Expression of apical membrane L-glutamate transporters in neonatal porcine epithelial cells along the small intestinal crypt-villus axis
Expression of apical membrane L-glutamate transporters in neonatal porcine epithelial cells along the small intestinal crypt-villus axis
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DOI：
10.1152/ajpgi.00232.2003
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发表时间：
2004-08-01
影响因子：
4.5
通讯作者：
Burrin, DG
中科院分区：
文献类型：
--
作者：
Fan, MZ;Matthews, JC;Burrin, DG
肠内L-谷氨酸被新生儿的肠粘膜广泛用作氧化燃料。为了确定主要的摄取途径，并了解摄取调节，我们研究了转运动力学和顶端膜L-谷氨酸转运蛋白的上皮细胞顺序分离沿着小肠隐窝绒毛轴从牛奶蛋白喂养，16日龄猪的分子身份。采用扩张肠囊法从小肠绒毛顶端到底部连续分离出12个细胞组分。L-谷氨酸摄取的初始速率和动力学用L-[G-H-3]谷氨酸通过在顶端膜囊泡中快速过滤来测量，所述顶端膜囊泡通过Mg 2+沉淀和差速离心制备，膜电位由SCN-钳位。初始L-谷氨酸摄取结果表明存在B-o和X-AG（-）转运系统，但X-AG（-）系统主要用于跨顶膜摄取。动力学数据表明，L-谷氨酸通过X-AG（-）系统的摄取与更高的最大转运活性，但较低的转运蛋白亲和力在隐窝细胞比在绒毛细胞。基于免疫印迹和RT-PCR分析，X-AG（-）谷氨酸转运蛋白的分子身份主要是确定的兴奋性氨基酸载体（EAAC）-1。EAAC-1表达随着细胞分化而增加，并在转录和翻译水平上从隐窝到上绒毛细胞进行调节。总之，通过改变X-AG（-）系统转运蛋白基因EAAC-1在转录和翻译水平的表达以及新生儿中沿肠隐窝-绒毛轴沿着的最大摄取活性和转运蛋白亲和力来调节跨顶膜的腔L-谷氨酸摄取的效率和能力。
Enteral L-glutamate is extensively utilized as an oxidative fuel by the gut mucosa in the neonate. To identify major uptake pathways and to understand uptake regulation, we examined transport kinetics and molecular identities of apical membrane L-glutamate transporters in epithelial cells sequentially isolated along the small intestinal crypt-villus axis from milk protein-fed, 16-day-old pigs. The distended intestinal sac method was used to isolate 12 sequential cell fractions from the tip villus to the bottom crypt. Initial rates and kinetics of L-glutamate uptake were measured with L-[G-H-3]glutamate by fast filtration in apical membrane vesicles prepared by Mg2+ precipitation and differential centrifugation, with membrane potential clamped by SCN-. Initial L- glutamate uptake results suggested the presence of B-o and X-AG(-) transport systems, but the X-AG(-) system was predominant for uptake across the apical membrane. Kinetic data suggested that L- glutamate uptake through the X-AG(-) system was associated with higher maximal transport activity but lower transporter affinity in crypt than in villus cells. Molecular identity of the X-AG(-) glutamate transporter, based on immunoblot and RT-PCR analysis, was primarily the defined excitatory amino acid carrier (EAAC)-1. EAAC-1 expression was increased with cell differentiation and regulated at transcription and translation levels from crypt to upper villus cells. In conclusion, efficiency and capacity of luminal L- glutamate uptake across the apical membrane are regulated by changing expression of the X-AG(-) system transporter gene EAAC-1 at transcription and translation levels as well as maximal uptake activity and transporter affinity along the intestinal crypt-villus axis in the neonate.