Epand R M, Stafford A R, Tyers M, Nieboer E
Mol Pharmacol. 1985 Mar;27(3):366-74.
Using model systems, we have studied the properties of a number of zinc-chelating agents which are known to cause diabetes in laboratory animals. The abilities to permeate membranes and to complex zinc inside liposomes with the release of protons are suggested as chemical properties that can enhance diabetogenicity. When such complexing agents are added to lipid vesicles at pH 6 containing entrapped zinc ions, they acidify the contents of these vesicles. We have demonstrated this effect by measuring intravesicular pH both with a fluorine-containing F NMR probe as well as with the fluorescent probe, quinine. For example, using quinine, we observed that 0.1 mM 8-hydroxyquinoline reduced the intravesicular pH of sonicated phospholipid vesicles containing entrapped Zn2+ (as sulfate) from pH 6.0 to 2.8. These diabetogenic chelating agents also solubilized zinc-insulin precipitates from unbuffered suspensions at pH 6.0. The solubilization results from the acidification of these suspensions. Dithizone and 8-hydroxyquinoline at 4 mM solubilized 97 and 42%, respectively, of the suspended insulin. We suggest that if such proton release occurs within the zinc-containing insulin storage granules of pancreatic beta-cells, solubilization of insulin would be induced. Such an event would lead to osmotic stress and eventually to rupture of the granule. The effects of diethyldithiocarbamate (DDC), an agent that has been found to protect rabbits against the induction of diabetes by some other zinc-chelating agents, were also studied. DDC caused a decrease of 3.5 units in the intravesicular pH of zinc-containing vesicles by a mechanism not involving the release of protons upon chelation of zinc. We have demonstrated several properties of DDC which may contribute to its ability to protect against the induction of diabetes. These include its ability to store zinc as a hydrophobic complex in membranes, its consumption of protons upon spontaneous decomposition, and the ability of one of its decomposition products, diethylamine, to accelerate the dissipation of pH gradients across lipid bilayers. Diethylamine is particularly effective in stimulating a rapid dissipation of such pH gradients, even at micromolar concentrations. We have attempted to estimate quantitatively the extent of proton liberation by various zinc-chelating agents. This analysis demonstrated that partitioning of the ligand between organic and aqueous phases, ligand acidity, and zinc complex stability determine the extent of proton release.
利用模型系统，我们研究了多种已知会在实验动物中引发糖尿病的锌螯合剂的特性。膜渗透能力以及在脂质体内与锌络合并释放质子的能力被认为是能够增强致糖尿病性的化学性质。当将此类络合剂添加到含有包封锌离子的pH 6脂质囊泡中时，它们会使这些囊泡的内容物酸化。我们通过使用含氟的F NMR探针以及荧光探针奎宁来测量囊泡内pH值，证实了这种效应。例如，使用奎宁时，我们观察到0.1 mM的8-羟基喹啉将含有包封Zn2 +（以硫酸盐形式）的超声处理磷脂囊泡的囊泡内pH值从6.0降低到2.8。这些致糖尿病性螯合剂还能在pH 6.0的未缓冲悬浮液中溶解锌 - 胰岛素沉淀物。溶解是由于这些悬浮液的酸化所致。4 mM的双硫腙和8-羟基喹啉分别溶解了97%和42%的悬浮胰岛素。我们认为，如果这种质子释放在胰腺β细胞含锌的胰岛素储存颗粒内发生，将会诱导胰岛素的溶解。这样的事件将导致渗透压应激并最终导致颗粒破裂。还研究了二乙基二硫代氨基甲酸盐（DDC）的作用，DDC是一种已被发现可保护兔子免受其他一些锌螯合剂诱导糖尿病影响的试剂。DDC通过一种不涉及锌螯合时质子释放的机制使含锌囊泡的囊泡内pH值降低了3.5个单位。我们已经证明了DDC的几种特性，这些特性可能有助于其预防糖尿病诱导的能力。这些特性包括它能够以疏水络合物的形式在膜中储存锌，它在自发分解时消耗质子，以及它的一种分解产物二乙胺能够加速跨脂质双层的pH梯度消散。即使在微摩尔浓度下，二乙胺在刺激这种pH梯度的快速消散方面也特别有效。我们试图定量估计各种锌螯合剂释放质子的程度。该分析表明，配体在有机相和水相之间的分配、配体酸度以及锌络合物稳定性决定了质子释放的程度。
