Relationships Among ATP Synthesis, K⁺ Gradients, and Neurotransmitter Amino Acid Levels in Isolated Rat Brain Synaptosomes

Abstract

Correlations were made among ATP synthesis, transmembrane K+ gradients, and leakage of three amino acid neurotransmitters, gamma-aminobutyric acid (GABA), aspartate, and glutamate, in rat brain synaptosomes incubated under normoxic and respiration-limited conditions. Even under normoxic conditions, a substantial proportion of total ATP synthesis (8%) was provided by glycolysis. Limitation of respiration by approximately 30% through addition of amobarbital (Amytal) caused a twofold decrease in the creatine phosphate/creatine ([CrP]/[Cr]) ratio, and consequently the [ATP]/[ADP] ratio, and a threefold increase in lactate production. There was a detectable decrease in intracellular [K+] and small rises in external GABA, aspartate, and glutamate concentrations. More severe limitations in ATP synthesis caused larger declines in the [CrP]/[Cr] ratio and progressive leakage of K+ and neurotransmitter amino acids. A comparison of delta GATP and delta GNa, K showed the former to be larger by 6 kcal, which indicates that the plasma membrane Na+/K+ pump operates at far from equilibrium. Under respiration-limited conditions, even when total ATP synthesis decreased by approximately 80% and [ATP] declined to less than 0.4 mM, delta GATP was still larger than delta GNa,K. It is suggested that during hypoxia and ischemia, the activity of the plasma membrane Na+/K+ pump in brain becomes limited by [ATP], which falls below the Km value for the low-affinity regulatory site on the enzyme. This failure of the pump and consequent collapse of the ion gradients may contribute to the leakage of neurotransmitter amino acids that occurs in these pathological states.

Relationships between the neuronal sodium/potassium pump and energy metabolism. Effects of K+, Na+, and adenosine triphosphate in isolated brain synaptosomes.

“…Fig. 6 shows, in agreement with our earlier studies (Dagani and Erecifiska, 1987), that Amytal at 1 mM inhibited oxygen uptake by -30% and at 2 mM by ~70%. Over 80% blockage was obtained with 10 ~tM rotenone.…”

“…Aliquots were withdrawn and used either for the measurement of O~ uptake or rapidly quenched by addition of cold perchloric acid (0.6 M final concentration). Oxygen uptake was assayed with a Clark-type electrode in a stirred chamber thermostated at 30 + I*C. The perchloric acid extracts were neutralized with 2.5 M KHCOs and centrifuged, and the clear supernates were used for the determination of metabolites by standard enzymatic techniques (Bergmeyer, 1974;and Dagani and Erecifiska, 1987 for details).…”

Secobarbital Attenuates Excitotoxicity but Potentiates Oxygen—Glucose Deprivation Neuronal Injury in Cortical Cell Culture

“…Despite other presumably favorable effects on metabolic rate, Oberpichler et al (1990) observed that meth ohexital failed to ameliorate the loss of ATP in chick cerebral cultures exposed to cyanide, and Duffy et al (1972) found no significant effect of pen tobarbital on ATP levels in mouse brains exposed to hypoxia. Furthermore, Dagani and Erecinska (1987) observed that amy tal intensified A TP deple tion in glucose-deprived synaptosomes.…”

Control of respiration and ATP synthesis in mammalian mitochondria and cells

“…The isolated Na+/K+-ATPase is relatively insensitive to ATP, but the apparent sensitivity measured in cells is higher, with activity roughly proportional to ATP concentration in rat kidney proximal tubules (Soltoff& Mandel, 1984;Tessitore et al, 1986), and cultured HeLa cells (Ikehara et al, 1984), and a greater sensitivity in brain synaptosomes (Dagani & Erecinska, 1987;Erecinska & Dagani, 1990 (Soltoff, 1986).…”