Regulation of norepinephrine uptake by adenine nucleotides and divalent cations: role for extracellular protein phosphorylation.
This study examined the hypothesis that ATP, released together with norepinephrine (NE) from brain noradrenergic nerve terminals, may serve as a cosubstrate for an extracellular protein phosphorylation system that regulates the reuptake of the transmitter, NE. The possible regulation of high-affinity uptake (uptake 1) of [3H]NE by divalent cations and ATP, both of which are involved in protein phosphorylation, was examined in rat cerebral cortical synaptosomes. A marked inhibition of uptake 1 by 5'-adenylylimidodiphosphate [App(NH)p], a nonhydrolyzable, competitive antagonist of ATP, was observed. A similar inhibition of uptake was observed when Ca2+ and Mg2+ were both omitted from the incubation medium. App(NH)p distinguished the actions of Ca2+ from those of Mg2+: Ca2+-stimulated uptake 1 was blocked by App(NH)p; Mg2+-stimulated uptake was not. In parallel experiments, the patterns of protein phosphorylation in crude and purified preparations of synaptosomes were examined under conditions similar to those used in uptake assays. A striking correlation was found between the inhibition of uptake 1, by either App(NH)p or Ca-omission, and inhibition of the phosphorylation of one specific, 39,000-dalton, Ca2+-dependent, protein component in synaptosomes. This 39K protein was distinct from the alpha subunit of pyruvate dehydrogenase, a mitochondrial protein of similar electrophoretic mobility. These findings are consistent with the possibility that an ectokinase on synaptosomes utilizes extracellular ATP and Ca2+ in phosphorylating a protein(s) associated with the regulation of NE uptake.
Hendley
E D
ED
Department of Physiology and Biophysics, University of Vermont, College of Medicine, Burlington 05405.
Whittemore
S R
SR
Chaffee
J E
JE
Ehrlich
Y H
YH
D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors / Q000502:physiology*; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology; D002413:Cations, Divalent; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism*; D003999:Dichloroacetic Acid / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008297:Male; D009638:Norepinephrine / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains; D012964:Sodium / Q000494:pharmacology; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism
D002147:Calmodulin; D002413:Cations, Divalent; D010750:Phosphoproteins; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D003999:Dichloroacetic Acid; D012964:Sodium; D008274:Magnesium; D002118:Calcium; D009638:Norepinephrine