Limitation of glycolysis by hexokinase in rat brain synaptosomes during intense ion pumping

Incubation of rat brain synaptosomes under conditions of either increased energy utilization (addition of Na+ channel opener, veratridine, or ionophores, monensin and nigericin) or inhibition of oxidative phosphorylation (addition of rotenone), or a combination thereof, decreased [ATP], increased [ADP] and stimulated glycolysis. The rates of lactate generation were linear over a 15-min interval in the presence of rotenone alone but decreased in the other two conditions. During the first 5 min, the amount of lactate formed with veratridine, monensin or nigericin was as high or higher than with rotenone, but it was lower in the last 10 min. With a combination of one of the stimulators of ion movements and rotenone the rate of glycolysis was always markedly lower than with each compound added singly. The stimulated rates of lactate formation correlated positively with the synaptosomal content of [ATP]. After 15 min, [ATP] was 0.9-1.0 nmol/mg with rotenone, 0.5-0.9 nmol/mg with veratridine (or ionophores), and <0.3 nmol/mg with a combination of the two. Under the conditions used, calcium did not affect glycolytic activity directly. The Lineweaver-Burk plot of the rate of lactate formation against [ATP] yielded a straight line with a Km for ATP of about 0.1 mM, which is very similar to the Km for this nucleotide of brain hexokinase bound to mitochondria. In C6 cells glycolytic rate measured with a combination of an ionophore and rotenone was higher than with each of these compounds added singly while [ATP] never declined below about 9 nmol/mg prot. It is concluded that in synaptosomes, the high rate of energy utilization required for intense ion movement decreases [ATP] to a level that limits hexokinase activity kinetically. This may contribute to a reduction in the rate of glycolysis and hence energy production in brain hypoxia and ischemia.