Michalke W, Langer R, Burger A
Institut für Pharmakologie und Toxikologie, Universität Würzburg, Federal Republic of Germany.
Naunyn Schmiedebergs Arch Pharmacol. 1990 Sep;342(3):312-22. doi: 10.1007/BF00169443.
Unidirectional inward transport into, accumulation by and loss of biogenic amines from ghosts of bovine chromaffin granules were studied to determine whether a carrier-mediated process contributes to the outward passage of amines across the granule membrane. In the presence of ATP-Mg2+, incubated ghosts (30 degrees C; pH 7.3) showed a reserpine-sensitive (IC50 2-5 nmol/l), unidirectional, inward transport of catecholamine (CA = 70% adrenaline/30% noradrenaline), 5-hydroxytryptamine (5-HT) and tyramine with different Km values (mumol/l: tyramine 2; 5-HT 5; CA 8) but with the same Vmax [20 nmol/(mg protein.min)] during the first 3 min of incubation. During longer incubation, the rate of unidirectional inward transport declined rapidly with time to about 30 to 40%, at which level it stayed nearly constant from 50 to 100 min of incubation. As the decline of unidirectional transport was independent of the amine accumulated in the ghosts, it is concluded that it reflects ageing of the membrane vesicles. During incubation for up to 100 min with CA, 5-HT or tyramine (10- to 30-fold Km) in the presence of ATP-Mg2+, the amine content of the ghosts increased, approaching a steady-state content (nmol amine/mg protein: CA 400-500, 5-HT 250, tyramine 60), which was negatively correlated with the lipid solubility of the amine (tyramine greater than 5-HT greater than CA), whereas the rate of approach to steady state (t1/2 in min: CA 20-30, 5-HT 7-10, tyramine less than 5) was positively correlated. Low concentrations of reserpine (greater than or equal to 25 nmol/l) caused net loss of amine from amine-loaded ghosts by inhibition of inward transport. However, reserpine did not reduce the fractional rate of loss (FRL) of CA-loaded ghosts induced by NH+4 or uncoupling agents. Accelerated exchange diffusion was not found to occur at the granule membrane, as addition of high concentrations of 5-HT or dopamine to CA-loaded ghosts did not result in a higher FRL of CA than did blockade of inward transport by reserpine. Analysis of steady-state kinetics revealed the following features of the granule transport. The approach to steady state (t1/2) was independent of the rate of inward transport. The steady-state amine content of ghosts approached a limiting value as the external concentration of the amine was increased; it was determined by the same kinetic constants (Km and the time-dependent Vmax) as found for the rate of carrier-mediated inward transport.(ABSTRACT TRUNCATED AT 400 WORDS)
研究了生物胺向牛嗜铬颗粒膜泡的单向内转运、在其中的积累以及从膜泡中的丧失，以确定载体介导的过程是否有助于胺穿过颗粒膜的外向转运。在ATP-Mg²⁺存在下，孵育的膜泡（30℃；pH 7.3）显示出对利血平敏感（IC₅₀ 2 - 5 nmol/L）的单向儿茶酚胺（CA = 70%肾上腺素/30%去甲肾上腺素）、5-羟色胺（5-HT）和酪胺内转运，不同的Km值（μmol/L：酪胺2；5-HT 5；CA 8），但在孵育的最初3分钟内具有相同的Vmax [20 nmol/(mg蛋白·分钟)]。在更长时间的孵育过程中，单向内转运速率随时间迅速下降至约30%至40%，在此水平上从孵育50至100分钟基本保持恒定。由于单向转运的下降与膜泡中积累的胺无关，得出结论认为这反映了膜泡的老化。在ATP-Mg²⁺存在下，用CA、5-HT或酪胺（Km的10至30倍）孵育长达100分钟，膜泡中的胺含量增加，接近稳态含量（nmol胺/mg蛋白：CA 400 - 500，5-HT 250，酪胺60），这与胺的脂溶性呈负相关（酪胺＞5-HT＞CA），而接近稳态的速率（t₁/₂，分钟：CA 20 - 30，5-HT 7 - 10，酪胺＜5）呈正相关。低浓度的利血平（≥25 nmol/L）通过抑制内转运导致胺从负载胺的膜泡中净丧失。然而，利血平并未降低NH₄⁺或解偶联剂诱导的负载CA膜泡的胺丧失分数率（FRL）。未发现颗粒膜发生加速交换扩散，因为向负载CA的膜泡中添加高浓度的5-HT或多巴胺不会导致CA的FRL高于利血平对内转运的阻断。稳态动力学分析揭示了颗粒转运的以下特征。接近稳态（t₁/₂）与内转运速率无关。随着胺的外部浓度增加，膜泡的稳态胺含量接近一个极限值；它由与载体介导的内转运速率相同的动力学常数（Km和时间依赖性Vmax）决定。（摘要截断于400字）
