Depolarization of neurons promotes Ca2+ influx through voltage-activated channels, raising the intracellular Ca2+ concentration ([Ca2+]i). The consequences of such changes in [Ca2+]i for mitochondrial function were assessed in single, freshly dissociated mammalian neurons. Microfluorimetric techniques were used to measure [Ca2+]i, mitochondrial membrane potential [DELTA-psi(m), Rhodamine 123 (Rh 123) fluorescence], NAD(P)H/NAD(P)+ autofluorescence and flavoprotein autofluorescence combined with whole-cell voltage-clamp techniques. Brief (100-500 ms) depolarization of the cell membrane by high K+ or by voltage commands raised [Ca2+]i and depolarized DELTA-psi(m). The change in DELTA-psi(m) was dependent on extracellular Ca2+. Under voltage-clamp control of the cell membrane, the voltage-dependence of the change in Rh 123 fluorescence reflected that of the Ca2+ current. The response was reduced by Ca2+ buffers introduced into the cell. The behaviour of this signal is thus consistent with a mitochondrial response to raised [Ca2+]i and does not reflect the change in cell membrane potential per se. Similar stimuli caused a rapid decrease of NAD(P)H autofluorescence, followed by an increase which could last several minutes. Flavoprotein fluorescence increased transiently, followed by a decrease lasting for several minutes. These signals indicate an initial oxidation of NAD(P)H and FADH, followed by a prolonged increase in the reduced state of both coenzymes. All these changes were dependent on extracellular [Ca2+]. Raising [Ca2+]i again during the period of NAD+ reduction caused an oxidizing response. Ruthenium Red applied to the cells (i) reduced both the Ca2+ current and the depolarization-induced [Ca2+]i transient and (ii) directly quenched Rh 123 fluorescence. When introduced into the cells with patch pipettes, it prevented the changes in autofluorescence without interfering with the Ca2+ conductance. Oligomycin blocked neither the response of DELTA-psi(m) nor of NADH autofluorescence, suggesting that the signals do not reflect a response to falling ATP/ADP.P(i) ratios as a consequence of the high [Ca2+]i. The changes in NADH autofluorescence were sustained in the presence of iodoacetic acid with pyruvate as substrate. Thus brief physiological elevations of [Ca2+]i depolarize DELTA-psi(m), probably through Ca2+ cycling across the mitochondrial inner membrane. The changes in autofluorescence are consistent with (i) increased respiration which could result from the depolarization of DELTA-psi(m), followed rapidly by (ii) increased activity of the Ca2+-dependent intramitochondrial enzymes. Changes in [Ca2+]i within a physiological range may thus promote significant and long-lasting changes in mitochondrial energy production.
