Stereospecific (--)-[3H]norepinephrine binding to bovine hypothalamus. Possible identification of the catecholamine uptake site in synaptic vesicles.
A (--)-[3H]norepinephrine binding site was identified in a crude synaptosomal fraction isolated from bovine hypothalamus which bound norepinephrine rapidly, reversibly, and stereospecificially. The results were most consistent with binding of (-)-[H]norepinephrine to the carrier molecule used to translocate biogenic amines into synaptic vesicles. The binding studies indicated that specific binding of (--)-[3H]norepinephrine to the crude synaptosomal fraction was greatly enhanced by 4 mM MgCl2 pand 1 mM ATP. The increased binding of (--)-[3H5norepinephrine also occurred in the presence of MgCl2 and GTP, but AMP, adenosine and adenyl-5'-yl imidodiphosphate would not substitute for ATP. Neither CaCl2 nor ZnSO4 could be substituted for the MgCl2. In the presence of MgCl2 and ATP, the dissociation constant for (--)-[3H]norepinephrine was 280 nM with a specific binding site density of 4.8 pmol/mg protein. Binding was stereospecific with ratios of 15, 4, and 6.5 for the affinities of (--)-isomers to (+)-isomers for norepinephrine, epinephrine and isoproterenol, respectively. Drug competition studies, conducted in the presence of Mg2+ and ATP, indicated that (--)-epinephrine, (--)-norepinephrine, dopamine and serotonin had inhibitory constants ranging from 0.25 to 0.8 micron with (--)-isoproterenol and tyramine having inhibitory constants around 2 micron. Reserpine was the most potent inhibitor having an inhibition constant of 8.6 +/- 0.3 nM. The binding data were not consistent with the specific site being the alpha- or beta-receptors for norepinephrine, the Uptake1 Site for norepinephrine into synaptosomes or the metabolizing enzymes for norepinephrine.
D000227:Adenine Nucleotides / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001679:Biogenic Amines / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D007031:Hypothalamus / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D009638:Norepinephrine / Q000378:metabolism*; D012110:Reserpine / Q000494:pharmacology; D013572:Synaptic Vesicles / Q000378:metabolism*
D000227:Adenine Nucleotides; D001679:Biogenic Amines; D002413:Cations, Divalent; D012110:Reserpine; D000255:Adenosine Triphosphate; D008274:Magnesium; D007545:Isoproterenol; D009638:Norepinephrine