Fink Aaron S, Wang Yuanhong, Mendez Tatiana, Worrell Roger T, Eaton Douglas, Nguyen Toan D, Lee Sum P
Department of Surgery, Atlanta VAMC and Emory University, Atlanta VAMC, Atlanta, Georgia 30033, USA.
Pancreas. 2002 Oct;25(3):290-5. doi: 10.1097/00006676-200210000-00012.
Calcium-activated chloride conductance has been identified in normal pancreatic duct cells. Recent in vitro evidence suggests that angiotensin II (AngII) stimulates pancreatic secretion in both cystic fibrosis (CFPAC) and transformed pancreatic cells.
To investigate calcium-mediated stimulatory effects of AngII in both nontransformed dog pancreatic duct epithelial (DPDE) and CFPAC cells.
Western blots were performed in both cells seeking AngII receptors. In additional studies, DPDE and CFPAC cells were grown on vitrogen-coated glass cover slips and loaded with Indo-1-AM dye. Cells were placed in a confocal microscope's perfusion chamber and perfused with 100 microM AngII or ATP (control). Cells were excited with UV light, and intracellular calcium ([Ca+2]i) was read using fluorescence emission at 405 and 530 nm. Finally, single channels in the DPDE cells were examined using cell-attached patch clamps. Current amplitude histograms provided estimates of the conductance and open probability of channels.
Western blots demonstrated presence of both AT and AT AngII receptors in DPDE and CFPAC cells; the density of AT receptors appeared lower than that of AT receptors. Basal intracellular calcium concentrations did not differ between DPDE (109 +/- 11 nM) and CFPAC (103 +/- 8 nM) cells. AngII significantly increased measured intracellular calcium concentrations in both DPDE (909 +/- 98 nM) and CFPAC (879 +/- 207 nM) cells, as did ATP (DPDE = 1722 +/- 228 nM; CFPAC = 1522 +/- 245 nM). In the patch clamp studies, a variety of different channels were observed; they appeared to be an 11pS nonselective cation (NSC) channel, a 4.6pS Na+ channel, a 3pS anion channel, and an 8pS chloride channel. The latter channel had characteristics similar to cystic fibrosis transmembrane conductance regulator (CFTR). Apical or basolateral application of AngII activated both the 11pS NSC and the 3pS channels.
In nontransformed DPDE and CFPAC cells, specific AngII receptors mediate increases in [Ca ]. The latter effect of AngII may elicit activation of calcium-mediated chloride channels, suggesting a role for AngII as an alternative mediator of pancreatic ductal secretion.
在正常胰腺导管细胞中已发现钙激活氯电导。最近的体外证据表明，血管紧张素II（AngII）可刺激囊性纤维化（CFPAC）和转化胰腺细胞中的胰腺分泌。
研究AngII在未转化的犬胰腺导管上皮（DPDE）细胞和CFPAC细胞中钙介导的刺激作用。
对两种细胞进行蛋白质免疫印迹以寻找AngII受体。在其他研究中，将DPDE细胞和CFPAC细胞培养在涂有vitrogen的玻璃盖玻片上，并加载Indo-1-AM染料。将细胞置于共聚焦显微镜的灌注室中，并用100微摩尔/升的AngII或ATP（对照）进行灌注。用紫外光激发细胞，并使用405和530纳米处的荧光发射读取细胞内钙（[Ca+2]i）。最后，使用细胞贴附式膜片钳检查DPDE细胞中的单通道。电流幅度直方图提供了通道电导和开放概率的估计值。
蛋白质免疫印迹显示DPDE细胞和CFPAC细胞中均存在AT1和AT2 AngII受体；AT1受体的密度似乎低于AT2受体。DPDE细胞（109±11纳摩尔）和CFPAC细胞（103±8纳摩尔）的基础细胞内钙浓度没有差异。AngII显著增加了DPDE细胞（909±98纳摩尔）和CFPAC细胞（879±207纳摩尔）中测得的细胞内钙浓度，ATP也有同样的作用（DPDE = 1722±228纳摩尔；CFPAC = 1522±245纳摩尔）。在膜片钳研究中，观察到了多种不同的通道；它们似乎是一个11皮西门子非选择性阳离子（NSC）通道、一个4.6皮西门子钠离子通道、一个3皮西门子阴离子通道和一个8皮西门子氯离子通道。后一种通道具有与囊性纤维化跨膜电导调节因子（CFTR）相似的特征。顶端或基底外侧应用AngII可激活11皮西门子NSC通道和3皮西门子通道。
在未转化的DPDE细胞和CFPAC细胞中，特定的AngII受体介导[Ca]的增加。AngII的后一种作用可能引发钙介导的氯通道的激活，表明AngII作为胰腺导管分泌的替代介质发挥作用。
