A method is described, based on the differential accumulation of Rb+ and methyltriphenylphosphonium, for the simultaneous estimation of the membrane potentials across the plasma membrane of isolated nerve endings (synaptosomes), and across the inner membrane of mitochondria within the synaptosomal cytoplasm. These determinations, together with measurements of respiratory rates, and ATP and phosphocreatine concentrations, are used to define the bioenergetic behaviour of isolated synaptosomes under a variety of conditions. Under control conditions, in the presence of glucose, the plasma and mitochondrial membrane potentials are respectively 45 and 148mV. Addition of a proton translocator induces a 5-fold increase in respiration, and abolishes the mitochondrial membrane potential. The addition of rotenone to inhibit respiration does not affect the plasma membrane potential, and only lowers the mitochondrial membrane potential to 128mV. Evidence is presented that ATP synthesis by anaerobic glycolysis is sufficient under these conditions to maintain ATP-dependent processes, including the reversal of the mitochondrial ATP synthetase. Addition of oligomycin under non-respiring conditions leads to a complete collapse of the mitochondrial potential. Even under control conditions the plasma membrane (Na+ + K+)-dependent ATPase is responsible for a significant proportion of the synaptosomal ATP turnover. Veratridine greatly increases respiration, and depolarizes the plasma membrane, but only slightly lowers the mitochondrial membrane potential. High K+ and ouabain also lower the plasma membrane potential without decreasing the mitochondrial membrane potential. In non-respiring synaptosomes, anaerobic glycolysis is incapable of maintaining cytosolic ATP during the increased turnover induced by veratridine, and the mitochondrial membrane potential collapses. It is concluded that the internal mitochondria must be considered in any study of synaptosomal transport.

Possible mechanisms are evaluated for the acute regulation of the hamster brown-fat mitochondrial protonconductance pathway which is active during non-shivering thermogenesis. Isolated mitochondria are incubated under conditions designed to approximate to the non-thermogenic state, and the effect of the steady infusion of fatty acids or acyl derivatives upon respiration, membrane potential and membrane proton conductance is monitored continuously. Fatty acids increase the proton conductance with no detectable threshold concentration, allowing the generated acyl carnitine to be rapidly oxidized. The extent of depolarization and of respiratory increase is a function of the rate of infusion. Immediately infusion is terminated the conductance decreases, the mitochondria repolarize and respiration returns to the initial rate. Infusion of acyl-CoA and acylcarnitine cause only a slight depolarization or respiratory increase after high concentrations of these derivatives have accumulated. Any factor which decreases the rate of conversion of fatty acid to acyl-CoA potentiates the conductance increase. An effect of acyl-CoA upon chloride permeability is not specific to brown-fat mitochondria. Fatty acids infused into rat liver mitochondrial incubations produced a small conductance increase, comparable to that of acyl-CoA or acylcarnitine. It is concluded that fatty acids are the most plausible acute regulators of the proton conductance. The relation to the brown-fat-specific 32000-M, protein is discussed.

The. effect of a-latrotoxin from black widow. spider venom upon guinea pig cerebral cortical synaptosomes is described. Plasma membranepotential t(A.p), in situ mitochondrial membrane potential (Aqkm) (1-4, 11, 12); moreover, it releases acetylcholine, noradrenaline, dopamine, and y-aminobutyrate from cerebral cortical slices (5-7), synaptosomes (8, 10, 13),. and one neurosecretory cell line (9). A small number ofhigh-affinity binding sites for the toxin are located specifically on the presynaptic plasma. membrane (6, 10), and occupancy of these. correlates well with the subsequent release of neurotransmitter (10). In addition, the toxin is able to create stable conductance channels in protein-free black lipid membranes (14).A number of aspects of a-latrotoxin action are -unclear. In particular, there is no consensus as to the role ofCa2+. Whereas .there is agreement that the release of acetylcholine and yaminobutyrate is independent of external Ca2+ (3, 4, 7, 12, 13), the Ca2e dependency of catecholamine release appears dependent upon the experimental conditions and target membrane (5,(7)(8)(9). In addition, there is a contradiction between the results obtained with neurosecretory PC.12 cells, in which a. primary effect ofthe toxin appears to be a massive.uptake of Ca2+ across the plasma membrane (9), and those obtained with rat cerebral cortical synaptosomes, in which.no effect on Ca2+ flux could be detected (13).The possibility of a-latrotoxin-induced massive Ca2+ uptake across the presynaptic membrane raises a number of unanswered questions about the effects of the toxin on the energy levels in the cell. In particular, it is unclear whether the intraterminal mitochondria would be able to cope with this Ca2+ influx. Indeed swollen and disorganized mitochondria can be observed in electron micrographs of presynaptic expansions both at neuromuscular junctions (1-4, 11) and in cortical slices (7) treated with a-latrotoxin in the presence of external Ca2 . Deenergization of the mitochondria resulting in a collapse of the mitochondrial membrane potential (Aqi(m) would not only prevent mitochondrial synthesis ofATP but also would destroy the ability of the mitochondria to regulate the upper limit of the cytosolic free Ca2+ concentration (for reviews, see refs. 15-17), with a resultant risk of cell death (18).Recent developments in the study of isolated synaptosomes have enabled Ca2+ transport, Aim, and the plasma membrane potential (Ai/5p) to be determined simultaneously (15,(18)(19)(20)(21).In this paper we use these techniques .and introduce the application. of.the tetraphenylphosphonium (Ph4P+)-selective electrode (22) for the monitoring of synaptosomal potentials in suspension. EXPERIMENTAL PROCEDURESSynaptosomes.' Synaptosomes were prepared from the cerebral cortices (including corpus striatum) of Duncan-Hartley strain guinea pigs age 4-8 wk as described (19)(20)(21)(22). The synaptosomes were stored as a pellet in 250 mM suqrose/5 mM 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethane sulfonate (T...

Mitochondria from guinea-pig cerebral cortex incubated in the presence of Pi or acetate are unable to regulate the extramitochondrial free Ca2+ at a steady-state which is independent of the Ca2+ accumulated in the matrix. This is due to the superimposition on kinetically regulated Ca2+ cycling of a membrane-potential-dependent reversal of the Ca2+ uniporter. The latter efflux is a consequence of a low membrane potential, which correlates with a loss of adenine nucleotide loss from the matrix, enable the mitochondria to maintain a high membrane potential and allow the mitochondria to buffer the extramitochondrial free Ca2+ precisely when up to 200 nmol of Ca2+/mg of protein is accumulated in the matrix. The steady-state extramitochondrial free Ca2+ is maintained as low as 0.3 microM. The Na+-activated efflux pathway is functional in the presence of ATP and oligomycin and accounts precisely for the change in steady-state free Ca2+ induced by Na+ addition. The need to distinguish carefully between kinetic and membrane-potential-dependent efflux pathways is emphasized and the competence of brain mitochondria to regulate cytosolic free Ca2+ concentrations in vivo is discussed.