Role of Ca 2+ in Metabolic Inhibition–Induced Norepinephrine Release in Rat Brain Synaptosomes

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Abstract
Ischemia and simulated ischemic conditions induce enhanced release of norepinephrine (NE) in the brain and the heart. Although studies with neuronal preparations demonstrated a rise in [Ca 2+ ] i under energy-depleted conditions, such release of NE in the heart appears to be predominantly Ca 2+ independent. Since Ca 2+ overload occurs in ischemia or energy depletion and since a rise in [Ca 2+ ] i triggers exocytosis without membrane depolarization, we tested the possibility, using brain synaptosomes, that increased NE release could be, at least in part, a consequence of raised [Ca 2+ ] i . Brain synaptosomes were incubated with Krebs-Henseleit medium, and ischemia was mimicked by treatment with metabolic inhibitors. NE content in incubation medium (supernatant) and synaptosomes was analyzed chromatographically. Treatment with metabolic inhibitors reduced ATP content by 75% and increased [Ca 2+ ] i by more than fourfold within minutes. Metabolic inhibition elicited NE release, which started within 10 minutes and reached a maximum after 30 minutes, with a corresponding 55% reduction in synaptosomal NE content after 40 minutes. NE release, together with a marked increase in [Ca 2+ ] i , was also induced in energy-depleted synaptosomes by Ca 2+ repletion after incubation with the Ca 2+ -free medium. Effects on NE release of various interventions to prevent Ca 2+ overload were tested. Omission of Ca 2+ from the incubation medium or loading synaptosomes with the Ca 2+ chelator BAPTA-AM (20 and 100 μmol/L) prevented NE release, indicating a Ca 2+ -dependent mechanism. Inhibition of Ca 2+ channels with ω-conotoxin, cadmium, or nifedipine had no effect on NE release during energy depletion. In contrast, nickel and 3,4-dichlorobenzamil, Na + -Ca 2+ exchange inhibitors, dose-dependently inhibited NE release. In conclusion, this study provides evidence that under energy-depleted conditions, Ca 2+ overload in synaptosomes of noradrenergic neurons from the brain is an important mechanism for the enhanced release of NE and that a reversal of Na + -Ca 2+ exchange may be the key pathway leading to intraneuronal Ca 2+ overload.