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(Circulation Research. 1997;80:179-188.)
© 1997 American Heart Association, Inc.

Articles

Role of Ca²⁺ in Metabolic Inhibition–Induced Norepinephrine Release in Rat Brain Synaptosomes

Xiao-Jun Du, Alex Bobik, Peter J. Little, Murray D. Esler, Anthony M. Dart

Alfred and Baker Medical Unit, Baker Medical Research Institute and Alfred Hospital, Melbourne, Victoria, Australia.

Correspondence to Dr X.-J. Du, Alfred and Baker Medical Unit, Baker Medical Research Institute, Commercial Road, Prahran, Victoria 3181, Australia. E-mail xiao jun du@baker.edu.au

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 [Ca²⁺]_i under energy-depleted conditions, such release of NE in the heart appears to be predominantly Ca²⁺ independent. Since Ca²⁺ overload occurs in ischemia or energy depletion and since a rise in [Ca²⁺]_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 [Ca²⁺]_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 [Ca²⁺]_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 [Ca²⁺]_i, was also induced in energy-depleted synaptosomes by Ca²⁺ repletion after incubation with the Ca²⁺-free medium. Effects on NE release of various interventions to prevent Ca²⁺ overload were tested. Omission of Ca²⁺ from the incubation medium or loading synaptosomes with the Ca²⁺ chelator BAPTA-AM (20 and 100 µmol/L) prevented NE release, indicating a Ca²⁺-dependent mechanism. Inhibition of Ca²⁺ channels with -conotoxin, cadmium, or nifedipine had no effect on NE release during energy depletion. In contrast, nickel and 3,4-dichlorobenzamil, Na⁺-Ca²⁺ exchange inhibitors, dose-dependently inhibited NE release. In conclusion, this study provides evidence that under energy-depleted conditions, Ca²⁺ 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⁺-Ca²⁺ exchange may be the key pathway leading to intraneuronal Ca²⁺ overload.

Key Words: norepinephrine • metabolic inhibition • intracellular Ca²⁺ • synaptosome • Na⁺-Ca²⁺ exchange

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