(31) MITOCHONDRIAL MEMBRANE POTENTIAL IN CULTURED NEURAL CELLS:  DISTINCTIVE RESPONSES TO APOPTOTIC STIMULI, NMDA AND AMPA RECEPTOR ACTIVATION AND MODELLING WITH A 'VIRTUAL CELL'

D.G. Nicholls¹, M.W. Ward², S. Chalmers¹, S. Vesce¹ and A.C. Rego^3
1Buck Institute, Novato 94945, CA
²Dept. of Pharmacology and Neuroscience, University of Dundee, UK
³Laboratory of Biochemistry, Faculty of Medicine and Center for Neurosciences of Coimbra, University of Coimbra

There is much confusion in the literature as to how to interpret the complex fluorescence responses obtained from cationic potentiometric probes at single cell resolution. Since the mitochondrial membrane potential is central to an understanding of the mechanisms underlying cell death, we have approached the behavior of probes such as TMRM+ and rhodamine 123 from first principles rather than empirically. Inputting a series of simple parameters into an Excel program has allowed us to reproduce the single-cell fluorescence responses of cells with considerable accuracy, enabling us to estimate the approximate rate and extent of changes in membrane potential in response to excitotoxic and apoptotic challenges. Some surprises have emerged: mitochondria retain the capacity to generate ATP for long periods during NMDA receptor activation but rapidly reverse their ATP synthase during AMPA receptor activation, even though the mitochondria only accumulate Ca2+ in the presence of NMDA. We have studied staurosporine-induced apoptosis in GT1-7 cells. Limitation of electron transport, rather than inhibition of adenine nucleotide exchange or mitochondrial hyperpolarization, precedes apoptosis, and this is absent in Bcl-2 over-expressing cells.

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