Extracellular ATP causes apoptosis and necrosis of cultured mesangial cells via P2Z/P2X(7) receptors

Mesangial cells undergo cell death both by apoptosis and necrosis during gl omerular disease. Since nucleotides are released from injured and destroyed cells in the glomerulus, we examined whether extracellular ATP and its rec eptors may regulate cell death of cultured mesangial cells. Addition of ext racellular ATP (300 mu M to 5 mM) to cultured rat mesangial cells for 90 mi n caused a 5.8-fold increase in DNA fragmentation (terminal deoxynucleotidy l transferase assay) and a 4.2-fold increase in protein levels of the tumor suppressor p53, which is thought to regulate apoptosis. Apoptotic DNA frag mentation was confirmed by the diphenylamine assay and by staining with the DNA-specific fluorochrome Hoechst 33258. The necrotic markers, release of lactate dehydrogenase and uptake of trypan blue, were not positive before 3 h of ATP addition. The effects of ATP on DNA fragmentation and p53 express ion were reproduced by the purinergic P2Z/P2X(7) receptor agonist, 3'-O-(4- benzoylbenzoyl)-ATP, and inhibited by the P2Z/P2X(7) receptor blocker, oxid ized ATP. Transcripts encoding the P2Z/P2X(7) receptor were expressed by cu ltured mesangial cells as determined by Northern blot analysis. P2Z/P2X(7) receptor-associated pore formation in the plasma membrane was demonstrated by the Lucifer yellow assay. We conclude that activation of P2Z/P2X(7) rece ptors by extracellular ATP causes apoptosis and necrosis of cultured mesang ial cells. Activation of purinergic P2Z/P2X(7) receptors may play a role