Johnson R G, Carty S E, Hayflick S, Scarpa A
Biochem Pharmacol. 1982 Mar 1;31(5):815-23. doi: 10.1016/0006-2952(82)90468-3.
The effects of the transmembrane pH gradient (delta pH) and the transmembrane potential gradient (delta psi) 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 delta psi of 80 mV, positive inside, as measured by [14C] methylamine distribution. When tyramine, 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 microM) 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 delta psi produced a stimulation of amine uptake in the order: serotonin greater than isoproterenol greater than tyramine; metaraminol accumulation was not enhanced by ATP addition. The relationship between the electrochemical proton gradient (delta micro H+) and the electrochemical gradient for each of the sympathomimetic amines (delta micro A) was investigated utilizing chromaffin ghosts devoid of endogenous matrix gradients or components. All amines were accumulated in the presence of delta pH alone. In the presence of delta psi alone, [14C] serotonin, (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 delta pH alone. Tyramine uptake proceeded by both mechanisms. The implications of the mechanism of accumulation of these potent physiologic and pharmacologic agents for their in vivo action are discussed.
利用等渗蔗糖中分离出的牛肾上腺嗜铬颗粒，研究了跨膜pH梯度（δpH）和跨膜电位梯度（δψ）对几种拟交感神经胺摄取的影响。如先前所述[R. 约翰逊和A. 斯卡尔帕，《生物化学杂志》254, 3750 (1979)]，通过[14C]甲胺分布测量，新鲜分离的嗜铬颗粒维持颗粒内pH为5.5，并且在ATP存在下，通过[14C]甲胺分布测量，产生80 mV的δψ，内部为正。当将酪胺、间羟胺和异丙肾上腺素（1 - 50 mM）添加到pH 7.0的颗粒缓冲良好的悬浮液中时，观察到颗粒内部呈剂量相关的碱化。对用放射化学标记（5 - 21 μM）的相同胺的时间分辨流入的研究表明，所有胺都逆着明显的浓度梯度积累。然而，虽然[14C]血清素和[3H]异丙肾上腺素的积累被利血平完全抑制，但[14C]酪胺的积累仅被抑制60%，[14C]间羟胺的摄取不受影响。ATP依赖性的δψ产生以血清素＞异丙肾上腺素＞酪胺的顺序刺激胺摄取；添加ATP并未增强间羟胺的积累。利用缺乏内源性基质梯度或成分的嗜铬颗粒空壳，研究了电化学质子梯度（δμH+）与每种拟交感神经胺的电化学梯度（δμA）之间的关系。仅在存在δpH时，所有胺都积累。仅在存在δψ时，[14C]血清素、[14C]酪胺和[3H]异丙肾上腺素积累，但未显示出[3H]间羟胺的摄取。结果表明，血清素和异丙肾上腺素通过利血平敏感机制在分离的嗜铬颗粒和颗粒空壳中积累，由电化学质子梯度的大小驱动。相反，间羟胺通过非极性脂质相渗透嗜铬颗粒膜，仅根据δpH分布。酪胺的摄取通过两种机制进行。讨论了这些强效生理和药理剂的积累机制对其体内作用的影响。
