against a combination of apoptotic and necrotic cell death in cerebellar granule neurons

Agonists at A₁ receptors and antagonists at A_2A receptors are known to be neuroprotective against excitotoxicity. We set out to clarify the mechanisms involved by studying interactions between adenosine receptor ligands and endogenous glutamate in cultures of rat cerebellar granule neurons (CGNs). Glutamate and the selective agonist N-methyl-d-aspartate (NMDA), applied to CGNs at 9 div (days in vitro), both induced cell death in a concentration-dependent manner, which was attenuated by treatment with the NMDA receptor antagonists dizocilpine, d-2-amino-5-phosphono-pentanoic acid (d-AP5) or kynurenic acid (KYA), but not by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Glutamate toxicity was reduced in the presence of all of the following: cyclosporin A (CsA), a blocker of the membrane permeability transition pore, the caspase-3 inhibitor, benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone (Z-DEVD-fmk), the poly (ADP-ribose) polymerase (PARP-1) inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone (DPQ), and nicotinamide. This is indicative of involvement of both apoptotic and necrotic processes. The A₁ receptor agonist, N ⁶-cyclopentyladenosine (CPA), and the A_2A receptor antagonist 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazo-5-yl-amino]ethyl)phenol (ZM241385) afforded significant protection, while the A₁ receptor blocker 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and the A_2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxyamidoadenosine (CGS21680) had no effect. These results confirm that glutamate-induced neurotoxicity in CGNs is mainly via the NMDA receptor, but show that a form of cell death which exhibits aspects of both apoptosis and necrosis is involved. The protective activity of A₁ receptor activation or A_2A receptor blockade occurs against this mixed profile of cell death, and appears not to involve the selective inhibition of classical apoptotic or necrotic cascades.