During repetitive stimulation of motor nerve terminals, mitochondrial Ca2+ uptake limits increases in free cytosolic [Ca2+] and helps ensure faithful neuromuscular transmission. Changes in cytosolic [Ca2+] and in mitochondrial [Ca2+] as well as changes in mitochondrial membrane potential (Psi m) were studied in mouse motor nerve terminals using Ca2+ sensitive indicator and potentiometric dyes, respectively. Trains of action potentials (APs) at 50 to 100 Hz produced a rapid increase in mitochondrial [Ca2+] followed by a plateau which usually continued beyond the end of stimulation. After stimulation, mitochondrial [Ca2+] decayed back to baseline over the course of tens of seconds to minutes. Increasing the Ca2+ load delivered to the terminal by increasing the number of stimuli (500-2000), increasing bath [Ca2+], or prolonging the AP with 3,4-diaminopyridine (3-4, DAP, 100 micromolar), prolonged the post-stimulation decay of mitochondrial [Ca2+] without increasing the amplitude of the plateau. Inhibiting openings of the mitochondrial permeability transition pore with cyclosporin A (5 micromolar) had no significant effect on the decay of mitochondrial [Ca2+]. Inhibition of the mitochondrial Na+-Ca2+ exchanger with CGP-37157 (50 micromolar) dramatically prolonged the post-stimulation decay of mitochondrial [Ca2+], reduced post-stimulation residual cytosolic [Ca2+], and reduced the amplitude of end-plate potentials evoked after the end of stimulation. Stimulation-induced mitochondrial Ca2+ uptake resulted in Psi m depolarizations that were small or undetectable at near-physiological temperatures (~30 degrees C). Their amplitude became larger at lower temperatures (~20 degrees C), or when AP duration was increased with 3,4-DAP (20 micromolar). Psi m depolarizations were inhibited by lowering bath [Ca2+] or by blocking P/Q-type Ca2+ channels with omega-agatoxin (0.3 micromolar). Partial inhibition of complex I of the electron transport chain (ETC) with rotenone (50 nM) increased the amplitude of stimulation-induced Psi m depolarizations. These findings suggest that: (1) Ca2+ extrusion from motor terminal mitochondria occurs primarily via the Na+-Ca2+ exchanger and helps sustain post-tetanic transmitter release, and (2) that the depolarization of Psi m that accompanies Ca2+ uptake is limited by accelerated proton extrusion via the ETC.
| Identifer | oai:union.ndltd.org:UMIAMI/oai:scholarlyrepository.miami.edu:oa_dissertations-1081 |
| Date | 20 April 2008 |
| Creators | Garcia-Chacon, Luis Ernesto |
| Publisher | Scholarly Repository |
| Source Sets | University of Miami |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Open Access Dissertations |
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