Astrocytes release adenine-based and guanine-based purines under physiological and, particularly, pathological conditions. Thus, the aim of this study was to determine if adenosine induced apoptosis in cultured rat astrocytes. Further, if guanosine, which increases the extracellular concentration of adenosine, also induced apoptosis determined using the TUNEL and Annexin V assays. Adenosine induced apoptosis in a concentration-dependent manner up to 100 microM. Inosine, hypoxanthine, guanine, and guanosine did not. Guanosine or adenosine (100 microM) added to the culture medium was metabolized, with 35% or 15%, respectively, remaining after 2-3 h. Guanosine evoked the extracellular accumulation of adenosine, and particularly of adenine-based nucleotides. Cotreatment with EHNA and guanosine increased the extracellular accumulation of adenosine and induced apoptosis. Inhibition of the nucleoside transporters using NBTI (100 microM) or propentophylline (100 microM) significantly decreased but did not abolish the apoptosis induced by guanosine + EHNA or adenosine + EHNA, respectively. Apoptosis produced by either guanosine + EHNA or adenosine + EHNA was unaffected by A(1) or A(2) adenosine receptor antagonists, but was significantly reduced by MRS 1523, a selective A(3) adenosine receptor antagonist. Adenosine + EHNA, not guanosine + EHNA, significantly increased the intracellular concentration of S-adenosyl-L-homocysteine (SAH) and greatly reduced the ratio of S-adenosyl-L-methioine to SAH, which is associated with apoptosis. These data demonstrate that adenosine mediates apoptosis of astrocytes both, via activation of A(3) adenosine receptors and by modulating SAH hydrolase activity. Guanosine induces apoptosis by accumulating extracellular adenosine, which then acts solely via A(3) adenosine receptors.