A H-1 SPIN-ECHO AND V-51 NMR-STUDY OF THE INTERACTION OF VANADATE WITH INTACT ERYTHROCYTES

The action of vanadate on intact human erythrocytes was studied by H-1 spin echo and V-51 NMR spectroscopy as a model for the behaviour of v anadium(V) complexes in experimental diabetes. Vanadate is reduced by the intact erythrocyte at the expense of intracellular glutathione whi ch rapidly depletes from the intracellular volume. Using the blocking agent 4,4'-diisothio-cyanatostilbene-2,2'-disulfonic acid (DIDS), whic h specifically blocks the anion transporter, vanadate reduction could be inhibited and glutathione depletion arrested. Thus, for the reactio n with the intact cell to occur, vanadium(V) must cross the cell wall, possibly via the anion transporter. Nitrofurantoin was used to inhibi t glutathione reductase in the erythrocyte suspensions. Under these co nditions, treatment of the cells with vanadate induced glutathione oxi dation prior to depletion, A study of the reaction of vanadate with ha emolysate indicates that, without the influence of the membrane, rapid oxidation of glutathione to glutathione disulfide by the vanadyl cati on occurs with no glutathione depletion, and that under these conditio ns vanadate reduction is incomplete. This study generates a model for the behaviour of vanadium complexes in vivo, providing a basis for the rational design and synthesis of new vanadium-based agents as insulin mimics. In essence, vanadium is transported across the membrane as va nadate(V), is reduced in situ by glutathione, and becomes complexed to a wide range of intracellular binding sites. Exchange reactions betwe en glutathione and sulfhydryl groups present on haemoglobin and membra ne lead to the depletion of glutathione from the cytosol.