Effects of Glycosylation on RBC Ca2+ Pump in Diabetes

描述(由申请人提供)：细胞内钙稳态对生理功能至关重要，需要一个功能正常的质膜钙泵(PMCA)。一些报告描述了在体外或体内(糖尿病患者)暴露于高葡萄糖浓度下的红细胞(RBC)PMCA的抑制，这与纯化的PMCA上赖氨酸残基的糖基化有关。我们发现PMCA Vmax在正常红细胞中广泛分布，少数细胞的PMCA Vmax非常低。在进一步的初步实验中，我们发现(I)正常红细胞糖化Hb(%Hb A1c)与PMCA Vmax呈负相关，提示泵Vmax的下降与年龄有关(Ii)我们在镰刀形红细胞和正常红细胞中发现的高Na+、低K*RBC(“CVres”RBC：当K-渗透钾被[Ca~(2+)+A23187]或瓦林霉素)时，也有高%Hb A1c，提示它们代表衰老的RBC；(Iii)CVRES红细胞表现出膜磷脂不对称性的丧失(PS暴露)，糖尿病患者的红细胞中也存在这种现象，与血管内皮细胞的黏附有关。基于这些结果，我们提出了以下研究：(1)通过测试控制不佳的2型糖尿病患者红细胞中Hb A1c的增加是否与Vmax降低和%Hb A1c增加相关，以及这是否通过改善血糖控制来归一化，来检验RBC PMCA糖基化是导致其Vmax降低的原因，而不是简单地反映细胞年龄；(2)糖尿病患者RBC运输蛋白糖基化增加是否也与CVRES RBC数量增加相关，从而解释他们寿命缩短；(3)糖尿病红细胞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细胞的过程有关。如果Hb糖基化增加和Vmax降低之间的相关性得到证实，未来的研究将测试从低Vmax红细胞纯化的PMCA是否显示相对于从高Vmax红细胞部分纯化的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.