Zhu Jin Xia, Yang Ning, Zhu Hu, Chung Yiu Wa, Chan Hsiao Chang
Epithelial Cell Biology Research Center, Department of Physiology, Faculty of Medicine, Rm 410, Basic Medical Science Building, The Chinese University of Hong Kong, Shatin, Hong Kong, China.
Cell Biol Int. 2007 May;31(5):521-5. doi: 10.1016/j.cellbi.2006.11.021. Epub 2006 Nov 28.
Our previous study demonstrated that NYD-SP27 is a novel inhibitory PLC isoform expressed endogenously in human pancreas and upregulated in CFPAC-1 cells. The present study investigated the effect of NYD-SP27 down-regulation on the ATP-stimulated and Ca(2+)-dependent pancreatic anion secretion by CFPAC-1 cell line using short-circuit current (I(SC)) recording. NYD-SP27 antisense-transfected CFPAC-1 (AT-CF) cells exhibited a significantly higher basal transmembrane potential difference and current than those of empty vector-transfected CFPAC-1 (VT-CF) cells. Cl(-) channel blocker, DPC or Glibenclamide (1mM), and inhibitor of Na(+)-K(+)-Cl(-) cotransporter, bumetanide (100 microM) significantly inhibited the basal current in AT-CF cells. The inhibitor of adenylate cyclase, MDL12330A (20 microM), and Ca(2+)-dependent Cl(-) channel (CaCC) blocker, DIDS (100 microM) also significantly reduced the basal current in AT-CF. Apical application of ATP (10 microM) stimulated a fast transient I(SC) increase in VT-CF cells, but a more sustained rise with slower decline in AT-CF cells. Pretreatment with BAPTA-AM (50 microM) reduced the ATP-induced I(SC) response in AT-CF cells by 77.9%. PMA (1 microM), a PKC activator, inhibited the ATP-stimulated current increase (the transient peak) in VT-CF cells, but had no effect on the AT-CF cells. However, PKC inhibitor, staurosporine (40 microM) could inhibit the ATP-induced I(SC) response in AT-CF cells. The present results confirm the previously proposed inhibitory role of NYD-SP27 in the PLC pathway and demonstrate that the suppression of its expression could result in an enhancement of ATP-stimulated Ca(2+) dependent pancreatic anion secretion.
我们之前的研究表明，NYD-SP27是一种新型的抑制性磷脂酶C同工型，在人胰腺中内源性表达，在CFPAC-1细胞中上调。本研究使用短路电流（I(SC)）记录法，研究了NYD-SP27下调对CFPAC-1细胞系中ATP刺激的和Ca(2+)依赖性胰腺阴离子分泌的影响。NYD-SP27反义转染的CFPAC-1（AT-CF）细胞表现出比空载体转染的CFPAC-1（VT-CF）细胞显著更高的基础跨膜电位差和电流。Cl(-)通道阻滞剂DPC或格列本脲（1mM）以及Na(+)-K(+)-Cl(-)协同转运体抑制剂布美他尼（100 microM）显著抑制了AT-CF细胞中的基础电流。腺苷酸环化酶抑制剂MDL12330A（20 microM）和Ca(2+)依赖性Cl(-)通道（CaCC）阻滞剂DIDS（100 microM）也显著降低了AT-CF中的基础电流。在顶端施加ATP（10 microM）刺激VT-CF细胞中快速短暂的I(SC)增加，但在AT-CF细胞中增加更持久且下降更缓慢。用BAPTA-AM（50 microM）预处理使AT-CF细胞中ATP诱导的I(SC)反应降低了77.9%。PKC激活剂PMA（1 microM）抑制了VT-CF细胞中ATP刺激的电流增加（瞬态峰值），但对AT-CF细胞没有影响。然而，PKC抑制剂星形孢菌素（40 microM）可以抑制AT-CF细胞中ATP诱导的I(SC)反应。目前的结果证实了之前提出的NYD-SP27在PLC途径中的抑制作用，并表明其表达的抑制可能导致ATP刺激的Ca(2+)依赖性胰腺阴离子分泌增强。
