Physiological roles of low molecular weight 20KG GTP-binding protein : Effects on secretion in pancreatic acinar cells.

小分子GTP结合蛋白的调节蛋白(SmgP25A)，GDI(GDP解离抑制因子)，是一种50 KG的单肽，能抑制SMG介导的生理过程。通过膜片钳玻璃吸管将GDI导入大鼠嗜铬细胞，并监测膜电容的增加。用阶跃电压去极化激活Ca~(2+)~(2+)通道后，膜电容持续增加。在几个这样的试验中，GDI为200 NM(由Takai，Y.教授从牛脑中制备)，在细胞透析的前20分钟，膜电容的增加减少了。然而，GDI的疗效差异很大，在使用GDI制剂的某些批次细胞中，没有观察到任何效果。GDI的纯度约为90%，因此我们不能排除污染磷脂使这些细胞的钙依赖性胞吐减少或膜电容增加的可能性。GDS是SmgP21B(GDS：GDP解离刺激因子)的另一种调节蛋白。GDS可能与A-激酶协同作用，激活smgP21B。在胰腺腺泡细胞中，VIP(血管肠多肽)被认为通过cAMP依赖的过程诱导酶的分泌。因此，预计SMG和/或GDS在VIP介导的胞吐中发挥作用。用含cAMP和GTPYS的膜片钳内透析液刺激胰腺腺泡细胞。在这种刺激下，一些细胞的膜电容增加，但一些细胞没有。可能我们遗漏了另一个辅助因素，因为外加VIP(10 NM)持续诱导膜电容增加。缺失的因子可能与花生四烯酸(AA)的形成有关，我们现在正在研究AA的抑制剂4-BPB对腺泡细胞对内部cAMP和GTPyS反应的影响。

The regulatory protein of small molecular GTP-binding protein(smgP25A), GDI(GDP dissociation inhibitor), is a 50 KG single peptide which inhibits smg mediated physiological processes. GDI is introduced into chromaffin cells of rats through patch-clamp glass pipettes and increases in membrane capacitance was monitored. Activation of Ca^<2+> channels with a step-voltage depolarization consistently induced an increase in membrane capacitance. In several such trials with GDI of 200 nM(prepared from bovine brain by Prof. Takai, Y.), the increases in membrane capacitance were reduced during first 20 min of cell dialysis. However, the efficacy of GDI was quite variable, and in some batches of cells using a GDI preparation, no effects were observed. The purity of GDI was about 90%, so we could not rule oul the possibility that contaminating phospholipid reduced Ca^<2+>-dependent exocytosis or increases in membrane capacitance in these cells. We now try to improve the purity of GDI.GDS is a another regulatory protein of smgP21B(GDS : GDP dissociation stimulator). It is possible that GDS synergistically acts- with A-kinase to activate smgP21B. In pancreatic acinar cells, VIP(vasointestinal polypeptide)is thought to induce enzyme secretion via cAMP-dependent process. Thus, it is expected that smg and/or GDS play roles in VIP mediated exocytosis. We tried to stimulate pancreatic acinar cells using patch-clamp internal dialysis with CAMP and GTPYS containing solution. With this stimulation, some cells showed increases in membrane capacitance but some did not. Probably we are missing an another cofactor since external application of VIP(10 nM)consistently induced an increase in membrane capacitance. The missing factor probably relates the formation of arachidonic acid(AA), and we are now studying the effects of a inhibitor of AA, 4-BPB, on the acinar cell response to internal CAMP and GTPyS.