Provost J J, Olmschenk S M, Metcalf A L, Korpi N, Thronson H, Liu M, Wallert M A
Department of Biology, Minnesota State University Moorhead, 56563, USA.
Biochem Cell Biol. 2005 Apr;83(2):123-32. doi: 10.1139/o04-132.
The activation of the Na+-H+ exchanger 1 (NHE1) and extracellular-signal regulated kinase (ERK) phosphorylation in Chinese hamster lung fibroblasts (CCL39) was characterized in response to the specific alpha1-adrenergic agonist, phenylephrine (PE). Addition of 100 micromol PE/L increased the steady-state intracellular pH (pHi) by 0.16 +/- 0.03 pH units, as well as increasing the phosphorylation of ERK. The response of NHE1 to PE in CCL39 cells was determined by the use of specific antagonists. Use of 2 specific chemical inhibitors of phosphoinositide-specific phospholipase C (PLC) reduced the ability of PE to activate either the exchanger or ERK. Studies were conducted in PLCbeta-deficient cell lines derived from parental CCL39 cells. NHE1 activity in both mutant cell lines was increased in response to phorbal esters or lysophosphatidic acid, whereas the addition of PE only caused a minimal change in either pHi or ERK phosphorylation. These results, combined with reconstitution experiments with exogenously expressed PLCbeta1, PLCbeta2, or PLCbeta3, revealed that stimulation of NHE1 activity by PE in CCL39 cells is a PLCbeta1-coupled event. Furthermore, the data indicate that alpha1-adrenergic signaling of PLCbeta is upstream of ERK activation. These data demonstrate that PLCbeta1 is primarily involved in the activation of NHE1 in CCL39 fibroblasts.
在中国仓鼠肺成纤维细胞（CCL39）中，研究了特异性α1-肾上腺素能激动剂去氧肾上腺素（PE）对钠氢交换体1（NHE1）激活及细胞外信号调节激酶（ERK）磷酸化的影响。添加100 μmol/L的PE可使细胞内pH稳态（pHi）升高0.16±0.03个pH单位，并增加ERK的磷酸化。通过使用特异性拮抗剂来确定CCL39细胞中NHE1对PE的反应。使用2种磷酸肌醇特异性磷脂酶C（PLC）的特异性化学抑制剂可降低PE激活交换体或ERK的能力。在源自亲代CCL39细胞的PLCβ缺陷细胞系中进行了研究。两种突变细胞系中的NHE1活性在佛波酯或溶血磷脂酸刺激下均增加，而添加PE仅引起pHi或ERK磷酸化的最小变化。这些结果与外源性表达PLCβ1、PLCβ2或PLCβ3的重组实验相结合，表明PE对CCL39细胞中NHE1活性的刺激是一种与PLCβ1偶联的事件。此外，数据表明PLCβ的α1-肾上腺素能信号传导在ERK激活的上游。这些数据证明PLCβ1主要参与CCL39成纤维细胞中NHE1的激活。
