(4) REGULATION OF NMDA MEDIATED PERMEABILITY TRANSITION

Gisela Beutner (1), Conrad C. Alano (1), Robert A. Gross (1,2), Shey-Shing Sheu (1)
(1) Department of Pharmacology and Physiology
(2) Department of Neurology, University of Rochester SMD, Rochester, NY 14642

Activation of the N-Methyl-D-Aspartate (NMDA) receptor leads to an increase in cytosolic Ca2+ concentration ([Ca2+]c) and therefore provides for a favorable condition for the opening of the permeability transition pore (PTP), an unspecific channel of the inner mitochondrial membrane. Opening of the PTP and the subsequent breakdown of the mitochondrial membrane potential are possible key events leading forward to apoptosis and necrosis.

Using primary cultures of rat pup striatum (E18), we were able to monitor an increase in [Ca2+]c as well as in mitochondrial Ca2+ concentrations ([Ca2+]m) upon activation of the NMDA receptor with fluorescent calcium indicators fura-2 and rhod-2, respectively. Around 2 h after exposure of the cells with 0.3 mM NMDA for 10 minutes we observed a gradual increase in [Ca2+]c measured with the Ca2+ indicator fura-2. In rhod-2 loaded striatal neurons, in which [Ca2+]m was recorded under similar experimental conditions, we observed a sudden decrease in rhod-2 fluorescence along the same time frame. These processes were inhibited by 100 nM cyclosporin A.

Exposure of cells with 0.3 mM NMDA for 10 minutes led to 50% apoptotic and 18% necrotic cells within 24 h compared to 33% apoptotic and 48% necrotic cells while treating the cells with 0.3 mM NMDA for 1 hour as proofed by staining with the Hoechst dye 33258. Exposure of the neurons with 1 mM NMDA for 10 minutes led to 72 % necrotic and 4 % apoptotic cells.

These results demonstrate that mild treatment with NMDA is one possible method for detecting PTP opening in intact cells and its function for inducing of the apoptotic cell death cascade. Supported by NIH grant 33333 and CTR grant 4299R1.