A kinetic study of the ouabain-induced efflux of norepinephrine from the dog saphenous vein

Dog saphenous vein strips were incubated with 3H]norepinephrine (3H]NE), 1.4 microM, after inhibition of the NE-metabolizing enzymes and extraneuronal uptake, and superfused for up to 290 min. From the 70th min onwards the strips were exposed to 10 microM ouabain, some of them being subject to electrical stimulation from the 140th min onwards. Other strips were exposed to either 1, 10 or 100 microM ouabain from the 70th min onwards. The spontaneous efflux of 3H]NE had a long half-time (156 min), and over 90% of the 3H]NE accumulated did not participate in efflux ("bound fraction"). Ouabain, 10 microM, induced a pronounced increase of the rate of efflux of 3H]NE, which was delayed in its onset and reached a maximum at t = 135 min of superfusion. Increasing the concentration of ouabain decreased both the delay to the beginning of the overflow and the time to maximum efflux and increased the maximum rate of efflux. In Ca(++)-free medium (during the superfusion period), the maximum rate of efflux was lower than in Ca(++)-containing medium, but was attained earlier. The bound fraction amounted to 22% when the efflux was induced by 10 microM ouabain in Ca(++)-containing medium, a value unnaffected by electrical stimulation but reduced markedly by omitting calcium. The results support the view that the efflux of 3H]NE induced by ouabain is delayed and that it is both carrier-mediated and due to exocytosis.