Effects of Glycosylation on RBC Ca2+ Pump in Diabetes

描述（由申请人提供）：细胞内Ca 2+稳态对生理功能至关重要，需要正常功能的质膜Ca 2+泵（PMCA）。几份报告描述了体外或体内（糖尿病患者）暴露于高葡萄糖浓度的红细胞（RBC）中PMCA的抑制，与纯化PMCA上赖氨酸残基的糖化相关。我们发现PMCA Vmax在正常RBC中分布广泛，少数细胞具有非常低的PMCA Vmax。在另外的初步实验中，我们发现（i）正常RBC中糖化Hb（%Hb A1 c）和PMCA Vmax之间呈负相关，这表明泵Vmax的下降与年龄有关（ii）我们在镰状RBC中发现的高Na+、低K * RBC的分数以及正常RBC中的少量（“CVres”RBC：当被[Ca 2 + + A23187]或缬氨霉素K-透化时抗脱水），也具有高的%Hb A1 c，表明它们代表衰老的RBC;和（iii）CVres RBC显示膜磷脂不对称性的丧失（PS暴露），这也在糖尿病患者的RBC中发现，与对血管内皮的粘附相关。基于这些结果，我们提出了以下研究：为了检验（1）红细胞PMCA的糖化是降低其Vmax的原因，而不是简单地反映细胞年龄的假设，通过检验来自控制不佳的2型糖尿病患者的红细胞中Hb A1 c的增加是否与具有降低的Vmax和较高的%Hb A1 c的红细胞分数增加相关，以及这是否通过改善血糖控制而正常化;（2）糖尿病患者RBC转运蛋白的糖基化增加是否也与CVres RBC数量增加相关，从而解释其寿命缩短;和（3）糖尿病RBC中RBC膜磷脂不对称性的丧失（PS暴露）是否与糖基化产生低PMCA Vmax和CVres细胞的过程相关。如果Hb糖基化增加与Vmax降低之间的相关性得到证实，未来的研究将测试从低Vmax RBC中纯化的PMCA是否显示相对于从高Vmax RBC组分中纯化的PMCA，赖氨酸残基的糖基化增加。

DESCRIPTION (provided by applicant): Intracellular Ca2+ homeostasis, crucial for physiological functions, requires a normal functioning plasma membrane Ca2+ pump (PMCA). Several reports described inhibition of the PMCA in red cells (RBCs) exposed to high glucose concentrations in vitro, or in vivo (in diabetics), correlated with glycation of lysine residues on the purified PMCA. We discovered a broad distribution of PMCA Vmax in normal RBCs, with a minority of cells having a very reduced PMCA Vmax. In additional preliminary experiments we found (i) an inverse correlation between glycated Hb (%Hb A1c) and PMCA Vmax in normal RBCs, suggesting that the decline in pump Vmax was age-related (ii) that the fraction of high- Na+, low-K* RBCs which we discovered in sickle RBCs and in small amounts among normal RBCs ("CVres" RBCs: resistant to dehydration when K-permeabilized by [Ca2+ + A23187] or Valinomycin), also had a high %Hb A1c, suggesting that they represent senescent RBCs; and (iii) that CVres RBCs show loss of membrane phospholipid asymmetry (PS-exposure), found also in diabetics' RBCs, associated with adherence to vascular endothelia. Based on these results we propose the following studies: To test (1) the hypothesis that glycation of the RBC PMCA is causative in lowering its Vmax, rather than simply reflecting cell age, by testing whether the increase in Hb A1c in RBCs from poorly controlled Type 2 diabetics correlates with increased fractions of RBCs with reduced Vmax and higher %Hb A1c, and if this is normalized by improved glycemic control; (2) whether increased glycation of diabetics' RBC transport proteins also correlates with increased numbers of CVres RBCs thus explaining their reduced life-span; and (3) whether the loss of RBC membrane phospholipid asymmetry (PS-exposure) in diabetic RBCs is related to the processes by which glycation generates low PMCA Vmax and CVres cells. If the correlation between increased Hb glycation and Vmax reduction is confirmed, future studies would test whether the PMCA purified from Iow-Vmax RBCs shows increased glycation of the lysine residues relative to that purified from high-Vmax RBC fractions.