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Ionic and nucleotide cofactor requirements for uptake of [3H]norepinephrine by rat brain synaptic vesicle preparations.

Norepinephrine uptake into a crude preparation of rat brain synaptic vesicles showed a marked dependence on Mg 2+ concentration. Mn 2+ or Co + could substitute for Mg 2+ , but displayed lower affinities. Zn 2+ , Ni 2+ and Ca 2+ stimulated uptake only slightly and other divalent cations were ineffective. ATP, GTP and UTP produced stimulation of norepinephrine uptake, but only ATP was fully effective. ADP and AMP inhibited the ATP-induced stimulation. The irreversible inhibitor of ATPases, N-ethylmaleimide (NEM), blocked norepinephrine uptake; the effect was enhanced by pre-incubation of the vesicle preparation with NEM prior to addition of the cofactors and the enhancement was partially prevented by addition of ATP-Mg 2+ during the pre-incubation. Replacement of K + by Na + in the medium did not alter norepinephrine uptake, but Li + inhibited uptake by competing with Mg 2+ . The use of hypertonic medium inhibited uptake, while hypotonic medium markedly enhanced only the nonspecific uptake component (not ATP or Mg 2+ -dependent, not reserpine-sensitive). The similarities of these data to those obtained with adrenomedullary or peripheral nerve vesicles validate the historical use of the peripheral preparations as models of central nervous system vesicles, and the simple and sensitive technique utilized in this study affords direct detailed evaluation of the effects of in vivo drug administration on neurotransmitter storage in the brains of small animals.