(45) VISUALIZATION AND MODIFICATION OF THE MITOCHONDRIAL PERMEABILITY TRANSITION PORE IN STRIATAL NEURONS

S-S. Sheu¹, G. Beutner¹, C.C. Alano¹, K.M. Piech-Dumas¹ and R.A. Gross^1,2
1Department of Pharmacology and Physiology
²Department of Neurology, University of Rochester, School of Medicine and Dentistry, Rochester, NY 14642

Activation of N-Methyl-D-Aspartate receptor (NMDAR) leads to an increase in cytosolic and mitochondrial calcium concentrations ([Ca2+]c and [Ca2+]m, respectively) and, therefore, provides a favorable condition for the opening of the mitochondrial permeability transition pore (MPTP).  Using fluorescence microscopy, changes in [Ca2+]c and [Ca2+]m upon activation of the NMDAR were monitored in cultured striatal neurons.  Exposure of neurons to 1 mM NMDA caused a sustained increase in [Ca2+]c as expected.  A secondary increase in [Ca2+]c occurred after a period of about 23.4 + 9.8 minutes of NMDA exposure, which was inhibited by pretreatment with 100 nM N-methyl/valin-cyclosporin (CsA).  Exposure of neurons to NMDA also resulted in an increase in [Ca2+]m, which was followed by a precipitous drop in a comparable time frame as the secondary increase in [Ca2+]c.  Preincubation of the neurons with CsA also prevented the decrease in [Ca2+]m.  These results indicate that using fluorescent Ca2+ indicators, MPTP can be detected in living neurons.

We then study the modulation of MPTP in striatal neurons by atractyloside (ATR), an inhibitor of the adenine nucleotide translocase (ANT).  In isolated mitochondria from adult rat brain, ATR caused a concentration-dependent swelling as measured by the decrease of absorbency at 540 nm.  Mitochondrial swelling was prevented by pre-incubation with 200 nM N-metVal-cyclosporin (CyA).  Incorporation of 0.5 to 5 mM ATR into striatal neurons via Lipofectin led to a concentration-dependent decrease in intracellular ATP concentration.  In neurons loaded with TMRE to monitor the mitochondrial membrane potential, ATR caused a transient hyperpolarization followed by a strong depolarization approximately after 90 minutes of exposure.  The depolarization was prevented with 200 nM CyA.  However, in neurons incubated with 1mM glucose and 2 mM pyruvate to enhance the activity of mitochondrial electron transport, the ATR-induced strong depolarization in the mitochondrial membrane potential occurred approximately after 30 minutes of exposure.  These results show that ATR modulates the opening of MPTP in intact neurons possibly through its action on ANT.

Supported by NIH grants HL-33333, NS37710, AHA affiliate grant 9920244T, ES07026, DA07232 and DA10514 .

© 1999 Wadsworth Center, All Rights Reserved. Legal & Privacy Notices