A(1) adenosine receptor signal and AMPK involving caspase-9/-3 activation are responsible for adenosine-induced RCR-1 astrocytoma cell death.

Extracellular adenosine reduced viability of RCR-1 rat astrocytoma cells in a dose (0.3-10mM)- and treatment time (24-72h)-dependent manner. In the apoptosis assay using propidium iodide (PI) and annexin V, treatment with adenosine (1mM) for 72h increased the population of PI-negative/annexin V-positive cells, that is related to early apoptosis, and that of PI-positive/annexin V-positive cells, that is related to late apoptosis/secondary necrosis. In addition, nuclei of cells treated with adenosine (1mM) for 72h were reactive to an antibody against single-stranded DNA. Adenosine activated caspase-3, -8 and -9, but mitochondrial membrane potentials were not affected. Adenosine-induced RCR-1 cell death was significantly inhibited by 8-CPT, an antagonist of A(1) adenosine receptors, and forskolin, an adenylate cyclase activator. SQ22536, an adenylate cyclase inhibitor, alternatively, exhibited an effect similar to adenosine. CHA, an agonist of A(1) adenosine receptors, activated caspase-3 and -9, but not caspase-8. Adenosine-induced cytotoxicity of RCR-1 cells was also significantly inhibited by dipyridamole, an inhibitor of adenosine transporter, and AMDA, an inhibitor of adenosine kinase. AICAR, an activator of AMP-activated protein kinase (AMPK), reduced RCR-1 cell viability, but synergistic effect was not obtained with co-treatment with adenosine and AICAR. AICAR activated caspase-3 and -9, but not caspase-8. An additive inhibition was found in the co-presence of 8-CPT and dipyridamole. Extracellular adenosine, thus, appears to activate caspase-9 followed by the effector caspase, caspase-3, at least via two independent pathways linked to A(1) adenosine receptor-mediated adenylate cyclase inhibition and adenosine uptake into cells/conversion to AMP/activation of AMPK, possibly regardless of mitochondrial damage, thereby leading to RCR-1 cell death, dominantly by apoptosis. Moreover, caspase-8 activation could again contribute to adenosine-induced cytotoxicity, although the underlying mechanism is currently unknown. Collectively, the results of the present study may represent a new pathway for caspase activation relevant to diverse adenosine signals in cell death.

Pipecolic acid induces apoptosis in neuronal cells.

Pipecolic acid, a lysine metabolite, is thought to be a factor responsible for hepatic encephalopathy; however, the underlying mechanism is far from understood. Twenty minutes treatment with D-, L-, and DL-pipecolic acid at concentrations ranging from 1 to 100 microM, except for 1 microM L-pipecolic acid, had no inhibitory effect on excitatory postsynaptic responses in the dentate gyrus of rat hippocampal slices. In a whole-cell voltage-clamp configuration, DL-pipecolic acid (10 and 100 microM) did not affect voltage-sensitive Na(+) channel currents and K(+) channel currents, but it potentiated voltage-sensitive Ca(2+) channel currents, but to a lesser extent, in cultured rat cortical neurons and Neuro-2A cells, a mouse neuroblastoma cell line. Notably, 72-h treatment with D-, L-, and DL-pipecolic acid reduced Neuro-2A cell viability in a dose-dependent manner at concentrations ranging from 1 to 100 microM in a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, in parallel with reactions to propidium iodide, a marker of cell death, and Hoechst 33,342, a marker of apoptosis in a fluorescent microscopic study, with DL-pipecolic acid being the most potent. The results of the present study suggest that pipecolic acid could cause hepatic encephalopathy by inducing neuronal cell death, perhaps apoptosis, rather than by depressing neurotransmissions.