Kopelman H, Ferretti E, Gauthier C, Goodyer P R
Department of Pediatrics, McGill University-Montreal Children's Hospital Research Institute, Quebec, Canada.
Am J Physiol. 1995 Sep;269(3 Pt 1):C626-31. doi: 10.1152/ajpcell.1995.269.3.C626.
Cystic fibrosis transmembrane conductance regulator (CFTR) is responsible for adenosine 3',5'-cyclic monophosphate (cAMP)-activated chloride transport in epithelial cells. Isolated rabbit pancreatic acini possess a cAMP-activated chloride efflux mechanism distinct from zymogen granule secretion. To determine whether CFTR is expressed in acini, we used polymerase chain reaction (PCR) to amplify a 480-base pair (bp) sequence from reverse-transcribed rabbit acinar RNA. The PCR product was consistent with a 480-bp band amplified in T84 cells, and its sequence was > 90% homologous to human CFTR. CFTR antibody M3A7 recognized a 180- and a 160-kDa protein from acinar membranes consistent with bands seen in Chinese hamster ovary (CHO) cells transfected with CFTR. To determine if CFTR was responsible for the cAMP-activated chloride efflux previously demonstrated in pancreatic acini, we incubated acinar cells for 20 h with 1.75 microM CFTR antisense or sense oligodeoxynucleotide. Chloride efflux, in response to 8-bromoadenosine 3',5'-cyclic monophosphate and phorbol ester but not to calcium ionophore, was selectively inhibited by CFTR antisense oligodeoxynucleotide. Antisense oligodeoxynucleotide did not inhibit acinar amylase secretion. These findings indicate that isolated pancreatic acini can be used for future studies of CFTR expression and function.
囊性纤维化跨膜传导调节因子（CFTR）负责上皮细胞中由3'，5'-环磷酸腺苷（cAMP）激活的氯离子转运。分离的兔胰腺腺泡具有一种与酶原颗粒分泌不同的cAMP激活的氯离子外流机制。为了确定CFTR是否在腺泡中表达，我们使用聚合酶链反应（PCR）从逆转录的兔腺泡RNA中扩增出一段480个碱基对（bp）的序列。该PCR产物与在T84细胞中扩增出的480-bp条带一致，其序列与人类CFTR的同源性>90%。CFTR抗体M3A7识别出腺泡膜上的一种180-kDa和一种160-kDa的蛋白质，这与用CFTR转染的中国仓鼠卵巢（CHO）细胞中看到的条带一致。为了确定CFTR是否负责先前在胰腺腺泡中证明的cAMP激活的氯离子外流，我们用1.75 microM的CFTR反义或正义寡脱氧核苷酸将腺泡细胞孵育20小时。CFTR反义寡脱氧核苷酸选择性地抑制了对8-溴-3'，5'-环磷酸腺苷和佛波酯的氯离子外流反应，但不抑制对钙离子载体A23187的反应。反义寡脱氧核苷酸不抑制腺泡淀粉酶的分泌。这些发现表明，分离的胰腺腺泡可用于未来对CFTR表达和功能的研究。
