Mechanisms of accumulation of tyramine, metaraminol, and isoproterenol in isolated chromaffin granules and ghosts

The effects of the transmembrane pH gradient (ΔpH) and the transmembrane potential gradient (ΔΨ) on the uptake of several sympathomimetic amines were investigated, using bovine adrenal chromaffin granules isolated in isotonic sucrose. As previously described [R. Johnson and A. Scarpa, J. biol. Chem. 254, 3750 (1979)], freshly isolated chromaffin granules maintain an intragranular pH of 5.5 as measured by [¹⁴C]methylamine distribution and, in the presence of ATP, generate a ΔΨ of 80 mV, positive inside, as measured by [¹⁴C]thiocyanate distribution. When tryamine, metaraminol, and isoproterenol (1-50 mM) were added to well-buffered suspensions of granules at pH 7.0, a dose-related alkalinization of the granule interior was observed. Study of the time-resolved influx of the same amines labeled radiochemically (5-21 μM) revealed that all the amines were accumulated against an apparent concentration gradient. However, while accumulation of [¹⁴C]serotonin and [³H]isoproterenol was totally inhibited by reserpine, [¹⁴C]tryramine accumulation was inhibited by only 60% and [¹⁴C]metaraminol uptake was unaffected. The ATP-dependent generation of a ΔΨ produced a stimulation of amine uptake in the order: serotonin > isoproterenol > tyramine; metaraminol accumulation was not enhanced by ATP addition. The relationship between the electrochemical proton gradient (Δ \ ̄gm_H+) and the electrochemical gradient for each of the sympathomimetic amines (Δ \ ̄gm_A) was investigated utilizing chromaffin ghosts devoid of endogenous matrix gradients or components. All amines were accumulated in the presence of ΔpH alone. In the presence of ΔΨ alone, [¹⁴serotonin, [¹⁴C]tyramine, and [³H]isoproterenol were accumulated, but no [³H]metaraminol uptake was demonstrable. The results indicate that serotonin and isoproterenol accumulated in isolated chromaffin granules and ghosts via a reserpine-sensitive mechanism, driven by the magnitude of the electrochemical proton gradient. Conversely, metaraminol permeated the membrane of the chromaffin granule through the apolar lipid phase and distributed according to the ΔpH alone. Tyramine uptake proceeded by both mechanisms. The implications of the mechanism of accumulation of these potent physiologic and pharmacologie agents for their in vivo action are discussed.