Perry Clint, Baker Olga J, Reyland Mary E, Grichtchenko Irina I
Department of Physiology and Biophysics, University of Colorado Denver, Aurora, 80045, USA.
Am J Physiol Cell Physiol. 2009 Dec;297(6):C1409-23. doi: 10.1152/ajpcell.00028.2009. Epub 2009 Sep 25.
We examined membrane trafficking of NBCe1-A and NBCe1-B variants of the electrogenic Na(+)-HCO(3)(-) cotransporter (NBCe1) encoded by the SLC4A4 gene, using confocal fluorescent microscopy in rat parotid acinar cells (ParC5 and ParC10). We showed that yellow fluorescent protein (YFP)-tagged NBCe1-A and green fluorescent protein (GFP)-tagged NBCe1-B are colocalized with E-cadherin in the basolateral membrane (BLM) but not with the apical membrane marker zona occludens 1 (ZO-1). We inhibited constitutive recycling with monensin and W13 and detected that NBCe1-A and NBCe1-B accumulated in vesicles marked with the early endosomal marker early endosome antigen-1 (EEA1), with a parallel loss from the BLM. We observed that NBCe1-A and NBCe1-B undergo massive carbachol (CCh)-stimulated redistribution from the BLM into early endosomes. We showed that internalization of NBCe1-A and NBCe1-B was prevented by the general PKC inhibitor GF-109203X, the PKCalphabetagamma-specific inhibitor Gö-6976, and the PKCdelta-specific inhibitor rottlerin. We verified the involvement of PKCdelta by blocking CCh-induced internalization of NBCe1-A-cyan fluorescent protein (CFP) in cells transfected with dominant-negative kinase-dead (Lys376Arg) PKCdelta-GFP. Our data suggest that NBCe1-A and NBCe1-B undergo constitutive and CCh-stimulated endocytosis regulated by conventional PKCs (PKCalphabetagamma) and by novel PKCdelta in rat epithelial cells. To help develop a more complete model of the role of NBCe1 in parotid acinar cells we also investigated the initial phase of the secretory response to cholinergic agonist. In an Ussing chamber study we showed that inhibition of basolateral NBCe1 with 5-chloro-2,3-dihydro-3-(hydroxy-2-thienylmethylene)-2-oxo-1H-indole-1-carboxamide (tenidap) significantly decreases an initial phase of luminal anion secretion measured as a transient short-circuit current (I(sc)) across ParC10 cell monolayers. Using trafficking and functional data we propose a model that describes a physiological role of NBC in salivary acinar cell secretion.
我们在大鼠腮腺腺泡细胞（ParC5和ParC10）中运用共聚焦荧光显微镜，研究了由SLC4A4基因编码的电中性Na(+)-HCO(3)(-)协同转运蛋白（NBCe1）的NBCe1-A和NBCe1-B变体的膜运输情况。我们发现，黄色荧光蛋白（YFP）标记的NBCe1-A和绿色荧光蛋白（GFP）标记的NBCe1-B与E-钙黏蛋白在基底外侧膜（BLM）中共定位，但不与顶端膜标记物闭合蛋白1（ZO-1）共定位。我们用莫能菌素和W13抑制组成型循环，并检测到NBCe1-A和NBCe1-B在标记有早期内体标记物早期内体抗原-1（EEA1）的囊泡中积累，同时从BLM中平行减少。我们观察到，NBCe1-A和NBCe1-B在卡巴胆碱（CCh）刺激下大量从BLM重新分布到早期内体中。我们发现，一般的蛋白激酶C（PKC）抑制剂GF-109203X、PKCalphabetagamma特异性抑制剂Gö-6976和PKCdelta特异性抑制剂rottlerin可阻止NBCe1-A和NBCe1-B的内化。我们通过在转染了显性负性激酶失活（Lys376Arg）PKCdelta-GFP的细胞中阻断CCh诱导的NBCe1-A-青色荧光蛋白（CFP）内化，验证了PKCdelta的参与。我们的数据表明，在大鼠上皮细胞中，NBCe1-A和NBCe1-B经历由传统PKCs（PKCalphabetagamma）和新型PKCdelta调节的组成型和CCh刺激的内吞作用。为了帮助建立一个更完整的NBCe1在腮腺腺泡细胞中作用的模型，我们还研究了对胆碱能激动剂分泌反应的初始阶段。在Ussing室研究中，我们发现用5-氯-2,3-二氢-3-(羟基-2-噻吩基亚甲基)-2-氧代-1H-吲哚-1-甲酰胺（替硝唑）抑制基底外侧NBCe1，可显著降低以跨ParC10细胞单层的瞬时短路电流（I(sc)）衡量的管腔阴离子分泌的初始阶段。利用运输和功能数据，我们提出了一个描述NBC在唾液腺泡细胞分泌中生理作用的模型。
