Forward operation of adenine nucleotide translocase during F0F1-ATPase reversal: critical role of matrix substrate-level phosphorylation.

In pathological conditions, F(0)F(1)-ATPase hydrolyzes ATP in an attempt to maintain mitochondrial membrane potential. Using thermodynamic assumptions and computer modeling, we established that mitochondrial membrane potential can be more negative than the reversal potential of the adenine nucleotide translocase (ANT) but more positive than that of the F(0)F(1)-ATPase. Experiments on isolated mitochondria demonstrated that, when the electron transport chain is compromised, the F(0)F(1)-ATPase reverses, and the membrane potential is maintained as long as matrix substrate-level phosphorylation is functional, without a concomitant reversal of the ANT. Consistently, no cytosolic ATP consumption was observed using plasmalemmal K(ATP) channels as cytosolic ATP biosensors in cultured neurons, in which their in situ mitochondria were compromised by respiratory chain inhibitors. This finding was further corroborated by quantitative measurements of mitochondrial membrane potential, oxygen consumption, and extracellular acidification rates, indicating nonreversal of ANT of compromised in situ neuronal and astrocytic mitochondria; and by bioluminescence ATP measurements in COS-7 cells transfected with cytosolic- or nuclear-targeted luciferases and treated with mitochondrial respiratory chain inhibitors in the presence of glycolytic plus mitochondrial vs. only mitochondrial substrates. Our findings imply the possibility of a rescue mechanism that is protecting against cytosolic/nuclear ATP depletion under pathological conditions involving impaired respiration. This mechanism comes into play when mitochondria respire on substrates that support matrix substrate-level phosphorylation.

A novel kinetic assay of mitochondrial ATP-ADP exchange rate mediated by the ANT.

A novel method exploiting the differential affinity of ADP and ATP to Mg(2+) was developed to measure mitochondrial ADP-ATP exchange rate. The rate of ATP appearing in the medium after addition of ADP to energized mitochondria, is calculated from the measured rate of change in free extramitochondrial [Mg(2+)] reported by the membrane-impermeable 5K(+) salt of the Mg(2+)-sensitive fluorescent indicator, Magnesium Green, using standard binding equations. The assay is designed such that the adenine nucleotide translocase (ANT) is the sole mediator of changes in [Mg(2+)] in the extramitochondrial volume, as a result of ADP-ATP exchange. We also provide data on the dependence of ATP efflux rate within the 6.8-7.8 matrix pH range as a function of membrane potential. Finally, by comparing the ATP-ADP steady-state exchange rate to the amount of the ANT in rat brain synaptic, brain nonsynaptic, heart and liver mitochondria, we provide molecular turnover numbers for the known ANT isotypes.

Identification of protease-activated receptor-4 (PAR-4) in puromycin-purified brain capillary endothelial cells cultured on Matrigel.

An improved method is described for culturing primary rat brain capillary endothelial cells (RBCEC) on glass, covered by Matrigel. The procedure using Matrigel yields spindle-shaped endothelial cells exhibiting close cell-cell appositions seen on electron microscopic sections. These cells permanently express tight junction proteins ZO-1, claudin-5 and the adherent junction protein beta-catenin, as revealed by immunofluorescence. Furthermore, glass coverslips covered with Matrigel provide a stable and low-background fluorescent base for microfluorimetric calcium measurements. By this method, hereby we show that the PAR-4 agonist peptide induces transient [Ca2+]i changes with different kinetics compared to that due to activation of the PAR-1 receptor. This indicates that RBCE cells grown on Matrigel express PAR-4 receptors.