Rapid turnover of the "functional" Na+-Ca2+ exchanger in cardiac myocytes revealed by an antisense oligodeoxynucleotide approach
Rapid turnover of the "functional" Na+-Ca2+ exchanger in cardiac myocytes revealed by an antisense oligodeoxynucleotide approach
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DOI：
10.1016/j.ceca.2004.10.006
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发表时间：
2005-03-01
期刊：
影响因子：
4
通讯作者：
Schwaller, B
中科院分区：
文献类型：
--
作者：
Egger, M;Porzig, H;Schwaller, B
将反义寡脱氧核苷酸（AS-ODNs）与心脏Na+-Ca2+交换器（NCXI）的瞬时功能表达联合使用，将Na+-Ca2+交换功能的抑制与NCXI蛋白表达的下调联系起来。在denovo表达系统（Sf9细胞）中，应用AS-ODN后NCXI mRNA和蛋白的降低与NCXI活性的降低是平行的，这是真正的“反义效应”的典型标志。虽然AS-ODN摄取在大鼠新生心肌细胞中也很有效，但在用AS-ODNs处理的这些细胞的全细胞提取物中，尽管NCXI功能迅速丧失，但定量结合试验中测定的NCXI蛋白量几乎保持不变。肌细胞的免疫细胞化学染色显示，大部分免疫反应性不局限于质膜，而是在细胞内，几乎不受AS-ODN治疗的影响。这些结果表明NCXI蛋白在新生儿心肌细胞质膜中的“功能半衰期”惊人地短，远短于其他膜蛋白约30小时的半衰期。(C) 2004 Elsevier Ltd.版权所有。
Antisense oligodeoxynucleotides (AS-ODNs) were used in combination with transient functional expression of the cardiac Na+-Ca2+ exchanger (NCXI) to correlate suppression of the Na+-Ca2+ exchange function with down-regulation of NCXI protein expression. In a denovo expression system (Sf9 cells), a decrease in both, NCXI mRNA and protein after AS-ODN application was paralleled by diminished NCXI activity, a typical hallmark of a true "antisense effect". Although AS-ODN uptake was also efficient in rat neonatal cardiac myocytes, in whole-cell extracts of these cells treated with AS-ODNs, the amount of NCXI protein determined in a quantitative binding assay remained almost unchanged, despite a prompt loss of NCXI function. Immunocytochemical staining of myocytes revealed that most of the immunoreactivity was not localized in the plasma membrane, but in intracellular compartments and was barely affected by AS-ODN treatment. These results indicate that the "functional half-life" of the NCXI protein in the plasma membrane of neonatal cardiac myocytes is surprisingly short, much shorter than reported half-lifes of about 30 h for other membrane proteins. (C) 2004 Elsevier Ltd. All rights reserved.